Micropropagation Methods in Temporary Immersion Systems (Methods in Molecular Biology, 2759) 1071636537, 9781071636534

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Methods in Molecular Biology 2759

Marco A. Ramírez-Mosqueda Carlos A. Cruz-Cruz  Editors

Micropropagation Methods in Temporary Immersion Systems

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-bystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

Micropropagation Methods in Temporary Immersion Systems Edited by

Marco A. Ramírez-Mosqueda Unidad Guerrero Negro, Centro de Investigaciones Biológicas del Noroeste S.C. (CIBNOR), Guerrero Negro, Baja California Sur, Mexico

Carlos A. Cruz-Cruz Facultad de Ciencias Químicas, Universidad Veracruzana, Orizaba, Veracruz, Mexico

Editors Marco A. Ramı´rez-Mosqueda Unidad Guerrero Negro Centro de Investigaciones Biolo´gicas del Noroeste S.C. (CIBNOR) Guerrero Negro, Baja California Sur, Mexico

Carlos A. Cruz-Cruz Facultad de Ciencias Quı´micas Universidad Veracruzana Orizaba, Veracruz, Mexico

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-3653-4 ISBN 978-1-0716-3654-1 (eBook) https://doi.org/10.1007/978-1-0716-3654-1 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A. Paper in this product is recyclable.

Preface This book presents the information necessary for the commercial micropropagation of plants. Currently, there are innovations in in vitro culture systems, including the use of temporary immersion systems (TIS). These systems allow the semi-automation and reduction of production costs during plant micropropagation; they are also considered to increase the biological performance and vigor of the plants. The search for an efficient protocol for commercial micropropagation of plant species in TIS requires long investigations that sometimes do not guarantee success in the process. This book seeks to recapitulate the efforts and main micropropagation protocols in different types of TIS already established in plant species with agricultural, medicinal, ornamental, and forestry interest, detailing the type of bioreactor and the optimal parameters necessary to guarantee obtaining the largest number of commercial propagules. Guerrero Negro, Baja California Sur, Mexico Orizaba, Veracruz, Mexico

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Marco A. Ramı´rez-Mosqueda Carlos A. Cruz-Cruz

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PART I

TEMPORARY IMMERSION SYSTEM DESIGNS AND APPLICATIONS

1 Temporary Immersion Systems in Plant Micropropagation . . . . . . . . . . . . . . . . . . . Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz 2 Types of Temporary Immersion Systems Used in Commercial Plant Micropropagation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ivonne N. Bravo-Ruı´z, Ma. Teresa Gonza´lez-Arnao, Fabiola Herna´ndez-Ramı´rez, Jaime Lo pez-Domı´nguez, and Carlos A. Cruz-Cruz 3 Use of Temporary Immersion Systems in the Establishment of Biofactories . . . . Marco Vinicio Rodrı´guez-Deme´neghi

PART II

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MICROPROPAGATION IN TIS OF AGROINDUSTRIAL SPECIES: PROTOCOLS

4 Large-Scale Micropropagation of Vanilla (Vanilla planifolia Jacks.) in a Temporary Immersion Bioreactor (TIB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Marco A. Ramı´rez-Mosqueda, Marco Vinicio Rodrı´guez-Deme´neghi, Heidi P. Medorio-Garcı´a, and Rube´n H. Andueza-Noh 5 Temporary Immersion Bioreactors for Sugarcane Multiplication and Rooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jose´ Luis Spinoso-Castillo, Marı´a Karen Serrano-Fuentes, Monserrat Sorcia-Morales, and Jerico Jabı´n Bello-Bello 6 Micropropagation of Stevia (Stevia rebaudiana Bert.) in RITA® . . . . . . . . . . . . . . Heidi P. Medorio-Garcı´a, Elizabeta Herna´ndez-Domı´nguez, Rube´n H. Andueza-Noh, David Rau´l Lo pez-Aguilar, and Marco A. Ramı´rez-Mosqueda 7 In Vitro Multiplication of Agave (A. marmorata and A. potatorum) by Temporary Immersion in SETIS™ Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . ´ lvarez, Marı´a del Rosario Moreno-Herna´ndez, Eucario Mancilla-A Daniel Aguilar-Jime´nez, and Jerico Jabı´n Bello-Bello 8 BioMINT: A Temporary Immersion System for Agave Micropropagation. . . . . . ´ ngel Herrera-Alamillo, Kelly M. Monja-Mio, Gabriel Ojeda, Miguel A Lorenzo Felipe Sa´nchez-Teyer, and Antonio Rescalvo-Morales 9 Plant Regeneration of Agave cupreata by Somatic Embryogenesis in a Temporary Immersion System with Silver Nanoparticles . . . . . . . . . . . . . . . . . Sandra Y. Martı´nez-Martı´nez, Amaury M. Arzate-Ferna´ndez, and Marı´a G. Gonza´les-Pedroza

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Contents

Micropropagation of Chayote (Sechium edule L.) var. virens levis in RITA® . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ˜ ez-Pastrana, Lizandro Ramı´rez-Trejo, Rosalı´a Nu´n and Anell Soto-Contreras

PART III 11

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MICROPROPAGATION IN TIS OF ORNAMENTAL SPECIES: PROTOCOLS

Direct Shoot From Root and True-to-Type Micropropagation of Limonium “Misty Blue” in Partially Immersed Culture on an Aluminum Mesh Raft . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Priyanka Raha, Gourab Saha, Ishita Khatua, and Tapas Kumar Bandyopadhyay Increased Multiplication Rates of Vriesea hieroglyphica (Carriere) E. Morren Through a Temporary Immersion System. . . . . . . . . . . . . . . . . . . . . . . . Carolina Rossi de Oliveira, Alice Noemi Aranda-Peres, Leonardo Soriano, Paulo Hercı´lio Viegas Rodrigues, and Adriana Pinheiro Martinelli Micropropagation of Encyclia cordigera (Kunth) Dressler in Ebb-and-Flow Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Obdulia Baltazar-Bernal and Evelia Guadalupe Mora-Gonza´lez Micropropagation of Guarianthe skinneri (Bateman) Dressler & W. E. Higging in Temporary Immersion Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . Elizabeta Herna´ndez-Domı´nguez, David Rau´l Lopez-Aguilar, ˜ o-Cruz, Pedro Zetina-Cordoba, Andre´s Ordun and Marco A. Ramı´rez-Mosqueda Alstroemeria Micropropagation in a RITA® Temporary Immersion System . . . . Lilia Castro Pereira, Leonardo Soriano, Carolina Rossi de Oliveira, Paulo Hercı´lio Viegas Rodrigues, and Adriana Pinheiro Martinelli

PART IV

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MICROPROPAGATION IN TIS OF WOODY SPECIES: PROTOCOLS

Proliferation of Axillary Shoots of Chestnut in Temporary Immersion Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Nieves Vidal, Conchi Sa´nchez, and Beatriz Cuenca 17 In Vitro Propagation of Plants via Organogenesis in Bambusa vulgaris Schrad. ex Wendl Using Temporary Immersion Systems . . . . . . . . . . . . . . . . . . . . . 183 Yudith Garcı´a-Ramı´rez, Mallelyn Gonza´lez-Gonza´lez, Marisol Freire-Seijo, Rau´l Barbon-Rodrı´guez, and Sinesio Torres-Garcı´a 18 Multiplication of Cupressus guadalupensis Using the RITA® Temporary Immersion System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193 Luis Alberto Go mez-Reyes, Esmeralda Judith Cruz-Gutie´rrez, Lorena Jacqueline Gomez-Godı´nez, Manuel de Jesu´s Bermu´dez-Guzma´n, Claudia Berenice Espitia-Flores, and Juan Manuel Pichardo Gonza´lez

Contents

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Secondary Embryogenesis of Linaloe in Temporary Immersion Bioreactor-Type RITA® . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Gregorio Arellano-Ostoa, Monica Gonza´lez-Orozco, Izaac Va´zquez-Cisneros, and Sandra Mitchelle Arellano-Gonza´lez

PART V

REVIEWS OF THE USE OF TEMPORARY IMMERSION SYSTEMS IN MICROPROPAGATION

Temporary Immersion System for Biomass Production of Salvia spp.: A Mini-Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 Yudith Garcı´a-Ramı´rez 21 Orchid Micropropagation Using Temporary Immersion Systems: A Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 Obdulia Baltazar-Bernal, Evelia Guadalupe Mora-Gonza´lez, and Marco A. Ramı´rez-Mosqueda

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PART VI 22

GENERAL CONCLUSIONS AND PERSPECTIVES

Conclusions and Perspectives on Plant Micropropagation in Temporary Immersion Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors DANIEL AGUILAR-JIME´NEZ • Educational Program of Agrobiotechnology, Technological University of Izucar de Matamoros, Izucar de Matamoros, Puebla, Mexico RUBE´N H. ANDUEZA-NOH • CONACYT-Instituto Tecnologico de Conkal, Conkal, Yucata´n, Mexico ALICE NOEMI ARANDA-PERES • Center for Nuclear Energy in Agriculture – Plant Biotechnology Lab, University of Sa˜o Paulo, Piracicaba, SP, Brazil ´ rea de Recursos SANDRA MITCHELLE ARELLANO-GONZA´LEZ • Departamento de Suelos, A Naturales Renovables, Universidad Autonoma Chapingo, Texcoco, State of Mexico, Mexico GREGORIO ARELLANO-OSTOA • Postgrado en Recursos Gene´ticos y Productividad – Fruticultura, Campus Montecillo, Colegio de Postgraduados, Texcoco, State of Mexico, Mexico AMAURY M. ARZATE-FERNA´NDEZ • Center for Research and Advanced Studies in Plant Breeding, Faculty of Agricultural Sciences, UAEMe´x, Toluca, State of Mexico, Mexico OBDULIA BALTAZAR-BERNAL • Laboratorio de Cultivo de Tejidos Vegetales, Colegio de Postgraduados Campus Cordoba, Amatla´n de los Reyes, Veracruz, Mexico TAPAS KUMAR BANDYOPADHYAY • Department of Molecular Biology and Biotechnology, Faculty of Science, University of Kalyani, Kalyani, West Bengal, India RAU´L BARBO´N-RODRI´GUEZ • Instituto de Biotecnologı´a de las Plantas, Universidad Central “Marta Abreu” de Las Villas, Santa Clara, Cuba JERICO´ JABI´N BELLO-BELLO • CONAHCYT-Postgraduate College-Campus Cordoba, Amatlan de los Reyes, Veracruz, Mexico MANUEL DE JESU´S BERMU´DEZ-GUZMA´N • Laboratorio de Biotecnologı´a, Campo Experimental Tecoma´n, Instituto Nacional de Investigaciones Forestales Agrı´colas y Pecuarias (INIFAP), Tecoman, Colima, Mexico IVONNE N. BRAVO-RUI´Z • Facultad de Ciencias Quı´micas, Universidad Veracruzana, Orizaba, Veracruz, Mexico CARLOS A. CRUZ-CRUZ • Facultad de Ciencias Quı´micas, Universidad Veracruzana, Orizaba, Veracruz, Mexico ESMERALDA JUDITH CRUZ-GUTIE´RREZ • Laboratorio Agrı´cola-Forestal, Centro Nacional de Recursos Gene´ticos, Instituto Nacional de Investigaciones Forestales Agrı´colas y Pecuarias (INIFAP), Tepatitlan de Morelos, Jalisco, Mexico BEATRIZ CUENCA • Maceda Nursery, Grupo Tragsa, Maceda, Ourense, Spain CAROLINA ROSSI DE OLIVEIRA • Center for Nuclear Energy in Agriculture – Plant Biotechnology Lab., University of Sa˜o Paulo, Piracicaba, SP, Brazil MARI´A DEL ROSARIO MORENO-HERNA´NDEZ • Postgraduate College-Campus Cordoba, Amatlan de los Reyes, Veracruz, Mexico CLAUDIA BERENICE ESPITIA-FLORES • Posgrado de Biociencias, Universidad de Guadalajara – Centro Universitario Los Altos, Tepatitlan de Morelos, Jalisco, Mexico MARISOL FREIRE-SEIJO • Instituto de Biotecnologı´a de las Plantas, Universidad Central “Marta Abreu” de Las Villas, Santa Clara, Cuba YUDITH GARCI´A-RAMI´REZ • Instituto de Biotecnologı´a de las Plantas, Universidad Central “Marta Abreu” de Las Villas, Santa Clara, Cuba

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Contributors

LORENA JACQUELINE GO´MEZ-GODI´NEZ • Laboratorio Agrı´cola-Forestal, Centro Nacional de Recursos Gene´ticos, Instituto Nacional de Investigaciones Forestales Agrı´colas y Pecuarias (INIFAP), Tepatitlan de Morelos, Jalisco, Mexico LUIS ALBERTO GO´MEZ-REYES • Laboratorio Agrı´cola-Forestal, Centro Nacional de Recursos Gene´ticos, Instituto Nacional de Investigaciones Forestales Agrı´colas y Pecuarias (INIFAP), Tepatitlan de Morelos, Jalisco, Mexico MARI´A G. GONZA´LES-PEDROZA • Department of Biotechnology, Faculty of Sciences, UAEMe´x, Toluca, State of Mexico, Mexico JUAN MANUEL PICHARDO GONZA´LEZ • Laboratorio Agrı´cola-Forestal, Centro Nacional de Recursos Gene´ticos, Instituto Nacional de Investigaciones Forestales Agrı´colas y Pecuarias (INIFAP), Tepatitlan de Morelos, Jalisco, Mexico MA. TERESA GONZA´LEZ-ARNAO • Facultad de Ciencias Quı´micas, Universidad Veracruzana, Orizaba, Veracruz, Mexico MALLELYN GONZA´LEZ-GONZA´LEZ • Departamento de Agronomı´a, Facultad de Ciencias Agropecuarias, Universidad Central “Marta Abreu” de Las Villas, Santa Clara, Cuba ´ MONICA GONZA´LEZ-OROZCO • Postgrado en Ciencias Forestales, Campus Montecillo, Colegio de Postgraduados, Texcoco, State of Mexico, Mexico ELIZABETA HERNA´NDEZ-DOMI´NGUEZ • Tecnologico Nacional de Me´xico/Instituto Tecnologico Superior de Acayucan, Acayucan, Veracruz, Mexico FABIOLA HERNA´NDEZ-RAMI´REZ • Facultad de Ciencias Quı´micas, Universidad Veracruzana, Orizaba, Veracruz, Mexico ´ NGEL HERRERA-ALAMILLO • Unidad de Biotecnologı´a, Centro de Investigacion MIGUEL A Cientı´fica de Yucata´n A.C., Me´rida, Yucata´n, Mexico ISHITA KHATUA • Department of Molecular Biology and Biotechnology, Faculty of Science, University of Kalyani, Kalyani, West Bengal, India DAVID RAU´L LO´PEZ-AGUILAR • Centro de Investigaciones Biologicas del Noroeste S.C., Unidad Guerrero Negro, Guerrero Negro, Baja California Sur, Mexico JAIME LO´PEZ-DOMI´NGUEZ • Facultad de Ciencias Quı´micas, Universidad Veracruzana, Orizaba, Veracruz, Mexico ´ LVAREZ • Postgraduate College-Campus Cordoba, Amatlan de los EUCARIO MANCILLA-A Reyes, Veracruz, Mexico ADRIANA PINHEIRO MARTINELLI • Center for Nuclear Energy in Agriculture – Plant Biotechnology Lab, University of Sa˜o Paulo, Piracicaba, SP, Brazil SANDRA Y. MARTI´NEZ-MARTI´NEZ • Center for Research and Advanced Studies in Plant Breeding, Faculty of Agricultural Sciences, UAEMe´x, Toluca, State of Mexico, Mexico HEIDI P. MEDORIO-GARCI´A • Facultad de Ciencias Quı´micas, Universidad Veracruzana, Coatzacoalcos, Veracruz, Mexico KELLY M. MONJA-MIO • Unidad CICY en el Centro de Estudios e Investigacion en Biocultura, Agroecologı´a, Ambiente y Salud, Acapulco de Jua´rez, Guerrero, Mexico EVELIA GUADALUPE MORA-GONZA´LEZ • Universidad Autonoma Metropolitana, Unidad Iztapalapa, Vicentina, Iztapalapa, Mexico ROSALI´A NU´N˜EZ-PASTRANA • Facultad de Ciencias Biologicas y Agropecuarias, Universidad Veracruzana, Amatla´n de los Reyes, Veracruz, Mexico GABRIEL OJEDA • Grupo Especializado de Apoyo a la Biofa´brica, Direccion de Gestion Tecnologica, Centro de Investigacion Cientı´fica de Yucata´n A.C., Me´rida, Yucata´n, Mexico ANDRE´S ORDUN˜O-CRUZ • Centro de Investigaciones Biologicas del Noroeste S.C., Unidad Guerrero Negro, Guerrero Negro, Baja California Sur, Mexico

Contributors

xiii

LILIA CASTRO PEREIRA • Center for Nuclear Energy in Agriculture – Plant Biotechnology Lab., University of Sa˜o Paulo, Piracicaba, SP, Brazil PRIYANKA RAHA • Department of Molecular Biology and Biotechnology, Faculty of Science, University of Kalyani, Kalyani, West Bengal, India MARCO A. RAMI´REZ-MOSQUEDA • Centro de Investigaciones Biologicas del Noroeste S.C., Unidad Guerrero Negro, Guerrero Negro, Baja California Sur, Mexico LIZANDRO RAMI´REZ-TREJO • Facultad de Ciencias Biologicas y Agropecuarias, Universidad Veracruzana, Amatla´n de los Reyes, Veracruz, Mexico ANTONIO RESCALVO-MORALES • Unidad CICY en el Centro de Estudios e Investigacion en Biocultura, Agroecologı´a, Ambiente y Salud, Acapulco de Jua´rez, Guerrero, Mexico PAULO HERCI´LIO VIEGAS RODRIGUES • Crop Science Departament – Tissue Culture and Ornamental Plants Lab, University of Sa˜o Paulo, Piracicaba, SP, Brazil MARCO VINICIO RODRI´GUEZ-DEME´NEGHI • Facultad de Ciencias Biologicas y Agropecuarias, Universidad Veracruzana, Amatla´n de los Reyes, Veracruz, Mexico GOURAB SAHA • Department of Molecular Biology and Biotechnology, Faculty of Science, University of Kalyani, Kalyani, West Bengal, India CONCHI SA´NCHEZ • Department of Plant Production, Mision Biologica de Galicia, Sede Santiago de Compostela, CSIC, Santiago de Compostela, Spain LORENZO FELIPE SA´NCHEZ-TEYER • Unidad de Biotecnologı´a, Centro de Investigacion Cientı´fica de Yucata´n A.C., Me´rida, Yucata´n, Mexico MARI´A KAREN SERRANO-FUENTES • Postgraduate College-Campus Cordoba, Amatlan de los Reyes, Veracruz, Mexico MONSERRAT SORCIA-MORALES • Sustainable and Protected Agriculture, Technological University of the Center of Veracruz, Cuitlahuac, Veracruz, Mexico LEONARDO SORIANO • Center for Nuclear Energy in Agriculture – Plant Biotechnology Lab, University of Sa˜o Paulo, Piracicaba, SP, Brazil ANELL SOTO-CONTRERAS • Facultad de Ciencias Biologicas y Agropecuarias, Universidad Veracruzana, Amatla´n de los Reyes, Veracruz, Mexico JOSE´ LUIS SPINOSO-CASTILLO • Postgraduate College-Campus Cordoba, Amatlan de los Reyes, Veracruz, Mexico SINESIO TORRES-GARCI´A • Departamento de Agronomı´a, Facultad de Ciencias Agropecuarias, Universidad Central “Marta Abreu” de Las Villas, Santa Clara, Cuba IZAAC VA´ZQUEZ-CISNEROS • Postgrado en Ciencias Forestales, Campus Montecillo, Colegio de Postgraduados, Texcoco, State of Mexico, Mexico NIEVES VIDAL • Department of Plant Production, Mision Biologica de Galicia, Sede Santiago de Compostela, CSIC, Santiago de Compostela, Spain PEDRO ZETINA-CO´RDOBA • Programa de Ingenierı´a Agroindustrial, Universidad Polite´ cnica de Huatusco, Huatusco, Veracruz, Mexico

Part I Temporary Immersion System Designs and Applications

Chapter 1 Temporary Immersion Systems in Plant Micropropagation Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz Abstract Temporary immersion systems (TIS) are technological tools that support plant micropropagation. Given their high efficiency in the in vitro propagation of shoots, a current goal is to update the protocols addressing micropropagation in semisolid culture systems to protocols involving TIS. To this end, different parameters have been evaluated, including TIS types and designs, immersion times, immersion frequencies, and volume of medium per explant, among other characteristics. This has resulted in the improved production of propagules of plants of economic interest and the production of physiologically upgraded plants with high percent survival during acclimatization. TIS are specialized culture flasks that provide countless advantages during the commercial micropropagation of plants. Key words Bioreactors, In vitro propagation, Commercial propagules, Mass propagation

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Introduction Currently, new methodologies are being sought to increase plant propagation and produce the largest number of commercial propagules possible [1]. Plant micropropagation allows massively cloning a genotype of commercial interest or with outstanding agronomic characteristics [2]. However, there are multiple limitations in the protocols of in vitro propagation of plants, including species, genotype, recalcitrance per se, physiological deficiencies resulting from acclimatization, high production costs, and lack of automated processes (protocols) [3]. Technological innovations have explored solutions to these issues. The use of liquid media is a technical approach to reduce the high costs of gelling agents [4]. However, the physiology of plants produced under this culture system is affected when it is applied continuously [5]. Temporary immersion systems (TISs) represent a technological innovation involving a liquid culture medium that allows the periodical immersion of plant material [6]. This book compiles basic, fundamental, and applied aspects of temporal immersion systems in massive plant micropropagation.

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_1, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz

Culture Systems for In Vitro Plant Propagation The composition of the culture medium is a central factor in the success of plant micropropagation. Micro- and macronutrients, carbon sources, plant growth regulators (PGRs), antioxidants, and inorganic and organic compounds are fundamental for plant growth and development [7]. Gelling agents are inert substances that support propagated plants. However, these agents determine the availability of nutrients for explants [8].

2.1 Semisolid System

Gellan gum and agar are two of the most commonly used gelling agents in most protocols for in vitro plant propagation [9]. However, their high costs make the production processes of commercial propagules more expensive and preclude automation in these processes, in addition to limiting the availability of nutrients in the culture medium, thus lowering shoot regeneration efficiency [10]. Most plant micropropagation processes are carried out under this system; however, improvements in in vitro culture systems now guarantee the mass propagation of the plants of interest [11]. Despite these limitations, the semisolid culture system is highly effective in the in vitro establishment stage due to factors, such as effective and inert support for the different types of explants, individualization of explants in culture containers (avoiding mass contamination), and an optimal system for studies on plant physiology due to the easy manipulation and evaluation, among others [12, 13].

2.2 Liquid System (Partial and Continuous Immersion)

Systems using liquid media were implemented to avoid the costs of gelling agents. In addition, greater availability of the substances in the culture medium has been demonstrated [14]. However, the continuous use (continuous immersion) of liquid media produced physiological alterations in the cells of cultivated plants, such as hyperhydricity [15]. Hyperhydricity can be defined as the physiological disorder that occurs in plant tissue cultures, characterized by high water retention due to adverse crop conditions. This physiological condition affects the viability of plant tissues and the survival of plants produced through micropropagation [16]. Therefore, partial immersion systems have been developed using liquid media [11]. This technique requires dosing a small amount of medium in culture flasks so that the explants are not fully submerged [11]. However, some plant species develop a high hydricity despite some cells not being in continuous contact with the culture medium [17].

2.3 Temporary Immersion Systems

Temporary immersion systems (TISs) combine the advantages of liquid culture media, thus avoiding the physiological disorders caused by these through scheduled immersions [6]. Explant

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immersions in the growth medium are temporary and programmable, adapted to each plant species, depending on its nutritional requirements [11, 18]. In addition, it has been shown that gas exchange under TIS improves the physiology of the plants obtained, mainly as regards stomatal morphology and functions [19]. Plants produced through TIS have shown better functionality by adapting to the outdoor environment during acclimatization [20]. TISs are mainly used to increase biological yield (number of shoots per explant) in the shoot multiplication/proliferation stage during micropropagation [21]. However, they can also be efficient during the elongation and rooting stages in various plant species [19] and when used for in vitro selection techniques [22] and plant physiology studies [23]. Today, the efficiency of this culture system compared to the semisolid, continuous immersion, and partial immersion systems has been demonstrated in the massive micropropagation of various plant species [18, 19]. The search and establishment of TIS in vitro propagation protocols in plant species for which these are not yet available have been recurrent goals in scientific research. However, these require setting fundamental parameters, such as type of TIS, immersion time, immersion frequency, and volume of medium per explant, among others, for each plant species targeted for mass micropropagation [24].

3

Applications of Temporary Immersion Systems in Plant Micropropagation TIS micropropagation protocols for use at the commercial level have been developed for various plant species of interest in food, agro-industrial, medicinal, and ornamental areas [6, 21] (Fig. 1). These systems are applied mainly to produce a large number of commercial propagules over a short period and in a reduced space. They have also been used to improve plant vigor and physiological functionality relative to those obtained in semisolid systems [25]. Besides, these systems produce plants with better stomatal functionality, resulting in higher percent survival during acclimatization [3, 26]. In this sense, TISs, as technological developments implemented in plant biotechnology, have allowed optimizing the micropropagation protocols already established in semisolid systems [27]. New TIS types are being designed constantly with different designs and manufacturing materials to enhance plant propagation [28]. However, their efficiency in the micropropagation of each plant species should be evaluated through experiments and scientific work to support their acceptance by the community interested in using this technological tool.

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Fig. 1 Sugar Cane Research Institute Biofactory (Cuba). (a) Sugarcane mother plants, (b) Micropropagation in SETISTM, (c) Commercial propagules in the process of acclimatization

Today, TISs are used in the generation of somatic embryogenesis to elite genotypes, and for obtaining secondary metabolites from the increase in biomass produced during the multiplication stage [24, 29]. Currently, TISs facilitate semi-automation in mass micropropagation processes in commercial laboratories. However, a 100% TIS in vitro propagation protocol has not yet been developed because it is recommended that the establishment stage be carried out in semisolid systems to avoid significant losses of plant material due to initial contamination [4, 30, 31].

4 Concluding Remarks As mentioned above, the primary use of TISs is the mass proliferation of shoots during micropropagation and the increase in the multiplication rate, as TISs produce a larger number of shoots per explant than semisolid and partial immersion systems [4]. These characteristics make of TISs indispensable tools in biological laboratories that produce propagules at commercial levels [27]. However, there is no single most effective TIS for the in vitro propagation of all plant species. Therefore, the TIS most suitable for propagation should be determined for each plant species, considering the particular production characteristics and the specific TIS design. This book compiles fundamental and relevant aspects, techniques, and protocols for the adequate micropropagation of plant species in temporary immersion systems.

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References 1. Sodre´ GA, Gomes ARS (2019) Cocoa propagation, technologies for production of seedlings. Rev Bras Frutic 41. https://doi.org/10. 1590/0100-29452019782 2. Rout GR, Jain SM (2020) Advances in tissue culture techniques for ornamental plant propagation. In: Reid M (ed) Achieving sustainable cultivation of ornamental plants. Burleigh Dodds Science Publishing, London, pp 1 4 9 – 1 8 8 . h t t p s : // d o i . o r g / 1 0 . 1 2 0 1 / 9781003047766 3. Monja-Mio KM, Olvera-Casanova D, HerreraHerrera G, Herrera-Alamillo MA, SanchezTeyer FL, Robert ML (2020) Improving of rooting and ex vitro acclimatization phase of Agave tequilana by temporary immersion system (BioMINT™). In Vitro Cell Dev Biol Plant 56:662–669. https://doi.org/10. 1007/s11627-020-10109-5 4. Le KC, Johnson S, Aidun CK, Egertsdotter U (2023) In vitro propagation of the blueberry ‘blue suede™’(Vaccinium hybrid) in semisolid medium and temporary immersion bioreactors. Plan Theory 12:2752. https://doi. org/10.3390/plants12152752 5. Hegele S, Hegele M, Wu¨nsche JN (2021) Low-cost gelling agents for tissue culture propagation of plantain. IV International Symposium on Horticulture in Europe-SHE2021 Acta Hortic 1327:341–348. https://doi.org/ 10.17660/ActaHortic.2021.1327.46 6. Mirzabe AH, Hajiahmad A, Fadavi A, Rafiee S (2022) Temporary immersion systems (TISs): a comprehensive review. J Biotechnol 357:56– 83. https://doi.org/10.1016/j.jbiotec.2022. 08.003 7. Phillips GC, Garda M (2019) Plant tissue culture media and practices: an overview. In Vitro Cell Dev Biol Plant 55:242–257. https://doi. org/10.1007/s11627-019-09983-5 8. Asayesh ZM, Aliniaeifard S, Vahdati K (2021) Stomatal morphology and desiccation response of persian walnut tissue culture plantlets influenced by the gelling agent of in vitro culture medium. J Nuts 12:41–52. https://doi.org/ 10.22034/jon.2021.1922749.1105 9. Al-Mayahi AMW, Ali AH (2021) Effects of different types of gelling agents on organogenesis and some physicochemical properties of date palm buds, Showathy cv. Folia Oecol 48: 110–117. https://doi.org/10.2478/foecol2021-0012 10. Sudheer WN, Praveen N, Al-Khayri JM, Jain SM (2022) Role of plant tissue culture medium components. In: Rai AC, Kumar A, Modi A,

Singh M (eds) Advances in plant tissue culture. Academic, pp 51–83. https://doi.org/10. 1016/B978-0-323-90795-8.00012-6 11. Leyva-Ovalle OR, Bello-Bello JJ, Murguı´a˜ ez-Pastrana R, Ramı´rez-MosGonza´lez J, Nu´n queda MA (2020) Micropropagation of Guarianthe skinneri (Bateman) Dressler et WE Higging in temporary immersion systems. 3 Biotech 10:1–8. https://doi.org/10.1007/ s13205-019-2010-3 12. Avelino Lea˜o JR, Raposo A, Lopes da Silva AC, Barbosa Sampaio PT (2020) Control of contaminants in the in vitro establishment of Guadua latifolia. Pesqui Agropecu Trop 50: e63541. https://doi.org/10.1590/198340632020v5063541 13. Ozudogru EA, Karlik E, Elazab D, Lambardi M (2023) Establishment of an efficient somatic embryogenesis protocol for giant reed (Arundo donax L.) and multiplication of obtained shoots via semi-solid or liquid culture. Horticulturae 9:735. https://doi.org/ 10.3390/horticulturae9070735 14. Manokari M, Priyadharshini S, Jogam P, Dey A, Shekhawat MS (2021) Meta-topolin and liquid medium mediated enhanced micropropagation via ex vitro rooting in Vanilla planifolia Jacks. ex Andrews. Plant Cell Tissue Organ Cult 146:69–82. https://doi.org/10. 1007/s11240-021-02044-z 15. Bayraktar M, Hayta-Smedley S, Unal S, Varol N, Gurel A (2020) Micropropagation and prevention of hyperhydricity in olive (Olea europaea L.) cultivar ‘Gemlik’. S Afr J Bot 128:264–273. https://doi.org/10.1016/ j.sajb.2019.11.022 16. Polivanova OB, Bedarev VA (2022) Hyperhydricity in plant tissue culture. Plan Theory 11: 3 3 1 3 . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / plants11233313 17. El-Mahrouk ME, El-Shereif AR, Dewir YH, Hafez YM, Abdelaal KA, El-Hendawy S, Migdadi H, Al-Obeed RS (2019) Micropropagation of banana: reversion, rooting, and acclimatization of hyperhydric shoots. HortScience 54:1384–1390. https://doi.org/10.21273/ HORTSCI14036-19 18. Monja-Mio KM, Olvera-Casanova D, Herrera´ , Sa´nchez-Teyer FL, Robert ML Alamillo MA (2021) Comparison of conventional and temporary immersion systems on micropropagation (multiplication phase) of Agave angustifolia Haw. ‘Bacanora’. 3 Biotech 11:1– 8. https://doi.org/10.1007/s13205-02002604-8

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19. Ramı´rez-Mosqueda MA, Bello-Bello JJ (2021) SETIS™ bioreactor increases in vitro multiplication and shoot length in vanilla (Vanilla planifolia Jacks. Ex Andrews). Acta Physiol Plant 43:52. https://doi.org/10.1007/s11738021-03227-z 20. Ramı´rez-Mosqueda MA, Cruz-Cruz CA, Cano-Rica´rdez A, Bello-Bello JJ (2019) Assessment of different temporary immersion systems in the micropropagation of anthurium (Anthurium andraeanum). 3 Biotech 9:307. https://doi.org/10.1007/s13205-0191833-2 21. Hwang HD, Kwon SH, Murthy HN, Yun SW, Pyo SS, Park SY (2022) Temporary immersion bioreactor system as an efficient method for mass production of in vitro plants in horticulture and medicinal plants. Agronomy 12:346. h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / agronomy12020346 22. Go´mez D, Herna´ndez L, Valle B, Martı´nez J, Cid M, Escalona M, Herna´ndez M, Yabor L, Beemster GTS, Tebbe CC, Papenbrock J, Lorenzo JC (2017) Salinity induces specific metabolic changes in sugarcane shoot explants in temporary immersion bioreactors. J Appl Bot Food Qual 90:354–358. https://doi.org/10. 5073/JABFQ.2017.090.044 23. Klimek-Szczykutowicz M, Dziurka M, Blazˇevic´ I, Ðulovic´ A, Granica S, KoronaGlowniak I, Ekiert H, Szopa A (2020) Phytochemical and biological activity studies on Nasturtium officinale (watercress) microshoot cultures grown in RITA® temporary immersion systems. Molecules 25:5257 24. Aguilar ME, Wang XY, Escalona M, Yan L, Huang LF (2022) Somatic embryogenesis of Arabica coffee in temporary immersion culture: advances, limitations, and perspectives for mass propagation of selected genotypes. Front Plant Sci 13:994578. https://doi.org/10.3389/ fpls.2022.994578

25. Rico S, Garrido J, Sa´nchez C, Ferreiro-Vera C, Codesido V, Vidal N (2022) A temporary immersion system to improve cannabis Sativa micropropagation. Front Plant Sci 13:895971. https://doi.org/10.3389/fpls.2022.895971 26. Martı´nez-Estrada E, Islas-Luna B, Pe´rez-Sato JA, Bello-Bello JJ (2019) Temporary immersion improves in vitro multiplication and acclimatization of Anthurium andraeanum Lind. Sci Hortic 249:185–191. https://doi.org/10. 1016/j.scienta.2019.01.053 27. Murthy HN, Joseph KS, Paek KY, Park SY (2023) Bioreactor systems for micropropagation of plants: present scenario and future prospects. Front Plant Sci 14:1159588. https:// doi.org/10.3389/fpls.2023.1159588 28. Georgiev V, Schumann A, Pavlov A, Bley T (2014) Temporary immersion systems in plant biotechnology. Eng Life Sci 14:607–621. https://doi.org/10.1002/elsc.201300166 29. Le KC, Dedicova B, Johansson S, Lelu-Walter MA, Egertsdotter U (2021) Temporary immersion bioreactor system for propagation by somatic embryogenesis of hybrid Larch (Larix× eurolepis Henry). Biotechnol Rep 32: e00684. https://doi.org/10.1016/j.btre. 2021.e00684 ˜ ez-Palenius HG, 30. Va´zquez-Martı´nez O, Nu´n Balch EMPM, Valencia-Posadas M, Pe´rezMoreno L, Ruiz-Aguilar GM, Go´mez-Lim M (2022) In vitro-propagation of Agave tequilana weber cv. azul in a temporary immersion system. Phyton 91:83. https://doi.org/10. 32604/phyton.2022.017281 31. San Jose´ MC, Bla´zquez N, Cernadas MJ, Janeiro LV, Cuenca B, Sa´nchez C, Vidal N (2020) Temporary immersion systems to improve alder micropropagation. Plant Cell Tissue Organ Cult 143:265–275. https://doi. org/10.1007/s11240-020-01937-9

Chapter 2 Types of Temporary Immersion Systems Used in Commercial Plant Micropropagation Ivonne N. Bravo-Ruı´z, Ma. Teresa Gonza´lez-Arnao, Fabiola Herna´ndez-Ramı´rez, Jaime Lo´pez-Domı´nguez, and Carlos A. Cruz-Cruz Abstract Technological innovation in the design and manufacture of temporary immersion systems (TIS) has increased in the past decade. Innovations have involved the size, fitting, and replacement of components, as well as manufacturing materials. Air replacement by compressor has also been substituted by air replacement by preset tilting/rotation of culture bottles. This design modification aims to increase the biological yield (number of shoots) produced in these bottles and reduce manufacturing costs. However, the operative principle has remained unchanged through time: promote an environment where explant immersions in the culture medium are programmable. The changes in the TIS design involve advantages and disadvantages, generating the efficiency of one type over another. However, validation to identify the most effective type of TIS should be carried out for each plant species. This chapter lists the different types of temporary immersion available on the market, emphasizing the advantages and disadvantages of each when used for plant micropropagation. Key words Bioreactors, Design innovations, Mass propagation

1

Introduction The global population demands the mass production of plant species involving food, agro-industrial and medicinal interests [1]. It is necessary to produce thousands and thousands of propagules for planting and cultivation in the field. However, propagation (reproduction) methods are inefficient in some cultures [2]. The conventional production of propagules in greenhouses and the field requires large amounts of natural resources (soil, water, nutrients and others) and the use of pesticides for phytosanitary control [3]. The use of these resources is directly proportional to the number of propagules produced; in this sense, it is

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_2, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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necessary to implement new technologies to improve the sustainability of the reproduction processes [4]. Plant biotechnology offers a series of technological tools that facilitate the manipulation of organisms to benefit humans [5]. Among these techniques, plant tissue culture (PTC) uses cellular totipotentiality to make any plant cell grow into a complete plant [4]. PTC has been widely used in plant micropropagation, a term that refers to the mass propagation of plants under in vitro (laboratory) culture conditions [6]. Although this in vitro propagation method has yielded satisfactory results, increasing the number of plants and boosting the reproductive rate, the commercialscale implementation of this technology is limited, given the high production costs related to time invested and highly qualified staff required [7, 8]. Therefore, it is necessary to update these protocols and adapt the technological advances in this area of knowledge. In this sense, temporary immersion systems (TIS) are technological adaptations to culture vessels to allow temporary immersion of explants in the culture medium [9] developed for the commercial scale-up of micropropagation, because they facilitate culture system automation from temporal tissue immersion [10]. Using bioreactors in temporal immersion media reduces production costs in large-scale cultures because the immersion time is generally shorter (a few minutes) while the air-exposure time is longer (several hours). This feature allows a higher availability of growth regulators and nutrients while preventing physiological alterations, like hyperhydricity and tissue oxidation reported for partial immersion systems, making TIS an efficient large-scale plant micropropagation system [11, 12]. Besides, TIS promotes physiological processes, such as photosynthesis, respiration, chlorophyll synthesis and stomata function for proper adaptation to ex vitro environments during acclimatization [13]. According to Georgiev [12], there is a wide variety of automated models and systems used for in vitro plant micropropagation. The design and manufacturing materials are two of the main characteristics that have been innovated in commercial brands, as well as size, leading to greater production of plant material; each model promises improved advantages in contrast to its competition (Fig. 1) [14]. Therefore, we usually follow the manufacturers’ recommendations or previous use experiences in a particular species reported in the literature. However, a review identifying the strengths and weaknesses of each TIS would be helpful when selecting a particular type of TIS. Therefore, this chapter addresses the different types of commercial TIS, mentioning the advantages and disadvantages of each. The aim is to provide a quick guide to aid users in selecting a particular design.

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Fig. 1 Variety of bioreactors used in plant micropropagation: (a) Recipient Immersion Temporaire Automatique (RITA®); (b) Temporary Immersion Bioreactor (BIT); (c) GreenTray®; (d) Ebb-and-Flow TIS (Gravity Immersion Bioreactor-BIG); (e) PLANTFORM; (f) Modular bioreactor (BioMINT®); (g) MATIS®; (h) SETIS™

2 Main TIS Operation All TIS currently marketed operate under the same principle that are exposing plant tissues to programmable alternating cycles of immersion in liquid culture media and exposure to a gaseous environment [9, 12]. TIS consists of cultures of plant cells, tissues or organs in semi-automated bioreactors under axenic and controlled conditions [15]. TIS generally have a support structure for the plant material, commonly a plastic mesh; this structure keeps explants out of the culture medium, favoring better aeration of the tissues when there is no immersion [11]. In many TIS designs, this structure is designed to be at a higher position relative to the bottom of the container. Immersion times are defined as the periods in which the culture medium is in contact with explants; after a time interval, the medium is drained back into the storage tank or the bottom of the container for reuse [12]. To this end, immersion is controlled by electrovalves connected to a programmable logic controller or, in less sophisticated cases, a timer. Depending on the plant species and its nutritional needs, the immersion time and the interval between immersions vary from a few seconds to a few hours [16]. In general, TIS includes a single container or two containers [17]. Some of the commercially available TIS are listed below.

3 Single-Container Temporary Immersion Systems 3.1

RITA®

RITA (Recipient Immersion Temporaire Automatique), marketed by VITROPIC, is a container specialized for plant micropropagation [18]. This TIS is manufactured with sterilizable polypropylene in a 500 mL presentation only; it has two compartments separated

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Fig. 2 Design and parts of RITA® system: (a) RITA® system; (b) Axial view of RITA® system components; (c) Parts of RITA® system: (1) Inlet/outlet air filters; (2) Screw cap and hermetic packaging; (3) Center pipe with hermetic packing; (4) Top compartment and mesh; (5) Bell and lower chamber

Fig. 3 Operational design of RITA® TIS: (a) Idle bioreactor; (b) Air inlet to the system; (c) Plants immersed in the culture medium; (d) Culture medium descends by gravity to the lower chamber

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by a mesh that serves as support and plastic tubing in the center (Fig. 2a). The sealing of this TIS consists of a screw cap and an airtight pack; the cap has two outlets/inlets: a central one and a lateral one. Membrane filters are affixed at the inlet/outlet; air enters via the central inlet connected to an air distribution tube controlled by a timer and an electromagnetic valve. The vent outlet is the lateral access and uses a membrane filter (0.22 μm). RITA® has two compartments; generally, the upper compartment is the culture chamber, while the bottom compartment stores liquid culture medium (Fig. 2b, c). During immersion, air is insufflated through the medium (Fig. 3), gently stirring the tissues and renewing the atmosphere inside the bottle, while overpressure is avoided through outlets located on the top of the apparatus [9]. Advantages 1. Reliable and simple operation due to its design, sealing and larges space in the container mouth.

2. Head space suitable for the growth of shoots of most plant species (except those of considerable height, such as sugar cane). 3. Proper handling of relative humidity as explants are separated from the culture medium. 4. Interconnected elements (assembled), allowing easy handling and subcultures (semi-automation).

Disadvantages 1. Internal assemblage with too many parts (some are easily lost during washing).

2. Difficulty in renewing the culture medium as the central column is difficult to handle. 3. Lack of options for air turnover (reduced head space). 4. Difficult application in plant species that produce high shoots (reduced upper chamber height). 5. Difficulty for the rooting of plant species with small-diameter roots (they get entangled in the support mesh, e.g., Stevia). 6. In some countries, the importation cost is high [12]. There is a variant of RITA® called MATIS® [19]. This novel TIS is a monobloc device of high transparency for optimal light dispersion. A detailed description of this system is available on the manufacturer’s website (http://www.bioreactor-matis.com; accessed on July 4, 2023). However, this system has failures in its airtight seal; besides, its wide mouth implies that it is hard to

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manipulate in a hood chamber, which increases the entry of potential contaminants. 3.2

Rocker TIS

These TIS are based on a mechanical programmable periodic tilt system (shelves) where rectangular containers are balanced, allowing the culture medium to be evenly distributed from one box end to the other (Fig. 4). Periodic tilting produces waves that favor alternating aeration and immersion of the explants. Generally, culture boxes are made of autoclavable polycarbonate, rectangular, with a wide lateral opening, and closed by a wide screwcap with a membrane filter [9]. Advantages 1. High volume of plant material that can be handled, given the large number of containers that can be fitted on the shelf.

2. Culture containers do not require additional connections. 3. Do not require a highly sophisticated infrastructure (tubing and electrovalves). Disadvantages 1. Culture boxes have inadequate air turnover due to the lack of forced ventilation.

2. There are no options to renew the liquid culture medium other than changing the container. 3. The mechanical platform can raise costs, depending on the production capacity. 4. It requires more space in the incubation chamber due to its complex structure. 3.3

BioMINT™ TIS

This TIS also works by mechanical tilt (rocker). It comprises two polycarbonate containers that can be cylindrical BioMINT™) or rectangular (BioMINT™ II), assembled by an adapter. One container will contain the plant material, and the other the culture medium (Fig. 5). The adapter has a perforation for the flow of liquid culture medium between the two containers, while the explants are held in place when the bioreactor changes position [20]. The containers have accessible orifices for optional forced ventilation. Advantages 1. This design improves the efficiency of Rocker TIS by having optional venting orifices.

2. Easy operation by tilting only. 3. Hyperhydricity is avoided since explants are contained away from the culture medium.

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Fig. 4 Operational design of Rocker TIS: (a) Container design; (b) Latent period where mechanical inclination the culture medium is positioned on the opposite side of the explants; (c) Immersion period where the explants are submerged in the liquid medium by mechanical inclination

Fig. 5 Operational design of BioMINT™ TIS: (a) Container design; (b) Latent period where mechanical inclination the culture medium is positioned on the opposite side of the explants; (c) Immersion period where the explants are submerged in the liquid medium by mechanical inclination

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Disadvantages 1. Depending on the production capacity, the mechanical platform can raise costs.

2. It requires more space in the incubation chamber due to its complex structure. 3. Its size makes autoclaving difficult. 4. The manufacturer has no official website to facilitate its distribution. 3.4

PLANTFORM™

This TIS operates on the same principle as RITA®. PLANTFORM™ consists of a transparent polycarbonate body measuring 180 × 150 × 150 mm that can withstand heating of 120 °C. Gas exchange is controlled through three inlets/outlets anchored to one side by nuts and clamps through orifices in the body and sealed with heat-resistant silicone O-rings. The bottom of the bioreactor contains an inner chamber with three slots on the long side and two on the short side. A basket containing the plant material is placed above the inner chamber. The body is closed by an airtight lid using a silicone seal inside a slot in the lid. The lid is a snap-on lid that is easy to affix and remove (Fig. 6). The body is usually filled with 500 mL of nutrient solution under the inner chamber. Sterile plant material is then placed inside the basket (Fig. 7) (http://www.plantform.se; accessed on 04 July 2023). Advantages 1. High-quality and durable manufacturing materials.

2. The size of the bioreactor allows more plants in each unit, reducing labor costs related to transferring plants to new containers. 3. Orifices in the basket prevent the accumulation of liquid, which is important to avoid hyperhydricity. 4. Easy to buy through the manufacturer’s website. Disadvantages 1. Its size makes conventional autoclaving difficult.

2. Its assembly requires instructions, in addition to having many parts that need to be screwed with a tool. 3. The airtight sealing system is sometimes ineffective (the seal wears away with constant use).

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Fig. 6 Design and parts of PLANTFORM™ system: (a) Basket; (b) Container; (c) Support; (d) Airtight lid; (e) Inlet/outlet air filters; (f) Bottom compartment with 3 slots on the long side and 2 short slots

Fig. 7 Operational design of PLANTFORM™ TIS: During the exposure period, the volume of the liquid medium is in the medium storage tank and by gas exchange action it rises to the upper container, temporarily submerging the explants, (a, b) Front view; (c, d) Side view

4 Twin-Flask Temporary Immersion Systems 4.1 Temporary Immersion Bioreactor (BIT)

This TIS was one of the first to be developed for mass micropropagation of plants. The BIT consists of two plastic or glass containers (bottles or flasks) of different volumes interconnected by a glass,

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Fig. 8 Operational design of BIT TIS: (a) Exposure period (the entire volume of the liquid medium is in the storage bottle); (b) Distribution of the liquid medium (a solenoid valve opens, and compressed air pushes the medium into the tissue container); (c) Immersion period (the tissues are immersed in the liquid medium, the storage bottle in the middle is empty and airlines for both vessels are closed, and solenoid valves open to atmosphere); (d) Drained (A second solenoid valve opens, and air pressure forces the medium back into the original container)

plastic or silicone tube [21]. One flask is a culture chamber and the other is a culture medium container. There are some modifications to the original design of the container serving as a culture chamber: it includes support for the plant material, which can be manufactured of metal, polyurethane or glass among other materials. Each flask contains a pressurized airline that increases the inner pressure; this action allows an aeration period that moves the culture medium to the flask with plant material (immersion time). Afterward, a reverse pressure returns the culture medium to its respective container (aeration cycle) (Fig. 8). The immersion frequency refers to the number of immersions performed in a day (time elapsed between consecutive immersions). It is usually easy to operate and its design can keep environments sterile for long culture periods. Advantages 1. Easy to assemble; homemade materials can be used to replace parts.

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2. The volume of flasks can vary, usually from 500 to 5000 mL, allowing adaptation to the requirements of most plant species. 3. The head space, mainly the height, is suitable for obtaining long shoots. 4. Rooting can take place with no roots getting entangled, unlike the supports of other TIS types. 5. Can be easily modified using parts made of various materials, inserting support for plant materials and adapting electronic sensors for monitoring atmospheric parameters, among others. Disadvantages 1. Does not allow full automation, as it depends on timers and electromagnetic valves that require programming.

2. No options for culture medium replacement. 3. It is not equipped with a port for the external supply of substances or gases such as CO2 during the aeration period. 4. When using glass flasks, these are frequently damaged during autoclaving or washing. 4.2 Ebb-and-Flow TIS

It is a twin-flask system in which the connection between the flasks is located at the bottom of the containers. The flasks (culture chamber and culture medium container) may be on the same or different levels. When the culture medium container is slightly higher than the nutrient container, it is known as a gravity system because airflow forces the culture medium into the plant growth chamber; when air pressure stops, the fluid returns to its original level by gravity in both containers (Fig. 9) [22, 23]. Advantages 1. The gravity TIS only needs an air inlet, unlike BIT, which means savings in infrastructure.

2. Easy to assemble and handle in the hood chamber. 3. Energy saving as it uses gravity to return the culture medium to its container. 4. It can be manufactured with homemade materials. 5. Modifications are easy to implement, such as using parts made of different materials and inserting a holder for the plant material. Disadvantages 1. When using glass flasks, making the orifices in the bottom of containers is very difficult.

2. As no aeration is used to return the culture medium to its original container, there is no extra air replacement, unlike BIT.

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Fig. 9 Operational design of Ebb-and-Flow TIS: (a) Exposure period (the entire volume of the liquid medium is in the storage bottle); (b) Liquid medium distribution by pressurized or gravimetric culture medium transfer system; (c) Immersion period (the tissues are immersed in the liquid medium, the storage bottle in the middle is empty); (d) drained (when air pressure stops) the fluid returns to its original level by gravity in both containers

3. As glass flasks are used, these are frequently damaged during autoclaving or washing. 4.3

SETIS™

This TIS, distributed by VERVIT, was designed for large-scale plant micropropagation using the twin-flask concept [24]. It has a standard design and home manufacturing is impossible; it is made of polycarbonate, which optimizes light dispersion and ensures its durability. It has a small number of parts for easy assembling: culture container (6000 mL), culture medium container (4000 mL), two polypropylene screw caps containing silicone seals, air filters and silicone tubing (Fig. 10). Its operation (Fig. 11) involves the following phases: 1. Stationary phase: No compressed air is supplied; the growth medium remains in the medium container and the plant material is in a gaseous environment. 2. Immersion phase: Compressed air is supplied to the medium container to transfer the medium to the upper culture container; the plant material remains in a liquid environment and nutrient absorption takes place.

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Fig. 10 Operational design of SETIS™ TIS: (a, b) Airtight screw caps; (c, d) Inlet/outlet air filters; (e, f) Hose connection; (g) Culture medium container; (h) Tissue container

Fig. 11 Operational design of SETIS™ TIS: During the exposure period, the volume of the liquid medium is in the medium storage tank and by action of compressed air it rises to the upper container, temporarily submerging the explants: (a, b) Front view; (c, d) Side view

3. Drainage phase: The culture medium returns to the medium container by gravity. 4. Ventilation phase: Compressed air is supplied to the culture vessel to replace its inner gaseous environment (https://setissystems.be; accessed on 4 July 2023).

22

Ivonne N. Bravo-Ruı´z et al.

Advantages 1. Fully autoclavable.

2. Minimum number of components. 3. Long service life due to its manufacturing materials. 4. Ideal volume for mass micropropagation of plants. 5. It can be stacked for maximum use of shelf space. 6. Easy assembling and handling, in addition to optimum use of light due to its horizontal configuration. 7. Excellent gravity drainage due to its design. Disadvantages 1. Its size makes autoclaving difficult (considering a conventional autoclave).

2. Cracks in containers are frequent due to repeated use and autoclaving; however, the official SETISTM website offers a repair kit. 3. In some countries, importation costs raise costs.

5

Concluding Remarks The TIS currently marketed offer various designs and types, each with different characteristics. The acceptability of a particular TIS depends primarily on its ease of use and installation in the operational facilities [12]. Most laboratories have conventional autoclaves (vertical autoclaves with a capacity of 40–60 L); however, the large size of some TIS require sterilization equipment of larger dimensions. Explants should be easily placed inside the growth chamber; that is, the different parts of the bioreactor should be easily assembled and disassembled, especially if incubation is to be carried out in a hood chamber, where the user’s hand has less room to maneuver [9]. The durability of manufacturing materials is a key element to be considered when selecting a TIS. However, using glass flasks remains a cost-effective option in the cost-benefit analysis. On the other hand, scientific validation of the plant micropropagation efficiency of each TIS type is necessary; however, it should be borne in mind that each plant species has specific nutritional requirements for its development and growth. Therefore, it is particularly important to understand the factors that affect the functioning of TIS components to make the best choice for their use in plant micropropagation. This chapter intends to familiarize the reader with the different types of TIS and their operation, as well as demonstrate the advantages and disadvantages of each type. This information will allow selecting the design that is compatible with the purposes of particular research/work.

Types of Temporary Immersion Systems for Micropropagation

23

Acknowledgments The authors thank Plant Tissue Culture Laboratory (Colegio de Postgraduados-Campus Co´rdoba, Me´xico) for access to Temporary Immersion Systems collection. Figures created with BioRender.com. References 1. Chandran H, Meena M, Barupal T, Sharma K (2020) Plant tissue culture as a perpetual source for production of industrially important bioactive compounds. Biotechnol Rep 26: e00450. https://doi.org/10.1016/j.btre. 2020.e00450 2. Campbell SM, Anderson SL, Brym ZT, Pearson BJ (2021) Evaluation of substrate composition and exogenous hormone application on vegetative propagule rooting success of essential oil hemp (Cannabis sativa L.). PLoS One 16:e0249160. https://doi.org/10.1371/jour nal.pone.0249160 3. Singh MK, Alam MK, Pandey MV, Singh MS, Kumar M (2023) Advanced technology of horticulture. Daya Publishing House, New Delhi, p 38 4. Twaij BM, Jazar ZH, Hasan MN (2020) Trends in the use of tissue culture, applications and future aspects. Int J Plant Biol 11:8385. https://doi.org/10.4081/pb.2020.8385 5. Eskandar K (2023) Revolutionizing biotechnology and bioengineering: unleashing the power of innovation. J Appl Biotechnol Bioeng 10:81–88. https://doi.org/10.15406/jabb. 2023.10.00332 6. Phillips GC, Garda M (2019) Plant tissue culture media and practices: an overview. In Vitro Cell Dev Biol Plant 55:242–257. https://doi. org/10.1007/s11627-019-09983-5 7. Le KC, Johnson S, Aidun CK, Egertsdotter U (2023) In vitro propagation of the blueberry ‘blue suede™’(Vaccinium hybrid) in semisolid medium and temporary immersion bioreactors. Plan Theory 12:2752. https://doi. org/10.3390/plants12152752 8. San Jose´ MC, Bla´zquez N, Cernadas MJ, Janeiro LV, Cuenca B, Sa´nchez C, Vidal N (2020) Temporary immersion systems to improve alder micropropagation. Plant Cell Tissue Organ Cult 143:265–275. https://doi. org/10.1007/s11240-020-01937-9 9. Mirzabe AH, Hajiahmad A, Fadavi A, Rafiee S (2022) Temporary immersion systems (TISs): a comprehensive review. J Biotechnol 357:56–

83. https://doi.org/10.1016/j.jbiotec.2022. 08.003 10. Aguilar ME, Wang XY, Escalona M, Yan L, Huang LF (2022) Somatic embryogenesis of Arabica coffee in temporary immersion culture: advances, limitations, and perspectives for mass propagation of selected genotypes. Front Plant Sci 13:994578. https://doi.org/10.3389/ fpls.2022.994578 11. Etienne H, Berthouly M (2002) Temporary immersion systems in plant micropropagation. Plant Cell Tissue Organ Cult 69:215–231. https://doi.org/10.1023/A:1015668610465 12. Georgiev V, Schumann A, Pavlov A, Bley T (2014) Temporary immersion systems in plant biotechnology. Eng Life Sci 14:607–621. https://doi.org/10.1002/elsc.201300166 13. Arago´n CE, Sa´nchez C, Gonzalez-Olmedo J, Escalona M, Carvalho L, Amaˆncio S (2014) Comparison of plantain plantlets propagated in temporary immersion bioreactors and gelled medium during in vitro growth and acclimatization. Biol Plant 58:29–38. https://doi.org/ 10.1007/s10535-013-0381-6 14. Murthy HN, Joseph KS, Paek KY, Park SY (2023) Bioreactor systems for micropropagation of plants: present scenario and future prospects. Front Plant Sci 14:1159588. https:// doi.org/10.3389/fpls.2023.1159588 15. De Carlo A, Tarraf W, Lambardi M, Benelli C (2021) Temporary immersion system for production of biomass and bioactive compounds from medicinal plants. Agronomy 11:2414. h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / agronomy11122414 16. Leyva-Ovalle OR, Bello-Bello JJ, Murguı´a˜ ez-Pastrana R, Ramı´rez-MosGonza´lez J, Nu´n queda MA (2020) Micropropagation of Guarianthe skinneri (Bateman) Dressler et WE Higging in temporary immersion systems. 3 Biotech 10:1–8. https://doi.org/10.1007/ s13205-019-2010-3 17. Hwang HD, Kwon SH, Murthy HN, Yun SW, Pyo SS, Park SY (2022) Temporary immersion bioreactor system as an efficient method for mass production of in vitro plants in

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horticulture and medicinal plants. Agronomy 1 2 : 3 4 6 . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / agronomy12020346 18. Alvard D, Cote F, Teisson C (1993) Comparison of methods of liquid medium culture for banana micropropagation: effects of temporary immersion of explants. Plant Cell Tissue Organ Cult 55–60. https://doi.org/10.1007/ BF00040116 19. Robert ML, Herrera-Herrera JL, HerreraHerrera G, Herrera-Alamillo MA, FuentesCarrillo P (2006) A new temporary immersion bioreactor system for micropropagation. In: Loyola-Vargas VM, Va´zquez-Flota F (eds) Plant cell culture protocols. Methods in molecular biology™, vol 318. Humana Press. https://doi.org/10.1385/1-59259-959-1: 121 20. Monja-Mio KM, Olvera-Casanova D, HerreraHerrera G, Herrera-Alamillo MA, SanchezTeyer FL, Robert ML (2020) Improving of rooting and ex vitro acclimatization phase of Agave tequilana by temporary immersion

system (BioMINT™). In Vitro Cell Dev Biol Plant 56:662–669. https://doi.org/10.1007/ s11627-020-10109-5 21. Escalona M, Lorenzo J, Gonza´lez B (1999) Pineapple (Ananas comosus L. Merr) micropropagation in temporary immersion systems. Plant Cell Rep 18:743–748. https://doi.org/ 10.1007/s002990050653 22. Tisserat B, Vandercook CE (1985) Development of an automated plant culture system. Plant Cell Tissue Organ Cult 5:107–117. https://doi.org/10.1007/BF00040307 23. Ducos JP, Labbe G, Lambot C, Pe´tiard V (2007) Pilot scale process for the production of pregerminated somatic embryos of selected robusta (Coffea canephora) clones. In Vitro Cell Dev Biol Plant 43:652–659. https://doi. org/10.1007/s11627-007-9075-0 24. Vervit (2023) SETIS™ Bioreactor Temporary immersion systems in plant micropropagation. http://www.setis-systems.be. Cited 31 de July 2023

Chapter 3 Use of Temporary Immersion Systems in the Establishment of Biofactories Marco Vinicio Rodrı´guez-Deme´neghi Abstract Companies dedicated to the large-scale propagation of plant species are known as biofactories or agricultural biotechnology companies. Globally, there are a large number of biofactories (large-scale production) or plant tissue culture laboratories (small-scale production) in charge of supplying commercial propagules of plants of economic interest. Each biofactory implements technological developments such as temporary immersion (TIS) systems that allow them to reduce costs. This chapter analyzes some of the biofactories established globally, the main plant species propagated, and whether or not they implement the use of TIS. Key words Large-scale micropropagation, TIS, Agricultural biotechnology companies, Cost reduction

1

Introduction The application of biotechnology in agriculture has achieved important advances in the improvement and propagation of plant species [1]. In this sense, plant tissue cultures have successfully produced commercial propagules on a large scale [2]. The industry has used these results to obtain an economic income, providing farmers with plants characterized by high vigor, high health, large production volume, and sometimes certified [2]. Industries dedicated to the mass micropropagation of plants are named biofactories [3]. In Mexico, this is a well-known term related to facilities dedicated to elaborating biofertilizers. However, in other countries, such as Cuba, there are multiple companies labeled as biofactories dedicated to the large-scale micropropagation of plants for agronomic purposes [4], while at a worldwide level, some biofactories are called agricultural biotechnology companies. The use of new technologies has positively impacted the in vitro propagation of plants, including the use of temporary immersion systems (TIS) [5]. These are culture containers that allow the use of

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_3, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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26

Marco Vinicio Rodrı´guez-Deme´neghi

liquid media while controlling the duration of explant immersions [6]. The main advantage of this technology is the reduction of costs associated with the use of gelling agents and the prevention of physiological disorders in plants, such as hyperhydricity, caused by liquid culture media [7]. However, a morphological and physiological improvement of plants produced through TIS during acclimatization has been reported. These improvements include stomatic functionality, the prevention of cellular dehydration, the greater synthesis of photosynthetic pigments, and a greater root system, among others [8]. The main advantage of using a TIS is the increase in shoot production in the proliferation stage [8]. In this sense, TISs have been extensively used in the establishment and operation of biofactories dedicated to the production and marketing of plant propagules with food, industrial, pharmaceutical, and ornamental interest [9]. This chapter provides examples and relevant information for the correct use of TISs in the operation of biofactories worldwide. It also outlines the scientific research that has been carried out in search of large-scale micropropagation protocols involving TISs for application by industry.

