Pollen Cryopreservation Protocols 1071628429, 9781071628423

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P. E. Rajasekharan M. R. Rohini Editors

Pollen Cryopreservation Protocols

SPRINGER PROTOCOLS HANDBOOKS

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Springer Protocols Handbooks collects a diverse range of step-by-step laboratory methods and protocols from across the life and biomedical sciences. Each protocol is provided in the Springer Protocol format: readily-reproducible in a step-by-step fashion. Each protocol opens with an introductory overview, a list of the materials and reagents needed to complete the experiment, and is followed by a detailed procedure supported by a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. With a focus on large comprehensive protocol collections and an international authorship, Springer Protocols Handbooks are a valuable addition to the laboratory.

Pollen Cryopreservation Protocols Edited by

P. E. Rajasekharan Division of Plant Genetic Resources, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India

M. R. Rohini Division of Flower and Medicinal Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India

Editors P. E. Rajasekharan Division of Plant Genetic Resources ICAR-Indian Institute of Horticultural Research Bangalore, Karnataka, India

M. R. Rohini Division of Flower and Medicinal Crops ICAR-Indian Institute of Horticultural Research Bangalore, Karnataka, India

ISSN 1949-2448 ISSN 1949-2456 (electronic) Springer Protocols Handbooks ISBN 978-1-0716-2842-3 ISBN 978-1-0716-2843-0 (eBook) https://doi.org/10.1007/978-1-0716-2843-0 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023 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.

Foreword

Cryopreservation, storage of biological materials in liquid nitrogen, is an ex situ, cost effective conservation method for plant genetic resources with minimal requirement of space and maintenance. Cryopreserved materials such as seeds, pollen grains, shoot apices, and vegetative buds maintain viability for years and thus provide national and international access to stored germplasm. Cryopreservation is probably the only convenient and effective conservation option for seedless species, species with recalcitrant seeds, and for cultivars with unique attributes that can be maintained only through vegetative propagation. Although the method of cryopreservation has been in practice for long, in recent years it is being used more widely and is being extended to a large number of species. Pollen grains, being the male partners in the sexual reproduction of seed plants, contain all the genetic information of the haploid nuclear genome of the species. They can be used directly for pollination without the need for raising plants. Thus, they form the most convenient components for effective use particularly in hybridization programs between parents which are spatially and/or temporally isolated. When compared to cryopreservation of other plant parts such as seeds, shoot apices, and vegetative buds, cryopreservation of pollen is much simpler; a large number of samples can be preserved in a limited space. Pollen grains can be cryopreserved for a number of years without loss of their fertilization ability. For example, it has been possible to cryopreserve pollen of oil palm for 23 years without a significant loss of viability (Rajesh Tandon et al., Delhi University, unpublished data). Pollen cryopreservation is also useful in the breeding program of species in which the flowering period is confined to a short period and requires staggered sowing of the male parent. Further, pollen grains of species belonging to a number of families such as Alismataceae, Poaceae, Cyperaceae, Commelinaceae, and Juncaceae are short-lived. Cryopreservation can provide viable pollen for years without the need to raise fresh plants. In the light of recent negative impact of human-induced environmental changes, particularly climate change on biodiversity, the need for long-term preservation of germplasm of agrobiodiversity has gained prominence. Plant breeding in the coming decades needs desirable genes, particularly for resistance/tolerance to biotic and abiotic stresses, for sustenance and improvement of yield in crop species to safeguard food, and for nutritional security of the expanding population. International exchange of accessions with elite genes, wherever they are discovered and conserved, is going to be important for future breeding programs. As pollen grains are subjected to less stringent quarantine restrictions, they become more convenient germplasm for international exchange than seeds or vegetative parts. As they can be used directly for crossing, it greatly improves breeding efficacy,

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particularly for species with long juvenile phase. However, cytoplasmic genes cannot be tapped through pollen; therefore, pollen banks should act as additional conservation approaches and not as substitutes for seed banks. Often, pollen exchange serves as an effective means for ex situ maintenance of rare, endangered, and precious germplasm. For example, double coconut (Lodoicea maldivica) is a dioecious species, endemic to Seychelles islands. It produces the largest seeds and is globally threatened. The trees are slow growing and are reported to live for up to 1000 years. At JC Bose Indian Botanical Garden in Howrah district in West Bengal has the only double coconut tree (female) in India planted in 1894. The tree started flowering after over 100 years, but there was no fruit set in the absence of any male tree. The authorities were able to obtain pollen from male trees growing in Thailand to carry out artificial pollination and were able to get some fruits (Curr Sci 2016, 110: 976–978). Pollen grains represent one of the simplest independent functional units among multicellular organisms. Pollen grains of flowering plants are made up of just two or three cells. As pollen germination and pollen tube growth are rapid and most of the experiments can be completed within a few hours without requiring aseptic conditions, they provide a model system to study a range of physiological and developmental processes at cellular and organism level. In recent years, they are being increasingly used for rapid analysis of a number of physical and chemical agents on plant species. Cryopreservation can provide pollen grains of the same genotype for years without the loss of viability so experiments need not be limited to the flowering period of the species. ICAR–Indian Institute of Horticultural Research, Bengaluru, is the leading Institute in the country working extensively on cryopreservation of pollen since many years. Pollen biologists of the institute, including both the editors, Dr P.E. Rajasekharan and Dr M.R. Rohini, of the present volume Pollen Cryopreservation Protocols, have been associated with this institute throughout their research career and have been working on the cryopreservation of pollen grains since long. They have standardized cryopreservation protocols for a number of species. I happen to know Dr. Rajasekharan’s work in this field for many decades. Both the editors have gained first-hand competence through their vast experience in the field to edit this present volume. As the contents indicate, introductory chapters written by the editors elaborate the scope, various tests used for pollen viability, and general methodologies of cryopreservation of pollen and its applications. Pollen cryopreservation protocols have been given for 13 fruit species, 15 vegetable species, 6 ornamentals, 11 medicinal plants, 2 plantation crops, 1 root crop, and 7 forest species. The protocols describe the steps involved from pollen collection, tests for viability, cryopreservation, pollen retrieval, and finally testing of their fertilizing ability to set fruits and seeds. Thus the book is going to be useful for all those who are interested in pollen biology, pollen storage, and their utility not only in practical aspects of plant breeding but also in a variety of other experimental studies. I congratulate both the editors for bringing out this useful volume. I hope the book would encourage young researchers to take up cryopreservation for their research so that the potential of the technique can be used more widely. K. R. Shivanna FNA, FASc, FNAAS, FNASc, FKSTA

Preface Pollen transmits the male genetic material in the sexual reproduction of all higher plants. The independent lifetime of flowering plant gametophytes is greatly abbreviated compared to that of gametophytes of more primitive plants, yet the angiosperm pollen grain must be able to survive at at least briefly free from the sporophytic plant and perform a number of specialized functions before fertilization is accomplished. Pollen has direct relevance in agriculture, horticulture, plant breeding, crop improvement, and biotechnology signifying the necessity for its conservation and use. Pollen cryopreservation offers a safe reliable technology for the ex situ conservation and sustained maintenance of viable pollen for various studies. Cryopreserved pollen grains conserve the Nuclear Genetic Diversity (NGD) of genotypes in limited space conveniently and have emerged as a potential tool assisting hybridization programs in difficult-to-hybridize and non-synchronous flowering species to support actions for genetic improvement in addition to conserving the male gene pool for long periods. Pollen cryopreservation methods have been standardized for around 200 species including fruit and forest trees, staple crops, vegetables, forage grasses, ornamental and medicinal plants including rare, endangered, and threatened (RET) species. This volume focuses upon pollen cryopreservation in various crops right from the most widely cultivated tomato to the lesser known medicinal species like Decalepis arayalpatra. Procedural details of the standard techniques involved such as pollen collection, cryopreservation, pollen culture, germination tests for viability, etc., which have been tested and improvised by the authors on specific crops are given in detail for better understanding. Step-by-step protocols are complemented by personal notes and precautions, specifying the reagents to be used in each step to ensure the repeatability of the procedure across labs. The volume comprises pollen cryopreservation protocols developed in 58 different horticultural and forest tree species. The first three chapters discuss a general introduction to the topic explaining the advances and prospects with regard to pollen cryopreservation, its implications in conservation and plant breeding, and the general procedures involved in establishing a pollen cryobank. Pollen cryopreservation in fruit crops has been highly successful in many of the commercially important crops like mango, papaya, citrus, grapes and has also shown promising results in minor fruits like jackfruit, dragon fruit, jamun, and annona which can have a greater impact in commercializing these minor crops. The chapters have included a detailed procedure with necessary notes regarding pollen cryopreservation in these important fruit crops. Although pollen conservation doesn’t accomplish whole genome conservation in fruit crops, it will be highly useful for the fruit breeders to make use of the conserved pollen in their breeding programs enabling the crossing of plants that flower at different times and that grow in different and distant locations. The technique can also be employed in crossing programs for developing cultivars resistant to diseases and pests where the vectors are pollinators. Similarly, in vegetable crops, many a times breeding for biotic and abiotic stress resistance requires crossing with wild species in which pollen availability and crossability may be a limitation. In such cases, cryopreserved pollen may assist in hybridization programs for getting improved cultivars. Pollen cryopreservation protocol in important vegetable crops has been successfully licensed to many private companies engaged in seed production by ICAR-Indian Institute of Horticultural Research

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(ICAR-IIHR), Bengaluru-89. Vegetable hybrid seed production using cryopreserved pollen is gaining popularity among private seed companies, and ICAR-IIHR, Bengaluru, has successfully licensed the protocol to many seed companies. Hence, the volume has covered the cryopreservation protocols developed in most of the commercially important vegetables like tomato, brinjal, onion, okra, pumpkin, ash gourd, bottle gourd, ridge gourd, and also in underutilized vegetables like teasel gourd, spine gourd, and wild relatives of brinjal. Ornamental crop improvement programs have always focused on to develop novel colors by hybridizing with different species or different genera in total. In such programs, it becomes highly essential to conserve and utilize the pollen as and when required. For aiding such events, cryopreservation protocols are developed in ornamentals like rose, tuberose, gladiolus, glory lily, and orchids. Most of the medicinal and forest tree species always come in the category of high conser vation priority, and in situ approaches are already in place for such species. Thus, pollen cryopreservation has served as a potent ex situ conservation method in most of the medicinal and forestry species especially the ones belonging to RET category like Oroxylum indicum, Decalepis hamiltonii, Decalepis arayalpatra, Salacia chinensis, Holostemma-kodien, Embelia ribes, Celastrus paniculatus, Saraca asoka and tree species like teak, sandalwood, Serbian spruce, Gmelina arborea, etc. Plantation crops like coconut, arecanut, and date palm and root crops like tuber crops hold a prominent place in the horticultural production system necessitating the need to conserve and enhance the germplasm for yield and quality traits. Pollen cryopreservation and utilization will provide a long-term availability of viable pollen for such long-term enhancement programs. Thus, we have tried our best to compile the information available in majority of the horticultural crops which occupy a significant place in the global horticulturtal production system. All chapters are written by experts who are recognized authorities in their respective fields. We hope that the volume will serve as an excellent reference work for researchers working in the area of plant breeding and plant conservation biology. Protocols can be adopted by breeders who are involved in improvement programs for difficult to hybridize species or can serve as a reference protocol for other unexplored crops. The editors are thankful to all authors for accepting our invitation and rendering their contributions toward the successful and timely compilation of this volume. We hope that the chapters will provide an in-depth understanding of the pollen cryopreservation in specified crops and protocols to be adopted for their long-term conservation, and finally, we are also grateful to Springer Nature for giving us an opportunity to compile this volume. ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India

P. E. Rajasekharan M. R. Rohini

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

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1 Pollen Cryopreservation: Advances and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . P. E. Rajasekharan and M. R. Rohini 2 Pollen Cryobanking—Implications in Genetic Conservation and Plant Breeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. E. Rajasekharan and M. R. Rohini 3 Establishment of a Pollen Cryobank and General Procedures of Pollen Cryopreservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. E. Rajasekharan and M. R. Rohini

1

PART I

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43

FRUITS

4 Pollen Cryopreservation Protocol in Annona Species. . . . . . . . . . . . . . . . . . . . . . . . Subhash Chander, P. E. Rajasekharan, Pradeep Kumar Vishwakarma, T. Sakthivel, and A. N. Ayesha 5 Pollen Cryopreservation in Aonla . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Linta Vincent and P. E. Rajasekharan 6 Cryopreservation of Pollen Grains of Carica papaya and Different Vasconcellea Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. E. Rajasekharan, S. Ganeshan, Pradeep Kumar Vishwakarma, and C. Vasugi 7 Cryopreservation of Pollen Grains of Citrus and Other Aurantioideae . . . . . . . . . Xiaoling Chen, Jinmei Zhang, Dong Jiang, Xinxiong Lu, Xia Xin, and Guangkun Yin 8 Pollen Cryopreservation Protocol for Dragon Fruit . . . . . . . . . . . . . . . . . . . . . . . . . G. S. Anilkumar and P. E. Rajasekharan 9 Cryopreservation of Grape Pollen (Vitis Species). . . . . . . . . . . . . . . . . . . . . . . . . . . . P. E. Rajasekharan, S. Ganeshan, and Pradeep Kumar Vishwakarma 10 Pollen Cryopreservation in Jackfruit (Artocarpus heterophyllus) for Crop Improvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. L. Navya, P. E. Rajasekharan, and Sridhar Gutam 11 Pollen Cryopreservation in Jamun (Syzygium cuminii Skeels) . . . . . . . . . . . . . . . . P. L. Anushma and P. E. Rajasekharan 12 Pollen Cryopreservation in Passiflora Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. L. Anushma and P. E. Rajasekharan 13 Pollen Cryopreservation Protocol for Poncirus trifoliata . . . . . . . . . . . . . . . . . . . . . P. E. Rajasekharan, S. Ganeshan, and R. Harsha

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135 147 155 167

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Contents

Cryopreservation of Pollen Grains of Different Psidium Species . . . . . . . . . . . . . . 175 Pradeep Kumar Vishwakarma, Subhash Chander, P. E. Rajasekharan, and C. Vasugi Cryopreservation of Pomegranate (Punica granatum L.) Pollen. . . . . . . . . . . . . . 189 P. E. Rajasekharan and Pradeep Kumar Vishwakarma Pollen Cryopreservation in Mango. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 G. L. Veena, Laxmi Mastiholi, and P. E. Rajasekharan

PART II 17 18 19 20

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29

VEGETABLES

Pollen Cryopreservation in Ash Gourd (Benincasa hispida (Thunb.) Cogn.) . . . Laxmi Mastiholi and P. E. Rajasekharan Pollen Cryopreservation in Bitter Gourd. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. S. Anilkumar, P. E. Rajasekharan, Swamini Bhoi, and B. Varalakshmi Pollen Cryopreservation in Bottle Gourd for Breeding and Conservation . . . . . . Laxmi Mastiholi and P. E. Rajasekharan Cryopreservation of Eggplant (Solanum melongena L.) Pollen for Utilization in Crop Improvement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Koushik Saha, P. E. Rajasekharan, and T. H. Singh Cryopreservation of Pollen Grains of Hot Pepper. . . . . . . . . . . . . . . . . . . . . . . . . . . Koushik Saha, P. E. Rajasekharan, K. Madhavi Reddy, and Rakesh C. Mathad Cryopreservation of Muskmelon (Cucumis melo) Pollen for Exploring Breeding Possibilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sourav Mahapatra, Soudamini Karjee, P. E. Rajasekharan, and E. Sreenivasa Rao Onion (Allium cepa L.) Pollen Cryopreservation Protocol . . . . . . . . . . . . . . . . . . . Ajay Kumar Pandav, Koushik Saha, and P. E. Rajasekharan Cryopreservation of Okra (Abelmoschus esculentus L.) Pollen . . . . . . . . . . . . . . . . . R. Gowthami, Neelam Sharma, K. K. Gangopadhyay, and Anuradha Agarwal Protocol for Pollen Viability and Cryopreservation of Pumpkin (Cucurbita moschata Duchesne ex Poir) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. S. Anilkumar and P. E. Rajasekharan Pollen Cryopreservation in Ridge Gourd (Luffa acutangula (Roxb.) L.) . . . . . . Pydi Roshni, Sourav Mahapatra, P. E. Rajasekharan, G. S. Anilkumar, and B. Varalakshmi Pollen Cryopreservation Protocol for Wild Species of Solanum . . . . . . . . . . . . . . . P. E. Rajasekharan, R. Harsha, and T. H. Singh Feasibility Studies on Pollen Cryopreservation in Spine Gourd (Momordica dioica Roxb.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. S. Anilkumar and P. E. Rajasekharan Cryopreservation Protocol for Tomato (Solanum lycopersicum L.) and its wild relatives Pollen for Utilization in Crop Improvement . . . . . . . . . . . . . Koushik Saha, P. E. Rajasekharan, and H. C. Prasanna

215 225 235

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293 301

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Pollen Cryopreservation Protocol for Teasel Guard (Momordica subangulata subsp. Renigera) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 G. S. Anilkumar and P. E. Rajasekharan 31 Cryopreservation of Watermelon Pollen to Explore New Breeding Possibilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 Sourav Mahapatra, Soudamini Karjee, P. E. Rajasekharan, and E. Sreenivasa Rao

PART III

ORNAMENTALS

Pollen Cryopreservation Protocol for Gladiolus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. E. Rajasekharan and T. M. Rao 33 Pollen Cryopreservation Protocol for Gloriosa superba L. . . . . . . . . . . . . . . . . . . . . P. E. Rajasekharan, M. R. Rohini, R. Harsha, and G. S. Anilkumar 34 Pollen Cryopreservation in Marigold (Tagetes spp.) . . . . . . . . . . . . . . . . . . . . . . . . . Laxmi Mastiholi, P. E. Rajasekharan, and Tejaswini Prakash 35 Pollinia Cryopreservation of Selected Orchid Species of Western Ghats, India. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. William Decruse, S. Ajeeshkumar, and Sayoogia Vargheese 36 Pollen Cryopreservation Protocol for Tuberose (Polianthes tuberosa L.) . . . . . . . G. S. Anilkumar and P. E. Rajasekharan 37 Pollen Cryopreservation Protocol for Rose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. E. Rajasekharan and S. Ganeshan 32

PART IV

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401 415 423

MEDICINAL PLANTS

Pollen Cryopreservation in Aloe vera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. E. Rajasekharan, R. Harsha, and G. S. Anilkumar 39 Pollen Cryopreservation Protocol for Cayratia pedata . . . . . . . . . . . . . . . . . . . . . . P. E. Rajasekharan and R. Harsha 40 Pollen Cryopreservation in Celastrus paniculatus . . . . . . . . . . . . . . . . . . . . . . . . . . . P. E. Rajasekharan, M. R. Rohini, N. R. Aysha, and R. Harsha 41 Pollen Cryopreservation Protocol for Decalepis arayalpathra (Apocyanaceae) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. Gokul, R. K. Divya, Anup S. Deshpande, P. Ravichandran, K. Ravikumar, M. K. Janarthanam, P. E. Rajasekharan, and M. R. Rohini 42 Pollinia Cryopreservation Protocol for Decalepis hamiltonii Wight & Arn . . . . . P. E. Rajasekharan and R. Harsha 43 Cryopreservation and In Vitro Pollen Germination Protocol in Embelia ribes Burm. f . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. M. Aparna, P. E. Rajasekharan, and H. N. Vachana 44 Pollinia Cryopreservation Protocol for Holostemma ada-kodien. . . . . . . . . . . . . . . P. E. Rajasekharan, M. R. Rohini, and R. Harsha 38

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Contents

Pollen Cryopreservation Protocol for Oroxylum indicum . . . . . . . . . . . . . . . . . . . . R. Harsha and P. E. Rajasekharan 46 Pollen Cryopreservation Protocol for Salacia chinensis Linn . . . . . . . . . . . . . . . . . P. E. Rajasekharan and R. Harsha 47 Pollen Cryopreservation Protocol For Saraca asoca (Roxb.) De Wilde . . . . . . . . P. E. Rajasekharan and R. Harsha 48 Pollen Cryopreservation in Stevia rebaudiana Bertoni. . . . . . . . . . . . . . . . . . . . . . . Laxmi Mastiholi and P. E. Rajasekharan

45

PART V 49

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501 509

PLANTATION CROPS

ROOT CROP

Pollen Cryopreservation in Cassava . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543 Vivek Hegde

PART VII 52

493

Methods for Cryopreserving of Date Palm Pollen. . . . . . . . . . . . . . . . . . . . . . . . . . . 519 Annie Carolina Arau´jo de Oliveira, Ana da Silva Le´do, MaryLou Polek, Robert R. Krueger, Ashley Shepherd, and Gayle M. Volk Cryopreservation of Coconut and Arecanut Pollen. . . . . . . . . . . . . . . . . . . . . . . . . . 527 Anitha Karun, K. S. Muralikrishna, K. K. Sajini, and M. K. Rajesh

PART VI 51

483

FOREST SPECIES

Pollen Cryopreservation Protocol for Neolamarckia cadamba (Roxb.) Miq . . . . 555 P. E. Rajasekharan and R. Harsha Pollen Cryopreservation of Coniferous Serbian Spruce (Picea omorika/Pancˇ./Purkyne) and Deciduous Pedunculate Oak (Quercus robur L.) Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561 Branislava Batos and Danijela Miljkovic´

Pollen Cryopreservation Protocol for Couroupita guianensis Aubl.. . . . . . . . . . . . G. S. Anilkumar, P. E. Rajasekharan, and R. Harsha 55 Pollen Cryopreservation Protocol for Gmelina arborea . . . . . . . . . . . . . . . . . . . . . . G. S. Anilkumar, B. L. Navya, P. E. Rajasekharan, and R. Harsha 56 Pollen Cryopreservation in Moringa concanensis for Crop Improvement . . . . . . . B. L. Navya and P. E. Rajasekharan 57 Cryopreservation of Sandalwood (Santalum album L.) Pollen. . . . . . . . . . . . . . . . G. S. Anilkumar and P. E. Rajasekharan 58 Cryopreservation of Pollen for Long-Term Storage in Teak (Tectona grandis) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. S. Anilkumar and P. E. Rajasekharan 54

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

575 583 591 601

609 617

Contributors ANURADHA AGARWAL • Tissue Culture and Cryopreservation Unit, Indian Council of Agricultural Research (ICAR)-National Bureau of Plant Genetic Resources (NBPGR), Pusa Campus, New Delhi, India S. AJEESHKUMAR • KSCSTE-Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram, India G. S. ANILKUMAR • Division of Flower and Medicinal Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India; Division of Plant Genetic Resources, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India P. L. ANUSHMA • Division of Flower and Medicinal Crops, Division of Fruit Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India P. M. APARNA • College of Horticulture, Kerala Agricultural University, Vellanikkara, Thrissur, Kerala, India A. N. AYESHA • Division of Plant Genetic Resources, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India N. R. AYSHA • Division of Flower and Medicinal Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India BRANISLAVA BATOS • Department of Genetics, Institute of Forestry, Belgrade, Serbia SWAMINI BHOI • Division of Vegetable Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India SUBHASH CHANDER • Punjab Agricultural University, RRS, Abohar, Punjab, India XIAOLING CHEN • National Crop Genebank of China, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China ANA DA SILVA LE´DO • Embrapa Coastal Tablelands, Aracaju, SE, Brazil ANNIE CAROLINA ARAU´JO DE OLIVEIRA • Federal University of Sergipe, Sa˜o Cristova˜o, SE, Brazil S. WILLIAM DECRUSE • KSCSTE-Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram, India ANUP S. DESHPANDE • Department of Botany, Goa University, Taleigao, Goa, India R. K. DIVYA • In-Vitro Conservation and Cryopreservation, Division of Plant Genetic Resources, Indian Institute of Horticultural Research, Bangalore, Karnataka, India S. GANESHAN • Division of Plant Genetic Resources, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India; Division of Flower and Medicinal Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India K. K. GANGOPADHYAY • Division of Germplasm Evaluation, ICAR-NBPGR, Pusa Campus, New Delhi, India S. GOKUL • Department of Plant Biology and Systematics, CSIR–Central Institute of Medicinal and Aromatic Plants Research Centre, Bangalore, Karnataka, India R. GOWTHAMI • Tissue Culture and Cryopreservation Unit, Indian Council of Agricultural Research (ICAR)-National Bureau of Plant Genetic Resources (NBPGR), Pusa Campus, New Delhi, India

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Contributors

SRIDHAR GUTAM • All India Co-ordinated Research Project on Fruits, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India R. HARSHA • Division of Flower and Medicinal Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India VIVEK HEGDE • ICAR-Central Tuber Crops Research Institute, Thiruvananthapuram, Kerala, India M. K. JANARTHANAM • Department of Botany, Goa University, Taleigao, Goa, India DONG JIANG • Citrus Research Institute, Southwest University, Chongqing, China SOUDAMINI KARJEE • Division of Flower and Medicinal Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India ANITHA KARUN • ICAR-Central Plantation Crops Research Institute, Kasaragod, Kerala, India ROBERT R. KRUEGER • USDA-ARS National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA, USA XINXIONG LU • National Crop Genebank of China, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China K. MADHAVI REDDY • Division of Vegetable Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India SOURAV MAHAPATRA • Division of Vegetable Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India LAXMI MASTIHOLI • Division of Flower and Medicinal Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India RAKESH C. MATHAD • Assistant Professor-Seed Science and Technology, UAS, Raichur, Karnataka, India DANIJELA MILJKOVIC´ • Department of Evolutionary Biology, Institute for Biological Research “SinisˇaStankovic´”–National Institute of Republic of Serbia, University of Belgrade, Belgrade, Serbia K. S. MURALIKRISHNA • ICAR-Central Plantation Crops Research Institute, Kasaragod, Kerala, India B. L. NAVYA • Division of Flower and Medicinal Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India AJAY KUMAR PANDAV • Division of Vegetable Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India MARYLOU POLEK • USDA-ARS National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA, USA TEJASWINI PRAKASH • Division of Flower and Medicinal Crops, ICAR – Indian Institute of Horticultural Research, Bangalore, India H. C. PRASANNA • Division of Vegetable Crops, ICAR- Indian Institute of Horticultural Research, Bangalore, India P. E. RAJASEKHARAN • Division of Plant Genetic Resources, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India; Division of Flower and Medicinal Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India; In-Vitro Conservation and Cryopreservation, Division of Plant Genetic Resources, Indian Institute of Horticultural Research, Bangalore, Karnataka, India M. K. RAJESH • ICAR-Central Plantation Crops Research Institute, Kasaragod, Kerala, India

Contributors

xv

T. M. RAO • Division of Flower and Medicinal Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, India P. RAVICHANDRAN • Department of Plant Science, Manonmaniam Sundaranar University, Tirunelveli, Tamil Nadu, India K. RAVIKUMAR • School for Conservation of Natural Resources, Repository for Medicinal Resources, Trans-Disciplinary University (TDU-FRLHT), Bangalore, Karnataka, India M. R. ROHINI • Division of Flower and Medicinal Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India; In-Vitro Conservation and Cryopreservation, Division of Plant Genetic Resources, Indian Institute of Horticultural Research, Bangalore, Karnataka, India PYDI ROSHNI • Division of Vegetable Crops, ICAR- Indian Institute of Horticultural Research, Bangalore, India KOUSHIK SAHA • Division of Vegetable Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India K. K. SAJINI • ICAR-Central Plantation Crops Research Institute, Kasaragod, Kerala, India T. SAKTHIVEL • Division of Plant Genetic Resources, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India NEELAM SHARMA • Tissue Culture and Cryopreservation Unit, Indian Council of Agricultural Research (ICAR)-National Bureau of Plant Genetic Resources (NBPGR), Pusa Campus, New Delhi, India ASHLEY SHEPHERD • USDA-ARS National Laboratory for Genetic Resources Preservation, Fort Collins, CO, USA T. H. SINGH • Division of Vegetable Science, ICAR- Indian Institute of Horticultural Research, Bangalore, Karnataka, India E. SREENIVASA RAO • Division of Vegetable Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India H. N. VACHANA • Division of Plant Genetic Resources, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India B. VARALAKSHMI • Division of Vegetable Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India SAYOOGIA VARGHEESE • KSCSTE-Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram, India C. VASUGI • Division of Fruit Crops, ICAR – Indian Institute of Horticultural Research, Bangalore, Karnataka, India G. L. VEENA • Indian Institute of Horticultural Research, Bangalore, Karnataka, India LINTA VINCENT • Division of Flower and Medicinal Crops, ICAR-Indian Institute of Horticultural Research, Bangalore, Karnataka, India PRADEEP KUMAR VISHWAKARMA • Krishi Vigyan Kendra, Sukhet, Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur, Bihar, India; Division of Fruit Crops and Division of Flower and Medicinal Crops, ICAR – Indian Institute of Horticultural Research, Bangalore, Karnataka, India GAYLE M. VOLK • USDA-ARS National Laboratory for Genetic Resources Preservation, Fort Collins, CO, USA

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Contributors

XIA XIN • National Crop Genebank of China, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China GUANGKUN YIN • National Crop Genebank of China, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China JINMEI ZHANG • National Crop Genebank of China, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China

Chapter 1 Pollen Cryopreservation: Advances and Prospects P. E. Rajasekharan and M. R. Rohini Abstract Cryopreservation is one of the most promising techniques applied for the long-term conservation of various plant genetic resources ex situ. Tremendous progress has been made in this field for the last 30–40 years. Cryobanks have been established in various parts of the world as a strategy to conserve difficult-to-conserve species. Currently, it is estimated that over 10,000 accessions of vegetatively propagated crops starting from in vitro cultures are safely conserved for the long term through cryopreservation. Pollen cryopreservation is considered to be a complementary conservation strategy for crop species, and tremendous progress has been made in this area in the last few years. Pollen conservation promotes improved efficiency in breeding programs and germplasm conservation and exchange in the case of clonally propagated crops. In this chapter, we try to chart out the history of plant cryopreservation and how it has evolved as a technique and how it is applied for pollen conservation. Key words Cryopreservation, Pollen, Cryobanks, Applications

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Introduction and Relevance of Plant Cryopreservation The ever-alarming irreversible loss of biodiversity in nature has provoked the thought and need for conservation in the minds of many plant biologists. At times, when the in situ conservation strategies failed due to uncontrolled circumstances like habitat loss and biotic and abiotic stresses, ex situ conservation programs were applied and put in place. Plant cryopreservation is one of the ex situ conservation strategies wherein plant tissues are stored in liquid nitrogen (LN) at -196 °C or in the vapor phase of LN at -135 °C in such a way that the viability of stored tissues is retained following re-warming [1, 2]. The technique is particularly adopted for species with recalcitrant (i.e., dehydration sensitive) seeds that are not storable by any other means, conservation of specific cultivars of vegetatively propagated crops, unique ornamental genotypes, and endangered plant species, particularly where seeds may be extremely scarce or of doubtful quality, and the species is threatened with imminent extinction [3–8]. Pence et al. [9]

P.E. Rajasekharan and M.R. Rohini (eds.), Pollen Cryopreservation Protocols, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-2843-0_1, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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highlighted the role of cryopreservation in securing the germplasm of threatened taxa that are declining in the wild. The technique has also proved to be of great value for conserving and using biotechnology products such as metabolite-producing cell lines and genetically engineered materials. More importantly, cryopreservation eliminates the need for regular renewal of the collection thus reducing the risk of genetic erosion caused by pests, diseases, weather conditions, pollution, and genetic variations [10, 11]. It also prevents the decline in culture productivity, which is usually associated with culture duration [12]. Such advantages are due to the effect of extremely low temperatures (usually the materials are stored in liquid nitrogen (LN) or its vapor phase at temperatures ranging from -140 °C to -196 °C) that arrest nearly all cell division and metabolic activities of the cells. Thus, the plant samples can be stored unaltered and they remain viable for a theoretically unlimited duration [11, 13, 14]. Cryopreservation enables the conservation of plant genetic resources cost-effectively for decades with minimal requirements of space and routine maintenance [15–17]. Unlike other conservation methods like low temperature storage and in vitro storage, cryopreservation eliminates the need for regular regeneration and viability monitoring thereby reducing the chances of genetic erosion. Such advantages are due to the effect of extremely low temperatures that arrest nearly all cell division and metabolic activities of the cells. Thus, the plant samples can be stored unaltered and they remain viable for a theoretically unlimited duration [11, 13, 14]. Modern-day breeding programs are well supported by cryopreservation by providing long-term conservation and easy international access to various genetic materials such as seeds, pollen, and meristematic apices and buds. Cryopreserved collections are valuable materials for future crop improvement and ecosystem restoration programs. They provide a safety backup of vegetatively propagated species and also species which have very narrow population distribution. Cryopreservation also enables the storage of unique or trait-specific genotypes at a much lower cost by obviating the need for field establishment or constant regeneration of seed collections [18]. Cryotherapy, the technique of eliminating plant pathogens from plant materials by exposing to ultra-low temperatures, is also emerging having a consequence on improved plant health and quality [19]. At present, cryogenic technology has evolved in an extensive mode finding its applications in different fields like medical, horticultural, agricultural, aquaculture, and forestry sectors [20]. Cryopreservation is a broader discipline encompassing various disciplines from biology, physics, physiology, to cryo physics, and a thorough understanding of these disciplines is required for developing a successful cryopreservation protocol. Time long, researchers have already proved the amenability of almost all plant parts such as seeds, somatic and zygotic embryos,

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vegetative material (e.g., roots, bulbs, tubers, buds, meristematic apices), pollen, suspension cultures, and callus for cryopreservation [21]. However, the choice of explant is determined by the objective of conservation. Seeds, embryos or embryonic axes are used when the goal is to conserve species diversity whereas the vegetative propagules like shoot tips and dormant buds are used to retain the specific genotype [22]. The first successful cryopreservation was reported in mulberry twigs exposed to liquid nitrogen. It was found that hardening on one hand and dehydration on the other was essential for survival. Dehydration and freezing are the two critical steps involved in cryopreservation, and the success of any protocol depends on the desiccation and freezing tolerance of the tissue. Cryopreservation is achieved by optimal drying of the explant material and immersing it in liquid nitrogen followed by rewarming and regrowth procedures. Sufficient and optimal drying of the explant is essential for avoiding ice crystal formation during the freezing process. Insufficient drying will result in residual water content which will immediately lead to intracellular ice formation upon freezing and cause injury to the tissues. Thus, based on different drying methods, there are different cryopreservation techniques available. The two broad categories of cryopreservation are traditional or classical method of slow freezing and the second is vitrification-based methods which are further divided into simple desiccation, encapsulation-dehydration, vitrification freezing, droplet vitrification, etc. Vitrification is the most widely adopted method of cryopreservation and is successfully applied to hundreds of plant species [23]. Vitrification methods are being continuously modified and improved over time to achieve greater success in cryopreservation. Few of the innovative approaches of vitrification methods include Vacuum Infiltration Vitrification (VIV), cryo-plate protocols, and a new cryo-mesh protocol [24]. VIV technique involves the application of vacuum to the explant material while it is treated with loading and cryoprotectant solutions. This will in turn remove the air bubbles from the surface of the plant material thereby increasing the interaction between the plant material and the cryoprotective solutions. This will result in a more uniform infiltration and a potentially greater intake of cryoprotective solutions. This method was successfully used in the cryopreservation of Carica papaya, Passiflora edulis, and Laurus nobilis seeds [25]. Cryo-plate protocol developed by Yamamoto et al. (2011) was successfully applied in Mentha spp. to achieve a higher post cryopreservation protocol. Funnekotter et al. [26] modified the cryo-plate method to develop cryo-mesh method which allows alginate to act as a glue between the mesh and the explant. Cryo-mesh protocol was found more efficient with less skill requirement than cryo-plate and droplet vitrification method, respectively.

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Cryopreservation has now emerged as the most potential longterm conservation method of different biological materials. Cryopreservation goes hand in hand with in vitro technologies, hence termed cryobiotechnology. Cryobiotechnology is defined as “the use of modern technologies to understand the response of biological systems to low temperature environments, whether natural or imposed, leading to the production of knowledge, goods and services, including the cryopreservation of organisms, cells and tissues for use by industry, agriculture, medicine and conservation” [27]. Cryopreservation is based on the dehydration of cells or tissues to prevent ice crystal formation, and thus, cryopreservation and dry preservation of cells and tissues are highly interdisciplinary fields of research requiring insights from biologists, chemists, physicists, medical scientists, as well as engineers at ultra-low temperatures below -150 °C, in liquid nitrogen tanks or mechanical freezers [28]. Validating the long-term viability and survival of biological material and developing genotype-specific protocols for cryo storage are critical for the widespread adoption of cryopreservation technique [29]. Panis et al. [30] in their latest review mentioned cryopreservation as a cost- and labor-efficient conservation method that ensures genetic stability over time.

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Historical Aspects of Plant Cryopreservation The history of cryopreservation dates back to the inception of the field of cryobiology in 1800s when the technique of conversion of compressed gases to the liquid state has been developed [31]. The use of ultra-low temperatures for protecting biological materials has been used since long, but further scientific investigations into the subject have led to the discovery of cryopreservation technique in the nineteenth century. Initially, the technique was applied to prokaryotic organisms and later to eukaryotes and further for the preservation of animal cells and plant cells. The history of plant cryopreservation dates back to the 1960s when it was initially applied to cell cultures and other unorganized tissues. The first applications of cryopreservation to organized tissues by Dr. Kutty Kartha and Dr. Akira Sakai showed the promise of the technique for the storage of plant diversity. By the 1980s, the method was extended to several temperate plant species, and with the advent of vitrification techniques in the 1990s, cryopreservation has been widely adopted for many tropical species also. Knowlton (1992) first reported the retention of viability in pollen grains even after freezing at -180 °C. Since then, numerous studies have been conducted to test the efficiency of pollen conservation in many plant species. The feasibility of using pollen as an ex situ conservation material was established by the studies conducted at the Agricultural University of Wageningen. They have examined the storability studies in pollen of approximately 1600 species.

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The survival of pollen when stored at room temperature differed significantly between the species and was also 10 to 100 times less than the seeds. The seeds of the studied species displayed recalcitrant or intermediate seed storage behavior which was however not correlated with the storage behavior of their pollen. Thus, pollen was found to be an effective means of conserving nuclear genetic diversity, particularly for vegetatively propagated species and species with recalcitrant seeds. However, the utilization of stored pollen necessitated the availability of a recipient female species for hybridization. As per the recent reports, large-scale implementation of cryostorage to conserve genetic resources is rising steadily [22, 32, 33], and today cryopreservation protocols are available for more than 200 species of plants. Even though the technique is mostly developed for clonally propagated and recalcitrant seed species, a number of global genebanks are routinely cryoconserving the orthodox seeds of endangered species, pollen of perennial horticultural crops, dormant buds of fruit and forestry species, in vitro cultures of vegetatively propagated plants, and biotechnology products. Some of the successful examples of germplasm cryobanking include that of Musa [10, 34], Allium [35, 36], mulberry [37, 38], Malus [39], and potato [23, 40]. Past decades have witnessed substantial progress in the understanding of biophysical processes involved in cryopreservation and also an increase in the adoption of cryopreservation as a long-term conservation strategy for many species. Cryopreserved collections, now located in several countries around the world, are a testament to the utility of cryopreservation. Now the challenge is to expand the utility of these techniques by making them available to laboratories that do not specialize in cryopreservation, but rather wish to use it as a safe backup for valuable plant materials. Figure 1 depicts how cryopreservation methods have evolved over time.

