Physiology and Biochemistry of Seeds in Relation to Germination: Volume 2: Viability, Dormancy, and Environmental Control [1 ed.] 978-3-642-68645-0, 978-3-642-68643-6

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Physiology and Biochemistry of Seeds in Relation to Germination In Two Volumes

21. D. Bewley· M. Black

Viability, Dormancy, and Environmental Control

Springer-¥erlag Berlin Heidelberg New York 1982

Dr. J. DEREK BEWLEY, Department of Biology, University of Calgary, 2500 University Drive, N.W. Calgary, Canada T2N IN4 Dr. MICHAEL BLACK, Department of Biology, Queen Elizabeth College, University of London, Campden Hill Road, London W8 7AH, Great Britain

With 153 Figures

e- ISBN -13 :978-3-642-68643-6 ISBN -13 :978-3-642-68645-0 DOl: 10.1007/978-3-642-68643-6 This work is subjet;t to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re·use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. Under § 54 of the German Copyright Law, where copies are made for other than private use, a fee is payable to "VerwertungsgeseUschaft Wort", Munich.

© by Springer·Verlag Berlin Heidelberg 1982 Sof tcover reprint of the hardcover 1st edition 1982 The use of registered names, trademarks, 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. 2131/3130-543210

"They also serve who only stand and wait" Milton

Dedicated to our wives and children, Christine, Alex and Janette Bewley, and Marianne, Pauline, Nicola and Martin Black, whose continued patience and encouragement contributed in no small way to the production of this book.

Acknowledgements

It might be thought appropriate that this volume, in which consider-

ations of dormancy occupy a major part, should emerge some years after Volume I! But like the dormant seed whose seeming torpor can conceal a vigorous metabolism we, too, have not been inactive. Readers who already have some familiarity with the subject matter of this book will be aware of the vast and relevant research literature that is available. We have attempted to gather from this the essential features of seed viability, dormancy and environmental control of germination. In doing so, we have inevitably omitted very many research contributions and we do not claim to present an encyclopedic account; but we hope that the result is a fair statement of modem knowledge of these areas of plant physiology, useful to advanced undergraduates, graduate students, teachers and established research workers. We are grateful to many who have contributed to the production of this book: to those who kindly allowed us to use their published material and to Profs. E. H. Roberts and E. B. Dumbroff for reading and commenting on certain sections. Grants from the British Council, the Canada Council, the Department of Biology and the University at Calgary, made possible in situ collaboration between the authors in Calgary. One of us (J. D. B.) was in receipt of an award from the Natural Sciences and Engineering Research Council of Canada which is gratefully acknowledged. Erin Smith in Calgary had the unenviable task of producing the typescript - a job which she did quickly and efficiently: for this she receives our deepest gratitude. Karen Larsen and Joanne Papp provided invaluable assistance with the indexing. Finally, to our publishers we say "thank you" for being so patient and helpful.

Contents

Chapter 1. Viability and Longevity .

1.1 1.1.1 1.1.2 1.2 1.2.1 1.2.2 1.2.3 1.2.4 1.3 1.4 1.5 1.5.1 1.5.2 1.6 1.7 1.7.1 1.8 1.9 1.10 1.11 1.11.1 1.11.2

The Life-Span of Seeds . . The Oldest Seeds - from the Pharoah's Tomb to the Incendiary Bomb . . . . . . . Life-Span of Seeds Buried in Soil Viability of Seeds in Storage Recalcitrant Seeds. . . . . . Orthodox Seeds. . . . . . . The Basic Viability Equations Improved Viability Equations. Microflora and Seed Deterioration The Biochemical Basis of Deterioration Respiration and the Production of ATP Non-Viable Seeds and Embryos. . . . Seed Populations with Reduced Viability and/or Vigour. Protein and RNA Synthesis . . . . . . . . Chromosome Aberrations and DNA Synthesis Chromosome Damage and Repair. Metabolism of Dry Seeds . . . . . . . . . Changes in Food Reserves. . . . . . . . . Free Fatty Acids and Interference with Metabolism Membrane Changes and Leakage . . . . . . . . Leakage of Metabolites and Integrity of the Bounding Membranes . . . . . . . . . . . . . . . The Nature and Cause of Membrane Damage Some Works of General Interest References. . . . . . . . . . . . . . . .