2 2.1

Applications of TISs in Large-Scale Micropropagation of Plants Agri-food Plants

The global population growth demands producing a greater amount of food [10]. To increase crop production, a larger number of propagules should be produced for planting them in agricultural plots [11]. However, this increased plant propagation requires novel techniques, such as plant tissue cultures [11]. The increase in the propagation of plants of agri-food interest is a recurring topic in scientific research focusing on ensuring food security [12]. At a global level, new technologies have been implemented, such as temporary immersion systems designed to improve the efficiency of commercial propagule production [6]. On the other hand, plant tissue culture laboratories are considered small-scale biofactories, with countless laboratories worldwide. However, most are linked to scientific research in universities or educational centers. For this reason, they are not intended for large-scale production and marketing of plants. There are laboratories established by private industries seeking to market the plants produced and achieve their genetic improvement. Worldwide, many companies are dedicated to the large-scale production of plant propagules of agri-food interest, as well as tissue culture laboratories that offer small-scale production services. Table 1 shows examples of commercially micropropagated agrifood species.

Use of Temporary Immersion Systems in the Establishment of Biofactories

27

Table 1 Agri-food species micropropagated on a large scale in companies with a biofactory seal Cultivation technique

Web page

Biofabrica – UNAHUR (Argentina)

Semisolid TIS

https://unahur.edu.ar/biofabrica/

Phytelligence (USA)

Semisolid

https://www.qualterraag.com/

Apple tree Rahan Meristem (Israel) (rootstock)

Semisolid

http://www.rahan.co.il/?page_id= 6&lang=en

Apple tree

Tissue-Grown (USA).

Semisolid

http://www.tissuegrown.com/

Artichoke

Plant Sciences Inc. (USA)

Semisolid

https://plantsciences.com/

Avocado

Rahan Meristem (Israel)

Semisolid

http://www.rahan.co.il/?page_id= 6&lang=en

Avocado

˜ edosVivero los Vin AGREXLABS (Peru)

Semisolid

https://vlv.pe/

Banana

Rahan Meristem (Israel)

Semisolid

http://www.rahan.co.il/?page_id= 6&lang=en

Banana

Phyto Biolabs (India)

Semisolid

Banana

Biofabrica Misiones S.A. (Argentina)

Semisolid TIS

https://biofabrica.misiones.gob. ar/

Banana

Institute of Plant Biotechnology (Cuba)

Semisolid TIS

https://www.ibp.co.cu/es/

Banana

Biofabrica- Yucatan Semisolid TIS Scientific Research Center (Mexico)

https://www.cicy.mx/servicio/ biofabrica

Banana

IribovSBW (Netherlands)

https://iribov.com/

Banana

NSIP (before AGROMOD) Semisolid TIS (Mexico)

https://mx.nsiplants.com/

Banana

Institute of Plant Biotechnology (Cuba)

https://www.ibp.co.cu/es/

Banana

Biofabrica Las Tunas (Cuba) Semisolid TIS

Banana

Bioplant Center (Cuba)

Semisolid TIS

Banana

Biofa´brica de semillas Mayabeque (Cuba)

Semisolid TIS

Banana

Bioplan In Vitro (Mexico)

Semisolid

http://www.bioplaninvitro.com/

Banana

DP-Deroose Plants (Belgium)

Semisolid TIS

https://derooseplants.com/

Banana

Plant Sciences Inc. (USA)

Semisolid

https://plantsciences.com/

Banana

Galiltec (Honduras)

Semisolid

https://galiltec.com/

Species

Company

Almond Apple tree

Semisolid

Semisolid TIS

http://www.bioplantas.cu/

(continued)

28

Marco Vinicio Rodrı´guez-Deme´neghi

Table 1 (continued)

Species

Company

Cultivation technique

Web page

Barley

KWS (Spain)

Semisolid

https://www.kws.com/es/es/

Berries

Agri-Starts (USA)

Semisolid

https://www.agristarts.com/

Berries

Hartmann’s Plant Company Semisolid TIS (USA)

https://hartmannsplantcompany. com/

Berries

North American Plants (USA)

Semisolid

https://www.naplants.com/

Berries

Phytelligence (USA)

Semisolid

https://www.qualterraag.com/

Berries

Plant Sciences Inc. (USA)

Semisolid

https://plantsciences.com/

Berries

Plant Sciences Inc. (USA)

Semisolid

https://plantsciences.com/

Berries

JRT Nurseries Inc (Canada) Semisolid

http://jrtnurseries.com/

Blueberry

Cultigar (Spain)

Semisolid TIS

https://www.cultigar.es/

Blueberry

˜ edosVivero los Vin AGREXLABS (Peru)

Semisolid

https://vlv.pe/

Cereals

Inari (E.U.)

Semisolid

https://inari.com/

Cherry tree

˜ edosVivero los Vin AGREXLABS (Peru´)

Semisolid

https://vlv.pe/

Cherry tree

Cultigar (Spain)

Semisolid TIS

https://www.cultigar.es/

Semisolid

http://www.rahan.co.il/?page_id= 6&lang=en

Citrus varieties Rahan Meristem (Israel)

Semisolid TIS Coconut palm Biofa´brica- Yucatan Scientific Research Center (Mexico)

https://www.cicy.mx/servicio/ biofabrica

Coffee

NSIP (before AGROMOD) Semisolid TIS (Mexico)

https://mx.nsiplants.com/

Corn

KWS (Spain)

Semisolid

https://www.kws.com/es/es/

Corn

Syngenta (USA)

Semisolid

https://www.syngenta.com/

Cucumber

PLANTFORT (Mexico)

Semisolid

http://www.plantfort.com.mx/ index.html

Dates

Kelumilla SpA (Chile)

Semisolid

https://www.kelumilla.cl/

Dragon Fruit

Agri-Starts (USA)

Semisolid

https://www.agristarts.com/

Eggplant

PLANTFORT (Mexico)

Semisolid

http://www.plantfort.com.mx/ index.html

Grapevine

Knight Hollow Nursery (USA)

Semisolid

https://knighthollownursery.com/

Grapevine

Phytelligence (USA)

Semisolid

https://www.qualterraag.com/ (continued)

Use of Temporary Immersion Systems in the Establishment of Biofactories

29

Table 1 (continued) Cultivation technique

Web page

Semisolid TIS

http://www.bioplantas.cu/

Species

Company

Guava

Centro de Bioplantas (Cuba)

Kiwi

JRT Nurseries Inc (Canada) Semisolid

http://jrtnurseries.com/

Papaya

Biofabrica- Yucatan Semisolid TIS Scientific Research Center (Mexico)

https://www.cicy.mx/servicio/ biofabrica

Papaya

Bioplan In Vitro (Mexico)

Semisolid

http://www.bioplaninvitro.com/

Pear

Rahan Meristem (Israel)

Semisolid

http://www.rahan.co.il/?page_id= 6&lang=en

Pear

Phytelligence (USA)

Semisolid

https://www.qualterraag.com/

Pepper

PLANTFORT (Mexico)

Semisolid

http://www.plantfort.com.mx/ index.html

Pineapple

Rahan Meristem (Israel)

Semisolid

http://www.rahan.co.il/?page_id= 6&lang=en

Pineapple

´ vila (Cuba) Ciego de A

Semisolid TIS

Pineapple

Institute of Plant Biotechnology (Cuba)

Semisolid TIS

https://www.ibp.co.cu/es/

Pineapple

Bioplant Center (Cuba)

Semisolid TIS

http://www.bioplantas.cu/

Pineapple

Produce Foundation Sinaloa Semisolid (Mexico)

https://www.fps.org.mx/

Pineapple

Galiltec (Honduras)

Semisolid

https://galiltec.com/

Pineapple

Plant Biotech (Australia)

Semisolid TIS

https://www.plantbiotech.com.au/

Pistachio

Tissue-Grown (USA)

Semisolid

http://www.tissuegrown.com/

Potato

Biofabrica Mayabeque (Cuba)

Semisolid TIS

Potato

Produce Foundation Sinaloa Semisolid (Mexico)

https://www.fps.org.mx/

Potato

Valley Tissue Culture (USA) Semisolid

https://potatoseed.com/

Potato

Perfect Plants (Canada)

Semisolid

https://perfectplants.nl/

Potato

Norwa Plants (Poland)

Semisolid TIS

https://norwa.eu/

Rice

RiceTec (USA)

Semisolid

https://www.ricetec.com/

Rye

KWS (Spain)

Semisolid

https://www.kws.com/es/es/

Strawberry

ValGenetics (Spain)

Semisolid

https://fito.valgenetics.com/ (continued)

30

Marco Vinicio Rodrı´guez-Deme´neghi

Table 1 (continued) Cultivation technique

Species

Company

Taro

Plant tissue Culture Semisolid TIS Laboratory of Colegio de Mixotrophism Postgraduados Campus Cordoba (Mexico)

Taro

Biofa´brica Las Tunas (Cuba) Semisolid TIS

Taro

Galiltec (Honduras)

Semisolid

https://galiltec.com/

Taro

Plant Biotech (Australia)

Semiso´lid SIT

https://www.plantbiotech.com.au/

Tobacco

Lifeasible (USA)

Semisolid

https://www.lifeasible.com/

Tomato

PLANTFORT (Mexico)

Semisolid

http://www.plantfort.com.mx/ index.html

Tomato

Produce Foundation Sinaloa Semisolid (Me´xico)

https://www.fps.org.mx/

Tubers

Biofa´brica – UNAHUR (Argentina)

Semisolid TIS

https://unahur.edu.ar/biofabrica/

Vegetables

IribovSBW (Paises Bajos)

Semisolid

https://iribov.com/

Walnut

Biofabrica Las Tunas (Cuba) Semisolid TIS

Watermelon

PLANTFORT (Mexico)

Semisolid

http://www.plantfort.com.mx/ index.html

Watermelon

DP-Deroose Plants (Belgium)

Semisolid TIS

https://derooseplants.com/

Wheat

KWS (Spain)

Semisolid

https://www.kws.com/es/es/

Yam

Rahan Meristem (Israel)

Semisolid

http://www.rahan.co.il/?page_id= 6&lang=en

Yam

Institute of Plant Biotechnology (Cuba)

Semisolid TIS

https://www.ibp.co.cu/es/

Yucca

Biofa´brica de semillas Mayabeque (Cuba)

Semisolid TIS

TIS temporary immersion systems

Web page https://www.colpos.mx/cp/ campus-cordoba/investigacion/ laboratorio-de-cultivos-detejidos-vegetales-campuscordoba

Use of Temporary Immersion Systems in the Establishment of Biofactories

31

2.2 Agro-industrial Plants

Agricultural production requires high productivity in the field, making it necessary to sow a large number of commercial propagules [13]. However, there are species for which conventional propagation (seeds or cuttings) is insufficient to meet agricultural demands. For this reason, plant micropropagation is an alternative to guarantee plant production [14]. The establishment of biofactories for the in vitro propagation of agro-industrial species has allowed to supply plants ready to be cultivated in field crops and produce large numbers of pathogenfree, vigorous plants [3]. Table 2 shows examples of commercially micropropagated agro-industrial species.

2.3

The large-scale micropropagation of plants with medicinal properties has been studied in several scientific research studies carried by pharmaceutical companies. However, the establishment of biofactories for producing plants of pharmaceutical interest focuses on the production of complete plants but also uses cultured suspension cells for the subsequent extraction of metabolites of interest [6]. There are a large number of species with medicinal properties that have been mass-propagated for marketing purposes. The number of biofactories for the large-scale reproduction of cannabis has increased in recent years due to changes in laws and regulations that now allow its medicinal and recreational use in some countries [15]. Table 3 shows examples of commercially micropropagated medicinal plants species.

Medicinal Plants

2.4 Ornamental Plants

The in vitro propagation of plant species of ornamental interest has been one of the most profitable economic activities due to the high market value of some species [16]. Orchids are highly valued plants in the ornamental plant industry because of the colors, sizes, and shapes of their flowers [17]. Therefore, orchids have been the most reproduced ornamental plants through plant tissue cultures. However, the market value of some plant species currently exceeds the high price of orchids, in addition to having a high demand; examples include species of the family Araceae, such as Monstera spp. [18]. Biofactories dedicated to the mass propagation of ornamental plants have been established around the world, taking advantage of the various species available in different regions. Table 4 shows examples of commercially micropropagated ornamental plants species.

32

Marco Vinicio Rodrı´guez-Deme´neghi

Table 2 Agro-industrial species micropropagated on a large scale in companies with a biofactory seal Cultivation technique

Web page

NSIP (before AGROMOD) (Mexico)

Semisolid TIS

https://mx.nsiplants.com/

Agave

Plant tissue Culture Laboratory of Colegio de Postgraduados Campus Cordoba (Mexico)

Semisolid TIS Mixotrophism

https://www.colpos.mx/cp/ campus-cordoba/ investigacion/laboratorio-decultivos-de-tejidos-vegetalescampus-cordoba

Agave

Bosky (Mexico)

Semisolid TIS

https://bosky.com.mx/

Agave

Biofa´brica- Yucatan Scientific Research Center (Mexico)

Semisolid TIS

https://www.cicy.mx/servicio/ biofabrica

Agave

Rancho Tissue Technologies (USA)

Semisolid

https://www.ranchotissue.com/

Annatto

Biofa´brica- Yucatan Scientific Research Center (Mexico)

Semisolid TIS

https://www.cicy.mx/servicio/ biofabrica

Canola

Syngenta (USA)

Semisolid

https://www.syngenta.com/

Cassava

Biofa´brica Misiones S.A. (Argentina)

Semisolid TIS

https://biofabrica.misiones.gob. ar/

Cocoa

DP-Deroose Plants (Belgium)

Semisolid TIS

https://derooseplants.com/

Coffee

Biofabrica- Yucatan Scientific Research Center (Mexico)

Semisolid TIS

https://www.cicy.mx/servicio/ biofabrica

Coffee

Tissue-Grown (USA)

Semisolid

http://www.tissuegrown.com/

Coffee

CIRAD (France)

Semisolid TIS

https://www.cirad.fr/

Coffee

United Agro Industries of Mexico (Mexico)

Semisolid TIS

Cotton

Syngenta (USA)

Semisolid

https://www.syngenta.com/

Date palm

Semisolid Date Palm Tissue Culture Laboratory (DPTCL) (United Arab Emirates)

https://www.uaeu.ac.ae/en/

Fig

Mountain Shadow Nursery (USA)

Semisolid TIS

https://mtshadow.com/

Grapevine

Rahan Meristem (Israel)

Semisolid

http://www.rahan.co.il/?page_ id=6&lang=en

Grapevine

˜ edosVivero los Vin AGREXLABS (Peru)

Semisolid

https://vlv.pe/

Hop

3 Rivers Biotech (USA)

Semisolid

https://3riversbiotech.com/

Olive

Rahan Meristem (Israel)

Semisolid

http://www.rahan.co.il/?page_ id=6&lang=en

Species

Company

Agave

(continued)

Use of Temporary Immersion Systems in the Establishment of Biofactories

33

Table 2 (continued)

Species

Company

Cultivation technique

Web page

Rice

Chi Botanic (USA)

Semisolid

https://www.chibotanic.com/

Seeds

Benson Hill Biosystems (USA)

Semisolid

https://bensonhill.com/

Soy

Calyxt (USA)

Semisolid

https://calyxt.com/

Soy

Syngenta (USA)

Semisolid

https://www.syngenta.com/

Stevia

Rahan Meristem (Israel)

Semisolid

http://www.rahan.co.il/?page_ id=6&lang=en

Stevia

Plant tissue Culture Laboratory of Colegio de Postgraduados Campus Cordoba (Mexico)

Semisolid TIS Mixotrophism

https://www.colpos.mx/cp/ campus-cordoba/ investigacion/laboratorio-decultivos-de-tejidos-vegetalescampus-cordoba

Stevia

Bioplan In Vitro (Mexico)

Semisolid

http://www.bioplaninvitro.com/

Sugar beet

KWS (Spain)

Semisolid

https://www.kws.com/es/es/

Sugar cane

Biofa´brica Misiones S.A. (Argentina)

Semisolid TIS

https://biofabrica.misiones.gob. ar/

Sugar cane

Plant tissue Culture Laboratory of Colegio de Postgraduados Campus Cordoba (Mexico)

Semisolid TIS Mixotrophism

https://www.colpos.mx/cp/ campus-cordoba/ investigacion/laboratorio-decultivos-de-tejidos-vegetalescampus-cordoba

Sugar cane

Biofa´brica- Yucatan Scientific Research Center (Mexico)

Semisolid TIS

http://www.cniaa.mx/cidca

Sugar cane

Sugar Cane Research Institute (INICA) (Cuba)

Semisolid TIS

http://www.inica.minaz.cu/

Vanilla

Plant tissue Culture Laboratory of Colegio de Postgraduados Campus Cordoba (Mexico)

Semisolid TIS Mixotrophism

https://www.colpos.mx/cp/ campus-cordoba/ investigacion/laboratorio-decultivos-de-tejidos-vegetalescampus-cordoba

Vanilla

Chi Botanic (USA)

Semisolid

https://www.chibotanic.com/

Vanilla

Mexican Vanilla Research Center Semisolid TIS (CEMIVAC)

TIS temporary immersion systems

https://www.gayamexico.com/ cemivac

34

Marco Vinicio Rodrı´guez-Deme´neghi

Table 3 Medicinal plants species micropropagated on a large scale in companies with a biofactory seal Cultivation technique

Web page

Institute of Plant Biotechnology (Cuba)

Semisolid TIS

https://www.ibp.co.cu/es/

Aloe

Chi Botanic (USA)

Semisolid

https://www.chibotanic.com/

Aloe

Rancho Tissue Technologies (USA)

Semisolid

https://www.ranchotissue. com/

Aloe

Plant Biotech (Australia)

Semisolid TIS

https://www.plantbiotech. com.au/

Cannabis

Biofa´brica Misiones S.A. (Argentina)

Semisolid TIS

https://biofabrica.misiones. gob.ar/

Cannabis

BioHarvest (Israel)

Cannabis

3 Rivers Biotech (USA)

Semisolid

https://3riversbiotech.com/

Cannabis

Conception Nurseries (USA)

Semisolid

https://conceptionnurseries. com/

Cannabis

Segra (Canada)

Semisolid

https://www.segra-intl.com/

Cannabis

THC Design (USA)

Semisolid TIS

https://thcdesign.com/

Cannabis

Willow Biosciences (Canada)

Semisolid

https://willowbio.com/

Cannabis

Perfect Plants (Canada)

Semisolid

https://perfectplants.nl/

Carqueja

Biofa´brica Misiones S.A. (Argentina)

Semisolid TIS

https://biofabrica.misiones. gob.ar/

Various species

Phyton Biotech (Germany)

Liquid Bioreactors https://phytonbiotech.com/

Eucalyptus

Biofabrica Las Tunas (Cuba)

Semisolid TIS

Eucalyptus

Galiltec (Honduras)

Semisolid

https://galiltec.com/

Eucalyptus

Biofa´brica Misiones S.A. (Argentina)

Semisolid TIS

https://biofabrica.misiones. gob.ar/

Ginger

Institute of Plant Biotechnology (Cuba)

Semisolid TIS

https://www.ibp.co.cu/es/

Mint

Biofa´brica Misiones S.A. (Argentina)

Semisolid TIS

https://biofabrica.misiones. gob.ar/

Stevia

Biofa´brica Misiones S.A. (Argentina)

Semisolid TIS

https://biofabrica.misiones. gob.ar/

Vetiver

Chi Botanic (USA)

Semisolid

https://www.chibotanic.com/

Species

Company

Aloe

TIS temporary immersion systems

https://bioharvest.com/

Use of Temporary Immersion Systems in the Establishment of Biofactories

35

Table 4 Ornamental plants micropropagated on a large scale in companies with a biofactory seal

Species

Company

Cultivation technique

Web page

African violet

Bosky (Mexico)

Semisolid TIS

https://bosky.com.mx/

Agapando

AG3 (USA)

Semisolid TIS

https://www.ag3inc.com/

Agapando

Mountain Shadow Nursery (USA)

Semisolid TIS

https://mtshadow.com/

Alocasia

Rancho Tissue Technologies (USA)

Semisolid

https://www.ranchotissue. com/

Anthurium

Institute of Plant Biotechnology (Cuba)

Semisolid TIS

https://www.ibp.co.cu/es/

Anthurium

Plant tissue Culture Laboratory of Colegio de Postgraduados Campus Cordoba (Mexico)

Semisolid TIS Mixotrophism

https://www.colpos.mx/cp/ campus-cordoba/ investigacion/laboratoriode-cultivos-de-tejidosvegetales-campus-cordoba

Anthurium

Oglesby Plants (USA)

Semisolid

https://www.oglesbytc.com/

Anthurium

Anthura (Netherlands)

Semisolid TIS

https://www.anthura.nl/

Azalea

Mountain Shadow Nursery (USA)

Semisolid TIS

https://mtshadow.com/

Azalea

JRT Nurseries Inc. (Canada)

Semisolid

http://jrtnurseries.com/

Bamboo

Semisolid TIS Crescent Innovation and Incubation Council (CIIC) (India)

https://ciic.ventures/

Begonia

Norwa Plants (Poland)

Semisolid TIS

https://norwa.eu/

Bougainvillea

Bioplants Center (Cuba)

Semisolid TIS

http://www.bioplantas.cu/

Bromeliad

Bioplants Center (Cuba)

Semisolid TIS

http://www.bioplantas.cu/

Bromeliad

DP-Deroose Plants (Belgium)

Semisolid TIS

https://derooseplants.com/

Bromeliad

Oglesby Plants (USA)

Semisolid

https://www.oglesbytc.com/

Bromeliad

Plant Biotech (Australia)

Semisolid TIS

https://www.plantbiotech. com.au/

Camellia

JRT Nurseries Inc. (Canada)

Semisolid

http://jrtnurseries.com/

Carnation

Bosky (Mexico)

Semisolid TIS

https://bosky.com.mx/

Chrysanthemum

Lifeasible (USA)

Semisolid

https://www.lifeasible.com/

Dieffenbachia

Bioplants Center (Cuba)

Semisolid TIS

http://www.bioplantas.cu/

Elm tree

North American Plants (USA)

Semisolid

https://www.naplants.com/ (continued)

36

Marco Vinicio Rodrı´guez-Deme´neghi

Table 4 (continued) Cultivation technique

Species

Company

Ferns

Trusts Established in Relation Semisolid to Agriculture (FIRA) (Mexico)

https://www.fira.gob.mx/ Nd/Tezoyuca.jsp

Ferns

AG3 (USA)

Semisolid TIS

https://www.ag3inc.com/

Gerbera

Bioplants Center (Cuba)

Semisolid TIS

http://www.bioplantas.cu/

Gerbera

Norwa Plants (Poland)

Semisolid TIS

https://norwa.eu/

Heliconia

Biofabrica Misiones S.A. (Argentina)

Semisolid TIS

https://biofabrica.misiones. gob.ar/

Hibiscus

Bioplants Center (Cuba)

Semisolid TIS

http://www.bioplantas.cu/

Hydrangea

Mountain Shadow Nursery (USA)

Semisolid TIS

https://mtshadow.com/

Hydrangea

North American Plants (USA)

Semisolid

https://www.naplants.com/

Insectivorous

RAPAXBIOTEC (Mexico)

Semisolid

https://rapaxbiotec.com/

Insectivorous

AG3 (USA)

Semisolid TIS

https://www.ag3inc.com/

Lily

Knight Hollow Nursery (USA)

Semisolid

https://knighthollownursery. com/

Lily

North American Plants (USA)

Semisolid

https://www.naplants.com/

Mandevilla

Oglesby Plants (USA)

Semisolid

https://www.oglesbytc.com/

Orchids

Biofa´brica Misiones S.A. (Argentina)

Semisolid TIS

https://biofabrica.misiones. gob.ar/

Orchids

Bosky (Mexico)

Semisolid TIS

https://bosky.com.mx/

Orchids

RAPAXBIOTEC (Mexico)

Semisolid

https://rapaxbiotec.com/

Orchids

Bioplants Center (Cuba)

Semisolid TIS

http://www.bioplantas.cu/

Orchids

Cultigar (Spain)

Semisolid TIS

https://www.cultigar.es/

Orchids

DP-Deroose Plants (Belgium)

Semisolid TIS

https://derooseplants.com/

Orchids

Anthura (Netherlands)

Semisolid TIS

https://www.anthura.nl/

Ornamental

Biofabrica – UNAHUR (Argentina)

Semisolid TIS

https://unahur.edu.ar/ biofabrica/

Ornamental species Darwin Colombia S.A.S (Colombia)

Semisolid

https://www. darwinperennials.com/

Ornamental trees and shrubs

Semisolid

https://www. agriforestbiotech.com/

AgriForest Bio-Technologies (Canada)

Web page

(continued)

Use of Temporary Immersion Systems in the Establishment of Biofactories

37

Table 4 (continued)

Species

Company

Cultivation technique

Web page

Pelargonium

Norwa Plants (Polonia)

Semisolid TIS

https://norwa.eu/

Phalaenopsis

Lifeasible (USA)

Semisolid TIS

https://www.lifeasible.com/

Philodendron

Oglesby Plants (USA)

Semisolid

https://www.oglesbytc.com/

Philodendron

Bioplants Center (Cuba)

Semisolid TIS

http://www.bioplantas.cu/

Rose

Bosky (Mexico)

Semisolid TIS

https://bosky.com.mx/

Rose

Bioplants Center (Cuba)

Semisolid TIS

http://www.bioplantas.cu/

Spathiphyllum

Bioplants Center (Cuba)

Semisolid TIS

http://www.bioplantas.cu/

Spathiphyllum

Trusts Established in Relation Semisolid to Agriculture (FIRA) (Mexico)

https://www.fira.gob.mx/ Nd/Tezoyuca.jsp

Spathiphyllum

Oglesby Plants (USA)

Semisolid

https://www.oglesbytc.com/

Succulents

Rancho Tissue Technologies (USA)

Semisolid

https://www.ranchotissue. com/

Syngonium

Oglesby Plants (USA)

Semisolid

https://www.oglesbytc.com/

Tulip

IribovSBW (Netherlands)

Semisolid

https://iribov.com/

TIS temporary immersion systems

3

Concluding Remarks on the Use of TIS in Biofactories The establishment of biofactories for large-scale production of plants is an economic activity involving the technological and scientific development of each country [19]. In this sense, some countries are committed to biotechnology to boost agricultural development through plant micropropagation (Fig. 1). As well as gaining profits by exporting plant material to countries demanding large numbers of propagules. The biofactories established in Latin America are mainly joint efforts with federal institutions (universities, government agencies, and others) and private institutions. The facilities and operation of an agrobiotechnology biofactory involve high initial costs, and it is mentioned that the return of these costs takes three to 5 years of operation [3, 9]. This depends entirely on the production volume for which the biofactory is designed. For this reason, alternatives that reduce production costs are always sought, such as eliminating gelling agents, implementing automated processes, using homemade materials, and others [6].

38

Marco Vinicio Rodrı´guez-Deme´neghi

Fig. 1 Sugar Cane Research Institute Biofactory (Cuba). (a) Large-scale micropropagation of banana in SETISTM biorector. (b) Large-scale micropropagation of sugar cane in SETISTM bioreactor. (c) Banana propagules obtained in TIS in acclimatization. (d) Sugar cane propagules obtained in TIS in acclimatization

The use of temporary immersion systems (TISs) is a strategy to reduce costs, increase plant production, generate plants with enhanced morpho-physiological characteristics, and facilitate semiautomated processes [20]. However, TISs are only applied in some of the biofactories already mentioned for the following reasons: high costs of infrastructure and temporary immersion systems, lack of knowledge in the technical operation, and lack of a research department in some biofactories. Some actions could ameliorate these difficulties: 1. The use of homemade materials for the manufacture of temporary immersion systems (twin flasks).

Use of Temporary Immersion Systems in the Establishment of Biofactories

39

2. Attend specialized courses on the installation and operation of TIS to learn how to install the necessary infrastructure at a low cost and properly operate TISs. 3. Partner with research/public education institutions to have the support of a research area at a low cost. 4. Seek funds from associations, calls, or government agencies. 5. Have specialized technical advice. On the other hand, the mass production of plants is a profitable business due to the high demand by the agricultural sector. There are ornamental species with a price per unit much higher than the one of other types of plants. New small-scale biofactories (in vitro culture laboratories) are emerging in Mexico, seeking to benefit from this situation. However, key factors that has prevented production scale-up are the lack of an adequate marketing strategy and the low survival rates during acclimatization. Issues that must be solved before establishing a biofactory include the identification of the local, national, and international markets, distribution methods, potential buyers, technical specialists, and others. In North America, the number of biofactories in charge of the large-scale propagation of cannabis has increased due to the modification of the regulations that now allow its medicinal and recreational use [15]. In this sense, there is an increasing number of companies that offer different cannabis varieties, which will probably reduce profits as a result of the market competition. In addition, in the United State, the technological development and scientific research for the production of berries are reflected in most biofactories being dedicated to producing and commercializing this crop. Research in ornamental plants is also leveraged by different biofactories around the world [21]. In summary, biofactories are companies dedicated to the largescale production of plant species of economic interest. In addition, there are countries such as Cuba and the United States where these companies have experienced sustained development for several decades, while in other countries, such as Mexico, their development is recent (except for some companies). Biofactories require strategies to reduce production costs, such as adopting temporary immersion systems; however, most biofactories still need to adapt their micropropagation protocols from semisolid media to TISs. Scientific research and technological development related to the use of TIS in plant micropropagation are some of the biotechnology topics that deserve attention from the scientific community.

40

Marco Vinicio Rodrı´guez-Deme´neghi

References 1. Steinwand MA, Ronald PC (2020) Crop biotechnology and the future of food. Nat Food 1: 273–283. https://doi.org/10.1038/s43016020-0072-3 2. Madzikane O, Gebashe FC, Amoo SO (2022) Use of alternative components in cost-effective media for mass production of clonal plants. In: Gupta S, Chaturvedi P (eds) Commercial scale tissue culture for horticulture and plantation crops. Springer, Singapore, pp 49–64. https://doi.org/10.1007/978-981-190055-6_3 3. Rodrı´guez-Deme´neghi MV, RamirezMosqueda MA, Armas-Silva AA, AguilarRivera N, Gheno-Heredia YA (2022) Biofa´bricas de vainilla (Vanilla planifolia Jacks.) en Me´xico como oportunidad de desarrollo agrario. Cuadernos de Biodiversidad 63:49–54. https://doi.org/10.14198/cdbio.21952 4. Dı´az NL, Pe´rez IB, Garcı´a JCS, Francesena DZV, Dı´az IA (2022) Diagno´stico del clima ˜ a de organizacional en la Biofa´brica de Can Azu´car de Villa Clara. Revista Cientı´fica Agroecosistemas 10:13–22. https://aes.ucf.edu.cu/ index.php/aes 5. San Jose´ MC, Bla´zquez N, Cernadas MJ, Janeiro LV, Cuenca B, Sa´nchez C, Vidal N (2020) Temporary immersion systems to improve alder micropropagation. Plant Cell Tissue Organ Cult 143:265–275. https://doi. org/10.1007/s11240-020-01937-9 6. Hwang HD, Kwon SH, Murthy HN, Yun SW, Pyo SS, Park SY (2022) Temporary immersion bioreactor system as an efficient method for mass production of in vitro plants in horticulture and medicinal plants. Agronomy 12:346. h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / agronomy12020346 7. Garcı´a-Ramı´rez Y (2023) Temporary immersion system for in vitro propagation via organogenesis of forest plant species. Trees 37:611– 626. https://doi.org/10.1007/s00468-02202379-w 8. Monja-Mio KM, Olvera-Casanova D, HerreraHerrera G, Herrera-Alamillo MA, SanchezTeyer FL, Robert ML (2020) Improving of rooting and ex vitro acclimatization phase of Agave tequilana by temporary immersion system (BioMINT™). In Vitro Cell Dev Biol Plant 56:662–669. https://doi.org/10. 1007/s11627-020-10109-5

˜a JC, Caamal Velazquez JH, 9. Alamilla Magan Criollo Chan MA, Vera Lopez JE, Reyes Montero JA (2019) Biofa´bricas y biorreactores de inmersio´n temporal: propagacio´n in vitro de Anthurium andreanum L., y su viabilidad econo´mica. Agro Productividad 12. https://doi. org/10.32854/agrop.vi0.1457 10. Fro´na D, Szendera´k J, Harangi-Ra´kos M (2019) The challenge of feeding the world. Sustain For 11:5816. https://doi.org/10. 3390/su11205816 11. Gulzar B, Mujib A, Malik MQ, Mamgain J, Syeed R, Zafar N (2020) Plant tissue culture: agriculture and industrial applications. In: Kiran U, Abdin MZ, Kamaluddin (eds) Transgenic technology based value addition in plant biotechnology. Academic, pp 25–49. https:// doi.org/10.1016/B978-0-12-818632-9. 00002-2 12. Lenaerts B, Collard BC, Demont M (2019) Improving global food security through accelerated plant breeding. Plant Sci 287:110207. https://doi.org/10.1016/j.plantsci.2019. 110207 13. Lal N (2021) Micropropagated plants as alternative planting material to sugarcane setts. Indian J Biotechnol 8:27–30 14. Martins M, Ribeiro MH, Almeida CM (2023) Physicochemical, nutritional, and medicinal properties of Opuntia ficus-indica (L.) mill. and its main agro-industrial use: a review. Plan Theory 12:1512. https://doi.org/10.3390/ plants12071512 15. Monthony AS, Page SR, Hesami M, Jones AMP (2021) The past, present and future of Cannabis sativa tissue culture. Plan Theory 10: 1 8 5 . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / plants10010185 16. Pyati AN (2022) In vitro propagation of some important medicinal and ornamental Dendrobiums (Orchidaceae): a review. J Appl Hortic 24:245–253. https://doi.org/10.37855/jah. 2022.v24i02.45 17. Setiaji A, Annisa RRR, Santoso AD, Kinasih A, Riyadi ADR (2021) In vitro propagation of Vanda orchid: a review. Comun Sci 12:e3427– e3427. https://doi.org/10.14295/cs.v12. 3427 18. Casanova Palomeque NM, Bertolini V, Donjuan LI (2021) In vitro establishment: Monstera acuminata Koch and Monstera deliciosa

Use of Temporary Immersion Systems in the Establishment of Biofactories Liebm. Trends Hortic 4:13–21. https://doi. org/10.24294/th.v4i1.1795 19. Bapat VA, Kavi Kishor PB, Jalaja N, Jain SM, Penna S (2023) Plant cell cultures: biofactories for the production of bioactive compounds. Agronomy 13:858. https://doi.org/10. 3390/agronomy13030858

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20. Mirzabe AH, Hajiahmad A, Fadavi A, Rafiee S (2022) Temporary immersion systems (TISs): a comprehensive review. J Biotechnol 357:56– 83. https://doi.org/10.1016/j.jbiotec.2022. 08.003 21. Lal N, Singh M (2020) Prospects of plant tissue culture in orchid propagation: a review. Indian J Biotechnol 7:103–110

Part II Micropropagation in TIS of Agroindustrial Species: Protocols

Chapter 4 Large-Scale Micropropagation of Vanilla (Vanilla planifolia Jacks.) in a Temporary Immersion Bioreactor (TIB) Marco A. Ramı´rez-Mosqueda, Marco Vinicio Rodrı´guez-Deme´neghi, Heidi P. Medorio-Garcı´a, and Rube´n H. Andueza-Noh Abstract The cultivation of vanilla (Vanilla planifolia) is of economic interest because vanillin is extracted from the fruits of this species. Vanillin is a natural flavoring highly valued in the food market. However, there is a short supply of propagules available for establishing commercial plantations and good-quality plants with phytosanitary certification. Plant tissue culture represents a viable option to supply large amounts of healthy plants to vanilla producers. In addition, the use of temporary immersion systems will allow commercial scale-up and the establishment of biofactories dedicated to in vitro vanilla propagation. This chapter describes a large-scale micropropagation protocol for vanilla using temporary immersion bioreactors (TIB). Key words Massive micropropagation, Commercial propagules, Temporary immersion system, TIB

1

Introduction Plant tissue culture is a tool used to produce vanilla plants (Vanilla planifolia Jacks.) [1]. However, new technologies are needed for large-scale micro-propagation to supply propagules [2]. The use of new technologies, such as temporary immersion systems (TIS), has increased the in vitro propagation of various plant species [3]. In this context, there are different types of TIS on the market that differ in terms of design, manufacturing materials, cost, and efficiency [4]. In V. planifolia, a few studies have compared the efficiency of different types of TIS during in vitro propagation. Among these, the temporary immersion bioreactor (TIB) is the current option that yields the highest number of shoots per explant [5]. However, other types of TIS designed more recently, such as SETIS™, have improved the height of cultivated plants [6]. The TIB consists of two interconnected twin flasks, in which one flask contains the explants (culture chamber) and the other the culture medium (storage container) [4]. In addition, this system

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_4, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

45

46

Marco A. Ramı´rez-Mosqueda et al.

can be assembled at a low cost and coupled to a CO2 injection system (photomixotrophic system) to increase the number of plants obtained [7, 8]. This type of TIS would be effective in establishing a biofactory dedicated to the large-scale micropropagation of vanilla. This chapter describes an effective protocol that uses a TIB for the large-scale micropropagation of V. planifolia. This protocol can be used to establish and operate biofactories to produce thousands of commercial propagules of this species. The use of a TIB for mass propagation reduces costs and produces a large number of vigorous plants of high phytosanitary quality.

2 2.1

Materials Plant Material

2.2 Surface Sterilization 2.3

Culture Media

Greenhouse-grown V. planifolia (Mansa morphotype) plants (Fig. 1a). The plants were collected from the Papantla region, Veracruz, Mexico. Disinfectant solution: 0.6% (v/v) sodium hydrochloride, Tween® 20 (Three drops per 100 mL solution), and commercial detergent. 1. Basal culture medium: the stock solutions of MS medium (Murashige and Skoog salts) [9] (see Table 1). 2. Medium additives used for various culture stages: MS medium supplemented with various additives according to the culture stage (Table 2), (1) establishment, (2) shoot multiplication, (3) shoot multiplication TIB, and (4) rooting. 3. pH adjustment solutions: 1 N sodium hydroxide (NaOH) and hydrochloric acid (HCl).

2.4

Equipment

1. Culture room: airflow cabinet, bead sterilizer. 2. Surgical tools: stainless steel tweezers of 25 cm, surgical scalpels, removable sterile surgical blades, sterile craft paper, cotton, and adherent film. 3. Medium preparation: analytical balance, spatula, hot stirrer plate, pH meter, micropipettes, magnetic stirrer, refrigerator, and autoclave. 4. Glassware: bottles (200 mL capacity), beakers (500 mL and 1 L capacity), reagent bottles, and measuring cylinders. 5. Culture vessels: glass culture jars (100 mL).

2.5 Bioreactor System

1. Temporary immersion bioreactors (TIB) (100 mL). 2. Platforms (metal shelf) equipped with silicone tubing/hoses, air compressor equipment, filters, and LED lighting system.

Vanilla Micropropagation Using TIB

47

Fig. 1 Establishment of Vanilla planifolia in TIB. (a) Vanilla mother plants, (b) nodal segments (containing an axillary bud), (c) disinfection of the explants (nodal segments) in the laminar flow hood

3. Timer (1 unit for platform) for control the photoperiod. 4. Cling film (3 cm width).

3 Methods 3.1

Culture Media

3.1.1 Preparation of MS Stocks

1. The stock solutions (1–6) of MS medium (Table 1) are prepared separately. 2. Weigh and dissolve the components of each stock in distilled water by using a magnetic stirrer, and make up the final volume to 1000 mL.

3.1.2 Plant Growth Regulators (PGR) Stock Solutions

1. Benzyladenine (BA, 0.1 mg/mL): Dissolve 10 mg of BA in 1 mL 1 N NaOH, and then add water to a volume of 100 mL.

3.1.3 Preparation Culture Media

1. Prepare the culture medium from mother stock solutions and distilled water. Except for the hormonal balance, the media

48

Marco A. Ramı´rez-Mosqueda et al.

Table 1 Salts components of MS medium

N° stock Compound Vitamins

1

Micronutrients 2

Macronutrients 3

4

5 EDTA-Fe

6

Amount required Concentration for 1 L stock of the final solution 100 X, medium (mg/L) mg/L

mL of stock/ L

Glycine Nicotinic acid Pyridoxine-HCl Thiamine-HCl Myoinositol

2 0.5 0.5 0.1 100

200 50 50 10 10,000

10

KI (potassium iodide) MnSO4.H2O (manganese sulfate monohydrate) H3BO3 (boric acid) ZnSO4.7H2O (zinc sulfate heptahydrate) NaMoO4.2H2O (sodium molybdate dihydrate) CuSO4.5H2O (copper sulfate pentahydrate) CoCl2.6H2O (cobalt chloride hexahydrate)

0.83 16.90

83 1690

10

6.2 8.6

620 860

0.25

25

0.025

2.5

0.025

2.5

KH2.PO4 (potassium phosphate monobasic) MgSO4.7 H2O (magnesium sulfate heptahydrate) KNO3 (potassium nitrate) NH4.NO3 (ammonium nitrate) CaCl2.2H2O (calcium chloride)

170

17,000

370

37,000

1820 400

182,000 40,000

10

440

44,000

10

3672

10

EDFS 36.72 (ethylenediaminetetraacetic acid iron (III) sodium salt)

10

have the same compositions for the three propagation stages (Table 2). 2. Add aliquot of hormones according to the culture stages (Table 2). 3. Using a pH meter, adjust the pH to 5.8 by adding one drop of 1 N NaOH or HCl while stirring continuously. 4. Distribute 250 mL of culture medium in glass bottles (in only one of the two bottles that make up the TIB) of 1000 mL capacity [5] (see Note 1). 5. Autoclave at 121 °C with a pressure of 15 psi for 20 min.

Vanilla Micropropagation Using TIB

49

Table 2 Substances that complement culture media Culture stage Media additives

Shoot multiplication medium

Shoot growth medium

Rooting medium

Stock 1–6

10 mL

10 mL

10 mL

2,4-Dichlorophenoxyacetic acid (2,4-D)

0.113 μM

0.113 μM

–

Benzylaminopurine (BAP)

44.4 μM

4.44 μM

–

Sacarose

30 g

30 g

30 g

6. After autoclaving, the glass bottles are kept in a clean room until use. 3.2 Explants: Plant Material

1. Nodal segments of 1–2 cm in length are used (establishment, shoot multiplication, and shoot multiplication TIS). 2. Shoots of 2 cm in length are used (rooting).

3.3

Establishment

1. Nodal segments 2 cm long (contents an axillary bud) should be extracted from greenhouse-grown plants (Fig. 1b). 2. Applying a 1 g/L solution of commercial fungicide and bactericide for 15 min. 3. Disinfect the laminar flow surface with 70% alcohol after UV light exposure for 15 min. 4. Nodal segments are rinsed for 10 min in a 0.6% (v/v) sodium hydrochloride solution with three drops of Tween® 20 per 100 mL of water (Fig. 1c). After, rinse three times with sterile distilled water and place in glass culture jars 100 mL containing 20 mL of medium (Table 2) (see Note 2). 5. After 45 days, the nodal segments are transferred for multiplication.

3.4 Shoot Multiplication

1. Nodal segments, 1–2 cm in length to be transferred to 100 mL glass culture jars containing 20 mL of medium (Table 2) [5]. 2. The medium pH is adjusted to 5.8, and 2.2 g/L Phytagel was added as a gelling agent. Then, the medium is sterilized in an autoclave for 15 min at 120 °C and 115 kPa. 3. The explants are incubated at 24 ± 2 °C, under 40 ± 5 μmol m-2 s-1 irradiance and a 16 h photoperiod. After two subcultures (30 days each), proceed to the multiplication stage using the TIB.

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3.5 Shoot Multiplication TIB

1. The TIB (1000 mL) are used. To sterilize these containers, take them to a laminar flow hood. Prepare a 0.3% (v/v) solution of sodium hydrochloride, and leave it still for 5 min. After rinsing the containers three times with sterile water, turn on the UV light in the laminar flow hood for 10 min (see Note 3). 2. Then, pour into the container 250 mL of medium previously described (Table 2) [5]. Subsequently, place ten explants at the top of the TIS. 3. Connect both containers to an air pump and apply air pressure to submerge the explants. After the immersion time, a solenoid valve prevents the passage of air, and the medium returns to the original container. Use an immersion frequency of 2 min every 8 h for 30 days. Maintain the explants under the light and temperature conditions previously mentioned (Fig. 2a, b) [5].

3.6

Rooting

1. After 30 days, vanilla shoots be individualized for better nutrient absorption. Divide agglomerate of shoots at base (shoots of 2 cm long). 2. Change the container for rooting medium containing 250 mL of half-strength MS medium with no PGR supplemented with 30 g/L sucrose (Table 2) [5]. 3. Use an immersion frequency of 2 min every 8 h for 30 days. Maintain the explants under the light and temperature conditions previously described [5].

3.7

Acclimatization

1. Prepare the substrate in trays (50 × 30 × 5 cm). The substrate consists of a mixture of peat and agrolite 1:1 (v/v) (see Note 4) [5]. 2. Wash the shoots rooting to remove adhering MS medium (Fig. 2c). 3. Prepare a 1 g/L fungicide and bactericide solution and submerge the seedlings for 10 min. 4. Before seeding, moisten the substrate. After seeding, cover the seedlings with a translucent dome. Maintain seedlings under greenhouse conditions with 60% shade and at 30 ± 2 °C, relative humidity of 60 ± 10%, and natural light with an irradiance of 80 ± 10 μmol m-2 s-1 for 1 month (Fig. 2d) (see Notes 5–7) [5].

4

Notes 1. For the establishment and shoot multiplication stage, it is necessary to use 2.2 g/L of Phytagel and dose 100 mL containing 20 mL of medium in glass culture jars.

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Fig. 2 Micropropagation of Vanilla planifolia in TIB. (a) Temporary immersion bioreactors (TIB). (b) In vitro proliferation of vanilla shoots in TIB. (c) In vitro rooting of vanilla. (d) Acclimatization of vanilla

2. To ensure asepsis, four seeds were established per flask. 3. It is recommended to assemble the bioreactors and verify with running water that the system works correctly. Verify that caps and hoses do not leak; this can be solved using safety straps. If the TIB bottle containing the explants has a curvature at the bottom, the bottle can be tilted to avoid stagnation of the culture medium that favors hyperhydricity (Fig. 2a). 4. Sterilize the substrate in the autoclave for 30 min at 120 °C and 115 kPa. 5. Maintain the pots closed for a week to avoid moisture loss. Sprinkle water on the dome to maintain a cool environment. 6. After a month, keep the trays at 30 ± 5 °C, with a relative humidity of 30% and natural light with an irradiance of

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250 ± 10 μmol m-2 s-1. If the temperature surpasses 30 °C, water daily and fertilize and fumigate once a week. 7. Maintaining the seedlings under open-sky conditions is suggested to prepare them for field transplant. References 1. Ramı´rez-Mosqueda MA, Bello-Bello JJ, ArmasSilva AA, Rodrı´guez-Deme´neghi MV, Martı´nezSantos E (2022) Advances in somatic embryogenesis in vanilla (Vanilla planifolia Jacks.). In: Ramı´rez-Mosqueda MA (ed) Somatic embryogenesis: methods and protocols. Humana Press, New York, pp 29–40. https://doi.org/10. 1007/978-1-0716-2485-2_3 2. Rodrı´guez-Deme´neghi MV, RamirezMosqueda MA, Armas-Silva AA, Aguilar-RiveraN, Gheno-Heredia YA (2022) Biofa´bricas de vainilla (Vanilla planifolia Jacks.) en Me´xico como oportunidad de desarrollo agrario. Cuadernos de Biodiversidad 63:49–54. https://doi. org/10.14198/cdbio.21952 3. De Carlo A, Tarraf W, Lambardi M, Benelli C (2021) Temporary immersion system for production of biomass and bioactive compounds from medicinal plants. Agronomy 11:2414. https://doi.org/10.3390/agronomy11122414 4. Mirzabe AH, Hajiahmad A, Fadavi A, Rafiee S (2022) Temporary immersion systems (TISs): a comprehensive review. J Biotechnol 357:56–83. https://doi.org/10.1016/j.jbiotec.2022. 08.003 5. Ramı´rez-Mosqueda MA, Iglesias-Andreu LG (2016) Evaluation of different temporary

immersion systems (BIT®, BIG, and RITA®) in the micropropagation of Vanilla planifolia Jacks. In Vitro Cell Dev Biol Plant 52:154– 160. https://doi.org/10.1007/s11627-0159735-4 6. Ramı´rez-Mosqueda MA, Bello-Bello JJ (2021) SETIS™ bioreactor increases in vitro multiplication and shoot length in vanilla (Vanilla planifolia Jacks. Ex Andrews). Acta Physiol Plant 43: 52. https://doi.org/10.1007/s11738-02103227-z 7. Georgiev V, Schumann A, Pavlov A, Bley T (2014) Temporary immersion systems in plant biotechnology. Eng Life Sci 14:607–621. https://doi.org/10.1002/elsc.201300166 8. Spinoso-Castillo JL, Jabı´n BBJ (2023) CO2enriched air in a temporary immersion system induces photomixotrophism during in vitro multiplication in vanilla. Plant Cell Tissue Organ Cult 1–11. https://doi.org/10.1007/ s11240-023-02546-y 9. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962. tb08052.x

Chapter 5 Temporary Immersion Bioreactors for Sugarcane Multiplication and Rooting Jose´ Luis Spinoso-Castillo, Marı´a Karen Serrano-Fuentes, Monserrat Sorcia-Morales, and Jerico´ Jabı´n Bello-Bello Abstract Sugarcane is used to produce sugar, ethanol, and other by-products, so it is considered one of the most important crops worldwide. Using temporary immersion systems for sugarcane micropropagation represents an alternative to reduce the labor force, increase plant development, and improve plant quality. Temporary immersion systems are semi-automated bioreactors designed for the large-scale propagation of tissues, embryos, and organs. These are temporarily exposed in a liquid culture medium under a specific time and immersion frequency. Using this protocol and a temporary immersion bioreactor, it is possible to achieve multiplication and rooting. The use of temporary immersion bioreactors improves the multiplication rate and the rooting of sugarcane, reducing the culture time, labor force, and reagents needed while maintaining high survival rates during acclimatization. Key words Plant tissue culture, Micropropagation, Shoot proliferation, Acclimatization

1

Introduction The importance of sugarcane (Saccharum spp. Hybrids) resides in the fact that it is the main source of sugar consumed worldwide. Additionally, this plant can be used to obtain different by-products and coproducts, such as bagasse, molasses, and bioethanol [1, 2]. Various reports describe using plant tissue culture techniques for the large-scale in vitro propagation of sugarcane, resulting in rejuvenated, pest-free, and disease-free plants [3]. However, conventional techniques use semisolid culture media and are labor intensive, which limits semi-automation and increases production costs. Implementing liquid culture media and bioreactors represents an alternative to reduce production costs, facilitate semiautomation, and increase the in vitro regeneration capacity [4]. The use of temporary immersion systems (TISs) for the in vitro propagation of sugarcane increases the availability of culture media

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_5, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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components, such as nutrients, vitamins, and growth regulators, among others [5]. TISs can increase the physiologic and morphologic performance of plants by providing the right conditions for optimal aeration and nutrition [6]. Furthermore, it has been shown that the photomixotrophic capacity of explants is due to the atmospheric renewal inside the container. This is because TISs allow a greater flow of gases (O2, CO2, and N2), inducing plants to go from a heterotrophic to an autotrophic state, also known as in vitro photomixotrophism. This effect influences survival during acclimatization [7]. The temporary immersion bioreactor (TIB) is of Cuban origin and consists of twin flasks, one for growing plants and the other for liquid media. Both flasks are connected by silicone tubes and use hydrophobic filters, as described by Escalona et al. [8]. In this chapter, we describe the in vitro propagation of sugarcane using a TIB. This system has improved the micropropagation of sugarcane in commercial biofactories, decreasing production costs and obtaining healthy, homogeneous, and reinvigorated seedlings. This chapter aims to show an efficient protocol for the micropropagation of sugarcane using a TIB for multiplication and rooting stages.

2 2.1

Materials Plant Material

2.2 Surface Sterilization 2.3

Culture Medium

Thirty-centimeter apices excised from mature (8 months old) fieldgrown sugarcane plants (Saccharum spp. cv MEX-69–290) growing in Veracruz, Mexico, were used as initial plant material. Disinfectant solution: 0.6% (v/v) sodium hydrochloride, Tween® 20 (three drops per 100 mL solution), and commercial detergent. 1. Basal Murashige and Skoog (MS) [9] culture medium is used (see Note 1). 2. Medium additives used for various culture stages: MS basal medium supplemented with various additives according to the culture stage, including culture initiation (CI), shoot multiplication (SM), temporary immersion system liquid medium (SML), and rooting medium (RM) (see Table 1). 3. pH adjustment solutions: 0.1 and 1 N NaOH and HCl.

2.4

Equipment

1. Culture room: thermostatic bath and laminar airflow hood. 2. Surgical tools: stainless steel forceps, surgical scalpels, removable sterile surgical blades, and sterile Petri dishes.

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Table 1 Culture medium additives used for the different culture stages of sugarcane Culture stage

Media additives

Culture initiation (CI)

Shoot multiplication on semisolid medium (SM)

Shoot multiplication in liquid medium (SML)

Rooting medium (RM)

Cysteine (mg/L)

50

–

–

–

Methylene blue (mg/L)

1

1

1

–

Kinetin (KIN) (mg/L)

–

1

1

–

Indoleacetic acid (IAA) (mg/L)

–

0.6

0.6

–

Benzylaminopurine (BAP) (mg/L)

–

0.6

0.6

–

1-Naphthaleneacetic acid (NAA) (mg/L)

–

–

–

2

Indole-3-butyric acid – (IBA) (mg/L)

–

–

2

Activated charcoal (g/L)

–

–

–

1

Sucrose (g/L)

30

30

30

30

Phytagel (g/L)

2.2

2.2

–

–

3. Medium preparation: weighing balances, magnetic stirrer, hot plate, pH meter, microwave, micropipettes, magnets, refrigerator, and autoclave. 4. Culture vessels: reagent bottles, flasks (500 mL and 1000 mL capacity), beakers, measuring cylinders, and culture tubes (25 × 150 mm) with polypropylene caps and recycled 1-gallon water bottles. 5. Glassware cleaning: Scrub glassware with a liquid detergent solution, thoroughly wash with tap water, and rinse glassware with distilled water and dry.

3

Methods

3.1 Stage 0: Plant Selection

1. Before cutting the sugarcane apices, experts must identify the variety (Fig. 1a) (see Note 2). 2. Once the variety has been identified, plants must be quarantined, applying a 1 g/L solution of fungicide and bactericide (see Note 3).