1800

• Conversion of compressed gases to the liquid state has been developed

1960’s

• Cryopreservation of unorganized plant tissues

1970’s

• Cryopreservation of organized plant tissues

• Cryopreservation using controlled rate cooling 1980’s • Cryopreservation in temperate plant species • Development of vitrification based cryopreservation methods 1990’s • Cryopreservation of tropical plant species

Fig. 1 Timeline showing evolution of plant cryopreservation methods

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Pollen Cryopreservation Conservation of pollen grains is an important aspect in the management of plant genetic resources because it provides access to genetic resources that can be readily used in breeding programs. There are different methods of storing pollen which include refrigeration (3–5 °C), freeze-drying and freeze-storage, vacuum drying, using organic solvents, and cryopreservation in liquid nitrogen (LN, -196 °C) [41]. Cryopreservation proved to be the most efficient method for the long-term conservation of pollen grains when compared with other methods. Cryopreservation offers a simple and effective method of storing pollen in liquid nitrogen for several years without losing its ability to pollinate, fertilize, and set fruits when used for controlled pollination [42]. The technique is useful for germplasm curators for the conservation of important alleles and for plant breeders to overcome asynchronous flowering for genetic improvement. Pollen grains can be easily collected and stored in a viable condition for long period enabling the conservation of more alleles in limited space. It has been reported that cryopreservation of pollen is simpler than any other explant because of its low moisture content, highly packed storage materials such as sugars, oil, and starch, the non-vacuolated nature, and also the highly resistant exine wall. Moreover, pollen grains do not require any specific rate of cooling and can be directly immersed in liquid nitrogen [41]. It has been found that in the majority of tropical plant species, fresh pollen grains are in ideal desiccated condition for direct cryopreservation [43]. Pollen cryopreservation is achieved through a sequence of steps from pollen collection, desiccation, viability testing, storage in liquid nitrogen, and longevity assessment. Like any other explant, it is necessary to reduce or adjust the water content in pollen to maintain pollen viability after storage at ultra-low temperatures. Increased pollen longevity can be obtained by using high-quality pollen desiccating it sufficiently in a rapid manner and subsequently storing it at very low temperatures. However, the drying should not be too rigorous, preferably not below about 6–7%, as this reduces storage life. Methods for pollen collection, desiccation, viability testing, and longevity assessment have been developed for many species of interest, and have revealed the critical importance of increased longevity by using high-quality pollen desiccating it sufficiently in a rapid manner and subsequently storing it at very low temperatures. Optimum pollen moisture level turns out to be the most critical factor in deciding the pollen viability after storage at ultralow temperatures. The moisture content should be adjusted such that the freezable water is removed; this further varies among the species, and different methods have been applied to control the moisture content. Apart from the pollen moisture content, other

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factors affecting the successful pollen conservation include the genotype, stage of pollen collection, physiological status of the plant, protocol followed, etc. The objective of any pollen cryopreservation protocol is to collect dry mature pollen from the desired species and conserve using methods which allow retention of its normal function, ultimately assessed by its ability to germinate in vivo and effect normal fertilization [44]. Thus, it is always imperative to choose the most effective method which will ensure the maintenance of high genetic integrity, vigor, and germination percentages. Barnabas and Kovacs [45] and Berthoud [46] stressed the importance and need for pollen conservation by cryopreservation, and Hoekstra [47] assessed the merits and demerits of pollen as a genetic resource. Pollen cryopreservation methods have been reported for at least 170 species, including fruit and forest trees, staple crops, vegetables, forage grasses, and ornamental economic and medicinal plants including rare, endangered, and threatened species [43]. Alexander and Ganeshan [48] reviewed the research carried out on pollen storage in fruit crops and Ganeshan and Rajasekharan [49] reviewed the work on ornamental species. Cryopreserved pollen can serve as the resource material for conservation and utilization of nuclear genetic diversity in the breeding of vegetable and ornamental crops [50]. 3.1 Why Pollen Cryopreservation?

Pollen is a useful source of diverse alleles within a genepool and is useful in a number of ways for the geneticists, breeders as well as embryologists. The cryopreservation of pollen plays an important role in hybridization programs and in the conservation of genetic resources in agricultural and forestry systems. It is considered the most simple, safest, and effective method for prolonged storage of genetic resources in comparison to other plant organelles. Pollen can be kept in LN for many years without losing its ability to pollinate and fertilize, as well as subsequent fruiting capabilities. This provides ample opportunities for the researchers to design their breeding programs. The technique has the potential to overcome many challenges of breeding programs including flowering asynchrony between different parent genotypes, production of insufficient pollen in nature, etc. Many a time breeder has to do multiple and staggered plantings in order to synchronize flowering for crossing desirable genotypes. This can be avoided by using cryopreserved viable pollen facilitating hybrids between genera, species, and genotypes. The use of cryopreserved pollen in artificial or assisted or supplementary pollination increases the amount and quality of fruit production in different crops under conditions in which natural pollination is limited [51]. This technique also overcomes the variability that may arise due to daily pollen collection [45]. It is of pertinent use in the male sterility-based hybrid production system where the male sterile populations can be perpetuated by cryostored maintainer pollen, thus avoiding frequent

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Fig. 2 Advantages of pollen as an explant

planting of maintainer line. Pollen storage also enables the transportation of a sufficient quantity of pollen to various locations for taking up the hybridization programs [49]. Conservation of recalcitrant pollen in many of the important fruit and forestry species for overcoming geographic, seasonal, and physiological barriers in hybridization is the foremost achievement of pollen cryopreservation. The technique has proved immensely useful to plant species, where the flowering period is very short and the pollen grains are very short-lived. Proper pollination is a very crucial factor in determining the production of many fruit crops, and therefore, supplementary pollination with cryopreserved pollen grains has stabilized the production of many fruit crops. It also avoids the interference of foreign or illegitimate pollen. Pollen cryopreservation extends the pollen viability for longer periods which enables a pollen biotechnologist to carry out basic studies over extended durations on viable and fertile pollen. These studies could include genetic characterization of pollen, which allows more discrete use of pollen stocks for breeding. Another area of study could be to introduce known sequences of alien DNA in pollen with extended viability and fertility which could have an impact on the understanding of its integration with the gametophyte prior to use in breeding. Figure 2 summarizes the advantages of using pollen as an explant for cryopreservation. The various applications of pollen cryopreservation are outlined as follows: • Helps the plant breeders to redesign the breeding strategies and undertake hybridization between asynchronously flowering and geographically isolated plants

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• Facilitates conservation of difficult to conserve species like wild, rare, threatened, and endangered plants. • Provides long-term supply of pollen grains for hybridization of species which has short-lived or recalcitrant pollen • Facilitates supplementary pollinations in orchard crops for an increased and stable crop production • Availability of pollen year-round enables the hybridization process twice or thrice a year depending on the duration of the crop. • Eliminates the need to grow male lines continuously in breeding programs thus saving land and labor resources • Provides a continuous supply of pollen grains for taking up pollen biology studies at any point in time. • Facilitates easy exchange of germplasm with less stringent quarantine restrictions • Enables fast breeding of tree species with a long juvenile phase by allowing introgression of characters at a much faster rate by using pollen directly on the seed parent 3.2

Pollen Cryobanks

Pollen banks were established with the objective of providing a constant supply of viable pollen for crop breeding programs. Flowering asynchrony in the cultivars and their wild relatives, geographical isolation of desired parent species, and lack of insufficient pollen in promising male parents have hindered the breeding program in many economically important plant species [52]. Moreover, haploid breeding procedures and biotechnology experiments which use the pollen as source material for achieving gene expression of the transformed pollen cells suggest the need for conservation of pollen thereby necessitating the establishment of pollen banks. The importance of pollen conservation was foreseen in the late 18th century as is evident from the words of William King in 1885, that “nothing would tend more toward the rapid termination of an experiment than control over the supply of pollen, so that we might use it when and where convenient to ourselves.” The establishment of “pollen banks,” in addition to fulfilling various other needs, considerably reduces the maintenance of orchards and nurseries for the regular supply of pollen [41]. Barnabas and Kovacs [45] reviewed the use of conserved pollen to eliminate the need for raising nurseries and greenhouses for plant growth and also providing the scientists and breeders with the pollen source irrespective of time and space. A pollen bank was established in Quebec in 1995 for the long-term conservation of a large quantity of tree pollen of coniferous trees on a large scale for up to five years [53]. Since then, numerous techniques are available for pollen collection, viability assessment, and cryopreservation, and as a result, pollen cryobanks for various species have been established in different countries like China, India, and USA [43, 54, 55].

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Methods of pollen cryopreservation and procedures needed for developing pollen genebanks were reviewed by Towill and Walters [56]. As per the review of Hoekstra [47] pollen collection is the most critical component for a pollen bank, and pollen collection procedures depend on the type of species, inflorescence, and peak anthesis period. Pollen grains are an effective unit for undertaking gene manipulation studies owing to their easiness in collection and availability in large quantities. Recently, they are also used in gene expression studies of transgenics as there is a stable expression of many proteins in them. In spite of its usefulness, pollen banks are maintained by very few organizations such as the forest industry for seed orchards and tree improvement programs [57] and [53]. Pollen banks should ensure the long-term conservation of viable pollen under cryogenic storage and should also have the facilities to collect, conserve, and distribute the stored pollen. Since genebanks operate on very limited budgets, it is difficult to incorporate new approaches, even if the overall germplasm use efficiency is improved. Considering the economic point of view, it is advisable either to integrate the pollen banks in the already existing national or international genebanks or to develop pollen banks only for high-value crops. Many private seed companies have established their own in-house conservation systems for pollen storage for use in breeding and hybrid development. The challenge for developing a pollen bank is to have a system that utilizes the advances made in pollen biology to fulfill the goal of providing pollen samples to the user community rather than developing pollen cryopreservation protocol. A better understanding of the system and subject is required to encourage the use of this methodology in germplasm banks and plant breeding programs. 3.3 Pollen Cryopreservation: Advantages and Disadvantages

The advantages of pollen cryopreservation outnumber its disadvantages. Some of the advantages include the following: • The spatial and temporal isolation of parental species that enforce cross pollination barriers can be overcome. It allows for wide hybridization across seasonal and geographical limitations and reduces the coordination required to synchronize flowering and pollen availability for use in crosses [41]. • Pollen storage can make recurrent and breeding lines immediately available as needed, regardless of the response of material to flowering and planting date. • In order to increase productivity, supplementary pollinations like pollen sprays can be implemented. • In breeding programs, there is no need to grow the pollen parent continuously, thus saving time, land, and labor. • Genetic property can be conserved in small sample sizes and can be a source for germplasm in international exchange programs.

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• Pollen also serves as a source of genetic diversity in collections where it is hard to maintain diversity with seeds (species of low fecundity, large seeds, or seeds that require an investment of labor to store). • Use of conserved pollen lowers the disease transmission when the vectors are pollinators. • In the study of pollen allergy and pollen biology, it can serve as a continuous source of pollen. • Exotic nuclear genetic diversity can be easily received and exchanged through pollen, thereby eliminating the need to go through a long juvenile phase, common in most fruit trees to produce pollen for hybridization at the desired location. Thus, stored pollen can be used to improve breeding efficiency. • Fruit tree pollen is generally required to be stored for controlled crossings, either to achieve the desired breeding objective or to overcome a constraint involved in commercial fruit production. The disadvantages of pollen cryopreservation include the following: • Only part of the genome (n) is conserved. • Cryopreservation protocols not available for recalcitrant pollen species. • Limited pollen production in some species hinders the pollen storage in gene banks due to difficulty in obtaining adequate quantities for storage. • Pollen collection, desiccation, and viability assessment are laborintensive processes. • Pollen processing and viability-testing methods have not been documented and standardized in a manner similar to that of seed testing. • Pollen regeneration requires the presence of associated mother plants to replenish pollen supplies when quantities are depleted or have deteriorated. • There is a need to test the genetic stability of the cryopreserved pollen. 3.4 Requirements and Practical Considerations in Pollen Cryopreservation

Pollen grains are considered as the simplest form amenable to cryostorage. Among the different cryopreservation methods, simple dehydration of pollen grains followed by LN freezing is the most routinely followed one. Dehydration is achieved through the use of silica gel, saturated salt solutions, airflow cabinet. or oven drying. Modern methodologies of vitrification by using Plant Vitrification Solution (PVS) can also be used alternatively for cryoprotection of pollen grains. As stated earlier, the success of pollen cryopreservation depends on various factors including the genotype, type of pollen, time of pollen collection, physiological status

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of the plant, moisture content of pollen, protocol followed, and environmental factors like humidity and temperature. Among these, the most important factor is the moisture content of the pollen. Like any other explants, the pollen should be sufficiently desiccated to moisture contents between 7% and 20% when using 80 to -196 °C temperatures. It is generally accepted that pollen moisture reduction improves long-term storage success, assuming that the pollen has the ability to fully tolerate the dehydration process. The tolerance of pollen to desiccation treatments is also related to pollen morphology. The pollen can be either binucleate or trinucleate depending on the number of nucleus present when the pollen is released from the anther, and it has been studied that about 70% of plant species disperse binucleate pollen, which has reduced metabolic activity, is longer-lived, and can tolerate desiccation. These factors make binucleate pollen readily amenable for cryopreservation without any treatments, whereas 30% of plant species including Poaceae possess trinucleate pollen which needs to be dehydrated and treated for successful cryostorage [58, 59]. The post-cryo thawing is yet another important factor determining the successful retrieval of stored pollen as this is directly related to pollen metabolism and the reactivation of postconservation metabolic processes. Pollen thawing can be done either slowly by exposing them to room temperature or quickly by dipping in a hot water bath for a few minutes depending on the species. 3.5 Uses and Applications

Uses of pollen cryopreservation are numerous, finding its applications in the conservation as well as well as crop improvement programs. Pollen conservation is useful in the breeding programs of species that have a long vegetative period or that bloom a few times a year, or for some plants that propagate vegetatively. Conserved pollen is useful to support reproduction in species with inefficient, ineffective, or non-existent pollinating agents [60]. Stored pollen can be exploited in the field of haploid production as well as in vitro pollination and in vitro fertilization in highly heterogeneous crops with a longer duration. Owing to the stable expression of certain genes in pollen, they can be very well used for gene transfer studies. Pollen cryopreservation ensures the availability of pollen throughout the year enabling biochemical and physiological studies. They are also potential explants for the conservation of genetic diversity in recalcitrant, wild, rare, and endangered species. Being simple to handle with less stringent quarantine regulation, stored pollen qualifies for easy exchange of germplasm across national and international borders. Figure 3 illustrates the various ways in which conserved pollen grains finds use in different fields.

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Fig. 3 Applications of pollen bank

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Present Status of Plant and Pollen Cryopreservation Plant cryopreservation offers considerable advantages in terms of conservation and utilization of genetic resources. As of today, cryopreservation protocols are readily available for a large range of plants ranging from temperate to tropical species. There are over 1750 genebanks worldwide, about 130 of which hold more than 10,000 cryopreserved accessions. The vitrification-based cryopreservation protocols are being rapidly developed for a large number of species globally. Species-specific cryoprotocols are developed using explants like seeds, embryos, embryonic axes, pollen, dormant buds, and vegetative meristems/shoot tips of difficult-toconserve species. Among these, pollen cryopreservation has proved immensely useful not only as an ex situ conservation strategy but also in assisting crop breeding programs. Continuous supply of viable pollen provided by pollen cryobanks removes seasonal, geographical, or physiological limitations of hybridization programs and supports hybrid development between genera and species. World Cryogene banks holding important horticultural species are as follows: 1. National Center for Genetic Resources (NCGRP), Fort Collins, Colorado, USA

Preservation

2. Association Forest Cellulose (AFOCEL), France 3. National Institute of Agrobiological Resources (NIAR), Japan 4. Institute of Plant Genetics and Crop Plant Research (IPK), Germany

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In India, cryobanking is being carried out at ICAR-NBPGR, New Delhi, ICAR-IIHR, Bengaluru, ICAR-CPCRI, Kasaragod, JNTBGRI, Palode, Thiruvanathapuram, ICAR-IISR, Kozhikode, and ICAR-CPRI, Shimla. ICAR-NBPGR is designated as the National Cryogenebank aiming to develop strong linkages with field genebanks of NBPGR Regional Stations to prioritize conservation. Tissue Culture and Cryopreservation Unit at NBPGR has already been designated as “Centre of Excellence” for imparting international training in the field of in vitro and cryopreservation where so far more than 100 trainees have been trained from more than 15 countries. Pollen, which is an interesting material for genetic resource conservation of various species, is stored by several institutes. NBPGR has conserved 576 germplasm accessions belonging to different species in the form of pollen grains (Cryodatabase, NBPGR); ICAR-Indian Institute for Horticultural Research (IIHR, Bangalore) conserves pollen of 700 accessions belonging to 100 species from 15 different families, some of which have been stored for over 15 years [61]. In the United States, the NCGRP (USDA-National Centre for Genetic Resource Preservation) conserves pollen of 13 pear cultivars and 24 Pyrus species [62]. In China, pollen of over 700 accessions of traditional Chinese flower species is conserved under cryopreservation [63]. The field of cryopreservation has advanced to the extent of analyzing and understanding different stresses that are imposed on the plant germplasm and also developing novel cryopreservation protocols to counteract them. This has greatly improved the scope for using this technique across species and genera. Despite the promising results and achievements, the success in the large-scale utilization of cryopreservation for long-term conservation of plant germplasm collections is limited as compared to veterinary or medical field. This is largely because plant conservationists are required to cryopreserve a very broad range of genetic diversity which behaves differentially under cryopreservation. There occurs species-to-species variation in the response of plant material to preand post-cryopreservation treatments, and even within the same species, different varieties and tissue types behave differently under cryopreservation. To date, there is no uniform method that can be applied with minor modifications to a broad range of plant materials. Hence, there is a need of continued research to develop viable and efficient cryopreservation protocols applicable to broad range of species in highly biodiverse and threatened ecosystems. In literatures pertaining to pollen conservation, there exists a gap in laying out of detailed methodology of the whole process which makes the replication of procedures difficult. Thus, there is an explicit need to have comprehensive protocols for pollen cryopreservation of each species so as to ensure repeatability across labs.

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Prospects and Way Forward Cryopreservation has emerged as an important complementary ex situ long-term conservation strategy for diverse germplasm with minimal requirements for cost and labor and maximum safety of stored samples. The method applies to the conservation of sexually and asexually propagated crops, crops with recalcitrant or shortlived seeds, crops with no seeds, plants in the RET category, unique genotypes or genetic stocks, etc. The success of cryopreservation also accounts to its amenability for using a plethora of explant material. Extending this method to the conservation of wild species has contributed immensely to the global breeding programs by ensuring quick availability of donor parents for the development of pre breeding lines. Increased adoption of cryopreservation methods to a large number of species and genera has substantially led to the development of many innovative approaches paving the way for cost- and labor-effective protocols. This will further enhance the acceptability of cryopreservation as a method for long-term storage. Pollen conservation through cryopreservation is a method of integration of traditional breeding method with modern biotechnology wherein pollen conserved through biotechnological approaches is used in traditional breeding for hybrid or progeny development. Pollen cryopreservation holds an important place in the germplasm conservation system ensuring the potential use of stored pollen in multiple fields of research. The establishment of pollen bank integrated with genebanks should be prioritized for ancillary germplasm activities. The response of plant material to the cryopreservation method varies with the species, and a thorough understanding of the mechanisms of damage to the explant upon freezing, maintenance of the structural and genetic integrity, maintenance of viability over different storage periods, etc. is crucial. Thus, the future line of studies should focus on the development of species-specific protocols of cryopreservation as well as pollen conservation that can be adopted by researchers, horticulturists, academicians, institutes, government agencies, and botanical gardens across the continents. Concerted efforts and collaboration between these groups are critical for progress and efficiency.

References 1. Day JG, Harding KC, Nadarajan J, Benson EE (2008) Cryopreservation. In: Molecular biomethods handbook. Humana Press, pp 917–947 2. Hamilton KN, Turner SR, Ashmore SE (2009) Cryopreservation. In: Offord CA, Meagher PF (eds) Plant germplasm conservation in

Australia: strategies and guidelines for developing, managing and utilising ex situ collections. Australian Network for Plant Conservation Inc., ISBN 978–0–9752191-1-9, Canberra, pp 129–128 3. Decruse SW, Seeni S, Pushpangadan P (1999) Cryopreservation of alginate coated shoot tips

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of in vitro grown Holostemma annulare (Roxb.) K. Schum, an endangered medicinal plant: influence of preculture and DMSO treatment on survival and regeneration. CryoLetters 20:243–250 4. Mallon R, Bunn E, Turner SR, Gonzalez ML (2008) Cryopreservation of Centaurea ultreiae (Compositae) a critically endangered species from Galica (Spain). CryoLetters 29:363–370 5. Mandal BB, Dixit-Sharma S (2007) Cryopreservation of in vitro shoot tips of Dioscoreadeltoidea Wall, an endangered medicinal plant: effect of cryogenic procedure and storage duration. Cryo Lett 28:461–470 6. Paunescu A (2009) Biotechnology for endangered plant conservation: a critical overview Romanian. Biotechnol Lett 14:4095– 4103 7. Sen-Rong H, Ming-Hua Y (2009) Highefficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of rare and endangered plant EmmenopteryshenryiOliv. Plant Cell Tissue Org Cult 99:217–226 8. Touchell DH, Turner SR, Bunn E, Dixon KW (2002) Cryostorage of somatic tissues of endangered Australian species. In: Towill LE, Bajaj YPS (eds) Cryopreservation of plant germplasm. Springer Verlag, ISBN 7055–8630, Berlin Heidelberg, pp 357–372 9. Pence VC, Ballesteros D, Walters C, Reed BM, Philpott M, Dixon KW et al (2020) Cryobiotechnologies: tools for expanding ex situ conservation to all species. Biol Conserv. (in press) 10. Panis B, Lambardi M (2005) Status of cryopreservation technologies in plants (crops and forest trees). Proceedings of the International Workshop on ‘The Role of Biotechnology’, pp 43–54, Villa Gualino, Turin, Italy, 5–7. March 2005 11. Wang B, Wang RR, Cui ZH, Bi WL, Li JW, Li BQ, Ozudogru EA, Volk GM, Wang QC (2014) Potential applications of cryogenic technologies to plant genetic improvement and pathogen eradication. Biotech Adv 32: 583–595 12. Reinhoud PJ, Versteege I, Kars I, van Iren F, Kijne JW (2000) Physiological and molecular changes in tobacco suspension cells during development of tolerance to cryopreservation by vitrification. In: Engelmann F, Takagi H (eds) Cryopreservation of tropical plant germplasm. Current research progress and application. International Plant Genetic Resources Institute, ISBN 92–9043–428-7, Rome, pp 57–66 13. Benson EE (2008) Cryopreservation of phytodiversity: a critical appraisal of theory &

practice. Crit Rev Plant Sci 27(3):141–219. h t t p s : // d o i . o r g / 1 0 . 1 0 8 0 / 07352680802202034 14. Harding K (2004) Genetic integrity of cryopreserved plant cells: a review. CryoLetters 25(1):3–22 15. Engelmann F (2004) Plant cryopreservation: Progress and prospects. In Vitro Cell Dev Biol Plant 40:427–433 16. Pence V (2011) Evaluating costs for the in vitro propagation and preservation of endangered plants. In Vitro Cell Dev Biol Plant 47:176–187 17. Volk GM, Jenderek M, Chao CT (2014) Prioritization of Malus accessions for collection cryopreservation at the USDA-ARS National Center for Genetic Resources Preservation. Acta Hortic 112:267–272 18. Engelmann F (2011) Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cell Dev Biol Plant 47:5–16 19. Wang QC, Panis B, Engelmann F, Lambardi M, Valkonen JPT (2009) Cryotherapy of shoot tips: a technique for pathogen eradication to produce healthy planting materials and prepare healthy plant genetic resources for cryopreservation. Ann Appl Biol 154:351–363 20. Benson EE (2008) Cryopreservation theory 2008. In: Reed BM (ed) Plant cryopreservation: a practical guide.ⓒ. Springer 21. Popova E, Shukla M, Kim HH (2020) Root cryobanking: an important tool in plant cryopreservation. Plant Cell Tiss Organ Cult (Online) 144:49–66. https://doi.org/10. 1007/s11240-020-01859-6 22. Reed BM (2008) Plant cryopreservation: a practical guide. Springer, ISBN 978–0–38772275-7, New York 23. Sakai A, Engelmann F (2007) Vitrification, encapsulation-vitrification and dropletvitrification: a review. Cryo Lett 28:151–172 24. Streczynski R, Clark H, Whelehan LM, Ang ST, Hardstaff LK, Funnekotter B, Mancera RL (2019) Current issues in plant cryopreservation and importance for ex situ conservation of threatened Australian native species. Aust J Bot 67(1):1–15 25. Nadarajan J, Pritchard HW (2014) Biophysical characteristics of successful oilseed embryo cryoprotection and cryopreservation using vacuum infiltration vitrification: an innovation in plant cell preservation. PLoS One 9(5):e96169 26. Funnekotter B, Mancera RL, Bunn E (2017) Advances in understanding the fundamental aspects required for successful cryopreservation

Pollen Cryopreservation: Advances and Prospects of Australian flora. In Vitro Cellular & Developmental Biology-Plant 53(4):289–298 27. Ballesteros D, Nebot A, Pritchard HW (2018, March) Cryobiotechnology for the long-term preservation of oak (Quercus sp.) genetic resources. In: III International Symposium on Plant Cryopreservation 1234, pp 37–46 28. Wolkers WF, Oldenhof H (2021) Principles underlying cryopreservation and freeze-drying of cells and tissues. In: Wolkers WF, Oldenhof H (eds) Cryopreservation and freeze-drying protocols. Methods in molecular biology, vol 2180. Humana, New York. https://doi.org/ 10.1007/978-1-0716-0783-11 29. Popova E, Shukla M, Kim HH, Saxena PK (2015) Plant cryopreservation for biotechnology and breeding. In: Al-Khayri JM, Jain SM, Johnson DV (eds) Advances in plant breeding strategies: breeding, biotechnology and molecular tools. Springer International Publishing, Berlin, pp 63–93 30. Panis B, Nagel M, Van den houwe, I. (2020) Challenges and prospects for the conservation of crop genetic resources in field Genebanks, in in vitro collections and/or in liquid nitrogen. Plan Theory 9:1634. https://doi.org/10. 3390/plants9121634 31. Stacey GN, Day JG (2007) Long-term ex situ conservation of biological resources and the role of biological resource centers. In: Cryopreservation and freeze-drying protocols, pp 1–14 32. Cruz-Cruz CA, Gonza´lez-Arnao MT, Engelmann F (2013) Biotechnology and conservation of plant biodiversity. Resources 2:73–95 33. Reed BM (2001) Implementing cryogenic storage of clonally propagated plants. Cryo Lett 22:97–104 34. Panis B, Thinh NT (2001) Cryopreservation of Musa germplasm. In: Escalant JV, Sharrock S (eds) INIBAP technical guideline 5. International Network for the Improvement of Banana and Plantain, Montpellier 35. Ellis D, Skogerboe D, Andre C et al (2006) Implementation of garlic cryopreservation techniques in the national plant germplasm system. Cryo Lett 27:99–106 36. Kim HH, Popova E, Shin DJ et al (2012) Cryobanking of Korean Allium germplasm collections: results from a 10 year experience. Cryo Lett 33:45–57 37. Atmakuri AR, Chaudhury R, Malik SK et al (2009) Mulberry biodiversity conservation through cryopreservation. In Vitro Cell Dev Biol Plant 45:639–649

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38. Fukui K, Shirata K, Niino T, Kashif IM (2011) Cryopreservation of mulberry winter buds in Japan. Acta Hort 908:483–488 39. Towill LE, Forsline PL, Walters C et al (2004) Cryopreservation of Malus germplasm using a winter vegetative bud method: results from 1915 accessions. Cryo Lett 25:323–334 40. Kaczmarczyk A, Rokka VM, Keller ERJ (2011) Potato shoot tip cryopreservation. A review. Potato Res 54:45–79 41. Bajaj YPS (1987) Cryopreservation of pollen and pollen embryos, and the establishment of pollen banks. Int Rev Cytol 107:397–420 42. Towill LE (1985) Low temperature and freezevacuum-drying preservation of pollen. In: Kartha KK (ed) Cryopreservation of plant cells and organs. CRC Press, Boca Raton, pp 171–198 43. Ganeshan S, Rajasekharan PE, Shashikumar S, Decruze W (2008) Cryopreservation of pollen. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, New York, pp 443–464 44. Hanna WW, Towill LE (1995) Long term storage of pollen. In: Janick J (ed) Plant breeding reviews, vol 13. Wiley, Oxford, UK, pp 179–207 45. Barnabas B, Kovacs G (1997) Storage of pollen. In: Shivanna KR, Sawney VK (eds) Pollen biotechnology for crop production and improvement. Cambridge University Press, Cambridge, UK, pp 293–314 46. Berthaud J (1997) Strategies for conservation of genetic resources in relation with their utilization. Euphytica 96:1–12 47. Hoekstra FA (1995) Collecting pollen for genetic resources conservation. In: Guarino L, Ramanatha Rao V, Ried R (eds) Collecting plant genetic diversity. Technical guidelines. CAB International, Wallingford, U.K., pp 527–550 48. Alexander MP, Ganeshan S (1993) Pollen Storage. In: Chadha KL, Pareek OP (eds) Advances in horticulture, vol I. Fruit Crops Part I. Malhotra Publishing House, New Delhi, pp 481–496 49. Ganeshan S, Rajashekharan PE (1995) Genetic conservation through pollen storage in ornamental plants. In: Chadha KL, Bhattacharjee SK (eds) Advances in horticulture, vol 12. Malhotra Publishing House, New Delhi, pp 87–108 50. Rajashekharan PE, Ganeshan S, Srinivasan VR (2003) Polbase: Digitalisation of information management system for pollen cryobanks.

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National Seminar on Bioinformatics & biodiversity data management.TBGRI 15–17 May,2003, Thiruvananthapuram. Book of Abstract-page-10 51. Pinillos V, Cuevas J (2008) Artificial pollination in tree crop production horticultural reviews, vol 34, pp 239–276. Edited by Jules Janick ISBN 9780470171530 _ 2008 J 52. Vithanage HLMV, Alexander DM (1985) Synchronous flowering and pollen storage techniques as aids to artificial hybridization in pistachio (Pistacia ssp). J Hortic Sci 60:107– 113 53. Mercier S (1995) The role of a pollen bank in the tree genetic improvement program in Quebec (Canada). Grana 34(6):367–370 54. Rajasekharan PE (2015) Gene banking for ex situ conservation of plant genetic resources. In: Plant biology and biotechnology. Springer, New Delhi, pp 445–459 55. Rajasekharan PE, Ramanatha Rao V (2019) An overview of horticultural genetic resources diversity, distribution and conservation. In: Conservation and utilization of horticultural genetic resources, pp 3–25 56. Towill LE, Walters C (2000) Cryopreservation of pollen. In: Cryopreservation of tropical plant germplasm, pp 115–129 57. Jett JB, Bramlett DL, Webber JE, Ericksson U (1993) Advances in Pollen Management. In: Bramlett DL, Askew GR, Blush TD, Bridgewater FE, Jett JB (eds) USDA-Forest Service agricultural handbook 698, pp 41–46

58. Hoekstra FA (2002) Pollen and spores: desiccation tolerance in pollen and spores of lower plants and fungi. In: Black M, Pritchard HW (eds) Desiccation and survival in plants. Drying without dying. CABI Publishing, Wallingford, UK, pp 185–205 59. Franchi GG, Piotto B, Nepi M, Baskin CC, Baskin JM, Pacini E (2011) Pollen and seed desiccation tolerance in relation to degree of developmental arrest, dispersal, and survival. J Exp Bot 62:5267–5281 60. Dinato NB, Santos IRI, Vigna BBZ, de Paula AF, Fa´vero AP (2020) Pollen cryopreservation for plant breeding and genetic resources conservation. In: EmbrapaRecursosGene´ticos e Biotecnologia-Artigoem periodic indexado (ALICE) 61. Rajasekharan PE, Ganeshan S (2018, March) Current perspectives on pollen cryopreservation in horticultural species. In: III international symposium on plant cryopreservation 1234, pp 47–56 62. Reed BM, DeNoma J, Chang Y (2000) Application of cryopreservation protocols at a clonal genebank. In: Engelmann F, Takagi H (eds) Cryopreservation of tropical plant germplasm – current research progress and applications. JIRCAS/IPGRI, Tsukuba/Rome, pp 246–249 63. Li DZ, Pritchard HW (2009) The science and economics of ex situ plant conservation. Trends Plant Sci 14:614–621

Chapter 2 Pollen Cryobanking—Implications in Genetic Conservation and Plant Breeding P. E. Rajasekharan and M. R. Rohini Abstract Cryopreservation offers a safe reliable technology for conservation and sustained maintenance of viable pollen. Pollen cryobanking has emerged as a potential tool assisting hybridization programs in difficult-tohybridize species. The supply of viable pollen from pollen cryobanks overcomes the seasonal, geographical, or physiological barriers in natural hybridizations and facilitates hybrid development between genera and species. In addition to assisting hybridization events, pollen cryopreservation also aids in the long-term complementary conservation of various threatened plant species. Although genetic conservation through pollen cryopreservation does not accomplish whole genome conservation, a breeder involved in the genetic enhancement of a given crop can have access to a pollen cryobank facility, for Nuclear Genetic Diversity (NGD) inputs in his amelioration program. Conservation of NGD contained in pollen is desirable for a variety of reasons. Cryopreserved NGDs can be a major source of useful genes for pre-breeding, genetic amelioration programs, hybridization of asynchronously flowering genotypes, biotechnology, and basic studies. Pollen grains also serve as reference material providing phylogenetic evidence in plant systematics. Key words Pollen, Cryopreservation, Nuclear Genetic Diversity, Conservation

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Introduction Pollen cryopreservation is considered the most efficient means of long-term conservation by many workers owing to many advantages like ease of its handling, transportation, storage, and other physiological parameters like low water content, highly resistant exine, and non-vacuolated cells [1]. Alexander and Ganeshan [2] reviewed the work on pollen storage in fruit crops. Hoekstra [3] has assessed the merits and demerits of pollen as a genetic resource. Grout and Roberts [4] detailed the methodology for pollen cryopreservation. Barnabas and Kovacs [5] and Berthoud [6] stressed the importance and need for pollen conservation. Rajasekharan and Ganeshan [7] have reviewed the current status of pollen cryopreservation research and its relevance to tropical horticulture. The method is a boon in the case of perennial tree species as pollen

P.E. Rajasekharan and M.R. Rohini (eds.), Pollen Cryopreservation Protocols, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-2843-0_2, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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grains act as potential germplasm material facilitating easy exchange eliminating long juvenile phase. Implications of pollen cryobanking for plant breeding and germplasm conservation are discussed below. 1.1 Germplasm Conservation

Pollen cryopreservation conserves NGD of genotypes in limited space conveniently. This method of conservation is useful for plants listed as rare and endangered category where no other alternatives are available. The conserved genetic diversity is utilized in further breeding or improvement programs. Pollen conservation is a supplementary strategy for seed or clone conservation of different crops and is not intended to replace them. The success of pollen cryopreservation for genetic conservation purposes depends on many factors, and it is essential that the chosen procedure will ensure the maintenance of high genetic integrity, vigor, and germination percentages [8]. Thus, it is essential to evaluate pollen viability before, during, and after long-term conservation [9].

1.2 Increasing Breeding Efficiency

In many plant species, natural hybridization is hindered by asynchronous flowering of male and female genotypes. This can be avoided when cryopreserved viable pollen is available, facilitating hybrids between genera, species, and genotypes. This could effectively conserve field and greenhouse space. A pollen cryobank for a given crop can provide a constant supply of viable and fertile pollen and can also allow supplementary pollinations for improving seed set. The variability due to daily pollen collection can be nullified [5]. Male sterile populations can be perpetuated by cryostored maintainer pollen, thus avoiding frequent planting of maintainer line. Large-scale consolidation of potential pollen from male parents will ensure an uninterrupted supply of the male gametophyte for hybrid seed production at a given location and pollen can be transported to different locations where seed parents are grown for crossing [10]. Overall, characteristics would be incorporated more rapidly and would improve efficiency in a plant breeding program. Breeding programs may profit from pollen selection strategies [11]. Increased longevity in cryopreservation would ensure goodquality pollen throughout the year for selection.

1.3 Exchange of Pollen (Nuclear Genetic Diversity)

Exchange and transfer of germplasm across international borders becomes easy when the material is dried pollen grains. This method of pollen transfer eliminates the need to raise plant populations and also pollen is subjected to less stringent quarantine restrictions. So, it can be easily shipped and used. Through exchange of pollen, desired crosses can be made directly on the seed parent, allowing introgression of characters at a much faster rate. This would find favor especially in breeding of species with long juvenile phase like Eucalyptus, palms, sugarcane, yam, and other species. A research work done at ICRISAT [12] shows that it is possible to use

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preserved pollen as a means of international transfer of germplasm in situations where seed transfer is not possible for particular accessions or wild species that do not produce flowers due to differences in environmental conditions, as has been observed for perennial Cicer species at ICRISAT. Pollen collected from USDA-ARS, Pullman, which was preserved for 15–20 days, following the method described above, was used in the crossing program at ICRISAT, Patancheru. Cultivated chickpea genotypes ICCV 10 and ICCV 92318 were used as the female parents and pollinated with C. oxyodon, C. microphyllum, C. nuristanicum, and C. macracanthum. 1.4 Integration of Conventional Breeding Methods with Modern Biotechnological Practices

Prolonged pollen viability conferred because of cryogenic storage enables a pollen biotechnologist to carry out basic studies over extended durations on viable and fertile pollen. These studies could include genetic characterization of the male gametophyte, which allows more discrete use of pollen stocks for breeding. Another area of study could be to introduce known sequences of alien DNA in pollen with extended viability and fertility which could have an impact on the understanding of its integration with the male gametophyte prior to use in breeding. Thus, cryopreservation will be beneficial wherever viable pollen is a limiting factor in such studies.

1.5 Genetic Amelioration

The availability of pollen with good quality in a pollen cryobank will provide a constant supply of the same for extended durations. Pollen in such a state can be termed value added by virtue of its potential extended life, for have been able to be kept viable and fertile for extended durations to perform its natural function of fertilization, leading to formation of fruit and seed set, e.g., pollen used in hybrid seed production. The process of genetic characterization further enhances the value of pollen, especially when it is established that it contains specific DNA sequences which are attributable to specific traits, e.g., pollen with trifoliate marker genes in citrus [13, 14].

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Uses of Cryopreserved Pollen in Production Fields Stored pollen can be used in production fields in the following circumstances: 1. Poor pollen producers: In some cases, the male in combination with the female parent yields an excellent hybrid but is difficult to produce because the male is a poor pollen producer and therefore consistency in hybrid seed yield is not obtained. Such male parents can be grown in a favorable environment, pollen

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collected and cryopreserved in advance so that in production plots there is no constraint on the availability of pollen. 2. When the male parent is difficult to grow or is susceptible to some disease or is poor in vigor, this technology can be used. This happens in contract production plots where due to the absence of prior information, the performance of a particular parent is poor, resulting in low pollen yield. This would reflect in low hybrid seed yield. 3. Genetic security for the male parent is ensured. In situations where there is no IPR protection for the parents, security for at least one of the parents is assured. 4. There can be more number of female plants in a production plot. Generally, a ratio of 3:1 female to male plants is maintained in the production plot. Thus, one-third of the plot with male plants would not provide any hybrid seeds. After pollination is completed, these plants are generally uprooted to prevent contaminations as well as to prevent loss of genetic material. 5. When the MS line is used, parental increase of the A line can be made easier by storing the pollen of the B line. The basis of a successful pollen cryopreservation protocol is the appropriate identification of the nature of pollen grains present in a species. The link between pollen and vegetation diversity is complex due to species-specific differences in pollen production and dispersal [15, 16]. The life span of pollen at ambient temperature is considerably shorter, which again varies with species, ranging from a few hours to several months. Thus, it is important to understand the pollen morphology thoroughly before adopting a cryopreservation protocol.