1 1

2 3 7 9 11 12 21 24 26 27 27 30 34 39 41 43 46 49 49 49 50 55 56

Chapter 2. Dormancy . . .

60

2.1 2.1.1 2.1.2

60 62 63 65 66 69 69 71

What is Dormancy? . Categories of Dormancy . Biological Significance of Seed Dormancy 2.1.3 Dormancy in Cultivated Plants . 2.1.4 Polymorphism and Heteroblasty 2.2 Dormancy Mechanisms . . . . Embryo Dormancy . . . . . . 2.3 2.3.1 Control Mechanisms in Embryo Dormancy.

Contents

VIII

2.3.2 The Role of the Cotyledons 2.3.3 The Role of Inhibitors. . 2.3.4 Embryo Immaturity. . . . Coat-imposed Dormancy. . 2.4 2.4.1 Interference with Water Uptake. 2.4.2 Interference with Gaseous Exchange . 2.4.3 Inhibitors in the Coat . . . . . . . 2.4.4 Prevention of the Escape of Inhibitors 2.4.5 The Coat as a Light Filter 2.4.6 Mechanical Restraint . . . Two Case Histories . . . . 2.5 2.5.1 Sinapis arvensis (Charlock) . 2.5.2 Xanthium pennsylvanicum (Cocklebur) Coat-imposed Dormancy - a Retrospective View 2.6 Relationships Between Coat-imposed and Embryo 2.7 Dormancy . . . . . . The Onset of Dormancy 2.8 2.8.1 Timing . . . . . 2.8.2 Control . . . . . . . 2.8.3 The Genetic Factor . . 2.8.4 Environmental Factors 2.8.5 Correlative Effects . . 2.8.6 Hormones . . . . . . 2.8.7 Secondary or Induced Dormancy 2.8.8 The Development of Hard Coats Endogenous Germination Inhibitors . 2.9 2.9.1 Chemical Nature of Inhibitors . Some Works of General Interest References . . . . . . . . . .

72 73 75 77

78 80 86 89 90 91 94 94 96 100 101 101 102 105 105 106 112 113 115 115 116 119 120 120

Chapter 3. The Release from Dormancy

126

3.1 3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.2.6 3.2.7 3.2.8 3.2.9 3.2.10 3.2.11 3.3

126 127 129 132 133 134 138 139 141 144 146 148 151 152

Light and Phytochrome. . . . The Phytochrome System. . . Spectral Sensitivity and PhotoreversibiIity Energies for Photoconversion The Escape Time. . . . . . Phytochrome Photoequilibria Chemistry of Phytochrome . The Pathway of Phytochrome Photoconversion The State of Phytochrome in Seeds. . . Seed Hydration and Sensitivity to Light. . . . Reversion of PCr in Darkness. . . . . . . . . Thermal Processes Connected with Phytochrome Action Phytochrome Location and the Photosensitive Site Phytochrome - an Overview . . . . . . . . . . . .

Contents

3.4 3.5 3.6 3.6.1 3.6.2 3.6.3 3.7 3.8 3.9 3.9.1 3.9.2 3.10 3.11 3.11.1 3.11.2 3.11.3 3.12 3.13 3.14 3.14.1 3.14.2 3.14.3 3.14.4 3.14.5 3.14.6 3.14.7 3.14.8 3.14.9 3.14.1 0

IX

Blue Light Effects . . . . . . . . . Response Types . . . . . . . . . . Temperature and the Action of Light . Constant Temperature . . . . . . Temperature Alternations and Shifts . Chilling.............. Temperature and the Release from Dormancy Termination of Dormancy by Temperature Alternations and Shifts . . . . . . . . . . . . . . . . . Termination of Dormancy by Low Temperature Response Types . . . . . . . . . . . . . . Temperature and Time Requirements. . . . . Termination of Dormancy by High Temperature Loss of Dormancy in Dry Seeds - After-ripening . Moisture Content Temperature. . . . . . . . . . . . . Oxygen. . . . . . . . . . . . . . . Finale - Replacements and Interactions. Hard-coated Seeds . . . . . . . . Removal of Dormancy by Chemicals Growth Regulators. Gibberellins. Cytokinins . . . . Ethylene . . . . . Plant and Fungal Products Respiratory Inhibitors . Oxidants . . . . . . . Nitrogenous Compounds Sulfhydryl Compounds . Various Other Chemicals Including Anaesthetics Some Works of General Interest References . . . . . . . .