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Fig. 1 Selection of mother plant and in vitro establishment of sugarcane. (a) Variety identification (donor plants), (b) explant preparation, (c) disinfestation with sodium hypochlorite, and (d) in vitro establishment

3. For the in vitro establishment, young apices of about 40 cm in length must be cut off, removing excess leaves before transferring to the laboratory (see Note 4). 3.2 Stage I: In Vitro Establishment

1. After cutting off the sugarcane plant, apices must be washed with water and commercial soap. Then, wrapped in paper bags and kept at 8 °C for 2–3 days. Then, apices must be cut to a length of 15 cm and subjected to hydrothermotherapy in a circulating thermostatic bath at 50 °C for 20 min (Fig. 1b) (see Notes 5–6). 2. Disinfect the laminar flow surface with 70% alcohol after UV light exposure for 15 min. 3. Apices are rinsed for 5 min in a 0.6% (v/v) sodium hydrochloride solution with three drops of Tween® 20 per 100 mL of water (Fig. 1c) (see Notes 7–8). Apices are placed in tubes containing MS medium without regulators (Fig. 1d) (see Note 1). 4. After 1 week, the apices are transferred for multiplication to MS medium.

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Fig. 2 In vitro shoot multiplication of sugarcane. (a) Multiplication using semisolid medium, (b) multiplication of shoots in temporary immersion bioreactor 3.3 Stage II: In Vitro Multiplication Using Semisolid Medium

1. For the multiplication in semisolid media stage, apices need to be transferred to 500 mL flasks containing 30 mL of MS medium supplemented with 30 g/L sucrose, 1 mg/L kinetin (KIN), 0.6 mg/L indoleacetic acid (IAA), and 0.6 mg/L benzylaminopurine (BAP) (Fig. 2a) (see Note 9) [10]. 2. The medium pH is adjusted to 5.8, and 2.2 g/L Phytagel was added as a gelling agent. Then, the medium is sterilized in an autoclave for 15 min at 120 °C and 115 kPa. 3. The explants are incubated at 24 ± 2 °C, under 40 ± 5 μmol m2 -1 s irradiance and a 16 h photoperiod. After four subcultures, proceed to the multiplication stage using the TIB.

3.4 Establishing Culture in TIB

1. The bioreactor consists of two flasks: one is used to culture the explants, and the second contains the culture medium [8]. Use recycled 1-gallon water bottles. To sterilize these containers, take them to a laminar flow hood. Prepare a 0.3% (v/v) solution of sodium hydrochloride and leave it still for 5 min. After rinsing the containers three times with sterile water, turn on the UV light in the laminar flow hood for 10 min (see Note 10). 2. Then, pour into one of the containers 500 mL of the multiplication culture medium previously described (see Note 9) [10]. In the empty container, place ten explants (Fig. 2b) (see Note 11). 3. Connect both containers to an air pump and apply air pressure to submerge the explants. After the immersion time, a solenoid valve prevents the passage of air, and the medium returns to the original container [8]. Use an immersion frequency of 2 min every 8 h for 30 days. Maintain the explants under the light and temperature conditions previously mentioned [10].

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3.5 Stage III: Elongation and Rooting

1. In vitro rooting is a strategy that improves seedling survival during the acclimatization phase [11]. During this phase, hormones are added to the culture medium to induce elongation and rooting (see Notes 12–13). 2. After 30 days in the multiplication medium, change the container for rooting medium containing 500 mL of MS medium supplemented with 30 g/L sucrose, 2 mg/L indole-3-butyric acid (IBA), and 2 mg/L 1-naphthaleneacetic acid (NAA) [3]. 3. Use an immersion frequency of 2 min every 8 h for 30 days. Maintain the explants under the light and temperature conditions previously described [10].

3.6 Stage IV: Acclimatization

1. Prepare the substrate in 50-cavity trays. The substrate consists of a mixture of compost, peat, and agrolite 1:1:1 (v/v) (see Note 14) [12]. 2. Separate seedlings individually and carefully wash the shoots and roots to remove adhering MS medium. 3. Prepare a 1 g/L fungicide and bactericide solution and submerge the seedlings for 10 min. 4. Before seeding, moisten the substrate. After seeding, cover the seedlings with a translucent dome. Maintain seedlings under greenhouse conditions with 60% shade and at 30 ± 2 °C, relative humidity of 60 ± 10%, and natural light with an irradiance of 80 ± 10 μmol m-2 s-1 for 1 month (Fig. 3a, b) (see Notes 15–17) [12].

Fig. 3 Acclimatization of sugarcane plantlets. (a) Plantlets at 30 days and (b) ready for field transfer after 60 days

Sugarcane Micropropagation Using TIB

4

59

Notes 1. Prepare separate MS stock A-F solutions. Then, weigh and dissolve the components of each stock in 50 mL of distilled water using a magnetic stirrer, and make up the final volume to 100 mL by using reverse osmosis water (prepared by purifying deionized water to attain a sensitivity of 18 MΩ-cm at 25 °C) and analytical grade reagents. Diligently follow all waste disposal regulations when disposing of waste materials. For 1 L semisolid MS medium elaboration, pour 750 mL of distilled water into a 1 L flask with a magnetic stirring bar, add 30 g of sucrose, and dissolve. Adjust the pH to 5.8 with a pH meter by adding 0.1 or 1 N NaOH or HCl with constant stirring. Then, add 2.2 g of Phytagel and make up the volume to 1 L with distilled water using a 1 L measuring cylinder. Heat the culture medium until it starts boiling, dispense 10 mL of medium per culture tube, and cap the tubes. Autoclave at 120 °C with a pressure of 115 kPa, for 15 min. Take out the culture medium from the autoclave, and allow it to cool and solidify at room temperature. 2. Identify the taxonomic variety by experts to avoid varietal mixing. 3. Cut off the apices during the morning and extract them from 3-month-old plants and a maximum of 8 or 9 months old. 4. Choose plants visibly free of lateral aerial shoots, pests, and diseases that cause biotic and abiotic stress. 5. Cut stems about 4–5 cm thick. In sugarcane, smaller thickness reflects poor development. 6. Leave thick layers of the stem to protect apices from thermal shock and prevent oxidation. Oxidation leads to a decrease in plant viability. 7. When sugarcanes are 3–5 months of age, thermotherapy at 50 ° C for 8 min is recommended and 20 min for sugarcanes older than 5 months. 8. It is suggested not to handle the apices in the thicker layers with a scalpel; this will prevent their oxidation. To prevent oxidation, cut off the apices around 2 cm long and keep them hydrated with a 50 mg/L cysteine solution. 9. For 1 L MS medium elaboration, pour 750 mL of distilled water into a 1 L flask with a magnetic stirring bar, add 30 g of sucrose, and dissolve. Add an aliquot of hormones, and methylene blue (1 mg/L) can be added to the multiplication medium to prevent phenolization. Adjust the pH to 5.8 with a pH meter by adding 0.1 or 1 N NaOH or HCl. Autoclave at 120 °C with a pressure of 115 kPa, for 15 min.

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10. To ensure asepsis, individualize the apices established in different flasks. It is recommended to assemble the bioreactors and verify with running water that the system works correctly. Verify that caps and hoses do not leak; this can be solved using safety straps. 11. Divide ten 2 cm long explants (2–3 shoots, each fused at the base). 12. Before rooting the sugarcane seedlings, shoots can be individualized for better nutrient absorption. 13. Activated carbon (1 g/L) can be added to the liquid culture medium. Activated carbon is an antioxidant molecule that absorbs phenols and has been shown to stabilize nutrients and growth hormones. Additionally, this molecule acts as a primary substrate in tissue culture to darken the environments of the culture medium [13, 14]. 14. Sterilize the substrate in the autoclave for 30 min at 120 °C and 115 kPa. 15. Maintain the trays closed for a week to avoid moisture loss. Sprinkle water on the dome to maintain a cool environment. 16. After a month, keep the trays at 30 ± 5 °C, with a relative humidity of 30% and natural light with an irradiance of 250 ± 10 μmol m-2 s-1. Prune leaves every 20 days. If the temperature surpasses 30 °C, water daily and fertilize and fumigate once a week. 17. Maintaining the seedlings under open-sky conditions is suggested to prepare them for field transplant (Fig. 3b). References 1. Shabbir R, Javed T, Afzal I, Sabagh AE, Ali A, Vicente O et al (2021) Modern biotechnologies: innovative and sustainable approaches for the improvement of sugarcane tolerance to environmental stresses. Agronomy 11(6): 1 0 4 2 . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / agronomy11061042 2. Saleem Y, Emad MZ, Ali A, Naz S (2022) Synergetic effect of different plant growth regulators on micropropagation of sugarcane (Saccharum officinarum L.) by callogenesis. Agriculture 12(11):1812. https://doi.org/ 10.3390/agriculture12111812 3. da Silva JA, Solis-Gracia N, Jifon J, Souza SC, Mandadi KK (2020) Use of bioreactors for large-scale multiplication of sugarcane (Saccharum spp.), energy cane (Saccharum spp.), and related species. In Vitro Cell Dev Biol Plant 56(3):366–376. https://doi.org/10. 1007/s11627-019-10046-y

4. Garcı´a-Ramı´rez Y (2023) Temporary immersion system for in vitro propagation via organogenesis of forest plant species. Trees 37:611– 626. https://doi.org/10.1007/s00468-02202379-w 5. Go´mez D, Herna´ndez L, Martı´nez J, Escalante D, Zevallos BE, Yabor L et al (2019) Sodium azide mutagenesis within temporary immersion bioreactors modifies sugarcane in vitro micropropagation rates and aldehyde, chlorophyll, carotenoid, and phenolic profiles. Acta Physiol Plant 41:114. https:// doi.org/10.1007/s11738-019-2911-0 6. Mirzabe AH, Hajiahmad A, Fadavi A, Rafiee S (2022) Temporary immersion systems (TISs): a comprehensive review. J Biotechnol 357:56– 83. https://doi.org/10.1016/j.jbiotec.2022. 08.003 7. Orozco-Ortiz C, Sa´nchez L, Araya-Mattey J, Vargas-Solorzano I, Araya-Valverde E (2023)

Sugarcane Micropropagation Using TIB BIT® bioreactor increases in vitro multiplication of quality shoots in sugarcane (Saccharum spp. variety LAICA 04-809). Plant Cell Tissue Organ Cult 152(1):115–128. https://doi. org/10.1007/s11240-022-02392-4 8. Escalona M, Lorenzo JC, Gonza´lez B, Daquinta M, Gonza´lez JL, Desjardins Y et al (1999) Pineapple (Ananas comosus L. Merr) micropropagation in temporary immersion systems. Plant Cell Rep 18:743–748. https://doi. org/10.1007/s002990050653 9. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473– 497. https://doi.org/10.1111/j.1399-3054. 1962.tb08052.x 10. Sorcia-Morales M, Go´mez-Merino FC, Sa´nchez-Segura L, Spinoso-Castillo JL, BelloBello JJ (2021) Multi-walled carbon nanotubes improved development during in vitro multiplication of sugarcane (Saccharum spp.) in a semi-automated bioreactor. Plan Theory 10: 2 0 1 5 . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / plants10102015 11. Custo´dio L, Slusarczyk S, Matkowski A, Casta˜ eda-Loaiza V, Fernandes E, Pereira C et al n

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(2022) A first approach for the micropropagation of the medicinal halophyte Polygonum maritimum L. and phenolic profile of acclimatized plants. Front Plant Sci 13:960306. https://doi.org/10.3389/fpls.2022.960306 12. Moreno-Herna´ndez MR, Spinoso-Castillo JL, Sa´nchez-Segura L, Sa´nchez-Pa´ez R, BelloBello JJ (2022) Arbuscular mycorrhizal fungi: inoculum dose affects plant development and performance of sugarcane (Saccharum spp.) plantlets during acclimatization stage. J Soil Sci Plant Nutr 22:4847–4856. https://doi. org/10.1007/s42729-022-00964-z 13. Belguendouz A, Kaide Harche M, Benmahioul B (2021) Evaluation of different culture media and activated charcoal supply on yield and quality of potato microtubers grown in vitro. J Plant Nutr 44(14):2123–2137. https://doi. org/10.1080/01904167.2021.1881545 14. Manokari M, Latha R, Priyadharshini S, Shekhawat MS (2021) Effect of activated charcoal and phytohormones to improve in vitro regeneration in Vanda tessellata (Roxb.) Hook. ex G. Don. Vegetos 34:383–389. https://doi. org/10.1007/s42535-021-00196-z

Chapter 6 Micropropagation of Stevia (Stevia rebaudiana Bert.) in RITA® Heidi P. Medorio-Garcı´a, Elizabeta Herna´ndez-Domı´nguez, Rube´n H. Andueza-Noh, David Rau´l Lo´pez-Aguilar, and Marco A. Ramı´rez-Mosqueda Abstract Stevia rebaudiana Bert. is a plant that contains noncaloric sweeteners highly appreciated in the food industry. However, there is a high demand for propagules to establish commercial plantations, and the conventional reproduction types for this species are inefficient. Micropropagation is a technique that allows obtaining a large number of plants and can be used to meet the demand in the field. However, it requires in vitro propagation techniques such as temporary immersion systems (SIT) to increase yield and reduce production costs. This chapter describes an effective protocol for the large-scale micropropagation of S. rebaudiana using a TIS. Key words RITA®, In vitro propagation, Sweeteners, Stevia

1

Introduction The use of noncaloric sweeteners in the food industry is a recurring topic in scientific research [1]. Stevia rebaudiana Bert. is a plant with leaves that contain noncaloric sweeteners widely used for food manufacturing [2]. The cultivation of this species has a high economic interest [3]. However, there is a high demand for commercial propagules, and conventional propagation techniques (seeds and stakes) are insufficient to meet this demand [4]. In the field, the seed germination rate of S. rebaudiana is extremely low, 10–15% [5]. For its part, stake rooting in this species produces a limited number of individuals [6]. Therefore, new propagation techniques are necessary to obtain large numbers of individuals over a short time and space; thus, the plant tissue culture represents a feasible option [7]. There is a large number of stevia micropropagation protocols. However, innovative techniques are currently sought to obtain

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_6, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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large numbers of vigorous plants with reduced cost, enabling the semi-automation of the propagation process [7]. The use of temporary immersion systems in the mass micropropagation of plants is a novel alternative involving liquid culture media, sparing the use of gelling agents [8]. The RITA® system consists of a single culture vessel that separates the plant material from the culture medium and produces programmable immersions through sterile air injection [8]. This chapter describes the in vitro propagation of S. rebaudiana using a RITA®. This system has improved the micropropagation of stevia in commercial biofactories, reducing production costs and yielding healthy, homogeneous, and reinvigorated seedlings.

2 2.1

Materials Plant Material

2.2 Surface Sterilization 2.3

Culture Medium

Seeds of S. rebaudiana variety Morita II from ecological greenhouses in Xalapa, Veracruz, Mexico, were used for the establishment of aseptic plants. Disinfectant solution: 0.6% (v/v) sodium hydrochloride, Tween® 20 (three drops per 100 mL solution), and commercial detergent. 1. Basal Murashige and Skoog (MS) [9] culture medium is used (see Note 1). 2. Medium additives used for various culture stages: MS basal medium supplemented with various additives according to the culture stage, including culture initiation medium (CIM), shoot multiplication medium (SMM), temporary immersion system medium (TISM), and rooting medium (RM) (see Table 1). 3. pH adjustment solutions: 0.1 and 1 N NaOH and HCl.

2.4

Equipment

1. Culture room: laminar airflow hood. 2. Surgical tools: stainless steel forceps, surgical scalpels, removable sterile surgical blades, and sterile Petri dishes. 3. Medium preparation: weighing balances, magnetic stirrer, hot plate, pH meter, microwave, micropipettes, magnets, refrigerator, and autoclave. 4. Culture vessels: reagent bottles, flasks (500 mL and 1000 mL capacity), beakers, measuring cylinders, glass culture jars (100 mL), and RITA® bioreactor (1000 mL). 5. Glassware cleaning: Scrub glassware with a liquid detergent solution, thoroughly wash with tap water, and rinse glassware with distilled water and dry.

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Table 1 Culture medium additives used for the different culture stages of S. rebaudiana Culture stage

Media additives

Shoot Culture initiation multiplication medium (SMM) medium (CIM)

Temporary immersion system medium (TISM)

Rooting medium (RM)

Medium MS (%)

100

100

100

50

Vitamins MS (%)

100

100

100

100

Benzylaminopurine – (BA) (mg/L)

2

2

–

Myoinositol (mg/L)

100

100

100

100

Sucrose (g/L)

30

30

30

30

Phytagel (g/L)

2.2

2.2

–

2.2

3

Methods

3.1 Stage I: In Vitro Establishment

1. Seeds washed with water and commercial soap. 2. Disinfect the laminar flow surface with 70% alcohol after UV light exposure for 15 min. 3. Seeds are rinsed for 10 min in a 0.6% (v/v) sodium hydrochloride solution with three drops of Tween® 20 per 100 mL of water (see Note 2). Seeds were rinsed three times with sterile distilled water and placed in glass culture jars 100 mL containing 20 mL of CIM (see Note 3). 4. The explants are incubated at 24 ± 2 °C, under 40 ± 5 μmol m2 -1 s irradiance and a 16 h photoperiod. 5. After 30 days, the nodal segments are transferred for multiplication to SMM.

3.2 Stage II: In Vitro Multiplication Using Semisolid Medium

1. For the multiplication in the semisolid media stage, nodal segments, 1–2 cm in length, are to be transferred to 100 mL glass culture jars containing 20 mL of SMM (Fig. 1a) (see Note 4) [7]. 2. The medium pH is adjusted to 5.8, and 2.2 g/L Phytagel was added as a gelling agent. Then, the medium is sterilized in an autoclave for 15 min at 120 °C and 115 kPa. 3. The explants are incubated at 24 ± 2 °C, under 40 ± 5 μmol m2 -1 s irradiance and a 16 h photoperiod. After two subcultures, proceed to the multiplication stage using the RITA®.

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Fig. 1 Micropropagation de Stevia rebaudiana in RITA®. (a) In vitro multiplication using semisolid medium. (b and c) Establishing culture in RITA®. (d) Acclimatization

3.3 Establishing Culture in RITA®

1. The TIS RITA® (1000 mL, 150 × 130 mm) (VITROPIC, Saint-Mathieu-de-Tre´viers) is used. To sterilize these containers, take them to a laminar flow hood. Prepare a 0.3% (v/v) solution of sodium hydrochloride, and leave it still for 5 min. After rinsing the containers three times with sterile water, turn on the UV light in the laminar flow hood for 10 min (see Note 5). 2. Then, pour into the container 200 mL of TISM previously described (Table 1) [7]. Subsequently, place ten explants at the top of the tis (Fig. 1b). 3. Connect the RITA® to an air pump and apply air pressure to submerge the explants. After the immersion time, a solenoid valve prevents the passage of air, and the medium returns to

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gravity the original site. Use an immersion frequency of 2 min every 8 h for 30 days (Fig. 1c). Maintain the explants under the light and temperature conditions previously mentioned [7]. 3.4 Stage III: Elongation and Rooting

1. After 30 days in the TISM, stevia shoots be individualized for better nutrient absorption. Divide agglomerate of shoots at base (shoots of 2 cm long). 2. Change the shoots at 100 mL glass culture flasks containing 20 mL of RM (see Note 6). Place ten shoots per flasks. Maintain the explants under the light and temperature conditions previously described [7].

3.5 Stage IV: Acclimatization

1. Prepare the substrate in plastic tube (15.8 cm long × 4.6 diameter). The substrate consists of a mixture of peat and agrolite 1:1 (v/v) (see Note 7) [7]. 2. Wash the shoots rooting to remove adhering MS medium. 3. Prepare a 1 g/L fungicide and bactericide solution and submerge the seedlings for 10 min. 4. Before seeding, moisten the substrate. After seeding, cover the seedlings with a translucent dome. Maintain seedlings under greenhouse conditions with 60% shade and at 30 ± 2 °C, relative humidity of 60 ± 10%, and natural light with an irradiance of 80 ± 10 μmol m-2 s-1 for 1 month (Fig. 1d) (see Notes 8–9) [7].

4

Notes 1. Prepare separate MS stock A-F solutions. Then, weigh and dissolve the components of each stock in 50 mL of distilled water using a magnetic stirrer, and make up the final volume to 100 mL by using distilled water and analytical grade reagents. Diligently follow all waste disposal regulations when disposing of waste materials. For 1 L semisolid MS medium elaboration, pour 750 mL of distilled water into a 1 L flask with a magnetic stirring bar, add 30 g of sucrose, and dissolve. Adjust the pH to 5.8 with a pH meter by adding 0.1 or 1 N NaOH or HCl with constant stirring. Then, add 2.2 g of Phytagel and make up the volume to 1 L with distilled water using a 1 L measuring cylinder (depends if the medium is semi-solid or liquid). Heat the culture medium until it starts boiling, dispense medium per glass culture flasks o TIS, and cap. Autoclave at 120 °C with a pressure of 115 kPa, for 15 min. Take out the culture medium from the autoclave, and allow it to cool and solidify at room temperature.

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2. The seeds must be recovered from the disinfectant solutions with the help of qualitative grade 1 filter paper (110 mm). 3. To ensure asepsis, four seeds were established per flask. 4. You must work quickly, due to the dehydration of the explants in the laminar flow hood. It is recommended to hydrate the explants in sterile distilled water. 5. It is recommended to assemble the bioreactors and verify with running water that the system works correctly. Verify that caps and hoses do not leak; this can be solved using safety straps. 6. The rooting of shoots in semisolid medium was required, because S. rebaudiana roots entangle in the pores of the RITA® support mesh. 7. Sterilize the substrate in the autoclave for 30 min at 120 °C and 115 kPa. 8. Maintain the pots closed for a week to avoid moisture loss. Sprinkle water on the dome to maintain a cool environment. 9. After a month, keep the trays at 30 ± 5 °C, with a relative humidity of 30% and natural light with an irradiance of 250 ± 10 μmol m-2 s-1. If the temperature surpasses 30 °C, water daily and fertilize and fumigate once a week. References 1. Agullo´ V, Garcı´a-Viguera C, Domı´nguez-Perles R (2022) The use of alternative sweeteners (sucralose and stevia) in healthy soft-drink beverages, enhances the bioavailability of polyphenols relative to the classical caloric sucrose. Food Chem 370:131051. https://doi.org/10.1016/ j.foodchem.2021.131051 2. Ahmad J, Khan I, Blundell R, Azzopardi J, Mahomoodally MF (2020) Stevia rebaudiana Bertoni.: an updated review of its health benefits, industrial applications and safety. Trends Food Sci Technol 100:177–189. https://doi. org/10.1016/j.tifs.2020.04.030 3. Dı´az-Gutie´rrez C, Hurtado A, Ortı´z A, Poschenrieder C, Arroyave C, Pela´ez C (2020) Increase in steviol glycosides production from Stevia rebaudiana Bertoni under organomineral fertilization. Ind Crop Prod 147: 112220. https://doi.org/10.1016/j.indcrop. 2020.112220 4. Miladinova-Georgieva K, Geneva M, Stancheva I, Petrova M, Sichanova M, Kirova E (2022) Effects of different elicitors on micropropagation, biomass and secondary metabolite production of Stevia rebaudiana Bertoni – a review. Plan Theory 12:153. https://doi.org/ 10.3390/plants12010153

5. Gantait S, Das A, Banerjee J (2018) Geographical distribution, botanical description and selfincompatibility mechanism of genus Stevia. Sugar Tech 20:1–10. https://doi.org/10. 1007/s12355-017-0563-1 6. Medina EL, Rivero AEG, Zavaleta AL (2016) Enraizamiento de esquejes de Stevia rebaudiana Bertoni (Asteraceae) “estevia”, aplicando dosis creciente de a´cido indolbutı´rico. Arnaldoa 23: 569–576 7. Ramı´rez-Mosqueda MA, Iglesias-Andreu LG, Ramı´rez-Madero G, Herna´ndez-Rinco´n EU (2016) Micropropagation of Stevia rebaudiana Bert. in temporary immersion systems and evaluation of genetic fidelity. S Afr J Bot 106:238– 243. https://doi.org/10.1016/j.sajb.2016. 07.015 8. Mirzabe AH, Hajiahmad A, Fadavi A, Rafiee S (2022) Temporary immersion systems (TISs): a comprehensive review. J Biotechnol 357:56–83. https://doi.org/10.1016/j.jbiotec.2022. 08.003 9. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962. tb08052.x

Chapter 7 In Vitro Multiplication of Agave (A. marmorata and A. potatorum) by Temporary Immersion in SETIS™ Bioreactor Marı´a del Rosario Moreno-Herna´ndez, Eucario Mancilla-A´lvarez, Daniel Aguilar-Jime´nez, and Jerico´ Jabı´n Bello-Bello Abstract In Mexico, wild agaves are important for the production of alcoholic beverages known as mezcal and pulque. However, the propagation of agave seeds and pups is not enough to satisfy the national demand. Temporary immersion systems represent an agave micropropagation alternative that reduces the labor force, increases development, and improves the quality of seedlings. The use of the SETIS™ bioreactor in A. marmorata and A. potatorum improves the multiplication rate and allows rooting. Additionally, this bioreactor reduces the culture time, labor force, and reagents needed while maintaining high survival rates during the acclimatization phase. Key words Micropropagation, Bioreactor, Shoot proliferation, Acclimatization

1 Introduction In Mexico, some species of Agave spp. (Asparagaceae) have ornamental, economic, and cultural importance due to the production of traditional alcoholic beverages, such as tequila, mezcal, and pulque. Additionally, some species have a high potential for bioethanol production [1, 2]. The recent rise in the national and international demand for agave to produce tequila and mezcal has increased the need to propagate plants to maintain and expand new culture zones [3]. Wild agaves like A. marmorata and A. potatorum have a very low reproduction rate. These species can reproduce sexually and asexually via seeds and pups, respectively. Sexual reproduction has significant disadvantages due to the mass extraction of plants before flowering, the long-life cycle of agave (8–10 years), and the lack of sustainable agricultural practices. As for asexual reproduction, these species have null or limited pups. Additionally,

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_7, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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the pups obtained from mother plants can propagate pests and diseases [4, 5]. These circumstances have pushed the search for biotechnological alternatives for the in vitro propagation of these species. Various studies have described the use of plant tissue culture techniques for the in vitro propagation of Agave spp. [3]. However, conventional techniques use gelling agents and require an intensive labor force, which limits semi-automation and increases production costs. Implementing liquid culture media using temporary immersion systems (TISs) is an alternative for the fast and efficient production of plants with high genetic and phytosanitary quality. Temporary immersion systems are semi-automated bioreactors designed for the large-scale propagation of tissues, embryos, or organs. These are temporarily exposed to a liquid culture medium for a specific time and immersion frequency. Additionally, TISs increase the in vitro regeneration capacity and the multiplication rate, reduce explant hyperhydricity, and decrease production costs compared to the conventional system in the semi-solid cultured medium [4]. The SETIS™ bioreactor is a TISs made from polycarbonate that consists of two containers, one for the growing explants and the second for the liquid medium. The containers are connected by silicone tubes and use hydrophobic filters. The SETIS™ bioreactor allows greater availability of light irradiation and is easy to handle due to the lack of interior accessories [8–10]. The features of this bioreactor make it an attractive alternative for the commercial micropropagation of Agave spp. with high genetic and phytosanitary quality [5]. This study aimed to develop an efficient protocol for the large-scale micropropagation of A. marmorata and A. potatorum.

2 2.1

Materials Plant Material

2.2 Surface Sterilization 2.3

Culture Medium

A. potatorum and A. marmorata pups of approximately 1 year were collected from their natural habitat in San Diego la Mesa, Puebla, Mexico. Disinfectant solutions: 0.6% (v/v) sodium hydrochloride, Tween® 20 (two drops per 100 mL of solution), and commercial detergent. 1. Basal Murashige and Skoog (MS) [8] culture medium is used (see Note 1). 2. Medium additives used for various culture stages: MS basal medium supplemented with various additives according to the culture stage, including culture initiation (CI), shoot

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Table 1 Culture medium additives used for the different culture stages of agave Culture stage Media additives

Culture initiation (CI)

Shoot multiplication on semisolid medium (SM)

Shoot multiplication in liquid medium (SML)

Cysteine (mg/L)

50

–

–

Ascorbic acid (mg/L)

100

–

–

Indoleacetic acid (IAA) (mg/L)

–

2

2

Benzylaminopurine (BAP) (mg/L)

–

3

3

Sucrose (g/L)

30

30

30

Agar (g/L)

7

7

–

multiplication (SM), and temporary immersion system (TISs) liquid medium (SML) (see Table 1). 3. pH adjustment solutions: 0.1 and 1 N NaOH and HCl. 2.4

Equipment

1. Culture room: laminar airflow hood. 2. Surgical tools: stainless steel forceps, surgical scalpels, removable sterile surgical blades, and sterile Petri dishes. 3. Medium preparation: weighing balances, magnetic stirrer, hot plate, pH meter, microwave, micropipettes, magnets, refrigerator, and autoclave. 4. Culture vessels: reagent bottles, conical flasks (500 and 1000 mL capacity), beakers, measuring cylinders, and culture tubes (25 × 150 mm) with polypropylene caps. 5. Scrub glassware with a liquid detergent solution and thoroughly wash with tap water. 6. Rinse glassware with distilled water and dry.

3

Methods

3.1 Stage 0: Plant Selection

1. Experts must correctly identify the species before selecting the agave plant (Fig. 1a) (see Note 2). 2. Once the species has been identified, plants must be quarantined for 2 months applying once a week a 1 g/L solution of fungicide and bactericide (see Notes 3–4).

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Fig. 1 Selection of mother plant and in vitro establishment of Agave. (a) Identification of donor plants, (b) explant preparation, (c) disinfestation with sodium hypochlorite, and (d) in vitro establishment

3. Use pups of 1 year of age. The plants were stripped of their developed leaves, and their basal and apical ends were cut off until just the stem was left (see Note 5). 3.2 Stage I: In Vitro Establishment

4. After cutting the agave stems, wash them with tap water and the surfactant solution made of two drops of Tween® 20 per 100 mL of distilled water for 5 min. Follow with five rinses with distilled water, and then submerge in 70% ethanol for 3 min (Fig. 1b) (see Note 5). 5. Disinfect the surface of the laminar flow hood with 70% alcohol after UV light exposure for 15 min. 6. Transport the stems to the culture room and place them inside the laminar flow hood. Then, submerge them in a 0.6% (v/v)

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sodium hypochlorite solution (100 mL) for 15 min. Rinse twice with sterile water (Fig. 1c) (see Note 6). Apices are placed in tubes containing MS medium without regulators (Fig. 1d). 7. After a week, apices are transferred into MS medium for multiplication (see Note 7). 3.3 Stage II: In Vitro Multiplication Using Semisolid Medium

1. For the multiplication in a semisolid medium, transfer the apices to 500 mL flasks containing 30 mL of MS medium supplemented with 30 g/L sucrose, 3 mg/L benzylaminopurine (BAP), and 2 mg/L IAA (indole-3-acetic acid) (see Notes 7–8) [4]. 2. The medium pH is adjusted to 5.8. Then, 7 g of micropropagation grade agar was added as a gelling agent. The medium is sterilized in an autoclave for 15 min at 120 °C and 115 kPa. 3. The explants are incubated at 24 ± 2 °C, under 40 ± 5 μmol m2 -1 s irradiance and a 16 h photoperiod. After three subcultures, proceed to the multiplication phase using the SETIS™ bioreactor.

3.4 Establishing Culture in SETIS™ Bioreactor

4. The bioreactors consist of two containers: one is used to culture the explants, and the second contains the culture medium [7–10]. To sterilize these containers, autoclave them for 20 min at 120 °C and 115 kPa (see Notes 9–10). 5. Then, pour into one of the containers 2000 mL of the multiplication culture medium previously described (see Note 10) [7]. Place 40 explants (2–3 shoots each fused at the base) 1.5–2 cm in length in the medium-free container (Fig. 2) (see Notes 11–13). 6. Connect both containers to an air pump, and apply air pressure to submerge the explants. After immersion, a solenoid valve prevents air passage, and the medium returns to the original container [4–7]. Use an immersion frequency of 2 min every 8 h for 45–60 days. Maintain the explants under the light and temperature conditions previously mentioned [4].

3.5 Stage IV: Acclimatization

1. Prepare the substrate in 72-cavity polypropylene trays. The substrate consists of a 3:1 v/v mixture of tezontle (volcanic rock with a particle size of 3–5 mm) and agrolite (see Note 14). 2. Separate seedlings individually and carefully wash the shoots and roots to remove adhering MS medium. 3. Prepare a 1 g/L fungicide and bactericide solution and submerge the seedlings for 10 min. 4. Before seeding, moisten the substrate. After seeding, cover the seedlings with a translucent dome. Maintain seedlings under greenhouse conditions with 60% shade and at 30 ± 2 °C,

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Fig. 2 In vitro shoot multiplication of agave using a SETIS™ bioreactor. (a, b) Multiplication of A. marmorata and (c, d) A. potatorum shoots

Fig. 3 Acclimatization of agave plantlets. (a) A. potatorum and (b) A. marmorata plantlets at 60 days

relative humidity of 60 ± 10%, and natural light with an irradiance of 80 ± 10 μmol m-2 s-1 for a month. Then, seedlings were transplanted into 32-cavity trays and exposed to light with an irradiance of 250 ± 10 μmol m-2 s-1. If the temperature surpasses 30 °C, water daily and fertilize and fumigate once a week (Fig. 3a, b) (see Note 15) [4].

Agave Micropropagation Using SETIS™ Bioreactor

4

75

Notes 1. Prepare separate MS stock A-F. Weigh and dissolve the components of each stock in 50 mL of distilled water using a magnetic stirrer, and make up the final volume to 100 mL by using reverse osmosis water (prepared by purifying deionized water to attain a sensitivity of 18 MΩ-cm at 25 °C) and analytical grade reagents. Diligently follow all waste disposal regulations when disposing of waste materials. Pour 750 mL of distilled water into a 1 L flask with a magnetic stirring bar, add 30 g of sucrose, and dissolve. Adjust the pH to 5.8 with a pH meter by adding 0.1 or 1 N NaOH or HCl with constant stirring. Add 7 g of micropropagation grade agar, and make up to 1 L with distilled water by using a measuring cylinder. Heat the culture medium until it starts boiling, pour 10 mL per culture tube, and cap the tubes. Autoclave for 15 min at 120 °C with a pressure of 115 kPa, take out the culture medium from the autoclave, and allow it to cool and solidify at room temperature. 2. To avoid varietal mixing, experts must identify the taxonomic variety. 3. Choose plants visibly free of pests and diseases that cause biotic stress. 4. Stems approximately 10–15 cm long are obtained for in vitro establishment. 5. To remove dirt or contaminants, stems must be washed in tap water for 5 min. 6. Cut stems approximately 2 cm in length for in vitro establishment. 7. For Agave marmorata, add 3 mg/L IAA (indole-3acetic acid). 8. Before sterilizing, the SETIS™ bioreactor containers must be placed inside poly paper bags to avoid contamination when transferring to the laminar flow hood. 9. To prevent contamination, the culture medium and the explants are placed into the bioreactors inside the laminar flow hood. 10. For 1 L MS medium elaboration, pour 750 mL of distilled water into a 1 L flask with a magnetic stirring bar, add 30 g of sucrose, and dissolve. Add aliquot hormones according to the culture stages (see Table 1). Adjust the pH to 5.8 with a pH meter by adding 0.1 or 1 N NaOH or HCl. Autoclave at 120 ° C with a pressure of 115 kPa, for 15 min.

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11. To secure asepsis, use partials (four sterilized Gerber flasks containing 10 mL of regulator-free MS medium where we place the ten explants per bioreactor). It is recommended to assemble the bioreactors and verify with running water that the system works correctly. Verify that caps and hoses do not leak; this can be solved using safety straps. 12. Sterilize the substrate in the autoclave for 30 min at 120 °C and 115 kPa. 13. Maintain the trays covered for 2 weeks to avoid moisture loss. Sprinkle water on the dome to maintain a cool environment. 14. Maintain the trays covered for a week to avoid the loss of moisture. Sprinkle water on the dome to maintain a cool environment. 15. It is suggested to maintain the seedlings under open sky conditions for a month to prepare them for field conditions (Fig. 3b). References 1. Santiago-Martı´nez A, Pe´rez-Herrera A, Martı´nez-Gutie´rrez GA, Meneses ME (2023) Contributions of agaves to human health and nutrition. Food Biosci:102753. https://doi. org/10.1016/j.fbio.2023.102753 2. Alducin-Martı´nez C, Ruiz MKY, Jime´nezBarro´n O, Aguirre-Planter E, Gasca-Pineda J, Eguiarte LE et al (2023) Uses, knowledge and extinction risk faced by agave species in Mexico. Plan Theory 12:1–124. https://doi.org/ 10.3390/plants12010124 3. Monja-Mio KM, Olvera-Casanova D, Herrera´ , Sa´nchez-Teyer FL, Robert ML Alamillo MA (2021) Comparison of conventional and temporary immersion systems on micropropagation (multiplication phase) of Agave angustifolia Haw. ‘Bacanora’. 3 Biotech 11:1– 8. https://doi.org/10.1007/s13205-02002604-8 4. Correa-Herna´ndez L, Baltazar-Bernal O, Sa´nchez-Pa´ez R, Bello-Bello JJ (2022) In vitro multiplication of agave tobala (Agave potatorum Zucc.) using ebb-and-flow bioreactor. S Afr J Bot 147:670–677. https://doi.org/ 10.1016/j.sajb.2022.03.009 5. Martinez-Rodriguez A, Beltran-Garcia C, Valdez-Salas B, Santacruz-Ruvalcaba F, Di Mascio P, Beltran-Garcia MJ (2022) Micropropagation of seed-derived clonal lines of the endangered Agave marmorata Roezl and their compatibility with endophytes. Biology

11(10):1423. https://doi.org/10.3390/ biology11101423 6. Bello-Bello JJ, Schettino-Salomo´n S, OrtegaEspinoza J, Spinoso-Castillo JL (2021) A temporary immersion system for mass micropropagation of pitahaya (Hylocereus undatus). 3 Biotech 11:437. https://doi.org/10.1007/ s13205-021-02984-5 7. Ramı´rez-Mosqueda MA, Bello-Bello JJ (2021) El biorreactor SETIS™ aumenta la multiplicacio´n in vitro y la longitud de los brotes en vainilla (Vanilla planifolia Jacks. Ex Andrews). Acta Physiol Plant 43:52. https://doi.org/10. 1007/s11738-021-03227-z 8. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473– 497 ˜ ez-Palenius HG, 9. Va´zquez-Martı´nez O, Nu´n Balch EMPM, Valencia-Posadas M, Pe´rezMoreno L, Ruiz-Aguilar GM et al (2022) In vitro-propagation of Agave tequilana weber cv. azul in a temporary immersion system. Phyton 91(1):83. https://doi.org/10.32604/phy ton.2022.017281 10. Vervit (2021) SETIS™ bioreactor temporary immersion systems in plant micropropagation. Available online http://www.setissystems.be. Accessed 23 June 2023

Chapter 8 BioMINT: A Temporary Immersion System for Agave Micropropagation Kelly M. Monja-Mio, Gabriel Ojeda, Miguel A´ngel Herrera-Alamillo, Lorenzo Felipe Sa´nchez-Teyer, and Antonio Rescalvo-Morales Abstract Agaves are cultivated in Mexico as a source of industrial products such as fibers, nutritional supplements, and alcoholic beverages. Due to the demand for plant material, its long-life cycle, and the need to avoid predation on its natural populations, in vitro micropropagation represents a good option for agaves. Plant tissue culture has been successfully used to micropropagate selected elite individuals from plants of various Agave species of economic interest. However, it is necessary to implement systems that lower production costs without losing the quality of the plantlets obtained. This chapter describes the BioMINT™ bioreactor as an alternative for the micropropagation of agaves in the different stages of the micropropagation process. Key words Plant micropropagation, Clonal lines, Temporary immersion system, BioMINT™ bioreactor

1

Introduction Agaves are plants with a long-life cycle (8–16 years), which are used to obtain a wide range of products, including liquors, fibers, cellulose, inulin, etc., due to which they present great industrial demand as raw materials [1, 2]. Some of its species are obtained from their natural environments, such as A. angustifolia “Bacanora” [3, 4], A. cupreata [5], and A. potatorum [6], among others, causing a loss of their natural populations. Micropropagation is of particular relevance for species with a long-life cycle, such as agaves, and that is why it represents an effective option to rescue and rapidly produce large numbers of pathogen-free plants while, at the same time, selecting elite, vigorous, fast-growing individuals [2, 7], avoiding the predation of species endemics. In agaves, efficient in vitro micropropagation systems have been developed and used for tequila and several other economically

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_8, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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important agave crops in Mexico, such as henequen and mezcal [2, 7–9], but they are all susceptible to improvements to reduce time and diminish costs. An option for this is the use of temporary immersion systems (TIS), which can be used in one or several stages of the micropropagation process. This type of system allows greater absorption of nutrients, since the explants are covered by the culture medium for a few minutes, and allows a passive renewal of the atmosphere inside the container [10, 11]. Currently, there are several types of TIS for the micropropagation of plants of commercial interest [10, 12]. Properly designed, the TIS can be a rapid, economical system that generates highquality plants that adapt more efficiently when transplanted to soil [13–15]. The culture conditions in TIS need, however, to be optimized for the species being cultured, the morphogenic pathway, and the phase of the micropropagation process used [16, 17]. The use of temporary immersion systems in micropropagation has proven to be effective in many species [10, 11]. Different reports show that the microenvironment in TIS in terms of nutrients, transfer, and better gas exchange is associated with a higher multiplication rate, greater biomass growth, and better plantlet physiology [10, 18]. However, to achieve reproducible and efficient protocols, it is necessary to standardize the main factors involved, such as the time and frequency of immersion, the density of the inoculum, the volume of culture medium used, the incubation time, and the size, and design of the bioreactor [10, 17, 19, 20]. Bioreactors have been tested at different stages of the micropropagation process for agaves [3, 8, 16, 17, 21], and it has been observed that help the plantlets to adapt to ex vitro environment [17]. This means that TIS can be used for micropropagation in different agave species. The temporary immersion modular bioreactors (BioMINT™) were developed at the Yucatan Scientific Research Center and designed to operate in the simplest and most economical manner to promote the growth and development of individual plants under inducing autotrophic conditions [14]. The structural simplicity and modular and independent nature of the bioreactors simplify their operation and reduce the amount of hand labor required for transfers, thereby reducing the cost of the whole micropropagation process [7]. They have been designed in two models (BioMINT™ I and II PA/a/2004/003837). The BioMINT™ I unit is a mid-sized (1.2 L) reactor that consists of two cylindrical vessels coupled together through a perforated adaptor piece, which permits the flow of the liquid media from one vessel to the other, while The BioMINT™ II unit is a rectangular container (5 L), divided by compartments [14]. BioMINT bioreactors have been used mainly in the micropropagation of different species of agave, such as A. fourcroydes,

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A. tequilana, and A. angustifolia [3, 8, 14, 17, 21]. However, they have also been used in the micropropagation of Capsicum chinense [22], Cedrela odorata [23], and Saccharum officinarum [24].

2 2.1

Materials Equipment

1. Culture room: airflow cabinet, bead sterilizer. 2. Surgical tools: stainless steel tweezers of 25 cm, surgical scalpels, removable sterile surgical blades, sterile craft paper, cotton, and adherent film. 3. Medium preparation: analytical balance, spatula, hot stirrer plate, pH meter, micropipettes, magnetic stirrer, refrigerator, and autoclave. 4. Glassware: bottles (200 mL capacity), beakers (500 mL and 1 L capacity), reagent bottles, measuring cylinders, and volumetric flask (100 mL and 1 L capacity). 5. Culture vessels: magenta culture boxes GA7 and temporary immersion bioreactors.

2.2

Culture Media

1. Basal culture medium: Prepare separate of the stock solutions (1–6) of MSB medium (Murashige and Skoog salts with reduced nitrogen) (Table 1). Weigh and dissolve the components of each stock in distilled water by using a magnetic stirrer, and make up the final volume to 1000 mL (see Note 1). 2. (0.1 mg/mL) 2,4-dichlorophenoxyacetic acid (2,4-D): Weigh 10 mg of 2,4-D and dissolve with a few drops of 1 N KOH. Add the remaining water to bring up to volume in a 100 mL volumetric flask (see Note 2). 3. (2 mg/mL) 6-benzyladenine (BA): Dissolve 200 mg of 6-BA in 10 mL 1 N KOH, and then, add distilled water to make up volume in a 100 mL volumetric flask. 4. Induction medium (IM): For prepare 1 L of IM, add 10 mL of each stock solutions (1–6), 0.113 μM 2,4-D, 44.4 μM BA, 30 g/L sucrose, and 0.8% agar. 5. Shoot multiplication medium (SMM): same as IM but with 0.2% agar and 0.2% Gelrite. 6. Shoot growth medium (SGM): same as SMM except plant growth regulators (PGRs). In this medium, add 0.113 μM 2,4-D and 4.44 μM BA as PGRs. 7. Rooting medium (RM): same as SMM but without PGRs. 8. For all culture media, adjust the pH to 5.8 using 0.1 N KOH or 0.1 N HCl.

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Table 1 Salts components of MSB medium

N° stock Compound Vitamins

1

Micronutrients 2

Macronutrients 3

4

5 EDTA-Fe

6

Amount required Concentration for 1 L stock of the final solution 100 X, medium (mg/L) mg/L

mL of stock/L

Glycine Nicotinic acid Pyridoxine-HCl Thiamine-HCl Myoinositol

2 0.5 0.5 0.1 100

200 50 50 10 10,000

10

KI (potassium iodide) MnSO4.H2O (manganese sulfate monohydrate) H3BO3 (boric acid) ZnSO4.7H2O (zinc sulfate heptahydrate) NaMoO4.2H2O (sodium molybdate dihydrate) CuSO4.5H2O (copper sulfate pentahydrate) CoCl2.6H2O (cobalt chloride hexahydrate)

0.83 16.90

83 1690

10

6.2 8.6

620 860

0.25

25

0.025

2.5

0.025

2.5

KH2.PO4 (potassium phosphate monobasic) MgSO4.7 H2O (magnesium sulfate heptahydrate) KNO3 (potassium nitrate) NH4.NO3 (ammonium nitrate) CaCl2.2H2O (calcium chloride)

170

17,000

370

37,000

1820 400

182,000 40,000

10

440

44,000

10

3672

10

EDFS 36.72 ethylenediaminetetraacetic acid iron (III) sodium salt

10

9. When the medium is liquid, dispense 200 mL of culture medium in glass bottles of 250 mL capacity. 10. Autoclave at 121 °C with a pressure of 15 psi for 20 min. 11. After autoclaving, the glass bottles are kept in a clean room until use.

BioMINT: A Temporary Immersion System for Agave Micropropagation

2.3 BioMINT Bioreactor System

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This bioreactor consists of two parts, the support, on which it is operated called SyB (“up” and “down” system), and the BioMINT unit (Fig. 1a, see Note 3). 1. SyB platforms: The SyB consists of several platforms, which can operate a variable number of BioMINT units (up to 36 bioreactors), and includes a lighting system, a drive system, and a control system (see Note 4). 2. BioMINT™ II bioreactor: The BioMINT units, which contain the shoots and the culture medium, are made of polycarbonate and are fully autoclavable, translucent, and reusable. This bioreactor consists of a 540 × 100 × 100 mm rectangular container, with a volume capacity of 5 L (Fig. 1b). The lid of the container has two openings that can be covered with cotton filters or 3 M micropore tape to allow passive aeration (Fig. 1c). It has four compartments – three for the plant tissues and one for the liquid culture medium, which is black to protect the medium from light (Fig. 1d–f). 3. The liquid media moves by gravity from the media compartment to the plant compartments, and vice versa, when placed in a rack (SyB) that moves in a seesaw manner (see Note 5). 4. The whole system is automatically controlled by a programmable control panel that regulates the timing and speed with which the platforms change positions.

2.4

Plant Material

Shoots of clonal lines of Agave, obtained using the protocol of Robert et al. [25], can be used as the source of explants. The protocol consists of the following: 1. Selection of mother plants: Apical offshoots from rhizomes from selected elite plants are generally a good source of explant (meristematic) tissue (Fig. 2a, b). 2. Induction: The small cubes (0.8 cm3) are incubated in baby food jars containing 20 mL of IM. The jars are incubated in a growth room at 27 ± 2 °C under a 16 h photoperiod for 8–12 weeks until new adventitious shoots are formed on the surface of the explants (Fig. 2c–e). 3. Multiplication: The individualized shoots or groups of two or three of them are then grouped by size and transferred to magenta boxes with 50 mL of SMM and incubated in a growth room at 27 ± 2 °C under a 16 h photoperiod for 4 weeks (Fig. 2f). 4. Growth: The individualized shoots are transferred to magenta boxes with 50 mL of SGM (Fig. 2g). 5. Rooting: The plantlets are placed in magenta boxes with 50 mL of RM (Fig. 2h).

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Fig. 1 The BioMINT™ II bioreactor. SyB platform (a). The BioMINT unit (b). The cover with two open ends to allow the exchange of passive atmosphere (c). The main body is a translucent rectangle divided into three sections (d). Black box that protects the medium nutrients that are susceptible to the action of light (e). Three removable accessories to divide the bioreactor into sections, each one has nine channels (2 mm each one) (f)

6. Acclimatization: The plants are transferred to soil in polystyrene trays under greenhouse conditions (Fig. 2i, j) and later be placed under shading conditions (Fig. 2k).

3 Methods 3.1 Preparation of Explants

1. The multiplication (Fig. 3), shoot growth, and rooting phases (Fig. 4) can be carried out in bioreactors (see Note 6). 2. In the multiplication and growth phases, shoots longer than 3 centimeters can be used, whereas in the rooting phase, shoots longer than 5 centimeters are utilized. 3. Disinfect the laminar flow surface with 70% alcohol and then UV light exposure for 20 min. 4. Before placing the plant material in the bioreactors, it must be divided and separated by micropropagation stage and placed in sterile containers. 5. The number of shoots per bioreactor varies according to micropropagation stage, species, and shoot size (Figs. 3 and 4). 6. The cultured plants in the multiplication stage that require separation can be easily removed from the bioreactor through its wide mouth and transferred to another container (Fig. 3).

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Fig. 2 Obtaining clonal lines of Agave spp. used as a source of plant material. Plant mother (a). Extraction of the meristematic tissue from young selected plants (b). Cutting the block meristematic into smaller cubes (c). Adventitious organogenesis induction (d). Adventitious shoots in the top level of the explant (e). Multiplication

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3.2 Sterilization of Bioreactors

1. BioMINT II units can be assembled and sterilized in an autoclave at 121 °C for 20 min or alternatively; the bioreactors can also be sterilized with 5% commercial bleach (see Note 7). 2. After either of the two sterilization methods, the bioreactors are sealed with cling film and stored until use.

3.3 Filling of Bioreactors

1. Disinfect the surface of the airflow cabinet with 70% alcohol and then UV light exposure for 20 min. 2. The assembled bioreactor, the glass bottle with the culture medium (according to the stage of micropropagation), and the vessel with the plant material are disinfected with 70% alcohol and placed inside the laminar cabinet. 3. Open the lid of the BioMINT and place the plant material in the corresponding compartments. Add the culture medium (200 mL) and cover again. 4. The vessel containing the plants and culture medium is then closed and sealed with cling film.

3.4

Running the SyB

1. The bioreactors are placed horizontally on the SyB platforms (Fig. 1a). 2. Care must be taken to place plant tissues on the side of the platform that will remain in the “up” position during the longer aeration phase. 3. The timer must be set for the length of time of both the immersion and the aeration stages. In agaves, the immersion and aeration cycles can vary according to the stage of micropropagation and the species, but in general, the immersion time is very short (1 min), while the aeration periods can vary depending on the stage, from 6 to 8 h for the multiplication stage, 8–12 h for the growth stage, and 12–24 h for the rooting stage. 4. Place the bioreactor system in a growth room at 24 ± 2 °C under a photoperiod of 16 h light/8 h dark for a period of 30–45 days.

3.5 Hardening and Acclimatization

1. Remove the plants from the bioreactors that are in the rooting phase, and wash them carefully under running tap water. 2. Place plantlets in a potting mixture of soil, peat moss, and agrolite in a 2:2:1 ratio, and cultivate them in the greenhouse

ä Fig. 2 (continued) stage of adventitious and axillary shoots (f). Growth stage of shoots (g). Rooting stage of shoots (h). Acclimatization stage of plantlets (i). Acclimatized and developed agave plant (j). Agave plants under shading conditions (k)

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Fig. 3 Agave multiplication and growth phase in the BioMINT bioreactor. Agave shoots in the BioMINT™ II bioreactor in the growth stage (a). Shoots of A. tequilana in multiplication stage (b). Shoots of A. angustifolia in multiplication stage (c)

under natural sunlight with 27 + 2 °C and 50–60% relative humidity for a period of 4 weeks. 3. The plants are maintained at a fairly high relative humidity (above 80%) during the first 14 days, and the trays are covered with a plastic top. 4. The plants were watered once a day for 15 days and then once a week (Fig. 4).

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Fig. 4 Agave rooting phase in BioMINT bioreactor. Rooting of plantlets of A. tequilana in the BioMINT™ II (a). Vitro plantlets roots grown in the bioreactor (b). Plantlets obtained in the rooting phase in the BioMINT ready for acclimatization (c)

4

Notes 1. Prepare all the stock solutions and tissue culture media with distilled or deionized water. 2. The stock solution (macronutrients, micronutrients, vitamins, and plant growth regulators) can be stored at 4 °C. 3. BioMINT stands for “modular temporary immersion bioreactor” in Spanish (PA/a2004/003837).

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4. SyB stands for “up” and “down” in Spanish, referring to the seesaw mechanism (PA/a2004/003837). 5. To avoid vitrification, it is imperative that the angle of inclination be sufficient to allow the entire liquid medium to drain to the lower vessel. 6. The shoots should be carefully inspected to discard any bacterial or fungal contamination, since both might compromise the process of micropropagation in the bioreactors. 7. The bioreactor parts are immersed in a 5% commercial bleach solution (containing 5% active sodium hypochlorite) for 15 min, assembled in the laminar flow cabinet and sealing the holes of the cover with 3 M micropore tape. References 1. Monja-Mio KM, Escalante-Erosa F, Eb-Puc XM et al (2019) Epicuticular wax analysis of wild and agronomically important Agave species. Phytochem Lett 34:103–107 2. Monja-Mio KM, Herrera-Alamillo MA, Sa´nchez-Teyer LF et al (2019) Breeding strategies to improve production of agave (Agave spp.). In: Al-Khayri J, Jain S, Johnson D (eds) Advances in plant breeding strategies: industrial and food crops. Springer, Cham 3. Monja-Mio KM, Pool FB, Herrera GH et al (2015) Development of the stomatal complex and leaf surface of Agave angustifolia Haw. “Bacanora” plantlets during the in vitro to ex vitro transition process. Sci Hortic (Amsterdam) 189:32–40 4. Sa´nchez A, Coronel-Lara Z, Gutie´rrez A et al (2020) Aclimatacio´n y trasplante de vitroplantas de Agave angustifolia Haw. en condiciones silvestres. Rev Mex Ciencias Agrı´colas 11: 1593–1605 5. Aguirre-Dugua X, Eguiarte LE (2013) Genetic diversity, conservation and sustainable use of wild Agave cupreata and Agave potatorum extracted for mezcal production in Mexico. J Arid Environ 90:36–44 ´ J, Caballero OG 6. Torres AGT, Rodrı´guez JA et al (2023) In vitro culture of Agave potatorum a threatened species, endemic to Mexico. Bot Sci 101:883–894 7. Robert ML, Herrera-Herrera JL, Castillo E et al (2006) An efficient method for the micropropagation of Agave species. Methods Mol Biol 318:165–178 8. Robert ML, Herrera-Herrera J, HerreraAlamillo MA et al (2004) Manual for the in vitro culture of Agaves. United Nations Industrial Development Organization, Vienna

9. Monja-Mio KM, Robert ML (2016) Somatic embryogenesis in agave: an overview. In: Loyola-Vargas VM, Ochoa-Alejo N (eds) Somatic embryogenesis: fundamental aspects and applications. Springer, pp 1–506 10. Georgiev V, Schumann A, Pavlov A et al (2014) Temporary immersion systems in plant biotechnology. Eng Life Sci 14:607–621 11. San Jose´ MC, Bla´zquez N, Cernadas MJ et al (2020) Temporary immersion systems to improve alder micropropagation. Plant Cell Tissue Organ Cult 143:265–275 12. Vidal N, Sa´nchez C (2019) Use of bioreactor systems in the propagation of forest trees. Eng Life Sci 19:896–915 13. Berthouly M, Etienne H (2005) Temporary immersion system: a new concept for use liquid medium in mass propagation. In: Hvoslef-Eide AK, Preil W (eds) Liquid culture systems for in vitro plant propagation. Springer, Dordrecht, pp 165–195 14. Robert ML, Herrera-Herrera JL, HerreraHerrera G et al (2006) A new temporary immersion bioreactor system for micropropagation. Methods Mol Biol 318:121–129 15. Ramı´rez-Mosqueda MA, Cruz-Cruz CA, Cano-Rica´rdez A et al (2019) Assessment of different temporary immersion systems in the micropropagation of anthurium (Anthurium andreanum). 3 Biotech 9:1–8 16. Monja-Mio KM, Olvera-Casanova D, HerreraAlamillo M et al (2021) Comparison of conventional and temporary immersion systems on micropropagation (multiplication phase) of Agave angustifolia Haw. ‘Bacanora’. 3 Biotech 11:1–8 17. Monja-Mio KM, Olvera-Casanova D, HerreraHerrera G et al (2020) Improving of rooting

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and ex vitro acclimatization phase of Agave tequilana by temporary immersion system (BioMINT™). Vitr Cell Dev Biol Plant 56: 662–669 18. Arago´n CE, Sa´nchez C, Gonzalez-Olmedo J et al (2014) Comparison of plantain plantlets propagated in temporary immersion bioreactors and gelled medium during in vitro growth and acclimatization. Biol Plant 58:29–38 19. Etienne H, Berthouly M (2002) Temporary immersion systems in plant micropropagation. Plant Cell Tissue Organ Cult 69:215–231 20. Paula W (2012) The status of temporary immersion system (TIS) technology for plant micropropagation. Afr J Biotechnol 11: 14025–14035 21. Monja-mio KM, Herrera-Alamillo MA, Robert ML (2016) Somatic embryogenesis in temporary immersion bioreactors. In: Loyola-Vargas VM, Ochoa-Alejo N (eds) Somatic embryogenesis: fundamental aspects and applications. Springer, Cham, pp 435–454 22. Bello-Bello JJ, Canto-Flick A, Balam-Uc E et al (2010) Improvement of in vitro proliferation

and elongation of habanero pepper shoots (capsicum chinense jacq.) by temporary immersion. HortScience 45:1093–1098 ˜ a-Ramı´rez YJ, Jua´rez-Go´mez J, Go´mez23. Pen Lo´pez L et al (2010) Multiple adventitious shoot formation in Spanish Red Cedar (Cedrela odorata L.) cultured in vitro using juvenile and mature tissues: an improved micropropagation protocol for a highly valuable tropical tree species. Vitr Cell Dev Biol Plant 46:149–160 24. Carrillo-Bermejo EA, Herrera-Alamillo MA, Gonza´lez-Mendoza VM et al (2019) Comparison of two different micropropagation systems of Saccharum officinarum L. and expression analysis of PIP2;1 and EIN3 genes as efficiency system indicators. Plant Cell Tissue Organ Cult 136:399–405 25. Robert ML, Herrera JL, Chan JL et al (1992) Micropropagation of agave spp. In: Bajaj YP (ed) Biotechnology in agriculture and forestry. Springer, Berlin/Heidelberg, pp 306–329

Chapter 9 Plant Regeneration of Agave cupreata by Somatic Embryogenesis in a Temporary Immersion System with Silver Nanoparticles Sandra Y. Martı´nez-Martı´nez, Amaury M. Arzate-Ferna´ndez, and Marı´a G. Gonza´les-Pedroza Abstract Somatic embryogenesis in Agave genus has been induced; however, it is desirable to increase the rate of growth to get a more efficient propagation system. In this chapter, we present in detailed a protocol for somatic embryogenesis in Agave cupreata and the use of silver nanoparticles in a temporary immersion system. This is an efficient method that can be used commercially to improve the production and germination of somatic embryos. Key words Agave cupreata, Temporary immersion system, Somatic embryogenesis, Silver nanoparticles

1

Introduction Throughout the world, climate change and the extension of markets for some agave products, such as fructans, inulin, biofuels, tequila, mezcal and fermented sap, etc., have caused a growing interest in these plants in the national and global context. This demand has been accompanied by the overexploitation of wild populations, illegal looting of plants, and the destruction of their habitat [1]. Naturally, these plants reproduce sexually and/or asexually; the first requires 7–35 years to reach maturity and the second is limited in some species [2]. An alternative to conventional propagation in agave is the use of tissue cultures and plant cells, to easily obtain new seedlings in a short time and on a large scale [3]. Agave seedlings obtained by somatic embryogenesis can improve their quality with the use of temporary immersion system (TIS) [4]. Recently, the use of TIS has allowed to reduce production costs and increase the multiplication rate gates in these plants

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_9, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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[5]. However, contamination is a serious problem in TIS during the micropropagation process [6]. The presence of endophytic contaminants causes high losses in the in vitro culture of plants [7]. In Agave, Bacillus licheniformis has been found as an endophytic organism, which affects in vitro propagation procedure [8]. An environmental friendly strategy for the control of microorganisms is the green synthesis of silver nanoparticles using Agave leaves [9, 10], which organs have been reported to have antimicrobial activity due to their saponin content [11, 12].