3

Pollen Morphology and Pollen Classification Pollen grains are microscopic particles representing male gametophyte. Pollen grains vary in size, shape, and surface characteristics depending on the plant species. In general, pollen grains have a double wall consisting of a thin inner wall composed of cellulose, termed the endospore (associated with pollen tube formation), and a thick outer wall comprised of sporopollenin, termed the exospore or exine providing protection. The shape and the external features of the exospore are highly variable and are often used to distinguish pollen grain produced by different species. The angiosperm pollen grain evolved to function in its particular environment and thus shows considerable diversity across species both in grain size, cell composition, architecture, and composition of the mature pollen grain wall and in the cytology of the vegetative nucleus and sperm

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Fig. 1 Microsporogenesis leading to bicellular and tricellular pollen production

cells [17]. A major feature that is used to describe angiosperm pollen grains is the number of cells within the grain at maturity or anthesis. Bicellular grains contain a generative cell and a vegetative cell. Tricellular grains have a vegetative nucleus plus two sperm cells formed from the mitotic division of the generative cell prior to pollen maturation (Fig. 1). Bicellular grains have a much longer life span because of their protective structure, low plasma water content, and reduced metabolic activity, whereas the trinucleate pollens are short-lived due to their less resistant wall construction and high moisture content, which can easily be lost by desiccation. This type of pollen has a high rate of metabolism. Bicellular pollens are found in more primitive families, and the occurrence of tricellular pollens in several orders within monocots and dicots suggests that the trait has evolved many times [18]. The longevity of pollen grains also varies considerably between different families as mentioned below: 1. Long-lived pollen (6 months to a year): Gingkoaceae, Pinaceae, Palmae, Saxifragaceae, Rosaceae, Leguminosae, Anacardiaceae, Vitaceae, and Primulaceae 2. Pollen with a medium life span (approximately 1–3 months): Liliaceae, Amaryllidaceae, Salicaceae, Ranunculaceae, Cruciferae, Rutaceae, Solanaceae, and Scrophulariaceae 3. Short-lived pollen (ranging from a few minutes to a couple of days): Alismataceae, Gramineae, Cyperaceae, Commelinaceae, and Juncaceae

4

Pollen Cryopreservation Protocol The objective of a useful pollen cryopreservation protocol is to collect mature pollen from the desired species and conserve using methods which allow retention of its normal function, ultimately assessed by its ability to germinate in vivo and effect normal fertilization [19]. In order to maintain the pollen viability as high as the

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fresh pollen during long-term conservation, it is necessary to follow standardized protocols of collection, drying, storage, and viability pollen tests. Different steps involved in cryopreservation of pollen are as below. 4.1 Pollen collection Methods

Collection and handling of pollen grains in the viable condition is a primary requirement for any experimental study on pollen. Pollen should be collected soon after anthesis from dehiscing anthers, usually in the morning hours [20]. Shelf life is short for pollen collected from immature, aged, or weather-damaged anthers [21]. It is usually more practical to collect anthers in the field and then separate the pollen grains from the anthers in a laboratory environment soon after collection. One should thus refrain from collecting on rainy days. Also, only physiologically mature pollen should be collected, which means one should avoid collecting closed anthers from young flower buds. Immature pollen may lack the capacity to germinate, have reduced vigor, or still be desiccation-sensitive. While collecting pollen for conservation of genetic resources and long-term storage, it is advisable to collect pollen from more than 60 randomly selected flowers, which are fully outcrossed, randomly mating species of the same population; such a sample usually contains 95% of the alleles that occurs at frequency greater than 0.05. Self-incompatibility is not likely to cause any problems when using such pollen samples in crosses. A population of strictly outbreeding, self-incompatible plants will carry S-allele, irrespective of whether the gametophyte or sporophytic system of self-incompatibility is operating; thus, pollinating a mother plant with the collected pollen will not lead to mass rejection [3]. Old pollen from previous sheddings should also be avoided. Outside, pollinators and wind may be serious competitors for the collector, necessitating the bagging of flowers, though temperature and humidity can become very high inside such bags. The collector must also be aware that the time of flower opening and anther dehiscence may be earlier than normal in bags because of the higher temperature [22]. Pollen should be collected as soon as it is shed. Collecting can be done by shaking flowers over folded sheets of paper or into vials or by using small aspirators. All pollen must be processed immediately (within hours) to ensure maximum potential longevity. Collected pollen serves to maintain and preserve the alleles of an individual or population. Sampling strategies have often recommended collecting a set number of individuals per population to ensure that the common alleles are captured. The exact number of individuals that most effectively captures the genetic variation is dependent upon the genetic diversity and life-history traits of the species. In a randomly outbreeding species, a sample of 59 random unrelated gametes from a population is sufficient to attain the

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objective of including in the sample at least one copy of 95% of the alleles that occurred there at frequencies greater than 0.05. This means that when collecting pollen, the benchmark criterion will be 59 individuals per population. Namkoong [23] suggests that collecting pollen from a single tree easily captures the alleles for that genotype; however, it is recommended that a minimum of 68 trees be sampled to represent a wild population. Pollen can also be collected from individual trees within a Field Gene Bank both to conserve alleles specific to each individual and to provide male gametes for breeding purposes. Although only small quantities of pollen are required to capture the genes of an individual, because of the challenges of pollen collection and processing, it might be more efficient to collect larger quantities to ensure its long-term availability to the user community. Pollen collection procedures are not available for many species. Although devices used for pollen collection are described (Stanley and Linskens, 1974), the manual extraction optimized in this study though cumbersome ensures highest purity levels of collected pollen. Extraction of non-sticky pollen by gentle tapping of flowers/ inflorescence was optimized and for species where the pollen is sticky, a camel brush was found more suitable to extract pollen without anther or tapetal tissue debris. 4.2 Pollen Viability Testing

Retention of pollen viability before and after cryopreservation is important for using it in breeding, conservation, and any other applications. Only viable pollen is considered valuable pollen. Assessment of pollen viability is critical for the study of the following aspects of pollination biology: monitoring pollen vigor during storage; genetics and pollen-stigma interaction; crop improvement and breeding programs; gene bank maintenance; incompatibility and fertility studies; and evaluation of pollen germinability after exposure to certain conditions. The terms viability, stainability, vigor, germinability, fertility, and fertilization ability indicate different aspects of pollen potential. There are essentially four approaches to pollen viability testing as follows (Fig. 2).

4.2.1 Staining Techniques

Acetocarmine and cotton blue are among the oldest stains used to assess pollen viability. Positive staining indicates the presence of cytoplasm and nuclei. Whereas lack of staining shows that pollen is non-viable. One commonly used vital stain for testing pollen viability is the fluorogenic ester, Fluorescein Diacetate (FDA). This test measures membrane integrity. Pollen grains fluoresce green when cellular esterase cleaves the FDA [24] (Fig. 3). Since this assay is dependent upon functional membranes, the osmoticum of the FDA staining solution is critical; stain is often dissolved in a 10% to 20% sucrose solution containing boric acid and calcium nitrate to minimize plasmolysis and membrane leakage.

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Fig. 2 Pollen viability testing methods

Fig. 3 FDA or FCR method of testing pollen viability

Procedure Stock solution of FDA is prepared in acetone (2 mg/ mL). Then drops of this stock solution are added to 2–5 mL of sucrose solution until the resulting mixture shows persistent turbidity. The mixture (FDA and Sucrose) should be used within 30 min from preparation. Several tetrazolium-based stains are available for testing pollen viability [25]. The 3-(4,5-dimethyl thiazolyl 1-2)-2,5-diphenyl tetrazolium bromide (MTT) test was shown to give the most

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Fig. 4 TTC method of testing pollen viability

dependable results in a comparison trial using plum pollen [25]. In general, tetrazolium tests measure the ability to reduce colorless tetrazolium to colored formazan, thus identifying pollen that has a capacity for oxidative metabolism [26]. Viable pollen grains turn red due to accumulation of formazan (Fig. 4). Comparisons of viability determined through the use of FDA or tetrazolium and those obtained using in vitro germination or in vivo fertilization tests reveal consistently high correlations, provided pollen is adequately rehydrated prior to testing [27]. FDA has occasionally been shown to give false negative results, where viable pollen appears dead [28]. The tetrazolium test differs from older staining methods such as the acetocarmine test in that extra information on the integrity of the pollen plasma membrane is obtained. The same is true for the vital stain, FDA [28]. Many other vital stains have been developed and proposed over the past 50 years. Stains such as Alexanders, potassium iodide, acetocarmine, aniline blue, and X-gal have been shown to be successful indicators of viability for relatively few species or under specialized conditions [29]. 4.2.2 In Vivo Pollen Germination

Pollen can be germinated in vitro by placing pollen grains onto a medium and measuring the elongation of the pollen tube after a few hours of incubation. Pollen tubes that elongate to a length that is at least the diameter of the pollen grain are considered viable [30]. Automated counting procedures using morphometry software result in pollen counts that are within 5% of visual observations and allow the determination of pollen tube length in addition to the data obtained by eye on tube presence or absence [31]. These automated systems may expedite time-consuming

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assays of in vitro pollen germination. As for viability testing, it is important to implement repeatable and standardized methods and to use dead pollen samples as controls. The optimal temperature for in vitro germination assays can be species dependent. The pollen from many species germinates well at 25 °C; however, differences exist. For example, cotton pollen has an optimum germination temperature of 28 °C to 31 °C [32]. Hence, for the purpose of pollen conservation, such information should be known for the target species. In vitro germination methods utilize pollen immersed in aerated solutions, “hanging” drops, or dispersed on solidified medium. The medium is often that described by Brewbacker and Kwack [33] or a slight modification thereof. Boric acid, calcium nitrate, and sucrose concentrations in the medium might have to be optimized according to species [34, 35]. Commonly used in vitro germination methods are depicted in Fig. 5 and explained as below: (a) Hanging drop culture (cavity slide method): The hanging drop culture method is used with liquid pollen germination medium. A drop of pollen germination medium is placed in a cavity slide, and pollen is dusted over and covered with coverslip with its periphery sealed with vaseline. (b) Sitting drop culture: The sitting drop culture method is one of the most commonly used methods and is simpler than the hanging drop method. In this method, a drop of the liquid

Fig. 5 In vitro pollen germination methods

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culture medium is placed on a slide, and pollen is dusted on the drop. The culture is then maintained in a humid chamber to prevent evaporation. (c) Suspension culture: This method is suitable for large samples. Pollen grains are dusted in a vial containing 2 to 10 mL culture medium and incubated on a shaker and observed for germination by placing a drop on a slide. (d) Surface culture: Pollen grains of many species germinate better on the surface of agar/agarose/gelatin medium or on cellophane placed in contact with culture medium than in liquid medium. After the prescribed duration of incubation (generally 3–6 h) in a humid chamber, record the germination percentage under the microscope for qualitative and quantitative viability estimates. Pollen showing tube lengths longer than the pollen diameter are scored as viable with 400–500 pollen in two replicate drops scored in total. Many a times, for testing the longevity and effectiveness of pollen, staining tests are not enough, some pollen may give reactions but such pollen may not germinate, or may germinate in vitro and produce short tubes. There are also cases when the rate of in vitro germination and the seed set are not correlated. Thus, the stored pollen should be germinated in vitro and in vivo; observation of the passage of pollen tubes through the stigma and style would be more desirable to determine their effectiveness to bring about fertilization and subsequent formation of seeds. Pollen viability estimation Viability or germination percentage of pollen is estimated and scoring is given as per Table 1. Observations are recorded in three replications wherein a minimum of 10 microscopic fields (i.e., approximately 500 pollen grains) are observed per replication. In the staining methods, pollen viability is calculated as per the formula: Pollen viability% =

Number of well stained pollen grains × 100 Total number of pollen

In the in vitro germination method, pollen germination % is calculated as: Pollen germination% =

Number of germinated pollen per field of view × 100 Total number of pollen per field of view

Seed set on male sterile or emasculated inflorescences after pollination with collected pollen is an easy method to determine pollen

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Table 1 Pollen scoring as given by Stanley and Linskens, 1974 Viability

Pollen germination %

Excellent

>80%

Good

60–80%

Moderate

40–60%

Low

20–40%

Poor

100) in the slide which ensures better germination.

References 1. Vikas Gautam M, Tandon R, Ram HYM (2009) Pollination ecology and breeding system of Oroxylum indicum (Bignoniaceae) in the foothills of the Western Himalaya. J Trop Ecol 25(1):93–96 2. Ravikumar K, Ved DK (2000) 100 red-listed medicinal plants of conservation concern in

southern India, 1st edn. Foundation for Revitalization of Local Health Traditions (FRLHT), Bangalore 3. Srithongchuay T, Bumrungsri S, Sripao-Raya E (2008) The pollination ecology of the latesuccessional tree, Oroxylum indicum (Bignoniaceae) in Thailand. J Trop Ecol 24(5):477–484

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4. Ganeshan S, Rajasekharan PE, Shashikumar S, Decruze W (2008) Cryopreservation of pollen. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, New York, pp 281–332. https://doi.org/10.1007/978-0387-72276-4_17 5. Ganeshan S, Rajasekharan PE (2000) Current status of pollen cryopreservation research: relevance to tropical horticulture. In: Engelmann F, Takagi H (eds) Cryopreservation of tropical plant germplasm programs and application. JIRCAS/IPGRI publication, pp 360–365 6. Rajasekharan PE, Ravish BS, Kumar TV, Ganeshan S (2015) Pollen cryo-banking for tropical

plant species. In: Normah M, Chin H, Reed B (eds) Conservation of tropical plant species. Springer, pp 65–75 7. Sharma BK, Jain AK (2016) Phenological studies on Oroxylum indicum in Sikkim. Bionature 36(1):25–29 8. Brewbaker JL, Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am J Bot 50:859–865 9. Alexander MP (1980) A versatile stain for pollen, fungi, yeast and bacteria. Stain Technol 55: 13–18

Chapter 46 Pollen Cryopreservation Protocol for Salacia chinensis Linn P. E. Rajasekharan and R. Harsha Abstract Salacia chinensis is a perennial woody shrub belonging to the family Celastraceae. In this species flowering occurs in the month of January with main pollinators being insects. Pollen is an important entity in breeding program which can be cryopreserved for many years without loss in its fertility. Hence, pollen cryopreservation was carried out to check the feasibility in this species. In vitro viability studies were carried out by hanging drop technique using Brewbaker and Kwack medium containing 25% sucrose solution. Pollen were cryopreserved by packing in butter paper which were enclosed in aluminum pouches, sealed and rapidly plunged into cyobiological system containing liquid nitrogen. Post-viability of cryopreserved pollen grains was carried out to evaluate the suitability for long term conservation of pollen and further use in breeding programs. The details of material and methods used in development of protocol are discussed in this chapter. Key words Salacia chinensis, Celastraceae, Pollen cryopreservation, Liquid nitrogen

1

Introduction Salacia chinensis also called as saptrangi belongs to the family Celastraceae. It is perennial woody climbing shrub with lots of medicinal uses and native to India. It is used in prevention and treatment of arthritis, inflammation, diabetes, obesity, and liver disorders [1]. The leaves are simple, usually opposite, petioled, being glabrous underneath and shiny on the upper face. Leaf shape varies from ovate, oblong, acuminate, elliptic-oblong with acute base and abruptly acuminate leaf apex. Salacia flowers are small and bisexual in nature, occurring as clusters of 2 to 8 units per leaf axil. The flower colors are typically greenish white to greenish yellow, with entire calyx lobes and anthers dehiscing transversely. Salacia fruits occur as pinkish-orange globes when ripe. Each fruit contains one to four almond shaped seeds. The color of the root bark is golden yellow [2]. The popularity of S. chinensis in traditional medicine has led to the indiscriminate harvest of this species in its natural habitat.

P.E. Rajasekharan and M.R. Rohini (eds.), Pollen Cryopreservation Protocols, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-2843-0_46, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Indiscriminative management has led to the status of “near threatened” according to the International Union for Conservation of Nature (IUCN) [3]. It is naturally propagated through seeds which is a slow process and fruit set is a major problem. The depletion of genetic pools and reduced natural resources is the major concern attracting international attention. Conservation of Nuclear Genetic Diversity is an attribute for the RET medicinal plants. Conventional methods of conservation for long term are difficult and non-economical. Low-temperature storage of plant materials can be achieved at freezing temperature with zero cell division and arrested metabolic state. Cryopreservation is highly effective in conserving pollen, as pollen is small in size and tolerant to desiccation because of which it can be cryopreserved for many years without any change in its abilities to pollinate and fertilize [4]. This technique can also be utilized in future breeding programs, where there is an easy access to the diverse gene pool. In this protocol, details regarding flowering, anther dehiscence, pollen collection, germination and cryopreservation studies have been described.

2

Materials

2.1 Laboratory Equipment

1. Compound microscope 2. Stereomicroscope, e.g., Leica MZ12 3. Scanning electron microscope 4. Laminar flow hood 5. Cryotank 6. Drying oven 7. Desiccator

2.2 General Laboratory Supplies

1. Dissection tools, namely, scalpels, forceps, scissors, needles, and paint brushes 2. Routine laboratory supplies such as flasks, disposable transfer pipette, and petri plates 3. Borosil glass measuring cylinders, glass bottles (10 mL) 4. Microscope slides and cover slips 5. Syringes 6. Cryovials (2 mL) 7. Aluminum foil 8. White butter papers 9. Whatman filer paper

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2.3 Chemicals and Solutions

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1. Alexander stain containing ethyl alcohol (95%), malachite green, glycerol, acid fuchsin, phenol, and distilled water for viability testing 2. Pollen medium [5] containing boric acid, 300 ppm; calcium nitrate, 100 ppm; magnesium sulfate, 300 ppm; potassium nitrate, 300 ppm; and sucrose, 15% 3. Starch powder (soluble) 10% 4. Sterilizing solution 0.3 and 0.6% sodium hypochlorite solutions 5. Ethanol, 70% 6. Liquid nitrogen

3

Methods The phenology of the species and the floral biology is to be known before proceeding to pollen collection. Flowering season was observed during January and February months. Peak flowering was observed during the last week of January (Fig. 1). Bright yellowish green coloured flowers are borne in clusters exposing red-colored pollen grains. Anthesis takes place in the morning hours and flowers are in full bloom by 9:00 a.m. Anther dehisces as soon as anthesis occurs. Fully opened flowers are collected to extract pollen grains.

Fig. 1 (A & B) S. chinensis inflorescence, (C) Flowers exposing orange coloured pollen grains

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3.1 Collection of Pollen

Flowers are collected between 9:00–10:00 a.m. in the morning when they are fully open. Red-coloured pollen grains are extracted using the needle and collected in butter paper. Pollen collected is bulked to get uniform in vitro germination. The fresh pollen collected at peak anthesis time (7:00–8:00 am) records the highest germination percentage (see Note 2).

3.2 Dehydration of Pollen

After collection and extraction of pollen, it is packed in the butter paper and kept in desiccator containing zeolite granules for an hour. Moisture content of the pollen grains has to be reduced to 10% to ensure better pollen germination (see Note 4).

3.3 Preparation of Pollen Germination Medium

Pollen medium composition for 100 mL. • Boric acid (10 mg) • Sucrose (15%) • Calcium nitrate (30 mg) • Potassium nitrate (10 mg) • Magnesium sulphate (20 mg) The above chemicals are weighed in micro balance and dissolved in distilled water to make up the volume to 100 mL. Pollen growth media can be stored in refrigerated condition as a stock, and sucrose is added during viability testing (see Note 8).

3.4 Viability Assessment of Pollen

Viability was assessed by initial germination using Brewbaker and Kwack (1963) [5] medium, consisting of 25% sucrose using the hanging drop method. Pollen dispersed in hanging drop position was incubated in a moist chamber at 25 ± 2 °C [6]. After germination, pollen was stained with Alexander stain [7] and viewed under microscope. Germinated pollen was counted and percentage germination computed. Five replicates were assessed with ten microscopic fields taken for count (see Notes 1, 3, 9, 10 and 11). Maximum pollen germination of 93% was observed when fresh pollen grains were used for germination (Fig. 2).

3.5 Cryopreservation of Pollen Grains

Fresh pollen is dried to reduce the moisture content and packed in butter paper covers and then enclosed in an aluminum foil. Presence of moisture crystal in the pollen grain leads to detrimental intracellular ice formation causing cell death. Therefore, moisture has to be reduced to avoid changes in the cell integrity. These covers are labelled, sealed with cellophane tape, and then rapidly plunged into the cryobiological system containing liquid nitrogen (see Notes 5, 6 and 7).

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Fig. 2 In vitro pollen germination of S. chinensis

3.6 PostCryopreservation Viability Assessment

The pollen grains are retrieved from the canisters containing liquid nitrogen at monthly intervals and rapidly thawed at room temperature 25–27 °C for 5 min. Thawing should be as rapid as possible to avoid the phenomenon of recrystallization. The post-viability assessment was carried out to evaluate the pollen germination percentage after cryopreservation. Maximum pollen germination of 87% was observed when cryopreserved pollen grains were used for germination.

3.7 Scanning Electron Microscopy Analysis

Fresh and cryopreserved pollen grains were dehydrated in zeolite granules for 1 h to reduce the moisture content. Further, these pollen were placed directly onto copper double sided tape on the disc surface of polished aluminum stabs and passed through a vacuum evaporator under the scanning electron microscope (Model No. TM 3030 Plus Scanning Electron Microscope). Pollen images were taken at different resolutions. Morphological measurements such as pollen length (μm), pollen width (μm), and pollen perimeter (μm) were recorded in FijiJ software (ImageJ win 64). (Fig. 3) Scanning micrograph images showed no significant variation in shape and size (equatorial length and equatorial width) of pollen after cryopreservation.

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Fig. 3 (A) SEM of fresh pollen (B) SEM of cryostored pollen

4

Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination 1. A stereoscopic microscope with a photo documentation system should be used in the evaluation 2. Pollen collection at peak anthesis time (7:00–8:00 am) is ideal for obtaining highest viability and in vitro germination 3. The procedures as to be carried out in a clean place possibly in a laminar flow chamber 4. The dehydration time can vary according to the size of the pollen grains, exine thickness, and ambient humidity. The moisture of the pollen grains should be between 15 and 30%. A sample of pollen grains after dehydration should be tested for viability [8, 9] 5. The envelopes should be closed carefully so as not to crush the pollen grains and then covered with aluminum foil by plastering, and inserted into canisters. Always leave some open space at the top of canister 6. Direct contact of liquid nitrogen with pollen tissue causes injuries to the exine of the pollen grains, impairing their viability. Therefore, the envelopes should be well sealed to prevent liquid nitrogen from entering 7. The process of immersion in liquid nitrogen and transfer to the cryogenic tank should be carried out as fast as possible, to avoid sudden temperature variations, which can harm the pollen grains

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8. Pollen media with sucrose concentration should be prepared at the time of slide preparation. Use the media within one day of its preparation to avoid contamination 9. The pollen grains should be uniformly distributed on the medium, preferably without puncturing. If the grains are agglomerated, it will be hard to count them 10. After slide preparation, the slides are kept in petri dishes covered with wet filter paper. It maintains the relative humidity and does not allow the pollen media to get dry 11. To ascertain the germination percentage, all the pollen grains in each photomicrograph should be counted. Pollen grains are considered germinated when they have a tube length greater than or equal to the grain diameter References 1. Paarakh PM, Patil LJ, Thanga SA (2008) Genus Salacia: a comprehensive review. J Nat Remed 8(2):116–131 2. Patwardhan A, Pimputkar M, Joshi R (2015) Salacia chinensis L. – utility and propagation techniques. ENVIS Newslett Med Plants 8(1) 3. Ravikumar K, Ved DK (2000) 100 Red-listed medicinal plants of conservation concern in southern India, 1st edn. Foundation for Revitalization of Local Health Traditions (FRLHT), Bangalore 4. Rajasekharan PE, Ravish BS, Kumar TV, Ganeshan S (2012) Pollen cryobanking for tropical plant species. In: Conservation of tropical species, pp 65–75 5. Brewbaker JL, Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am J Bot 50:859–865

6. Ganeshan S, Rajasekharan PE, Shashikumar S, Decruze W (2008) Cryopreservation of pollen. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, New York, pp 281–332. https://doi.org/10.1007/978-0387-72276-4_17 7. Alexander MP (1980) A versatile stain for pollen, fungi, yeast and bacteria. Stain Technol 55: 13–18 8. Silva RL, Souza EH, Vieira LJ (2017) Cryopreservation of pollen of wild pineapple accessions. Sci Hortic 219:326–334. https://doi.org/10. 1016/j.scienta.2017.03.022 9. Souza EH, Souza FVD, Rossi ML (2015) Viability, storage and ultrastructure analysis of Aechmea bicolor (Bromeliaceae) pollen grains, an endemic species to the Atlantic forest. Euphytica 204:13–28. https://doi.org/10.1007/s10681014-1273-3

Chapter 47 Pollen Cryopreservation Protocol For Saraca asoca (Roxb.) De Wilde P. E. Rajasekharan and R. Harsha Abstract Saraca asoca is a perennial tree bearing bisexual, staminate and hermaphrodite flowers which are actinomorphic and borne in axillary corymbs. Flowering in this species begins from early December and extends up to May. Fully opened flowers are collected at 5:30 a.m. in the morning and pollen was extracted. Pollen viability was observed to be maximum in newly opened flowers and decreases progressively upon closure. Viability by in vitro germination assessment was carried out by hanging drop technique using Brewbaker and Kwack medium containing 15 percent sucrose solution. Cryopreservation was carried out by immersing fresh pollen grains in the cryobiological system containing liquid nitrogen (-196 °C). Post-viability assessment was also carried out to check the fertility status of pollen grains after cryopreservation. To use the pollen over extended period, there is a need to store pollen by maintaining its viability. The present study explains the feasibility of pollen cryopreservation in Saraca asoca. Key words Saraca asoca, Pollen germination, Cryopreservation, Liquid nitrogen

1

Introduction Saraca asoca is a perennial evergreen tree belonging to the family Caesalpinaceae. The species is native to Western Ghats and Deccan plateau. This indigenous tree is cultivated for its evergreen foliage and decorative yellow flowers and is used in many Ayurvedic drugs for the treatment of several feminine disorders [1]. The tree is 6–9 m high with glabrous branching, stem bark is rough and uneven due to presence of projecting lenticels. Inflorescence is numerous axillary corymbs with actinomorphic flowers and flowers are bisexual, staminate, and hermaphrodite with or without bracts which persists until maturity (Fig. 1). The calyx is petaloid with four yellow to orange sepals. Corolla consists of four yellow-colored petals which after pollination turns into vermilion. The flowers contain filiform filamentous stamens with purple anthers and ovary is pubescent. The pollinators are majorly the honey bees,

P.E. Rajasekharan and M.R. Rohini (eds.), Pollen Cryopreservation Protocols, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-2843-0_47, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Fig. 1 (A) S. asoca twig with inflorescence, (B) Inflorescence

butterflies, and wasps. The flowers are self-incompatible and exhibits facultative geitonogamy [2]. The tree has immense respect in literatures since it is a sacred tree for Hindus. Also it has potential benefits in Indian system of medicine. The overexploitation and unscientific harvesting from the wild are the alarming causes for the gradual loss of its diversity in its natural habitat. Now, the genus has the status of “globally vulnerable” in the red listed species of International Union for Conservation of Nature and Natural Resources (IUCN) [3]. Hence, there is an urgent need for conservation of the germplasm for the sustainability of the species. Development of multiple seedlings has been reported in Saraca through polyembryony [4]. This species exhibits high frequency of cleavage polyembryony where multiple embryos frequently form from the single zygote. Conservation of this species can be fulfilled through pollen cryopreservation where pollen grains can be conserved for unlimited time interval with all capabilities to pollinate, fertilize, and produce seedlings. These pollen grains can be used in breeding programs and eco-restoration programs.

2

Materials

2.1 Laboratory Equipment

1. Compound microscope 2. Stereomicroscope, e.g., Leica MZ12 3. Scanning Electron Microscope 4. Laminar flow hood 5. Cryotank 6. Drying oven 7. Desiccator

Pollen Cryopreservation Protocol For Saraca asoca (Roxb.) De Wilde

2.2 General Laboratory Supplies

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1. Dissection tools, namely, scalpels, forceps, scissors, needles, and paint brushes 2. Routine laboratory supplies such as flasks, disposable transfer pipette, and petri plates 3. Borosil glass measuring cylinders, glass bottles (10 mL) 4. Microscope slides and cover slips 5. Syringes 6. Cryovials (2 mL) 7. Aluminum foil 8. White butter papers 9. Whatman filer paper

2.3 Chemicals and Solutions

1. Alexander stain containing ethyl alcohol (95%), malachite green, glycerol, acid fuchsin, phenol and distilled water for viability testing 2. Pollen medium (Brewbaker and Kwack) containing boric acid, 300 ppm; calcium nitrate, 100 ppm; magnesium sulfate, 300 ppm; potassium nitrate, 300 ppm; and sucrose 3. Agarose, 1% 4. Starch powder (soluble), 10% 5. Sterilizing solution 0.3 and 0.6% sodium hypochlorite solutions 6. Ethanol, 70% 7. Liquid nitrogen

3

Methods The understaning of phenological events such as bud break, peak flowering period, anther dehiscence, and time taken for flowering are prerequisite for the pollen collection. The flowering primordial in the leaf axis appeared in early December and flower clusters appeared towards the end of May. Peak flowering was observed between February and March. The tree remained in bloom for 3 months [5]. The detailed methods involved in flower collection, pollen extraction, dessication and cryopreservation is given in Fig. 2. In an inflorescence, the flowers are arranged in an acropetal succession and anther dehiscence occurred in the same order. Both hermaphrodite and staminate flowers are observed in the same inflorescence. Anthesis and anther dehiscence is the most important event in the process of flower development. Anthesis was observed in the early morning from 3:00 a.m. and lasted till 5:30 a.m. Only few flowers opened at 7:00–8:00 a.m. The anther dehiscence

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Fig. 2 (A) Inflorescence collected for extraction of pollen, (B) Pollen extracted in the butter paper, (C) Desiccator containing zeolite beads, (D) Canisters plunging into the cryobiological system, (E) Cryotank

coincides with the time of anthesis. Anthers are dithecous and dehiscence occurs from the longitudinal slit. Dehiscence is facilitated by radially enlarged endothecial cells with characteristic fibrous thickenings on their radial walls. Stigma receptivity was observed between 6:30 and 7:30 a.m. soon after the anthesis [6]. 3.1 Collection of Pollen Grains

Flowers are collected at 5:30 am in the morning when they are fully open. Pollen grains are collected immediately within 2 h of anthesis to ensure maximum pollen viability and germination percentage. Pollen grains are extracted from the longitudinal slit end of the anther using the needle and packed in butter paper. Pollen collected is bulked to get uniform in vitro germination (See Notes 2–4).

3.2 Dehydration of Pollen Grains

After extraction, it is labelled and stored in the desiccator containing zeolite beads for 1 h to reduce the moisture content of pollen grains. Moisture content of the pollen grains has to be reduced to 10% to ensure better pollen germination (See Notes 5 and 6).

3.3 Preparation of Pollen Medium

Preparation of Brewbaker and Kwack(100 mL) medium is done by dissolving the different chemicals (boric acid, 300 ppm; calcium nitrate, 100 ppm; magnesium sulfate, 300 ppm; potassium nitrate, 300 ppm) in double-distilled water. 1 mL of this stock solution is used for in vitro germination of pollen grains using different concentrations of sucrose, i.e., 5, 10, 15, 20, 25, and 30 percent [7] (See Note 10).

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Fig. 3 (A) Fresh pollen germination, (B) cryopreserved pollen germination 3.4 Viability Assessment of Pollen

Initial viability was assessed using Brewbaker and Kwack medium containing sucrose using the hanging drop method. Pollen dispersed in hanging drop position was incubated in a moist chamber at 25 ± 2°C for 2 h to ensure maximum pollen germination and pollen tube growth [8]. Maximum pollen germination percentage and pollen tube length was observed at 15% sucrose concentration (See Notes 1, 11–14). After germination, pollen was stained with Alexander stain [9] and viewed under microscope. Germinated pollen was counted and percentage germination computed. Five replicates were assessed with 10 microscopic fields taken for count (Fig. 3). The tree produces large number of pollen grains per flower. The pollen grains were oval to spherical in shape, tricolporate with reticulate exine. The pollen viability was higher when pollen grains were collected 2 h after anthesis. However, the pollen viability declined gradually after 6–8 h of anthesis.

3.5 Cryopreservation of Pollen Grains

After the initial viability assessment, the desiccated pollen were packed in butter paper covers and enclosed in sealed aluminum pouches, which were then rapidly plunged into the cryobiological system containing liquid nitrogen (See Notes 7–9).

3.6 PostCryopreservation Viability Assessment

After cryopreservation, the pollen grains were retrieved from liquid nitrogen and rapidly thawed at room temperature for 3–5 min. The germination test was carried out using Brewbaker and Kwack medium consisting of 15% sucrose using the hanging drop method to know the percentage of germination of cryopreserved pollen. Germination percentage was carried out by counting the number of germinated pollen grains and aborted pollen grains. Difference in the germination profiles of fresh pollen and cryopreserved pollen was estimated.

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Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination 1. A stereoscopic microscope with a photo documentation system should be used in the evaluation. 2. Pollen collection at peak anthesis time is ideal for obtaining highest viability and in vitro germination. 3. Separation of two different male reproductive organs immediately after harvest is important to avoid the mixing up of pollen grains. 4. The procedures should be carried out in a clean place possibly in a laminar flow chamber. 5. The dehydration time can vary according to the size of the pollen grains, exine thickness and ambient humidity. 6. The moisture of the pollen grains should be between 15 and 30%. A sample of pollen grains after dehydration should be tested for viability [10, 11]. 7. Always leave some open space at the top of canister. 8. Direct contact of liquid nitrogen with pollen grains causes injuries to the exine of the pollen grains, impairing their viability. Therefore, the envelopes should be well sealed to prevent liquid nitrogen from entering. 9. The process of immersion in liquid nitrogen and transfer to the cryogenic tank should be carried out as fast as possible, to avoid sudden temperature variations, which can harm the pollen grains. 10. Pollen media with sucrose concentration should be prepared at the time of slide preparation to avoid contamination. 11. The pollen grains should be uniformly distributed on the medium; if the grains are agglomerated, it will be hard to count them. 12. After slide preparation, the slides are kept in petri dishes lined with wet filter paper to maintain relative humidity, and do not allow the pollen media to get dry. 13. To ascertain the germination percentage, the pollen grains in each photomicrograph should be counted. Pollen grains are considered germinated when they have a tube length greater than or equal to the grain diameter. 14. To confirm any morphological variations on surface of pollen grains, observations of freshly collected and cryopreserved pollen have to be done under scanning electron microscope (SEM).

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References 1. Bhalerao SA, Verma DR, Didwana VS, Teli NC (2014) Saraca asoca (Roxb.) De Wilde: an overview. Ann Plant Sci 3(7):770–775 2. Borokar AA, Pansare TA (2017) Plant profile, phytochemistry and pharmacology of Ashoka (Saraca asoca (Roxb.) De Wilde) – a comprehensive review. Int J Ayurvedic Herbal Med 7(2):2524–2541 3. Ravikumar K, Ved DK (2000) 100 Red-listed medicinal plants of conservation concern in southern India, 1st edn. Foundation for Revitalization of Local Health Traditions (FRLHT), Bangalore 4. Singh BG, Sivalingam R, Mahadevan NP, Chevanan G, Warrier RR, Anandalakshmi R, Sivakumar V (2005) High frequency polyembryony in Saraca asoca (Roxb.) De Wilde (Caesalpiniaceae) – a red listed medicinal tree. MyForest 41(4):573–582 5. Chauhan S (2019) Floral and pollination biology of Saraca asoca (Roxb.) De Wilde (Caesalpinioideae). Int J Plant Reprod Biol 11(1): 31–37 6. Smitha GR, Thondaiman V (2025) reproductive biology and breeding system of Saraca

asoca (Roxb.) De Wilde: a vulnerable medicinal plant. Springerplus 5:1–15 7. Brewbaker JL, Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am J Bot 50:859–865 8. Ganeshan S, Rajasekharan PE, Shashikumar S, Decruze W (2008) Cryopreservation of pollen. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, New York, pp 281–332. https://doi.org/10.1007/978-0387-72276-4_17 9. Alexander MP (1980) A versatile stain for pollen, fungi, yeast and bacteria. Stain Technol 55: 13–18 10. Silva RL, Souza EH, Vieira LJ (2017) Cryopreservation of pollen of wild pineapple accessions. Sci Hortic 219:326–334. https://doi. org/10.1016/j.scienta.2017.03.022 11. Souza EH, Souza FVD, Rossi ML (2015) Viability, storage and ultrastructure analysis of Aechmea bicolor (Bromeliaceae) pollen grains, an endemic species to the Atlantic forest. Euphytica 204:13–28. https://doi.org/10. 1007/s10681-014-1273-3

Chapter 48 Pollen Cryopreservation in Stevia rebaudiana Bertoni Laxmi Mastiholi and P. E. Rajasekharan Abstract This chapter focuses on pollen germination in vitro and cryopreservation of Stevia rebaudiana. At the time of anthesis, pollen were collected from the field and pre-storage germination assessment was carried by hanging drop technique using modified Brewbaker and Kwack medium containing different combination and concentration of sucrose and PEG (Polyethylene Glycol). Pollen were then packed in butter paper covers enclosed in sealed aluminum pouches which were rapidly plunged into the cryobiological system containing liquid nitrogen. After 1 week of cryopreservation, the germination test was carried out to know the percentage of germination of cryopreserved pollen. SEM (Scanning Electron Microscopy) analysis of pollen was carried out to detect any morphological changes in pollen structure. The results indicated that there is no significant decline in viability of cryopreserved pollen. Therefore, it can be concluded that cryopreservation of Stevia rebaudiana pollen can be achieved effectively without loss of its viability and can be conserved successfully to pollinate and use in breeding programs to broaden the genetic base through pollen. Key words Stevia rebaudiana, Pollen cryopreservation, In vitro germination, SEM studies

1

Introduction Stevia [Stevia rebaudiana (Bertoni)] commonly referred as sweet leaf, sugar leaf, candy leaf, sweet weed, and honey leaf belongs to the family Asteraceae [1–3] and is an important sugar substitute plant recognized throughout the globe. It has high economic and medicinal potential due to presence of sweetener compounds, i.e., stevioside and rebaudioside. This species constitutes a safe sweetener for diabetic patients. In Stevia rebaudiana there has been limited studies on the pollen. Poor seed germination (10%) posed obstacles towards large scale establishment [4]. Pollen play a fundamental role in fertilization and seed set. Studying pollen in vitro germination and growth of pollen tube is essential for explaining the lack of fertility in spermatophytes. Optimization of in vitro pollen germination protocol is the fundamental requirement to study the pollen

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viability [5]. Pollen viability and germinability in Stevia accessions were important for germplasm conservation as well as conventional breeding. Cryopreservation of pollen offers a simple and effective method of long-term storage. Pollen may be stored in liquid nitrogen for many years without loss of its essential capabilities to pollinate, fertilize, set normal fruit and seed when used in breeding, for controlled pollinations or for the conservation of plant genetic resources [6, 7]. The objective of this chapter is to describe a method for viability assessment and cryopreservation of Stevia pollen.

2 2.1

Material and Methods Plant Material

2.2 Laboratory Equipment Required to Carry Out Experiments

Healthy Stevia rebaudiana Bertoni plants established in the field gene bank of ICAR-IIHR, Bangalore are selected for pollen collection, viability assessment and cryopreservation and field pollination (Fig. 1). 1. Stereomicroscope (Olympus BX43 with DP22 and cellsens standard software) 2. Compound microscope (Olympus binocular) 3. Cryotank 4. Drying oven 5. Zeolite beads

Fig. 1 (A) Stevia plant at full bloom stage, (B) Perfect (hermaphrodite) flower having both male and female organs, (C) Collection of flowers for pollen extraction, (D) Pollen grains extraction from flowers using forceps, (E) Desiccation of pollen grains using zeolite beads, (F) Pollen viability assessment in Brewbaker and Kwack media, (G) Pollen grains are smeared on Brewbaker and Kwack media for germination

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2.3 General Laboratory Supplies

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1. Dissection tools, i.e., scalpels, forceps, scissors, and needles 2. Routine laboratory supplies such as disposable transfer pipette and tips and petri plates 3. Microscope slides (cavity and plane slides) and cover slips 4. Cryovials (2 mL) 5. Aluminum foil or pouches 6. Butter paper

2.4 Chemicals and Solutions Required for Viability Assessment

1. Sucrose 2. Polyethylene glycol (PEG) 3. Boric acid 4. Calcium nitrate 5. Potassium nitrate 6. Magnesium sulfate 7. Distilled water 8. Liquid nitrogen (LN)

2.5 Collection of Pollen

Preliminary information on flowering period of the genotype/variety/accession, anthesis time, pollen viability in natural conditions, stigma receptivity duration, and stage of the flower for pollen collection and emasculation is required to be determined, before standardization of cryopreservation protocol.

2.5.1 Procedure for Pollen Collection

Equipment Brush, butter paper, scissors, marker pen, forceps, needle, petri plate. • Cover the unopened flower buds on the previous day with perforated butter paper bags. • Collect the covered freshly opened flowers from healthy plants in the field between 9 and 11.00 a.m. (Bangalore, India conditions) (see Notes 1 and 2). • Place the collected flowers in butter paper bag, label properly, and bring to the laboratory. • Take a clean dry petri dish and place a butter paper in it. • Gently tap the fully opened anthers on butter paper using forceps. • Remove flower and anther debris and tissues as much as possible (see Note 3). • The extracted pollen was kept 1 h in zeolite beads for desiccation to remove excess moisture.

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Table 1 Composition of Brewbaker and Kwack medium for Stevia rebaudiana pollen viability assessment [8] Compound

Concentration

Sucrose

25%

Polyethylene glycol (PEG)

25%

Boric acid [(H3BO3)]

10 mg/100 ml

Calcium nitrate [Ca(NO3)2·4H2O]

30 mg/100 ml

Magnesium sulfate [MgSO4·7H2O]

20 mg/100 ml

Potassium nitrate KNO3

10 mg/100 ml

2.6 In Vitro Pollen Germination Medium

Equipment Analytical balance, pH meter, refrigerator, beakers, conical flasks (100 mL), glass rods, measuring cylinders (100 mL), petri dish (90 mm dia), spatulas, and tissue paper.

2.6.1 Brewbaker and Kwack Medium (100 mL) Stock Solution Preparation

• Pour 50 ml distilled water in a 100 ml beaker with a magnetic stirring bar. • Add 10 mg of boric acid (100 ppm), 30 mg of calcium nitrate (300 ppm), 20 mg of magnesium sulfate (200 ppm), 10 mg of potassium nitrate (100 ppm) one by one, and dissolve (Table 1). • Make up the volume to 100 mL. • Pour in an autoclaved reagent bottle and autoclave at 121 °C with a pressure of 15 psi (1.06 kg/cm2) and for 15 min (optional). • Take out the medium from autoclave, and allow to cool at room temperature. • Store base solution in refrigerator (4 °C), which can be used up to 1 month.