Chapter 4. The Control of Dormancy . 4.1 4.2 4.2.1 4.2.2 4.2.3 4.2.4 4.3 4.3.1 4.3.2 4.3.3 4.4

154 156 158 160 163 168 169 170 173 174 175 178 179 181 181 182 183 185 186 188 188 188 189 190 191 191 191 192 192 193 193 199

Introduction........ 199 Dormancy - Events and Causes. 200 Metabolism of Dormant and After-ripened Seeds 200 Dormancy and Maturation. . . . . 204 204 Chemical Inhibition. . . . . . . . . . . Membrane Properties and Dormancy . . . 207 Primary Events in the Release of Dormancy 212 Phytochrome Action . . . . . . . . . . 212 Chilling Action. . . . . . . . . . . . . 214 Alternating Temperatures and After-ripening 217 Secondary Events in the Release from Dormancy Physiological Considerations . . . . . . . . . . . . 217

x 4.4.1 4.4.2 4.4.3 4.4.4 4.4.5 4.5 4.5.1 4.5.2 4.5.3 4.5.4 4.5.5 4.5.6 4.5.7

Contents

Hormones and Dormancy . . . . . . . . . . . Hormones and Light-terminated Dormancy Hormones and the Low-temperature Release from Dormancy . . . . . . . . . . . . . Ethylene and Dormancy . . . . . . . . . . . . Water Relations and Growth Potentials . . . . . Secondary Events in the Release from Dormancy Metabolic Considerations . . . . . . . Perpetuated Misinterpretations of Studies on Dormancybreaking Mechanisms . . . Hormonal Effects on Nucleic Acid and Protein Synthesis Fusicoccin and Cell Elongation. . . . . . . . . . . Hormonal Effects on Respiration . . . . . . . . . . The Pentose Phosphate Pathway - a Role in Dormancy Breaking? . . . . . . . . . . . . . . . . . . Phytochrome-induced Changes in Metabolism Low temperature-induced Changes in Metabolism . Some Works of General Interest References . . . . . . . . . . . . . . . . . .

218 219 223 231 233 237 238 238 243 243 244 255 258 264 264

Chapter 5. Perspective on Dormancy .

270

References. . . . . . . . .

275

Chapter 6. Environmental Control of Germination

6.1 6.2 6.2.1 6.2.2 6.3 6.3.1 6.3.2 6.3.3 6.3.4 6.4 6.5 6.6 6.6.1 6.6.2 6.6.3 6.6.4 6.6.5 6.6.6

Introduction.... Light . . . . . . . Light-inhibited Seeds Dual Effects of Light Spectral Effects in Photoinhibition Short-Duration and Intermittent Far-Red Light. Prolonged or High-Irradiance Far-Red Light . Inhibition by Prolonged Blue Light . . . . . Suppression of Germination by White Light A Re-Examination . . . . . Light and Seed Burial . . . . Shade and Seed Germination. Temperature........ Temperature Minima, Optima and Maxima Temperature and Germination Rate. . The Action of Temperature . . . . . . . Temperature and Germination Ecology . . Geographical Adaptation and Plant Distribution Chilling Injury . . . . . . . . . . . . . . .