2

Materials

2.1

Plant Material

1. Agave cupreata seeds.

2.2

Reagents

1. Seed sterilization solution: 20 mL anti-benzyl surgical soap and two drops Tween 20. 2. Ethanol: 70% (v/v) solution in water. 3. Sodium hypochlorite: 1% (v/v) solution in water. 4. Silver nanoparticles (AgNP) biosynthesized from Agave cupreata with a particle size of 2.1 ± 0.8 nm.

2.3

Instrumentation

1. 50 mL sterile Falcon tubes. 2. Scalpel and forceps. 3. Sterile plastic Petri dishes (100 mm × 15 mm).

2.4

Equipment

1. Magnetic stirrer. 2. Fridge. 3. Stereo microscope. 4. Twin Bottle-type temporary immersion system (see Fig. 1).

2.5

Media

The culture media commonly used are based on modifications of the MS medium [13]. Supplements such as growth regulators, gelling agents, and other additives are specifically indicated for each stage in the protocol (Table 1). 1. Embryogenic callus induction medium (ECIM): modified MS medium supplemented with 5 mg/L 2,4-D, 3 mg/L BA, and 60 g/L sucrose. Adjust the pH to 5.7 with 0.1 N NaOH or 0.1 N HCl. Add 8.0 g/L of agar and then autoclave at 121 °C for 20 min. Pour 20 mL of culture medium into Petri dishes (100 mm × 15 mm). 2. Somatic embryo maturation and expression medium (SEMEM): modified MS medium supplemented with 100 mg/L putrescine and 30 g/L sucrose. Adjust the pH to 5.7 with 0.1 N NaOH or 0.1 N HCl. Add 8.0 g/L of agar and

Agave Somatic Embrygenesis in a Temporary Immersion System

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Fig. 1 Parts of a twin vial temporary immersion system (TIS). (a) TIS with two twin flasks, one is used as a deposit of the plant material and the other contains medium culture, (b) air compressor that allows the flow of air from one container to another, (c) programmable logic controller (PLC) for establish the times and frequencies of immersion, (d) filters and manometer to purify and regulate the flow of air, (e) solenoid valves to control the flow of air between containers

then autoclave at 121 °C for 20 min. Pour 20 mL of culture medium into Petri dishes (100 mm × 15 mm). 3. Somatic embryo germination and growth medium (SEGGM): modified MS liquid medium supplemented with 30 g/L sucrose. Adjust the pH to 5.7 with 0.1 N NaOH or 0.1 N HCl. Autoclave at 121 °C for 20 min.

3 Methods 3.1

Seed Sterilization

1. In the laminar flow hood, transfer seeds to a Falcon tube with the seed sterilization solution, and shake with a magnetic stirrer for 15 min. 2. Discard the solution anti-benzyl surgical soap and Tween 20, and add 70% ethanol solution and shake for 1 min. 3. Discard the 70% ethanol solution, add 1% sodium hypochlorite solution, and shake for 1 min. 4. Discard the 1% sodium hypochlorite solution, and rinse three times with sterile distilled water. 5. Store the seeds in a Falcon tube with sterile distilled water in a refrigerator at 4 °C for 24 h (see Note 1).

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Table 1 Components of the media culture used in each phase of the somatic embryogenesis in Agave cupreata, including mineral salts, growth regulators, vitamins, and pH

Component

Somatic embryo Embryogenic callus maturation and induction medium expression medium (mg/L) (mg/L)

Somatic embryo germination and growth medium (mg/L)

Ammonium nitrate

412.5

825

825

Potassium nitrate

475

950

950

Calcium chloride

110

220

220

Magnesium sulfate

92.5

185

185

Potassium phosphate

42.5

85

85

EDTA disodium salt

9.325

18.65

18.65

Ferrous sulfate

6.95

13.9

13.9

Manganese sulfate

5.575

11.15

11.15

Zinc sulfate

2.15

4.3

4.3

Boric acid

1.55

3.1

3.1

Potassium iodide

0.2075

0.415

0.415

Sodium molybdate

0.0625

0.125

0.125

Cupric sulfate

0.00625

0.0125

0.0125

Cobalt chloride

0.00625

0.0125

0.0125

2,4-Dichlorophenoxyacetic (2,4-D)

5

6-Benzylaminopurine (BA)

3

Putrescine

100 30a

30a

250

50

50

Thiamine

2.5

0.05

0.05

Pyridoxine

0.5

0.25

0.25

0.25

0.25

Sucrose

60

Myoinositol

a

Nicotinic acid

a

Agar

8a

8a

pH

5.7

5.7

g/L

5.7

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3.2 Induction of Embryogenic Callus from Zygotic Embryos

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1. In the laminar flow hood, under aseptic conditions, extract the zygotic embryo from the seeds with forceps and a scalpel, with the aid of a stereoscopic microscope (see Note 2). 2. Establish 10 zygotic embryos per Petri dish containing ECIM, and seal with parafilm or plastic wrap. 3. Incubate in the dark at 25 ± 2 °C in the growth room for 30 days (see Note 3). 4. Transfer callus to fresh ECIM. 5. Incubate in the dark at 25 ± 2 °C in the growth room for additional 30 days (see Note 4).

3.3 Expression and Maturation of Somatic Embryos

1. Transfer embryogenic callus to SEMEM. 2. Incubate in the dark at 25 ± 2 °C in the growth room for 30 days (see Note 5). 3. Transfer callus to fresh SEMEM (see Note 6). 4. Incubate in the dark at 25 ± 2 °C in the growth room for additional 30 days (see Note 7).

3.4 Germination and Growth of Somatic Embryos

1. Transfer scutellar stage somatic embryos to the twin bottle temporary immersion system plant jar and SEGGM medium (see Notes 8, 9, and 10). 2. After medium sterilization, it was added to different concentrations (0, 100, and 200 mL/L) of biosynthesized silver nanoparticles from Agave cupreata to stop microbial growing (see Note 11). 3. Program the PLC to have an immersion frequency of 2 min every 8 h. 4. Incubate at 25 ± 2 °C with a photoperiod of 16 h in the growth room, for 45 days.

4

Notes 1. It is necessary to keep the seeds in sterile distilled water to favor the softening of the testa. 2. Extract the entire zygotic embryo, without causing mechanical damage (see Fig. 2a). 3. Callus is induced in the zygotic embryo after 3 days in ECIM medium. Auxins, mainly 2,4-D, are important for the acquisition of embryogenic capacity and for the initial stimulation of somatic embryogenesis (see Fig. 2b). 4. The callus proliferates and is maintained in the same ECIM medium (see Fig. 2c).

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Fig. 2 Somatic embryogenesis process in a twin vial temporary immersion system. (a) Extraction of the zygotic embryo from the seed (arrow), (b) callus (arrow) obtained from the zygotic embryo after 3 days in ECIM medium, (c) callus obtained after 60 days in ECIM medium, (d) somatic embryos obtained after 60 days in SEMEM medium, (e and f) regenerated plantlets of A. cupreata from somatic embryos in a twin flask temporary immersion system

5. Expression of somatic embryos is observed after 15 days in the SEMEM medium. 6. This promoting action of putrescine on somatic embryogenesis may be due to efficient conversion of competent cells into embryos [14].

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Fig. 3 Effect of silver nanoparticles (AgNP) biosynthesized from Agave cupreata extract on the germination and growth of A. cupreata somatic embryos in TIS. (a) AgNP biosynthesized from A. cupreata extract, (b) AgNP image obtained from a transmission electron microscope (TEM 2010 JEM at 200 kV acceleration in brightfield mode) (Bar = 20 nm), (c) seedlings regenerated from somatic embryos in a TIS supplemented with biosynthesized AgNP

7. Mature embryos can be obtained at 45–60 days on SEMEM medium (see Fig. 2d). 8. Selection the somatic embryos in the scutellar stage with whitish coloration. 9. The twin flask temporary immersion system is efficient in somatic embryo germination (see Fig. 2e). 10. Germination of the somatic embryos is a similar process that those happened in zygotic embryos; thus, in somatic embryogenesis, an apical part and a root part are generated without the need for a rooting stage (see Fig. 2f). 11. Silver nanoparticles biosynthesized from Agave cupreata extract reduce contamination in TIS, and with a concentration of 200 μL/L, they completely inhibit its growth (see Fig. 3). 12. The effectiveness of AgNP in the stop growing microbial contaminants from in vitro cultures depends on AgNP size, shape, and type of coating [15]. 13. AgNP favor the germination percentage of somatic embryos, without affecting their growth rate. 14. Silver nanoparticles are more effective in a liquid medium; however, using them in a semisolid medium makes their diffusion difficult [6].

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15. The use of polyethylene foam as a support in the TIS promotes hyperhydricity of plants; therefore, it is convenient to place a mesh in the glass tube of the container of plant material, to prevent somatic embryos from being transferred to the container of medium. 16. Using this protocol, high-quality, easy-to-handle plantlets are regenerated, and the oxygenation of the medium and gas exchange through the stationary medium are kept under control. References 1. Torres-Garcı´a I, Garcı´a-Mendoza AJ, Sandoval-Gutie´rrez D, Casas A (2020) Agave cupreata, Maguey Papalote. IUCN Red List Threat Species:1–8. https://doi.org/10. 2305/IUCN.UK.2020-1.RLTS. T114979361A116353713 2. Garcı´a Mendoza AJM (2007) Los agaves de Me´xico. Ciencias 87:14–23 3. Bautista-Montes E, Hernandez-Soriano L, Simpson J (2022) Advances in the micropropagation and genetic transformation of Agave species. Plan Theory 11:1–12. https://doi. org/10.3390/plants11131757 4. Martı´nez-Martı´nez SY, Arzate-Ferna´ndez AM, Alvarez-Arago´n C, Martı´nez-Velasco I, Norman-Mondrago´n TH (2021) Regeneration of Agave marmorata roelz plants in temporary immersion systems, via organogenesis and somatic embryogenesis. Trop Subtrop Agroecosyst 24:1–13. https://doi.org/10.56369/ tsaes.3472 5. Portillo L, Santacruz-Ruvalcaba F (2006) Factibilidad de uso de un nuevo sistema de inmersio´n temporal (orbitabion ®1 ) para embrioge´nesis soma´tica de Agave tequilana Weber Cultivar Azul. Boletin Nakari 17:43–48 6. Spinoso-Castillo JL, Chavez-Santoscoy RA, Bogdanchikova N, Pe´rez-Sato JA, MoralesRamos V, Bello-Bello JJ (2017) Antimicrobial and hormetic effects of silver nanoparticles on in vitro regeneration of vanilla (Vanilla planifolia Jacks. ex Andrews) using a temporary immersion system. Plant Cell Tissue Organ Cult 129:195–207. https://doi.org/10. 1007/s11240-017-1169-8 7. Abreu E, Sosa M, Ascunce Del Sol G, Gonza´lez G (2016) Efecto de antibio´ticos en la propagacio´n in vitro de Agave fourcroydes Lem. Biotecnologı´a Vegetal 16(1):31–36 8. Martinez-Rodriguez A et al (2019) Agave seed endophytes: ecology and impacts on root architecture, nutrient acquisition, and cold stress tolerance. In: Verma S, White J Jr (eds)

Seed endophytes. Springer, Cham. https://doi. org/10.1007/978-3-030-10504-4_8 9. Moreno-Luna FB, Tovar-Corona A, HerreraPerez JL, Santoyo-Salazar J, Rubio-Rosas E, Va´zquez-Cuchillo O (2019) Quick synthesis of gold nanoparticles at low temperature, by using Agave potatorum extracts. Mater Lett 235:1–15. https://doi.org/10.1016/j.matlet. 2018.09.122 10. Moreno-Luna FB, Herrera-Pe´rez JL, BautistaHerna´ndez A, Meraz-Melo MA, SantoyoSalazar J, Va´zquez-Cuchillo O (2022) Biosynthesis of gold nanoparticles from Agave potatorum extracts: effect of the solvent in the extraction. Mater Today Sustain 20:1–8. https://doi.org/10.1016/j.mtsust.2022. 100231 11. Leal-Dı´az AM, Santos-Zea L, Martı´nez-Escobedo HC, Guajardo-Flores D, Gutie´rrez-Uribe JA, Serna-Saldivar SO (2015) Effect of Agave americana and Agave salmiana ripeness on Saponin content from Aguamiel. J Agric Food Chem 63:3924–3930. https://doi.org/10. 1021/acs.jafc.5b00883 12. Sidana J, Singh B, Sharma OP (2016) Saponins of Agave: chemistry and bioactivity. Phytochemistry 130:22–46. https://doi.org/10. 1016/j.phytochem.2016.06.010 13. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473– 497 14. Bajaj S, Rajam MV (1996) Polyamine accumulation and near loss of morphogenesis in longterm callus cultures of rice. Plant Physiol 112: 1343–1348 15. Ramı´rez-Mosqueda MA, Sa´nchez-Segura L, Herna´ndez-Valladolid SL, Bello-Bello E, Bello-Bello JJ (2020) Influence of silver nanoparticles on a common contaminant isolated during the establishment of Stevia rebaudiana Bertoni culture. Plant Cell Tissue Organ Cult 143:609–618. https://doi.org/10.1007/ s11240-020-01945-9

Chapter 10 Micropropagation of Chayote (Sechium edule L.) var. virens levis in RITA® Lizandro Ramı´rez-Trejo, Rosalı´a Nu´n˜ez-Pastrana, and Anell Soto-Contreras Abstract Chayote (Sechium edule) belongs to the Cucurbitaceae family, an important family at the nutritional and medicinal levels, that has been covering international markets. Having vigorous and healthy plants is important for producers, who are very interested in cultivating chayote plants obtained from in vitro tissue culture in their orchards. Bioreactors have become an alternative with high potential for plant propagation, showing significant advantages over micropropagation in semisolid medium, by generating more plant material, larger, and more vigorous. In this chapter, a micropropagation protocol of S. edule in RITA® bioreactors is reported. Key words RITA®, Micropropagation, Sechium edule, Cucurbitaceae

1

Introduction Sechium edule is a cucurbit with high demand in the international market [1]. The nutritional and nutraceutical properties of this species are important, classifying it as a functional food with a wide diversity of bioactive compounds [2]. In addition, it is recognized for its medicinal properties, and various studies have shown its anticancer activity, especially the activity of its secondary metabolites, the cucurbitacins [3]. This species is propagated by seed that can spread diseases; furthermore, being a viviparous plant, with embryos covered by a thin and soft testa, the seed cannot be stored for a long time, and its transport can be limited [4]. There are several protocols for chayote in vitro culture in semisolid medium, where indirect organogenesis is a way of obtaining elite genotypes; however, massive multiplication in short time and space is limited using traditional in vitro propagation [5]. After the advent of temporary immersion systems (TISs), process, costs, space, and time have been more efficient, in addition to

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considerably increasing plant vigor at the acclimatization stage compared to other micropropagation systems [6–8]. TISs have demonstrated in several plant species to produce a greater number of plants compared to multiplication in semisolid medium [9]. During bioreactors functioning there are different parameters that can be manipulated to make propagation more efficient, such as type of reactor, culture medium volume, explant density, culture media, and plant growth regulators; moreover, there is also a great diversity of interval and immersion times [9]. This chapter describes the methodology to multiply S. edule in a RITA®-type TIS, which can allow the massive clonal micropropagation of genotypes with outstanding agronomic characteristics.

2 2.1

Materials Plant Material

2.2 Surface Sterilization 2.3

Culture Medium

Zygotic embryos extracted from physiologically mature fruits of S. edule var. virens levis, from commercial plantations in Huatusco, Veracruz, Mexico, are used for in vitro establishment. Disinfectant solutions: commercial detergent, 70% (v/v) ethanol and 3% (v/v) sodium hypochlorite (NaClO). 1. In all stages, Murashige and Skoog (MS) basal culture medium is used [10] (see Note 1). 2. Supplements of culture medium: MS medium is supplemented with different additives depending on the stage of culture. Culture media in different stages are as follows: establishment culture medium (ECM), shoot multiplication medium (SMM), temporary immersion system medium (TISM), and rooting medium (RM) (Table 1). 3. Stock solutions of plant growth regulators (see Note 2). 4. pH is adjusted with 0.1 and 1 N NaOH and HCl.

2.4

Equipment

1. Laboratory equipment: analytical balance, electronic balance, magnetic stirrers, heating plate, potentiometer, microwave, refrigerator, and autoclave. 2. Crystal material: volumetric flasks (100 mL), Erlenmeyer flasks (250 mL), beakers (1 L), graduated cylinder (100, 500 and 1000 mL), and micropipettes (500 y 1000 μL). 3. Culture vessels: glass containers (100 mL) (see Note 3) and RITA® bioreactor (1000 mL). 4. Laminar flow hood for working in aseptic conditions. 5. Culture instruments: stainless steel forceps, surgical scalpel, scalpel blades, and sterilized glass Petri dishes (150 × 15 mm). 6. Other materials: adhesive plastic wrap, paper towels and aluminum foil.

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Table 1 Culture media used for the different culture stages of S. edule Culture stage

Media additives

Culture establishment medium (CEM)

Shoot multiplication medium (SMM)

Rooting Temporary immersion medium system medium (TISM) (RM)

MS salts (%)

50

100

100

100

MS vitamins (%)

100

100

100

100

6-Benzylaminopurine (BAP) (mg/L)

–

1

1

–

1-Naphthaleneacetic acid (mg/L)

–

–

–

1

Myoinositol (mg/L)

100

100

100

100

Sucrose (g/L)

30

30

30

30

Phytagel (g/L)

2.5

2.5

–

2.5

3

Methods

3.1 Stage I: In Vitro Establishment

1. Wash the fruits with plenty tap water and commercial soap. 2. Extract the zygotic embryos, following the methodology of Cruz-Martı´nez et al. [11]. The exocarp is removed and cubes of approximately 5 × 4 × 5 (width, depth, height) cm are obtained as explants, containing the embryos, cotyledons, and endosperm, covered with a layer of mesocarp (Fig. 1a). This activity can be performed on a conventional laboratory bench. 3. Disinfect the surface of the laminar flow hood with 70% ethanol. 4. The explants are surface sterilized by immersion in 70% ethanol for 3 min and then disinfected with 3% (v/v) NaClO for 5 min. Subsequently, the explants are rinsed three times with sterile distilled water. 5. Using forceps and scalpel, cotyledons and embryos are removed using a sterile glass Petri dish as support and placed in CEM for 15–20 days, until embryos germinate and seedlings have 4–6 well-developed leaves (Fig. 1b, c). 6. The explants are incubated at 25 ± 2 °C, under an irradiance of 50 μmol m2 s-1 and a 16/8 h photoperiod (light/dark). 7. After 15–20 days, explants are multiplied in SMM.

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Fig. 1 Establishment and multiplication in vitro of chayote. (a) Fruits in physiological maturity. (b) Establishment of zygotic embryos. (c) Zygotic embryos germinated at 20 days of culture. (d) Multiplication in semisolid medium

3.2 Stage II: In Vitro Multiplication Using Semisolid Medium of Plant Material That Will Be Introduced into RITA® Bioreactor

1. For multiplication on semisolid medium, place 2 cm-long nodal segments containing a node from established seedlings in 100 mL glass containers with 20 mL of SMM, and place four nodal segments per container (Fig. 1d).

3.3 Multiplication in RITA®

1. RITA® TISs (1000 mL, 150 × 130 mm) (VITROPIC, SaintMathieu-de-Tre´viers) are used. Wash the RITA® bioreactor and polypropylene hoses with detergent and tap water, then rinse them with distilled water, immerse them in a 5% (v/v) NaClO solution for 1 h, and rinse them three times with sterile distilled water (see Note 4).

2. Incubate explants under the same conditions indicated in the establishment stage for 30 days. After two subcultures, continue with the multiplication stage using the RITA® system.

2. Use new polytetrafluoroethylene (PTFE, 0.20 μm) filters (see Note 5). 3. Assemble the RITA® system (see Note 6) (Fig. 2). Pour 250 mL of TISM (Table 1) into the bioreactor. Seal the filter holes with aluminum foil, then place the RITA® in two nylon

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Fig. 2 Assembly of RITA® system

Fig. 3 Multiplication of chayote in RITA system. (a) RITA® system coupled to the pneumatic system. (b and c) Multiplication in RITA® system (scale bar = 1 cm)

bags, and autoclave at 120 °C with a pressure of 115 kPa for 20 min. 4. Prepare the laminar flow hood, then culture 2 cm-long nodal segments in the RITA® system, and place 10 nodal segments per bioreactor.

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5. Install the RITA® system: Connect the air outlet of the pneumatic system to the central filter orifice of the RITA® system, and set the timer to a frequency of 8 h with a 2 min immersion for 30 days (Fig. 3a). 6. Maintain the bioreactors under the light and temperature conditions described above. 3.4 Stage III: Elongation and Rooting

1. After 30 days of culture in TISM, disassemble the RITA® system from the air outlet of the pneumatic system, and take it to the laminar flow hood. Subsequently, separate and cut the chayote shoots (Fig. 3b, c) into 2 cm explants. 2. Culture explants in 100 mL glass containers containing 20 mL of RM (Table 1), place four explants in each container, and keep them under the light and temperature conditions described above for 30 days.

3.5 Stage IV: Acclimatization

1. Place a mixture of substrate, peat moss and agrolite (1:1) in nylon bags, and autoclave at 120 °C with a pressure of 115 kPa for 20 min (see Note 7). Once cooled, fill the cavities of the culture tray with the previously sterilized substrate. 2. Carefully remove the seedlings from the RM, wash, and remove the culture medium attached to the roots with distilled water (see Note 8). 3. Prepare a solution of commercial fungicide and bactericide at the dose recommended by the manufacturer, and soak the seedlings for 5 min to prevent any contamination. Then, apply a commercial rooter to the roots of the seedlings. 4. Plant chayote seedlings in the growing tray, and place a transparent lid over the tray to maintain high relative humidity, taking care that the lid is high enough so that the seedlings do not rub against it (Fig. 4a). 5. Maintain seedlings in greenhouse conditions with 50% shade and 50 ± 10% relative humidity, for 30 days (see Note 9). 6. From day 1 to 15, water once a week and keep the culture tray covered. 7. From day 15 onward, irrigate every third day, and apply a commercial fertilizer every 15 days. Remove the lid for prolonged periods of time during irrigation, until 30 days of acclimatization, and finally remove the lid completely (Fig. 4b).

4

Notes 1. The medium can be prepared using the commercial MS formulation (Murashige and Skoog 1962) or individually prepared stock solutions A (macronutrients), B (micronutrients), F (iron

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Fig. 4 Acclimatization of chayote seedlings. (a) Seedlings grown in the acclimatization tray with a transparent plastic lid. (b) Thirty-days old, acclimatized seedlings

source), and G (vitamins). To make 1 L of MS medium, pour 800 mL of distilled water into a 1 L flask with a magnetic stir bar; add 30 g of sucrose, the salts, and vitamins of the MS medium; and dissolve. Adjust the pH to 5.8 with a potentiometer, adding 0.1 or 1 N NaOH or 0.1 or 1 N HCl under continuous stirring, and then make up to 1 L with distilled water using a graduated cylinder (1000 mL). If the medium is liquid, do not add phytagel; otherwise, preheat and add the amount indicated in Table 1. Boil the medium and pour 20 mL into 100 mL glass containers. Finally, sterilize in autoclave at 115 KPa at 120 °C for 20 min. 2. Stock solutions of plant growth regulators are made by weighing 10 mg of BAP and 10 mg of ANA; dissolve each regulator individually with drops of 1 N NaOH, and make up to a final volume of 100 mL with distilled water. 3. To avoid contamination, wash glassware with detergent and tap water, and then rinse with distilled water. 4. Before sterilization, check with tap water the correct functioning of the RITA® system already assembled and connected to the pneumatic system; the use of metal clamps on the polypropylene hoses connecting the bioreactor is recommended. 5. If the PTFE membrane filters are not new, clean them by injecting 96% (v/v) ethanol with a needle-free syringe, and dry them at room temperature. 6. Cap assembly: Place the cap ring, install the PTFE filters to the polypropylene hoses and secure them with metal clamps, and then connect them to the air inlet and outlet of the cap. Internal assembly of the system: Install the center tube ring at the top and then at the bottom (where the thread is), and assemble the netting, spacer ring, culture basket, and bell.

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Container assembly: Place the internal assembly inside the container, and close it with the cap assembled. 7. The prepared substrate should be uniformly moistened before sterilization. The sterile substrate should not be stored for long periods of time, as it could become contaminated. 8. Chayote seedlings are fragile and dehydrate quickly, so it is recommended to wash them gently with the fingertips and store them in Petri dishes with distilled water to keep them hydrated. 9. Follow all waste disposal regulations when disposing waste materials. References ˜ iguez J (2010) El chayote (Sechium 1. Cadena-In edule (Jacq.) SW.), importante recurso fitogene´tico mesoamericano. Agroproductividad 3:3– 11 2. Vieira E, Pinhob O, Ferreira I, Delerue C (2019) Chayote (Sechium edule): a review of nutritional composition, bioactivities and potential applications. Food Chem 257:557– 568. https://doi.org/10.1016/j.foodchem. 2018.09.146 3. Salazar-Aguilar S, Ruiz-Posadas LDM, ˜ iguez J, Soto-Herna´ndez M, Cadena-In ˜ iga-Sa´nchez I, RivSantiago-Osorio E, Aguin era-Martı´nez AR, Aguirre-Medina JF (2017) Sechium edule (Jacq.) Swartz, a new cultivar with antiproliferative potential in a human cervical cancer HeLa cell line. Nutrients 9:798. https://doi.org/10.3390/nu9080798 4. Veigas GJ, Bhattacharjee A, Hegde K, Shabaraya AR (2020) A brief review on Sechium edule. Int J Pharm Sci Rev Res 65:165–168. https:// doi.org/10.47583/ijpsrr.2020.v65i02.026 ˜ ez-Pastrana R, Rodrı´5. Soto-Contreras A, Nu´n guez-Deme´neghi M, Aguilar-Rivera N, Galindo-Tovar M, Ramı´rez-Mosqueda M (2022) Indirect organogenesis of Sechium edule (Jacq.) Swartz. In Vitro Cell Dev Biol Plant 58:903–910. https://doi.org/10. 1007/s11627-022-10304-6 6. Etienne H, Berthouly M (2002) Temporary immersion systems in plant micropropagation.

Plant Cell Tissue Cult 69:215–231. https:// doi.org/10.1023/A:1015668610465 7. Georgiev V, Schumann A, Pavlov A, Bley T (2014) Temporary immersion systems in plant biotechnology. Eng Life Sci 14:607–621. https://doi.org/10.1002/elsc.201300166 8. Steingroewer J, Bley T, Georgiev V, Ivanov I, Lenk F, Marchev A, Pavlov A (2013) Bioprocessing of differentiated plant in vitro systems. Eng Life Sci 13:26–38. https://doi.org/10. 1002/elsc.201100226 9. Mirzabe AH, Hajiahmad A, Fadavi A, Rafiee S (2022) Temporary immersion systems (TISs): a comprehensive review. J Biotechnol 357:56– 83. https://doi.org/10.1016/j.jbiotec.2022. 08.003 10. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473– 497. https://doi.org/10.1111/j.1399-3054. 1962.tb08052.x 11. Cruz-Martı´nez V, Castellanos-Herna´ndez OA, Acevedo-Herna´ndez GJ, Torres-Mora´n MI, Gutie´rrez-Lomelı´ M, Ruvalcaba-Ruiz D, Rodrı´guez-Sahagu´n A (2017) Genetic fidelity assessment in plants of Sechium edule regenerated via organogenesis. S Afr J Bot 112:118– 122. https://doi.org/10.1016/j.sajb.2017. 05.020

Part III Micropropagation in TIS of Ornamental Species: Protocols

Chapter 11 Direct Shoot From Root and True-to-Type Micropropagation of Limonium “Misty Blue” in Partially Immersed Culture on an Aluminum Mesh Raft Priyanka Raha, Gourab Saha, Ishita Khatua, and Tapas Kumar Bandyopadhyay Abstract Commercial plant tissue culture now primarily serves the ornamental horticulture industry. The main pillars of the commercial tissue culture business are scalability of production, cost reduction, limited labor involvement, high quality, and genetic homogeneity of propagated plants. Based on these requirements, the current protocol employs a partially immersed liquid culture medium supported by a flexible aluminum mesh raft with a wire stand to facilitate shoot organogenesis from the horizontally placed root explants and hold the plants upright for shoot multiplication and rooting of Limonium Misty Blue. It is a florist crop that is in high demand as both dried and fresh flower fillers in various floral decorations. The majority of cultivated Limonium or statice cultivars are heterozygous in nature and propagate commercially through in vitro propagation to cater to the huge demand for planting materials needed for flower production. This is the first protocol to describe direct shoot organogenesis from the roots in a liquid half-component of Murashige and Skoog’s (1962) (MS) basal medium supplemented with 1.6 μM NAA and 1.1 μM BA. The regenerated shoots are multiplied and rooted at the same time on the raft in a MS-based liquid culture medium that included 0.44 μM BA and 1.07 μM NAA. In comparison to agar-gelled medium, plants cultured in liquid medium grow more quickly without any signs of hyperhydricity. In liquid medium, a clump of 4–5 shoots is formed from a single shoot explant within 4 weeks and are rooted simultaneously within 6 weeks. On average, seven explants may fit on each raft, so on average, 25 healthy plants are produced from a single bottle. The regenerated plants are easily hardened in the greenhouse, and using ISSR-based molecular markers, the genetic homogeneity of the randomly selected hardened plants can be determined. Key words Statice, In vitro propagation, Liquid culture, Organogenesis, Shoot proliferation, Rooting, ISSR, Hardening

1

Introduction Floriculture, ornamental horticulture, and landscape horticulture are three important sectors are coming up with the profitable ventures in the agribusiness. It is concerned with the cultivation

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_11, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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and commercialization of flowers, flowering plants, foliage plants, and landscape plants. According to the “Cut flower global market report 2023” (https://www.researchandmarkets.com/reports/ 5820017/cut-flowers-global-market-report#src-pos-1), the cut flower market is predicted to grow at a compound annual growth rate (CAGR) of 5.7% from $32.00 billion in 2022 to $33.83 billion in 2023, which would reach $41.53 billion in 2027 with a CAGR of 5.3%. The improvement of growing technology under protective cultivation requires the supply of new, high-quality planting materials for expansion of horticulture business with its demanding markets in recent era. In ornamental horticulture, tissue culture techniques are widely used for the propagation of uniform, true-totype, season-independent, and disease-free seedlings. Limonium is a well-known summer flowers among the cut flower used as filler in bouquets and has the capacity to sustain salt and drought stress due to their Mediterranean origin. The Misty series of Limonium hybrids are very popular in international floriculture market, Limonium “Misty Blue,” a hybrid of Limonium latifolium and Limonium bellidifolium, is very popular for their stunning purple-blue-colored flowers and long vase life. Moreover, due to its persistent colored calyx, Limonium can be dried easily and has a huge market in dry flower business. According to Reno Wholesale Flowers, a bunch of seven stems of Limonium costs is $16.57 (https://renowholesaleflowers.com accessed on 14.06.2023). The heterozygous Limonium plants are sexually incompatible to produce viable seeds and asexual reproduction through root cutting takes much more time. Therefore, the micropropagation technique was adapted commercially to fulfill the huge demand in the cut flower market [1]. In general, semi-solid medium is commonly used for micropropagation to provide support for the developing plants, but solid media leads to irregular distribution of nutrients including growth regulators to the plants [2] and also hinder intact root recovery during field transplantation [3–7]. The other important aspect in commercial tissue culture is a huge cost involvement to prepare semi-solid media, as agar-agar and Gelrite are expensive solidifying agent. If we eliminate the solidifying agent, the cost of 1 L medium will be reduced approximately US $ 1.5, which will be beneficial for commercial tissue culture industries in a competitive market. The use of liquid media may be an alternative approach, because it is easy to handle, evenly distributes nutrients, and promotes growth regulators to all the plants, which reduces the oxidative stress of the in vitro propagules [8] and ultimately reduces cost of production. In comparison to traditional gel-based micropropagation procedures, several workers reported that the liquid medium is generally more productive and results higher multiplication ratio in Stevia rebaudiana [9], Bamboo [10], Hippeastrum chmielii

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[11], Gladiolus [12], Narcissus sp. [13], Lilium longiflorum [14], and garlic [15], roses [16], and conifers [17], etc. Among the different liquid culture methods, the submerged culture exhibits unique apoplastic water buildup, resulting in physiological, anatomical, and gross morphological abnormalities; temporary immersion system requires a big investment, sophisticated technical backing, and the risk of large-scale plant contamination. Moreover, during a complete submerged or temporary immersion system, there is no provision to keep the explants erect, which is required for axillary shoot proliferation and rooting, and it ultimately leads to abnormal foliar development [18, 19]. Apart from temporary immersion [20–25] and thin film cultures [26], the use of inert support materials, such as cotton fiber, cellulose, florialite, glass beads, polyurethane foam, rock wool, and sugarcane bagasse, has been suggested for efficient liquid culture in a variety of plant species to avoid or minimize hyperhydricity [27–30]. In vitro shoot multiplication using the membrane raft (MR) system was reported in Cyclopia genistoides [31]. An improved root induction system by using a 2-scaffold system in static liquid medium was reported in Malus domestica, Betula lenta, and Musa sp. [32], and shoot regeneration from leaf disks was also reported in Limonium peregrinum [33]. The shoot organogenesis from the root was previously described in Limonium “Misty Blue” [1], but the 3-month-old Limonium plants were grown in sterile, submerged Hoagland liquid medium [34] along with 4.92 μM IBA for root-to-root formation. No efforts were made then to regenerate the plants from the root in the liquid culture. The protocol which is involved in commercial tissue culture must follow the standard procedure to assess the genetic homogeneity before handing over the plants to the growers. Presently, the ISSR-based molecular markers are frequently used to study the genetic fidelity of regenerated plants [1, 35–37]. On the above background, the current methodology includes a raft system, where a flexible aluminum mesh supported by a metal wire stand was utilized in a jam bottle for liquid culture to induce shoot buds from the root and their subsequent multiplication and rooting to produce in vitro plantlet. Operators of the commercial laboratory who are truly trained in culturing plants in solid medium may easily handle the inoculation operation during shoot multiplication and rooting in this aluminum mesh wire rafting system. In order to skip a separate rooting step and reduce the chance of contamination of a large number of plants at a time, a procedure was developed here, where shoot multiplication and rooting were done simultaneously. Because the high-quality plants are generated more quickly and at a lower cost of production than conventional solid media culture, this method will be economically feasible for large-scale in vitro propagation of Limonium. This chapter outlines a procedure for partially immersed culture with an aluminum mesh

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raft support for root-to-shoot organogenesis, single-step shoot multiplication and rooting, hardening, and random genetic homogeneity assessment of secondary hardened plants of Limonium “Misty Blue”.

2

Materials All the chemicals required for the experiment must be of analytical grade. Solutions, which are needed for this experiment, must be prepared with sterilized double-distilled water (ddH2O) and kept in the refrigerator at 4 °C to avoid fungal and bacterial growth. Always keep your laboratory clean and dispose of the laboratory waste in a particular place as directed by the institute. All the contaminated bottles should be handled with gloves and masks, and they must be decontaminated by autoclaving before discarding.

2.1 Mother Plant Collection, Maintenance, and Management of Microplantlets

1. Healthy mature 1-year-old Limonium “Misty Blue” plants (see Note 1). 2. Purchase clay pots with dimensions of 35 × 30 × 35 cm and wash them properly before use (see Note 2). 3. Pro-trays and net pots: 98 holes pro-trays, 1-inch net pots. 4. Soilrite: Sterilization was carried out before use (see Note 3). 5. Soil mix: Mix 1 part of soil, 1 part of well-rotten Farm yard manure (FYM), and 1 part of sand on a (v/v) basis. 6. Add 1 kg of single superphosphate (SSP) and 1 kg of magnesium sulfate to 500 kg of soil mix. 7. The soil mix should be sterilized with 1 part of a 37% formaldehyde solution and 50 parts of tap water, covered with transparent low-density polyethylene (LDPE) for 3 days, and dried in the sun for 7 days with occasional stirring every day before use. Avoid the soil mix, if any smell of formalin is retained, and leave it for another few days in open air. 8. Drench of fertilizer N: P: K in 19:19:19 at 2 g/L weekly twice before 8 am for vegetative growth. 9. Freshly prepared micronutrient Tracel solution at 0.5 g/L water should be sprayed weekly, one time before 8 am. 10. Antibiotic streptocycline: At the time of treatment, an antibiotic solution should be freshly prepared by dissolving 250 mg of streptocycline powder in 1 L of water. 11. Fungicide Bavistin (50% carbendazim WP): Prepare a 0.2% Bavistin solution by dissolving 2 g of Bavistin in 1 L of water and stirring repeatedly. Use as a freshly prepared solution.

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12. Fungicide Aliette fosetyl: Just before using, dissolve 2.5 g of fungicide in 1 L of water and spray in the morning. 13. Fungicide mancozeb: Freshly prepare the solution by dissolving 2 g of mancozeb powder in 1 L of water. 14. Pesticide spinosad: Take 0.3 mL of solution and dissolve in 1 L of water just before spraying on plants. 15. Bamboo stick. 2.2 Composition of the Culture Medium

In the present study, the most common Murashige and Skoog (1962) [38] basal medium, frequently known as MS basal medium (Table 1), has been used with slight modifications. 1. Macronutrient stock solution (5×): Take about 200 mL of ddH2O in a graduated cylinder or a glass conical flask, and add 9500 mg of potassium nitrate (KNO3), 8250 mg of ammonium nitrate (NH4NO3), 2200 mg of calcium chloride dihydrate (CaCl2.2H2O), 1850 mg of magnesium sulfate heptahydrate (MgSO4.7H2O), and 850 mg of potassium dihydrogen phosphate (KH2PO4) to the water one by one and mix thoroughly with a glass rod. One component should be well dissolved in water before the next is added to the solution mixture. Some water can be added to the solution to dissolve each chemical and finally bring the volume up to 500 mL. The solution should be labelled and stored at 4 °C (see Notes 4 and 5). 2. Micronutrient stock solution (50×): Prepare the micronutrient stock solution in two parts. In the first part, take 5 mg of copper (II) sulfate pentahydrate (CuSO4.5H2O) and 5 mg of cobalt chloride hexahydrate (CoCl2.6H2O), and add them one by one to 50 mL of autoclaved ddH2O in a 250 mL conical flask. Dissolve each of the chemical properly and make up the volume to 100 mL, label it as stock-I, and keep it at 4 °C. For the second part, take 25 mL of stock-I in a conical flask, and add 1115 mg of manganese sulfate tetrahydrate (MnSO4.4H2O), 430 mg of zinc sulfate heptahydrate (ZnSO4.7H2O), 310 mg of boric acid (H3BO3), 41.5 mg of potassium iodide (KI), and 12.5 mg of sodium molybdate (Na2MoO4.2H2O) one by one, and make it 500 mL as per the previous step. Label it and store it at 4 °C (see Note 6). 3. Iron stock solution (100×): Take 3725 mg of ethylenediaminetetraacetic acid disodium dihydrate (Na2EDTA.2H2O) and 2785 mg of ferrous sulfate heptahydrate (FeSO4.7H2O) separately in 500 mL and 1 L conical flasks, respectively, and add 200 mL of boiled autoclaved ddH2O to each conical flask to dissolve each chemical properly. Then, slowly add the hot Na2EDTA.2H2O solution to the FeSO4.7H2O solution and make up the volume to 500 mL, and quickly the solution

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Table 1 Components and concentration of MS (1962) basal medium Components

Concentration (mg/L)

Macronutrients KNO3

1900

NH4NO3

1650

CaCl2.2H2O

440

MgSO4.7H2O

370

KH2PO4

170

Micronutrients MnSO4.4H2O

22.3

ZnSO4.7H2O

8.6

H3BO3

6.2

KI

0.83

Na2MoO4.2H2O

0.25

CuSO4.5H2O

0.025

CoCl2.6H2O

0.025

Iron source Na2EDTA.2H2O

37.25

FeSO4.7H2O

27.85

Vitamins and glycine Nicotinic acid

0.5

Pyridoxine HCl

0.5

Thiamine HCl

0.1

Glycine

2

Myo-inositol

100

Other constituents Sucrose

30,000

Agar-Agar

7000–8000

changes into a straw color. Place the conical flask on a magnetic stirrer for 6 h, and then keep it at 4 °C after wrapping it with aluminum foil. 4. Vitamin and glycine stock solution (50×): Weigh 25 mg of nicotinic acid, 25 mg of pyridoxine hydrochloride, 5 mg of thiamine hydrochloride, and 100 mg of glycine, and add them

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one by one to 100 mL of autoclaved ddH2O. Mark it and store it at 4 °C (see Note 7). 5. Myo-inositol: Weigh 100 mg of myo-inositol and add directly to 1 L of medium. 6. Sucrose: Weigh 30 g of sucrose, mix it with 250 mL of ddH2O, and finally add it to 1 L of medium. 7. Agar-agar: Add 7 g/L agar directly into the boiled culture medium after maintaining the pH. 8. Plant growth regulator (PGRs) stock: Weigh 25 mg of 6-benzyladenine (BA) (Sigma-Aldrich, USA) into a dry, sterile falcon tube, and pour 1 mL of 1 N HCl solution into it and shake properly to dissolve. Finally, add sterile ddH2O to adjust the volume to 50 mL. Weigh 5 mg of 1-naphthalene acetic acid (NAA) (Sigma-Aldrich, USA) and dissolve in 1 mL of 1 N NaOH, and finally make the volume up to 50 mL. Mark both the solution with the date and store at 4 °C (see Note 8). 2.3 Media Composition for Shoot Bud Induction, Shoot Bud Proliferation, and One-Step Shoot Multiplication-Rooting of Plantlets

2.4 Surface Sterilization for Culture Establishment

1. Shoot bud induction: Add half strength of macronutrient, half strength of micronutrient stock, half strength of iron stock, half strength of vitamin and glycine stock of MS basal medium, 50 mg/L myo-inositol, 1.6 μM NAA, 1.11 μM BA, and 15 g/L sucrose, and adjust the pH of the solution to 5.6–5.8 by using 1 N HCl or 1 N NaOH (see Note 9). 2. Shoot bud proliferation, shoot multiplication, and rooting: Add full strength of all the stock solutions of MS basal medium, like macronutrients, micronutrients, iron stock, vitamins, and glycine stock, 100 mg/L myo-inositol, 30 g/L sucrose, 1.07 μM NAA, and 0.44 μM BA, and adjust the pH in the manner described above. All the chemicals should be prepared freshly before use. 1. Bavistin solution: Weigh 500 mg of powder and mix properly with a glass rod in 1 L of water. 2. Streptocycline solution: Take 250 mg and mix in 1 L of water. 3. Tween-20 (Merck, India): Add 1 mL in 99 mL of water. 4. Sodium hypochlorite solution (1%) (NaOCl) (4% active chlorine): Measure 25 mL of NaOCl, and add it to 375 mL of autoclaved ddH2O. 5. 0.1% mercuric chloride (HgCl2) solution: Take 100 mg into a conical flask, and add a little absolute ethanol for better dissolution. Finally, make the volume up to 100 mL with sterile distilled water.

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2.5 DNA Isolation and PCR Amplification

In the present study, the most common genomic DNA isolation protocol using the CTAB method [39] has been used. 1. 1 M Tris-HCl solution: Weigh 121.8 g of Tris-HCl and dissolve it in 700 mL of autoclaved ddH2O. Adjust the solution pH to 8.0 with 1 N HCl or 1 N NaOH, and after adjusting the volume to 1000 mL, the solution should be autoclaved for 15 min (see Note 10). 2. 5 M sodium chloride (NaCl) solution: Add 292.2 g of NaCl to a small amount of ddH2O, and make the volume up to 1000 mL, followed by autoclaving for 15 min (see Note 11). 3. 5 M ethylenediaminetetraacetic acid (EDTA) solution: Adjust the pH of 80 mL of ddH2O to 7.5 with NaOH pellets, followed by adding 18.6 g of EDTA, and place it on a magnetic stirrer. Once EDTA has dissolved completely in water, adjust the final volume to 100 mL and keep the pH at 8.0 (see Note 12). 4. CTAB buffer: Take 400 mL of boiling water into a 1000 mL conical flask, and add 100 mL of 1 M Tris-HCl (pH 8), 280 mL of 5 M NaCl, 40 mL of 0.5 M EDTA, and 20 g of CTAB (Merck, India). Mix well with a glass rod. After making the volume up to 1000 mL, autoclave it for 15 min (see Note 13). 5. Chloroform-isoamyl alcohol (24:1) (Merck, India): Measure 96 mL of chloroform and 4 mL of isoamyl alcohol in a 100 mL graduated cylinder, mix well, and keep it in an amber-colored bottle (see Note 14). 6. Tris-EDTA buffer (TE buffer): Measure 10 mL of 1 M tris base and 2 mL of 0.5 M EDTA, and add to 900 mL of autoclaved ddH2O water. Finally, make up the volume to 1000 mL, and sterilize through a 0.1 μM filter (see Note 15). 7. Liquid nitrogen. 8. Polyvinylpyrrolidone (PVP). 9. β-Mercaptoethanol. 10. TAE stock (10×) solution: Take 600 mL of deionized ddH2O in a 2 L conical flask and dissolve 48.4 g of Tris base, followed by adding 11.4 mL of glacial acetic acid and 20 mL of 0.5 M EDTA into it. The final volume should be up to 1000 mL. It does not need to be autoclaved and should be kept at room temperature. 11. Ethidium bromide (EtBr): Weigh 1000 mg and add it to 100 mL of ddH2O. The solution should be kept on a magnetic stirrer for 2–3 h until the dye has dissolved completely. 12. 6× gel loading dye: To prepare 10 mL of loading dye, take 5 mL of glycerol, 120 μL EDTA (6 mM), 600 μL of Tris HCl (60 mM), 100 μL of SDS (0.01%), 25 mg of bromophenol

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blue, and 4 mL of 180 μL ddH2O in a 15 mL falcon tube. Mix well by inverting or by using a vortexer until all components dissolve properly (see Note 16). 13. 10× Taq buffer (NEB, USA). 14. 2.5 mM dNTPs (NEB, USA). 15. Taq DNA polymerase (NEB, USA). 16. 4% DMSO. 17. MgCl2. 18. Autoclaved mili-Q water. 19. ISSR primer [(AC)8AT] (10 μM). 20. 100 bp DNA ladder. 21. RNase A. 22. Ethanol. 23. Low EEO agarose: For DNA detection, mix 800 mg of agarose in 100 mL of ddH2O (0.8%). For PCR product assessment, weigh 540 mg of agarose and mix it with 30 mL of autoclaved ddH2O (1.8%). Boil it in the microwave oven until it appears to be a clear solution. 2.6 Glasswares and Plasticwares

1. 400 mL screw-cap culture bottles. 2. Airtight glass containers. 3. Conical flask: ranges from 100 mL–2000 mL size. 4. 1000 mL reagent bottles. 5. Graduated glass measuring cylinders ranges from 100 to 1000 mL size. 6. Graduated glass pipette of different sizes (1 mL, 2 mL, 5 mL, 10 mL, and 25 mL). 7. Variable range of micropipettes (0.5–10 μL, 10–100 μL, 20–200 μL, 100–1000 μL). 8. Sterile plastic or glass petri dishes (90 mm). 9. Plastic wash bottle 500 mL. 10. A range of microcentrifuge tubes (0.2 mL, 1.5 mL, 2 mL). 11. Mortar and pestle. 12. Floating rack. 13. Glass rods. 14. Plastic beaker (100–2000 mL). 15. Heat-resistant gloves (1 pair). 16. Cryogenic protective gloves (1 pair). 17. Gel casting tray. 18. Gel running tank.

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19. Comb for the gel lane. 20. Funnel. 21. Rose can. 22. Other common laboratory items like absorbent and nonabsorbent cotton, polypropylene plastic bags, scalpel holders, blades, forceps, etc. 2.7 Apparatus, Equipment, and Infrastructure

1. Laminar airflow (LAF) cabinet. 2. An apparatus for explant culture: Prepare a raft system where a flexible aluminum mesh (0.5 mm) will be tied up with a “U”-shaped metal wire stand (2 mm) (Fig. 1a), and finally place in a jam bottle for liquid culture. The diameter of the raft should be smaller than the bottle’s mouth (diameter—5 cm) and the raft’s height should not exceed 2–2.5 cm. (Fig. 1b) (see Note 17). 3. Magnetic stirrer. 4. pH meter. 5. Autoclave. 6. Refrigerator (-20 °C, 4 °C). 7. Instruments that should be used in an established tissue culture facility. 8. Slotted angle racks with cool white fluorescent lights at photon flux density of 55 μmol m-2 s-1 and a Leyden programmable electronic timer for incubation of cultures. 9. Stereo zoom microscope (Olympus, Japan). 10. Indoor digital thermometer with temperature and humidity gauge. 11. Greenhouse with fan pad cooling system along with misting and shade net (movable 50% agro shade net). 12. Centrifuge (Eppendorf, Germany). 13. Hot water bath. 14. Nanodrop 2000 (Thermo Scientific, USA). 15. Thermal cycler (Applied Biosystems, USA). 16. Power pack for gel apparatus. 17. Gel-documentation system (Bio-Rad XRS+, Bio-Rad, USA). 18. Microwave oven.

3

Methods Plant regeneration micropropagation method in immersion culture system consists of several steps: (1) maintenance of the mother

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Fig. 1 Preparation of an aluminum mesh wire rafting system for liquid culture of Limonium “Misty Blue” (a) basic materials used to make the raft system; (b) the raft is ready before implanting in the bottle; (c) liquid culture medium with the raft system in a jam bottle; (d) surface sterilized root explants cultured on raft (bar represents 1 cm)

plant, (2) media preparation, (3) in vitro culture establishment from the root, (4) one-step shoot multiplication and rooting, (6) hardening of plants, (7) DNA isolation, and (8) genetic homogeneity assessment through ISSR primers. 3.1 Maintenance of Mother Plant

1. Healthy plantlets should be procured from a nursery and placed under greenhouse conditions for at least 7–10 days. Then, transfer the plantlets into earthen pots containing soil, FYM, and sand mixture in a 1:1:1 ratio, and keep it in green house conditions (temperature and humidity) for 1 year under a 50% agro shade net to avoid contamination during culture initiation (see Notes 18, 19 and 20). 2. Fertilize the plantlets with NPK (19:19:19) as a foliar spray every alternate day during the vegetative stage and spray micronutrient Tracel at 0.5 g/L concentration with water. A broad-

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spectrum fungicide and antibiotic treatment should be done every 15 days. For this purpose, the following chemicals should be used: Bavistin, malathion, Aliette, streptocycline, etc. For pest control, use spinosad. 3. After 12–18 months of plantation, the best time to initiate culture is the beginning of the winter season, when fresh leaves first appear. Uproot healthy plants and thoroughly wash the root portion under running tap water. Take some young, healthy roots for culture initiation, put the mother plant again in the earthen pot, and keep it in the same condition. 3.2 Culture Medium Preparation

1. During media preparation, add an adequate amount of each stock solution one by one, i.e., if you are making 1 L of MS medium, you will need to add 100 mL of macronutrients, 10 mL of micronutrients, 5 mL of iron stock solution, 10 mL of vitamins and glycine stock, 100 mg of myo-inositol, and 30 g of sucrose, and pour some water into the conical flask after adding each ingredient. Again, if you have to prepare 1 L of medium containing ½ MS and ½ sucrose, add half of the amount of all stock solutions, 50 mg of myo-inositol, and 15 g of sucrose to it. Add the required amount of PGRs to the medium, and stir well with a clean, sterile glass rod. Make the final volume up to 1 L (see Note 21). 2. The pH of tissue culture medium should be adjusted to 5.6–5.8 by using 1 N NaOH or 1 N HCl (see Note 22). 3. For support of liquid culture, insert the aluminum mesh rafting system apparatus into the bottles (Fig. 1c) (see Note 23). 4. Dispense 50 mL of culture medium in each of the 400 mL culture bottles. Cap them properly and cover with plain paper, followed by autoclaving at 15 lb. pressure and 121 °C temperature for 15 min (see Note 24). 5. After autoclaving, the cap of each bottle should be checked properly and made tight if required. Keep the bottles at room temperature for at least a few days before use.

3.3 In Vitro Culture Establishment

1. Collect the root from the uprooted mother plant on the day of the experiment, and wash thoroughly with running tap water to remove the soil. Then, the roots should be dipped in a conical flask having a solution of 0.5% (w/v) Bavistin and 0.25% (w/v) Streptocycline and kept for 2 h (see Notes 25, 26 and 27). 2. Then, wash thoroughly with running tap water and rub the root surface with a tween-20 solution. Soak them in a 1% (v/v) aqueous solution of tween-20 for the next 15 min, and wash properly with running tap water to remove all detergent from the explants (see Note 28).

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3. In the LAF cabinet, cut the root into small pieces (8–10 mm) with the help of a sterile scalpel, and wash properly with autoclaved ddH2O. Dip the root segments rapidly into a 1% (v/v) NaOCl solution for 5–7 min, and rinse the treated root pieces 5 times with sterile ddH2O (see Note 29). 4. Disinfect the roots further with 0.1% (w/v) HgCl2 for 2–3 min, and wash 5 times with sterile ddH2O (see Note 30). 5. At the last stage of explant preparation, take the roots on a sterile aluminum plate and trim slightly from both sides. Prick the whole surface of the root with a sterile scalpel, and place the explants horizontally on a raft-like stage (Fig. 1d) of the culture bottle, containing liquid medium. 6. Carefully place all the culture bottles on the culture rack, with cool white fluorescent lamps at a photon flux density of 55 μmol m-2 s-1 and a 16-h photoperiod for 4–5 weeks for callus-mediated shoot bud initiation. Maintain the culture at 25 ± 2 °C and 60–65% relative humidity. 3.4 Shoot Bud induction Through Callus

1. You can notice the first sign of response by swelling of the explants at the cut portion after 2 weeks of culture in shoot bud induction medium (Fig. 2a) (see Notes 31 and 32). 2. The appearance of some red-colored callus can be seen along the entire length of the root. Especially the pricked portion of the root exhibits more organogenic calluses, which produce small shoot buds.