2.6.2 Preparation of Pollen Germination Media

• For pollen viability assessment, take 1 ml stock solution in a small bottle (see Note 4) • Add 0.25 g sucrose and 0.25 g PEG; dissolve one by one. (25% sucrose and 25% PEG).

2.7 Pollen Germination (Hanging Drop Method )

Equipment Compound microscope, cover slip, filter paper, cavity slide, needle, and petri dish. 1. Place a drop (2 μL) of pollen germination medium solution on a clean cover slip 2. Place a small amount of pollen on the drop using needle (see Note 5) 3. Spread the pollen grains uniformly on a cover slip with a needle; make a homogenous distribution of pollen in the medium 4. Put vaseline on edge of the cover glass, then inverted and rested on the cavity slide

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Fig. 2 (A) Germination of fresh pollen grains, (B) Germination of cryostored pollen grains, (C) SEM (Scanning Electron Microscope) images of fresh pollens, (D) SEM images of cryopreserved pollens

5. Place slides in a petri dish with moist filter paper and cover with lid. (see Note 6) 6. Incubate for some time at room temperature 7. Assess pollen germination and pollen tube growth under a compound microscope (Fig. 2) 8. Pollen germination (%) is determined by dividing the number of germinated pollen grains per microscopic field view by the total number of pollen per field view and expressed as a percentage. Pollen grains were considered germinated if the pollen tube growth exceeds twice the diameter of the pollen grain. 9. At least 10 microscopic field view has to be counted and average was taken for percent pollen germination calculation (see Note 7). 10. Stain germinated pollen with alexander stain by placing a drop of alexander stain and glycerol to germinated pollen grains, and cover slip was then inverted and rested on the plain slide and incubate for 24 h for better visualization under stereomicroscope [9] (see Note 12).

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2.8 Fresh Pollen Moisture Content Estimation

The desiccation period needs to be standardized depending upon the initial and desired moisture levels for pollen cryopreservation. Equipment Analytical balance, hot air oven, moisture meter. • Weigh the empty butter paper on analytical balance (Wa). • Place fresh pollen grains in the pre-weighed butter paper and weigh (Wb). • Keep the petri dish containing above weighed butter paper with pollen in a hot air oven (Wc) and weigh dried pollen at different time intervals until it reaches to constant weight. We can also use moisture meter for percent moisture estimation. • Moisture content (MC) of fresh pollen is calculated using the following equation: MC of fresh pollen = ½ðW b - W c Þ=ðW b - W a Þ × 100:

2.9 Desiccation of Pollen

Fresh pollen collected in butter paper cover is placed in zeolite beads for 1 h to remove excess moisture, and these desiccated pollen were used for germination and cryopreservation (see Note 8).

2.10 Cryopreservation

Equipment Aluminum foil strips, butter paper, tape, labels, marker pen, forceps, cryotank, canisters, and liquid nitrogen (LN). • Place the desiccated pollen on butter paper pouches, and they are sealed in aluminum pouches and packed with tape and labelled with crop name, variety, and date and time of pollen collection (see Note 10). • Place the pouches with desiccated pollen in a canister; close the lid of the cryocans before plunging into LN (see Note 9). • Place the canisters in a cryotank till further use.

2.11 Retrieval and Regrowth of Cryopreserved Pollen

• Remove the cryostored pollen samples from cryotank and thaw (slow thawing) by keeping at room temperature (25 °C) for 30 min. • Assess germination of cryopreserved pollen (post-storage viability assessment) as described in Subheading 2.7.

2.12 Fertility Assessment in Field

• In Stevia tiny white florets are perfect (hermaphrodite) flowers having both male and female organs; emasculate the unopened flower bud by removing sepals and petals and un-dehisced anthers using forceps, between 4.00 and 5.00 p.m. • Cover the emasculated buds with perforated butter paper bags (see Note 16). • Next day morning, take out the crystored pollen from cryotank and thaw (as described in Subheading 2.10). • In field, take out the pollen from butter paper, and dust the cryopreserved pollen on the receptive stigma of emasculated

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buds during the peak anthesis period (i.e., between 9 to 11 a.m.) using soft camel brush (see Note 14). • Bag the pollinated buds with perforated butter paper and label it properly. • For controls, dust the fresh pollen on the stigmas of emasculated buds. • Periodically monitor and record observations on number of fruits set to maturity, number of seeds per fruit and number of aborted seeds etc. 2.13 Scanning Electron Microscopy Analysis

3

Fresh pollen and cryopreserved pollen were dehydrated in zeolite granules for 1 hour to reduce the moisture content. Further, these pollens were placed directly onto the copper double sided tape on the disc surface of polished aluminium stabs and passed through a vacuum evaporator under the SEM (Model No. TM 3030 Plus Scanning Electron Microscope). Pollen images were taken at different resolutions. Morphological measurements such as pollen length (μm), pollen width (μm) and pollen perimeter (μm) were recorded in Fiji J software (Image J win 64). Scanning micrograph images showed no significant variation in shape and size (equatorial length and equatorial width) of pollen after cryopreservation (Fig. 2c, 2d).

Notes 1. Pollen should be harvested during peak flowering period and soon after anthesis, usually in the morning hours (8–10 a.m.) on a bright sunny day 2. Pollen should not be collected from infected or insect pestdamaged flowers, on a rainy day or if it had rained overnight 3. Pollen should not be forced from anther and pollen should be free of anther debris 4. Check for the contamination before using the stock solution 5. Excess amount of pollen per drop of medium may lead to competition and result in low pollen viability 6. Moist filter paper should be placed in a petri dish to avoid drying of medium 7. A minimum of three replications with 10 microscopic fields per replication and a minimum of 300 pollen grains to be counted 8. During desiccation, monitoring of the sample should be done at different intervals to identify optimum desiccation period to ensure high pollen viability with low moisture content. Excessive desiccation can lead to a loss in viability 9. Direct contact of pollen grains with LN may causes injuries to the exine of the pollen grains, impairing their viability. Hence,

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canisters lid should be closed properly before plunging into the LN 10. The aluminum foil pouches containing butter paper (filled with pollen) should be sealed with tape carefully to prevent liquid nitrogen entering to avoid any tissue injuries 11. Use stereoscopic microscope with a photo documentation system for better interpretation 12. Stored alexander stain has to use for staining for better visualization 13. The cryopreserved pollen grains should be removed from the liquid nitrogen and taken to the place for pollination immediately 14. To perform the emasculations and pollination, the tweezers and brush should be cleaned with 70% ethanol to prevent mixing of pollen grains of different genotypes 15. The date of pollination and the pollinated flowers should be labeled properly 16. The female flower (receptor) should be protected one day prior to anthesis, to avoid contamination with undesirable pollen grains 17. Crytanks have to filled regularly with liquid nitrogen at 15 days interval to avoid drying of pollen

Acknowledgments I am very much grateful to the ICAR-IIHR, Bangalore, for providing all the essential laboratory and field facilities to conduct pollen viability assessment and cryopreservation work. References 1. Brandle JE, Telmer PG (2007) Steviol glycoside biosynthesis. Phytochemistry 68(14): 1855–1863 2. Madan S, Ahmad S, Singh GN, Kohli K, Kumar Y, Singh R, Garg M (2010) Stevia rebaudiana(Bert.) Bertoni - A review. Indian J Nat Prod Resour 1(3):267–286 3. Soejatro DD (2002) Botany of stevia and Stevia rebaudiana. In: Stevia, Kinghorn AD (eds) Medicinal and aromatic plants-industrial profiles, vol 19. Taylor & Francis, London, p 18–40 4. Abdullateef RA, Zakaria NH, Hasali NH, Osman M (2012) Studies on pollen viability and germinability in accessions of Stevia rebaudianaBertoni. Int J Biol 4(3):72–79 5. Vishwakarma PK, Linta V, Vasugi C, Rajasekharan PE (2020) Effect of cryopreservation

on pollen viability, fertility and morphology of different Psidium species. Cryobiology:1–7 6. Ganeshan S, Rajasekharan PE, Shashikumar S (2008) Cryopreservation of pollen. In: Plant cryopreservation, pp 443–464 7. Rajasekharan PE, Ganeshan S (2018) Current perspectives on pollen cryopreservation in horticultural species. Acta Hortic:47–56 8. Brewbaker JL, Kwack BH (1964) The calcium ion and substances influencing pollen growth. In: Linskens HF (ed) Pollen physiology and fertilization. Elsevier North Holland, Amsterdam, pp 145–151 9. Alexander MP (2009) A versatile stain for pollen fungi, yeast and bacteria. Stain Technol 55(1): 13–18

Part V Plantation Crops

Chapter 49 Methods for Cryopreserving of Date Palm Pollen Annie Carolina Arau´jo de Oliveira, Ana da Silva Le´do, MaryLou Polek, Robert R. Krueger, Ashley Shepherd, and Gayle M. Volk Abstract On the matter of conservation, pollen is a secure alternative to maintain the variability of date palm (Phoenix dactylifera L.) genetic resources. This chapter describes a protocol developed to cryopreserve pollen from trees in the USDA-ARS National Plant Germplasm System (NPGS). Pollen viability levels were higher prior moisture adjustment content over saturated salts solutions of Ca(NO3)2(46% RH) or MgCl2(33% RH) at 23
C. After storage in liquid nitrogen, hydration over H2O for 2 h, inoculation on Marquard medium and incubation at 23
C overnight were identified as optimized in vitro germination conditions. These data suggest that date palm pollen can be successfully stored in genebanks using cryopreservation technique. Key words Phoenix dactylifera L., Liquid nitrogen, Germination medium, Viability assessment

1

Introduction Date palm (Phoenix dactylifera L.), Arecaceae, is a traditional crop in certain hot and humid areas, growing in many countries around the world, such as Egypt, Iran, Argelia, Saudi Arabia, and Iraq [1]. In the late eighteenth century, it was also introduced in the United States and Mexico, where they are well established [2]. The species is cultivated for its edible fruits, rich in carbohydrates, especially sugars and antioxidants [3]. Dates are the major source for commercial production. However, the industry is comprised of only a few elite cultivars which has been increasing genetic erosion [4]. The National Clonal Germplasm Repository for Citrus and Dates (NCGRCD) in Thermal, CA, and the National Laboratory for Genetic Resources Preservation (NLGRP) in Fort Collins, CO, are part of the USDA-ARS National Plant Germplasm System (NPGS) [5, 6]. Both play a critical role in maintaining date palm important alleles through field collections and tissue culture,

P.E. Rajasekharan and M.R. Rohini (eds.), Pollen Cryopreservation Protocols, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-2843-0_49, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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respectively. Nevertheless, a few challenges towards in vitro approaches need to be overcome, such as the possibility of somaclonal variation. Meanwhile, date palm pollen has demonstrated to be a valuable resource for conservation. Due to the lack of flowering synchrony during the breeding season, male flowers usually emerge before female flowers, making it necessary to store pollen to perform crosses [7]. Hence, genebanks and date palm breeders benefit by having clearly methods for the preservation of pollen. Conventional methods, involving room temperature (25–30
C) and low-temperature (4
C, 20
C, 80
C) storage, have been successfully reported by Shaheen et al. [8], Boughediri et al. [9], Maryam et al. [10], Anushma et al. [11], and El Kadri and Ben Mimoun [12]. Cryostorage of date palm pollen was achieved by Tisserat et al. [13], Mortazavi et al. [14] and Anushma et al. [11]. Herein, the water levels of the tissues when exposed to low temperatures is known for influence pollen longevity. Connor and Towill [15] reported that these levels must be reduced to 15%. Moisture content adjustments can be achieved by desiccating the material over saturated salts or by placement in low-humidity environmental chambers [16, 17]. Germination in vitro is one of the most practical techniques for assess pollen viability. The germinability is measured by the elongation of the pollen tube in a specific culture medium under controlled conditions [18]. The addition of nutrients, such as sucrose and boron, are often required [14]. According to Maryam et al. [10], pollen germinates well at 25
C. In general, previous analyses regarding these factors can be helpful while implement a new pollen conservation program. The aim of this chapter is to describe a method for cryopreservation of date palm pollen.

2

Materials Tools such as tweezers, scalpels, and spatulas are necessary. Equipment is listed below. Collection

1. Trays, filter paper sheets, sieves, and sealable storage containers.

2.2 Determination of Moisture Content

1. Saturated salts of calcium nitrate (Ca(NO3)2; 46% RH) and magnesium chlorite (MgCl2 C; 33%RH), desiccators, and petri dishes (30  15 mm).

2.1

2. Aluminum foil envelopes (2  2 cm), drying oven (90
C), and precision scale balance.

Methods for Cryopreserving of Date Palm Pollen

2.3

Cryostorage

2.4 Viability Assessment 2.4.1

Culture Medium

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1. Polypropylene cryotubes (4 mL), cryoboxes, and liquid nitrogen. 1. Marquard medium [19]: calcium nitrate dihydrate (0.03%Ca (NO3)2• 2H2O), boric acid (0.01% H3BO3), potassium nitrate (0.01% KNO3), magnesium sulfate heptahydrate (0.02% MgSO4• 7H2O), 15% sucrose, 2% agarose, and distilled water. 2. Precision scale balance, beaker, magnetic stirrer, graduated cylinder, Erlenmeyer flask, aluminum foil, autoclave, disposable transfer pipette, petri dishes (30  15 mm), laminar flow chamber.

2.4.2 Hydration/ Rehydration Conditions

1. Petri dishes (150  15 mm and 30  15 mm), filter paper sheets, distilled water.

2.4.3 Germination In Vitro

1. Petri dishes with Marquard medium, brush.

3

2. Microscope. In our studies, a 10 objective in a Leica DM LM (Buffalo Grove, IL, USA) was used.

Methods The following procedures should be carried out on a bench disinfected with 70% ethanol or, when described, in a laminar flow chamber.

3.1

Collection

1. Keep mature unopened spathes at room temperature (23
C) or in a dry outdoor environment (we have had good success with spathes that opened in California in a “garage” type setting) until their natural opening (Fig. 1a). 2. Shake the inflorescences over trays lined with filter paper sheets to release anthers with pollen (Fig. 1b). 3. Sieve the collected material to separate pollen from anthers. Pollen is then collected in sealed plastic tubes and prepared for long-term preservation. In our studies, we observed that pollen can be harvested up to 4 days after spathe opening if processing delays are encountered without any significative viability lost. (Fig. 1c)

3.2

Moisture Content

1. Transfer pollen to petri dishes (30  15 mm) and adjust the moisture content over saturated salts of Ca(NO3)2(46% RH) or MgCl2(33% RH) in desiccators for 18 h at room temperature (23
C) (Fig. 1d). 2. After 18 h, set small amounts (1.0–5.0 mg) of pollen in aluminum foil envelopes (2  2 cm), and weigh before and after

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Fig. 1 (a) Spathe after natural cracking; (b) anthers with pollen on a tray lined with filter paper sheet; (c) sifting pollen from anthers; (d) pollen in petri dishes placed over saturated salts of Ca(NO3)2 or MgCl2 for moisture adjustment; (e) drying oven at 90
C; (f) precision scale balance for moisture content determination

4 days of drying (90
C), on a precision scale balance (Fig. 1e– f). 2. Moisture content (MC) is calculated according to the equation MC ¼ [(FW  DW)/(DW  TW)], where MC is the moisture content (g g1dw), FW is the fresh weight (g), DW is the dry weight (g), and TW is the tare weight (g) (see Note 1). 3.3

Storage

1. Moisture-adjust fresh pollen over saturated salt solutions of Ca (NO3)2 or MgCl2 for 18 to 24 h at room temperature (23
C). 2. After moisture-adjustment, place pollen into cryotubes (4 mL), and store the cryotubes into cryoboxes. 3. Immerse the cryoboxes into the cryotanks filled with liquid nitrogen at 196
C. Precautions Identify your material while dealing with one or more samples. Seal the cryotubes and cryoboxes well to prevent the entering of liquid nitrogen.

Methods for Cryopreserving of Date Palm Pollen

3.4 Retrieval and Rewarming 3.5 Viability Assessment 3.5.1 Medium Preparation

523

1. Warm cryotubes for 15 min at room temperature (23
C).

To prepare 250 mL of Marquard medium: 1. In a 250 mL beaker, add 0.075 g of Ca(NO3)2.2H2O, 0.025 g of H3BO3, 0.025 g of KNO3, 0.05 g of MgSO4.7H2O, and 37.5 g of ultrapure sucrose. Use a precision scale balance. 2. Add a small volume of distilled water almost to 250 mL and stir the solution until homogeneous. 3. Complete the volume to 250 mL with distilled water using a graduated cylinder. 4. Add 5 g of agarose to a 500 mL Erlenmeyer flask. Then, mix in the previously solution. It is not necessary to adjust solution pH. 5. Seal the Erlenmeyer flask containing the medium with aluminum foil and sterilize by autoclaving at 121
C for 20 min. 6. In a laminar flow chamber, dispense 5 mL of the culture medium in petri dishes (30  15 mm) with the help of a disposable transfer pipette (see Note 2)

3.5.2 Hydration/ Rehydration Conditions

Pollen must be hydrated/rehydrated in a saturated H2O (100% RH) environment. 1. Line petri dishes (150  15 mm) with filter paper sheets moisten with distilled water. 2. Add small amounts of pollen in petri dishes (30  15 mm). Leave them open. 3. Distribute the small petri dishes into the large ones. Keep them closed. 4. Incubate the petri dishes for 2 h at room temperature (23
C).

3.5.3 In Vitro Germination

After incubation, pollen is germinated on culture medium. 1. With the aid of a brush, dust the rehydrated pollen on the Marquard medium in petri dishes. 2. Keep the petri dishes at room temperature (23
C) overnight in the dark. Precautions Distribute the pollen uniformly on the culture medium. If the grains are agglomerated, it will be hard to count.

3.5.4 Scoring Pollen Viability

1. Determine pollen viability by counting 100 pollen grains, and determine the number that had pollen tubes that were at least the length of the pollen grain, using a microscope (Fig. 2).

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Fig. 2 In vitro germination of Date palm pollen. 2

Precautions Germination must be evaluated in the interval of 18–24 h after inoculation on the culture medium; otherwise, microorganism growth may occur, as a result of the high sucrose concentrations and application of non-sterile pollen.

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Notes 1. According to Oliveira et al. [20], pollen desiccated for 18 h at 23
C, over a saturated salt solution of MgCl2, had a lower moisture content (4.65%) than that of pollen desiccated over a saturated salt solution of Ca(NO3)2(6.22%). 2. Use the culture medium only after complete cooling, and within 1 month after preparation. Storage can be done at 4
C.

Acknowledgments We thank Vince Samons for the assistance in date palm spathe collection and orchard maintenance. We acknowledge the Coordination for the Improvement of Higher Education Personnel (CAPES) for granting the doctoral scholarship to A.C.A de Oliveira. USDA is an equal opportunity employer. Any mention of trade names of commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.

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References 1. FAOSTAT Food and Agriculture Organization Corporate Statistical Database (2020) Domains: production, crops, elements: production quantity, item: dates. http://www.fao. org/faostat/en/#data. Accessed 19 Nov 2020 2. Wright GC (2016) The commercial date industry in the UnitedStates and Mexico. Hort Sci 51(11):1333–1338. https://doi.org/10. 21273/HORTSCI11043-16 3. Ghnimi S, Umer S, Karim A, Kamal-Eldin A (2017) Date fruit (Phoenixdactylifera L.): an underutilized food seeking industrial valorization. NFS J 6:1–10. https://doi.org/10. 1016/j.nfs.2016.12.001 4. Chao CT, Krueger RR (2007) The date palm (Phoenix dactyliferaL.): overview of biology, uses, and cultivation. Hort Sci 42(5): 1077–1082. https://doi.org/10.21273/ HORTSCI.42.5.1077 5. Krueger R (2015) National date palm germplasm repository. Plant Anim Genome 1:560 6. Byrne PF, Volk GM, Gardner C, Gore MA, Simon PW, Smith S (2018) Sustaining the future of plant breeding: the critical roleof the USDA-ARS National Plant Germplasm System. Crop Sci 58:451–468. https://doi.org/ 10.2135/cropsci2017.05.0303 7. Mesnoua M, Roumani M, Bensalah MK, Salem A, Benaziza A (2018) Optimization of conditions for in vitro pollen germinationand pollen tube growth of date palm (Phoenix dactyliferaL.). J Fundam Appl Sci 10(1):158–167. https://doi.org/10.4314/jfas.v10i1.11 8. Shaheen MA, Nasr TA, Bacha MA (1986) Date palm pollen viability in relation to storage conditions. In: Proceedings of thesecond symposium on the date palm in Saudi Arabia, vol 1, pp 331–336 9. Boughediri L, Cerceau-Larrival M-T, Dor J-C (1995) Significanceof freeze-drying in long term storage of date palm pollen. Grana 34(6):408–412. https://doi.org/10.1080/ 00173139509429470 10. Maryam M, Jaskani MJ, Fatima B, Haider MS, Naqvi AS, NafeesM AR, Khan IA (2015) Evaluation of pollen viability indate palm cultivars under different storage temperatures. Pak J Bot 47(1):377–381 11. Anushma PL, Vincent L, Rajesekharan PE, Ganeshan S (2018) Pollenstorage studies in

date palm (Phoenix dactylifera L.). Int J Chem Stud 6(5):2640–2642 12. El Kadri N, Ben Mimoun M (2020) In vitro germination of different date palm (Phoenix dactylifera L.) pollen sources from southern Tunisia under the effect of three storage temperatures. Int J Fruit Sci:1–11. https://doi. org/10.1080/15538362.2020.1815116 13. Tisserat B, Ulrich JM, Finkle BJ (1983) Survival of Phoenix pollengrains under cryogenic conditions. Crop Sci 23(2):254–256. https:// doi.org/10.2135/cropsci1983. 0011183X002300020017x 14. Mortazavi SMH, Arzani K, Moieni A (2010) Optimizing storage and in vitro germination of date palm (Phoenix dactylifera) pollen. J Agric Sci Technol 12:181–189 15. Connor KF, Towill LE (1993) Pollen-handling protocol and hydration/dehydration characteristics of pollen for application to longtermstorage. Euphytica 68(1–2):77–84. https://doi.org/10.1007/BF00024157 16. Volk GM (2011) Collecting pollen for genetic resources conservation. In. In: Guarino L, Ramanatha VR, Goldberg E (eds) Collecting plantgenetic diversity: technical guidelines. Bioversity International, Rome, pp 1–10 17. Walters C (2015) Orthodoxy, recalcitrance and in-between: describing variation in seed storage characteristics using threshold responsesto water loss. Planta 242:397–406. https://doi. org/10.1007/s00425-015-2312-6 18. Franzon RC, do Raseira MCB (2006) In vitro pollen germination of Rio-Grande-Cherry (Eugenia involucrata DC). Rev Bras Frutic 28(1):18–20. https://doi.org/10.1590/ S0100-29452006000100008 19. Marquard RD (1992) Pollen tube growth in Carya and temporalinfluence of pollen deposition on fertilization success in pecan. J Am Soc Hortic Sci 117(2):328–331. https://doi.org/ 10.21273/JASHS.117.2.328 20. de Oliveira ACA, da Silva LA, Polek M, Krueger R, Shepherd A, Volk GM (2020) Optimization of in vitro germination and cryopreservation conditions for preserving date palm pollen in the USDA National Plant Germplasm System. Plant Cell Tissue Organ Cult 1–10. https://doi.org/10.1007/ s11240-020-01907-1

Chapter 50 Cryopreservation of Coconut and Arecanut Pollen Anitha Karun, K. S. Muralikrishna, K. K. Sajini, and M. K. Rajesh Abstract Coconut (Cocos nucifera L.) and arecanut (Areca catechu L.) are important commercial crops sustaining the life of millions of small and marginal farmers. The coconut palm (Arecaceae family), cultivated across the tropical regions of the world, is monotypic and the genetic diversity is distributed amongst various ecotypes and landraces. Areca catechu is one of the commercially important palms, also belonging to the family Arecaceae and it is the only cultivated species in the genus Areca. There exists a vast potential for implementation of in vitro technologies for conservation of coconut and arecanut genetic resources in addition to various exsitu and insitu approaches. Through pollen cryopreservation, pollen grains from desirable palms can be stored over a long period of time in addition to storage of multiple genotypes in small space for their future utility in case of threat from biotic and abiotic factors. The protocol for coconut cryopreservation, developed at Indian Council of Agricultural Research-Central Plantation Crops Research Institute (ICAR-CPCRI), involves desiccation of male flowers at 40°C for 24 h followed by pollen extraction. The protocol is simple and highly effective for the implementation of pollen cryobank to complement the field germplasm collections as well as for its utilization in hybridization programs. Besides this, a protocol has also been developed for pollen collection and its desiccation, in vitro germination, pollen cryopreservation, and its viability and fertility assessment in arecanut. Using this procedure, pollen extraction could be done from fully opened male flowers following desiccation at room temperature. Key words Arecanut, Coconut, Cryopreservation, Desiccation, Moisture content, Pollen, Viability

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Coconut Coconut palm (Cocos nucifera L.; Arecaceae; 2n = 32) is monoecious and carries numerous male flowers and few female ones. Each coconut male flower contains around 272 million pollen grains [1]. Fresh coconut pollen grains are spherical and smooth, but after a few seconds, they turn elliptical with a longitudinal groove in the middle on exposure to dryness [2]. Under natural conditions, the lifespan of fresh coconut pollen is only few days [3]. Coconut pollen is mainly used for breeding programs for production of hybrids and also for conservation of diverse

P.E. Rajasekharan and M.R. Rohini (eds.), Pollen Cryopreservation Protocols, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-2843-0_50, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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genotypes. Even though partial dehydration can prolong coconut pollen viability for a short period, cryopreservation is the only available option for its long-term storage [4]. Cryopreservation facilitates long-term storage of coconut pollen from palms of distinguishable characteristics that can be utilized for future breeding programs and also for conservation of diverse genotypes. Pollen collection and processing are important components for a pollen bank [5]. Moisture content of pollen grains at the time of dispersion varies among different families with most recorded value in the range of 15–35% fresh weight [6]. It is necessary to desiccate pollen to a minimal value to retain its viability after storage in liquid nitrogen [7]. Coconut pollen cryopreservation protocol has been standardized at ICAR-CPCRI [8]. The key elements of this protocol are collection of inflorescence with pollen at dehiscence stage (few opened male flowers at the tip of spikes), drying of male flowers, pollen recovery, and germination test and fertility assessment. No significant variation was observed for pollen germination and tube length in desiccated and cryopreserved pollen from West Coast Tall (WCT) and Chowghat Orange Dwarf (COD) palms [8]. Moreover, germination and vigour of cryopreserved pollen was found to be higher than in non-cryopreserved pollen. The efficacy of the protocol was confirmed by the production of normal seed test by using pollen cryopreserved for 7 years. Report on coconut pollen cryopreservation is scarce. Research group from Brazil stored the pollen grains of Brazil Green Dwarf (BGD), Brazilian Tall (BRA) and Cameroon Red Dwarf (CRD) coconut accessions under different conditions such as refrigerator (-4 °C); freezer (-20 °C); ultra-freezer (-80 °C) and liquid nitrogen (-196 °C). Pollen grain viability was promoted under refrigerator (-4 °C), freezer (-80 °C) and liquid nitrogen (-196 °C) conditions for a storage period up to 60 days [9].

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Materials and Methods

2.1 Male Flower Extraction

1. Sharp knife or scissors 2. Pollination bag 3. Polythene bag 4. Marking pen

2.2 Drying of Male Flowers

1. Petri dish 2. Aluminum foil 3. BOD incubator 4. 2 Sieve (0.2 mm pore size) for collecting pollen from the dried male flower.

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2.3 Pollen Germination Studies

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1. Micro slides 2. Beaker 3. Measuring cylinder 4. Cotton 5. Petri dish 6. Germination medium 7. Compound microscope with image analyzer 8. Micro-balance

2.4

Cryopreservation

1. Cryovials 2. Canisters 3. Cryostick 4. Cryopen 5. Cryotank with liquid nitrogen

2.5 Male Flower collection and Extraction of Pollen

In coconut, once in every 20–25 days, a fresh inflorescence emerges out and each spikelet sheds pollen continuously for 18–25 days depending upon the variety and season. Procedure for extraction and collection of pollen for cryopreservation: (i) The inflorescence is bagged to prevent contamination from foreign pollen once it completely emerges out of the leaf axil and just before the natural opening of the male flowers starts. Normally, it is 6–7 days before the collection of the spikelets. (ii) When the male flowers are about to open from the tip, the spikelets are collected in a polythene bag on a bright sunny day between 8 and 10 AM and label (Fig. 1a, b). (iii) The male flowers are stripped from the spikelets to an aluminum foil placed on a petri dish (Fig. 1c).

2.6 Dehydration of Pollen

(i) The petri dish with male flowers is placed in an incubator set at 40 °C for 24 h (Fig. 2a). (ii) The pollen is extracted by sieving (sieve of 0.2 mm pore sieve) the dried male flowers (Fig. 2b, c).

2.7 Assessment of Pollen Viability Before Storage in Liquid Nitrogen

Preparation of medium for germination of pollen: (i) Sucrose (8%), 1% agar, 1% gelatin, and 0.01% boric acid are dissolved in distilled water. (ii) The mixture is boiled until the agar and gelatin gets fully dissolved.

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Fig. 1 Collection of male flowers from coconut inflorescence. (a) Spikes in inflorescence with male flowers ready for the sampling, (b) spikelets with staminate flowers, (c) stripped male flowers collected on a petri plate

(iii) The medium is allowed to cool for a while and smeared uniformly on a clean microscopic slide and left for a while to solidify. 2.8 Pollen Germination Procedure

(i) With the help of a small cotton ball, desiccated pollen grains are dispersed evenly over the medium on the slide by gently tapping it and holding between the fingers (Fig. 3a). (ii) Slides are placed in a petri dish lined with moist filter paper and covered to maintain humidity inside the chamber (Fig. 3b). (iii) Slides are incubated at ambient condition in dark for 90 min. (How it is done? In moist chamber/give details).

Note: If the ambient temperature is very low (below 25 °C), an incubator can be used with temperature set to 30 °C. Scoring of pollen for germinability: (i) Slides are observed under a compound microscope. (ii) Pollen germination is scored in ten randomly selected microscopic fields (Fig. 3c). (iii) Pollen germination percentage is calculated using the formula Percentage germination = ½No:of germinated pollenin the field=Total number of pollen in the field × 100

Note: The accepted quality norm for pollen germination is found to be above 25%. The pollen with poor viability may be discarded [10].

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Fig. 2 Pollen extraction from desiccated male flowers. (a) Desiccation of male flowers at 40 °C for 24 h in oven, (b) sieving apparatus having 0.2 mm sieve, (c) extracted desiccated pollen

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Fig. 3 Viability check in desiccated pollen of coconut through in vitro germination assay. (a) Pollen dusted on a microscope slide with in vitro germination medium, (b) incubation of pollen at room temperature for 90 min with maximum humidity, (c) microscopic image of germinating pollen with pollen tube 2.9 Cryopreservation Procedure

(i) Desiccated pollen grains are filled in the aluminum foil pouches and inserted in to the cryovials (Fig. 4a), which are then affixed on to cryosticks (Fig. 4b). (ii) Canisters, consisting of cryosticks, are then plunged in to cryotanks containing liquid nitrogen (Fig. 4c).

2.10 Retrieval of Cryostored Pollen

(i) The canister is lifted from the liquid nitrogen and left in the room temperature for 1 h for thawing.

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Fig. 4 Cryo-conservation of pollen in coconut. (a) Desiccated pollen filled in cryovials, (b) cryovials affixed to cryostick, c plunging of cryostick with cryovial into cryo-tanks containing liquid nitrogen

(ii) After cryostorage, the viability of pollen grains is reassessed as in case of desiccated pollen for its use in artificial pollination process. 2.11 Assessment Fertility of Cryopreserved Pollen 2.11.1 Collection and Preparation of Pollen

(i) Cryovials are retrieved from the cryotank and placed at room temperature for thawing for 1 h. (ii) Pollen grains are mixed with talcum powder (1:9) to protect it from heat and becoming too moist. Isolation and Pollination of Female Flowers

(i) Target inflorescence is emasculated by removing male flowers 3–4 cm above the female flowers. Care is to be taken to remove one or two male flowers found at the base of the female flowers. (ii) Inflorescence is bagged for the entire female phase. (iii) Cryo-conserved pollen mixed with talc is dusted on the receptive stigma up to the 5th day starting from the day when the first female flower comes to receptivity in tall palms and up to 7th–9th day in dwarf palms depending on season. (iv) Cloth bag is removed three days after the start of receptive period of the last female flower. (v) Pollinated bunches should be marked properly. (vi) The percentage of nut set is recorded (Fig. 5).

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Fig. 5 Fertility test of cryopreserved pollen in coconut. (a, c) Nut set in tall (WCT) palm pollinated with dwarf (COD) pollen cryo-conserved for 6 months and 4 years. (b, d, e) Nut set in dwarf (COD) palm pollinated with WCT pollen cryo-conserved for 6 months, 4 years, and 6 years

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Arecanut The arecanut palm (Areca catechu L.; Arecaceae; 2n = 32) is monoecious, and its pollen remains viable only for 8–9 h under ambient conditions [11]. For the breeding programs, it is essential to have quality pollen from good pollen parent. The palm should attain 12 years with consistent high yield, free from diseases and pest, high pollen recovery and higher pollen germination rate. Increased longevity of pollen, from 15 to 21 days by storing in desiccators at room temperature, was reported by Bhat et al. [12]. Since pollen is a useful source of diverse alleles within gene pool storage of pollen, it is highly essential for germplasm conservation as well as for hybrid seed production and also for assisted

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pollination. Pollen cryopreservation is the only large-scale, longterm option for the ex situ conservation of areca nut pollen. The protocol developed at ICAR-CPCRI [13] provides an avenue to store desiccated pollen for long duration without compromising on its viability and fertility in arecanut. 3.1 Materials and Methods

A. Laboratory Equipment Required to Carry Out Experiments 1. Compound microscope, e.g., Leitz Optilux 2. Desktop computer with image analyzer software such as Leica Q Win 3. Cryotank 4. Weighing balance 5. Oven B. General Laboratory Supplies 6. Routine laboratory supplies such as flasks, micropipette, and Petri plates 7. Chemicals such as sucrose (SRL) and boric acid (SIGMA) 8. Distilled water 9. Microscope slides 10. Cryovials (2 mL) 11. Cryosticks 12. Cryomarker and cryobabies (Tarsons) 13. Cryogloves and cryo apron (Tarsons) 14. Aluminum foil, paint brush 15. Sieving apparatus with 0.2 mm pore size mesh

Collection

Selection of good pollen parents based on age, health, and yielding behavior of the palm is important. The spikes with staminate (male) flowers are excised from fully opened inflorescences. Fresh pollen grains are collected by gently tapping the opened male flowers. Opened flowers and flowers to be opened next day are separated from spikes. Flowers placed on a dry paper are crushed gently with a wooden roller and placed on aluminum foil in a Petri plate (Fig. 6). Note: Inflorescence infested with mealy bugs and other biotic pests and young unopened staminate flowers should be avoided. Optimum time for collection of pollen is morning with low relative humidity and temperature, which maintains pollen moisture content and viability.

3.3 Desiccation, Extraction, and Determination of Moisture Content of Pollen

Desiccation of staminate flowers for higher pollen yield is carried out in room temperature. Petri plates with slightly crushed male flowers are kept in room temperature for 24 h. Desiccated flowers are then sieved through 0.2 mm sieving mesh to collect pollen

3.2

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Fig. 6 Collection of male flowers from arecanut inflorescence

(Fig. 7). Moisture content is measured by keeping known quantity of pollen at 100 °C for 24 h in an oven. Desiccation of staminate flowers at room temperature for 24 h results in the yield of pollen with approximately 6–7% moisture content. Note: Constant temperature in incubator affects the pollen yield and viability and desiccation in room temperature can be preferred. Selection of fully matured staminate flowers within the inflorescence is essential as the pollen is scattered around the anther lobes because of the complete breakage of sporangia; this improves the pollen yield.

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Fig. 7 (a) Desiccation of male flowers at room temperature and (b) pollen extraction 3.4 Viability Tests In Vitro and Documentation

In a microscopic slide, an in vitro medium consisting of 0.01% boric acid, 1% agar, 1% gelatin and 2.5% sucrose is poured (1 mL). Using another microscopic slide, the medium is smeared uniformly and fresh and desiccated pollen were dusted with the help of a cotton ball. Slides are placed on a petri plate lined with a moist blotting paper and they are kept in dark condition to avoid light. Slides were incubated at room temperature for 90 min. The germination % is calculated by counting the germinated pollen in three randomly selected fields on the slide with a minimum of 30–50 pollen grains per field (Fig. 8). Pollen grains were considered viable if the pollen tube growth exceeds the pollen grain diameter and germination % are calculated. The length of pollen tubes is measured using the Leica Qwin software with 60–80 pollen tubes in 10 randomly selected fields and average is calculated. Note: Surface of the media on the slides should be uniform as it interferes in image documentation. Covering the entire setup also protects the slides from ants. Humidity should be maintained nearly at 100% for better germination of pollen. Dusting of pollen by gently tapping the cotton balls helps in avoiding aggregated lumps of pollen as it hampers in viability assessment.

3.5 Cryopreservation and Rewarming

Desiccated pollen is wrapped in a piece of aluminum foil and placed in cryovials (2 mL) and affixed to the cryostick. Cryostick is plunged to the liquid nitrogen in a cryotank. Pollen is retrieved at frequent intervals of 24 h, 1 year and 2 years. Thawing or rewarming is done in a slow manner. Retrieved cryovials are left in room temperature for 1 h to bring the temperature slowly to normal.

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Fig. 8 In vitro germination of arecanut pollen grains

Viability of the cryo-conserved pollen is carried out following the procedure explained in Subheading 2.3. 3.6 Fertility Assessment

4

Emasculation of the inflorescence in female parent is carried out by removing the rachillae having male flowers once it has completely emerged out from the spathe. The inflorescences are covered with a cotton bag to avoid the entry of foreign pollen. Receptivity in the female flower is conspicuous as a minute slit in the corolla and widening of the gap in 5–6 days at the tip of the free petals exposing the stigma. With the help of a paint brush, cryostored pollen is dusted on the receptive stigma. The maximum receptivity of stigma is observed to be on days 1–3, after which it declines rapidly. For the entire inflorescence, it usually extended up to 3 to 10 days. The nut set % is recorded after 4–5 months (Fig. 9).

Conclusion Employing the protocol developed at ICAR-CPCRI, pollen from coconut and arecanut could be collected, desiccated, and cryostored. Maintenance of viability and field level fertility of pollen post cryopreservation indicate the validity of the protocol.