,

276 276 276 277 281 282 282 283 288 290 291 294 297 297 300 302 304 305 306

Contents

XI

6.6.7 Alternating Temperatures 6.7 Oxygen and Carbon Dioxide 6.8 Secondary Dormancy . . . 6.8.1 Mechanism of Secondary Dormancy . 6.8.2 Secondary Dormancy in Nature. . . 6.9 Water Stress . . . . . . . . . . . 6.9.1 Drought During Seed Development and Maturation. 6.9.2 Germination Under Stress . . . . . . . . . . " 6.9.3 Dehydration and Rehydration Following Imbibition Effects on Germination . . . . . . . . . . . . . . 6.9.4 Dehydration and Rehydration Following Imbibition Effects on Growth, Yield and Tolerance: DroughtHardening. . . . . . . . . . . . . . . . 6.9.5 Osmotic Pretreatment - the Priming of Seeds . 6.9.6 Salinity Stress . . . . . . . . . . 6.9.7 Cellular Changes Associated with Dehydration-Rehydration Treatments 6.9.8 Changes to Membranes Induced by Desiccation-Rehydration Treatments 6.9.9 Desiccation-Induced Changes to Metabolism and Structure . . . . . . . . . . Some Works of General Interest References. . . . . . . . . . Glossary and Index of English and Botanical Names

307 307 308 311 312 313 313 315 317 318 320 323 326 326 330 333 334

. . . . . 341

Author Index . . . . . . . . . . . . . . . . . . . . . . 349

Subject Index . . . . . . . . . . . . . . . . . . . . . . 359

Contents of Volume 1

Chapter 1. Introduction Chapter 2. The Structure of Seeds and Their Food Reserves Chapter 3. The Legacy of Seed Maturation Chapter 4. Imbibition, Germination, and Growth Chapter 5. Biochemistry of Germination and Growth Chapter 6. Mobilization of Reserves Chapter 7. Control Processes in the Mobilization of Stored Reserves Glossary and Index of English and Botanical Names Author Index Subject Index

Chapter 1. Viability and Longevity

1.1. The Life-Span of Seeds In his publication of 1908, Ewart [88] divided seeds into three categories on the basis of their life-span under optimum conditions. These categories were (l) microbiotic - seeds whose life-span does not exceed 3 years; (2) mesobiotic - those whose life-span ranges from 3-15 years; and (3) macrobiotic - whose life span ranges from 15 to more than 100 years. This classification oflongevity is not particularly satisfactory, however, and it has not been widely adopted. For many seeds the most favourable storage environment has not been determined, and until we possess this information (for different cultivars and harvests also) the categories have little meaning. As storage conditions are improved for any given seed, it may change from micro- to mesobiotic, or even to the macrobiotic class. For a comprehensive list of seeds whose known viability range (not necessarily under optimal conditions) extends up to a hundred or more years, see the review by Harrington [11]. A few examples are presented in Table 1.1. A fuller discussion of factors affecting longevity in storage is to be found in Section 1.2. Here we will first answer the intriguing question: how old are the oldest viable seeds? We will then consider the longevity of seeds buried in soil.

Table 1.1. Viability-span of some orthodox a seeds in storage Plant species

Longevity and (% germinated)

Papaver rhoeas-corn poppy Brassica napus-rape Cucurbita pepo-squash, marrow Glycine max-soybean Picea glauca-white spruce Cannabis sativa-hemp Nicotiana tabacum-tobacco Lactuca sativa-lettuce Phaseolus vulgaris-dwarf bean Allium cepa-onion Fragaria spp.-strawberry Hordeum vulgare-barley M edicago sativa-alfalfa Anthryl/is vulneraria-kidney vetch

10 years (53) 10 years (12) 10 years (55) l3 years 15 years (40) 19 years 20 years (92) 20 years (86) 22 years (30) 22 years (33) 23 years (89) 32 years (96) 78 years (22) 90 years (4)

See Section 1.2.2 Excerpt from Harrington, 1972 [11]

a

Storage environment

Laboratory Laboratory Laboratory, sealed Storage - 4 DC, sealed Laboratory, sealed Laboratory, sealed _4 C, 8% R.H. Laboratory Dry, laboratory ome are founded in fact, such as the famous case of the

Raspberrv-seed taken along with the coins of the Emperor "Hadrian from an ancient barrow in Dorsetshire, the offspring of which is now to be seen in the