3.5 Shoot Bud Proliferation, Multiplication, and Rooting in Liquid Media

1. After 4–6 weeks, once the calluses start to differentiate into shoot buds (Fig. 2b), they should be subcultured again in the shoot proliferation and rooting medium. During this step, isolate the regenerated shoot very carefully with sterile forceps, and place them erectly in the small hole of aluminum mesh on the raft (Fig. 2c) in a liquid medium containing full strength of MS basal medium containing 1.07 μM NAA, 0.44 μM BA, and 30 g/L sucrose (see Note 33). 2. Incubate the cultures again under cool white fluorescent lamps with a photon flux density of 55 μmol m-2 s-1 at 25 ± 2 °C, and maintain the light and dark photoperiod (16 h/8 h) with a Leyden programmable electronic timer. 3. After 15 days, you can notice the shoot multiplication along with good vegetative growth and root initials at the base of the culture (Fig. 2d). In the next 4 weeks, you will notice the vigorous growth of the plants in liquid medium and much better performance than in the traditional solid culture medium (Fig. 2e) with the same chemical constituents. 4. At the end of 8 weeks of culture, remove the rooted clumps along with the raft system from the bottle (Fig. 2f), where each

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Fig. 2 Different steps of in vitro liquid culture supported by aluminum mesh wire rafting system of Limonium “Misty Blue” from lab to field (a) formation of organogenic calli and shoot bud differentiation (inset) after 1-month of culture in liquid medium containing half strength of MS basal medium with 1.6 μM NAA, 1.11 μM BA, and 15 g/L sucrose; (b) regeneration of a large number of shoots in the same constituents of culture medium after second subculture of 1 month; (c) regenerated shoots are cultured in liquid medium containing full strength of MS basal medium containing 1.07 μM NAA, 0.44 μM BA, and 30 g/L sucrose for shoot

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clump normally produces 4–6 plants along with a good root system, and you can easily separate them without damaging their root system (Fig. 2g). The plants should be thoroughly cleaned in running tap water and go for hardening (see Note 34). 3.6 Hardening of Plantlets

1. You treat the plants with a solution of 0.2% Bavistin and 0.25% (w/v) streptocycline for 15 min, and the extra solution is soaked in blotting paper or newsprint. 2. For primary hardening, take the portrays of 98 holes, place a single net-pot at each hole, and fill it up with sterilized soilrite. Carefully implant the plantlets in the middle of the net pot after making a small hole with a bamboo stick, and tightly press the base of the plants inside the soilrite (Fig. 2h). 3. Immediately after spraying with water, you must place the trays in an enclosed polytunnel inside a green house. During this time, maintain an approximately 25–30 °C temperature, along with 70–90% relative humidity, and a photosynthetic photon flux density of 100 μmol m-2 s-1. A hand sprayer or overhead sprinkler can be used to keep the leaf surface moist for at least 15 days (see Note 35). 4. After 2 weeks, when the new roots are emerging and you feel the plants are tight against pulling, you remove the polythene every day for 1 to 2 h temporarily to adapt the plantlets to the conditions outside the tunnel. Gradually extend the time of exposure to acclimatize the plants to the greenhouse conditions. Under favorable conditions, new roots develop from the plantlets, forming root balls surrounding the net pot. From this time on, you have to start the application of the foliar feed every week with NPK (19:19:19) at 1 g/L concentration. Every day, you must observe each and every plant carefully. Taking care of the plantlets is necessary from early morning to late night, and if any of the plants show signs of infection, you must remove them from the tray and drench the whole tray with Bavistin solution at 1 g/L. 5. Transfer the primary hardened plants into polythene packets (15 cm × 10 cm) containing an equal amount of soil, sand, and farmyard manure at the sixth week of the plantation in portrays

ä Fig. 2 (continued) multiplication and rooting; (d) shoot multiplication along with good vegetative growth noticed in 15 days culture period; (e) visual comparison of rooted plants in both solid medium (left) and liquid medium (right); (f) rooted clumps with raft system removed from the bottle, each raft accommodate 5–7 clumps; (g) four plants having good shoot and root growth were easily isolated from a clump (inset) of the raft, (h) primary, (i) secondary hardening of regenerated plants; (j) a representative ISSR band pattern of randomly selected regenerated hardened plants of Limonium “Misty Blue” using primer (AC)8AT showed genetic homogeneity (MO—mother plant, F1–F6 regenerated plants, M—100 bp DNA marker) (bar represents 1 cm)

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(Fig. 2i). Before transplantation, pick and cut the upper ring of the net pot with a scissor, and set the plant without disturbing the root ball in the middle of the previously filled soil mix in a polythene packet. During plantation, keep in mind that the upper ring of the net pot should be set above the soil level. Immediately shower the plants with fresh water with a rose can, and keep the plant for at least 7 days under 100% shade conditions by placing two 50% agro shade nets at the top of the plants. Regularly observe each and every plant, and when you observe the initiation of growth in the maximum number of plants, you must remove one 50% agro shade net from the top and expose the plants to 50% light. After 15 days, when you will see the new leaves coming out, start fertigation with foliar grade NPK (19:19:19) at 100 mg/plant after mixing with water, maintaining a pH of 6.5–7.5 before application, and feeding them every alternate day. Spray the micronutrient solution (Tracel at 0.5 g/L) on the plants once a week. Based on the sign and symptom, apply pesticide or fungicide according to the recommended dose written on the label. The 2-monthold secondary hardened plants are now ready for plantation in the main field. 3.7

DNA Isolation

Isolate the total genomic DNA from the mother plant and six secondary hardened plants by using the CTAB DNA extraction method [39]. 1. Take 100–200 mg of leaf in an autoclaved mortar and pestle, and crush it into a fine powder with liquid nitrogen (see Note 36). 2. Add a pinch (10–20 mg) of PVP to the fine powder in the mortar and pestle and mix well. 3. Immediately add 1 mL of CTAB buffer and 0.5 μL of β-mercaptoethanol into the powder and mix well to make a paste. Take the solution into a sterile microcentrifuge tube of 2 mL and mix well by vortexing. 4. Place the 2 mL microcentrifuge tube in a floating rack, and keep it at 65 °C in a hot water bath for 2 h (see Note 37). 5. After 2 h of incubation, take the microcentrifuge tube and centrifuge it at 14,549 ×g force for 10 min. Transfer the supernatant into another microcentrifuge tube and discard the cell debris. 6. Divide the supernatant in equal volume into two microcentrifuge tubes of 2 mL and add 1 mL chloroform:isoamyl alcohol solution to each tube. Mix the solution by inverting the tube, and spin the tubes again at 14,549 ×g force for 10 min.

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7. Transfer only the aqueous layer of the supernatant carefully by using a micropipette into another sterile, microcentrifuge tube. 8. To each tube, add 400 μL 5 M NaCl and 800 μL ice-cold ethanol, followed by slowly inverting the tubes for several times. 9. Keep the tubes at -20 °C for 2 h or overnight. 10. After the incubation time, centrifuge it at 14,549 ×g force for 10 min to precipitate the DNA, and discard the supernatant. Wash the pellet repeatedly (3–5 times) with 70% ice-cold ethanol by centrifuging at 14,549 ×g force for 10 min. 11. After the last centrifugation, carefully discard the supernatant, and the pellet should dry at LAF. 12. Pellet is again resuspended in 50 μL of 1× TE buffer. 13. Then, treat the whole genomic DNA with 4 μL of RNAse and incubate at 37 °C for 2 h. 14. Assess the quality, integrity, and size of genomic DNA through a 0.8% agarose gel, and measure the quantity of DNA with a spectrophotometer. 15. Dilute the samples to 125 ng/μL with 1× TE buffer and store at 4 °C for the ISSR experiment. 3.8 Genetic Homogeneity Assessment Through ISSR Markers

For the genetic homogeneity assessment, a PCR amplification should be carried out with the isolated genomic DNA and ISSR primers by following steps: 1. You should perform the PCR reaction in a 25 μL reaction tube containing a PCR mixture, including 2.0 μL of DNA (75 ng) and 1 μL of ISSR primer [(AC)8AT] [40]. 2. To make a 25 μL reaction PCR mixture, take 2.5 μL of 10× Taq buffer, 2 μL (2.5 mM) of dNTPs, 0.2 μL 500 U of Taq DNA polymerase, 1.0 μL of ISSR primers (10 μM), 2.0 μL of DNA template (75 ng), 1.0 μL of (4%) DMSO, 1.25 μL of MgCl2 (50 mM), and 15.05 μL of autoclaved mili-Q water, and mix well by using a micropipette (see Note 38). 3. The PCR reactions should be carried out in a 2720 thermal cycler. For PCR amplification, the initial denaturation should be carried out at 94 °C for 4 min, followed by 40 cycles of PCR reaction at 94 °C for 1 min, primer annealing at the specified temperature for 1 min, elongation at 72 °C for 2 min, and final extension at 72 °C for 7 min (see Note 39). 4. Combine the PCR products with 4 μL of 6× DNA loading dye, and resolve on a 1.8% agarose gel matrix containing 0.5 μg/L of ethidium bromide in the presence of a 100 bp DNA ladder. Analyze the electrophoresed fragments of the gel by using the gel-documentation system (Fig. 2j) (see Note 40).

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Notes 1. It is recommended to buy flowering, healthy, authenticated plants from a reliable nursery that can provide the varietal features. Purchases should preferably be made in the summer for easier acclimatization to a new environment. 2. The earthen pots with three holes at the bottom should be preferred because a one-hole pot may be clogged and the plants suffer from waterlogging. 3. The soilrite should be put in a cotton bag and autoclaved for at least 30 min at 121 °C and 15 lb. pressure. After autoclaving, the sterilized soilrite must be stored in polypropylene bags, if necessary, for future use. 4. Firstly, prepare a list of chemicals that are used for the preparation of stock solution, and mark them properly after the addition of each of them. On the glass jar, note the date of preparation as well as the date after every use. 5. For basal medium preparation, always use autoclaved glassdistilled water. Do not use tap water or tap-distilled water. In the case of all nitrate stock, sometimes it may precipitate out. So, before using the stock, the solution must be heated until it dissolves completely. Always make some aliquots of the total volume of the solution in a different glass container to reduce the chances of contamination. Discard any stock that seems unclear or has precipitation at the bottom. 6. For stock-I preparation, weigh the chemical at 200× because a very small amount is necessary for this, which is non-measurable. 7. Ready-made sachets of MS basal salts and other media are available in the market (like HiMedia Company, India) to eliminate the time for stock solution preparation. The sachet contains all the instructions needed to prepare 1 L of medium; for example, for 1 L of medium, 4.4 g of this ready-made basal salt is needed. 8. NAA may also be dissolved in 95% ethanol, but it should be avoided because ethanol is very harmful for plant cells. Always mention the name, date of preparation, and concentration of the hormone stock on the falcon tube. Always use reagentgrade chemicals to get the maximum purity. 9. Do not use a solidifying agent (agar powder) in the case of liquid culture. 10. The pH of the solution should be checked again on the next day, and if the pH value changes, then it should be adjusted by following the abovementioned procedure.

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11. You can use hot ddH2O and a magnetic stirrer for a better dissolution of the salts. 12. Dissolving the entire quantity of EDTA is really challenging, so you have to use boiled ddH2O, stir continuously on a magnetic stirrer, and add NaOH pellets to the solution until the pH reaches 8.0. At this pH, the entire quantity of EDTA dissolves into the solution. At last, adjust the final volume to 100 mL. 13. The addition of CTAB powder makes the solution frothy. You should remove the froth from the solution to get the actual volume. So, keep the solution in a 37 °C incubator for 6–12 h or overnight. After this interval, slowly pour the buffer into the measuring cylinder and add ddH2O, for making the volume, up to 1000 mL. Do not pour quickly because CTAB may get foamy again. 14. It should be freshly prepared during every use. It is very much light sensitive; so, keep it in amber colored bottle for avoiding light. 15. Do not sterilize through autoclave because high temperature may destroy its components. 16. To eliminate the long processing time, you may buy the readymade 6× gel loading dye, which is available in the market. 17. The apparatus is self-designed and self-made. The material should be autoclavable and rust-free. It can be used repeatedly for liquid medium. 18. After 7–10 days of procurement from the nursery, the whole plant should be uprooted and washed with running tap water and soak them in 1 mL/L Tween-20 solution for at least 1 h before plantation. 19. The basic potting mixture should be sterilized with 1:50, 37% formaldehyde and water. Keep the potting mixture for at least 4–5 weeks to reduce the harmful effect and smell of sterilizing chemicals. Plantlets transferred into earthen pots filled with the abovementioned potting mixture, and give sufficient amount of overhead water to moisturize the root portion, sufficiently. During the first 1 month, plants should be kept under 100% agro shade net. 20. The plants will be treated periodically with fungicide, insecticide, and bactericide at an interval of 3–5 days from the start of the new season and should be stopped at least 1 month before the collection of roots to initiate culture. If the moisture level is very high for the plants, it may cause botrytis or downy mildew. The green house should be kept very clean to avoid this type of fungus. 21. If an excess amount of stock solution is taken, it should be discarded. Do not pour it back into the main stock. The same

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thing should be maintained in the case of sucrose and agar. Always clean the digital balance machine after using it. 22. Always measure the pH of the culture medium carefully. It should be between 5.6 and 5.8. If the pH of the media is less than 5.5 or above 6.0, it will hamper the explant’s response. In the case of solid agar media, below pH 5.5, the agar will not gel properly, and above pH 6.0, the gel may be too hard. Always check the pH of the medium before autoclaving because it will drop by 0.6 to 1.3. In the case of agar medium, pH should be measured before adding agar to the medium. 23. For agar-free medium, there is no need to boil the medium. In the case of liquid medium, a stage for holding the explants is required in the medium. 24. If autoclaved for a very long time, sugars turn caramelized and undergo the Maillard process. When sugars are heated excessively, they break down and produce melanoidin, which is a brown, high-molecular-weight molecule and has the ability to stop cell growth. An autoclaved medium’s yellow to light brown color is a sign that it spent too much time in the autoclave. The medium needs to be thrown away. 25. Follow and note the young stage of the root, appropriate climatic conditions at the time of explant collection, and other specified criteria, as these will affect the endogenous plant hormone concentrations, which again regulate the explant’s ability for regeneration. 26. Streptocycline, an antibiotic, and carbendazim, a systemic fungicide, may be used to kill some of the endogenous and exogenous bacteria and fungi, respectively, which aid in the development of sterile cultures. 27. Remember that you should collect the root explants from a single mother plant on the day of inoculation. Do not handle multiple plants at once. Always label the culture bottle of the mother plant with a designated code number, and label the mother plant with the same code. Comparing the genetic homogeneity of your cloned plants to that of their mother plant will be beneficial. 28. It is good to perform a final wash with double-distilled water in a sterile container. Use a different washbasin if you normally wash bottles in that one. Use a sterile lab coat and wash your hands with liquid soap before starting any procedure in the culture lab. 29. Alcohol-soaked cotton swabs should be used to sterilize the LAF cabinet’s floor and sidewall. After cleaning with alcohol, sterilize all the usable glass containers, forceps, and scalpels with direct flames. Remember that the alcohol is highly

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inflammable, so it requires expert hands. Keep a bucket of water near the LAF to get rid of any unwanted flaming incidents. 30. The double-distilled water, all required glass goods, and small equipment like scalpels, forceps, etc. should be sterilized by autoclaving for 1 h at 121 °C with 15 lb. pressure. Carry out all the sterilization procedures 1 day before the experiment day. Carefully check the autoclaved water on the day of inoculation; it may have fungal growth. Do not use 3-day-old or older distilled water. During the sterilization procedure, repeated shaking and flaming at the mouth of the conical flask can reduce the chance of contamination. In LAF, there should be an autoclaved 2 L conical flask or beaker where all the used chemicals and waste water can be discarded. Do not expose the container outside the LAF during the sterilization procedure, which increases contamination chances. 31. After 4 weeks of culture in our lab, we were able to get 61.34% of the explants to produce calluses; however, not all the explants experienced callus initiation at the same time. Carefully monitor the culture on every alternate day, because some of the explants require more time to initiate callus formation. 32. Use the hand lens to check for contamination; if found, keep the bottles away from the culture room and autoclave them without opening the cap. Do not lose hope; because mother plants are produced in low-light and high-humid environment, the contamination percentage during culture establishment may be substantial. Your sterilization method is almost flawless if you obtain 50% contamination-free cultures. Aim to inoculate more cultures at once whenever possible. 33. You must enlarge the pores between the aluminum mesh to put the shoot upright. Now place the aluminum mesh raft inside the bottle, pour the liquid medium carefully so that it just touches the lower surface of the mesh, and autoclave at 121 ° C at 15 lb. pressure for 15 min. At the time of transfer in the LAF, carefully hold the plant with sterile forceps, and place it inside the pore so that the shoots remain erect but not too deep inside the medium to avoid early rooting. Place the bottle carefully on the rack. 34. In our investigation, the shoot multiplication ratio was average (1:4), along with a good root system that can be directly transferred for hardening. In such a way, we can eliminate the pre-hardening step of micropropagation and therefore reduce the cost of production in terms of labor and medium. 35. Keep in mind that overwatering the substrate may lead to fungal or bacterial growth. To avoid this condition, the spraying should be carried out only for a few seconds. Select the

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number and time interval between two sprayings after assessing the climatic condition and microenvironment surrounding the plants inside the tunnel. If the green house is fitted with a fan-pad cooling system, it should be run from the early morning to the late evening. 36. Always wear a pair of cryogenic protective gloves when handling the liquid nitrogen. 37. After a certain interval, the sample should be mixed by inverting the tube repeatedly. 38. We can avoid the preparation of PCR mix by using PCR master mix (Takara, Japan). So, in this case, to make 25 μL of PCR mixture, take 12.5 μL of master mix, 1.0 μL of DNA (75 ng), 0.5 μL of ISSR primer, and 11 μL of autoclaved mili-Q water. 39. Ding et al. (2013) reported 30 microsatellite-based ISSR primers used in Limonium sinense. In our experiment, we have used one primer [(AC)8AT] from this list to prove the genetic homogeneity. 40. To ensure the repeatability of the results for the ISSR analysis, the PCR reactions should be carried out three times for each primer. Only distinct and repeatable bands should then be scored for the data.

Acknowledgments This work was financially supported by WBHESTB, Govt. of West Bengal [846 (Sanc.)/ST/P/S and T/2G-11/2018; June 19, 2019]; DST-PURSE PROGRAMME, GOI; Savitribai Jyotirao Phule Fellowship for Single Girl Child (UGCES-22-GE-WES-FSJSGC-16026, UGCES-22-GE-WES-F-SJSGC-16035), RUSA 10 Component (CH&E) [IP/RUSA(C-10)/17/2021 dated 26.11.2021]; and Personal Research Grants (PRG) for teachers and URS provided by the University of Kalyani, West Bengal, India. The assistance of Purbasa Kole during the experiment is gratefully acknowledged. References 1. Bose S, Karmakar J, Fulzele DP et al (2017) In vitro shoots from root explant, their encapsulation, storage, plant recovery and genetic fidelity assessment of Limonium hybrid ‘Misty Blue’: a florist plant. Plant Cell Tissue Organ Cult 129:313–324 2. Scholten HJ, Pierik RLM (1998) Agar as gelling agent: differential biological effects in vitro. Sci Hortic 77:109–116

3. Pati PK, Kaur J, Singh P (2011) A liquid culture system for shoot proliferation and analysis of pharmaceutically active constituents of Catharanthus roseus (L.) G. Don Plant Cell Tissue Organ Cult 105:299–307 4. Wawrosch C, Kongbangkerd A, Ko¨pf A et al (2005) Shoot regeneration from nodules of Charybdis sp.: a comparison of semisolid, liquid and temporary immersion culture systems. Plant Cell Tissue Organ Cult 81:319–322

Limonium In Vitro Propagation in Partially Immersed Culture 5. Latawa J, Shukla MR, Saxena PK (2016) An efficient temporary immersion system for micropropagation of hybrid hazelnut. Botany 94:1 6. Jones MP, Yi Z, Murch SJ et al (2007) Thidiazuron induced regeneration of Echinacea purpurea L.: micropropagation in solid and liquid culture systems. Plant Cell Rep 26:13–19 7. Simonton W, Robacker C, Krueger S (1991) A programmable micropropagation apparatus using cycled liquid medium. Plant Cell Tissue Organ Cult 27:211–218 8. Caplin S, Steward F (1949) A technique for the controlled growth of excised plant tissue in liquid media under aseptic conditions. Nature 163:920–924 9. Javed R, Yu¨cesan B (2022) Impact of Stevia rebaudiana culturing in liquid medium: elevation of yield and biomass, mitigation of steviol glycosides: comparative analysis of culturing of Stevia rebaudiana in solid and liquid media. Proc Pak Acad Sci B 59:69–75 10. Ara MT, Nomura T, Kato Y et al (2020) A versatile liquid culture method to control the in vitro development of shoot and root apical meristems of bamboo plants. Am J Plant Sci 11(02):262–275 11. Ilczuk A, Winkelmann T, Richartz S et al (2005) In vitro propagation of Hippeastrum× chmielii Chm.–influence of flurprimidol and the culture in solid or liquid medium and in temporary immersion systems. Plant Cell Tissue Organ Cult 83:339–346 12. Nhut DT, da Silva JAT, Huyen PX et al (2004) The importance of explant source on regeneration and micropropagation of Gladiolus by liquid shake culture. Sci Hortic 102:407–414 13. Chen J, Ziv M (2004) Ancymidol-enhanced hyperhydric malformation in relation to gibberellin and oxidative stress in liquid cultured Narcissus leaves. In Vitro Cell Dev Biol Plant 40:613–616 14. He Han B, Yu JJ, Yae BW et al (2004) In vitro micropropagation of Lilium longiflorum (Georgia) by shoot formation as influenced by addition of liquid medium. Sci Hortic 103:39– 49 15. Kim EK, Hahn EJ, Murthy HN et al (2003) High frequency of shoot multiplication and bulblet formation of garlic in liquid cultures. Plant Cell Tissue Organ Cult 73:231–236 16. Wong MY, Chu CY (1995) Effect of medium phase on the growth of rose explants in vitro. J Agric For 44:71–77 17. Paques M, Bercetche J, Dumas E (1992) Liquid media to improve and reduce the cost of in vitro conifer propagation. In: Abstracts of

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the international symposium on transplant production systems, pp 95–100, Yokohama, Japan, October 1992 18. Gangopadhyay G, Das S, Mitra SK et al (2002) Enhanced rate of multiplication and rooting through the use of coir in aseptic liquid culture media. Plant Cell Tissue Organ Cult 68:301– 310 ´ lvarez C (2009) 19. Santos Dı´az MS, Carranza A Plant regeneration through direct shoot formation from leaf cultures and from protocormlike bodies derived from callus of Encyclia maria (Orchidaceae), a threatened Mexican orchid. In Vitro Cell Dev Biol Plant 45:162– 170 20. Leyva-Ovalle OR, Bello-Bello JJ, Murguı´aGonza´lez J et al (2020) Micropropagation of Guarianthe skinneri (Bateman) Dressler et WE Higging in temporary immersion systems. 3 Biotech 10:1–8 21. Ruta C, De Mastro G, Ancona S et al (2020) Large-scale plant production of Lycium barbarum L. by liquid culture in temporary immersion system and possible application to the synthesis of bioactive substance. Plan Theory 9:844–854 22. Bello-Bello JJ, Cruz-Cruz CA, Pe´rez-Guerra JC (2019) A new temporary immersion system for commercial micropropagation of banana (Musa AAA cv. Grand Naine). In Vitro Cell Dev Biol Plant 55:313–320 23. Ramı´rez-Mosqueda MA, Cruz-Cruz CA, Cano-Rica´rdez A et al (2019) Assessment of different temporary immersion systems in the micropropagation of anthurium (Anthurium andraeanum). 3 Biotech 9:1–7 24. Ramı´rez-Mosqueda MA, Iglesias-Andreu LG, Favia´n-Vega E et al (2019) Morphogenetic stability of variegated Vanilla planifolia Jacks. plants micropropagated in a temporary immersion system (TIB®). Rend Lincei Sci Fis Nat 30:603–609 25. Afreen F (2006) Temporary immersion bioreactor: engineering considerations and applications in plant micropropagation. In: Dutta Gupta S, Ibaraki Y (eds) Plant tissue culture engineering. Springer, Berlin/Heidelberg/ New York 26. Adelberg J (2006) Agitated thin-films of liquid media for efficient micropropagation. In: Dutta Gupta S, Ibaraki Y (eds) Plant tissue culture engineering. Springer, Berlin/Heidelberg/ New York 27. Soccol CR, Mohan R, Quoirin M et al (2004) Use of sugarcane bagasse as an alternative low-cost support material during the rooting

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stage of apple micropropagation. In Vitro Cell Dev Biol Plant 40:408–411 28. Prakash S, Hoque MI, Brinks T (2004) Culture media and containers. In: Low cost options for tissue culture technology in developing countries. Proceedings of a technical meeting organized by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Vienna 29. Afreen-Zobayed F, Zobayed SMA, Kubota C et al (2000) Supporting material affects the growth and development of in vitro sweet potato plantlets cultured photoautotrophically. In Vitro Cell Dev Biol Plant 35:470–474 30. Afreen-Zobayed F, Zobayed SMA, Kubota C et al (1999) Mass propagation of Eucalyptus camaldulensis in a scaled-up vessel under in vitro photoautotrophic condition. Ann Bot 85:587–592 31. Kokotkiewicz A, Bucinski A, Luczkiewicz M (2015) Xanthone, benzophenone and bioflavonoid accumulation in Cyclopia genistoides (L.) Vent.(honeybush) shoot cultures grown on membrane rafts and in a temporary immersion system. Plant Cell Tissue Organ Cult 120: 373–378 32. Shukla MR, Piunno K, Saxena PK et al (2020) Improved in vitro rooting in liquid culture using a two piece scaffold system. Eng Life Sci 20:126–132 33. Seelye JF, Maddocks DJ, Burge GK et al (1994) Shoot regeneration from leaf discs of Limonium peregrinum using thidiazuron. N Z J Crop Hortic 22(1):23–29

34. Hoagland DR, Arnon DI (1950) The waterculture method for growing plants without soil. In: Agricultural experiment station, 2nd edn. Circular, California 35. Biswas P, Kumar N (2023) Application of molecular markers for the assessment of genetic fidelity of in vitro raised plants: current status and future prospects. In: Molecular marker techniques: a potential approach of crop improvement. Springer 36. Konar S, Karmakar J, Ray A et al (2018) Regeneration of plantlets through somatic embryogenesis from root derived calli of Hibiscus sabdariffa L. (Roselle) and assessment of genetic stability by flow cytometry and ISSR analysis. PLoS One 13(8):e0202324 37. Konar S, Adhikari S, Karmakar J et al (2019) Evaluation of subculture ages on organogenic response from root callus and SPAR based genetic fidelity assessment in the regenerants of Hibiscus sabdariffa L. Ind Crop Prod 135: 321–329 38. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473– 497 39. Doyle JI, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15 40. Ding G, Zhang D, Yu Y et al (2013) Analysis of genetic variability and population structure of the endemic medicinal Limonium sinense using molecular markers. Gene 520:189–193

Chapter 12 Increased Multiplication Rates of Vriesea hieroglyphica (Carriere) E. Morren Through a Temporary Immersion System Carolina Rossi de Oliveira , Alice Noemi Aranda-Peres, Leonardo Soriano, Paulo Hercı´lio Viegas Rodrigues , and Adriana Pinheiro Martinelli Abstract The main difficulty for the cultivation and conservation of bromeliad species is the reduced number of propagules and slow growth of many of the species, resulting in a low propagation efficiency. Bromeliad plants are hardy and relatively easy to cultivate, with a high ornamental and ecological importance. Aiming at efficient micropropagation rates of V. hieroglyphica, a highly valued bromeliad, with very low propagation efficiency, a temporary immersion system was used and compared to semisolid and liquid static medium. Cultures obtained from in vitro germinated seeds were used as explants, maintaining their genetic diversity. Micropropagation with this simple temporary immersion system, composed of two autoclavable flasks, each with one opening for the attachment of 22 μm syringe filters, connected by a rubber stopper and an inner glass tube. In the bottom flask, an air valve is attached to the filter, which is subsequently connected to an aquarium pump and a timer and plugged to an outlet. This simple temporary immersion system showed improved micropropagation efficiency and is a method that can also be evaluated for other species. Key words Bromeliad, Liquid medium, Micropropagation, Semisolid medium, Static medium, Temporary immersed medium

1

Introduction The genus Vriesea is composed of approximately 223 species, distributed in the American continent. They are epiphytic plants, with an open rosette forming a tank, leaves without spines, long, erect, or pendular inflorescence [1]. Vriesea hieroglyphica (Carriere) E. Morren is a bromeliad of ornamental importance and is vulnerable due to indiscriminate extractivism in their natural habitat. Bromeliads have ornamental potential, as well as ecological

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importance. The efficient propagation of the species for commercial availability can prevent, or at least reduce, the indiscriminate extractivism of bromeliads. Germplasm conservation, through conventional and in vitro propagation, results in rapid and uniform multiplication of ornamental plants [2, 3]. The asexual propagation of bromeliads by separation of shoots is, however, not very productive, since bromeliads produce lateral shoots mostly after flowering, due to a strong apical dominance, and in small numbers per plant. However, the greatest difficulty in the in vitro propagation of certain species of bromeliads is the intense oxidation and slow multiplication rates [3]. Among the most difficult species to micropropagate is V. hieroglyphica [3], an endemic species with occurrences in the states of Espı´rito Santo, Rio de Janeiro, Sa˜o Paulo, and Parana´, in Brazil [4]. The use of liquid culture medium in the micropropagation of plants can decrease the oxidation of cultures, reduce the growing period and production costs, and minimize contamination [5]. Agar can affect the matric potential of the culture medium by changing the availability of water, nutrients, and growth regulators, in addition to potentially altering its chemical composition [6]. Temporary immersion bioreactors are commonly used to increase the multiplication rate, decrease the cost of seedling production, and reduce contamination, which can be increase through subcultures [7–10]. This method is based on the principle that seedlings develop better and more quickly when cultivated at regular intervals of immersion in liquid medium, which can be static or under agitation, providing plant culture medium for definite times [9]. The contact of seedlings with the culture medium considerably increases absorption, since nutrients can be absorbed by leaves, stems and roots and, consequently, plants absorb more nutrients in the immersion system than in the traditional one and, consequently, produce more biomass [11]. In studies involving banana stem tips, obtained a higher rate of multiplication in liquid medium with temporary immersion of explants [7]. Many positive results have been obtained using the temporary immersion system for micropropagation of numerous cultures, such as ornamentals, fruits, crops, and forest species [10]. Considering the micropropagation of bromeliads through the temporary immersion system as a potential tool for micropropagation of plants with ornamental and ecological importance, the objective of this chapter is to optimize multiplication in vitro, of Vriesea hieroglyphica through a temporary immersion system.

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Materials Plant Material

2.2 Material and Equipment

1. Seeds of V. hieroglyphica. 1. Sterile polystyrene Petri dishes (90 mm × 15 mm and 100 mm × 15 mm). 2. Magenta™ boxes. 3. Glass flasks. 4. Agar. 5. Commercial sodium hypochlorite solution. 6. Parafilm™ or plastic wrap. 7. Growth room at 27 ± 2 °C. 8. Culture room: laminar airflow hood. 9. Surgical tools: stainless steel forceps, surgical scalpels, removable sterile surgical blades, and sterile Petri dishes.

2.3 Bioreactor System 2.4

1. In-lab-made temporary immersion system (see Note 1) (Fig. 1).

Culture Media

Fig. 1 Temporary immersion system (TIS), showing its assemblage with two autoclavable glass flasks (1), the top one upside down and an inner glass tube (2) connecting both flasks and a rubber stopper (3) securing them by the flak mouths. Two openings were made, one in each flask, for air filtration, and silicone tubes were attached. Air filtration is done by two 22 μm syringe filters (4) attached to these silicone tubes (4). The bottom flask, an air valve (5), is connected to an aquarium pump (6) and a timer (7) and plugged into an outlet

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1. Prepare stock solutions of vitamins and macro- and micronutrients of MS medium [12], dissolving the components in deionized H2O, and store at 4 °C. 2. 6-Benzylaminopurine (BAP) stock solution (1 mg/mL): Dissolve the BAP powder in a few drops of 5 M NaOH, bring to final volume with H2O, and store at 4 °C. 3. Introduction and multiplication medium (½ MS 2BAP): Using the stock solutions above, prepare the culture medium at half the concentration of salts and vitamins of MS medium [2]: ½ MS supplemented with 2 mg L-1 BAP.

3

Methods

3.1 Seed Germination

1. The seeds washed with water and commercial soap. 2. Seeds are cleaned with 25% (v/v) of commercial sodium hypochlorite solution (2.5% active chlorine). Seeds were placed in Magenta™ boxes containing culture medium. 3. Germinate and cultivate the seedlings of V. hieroglyphica in culture medium (½ MS 2 mg/L BAP). 4. Subculture every 3 weeks for approximately 5 months (see Note 2).

3.2 Temporary Immersion System

1. In a laminar flow hood, introduce the seedings into the temporary immersion system. 2. Maintain the seedlings in the upper flask and the culture medium in the lower flask (Fig. 2a).

Fig. 2 In vitro culture of Vriesea hieroglyphica using temporary immersion system. (a) Temporary immersion system in which cultures are in the dry phase and (b) immersion phase

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3. When a peristaltic pump is activated, the filtered air enters the upper compartment. 4. When the pressure increases, the liquid medium is transferred to the upper compartment through a glass tube (see Note 3). 5. The air entering the system causes the culture medium to aerate. 6. The pump is deactivated, and the liquid medium migrates from the upper to the lower compartment through the same glass tube (see Note 4).

4

Notes 1. The system basically consists of two 500 mL autoclavable glass flasks, joined by a rubber stopper and containing filters attached to each flask for air filtration. The system runs using an aquarium pump to move the liquid medium from one flask to the other allowing the temporary immersion of plants in culture medium, with the aid of a timer (Fig. 1). The period of immersion time is defined according to the culture. 2. All the cultures are maintained in a growth room, at 27 ± 2 °C. 3. This movement is possible with the entrance of external air through an attached filter (22 μm) to avoid contamination, in the upper flask, by the pressure exerted by the pump (Fig. 2). 4. This process occurs by gravity and the air escapes through a valve connected to the system.

Acknowledgments All authors acknowledge Fundac¸˜ao de Amparo a` Pesquisa do Estado de Sa˜o Paulo, FAPESP, SP, Brazil, for a doctoral scholarship to ANAP (2000/07987-5); CNPq, DF, Brazil, for a research fellowship to APM (304659/2005-3); and Joa˜o Geraldo Brancalion, CENA/USP for graphics assistance. References 1. De Paula CC (2000) Cultivo de Brome´lias. Editora Aprenda Fa´cil, p 140 2. Ramı´rez-Mosqueda MA, Cruz-Cruz CA, Cano-Rica´rdez A, Bello-Bello JJ (2019) Assessment of different temporary immersion systems in the micropropagation of anthurium (Anthurium andraeanum). 3 Biotech 9:307. https://doi.org/10.1007/s13205-0191833-2

3. Carneiro LA, Candido MSD, Gagliardi RF et al (1998) Clonal propagation of Cryptanthus sinuosus L.B. Smith, and endemic stoloniferous Bromeliaceae species from Rio de Janeiro, Brazil. Plant Tissue Cult Biotechnol 4:152–158 4. Aranda-Peres AN, Rodriguez APM (2006) Bromeliads. In: Teixeira da Silva JA (ed) Floriculture, ornamental and plant

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biotechnology, vol IV. Global Science Books, London 5. Aranda-Peres AN, Peres LEP, Higashi EM, Martinelli AP (2009) Adjustment of mineral elements in the culture medium for the micropropagation of three Vriesea bromeliads from the Brazilian Atlantic Forest: the importance of calcium. HortSci 44:106–112. https://doi. org/10.21273/HORTSCI.44.1.106 6. Costa AF, Moura RL, Neves B et al (2023) Vriesea hieroglyphica (Carrie`re) E. Morren var. hieroglyphica in Flora e Funga do Brasil. Jardim Botaˆnico do Rio de Janeiro. Available in: https://floradobrasil.jbrj.gov.br/FB121787. Access in: 6 Sept 2023 7. Alvard D, Coˆte F, Teisson C (1993) Comparison of methods of liquid medium culture for banana micropropagation. Plant Cell Tissue Organ Cult 32:55–60. https://doi.org/10. 1007/BF00040116

8. Grattapaglia D, Machado MA (1990) Micropropagac¸˜ao. In: Torres AC, Caldas LS ˜ es da cultura de tecidos (eds) Te´cnicas e aplicac¸o de plantas. EMBRAPA, Brasilia 9. Gianguzzi V, Inglese P, Barone E, Sottile F (2019) In vitro regeneration of Capparis spinosa L. by using a temporary immersion system. Plan Theory 8:177. https://doi.org/10. 3390/plants8060177 10. Vidal N, Sa´nchez C (2019) Use of bioreactor systems in the propagation of forest trees. Eng Life Sci 19(12):896–915. https://doi.org/10. 1002/elsc.201900041 11. George EF (1993) Plant propagation by tissue culture. The Technology Exegetics, London 12. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473– 497. https://doi.org/10.1111/j.1399-3054. 1962.tb08052.x

Chapter 13 Micropropagation of Encyclia cordigera (Kunth) Dressler in Ebb-and-Flow Bioreactor Obdulia Baltazar-Bernal and Evelia Guadalupe Mora-Gonza´lez Abstract The use of new technologies for micropropagation such as temporary immersion systems (TISs) is important, because it reduces costs by 40% lowering labor, agar and containers. TISs are containers designed for large-scale, semiautomatic production of plants in a liquid medium, which has been used in propagation of commercial orchids. This tool has high potential for application in micropropagation of medicinal and endangered orchids for conservation and commercial purposes. In this chapter, we describe a detailed protocol for propagation and development of Encyclia cordigera to be used in research projects for small-scale production. This protocol comprises all steps from explant preparation to the establishment orchids plantlets. Key words Asymbiotic germination, Fragrant orchid, Gravity immersion bioreactor, Mexican orchid, Orchid plantlets

1

Introduction Orchids are very important in the ecological context for feeding pollinators [1] and in the economic context for the commercialization of ornamental plants [2]. E. cordigera has been heavily extracted from its natural habitat, for being a fragrant and colorful orchid with a medium-sized inflorescence. This pressure has resulted in having only 2% of fruit set in the Soconusco region, Chiapas, Me´xico [3]. Furthermore, in the region of the high mountains of Veracruz, Mexico, fruit formation doesn’t occur [4]. To increase the production of plantlets of this species for conservation, reintroduction, and commercialization purposes in less time, it is necessary to use biotechnological tools, such as plant tissue culture (PTC) [5]. Temporary Immersion Systems (TISs) are one of the PTC tools for the commercial micropropagation of plant species. The temporary immersion (TI) consists of immersing the explant in a liquid

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_13, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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medium for periods of time using automated bioreactors. The immersion helps the plant to absorb nutrients and components of the medium [6]. This system improves the oxygenation of the explant, reduces asphyxia, and favors the multiplication rate [7]. TI favors the processes of photosynthesis and respiration and increases the chlorophyll content, stomatal function, and nutrition of plants, benefiting in vitro development and the acclimatization process [8]. There are different models of TISs, one of them is the Ebb-and-Flow bioreactor, also known as gravity immersion bioreactor (GIB) [9, 10]. The use of TISs for the propagation of ornamental orchids has been implemented as an improved technique. In this sense, micropropagation of E. cordigera in bioreactor is a process controlled by the immersion frequency and the medium volume per explant; a better knowledge of these steps could help to optimize the process to produce high quality plantlets on a large scale. Therefore, the establishment of an efficient and detailed protocol of micropropagation of E. cordigera in Ebb-and-Flow bioreactor can improve the quality and reduce process costs of plantlets. This chapter describes an easy and effective protocol for micropropagation of E. cordigera in Ebb-and-Flow bioreactor in research.

2 2.1 2.1.1

Materials Germination Plant Material

2.1.2 Capsule Disinfection

A mature, hand-pollinated E. cordigera capsule is harvested in the greenhouse at Campus Cordoba, Colegio de Postgraduados (Fig. 1a, b). The capsule is stored at 4 °C, inside a paper bag for no more than 5 days. – Commercial liquid soap. – Commercial sodium hypochlorite 1.2% solution. – 96% ethanol. – Systemic fungicide Ridomil (mefenoxam) (0.5 g/L). – Sterile distilled water. – Orbital shaker.

2.1.3 Growing Culture Medium for Sowing

– Stock solutions for Murashige-Skoog [11] medium. – Sterile distilled water. – Sucrose. – HCl (1 N) solution. – NaOH (1 N) solution. – Gellan gum (gelling agent). – Magnetic stirrer.

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Fig. 1 Different parts of E. cordigera: (a) Inflorescence; (b) mature capsule; (c) cut open capsule and exposed seeds; (d) 5-month-old plantlets for bioreactor culture

– Microwave oven. – Autoclaved scalpel, forceps, and Petri dishes. – The 120 mL glass culture jars and transparent caps. – Alcohol burner. 2.2 Ebb-and-Flow Bioreactor Growth and Development

Ebb-and-Flow bioreactor (Temporal Gravity Immersion: GIB) System which consist of two 1-liter glass vessels, one of which contains explants and the second contains culture medium as seen in Fig. 2.

2.3

– Commercial transparent plastic trays (recommended size: 23 × 23 × 11 cm; LxWxD).

Acclimatization

– Systemic fungicide Ridomil (mefenoxam) (0.5 g/L). – Sphagnum moss. – Masking tape. 2.4 Laboratory Structure

– Autoclave. – Greenhouse or place to grow plants. – Growth chamber. – Laminar flow chamber.

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Fig. 2 Bioreactor. (a and b) Partial cultures in 10 mL MS liquid medium. (c) Upper part of the Ebb-and-Flow bioreactor containing 500 mL of MS liquid medium and 100 plantlets (5 mL/plantlet)

3 3.1

Methods Germination

3.1.1 Capsule Disinfection

1. The capsule (Fig. 1b) is washed with tap water and liquid soap using a soft brush (see Note 1). 2. The capsule is immersed in sodium hypochlorite 1.2% and is constantly stirred for 20 min and then is washed with tap water. 3. Then, it is immersed in a systemic fungicide (mefenoxam) (0.5 g/L) solution and kept in constant stirring for 15 min. 4. The capsule is washed three times with sterile distilled water in a laminar flow chamber.

3.1.2 Growing Culture Medium for Sowing

1. The culture medium is prepared by dissolving 5 mL/L of each Murashige-Skoog (MS) solutions in sterile distilled water (Table 1). Add 15 g/L sucrose. 2. The pH of the culture medium must be adjusted to 5.7 ± 0.1 with 1 N NaOH or 1 N HCl.

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Table 1 Medium composition (1/2 MS) Solution I Compound

Amount (g/L)

Add (mL/L)

NH4NO3 KNO3

165 190

5

37 1.688 0.86 0.0024

5

44 0.08 0.0024

5

17 0.62 0.0248

5

2.784 3.724

5

0.1 0.5 0.5 0.04 0.002

5

Solution II MgSO4 · 7H2O MnSO4 · H2O ZnSO4 · 7H2O CuSO4 · 5H2O Solution III CaCl2 · 2H2O KI CoCl2 · 6H2O Solution IV KH2PO4 H3BO3 Na2MoO4 · 2H2O Solution V FeSO4 C10H18N2O10Na2 · 2H2O Vitamins Inositol Pyridoxine Nicotinic acid Thiamine Glycine

Add (g/L) Sucrose Gelling agent

a

15 2.5

a

For germination culture only

3. Then, 2.5 g/L gellan gum (gelling agent) is added to the solution and is stirred until removing all clumps. 4. After that, the medium is boiled in a microwave and is stirred occasionally. 5. In 100 mL glass culture jars, 15 mL of MS medium is poured. The culture glass is closed. 6. Medium is sterilized by autoclaving it at 121 °C for 15 min (Table 2, germination).

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Table 2 E. cordigera, germination, multiplication, partial, and bioreactor medium or substrate Germination (semisolid) Component

Amount

mL/flask

Autoclaving time (minutes)

½ MS Sucrose Gelling agent

30 mL/L 15 g/L 2.5 g/L

15 mL

30 mL/L 15 g/L

10 mL

15′

500 mL

30′

500 mL per tray

25′

15′

Partial culture ½ MS Sucrose

Multiplication (ebb-and-flow bioreactor) ½ MS Sucrose

30 mL/L 15 g/L

Acclimatization substrate Sphagnum moss Tap water

Up to 20 L 200 mL/L (approximately)

7. The medium is cooled at room temperature and stored at room temperature. 3.1.3

Seed Culture

1. In a flow chamber, sterile distilled water is used to wash any remnants of fungicide (mefenoxam). 2. The previously washed capsule is sprayed with 70% ethanol and flamed briefly (see Note 2). 3. The capsule is cut longitudinally to expose the seeds (Fig. 1c) (see Note 1). 4. A sterile scalpel is used to take approximately 100 seeds and spread them evenly inside the culture glass jar containing germination MS medium (Table 2). 5. The jar is closed, sealed, and incubated at 24 °C with a 16-h photoperiod at 57 μM m-2 s-1.

3.2 Ebb-and-Flow Bioreactor System Growth and Development 3.2.1 Partial Culture Preparation

1. MS medium is prepared as previously mentioned (do not add gellant gum (gelling agent) (Table 2, partial culture). 2. Then, 10 mL of MS medium is poured into a 100 mL glass jar. 3. Plantlets that are at least 5 months old and have two 0.5-cmlong rhizoids and two leaves are selected and transferred to jars (Fig. 1d); 4. Partial cultures are incubated during a week-long period to discard possible contamination.

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1. For each Ebb-and-Flow bioreactor system, 500 mL MS liquid medium is prepared (Table 2). 2. Previously washed bioreactor is assembled and put inside polyethylene bags (Fig. 3) (see Notes 3 and 4). 3. The Ebb-and-Flow bioreactor system is sterilized by autoclaving at 121 °C for 20 min. 4. Then, it is cooled at room temperature and put it inside a laminar flow chamber. 5. A small amount of the liquid medium is poured inside the glass jar containing the sponge (3 cm wide). The liquid medium must flow down correctly to the second jar. 6. Once the medium flows correctly, the rest of the liquid medium is poured, and the content of the 100 mL glass jar is added (partial culture step) (see Note 5). 7. Ebb-and-Flow bioreactor is therefore sealed and incubated at 24 °C with a 16-h photoperiod at 57 μM m-2 s-1. 8. Medium irrigation for 2 min every 8 is viable (see Note 6) and 5 mL per explant (see Note 7).

Fig. 3 Diagram of the Ebb-and-Flow bioreactor (a): (1) output sterile 0.22 μm pore-size filter; (2) metallic pipe, helps the sponge to maintain its position on the bottom; (3) sponge, prevents the obstruction of the nozzle; (4) jar nozzle, allows the flow of the MS liquid medium; (5) plastic pipe attach to the lid nozzle; (6) sterile 0.22 μm pore-size filter, allows air pumping inside the system; (7) medium containing jar. Complete Ebb-andFlow bioreactor assembled (b)

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Acclimatization

1. For each Ebb-and-Flow bioreactor, the sponge and plantlets are carefully removed from the jar. Each plantlet is placed in a small tray and washed with water for 10 min (Fig. 4) (see Note 8). 2. Twenty-five plantlets are immersed in 100 mL of systemic fungicide Ridomil (0.5 g/L), in constant shaking for 10 min (see Note 9). 3. A transparent plastic box container is prepared by making small, well-distributed holes at the lid and base of the container. 4. Up to 20 L of sphagnum moss are prepared at field capacity by adding sufficient water (approximately 100 mL every half liter) and mixing properly (see Note 10). 5. The 20 L of sphagnum moss at field capacity is placed inside a polyethylene bag and sterilized by autoclaving at 121° for 25 min. 6. The clear plastic box container is filled up with half a liter of previously sterilized sphagnum moss. The sphagnum moss is evenly placed in the container (Fig. 5a, b); 7. The plantlets are equidistantly placed on the surface of the sphagnum moss. Up to 50 medium-sized plantlets can be placed on a single container (Fig. 5c); 8. The container’s lid is closed, and the surface holes are sealed using masking tape (Fig. 5b). 9. The container is placed away from sunlight and on a wet surface for 2 weeks. 10. After 2 weeks, the masking tape is removed, but the lid is kept closed for another week; when uncovering the trays, water by sprinkler daily 1 min every hour from 10 a.m. to 4 p.m. 11. After 12 weeks, the plants are ready to go outside.

Fig. 4 E. cordigera plantlets after 3 months of multiplication culture

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Fig. 5 Acclimatization of E. cordigera. (a) Half a litter of Sphagnum substrate at field capacity. (b) Container is filled with half a litter of Sphagnum to establish the plantlets and seal the container. (c and d) E. cordigera plantlets in acclimatization

4 Notes 1. Before washing, the peduncle must be removed using a blade. This is done to prevent further sources of contamination. The blade or scalpel must be flamed or changed after cutting the capsule and before touching the seeds. 2. For E. cordigera, the capsule should be sprayed and flamed for 2 s, making sure to not overheat the seeds, since it is a mediumsized capsule. 3. A complete Ebb-and-Flow bioreactor consists of two sterile filters, 0.22 μm pore size; three 4 cm-long silicon tubes (6 mm inside and 9 outside diameter); one 18 cm-long silicon tube; one 45 cm-long silicon tube; one lid with a plastic nozzle and one lid with two plastic nozzles; two 1-liter glass vessels, one of which must have a nozzle at the lower part; one 20-cmlong metallic pipe; one circular fit-size sponge; one air pump, 150 psi; and digital programmer for immersion frequency.

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4. The completed assembled Ebb-and-Flow bioreactor must be tested by connecting it to the air pump and filling the system with 0.6% sodium hypochlorite to prevent or detect leakage, before sterilization. 5. If any leakage is detected at this stage, the Ebb-and-Flow bioreactor system must remain inside the laminar flow chamber until the leakage is repaired. The nozzles can be secured by using nylon cable ties. 6. Immersion frequency of 8 h has shown better overall plantlet development (Fig. 4a). 7. Density of 5 mL per explant is recommended for roots, leaves, and overall plant development. 8. Roots are sometimes attached to the sponge, so careful removal must be performed. To prevent root damage, small fragments of the sponge may remain attached to the root for the acclimatization. 9. The plantlets are kept in fungicide solution for 10 min. The fungicide is not washed away before the plantlets are placed in the sphagnum mos. 10. Sphagnum moss must be moisturized at field capacity before autoclaving. Field capacity is obtained by adding sufficient water to the substrate, until an evenly wet moss is achieved. Excess humidity is detected if the substrate pours water when pressed (Fig. 5a). References 1. Ackerman JD, Phillips RD, Tremblay RL, Karremans A, Reiter N, Peter CI, Bogarı´n D, Pe´rez-Escobar OA, Liu H (2023) Beyond the various contrivances by which orchids are pollinated: global patterns in orchid pollination biology. Bot J Linn Soc 20:1–30. https://doi. org/10.1093/botlinnean/boac082 2. Hinsley A, De Boer HJ, Fay MF, Gale SW, Gardiner LM, Gunasekara RS, Kumar P, Masters S, Metusala D, Roberts DL, Veldman S, Wong S, Phelps J (2018) A review of the trade in orchids and its implications for conservation. Bot J Linn Soc 186:435–455. https://doi.org/10.1093/botlinnean/ box083 3. Damon A, Roblero PS (2007) A survey of pollination in remnant orchid populations in Soconusco Chiapas, Mexico. Trop Ecol 48:1– 14 4. Baltazar-Bernal O, De-la-Cruz-Martı´nez VM, Herna´ndez-Garcı´a A, Zavala-Ruiz J (2023) An exploratory study of orchidaceae species fruits in the central zone of Veracruz state, Mexico.

Agrociencia 57:En prensa. https://doi.org/ 10.47163/agrociencia.v57i5.2860 5. Zhang B, Niu Z, Li C, Hou Z, Xue Q, Liu W, Ding X (2022) Improving large-scale biomass and total alkaloid production of Dendrobium nobile Lindl. using a temporary immersion bioreactor system and MeJA elicitation. Plant Methods 18:10. https://doi.org/10.1186/ s13007-022-00843-9 6. Baoqian Z, Sarsaiya S, Pan X, Jin L, Xu D, Benhou Z, Duns GJ, Shi J, Chen J (2018a) Optimization of nutritional conditions using a temporary immersion bioreactor system for the growth of Bletilla striata pseudobulbs and accumulation of polysaccharides. Sci Hortic 240:155–161. https://doi.org/10.1016/j. scienta.2018.06.010 7. Wu HC (2016) In vitro culture of Protea cynaroides L. microshoots in a temporary immersion system. Acta Hortic 1113:67–72. https://doi.org/10.17660/ActaHortic.2016. 1113.9

Encyclia cordigera Micropropagation Using SIT 8. Ramı´rez-Mosqueda MA, Bello-Bello JJ (2021) SETIS™ bioreactor increases in vitro multiplication and shoot length in vanilla (Vanilla planifolia Jacks. Ex Andrews). Acta Physiol Plant 43:52. https://doi.org/10.1007/s11738021-03227-z 9. Georgiev V, Schumann A, Pavlov A, Bley T (2014) Temporary immersion systems in plant biotechnology. Eng Life Sci 14:607–621. https://doi.org/10.1002/elsc.201300166

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10. Bello-Bello JJ, Ruiz JZ, Cruz-Huerta N, Baltazar-Bernal O (2020) In vitro germination and development of the trumpetist orchid (Myrmecophila grandiflora Lindl.) using ebband-flow bioreactor. Propag Ornam Plants 20: 88–95 11. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473– 497. https://doi.org/10.1111/j.1399-3054. 1962.tb08052.x

Chapter 14 Micropropagation of Guarianthe skinneri (Bateman) Dressler & W. E. Higging in Temporary Immersion Bioreactors Elizabeta Herna´ndez-Domı´nguez, David Rau´l Lo´pez-Aguilar, Andre´s Ordun˜o-Cruz, Pedro Zetina-Co´rdoba, and Marco A. Ramı´rez-Mosqueda Abstract Guarianthe skinneri (Bateman) Dressler & W. E. Higgins is an orchid valued for its ornamental characteristics. However, it is an orchid classified as threatened with extinction due to the illegal extraction from its natural habitat. In addition, its propagation through seed germination is very low, as is the case with most members of the family Orchidaceae. Its asexual propagation through pseudobulb separation is slow and produces a few propagules. For this reason, in vitro propagation techniques are an alternative to increase the number of plants obtained and thus be able to recover this valuable plant genetic resource. Temporary immersion systems (TIS) offer the advantage of mass-propagating plants for different purposes. This chapter describes a large-scale micropropagation protocol for Guarianthe skinneri using temporary immersion bioreactors (TIB). Key words Micropropagation, Orchid, Ornamental, Threatened species

1

Introduction The members of the family Orchidaceae are appreciated for their ornamental characteristics, mainly related to their flowers (size, color, and type). However, this makes orchids vulnerable to illegal looting of their natural populations for marketing [1]. In addition, the fragmentation of their natural habitat and the change in land use have reduced the number of wild individuals, threatening some orchid species with extinction [2]. The reproductive issue of orchids is due to their seed morphology (lack of endosperm), which limits natural seed germination [3]. On the other hand, asexual reproduction (vegetative shoots) is a time-consuming technique that produces a limited number of

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_14, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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propagules [4]. Therefore, biotechnological strategies such as plant tissue culture have allowed, in various orchid species, asymbiotic seed germination, or somatic organogenesis or embryogenesis, which have led to increased propagation [5]. Guarianthe skinneri (Bateman) Dressler & W. E. Higgins is an orchid distributed from Mexico to Panama; however, its natural populations have been decimated due to overexploitation for ornamental purposes [6]. Therefore, conservation and propagation strategies for this important species should be implemented to recover the currently threatened populations [7]. Temporary immersion systems are an effective tool for the mass propagation of orchids [8]. In this context, temporary immersion bioreactors (TIB) or twin-flask bioreactors offer advantages over other systems due to their different capacities, easy assembling, and wide availability of their components [9]. This chapter aims to develop a production system for G. skinneri using a TIB to contribute to the recovery of this important species.

2 2.1

Materials Plant Material

2.2 Surface Sterilization 2.3

Culture Medium

Capsules mature (4 months) in G. skinneri collected in Veracruz, Mexico, were used for the establishment of aseptic plants. Disinfectant solution: 0.6% (v/v) sodium hydrochloride, Tween® 20 (three drops per 100 mL solution), and commercial detergent. 1. Basal Murashige and Skoog (MS) [10] culture medium is used (see Note 1). 2. Medium additives used for various culture stages: MS basal medium supplemented with various additives according to the culture stage, including seed germination medium (SGM), shoot multiplication medium (SMM), shoot multiplication medium in TIS (SMMTIS), and rooting and elongation medium in TIS (REMTIS) (see Table 1). 3. pH adjustment solutions: 0.1 and 1 N NaOH and HCl.

2.4

Equipment

1. Culture room: laminar airflow hood. 2. Surgical tools: stainless steel forceps, surgical scalpels, removable sterile surgical blades, and sterile Petri dishes. 3. Medium preparation: weighing balances, magnetic stirrer, hot plate, pH meter, microwave, micropipettes, magnets, refrigerator, and autoclave.

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Table 1 Culture medium additives used for the different culture stages of S. rebaudiana Culture stage

Media additives

Seed Shoot germination multiplication medium (SGM) medium (SMM)

Shoot multiplication Rooting and medium in TIS elongation medium (SMMTIS) in TIS (REMTIS)

Medium MS (%)

100

100

100

50

Vitamins MS (%)

100

100

100

100

Benzylaminopurine – (BA) (mg/L)

3

3

–

Gibberellic acid (GA3) (mg/L)

–

–

–

1

Myoinositol (mg/L)

100

100

100

100

Sucrose (g/L)

30

30

30

30

Phytagel (g/L)

2.2

2.2

–

–

4. Culture vessels: reagent bottles, flasks (500 mL and 1000 mL capacity), beakers, measuring cylinders, and glass culture jars (100 mL). 5. Glassware cleaning: scrub glassware with a liquid detergent solution, thoroughly wash with tap water, and rinse glassware with distilled water and dry. 2.5 Bioreactor System

1. Temporary immersion bioreactors (TIB) (100 mL). 2. Platforms (metal shelf) equipped with silicone tubing/hoses, air compressor equipment, filters, and LED lighting system. 3. Timer (1 unit for platform) for control the photoperiod. 4. Cling film (3 cm width).

3

Methods

3.1 Stage I: Seed Germination

1. The capsule washed with water and commercial soap [8] (Fig. 1a) (see Note 2). 2. Disinfect the laminar flow surface with 70% alcohol after UV light exposure for 15 min. 3. Capsules are cleaned with 75% ethanol and flamed with the aid of an alcohol lamp four times in the laminar flow hood (Fig. 1b); afterwards, these are cut transversally and immature

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Fig. 1 In vitro establishment of Guarianthe skinneri in TIB. (a) Washing of the non-dehiscent capsule. (b) Disinfection and flaming of the non-dehiscent capsule. (c) Longitudinal cut of the capsule. (d) Seeding of the seeds in a semisolid system

seeds placed in glass culture jars 100 mL containing 20 mL of SGM using sterile scalpels (Fig. 1c, d) (see Note 3). 4. For dehiscent capsules, seeds are rinsed for 10 min in a 0.6% (v/v) sodium hydrochloride solution with three drops of Tween® 20 per 100 mL of water (see Note 4). Seeds were rinsed three times with sterile distilled water and placed in glass culture jars 100 mL containing 20 mL of SGM (see Note 3). 5. The explants are incubated at 24 ± 2 °C, under 40 ± 5 μmol m2 -1 s irradiance and a 16 h photoperiod. 6. After 30 days, the seedlings (germinated seeds) are transferred for multiplication to SMM. 3.2 Stage II: In Vitro Multiplication Using Semisolid Medium

1. For the multiplication in semisolid media stage, seedlings (1 cm in length) are transferred to 100 mL glass culture jars containing 20 mL of SMM (Fig. 2a) [8].

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Fig. 2 Micropropagation of Guarianthe skinneri in TIB. (a) Multiplication in semisolid medium. (b) Proliferation of shoots in TIB. (c) Rooting of shoots. (d) Acclimatization process

2. The medium pH is adjusted to 5.8, and 2.2 g/L Phytagel was added as a gelling agent. Then, the medium is sterilized in an autoclave for 15 min at 120 °C and 115 kPa. 3. The explants are incubated at 24 ± 2 °C, under 40 ± 5 μmol m2 -1 s irradiance and a 16 h photoperiod. After two subcultures (30 days each), proceed to the multiplication stage using the BIT. 3.3 Establishing Culture in BIT®

1. The TIB (1000 mL) is used. To sterilize these containers, take them to a laminar flow hood. Prepare a 0.3% (v/v) solution of sodium hydrochloride, and leave it still for 5 min. After rinsing the containers three times with sterile water, turn on the UV light in the laminar flow hood for 10 min (see Note 5).