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Fig. 9 Emasculation and nut set after artificial pollination with cryopreserved arecanut pollen

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References 1. Aldaba VC (1921) The pollination of coconut. Philip Agric 10:195–210 2. Menon KPV, Pandalai KM (1958) Floral biology. In: The coconut palm. A monograph. Indian Central Coconut Committee, Ernakulam, pp 39–85 3. Patel JS (1938) The coconut-A monograph. Govt. Press, Madras, pp 90–133 4. Engelmann F (1997) in vitro conservation methods. In: Ford Lloyd BV, Newbury HJ, Callow JA (eds) Biotechnology and plant genetic resources: conservation and use. CABI, UK, pp 119–162 5. Hoekstra FA (1995) Collecting pollen for genetic resources conservation. In: Guarino L, Rao VR, Reid R (eds) Collecting plant genetic diversity. Technical Guidelines. IPGRI/FAO/ UNEP/IUCN. CABI publishing, Wallingford, UK, pp 527–550 6. Heslop-Harrison J (1979) An interpretation of the hydrodynamics of pollen. Am J Bot 66: 737–743 7. Barnabas B, Rajki E (1981) Fertility of deepfrozen maize (Zea mays L.) pollen. Ann Bot 48: 861–864

8. Karun A, Sajini KK, Niral V, Amarnath CH, Remya P, Rajesh MK, Samsudeen K, Jerard BA, Engelmann F (2014) Coconut (Cocos nucifera L.) pollen cryopreservation. CryoLetters 35(5):407–417 9. Caroline de AM, Moura CRF, Pinto de Lemos EE, Ramos SRR, Ribeiro FE, Ana da Silva L (2014) Pollen grain viability of coconut accessions at low temperatures. Acta Sci Agron 36(2):227–232 10. https://www.bioversityinternational.org/ fileadmin/bioversity/publications/Web_ver sion/108/begin.htm#Contents 11. Raghavan V, Baruah HK (1956) On factors influencing fruit-set and sterility in arecanut (Areca catechu Linn.): I. Studies on pollen grains. J Indian Bot Soc 35:139–151 12. Bhat SK, Krishna Murthi S, Madhava Rao VN (1962) South Indian Hortic 10:22–34 13. Karun A, Sajini KK, Muralikrishna KS, Rajesh MK, Engelmann F (2017) Cryopreservation of arecanut pollen. CryoLetters 38(6):463–470

Part VI Root Crop

Chapter 51 Pollen Cryopreservation in Cassava Vivek Hegde Abstract Crossing between the parental lines is very difficult in cassava since the flowering depends on the genotype and environmental conditions. Poor and irregular flowering, flowering asynchronization between parents, and monoecious and protogynous nature also cause difficulty in cassava breeding. This flowering behavior slows down the cassava improvement through breeding, and in such conditions, it is convenient to cryopreserve the pollen from the desired male parent for later use in hybridization with the female parent when the female flowers are available. The cryopreservation of cassava pollen could be the best strategy for efficient conservation of nuclear genetic diversity and used for pollination in the hybridization program. Cryopreserved cassava pollen assures the availability of pollen for hybridization program during the same season, in the succeeding seasons and also in different locations. This chapter describes the detailed method of cryopreservation of cassava pollen, viability assessment of stored pollen and production of hybrid seeds after hand pollination with the female parent using cryopreserved pollen. This protocol would help the breeders to plan the hybridization programs in cassava and make the exchange of genetic material among the researchers easier because of less stringent restrictions on the exchange of stored pollen. Key words Cassava, Pollen conservation, Manihot esculenta, Pollen germination, Acetocarmine test, Asynchronization, Hybridization, Germplasm storage

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Introduction Cassava/tapioca (Manihot esculenta Crantz.) is a very important food crop after rice, wheat, and maize. Billions of people of tropical countries belonging to Africa, Asia, and South America depend on cassava as a source of calories [6]. Cassava has been evidenced to be a reliable source of food during natural calamities and disasters. It was adopted as a popular famine reserve food crop due to its ability to grow and yield well in marginal as well as in wasteland. Presently, this crop has emerged as food and a commercial crop of industrial significance [2]. The productivity of many crops increased remarkably during the twentieth century because of the genetic gains achieved through crop breeding. Hybridization/crossing between selected

P.E. Rajasekharan and M.R. Rohini (eds.), Pollen Cryopreservation Protocols, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-2843-0_51, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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elite genotypes and subsequent selection (mass selection) of superior plants within F1 progenies based on phenotype and following clonal generations is the widely followed method of developing new varieties in cassava [4]. Since the flowering in cassava depends on genotype and environmental conditions, the crossing of elite clones has become very difficult. Flowering asynchronization between female and male parents is also a major problem in cassava breeding. Some clones, flower relatively early at 4 to 5 months after planting whereas others flower at 8–10 months. Besides, cassava is monoecious and protogynous; its female flowers open 10–14 days before the opening of male flower, on the same inflorescence. This kind of flowering behavior of cassava hinders its improvement through hybridization, and in such circumstances, it is convenient to store pollen from the desired male parents for later hybridization with desired female parents. Cryopreservation guarantees the optimal retention of viability and genetic stability of stored tissues [5]. For the conservation of genetic resources, in the long run, cryopreservation is a safe and cost-effective method among the techniques available. Different storage conditions have been studied to store the cassava pollen and reported cryopreservation is the best storage strategy for efficient conservation of nuclear genetic diversity [10]. A simple, inexpensive, fast, and safe method for cryopreservation of cassava pollen grains is explained in this chapter. This method would help the breeders to plan the hybridization program and long-term storage of nuclear genetic diversity in cassava.

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Materials Cryopreservation of cassava pollen grains needs care, because of its fragile nature. The required reagents and solutions are prepared with high analytical purity reagents/chemicals and in ultrapure water/solvent.

2.1 Laboratory Equipment

• Stereomicroscope • Cryotank • Fluorescent light • Laminar airflow hood • Refrigerator

2.2 General Laboratory Supplies

• Cryovials • Microscope slides • Dissection tools, i.e., scalpels, forceps, scissors, and needles.

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• Routine laboratory supplies such as petri plates, flasks, disposable transfer pipettes, syringes, glass bottles. • Aluminum foil • Tissue paper/cotton 2.3 Chemicals and Solutions

• Ethanol (70%) • Liquid nitrogen (LN) • Acetocarmine (2%) • Cassava pollen germination medium [7]: containing sucrose 5%, boric acid 100 mg/L, calcium nitrate 300 mg/L, magnesium sulfate 200 mg/L, potassium nitrate 100 mg/L (pH 5.8).

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Methods Before starting pollen cryopreservation, it is essential to understand the details of the reproductive system, the viability of the fresh pollen grains, the timing of anthesis, and stigma receptivity of the species under study. Cassava flower profusely (Fig. 1) but highly depends on the genotype and environmental conditions. Cassava is a monoecious plant; it bears both female (Fig. 2a) and male (Fig. 2b) flowers on the same plant. Normally, cassava is a highly cross-pollinating crop and pollination is accomplished by the insects. In cassava, there is up to 94.3% pollen viability reported through acetocarmine staining viability test [10]. The pollen is highly susceptible to desiccation, and there is rapid moisture loss when stored at room temperature. Whereas total loss of pollen viability was observed after 24 h at room temperature [9], and stigma remains receptive for up to three days after anthesis [1]. Up to 80% fruit set can be obtained when pollination is carried out with fresh pollen on the freshly opened female flower [11].

3.1 Collection of Male Flowers

• Select the inflorescences having male flower buds, which are ready to open on the next day and protect them by covering with cloth bags (Fig. 3a). • On the day of collection, male flowers are collected in the morning hours, between 9.00 AM and 10.00 AM before the anthesis (Fig. 3b) and placed in petri dishes/bottles lined with moist paper.

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Fig. 1 Mature cassava plant in full-bloom (a, b)

Fig. 2 Inflorescence of cassava (a) Female flowers, (b) Male flowers

Fig. 3 (a) Protected cassava inflorescence for collection of male flower, (b) Collected male flowers for cryopreservation

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3.2 Immersion and Storage of Male Flowers with Pollen Grains in Liquid Nitrogen

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• Freshly collected, unopened male flowers are placed in cryovials with a capacity of 1.5 mL or 2.0 mL. Each cryovial accommodates approximately 6–10 flowers. • Place the cryovials containing male flowers in cryo-rack, immerse, and store the samples in liquid nitrogen (at 196  C). • The male flowers stored in liquid nitrogen can be conserved with no change for an indefinite period. Whenever required, these flowers can be taken out and used for pollination.

3.3 Preparation of Medium for In Vitro Germination of Pollen

• Cassava pollen can be germinated successfully on modified Brewbaker and Kwack [3] medium. The in vitro pollen germination medium is prepared in double-distilled water by adding 50 g/L sucrose, 300 mg/L calcium nitrate, 200 mg/L magnesium sulphate, 100 mg/L boric acid, and 100 mg/L potassium nitrate and adjusted to pH 5.8 [7]. • To prepare 100 mL medium, add 5 g/L sucrose, 30 mg/L calcium nitrate, 20 mg/L magnesium sulfate, 10 mg/L boric acid, and 10 mg/L potassium nitrate in a 250 mL beaker or conical flask. Dissolve in a small volume (approximately 25–30 mL) of sterile deionized or double-distilled water and makeup to 100 mL by adding sterile deionized or doubledistilled water. • Adjust the pH to 5.8 using a pH meter. • Sterilize the in vitro pollen germination medium by autoclaving at 121  C for 20 min, and later store it in the refrigerator (at 4  C).

3.4 Pollen Viability Test Through In Vitro Germination

• Cassava pollen can be germinated under lab conditions using the sitting drop method [8]. • Place one or two drops (25–30 μL each) of pollen germination medium on a clean, dry microscope glass slide. • Since the cassava pollen is sticky, pollen grains are collected from the anther with the help of a clean brush. Uniformly distribute a suitable quantity of pollen grains over the drops with a clean brush. Pollen grains tend to clump and settle along the periphery of the drop. It is difficult to count germinated pollen grains if they are clustered. Hence, make a uniform distribution of pollen over the drop of germination solution. • Label the sample slide including the name of the crop, variety, storage duration, culture incubation time, and other details of the exercise. • A pair of large petri plates (15 cm diameter) with a moist filter paper lining on the lower plate serves as a humidity chamber. • The sample slides should be placed over two glass rods or glass slides placed parallel to 3–4 cm apart on the moist filter paper.

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Fig. 4 Cassava pollen viability (cryopreserved) assessed by acetocarmine test and in vitro germination tests (a) pollen staining, (b) germinating pollen [10]

• Incubate the slides at 25  2  C in a humid chamber to prevent evaporation and drying of the medium for 6 to 8 h. • Germinated pollen grains are recorded through a stereomicroscope directly or after the addition of a drop of acetocarmine solution to facilitate observation by better contrast (Fig. 4b). 3.5 Pollen Viability Test Through Acetocarmine Staining Assay

• Place a drop of 2% acetocarmine solution on a clean and dry microscope glass slide. • Suspend a small amount of cassava pollen in a drop of acetocarmine solution and distribute them uniformly. • Apply a cover glass carefully over the sample. • Observe the samples under a microscope to score viable and nonviable pollen grains. • Pollen grains with larger size and which are deeply stained (red) are classified as viable while those stained in a light color, abnormal, and reduced size are considered nonviable (Fig. 4a; [10]).

3.6 Evaluation of Pollen Fertility by Fruit and Seed Set Test

• Fertility/crossability of the stored pollen is to be tested by the controlled field pollination with a compatible female parent. • To avoid open pollination, inflorescences having female buds, which are ready to open in the day after of the desired female parents, are selected and protected by covering with cloth bags. • Preferably female flowers are covered with red-colored bags (red cloth bags to identify the unpollinated female flower) after removal of male flowers and already opened female flowers. • Male flowers stored in liquid nitrogen are removed from the storage tank and thawed by placing vials upright at room temperature (25  2  C) for 8–10 min (warming in air). Samples are

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Fig. 5 (a) Pollination of female parent with cryo-stored pollen grains, (b) Bagging of hand-pollinated female flowers to prevent the cross pollination

Fig. 6 (a, b) Fruit set by the cryo-stored pollen with proper labelles

not touched or agitated during this process. Immediately after thawing, male flowers are taken to the place for pollination. • Carefully open the petal-like bracts of cryopreserved male flowers, and remove the anthers without disturbing the nectar gland. • Carry out the pollination between 10.00 AM and 12.00 PM by rubbing the anthers on stigmas of the freshly opened female flower until the stigmas turn yellowish or place the male flower on the stigma (Fig. 5a). • Immediately cover the pollinated flowers with cloth bags (preferably cover with a white-colored cloth bag to identify the pollinated female flower) to prevent the open-pollination (Fig. 5b). • Properly label the pollinated flowers with details like the date of pollination, duration of pollen storage, and the identity of the male and female parents (Fig. 6).

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Fig. 7 Successful hybridization with cryopreserved cassava pollen (a) developing seeds, (b) matured seeds

Fig. 8 (a, b) Germinating cassava seeds obtained from cryopreserved pollen

• Remove the cloth bags after the fruit set (Fig. 6). • Record the observations on the percentage of fruit formation and seed development in all the crosses after allowing it to go through normal development and maturity (Fig. 7). • Collect the crossed seeds and sown in the soil to record the germination (Fig. 8).

4

Notes Do’s • Throughout the growing period, follow the normal cultivation practices to raise healthy cassava plants.

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• Select the healthy plants for collection of male flower for cryopreservation and healthy female patent to test the viability of the cryopreserved pollen. • Collect the male flowers just before the anthesis. • The process of collection of male flowers, transfer to the cryogenic tubes, and immersion in liquid nitrogen should be carried out as quickly as possible. • While placing the male flowers in the cryovials, the vial should be closed carefully leaving some space in the tube so that the flowers do not get crushed. • Use sterile double-distilled water for the preparing pollen germination solution. • Distribute the pollen grains uniformly over a drop of germination solution and acetocarmine solution to make it convenient to count the germinated and stained pollen grains, respectively. • During incubation of pollen for in vitro germination, maintain humidity in a humid chamber to prevent evaporation and drying of the medium. • Protect the female flower buds by covering with red cloth bags to identify the unpollinated female flower. Similarly, cover the pollinated female flowers with white-colored cloth bags. The color code will be followed depending on the researcher for easy identification. Don’ts • Don’t collect the male flowers from pest-infested and diseaseinfected cassava plants for cryopreservation. • Don’t select the opened and damaged male flowers for cryopreservation. • Don’t cryopreserve the pollen, which is already cryopreserved and thawed. • Don’t touch or agitate the cryopreserved samples during the thawing process. • Don’t incubate the pollen over 6–8 h on in vitro germination medium. Incubation of pollen over 8 h on medium may allow the fungus to grow; hence it is difficult to record the germinated pollen. References 1. Abril LNR, Pineda LM, Wasek I, Wedzony M, Ceballos H (2019) Reproductive biology in cassava: stigma receptivity and pollen tube growth. Commun Integr Biol 12(1):96–111.

https://doi.org/10.1080/19420889.2019. 1631110 2. Aerni P (2006) Mobilizing science and technology for development: the case of the

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Cassava Biotechnology Network. Ag Bio Forum 9:1–14 3. Brewbaker JL, Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am J Bot 50(9):747–758 4. Ceballos H, Carlos AI, Juan CP, Alfred GOD (2004) Cassava breeding: opportunities and challenges. Plant Mol Biol 56:503–516 5. Day JG, Harding KC, Nadarajan J, Benson EE (2008) Cryopreservation, conservation of bioresources at ultra low temperatures. In: Walker JM, Rapley R (eds) Molecular biomethods handbook. Humana Press, Totowa, pp 917–947 6. FAO (2008). http://www.fao.org/ag/agp/ agpc/gcds/index_en.html 7. Mary B, Robert SK, Arthur KT, Jacinta A, Stephen M, Hellen A, Erwin H, Maria W, Hernan C, Clair H, Yona B (2015) In vitro embryo rescue and plant regeneration

following self-pollination with irradiated pollen in cassava (Manihot esculenta Crantz). Afr J Biotec 14:2191–2201 8. Shivanna KR, Rangaswamy NS (1992) In Vitro germination methods. In: Pollen biology: a laboratory manual. Springer, Verlag, Berlin Heidelberg, p 13. (9-20) 9. Vieira LJ, Santana JRF, Alves AAC, Ledo CAS, Souza FED (2015) Use of aniline blue stain to observing pollen tubes development in different Manihot Mill. Species. Afr J Agric Res 10: 1805–1809 10. Hegde V, Koundinya AVV, Sheela MN, Visalakshi Chandra C, Mukherjee A (2019) Storage of cassava pollen for conservation of nuclear genetic diversity and overcoming hybridization barriers. Indian J Hortic 76(1):104–111 11. Wania MGF, Sebastiao O, Silva CI (2002) Cassava breeding. Crop Breed Appl Biotechnol 2(4):617–638

Part VII Forest Species

Chapter 52 Pollen Cryopreservation Protocol for Neolamarckia cadamba (Roxb.) Miq P. E. Rajasekharan and R. Harsha Abstract Neolamarckia cadamba is a potential tree in agroforestry system belonging to the family Rubiaceae. It is commonly called as Kadamba and considered sacred in Hindu religion. This tree bears globose head shaped inflorescence where flowers are arranged in clusters. They appear during May–July and are fragrant, orange to yellow in color. It has several medicinal uses and possesses religious importance. Pollen cryopreservation was attempted in this species. Initial and post-cryopreservation viability assessment was carried out by hanging drop technique using Brewbaker and Kwack medium containing sucrose solution. The germination profiles of fresh pollen and cryopreserved pollen were observed to check the feasibility of the species to cryogenic temperature. The details of material and methods used in development of protocol are discussed in this chapter. Key words Neolamarckia cadamba, Rubiaceae, Conservation, Pollen cryopreservation

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Introduction Neolamarkia cadamba, which is commonly called as Kadamba, belongs to the family Rubiaceae. This tree possesses religious importance and considered as a sacred tree to Lord Krishna. It is a miracle tree species with considerable economic potential which is used as timber and possess medicinal use in Ayurveda. It is wellknown for its rapid growing nature and fast decomposition rate and considered to be useful in agroforestry system and carbon sequestration [1]. The tree is native to S-E Asia and distributed in the deciduous forests. The tree is medium to large sized attaining height of 20–40 m with cylindrical branches and rounded crown. The leaves are opposite and dark green in color with pinnate venation. Inflorescence is terminal globose heads and appears in clusters. Flowers are bisexual, subsessile, and fragrant. They are light orange to yellow in color with gamopetalous corolla. Five stamens are present in the corolla

P.E. Rajasekharan and M.R. Rohini (eds.), Pollen Cryopreservation Protocols, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-2843-0_52, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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tube, filaments are short and anthers are basified. Pollen grains were three colporate and spheroidal in shape. The ovary is inferior, bilocular with spindle shaped stigma. Fruits contain numerous fruitlets in solid structures with 8000 tiny seeds [2]. Lack of information on the gene pool hampers the breeding and genetic improvement of the species. Since the tree species has multiple benefits such as in agroforestry system, therapeutic value and religious importance, genetic improvement and breeding become essential. The presence of small seeds is a key limiting factor for regeneration. Hence, conservation through male gametophyte can be attempted through cryogenic techniques. Pollen cryopreservation facilitates germplasm conservation where pollen viability and fertility are maintained for many years. This technique helps in conserving pollen, thus facilitating in breeding programs, developing wide crosses and periodic replenishment of pollen for pollinator lines. Flowering season, time of anthesis, anther dehiscence, pollen collection, germination, and cryopreservation studies have been discussed in this protocol.

2

Materials

2.1 Laboratory Equipment

1. Compound microscope 2. Stereomicroscope, e.g., Leica MZ12 3. Scanning electron microscope 4. Laminar flow hood 5. Cryotank 6. Drying oven 7. Desiccator

2.2 General Laboratory Supplies

1. Dissection tools, namely, scalpels, forceps, scissors, needles, and paint brushes 2. Routine laboratory supplies such as flasks, disposable transfer pipette, and petri plates 3. Borosil glass measuring cylinders, glass bottles (10 mL) 4. Microscope slides and cover slips 5. Syringes 6. Cryovials (2 mL) 7. Aluminum foil 8. White butter papers 9. Whatman filer paper

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2.3 Chemicals and Solutions

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1. Alexander stain containing ethyl alcohol (95%), malachite green, glycerol, acid fuchsin, phenol, and distilled water for viability testing 2. Pollen medium (Brewbaker) containing boric acid, 300 ppm; calcium nitrate, 100 ppm; magnesium sulfate, 300 ppm; potassium nitrate, 300 ppm; and sucrose, 10% 3. Agarose, 1% 4. Starch powder (soluble), 10% 5. Sterilizing solution 0.3 and 0.6% sodium hypochlorite solutions 6. Ethanol, 70% 7. Liquid nitrogen

3

Methods Flowering and fruiting starts at an early age from 6 to 7 years. Flowering season was observed during May–July and extends up to September (Fig. 1). Fruits appear from August to October and fall in January to February. Inflorescence is harvested, and flowers are collected from the globose head. Anthesis takes place in the early morning from 4:00 to 6:00 a.m. Anther dehiscence was observed prior to anthesis [3].

3.1 Collection of Pollen

Flowers are collected between 4:00 and 5:00 a.m. before anthesis. Anther dehiscence was observed from the longitudinal slit end of anther. Hence, pollen was extracted from the longitudinal slit end of anther and collected in the clean butter paper (See Notes 2 and 3).

3.2 Dehydration of Pollen Grains

After extraction, it is labeled and stored in the desiccator containing zeolite beads for 1 h to reduce the moisture content of pollen grains (Fig. 2). Moisture content of the pollen grains has to be reduced to 10% to ensure better pollen germination (See Notes 4 and 5).

Fig. 1 (A) Tree, (B) Tree at its peak flowering

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Fig. 2 (A) Unopened flower (B) Flower collection, pollen extraction, (C) desiccation, and (D) cryopreservation of pollen grains of Neolamarckia cadamba

3.3 Preparation of Pollen Medium

Preparation of Brewbaker and Kwack (100 mL) medium by dissolving the different chemicals (boric acid, 300 ppm; calcium nitrate, 100 ppm; magnesium sulfate, 300 ppm; potassium nitrate, 300 ppm) in double-distilled water. 1 mL of this stock solution is used for in vitro germination of pollen grains using different concentrations of sucrose, i.e., 5, 10, 15, 20, 25, and 30 percent [4] (See Note 8).

3.4 Viability Assessment of Pollen Grains

Viability was assessed by initial germination using Brewbaker and Kwack medium, supplemented with sucrose using the hanging drop method. Pollen dispersed in hanging drop position was incubated in a moist chamber at 25 ± 20 °C [5]. After germination, pollen was stained with Alexander stain [6] and viewed under a microscope (Fig. 3). Germinated pollen was counted and the percentage germination was computed. Five replicates were assessed with ten microscopic fields taken for count (See Notes 1, 9–12).

3.5 Cryopreservation of Pollen Grains

After the initial viability assessment of pollen, the desiccated pollen were packed in butter paper covers and enclosed in sealed aluminum pouches, which were then rapidly plunged into the cryobiological system containing liquid nitrogen (Fig. 2) (See Notes 6 and 7).

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Fig. 3 In vitro pollen germination of Neolamarckia cadamba. (A) Fresh pollen. (B) Cryopreserved pollen 3.6 PostCryopreservation Viability Assessments

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After cryopreservation, the pollen grains were retrieved from liquid nitrogen and rapidly thawed at room temperature for 3–5 min. The germination test was carried out as initial viability assessment to know the percentage of germination of cryopreserved pollen. Germination percentage was calculated by counting the number of germinated pollen grains and aborted pollen grains. Difference in the germination profiles of fresh pollen and cryopreserved pollen was estimated.

Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination 1. A stereoscopic microscope with a photo documentation system should be used in the evaluation. 2. Pollen collection at peak anthesis time is ideal for obtaining highest viability and in vitro germination. 3. The procedures should be carried out in a clean place possibly in a laminar flow chamber. 4. The dehydration time can vary according to the size of the pollen grains, exine thickness and ambient humidity. 5. The moisture of the pollen grains should be between 15 and 30%. A sample of pollen grains after dehydration should be tested for viability [7, 8]. 6. Direct contact of liquid nitrogen with pollen grains causes injuries to the exine of the pollen grains, impairing their viability. Therefore, the envelopes should be well sealed to prevent liquid nitrogen from entering. 7. The process of immersion in liquid nitrogen and transfer to the cryogenic tank should be carried out as fast as possible, to avoid sudden temperature variations, which can harm the pollen grains.

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8. Pollen media with sucrose concentration should be prepared at the time of slide preparation to avoid contamination. 9. The pollen grains should be uniformly distributed on the medium, if the grains are agglomerated it will be hard to count them. 10. After slide preparation, the slides are kept in petri dishes lined with wet filter paper to maintain relative humidity, and do not allow the pollen media to get dry (Fig. 3). 11. To ascertain the germination percentage, the pollen grains in each photomicrograph should be counted. Pollen grains are considered germinated when they have a tube length greater than or equal to the grain diameter. 12. To confirm any morphological variations on surface of pollen grains, observations of freshly collected and cryopreserved pollen have to be done under Scanning Electron microscope (SEM). References 1. Bijalwan A, Dobriyal MJR, Bhartiya JK (2014) A potential fast growing tree for agroforestry and carbon sequestration in India: Anthocephalous cadamba (Roxb.) Miq. Am J Agric Forest 221: 296 2. Mondal S, Bhar K, Mahapatra AS, Mukherjee J, Mondal P, Rahaman ST, Nair AP (2020) “Haripriya” god’s favorite: Anthocephalus cadamba (Roxb.) Miq. - at a glance. Phcog Res 12:1–16 3. Bhattacharya A, Mondal S, Mandal S (1999) Entomophilous pollen incidence with reference to atmospheric dispersal in eastern India. Aerobiologia 15:311–315 4. Brewbaker JL, Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am J Bot 50:859–865 5. Ganeshan S, Rajasekharan PE, Shashikumar S, Decruze W (2008) Cryopreservation of

pollen. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, New York, pp 281–332. https://doi.org/10.1007/978-0387-72276-4_17 6. Alexander MP (1980) A versatile stain for Pollen, fungi, yeast and bacteria. Stain Technol 55: 13–18 7. Silva RL, Souza EH, Vieira LJ (2017) Cryopreservation of pollen of wild pineapple accessions. Sci Hortic 219:326–334. https://doi.org/10. 1016/j.scienta.2017.03.022 8. Souza EH, Souza FVD, Rossi ML (2015) Viability, storage and ultrastructure analysis of Aechmea bicolor (Bromeliaceae) pollen grains, an endemic species to the Atlantic forest. Euphytica 204:13–28. https://doi.org/10.1007/s10681014-1273-3

Chapter 53 Pollen Cryopreservation of Coniferous Serbian Spruce (Picea omorika/Pancˇ./Purkyne) and Deciduous Pedunculate Oak (Quercus robur L.) Species Branislava Batos and Danijela Miljkovic´ Abstract One of the alternative strategies to conserve and preserve the genetic variation of many plant species is pollen cryopreservation. The aim of this study was to develop protocols for preserving pollen from coniferous Serbian spruce (Picea omorika/Pancˇ./Purkyne) and deciduous pedunculate oak (Quercus robur L.) species. Although these species have different forms (coniferous vs. deciduous), both of them are monoecious and anemophilous. Samples from different localities over a few successive years were collected and stored at four temperatures (+23 °C, +5 °C, -15 °C, and -20 °C). Pollen viability analysis methods include protocols for collecting fresh pollen, storing it under controlled temperatures and light conditions, and microscopic analysis of pollen (viability %, germination %, germination energy-pollen tube length μm). Experiments to analyze the viability of stored pollen were set up every month during the first year of storage, every 3 months during the second year of storage, and once a year during the third and subsequent years of storage. The pollen vitality was observed by staining test (TTC-2,3,5-triphenyl tetrazolium chloride). Pollen viability was evaluated by pollen germination percentage and pollen tube length on six sucrose medium (0%, 5%, 10%, 15%, 20%, 25%) for fresh pollen and periodically for pollen after cryopreservation. Key words Pollen conservation, Picea omorika, Quercus robur, Cryopreservation methods

1

Introduction The time variability of pollen viability is species specific [1]. Depending on environmental conditions (humidity and temperature), pollen viability is limited by/in time varying from a few minutes to a few hours. The fine powdery structure of pollen consists of pollen grains of male plant microgametophytes, which are the source of life in the plant world. After release from the pollen, the viability of pollen grains is short-lived, so the definition of pollen storage methods for plant species is complementary to seed storage (genobank). Cryopreservation of pollen allow us to

P.E. Rajasekharan and M.R. Rohini (eds.), Pollen Cryopreservation Protocols, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-2843-0_53, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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overcome primarily oscillations of environmental conditions of seasonal, asynchronous flowering, and geographically remote regions, and it preserves the possibility of using pollen in hybridization and breeding programs of plant species as well as preserving genetic variability and biodiversity. These protocols ensure the possibility of studying pollen biology, morphology and germination as an assessment of pollen viability. Long-term pollen storage is very important in the field of silviculture, agriculture, horticulture, evolutionary ecology, urban ecology, and the conservation of endangered plant species endangered by rapid and major climate change. For these reasons, it is important to present as many methodological approaches as possible for different numbers and forms of plant species [2–10]. In forestry, cryopreservation of woody species pollen is important, especially those that are exposed to stress today, due to changes in environmental conditions in their native ranges, global, and rapid climate change [11]. Genetic resources of plant species are maintained by cryopreservation techniques, preparing seeds, pollen, embryos, and meristems [12]. As a long-term ex situ preservation method for plant species, cryopreservation is of great importance in order to preserve species and their genetic variability. Reduction of optimal species’ habitats and habitat fragmentation is the main factor negatively influencing biodiversity, leading to the loss of plant biodiversity, and even the extinction of some species [13]. Trees can be grouped as deciduous and coniferous according to the morphological traits. For deciduous trees these are their broad leaves, male and female flowers, with seeds inside the fruit, whereas coniferous trees have long needle-shaped leaves, male and female cones, with seeds between the scales of cones. The morphological and physiological properties of pollen, the quality of pollen grains (i.e., percentage of germination and pollen tube length), fertilization, seed quality, survival of the species and preservation of biodiversity are all influenced by environmental factors and are primarily genetically determined. Plant genetic variability is confirmed by the study results, which show interpopulation and interindividual differences in pollen traits [7, 14–16]. Variability of pollen traits within a species can be used to identify donor trees with good pollen quality, over several growing seasons. Marking of such trees is of great importance in breeding programs for important plant species [7]. In this research, the pollen from coniferous Serbian spruce (Picea omorika/Pancˇ./Purkyne) and deciduous pedunculate oak (Quercus robur L.) species were cryopreserved (Fig. 1). According to the International Union for Conservation of Nature, Serbian spruce is on the Red List of Endangered Plants [17, 18]. The distribution of Serbian spruce (Picea omorika [Pancˇic´] Purkine) is limited to the Balkan Peninsula. Populations

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Fig. 1 Spruce seed plantation (Picea omorika/Pancˇ./Purkyne), Bela Zemlja, Zlatibor, Serbia (a); pedunculate tree (Quercus robur L.), Monosˇtorske sˇume, Sombor, Serbia (b)

of this endemic tertiary relict species are small and scattered on inaccessible terrains up to 1700 m above sea level. The species is characterized by high adaptive potential and degree of genetics, variability of analyzed allozyme markers and nuclear EST-SSR, despite their spatially limited distribution [19]. The flowering of the genus Picea is characterized by periodicity, more extensive flowering (occurs every second, third or fifth year) in the volume of flowering [20] and is adapted to wind pollination, as with most of the coniferous species. The two air-filled bladders develop laterally from the body of the P. omorika pollen grain, unlike broad leaved species’ pollen grains which have no air bladders. Another important statement is that coniferous species characteristically have significantly larger pollen than broad leaved species [21] (Figs. 2 and 3). In addition to the interindividual variability of flowering phenology (due to genetic differences between trees), this monoecious species is also characterized by intraindividual variability of flowering phenology of male flowers (microstrobiles) and female flowers (macrostrobiles) (Fig. 4). The tree is able to maintain genetic variability of traits and at the same time prevent inbreeding depression, characteristic for small isolated populations [9, 22]. The genus of oaks (Quercus) is one of the most numerous within the family Fagaceae, with a wide spectrum of distribution in the temperate zone of the northern hemisphere. Areas of

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Fig. 2 Spruce pollen (Picea omorika/Pancˇ./Purkyne) (a and b)

Fig. 3 Pedunculate oak pollen (Quercus robur L.) (a and b)

Fig. 4 Male flowers—microstrobiles (Picea omorika/Pancˇ./Purkyne) (a); male flowers—tassels (Quercus robur L.) (b)

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pedunculate oak (Quercus robur L.) have significantly decreased due to global changes in habitat climate factors [7, 23]. The flowers of this monoecious anemophilous species are located in separate inflorescences. Male flowers develop on branches from the previous growing season (Fig. 4) and female flowers on branches from the current one. Flowering is abundant every 2–3 years, usually in the April–May period. This species is also characterized by summer flowering in July and August. Pollen morphology and viability differ; pollen in summer flowering is usually dysfunctional [3, 6, 7].

2

Materials Pollen research requires timely and synchronized activities in the field and in the laboratory, as well as a chamber with controlled conditions. The process of collecting and preparing pollen for viability analysis is carried out through several phases. The first phase is preparatory and refers to the regular observation of the flowering phenophase in the field and determining the time of pollen collection [3, 6] (Fig. 4). The next phase is only the collection, which is done either directly in the field by collecting pollen from mature pollen in the pollination phase or by collecting pollen from anthers ripening in the laboratory—the method of “water cultures” [24] (Figs. 4 and 5). Then the collected pollen is purified, dried, and placed in test tubes in a desiccator. One part of the pollen sample prepared in this way is immediately analyzed for the viability of fresh pollen, and the other part is placed in the refrigerator and freezer to analyze the viability of stored pollen [25–27]. The viability of stored pollen is analyzed every month in the first year and in later at least once a year, always for the same duration. Thawing of stored pollen from lower temperature lasts in 2 h at room temperature (+23 °C). Samples with a germination rate less than 5% are treated as if there were no germination. Pollen viability analysis was performed: (1) by staining test (ability of living cells to change color in a solution with TTC (2,3,5-triphenyl tetrazolium chloride (see Note 2) in the dark, in a thermostat [28, 29], and (2) by an in vitro test of pollen germination on a nutrient medium MS medium [30] in a laboratory chamber under controlled conditions [31] (Fig. 6).

2.1 Collection of Pollen Grains

1. Spruce pollen (Fig. 5a, b) was collected by the “water culture” method (see Note 3). 2. Pedunculate pollen (Fig. 5c, d) was collected by the “water culture” method and by cutting off male flowers (fringes) from tress in the field during the pollination phase (see Note 4). 3. Pollen cleaning was done with a series of sieves.

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Fig. 5 Pollen collection by the “water culture” method (Picea omorika/Pancˇ./Purkyne) (a and b) and (Quercus robur L.) (c and d)

4. The collected purified pollen was spread in a thin layer on clean paper and thus dried in a thermostat at 30 °C for 48 h. 5. The dried pollen was packed in tubes/vials with rubber stoppers and placed in a desiccator with silica gel (to maintain the necessary minimum moisture) for various temperature treatments and cryopreservation (see Note 5). 2.2 Laboratory Equipment and Materials

1. 30–50 cm paper bags, pencil, and identification stickers. 2. Vases with water, clean A4 paper, laboratory sieves for separating larger, and smaller impurities with a final diameter of 0.2 mm. 3. Thermostat 4. Tubes: smaller for pollen storage (external dimensions (L * Ø) 75 * 12 mm), larger for staining test (external dimensions (L * Ø) 130 * 16 mm) with appropriate rubber closures. 5. Tube rack 6. Desiccator with silica gel SiO2 (see Note 1) 7. Refrigerator with freezer

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Fig. 6 Pollen analysis experiment: upstairs pollen incubation in the laboratory chamber (a and b), downstairs microscope/camera system and computer (c)

3

Method

3.1 Viability of Pollen Laboratory Equipment, Material, and Reagents

1. Libra 2. Magnetic stirrer 3. Light microscopy LM Leica Galen III (magnification × 40, ×100), and Scanning Electron Microscopy SEM JEOL JSM-6460 LV. 4. Camera (CCD Camera Topica TP/5001) 5. Laboratory accessories: instruments (tweezers, wand, pipettes, dropper) 6. Funnel 7. Erlenmeyer (volume 300 ml (L * Ø neck * Ø base) 156 * 34 * 87 mm) 8. Petri dishes (Ø 19 cm) 9. Microscope slides and cover plates 10. Metal aluminum brackets for slides 11. Cotton wool

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12. Distilled water 13. Alcohol 95% 14. Reagent-TTC (see Note 2) 15. Sucrose 3.2 Pollen Viability Analysis

1. Staining test—assessment of pollen vitality. (a) A small amount of pollen is first transferred into the tubes with a laboratory stick, and then a reagent solution (0.5% solution of TTC in distilled water) is prepared. (b) Tubes with pollen and reagent are placed in a thermostat at 30 °C for 8 h. (c) When the tube is removed from the thermostat on the mixer, the sample is equalized for a few seconds. (d) After that, a solution of reagent with pollen is applied with a dropper on an alcohol-coated glass plate (see Note 6). (e) On a sample of five drops of solution = 5 repetitions = 5 fields of view, intensely colored red pollen grains, and the total number of grains in the field view of the microscope is counted (see Note 7) (Fig. 7). (f) The estimation of pollen vitality is expressed as the mean value of all shoots and represents the percentage value of colored grains and the total number of grains in the field of view (Fig. 7).

Fig. 7 Vitality of spruce pollen—TTC method

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1. A nutrient medium is prepared (see Note 8). 2. A small amount of pollen is applied with a laboratory stick onto an alcohol-wiped microscope slides (see Notes 9 and 10). 3. A nutrient medium is applied over the pollen with a dropper (see Notes 6 and 11). 4. Pollen incubation takes place in the laboratory chamber in dayand night-light (16/8 h) at room temperature (23 °C) and lasts 24–48 h. During this period, pollen grains germinate; i.e., the pollen tube grows (Figs. 8 and 9).

Fig. 8 In vitro test—spruce (Picea omorika/Pancˇ./Purkyne) pollen germination on six sucrose media (0%, 5%, 10%, 15%, 20%, 25%)

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Fig. 9 In vitro test—germination of pedunculate oak (Quercus robur L.) pollen (a) dry pollen grains, (b) on 5% sucrose media, (c) on 10% sucrose media, (d) on 15% sucrose media

5. The percentage of germination represents the mean value from three drops (three repetitions) of the number of “normally” germinated pollen grains (see Note 12) in relation to the total number of grains in the field view of the microscope (see Notes 7, 12, and 13). 6. The length of the pollen tube represents the mean value from 25 randomly selected pollen tubes from three drops for each nutrient medium and is expressed in micrometers μm (1 × 10-6 m).

4 Notes 1. Silica gel is placed in an oven at 60 °C/24 h before use for dehydration. 2. Reagent TTC (color of living cells red): 2,3,5-triphenyl tetrazolium chloride.

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3. Male flowers (microstrobiles) in the phase immediately before pollination with part of their shoots (twigs) 15–30 cm long are cut from the tree. The twigs were wrapped in the wet paper in the lower part and transported to the laboratory on the same day in paper bags with an identity label (date, location, genotype). In the laboratory, twigs with microstrobiles were placed in vases with water on clean white paper. After 24–48 h, the anthers burst and pollen was released. 4. The tassels were packed into clean envelopes and on the same day, pollen was collected by shaking on clean paper in the laboratory. 5. The following temperatures were used as treatments: room temperature +23 °C; refrigerator temperature +5 °C; and cryopreservation at -15 and -20 °C. Before placing the pollen at “t” -15 and -20 °C, the pollen was kept at “t” +5 °C for 2 weeks. 6. The dropper must be rinsed with distilled water if it is transferred from one sample to another, or from the reagent to another solution, i.e., to another nutrient medium (sucrose concentration). 7. When viewing drops of reagent or pollen medium through a microscope, a cover plate was placed over to fix the pollen grains. 8. Modified MS medium was used (sucrose solution in distilled water: 0%, 5%, 10%, 15%, 20%, and 25%) [30]. 9. Before each new pollen sample, the laboratory stick was disinfected with 95% alcohol. 10. Before each pollen sampling for analysis, the tube must be carefully shaken to equalize the sample. 11. Three drops of each concentration of sucrose were place on microscope slides size: 25.4 × 76.2 mm (1′′ × 3′′), separated for each of concentrations, and put in the petri dish which was prepared with metal support on which the microscope slides were lifted from the bottom of the vessel (Fig. 6b). The petri dish bottoms were covered with 5–10 mL of water to maintain moisture. 12. Germinated pollen grains are those that are “normally” germinate: those that have developed a single pollen tube greater than or equal to ½ the width of the pollen body [32] poles of the germinal zone [33–37] (Fig. 10). “Abnormally” germinated pollen grains are characterized by some malformations: tube growth with more than one tube (poly-tube growth), tube tip thickening, and tube growth at opposite poles of the germinal zone [33].

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Fig. 10 Abnormal tube growth (Picea omorika/Pancˇ./Purkyne)

13. Counting of pollen grains and measuring the pollen tube length was carried out in the same experiment and they had to be done in the shortest possible time (24–48 h), because after that there is bursting of the pollen tubes and accelerated development of mycelium—phytoinfection.