Gardens of the Horticultural Society_ None among the ｾ｣｡ｬ･､＠ inslances of this excessive !ouge\·ity ha\'e excited more doubt and discussion than what is

called iI1ummy-'Vheat; that is to say, Corn taken from mnrnmies,31l11 therefore of the highest antiquity, which has ｾｯｷｮ＠ when sown. Every year prodnces cases of this sort about the harvest season, and e\"en this season at least 20 specimens have been sent us of "'heat.ears. purporting to have had a mummial -pardon the word-a mummial origin; and strange

to say, they have allproved to belong to the Egyptian 'Vheat, or Ble de :Miracle, caned by Botanists 1'rilicum COf1l1Josifum. 'Ve ba\"e never, however, 8ucceerlt!J in -satisfying ourselves that the Corn from which

such 'Vheat i. said to have been produced was really taken from mummy·cases" T'berc is alwa) 5 some ､･ｦｾｴ＠ in the evidence i as was the case with the

Tynuingham "'heat, mentioned in the :JJJark La;,e EZl'ress of Oct. 9, 1842, which had been raised from "ed .aid to have beenlltoduced in Egypt, from plants soid to have grown from grains said to have been taken from a mummy·case. Now all such statements may be true, but there is no proof that they are so; and when we are told that Onions taken from similar receptacles ha,"e also grown, wltich is impossible, we

may be pardoned for requiring very decisive evidence before we accord our belief in those prodigies_ N everthel..s they may be true; because we have before us an instance, in the evidence concerningwhichwefindno flaw whatever. ''Vellavebaditon our table for some the months,and produce it now, in order to ｾｳ｡ｴｩｦｹ＠ many inquiries that are made about such things_ The history of this Wheat was given by .IIlr_ Martin Farquhar Tupper, a most exact and conscientious man, in tbe Tintes of September, 1840; and to that gentleman we are indebted for the additional facts which we are DOlV able to communicate. Sir GardinerWilkinson, when in the Thebaid, opened an ancient tomb (which had probably remained unvisited by man during the greater part of :1000 years), and from 60me alabaster sepul.:kralvasc6 therein took with his. own hands a quantity of 'Vheat and Barley that had been there preserved. Portions of this grain Sit G. Wilkinson had given to lIfr/Pettigrew, who presented II1r. Tupper with 12 grains of the venerable_harvest. In 1840 .Ilk Tupper SOlVed these 12 grains, and to show the care with which he preserved their identity we shall quote hi. OWIl account of his proceedings thereupon. cc I ordered," he says, "four gardenpots of well-sifted loam, and, not content "ith my gardener's care in sifting,l emptied each pot successively into an open newspaper and put the earth back again, morsel by morsel, with my own fingers. It i. next to impossible that any other .ced should have been there_ I then (on the 7th of March, 1840), planted my grains, three in each pot, at the angles of an equilateral triangle, so as to be sure of the spota where the sprouts would probably come up, by way of additional security against any chance seed

ｵｾｳ･ｮ＠ lurking in the soil. Of the 12 one only germinated, ｾｨ＠ blade first becoming visible on Apri122; the rem:unmg II, ｡ｦｴｾｲ＠ long patience, I picked out agam; and found m every instance tloat they were rott!ng in ｴｨｾ＠ earth. being ･｡ｾｮ＠ away by a number of ｭＱｬｵｾ･＠ wl!lte worms. 1\ly Interesting p]ant of Wheat remamed III the atmosphere of my usual sittingroom until change of place and air seemed nec.... ry for its health, when I had i.t carefuny trallsplanted to the open ｦｩｯｷ･ｲｾ｢､Ｌ＠ where It bas prospered ever ＢＬｩｮ｣･ｾ＠ The first ear began to be developed on the 5th of July a second ear made its appearance. and both assumed ｾ＠

character somewhat different from all our known

varieties. 'fheir small size and weakness may, in one light, be regarded as collateral evidence of so great an age, for assuredly the energies of life would he but sluggish after baving slept so long; however, the season of the i-owing-spring insteacl of autumn-will furnish another sufficient cause_ The two ears on separate stalks were respectively Ｒｾ＠