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2. Then, pour into the container 200 mL of SMMTIS (Table 1) [8]. Subsequently, place ten explants in one of the two containers (Fig. 2b). 3. Connect both containers to an air pump and apply air pressure to submerge the explants. After the immersion time, a solenoid valve prevents the passage of air, and the medium returns to the original container. Use an immersion frequency of 2 min every 8 h for 30 days. Maintain the explants under the light and temperature conditions previously mentioned [8]. 3.4 Stage III: Elongation and Rooting in BIT

1. After 30 days, orchid shoots should be individualized for better nutrient absorption. Divide agglomerate of shoots at base (shoots of 2 cm long). 2. Change the container with containing 200 mL of REMTIS (half-strength MS medium with no PGR supplemented with 1 mg/L AG3) (Table 1) [8]. 3. Use an immersion frequency of 2 min every 8 h for 30 days. Maintain the explants under the light and temperature conditions previously described (5).

3.5 Stage IV: Acclimatization

1. Prepare the substrate in individual pots (4 in). The substrate consists of a mixture of peat and agrolite 1:1 (v/v) (see Note 6) [8]. 2. Wash the shoots rooting to remove adhering MS medium (Fig. 2c). 3. Prepare a 1 g/L fungicide and bactericide solution, and submerge the seedlings for 10 min. 4. Before seeding, moisten the substrate. After seeding, cover the seedlings with a translucent dome. Maintain seedlings under greenhouse conditions with 60% shade and at 30 ± 2 °C, relative humidity of 60 ± 10%, and natural light with an irradiance of 80 ± 10 μmol m - 2 s - 1 for 1 month (Fig. 2d) (see Notes 7 and 8) [8].

4

Notes 1. Prepare separate MS stock A-F solutions. Then, weigh and dissolve the components of each stock in 50 mL of distilled water using a magnetic stirrer, and make up the final volume to 100 mL by using distilled water and analytical grade reagents. Diligently follow all waste disposal regulations when disposing of waste materials. For 1 L semisolid MS medium elaboration, pour 750 mL of distilled water into a 1 L flask with a magnetic stirring bar, add 30 g of sucrose, and dissolve. Adjust the pH to 5.8 with a pH meter by adding 0.1 or 1 N NaOH or HCl with

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constant stirring. Then, add 2.2 g of Phytagel, and make up the volume to 1 L with distilled water using a 1 L measuring cylinder (depends if the medium is semisolid or liquid). Heat the culture medium until it starts boiling, dispense medium per glass culture flasks o TIS, and cap. Autoclave at 120 °C with a pressure of 115 kPa, for 15 min. Take out the culture medium from the autoclave, and allow it to cool and solidify at room temperature. 2. It is recommended to use the non-dehiscent orchid capsule, washed with tap water and liquid detergent for 10 min. 3. To ensure asepsis, ≈0.5 g seeds established per flasks. 4. In case of having a dehiscent capsule recovered from the disinfectant solutions with the help of qualitative grade 1 filter paper (110 mm). 5. It is recommended to assemble the bioreactors and verify with running water that the system works correctly. Verify that caps and hoses do not leak; this can be solved using safety straps. 6. Sterilize the substrate in the autoclave for 30 min at 120 °C and 115 kPa. 7. Maintain the pots closed for a week to avoid moisture loss. Sprinkle water on the dome to maintain a cool environment. 8. After a month, keep the trays at 30 ± 5 °C, with a relative humidity of 30% and natural light with an irradiance of 250 ± 10 μmol m-2 s-1. If the temperature surpasses 30 °C, water daily and fertilize and fumigate once a week. References 1. Vitt P, Taylor A, Rakosy D, Kreft H, Meyer A, Weigelt P, Knight TM (2023) Global conservation prioritization for the Orchidaceae. Sci Rep 13:6718. https://doi.org/10.1038/ s41598-023-30177-y 2. Jain A, Sarsaiya S, Chen J, Wu Q, Lu Y, Shi J (2021) Changes in global Orchidaceae disease geographical research trends: recent incidences, distributions, treatment, and challenges. Bioengineered 12:13–29. https://doi. org/10.1080/21655979.2020.1853447 3. Meng YY, Shao SC, Liu SJ, Gao JY (2019) Do the fungi associated with roots of adult plants support seed germination? A case study on Dendrobium exile (Orchidaceae). Glob Ecol Conserv 17:e00582. https://doi.org/10. 1016/j.gecco.2019.e00582 4. van Tongerlo E, van Ieperen W, Dieleman JA, Marcelis LF (2021) Vegetative traits can predict flowering quality in Phalaenopsis orchids despite large genotypic variation in response

to light and temperature. PLoS One 16: e0251405. https://doi.org/10.1371/journal. pone.0251405 5. Cardoso JC, Zanello CA, Chen JT (2020) An overview of orchid protocorm-like bodies: mass propagation, biotechnology, molecular aspects, and breeding. Int J Mol Sci 21:985. https://doi.org/10.3390/ijms21030985 ˜a C 6. Bertolini V, Damon A, Ibarra-Cerden (2016) Atlas de las orquı´deas del Soconusco: modelos digitales de nichos ambientales entre Centro y Sudame´rica. El Colegio de la Frontera Sur, Tapachula, p 56 7. Fatahi M, Anghelescu NE, Vafaee Y, Khoddamzadeh A (2023) Micropropagation of Dactylorhiza umbrosa (Kar. & Kir.) Nevski through asymbiotic seed germination and somatic embryogenesis: a promising tool for conservation of rare terrestrial orchids. S Afr J Bot 159: 492–506. https://doi.org/10.1016/j.sajb. 2023.06.036

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8. Leyva-Ovalle OR, Bello-Bello JJ, Murguı´a˜ ez-Pastrana R, Ramı´rez-MosGonza´lez J, Nu´n queda MA (2020) Micropropagation of Guarianthe skinneri (Bateman) Dressler et WE Higging in temporary immersion systems. 3 Biotech 10:1–8. https://doi.org/10.1007/ s13205-019-2010-3 9. Mirzabe AH, Hajiahmad A, Fadavi A, Rafiee S (2022) Temporary immersion systems (TISs):

a comprehensive review. J Biotechnol 357:56– 83. https://doi.org/10.1016/j.jbiotec.2022. 08.003 10. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473– 497. https://doi.org/10.1111/j.1399-3054. 1962.tb08052.x

Chapter 15 Alstroemeria Micropropagation in a RITA® Temporary Immersion System Lilia Castro Pereira , Leonardo Soriano, Carolina Rossi de Oliveira, Paulo Hercı´lio Viegas Rodrigues , and Adriana Pinheiro Martinelli Abstract The use of temporary immersion systems (TIS) for plant micropropagation is an efficient technique for plant production, and we have applied it for the production of alstroemerias. This method involves the cultivation of explants such as rhizomes and axillary buds in a nutrient medium to stimulate shoot growth. TIS offer advantages such as accelerated multiplication processes, uniform production, and cost reduction. This process has shown promise in meeting the growing demand for alstroemeria plants in the market. This chapter describes a specific protocol for temporary immersion bioreactor micropropagation of the “Albatroz” cultivar, with the potential for large-scale automation. Key words Alstroemeria, Bioreactor, Micropropagation

1

Introduction Alstroemerias belong to the Alstroemeriaceae family and comprise approximately 201 species [1]. The genus Alstroemeria L. is endemic to South America, with 75 species restricted to this continent. Propagation can be done through seed or vegetative methods, such as rhizome division. Both methods have advantages and disadvantages, including seed dormancy or even seed viability issues [2]. While vegetative propagation can produce clonal offspring [3] and can be successful in plants with approximately 3 years of cultivation, it has shown a low rate of micropropagation through traditional in vitro culture, making it inefficient plant propagation and new commercial plantings [4]. Micropropagation has been proven effective for large-scale high-quality seedling production for various species, minimizing the risk of disease propagation. It offers advantages, such as producing propagative materials with guaranteed genetic and phytosanitary quality in a reduced space and shorter period, unaffected by

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_15, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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external climatic variations due to the controlled environmental conditions, which also allows for plant propagation throughout the year [5]. For alstroemeria plant production, micropropagation has been widely used commercially. Research data suggest that various explants can be used, including rhizomes [6–8] and axillary buds [9], as well as leaves, shoots, and stems [10]. All explants are cultured in semisolid media containing the necessary nutrients for their development, supplemented with growth regulators [11, 12], to induce adventitious buds from the explants [8, 13]. For better results in alstroemeria propagation, micropropagation can be further enhanced by using bioreactors during specific multiplication stages, either through temporary or permanent immersion in a nutrient medium [14], with the potential of higher number of propagules, compared to the traditional micropropagation. Bioreactors consist of an interconnected flask system containing liquid nutrient medium, with or without the addition of plant growth regulators, which need to be defined case by case. In the case of alstroemeria multiplication rates can vary according to the genotype [10]. Advantages include accelerated multiplication processes, uniform production, simplified assembly and operation, adaptability to various plant species, and reduced labor costs [14, 15]. This chapter describes the procedures of a temporary immersion bioreactor micropropagation protocol for Alstroemeria cv. “Albatroz.” This protocol can be used to automate alstroemeria micropropagation on a large scale, with possible adaptations for different genotypes, using the temporary immersion system.

2 2.1

Materials Plant Material

2.2 Material and Equipment

1. Rhizomes of Alstroemeria cv. “Albatroz” plants cultivated in the greenhouse (Fig. 1). 1. Magenta™ boxes, glass flasks, or test tubes. 2. Surgical tools: stainless steel forceps, surgical scalpels, removable sterile surgical blades, and tweezers. 3. Pasteur pipettes. 4. 500 or 1000 mL autoclavable bottles (blue cap). 5. Parafilm™ or plastic wrap. 6. Commercial sodium hypochlorite solution (2.5% active chlorine). 7. Sucrose. 8. Phytagel®.

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Fig. 1 Alstroemeria explant preparation and in vitro introduction. (a) Shoot buds from the rhizome used as explants for in vitro culture establishment. (b and c) Explant disinfestation followed by three rinses (b) and a detail of the explants immersed in the sodium hypochlorite solution. (d) Explant with several buds, recently introduced in vitro. Bars: a, c = 1 cm, b = 2 cm, d = 0.5 cm

9. Agar. 10. Autoclave. 11. Laminar airflow hood. 12. Growth room at 27 ± 2 °C. 2.3 Bioreactor System

1. RITA® bioreactor system.

2.4

1. Prepare stock solutions of vitamins and macro- and micronutrients of MS medium [16], dissolving the components in deionized H2O, and store at 4 °C, or use commercial MS medium powder with vitamins.

Culture Media

2. 6-Benzylaminopurine (BAP) stock solution (1 mg/mL): Dissolve the BAP powder in a few drops of 1 M NaOH, bring to final volume with deionized H2O; store at 4 °C.

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3. Introduction medium (IM): MS medium [16] with vitamins, supplemented with 30.0 g/L sucrose, 1.0 mg/L 6-benzylaminopurine (BAP). Adjust pH to 5.8 with 0.1 N KOH or 0.1 N HCl. Add 2.0 g L-1 Phytagel®. Pour 40 mL of the medium into Magenta™ boxes or glass flasks, and autoclave at 121 °C, for 25 min. 4. Solid multiplication medium (SM): MS medium [16] with vitamins, supplemented with 30.0 g/L sucrose, 1.25 mg/L 6-benzylaminopurine (BAP), and 0.1 mg/L 3-indoleacetic acid (IAA). Adjust pH to 5.8 with 0.1 N KOH or 0.1 N HCl. Add 2.0 g/L Phytagel®. Pour 40 mL of the medium into each Magenta™ box or glass flask and autoclave at 121 ° C for 25 min. 5. Liquid multiplication medium (LM): MS medium [16] with vitamins, supplemented with 30.0 g/L sucrose, 1.25 mg/L 6-benzylaminopurine (BAP), and 0.1 mg/L 3-indoleacetic acid (IAA). Adjust pH to 5.8 with 0.1 N KOH or 0.1 N HCl; then, autoclave at 121 °C for 25 min. 6. Elongation and rooting medium (ER): MS medium [16] with vitamins, supplemented with 30.0 g/L sucrose and 0.5 mg/L naphthaleneacetic acid (NAA). Adjust pH to 5.8 with 0.1 N KOH or 0.1 N HCl. Add 6.0 g/L agar. Pour 40 mL of the medium into each Magenta™ box or glass flask and autoclave at 121 °C for 25 min.

3

Methods

3.1 Preparation of Explants and Culture Establishment and Initial Multiplication

1. Extract shoot buds from rhizomes (Fig. 1a) and rinse them with abundant running water, removing soil residues and roots (see Note 1). 2. Place them in a 500 mL beaker containing a 50% commercial sodium hypochlorite solution (2.5% active chlorine); stir with a magnetic stirrer for 15 min. 3. In a laminar flow hood, discard the hypochlorite solution and rinse three times with autoclaved deionized water (Fig. 1b, c). 4. Using a scalpel and tweezers, cut and reduce the shoot buds to approximately 5 to 10 mm (Fig. 1d). 5. Transfer the apices to individualized flasks with IM medium; seal with plastic wrap. 6. Cultivate in a growth room for 20 days at 25 °C ± 2, and a photoperiod of 16 h, at 50 μmol/m2/s-1. 7. After 3 weeks, transfer the cultures to the SM medium, and culture in a growth room at a temperature of 25 °C ± 2, and a photoperiod of 16 h, at 50 μmol/m2/s-1.

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8. Subculture every 2 weeks, for 6–8 weeks, until obtaining the necessary number of explants to initiate the cultivation in the temporary immersion bioreactor. 3.2 Temporary Immersion System

1. Pour 250 mL of LM medium into the lower part of the RITA® TIS (Fig. 2a, b). 2. Divide the shoot buds into small clusters with at least three shoots together; then, place them on the top part of the RITA® TIS (Fig. 3a). 3. Incubate under a 16-h photoperiod at 25 ± 1 °C, in a growth room. 4. Use an immersion frequency of 2 min every 12 h for a 35–40 days cultivation period (see Note 2). (Fig. 3b). 5. Connect the bioreactor to an air pump, which will inject air, creating a pressure gradient, causing the culture medium to move to the upper compartment, coming into direct contact with all parts of the plantlets. After the immersion time (2 min) of air injection, the medium returns to the lower compartment by gravity after the internal release of pressure through the air outlet filter. (see Notes 3 and 4). 6. Transfer the plantlets to ER medium after 35–40 days, and culture them under a 16-h photoperiod, at 25 ± 2 °C, for 20–30 days (see Note 5) (Fig. 3).

3.3

4

Acclimatization

Acclimatization is done by removing rooted plantlets from the flasks and carefully rinsing them in tap water to remove all the culture medium. Transfer the plantlets to trays with a mixture of horticulture substrate and coconut fiber (1:1), placing them under decreasing mist conditions for 25–30 days. Acclimatized plants can be transplanted into individual pots including fertilization and drip irrigation for further growth.

Notes 1. Since these are underground explant sources, rhizomes may have a high contamination rate during in vitro introduction. Alternatives, such as replanting the mother plants in inert substrate (coconut fiber and vermiculite or autoclaved sand), help reduce the microbial load present in the explant. Weekly applications of terpenoids are also effective, facilitating the in vitro introduction stage. 2. It is recommended to autoclave test tubes, glass flasks, and all RITA® bioreactor parts before use to prevent any possible contamination during in vitro plant cultivation.

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Fig. 2 Established cultures in the RITA® temporary immersion system. (a and b) RITA® temporary immersion system (a) and the system with Alstroemeria “Albatroz” under cultivation (b). Bars = 1 cm

3. The ideal immersion time may vary according to the genotype. For this reason, a preliminary test is required to adjust the immersion time and avoid potential undesirable problems, such as hyperhydration and subsequent necrosis. 4. Careful handling during bioreactor assembly is necessary to avoid potential contamination sources, which may appear after a few days of system installation. Both air inlet and outlet have filters to prevent potential external air contamination and enable the necessary gas exchange. Despite promising results, shoots cultured with temporary immersion systems may exhibit hyperhydration and contamination. These difficulties should be considered before using bioreactors for commercial production. If contamination is observed, these bioreactor cultures should be discarded. 5. After 40 days of cultivation in the bioreactor, the plants need to go through another in vitro stage, known as elongation and rooting, as they do not develop roots during multiplication. Only after this stage will the plants be ready for acclimatization.

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Fig. 3 Alstroemeria micropropagation using a temporary immersion system (RITA®) during the multiplication stage. (a) Plantlets after 8 weeks of cultivation in solid multiplication medium (SM), ready to begin multiplication in the bioreactor. (b) Plantlets after 20 days of multiplication in the bioreactor. (c) Plantlets after 40 days of multiplication in the bioreactor, ready to be transferred to elongation and rooting medium and subsequent acclimatization. Bars = 0.5 cm

Acknowledgments This study was financed in part by the Coordenac¸˜ao de Aperfeic¸oamento de Pessoal de Nı´vel Superior – Brasil (CAPES) – Finance Code 001. APM acknowledges Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico, CNPq, DF, Brazil, for a research fellowship to APM (312602/2019-7). References 1. Assis MC (2012) Alstroemeriaceae na Regia˜o Sul do Brasil. Rodriguesia 63(4):1117–1132. h t t p s : // d o i . o r g / 1 0 . 1 5 9 0 / S2175-78602012000400022 2. King JJ, Bridgen MP (1990) Environmental and genotypic regulation of Alstroemeria seed germination. HortSci 25:1607–1609

3. Petry C (2008) Plantas ornamentais: Aspectos para a produc¸˜ao. Universidade de Passo Fundo, Passo Fundo 4. Din A, Wani MA, Malik SA et al (2017) Micropropagation of Alstroemeria Hybrida Cv. Pluto. Int J Environ Agric Biotech 2:662– 680. https://doi.org/10.22161/ijeab/2.2.13 5. Rocha PSG, Oliveira RP, Scivittaro WB (2013) Uso de LEDs na multiplicac¸˜ao e enraizamento

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in vitro de framboeseiras. Pesq Agrop Gau´cha 19:98–105 6. Aros D, Va´squez M, Rivas C, Prat ML (2017) An efficient method for in vitro propagation of Alstroemeria pallida Graham rhizomes. Chil J Agric Res 77:95–99. https://doi.org/10. 4067/S0718-58392017000100012 7. Khaleghi A, Khalighi A, Sahraroo A et al (2008) In vitro propagation of Alstroemeria cv. “Fuego”. AEJAES 3:492–497 8. Yousef H, Sahar B, Abdollah H (2007) In vitro propagation of Alstroemeria using rhizome explants derived in vitro and in pot plants. Afr J Biotechnol 6:2147–2149 9. Lin HS, De Jeu MJ, Jacobsen E (1998) Formation of shoots from leaf axils of Alstroemeria: the effect of the position on the stem. Plant Cell Tissue Organ Cult 52:165–169. https:// doi.org/10.1023/A:1006063105940 10. Guzma´n C, Prat L, Rivas C, Aros D (2018) Induction of direct organogenesis from aerial explants of scented Alstroemeria genotypes. Cienc Inv Agr 45:158–168. https://doi.org/ 10.7764/rcia.v45i2.1918 11. Gaspar T, Keveks C, Penel C et al (1996) Plant hormones and plant growth regulators in plant tissue culture. In Vitro Cell Dev Biol Plant 32:

2 7 2 – 2 8 9 . h t t p s : // d o i . o r g / 1 0 . 1 0 0 7 / BF02822700 12. Smith RH (2013) Media components and preparation. In: Smith RH plant tissue culture, 3rd edn. Elsevier, Amsterdam. https://doi. org/10.1016/B978-0-12-415920-4. 00003-7 13. Shahriari AG, Bagheri A, Sharifi A, Moshtaghi N (2012) Efficient regeneration of Caralis Alstroemeria cultivar from rhizome explants. Not Sci Biol 4:86–90. https://doi.org/10. 15835/nsb.4.2.7336 14. Teixeira JB (2002) Biorreatores para ce´lulas, tecidos e o´rga˜os vegetais – Produc¸˜ao de mudas em larga escala. Biotecnologia Cieˆncia e Desenvolvimento 24:36–41 15. Rout GR, Jain SM (2020) Advances in tissue culture techniques for ornamental plant propagation. Burleigh Dodds Science Publishing Limited, Cambridge. https://doi.org/10. 19103/AS.2020.0066.04 16. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473– 497. https://doi.org/10.1111/j.1399-3054. 1962.tb08052.x

Part IV Micropropagation in TIS of Woody Species: Protocols

Chapter 16 Proliferation of Axillary Shoots of Chestnut in Temporary Immersion Systems Nieves Vidal, Conchi Sa´nchez, and Beatriz Cuenca Abstract Shoots from European chestnut and hybrids of European and Asian chestnuts can be efficiently proliferated in liquid medium by temporary immersion (TIS) using RITA® and Plantform™ bioreactors. The main challenges of applying TIS to these species were the lack of growth and hyperhydricity; problem solutions included the manipulation of the hormone concentration, the explant type, immersion frequency, and a support for maintaining shoots in a vertical position. After protocol optimization, explants cultured by TIS produced more rootable shoots than explants growing in semisolid medium, enabling increased number of rooted and acclimatable shoots. In this chapter, we will describe the protocols for proliferating chestnut by TIS in RITA® and Plantform™ bioreactors, together with tips for avoiding the main pitfalls of the technique. The strategies applied to chestnut can be useful for culturing other woody plants in bioreactors. Key words Bioreactors, Castanea crenata, Castanea sativa, Hyperhydricity, RITA®, Plantform™

1

Introduction Chestnuts are multipurpose trees grown worldwide for nuts and timber. Currently, chestnut trees are threatened because of diseases caused by fungal pathogens and insects [1, 2]. Breeding programs have produced disease-tolerant trees by crossing susceptible with tolerant species [3, 4]; hybrids obtained from controlled crosses need to be propagated vegetatively to maintain their tolerance to the disease [5]. Chestnut species are highly recalcitrant to conventional vegetative propagation, agar-based micropropagation protocols have problems regarding rooting and acclimation, and success is hampered by genotypic effects [5–10]. The consequence is that commercial vegetative propagation of chestnut species is still a bottleneck for the forest industry. Temporary immersion in liquid medium has several advantages for large-scale micropropagation, including increased absorption of nutrients and improved physiological status of the cultures due to gas exchange [11, 12]. We have

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_16, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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evaluated the use of a temporary immersion system (TIS) for chestnut micropropagation and reported successful proliferation, rooting, and acclimation of shoots of eight genotypes selected for their tolerance to ink disease (caused by the oomycete Phytophthora cinnamomi), suggesting the feasibility of culturing chestnut tissues without the use of gelling agents [13, 14]. In this chapter, we describe the steps for preparing and using commercial RITA® [15] and Plantform™ [16] bioreactors for in vitro proliferation of axillary shoots of Castanea sativa Mill and hybrids of C. sativa × C. crenata. Good multiplication rates can be obtained with both types of bioreactors, but the larger size of Plantform™ allows more explants, and these can grow larger. Additional air inlets in Plantform™ allow aeration at times not concurrent with immersions, which can reduce hyperhydricity in especially sensitive genotypes. Shoots cultured by TIS typically exhibit about twofold success on rooting and acclimation, which shows the potential of this approach for large-scale propagation of this species and for exploring emergent techniques like photoautotrophic micropropagation [14, 17, 18]. We will describe the main parameters influencing a successful outcome, such as plant growth regulator concentrations, explant type, immersion frequencies and duration. Tips for avoiding the main pitfalls of the technique (contamination and hyperhydricity) will be described in the Subheading 4.

2

Materials

2.1 General Equipment

1. Water purification system, refrigerator, weighing balances, microwave, hot plate, magnetic stirrer and bars, micropipettes, pH meter, dishwasher, oven and autoclave. 2. General laboratory glassware (e.g., selection of 25, 50, 100, 500, 1000 mL graduated cylinders, beakers, glass tubes, pipettes). 3. Deionized water, NaOH, HCl.

mild

detergent,

commercial

bleach,

4. Culture medium and plant growth regulators. 5. Work room with laminar airflow hood equipped with a glass bead sterilizer. 6. Surgical tools: stainless steel forceps, surgical scalpels, removable sterile surgical blades. 7. Cotton, ethanol, sterilized filter paper, plastic kitchen film, aluminum foil.

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1. RITA® or Plantform™ bioreactors. Bioreactors can be purchased from their manufacturers [15, 16], which also supply the required pumps, solenoid valves, timers, pipes, connectors, silicon tubes of various diameters and 0.22 μm hydrophobic filters. Alternatively, you might buy some of these elements (filters, silicon tubes, etc.) from your local distributors. 2. Rockwool cubes (optional). Available from many horticultural suppliers. 3. Eppendorf vials (several sizes) for protecting inlets, outlets, and filters during autoclaving.

2.3

Culture Media

Chestnut can be grown in several media [5–7]. We recommend MS (Murashige and Skoog 1962) [19] with half-strength nitrates (MS N ½) and GD medium (Gresshoff and Doy 1972) [20], using their respective vitamin formulations. Frequently better results are obtained with MS N ½ medium, but when hyperhydricity is high (see Note 1), GD can offer some advantages. There are several commercial GD media, the one we use is based on the DBM1 described by Gresshoff and Doy [20] with minor modifications in the concentration of ZnSO4, CuSO4, and CoCl2 micronutrients. See Table 1 for detailed media composition. Media is supplemented with 0.22 or 0.44 μM N6-benzyladenine (BA) and 3% sucrose. pH is adjusted to 5.6–5.8 before autoclaving at 120 °C for 20 min. For liquid medium preparation, follow these steps: 1. Prepare the medium following standard micropropagation laboratory methods. After dissolving macro- and micronutrients, vitamins and BA, add sucrose and stir till it is completely dissolved. Add deionized water to complete the required volume, and adjust the pH to 5.6–5.8 with a pH meter by adding 0.1 or 1 N NaOH or HCl as required. 2. The required media volume is 150 mL for RITA® and 500 mL for Plantform™ bioreactors. Media can be autoclaved in the bioreactors, but for RITA® vessels, we recommend autoclaving it separately in flasks or bottles marked at intervals of 150 mL of volume. Leave an air space of at least the same volume added to the flask or bottle and do not close tightly. Protect the lid and neck of the container with aluminum foil. 3. Autoclave at 120 °C for 20 min.

2.4 Plant Material and Growth Conditions

1. Use established in vitro cultures of chestnut that have achieved the stabilization stage (if necessary, follow the protocols described in Vieitez et al. [7]). 2. Select vigorous 25–40 mm shoots obtained after 4–5 weeks of culturing, and cut 15 mm apical, nodal, or basal segments attached to basal callus (Fig. 1). Proliferation and vigor of

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Table 1 Composition of the media used for the proliferation of chestnut shoots in temporary immersion bioreactors (MS N , Murashige and Skoog medium [19] with half-strength nitrates; GD, Gresshoff and Doy medium [20]) MS N

GD

Macroelements

Molarity (mM)

Macroelements

Molarity (mM)

CaCl2 KH2PO4 KNO3 MgSO4 NH4NO3

2.99 1.25 9.40 1.50 10.30

CaCl2·2H2O KCl KNO3 MgSO4·7H2O Na2HPO4 NaH2PO4·H2O (NH4)2·SO4

1.02 4.02 9.89 1.01 0.21 0.65 1.51

Microelements

Molarity (μM)

Microelements

Molarity (μM)

CoCl2·6H2O CuSO4·5H2O FeNaEDTA H3BO3 KI MnSO4·H2O Na2MoO4·2H2O ZnSO4·7H2O

0.11 0.10 100.00 100.27 5.00 100.00 1.03 29.91

CoCl2·6H2O CuSO4·5H2O FeNaEDTA H3BO3 KI MnSO4·H2O Na2MoO4·2H2O ZnSO4·7H2O

1.05 1.00 100.00 48.50 4.52 59.16 1.03 10.43

Vitamins

Molarity (μM)

Vitamins

Molarity (μM)

Glycine Myoinositol Nicotinic acid Pyridoxine HCl Thiamine HCl

26.64 554.94 4.06 2.43 0.30

Glycine Myoinositol Nicotinic acid Pyridoxine HCl Thiamine HCl

5.33 56.00 0.81 0.48 2.96

Media was supplemented with 0.22 or 0.44 μM BA and 3% sucrose

chestnut explants cultured by TIS are higher with basal segments, followed by apical sections. 3. Semisolid and liquid cultures can be incubated under a 16 h photoperiod provided by cool-white fluorescent lamps (photosynthetic photon flux density 50–60 μmol m-2 s-1) at 25 °C light/20 °C dark. Alternatively, white LED lamps providing similar light intensity can be used.

3

Methods RITA® and Plantform™ bioreactors share the same operation principle but have a different shape and parts. More detailed instructions regarding the operation and assembly of these bioreactors can be found in the manufacturers’ websites [15, 16].

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Fig. 1 Chestnut initial explants used in bioreactors: apical sections, nodal sections, and basal sections attached to basal callus 3.1 Assembling and Autoclaving of RITA® Bioreactors and Filters

1. Choose a clean vessel and check that the lid (with the 11 cm diameter red ring) fits smoothly and there are not leakages (see Note 2). 2. Assemble the inner section of the bioreactor by connecting (in this order) the bell, the basket with holes, the black ring, the net, and the central tube. Place the 1 cm diameter red ring in the upper section of the central tube (Fig. 2a). 3. Insert the inner section of the bioreactor inside the vessel (Fig. 2b). Add 20–50 mL of water. When using a support for the explants, go to Note 3. 4. Protect the inlet and outlet lid holes. We recommend using 40–45 mm silicon tubes (6 mm internal diameter) closed in one of its ends with a 0.5 mL Eppendorf vial wrapped with aluminum foil (Fig. 2c). These protective tubes can be easily removed and reused in subsequent experiments. 5. Partially close the lid to prevent deformations during autoclaving, and protect with aluminum foil (optional, Fig. 2d). 6. Protect the 0.22 μm filters for autoclaving. Fit the filter in a 25 mm silicon tube, and protect the tube end and the filter end with aluminum foil (Fig. 2e, see Note 4).

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Fig. 2 RITA® assembling. (a) RITA® internal parts (bell, basket, black ring, net, central tube, and red ring). (b) RITA® internal parts assembled inside the vessel. (c) Silicon tubes with 0.5 mL Eppendorf vials for protecting inlet and outlet lid holes. (d) RITA® prepared for autoclaving. (e) Filter protection for autoclaving. (f) Autoclaved filters drying in the oven

7. Autoclave bioreactors and filters at 115 °C for 15–20 min. Excessive pressure can damage the bioreactors. Let bioreactors cool in a flat shelf. Take the filters to an oven set between 40 and 60 °C for drying, lift the aluminum foil of the container, and let dry for 16–24 h (Fig. 2f). 3.2 Assembling and Autoclaving of Plantform™ Bioreactors and Filters

1. Take an outer polycarbonate vessel, and check that the polypropylene lid (with the blue silicon ring) fits smoothly and can be easily closed and opened. A coin can be used to help locate the ring in place. 2. Assemble the threaded tube connecters (TTC) in the three holes that will hold the filters using the white silicon rings on both sides and screwing the nuts on the TTC (Fig. 3a). 3. Add 500 mL of medium. 4. Put the inner container in the bottom of the vessel and use the 9 cm silicon tube to connect it with the central inlet TTC. Put the basket above the inner container so the silicon tube can pass through the cutout of the basket (Fig. 3b). When using a support for the explants, go to Note 5. 5. Put the four-legged frame above the basket, and close the container with the lid. Open two of the four flaps to avoid

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Fig. 3 Plantform™ assembling. (a) Assembling of the threaded tube connecter in the holes for inlet and outlet of the Platform™ vessel using silicon rings and a nut. (b) Adjustment of the inlet tube with the internal container. (c, d) Protection of the inlet and outlet holes using 4 mm internal diameter silicon tubes and 0.2 mL Eppendorf tubes. (e) Protection of the filters for autoclaving using silicon tubes, Eppendorf tubes, and aluminum

deformation during autoclaving (see Note 6), and protect the edges with aluminum foil as in step 5 in Subheading 3.1. 6. Protect the inlet and outlet holes. As with the RITA® system, we recommend using reusable protective silicon tubes. In this case, use 30–35 mm long tubes (4 mm internal diameter), closing off one of its ends with a 0.2 mL Eppendorf vial wrapped with aluminum foil (Fig. 3c, d). The time invested in preparing these protections will facilitate the handling in the laminar flow hood. 7. Protect the 0.22 μm hydrophobic filters for autoclaving. Fit the filter in a 30–35 mm-long silicon tube, and protect the tube end that will be connected to the bioreactor with aluminum foil (Fig. 3e) and the end that will be connected to the air supply in the growth chamber with a 0.2 mL Eppendorf tube as in step 6 in Subheading 3.2 (see Note 7). 8. Autoclave bioreactors and filters at 115 °C for 15–20 min. Let bioreactors cool on a clean flat shelf before closing the two flaps that had been open during the autoclave process. Put the filters in an oven set at 40–60 °C for drying, lifting one edge of the aluminum foil of the container as in Fig. 2f, and let dry for 16–24 h.

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3.3 Inserting the Plant Material in Bioreactors in the Flow Cabinet

1. Prepare the flow cabinet following the standard aseptic procedures. Set the bead sterilizer to 240–250 °C. Take the boxes with filters from the oven, and place them in the cabinet along with sterile filter paper, forceps, and scalpels (see Note 8). 2. In the case of RITA®, take a 500 mL sterile container to be used as a waste container. Clean the bioreactor by spraying with 70% ethanol and aseptically remove the aluminum foil off the bioreactor. Open the lid and place down inverted (so the open side is uppermost) on a clear space near the back of the flow cabinet. Use forceps to lift the inner section, and with the other hand, lift the vessel and drain the water into the waste container. 3. In the case of Plantform™, clean the bioreactor as in previous step and remove the aluminum foil (see Note 9). 4. Take the jars or tubes with chestnut shoots (Subheading 2.4), and check carefully for any kind of bacterial or fungal contamination. Do not take any risk. In liquid medium, microorganisms will grow and spread quickly, contaminating the whole bioreactor. 5. Refer to Subheading 2.4 and prepare 12 uniform apical, nodal, or basal explants for RITA® and 24 for Plantform™ (see Note 10). Rotate forceps and scalpels frequently to maintain sterility. 6. Open the lid of the bioreactor and place down inverted. Carefully insert the explants directly on the basket (Fig. 4a, b) or between the rockwool cubes (Fig. 4c, d). See Notes 3 and 5 for further clarification. 7. In the case of RITA®, add 150 mL of sterile liquid medium (Subheading 2.3, step 2), wetting the explants evenly (see Note 11). 8. Close the lids and wrap with plastic kitchen film. Label the bioreactor. 9. Connect the filters. In the case of RITA®, there are only two filters, one for inlet and other for outlet. Remove the protective silicon tube (Fig. 2c) from the central hole of the lid. Do not discard it as it can be used in subsequent experiments. Remove the aluminum foil from the inlet filter, and connect the filter with the bioreactor. Do the same with the outlet filter in the exterior hole of the lid. In the case of Plantform™, follow the same approach to insert the two inlet filters (one in the middle, other in one of the extremes) and the outlet filter in the other extreme (see Note 12).

3.4 Connecting the Bioreactors to the System

1. Take the bioreactors to a growth chamber with the growth conditions specified in Subheading 2.4 and with the required equipment (electric power points, pump, timers, solenoid

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Fig. 4 Placing the explants into the bioreactors. (a) Basal sections in RITA® without support. (b) Apical sections in Plantform™ without support. (c) Basal sections placed between rockwool cubes. (d) Apical sections placed between rockwool cubes. (e) Basal sections in Plantform™ after 5 weeks of culture with rockwool cubes. (f) Handling a Plantform™ lid in aseptic conditions

valves, rigid, and flexible connections for air supply). A scheme is available in Fig. 5 (see Note 13). 2. Connect the inlet filters to the air supply pipes (see Note 14). 3. Set the immersion/aeration conditions (number immersion, frequency). We recommend 3–6 daily immersion of 1–3 min, depending on the genotype (see Note 15). In RITA® bioreactors, when air enters the vessel, this always results in an immersion, but the additional inlet on Plantform™ allows air introduction in the bioreactor without causing an immersion. For Plantform™, we recommend 16–24 additional aerations of 1 min/day. 4. Check media movement and if the explants are getting appropriate wetting during the immersion cycle (see Notes 16 and 17).

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Fig. 5 Scheme of connections required for RITA® (a) and Plantform™ (b) bioreactors. Arrows indicate airflow direction

3.5

Subculturing

1. After 5 weeks of culture, basal shoots should reach about 5–7 cm length, whereas apical sections can average about 3–4 cm length, depending on the genotype. Shoots can be used for multiplication in new bioreactors or for either in vitro or ex vitro rooting. 2. Before opening the bioreactor, check carefully for microorganisms (see Note 18). 3. Before subculturing, discard hyperhydric explants, as they will not develop properly (see Note 1), and proceed as in Subheading 3.3.

3.6 Rooting of Shoots Grown in Bioreactors

1. Select healthy shoots 3–4 cm or longer and actively growing, as they can be considered rootable explants (see Note 19).

3.7 Cleaning and Reusing the Bioreactors

1. To clean the used bioreactor, remove the filters (which can be autoclaved and reused about ten times); keep them in a clean dry place.

2. For rooting, remove the basal callus that has developed, and apply standard methods for in vitro and ex vitro rooting. If necessary, use those described in [7].

2. Disassemble the bioreactor and carefully clean with mild detergent and water. 3. RITA® nets and Plantform™ baskets may need to be cleaned with a brush to completely remove plant tissues. Soaking them in water with some bleach for 2–4 h will help remove phenolic stains. 4. Rinse with deionized water and dry before assembling again.

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Notes 1. Hyperhydricity is a physiological disorder that can appear during cultivation in liquid medium [12, 21]. Briefly, hyperhydric tissues (previously described as “vitrified”) have a translucent appearance, show reduced cuticle and cuticular wax, low lignification, high water content, and very poor regeneration competence. 2. RITA® bioreactors are made of polysulfone, and after some uses, this material can develop cracks and produce leakages. Before assembling the bioreactors, check the vessel carefully for leakages. With small cracks that are only superficial, the vessel can still be used. You can check for leakages by pouring water into the vessel, and let it rest on filter paper for some minutes. Discard the vessel if the paper is wet. Also, adding water during autoclaving will allow you to detect leakages that developed during this process. 3. Micropropagated chestnut shoots can easily become hyperhydric; this problem can be more serious in liquid medium. In many chestnut genotypes, apical and nodal explants will not show this disorder when maintained in an upright position. We recommend placing these explants between 1 cm3 rockwool cubes. Before use, cubes can be pre-cleaned by the following process: place 200–300 g of cubes in a 5–6 L plastic box. Sprinkle liberally with water and autoclave at 120 °C for 20 min. Place the box (without lid) inside an oven (80–90 ° C) for 24–48 h or till the cubes are dried. Let them cool and cover with the lid till use. When preparing the bioreactor, weigh 3–4 g of dry rockwool cubes, and place them in the vessel on the net before autoclaving. Cubes can be also used for basal sections in genotypes especially prone to developing hyperhydricity. 4. Mark the filters that will be used for inlet and for outlet vents, and place them in different containers. Using the filters in one flow direction will help reduce potential contamination. Wrap with aluminum foil before autoclaving to avoid an excess of water entering the filters. 5. As indicated for RITA® bioreactors, in many chestnut genotypes, apical and nodal explants will not develop hyperhydric symptoms if it can be maintained in an upright position. When using rockwool cubes for that purpose, please follow instructions detailed in Note 3 but using 10 g of cubes per bioreactor instead of 3–4 g. In this case, cubes can be also used for basal sections in genotypes especially prone to developing hyperhydricity (Fig. 4e).

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6. Plantform™ lids can be deformed during autoclaving, and this can make them difficult to close properly. We recommend to autoclave some lids (wrapped in aluminum folder), and store them to replace deformed ones as required. 7. We recommend to use a silicon tube closed with an Eppendorf for the exterior extreme of the filter (which will be connected to the air supply), because filters recommended for Plantform™ are smaller than those used for RITA®. In small filters, to maintain a layer of aluminum foil to protect the exterior extreme in the right position can be difficult, and we consider that a silicon tube fits well, improves handling, and can be easily reused. As in RITA®, maintain inlet and outlet filters separately (see Note 4). 8. Prepare more forceps than normal as you may need to use two forceps at the same time to place the explants in the right position inside the bioreactor. Choose long forceps to avoid touching the components of the bioreactor with the gloved hands. 9. It is easier to work with two Plantform™ containers at the same time, as one of them will serve as a support for the lid of the other one during manipulation (Fig. 4f). Otherwise, use a couple of jars to improve ease of handling and help maintaining asepsis. 10. To avoid desiccation during preparation of the required number of explants, they can be placed inside sterile empty jars once they are cut from the donor shoot. Use different jars for each type of explant (apical, nodal, basal). 11. Remember that RITA® bioreactors have been autoclaved with water inside to protect the polysulfone, so it is necessary to remove this water add the liquid media. Also, wetting the explants in the moment they are placed in the bioreactors will protect them from excessive desiccation till they are connected to the temporary immersion system in the growth chamber. This is especially relevant for apical/nodal explants. Following the same idea, a small amount of media can be added over the explants in Plantform™ bioreactors after explant inoculation. 12. It is advisable to use always the same extreme (right or left) for the additional aeration inlet filter. This will avoid mistakes during the connection to the air pipes in the growth chamber. We also recommend to remove the protective silicon tube with the Eppendorf (Fig. 3c) when the bioreactor is still in the hood, this will avoid accidental disconnection of the whole filter from the bioreactor in non-sterile conditions. 13. For connection of pump, pipes, solenoid valves, and timer, refer to manufacturers’ websites [15, 16]. If it is the first time you are using a temporary immersion system, we recommend

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you check the whole system without plant material and using water instead of media. Put the appropriate volume of liquid in the bioreactors and check its movement; manipulate the conditions (number and frequency of immersion, additional aeration) till you are familiar with the system. 14. RITA® bioreactors have a single inlet (the central hole) and only need one pump, a solenoid valve, and a timer. In the case of Plantform™, there is an additional inlet for additional aerations, so it is necessary to install two pipelines with air supply, each one with a timer and solenoid valve [16]. 15. Increasing immersion duration and frequency can enhance growth but can induce hyperhydricity problems. We recommend to place agar-based tubes or jars close to bioreactors with the same type of explant and genotype to monitor differences in growth. Reduce the immersion time and frequency if you observe hyperhydricity; increase the time if growth is poor. Other ways of reducing hyperhydricity include lowering the cytokinin concentration, using media with less ammonium than MS N ½, as GD or Woody Plant Medium (Lloyd G, McCown [22]), increasing additional aerations (when using Plantform™), as well as maintaining a lower temperature. Depending on your growth room lighting arrangements, this may include using mesh rather than solid shelves. 16. Keep an eye on some frequent pitfalls and possible solutions. For solving some of them, it is advisable to keep 2–3 additional sterile bioreactors and some media as a backup. (a) The liquid medium is not going up in a bioreactor: Revise the connections and check if air is flowing homogeneously in all the tubes (a tube can be inadvertently bent preventing the airflow). Review if the container has the right amount of liquid or if there is a leakage. Review the assembly of the bioreactor, the media will not move properly if some seals and rings are not set in the right place or the lid is not properly closed (in this situation, you can prepare some extra bioreactors to remedy this, go back to the flow cabinet, and transfer the cultures to them). Review the inlet an outlet filters; they can be clogged with dirt or water and prevent air coming in or out the bioreactor (keep some extra sterilized filters, change them in the flow cabinet, and try again). (b) The liquid medium does not go down after immersion: Maybe there is an overpressure in the system; in that case, use one of the tubes that supply air and connect to a filter instead to a bioreactor. Doing so, it is possible you observe the opposite problem (the medium does not go up). In that case, reduce the flow in the tube with the filter

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by making a loose overhand bowknot. We recommend using a standard number of bioreactors connected in the system to prevent changes in the pressure that goes to every bioreactor. (c) The liquid medium reaches the explants with too much strength, causing bubbling: Reduce the pressure of the pump; too much pressure can damage the filters and cause fungal contamination. 17. Take note of the next expected time of immersion, and go to the growth chamber to check the system is working properly. If not, review the program of the timer, the electric valve, electric connections, and the assembly of all the components. 18. If any contamination is detected, filters should be discarded and the bioreactor disinfected with diluted bleach (10–20% for 20 min), autoclaved, and cleaned thoroughly. 19. Normally, basal explants attached to basal calli show multiplication coefficients about three times higher than apical/nodal explants. Also, they produce longer shoots and more rootable shoots. For this reason, basal shoots are the best choice for producing shoots for rooting, whereas apical/nodal explants are more suitable for maintenance of stock.

Acknowledgments This research was partially funded by Xunta de Galicia (Spain) through projects 09MRU016E and IN607A 2021/06 and by FEDER INNTERCONECTA 2013/2014 program (Project INTEGRACASTANEA EXP00064828/ITC-20133040). The ˜ a Correa, Anxela Aldrey, authors thank Blandina Blanco, Begon ˜ a Pato for technical assistance and Bruce Maite Garcı´a and Begon Christie for critical reading of the manuscript. References 1. Vettraino AM, Morel O, Perlerou C, Robin C, Diamandis S, Vannini A (2005) Occurrence and distribution of Phytophthora species in European chestnut stands, and their association with Ink Disease and crown decline. Eur J Plant Pathol 111:169–180. https://doi.org/ 10.1007/s10658-004-1882-0 2. Gonthier P, Robin C (2019) Diseases. In: Beccaro G, Alma A, Bounous G, GomesLaranjo J (eds) The chestnut handbook: crop and forest management. CRC Press, Taylor & Francis Group, Boca Raton, pp 297–316. https://doi.org/10.1201/9780429445606

3. Vie´itez FJ, Merkle SA (2005) Castanea spp. chestnut. In: Lizt RE (ed) Biotechnology of fruit and nut crops. CABI Publishing, Wallingford, pp 265–296 4. Santos C, Machado H, Correia I, Gomes F, Gomes-Laranjo J, Costa R (2015) Phenotyping Castanea hybrids for Phytophthora cinnamomi resistance. Plant Pathol 64:901–910. https://doi.org/10.1111/ppa.12313 ˜ a ME, Ferna´ndez-Lo´pez J 5. Miranda-Fontaı´n (2001) Genotypic and environmental variation of Castanea crenata x C. sativa and Castanea sativa clones in aptitude to micropropagation. Silvae Genet 50(3–4):153–162

Chestnut in Temporary Immersion 6. Sanchez MC, Vieitez AM (1991) In vitro morphogenetic competence of basal sprouts and crown branches of mature chestnut. Tree Physiol 8:59–70. https://doi.org/10.1093/ treephys/8.1.59 7. Vieitez AM, Sa´nchez C, Garcı´a-Nimo ML, Ballester A (2007) Protocol for micropropagation of Castanea sativa. In: Jain SM, H€aggman H (eds) Protocols for micropropagation of woody trees and fruits. Springer, Heidelberg, pp 299–312. https://doi.org/10.1007/978-14020-6352-7_28 8. Fernandes P, Tedesco S, Da Silva IV, Santos C, Machado H, Costa RL (2020) A new clonal propagation protocol develops quality root systems in chestnut. Forests 11:826. https://doi. org/10.3390/f11080826 9. Fernandes P, Amaral A, Colavolpe B, Balonas D, Serra M, Pereira A, Costa RL (2020) Propagation of new chestnut rootstocks with improved resistance to Phytophthora cinnamomi—new cast rootstocks. Silva Lusit 28:15–29. https://doi.org/10. 1051/silu/20202801015 10. Beccaro G, Bounous G, Cuenca B, Bounous M, Warmund M, Xiong H, € Akyu¨z B et al Zhang L, Zou F, Serdar U, (2019) Nursery techniques. In: Beccaro G, Alma A, Bounous G, Gomes-Laranjo J (eds) Chestnut handbook. CRC Press, Boca Raton, pp 119–154. https://doi.org/10.1201/ 9780429445606 11. Etienne H, Berthouly M (2002) Temporary immersion systems in plant micropropagation. Plant Cell Tissue Organ Cult 69:215–231. https://doi.org/10.1023/A:1015668610465 12. Preil W (2005) General introduction: a personal reflection on the use of liquid media for in vitro culture. In: Hvolslef-Eidee AK, Preil W (eds) Liquid culture systems for in vitro plant propagation. Springer, Dordrecht, pp 1–18. https://doi.org/10.1007/1-4020-3200-5_1 13. Vidal N, Blanco B, Cuenca B (2015) A temporary immersion system for micropropagation of

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axillary shoots of hybrid chestnut. Plant Cell Tissue Organ Cult 123:229–243. https://doi. org/10.1007/s11240-015-0827-y 14. Gago D, Bernal MA, Sa´nchez C, Aldrey A, Cuenca B, Christie CB, Vidal N (2022) Effect of sucrose on growth and stress status of Castanea sativa x C. crenata shoots cultured in liquid medium. Plan Theory 11:965. https:// doi.org/10.3390/plants11070965 15. https://www.vitropic.fr/en/rita. Accessed 24 July 2023 16. https://www.plantform.se/pub/. Accessed 24 July 2023 17. Xiao Y, Niu G, Kozai T (2011) Development and application of photoautotrophic micropropagation plant system. Plant Cell Tissue Organ Cult 105:149–158. https://doi.org/10. 1007/s11240-010-9863-9 18. Vidal N, Sa´nchez C (2019) Use of bioreactor systems in the propagation of forest trees. Eng Life Sci 19:896–915. https://doi.org/10. 1002/elsc.201900041 19. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473– 497. https://doi.org/10.1111/j.1399-3054. 1962.tb08052.x 20. Gresshoff PM, Doy CH (1972) Development and differentiation of haploid Lycopersicon esculentum. Planta 107:161–170. https://doi. org/10.1007/BF00387721 21. Takayama S, Akita M (2005) Practical aspects of bioreactor application in mass propagation of plants. In: Hvolslef-Eidee AK, Preil W (eds) Liquid culture systems for in vitro plant propagation. Springer, Dordrecht, pp 61–78. https://doi.org/10.1007/1-4020-3200-5_4 22. Lloyd G, McCown B (1980) Commerciallyfeasible micropropagation of mountain laurel (Kalmia latifolia) by use of shoot-tip culture. Comb Proc Int Plant Propag Soc 30:421–437

Chapter 17 In Vitro Propagation of Plants via Organogenesis in Bambusa vulgaris Schrad. ex Wendl Using Temporary Immersion Systems Yudith Garcı´a-Ramı´rez, Mallelyn Gonza´lez-Gonza´lez, Marisol Freire-Seijo, Rau´l Barbo´n-Rodrı´guez, and Sinesio Torres-Garcı´a Abstract The low multiplication and ex vitro survival rates during acclimatization in the culture house limit the in vitro mass propagation of B. vulgaris. Several scientific studies have described the development of different protocols for bamboo; however, not all of them address the effects of these systems on plant morphology, physiology, and biochemistry in vitro. In this chapter, a complete and optimized protocol is described for plants propagated via organogenesis in temporary immersion systems. In addition, the morphophysiological and biochemical characterization of the plants as well as the survival rates of the obtained plants under ex vitro conditions are analyzed. The obtained results will be the basis for the development of a technology for in vitro propagation as an alternative for the production of plants of the species. Key words Bamboo, Ex vitro survival, Temporary immersion systems

1

Introduction Bambusa vulgaris Schrad. ex Wendl (B. vulgaris) is one of the most ecologically important bamboo species and plays a crucial role in the habitat in which it grows. Its roots and rhizomes support the substrate, prevent erosion, and eliminate gullies. They also sequester atmospheric carbon dioxide (CO2) much more efficiently than tropical forest trees. In addition, they have great importance in conserving water resources worldwide and are considered an ideal material for building furniture and handicrafts [1]. Natural reproduction of this species is affected by sporadic flowering and low seed viability. Vegetative propagation by rhizome division is also hindered by low rooting rate and low availability of cuttings [2]. The above reasons affect the availability of seedlings for reforestation in Cuba [3]. As an alternative to the limitations of

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_17, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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vegetative propagation of this species, biotechnological methods allow obtaining a larger number of plants in a short time [4]. In research on in vitro propagation of B. vulgaris using semisolid culture media, organogenesis is the most commonly used method. Protocols for this species have been described in the scientific literature [5–7]. However, results have been obtained with a low multiplication coefficient and a low percentage of surviving plants under ex vitro conditions. On the other hand, scientific studies report that the use of temporary immersion systems (TIS) favors the increase of the multiplication coefficient in different plant species [8]. In addition, culture conditions at TIS allow improvement of plant morphophysiology and anatomy before transferring them to the ex vitro acclimatization phase [9]. To date, scientific studies have focused only on the effects of TIS on shoot morphology of bamboos such as Dendrocalamus latiflorus Munro [10] and Guadua angustifolia Kunth [11, 12]. However, in bamboo, the effects of these systems on shoot morphophysiology and biochemistry that might influence subsequent ex vitro adaptation have not yet been described in detail. This chapter presents a protocol for plant regeneration by organogenesis of B. vulgaris that can be used for mass propagation of plants (Fig. 1).

2 2.1

Materials Plant Material

2.2 Disinfection of Axillary Buds

To obtain and select stem segments, a bank of mother plants was previously established in a culture house from canes and stems of adult B. vulgaris plants. 1. Liquid detergent. 2. 70% (v/v) ethanol. 3. Sterile scalpel.

2.3 In Vitro Establishment

1. Sterile forceps. 2. No. 11 scalpel with sterile handle. 3. Facilities for tissue culture and instruments (laminar flow cabinet, electrical sterilization for instruments). In addition to personal protective equipment for work under aseptic conditions. 4. Culture medium: starch MS salts [13] (Table 1 MS-I) (see Note 1). 5. 100 mg/L myo-inositol (see Note 1). 6. Growth regulator: 3.0 μM 6-benzylaminopurine (BA) (see Note 2). BA: Dissolve 250 mg of BA in 10 mL of hot 0.5 N

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Fig. 1 Schematic representation of the protocol for in vitro propagationIn vitro propagation by organogenesisOrganogenesis plant in Bambusa vulgaris Schrad. ex Wendl. a) Bank of mother plants. b) Axillary buds. c) In vitro establishment after 20 days. d) In vitro multiplication of shoots of B. vulgaris in static liquid culture medium after 20 days. e) Shoots cultured after six subcultures each 20 days. f) Shoots cultured after 3 weeks. g) Shoots cultured in TIS after 30 days. h) Preparation of shoots for ex vitro acclimatization. i) Plants in greenhouse after 30 days

HCl, and make up to 500 mL with deionized water. Store in a closed bottle at 4 °C. 7. 30 g/L sucrose. 8. Test tubes with a total volume of 22 × 150 mm containing 7.0 mL static liquid culture medium. 9. 0.5 N potassium hydroxide. 10. 0.5 N hydrochloric acid. 11. Facilities for tissue culture and instrumentation (laminar flow cabinet, electrical sterilization for instruments, autoclave, culture room). In addition to the personnel with protective equipment for work under aseptic conditions. 2.4 In Vitro Multiplication of Shoots of B. vulgaris in Static Liquid Culture Medium

1. Culture medium: starch MS salts [13] (Table 1 MS-II) (see Note 1). 2. 100 mg/L myo-inositol (see Note 1). 3. Growth regulator: 12.0 μM BA (see Note 2). 4. 30 g/L sucrose. 5. Polycarbamate culture flasks with a total volume of 500 mL containing 70 mL of culture medium. Three groups of shoots are placed in 500 mL polycarbamate culture flasks containing

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Table 1 The basal media used in vitro propagation via organogenesis of shoots Bambusa vulgaris Schrad. ex Wendl Component (mg/L)

MS-I

MS-II

MS-III

MS-IV

CaCl2

332.02

332.02

332.02

332.02

KH2PO4

170.00

170.00

170.00

170.00

KNO3

1900.00

1900.00

1900.00

1900.00

MgSO4

180.54

180.54

180.54

180.54

NH4NO3

1650.00

1650.00

1650.00

1650.00

CoCl2·6H2O

0.025

0.025

0.025

0.025

CuSO4·5H2O

0.025

0.025

0.025

0.025

Na2EDTA·2H2O

36.70

36.70

36.70

36.70

H3BO3

6.20

6.20

6.20

6.20

KI

0.83

0.83

0.83

0.83

MnSO4·H2O

16.90

16.90

16.90

16.90

Na2MoO4·2H2O

0.25

0.25

0.25

0.25

ZnSO4·7H2O

8.60

8.60

8.60

8.60

Myoinositol

100.00

100.00

100.00

100.00

Sucrose

30,000

30,000

30,000

30,000

6-BAP (μM)

3.0

12.0

6.0

–

Indole-3-butyric acid (μM)

–

–

–

10.0

propagation culture medium. The pH is adjusted to 5.8, as in formation, prior to sterilization in Vitrofural® according to the manufacturer’s instructions [14]. 6. 0.5 N potassium hydroxide. 7. 0.5 N hydrochloric acid. 8. Facilities for tissue culture and instrumentation (laminar flow cabinet, electrical sterilization for instruments, autoclave, culture room). In addition to personnel with protective equipment for work under aseptic conditions. 2.5 In Vitro Multiplication of Shoots of B. vulgaris in TIS

1. Culture medium: starch MS salts [13] (Table 1 MS-III) (see Note 1). 2. 100 mg/L myo-inositol (see Note 1). 3. Growth regulator: 6.0 μM BA (see Note 2). 4. 30 g/L sucrose.

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5. TIS consisted of two glass flasks with a capacity of 1500 mL, one of which served as a nutrient reservoir and the other as a culture flask for the growth of plant material. 6. For TIS, 12 explants per TIS and 250 mL of culture medium per vessel were used, and the immersion time and frequency were 2 min every 6 h [15]. 7. 0.5 N potassium hydroxide. 8. 0.5 N hydrochloric acid. 9. Facilities for tissue culture and instruments (laminar flow cabinet, electrical sterilization for instruments, autoclave, culture room). In addition to personnel with protective equipment for work under aseptic conditions. 2.6 Preparation of B. vulgaris Shoots for Ex Vitro Acclimatization

1. Culture medium: ½ starch MS salts [13] (Table 1 MS-IV) (see Note 1). 2. Growth regulator: 10 μM indole-3-butyric acid (IBA) (see Note 2). 3. TIS consisted of two glass flasks with a capacity of 1500 mL, one of which served as a culture medium reservoir and the other as a culture flask for the growth of plant material. 4. For TIS, 12 explants per TIS and 250 mL of culture medium per vessel were used, and the immersion time and frequency were 2 min every 6 h [15]. 5. 0.5 N potassium hydroxide. 6. 0.5 N hydrochloric acid. 7. Facilities for tissue culture and instrumentation (laminar flow cabinet, electrical sterilization for instruments, autoclave, culture room). In addition to the personnel with protective equipment for work under aseptic conditions.