Acknowledgments This paper was financed by the Ministry of Education and Science of the Republic of Serbia (contract number 451-03-68/2020-14/ 200007). Gratitude: to the technical associate Milosˇ Bokorov, Faculty of Sciences, Department of Biology and Ecology, University Center for Electronic Microscopy, Novi Sad (UCEM-NS), who took Figs. 2 and 3. The other attached photos were taken by the author of the paper Dr. B. Batos, Institute for Forestry, Belgrade. The authors wish to thank the college Dr. Darka Sˇesˇlija Jovanovic´ and English language editor, native speaker, and teacher of English Mrs. Esther Grace Helajzen for proofreading and revising text correction. References 1. Cope J, Singletary G, Krone T, Etter SK (2020) U.S. Patent No. 10,575,517. Washington, DC: U.S. Patent and Trademark Office 2. Engelmann F (2011) Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cell Dev Biol Plant 47(1):5–16 3. Batos B, Miljkovic´ D, Bobinac M (2012) Some characters of the pollen of spring and summer

flowering common oak (Quercusrobur L.). Arch Biol Sci 64(1):89–95 4. Batos B (2013) Picea omorika /Pancˇ./ Purkyne – Balkan endemic and tertiary relict (Serbian spruce – flowering, pollen, seed). Monography, LAP LAMBERT Academic Publishing, Germany, GmbH & Co. KG, pp 1–129. ISBN: 978-3-659-47564-1

Pollen Cryopreservation of Coniferous and Deciduous Species 5. Batos B, Nikolic´ B (2013) Variability of in vitro germination of Picea omorika pollen. Dendrobiology 69:13–19 6. Batos B, SˇesˇlijaJovanovic´ D, Miljkovic´ D (2014) Spatial and temporal variability of flowering in the pedunculate oak (Quercusrobur L.). Sˇumarski list 7-8:371–379 7. Batos B, Miljkovic´ D (2017) Pollen viability in Quercusrobur L. Arch Biol Sci 69(1):111–117 8. Batos B, Veselinovic´ M, Rakonjac L, Miljkovic´ D (2019) Morphological properties of pollen as bioindicators of deciduous woody species in Belgrade parks (Serbia). Topola 203:19–30 9. Batos B, Miljkovic´ D (2019) The vitality of the Serbian spruce (Piceaomorika) pollen during the long-term cryopreservation. Grana 58(6): 433–446 10. Agrawal A, Gowthami R, Srivastava V, Malhotra EV, Pandey R, Sharma N, Gupta S, Bansal S, Chaudhury R, Rana JC, Tyagi RK, Singh K (eds) (2019) Laboratory Manual for Eighth International Training Course on In Vitro and Cryopreservation Approaches for Conservation of Plant Genetic Resources. ICAR-National Bureau of Plant Genetic Resources (NBPGR), New Delhi, Biodiversity International-India, Delhi, Asia Pacific Association for Agricultural Research Institutions (APAARI)/Asia-Pacific Consortium on Agricultural Biotechnology and Bioresources (APCoAB), Bangkok, 5–19 Nov 2019, xviii +83 p 11. Loo J, Fady B, Dawson I, Vinceti B, Baldinelli G (2011) Climate change and forest genetic resources – State of knowledge, risks and opportunities. Background study paper no. 56. https://hal.inrae.fr/hal-02808387/ document 12. Blakesley D, Pask N, Henshaw GG, Fay MF (1996) Biotechnology and the conservation of forest genetic resources: in vitro strategies and cryopreservation. Plant Growth Regul 20(1): 11–16 13. Edesi J, Tolonen J, Ruotsalainen AL, Aspi J, H€aggman H (2020) Cryopreservation enables long-term conservation of critically endangered species Rubushumulifolius. Biodivers Conserv 29(1):303–314 ˇ , Bogdan S, Franjic´ J, 14. Sever K, Sˇkvorc Z Krstonosˇic´ D, Alesˇkovic´ I, Keresˇa S, Fruk G, Jemric´ T (2012) In vitro pollen germination and pollen tube growth differences among Quercus robur L. clones in response to meteorological conditions. Grana 51(1):25–34 15. Wron´ska-Pilarek D, Wiatrowska B, Bocianowski J (2019) Pollen morphology and variability of invasive Spiraea tomentosa

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L. (Rosaceae) from populations in Poland. PLoS One 14(8):e0218276 16. Batos B, Miljkovic´ D (2019) b. the phenotypic plasticity of Piceaomorika/Panc./Purkyne morphological pollen traits. Genetika 51(1): 121–136 17. IUCN 2018 The IUCN red list of threatened species. Version 2018-2. http://www. iucnredlist.org 18. Dell’Oro M, Mataruga M, Sass-Klaassen U, Fonti P (2020) Climate change threatens on endangered relict Serbian spruce. Dendrochronologia 59:125651 19. Aleksic´ JM, Geburek T (2014) Quaternary population dynamics of an endemic conifer, Picea omorika, and their conservation implications. Conserv Genet 15:87–107 20. Jovanovic´ B (1971) Dendrology and phytocenology, 2nd unchanged edn. Scientific books University of Belgrade, pp 84–96 21. Erdtman G (1957) Pollen and spore morphology/plant taxonomy. Gymnospermae, Pteridophyta, Bryophita (illustrations). Almquist & Wiksell, Stockholm, p 151 22. Grbovic´ B (1998) Individual variability of regularity, abundance and morphometric properties of Serbian spruce (Piceaomorika/Pancˇ./ Purkyne) microstrobiles. Manuscript in the book Progress in Botanical Research by IoannesTsekos. Michael Moustakas – Kluwer Academic Publishers Dordrecht Hardbound, Proceedings of the 1st Balkan Botanical Congress, pp 493–96 23. Batos B (2012) Diversity of pedunculate oak (Quercus robur L.). Monograph, Foundation Andrejevic´, Belgrade, Serbia, pp 1–102. ISSN 1450-801X, ISBN 978-86-525-0057-4 24. Pulkkinen P, Rantio-Lehtimaki A (1995) Viability and seasonal distribution patterns of Scots pine pollen in Finland. Tree Physiol 15: 515–518 25. Lanteri S, Belletti P, Lotito S (1993) Storage of pollen of Norway spruce and different pine species. Silvae Genet 42:104–109 26. Fernando DD, Richards JL, Kikkert JR (2006) In vitro germination and transient GFP expression of American chestnut (Castaneadentata) pollen. Plant Cell Rep 25:450–456 27. Kormutak A, Bohovicˇova J, Vookova B, Gomory D (2007) Pollen viability in hybrid swarm populations of PinusmugoTurra and P. sylvestris L. Acta Biol Cracov Bot 49:61–66 28. Grbovic´ B, Isajev V (1997) Variability of pollen viability of 25 Serbian spruce (Piceaomorika/ Pancˇ./Purkyne) test tress. Proceedings of the 3th International Conference on the

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development of forestry and wood science/ technology. ICFWST ‘97 Belgrade Mt. Gocˇ Serbia/Yugoslavia II, pp 64–74 29. Rajora PO, Zsuffa L (2011) Pollen viability of some Populus species as indicated by in vitro pollen germination and tetrazolium chloride staining. Can J Bot 64(6):1086–1088 30. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15:473– 497 31. Kirby EG, Stanley RG (1976) Pollen handling techniques in forest genetics, with special reference to incompatibility. In: Miksche JP (ed) Modern methods in Forest genetics. Springer, Berlin, pp 229–241 32. Arista M, Talavera S (1994) PollenDispersal capacity and pollen viability of Abies pinsapo Boiss. Silvae Genet 43(2–3):155–158 33. Caliskan B, Colgecen H, Pehlivan S (2009) Pollen characteristics and in vitro pollen

germination of Cedrus libani A. Rich. Afr J Biotechnol 8(21):5696–5701 34. Lin L, Yao Q, Huanwen X, Huaizhi M, Jiang J (2013) Characteristics of the staminate flower and pollen from autotetraploidBetulaplatyphylla. Dendrobiology 69:3–11 35. Giordani E, Ferri A, Trentacoste E, Radice S (2014) Viability and in vitro germinability of pollen grains of olive cultivars grown in different environments. Acta Hortic 1057:65–71 36. Hechmi M, Khaled M, Echarari F (2015) In vitro pollen germination of four olive cultivars (Olea europea L.): effect of boric acid and storage. Am J Plant Physiol 10(2):55–67 D, Danielewicz W, 37. Wron´ska-Pilarek Bocianowski J, Malin´ski T, Janyszek M (2016) Comparative pollen morphological analysis and its systematic implications on three European Oak (Quercus L, Fagaceae) species and their spontaneous hybrids. PLoS One 11(8): e0161762

Chapter 54 Pollen Cryopreservation Protocol for Couroupita guianensis Aubl. G. S. Anilkumar, P. E. Rajasekharan, and R. Harsha Abstract Couroupita guianensis which is commonly called as “cannon ball tree” belongs to the family Lecythidaceae. It has several medicinal uses and possesses religious importance. The floral structure is modified, and there are two types of male reproductive organs. Both produce the pollen grains but the fertility status has to be determined. In this study, in vitro viability assessment was carried out for both the pollen grains by hanging drop technique using Brewbaker medium with 20% sucrose solution. Cryopreservation of pollen grains has been carried out for long-term storage of germplasm and breeding programs. Post-cryopreservation viability was also assessed to compare the germination profiles of fresh pollen and cryopreserved pollen grains. The details of material and methods used in development of protocol are discussed in this chapter. Key words Pollen cryopreservation, Couroupita guianensis

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Introduction Couroupita guianensis is one of the cauliflorous species in the Lecythidaceae family and native to Central and Southern America. It commonly called as cannonball tree because of the shape of the fruit. The presence of tree is a good indicator of healthy ecosystem. It is distributed in India as ornamental tree. It has religious importance in temples of lord Shiva and hence called “Kailashpatti” or “Nagalingapushpa.” It is a large deciduous evergreen tree growing up to the height of 20 m and found in tropical and subtropical regions. It is enlisted as “least concern” by IUCN Red List of Threatened Species [1]. Different parts of the tree such as leaf, flower, and fruit are known to have several therapeutic uses. Leaf juice are used to cure skin diseases and in treating malaria. The trees possess antibiotic, antifungal, antiseptic and anti-analgesic properties. The parts of the tree are reported to contain volatile oils, keto-

P.E. Rajasekharan and M.R. Rohini (eds.), Pollen Cryopreservation Protocols, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-2843-0_54, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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steroids, glycosides, couroupitone, indirubcin, isatin, and phenolic substances [2]. It is a remarkable tree with massive trunk bearing cannon balllike fruits with beautiful reddish yellow showy flowers. The leaves are in clusters and inflorescence is cymose. Fruit is globose, a woody capsule containing 200–300 seeds. The flowers are odoriferous and nectarless, and corolla has 6–8 petals. It possesses zygomorphic androecia and modified with two types of male reproductive organs, the hood staminode and the staminal ring. Both the structures contain dimorphic pollen grains with difference in their genetic mechanism [3]. The filaments of anther present in the staminal hood are greater than the staminal ring. The ring stamen contains more number of pollen grains compared to staminal hood. The pollen from the staminal hood are a rich source of nutrition for pollinators. When observed in microscope, the staminal ring pollen appeared as monads and staminal hood pollen as tetrads. In spite of modified and complex floral structure, the flower attracts the pollinators with its floral scent and are mostly bees [4]. Staminal hood pollen is released within few hours of flower opening whereas the staminal ring pollen remains in the flower till mid-day (since it is not easily accessible to the pollinators). Despite producing great quantity of flowers and having high fertilization rate, young fruits are aborted in large numbers [5]. Pollen grains are released from the pollen sac with different developmental stages that cause morphological, cytological and physiological change during anther dehiscence. There are no morphological differences among the pollen grains, but they do differ in their performances with respect to germination rate, vigor, and pollen tube length [6]. To restore the capabilities of pollen grains for long-term benefit and to exploit pollen grains in future breeding programs, cryopreservation is the only way [7]. Pollen cryopreservation in liquid nitrogen is the efficient technique at reduced cost to maintain the male gametophyte for extended use [8]. In this protocol a brief description on pollen viability, germination, and pollen cryopreservation is elaborated.

2

Materials

2.1 Laboratory Equipment

1. Compound microscope 2. Stereomicroscope, e.g., Leica MZ12 (see Note 1) 3. Scanning Electron Microscope 4. Laminar flow hood (see Note 4) 5. Cryotank 6. Drying oven 7. Desiccator

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2.2 General Laboratory Supplies

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1. Dissection tools, namely - scalpels, forceps, scissors, needles, and paint brushes 2. Routine laboratory supplies such as flasks, disposable transfer pipette, and petri plates 3. Borosil glass measuring cylinders, glass bottles (10 mL) 4. Microscope slides and cover slips 5. Syringes 6. Cryovials (2 mL) 7. Aluminum foil 8. White butter papers 9. Whatman filer paper

2.3 Chemicals and Solutions

1. Alexander stain containing ethyl alcohol (95%), malachite green, glycerol, acid fuchsin, phenol and distilled water for viability testing 2. Pollen medium [11] containing boric acid, 300 ppm; calcium nitrate, 100 ppm; magnesium sulfate, 300 ppm; potassium nitrate, 300 ppm; and sucrose, 10% 3. Agarose, 1% 4. Starch powder (soluble), 10% 5. Sterilizing solution 0.3% and 0.6% sodium hypochlorite solutions 6. Ethanol, 70% 7. Liquid nitrogen

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Methods The reproductive phenology of the tree is important to study the pollen biology. Flowering season was observed from November to mid-May and peak flowering in summer, i.e., April–May exhibiting reddish yellow tinge flowers (Fig. 1). The peak anthesis of cannon ball tree flower representing pollen was reported at 9:00 a.m. in the morning [9, 10] (see Note 2).

3.1 Collection of Pollen

The modified androecia structure develops microclimate inside the flower which inhibits the anther dehiscence. Therefore, there is delay in the anther dehiscence (9:00 a.m.) after anthesis (3: 00 a.m.). Fully opened flowers were harvested using a long wooden stick. During harvest, the staminal hood and the ring stamen is gently separated and placed in a butter paper bag separately to avoid mixing up of their pollen. Pollen grains from both the staminal hood and the staminal ring are extracted separately using a needle in a clean butter paper (Fig. 2d) (see Note 3).

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Fig. 1 (A) Tree bearing inflorescence, (B) unopened flower bud

Fig. 2 (A) Bagging of inflorescence to avoid contamination, (B) flower (front view), (C) flower (side view), (D) tapping of anthers to release pollen, (E) pollen collected in petri plate, (F) dehydration of pollen in desiccator 3.2 Dehydration of Pollen

After extraction, it is labeled and stored in the desiccator containing zeolite beads for 1 h to reduce the moisture content of pollen grains (Fig. 2f). Moisture content of the pollen grains has to be reduced to less than 10% to ensure better pollen germination (see Notes 5 and 6).

3.3 Preparation of Pollen Medium

Preparation of Brewbaker (100 mL) medium by dissolving the different chemicals (boric acid, 300 ppm; calcium nitrate, 100 ppm; magnesium sulfate, 300 ppm; potassium nitrate, 300 ppm) in double-distilled water (see Note 10). 1 mL of this stock solution is used for in vitro germinating of pollen grains using different concentrations of sucrose, i.e., 5%, 10%, 15%, 20%, 25%, and 30% [11].

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Fig. 3 (A, B) In vitro pollen germination of Couroupita guianensis 3.4 Viability Assessment of Pollen

Initial viability was assessed using Brewbaker and Kwack medium with added sucrose using the hanging drop method. Pollen dispersed in hanging drop position was incubated in a moist chamber at 25 ± 2°C for 2 h to ensure maximum pollen germination and pollen tube growth [12] (see Notes 11 and 12). Before staining, both pollen grains were observed under microscope to study the shape of pollen grains. Majority of the staminal ring pollen showed viability at different concentrations of sucrose and staminal hood pollen exhibited sterility. Staminal hood pollen did not germinate even in the presence of promotive sucrose concentrations. Pollen viability and length of pollen tube increased gradually with the increase in the sucrose concentration. No germination was observed in the pollen medium without the addition of sucrose. Maximum pollen germination percentage and pollen tube length was observed at 20% sucrose concentration (Fig. 3) (see Note 13). After germination pollen was stained with Alexander stain [13] and viewed under microscope. Pollen grains appeared in prolate shaped before staining, and after staining it was spheroidal. And the diameter of the staminal ring pollen is higher than the staminal hood pollen [9]. Germinated pollen was counted and percent germination was computed. Five replicates were assessed with ten microscopic fields taken for count. Alexander stain preparation: ethyl alcohol (95%), malachite green, glycerol, acid fuchsin, phenol, and distilled water.

3.5 Cryopreservation of Pollen

After the initial viability assessment of pollen, the desiccated pollen were packed in butter paper covers and enclosed in sealed aluminum pouches, which were then rapidly plunged into the cryobiological system containing liquid nitrogen (see Notes 7–9).

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3.6 PostCryopreservation Viability Assessment

4

After cryopreservation, the pollen grains were retrieved from liquid nitrogen and rapidly thawed at room temperature for 3–5 min. The germination test was carried out using Brewbaker’s and Kwack medium consisting of 20% sucrose using the hanging drop method to know the percentage of germination of cryopreserved pollen. Germination percentage was carried out by counting the number of germinated pollen grains and aborted pollen grains. Difference in the germination profiles of fresh pollen and cryopreserved pollen was estimated (see Note 14).

Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination 1. A stereoscopic microscope with a photo documentation system should be used in the evaluation [12]. 2. Pollen collection at peak anthesis time (9:00 am) is ideal for obtaining highest viability and in vitro germination [10]. 3. Separation of two different male reproductive organs immediately after harvest is important to avoid the mixing up of pollen grains [9]. 4. The procedures should be carried out in a clean place possibly in a laminar flow chamber (Fig. 2d). 5. The dehydration time can vary according to the size of the pollen grains, exine thickness, and ambient humidity [12]. 6. The moisture of the pollen grains should be between 15% and 30%. A sample of pollen grains after dehydration should be tested for viability [12]. 7. Always leave some open space at the top of canister [12]. 8. Direct contact of liquid nitrogen with pollen grains causes injuries to the exine of the pollen grains, impairing their viability. Therefore, the envelopes should be well sealed to prevent liquid nitrogen from entering [12]. 9. The process of immersion in liquid nitrogen and transfer to the cryogenic tank should be carried out as fast as possible, to avoid sudden temperature variations, which can harm the pollen grains [12]. 10. Pollen media with sucrose concentration should be prepared at the time of slide preparation to avoid contamination [12]. 11. The pollen grains should be uniformly distributed on the medium, if the grains are agglomerated it will be hard to count them [12]. 12. After slide preparation, the slides are kept in petri dishes lined with wet filter paper to maintain relative humidity and do not allow the pollen media to get dry [12].

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13. To ascertain the germination percentage, the pollen grains in each photomicrograph should be counted. Pollen grains are considered to be germinated when the pollen tube length is greater than or equal to the grain diameter [12]. 14. To confirm any morphological variations on surface of pollen grains, observations of freshly collected and cryopreserved pollen have to be done under a Scanning Electron Microscope (SEM) [12]. References 1. Ravikumar K, Ved DK (2000) 100 red-listed medicinal plants of conservation concern in southern India, 1st edn. Foundation for Revitalization of Local Health Traditions (FRLHT), Bangalore 2. Kumar CS, Naresh G, Sudheer V, Veldi N, Elumalai A (2011) A short review on therapeutic uses of Couroupita guianensis Aubl. Int Res J Pharm App Sci 1(1):105–108 3. Yarsick S, de Enrech NX, Ramirez N, Agostini G (1986) Notes on the floral biology of Couroupita guianensisAubl. (Lecythidaceae). Ann Mo Bot Gard 73(1):99–101 4. Knudsen JT, Mori SA (1996) Floral scents and pollination in Neotropical Lecythidaceae. Biotropica 28(1):42–60 5. Ormond WT, Pinheiro MCB, Castells A (1981) A contribution to the floral biology and reproductive system of Couroupita guianensis Aubl. (Lecythidaceae). Ann Mo Bot Gard 68(4):514–523 6. Pacini E, Franchi GG (2020) Pollen biodiversity – why are pollen grains different despite having the same function? A review. Bot J Linn Soc 193:1–24 7. Silva RL, Souza EH, Vieira LJ (2017) Cryopreservation of pollen of wild pineapple accessions. Sci Hortic 219:326–334. https://doi. org/10.1016/j.scienta.2017.03.022

8. Souza EH, Souza FVD, Rossi ML (2015) Viability, storage and ultrastructure analysis of Aechmea bicolor (Bromeliaceae) pollen grains, an endemic species to the Atlantic forest. Euphytica 204:13–28. https://doi.org/10. 1007/s10681-014-1273-3 9. Bodhipadma K, Noichinda S, Permchalad K, Changbandist S, Phanomchai S (2016) A study of cannonball trees in Thailand: hood staminodes are larger than ring stamens but only germination of stamina ring pollen can be stimulated by exogenous sucrose. Appl Sci Eng Prog 9(3):167–173 10. Thompson JM (1921) Studies in floral morphology and the staminal zygomorphy of Couroupita guianensis, Aubl. Trans R Soc Edinb 3(1):1–15 11. Brewbaker JL, Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am J Bot 50:859–865 12. Ganeshan S, Rajasekharan PE, Shashikumar S, Decruze W (2008) Cryopreservation of pollen. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, New York, pp 281–332. https://doi.org/10.1007/978-0387-72276-4_17 13. Alexander MP (1980) A versatile stain for pollen, fungi, yeast and bacteria. Stain Technol 55: 13–18

Chapter 55 Pollen Cryopreservation Protocol for Gmelina arborea G. S. Anilkumar, B. L. Navya, P. E. Rajasekharan, and R. Harsha Abstract Gmelina arborea is a medium to large-sized tree native to tropical forests of South East Asia. The species is fast growing with short vegetative phase and exploited for both timber and medicinal properties. The inflorescence is cyme and flowering season starts from February till Mid–May. The reproductive success and its availability are under pressure due to self-incompatibility and pollen limitation which in turn reduces the seed set and regeneration capacity. In vitro pollen germination studies were carried out using Brewbaker’s and Kwack pollen germination medium at 15% sucrose solution and cryopreservation in cryobiological system containing liquid nitrogen. Post-cryopreservation studies were achieved to compare the germination profiles of the fresh pollen and cryopreserved pollen. The details of the materials used and method developed for the protocol are discussed in this chapter. Key words Gmelina arborea, Pollen, Brewbaker’s and Kwack medium, Cryopreservation

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Introduction Gmelina arborea is a medium to large-sized tree, belonging to the family Lamiaceae and native to tropical and subtropical regions of South East Asia. It is found in the evergreen forests of Myanmar and Bangladesh and in relatively dry mixed deciduous types [1]. It is fast growing species, yields excellent timber for household fixtures, and hence popularly is called as “white teak” [2]. The tree is self-incompatible and majorly pollinated by bees. It is resistant to pests and diseases. The species has some good medicinal properties and used in Ayurvedic formulations such as “Dashmoola” and “Chyawanprash” [3]. Roots provide a bitter tonic for stomach ache and fruits are used to treat ulcer, anemia, leprosy, and constipation. It is also used as nerve tonic and reported to have wide range of pharmacological activities. Gmelina attains reproductive phase within 3–4 years of age. Leaves are simple, opposite 10–25 cm long and flowers are brownish yellow arranged in panicle cymes 15–30 cm long, which appear after leaf-fall. Flowers are bisexual, complete and zygomorphic.

P.E. Rajasekharan and M.R. Rohini (eds.), Pollen Cryopreservation Protocols, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-2843-0_55, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Calyx is tubular or campanulate and corolla irregular and infundibular. Corolla tube funnel shaped arranged in large terminal panicled cymes. Calyx and corolla are pubescence with leathery appearance. The flower consists of four stamens: bright yellow, two short and two long inserted in the base of corolla tube, the style is slender, located in between anthers. The ovary is four celled and fourovuled [4]. This tree species is threatened due to pollen limitation, maturation of male and female organs at different time, flower abortion and lower seed set [5]. Furthermore, habitat fragmentation and degradation has disrupted the plant-pollinator interactions and lead to decline in the pollinator populations which causes severe threat to its availability [6]. It is a cross-pollinated plant and propagated through seeds. The reproductive success of the plant is limited due to self-incompatible nature of the plant and less pollen availability which is a major problem in tropical forest species [4]. Conservation of Nuclear genetic diversity is important to retain the gene pool of the species and to maintain diverse germplasm. Cryopreservation has gaining considerable importance in breeding and hybridization programs. It is also reported that pollen supplementation increases female fertility and seed set in self-incompatible species [7]. Since the pollen grains are short lived, preservation becomes necessary. Pollen cryopreservation in liquid nitrogen can be carried out to extend the shelf life of pollen grains which can be further used in storage, maintenance, breeding, and germplasm exchange [8]. This protocol helps in determining the pollen viability, pollen germination and cryopreservation in liquid nitrogen.

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Materials

2.1 Laboratory Equipment

1. Compound microscope 2. Stereomicroscope, e.g., Leica MZ12 3. Scanning Electron Microscope (see Note 10) 4. Laminar flow hood 5. Cryotank 6. Drying oven 7. Desiccator

2.2 General Laboratory Supplies

1. Dissection tools, namely - scalpels, forceps, scissors, needles, and paint brushes 2. Routine laboratory supplies such as flasks, disposable transfer pipette, and petri plates 3. Borosil glass measuring cylinders, glass bottles (10 mL) 4. Microscope slides and cover slips

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5. Syringes 6. Cryovials (2 mL) 7. Aluminum foil 8. White butter papers 9. Whatman filer paper 2.3 Chemicals and Solutions

1. Alexander stain containing ethyl alcohol (95%), malachite green, glycerol, acid fuchsin, phenol and distilled water for viability testing 2. Pollen medium [12] containing boric acid, 300 ppm; calcium nitrate, 100 ppm; magnesium sulfate, 300 ppm; potassium nitrate, 300 ppm; and sucrose, 10% 3. Agarose, 1% 4. Starch powder (soluble), 10% 5. Sterilizing solution 0.3% and 0.6% sodium hypochlorite solutions 6. Ethanol, 70% 7. Liquid nitrogen

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Methods The flowering season starts from February till mid-May, and period of flowering can occur 2–3 times in a year [9]. The time duration required from the flower bud towards bloom is 2–13 days. Anther dehiscence occurs in three different stages; pre-anthesis, anthesis and post-anthesis (Fig. 1). In the first case, pollen is hidden and not accessible to pollinators. In the next two cases, pollen and nectar are readily available for pollination [10]. Anthesis which means opening of lower for full extension of stamens occurred in the morning hours, i.e., between 6:00 am and 11:00 am. Anther dehiscence was observed after 1 h of anthesis. The stigma receptivity coincides the anther dehiscence and was observed after 2 h of anthesis and receptivity continued up to 18 h [11].

3.1 Collection of Pollen

Flowers are collected at 8:00–9:00 a.m. in the morning when they are fully open (see Note 1). The flower contains four stamens from where pollen is extracted. Petals are separated and pollen is extracted on the butter paper from the longitudinal slit end of anther using needle (Fig. 1e). Pollen collected is bulked to get uniform in vitro germination.

3.2 Dehydration of Pollen

After extraction, pollen is packed in butter paper covered with aluminum foil and stored in desiccator containing zeolite beads for 2 h (Fig. 1f) (see Notes 2–4). Moisture content of the pollen

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Fig. 1 (A) G. arborea tree with inflorescence. (B) Twig bearing unopened flower bud, (C) flowers in full bloom, (D) dried anthers, (E) pollen extracted in petri plate, (F) pollen placed in desiccator containing zeolite beads for dehydration

grains has to be reduced to 10% to ensure better pollen germination. 3.3 Preparation of Pollen Medium

Preparation of Brewbaker (100 mL) medium by dissolving the different chemicals (boric acid, 300 ppm; calcium nitrate, 100 ppm; magnesium sulfate, 300 ppm; potassium nitrate, 300 ppm) in double-distilled water (see Note 7). 1 mL of this stock solution is used for in vitro germinating of pollen grains using different concentrations of sucrose, i.e., 5%, 10%, 15%, 20%, 25%, and 30% [12].

3.4 Viability Assessment of Pollen

Viability was assessed by initial germination using Brew-bakers and Kwack medium, consisting of 15% sucrose using the hanging drop method. Pollen dispersed in hanging drop position (see Note 11) was incubated in a moist chamber at 25 ± 2°C for 2 h to ensure maximum pollen germination and pollen tube growth [8, 13] (see Notes 8 and 9). After germination pollen was stained with Alexander stain [14] and viewed under microscope (Fig. 2). Germinated pollen was counted and percentage germination was computed. Five replicates were assessed with ten microscopic fields taken for count.

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Fig. 2 Microscopic images of in vitro pollen germination of G. arborea

Alexander stain preparation: ethyl alcohol (95%), malachite green, glycerol, acid fuchsin, phenol and distilled water. 3.5 Cryopreservation of Pollen Grains

After the initial viability assessment of pollen, the desiccated pollen were packed in butter paper covers and enclosed in sealed aluminum pouches which were then rapidly plunged into the cryobiological system containing liquid nitrogen (see Note 5).

3.6 PostCryopreservation Viability Assessment

After cryopreservation, the pollen grains were retrieved from liquid nitrogen and rapidly thawed at room temperature for 3–5 min [19] (see Note 6). The germination test was carried out as initial viability assessment to know the percentage of germination of cryopreserved pollen. Germination percentage was carried out by counting the number of germinated pollen grains and aborted pollen grains. The difference in the germination profiles of fresh pollen and cryopreserved pollen was estimated.

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Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination 1. The flowers are bagged prior to pollen collection before to avoid contamination since it is a cross-pollinated crop [13]. 2. Zeolite granules should be recharged at 60 °C for 24 h before using it for dehydration. The recharged granules can be used

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for 25–30 times depending on the type of pollen used for dehydration [15, 16]. 3. The zeolite granules should be stored in airtight gas jar to prevent it from absorbing the surrounding moisture (Fig. 1f). 4. The duration of pollen dehydration depends on the weather conditions as well. If the weather is highly humid, the pollen is dehydrated for longer durations [8]. 5. The pollen should be sealed airtight to prevent the direct contact of pollen with liquid nitrogen which otherwise damages the pollen tissues which ultimately affects the viability of cryopreserved pollen [8]. 6. Pollen grains are subjected to sudden fluctuations in temperature after cryopreservation which will directly affect the metabolic process of pollen. Hence, thawing is a crucial step after cryopreservation for pollen retrieval which is often after cryopreservation neglected [17]. 7. The prepared Brewbaker and Kwack medium can be stored under refrigerated condition without loss of its action provided sucrose added only at the time of conducting in vitro germination tests otherwise favors development of fungal growth [12]. 8. The optimum relative humidity of pollen should be maintained throughout the incubation period which otherwise inhibits the growth of pollen tube [18]. 9. Pollen grains are said to be germinated when the pollen tube length is greater or equal to the diameter of the pollen grain. The scored pollen is expressed in percentage by counting the total number of pollen to the number of pollen germinated [8]. 10. For better observation of pollen structure and morphology, the individual pollen is studied under a Scanning Electron Microscopy (SEM) [8]. 11. While mounting the micro glasses care should be taken to avoid the contact of pollen media and pollen grains with cavity of the slides [8]. References 1. Stock J, Vargas M, Angarita K, Lez RG (2004) Seed production of Gmelina arborea by controlled pollination. New For 28:167–177 2. Dvorak WS (2004) World view of Gmelina arborea: opportunities and challenges. New For 28:111–126 3. Ravikumar K, Ved DK (2000) 100 red-listed medicinal plants of conservation concern in southern India, 1st edn. Foundation for Revitalization of Local Health Traditions (FRLHT), Bangalore

4. Kumar, KS.,Khanduri, VP. And Tripathi, SK (2021) Reproductive adaptations and the availability of pollinating vectors in white Indian teak (Gmelina arborea Roxb.) in tropical rain forest of Indo-Burma Hotspot. Trees For People (3) 100058 https://doi.org/10.1016/j. tfp.2020.100058 5. Bolstad PV, Bawa KS (1982) Self incompatibility in Gmelina arborea L. (Verbenaceae). Silvae Genet 31(1):19–21

Pollen Cryopreservation Protocol for Gmelina arborea 6. Kearns CA, Inouye DW, Waser NM (1998) Endangered mutualisms: the conservation of plant-pollinator interactions. Annu Rev Ecol Evol Syst 29:83–112 7. Roger JG, Ellis AG (2018) Distinct effects of pollinator dependenceand self-incompatibility on pollen limitation in SouthAfrican biodiversity hotspots. Biol Lett 12:20160253. https:// doi.org/10.1098/rsbl.2016.0253 8. Ganeshan S, Rajasekharan PE, Shashikumar S, Decruze W (2008) Cryopreservation of pollen. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, New York, pp 281–332. https://doi.org/10.1007/978-0387-72276-4_17 9. Pramono AA, Rustam E, Syamsuwida D, Putri KP, Djaman DF, Pujiastuti E (2020) Flower and fruit development and flower-visiting insects of gmelina (Gmelina arborea Roxb). Earth Environ Sci 533:012037. https://doi. org/10.1088/1755-1315/533/1/012037 10. Raju AJS, Rao SP (2006) Anthesis schedules and pollen release in some forage plants of bees. In: Solomon Raju AJ (ed) Advances in pollen spore research, vol 114. Today and Tomorrow’s Printers & Publishers, pp 149–156 11. Khanduri VP, Kumar KS, Sharma CM, Riyal MK, Kar K (2019) Pollen limitation and seed set associated with year-to-year variation in flowering of Gmelina arborea in a natural tropical forest. Grana 58:133–143. https://doi. org/10.1080/00173134.2018.1536164 12. Brewbaker JL, Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am J Bot 50:859–865

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13. Rajasekharan PE, Ganeshan S, Anand M (2015) Pollen cryopreservation in RET medicinal plants of Indian origin. International conference on low temperature science and biotechnological advances. ICAR-NBPGR, New delhi 14. Alexander MP (1980) A versatile stain for pollen, fungi, yeast and bacteria. Stain Technol 55: 13–18 15. Silva RL, Souza EH, Vieira LJ, Pelacani CR, Souza FV (2017) Cryopreservation of pollen of wild pineapple accessions. Sci Hortic 219:326– 334 16. Souza EH, Souza FVD, Rossi ML (2015) Viability, storage and ultrastructure analysis of Aechmea bicolor(Bromeliaceae) pollen grains, an endemic species to the Atlantic forest. Euphytica 204:13–28. https://doi.org/10. 1007/s10681-014-1273-3 17. Cuchiara CC, Souza SA, Silva SD, Bobrowski VL (2012) Effect of the thawing time of castor bean pollen grains stored at different temperatures. Biotemas 25:65–73 18. Leech L, Simpson DW, Whitehouse AB (2002) Effect of temperature and relative humidity on pollen germination in four strawberry cultivars. Acta Hortic 567:261–263. https://doi.org/ 10.17660/ActaHortic.2002.567.53 19. Silva RL, Souza EH, Vieira LJ (2017) Cryopreservation of pollen of wild pineapple accessions. Sci Hortic 219:326–334. https://doi. org/10.1016/j.scienta.2017.03.022

Chapter 56 Pollen Cryopreservation in Moringa concanensis for Crop Improvement B. L. Navya and P. E. Rajasekharan Abstract Moringa concanensis Nimmo is a wild relative of cultivated species Moringa oleifera of family Moringaceae. Its leaves are rich source of vitamin A and C, antioxidants and also a good source of non-desiccating oil called Ben oil. The pollen of this species can be used in breeding programs for transfer of traits to cultivated species. Though a potential species with multiple uses, its availability is restricted due to its limited distribution in certain patches. A protocol for in vitro pollen germination and cryopreservation needs to be standardized to assess the viability of pollen and to make its availability as and when needed. This study was taken up to check the feasibility of pollen cryopreservation in this species. At the time of anthesis, pollen were collected by holding the anthers with forceps and were taken out using a needle; the collected pollen were dried by keeping them in zeolite granules. Pre-storage germination assessment was carried by hanging drop technique using modified Brewbaker and Kwack medium containing 15% sucrose. The pollen was packed in butter paper covers enclosed in sealed aluminum pouches which were rapidly plunged into the cryobiological system containing liquid nitrogen. After pollen cryopreservation was taken out, post-storage germination test was carried out similar to prestorage germination test and the pollen was also observed under SEM (Scanning Electron Microscopy). Cryopreservation of Moringa concanensis pollen can be used to eliminate the barrier of region, season and availability in breeding programmes. Key words Moringa concanensis, Cryopreservation, In vitro pollen germination, Pollination

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Introduction The genus Moringa belongs to the family Moringaceae also known as drumstick or horseradish family. It comprises of 13 species distributed through Southwest Asia, Southwest Africa, Northeast Africa and Madagascar [1]. Moringa concanensis is gaining importance due to its varied uses as one of the best natural coagulants, known to possess antimicrobial and antipyretic activities (Fig. 1). Seeds are effectively utilized for treating and purifying highly turbid water and are also a good source of non-desiccating oil, known as Ben oil [2]. Its leaves are rich source of vitamins A and C which act as a good source of antioxidants [2, 3].

P.E. Rajasekharan and M.R. Rohini (eds.), Pollen Cryopreservation Protocols, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-2843-0_56, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Fig. 1 (A) Tree (B) Slender pods, (C, D) Fully opened flowers displaying yellow anthers

Wild relatives of cultivated plants are an important source of many traits which are absent in cultivated species. They act as a good source for providing resistance to biotic stress and abiotic stress for improving quality and nutrient content. Hence, these species can be used in many breeding programs. Since, Moringa concanensis is a rich source of vitamins and antioxidants, it can be used for biofortification of other cultivated species through breeding for which pollen can act as an important and basic entity for transferring of traits. Long-term storage of pollen is essential as population of M. concanensis is restricted to specific regions, and flowering is seasonal [4, 5]. In order to eliminate the restriction on availability of pollen to use in any breeding strategy, cryopreservation is vital. Cryopreservation is an efficient way for long-term storage of pollen as it lifts the barrier on time, season, and regional constraint without damaging the genetic material. Prior knowledge on viability of pollen helps in better prediction of results in breeding. Factors such as season, flowering period and time of collection affect the viability of pollen. Hence knowledge about floral and pollen biology is crucial to obtain quality pollen. Pollen germination tests determine the viability of pollen which determines the success of cryopreservation. Thus, there is a need to develop protocol for germination of pollen and for cryopreservation.

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Materials

2.1 Laboratory Equipment Required to Carry Out Experiments

1. Stereomicroscope 2. Cryotank 3. Hot air oven 4. Electronic weighing balance 5. Dessicator

2.2 General Laboratory Supplies

1. Dissection tools, i.e., scalpels, forceps, scissors, and needles 2. Routine laboratory supplies such as flasks, disposable transfer pipette, and petri plates 3. Microscope slides 4. Cryovials (2 mL) 5. Aluminum foil 6. Filter papers 7. Butter paper covers

2.3 Chemicals and Solutions

1. Sucrose (15%) 2. Boric acid (100 ppm) 3. Calcium nitrate (300 ppm) 4. Potassium nitrate (100 ppm) 5. Magnesium sulphate (200 ppm) 6. Distilled water 7. Liquid nitrogen 8. Glycerol 9. Alexander’s stain 10. Zeolite granules

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Methods The details on floral biology of M. concanensis should be known for collection of pollen such as time and period of anthesis, peak anthesis, and viability of pollen in natural condition.

3.1

Pollen Collection

3.1.1

Inventory

3.1.2

Procedure

Butter paper covers, forceps, scissors, butter paper, hand gloves.

The flowering in M. concanensis occurs during January to April with peak during February. Anthesis takes place between 6.00 am and 12.00 pm with peak anthesis between 7.00 and 9.30 am [6].

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Fig. 2 Procedure for pollen germination. (A) Collection of flowers from the field, (B) extraction of pollen grains using needle, (C) Drop of pollen medium placed over a slide, (D) Pollen grains dusted over the medium, (E) Pollen placed in a desiccator, (F) desiccator containing zeolite beads

Flowers are collected at peak anthesis using scissors and transferred into a butter paper cover (Fig. 2). Pollen extraction is done by holding the flowers using forceps with the anthers exposed in one hand and extracting the pollen on to a butter paper by using a needle (see Note 1). 3.1.3

Precautions

1. The flowers of M. concanensis are delicate. The flower structure is such that pollen grains are exposed to the surface after anther dehiscence so while collecting the flowers care should be taken not to disturb the pollen grains. 2. The flowers wither quickly and pollen grains absorb moisture hence, the pollen is extracted immediately after flower collection.

3.2 Dehydration and Determination of Moisture Content of Pollen 3.2.1

Inventory

3.2.2

Procedure

Butter paper, zeolite granules, hot air oven.

The moisture content of pollen is one of the factors which determine the efficiency of cryopreservation. Pollen with high moisture content when subjected to cryopreservation induce ice crystal formation thereby losing its viability [7]. Hence, moisture determination and dehydration of pollen is crucial step. The moisture

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determination of M. concanensis pollen is done by oven dry method. The initial weight of the extracted pollen is recorded. Then the pollen are dried in hot air oven for 2 h at 60 °C temperature. After oven drying, the final weight of the pollen is recorded. The difference between the initial and final weight of the pollen expressed in percentage gives the moisture content of the pollen. The moisture content of M. concanensis is 23 ± 3% depending on the weather and time of collection. The dehydration of pollen is done by drying them in zeolite granule desiccators kept in glass jar for 2.5 h (see Notes 2–4). 3.2.3

Precautions

1. The pollen of Moringa concanensis are very sticky hence only butter paper was used for extraction and not petriplates.

3.3

Cryostorage

Cryocans, canisters, liquid nitrogen, aluminum pouches, butter paper.

3.3.1

Inventory

3.3.2

Cryopreservation

3.3.3

Precautions

The dehydrated pollen are packed in butter paper, enclosed in aluminum pouches and sealed. The packed pollen are placed in canisters and are rapidly plunged into the cryobiological system containing liquid nitrogen at -196 °C [8]. 1. The pollen should be sealed airtight to prevent the direct contact of pollen with liquid nitrogen (see Note 5). 2. The pollen grains are plunged into the cryobiological system immediately after extraction and dehydration to avoid the viability loss.

3.4 Thawing and Retrieval

After cryopreservation, the pollen grains were removed from the liquid nitrogen and rapidly thawed at room temperature (27 ± 3 ° C) for 2–3 min [9, 10]. The pollen from pouches are removed and can be used for in vitro germination and pollination (see Note 6).