and 3

inches long, the former beine much blighted, and the stalk about 3 feet in height. "If. and I see no rEason to disbelieve it," says l\fr. Tupper, in conclusion, "if this plant of Wheat be indeed tbe product of a grain preserved since the time of the Pharaohs, we moderus may, within a little year, eat bread made of Corn which Joseph might bave reasonably thought to store in his granaries, and almost literally snatch a meal from the kneadingtroughs of departing Israel/' Here we have no link lost in the chain of evidence_ Sir Gardiner Wilkinson himself opelled the tomb, and with his own hands emptied the alabaster .. ase ; of its contents he gal'e a portion to Mr. Pettigrew, who gave it to Mr_ Tupper, who himself sowed it, watched it, and reared it_ What better proof can we require? U flless it be alleged that ti,e grains, after all, may have been changed somewhere on ti,e roall between the Thebaid and Mr. Tupper's garden. Dut, upon this point, Mr_ Tupperexpress1y says, in a PlUlsage that we have not quoted, that the grains which he sowed were brown and shrunk; which is a just description of some that we too have seen from Sir Gardiner Wilkinson, hut which would not apply to any modem'Vheat_ They looked, indeed, as if they had been scorched_

The Life-Span of Seeds

5

But there are other proofs, less direct, but equally conclusive, as to the antiquity of the seed 50wn by IIfr. Tupper. Out of twelve grains one only grew; that one produced but t ... o ears-.mall, blighted at the .b..e, and yielding altogether only 27 grains. JIIr. Tupper has favoured us with a draw· ing of one of them. But in 18"'1, the second year, whell the 'Vheat was recovering its ｣ｯｮｾｴｩｵ｡ｬ＠ vigour, the ears were perfect, and averaged H inches each. In -1842, the renovation being ｣ｯｭｰｬｾｴ･Ｌ＠ some

of the ears measured 1! inches ill length. This, as Mr. Tupper obseT;' es, corroborates the idea of a re-

awakening from so long a sleep, as

if the Wheat had been gradually returning to its pristine vigour. One of these ears of 1812 i. no'v before

us, and is so like a good sample of Colonel Le Couleur'. Bellevue Tala· vera, that e'·en tbe experienced eye of that gentleman is unable to detect It proved a most a : difference. abundant bearer: III grains in Mr. Mitchell's Nursery Garden,Brighton, having produced 625 ears, which IIfr. Hallett of Brighton considers to have contained on an average 55

grains. AmI this (685, muhiplied by 55, divided by IS) gives a pro· ductiveness equal to two thousand and ninety-three fo\(!. But with the quality of this \Vheat we do not wish to concern ourselves just now. The important question is, what were the circumstances which preserved the

growing power of Sir Gardiner 'ViIkinson's Wheat from tile clays of the Pharaohs down to our own time-.

For if that can he ascertained, a light will necessarily be thrown upon the very important art of preserving seeds artificially. To us it appears Mummy-Wbeat in that we must ascribe f.h e result the first )"nr of its entirely to the DllYNF.SS of the air re\·j\"iticalion.

where the 'Vheat was kept. And we believe that dryness will have been the true cause of similar results in all other instances. Such i. ti,e conclusion at which we long .ince arrived. (" Theory of Horticulture," Pl'. 79 and 189). Daily experience confirms ot1r opinion; and reasoning. in the absence of experience, would almost have led to it. Decomposition, which in seeds is the cause of death, can only occur in a damp atmosphere; therefore to keel' 0.11' a damp atmosphere is to prevent decomposition, antI consequently to arrest the A",I yet how little is this .pproach of death. rcO"anletl by persons interested ill such matters. In a :lamp country like England no precaution should be llE-glected to ventilate, at least seed-rooms, if not seeds themselves. And yet what i. the proctice? The Eeetlsmen pack them in large sacks or huge casks, in

close iII·ventil.ted granaries; and gardeners place them in drawers or bag. in the damp and miserable sheds with which some masters sOlhoughtlessly provide them' farmer. in damp barn. or ol1thouse.. lVhat happen with such management except can ｰｾｳｩ｢ｬｶ＠ the speedY de.truction of vitality, especially when we kllo,v -how badly our' home-grown seeds are in almost all seasons ripened, anel how much free moisture

they necessaril), contain. 'Vbat wonder that French .eeds, ripened m a dry climate ami preserved in dry building., should often be found 80 much better tban English seed? Our climate offers 80 many impedimenta to the preservation of aeeds that we cannot afford to neglect a aingle precaution; ami we trust lIIr. Tupper's Pharaonic Wheat will have the effect of turning tho.e whom these observatiolls may concern to wiser and better w.ys.