2.7 Ex Vitro Acclimatization

1. Substrate: a 4:1 mixture of humus and zeolite is used. 2. Plastic trays with 60 holes of 76 cm3 capacity. 3. Irrigation system with micro-sprinklers. 4. Shade mesh 70%.

3 3.1

Methods Plant Material

Axillary buds obtained from a bank of mother plants previously grown in a culture house from rods and stems of adult B. vulgaris plants.

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3.2 Disinfection of Axillary Buds

1. Wash the axillary buds with detergent dissolved in water and rinse under running water. 2. Disinfect the laminar flow cabinet with 70% ethanol. 3. Disinfect the axillary buds with 70% ethanol for 3 min and rinse three times with sterile distilled water. 4. Immerse the axillary buds in 2% (v/v) sodium hypochlorite solution (NaClO) for 20 min, and rinse three times with sterile distilled water. 5. Place the individual segments in 22 × 150 mm test tubes containing 10 mL of the static liquid medium in the laminar flow cabinet.

3.3 In Vitro Establishment

1. Place the axillary buds in in vitro culture medium. The pH should be adjusted to 5.8 for all culture media before autoclaving. The culture media and culture vessels used should be sterilized in an autoclave at 121 °C and 101.3 kPa according to the manufacturer’s instructions (Sigma-Aldrich, USA) (see Note 5). 2. The test tubes containing axillary buds are placed in a growth chamber at 28 ± 2 °C with a photoperiod of 14 h and an irradiation of 38.0–45.7 μmol m-2 s-1. 3. After 4 weeks of culture, the new shoots are planted in groups of 2–3 (see Note 3).

3.4 In Vitro Multiplication of Shoots of B. vulgaris in Static Liquid Culture Medium

1. The formed shoot groups are divided into small groups of three shoots. 2. Three groups of shoots are placed in 500 mL polycarbamate culture flasks containing propagation culture medium. The pH is adjusted to 5.8, as in formation, prior to sterilization in Vitrofural® according to the manufacturer’s instructions [14]. 3. The required culture conditions are a growth chamber at 28 ± 2 °C with a photoperiod of 14 h at irradiation of 38.0–45.7 μmol m-2 s-1. 4. After 4 weeks of culture, the shoots that have multiplied are again divided into small groups (as in the previous culture). This procedure can be performed for five to six subcultures.

3.5 In Vitro Multiplication of Shoots of B. vulgaris in TIS

1. After six subcultures (20 days each), shoot tips were grown in TIS (Fig. 2). 2. Twelve groups of three shoots are placed in TIS with 250 mL propagation medium per vessel, and the immersion time and frequency were 2 min every 6 h [15]. The frequency and immersion time were controlled by a timer that controlled two solenoid valves with three paths that allowed air circulation

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Fig. 2 Temporary immersion system used for the multiplication of Bambusa vulgaris Schrad. ex Wendl shoots

and thus the passage of culture medium from one culture vessel to another (see Note 4). 3. The pH is adjusted to 5.8 prior to autoclaving, as in the case of formation. The culture media and culture vessels used are sterilized in an autoclave at 121 °C and 101.3 kPa according to the manufacturer’s instructions (Sigma-Aldrich, USA). 4. The required culture conditions are a growth chamber at 28 ± 2 °C with a photoperiod of 14 h and irradiation of 38.0–45.7 μmol m-2 s-1. 5. After 15 days of culture, the culture medium is renewed in a laminar flow cabinet. The flask containing the culture medium was removed, and a new one was inserted. This procedure can be performed for three to four cycles. 3.6 Preparation of B. vulgaris Shoots for Ex Vitro Acclimatization

1. The vessel containing the shoots cultured in TIS was provided with new culture medium in a laminar flow cabinet. The vessel with the culture medium was removed, and a new vessel with the prepared culture medium was used for 3 weeks. The pH of the culture medium is adjusted to 5.8 before autoclaving. The culture media and culture vessels used are sterilized in an autoclave at 121 °C and 101.3 kPa according to the manufacturer’s instructions (Sigma-Aldrich, USA). 2. The required culture conditions are a growth chamber at 28 ± 2 °C with a photoperiod of 14 h and irradiation of 38.0–45.7 μmol m-2 s-1. 3. After 3 weeks of culture, they are transplanted to a greenhouse.

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3.7 Ex Vitro Acclimatization

1. Remove the shoots from the culture vessel, and wash them to remove residues of the culture medium (see Notes 6 and 7). 2. Once the plants are separated by large size >3 cm, they are placed in black polyethylene trays (200 cm3) with 28 holes with the substrate, 4:1 mixture of humus and zeolite. 3. Watering is done twice a day with nebulizers. The seedlings were maintained at a relative humidity of 72% and a temperature of 29.73 ± 2 °C. 4. After 30 days of culture, the in vitro plants of B. vulgaris reached a height of 19.0–20.77 cm with four to five open leaves and are ready for planting in the field. 5. Schematic representation of the protocol for in vitro propagation of Bambusa vulgaris Schrad. ex Wendl by organogenesis.

4

Notes 1. Prepare stock solutions of macro- and hormones at 4 °C and of micro- and myoinositol. 2. Overheating of the medium should be avoided as this will lead to degradation of the PGRs. 3. During axillary bud establishment, the percent of shoot is more than 96%, after 7 days. The shoots grow fast and develop several buds in the establishment. After 3 weeks, the shoots can be multiplicated in static liquid culture medium. 4. Prior RITA and TIS assembling, wash all pieces with deionized water. Connect all components according to the maker’s instructions. 5. The TIS consists of two glass flasks with a capacity of 1500 mL, one of which served as a culture medium reservoir and the other as a culture flask for the growth of plant material. The flasks were connected by silicone tubing inserted into the lid of each flask and extending to the bottom to allow flow of the culture medium. Another connection was made through hydrophobic 0.2 μM filters that guaranteed the sterility of the inlet and outlet air, the pressure of which was controlled by a manometer. 6. All glassware and instruments should be properly sterilized to avoid any contamination. 7. During the curing process, care should be taken to maintain the water content of the plants. The plants should not dry out.

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References 1. Borpuzari P, Bisht S (2019) Enhanced rhizome induction and fast regeneration protocol in liquid culture of Dendrocalamus longispathus Kurz: a single step culture. Trop Plant Res 6:18–23 2. Catasu´s Guerra L (2003) Estudio de los bambu´es arborescentes cultivados en Cuba. ACTAF, La Habana-Cuba, p 56 3. Cordero-Miranda E (2010) Propuesta para el manejo sostenible de Bambusa vulgaris Schrader ex Wendl con objetivo protector en diferentes condiciones ecolo´gicas del rı´o Cuyaguateje, Pinar del Rı´o. Tesis en opcio´n al grado cientı´fico de doctor en Ciencias Ecolo´gicas, Universidad de Pinar del Rı´o, Pinar del Rı´o, pp 11–20 4. Goyal A, Pradhan S, Basistha B, Sen A (2015) Micropropagation and assessment of genetic fidelity of Dendrocalamus strictus (Roxb.) nees using RAPD and ISSR markers. 3 Biotech 5:473–482 5. Gielis J, Peeters H, Gillis K, Oprins J, Debergh P (2001) Tissue culture strategies for genetic improvement of bamboo. Acta Hortic 552: 195–203 6. Ndiaye A, Diallo M, Niang D, Gassama-Dia Y (2006) In vitro regeneration of adult trees of Bambusa vulgaris. Afr J Biotechnol 5:1245– 1248 7. Gajjar H, Raval A, Raval H, Patel H, Ramchandra S (2017) In vitro propagation of Bambusa vulgaris through branch node. Int J Curr Res Acad Rev 5:12–17 8. Gonza´lez N, Enmanuel J (2019) Micropropa˜ a de azu´car (Saccharum officigacio´n de can narum L.) del cultivar CCO6-791. Doctoral dissertation, Universidad Nacional Agraria, p 13 9. Carvalho L, Ozudogru E, Lambardi M, Paiva L (2019) Temporary immersion system for

micropropagation of tree species: a bibliographic and systematic review. Not Bot Horti Agrobot Cluj Napoca 47:269–277 10. Mongkolsook Y, Tanasombut M, Sumkaew R, Likitthammanit P, Wongwean P (2005) Temporary immersion system (TIS) for micropropagation of Dendrocalamus latiflorus in commercial production. Kasetsart Agricultural and Agro-Industrial Product Improvement Institute, Kasetsart University, Bangkok, Tailandia, pp 33–40 11. Holst Sanjua´n A (2010) Efecto del sistema de inmersio´n temporal (RITA®) sobre el desarrollo de pla´ntulas in vitro de Guadua angustifolia kunth (Poaceae: Bambusoideae) y su posterior aclimatizacio´n. Tesis (licenciatura en ingenierı´a agrono´mica), Universidad de Costa Rica, pp 21–29 12. Gutie´rrez L, Lo´pez-Franco R, Morales-Pinzo´n T (2016) Micropropagation of Guadua angustifolia Kunth (Poaceae) using a temporary immersion system RITA®. Afr J Biotechnol 15:1503–1510 13. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473– 497 14. Garcı´a-Ramı´rez Y (2021) Obtencio´n de plantas de Bambusa vulgaris Schrad. ex Wendl con calidad morfo-fisiolo´gica en medio de cultivo lı´quido. Tesis doctoral, Universidad Central “Marta Abreu” de Las Villas, pp 52–55 15. Garcı´a-Ramı´rez Y, Gonza´lez-Gonza´lez M, Torres Garcı´a S et al (2016) Efecto de la densidad de ino´culo sobre la morfologı´a y fisiologı´a de los brotes de Bambusa vulgaris Schrad. ex Wendl cultivados en Sistema de Inmersio´n Temporal. Biotecnol Veg 16:231–237

Chapter 18 Multiplication of Cupressus guadalupensis Using the RITA® Temporary Immersion System Luis Alberto Go´mez-Reyes, Esmeralda Judith Cruz-Gutie´rrez, Lorena Jacqueline Go´mez-Godı´nez, Manuel de Jesu´s Bermu´dez-Guzma´n , Claudia Berenice Espitia-Flores , and Juan Manuel Pichardo Gonza´lez Abstract The Guadalupe cypress (Cupressus guadalupensis S. Watson) is an endangered species included in the list of the NOM-059-SEMARNAT-2010. The presence of wild goats in the habitat has been the greatest threat to the propagation and survival of this species. Therefore, there is a need to generate propagation protocols that facilitate the regeneration of the species. Plant tissue culture offers various possibilities that can facilitate the regeneration of species under some risk. Temporary immersion systems have proven to be an option with various advantages in plant tissue culture, such as increasing the number of seedlings generated and reducing production times, compared to semisolid media. The objective of this chapter is to describe a protocol to propagate Guadalupe cypress tissues in a RITA® temporary immersion system. Key words Organogenesis, Asexual reproduction, Plant tissue culture, Temporary immersion systems

1

Introduction The Guadalupe cypress (Cupressus guadalupensis S. Watson) is an arboreal species of the Cupressaceae family, which is endemic to Guadalupe Island in Baja California, Mexico [1]. Due to various factors, the Guadalupe cypress is a species classified as endangered within the NOM-059-SEMARNAT-2010 [2]. Since it is known that there are only around 200 specimens of reproductive age on the island [1], it is essential to look for alternatives that facilitate the propagation of the species. There are different biotechnological tools, such as plant tissue culture (PTC), which can be used to develop of mass propagation protocols that benefit the conservation and repopulation activities of forest species under some risk, such as Guadalupe cypress [3].

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_18, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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Because it has been shown that, in general, in conifers it is more complicated to generate somatic embryos than to obtain new seedlings by direct organogenesis [4], this way is used for the propagation of Guadalupe cypress tissues. In the case of several species, direct organogenesis is one of the ways by which clones can be obtained [5, 6] without the need to go through tissue dedifferentiation (callogenesis) to differentiate them later again [7, 8]. Various systems, such as temporary immersion systems (SIT) can be use in plant tissue culture. The SITs haves been shown to have various advantages when compared to semisolid culture media. Among the main ones are the reduction of hyperhydricity problems and the accumulation of toxic gases so that the tissues show more vigor, a more significant amount of shoots, superior rooting, and others [9–11]. The objective of this chapter is to provide a method of Guadalupe cypress tissues propagation.

2

Materials

2.1

Plant Material

2.2

Sanitizing Agents

Germplasm must be tissue from a healthy, vigorous, pest-free juvenile donor plant. For establishment, young shoots (apical with one or two buds) are used. 1. Liquid detergent (See Note 1). 2. 70% ethanol solution (See Note 2). 3. 1.5% sodium hypochlorite solution (See Note 2). 4. Sterile distilled water (See Note 3).

2.3

Culture Medium

2.4

Equipment

The culture medium used in the different stages are described in Table 1. 1. Culture room: laminar airflow hood. 2. Surgical tools: stainless steel forceps, surgical scalpels, removable sterile surgical blades, and sterile Petri dishes. 3. Medium preparation: weighing balances, magnetic stirrer, hot plate, pH meter, microwave, micropipettes, magnets, refrigerator, and autoclave. 4. Culture vessels: reagent bottles, flasks (500 and 1000 mL capacity). 5. Glassware cleaning: Scrub glassware with a liquid detergent solution, thoroughly wash with tap water, and rinse glassware with distilled water and dry.

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Table 1 Culture medium additives used for the different culture stages of Cupressus guadalupensis Culture medium

Components

Establishment medium

Woody plant medium [12] basal medium with 30 g/L of sucrose, 3 g/L of activated charcoal, 10 g/L of agar, and a pH of 5.8

Propagation medium

Woody plant medium [12] basal medium with 30 g/L of sucrose, 1 mg/L of Kinetin, 0.2 mg/L of ANA, and a pH of 5.8

Rooting medium

Woody plant medium [12] basal medium with 30 g/L of sucrose, 4 mg/L of indole3-acetic acid and a pH of 5.8

2.5 Temporal Immersion System (TIS)

3

RITA® system, a registered trademark of the French company Vitropic S.A., is based on a one-liter container with two compartments: the upper one contains the explants and the lower one the culture medium. The air pressure applied in the lower compartment drives the medium to the upper compartment, irrigating the explants.

Methods

3.1 Disinfection Plant Material

1. Plant material is collected from a wild pest and disease-free plant. Make it a vigorous and well-nourished plant. 2. Wash the tissues with liquid detergent (see Note 1) and running water to remove impurities, foreign objects, and possible sources of contamination. 3. Immerse the tissues in a 70% ethanol solution for 2 min. 4. Immerse the tissues in a 1.5% sodium hypochlorite solution for 15 min. 5. Wash three or four times the tissues with sterile distilled water to remove residues of disinfectant solutions (see Notes 3 and 4). 6. Establish the tissues in the culture medium (see Note 5). 7. The shoots in vitro, free of microorganisms and the most vigorous tissue, is selected to be established in the TIS (Fig. 1a).

3.2 Proliferacion de brotes en RITA

1. The shoots with a size of 15 mm are established in the RITA® system (see Note 6) using a frequency of 24 h between immersions and with a time of 2 min per immersion, with a period of 40 days of incubation (Fig. 1b). 2. The shoots are incubated at a temperature of 24 °C ± 2 and a photoperiod of 16 h of light for 8 h of darkness at an intensity of 2000 lux.

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Fig. 1 Micropropagation de Cupressus guadalupensis in RITA®. (a) Apical bud explants, (b) Guadalupano cypress in RITA®, and (c) Formation of new shoots in RITA®

3. After 40 days of incubation, new shoots per explant are obtained subsequent processes (Fig. 1c). 3.3

Rooting

1. Put the shoots on the rooting media (Table 1). 2. The shoots are incubated with a photoperiod of 16 h of light and 8 h of darkness, at an intensity of 2000 lux and at an average temperature of 24 °C, for 90 days until complete (with roots) in vitro plants are obtained.

3.4

Acclimatization

1. Use 750 mL capacity containers, with a volume of substrate content of 500 mL (see Note 7). 2. The seedlings are extracted from the tubes, washed with running water to remove the culture medium, and established in the containers with substrate. 3. They are taken to a greenhouse, where relative humidity conditions between 50% and 95% and temperatures between 15 and 32 °C are maintained. 4. A daily irrigation is applied, with micro-aspersion.

4 Notes 1. The detergent is a commercial brand for washing dishes. 2. Disinfectant solutions are prepared with distilled water.

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3. The water is sterilized in an autoclave at 1.5 kg/cm2 pressure at a temperature of 121 °C for 15 min. 4. All the disinfection protocol is carried out inside the laminar flow clean bench. 5. The tissues are dissected, with a size of 15 mm, inside the laminar flow clean bench, and established in the culture medium (Table 1). 6. The RITA® system consists of a container made of polycarbonate and divided into two compartments separated by a mesh with a 400 μm pore. Plant tissues are located in the upper compartment, while the culture medium is concentrated in the liquid phase in the lower one. Its operation consists of injecting pressure using a compressor, which generates the flow of the medium from the bottom of the container to the area, where the tissues are located. Thus, they are exposed to the medium for short periods. 7. Substrate is made up of a mixture of peat moss: agrolite: vermiculite (1:1:1).

Acknowledgments This work was supported by the National Genetic Resources Center of National Institute of Forestry, Agriculture, and Livestock Research. References 1. Farjon A (2010) A handbook of the world’s conifers. Brill, London 2. SEMARNAT (2010) Norma Oficial Mexicana NOM-059-SEMARNAT-2010, Proteccio´n ambiental-Especies nativas de Me´xico de flora y fauna silvestres-Categorı´as de riesgo y especificaciones para su inclusio´n, exclusio´n o cambio-Lista de especies en riesgo. SEMARNAT, CDMX 3. Mroginski L, Roca W (1991) Establecimiento de cultivos de tejidos vegetales in vitro. In: Roca WM, Mroginski LA (eds) Cultivo de tejidos en la agricultura. Fundamentos y aplicaciones. CIAT, Cali 4. Gonza´lez N (2018) Mejora de protocolos de regeneracio´n por embrioge´nesis soma´tica en ˜onero. pino pin Doctoral dissertation, Agronomica ˜ ez V (2007) Propagacio´n 5. Ocampo F, Nu´n in vitro de Psidium guajaba mediante organoge´nesis directa a partir de segmentos nodales. Cienc Tecnol Agropecu 8:22–27

6. Cesty C, Saenz E, Pereira G (2007) Micropropagacio´n de Pothomorphe umbellata (L.) Miq. vı´a organoge´nesis directa. Rev Cubana Plant Med 12:1–12 7. Rodrı´guez M, Latsague M, Chaco´n M, Astorga P (2014) Induccio´n in vitro de calloge´nesis y organoge´nesis indirecta a partir de explantes de cotiledo´n, hipoco´tilo y hoja en Ugni molinae. Bosque (Valdivia) 35:111–118 8. Reyes S, Lecona C, Barredo F, Caldero´n J, Abud M, Rinco´n-Rosales R, Gutie´rrez-Miceli FA (2016) Plant growth regulators optimization for maximize shoots number in Agave americana L. by indirect organogenesis. Gayana Bot 73:124–131 9. Del Rivero N, Quiala E, Agramante D, Barbo´n R, Camacho W, Morejo´n L, Pe´rez M (2004) Empleo de sistemas de inmersio´n temporal para la multiplicacio´n in vitro de brotes de Anthurium andraeanum Lind. var. Lambada. Biotecnol Veg 4:2

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˜ iga T (2019) Multiplicacio´n 10. Ramı´rez J, Zu´n in vitro de Zingiber officinale Roscoe cv. “Gran Caima´n” en Sistema de Inmersio´n Temporal. Biotecnol Veg 19:297–306 11. Cha´vez J, Andrade M, Jua´rez P, Villegas O, Sotelo H, Perdomo F (2018) Evaluacio´n de tres sistemas de cultivo in vitro para la multiplicacio´n de microcormos de gladiolo. Rev Fitotec Mex 41:551–554

12. Lloyd G, McCown B (1981) Woody plant medium: a mineral nutrient formulation for microculture of woody plant species. HortScience 16:453

Chapter 19 Secondary Embryogenesis of Linaloe in Temporary Immersion Bioreactor-Type RITA® Gregorio Arellano-Ostoa , Mo´nica Gonza´lez-Orozco, Izaac Va´zquez-Cisneros, and Sandra Mitchelle Arellano-Gonza´lez Abstract The linaloe [Bursera linanoe (La Llave) Rzed, Calderon and Medina] is an endemic species of Mexico, representative of the low deciduous forest of the states of Guerrero, Puebla, Morelos, and Oaxaca, and has been of great economic importance for the people, mainly for the artisanal use of its aromatic wood that is used to make boxes, trunks, and furniture that are manufactured in Olinala, Guerrero, Mexico; and industrial, thanks to the fine aroma of its essential oil (linalool), which is used in the manufacture of perfumes and pharmaceuticals. Overexploitation has endangered the species in recent years, and propagation by seed and/or cuttings has produced very poor results compared to those obtained with other recalcitrant Bursera species. The protection of endangered species makes urgent the need to propose new alternatives for its propagation. Somatic embryogenesis is a reliable and feasible technique, including induction, maintenance, multiplication, and maturation of embryos, often in semisolid culture media; however, the recent use of liquid media has allowed semi-automation in temporary immersion bioreactors, for example, the RITA® system, which favors both the multiplication rate and the final conversion to seedlings. Key words Repetitive embryogenesis, Burseraceae, In vitro culture, Bioreactors

1

Introduction In Mexico, the linaloe Burseraceae is a threatened deciduous species, due to overexploitation of the native material, and has historical, cultural, and economic importance for the Alto Balsas area. It has artisanal, industrial, and medicinal uses and has become the economic support of many rural communities. They are generally low to medium-sized trees (5–15 m) and are characteristic of mature communities [1, 2]. There are few published studies on the propagation of the genus Bursera in Mexico, and it is only possible to obtain information on vegetative propagation by cuttings of some economically important species, such as white copal Bursera bipinnata, holy copal B. copallifera, and citronella

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_19, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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B. citronella [3, 4]. On the other hand, Bonfil-Sanders et al. [4] mention that studies on the vegetative propagation of this species would be important to accelerate succession in disturbed sites and thus help restore the composition and structure of natural communities. In species such as Quercus robur L. [5], mention that the use of epicormic stems, which have juvenile characteristics [6–8], promotes vegetative propagation in other woody plants; however, in linaloe these stems are infrequent [9]. Somatic embryogenesis (SE) is a reliable and feasible technique to be used in the in vitro propagation of linaloe [10], which comprises the induction, maintenance, proliferation, and maturation of embryos [11]; it is considered the best form of in vitro regeneration of woody plants [12]. Likewise, embryogenic cultures allow the application of biotechnology techniques, such as low-temperature storage or cryopreservation, the creation of germplasm banks, and could also allow the production of secondary metabolites synthesized during mass propagation using bioreactors [13, 14]. Traditional embryogenic cultures are prepared in semisolid medium; however, the liquid medium favors the absorption of nutrients originated by osmosis, which leads to an increase in cell proliferation [15]. The above has allowed the development of new biotechnological techniques, for example, the use of automated temporary immersion vessels, called RITA® [16–18]. This system consists of polycarbonate, glass, or polyethylene, semiautomated bioreactors designed for large-scale propagation of embryos or organs. The temporary exposure of the explants to the liquid culture medium can be for a short time (1–2 min), with a frequency of immersion between 2 and 6 h [19]. They have several advantages such as rapid and efficient production of plants, with high genetic and phytosanitary quality, reduction of hyperhydrated explants, and lower production costs, compared to the conventional system in semisolid culture media [16]. On the other hand, with the use of LED lamps of different wavelengths, the morphogenetic growth of embryos in vitro is favored, facilitating the adaptation and growth of seedlings in the greenhouse [20]. The RITA® temporary immersion system was developed at CIRAD, Montpellier, France (https://www.vitropic.fr), and is made of polycarbonate and consists of individual 1 L containers, which are divided into two parts; the lower part contains the liquid culture medium and the upper part the plant material or explants interconnected by a mesh. The system is composed of modules or racks of 90 × 40 × 60 cm with two or three levels, which are supplemented with LED lamps emitting an intensity of 47 μmol m-2 s-1, with a photoperiod of 16 h of light for 8 h of darkness, arranged in an incubation room at 25 ± 2 °C and 80% R.H. The vessel is connected to an air inlet, through silicone hoses to an autoclavable hydrophobic filter of 0.22 μm and 50 mm diameter. The air is supplied by an oil-free compressor, which allows

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the solution to rise toward the plant material when the compressor is on, and once the action of the compressor ceases, the liquid medium descends by gravity to the lower compartment. The frequency and duration of the immersions are given by a timer or by a software (Registration Number 03-2016-040510454800-011), which infers advantages, given that it allows to have a semiautomatic system that records the operation [16, 21]. The objective of this chapter is to establish a regeneration protocol using somatic embryos of linaloe, to obtain sufficient quality vegetative material to establish commercial plantations in Mexico in the medium period.

2 2.1

Materials Plant Material

2.2 Chemical Products

2.3

Culture Media

Fruits are collected from native linaloe trees, approximately 30 years old, in the town of Chiautla, Puebla, Mexico (Fig. 1a). The fruits are washed with running water and biodegradable soap to later remove the pulp and release the seed. Scarification (Fig. 1b) (see Note 1) and seed disinfestation (see Note 2), sulfuric acid (H2SO4) at 95.6% (see Note 1), calcium hypochlorite at 1.8% (v/v) (see Note 3), and ethanol at 70% (v/v) (see Note 4). Disinfection of surfaces and equipment: calcium hypochlorite a.i. 1.8% (v/v) (see Note 3) and 70% alcohol (see Note 4). pH adjustment solutions: sodium hydroxide NaOH 1 N (see Note 5) and hydrochloric acid HCl 0.1 N (see Note 6). Solvents for growth regulators: potassium hydroxide (KOH) 0.1 N volumetric solution and absolute ethanol. 1. MS culture medium [22] (Table 1), and MS at 50% of the concentration of the macrosalts (see Note 7). 2. Medium additives (Table 2) used for the different culture stages: seed germination (MG), induction (M1), initiation (M2), and expression of embryogenesis (M3), multiplication, and maintenance of proembryogenic masses (PEM’s) semisolid media (M4), multiplication by secondary embryogenesis in temporary immersion systems (M5), and maturation and germination stages of somatic embryos (M6) (see Notes 8–13).

2.4

Equipment

1. Transfer Area: laminar flow hood, dry heat sterilizer for surgical instruments. 2. Incubation area: shelves, LED lamps, oil-free air compressor, temporal RITA immersion system, air conditioning system, timers, having a minimum of seven events, or an automation system with software [21] (Fig. 2).

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Fig. 1 (a) 30-year-old native linaloe tree. (b) In vitro germination of scarified seeds. (c) Induction of somatic embryogenesis (SE) from cotyledons and expanding leaves. (d) Secondary embryos at developed cotyledon stage. (e) Embryos formed from cotyledons, 12 weeks after induction. (f) Individualized somatic embryo and (g) Somatic embryos showing the cauline apex

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Table 1 Complete MS culture medium and 50% in the concentration of macrosalts used in the different stages of in vitro culture of linaloe (mg/L) MS 100%

(mg/L) MS 50%

NH4NO3

1650

825

KNO3

1900

950

CaCl2·2H2O

440

220

MgSO4·7H2O

370

185

KH2PO4

170

85

H3BO3

6.2

6.2

MnSO4·4H2O

22.3

22.3

ZnSO4·7H2O

8.6

8.6

KI

0.83

0.83

Na2MoO4·2H2O

0.25

0.25

CoCl2·6H2O

0.025

0.025

CuSO4·5H2O

0.025

0.025

FeNa-EDTA

36.7

36.7

FeSO4·7H2O

27.8

27.8

Nicotinic acid

0.5

0.5

Pyridoxine

0.5

0.5

Thiamine

0.1

0.1

Myo-inositol

100

100

Glycine

2

2

Components Macronutrients

Micronutrients

Vitamins

3. Surgical instruments: stainless steel forceps, surgical scalpels, removable sterile surgical blades (number 10, 11, and 21), and sterile 9 cm diameter Petri dishes. 4. Preparation of the medium: granatary and analytical balances for weighing, weighing substances, spatulas, magnetic stirrers, hot plate with stirring, potentiometer, microwave oven, micropipette set, graduated test tubes and beakers, autoclave, and reverse osmosis water distiller.

– –

4 0.4

7 1 – –

–

–

30

7

–

–

1

1

ANA (mg/L)

BAP (mg/L)

Sucrose (g/L)

Agar (g/L)

AGP (g/L)

ABA (mg/L)

BR (mg/L)

AC (g/L)

1

1

7

30

0.4

0.4

–

1

–

–

–

7

30

–

–

–

100

SE expression (M3)

3

1

– 1

0.002

–

–

0.01

–

–

7

– 1

40

–

–

–

50

6 SE Maturation and germination (M6)

30

0.01

0.01

–

100

5 Multiplication in TIS (M5)

–

1

7

30

0.01

0.01

–

50

4 Multiplication and maintenance PEM’s (M4)

SE Somatic embryos, TIS temporary immersion system, MS Murashige and Skook media, HC hydrolyzed casein, ANA 1-naphthaleneacetic acid, BAP benzyl amino purine, AGP arabinogalactan, ABA abscisic acid, BR brassinosteroid, AC active carbon

1

30

500

–

HC (mg/L)

100

100

100

SE initiation (M2)

Media MS (%)

SE induction (M1)

2

Seed germination (MG)

1

Media additives

0

Stages

Table 2 Additives of the culture media used during the different stages of somatic embryogenesis of linaloe

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Fig. 2 (a) Temporary immersion system, (b) planting of proembryogenic masses inside the laminar flow chamber in RITA temporary immersion bioreactors, (c) incubation conditions and (d) software developed to control the temporary immersion system

5. Preparation of growth regulators: volumetric flasks (*100 and 500 mL capacity), funnels, granatary and analytical balances, weighing substances, and refrigerator. 6. Culture vessels: amber flasks for reagents, Erlenmeyer flasks (500 and 1000 mL capacity), beakers, culture tubes (25 × 150 mm) with polypropylene stoppers, 9 cm diameter Petri dishes, and vessels for temporary immersion system-type RITA®. 7. Miscellaneous materials: for cleaning glassware, commercial liquid detergent solution, and tap water. Cotton cloths and filter paper, polypropylene bags, aluminum foil, film, or plastic film for food packaging.

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Methods

3.1 Stage 0: Seed Germination

1. Wash the linaloe fruits with soap and tap water, and remove the pulp with a scalpel to extract the seeds (Fig. 1b). Perform chemical scarification of the seeds to soften the seed coat (see Note 1). 2. In the sowing area, in the laminar flow hood previously disinfected (see Notes 14 and 15). The seeds are treated with a solution of calcium hypochlorite i.a. 1.8% (v/v) and 70% ethanol to be disinfested (see Note 2). 3. Removal of the seed coat. A paper towel is placed at the bottom of the laminar flow hood, and five seeds are placed so that they lose moisture. A piece of filter paper is taken, and a seed is placed on it to which the seminal cover must be removed, being very careful not to damage the embryo, you can use tweezers to hold the seed and the scalpel to cut and remove the cover or help yourself with tweezers and pliers. 4. Seeding of embryos with cotyledons in vitro: one embryo per test tube is placed in MG medium (Table 2). The rack with the culture tubes is transferred to the incubation room in dark conditions, at a temperature of 25 ± 2 °C, until the emergence of the seedlings from 30 to 45 days. Seedlings are incubated at 25 ± 2 °C with a 16 h photoperiod using LED lamps providing a light intensity of 47.3 μmol m-2 s-1.

3.2 Stages 1, 2, and 3: Induction, Initiation, and Expression of Embryogenic Cultures [10, 23]

3.3 Stage 4: Multiplication and Maintenance of Proembryogenic Masses (PEM’s)

1. From the germinated seedlings, select the cotyledons and the young expanding leaves as original explants for embryogenic induction (Fig. 1c). 2. Place the cotyledons and expanding leaves in Petri dishes with the abaxial side in contact with the M1 culture medium (Tables 1 and 2) to which 500 mg/L of hydrolyzed casein is added when preparing the medium. The Petri dishes are placed in darkness at 25 ± 2 °C for 9 weeks. After this time, the explants are transferred to M2 medium for embryogenic induction (Fig. 1c, d, f; Table 2). In this second stage, the cultures are maintained for 4 weeks under conditions of 16 h of photoperiod at 25 ± 2 °C. The explants are then transferred to expression medium (M3) without growth regulators (Table 2) and maintained under the same light and temperature conditions with subcultures every 4 weeks (Fig. 1) (see Notes 7–11). Proembryogenic masses are transferred to modified MS [22] semisolid culture medium, reducing the macronutrients to half their concentration, medium M4 (Table 2; Fig. 1g) (see Note 7). In this medium, subcultures are performed every 30 days.

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3.4 Stage 5: Multiplication by Secondary Embryogenesis in Temporary Immersion System RITA®

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1. In the laminar flow chamber, 500 mg of plant material maintained in vitro in liquid medium M4 are weighed and placed in a temporary immersion vessel (see Notes 7 and 15–19) (Fig. 2b). 2. The RITA®-type temporary immersion system is used, which contains 150 mL of M5 liquid culture medium (Tables 1 and 2) (see Notes 16–19) (Fig. 2a). 3. The system is programmed with six immersions per day of 1 min each (Fig. 2d). Incubation conditions are 25 ± 2 °C, with a photoperiod of 16 h light provided by LED lamps, emitting 54 μmol m-2 s-1 intensity (Fig. 2c). 4. After 30 days, the formed embryos, which will be at different stages of development, are collected. The most developed embryos (torpedo and heart) will be used in stage 6 of maturation and in vitro germination.

3.5 Stage 6: Maturation and In Vitro Germination

1. Embryos are transferred to modified MS [22] culture medium, reducing the macronutrients (M6) to half their concentration (see Note 7), with the addition of 3 mg/L AgNO3, 0.01 mg/L ABA, 0.002 mg/L BR, and 4% sucrose (Table 2; Fig. 3a–c).

Fig. 3 (a) Maturation and germination, (b, c) secondary embryogenesis generated in temporary immersion RITA® bioreactors and (d) acclimatization of plants in greenhouses

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Fig. 4 Linaloe seedlings derived from somatic embryogenesis (SE) at different ages (a) ES germinated in SIT; (b) 3 months in greenhouse; (c) 6 months in greenhouse; (d) 1 year and (e) 3 years old

2. Incubation conditions are 25 ± 2 °C, with a photoperiod of 16 h light provided by LED lamps emitting 54 μmol m-2 s-1 intensity. 3. At stage 6, embryos are individualized in 25 × 150 mm test tubes in M6 culture medium (Table 2) for 4–6 weeks, until the appearance of radicle and developed cauline apex is observed. 3.6 Stage 7: Greenhouse Acclimatization

1. The somatic embryos that manage to mature and those present formed cotyledons and a developed radicle (Fig. 4a) with true leaves and that have a height of approximately 3–5 cm, which is achieved in approximately 90 days (Fig. 4a, c). They are transplanted into a substrate composed of perlite, pine bark, and peat moss (30:30:40, v/v/v) (see Notes 20 and 21) and kept in the incubation room for 30 days. 2. The seedlings are moved to the greenhouse for another 30 days; the box is gradually uncovered so that the seedlings can acclimatize without stress; at the end survival, height increase and appearance of new leaves and hardening are evaluated (Fig. 4b, c).

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Notes 1. Chemical scarification. To soften the testa of the seeds, in a flask of 500 mL, put a group of 70 seeds, add sulfuric acid to 95.6%, enough to cover only the seeds; cover the mouth of the flask with a fine polyester cloth, holding it perfectly with a rubber band, taking care not to splash; and shake it slowly and manually during 5 min. Without removing the cloth stopper, in case there is a reaction when rinsing, the mouth of the flask is directed toward the strainer and the seeds are rinsed with tap water. The seeds are put in a mesh and rubbed to remove the impurities that are still present. The seeds are washed with detergent and rinsed three times under running water. 2. Disinfestation of seeds. The seeds are placed in a closed flask in calcium hypochlorite solution i.a. 18% (v/v) (see Note 3) and are shaken for 18 min. The flask is cleaned with calcium hypochlorite solution i.a. 18% (v/v) and is introduced into the laminar flow hood. The seeds are rinsed with sterile distilled water using a strainer, later the seeds are placed in ethanol at 70% 1 min and rinsed three times with sterile distilled water; the water of the rinses is recovered inside the hood, in a sterile container to be discarded. 3. Calcium hypochlorite solution. In a 200 mL container. Prepare 100 mL of a solution of calcium hypochlorite i.a. 1.8% (v/v). From a commercial product (depending on the locality make sure of the concentration of the active ingredient of the product), e.g., in Mexico, the brand Cloralex® has 6% of active chlorine. In a 100 mL flask, 30 mL of the commercial product is added, and 100 mL is calibrated with distilled water. 4. Alcohol solution. The 70% ethanol solution is prepared by transferring 700 mL of ethanol (96°) into a 1 L graduated cylinder and is calibrated to 1000 mL with distilled water. 5. Preparation of the NaOH to 1 N. To weigh in a granatary balance 4.08 g of NaOH (purity of 98%) to dissolve the reagent in a beaker with 50 mL of distilled water, to add the dissolution in a volumetric flask of 100 mL, and to calibrate. 6. Preparation HCL to 0.1 N. In a 100 mL volumetric flask, put 50 mL of distilled water with a 1 mL pipette, take 0.19 mL of HCl (purity of 99% and density of 1.18 g/mL), add to the 100 mL volumetric flask, and make up to 100 mL. 7. Preparation of culture medium [22]. For 1 L of medium, weigh separately each one of the components of the medium (Table 1), add them one by one in a beaker of 1 L containing 300 mL of distilled water with the aid of magnetic agitator (Mixture 1). Weigh 7 g of agar-agar and dissolve it separately in

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500 mL distilled water (Mixture 2), with the aid of a grill with heat or in the microwave (boil twice, taking care that it does not spill). Mixture 2 is added to the beaker with mixture 1, and the medium additives and growth regulators (Table 2) of each of the stages are weighed. Make up to 1000 mL. Adjust the pH to 5.7 with HCl or NaOH 0.1 and 1 N (see Notes 5 and 6). In step 0, pour 10 mL of prepared medium into 25 × 150 mm test tubes and autoclave. In stages 1, 2, and 3, prepare the medium and autoclave in a flask; in a laminar flow hood, pour 20 mL of medium in 9 cm petri dishes. In stage 4 of multiplication and maintenance of proembryogenic masses (PEM’s), prepare the medium with agar and pour 10 mL in 100 mL glass flasks. In the multiplication of secondary embryos in the SIT temporary immersion system (stage 5), pour 150 mL of the liquid medium in RITA-type temporary immersion vessels with a capacity of 1 L. Finally, sterilize in autoclave for 20 min at 120 °C and 115 kPa. In stages 0, 1, 2, 3, 4, and 6, 1 g/L of activated carbon is added to the medium, at the end of the preparation, by means of agitation for its incorporation. 8. Preparation of NAA. Weigh in an analytical balance 10 mg of naphthalene acetic acid (NAA), in a precipitate glass of 10 mL, add a few drops of potassium hydroxide KOH to 0.1 N until dissolving the reagent. In a 100 mL volumetric flask, put 50 mL distilled water, place the dissolved reagent, and calibrate to 100 mL. The solution is stored in an amber-colored flask at a temperature of 4 °C. 9. Preparation of BAP weigh in an analytical balance 10 mg of benzyl amino purine (BAP), in a precipitated glass of 100 mL, dissolve the reagent with 50 mL of warm distilled water. In a 100 mL volumetric flask, place the dissolved reagent and calibrate to 100 mL. The solution is stored in an amber bottle at 4 °C. 10. Preparation of BR. Weigh in an analytical balance 2 mg of brassinosteroids (BR) in a precipitated glass of 100 mL, and dissolve the reagent with 50 mL of warm distilled water. In a 100 mL volumetric flask place the dissolved reagent and calibrate to 100 mL. The solution is stored in an amber flask at a temperature of 4 °C. 11. Preparation of arabinogalactan (AGP). To weigh in an analytical balance 1 g of arabic gum, in a precipitated glass of 1000 mL, dissolve the reagent with 500 mL of distilled warm water. In a 1000 mL volumetric flask, place the dissolved reagent and calibrate to 1000 mL. The solution is stored in an amber-colored flask at a temperature of 4 °C. 12. Preparation of ABA. Weigh in an analytical balance 2 mg of abscisic acid (ABA), in a precipitated glass of 100 mL, and

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dissolve the reagent with 50 mL of warm distilled water. In a 100 mL volumetric flask, place the dissolved reagent and calibrate to 100 mL. The solution is stored in an amber-colored flask at a temperature of 4 °C. 13. Preparation of the silver nitrate. To weigh in the analytical balance 10 mg of silver nitrate (AgNO3), in a precipitated glass of 100 mL, dissolve the reagent with 50 mL of warm distilled water. In a 100 mL volumetric flask, place the dissolved reagent and calibrate to 100 mL. The solution is stored in an amber-colored flask at a temperature of 4 °C. 14. Preparation of the laminar flow chamber. The laminar flow chamber should be turned on 15 min prior to use, with ultraviolet light; after the time, turn off the light. Use an atomizer with an i.a. 1.8% (v/v) chlorine solution, and clean it by passing a sterile cotton cloth over the entire surface, and finally, atomize with 70% alcohol and let it dry. The following materials to be used will be placed inside the chamber, disinfecting them before introducing them with a cloth and chlorine i.a. 1.8% (v/v): distilled water, the rack of tubes with basal culture medium, a package of paper towels, long and short tweezers, scalpels, forceps, pliers, and an empty 2 L container (to recover the water from the last rinses), all of them previously sterilized with autoclave and a stereoscopic microscope, as well as the containers with the seeds. It is recommended to place a disinfected garbage container as close as possible to the laminar flow chamber in order not to take the hands too much out of the aseptic area when removing the waste and reduce the risk of contamination. 15. About autoclave sterilization. All the materials that are introduced into the laminar flow hood must be previously sterilized in an autoclave for 20 min at 120 °C and 115 kPa. For this purpose, they must be perfectly wrapped in aluminum foil and/or placed inside polypropylene bags and sealed. Once sterilized, place them inside the aseptic area (seeding area). To sterilize the water, introduce the bottles with the lid slightly screwed on and tighten the lid once sterilization is finished. In the case of using aluminum foil (thick), two layers should be used that perfectly cover the mouth of the containers and seal with a rubber band. 16. The bioreactors are thoroughly washed with soap and running water. They are given a second rinse with 1.8% v/v chlorine solution and allowed to drain for 24 h. Carefully assemble all parts and add 150 mL of liquid medium M5 with 0.01 mg/L ANA, 0.01 mg/L BAP, and 1 mg/L AGP. The 0.22-micron hydrophobic filters are covered with aluminum paper, and the entire bioreactor is covered with a polypropylene bag fastened with a rubber band (Fig. 2a, c).

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17. The RITA® are autoclaved for 20 min at 120 °C and 115 kPa. 18. The RITA® are taken to the inoculation room, and inside the laminar flow chamber, carefully remove the lid of the vessel, place 500 mg of proembryogenic callus in the upper cavity of the vessel, and screw on the vessel lid, making sure that all the gaskets are well in place. The lid of the vessel is screwed on, making sure that all gaskets are securely in place and sealing with parafilm. 19. The bioreactors are transported with the filters wrapped in aluminum paper and with their polypropylene bag, where they were sterilized, to the incubation room. They are placed on the shelves prepared for this purpose under aseptic conditions, and the filter wrappings and the polypropylene bag are carefully removed to connect the hoses that conduct the air. 20. The tubes are filled with the substrate mixtures to three quarters of their capacity. The substrate is sterilized in autoclave, at a temperature of 121 °C 115 kPa, for 45 min, and left to stand for 2 days. After this, controlled release fertilizer (Osmocote®) (18-6-12 of N-P2O5-K2O) 1.5 g/L is added. 21. The 3–5 cm well-developed seedlings are carefully removed from the semisolid medium with the help of tweezers and washed with a solution of fungicide and bactericide, carefully removing the agar from the roots. They are placed in the center of the tube in the substrate, watered with the solution of fungicide and bactericide. They are placed for 30 days inside a transparent plastic box with a lid to maintain a high relative humidity. For transplanting, 100 cm3 (117 × 46 × 32 mm) of black polypropylene tubes are placed in 42-cavity (375 × 373 × 234 mm) tube-holder tables. References 1. Rzedowski J, Kruse H (1979) Algunas tendencias evolutivas en Bursera (Burseraceae). Taxon 28:103–116 2. Rzedowski J, Medina Lemus R, de Rzedowski GC (2004) Las especies de Bursera (Burseraceae) en la cuenca superior del rio Papaloapan (Me´xico). Acta Bot Mex 66:23–151 3. Rzedowski J, Medina Lemus R, de Rzedowski GC (2005) Inventario del conocimiento taxono´mico, ası´ como de la biodiversidad y del endemismo regionales de las especies de Bursera (Burseraceae). Acta Bot Mex 70:85–111 4. Bonfil-Sanders C, Mendoza-Herna´ndez P, Ulloa-Nieto J (2007) Enraizamiento y formacio´n de callos en estacas de siete especies del ge´nero Bursera. Agrociencia 41:103–109

5. Vidal N, Arellano G, San Jose´ MC, Vieitez AM, Ballester A (2003) Developmental stages during the rooting of in-vitro-cultured Quercus robur shoots from material of juvenile and mature origin. Tree Physiol 23:1247–1254. https://doi.org/10.1093/treephys/23.18. 1247 6. Bonga JM (1982) Vegetative propagation in relation to juvenility, maturity and rejuvenation. In: Bonga JM, Durzan DJ (eds) Tissue culture in forestry. Martinus Nijhoff/W. Junk Publishers, The Hague, pp 387–412 7. Hackett WP (1985) Juvenility, maturation and rejuvenation in woody plants. Hortic Rev 7: 109–155

Secondary Embryogenesis of Linaloe in Temporary 2 Immersion Bioreactor-Type RITA® 8. Vieitez AM, San-jose´ MC, Vieitez E (1985) In vitro plantlet regeneration from juvenile and mature Quercus robur, L. J Hortic Sci 60:99– 106 9. Arellano-Ostoa G, Gonza´lez-Bernal S, Arellano-Herna´ndez G (2014) El Lina´loe Bursera linanoe (La Llave) Rzedowski, Caldero´n & Medina, Especie Maderable Amenazada: una estrategia para su conservacio´n. AGROProductividad 7(3):42–51 10. Arellano-Ostoa G, Gonza´lez-Bernal S, Garcı´aVillanueva E (2011) Lina´loe cultivo y aprovechamiento. In: Propagacio´n in vitro y por estacas de linanoe Bursera aloexylon procedentes de los estados de Guerrero, Puebla y Morelos. Fundacio´n Produce Puebla, A.C., Me´xico 11. Toribio M, Ferna´ndez C, Celestino C, Martı´nez MT, San-Jose´ MC, Vieitez AM (2004) Somatic embryogenesis in mature Quercus robur trees. Plant Cell Tissue Organ Cult 76: 283–287 12. Braciela J, Vieitez AM (1993) Anatomical sequence and morphometric analysis during somatic embryogenesis on cultured cotyledon explants of Camellia japonica L. Ann Bot 71: 395–404 13. Corredoira E, Ballester A, Vieitez AM (2003) Proliferation, maturation and germination of Castanea sativa Mill. somatic embryos originated from leaf explants. Ann Bot 92:129–136 14. Vieitez AM, Corredoira E, Martı´nez T, San-Jose´ MC, Sa´nchez C, Valladares S, Vidal N, Ballester A (2012) Application of biotechnological tools to Quercus improvement. Eur J For Res 131:519–539. https://doi.org/10. 1007/s10342-011-0526-0 15. Corredoira E, Valladares S, Vieitez AM, Ballester A (2008) Improved germination of somatic embryos and plant recovery of European chestnut. In Vitro Cell Dev Biol Plant 44:307–315

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16. Etienne H, Berthouly M (2002) Temporary immersion systems in plant micropropagation. Plant Cell Tissue Organ Cult 69:215–231 17. Mallo´n R, Covelo P, Vieitez AM (2011) Improving secondary embryogenesis in Quercus robur: application of temporary immersion for mass propagation. Trees 26:731–741 18. Larque´ H, Sa´nchez-Arreola E, Arellano-OstoaG, Herna´ndez LR, Bach H, Cha´vez-Montes A (2023) Linalol and linalyl acetate quantitation in the essential oil of somatic embryos of Bursera linanoe R. Indian J Pharm Educ Res 57(1): 107–112 19. Etienne H, Lartuad M, Michaux-Ferrie´re N, Carron MP, Berthouly M, Teisson C (1997) Improvement of somatic embryogenesis in Hevea brasiliensis (Mu¨ll. Arg.) using the temporary immersion technique. In Vitro Cell Dev Biol Plant 33:81–87 20. Murillo-Talavera MM, Pedraza-Santos ME, Gutie´rrez-Rangel N, Rodrı´guez-Mendoza MN, Lobit P, Martı´nez-Palacios A (2016) Calidad de la luz led y desarrollo in vitro de Oncidium tigrinum y Laelia autumnalis (orchidaceae). Agrociencia 50(8):1065–1080 21. Lugo EO, Arellano OG, Herna´ndez CD (2017) Automatizacio´n de un sistema de inmersio´n temporal con base en plataformas abiertas de hardware y software. Terra Latinoam 35(3):269–277 22. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473– 497 23. Cuenca B, San-Jose´ MC, Martı´nez MT, Ballester A, Vieitez AM (1999) Somatic embryogenesis from stem and leaf explant of Quercus robur L. Plant Cell Rep 18:538–543

Part V Reviews of the Use of Temporary Immersion Systems in Micropropagation

Chapter 20 Temporary Immersion System for Biomass Production of Salvia spp.: A Mini-Review Yudith Garcı´a-Ramı´rez Abstract Salvia is a very valuable medicinal plant for the pharmaceutical industry. Tissue culture techniques can be used to increase the number of plants in a shorter time. Although protocols for in vitro propagation of more than 15 plant species have been developed, they are not yet efficient enough to increase mass propagation of plants. Therefore, the use of temporary immersion systems is necessary to increase the morphological quality of plants as well as their biomass in several Salvia species. In this chapter, progress in in vitro propagation in several Salvia species using liquid medium and automation is described. Key words Medicinal plant, Organogenesis, Temporary immersion systems, Salvia

1

Pharmaceutical Use and Conventional Propagation The genus Salvia includes nearly 900 species distributed in various regions, such as South and North America, South Africa, Southeast Asia, and Central America. Chia is also cultivated in areas such as Bolivia, Colombia, Peru, Argentina, and Europe [1]. These medicinal and aromatic plants contain active ingredients with great pharmaceutical potential. Their leaves are rich in essential oils and secondary metabolites such as phenols and terpenoids. The aerial parts are mainly rich in polyphenolic derivatives, such as phenolic acids, phenylethanoids, and flavonoids, with rosmarinic acid, verbascoside, and glycosides of apigenin and luteolin dominating [2]. Polyphenolic acids and diterpene derivatives predominate in the roots of the species, with small amounts of flavonoids and phenylethanoids also present [3]. Therefore, in vitro culture of Salvia spp. would be a viable alternative to increase the production of bioactive secondary metabolites on a large scale. These chemical compounds are believed to have broad antioxidant, antiviral, antithrombotic, antihypertensive, anticancer, anti-inflammatory, and antitumor effects.

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_20, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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The natural regeneration of Salvia species is by seeds. However, this method is not very practical and prevents large-scale production of plants due to low germination rate. Instead, propagation by stem cuttings has proven to be a suitable method for commercial production. However, success depends on the time of year and weather conditions. Thus, in vitro propagation of plants would solve this limitation by increasing the number of plants with constant biosubstances regardless of the season [4, 5].

2

Somatic Embryogenesis Somatic embryogenesis is the process by which we can produce embryos that have become complete plants. These somatic embryos (the process of which usually takes place in three steps: induction, maturation, and germination) can be induced directly on explants or through callus, depending on media optimization, growth regulators, and other factors. These somatic embryos are required for in vitro propagation, preservation, secondary metabolite production, and genetic transformation [6]. Somatic embryogenesis has been described in several species of medicinal plants [7]. However, there are few studies that have thoroughly investigated the success of this technique in Salvia species. In this sense, Modarres et al. [8] successfully established callus cultures and cell suspensions of Salvia leriifolia Benth (S. leriifolia) for the biotechnological production of phenolic acids. Moreover, they suggested that the application of growth regulators and sucrose could lead to the commercial production of valuable secondary metabolites, such as phenolic acids. They reported that further commercially oriented studies are needed to investigate the potential of somatic embryogenesis in Salvia plant species for enhanced phenolic acid production through stimulation, precursor feeding, and metabolic engineering, which has potential value for secondary metabolite research and development.

3

In Vitro Propagation by Organogenesis of Salvia spp. In vitro propagation by organogenesis of Salvia spp. began with the aim of developing protocols to increase biomass and production of chemical bioactives of pharmaceutical interest in plants [9]. The main advantages of in vitro culture over traditional plant propagation for secondary metabolite production are the ability to control environmental conditions and significantly increase the yield of specific metabolites [10]. In vitro culture techniques offer the possibility of obtaining new molecules that are not produced by plants in their natural habitat [11]. In addition, costs can be reduced and productivity increased; there are defined production

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systems and the possibility of establishing strict quality control systems for the product. Therefore, to achieve optimization of large-scale cultivation, the use of temporary immersion systems is necessary to increase the biomass of the Plants [12].

4

Use of Semisolid Media in In Vitro Propagation of Salvia spp. To date, most of the described in vitro propagation protocols for the cultivation of Salvia are based on the use of semisolid culture media. The main disadvantage of this cultivation system is the high labor input, which entails high labor costs, low multiplication coefficients compared to other regeneration systems, and the low possibility of automating the production process. In addition, the use of gelling agents contributes significantly to higher costs and limits the possibility of automation in commercial propagation [13]. Different morphological responses were observed in several Salvia species when different semisolid media were used. However, high concentrations of Cytokinin can also cause hyperhydricity in plants (Table 1). Mohamed et al. [22] succeeded in establishing an in vitro propagation protocol on organogenesis in Salvia officinalis L. (S. officinalis) in a semisolid culture medium. These authors used shoot segments and different concentrations of growth regulators as explants during in vitro propagation of the shoots. However, they observed the presence of callus at the base of the explant in all treatments studied. They also reported the presence of plants with low morphological quality. Eris¸en et al. [11] investigated the effect of different concentrations of meta-topolin on in vitro propagation of Salvia sclarea L. (S. sclarea) in a semisolid nutrient medium from seeds. These authors achieved shoot multiplication in this species. However, they observed a lower number of shoots per explant, which could affect the massive multiplication of this species for the production of secondary metabolites of pharmaceutical interest. Heydari and Chamani [15] developed a high-frequency protocol for in vitro regeneration from explants of leaves of Salvia nemorosa L. (S. nemorosa) in a semisolid culture medium. They reported a high number of shoots and leaves per explant during in vitro propagation. Gokdogan and Burun [23] reported that most of the research studies conducted on different Salvia species have focused on plant regeneration by organogenesis to produce secondary metabolites and synthetic seeds. However, these efforts have not been successful. Papafotiou et al. [5] developed a protocol for in vitro propagation in five Salvia species, such as the following: Salvia fruticose Mill. (S. fruticose), S. officinalis, Salvia ringens Sibth. & Sm. (S. ringens), Salvia tomentosa Mill. (S. tomentosa), and Salvia pomifera spp. pomifera (S. pomifera) from shoot segments in semisolid culture medium. However, they observed the

Seeds

Shoot tip and single node explants

Nodal stem segments

Stem segments

Salvia bulleyana L.

Salvia spp.

Salvia miltiorrhiza Bunge

Salvia splendens Sellow ex Schult., 1822

Semisolid medium

Semisolid medium

Semisolid medium

Semisolid medium

Semisolid medium

Fresh aerial Salvia parts and corrugata Vahl. roots

Seeds

RITA (TIS)

RITA (TIS) Plantform (TIS)

Seeds

Salvia bulleyana L.

Semisolid medium

Salvia apiana Jeps.

Seeds

Salvia nemorosa L.

Semisolid medium

RITA (TIS)

Seeds

Salvia runcinata L. f.

Culture systems

Salvia Young leaves tomentosa Mill.

Explant

Species

High presence of hyperhydricity

High concentration of cytokinins were also shown to induce hyperhydric shoot proliferation

Adenine sulfate was used for proliferation without the presence of hyperhydric plants

Transgenic roots exhibited a faster growth

Observation

[5]

[16]

[12]

[15]

[14]

References

High production of hairy roots and bioactive phenolic content

High production of biomass in TIS type RITA

High production of hairy roots

High production of plant

[21]

[20]

[19]

[18]

Highs agronomic traits, including the size Highs bioactive compounds was determined [17] of stomata, leaflet, pollen, and seed as well such as salvianolic acid B, tanshinone I, as shoot length, root diameter, number of tanshinone IIA, dihydrotanshinone I, and leaves, and fresh weight of plant cryptotanshinone, as well as total tanshinones

High shoot multiplication rate

High biomass and bioactive phenolic acid content

High shoot multiplication rate. Organogenic shoot lines revealed high level of genetic stability

High shoot multiplication rate. The molecular analysis revealed that direct organogenesis produced true-to-type plantlets

High root production

Result

Table 1 In vitro propagation of Salvia spp. in temporary immersion system (TIS) 220

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presence of hyperhydricity in the shoots and a lower number of shoots and leaves per explant. The authors pointed out the need to deepen the solution of these problems to facilitate their sustainable use in the pharmaceutical industry and floriculture. Therefore, it is necessary to apply other strategies to increase biomass for secondary metabolite production. In other species, the solution has been found through the use of liquid culture media, which allow a reduction in production costs and facilitate the automation of the in vitro propagation process [13].

5

Use of Liquid Media in the In Vitro Propagation of Salvia spp.

5.1 Temporary Immersion Systems for In Vitro Propagation of Salvia spp.

The production of plant biomass obtained in large-scale temporary immersion systems is an alternative and valuable method for the production of substances of great pharmaceutical interest with the possibility of controlling production according to product demand [24]. The reasons for its limited commercial success lie in the challenges associated with large-scale production aspects. The system allows intermittent and brief contact of the liquid nutrient medium with the plant material and a reduction in production costs compared to conventional forms of micropropagation [25]. The renewal of the atmosphere and the nutrient medium reduces the relative humidity and increases the assimilation of water and nutrients. On the other hand, immersion covers the plant explants with a thin film of liquid nutrient medium that prevents drying without hindering the diffusion of gases. It is also easy to handle and use [13]. Several investigations are being conducted with the aim of studying the main culture parameters in temporary immersion systems that allow better physiological response and higher biomass content [24]. The duration and frequency of immersion are important for both assimilation of nutrients and control of hyperhidicity [26]. In addition, they allow control of the internal atmosphere of the culture vessel [27]. Forced aeration, culture media composition and volume, and flask volume are some of the variables that can be controlled to prevent hyperhydration. Forced aeration is one aspect that provides benefits from the perspective of plant morphological quality [28].