3.5 Pollen Germination Medium

Sucrose (15%), boric acid (100 ppm), calcium nitrate (300 ppm), potassium nitrate (100 ppm), magnesium sulfate (200 ppm), distilled water, conical flasks, beakers, measuring cylinders, micropipette, digital weighing balance.

3.5.1

Inventory

3.5.2 Preparation of Pollen Germination Medium

The medium used for in vitro germination of M. concanensis is Brewbaker and Kwack medium [11]. It is prepared by adding 2.5 mg boric acid, 7.5 mg calcium nitrate, 5 mg magnesium sulfate and 2.5 mg potassium nitrate to some quantity of water; then the final volume is made up to 25 mL. 15% sucrose is added to the pollen media at the time of germination test (see Note 7).

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1. The final volume of medium is made up to 25 mL only after the chemicals are completely dissolved. 2. Sucrose is added at the time of conducting in vitro test; otherwise, it causes contamination and development of fungus in the medium.

3.6 In Vitro Germination Test 3.6.1

Cavity slides, micro cover glasses, needle, tissue paper, forceps, pipette, Vaseline, petri plates, filter paper, and distilled water.

Inventory

3.6.2 Hanging Drop Technique

Pollen is germinated using hanging drop technique. The dehydrated pollen taken out from the zeolite granules are used for in vitro germination tests. In this technique, the cavity slides are cleaned using distilled water. The edges of micro cover glasses are smeared with adhesive (Vaseline) to avoid its displacement. A small drop of pollen medium is put on the micro cover glass using a pipette. The pollen is taken from the tip of the needle; it is spread on the pollen medium placed on the cover glass. The micro cover glass is inverted on top of the cavity slide [12].

3.6.3

Incubation

The pollen grains are incubated for 12 h at room temperature in a petri plates covered with filter paper [13]. Distilled water is sprinkled on filter paper in order to maintain relative humidity (100%) [14] (see Note 8).

3.6.4

Precautions

1. The pollen don’t get evenly distributed on the pollen medium due its sticky nature. Hence, the pollen are evenly spread on the pollen media with the aid of a needle. 2. While mounting the micro glasses, care should be taken to avoid the contact of pollen media and pollen grains with cavity of the slides.

3.7 Staining and Scoring of Pollen 3.7.1

Inventory

3.7.2

Staining

Alexander’s stain, glycerol, stereomicroscope, needle, slides.

The germinated pollen grains are stained using Alexander’s stain [15]. The micro cover glasses are taken out from incubated cavity slides. The corners of micro glasses are wiped with tissue paper to remove adhesive (Vaseline). A drop of Alexander’s stain and a drop of glycerol are added onto the pollen medium containing germinated pollen grains with the aid of needle. The micro cover glasses are inverted onto a clean plain slide for further observation under microscope.

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Fig. 3 Microscopic images of in vitro pollen germination of M. concanensis. (A) Fresh pollen, (B) Cryopreserved pollen 3.7.3

Scoring

3.7.4

Precautions

3.8 Scanning Electron Microscopy Analysis

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The scoring of pollen is done by counting the total number pollen to the numbers of pollen germinated when observed under a microscope (Fig. 3). It is expressed in percentage (see Note 9). 1. While staining the germinated pollen, care should be taken to not to disturb the pollen; otherwise, the pollen count changes. Freshly dehisced pollen grains were placed onto double-sided copper tape on the disk surface of polished aluminum stabs and passed in a vacuum evaporator under SEM microscope version, HITACHI-TM3030Plus. The pollen grains were observed and photographed at 1000–5000× resolution for whole grain (Fig. 4). The grain size was estimated by measuring the length of the equatorial diameter (E) and polar axis (P), and exine sculpture ornamentation was observed (see Note 10).

Notes 1. The selected flowers should be bagged 1 day prior to pollination in pre-opening bud stage to avoid contamination. 2. Zeolite granules should be recharged at 60 °C for 24 h before using it for dehydration. The recharged granules can be used for 25–30 times depending on the type of pollen used for dehydration [16]. 3. The zeolite granules should be stored in airtight glass jar to prevent it from absorbing the surrounding moisture. 4. The duration of pollen dehydration depends on the weather conditions as well. If the weather is highly humid, the pollen are dehydrated for longer durations.

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Fig. 4 Scanning Electron Microscopy analysis. (A, B) Fresh pollen, (C, D) cryopreserved pollen

5. The direct contact of liquid nitrogen with the pollen sample damages the pollen tissues which ultimately affects the viability of cryopreserved pollen. 6. Thawing is a crucial step after cryopreservation for pollen retrieval which is often neglected. Thawing after cryopreservation directly affects the metabolic process of pollen after cryopreservation as the pollen grains are subjected to sudden fluctuations in temperature [17]. 7. The prepared Brewbaker and Kwack medium can be stored under refrigerated condition without loss of its action provided sucrose added only at the time of conducting in vitro germination tests; otherwise, it favors development of fungal growth. 8. The optimum relative humidity of pollen should be maintained throughout the incubation period which otherwise inhibits the growth of pollen tube [14]. 9. Pollen are said to be germinated only when the pollen tube length is greater or equal to the diameter of the pollen grain. The scored pollen grains are expressed in percentage by

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counting the total number of pollen to the number of pollen germinated. 10. For better observation of pollen structure and morphology the individual pollen are studied under Scanning Electron Microscopy (SEM). Since the pollen of M. concanensis are sticky good quality images are obtained when the pollen are inserted in cryo vials containing distilled water for 5–10 min, then water is filtered to obtain which can be observed under SEM. References 1. Abd Rani NZ, Husain K, Kumolosasi E (2018) Moringa genus: a review of phytochemistry and pharmacology. Front Pharmacol 9:108 2. Anwar F, Latif S, Ashraf M, Gilani AH (2007) Moringa oleifera: a food plant with multiple medicinal uses. Phytother Res 21(1):17–25 3. Siddhuraju P, Becker K (2003) Antioxidant properties of various solvent extracts of total phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa oleifera Lam). J Agric Food Chem 15: 2144–2155 4. Manzoor M, Anwar F, Iqbal T, Bhanger MI (2007) Physicochemical characterization of Moringa concanensis seeds and seed oil. J Am Oil Chem Soc 84:413419 5. Anwar F, Bhanger MI (2003) Analytical characterization of Moringa oleifera seed oil grown in temperate regions of Pakistan. J Agric Food Chem 51:6558–6563 6. Bhattacharya A, Mandal S (2004) Pollination, pollen germination and stigma receptivity in Moringa oleifera Lam. Grana 43:48–56 7. Almeida C, ALdo A, Barbosa JF (2011) Braz. J Bot 34:493–497 8. Malik SK, Chaudhury R (2019) Cryopreservation techniques for conservation of tropical horticultural species using various explants. In: Rajasekharan P, Rao V (eds) Conservation and utilization of horticultural genetic resources. Springer, Singapore, pp 579–594 9. Towill LE (2002) In: Towill LE, YPS B (eds) Cryopreservation of plant germplasm II, biotechnology in agriculture and foresty, vol 50. Springer, Berlin, pp 3–21

10. Vendrame WA, Carvalho VS, Dias JMM, Maguire I (2008) Pollination of Dendrobium hybrids using cryopreserved pollen. HortScience 43:264–267 11. Brewbaker JL, Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am J Bot 50:859–865. https://doi.org/10.1002/j.1537-2197.1963. tb06564.x 12. Ganeshan S, Rajasekharan PE, Shashikumar S, Decruze W (2008) Cryopreservation of pollen. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, New York, pp 443–464. https://doi.org/10.1007/978-0387-72276-4_17 13. Connor KF, Towill LE (1993) Pollen-handling protocol and hydration/dehydration characteristics of pollen for application to long-term storage. Euphytica 68:77–84. https://doi. org/10.1007/BF00024157 14. Leech L, Simpson DW, Whitehouse AB (2002) Effect of temperature and relative humidity on pollen germination in four strawberry cultivars. Acta Hortic 567:261–263. https://doi.org/ 10.17660/ActaHortic.2002.567.53 15. Alexander MP (1969) Differential staining of aborted and non-aborted pollen. Stain Technol 44:117–122 16. Silva RL, Souza EH, Vieira LJ, Pelacani CR, Souza FV (2017) Cryopreservation of pollen of wild pineapple accessions. Sci Hortic 219:326– 334 17. Cuchiara CC, Souza SA, Silva SD, Bobrowski VL (2012) Effect of the thawing time of castor bean pollen grains stored at different temperatures. Biotemas 25:65–73

Chapter 57 Cryopreservation of Sandalwood (Santalum album L.) Pollen G. S. Anilkumar and P. E. Rajasekharan Abstract Santalum album L. (Santalaceae) occupies a prime position in Indian forestry and has been rated as the most precious and valuable tree. For successful conservation of sandalwood germplasm, a detailed knowledge on reproductive biology is required. Pollen cryopreservation of sandalwood was attempted in view of long-term conservation of genetic resources. Before cryopreservation, the in vitro viability and fertility assessments of freshly collected pollen were done after desiccation for 1 h in zeolite beads. Modified Brewbaker and Kwack (1963) medium which was prepared by using following protocol. The bas medium constituted of sucrose (10%) supplemented with boric acid (300 ppm), calcium nitrate (100 ppm) and magnesium sulfate (300 ppm) was used for pollen germination. The concentrations were altered based on the response of pollen germination percentage (%). The germination percentage of fresh pollen was recorded to be 84.50% after 4 h of incubation. The dried anthers with pollen were packed in butter paper covers enclosed in sealed aluminum pouches which were rapidly plunged into the cryobiological system containing liquid nitrogen. Post-cryopreservation assessments (after 1 month of cryopreservation) were done to check viability and fertility status of cryopreserved pollen. The results indicated that there was no reduction in the germination percent (84.20%) of cryopreserved pollen when compared to fresh desiccated pollen (84.50%). The materials used and procedures followed in the development of protocol are discussed in detail in this chapter. Key words Santalum album, Pollen cryopreservation, In vitro germination, Brewbaker and Kwack medium

1

Introduction Santalum (sandal wood) is a genus of semi-parasitic tree species occurring throughout South and SouthEast Asia, Australia and the Pacific. The heartwood of several species produces valuable aromatic oil widely used in perfumery, medicines and incense. Throughout the world, Sandalwood products are being sourced from declining natural stands and the international price for natural sandalwood products continues to increase. Therefore, significant opportunity exists to establish commercial sandalwood agro

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forests, to reduce pressure on wild stands. The improvement of any Sandalwood species will depend upon knowledge of its breeding system and its cross-compatibility with related species that are a source for potentially useful characters [1]. Santalum album L. is one of the most primitive precious species since time immemorial. This plant was domesticated due to its multifarious usefulness. Sandalwood occupies a pre-eminent place among the forest crops which are of great economic value [2]. It is a source of East Indian sandal oil which underpins Indian culture. The oil extracted from the heart wood of Sandalwood tree has over 2000 years of uninterrupted history in the perfumery trade. An earlier study indicates that sandal is a polymorphic species [3]. Apart from natural impediments, the wanton destruction of tree by unscrupulous elements has been so devastating that this tree which underpins Indian culture is facing extinction and listed as an endangered species. Therefore, there is an imperative need to save the species from total annihilation and enrich the debilitated population with superior and economically viable techniques of conservation. Though, it has the natural regeneration capability, but the occurrence of Sandalwood species is very less in wild habitats due to the recalcitrant nature of the seeds and continuous inbreeding. Sandalwood crop improvement can be done by spontaneous interspecific hybridization for which continuous availability of quality pollen should be ensured. To make sure the supply of pollen for indefinite time period, pollen storage is very important. Cryopreservation method can be achieved for long-term storage, especially for those which are having recalcitrant seeds like sandalwood, which are difficult to store due to high moisture content, high desiccation and freezing sensitivity. Natural pollination and fruit set in Santalum album are usually low with high heterozygosity, genetic variation and abnormality in succeeding generations [4]. It is predominantly outbreeding and self-incompatible species, although its flower structure was designed for self-pollination, suggesting that pollination system depends on the agent of pollination. Hence, to get higher fruit set when crossing studies and hybridization program was carried out, sufficient quantity of quality pollen should be available which is achieved through pollen cryopreservation. The purpose of this chapter is to brief in detail about the materials and methods used for protocol development of Sandalwood pollen cryopreservation.

2

Materials Required

2.1 Collection of Pollen Grains

Inflorescence of Sandalwood (Fig. 1c), dissection tools, namely scalpels, forceps, scissors, Camlin paint brush, butter paper and needles, writing pens, label stickers for identification.

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Fig. 1 (A) Sandalwood tree, (B) Flowering twig of Sandalwood, (C) Inflorescence of Sandalwood, (D) Opened flowers of Sandalwood with anthers 2.2 Dehydration of Pollen

Aluminum foil sheet measuring 4 × 4 cm, ruler, white butter paper, zeolite beads and desiccator.

2.3 Cryopreservation of Pollen in Liquid Nitrogen

White butter papers, aluminum foil, cryogenic tubes with volume of 2 mL, cryogenic container equipped with canisters and liquid nitrogen.

2.4 In Vitro Germination of Pollen

Brewbaker and Kwack (100 mL) [5] medium containing boric acid, 300 ppm; calcium nitrate, 100 ppm; magnesium sulfate, 300 ppm; potassium nitrate, 300 ppm in double-distilled water. 1 mL of this stock solution is used for in vitro germinating of pollen grains using different concentrations of sucrose, i.e., 10%, 15%, 20%, and 25% [6]. Precision scale, beaker, spatulas, petri dishes, routine laboratory supplies such as flasks, disposable transfer pipette, petri plates, glass bottles (10 mL), microscopic cavity slides, and cover slips and Whatman filter paper. Alexander stain containing ethyl alcohol (95%), malachite green, glycerol, acid fuchsin, phenol, and distilled water for viability testing. Binocular microscope, photomicroscope, stereomicroscope, e.g., Leica MZ12 and Scanning Electron Microscope.

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Methods Reproductive behavior and floral characters should be understood before the start of any experiment. Sandalwood flowers arranged in inflorescences consisting of 20–40 single flowers (Fig. 1). Flowers are bisexual, actinomorphic and epigynous, borne on axillary or terminal panicles. A flower lasts for about 3 days and its color gradually changes from pale green or white to dark red with age. Though the ovary has 2–4 embryo sacs, only one matures and sets fruit [7].

3.1 Collection of Flowers

The inflorescence of the Sandalwood is collected during its anthesis time (9:00–10:00 am) (see Notes 1 and 2). The inflorescence consists of flowers with different growth stages. The fresh flowers which are opened during anthesis time will have white petals and anthers with viable pollen, such flowers are separated from inflorescence for pollen extraction. The flowers with brown petals have already shed their anthers and pollen, such kind of flowers should not be selected (Fig. 1c, d).

3.2 Collection and Dehydration of Anthers

The fresh anthers are dissected from the flowers using forceps and collected in the white butter paper and keep it for dehydration in desiccator containing silica gel or zeolite beads for 2 h for reduction of moisture content in pollen. Since the pollen is too small in size, it is difficult to extract; hence, anthers containing pollen are directly used for in vitro germination (see Notes 3 and 4).

3.3 Preparation of Pollen Medium

Pollen Brewbaker’s and Kwack medium (100 mL) was prepared by dissolving the different ingredients (boric acid, 300 ppm; calcium nitrate, 100 ppm; magnesium sulfate, 300 ppm; potassium nitrate, 300 ppm) in double-distilled water (see Note 6). 1 mL of this stock solution is used for in vitro germinating of pollen grains using different concentrations of sucrose, i.e., 10%, 15%, 20%, and 25% [6].

3.4 Viability Assessment of Pollen

Viability by in vitro germination assessment was carried out by hanging drop technique. A single drop of Brewbaker’s medium was smeared on coverslip with sufficient number of anthers containing pollen grains spread on the media and the coverslip was inversed on microscopic slide with single cavity. After this, the slides were placed on wet filter paper in a closed petri dish kept at room temperature (25–27 °C) for 24 h (see Notes 5, 7, and 8). Germination percentage was determined by microscopic examination, and the grains were scored as germinated when the pollen tube length exceeded the grain diameter (see Note 9). The fresh anthers containing pollen grains are germinated in pollen medium

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Fig. 2 (A) Flowers were taken to the laboratory for pollen extraction, (B) Desiccation of anthers in zeolite beads, (C) In vitro germination of Sandalwood fresh anthers containing pollen in Brewbaker medium, (D) Cryopreservation of Sandalwood anthers, (E) Post-cryopreservation viability assessment of Sandalwood pollen

containing 20% sucrose after 4 h of incubation. The fresh pollen grains have recorded maximum germination percentage of 84.50% (see Note 10). The slides of the germinated pollen were stained with alexander stain [8] and observations were recorded using stereomicroscope (Fig. 2). 3.5 Cryopreservation of Pollen

The desiccated anthers containing viable pollen are packed in butter paper covers enclosed in sealed aluminum pouches which were rapidly plunged into the cryobiological system containing liquid nitrogen (see Notes 11–13).

3.6 PostCryopreservation Viability and Fertility Assessment

The anthers are taken out from liquid nitrogen after 1 week of cryopreservation and rapidly thawed at room temperature for 3–5 min. The germination test was carried out to know the percentage of germination of cryopreserved pollen. The highest germination percentage (84.20%) of pollen grains present in anthers was recorded with 20% of sucrose concentration in Brewbaker medium after 1 week of cryopreservation. The results indicated that there was no reduction in the germination percent (84.20%) of cryopreserved pollen when compared to fresh desiccated pollen (84.50%) indicating the feasibility of anther/pollen cryopreservation.

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4 Notes: Precautions, Dos and Don’ts to Get Good-Quality Pollen and In Vitro Germination 1. The selection of flowers is very important, the flowers opened during the anthesis time (9:00–10:00 am) having anthers with viable pollen are selected [1]. 2. The selected inflorescence should be covered by bagging 1 day prior to collection [1]. 3. The size of the pollen is very small, which makes difficult for pollen extraction from the anthers [1]. 4. The anthers containing pollen grains should be dried for 2 h; the dried anthers were shaken in petri plates to obtain pollen [9]. 5. Since the pollen grains are not disinfested, the procedures should be carried out in a clean place and, if possible, in a laminar flow chamber [9]. 6. Pollen media with sucrose concentration should be prepared at the time of slide preparation. Use the media within 1 day of its preparation to avoid contamination [5]. 7. The pollen grains should be uniformly distributed on the medium, preferably without puncturing. If the grains are agglomerated, it will be hard to count them (Fig. 2c). 8. After slide preparation, the slides are kept in petri dishes covered with wet filter paper. It maintains the humidity and does not allow the pollen media to get dry [9]. 9. To ascertain the germination percentage, all the anthers which released pollen grains in each photomicrograph should be counted. Pollen grains are considered to be germinated when the pollen tube length is greater than or equal to the grain diameter [9]. 10. A stereoscopic microscope with a photo documentation system should be used in the evaluation of pollen germination [9]. 11. Direct contact of liquid nitrogen with anthers/pollen grains causes injuries to the exine of the pollen grains, impairing their viability. Therefore, the envelopes should be well sealed to prevent liquid nitrogen from entering [9]. 12. The envelopes should be closed carefully so as not to crush the anthers and then covered with aluminum foil by plastering and inserted into canisters. Always leave some open space at the top of canister (Fig. 2d). 13. The process of immersion in liquid nitrogen and transfer to the cryogenic tank should be carried out as fast as possible, to avoid sudden temperature variations, which can harm the anthers/ pollen grains (Fig. 2d).

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References 1. Page T, Tate H, Bled C (2012) Breeding behaviour of three sandalwood species (Santalum album, S. austrocaledonicum and S. lanceolatum). James Cook University, pp 102–108 2. Sundararaj R (2008) Distribution of predatory arthropod communities in selected sandal provenances of south India. J Biopest 4(1):86–91 3. Krishnakumar N, Parthiban KT (2017) Flowering phenology and seed production of Santalum album L. Int J Curr Microbiol App Sci 6(5): 963–974 4. Baskorowati L (2011) Flowering intensity and flower visitors of Santalum album L. at ex-situ conservation plot, Watusipat, Gunung Kidul, Yogyakarta. Indones J For Res. J For Res 8(2): 130–143 5. Brewbaker JL, Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am J Bot 50(9):859–865

6. Metz C, Nerd A, Mizrahi Y (2000) Viability of pollen of two fruit crop cacti of the genus Hylocereus is affected by temperature and duration of storage. Hort Sci 35(2):199–201 7. Ratnaningrum YW, Faridah E, Indrioko S, Syahbudin A (2016) Flowering and seed production of sandalwood (Santalum album; Santalaceae) along environmental gradients in Gunung Sewu Geopark, Indonesia. Nusantara Biosci 8(2):180–191 8. Alexander MP (1980) A versatile stain for pollen, fungi, yeast and bacteria. Stain Technol 55: 13–18 9. Ganeshan S, Rajasekharan PE, Shashikumar S, Decruze W (2008) Cryopreservation of pollen. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, New York, pp 281–332. https://doi.org/10.1007/978-0387-72276-4_17

Chapter 58 Cryopreservation of Pollen for Long-Term Storage in Teak (Tectona grandis) G. S. Anilkumar and P. E. Rajasekharan Abstract Studies on in vitro pollen viability and feasibility of pollen cryopreservation in teak (Tectona grandis L.) was conducted at ICAR-Indian Institute of Horticultural Research, Hessarghatta Lake Post, Bengaluru, during September 2021. Pollen germination was estimated by hanging drop technique using modified Brewbaker’s and Kwack (1963) medium which was prepared by using following protocol. The base medium constituted of sucrose (10%) supplemented with boric acid (100 ppm), calcium nitrate (300 ppm) and magnesium sulphate (200 ppm). The concentrations were altered based on the response of pollen germination percentage (%). The dried anthers with pollen were packed in butter paper covers enclosed in sealed aluminum pouches which were rapidly plunged into the cryobiological system containing liquid nitrogen. After 2 days of post-cryopreservation, the germination test was carried out to know the percentage of germination of cryopreserved pollen. The fresh pollen recorded germination percentage of 58.25% and it was 52.18% after 2 days of cryopreservation with 15% sucrose in Brewbaker and Kwack medium. The results indicated that there is no significant decline in the viability of cryopreserved pollen. The cryopreservation of pollen provides a constant supply of viable and fertile pollen to allow supplementary pollinations for crop improvement programs and can become an important component of national gene banks. The purpose of this chapter is to describe in detail a simple, fast, inexpensive, and widely applicable method for cryopreservation of pollen grains of teak. Key words Tectona grandis, Pollen cryopreservation, In vitro germination, Brewbaker media

1

Introduction Teak (Tectona grandis L.) is a tropical tree of commercial value due to the high demand of its high-quality wood and rapid growth. Teak has become one of the most widely planted species in tropical regions of the world, given its high price for its timber in international markets. Due to its strength, resistance, workability and aesthetic qualities, teak is considered to be one of the most valuable woods for furniture, shipbuilding, docks or piers, freshwater bridges and floodgates, railway ties, parquet floors, poles for electric transmission lines, and fences, as well as for musical instruments

P.E. Rajasekharan and M.R. Rohini (eds.), Pollen Cryopreservation Protocols, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-2843-0_58, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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and toys [3]. The great economic importance that teak currently has due to its wood value has stimulated the development of breeding programs. This has motivated the development of tree improvement programs in the Latin American region and in the tropical world in general. Genetic improvement programs for this species have resulted in seeds of better quality and at the same time, improvement in the quality of plantations. To preserve genetic diversity and guarantee the raw material for improvement programs and for future reproduction, seeds are kept under conventional seed banks conditions with temperature ranging between 4 and -20 °C. However, there are other means to conserve this valuable germplasm. Recently, efforts are being made to advance towards the next breeding generation, since knowledge about floral biology and pollen management have become important issues. In order to advance into the next breeding generations, building capacities in topics such as pollen banks requires pollen quality, thus enabling the exchange of pollen among cooperative members. Pollen fertility studies are of considerable value in breeding programs, in order to determine pollen viability and germination in collections of genotypes, before going into mating operational activities [4]. Cryopreservation is the storage of plant material in liquid nitrogen (-196 °C) and its major advantage is the conservation for indefinite periods of time, under high genetic stability conditions. Pollen cryopreservation is required for carrying out investigations in both fundamental and applied aspects of pollen biology. Besides the already existing role of pollen cryobanks in breeding, there are many promising applications which have come to focus with the recent advances in allied bio-scientific areas. Crossing desirable genotypes involves multiple and staggered plantings in order to synchronize flowering. This can be avoided when cryopreserved viable pollen is available, facilitating hybrids between genera, species, and genotypes. This could effectively conserve field and greenhouse space. Moreover, this will eliminate the need to grow plant populations to produce pollen. The determination of moisture content at which pollen grains tolerate dehydration is between 35% and 27%, which are reached at 2 and 4 h of dehydration in zeolite beads. Furthermore, immediately after harvesting, different authors have recommended determining a percentage of moisture content close to 30%, at which the pollen grain is tolerant to dehydration and can be managed and stored. On the other hand, it must be recognized that field conditions and relative humidity at the time of flower plucking will affect pollen moisture content, making it necessary to develop a methodology for the determination of the moisture content of the pollen grains at the moment of harvesting and hours after harvesting. However, in tropical species such as teak, fresh and dry pollen grains extracted directly from the anthers are in physiologically ideal conditions to be collected and stored [2]. The purpose of this chapter is to describe in detail a

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simple, fast, inexpensive, and widely applicable method for cryopreservation of pollen grains of teak. In this chapter, we optimized pollen quality analysis protocol in terms of the viability and germination of fresh and cryopreserved teak pollen.

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Materials

2.1 Laboratory Equipment

1. Binocular microscope 2. Stereomicroscope, e.g., Leica MZ12 (see Note 1) 3. Scanning Electron Microscope 4. Cryocan 5. Drying oven 6. Desiccator

2.2 General Laboratory Supplies

1. Dissection tools, namely - scalpels, forceps, scissors, and needles 2. Routine laboratory supplies such as flasks, disposable transfer pipette, and petri plates 3. Borosil glass measuring cylinders, glass bottles (10 mL) 4. Microscope slides and cover slips 5. Syringes 6. Cryovials (2 mL) 7. Aluminum foil 8. White butter papers 9. Whatman filer paper

2.3 Chemicals and Solutions

1. Alexander stain containing ethyl alcohol (95%), malachite green, glycerol, acid fuchsin, phenol, and distilled water for viability testing. 2. Pollen medium (Brewbaker) containing boric acid, 100 ppm; calcium nitrate, 300 ppm; magnesium sulfate, 200 ppm; potassium nitrate, 100 ppm; and sucrose, 5%, 10%, 15%, and 20%. 3. Polyethylene glycol 5%, 10%, 15%, and 20%. 4. Ethanol, 70%. 5. Liquid nitrogen.

3

Methods It is necessary to know the viability of the pollen grains in natural conditions before starting the cryopreservation procedure. This requires the complete knowledge on the timing of floral anthesis,

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Fig. 1 (A) Tectona grandis at flowering stage. (B) Inflorescence of teak. (C) Close view of inflorescence

stigma receptivity and the details of the reproductive system of the species under study (see Note 2). The images showing tree at its peak flowering and inflorescence is pictured in Fig. 1. In teak, pollen grains had the highest percentage of viability between 12.00 and 14.00 h. 3.1 Collection and Dehydration of Anthers

The peak anthesis time of teak flowers is from 10:00 am to 12:00 pm, and the flowers are collected during the above mentioned time (see Note 3), and the anthers were separated from the flowers by using forceps and packed in butter paper covered with aluminum foil and stored in desiccator containing zeolite beads for 2 h (Fig. 2f) (see Note 5).

3.2 Preparation of Pollen Medium

Pollen media/Brewbaker (100 mL) medium was prepared by dissolving the different chemicals (boric acid, 300 ppm; calcium nitrate, 100 ppm; magnesium sulfate, 300 ppm; potassium nitrate, 300 ppm) in double-distilled water (see Note 9). 1 mL of this stock solution is used for in vitro germinating of pollen grains using different concentrations of sucrose, i.e., 10%, 15%, 20%, and 25% [8].

3.3 Viability Assessment of Pollen

Hanging drop technique was used to check the viability of pollen by in vitro germination assessment. A single drop of pollen media was smeared on cover slip with sufficient number of anthers were spread on the media to release the pollen grains and the cover slip was inversed on microscopic slide with single cavity (see Notes 10 and 11). After this, the slides were placed on the wet filter paper in a

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Fig. 2 (A) Completely opened flowers of teak with anthers. (B) Flowers removed from panicle. (C) and (D) Anthers extracted from flowers and collected in petri dish. (E) and (F) Anthers in the desiccator with zeolite beads. (G) Immersion of desiccated anthers into liquid nitrogen inside the cryotank

closed petri dish kept at room temperature (25–27 °C) for 24 h (see Note 4). Germination percentage was determined by microscopic examination of at least 500 randomly selected pollen grains per sample. Grains were scored as germinated when the pollen tube length exceeded the grain diameter (see Note 12). The pollen grains present in the anthers have recorded maximum germination percentage of 58.25 in 15% sucrose concentration after 2 h of incubation (Fig. 3a), while there was no germination observed in 10% and 25% sucrose concentrations. The slides of the germinated pollen were stained with Alexander stain [1], and observations were recorded using stereomicroscope. 3.4 Cryopreservation of Anthers in Liquid Nitrogen

The dehydrated anthers were packed into three different samples in butter paper covers enclosed in sealed aluminum pouches which were rapidly plunged into the cryobiological system containing liquid nitrogen (Fig. 2g) (see Note 6–8).

3.5 PostCryopreservation Viability Assessment

After 2 days of post cryopreservation, the anthers are taken out from liquid nitrogen and rapidly thawed at room temperature for 3–5 min. The germination test was carried out to know the percentage of germination of cryopreserved pollen. The highest germination percentage (52.18%) of pollen grains present in anthers was recorded with 15% of sucrose + 15% polyethylene glycol (PEG)

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Fig. 3 (A) Germination of fresh pollen grains showing good uniformity. (B) Germination of cryopreserved pollen grains stained with Alexander stain

concentration in Brewbaker medium after 48 h of cryopreservation (Fig. 3b). The results indicate that there was no significant decline in viability of cryopreserved pollen after 48 h of postcryopreservation. 3.6 Scanning Electron Microscopy Analysis

4

Freshly dehisced pollen grains and cryostored pollen were placed onto double-sided copper tape on the disc surface of polished aluminum stabs and passed in a vacuum evaporator under SEM microscope version, HITACHI-TM3030Plus. The pollen grains were observed and photographed at 1000–5000× resolution for whole grain. The grain size was estimated by measuring the length of the equatorial diameter (E) and polar axis (P), and exine sculpture ornamentation was observed (Fig. 4).

Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination 1. A stereoscopic microscope with a photo documentation system should be used in the evaluation [2]. 2. The inflorescences should be protected before (pre-anthesis) and during anthesis to prevent contamination with undesirable pollen grains brought by pollinators or wind [3]. 3. Time of pollen collection is very important. Pollen collection at peak anthesis time (10:00 am to 12:00 pm) is ideal for obtaining highest viability and in vitro germination [4]. 4. Since the pollen grain are not disinfested, the procedures should be carried out in a clean place and, if possible, in a laminar flow chamber [2].

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Fig. 4 (A) SEM image of teak fresh pollen. (B) length of the fresh pollen (43.1 μm). (C) Width of the fresh pollen (22.0 μm). (D) SEM image of cryopreserved pollen. (E) Length of the cryopreserved pollen (42.5 μm). (F) Width of the cryopreserved pollen (21.7 μm)

5. The dehydration time can vary according to the size of the anthers or pollen grains, exine thickness and ambient humidity. Also, according to the findings of Souza et al. [5] and Silva et al. [6], the moisture of the pollen grains should be between 15% and 30%. A sample of pollen grains after dehydration should be tested for viability. 6. The envelopes should be closed carefully so as not to crush the anthers and then covered with aluminum foil by plastering and inserted into canisters. Always leave some open space at the top of canister (Fig. 2g). 7. Direct contact of liquid nitrogen with anthers/pollen grains causes injuries to the exine of the pollen grains, impairing their viability. Therefore, the envelopes should be well sealed to prevent liquid nitrogen from entering [2]. 8. The process of immersion in liquid nitrogen and transfer to the cryogenic tank should be carried out as fast as possible, to avoid sudden temperature variations, which can harm the pollen grains [2].

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9. Pollen media with sucrose concentration should be prepared at the time of slide preparation. Use the media within 1 day of its preparation to avoid contamination [2]. 10. The pollen grains should be uniformly distributed on the medium (Fig. 3a), preferably without puncturing. If the grains are agglomerated, it will be hard to count them. 11. After slide preparation, the slides are kept in petri dishes covered with wet filter paper. It maintains the humidity and does not allow the pollen media to get dry [2]. 12. To ascertain the germination percentage, all the anthers which released pollen grains in each photomicrograph should be counted. Pollen grains are considered to be germinated when the pollen tube length is greater than or equal to the grain diameter [2]. References 1. Alexander MP (1980) A versatile stain for pollen, fungi, yeast and bacteria. Stain Technol 55: 13–18 2. Ganeshan S, Rajasekharan PE, Shashikumar S, Decruze W (2008) Cryopreservation of pollen. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, New York, pp 281–332. https://doi.org/10.1007/978-0387-72276-4_17 3. Gomez A, Vargas Castillo P, Abdelnour Esquivel A (2013) Cryopreservation of teak (Tectona grandis L) seeds. Agron Costarric 37(1):51–60 4. Hine A, Rojas A, Suarez L, Murillo O, Espinoza M (2019) Optimization of pollen germination in Tectona grandis (teak) for breeding programs. Forests 10(10):908

5. Souza EH, Souza FVD, Rossi ML (2015) Viability, storage and ultrastructure analysis of Aechmea bicolor (Bromeliaceae) pollen grains, an endemic species to the Atlantic forest. Euphytica 204:13–28. https://doi.org/10.1007/s10681014-1273-3 6. Silva RL, Souza EH, Vieira LJ (2017) Cryopreservation of pollen of wild pineapple accessions. Sci Hortic 219:326–334. https://doi.org/10. 1016/j.scienta.2017.03.022 7. Brewbaker JL, Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am J Bot 50(9):859–865 8. Metz C, Nerd A, Mizrahi Y (2000) Viability of pollen of two fruit crop cacti of the genus Hylocereus is affected by temperature and duration of storage. HortScience 35(2):199–201

INDEX A Abelmoschus esculentus................................. 281, 282, 289 Aborted pollen grains ................................ 380, 389, 436, 505, 559, 580, 587 Accession ..............................................13, 14, 21, 44, 47, 49, 86, 99, 100, 108, 110, 124, 191, 192, 196, 283, 288, 289, 424, 510, 511, 528 Accessories .............................................................. 45, 567 Acetocarmine staining.........................101, 104, 545, 548 Agriculture.................................................................4, 562 Alexander stain .......................................67, 80, 115, 117, 295–297, 312–329, 333, 335, 336, 357, 359, 375, 386–388, 395, 417, 418, 425, 427, 435, 436, 443, 444, 449, 451, 458, 465, 467, 472, 477, 485, 488, 489, 495, 496, 503, 505, 513, 516, 557, 558, 577, 579, 585–587, 603, 605, 611, 613, 614 Alismataceae .................................................................... 23 Alleles ............................................. 6, 7, 24, 25, 100, 114, 236, 281, 294, 342, 364, 394, 416, 519, 534 Allium cepa........................................................... 273–279 Almond ............................................................................ 32 Aloe vera ...................................................... 433, 434, 438 Aluminum pouches .................................... 118, 127, 239, 297, 335, 360, 377, 388, 397, 398, 419, 427, 436, 450, 452, 459, 479, 480, 490, 491, 505, 514, 558, 579, 587, 595, 605, 613 Amaryllidaceae........................................................ 23, 415 Ambient temperature............................... 22, 67, 91, 100, 101, 127, 162, 171, 180, 194, 206, 207, 221, 241, 257, 277, 367, 398, 427, 530 Amenability .................................................................2, 15 Amruthaphala ................................................................ 455 Anacardiaceae .................................................................. 23 Angiosperm ........................................................ 22, 23, 35 Anhydrous CaCl2 .......................................................... 100 Annona atemoya.................................... 61–64, 66, 68–70 Annona cherimola ................................. 62, 64, 66, 68–70 Annona glabra ...................................... 63, 64, 66, 68–70 Annona muricata ........................................64, 66, 68–70 Annona reticulata................................. 62, 63, 66, 68–70 Annona species..........................................................61–71 Annona squamosa ................................. 61, 64, 66, 68–70

Annonaceae ..................................................................... 61 Anther dehiscence ................................24, 66, 76, 78, 90, 178, 201, 208, 221, 241, 246, 257, 274, 276, 283, 311, 312, 375, 384, 387, 399, 417, 443, 456, 465, 470, 477, 486, 494, 503, 556, 557, 576, 577, 585, 594 Anthers ................................... 12, 24, 25, 31, 32, 36, 50, 51, 54, 57, 62, 66, 67, 69, 71, 78, 84, 90, 91, 101, 103, 104, 108, 109, 113, 126, 127, 137–140, 142, 148, 150–153, 156, 160, 164, 168, 178, 180, 192, 195, 201–203, 208, 221, 222, 227, 228, 238, 241, 246, 247, 249, 256, 257, 259, 266, 276, 279, 283, 287, 288, 295, 296, 304, 312, 333, 347–349, 351, 356, 366, 374, 375, 377, 384, 385, 387, 399, 419, 426, 435, 436, 443, 456, 476, 484, 486, 487, 493, 495, 501, 504, 511, 514, 515, 521, 522, 536, 549, 556, 557, 565, 571, 576, 578, 584–586, 592, 594, 603–606, 610, 612, 613, 615, 616 Anthesis .....................................8, 23, 24, 50, 65, 66, 68, 71, 77–79, 82, 90, 97, 116–118, 120, 126, 128, 131, 137, 138, 148, 150, 153, 156, 159, 163, 168, 172, 178, 179, 185, 192, 196, 201, 208, 217, 221, 236, 237, 241, 245, 246, 250, 256, 259, 260, 270, 274, 275, 279, 283, 295, 299, 302, 304, 307, 311–314, 333, 334, 337, 347, 350–352, 357, 360, 368, 375, 381, 384, 387, 389, 395, 399, 417, 420, 435, 438, 443, 445, 449, 459, 460, 465, 467, 470, 471, 477, 481, 486, 495, 496, 498, 503–506, 511, 515, 516, 545, 551, 556, 557, 559, 577, 580, 585, 593, 594, 604, 606, 611, 612, 614 Antibacterial .................................................................. 475 Antidiabetic .......................................................... 147, 475 Anti-inflammatory......................................................... 475 Antioxidant.................................147, 475, 519, 591, 592 Antipyretic ............................................................ 475, 591 Aphrodisiac ........................................................... 331, 475 Apocyanaceae ....................................................... 455–461 Areca catechu................................................................. 534 Arecaceae .............................................................. 519, 527 Artificial pollination ..................................... 56, 101, 113, 114, 216, 264, 332, 356, 384, 533, 539 Artocarpus heterophyllus .............................. 135, 137, 144

P.E. Rajasekharan and M.R. Rohini (eds.), Pollen Cryopreservation Protocols, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-2843-0, © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

617

POLLEN CRYOPRESERVATION PROTOCOLS

618 Index

Asphodelaceae ............................................................... 433 Asphyxiation .................................................................... 49 Association Forest Cellulose (AFOCEL) ...................... 13 Asteraceae ............................................................. 393, 509 Asynchronous flowering .......................... 6, 20, 100, 114, 136, 176, 190, 200, 236, 264, 273, 342, 364, 394, 401, 562 Aurantioideae ..........................................................99–110 Avena ............................................................................... 32

B Benincasa hispida .......................................................... 215 Bicellular pollen..............................................23, 281, 282 Bignoniaceae.................................................................. 483 Binucleate pollen.......................................................12, 35 Biochemical ............................................... 12, 36, 86, 332 Biofortification .............................................................. 592 Biological oxygen demand (BOD) incubator ............. 103 Birdfoot grapevine ........................................................ 441 Boric acid ....................................... 25, 28, 51, 65, 67, 89, 92, 93, 101, 102, 104, 109, 115, 116, 125, 137, 140, 168, 170, 178, 180–182, 192, 193, 195, 201, 204, 217, 218, 227, 237, 245, 247, 255, 257, 265, 270, 275, 276, 284, 295, 304, 312, 313, 333, 344, 350, 357, 365, 368, 375, 376, 386, 387, 394, 395, 417, 425, 435, 443, 444, 449, 458, 459, 465, 466, 470, 471, 477, 479, 485, 488, 495, 496, 503, 504, 511, 512, 521, 529, 535, 537, 545, 547, 557, 558, 577, 578, 585, 586, 593, 595, 603, 604, 611, 612 Breeding ...................................... 2, 6–13, 15, 19–39, 56, 57, 86, 99, 114, 123, 124, 136, 155, 168, 176, 189, 190, 200, 216, 225–227, 236, 244, 254, 264, 294, 299, 302, 303, 310, 311, 342, 343, 356, 363–369, 373, 374, 384, 401, 402, 424, 434, 442, 448, 469, 485, 494, 502, 510, 520, 527, 528, 534, 543, 544, 556, 562, 576, 584, 592, 602, 610 Brewbacker and Kwack medium .................................... 28

C Cactaceae ....................................................................... 113 Calcium nitrate........................................... 25, 28, 32, 51, 101, 102, 104, 109, 115, 116, 137, 140, 168, 170, 201, 204, 217, 218, 237, 245, 247, 255, 257, 275, 276, 284, 295, 312, 313, 333, 344, 350, 357, 375, 376, 386, 387, 394, 395, 417, 425, 435, 443, 444, 449, 458, 459, 465, 466, 470, 477, 479, 485, 495, 496, 503, 504, 511, 512, 520, 521, 545, 547, 557, 558, 577, 578, 585, 586, 593, 595, 603, 604, 611, 612 Canisters ........................................ 45, 46, 48, 54, 65–67, 79, 90, 91, 96, 102, 121, 125, 127, 129, 130,

139, 153, 162, 163, 171, 178, 180, 192, 194, 204, 206, 221, 238–241, 247, 249, 257, 294, 313, 333, 338, 349, 350, 357, 361, 378, 381, 390, 397, 398, 410, 411, 421, 427, 439, 445, 450, 460, 467, 470, 479, 480, 497, 498, 504, 506, 514, 515, 529, 532, 580, 595, 603, 606, 615 Capsicum frutescens ....................................................... 253 Caricaceae ........................................................................ 85 Carica papaya ...................................................... 3, 85–97 Caryophylaceae................................................................ 35 Cavity slides ..........................................28, 67, 78, 81, 82, 92, 93, 128, 140, 141, 149, 152, 157, 158, 161, 170, 181, 193, 194, 204, 217, 220, 227, 236, 238, 255, 265, 270, 296, 304, 306, 365, 368, 394, 395, 405, 407, 408, 427, 450, 471, 479, 512, 596, 603 Cayratia pedata ................................................... 441–445 Cayratia pedate .................................................... 442, 444 Celastraceae .......................................................... 447, 493 Celastrus paniculatus ........................................... 447–451 Cellophane method ..................... 53, 193, 313, 377, 379 CFC ................................................................................. 37 Chenopodiaceae .............................................................. 35 Chickpea .......................................................................... 21 Chyawanprash ............................................................... 583 Cicer macracanthum....................................................... 21 Cicer microphyllum.......................................................... 21 Cicer nuristanicum ......................................................... 21 Cicer oxyodon ................................................................... 21 Cicer species .................................................................... 21 Citrullus lanatus ........................................................... 363 Citrus aurantiifolia ...................................................... 100 Citrus limetta ................................................................ 100 Citrus limon ................................................ 100, 108, 170 Citrus maxima ..................................................... 100, 293 Citrus sinensis ....................................................... 100, 101 Clone ....................................................... 20, 36, 114, 544 Cocos nucifera ................................................................ 527 Colchicaceae .................................................................. 383 Commelinaceae ............................................................... 23 Compound microscope ............................... 92, 115, 168, 180, 181, 200, 220, 227, 229, 237, 238, 268, 283, 285, 304, 306, 311, 367, 374, 386, 394, 395, 424, 434, 442, 448, 458, 464, 476, 485, 494, 502, 510, 512, 513, 529, 530, 535, 556, 576, 584 Conservation ...................................... 1–9, 12–15, 19–39, 57, 63, 75, 77–79, 86, 124, 147, 155, 156, 168, 176, 199, 200, 216, 226, 236, 244, 254, 269, 303, 311, 332, 356, 364, 368, 374, 384, 394, 433, 434, 448, 458, 464, 469, 476, 484, 488, 494, 502, 510, 520, 527, 528, 544, 556, 562, 584, 602, 610 Conservationist ............................................................... 14

POLLEN CRYOPRESERVATION PROTOCOLS Index 619 Controlled pollination ....................................6, 206, 236, 274, 448, 469, 510 Conventional breeding ........................... 21, 76, 394, 510 Corpusculum ........................................................ 456, 478 Couroupita guianensis Aubl. ........................................ 575 Cover slip..............................................67, 68, 78, 81, 82, 92, 115, 117, 125, 149, 151, 152, 157, 158, 160, 161, 169, 170, 180, 192, 200, 217, 236, 245, 248, 255, 258, 265, 276, 277, 283, 285, 295, 311, 333, 334, 350, 357, 359, 365, 374, 378, 386, 394, 416, 417, 425–427, 434, 443, 449, 450, 458, 459, 465, 470, 477, 479, 485, 488, 494, 503, 511–513, 556, 577, 584, 603, 604, 611, 612 Crop improvement....................................... 2, 12, 25, 75, 135–144, 148, 190, 236, 244, 245, 254, 256, 265, 274, 281, 297, 310, 343, 347, 364, 393, 476, 602 Cross pollination ....................................10, 94, 114, 124, 235, 279, 355, 384, 483 Cruciferae ..................................................................23, 35 Cryobank .........................50, 51, 57, 124, 279, 374, 402 Cryobiological system................................ 54, 65, 66, 78, 79, 82, 90, 118, 125, 127, 139, 148, 150, 153, 157, 158, 162–164, 170, 178, 192, 194, 240, 256, 297, 335, 344, 360, 377, 388, 397, 398, 427, 436, 444, 448, 450, 459, 467, 470, 479, 489, 496, 504, 505, 558, 579, 587, 595, 605, 613 Cryobiotechnology ........................................................... 4 Cryocane.......................................................................... 45 Cryodatabase ................................................................... 14 Cryoequipments........................................................44–47 Cryoflask ......................................54, 127, 129, 130, 171, 221, 239, 240, 247, 249, 276, 279, 313, 349, 350, 397, 427 Cryogenic preservation................................................... 32 Cryogenic temperature .............................. 34, 43, 49, 55, 204, 206, 221, 458, 480 Cryo-mesh protocol.......................................................... 3 Cryo-plate.......................................................................... 3 Cryopreservation.................................... 1–15, 20–22, 24, 25, 32, 33, 37, 39, 43, 45, 54–57, 61–71, 75–83, 86, 90, 91, 97, 100, 101, 113–121, 123–131, 135, 136, 138, 139, 142, 144, 148, 158, 159, 162, 163, 168, 171, 176, 178, 185, 190, 192–194, 196, 200, 203, 204, 217, 221, 226, 228, 231, 238, 240, 241, 244, 247, 249, 254, 256, 257, 260, 264–266, 269, 273, 274, 276, 281–283, 286, 287, 289, 294, 295, 297–299, 303, 304, 313, 332–335, 337, 342, 343, 347, 349, 351, 356–358, 360, 363–369, 373, 374, 377, 383, 384, 388, 389, 393–399, 401–421, 423, 424, 427, 433, 436, 441–445, 447, 450, 455–461, 463, 464, 467, 469–472, 475–481, 483, 485, 488, 493–499, 501–506, 509–516,

520, 527–538, 543, 544, 551, 555, 556, 558, 559, 561, 562, 566, 571, 572, 575, 576, 579, 580, 583–588, 592, 594, 595, 598, 601–606, 609–611, 613, 614, 616 Cryoprotectant ............................................ 3, 32, 56, 402 Cryoprotocols ................................................................. 13 Cryo storage .............................................. 4, 86, 170, 426 Cryotanks ............................................. 45–47, 49, 50, 65, 78, 79, 89, 102, 115, 117, 125, 136, 148, 150, 157, 158, 168, 177, 191, 200, 204, 223, 241, 277, 286, 287, 311, 343, 374, 378, 386, 399, 416, 424, 434, 442, 448, 458, 465, 476, 485, 494, 502, 504, 510, 514, 522, 529, 533, 535, 537, 544, 556, 576, 584, 593, 613 Cryotherapy....................................................................... 2 Cryotransit.................................................................47–48 Cryovial.............................................45, 47, 49, 102–104, 108, 109, 115, 136, 149, 150, 152, 153, 157, 158, 162, 169, 200, 201, 283, 286–289, 311, 374, 386, 406, 410, 425, 435, 443, 449, 458, 465, 470, 477, 485, 494, 503, 511, 529, 532, 533, 535, 537, 544, 547, 551, 556, 577, 585, 593, 611 Cucumis melo................................................................. 264 Cucurbitaceae ............................................... 31, 215, 235, 293, 331 Cucurbita moschata.............................................. 293, 294 Cyperaceae ....................................................................... 23

D Dashmoola............................................................ 483, 583 Decalepis arayalpathra .................................455–461, 463 Decalepis hamiltonii Wight & Arn............................... 463 Decalepis khasiana ......................................................... 463 Decalepis nervosa ........................................................... 463 Decalepis salicifolia........................................................ 463 Desiccation ............................................... 3, 6, 11, 12, 23, 31, 32, 35, 56, 100, 117, 139, 159, 200, 203, 217, 219, 276, 281–283, 286, 289, 295, 297, 334, 336, 343, 358, 359, 403, 405, 406, 420, 485, 488, 494, 510, 511, 514, 515, 531, 535–537, 545, 558, 602, 605 Desiccation-sensitive .......................................... 24, 31, 32 Desiccation-tolerant............................................... 31, 448 Dewar......................................................45, 54, 129, 283, 286, 406, 410 Dicot ................................................................................ 23 Dimorphic pollen grains ............................................... 576 Donor parents ................................................................. 15 Droplet vitrification .......................................................... 3 Drying oven....................................... 102, 115, 168, 200, 311, 374, 386, 416, 424, 434, 442, 448, 458, 465, 477, 485, 494, 502, 510, 520, 522, 556, 576, 584, 611 Dry shippers ..............................................................47, 48

POLLEN CRYOPRESERVATION PROTOCOLS

620 Index E

Ecofriendly ...................................................................... 37 Eco-rehabilitation ......................................................... 448 Emasculation ................................. 71, 97, 108, 120, 128, 131, 153, 185, 196, 248, 250, 254, 258, 260, 270, 278, 283, 347, 350, 352, 481, 511, 516, 538, 539 Embelia ribes Burm .............................................. 469–472 Encapsulation .................................................................... 3 Endangered ................................... 1, 5, 7, 9, 12, 20, 303, 332, 383, 384, 441, 455, 463, 562, 602 Endemic.................................................38, 455, 463, 563 Equatorial length of pollen ......................... 71, 119, 185, 207, 231, 298, 337, 360, 388, 451, 490, 497 Equatorial width of pollen........................... 71, 119, 185, 231, 298, 337, 360, 388, 451, 490, 497 Eucalyptus ........................................................................ 20 Euphorbiaceae ...........................................................75, 78 Exchange of pollen ................................................ 20, 610 Exine ................................................ 6, 19, 22, 35, 67, 71, 77, 91, 108, 121, 127, 148, 172, 180, 195, 205, 206, 232, 241, 249, 260, 270, 289, 299, 315, 338, 351, 360, 361, 368, 381, 387, 389, 390, 438, 439, 445, 460, 467, 481, 498, 505, 506, 515, 559, 580, 606, 615 Exine sculpture ornamentation .................. 205, 597, 614 Expectorant ................................................................... 475 Explant........................................................ 3, 6, 8, 12, 13, 15, 31, 46, 57 Ex situ conservation ........................................................... 1

F FDA staining ................................................................... 25 Fertile...........................................8, 20, 21, 54, 148, 200, 225, 254, 274, 310, 342, 448, 469, 502 Fertility assessment ................................33, 54, 118, 119, 125, 127, 171, 207, 221, 240, 258, 287, 313, 336, 344, 350, 352, 360, 380, 381, 398, 419–421, 427, 451, 480, 514, 515, 528, 538, 605 Fertilization ..................................... 7, 21, 23, 25, 27, 29, 30, 33, 114, 189, 190, 199, 274, 332, 384, 412, 416, 451, 509, 562, 576 Field gene bank ............................ 25, 147, 244, 254, 510 Field pollination ................................ 33, 54, 82, 93, 101, 125, 127, 128, 142, 153, 156, 163, 171, 182, 195, 206, 208, 221, 230, 240, 241, 380, 394, 398, 399, 406, 411–412, 427, 451, 510, 548 Floral biology ................................ 76, 78, 137, 216, 227, 245, 256, 265, 275, 295, 332, 347, 356, 365, 417, 593, 610 Forage grasses............................................................7, 282 Forest trees ......................................................... 7, 32, 282

Forestry........................................................ 2, 5, 7, 8, 562 Freezing .......................................3, 4, 11, 15, 31, 32, 35, 63, 109, 114, 168, 223, 226, 286, 294, 303, 364, 399, 403, 410, 416, 427, 434, 464, 494, 602

G Galactagogue ................................................................. 475 Gametophyte ................................. 8, 20–22, 24, 30, 168, 311, 332, 394, 433, 434, 448, 484, 556, 576 Gelatin capsules ....................................47, 54, 71, 78, 79, 90, 96, 125, 127, 130, 158, 162, 171, 178, 183, 194, 196, 204, 221, 239, 245, 247, 255, 257, 275, 313, 344, 349, 397, 427, 459, 470, 478–480 Gene bank..................................... 11, 25, 39, 43, 55, 342 Gene pool conservation .................................76, 332, 374 Genetic amelioration.............................................. 21, 200 Genetic diversity...................................11, 12, 14, 20, 24, 56, 99, 199, 342, 355, 356, 384, 456, 476, 610 Genetic erosion ....................................2, 75, 86, 99, 124, 199, 236, 254, 342, 448, 458, 464, 484, 519 Genetic integrity ......................................... 7, 15, 20, 303 Genetic security ............................................................... 22 Genetic stocks ................................................................. 15 Genotype ....................................... 1–4, 7, 11, 15, 20, 21, 25, 36, 48, 57, 76, 86, 97, 109, 124, 131, 136, 148, 176, 185, 189, 190, 192, 196, 200, 203, 216, 236, 282, 283, 288, 289, 364, 373, 374, 384, 416, 424, 481, 511, 516, 527, 528, 544, 545, 571, 610 Germination .....................................7, 20, 27–30, 33–36, 39, 55, 56, 62, 63, 66–69, 71, 77–83, 90–93, 97, 100–102, 104–107, 114, 115, 117, 118, 120–121, 127, 128, 130, 131, 139–141, 148, 151–153, 156, 157, 159, 160, 162–164, 168, 172–173, 176, 178, 180, 181, 184, 185, 192–194, 196, 203, 204, 206, 207, 216, 217, 219, 220, 227, 229, 232, 236, 238, 245, 247–248, 250, 255, 257, 258, 260, 265, 268, 270, 275–278, 284–287, 289, 295–299, 303–306, 313–330, 332, 335–338, 342–346, 350, 352, 357, 359–362, 364, 365, 367, 368, 376, 379–382, 384, 387–390, 394, 395, 397, 402, 405–409, 412, 416, 418–420, 424, 425, 427, 435, 436, 438–439, 443–445, 450, 452, 459–461, 466–468, 471, 472, 478–481, 487–489, 491, 494, 496–499, 504–506, 509, 510, 512–514, 520, 521, 523, 524, 528–530, 532, 534, 537, 538, 547, 550, 551, 556–560, 562, 565, 569, 570, 576, 578–581, 584–588, 592, 594–596, 598, 603–606, 610–616 Germplasm .......................................2, 5, 6, 8–10, 12, 14, 15, 20, 21, 47–49, 75, 86, 99, 100, 124–126, 147, 176, 190, 216, 225, 236, 263, 273, 303, 310, 311, 342, 343, 355, 384, 394, 406, 424, 433,

POLLEN CRYOPRESERVATION PROTOCOLS Index 621 434, 442, 448, 458, 469, 484, 485, 502, 519, 584, 610 Germplasm conservation .......................15, 20, 148, 216, 225, 236, 244, 294, 297, 311, 342, 416, 442, 456, 464, 510, 534, 556 Germplasm exchange .................................. 282, 476, 584 Gingkoaceae .................................................................... 23 Glacial acetic acid ................................................. 470, 488 Gladiolus hybridus ......................................................... 373 Gloriosa superba L. ........................................................ 383 Gmelina arborea................................................... 583–588 Gramineae.................................................... 23, 31, 35, 36

H Hanging drop culture (Cavity slide method)............... 28, 217, 512, 513 Haploid ............................... 8, 12, 56, 86, 124, 236, 254, 264, 332, 342, 374, 424, 485 Heterogeneous ................................................................ 12 HITACHI-TM3030 Plus ...............................70, 96, 183, 205, 451, 597, 614 Holostemma ada-kodien ....................................... 475–481 Horticulture ........................................................... 19, 562 Humidity ....................................... 12, 24, 34–36, 45, 71, 101, 104, 108, 121, 152, 161, 172, 236, 299, 315, 338, 360, 361, 365, 381, 389, 405, 407, 421, 438, 445, 460, 467, 471, 479, 481, 498, 506, 530, 532, 537, 547, 551, 559, 561, 580, 606, 615, 616 Hybrid ...................................7, 8, 13, 15, 20–22, 35, 37, 43, 55, 56, 61, 64, 83, 100, 114, 136, 216, 225, 226, 236, 244, 254, 264, 274, 282, 302, 332, 342, 364, 374, 394, 401, 402, 424, 527, 534, 610 Hybridization .....................................5, 7–11, 13, 20, 56, 63, 76, 83, 114, 124, 128, 156, 168, 176, 190, 216, 226, 236, 254, 281, 282, 294, 302, 310, 311, 342, 356, 374, 384, 401, 416, 423, 433, 434, 448, 488, 543, 544, 562, 584, 602 Hydration ..................................... 33, 406, 487, 521, 523 Hylocereus undatus ........................................................ 113 Hypoglycemic................................................................ 475

I ICAR-CPCRI, Kasaragod .............................................. 14 ICAR-CPRI, Shimla ....................................................... 14 ICAR-IIHR, Bengaluru..................................... 14, 54–55 ICAR-IISR, Kozhikode .................................................. 14 ICAR-NBPGR, New Delhi ............................................ 14 Ice crystal....................................... 3, 4, 31, 63, 100, 114, 138, 226, 282, 364, 594 ICRISAT....................................................................20, 21 Illegitimate pollen .....................................................8, 448 In situ conservation .......................................................... 1

In vitro assay.................................................................... 51 In vitro fertilization......................................................... 12 In vitro pollen germination ....................... 28, 68, 69, 92, 93, 128, 136, 140, 156, 161, 163, 170, 181, 193, 194, 196, 200, 204, 205, 220, 236–238, 240, 282–284, 288, 289, 311, 313, 394, 408, 416, 420, 421, 469–472, 512, 597 In vitro pollination.......................................................... 12 In vitro storage.................................................................. 2 In vivo assay ..................................................................... 51 In vivo fertility ........................................................ 82, 195 In vivo viability .......................................78, 82, 103, 105, 128–129, 195, 227, 229, 232, 245, 248, 250, 255, 260, 265, 268, 270, 275, 278, 304, 307, 365, 367, 368 Incubation period ...............................144, 289, 588, 598 Institute of Plant Genetics and Crop Plant Research (IPK) .................................................... 13 Inter-generic hybridization........................................... 114 International Air Transport Association (IATA) ........... 48 International Union for Conservation of Nature (IUCN) .................................. 383, 441, 455, 476, 483, 494, 502, 562, 575 Intestinal disorders........................................................ 475 Intra-cellular ice crystal formation ............................... 364 Introgression .................................... 9, 20, 226, 302, 310 IPR .............................................................................22, 38 Iridaceae......................................................................... 373

J Jivanthi........................................................................... 475 JNTBGRI ........................................................................ 14 Juglans regia ........................................................... 36, 100 Juncaceae ......................................................................... 23 Jyothishmati .................................................................. 447

K Kadamba ........................................................................ 555 Kailashpatti .................................................................... 575 Kiwi ............................................................................35, 63

L Lagenaria siceraria ....................................................... 235 Lamiaceae ...................................................................... 583 Laminar flow hood..................................... 115, 168, 200, 255, 260, 311, 374, 386, 416, 420, 424, 434, 442, 448, 458, 465, 470, 476, 485, 494, 502, 556, 576, 584 Least concern ................................................................ 575 Lecythidaceae ................................................................ 575 Leguminosae ................................................................... 23 Liliaceae ........................................................................... 23 Lily .......................................................................... 35, 383

POLLEN CRYOPRESERVATION PROTOCOLS

622 Index

Liquid nitrogen (-196°C) ........................ 1–4, 6, 33, 35, 36, 43–50, 54, 55, 65–71, 78–80, 86, 89–92, 95–97, 102–105, 109, 116–119, 121, 125, 127, 129, 130, 137, 139, 144, 150, 153, 164, 168, 170–172, 178, 180, 183–185, 192, 194–196, 201, 203, 204, 210, 219, 221, 223, 227, 232, 238–241, 245–247, 249, 255–257, 260, 264, 270, 274–279, 283, 294, 297, 299, 304, 312, 313, 315, 333, 335, 336, 338, 344, 349–351, 357, 359–361, 365, 368, 375, 377, 381, 386, 389, 390, 397–399, 402, 410, 411, 417–419, 425, 427, 434–436, 439, 443–445, 448–450, 452, 458–461, 464, 465, 467, 469, 470, 477, 479, 480, 485, 488–491, 495–498, 503, 505, 506, 510, 511, 514, 516, 521, 522, 528, 529, 532, 533, 537, 545, 547, 548, 551, 557–559, 576, 577, 579, 580, 584, 585, 587, 588, 593, 595, 598, 603, 605, 606, 610, 611, 613, 615 Long term storage ..................................... 12, 24, 30–32, 36, 45, 86, 279, 303, 424, 510, 528, 544, 592, 602 Long-term ex situ preservation method ...................... 562 Long-Term Storage ............................................. 609, 610 Low temperature storage............... 2, 281, 282, 364, 494 Luffa acutangula ................................................. 301, 302

M Magnesium chloride ....................................................... 32 Maintainer pollen ........................................................7, 20 Makaliberu..................................................................... 463 Male and female sterlity ................................................ 254 Male sterility-based hybrid production............................ 7 Malus × micromalus ........................................................ 57 Malvaceae....................................................................... 281 Mangifera indica ................................................. 199, 206 Manihot esculenta.......................................................... 543 Massulae ........................................................................ 456 Meiosis .................................................................. 424, 434 Metabolic activity .................................... 2, 12, 23, 32, 36 Microscopic slides ..................................81, 82, 117, 152, 154, 161, 162, 164, 334, 359, 417, 530, 537, 604, 612 Momordica dioica ................................................ 331, 332, 355, 356 Momordica subangulata subsp. Renigera........... 355–362 Monocot .......................................................................... 23 Moraceae ....................................................................... 135 Moringaceae .................................................................. 591 Moringa concanensis .................................. 591, 592, 595 Moringa oleifera ............................................................ 591 Morphometry .................................................................. 27 M. spectabilis var. riversii................................................. 57 Mulberry........................................................................ 3, 5 Myrtaceae ...................................................................... 175

N Nagalingapushpa ........................................................... 575 National Center for Genetic Resources Preservation (NCGRP).......................................................13, 14 National Institute of Agrobiological Resources (NIAR)................................................................. 13 Neolamarckia cadamba ................................................ 555 Non-aborted pollen grains .................................. 279, 488 Nuclear genetic diversity.................................5, 7, 54, 57, 86, 147, 155, 190, 199, 332, 448, 476, 494, 544

O Orchidaceae ..................................................................... 55 Orchids .................................................... 55, 56, 401–414 Ornamental crops......................................................7, 393 Oroxylum indicum ..............................483, 484, 486, 488 Orthodox pollen ............................................................. 36 Outbreeding ........................................................... 24, 602 Oven ..........................................11, 30, 32, 45, 109, 136, 138, 232, 249, 260, 269, 279, 283, 285, 289, 351, 368, 487, 514, 531, 535, 536, 570, 593–595

P Palmae.............................................................................. 23 Palode .............................................................................. 14 Passiflora edulis...................... 3, 155, 156, 159, 160, 163 Pennisetum ...................................................................... 32 Perennial ....................................5, 19, 21, 167, 176, 332, 356, 373, 433, 455, 475, 493, 501 Petri plate....................................... 65, 66, 78, 79, 82, 89, 90, 115, 116, 125, 136, 149, 152, 157, 159, 161, 162, 168, 171, 177, 178, 191, 200, 201, 204, 220, 229, 268, 270, 282, 295, 306, 311, 333, 357, 367, 374, 386, 416, 425, 427, 434, 443, 449, 458, 465, 477, 485, 494, 503, 511, 530, 535, 537, 545, 547, 556, 577, 578, 584, 586, 593, 595, 596, 603, 606, 611 Phoenix dactylifera ........................................................ 519 Physiological................................ 7, 8, 11–13, 19, 30, 36, 86, 101, 114, 167, 199, 200, 282, 294, 332, 416, 448, 562, 576 Picea omorika ....................................................... 561–572 Pinaceae .....................................................................23, 36 Pinus ................................................................................ 36 Piracy ............................................................................... 38 Plant genetic resources .............................. 2, 6, 176, 274, 469, 488, 510 Plant vitrification solution (PVS) ................................... 11 Poaceae ......................................................................12, 32 Poaching .......................................................................... 38 Polianthes tuberosa L. ........................................... 415–421 Pollen ......................................... 2–15, 19–39, 43, 46–48, 50, 51, 54–57, 61–71, 75–97, 99–110, 113–121,

POLLEN CRYOPRESERVATION PROTOCOLS Index 623 123–131, 135–144, 147, 148, 150–164, 168, 170–173, 176, 178, 180–185, 190, 192–196, 199–201, 203–208, 210, 216–223, 226–232, 236–241, 244–250, 254–260, 264–266, 268–270, 273–279, 281–289, 294–299, 302–307, 311–330, 332–338, 342–352, 356–369, 373–390, 393–399, 401–409, 412, 415–421, 423–428, 433–439, 441–445, 447–452, 455–461, 463–472, 475–481, 483–491, 493–499, 501–506, 509–516, 519–524, 527–539, 543–545, 547–549, 551, 555–572, 575–581, 583–588, 592–599, 601–606, 609–616 Pollen bank................................................... 8, 13, 15, 57, 469, 528, 610 Pollen biology ............................... 8, 9, 11, 63, 176, 190, 282, 476, 562, 577, 592, 610 Pollen collection.................................6–8, 11, 20, 24, 25, 31, 37, 50, 62, 63, 66, 71, 79, 90, 118, 120, 125, 126, 137–139, 160, 163, 168, 172, 176, 178, 190, 192, 201, 217, 218, 227, 237, 238, 275, 276, 283, 288, 289, 304, 312, 314, 337, 343, 347, 360, 368, 381, 386, 389, 394, 395, 425, 438, 443, 445, 449, 452, 459, 460, 464, 465, 467, 470, 472, 477, 486, 491, 494, 495, 498, 503, 506, 510, 511, 514, 528, 529, 556, 559, 565, 566, 580, 587, 593–594, 614 Pollen cryobank......................................8, 13, 20, 21, 43, 51, 54–55, 86, 176, 190, 200, 374, 448, 610 Pollen cryopreservation .................................. 6–8, 10–15, 19, 20, 22–34, 37–39, 43–57, 61–71, 75–83, 86, 100, 113–121, 123–131, 135–144, 148, 155, 157, 168, 176, 190, 200, 216, 221, 226, 227, 236, 245, 256, 265, 275, 279, 281, 282, 286, 289, 297, 303, 311, 356, 357, 364, 373, 383, 384, 393–399, 415–421, 423, 433–439, 441–445, 447, 448, 455–461, 463, 464, 475–481, 483, 485, 493, 501–506, 509–516, 528, 535, 543, 545, 555, 556, 561, 572, 575, 576, 583–588, 591, 602, 605, 609, 610 Pollen desiccation........................... 31, 32, 238, 395, 398 Pollen dimensions ........................................................... 96 Pollen dryers..............................................................31, 32 Pollen extraction .................................................... 90, 178 Pollen germination media ................................28, 92, 93, 125, 127, 140, 149, 151, 152, 158, 161, 162, 181, 195, 210, 217, 237, 282–284, 394, 512, 545, 547, 595 Pollen moisture ................................. 6, 12, 31, 116, 156, 283, 285, 286, 288, 289, 295, 417, 487, 514, 535, 610 Pollen moisture determination..................................... 487 Pollen morphology .............................. 12, 22–23, 77, 78, 83, 199, 394, 565

Pollen perimeter ................................ 118, 231, 298, 336, 360, 388, 497 Pollen rehydration........................................................... 33 Pollen scoring......................................................... 30, 488 Pollen tetrads..............................402, 403, 409, 456, 464 Pollen thawing ..........................................................12, 33 Pollen tube .................................... 22, 27, 29, 30, 33, 51, 56, 62, 68, 144, 170, 204, 205, 220, 258, 279, 285, 296, 332, 350, 369, 384, 387, 407–409, 412, 416, 427, 450, 452, 459, 471, 480, 488, 491, 505, 509, 513, 520, 523, 532, 537, 569–572, 579, 586, 588, 598 Pollen tube length.................................... 27, 71, 96, 110, 130, 154, 184, 196, 240, 296, 397, 480, 505, 562, 569, 572, 576, 579 Pollen viability assessment ..............................51–53, 127, 128, 217–221, 241, 283, 285, 396, 398, 510, 512 Pollen vitality............................................... 109, 168, 568 Pollinarium .................................................................... 456 Pollinating agents................................................... 12, 442 Pollination ...................................7, 8, 10, 22, 25, 28, 33, 57, 61, 62, 67, 69–71, 82, 83, 91, 93–95, 97, 105, 107, 108, 110, 114, 119–121, 128, 131, 139, 142, 144, 153, 156, 163, 171, 180, 182–185, 189, 190, 195, 196, 199, 200, 207, 221, 222, 225, 226, 230, 232, 236, 240, 241, 248–250, 254, 258–260, 265, 268–270, 273, 274, 278, 279, 288, 302, 303, 307, 308, 313, 332, 342, 347, 350, 352, 356, 364, 367, 369, 373, 374, 381, 384, 385, 394, 398, 411, 412, 424, 427, 443, 451, 480, 481, 483, 501, 516, 528, 533, 534, 545, 547–549, 563, 565, 571, 585, 595, 597, 602 Pollinium ............................................................. 404, 456, 477–480 Polyethylene glycol (PEG) .......................... 51, 115, 118, 158, 160, 204, 295, 333, 336, 357, 359, 360, 394–396, 417, 425, 435, 449, 458, 470, 471, 477, 485, 511, 512, 611, 613 Poncirus trifoliate ........................................ 167, 168, 170 Potassium iodide staining ............................................. 101 Pre-breeding..............................................................15, 56 Pre-freezing ...............................................................35, 57 Primulaceae ...............................................................23, 36 Production fields .......................................................21–22 Prospects................................................................. 1, 5, 15 Protection ........................................................... 22, 38, 50 Psidium guajava .................................175, 177–181, 184 Punicaceae ..................................................................... 189 Punica granatum ........................................ 189, 192, 193

Q Quarantine...........................................9, 12, 20, 124, 190 Quercus robur ......................................561–566, 570, 572

POLLEN CRYOPRESERVATION PROTOCOLS

624 Index R

Ranunculaceae ................................................................. 23 Rapid climate change .................................................... 562 Rare, Endangered and Threatened medicinal plants .................................................................. 332 Recalcitrant pollen .................................... 8, 9, 11, 31, 36 Refrigerator .................................33, 108, 157, 204, 218, 282, 284, 351, 395, 407, 512, 528, 544, 547, 565, 566, 571 Regeneration .............................................. 2, 11, 38, 342, 464, 556, 602 Rehydrated pollen .................................................. 33, 523 Relative humidity (RH) ............................. 31–36, 57, 82, 101, 114, 140, 144, 162, 172, 200, 264, 274, 288, 316, 364, 381, 390, 416, 427, 439, 445, 461, 468, 499, 506, 520, 521, 523, 535, 560, 580, 588, 596, 598, 610 Repeatability ..............................................................14, 37 Reproductive phenology............................................... 577 Respiration.................................................................36, 56 RET.........................................................15, 55, 384, 476, 483, 484, 494 Rewarming .................................... 3, 49, 67, 82, 91, 101, 153, 163, 180, 402, 523, 537 Rosa spp......................................................................... 423 Rosaceae ....................................................................23, 36 Rubiaceae....................................................................... 555 Rutaceae............................................................23, 99, 167

S Saccharum ....................................................................... 32 Salacia chinensis Linn. ......................................... 493–499 Salicaceae ......................................................................... 23 Santalaceae ..................................................................... 601 Santalum album................................................... 601–606 Saraca asoca (Roxb.) De Wilde........................... 501–506 Saxiferacea........................................................................ 36 Saxifragaceae.................................................................... 23 Scanning electron microscope (SEM) ................... 70, 71, 96, 97, 115, 118, 119, 143, 144, 168, 173, 183–185, 200–202, 205, 206, 230, 231, 295, 298, 311, 314–330, 333, 336–338, 357, 360, 361, 388–390, 428, 434, 438, 442, 451, 458, 465, 476, 485, 489, 490, 494, 497, 506, 513, 556, 567, 588, 597, 599, 614, 615 Scrophulariaceae.............................................................. 23 Secale ................................................................................ 32 Seed................................. 1–3, 5, 8, 9, 11, 13, 15, 20–22, 29, 31, 33, 36, 38, 39, 43, 54–57, 69, 70, 94, 95, 97, 100, 110, 114, 129, 131, 136, 142, 147, 171, 172, 176, 183, 185, 189, 195, 196, 216, 230, 236, 244, 254, 264, 269, 273, 274, 282, 287, 301, 308, 332, 342, 363, 368, 373, 374, 384,

394, 399, 402, 412–416, 433, 441, 447, 464, 469, 476, 481, 483, 484, 493, 494, 509, 510, 515, 528, 534, 550, 556, 561–563, 576, 584, 591, 602, 610 Seed set ................................... 20, 21, 28, 29, 34, 51, 54, 82, 94, 119, 129, 130, 142, 153, 163, 171, 183, 195, 200, 208, 221, 241, 248, 259, 278, 313, 351, 374, 381, 384, 398, 399, 411, 448, 480, 509, 548, 584 Self-incompatibility ...................................... 24, 110, 113, 156, 168, 216, 415, 433, 464 Shipment....................................................................47–48 Silica gel ................................................11, 32, 63, 66, 71, 115, 158, 244, 246, 247, 255, 257, 265, 275, 276, 279, 283, 286, 289, 299, 312, 344, 348, 349, 365, 566, 570, 604 Sitting drop culture........................................28, 285, 459 Slow thawing ..................................................33, 287, 514 Solanaceae..............................................23, 253, 309, 342 Solanum lycopersicum ............................................. 56, 341 Solanum melongena L ................................................... 243 Species............................................... 1–15, 19–25, 27–39, 55–57, 62–66, 68–70, 75, 78, 85, 90, 93, 95, 99–101, 105, 108–110, 114, 116, 119, 123–131, 155–157, 159, 164, 175–179, 181, 183–185, 190, 192, 195, 199, 205, 206, 215, 216, 225, 226, 236, 243, 248, 253, 254, 263, 264, 273, 274, 281, 282, 289, 293–295, 303, 311, 331, 332, 341, 342, 355–357, 364, 368, 374, 383, 384, 386, 401–414, 423, 424, 441–443, 448, 455, 456, 458, 463–465, 469, 471, 475–477, 479, 483–486, 493–495, 501, 502, 509, 519, 545, 556, 561–572, 575, 583, 584, 591, 592, 601, 602, 609, 610, 612 Species amplification ..................................................... 448 Species restoration ........................................................ 484 Sporophytic system ......................................................... 24 Staining ...............................................25–29, 51, 82, 101, 127, 141–142, 148, 151, 152, 157, 160, 161, 164, 220, 248, 258, 279, 304, 313, 378, 406, 408–409, 412, 488, 516, 565, 566, 568, 579, 596–597 Staminal hood pollen ........................................... 576, 579 Staminal ring pollen ............................................. 576, 579 Stereomicroscope ...................................65, 89, 115, 136, 141, 168, 177, 200, 295, 311, 333, 335, 357, 359, 374, 386, 405, 408, 409, 416, 424, 434, 442, 448, 458, 464, 476, 485, 494, 502, 510, 513, 544, 548, 556, 576, 584, 593, 596, 603, 605, 611, 613 Stevia rebaudiana Bertoni................................... 509–516 Stigma ..................................... 29, 30, 33, 69, 71, 77, 82, 97, 105, 113, 114, 119, 127, 142, 148, 153, 156, 163, 171, 184, 192, 196, 207, 208, 221, 241,

POLLEN CRYOPRESERVATION PROTOCOLS Index 625 248, 254, 259, 269, 274, 288, 306, 312, 313, 347, 350, 367, 381, 398, 405–409, 411, 412, 427, 435, 451, 464, 476, 480, 484, 514, 533, 538, 545, 549, 556 Stigma receptivity.................................54, 65, 66, 77, 78, 90, 116, 119, 126, 142, 159, 178, 192, 256, 283, 295, 311, 312, 335, 357, 369, 373–375, 384, 387, 417, 435, 465, 477, 504, 511, 545, 585, 611 Storage temperature........................................................ 34 Sucrose.....................................25, 26, 51, 65, 67–69, 71, 78, 79, 81, 89, 92, 93, 96, 101, 102, 104, 109, 115, 117, 118, 125, 127, 128, 130, 137, 140, 151, 157, 158, 160, 163, 168, 170, 178, 180–182, 184, 192, 193, 195, 196, 201, 204, 217, 219, 227, 228, 237, 238, 245, 247, 255, 257, 258, 265, 267, 270, 274–276, 284, 289, 295, 296, 304, 305, 312, 313, 333, 336, 344, 350, 357, 359, 360, 365, 366, 368, 375, 376, 378, 379, 386, 387, 389, 394–396, 402–404, 406, 407, 412, 417, 418, 425, 426, 435, 436, 443, 444, 449, 450, 458, 459, 465, 466, 470, 471, 477, 479, 480, 485, 488, 495, 496, 503–505, 511, 512, 520, 521, 523, 529, 535, 537, 545, 547, 557, 558, 568–571, 577–580, 585, 586, 588, 593, 595, 596, 598, 603–605, 611–613 Sucrose concentration.................................. 28, 121, 172, 297, 299, 316, 335, 338, 361, 381, 390, 402, 439, 445, 461, 468, 488, 499, 505, 506, 524, 560, 571, 579, 580, 605, 606, 613, 616 Sugarcane................................................................ 20, 100 Supplementary pollination ...........................7–9, 20, 190, 200, 216, 236, 364, 448 Surface culture................................................................. 29 Suspension culture ......................................................3, 29 Swallow root.................................................................. 463 Syzygium cuminii .......................................................... 147

T Tagetus erecta................................................................. 100 Tectona grandis..................................................... 609, 612 Tetrazolium staining ....................................................... 27 Thawing and pollen retrieval........................................ 588 Thiruvanathapuram......................................................... 14 Threatened ...........................................1, 2, 7, 9, 14, 332, 448, 483, 494, 575, 584 Three-celled pollen ......................................................... 36 Tomato ............................................ 54, 56, 57, 101, 310, 341–343, 345–352, 363

Tricellular pollens ............................................................ 23 Tricosecale ........................................................................ 32 Trinucleate pollen .............................................. 12, 23, 35 Triticum........................................................................... 32

U Ultra-low temperatures.............................. 2, 4, 6, 63, 86, 124, 226, 364 USDA-National Centre for Genetic Resource Preservation ......................................................... 14

V Vacuum infiltration vitrification (VIV) ............................ 3 Variation .......................................... 2, 14, 24, 30, 67, 71, 91, 101, 109, 114, 118, 121, 127, 136, 172, 173, 180, 226, 231, 236, 254, 298, 315, 330, 337, 338, 342, 360, 368, 381, 382, 388, 390, 394, 411, 421, 424, 433, 439, 445, 461, 468, 497, 498, 506, 520, 528, 559, 560, 580, 581, 602, 606, 615 Vasconcellea species....................................................85–97 Vegetative nucleus .....................................................22, 23 Vegetatively propagated species ................................... 2, 5 Vigour................................................................................ 7 Vitaceae.............................................................23, 36, 441 Vitis vinifera ......................................................... 124–128 Vitrification.......................................................3, 4, 11, 56 Vitrification freezing ......................................................... 3

W Walnut..................................................................... 36, 100 Water bath ............................................12, 33, 45, 67, 91, 100, 102, 104, 109, 180, 379, 406, 410, 411 Wild species of Solanum ............................................... 313

Y Yam .................................................................................. 20

Z Zea.................................................................................... 32 Zeolite beads ..................................... 116, 117, 150, 168, 170, 203, 217, 219, 232, 238, 239, 246, 249, 260, 269, 294, 295, 297, 299, 333, 334, 336, 351, 357, 359, 375, 378, 387, 395, 398, 417, 420, 425, 459, 478, 504, 510, 511, 514, 557, 578, 585, 586, 594, 603–605, 610, 612, 613