5.2 Types of TIS, Used for In Vitro Propagation of Salvia spp.

Different types of TIS have been used for propagation of Salvia spp. The most commonly used models are RITA® and Plantform®. The RITA® system consists of an upper chamber where explants are placed and a lower chamber containing the liquid culture medium. These are interconnected such that the medium is forced into the upper chamber when positive pressure is applied and into the lower chamber when the positive pressure is released. Consequently, the cultures in the upper chamber are temporarily flooded when the

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upper chamber is flooded with the liquid medium, and the frequency and duration of flooding can be regulated. However, this system has disadvantages such as the lack of forced aeration, the absence of CO2 enrichment, and the impossibility to renew the culture medium [13]. In the Plantform™ system, explants are placed separately from the liquid medium, either in a different compartment or in a section of the same flask. The explants can be placed directly on the inner surface of the flask or using different support materials (meshes, glass beads, rock wool cubes, polyurethane foam, etc.). 5.3 Utilization of TIS in Salvia spp. for Biomass and Bioactive Compound Production

The production of secondary metabolites in large-scale bioreactors is an alternative and valuable method for the production of substances of great interest with the possibility of controlling production according to the demand for the product. The reasons for the low commercial success are the challenges associated with aspects of large-scale production [29]. Several scientific papers show that organ culture, although a better source for obtaining secondary metabolites, presents serious problems (physical properties of the organs) when scaled up in costly conventional bioreactors. The scientific literature points out that organ culture generally has low sensitivity to stress due to mechanical damage compared to cell suspension culture [13]. It then points out the changes that occur in the in vitro environment when scaling shaken cultures in bioreactors and that productivity generally decreases. This has led to the development of numerous bioreactor modifications with the goal of implementing the processes on a commercial scale [25, 27]. It was found that biomass production differed when different types of TIS were used in Salvia species (Table 1). Kentsop et al. [21] described that cultivation of hairy roots of Salvia corrugata Vahl. (S. corrugata) in RITA® submerged systems. These authors found a significant increase in biomass in liquid medium without hormones using the temporal immersion system. GrzegorczykKarolak et al. [12] studied the optimization of large-scale cultivation of sage shoots in Salvia viridis L. (S. viridis) in a plantform bioreactor. These authors demonstrated for the first time that effective cultivation of S. viridis shoots in a plantform bioreactor using a temporary immersion system is possible. They showed that the culture duration and technique (batch or fed-batch), as well as the frequency of immersion in the liquid medium, affect biomass, multiplication rate, and secondary metabolite production. These figures demonstrate the potential of such cultivation methods, both in industry to produce medically important compounds and in research to obtain large amounts of valuable biomass for future research. Krol et al. [20] developed a protocol to establish an in vitro system of Salvia apiana Jeps. (S. apiana). These authors reported that the biomass of S. apiana shoots is affected by the type of TIS.

Temporary Immersion System for Biomass Production of Salvia spp.: A Mini-Review

223

They found that the fastest biomass growth was recorded for the RITA® immersion system compared to Plantform®.

6

Conclusions and Perspectives From TIS in Salvia spp. In this chapter, progress in vitro propagation in Salvia spp. was described. However, there is still no efficient protocol for mass propagation of these species to increase in vitro propagation and pharmacologically interesting compounds. It is important to emphasize that the results presented in this chapter demonstrate the importance of developing future strategies to standardize in vitro propagation by organogenesis and somatic embryogenesis, as well as the use of new TIS, which allow to increase the biomass in plants. Therefore, it is necessary to intensify the studies on the different types of explants to be used for each species. Likewise, it is necessary to study the effect of new growth regulators on the morphology of shoots obtained in vitro. It is also necessary to standardize the parameters that influence the efficacy of the cultivated shoots on TIS, such as the following: frequency and duration of immersion, density of inoculum, volume of culture medium, and forced aeration. Finally, it is necessary to deepen the morphological, physiological, anatomical, and biochemical response of the plants obtained in TIS and their response during ex vitro acclimatization in the culture house.

References 1. Nyingi Wambua J, Mburu M (2021) Chia (Salvia hispanica L.) seeds phytochemicals, bioactive compounds, and applications: a review. Eur J Agric Food Sci 3:1–12. https://doi.org/10. 24018/ejfood.2021.3.6.381 2. Grzegorczyk-Karolak I, Kiss AK (2018) Determination of the phenolic profile and antioxidant properties of Salvia viridis L. shoots: a comparison of aqueous and hydroethanolic extracts. Molecules 23:1468. https://doi. org/10.3390/molecules23061468 3. Zengin G, Mahomoodally F, Picot-Allain C, ˝ J, Czia´ky Z, Cvetanovic´ A, Diuzheva A, Jeko Aktumsek A, Zekovic´ Z, Rengasamy KRR (2019) Metabolomic profile of Salvia viridis L. root extracts using HPLC–MS/MS technique and their pharmacological properties: a comparative study. Ind Crop Prod 131:266– 280. https://doi.org/10.1016/j.indcrop. 2019.01.060 4. Zayova E, Nikolova M, Dimitrova L, Petrova M (2016) Comparative study of in vitro, ex

vitro and in vivo propagated Salvia hispanica (Chia) plants: morphometric analysis and antioxidant activity. AgroLife Sci J 5:166–174 5. Papafotiou M, Vlachou G, Martini AN (2023) Investigation of the effects of the explant type and different plant growth regulators on micropropagation of five Mediterranean Salvia spp. native to Greece. Horticulturae 9:96. h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / horticulturae9010096 6. Salau¨n C, Lepiniec L, Dubreucq B (2021) Genetic and molecular control of somatic embryogenesis. Plan Theory 10:1467. https://doi.org/10.3390/plants10071467 7. Simonovic´ ADM, Trifunovic´-Momcˇilov M, Filipovic´ BK, Markovic´ MP, Bogdanovic´ MD, Subotic´ AR (2020) Somatic embryogenesis in Centaurium erythraea Rafn—current status and perspectives: a review. Plan Theory 10:70. https://doi.org/10.3390/plants10010070 8. Modarres M, Esmaeilzadeh Bahabadi S, Taghavizadeh Yazdi ME (2018) Enhanced

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production of phenolic acids in cell suspension culture of Salvia leriifolia Benth. using growth regulators and sucrose. Cytotechnology 70: 741–750. https://doi.org/10.1007/s10616017-0178-0 9. Grzegorczyk-Karolak I, Hnatuszko-Konka K, Krzemin´ska M, Olszewska MA, Owczarek A (2021) Cytokinin-based tissue cultures for stable medicinal plant production: regeneration and phytochemical profiling of Salvia bulleyana shoots. Biomol Ther 11:1513. https:// doi.org/10.3390/biom11101513 10. Chandran H, Meena M, Barupal T, Sharma K (2020) Plant tissue culture as a perpetual source for production of industrially important bioactive compounds. Biotechnol Rep 26: e00450. https://doi.org/10.1016/j.btre. 2020.e00450 11. Eris¸en S, Kurt-Gu¨r G, Servi H (2020) In vitro propagation of Salvia sclarea L. by metaTopolin, and assessment of genetic stability and secondary metabolite profiling of micropropagated plants. Ind Crop Prod 157: 112892. https://doi.org/10.1016/j.indcrop. 2020.112892 12. Grzegorczyk-Karolak I, Staniewska P, Lebelt L, Piotrowska DG (2021) Optimization of cultivation conditions of Salvia viridis L. shoots in the Plantform bioreactor to increase polyphenol production. Plant Cell Tissue Organ Cult 149:269–280. https://doi.org/10.1007/ s11240-021-02168-2 13. De Carlo A, Tarraf W, Lambardi M, Benelli C (2021) Temporary immersion system for production of biomass and bioactive compounds from medicinal plants. Agronomy 11:2414. h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / agronomy11122414 14. Figlan S, Makunga NP (2017) Genetic transformation of the medicinal plant Salvia runcinata L. f. using Agrobacterium rhizogenes. S Afr J Bot 112:193–202. https://doi.org/10. 1016/j.sajb.2017.05.029 15. Heydari HR, Chamani E (2022) Highfrequency plant regeneration and genetic fidelity assessment of regenerants by molecular and biochemical markers in Woodland Sage (Salvia nemorosa L.). Ind Crop Prod 187:115404. https://doi.org/10.1016/j.indcrop.2022. 115404 16. Grzegorczyk-Karolak I, Krzemin´ska M, Kiss AK, Owczarek-Januszkiewicz A, Olszewska MA (2023) Role of phytohormones in biomass and polyphenol accumulation in Salvia bulleyana in vitro culture. Biomol Ther 13:227. https://doi.org/10.3390/biom13020227 17. Chen EG, Tsai KL, Chung HH, Chen JT (2018) Chromosome doubling-enhanced

biomass and dihydrotanshinone I production in Salvia miltiorrhiza, a traditional Chinese medicinal plant. Molecules 23:3106. https:// doi.org/10.3390/molecules23123106 18. Sharma S, Shahzad A, Kumar J, Anis M (2014) In vitro propagation and syn seed production of scarlet salvia (Salvia splendens). Rend Lincei 25:359–368. https://doi.org/10.1007/ s12210-014-0308-y 19. Marchev A, Georgiev V, Ivanov I, Badjakov I, Pavlov A (2011) Two-phase temporary immersion system for Agrobacterium rhizogenes genetic transformation of sage (Salvia tomentosa Mill.). Biotechnol Lett 33:1873–1878. https://doi.org/10.1007/s10529-0110625-5 20. Krol A, Kokotkiewicz A, Gorniak M, Naczk AM, Zabiegala B, Gebalski J, Luczkiewicz M (2023) Evaluation of the yield, chemical composition and biological properties of essential oil from bioreactor-grown cultures of Salvia apiana microshoots. Sci Rep 13:7141. https://doi.org/10.1038/s41598-02333950-1 21. Kentsop RAD, Iobbi V, Donadio G, Ruffoni B, De Tommasi N, Bisio A (2021) Abietane diterpenoids from the hairy roots of Salvia corrugata. Molecules 26(17):5144. https://doi. org/10.3390/molecules26175144 22. Mohamed MAH, Aly MK, Ahmed ET, Abd El-latif SAH (2019) Effect of plant growth regulators on organogenesis of Salvia officinalis L. plants. Minia J Agric Res Dev 39(3): 401–414 23. Gokdogan EY, Burun B (2022) The studies on seed germination and in vitro cultures of Salvia L. species from Turkish Flora. Nat Prod Biotechnol 2:60–73 24. Zhang B, Niu Z, Li C, Hou Z, Xue Q, Liu W, Ding X (2022) Improving large-scale biomass and total alkaloid production of Dendrobium nobile Lindl. using a temporary immersion bioreactor system and MeJA elicitation. Plant Methods 18:10. https://doi.org/10.1186/ s13007-022-00843-9 25. Wongsa T, Kongbangkerd A, Kunakhonnuruk B (2023) Optimal growth and biomass of Centella asiatica using a twin-bottle temporary immersion bioreactor. Horticulture 9:638. h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / horticulturae9060638 26. San Jose´ MC, Bla´zquez N, Cernadas MJ, Janeiro LV, Cuenca B, Sa´nchez C, Vidal N (2020) Temporary immersion systems to improve alder micropropagation. Plant Cell Tissue Organ Cult 143:265–275. https://doi. org/10.1007/s11240-020-01937-9

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Chapter 21 Orchid Micropropagation Using Temporary Immersion Systems: A Review Obdulia Baltazar-Bernal, Evelia Guadalupe Mora-Gonza´lez, and Marco A. Ramı´rez-Mosqueda Abstract Temporary immersion systems (TIS) have been used for orchid micropropagation. The main advantage of TIS use for micropropagation is that the explant is periodically immersed in nutrient media, and then, the nutrient solution is drained, which allows the explant tissue to stay in air. The current review resumes the application of TIS in orchid propagation. Fifty-three papers are discussed considering: explant, culture media, TIS bioreactor type, frequency and immersion time, and the TIS effects in acclimatization phase. Key words Orchidaceae, PLBs, Immersion frequency, Wild orchid, Large-scale production

1

Introduction The Orchidaceae family is the second largest worldwide with nearly 30,000 species [1]. Orchids have a very important ecological role as pollinators feed [2]. Nevertheless, orchid face intense population reduction in their natural habitats mainly due to looting and unregulated commercialization, deforestation, and accelerated habitat destruction [3]. The decline of orchid populations justifies the entire family being in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora [4]. There are more than 100,000 orchid hybrids [5], which have a big economic value due to their commercialization as ornamental plants [6]; hybrids of commercial genera, such as Phalaenopsis, Dendrobium, Epidendrum, Cymbidium, Cattleya, Oncidium, Vanda, and Paphiopedilum, are induced to blossom independently of the time of year, for being sold as cut and potted flowers. Besides, some species of Anoectochilus [7], Dendrobium [8], and Bletilla [9] genera are important for medical purposes, and some Vanilla species are used for flavor industry [10].

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_21, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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The semisolid (SS) culture system has been used for orchid propagation, seed germination, protocorm and protocorm-like body (PLB) growth, tissue differentiation, and multiplication. This culture system presents some limitations as low multiplication rate and scarcity of photosynthesis. These problems are associated with a low survival percentage at the acclimatization phase. SS culture system has a high production cost because it requires highly skilled hand labor and agar. In this sense, the use of technologies for micropropagation such as temporary immersion systems (TIS) is important since it reduces production costs by 40% [11, 12]. TIS are containers designed for large-scale semiautomatic production of plants in a liquid medium, which has been used in propagation of commercial orchids [13–15]. Therefore, this chapter review focuses on the application of TIS for orchid propagation. Fifty-three papers are discussed; they mainly focus on the effects of the culture medium, TIS bioreactor type, immersion time and frequency, and the advantages of using plants cultivated on TIS for the acclimatization phase (Table 1).

2

Temporary Immersion Systems Temporary immersion systems (TIS) are defined as self-contained sterile devices used for large-scale micropropagation, in which the explant is periodically immersed in culture media, and then the nutrient solution is drained, allowing the explant tissue to stay in the air [12]. Temporary immersion (TI) consists of submerging the explant in a liquid medium for short periods of time, which varies from a few minutes to a few hours. The immersion is enough for the plant to absorb its nutrients, growth regulators, and other compounds [12]. One of the main advantages of using TIS is the improvement of explant oxygenation, which occurs during the period without immersion; this reduces the incidence of asphyxiation and hyperhydricity and increases multiplication rates [12, 51, 52]. Therefore, they have been proposed for large-scale micropropagation of economically important species such as orchids [9]. TIS are used for production of secondary metabolites, expression of complex proteins, and micropropagation of plant material on a large scale [52]. It favors physiological processes, such as photosynthesis, respiration, chlorophyll synthesis, stomatal functioning, and the accumulation of dry matter in seedlings; as result, the seedlings are more vigorous for the acclimatization process [32]. The design of the TIS influences the physiology and development of in vitro plants; the different TIS models used in orchid propagation are as follows: Ebb and Flood bioreactor [42], RITA® [53], TIB® [54], MATIS® [55], and SETIS™ bioreactors [56].

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Table 1 Orchid species cultured in TIS with different bioreactors and immersion times Species or hybrids

Bioreactor

Anoectochilus formosanus

Explant

Immersion Main results

Reference

BTBB, Ebb Shoot and Flood

Not Plantlet growth was better in a reported continuous-immersion bioreactor with an air supply than in Ebb and Flood

[16]

Anoectochilus formosanus

Ebb and Flood

Shoot

15 min/ 1h

[17]

Anoectochilus formosanus

Ebb and Flood

Plantlets

Not Ebb and Flood and SS cultures [20] reported resulted in fewer roots than raft and CIS

Anoectochilus formosanus

Ebb and Flood

Shoot

30 min/ 6h

Bletilla striata

TIBS

Seedling

[9] 3 min/2 h. TIBS showed an improved micropropagation and better growth than SS

Bletilla striata

TIBS

Seedling

1, 3, or 9 min/ 2, 4 or 6h

High-quality seedlings were obtained by the use of TIBS

[21]

Brassavola nodosa (L.) Lindl.

SETIS™

Shoot

2 min/2, An immersion of 2 min/2 h produces the highest 4, or 8 h multiplication rates, 4.6 shoots per explant using SETIS™ compared with 2.8 shoots per explant in SS

[22]

Cattleya forbesii Lindl

RITA®

PLB

1 min/4 or 8 h

[23]

Cattleya tigrina

RITA®

Plantlets

Not RITA® was found more suitable [24] reported for PLB multiplication compared to CI

Cattleya walkeriana

TIB®

Seedling

3 min/ 90 min

The shoot length was more efficient in SS. However, the growth of plantlets was also higher in CIS with net

Shoot proliferation and biomass [7] accumulation were more efficient when culturing was performed under CIS

The highest number of PLBs per explant was 21 times higher than those from SS, and the highest number of shoots per explant was 95 times higher than those grown on SS

TIB® is the best [25] micropropagation system for the C. walkeriana (continued)

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Table 1 (continued) Species or hybrids

Bioreactor

Explant

Immersion Main results

Reference

Cymbidium sinense

BTBB

Rhizome

60 min/ 60 min

[26]

Dendrobium candidum

Ebb and Flood

Protocorm Not An inoculums density of 50 g/L [8] reported was appropriate for production of biomass and bioactive compounds

Dendrobium nobile Lindl.

TIBS

Protocorm 5 min/2, 4, 6 or 8h

The 5 min/6 h was the optimal [27] in culturing plantlets

Dendrobium nobile Lindl.

TIBS

Plantlets

5 min/2, 4, 6, or 8h

An immersion frequency 5 min/6 h in TIBS+MeJA is suitable for large-scale production of alkaloid in seedlings

Doritaenopsis

API

PLB

5 min/4 The API bioreactor is superior [29] or 24 h; to SS or liquid culture on PLB 15 min/ proliferation rates 4h

Epidendrum fulgens

Plantform™ Plantlets

3 min/3 h The plantlets grown in TIS presented a higher number of leaves, roots, shoots, and fresh weight than plantlets grown SS. The leaves developed thicker cuticles

[]58

Epipactis flava Seidenf

TIB®

Bud clusters

5 min/4 h TIB® was found to be the suitable to produce shoots and shoot bud formation than CIS and SS

[30]

Guarianthe skinneri

TIB®

Shoot

2 min/4 h The best growth was in TIB® at 2 min/4 h

[31]

Seedling

3 min/4, 6, 8, or 12 h

[32]

Lycaste aromatica Ebb and (Graham) Flood Lindl.

Bioreactors are to produce quality propagules and plantlets for the industrial

Seedlings in SS developed higher number of shoots (1.6 shoots) and root length (2 cm). Ebb and Flood showed the highest photosynthesis rate (0.74 μmol CO2 g-1 s-1) and stomatal function

[28]

(continued)

Orchid Micropropagation in TIS

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Table 1 (continued) Species or hybrids

Bioreactor

Explant

Immersion Main results

Reference

Mokara Leuen Berger Gold

Plantima

Shoot or cluster

1 min/1 or 2 h

An immersion of 1 min/2 h produce optimum shoots proliferation

[33]

Myrmecophila grandiflora Lindl.

Ebb and Flood

Seedling

2 min/4, 8, or 12 h

The best development occurred [3] with an immersion of 2 min/ 4 h, with 10 mL medium per explant

PLBs

60 min/ 60 min

Liquid bioreactor and Ebb and [34] Flood were better than SS and liquid agitate

Seedling

30 min/ 2h

Explants grown in BIB® showed [35] higher shoot number per explant (6.56), more fresh mass increment (5.7 g), more dry mass (0.59 g) and aerial part height (1.87 cm) compared to explants cultured at RITA and liquid medium

Paphiopedilum RITA® rothschildianum

Callus

5 min/ Callus proliferation in RITA® 125 min showed threefold increase in fresh weight than that on SS

[36]

Paphiopedilum RITA® rothschildianum

Callus

5 min/ Callus proliferation in RITA 125 min showed threefold increase in fresh weight than SS

[37]

Oncidium “Sugar Ebb and Sweet” Flood Oncidium leucochilum

RITA®; BIB®

Phalaenopsis

RITA®

Protocorm 1 min/4 or 8 h

The best proliferating was reached in a 15 g/L of sucrose with 1 min/4 h, resulting in 8.2 adventitious protocorms per protocorm per month

[38]

Phalaenopsis

TIB

Shoot

10 min/ 3h

TIB® is an efficient bioreactor for multiplication of Phalaenopsis, due to the uptake of nutrients and hormones over the whole plant surface

[39]

Phalaenopsis

BTBB

PLB

5 min/2 h BTBB with activated charcoal filter attached was most suitable to multiplication of PLBs

[40]

(continued)

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Table 1 (continued) Species or hybrids

Bioreactor

Explant

Immersion Main results

Reference

Phalaenopsishybrids

TIB

Shoot

Not TIB® enhances the biomass [41] reported production and multiplication rates compared to SS

Potinara sp.

APCS

Shoot

1 or Orchid tips in APCA produced [42] 2 min/ four times more tissue than in 1, 4, 12, SS and 24 h

RITA® Stanhopea hernandezii (Kunth) Schltr.

Protocorm 1 min/6 h Chitosan and RITA® showed benefits on orchids propagation for protocorms and seedling development

[43]

Stanhopea tigrina Ebb and Flood

Protocorm Not Ebb and Flood bioreactor has reported better growth than SS

[44]

RITA®

Shoot

5 or Shoots culture in RITA® is 10 min/ better than the thin-layer 12 h system

Vanilla planifolia RITA®

Shoot

2 min/4, 8, or 12 h

Vanilla planifolia Ebb and Flood Jacks. ex Andrews

Shoot

Not Ebb and Flood reached a higher [19] reported rate of multiplication (12 shoots/explant) in 30 days of cultivation, than in SS (7 shoots/explant)

Vanilla planifolia TIB® Jacks. ex Andrews

Shoot

1 min/4 h TIB® produced more shoot growth than SS

Vanilla planifolia TIB®, Ebb and Jacks. ex Flood, Andrews RITA®

Nodal 2 min/4 h TIB® generated vigorous plants [47] segment than BIG and RITA®

Vanilla planifolia TIB® Jacks. ex Andrews

Nodal 2 min/8 h Plants showed 100% segment monomorphism and morphological stability over all four subcultures in TIB®

Vanda tricolor

[45]

RITA® produced the maximum [10] multiplication rate (14.27 shoots per explant) whit 2 min/4 h and 25 mL per explant

[46]

[48]

(continued)

Orchid Micropropagation in TIS

233

Table 1 (continued) Species or hybrids

Bioreactor

Explant

Immersion Main results

Reference

Vanilla planifolia SETIS™ Jacks. ex Andrews

Shoot

2 min/4 h Shoot per explant in SETIS™ [49] was 11.41, followed by PI with 6.53, and SS with 3.76; the driest weights were obtained in SETIS™, followed by PI and SS. The most percent survival during acclimatization was in SETIS™

Vanilla planifolia RITA® Jacks. ex Andrews

Shoot

2 min/6 h RITA® and 50 mg/L of Argovit [50] in the culture media had an antimicrobial and hormetic effect on the shoots

The number of publications that addresses issues related to orchid micropropagation using TIS is 41; the publications on this topic began in 1985. Regarding to the countries, Fig. 1 shows that Mexico was the country with the highest percentage of contributions (28%); China showed the second highest percentage of contributions (17%), followed by Brazil, South Korea, Malaysia, Taiwan, and the United States. Most publications are in the research phase, which forces researchers to collaborate with the private initiative for using the advantages of the TIS to produce on a large scale [21]. The use of bioreactors for orchid micropropagation was first reported in 1985 for Anoectochilus formosanus [42]. However, the first published paper using Ebb and Flood bioreactor was in 2003 [18] and RITA® in 2005 [38]. Papers published with these bioreactors have the highest percentage of contributions (25%), followed by TIB®, BTBB, SETIS™, and Plantima (Fig. 2).

3 3.1

Orchid Micropropagation Using TIS Explants

Shoot explants for TIS culture can be obtained from seedlings or adult plants; the selection of a competent explant is crucial for the success of TIS micropropagation. Shoot explants, from either somatic or zygotic origin, were the most common source of explant out of the 41 revisited papers (Figs. 3 and 4). Formation of vitrified bodies in liquid culture is a common problem regarding TIS culture [57]. However, as TIS environment is often characterized by high relative humidity and poor gaseous exchange, hyperhydricity

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Fig. 1 Percentage of published papers by country (out of 41 papers); review papers are excluded

Fig. 2 TIS bioreactors application percentage in the reported papers for orchid propagation

remains a problem regardless of the explant source and can be managed by determining the appropriate immersion time and frequency [23, 27, 36]. The use of explants taken from in vitro germination is limited to capsule (Fig. 5a) availability and germination rate; however, since the products are young explants with greater regeneration potential than mature tissues from adult plants, the obtention of seedlings with leaves and roots present a significant advantage (Fig. 5b, c). For clonal propagation of an already established cultivar, explants, such as axillary buds, nodal segments, and stems, can be obtained from adult plants [10, 19, 49, 50]. Leaves and roots can be used to obtain protocorm-like bodies (PLBs); this way avoids damaging the mother plant. Nevertheless, flower stalks and stems have also been used for PLB propagation. PLB formation has been induced using BA and NAA at concentrations (1.2, 2, and 15 mg/

Orchid Micropropagation in TIS

235

Fig. 3 The application of TIS for orchid micropropagation

L for BA and 0.3, 1, and 1.2 mg/L for NAA). PLB induction in Knudson C medium supplemented with 2% (w/v) sucrose and 1.2 mg/L BA and NAA was reported [23]. Contamination risk must also be considered when deciding the source of the explant. Greenhouse or field collected explants must be carefully decontaminated since fungi or microbial pathogens may not be visible only after several weeks of in vitro culture. Surface sterilization can be carried out using fungicides or bactericides and tap water. Further disinfection may be performed with 70% ethanol (v/v), surfactant solution (Tween 20), sodium hypochlorite solution, and diluted ascorbic acid [19]. Thus, in vitro explants are used as a strategy to avoid field bacteria or fungi. 3.2

Culture Medium

The use of liquid medium in TIS propagation for commercial production reduces costs, since gelling agent is no longer required. Liquid culture in TIS allows full medium-explant contact, improving nutrient absorption. Murashige-Skoog (MS) is the most common medium used in TIS, although nutrient requirements may vary depending on the orchid species. Hyponex medium has proven effective for production of Anoectochilus formosanus [7, 19, 20], Cymbidium sinense [26], and Phalaenopsis spp.

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Fig. 4 Orchid explants used by each type of TIS

Fig. 5 Micropropagation of Stanhopea tigrina: (a) orchid seed, (b) shoot and root development of Encyclia cordigera in Ebb and Flow, (c) shoot and root development in RITA®

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[40]. Meanwhile, Knudson C (KC) medium and Vacin and Went (VW) medium have been used for Cattleya forbesii and Guarianthe skinneri, respectively [23, 31]. Regardless the origin of the explant used for TIS propagation, plantlet differentiation is affected controlled by the addition of plant growth regulators (PGRs) or sucrose concentration. Zhang [21] reported that 40 g/L sucrose favored pseudobulbs developed in Bletilla striata. Cattleya forbesii [23] reported shoot regeneration from PLB TIS culture by using KC medium supplemented with 2% w/v sucrose, 15% (v/v) coconut water (CW), and 2.5 mg/ L BA, while medium containing 1 mg/L BA and 15% (v/v) CW and 2% (w/v) sucrose promoted PLBs from PLBs TIS culture. In addition, Gao [26] used different NAA to promote Cymbidium sinense plantlet regeneration from rhizomes’ explants. Along with medium composition, immersion time and frequency are also an important factor to consider for plantlet regeneration. 3.3 Frequency and Time Immersion

The frequency and time of immersion are critical parameters when using TIS; in the liquid phase, they are crucial for nutrient intake, growth regulators, and control of hyperhydricity and asphyxia [15, 51, 57]; in the gas (dry) phase, the explant absorbs oxygen, carbon dioxide, and ethylene [11, 51]. Immersion frequency varies from 1 to 60 min among orchid species, but 1, 2, and 5 min are more used. Immersion frequency is normally constant, which varies in the frequency time from 2, 4, and 8 h in Phalaenopsis, Cattleya forbesii, and Mokara Leuen Berger Gold [23, 33, 38]. Tirado et al. [38] mentioned the presence of hyperhydration when using an immersion frequency of 1 min every 4 h and media with lower sucrose concentration; Also, Zhang et al. [58] reported that immersion frequencies of 5 min every 6 and 8 h were beneficial to the accumulation of total alkaloid and biomass in plantlets, whereas immersion frequencies of 5 min every 2 and 4 h had the opposite effect. Higher immersion frequencies cause hyperhydricity of explants because oxygen and carbon dioxide concentrations in the system are often insufficient to make physiological processes [12]. However, hyperhydricity and asphyxia doesn’t seem to be a problem in orchids.

3.4 Medium Volume Per Explant

Medium volume per explant is a very important factor in TIS. Decreasing the medium volume per explant appears to reduce the production costs; however, increasing the number of explants developed in a container with the same medium volume possibly leads to poor aeration that slows the growing period, which also reduces the plant vigor in acclimatization phase [11]. There are differences between medium volumes per explant, even in the same type of TIS and species of orchid. The use of 100 mL of medium volume has been reported in Vanilla planifolia

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Jacks. ex Andrews in Ebb and Flood, and it produced 12 shoots per explant [19]; the addition of 25, 33.3, and 50 mL of medium volume per explant has been tested in in RITA® for V. planifolia; 25 mL produced the most shoots per explant (14.27) [10]; and 25 mL of medium volume was used in V. planifolia in TIB®, Ebb and Flood, and RITA®; however, TIB® generated more vigorous plants than Ebb and Flood and RITA® [47]. Different volumes of medium per explants have been used for V. planifolia, for example, 20 mL per explant in RITA® [50], 17.5 mL per explant in TIB® [46], and 25 mL per explant in TIB® [48]. The 50 mL in SETIS™ produced 11.41 shoots per explant [49]. Thus, 25 mL medium volume per explant for V. planifolia in TIB® could be the best combination to produce the maximum multiplication rate. Anoectochilus formosanus plantlets grew satisfactory in Ebb and Flood with a medium volume per explant of 125 mL [7]. Wu [19] reported 8.3 and 12.5 mL per explant; also, Wu [20] reported 6.25, 8.3, and 12.5 mL per explant for the same plant. However, A. formosanus grew better in a continuous immersion bioreactor with an air supply than in TIS. The same medium volume per explant (20 g/2 L) was used in Phalaenopsis to develop PLBs in temporally immersion system in Balloon-type bubble bioreactor (BTBB) with charcoal filter [40] and protocorms in (TIB®) [39]; bioreactors were efficient to multiply PLBs and protocormos compared with SS. The medium volume per explant, 10 protocorm/175 mL, was utilized in RITA® with various sugar concentration; the 15 g/L of sucrose with 1 min every 4 h produced 8.2 adventitious protocorms per protocorm per month [38]. Besides, Phalaenopsis shoots grown in TIB® enhanced the biomass production and multiplication rates compared to SS. Development of Myrmecophila grandiflora Lindl. in Ebb and Flood under medium volume per explant of 5, 10, and 20 mL was evaluated. Volumes of 10 and 20 mL medium per explant had an increase in all development variables, but they decreased with volume of 5 mL. This effect could be caused by a smaller volume of medium; there is less availability of nutrients and sugars, as they are burnt out more quickly [3]. 3.5

Acclimatization

Most of the reported papers agree that TIS plants have greater survival rate at ex vitro conditions when compared to SS culture, presenting yet another advantage over semisolid culture. TIS culture significantly reduces hyperhydricity, caused by humidity excess during in vitro culture often observed in SS and liquid phase bioreactor culture. Chlorophyll content plays an important role in plant acclimatization as reported by Yang et al. [34]. In this study, plants from TIS showed higher photosynthetic rates than plants from SS medium, ultimately resulting in higher leaf area when compared to plants from SS culture after 65 days in greenhouse conditions [34]. As for water content, Moreira et al. [25] observed

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Fig. 6 SETIS® bioreactor consists of two containers place on top of each other

Fig. 7 Plantform bioreactor, a type of TIS consisting of a single container

that water loss after 120 min in ex vitro conditions was lower in TIS versus continuous immersion system while plants culture in liquid medium had greater water loss after 120 min at the same conditions [25]. Temporary immersion system may promote tissue foliar organization, which can explain this phenomenon as well as stomata index and close stomata, since morphological differences between SS culture and TIS were observed by Ramirez-Mosqueda et al. [47] in Vanilla planifolia and Yang et al. [34] in Calathea orbifolia. Survival rates when plants are transferred from TIS to greenhouse conditions rages from 100% for G. skinneri and V. planifolia [31, 48] to 33.3% at 8 h immersion frequency in SETIS™. SETIS™ allows a massive orchid multiplication (Fig. 6) same as MATIS® (Fig. 7); both are useful on the private sector.

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Fig. 8 Acclimatization (a, b) of Stanhopea tigrine and (c, d) Encyclia cordigera

A rooting step before transfer to soil is recommended by transferring 2 cm shoots to MS medium free of PGR [48]. However, TIS culture has shown to promote rooting, avoiding this previous step [10]. Peat moss is commonly used as soil substrate along with agrolite or perlite. Keeping a high relative humidity (60–95%) has proven important, as has low light incidence. Individual or tray pots containing 3–10 cm long plants may be covered with a PVC plastic bag or sealed to maintain appropriate humidity (Fig. 8) [31, 34].

4

Conclusion TISs are ideal for regenerating axillary and adventitious shoots and their subsequent rooting, producing massive quantities of explants. In vitro propagation of wild orchids requires seed germination under semisolid medium before putting the plantlets on a TIS. The optimal medium volume per explant, frequency, and time of immersion need to be determined for each orchid species for the best results. Also, i is necessary to increase the level of carbohydrates

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and to inject adequate supplementation of essential gases (oxygen and carbon dioxide) to get more member of shoots and quality of plantlets. The use of TIS for orchid propagation is useful to reduce the production costs for commercial and conservational purposes. Further research must be done to know the optimal parameters in which orchids can thrive and multiplicate faster with the use of TIS. References 1. Chase MW, Cameron KM, Freudenstein JV, Pridgeon AM, Salazar G, Van den Berg C, Schuiteman A (2015) An updated classification of Orchidaceae. Bot J Linn Soc 177(2): 151–174. https://doi.org/10.1111/boj. 12234 2. Ackerman JD, Phillips RD, Tremblay RL, Karremans A, Reiter N, Peter CI, Bogarı´n D, Pe´rez-Escobar OA, Liu H (2023) Beyond the various contrivances by which orchids are pollinated: global patterns in orchid pollination biology. Bot J Linn Soc 20(3):1–30. https:// doi.org/10.1093/botlinnean/boac082 3. Bello-Bello JJ, Ruiz JZ, Cruz-Huerta N, Baltazar-Bernal O (2020) In vitro germination and development of the trumpetist orchid (Myrmecophila grandiflora Lindl.) using ebband-flow bioreactor. Propag Ornamental Plants 20(3):88–95 4. Fay MF (2018) Orchid conservation: how can we meet the challenges in the twenty-first century? Bot Stud 59:16 5. RHS (2023) The International Orchid Register. 20 August 2023. https://apps.rhs.org.uk/ h o r t i c u l t u r a l d a t a b a s e / o r c h i d re g i s t e r / orchidregister.asp 6. Hinsley A, De Boer HJ, Fay MF, Gale SW, Gardiner LM, Gunasekara RS, Kumar P, Masters S, Metusala D, Roberts DL, Veldman S, Wong S, Phelps J (2018) A review of the trade in orchids and its implications for conservation. Bot J Linn Soc 186(4):435–455. https://doi.org/10.1093/botlinnean/ box083 7. Yoon YJ, Murthy HN, Eun JH, Kee YP (2007) Biomass production of Anoectochilus formosanus Hayata in a bioreactor system. J Plant Biol 50:573–576. https://doi.org/10.1007/ BF03030711 8. Cui HY, Murthy HN, Moh SH, Cui YY, Lee EJ, Paek KY (2014) Production of biomass and bioactive compounds in protocorm cultures of Dendrobium candidum Wall ex Lindl. using balloon type bubble bioreactors. Ind Crop Prod 53:28–33. https://doi.org/10.1016/j. indcrop.2013.11.049

9. Zhang B, Song L, Bekele LD, Shi J, Jia Q, Zhang B, Chen J (2018a) Optimizing factors affecting development and propagation of Bletilla striata in a temporary immersion bioreactor system. Sci Hortic 232:121–126. https:// doi.org/10.1016/j.scienta.2018.01.007 10. Ramos-Castella´ A, Iglesias-Andreu LG, BelloBello J, Lee-Espinosa H (2014) Improved propagation of vanilla (Vanilla planifolia Jacks. ex Andrews) using a temporary immersion system. In Vitro Cell Dev Biol Plant 50:576–581. https://doi.org/10.1007/s11627-0149602-8 11. Mirzabe AH, Hajiahmad A, Fadavi A, Rafiee S (2022) Temporary immersion systems (TISs): a comprehensive review. J Biotechnol 357:56. https://doi.org/10.1016/j.jbiotec.2022. 08.003 12. Murthy HN, Joseph KS, Paek KY, Park SY (2023) Bioreactor systems for micropropagation of plants: present scenario and prospects. Front Plant Sci 14:1159588. https://doi.org/ 10.3389/fpls.2023.1159588 13. Khatun K, Nath UK, Rahman MS (2020) Tissue culture of Phalaenopsis: present status and prospects. J Adv Biotechnol Exp Ther 3:273– 285. https://doi.org/10.5455/jabet.2020. d135 14. Cardoso JC, Zanello CA, Chen JT (2020) An overview of orchid protocorm-like bodies: mass propagation, biotechnology, molecular aspects, and breeding. Int J Mol Sci 21(3): 9 8 5 . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / ijms21030985 15. Nongdam P, Beleski DG, Tikendra L, Dey A, Varte V, El Merzougui S, Vendrame WA (2023) Orchid micropropagation using conventional semi-solid and temporary immersion systems: a review. Plan Theory 12(5):1136. https://doi.org/10.3390/plants12051136 16. K. Y., Paek D., Chakrabarty E J., Hahn (2005) Application of bioreactor systems for large scale production of horticultural and medicinal plants Plant Cell Tissue and Organ Culture 81(3) 287-300 10.1007/s11240-004-6648-z

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17. Wu R.Z. Chakrabarty D, Hahn EJ, Paek KY (2007). Micropropagation of an endangered jewel orchid (Anoectochilus formosanus) using bioreactor system. Horticulture Environment and Biotechnology 48(6):376–380 18. Ket N, Chakrabarty D, Hahn EJ, Paek KY (2003) Micropropagation of an endangered jewel orchid (Anoectochilus formosanus) using bioreactor system (unpublished) 19. Bello-Bello JJ, Espinoso-Castillo J, IglesiasAndreu LG (2014) Establecimiento de un sistema de biorreactores para la micropropagacio´n de Vainilla (Vanilla planifolia Jacks. ex Andrews). AGROPrductividad 7(3):63–68 20. Wu RZ, Baque MA, Paek KY (2010) Establishment of a large-scale micropropagation system for Anoectochilus formosanus in bioreactors. Acta Hortic 878:167–173. https://doi.org/ 10.17660/ActaHortic.2010.878.18 21. Zhang B, Sarsaiya S, Pan X, Jin L, Xu D, Zhang B, Duns GJ, Shi J, Chen J (2018) Optimization of nutritional conditions using a temporary immersion bioreactor system for the growth of Bletilla striata pseudobulbs and accumulation of polysaccharides. Sci Hortic 240: 155–161. https://doi.org/10.1016/j.scienta. 2018.06.010 22. Vendrame WA, Xu J, Beleski DG (2023) Micropropagation of Brassavola nodosa (L.) Lindl. using SETIS™ bioreactor. Plant Cell Tissue Organ Cult 153(1):67–76. https:// doi.org/10.1007/s11240-022-02441-y ¨ , Gu¨rel A 23. Ekmekc¸igil M, Bayraktar M, Akkus¸ O (2019) High-frequency protocorm-like bodies and shoot regeneration through a combination of thin cell layer and RITA® temporary immersion bioreactor in Cattleya forbesii Lindl. Plant Cell Tissue Organ Cult 136:451–464. https:// doi.org/10.1007/s11240-018-01540-z 24. Fritsche Y, Deola F, da Silva DA, Holderbaum DF, Guerra MP (2022) Cattleya tigrina (Orchidaceae) in vitro regeneration: main factors for optimal protocorm-like body induction and multiplication, plantlet regeneration, and cytogenetic stability. S Afr J Bot 149:96–108. https://doi.org/10.1016/j.sajb.2022.05.059 25. Moreira AL, Silva ABD, Santos A, Reis COD, Landgraf PRC (2013) Cattleya walkeriana growth in different micropropagation systems. Cienc Rural 43:1804–1810. https://doi.org/ 10.1590/S0103-84782013001000012 26. Gao R, Wu SQ, Piao XC, Park SY, Lian ML (2014) Micropropagation of Cymbidium sinense using continuous and temporary airlift bioreactor systems. Acta Physiol Plant 36:117– 124. https://doi.org/10.1007/s11738-0131392-9

27. Zhang B, Niu Z, Zhou A, Zhang D, Xue Q, Liu W, Ding X (2019) Micropropagation of Dendrobium nobile Lindl. plantlets by temporary immersion bioreactor. J Biobaased Mater Bioenergy 13(3):395–400. https://doi.org/ 10.1166/jbmb.2019.1852 28. Zhang B, Niu Z, Li C, Hou Z, Xue Q, Liu W, Ding X (2022) Improving large-scale biomass and total alkaloid production of Dendrobium nobile Lindl. using a temporary immersion bioreactor system and MeJA elicitation. Plant Methods 18:10. https://doi.org/10.1186/ s13007-022-00843-9 29. Liu THA, Kuo SS, Wu RY (2002) Mass micropropagation of orchid protocorm-like bodies using air-driven periodic immersion bioreactor. Acta Hortic 578:187–191. https://doi.org/ 10.17660/ActaHortic.2002.578.22 30. Kunakhonnuruk B, Inthima P, Kongbangkerd A (2019) In vitro propagation of rheophytic orchid, Epipactis flava Seidenf.—a comparison of semi-solid, continuous immersion and temporary immersion systems. Biology 8(4):72. https://doi.org/10.3390/biology8040072 31. Leyva-Ovalle OR, Bello-Bello JJ, Murguı´a˜ ez-Pastrana R, Ramı´rez-MosGonza´lez J, Nu´n queda MA (2020) Micropropagation of Guarianthe skinneri (Bateman) Dressler et WE Higging in temporary immersion systems. 3 Biotech 10:1–8. https://doi.org/10.1007/ s13205-019-2010-3 32. Solı´s-Zanotelli FY, Baltazar-Bernal O, CruzHuerta N, Hidalgo-Contreras JV, Pe´rez-Sato JA (2022) In vitro germination and development of “Canelita” (Lycaste aromatica (Graham) Lindl.) in gravity immersion bioreactors. In Vitro Cell Dev Biol Plant 58(6):1117–1125. https://doi.org/10.1007/s11627-02210314-4 33. Minh TV (2022) Micropropagation of Mokara orchid by temporary immersion system technique. Int J Res Innov Appl Sci 7:54–58. https://doi.org/10.51584/IJRIAS.2022. 7502 34. Yang JF, Piao XC, Sun D, Lian ML (2010) Production of protocorm-like bodies with bioreactor and regeneration in vitro of Oncidium “Sugar Sweet”. Sci Hortic 125:712–717. https://doi.org/10.1016/j.scienta.2010. 05.003 35. Scheidt GN, da Silva ALL, Dronk AG, Biasi LA, Arakaki AH, Soccol CR (2009) Multiplicac¸˜ao in vitro de Oncidium leucochilum (Orchidaceae) em diferentes sistemas de cultivo. Biociencias 17(1):82–85 36. Masnoddin M, Repin R, Abd Z (2016) Micropropagation of an endangered Borneo Orchid, Paphiopedilum rothschildianum Callus using

Orchid Micropropagation in TIS temporary immersion bioreactor system. Thai Agric Res J 34:161–171. https://doi.org/10. 14456/thaidoaagres.2016.12 37. Masnoddin M, Repin R, Aziz ZA (2018) PLB regeneration of Paphiopedilum rothschildianum using callus and liquid culture system. J Trop Biol Conserv 15:1–14. https://doi.org/ 10.51200/jtbc.v15i0.1469 38. Tirado JM, Naranjo EJ, Atehortu´a L (2005) Propagacio´n in vitro de Phalaenopsis (Orchidaceae) a partir de protocormos, mediante el sistema de inmersio´n temporal “RITA”. Rev Colomb Biotecnol 7(1):25–31 39. Hempfling T, Preil W (2005) Application of a temporary immersion system. In: Hroslef-Eide AK, Preil W (eds) Liquid culture systems for in vitro plant propagation. Springer, Berlin, pp 197–211 40. Park SY, Murthy HN, Paek KY (2000) Mass multiplication of protocorm like bodies using bioreactor system and subsequent plant regeneration in Phalaenopsis. Plant Cell Tissue Organ Cult 63:67–72 41. Pisowotzki C, Saare-Surminski K, Lieberei R (2008) Micropropagation of Phalaenopsishybrids in temporary immersion system (TIS)—effects of exudated phenolic substances on plant development. Propag Ornamental Plants 8(1):221–223 42. Tisserat B, Vandercook CE (1985) Development of an automated plant culture system. Plant Cell Tissue Organ Cult 5:107–117 43. Arellano-Garcı´a J, Enciso-Dı´az O, FloresPalacios A, Valencia-Dı´az S, Flores-Morales A, Perea-Arango I (2020) Asymbiotic germination, effect of plant growth regulators, and chitosan on the mass propagation of Stanhopea hernandezii (Orchidaceae). Bot Sci 98(4): 524–533. https://doi.org/10.17129/botsci. 2559 44. Martı´nez-Estrada E, Bell-Bello JJ, Morales RV (2015) Uso de Biorreactores de Inmersio´n por Gravedad (BIG) para la propagacio´n in vitro de Stanhopea tigrina: Una orquı´dea ende´mica amenazada. El Agro Veracruzano 15:211–217 45. Esyanti RR, Adhitama N, Manurung R (2016) Efficiency evaluation of Vanda tricolor growth in temporary immerse system bioreactor and thin layer culture system. J Adv Agric Technol 3(1):63 ˜ os O, Cue´llar-Zometa JF, 46. Flores-Castan Montes-de Godoy ME, Ga´mez-Pastrana MR, Gonza´lez-Arnao MT, Guevara-Valencia M, Aguilar-Rivera N (2017) Germinacio´n in vitro de semillas de Vanilla planifolia Jacks y comparacio´n de me´todos de micropropagacio´n. Av Investig Agropecu 21(2):69–83

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Part VI General Conclusions and Perspectives

Chapter 22 Conclusions and Perspectives on Plant Micropropagation in Temporary Immersion Systems Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz Abstract In vitro propagation protocols that include temporary immersion systems are available for the most economically important plant species. However, these have not been established yet for multiple species. Having protocols validated by the scientific community guarantees the success of the mass production of commercial propagules. Besides, adequate TIS parameters should be established for each plant species to improve the efficiency of micropropagation processes. This book compiles basic and applied aspects of temporal immersion systems used for in vitro plant micropropagation, along with several detailed protocols already established, which may be used as a guide by those interested in this technique, including laboratory technicians, scientists, and other professionals. Key words Bioreactors, Micropropagation protocols, Commercial scale-up

1

Introduction Temporary immersion systems (TISs) represent a technological advance in plant micropropagation by using specialized culture containers to increase biological yield [1]. These specialized containers are available in various designs and materials. They have different elements and structures that favor gas exchange, temporary immersions, easy handling of culture containers, and semi-automation of processes [2]. These systems have areas of opportunity regarding design, manufacture, and functioning innovations to improve their efficiency [3]. However, all these containers work under the same principle: carry out programmed and temporary immersion of explants in the culture medium [4]. This has led to the development of about ten commercial and multiple household systems, leading to a broad range of options when an in vitro propagation protocol of a plant species is scaled-up commercially [1]. Therefore, compiling TIS plant micropropagation protocols that have been successfully tested provides a valuable

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1_22, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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tool in decision-making for the commercial scaling-up of propagule production. In addition, this book contributes both basic and advanced information about the concepts for the correct application of TISs in plant propagation.

2

Conclusions Scale-up for the commercial production of plant propagules has been achieved using semi-solid culture systems. However, production processes are permanently seeking cost reduction through novel strategies [5]. Mass micropropagation protocols involving semi-solid culture systems have been gradually replaced by protocols that use TISs. This requires research, testing, and scientific validation of a particular TIS effective for the in vitro propagation of a given plant species of interest. As highlighted in this compilation, adequate parameters should be established, such as immersion time and frequency and volume of medium per explant, according to the flask type and design. The techniques gathered and exemplified in this book will facilitate the correct use of temporary immersion systems in plant micropropagation, adapting, for each plant species, the type of flask to be used and the culture parameters described above, along with examples of the results that should be obtained and the step-bystep method to be carried out. Additionally, practical advice is provided for the successful commercial production of propagules when using TIS. The key steps detailed in these published protocols are the most appealing element of this series of books. These guarantee increased plant propagation of different species of interest in the food, agroindustrial, medicinal, and ornamental areas. TIS micropropagation is a cutting-edge technology for use in the functionality of biological laboratories. In this sense, there is a constant search for micropropagation protocols through TIS for plant species for which effective alternatives have not been established yet. There are highly efficient protocols involving TIS applicable for food species, such as sugar cane (Saccharum sp.), agro-industrial species such as agave (Agave sp.), stevia (Stevia rabaudiana), and ornamental species such as orchids. However, commercial scaling for woody species is complex despite the use of TISs. On the other hand, using TIS combined with somatic embryogenesis is considered the most effective morphogenetic pathway and technology for producing commercial propagules [6]. There is an innovation in SIT technology called photomixotrophism, which consists of adding carbon dioxide (CO2) to the culture flask, making the traditional system more efficient, in addition to achieving stomatal improvement in the plants generated, as well the metabolic functionality of the photosynthetic system [7].

Conclusions and Perspectives on Plant Micropropagation in Temporary. . .

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249

Perspectives There is interest in developing TIS micropropagation protocols for most plant species, aiming to produce propagules at a commercial scale. An adequately established protocol allows for large-scale in vitro production of plants to achieve better biological yield than the semisolid culture system, in addition to reducing production costs and facilitating process semi-automation. In this sense, innovation in the design, manufacturing materials, and components of temporary immersion systems could be an exciting topic to double or triple the yield attained with the current techniques. Besides, a design allowing total process automation would open up the possibility of establishing biological plants with large production volumes of propagules to meet the current demands in the field, industry, and research.

References 1. Georgiev V, Schumann A, Pavlov A, Bley T (2014) Temporary immersion systems in plant biotechnology. Eng Life Sci 14:607–621. https://doi.org/10.1002/elsc.201300166 2. Mirzabe AH, Hajiahmad A, Fadavi A, Rafiee S (2022) Temporary immersion systems (TISs): a comprehensive review. J Biotechnol 357:56–83. https://doi.org/10.1016/j.jbiotec.2022. 08.003 3. Lotfi M, Werbrouck SPO (2018) SETIS™, a novel variant within the temporary immersion bioreactors. In XXX International Horticultural Congress IHC2018: II International Symposium on Micropropagation and In Vitro Techniques. Acta Hortic 1285:253–258. https://doi. org/10.17660/ActaHortic.2020.1285.37 4. Murthy HN, Joseph KS, Paek KY, Park SY (2023) Bioreactor systems for micropropagation of plants: present scenario and future prospects. Front Plant Sci 14:1159588. https://doi.org/ 10.3389/fpls.2023.1159588

5. Nongdam P, Beleski DG, Tikendra L, Dey A, Varte V, El Merzougui S, Pereira VM, Barros PR, Vendrame WA (2023) Orchid micropropagation using conventional semi-solid and temporary immersion systems: a review. Plan Theory 1 2 : 1 1 3 6 . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / plants12051136 6. Aguilar ME, Wang XY, Escalona M, Yan L, Huang LF (2022) Somatic embryogenesis of Arabica coffee in temporary immersion culture: advances, limitations, and perspectives for mass propagation of selected genotypes. Front Plant Sci 13:994578. https://doi.org/10.3389/fpls. 2022.994578 7. Spinoso-Castillo JL, Bello-Bello JJ (2023) CO2-enriched air in a temporary immersion system induces photomixotrophism during in vitro multiplication in vanilla. Plant Cell Tissue Organ Cult 155:1–11. https://doi.org/10.1007/ s11240-023-02546-y

INDEX A

H

Acclimatization........................................... 3, 5, 6, 10, 26, 38, 39, 50, 51, 54, 58, 66, 74, 82, 84, 86, 98, 101, 103, 138, 139, 142, 144–146, 153, 161–163, 184, 196, 208, 223, 228, 233, 237, 238, 240 Agave cupreata ................................................... 77, 89–96 Agricultural biotechnology companies .......................... 25 Alstroemeria.......................................................... 157–163 Asexual reproduction .....................................69, 108, 149 Asymbiotic germination ............................................... 150

Hardening .................................................... 84, 110, 117, 121–122, 127, 208 Hyperhydricity ............................................. 4, 10, 26, 51, 70, 96, 109, 168, 169, 177, 179, 194, 219–221, 228, 233, 237, 238

B Bamboo ............................... 35, 108, 111, 121, 183, 184 BioMINTTM bioreactor................. 11, 78, 80, 82, 85, 86 Bioreactors.............................................. 6, 10–12, 16, 21, 34, 46, 51, 53–60, 64, 68–76, 78–80, 82, 84–87, 98, 100–103, 132, 133, 137–146, 149–155, 158, 159, 161–163, 168–180, 200, 205, 207, 211, 212, 222, 228–234, 238, 239 Bromeliad .......................................................35, 131, 132

I Immersion frequency ................................... 5, 18, 50, 57, 58, 67, 70, 73, 93, 138, 145, 146, 154, 161, 168, 230, 237, 239 In vitro culture ............................................. 4, 39, 90, 95, 117, 134, 157, 159, 169, 188, 203, 217, 218, 235, 238 In vitro propagation..........................................3, 5, 6, 10, 25, 31, 45, 53, 54, 64, 70, 90, 97, 109, 132, 184, 186, 190, 200, 218–221, 223, 239, 247, 248 ISSR ............................................115, 117, 121, 123, 128

L

Design innovations .............................................. 247, 249

Large-scale micropropagation ...................................6, 25, 26, 31, 46, 70, 167 Large-scale production .....................................26, 37, 39, 218, 221, 222, 230 Liquid culture..................................................3, 4, 11–13, 26, 53, 60, 64, 70, 80, 109, 116–118, 120, 124, 132, 170, 185, 190, 200, 207, 221, 230, 233, 234 Liquid medium................................................3, 4, 15, 17, 18, 20, 21, 25, 54, 70, 71, 78, 80, 87, 91, 95, 108, 109, 119–121, 125–127, 132, 135, 137, 140, 143, 167, 174, 177–180, 188, 200, 201, 207, 210, 211, 222, 228, 231, 234, 239

E

M

Ex vitro survival............................................................. 184

Massive micropropagation................................................ 5 Mass propagation ................................................ 4, 10, 31, 46, 150, 184, 193, 200, 223 Medicinal plant.................................................31, 34, 218 Micropropagation ............................................. 3–6, 9–21, 25–31, 45–52, 54, 63–68, 70, 73, 75, 77–87, 97–104, 107–128, 132, 137–146, 149–155, 157–162, 168, 169, 196, 221, 227–241, 247–249

C Castanea crenata........................................................... 168 Castanea sativa ............................................................. 168 Clonal lines ................................................................80, 83 Commercial propagules ..................................3–5, 25, 26, 31, 38, 46, 63, 248 Commercial scale-up....................................................... 10 Cost reduction .............................................................. 248

D

F Fragrant orchid ............................................................. 137

G Gravity immersion bioreactor (GIB) .................. 138, 139

Marco A. Ramı´rez-Mosqueda and Carlos A. Cruz-Cruz (eds.), Micropropagation Methods in Temporary Immersion Systems, Methods in Molecular Biology, vol. 2759, https://doi.org/10.1007/978-1-0716-3654-1, © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

251

MICROPROPAGATION METHODS IN TEMPORARY IMMERSION SYSTEMS

252 Index

Micropropagation protocols......................................5, 39, 63, 158, 167, 247–249

O Orchid.....................................................31, 36, 137, 138, 149, 150, 154, 155, 227–229, 232–241, 248 Orchidaceae .......................................................... 149, 227 Orchid plantlets.................................................... 137, 138 Organogenesis .............................................. 97, 109, 110, 150, 183–190, 194, 218–220, 223 Ornamental ......................................................... 5, 26, 31, 35, 36, 39, 69, 107, 108, 131, 132, 137, 138, 149, 150, 227, 248

P Plant micropropagation .................................... 3–6, 9–21, 31, 37, 39, 247–249 Plant tissue culture (PTC) ...................................... 10, 23, 26, 30, 32, 33, 35, 45, 53, 63, 70, 137, 150, 193, 194 Plantform™........................................ 168–170, 172–176, 178, 179, 222, 230 Protocorm like bodies (PLBs)............................ 228–231, 234, 235, 237, 238

R RITA® .................................................. 11–13, 16, 63–68, 97–104, 157–163, 168–179, 193–197, 199–212, 221–223, 228, 229, 231–233, 236, 238 Rooting.............................................5, 13, 19, 46, 48–51, 53–60, 63–65, 67, 68, 79, 80, 82, 84, 86, 95, 98,

99, 109, 110, 112, 117, 119, 121, 127, 150, 151, 153, 154, 160, 162, 163, 167, 168, 175, 180, 183, 194–196, 239, 240

S Salvia ..................................................................... 217–223 Sechium edule...........................................................97–104 Semi-solid medium ...........................................57, 66, 68, 73, 108, 200, 212, 220 Shoot proliferation ..............................109, 119, 220, 229 Silver nanoparticles (AgNPs)....................................89–96 Somatic embryogenesis (SE) .............................. 6, 89–96, 200, 202, 204, 208, 218, 223, 248 Static medium ............................ 109, 185–186, 188, 190 Stevia.................................... 13, 33, 34, 63–68, 108, 248 Sweeteners ....................................................................... 63

T Temporary immersed medium ..................................... 167 Temporary immersion bioreactor (TIB)................ 11, 17, 45–60, 86, 149–155, 158, 161, 228–233, 238 Temporary immersion systems (TISs) ........................ 3–6, 9–23, 25–39, 45, 46, 48, 50, 53, 54, 64, 66, 67, 70, 71, 77–87, 89–98, 100, 109, 131–135, 137, 138, 150, 151, 155, 157–163, 167–180, 183–190, 193–197, 200, 204, 205, 207, 210, 219–223, 227–241, 247–249 Threatened species ...................................... 149, 150, 199

W Wild orchid.................................................................... 239