======

Fig. 1.1. An excerpt from The Gardener's Chronicle of Nov. 11, 1843 on "mummy" wheat

6

Viability and Longevity

Table 1.2. Becquerel's record of old seeds Species

Date Seeds growing collected in 1906

1853 1851 Astragalus massiliensis 1848 1843 Cytisus austriacus Lavatera pseudolobia 1842 1841 Dioclea pauciflora 1841 Ervum lens 7Nfolium arvense 1838 Leucaena leucocephala 1835 1829 Stachys nepetiJolia 1822 Cytisus biflorus Cassia bicapsularis 1819 Cassia mUltijuga 1776

Mimosa glomerata M elitotus lutea

5 out of 10 3 out of 10 o out of 10 lout of 10 2 out of 10 lout of 10 lout of 10 2 out of 10 2 out of 10 lout of 10 2 out of 10 3 out of 10

Seeds growing in 1934

Determined Probable longevity longevity (years) (years)

5 out of 10

81 55 86 63 64 93 65 68 99 77 84 115 158

o out of 10 lout of 10 o out of 10 oout of 10 2 out of 10 o out of 10 o out of 10 3 out of 10 o out of 10 o out of 10 4 out of 10 2 out of 2

221 100 121 155 199

Translated from Becquerel, 1934 [56]

thus maintaining the lupin seeds in a dry and continually frozen state. Alternative geologica] explanations were not considered, and prooffor such longevity of these lupin seeds obviously is missing. Doubt must also surround the claims for longevity of one to three thousand years for the seeds of the Indian or Sacred lotus, Nelumbium nucifera. Many of these seeds (or more strictly, fruits, which are extremely hard-coated) were recovered from peat under drained lakes of the Pulantien basin of South Manchuria. The age of the seeds was initially estimated by Ohga as 160-250 years using indirect evidence, particularly the known history of the drained lakes, although others have taken the view that the geological history of the Pulantien basin indicates an age of many thousands of years. But direct carbon dating of the seeds shows them to be indistinguishable in age from modern seeds! [94, 95]. Viable lotus seeds also have been found associated with the remains of a prehistoric boat, some 20 feet below the surface of a lake at Kemigawa, near Tokyo. The wood from the boat has been dated at about 3000 years, but this yields no evidence for the age of the lotus seeds, which could have been from modern lilies buried by natural sinking into soft lake sediments moved by the action of currents. 0dum [134] surveyed a considerable number of archaeological sites in Denmark and in Skane, Sweden and noted the appearance in recently excavated soils of plants which have not grown in such regions for many years previously. Using archaeological dating he claims that some weed seeds must have remained viable for between 100 and 600 years, and seeds of Chenopodium album and Spergula arvensis for about 1700 years. Evidence of seed age is indirect and weak, and until dating of the seeds themselves is obtained, the claims must be treated with due scepticism. To quote Ewart [88], such "observations are good evidence of the readiness of dispersal of certain seeds, but as evidence of their longevity are quite untrustworthy".

Viability of Seeds in Storage

7

A number of buried seed projects have demonstrated survival for considerably shorter periods of time. These have been discussed in detail by Barton in her book on longevity, and in Crocker's review of the same subject [4,10]. A study of buried weed seed, due to last for 50 years, was initiated in 1972, and the results of germination and viability tests after 2.5 years of burial have been published [84]: of 20 species buried, only 4 species maintained over 50% viability. A much earlier experiment, started by Duvel in 1902, involved the burying of seeds of 107 species of wild and cultivated plants. Of these, 71 species germinated after 1 year, 61 after 3 years, 68 after 6 and 10 years, 51 after 16 and 20 years, 44 after 30 years and 36 after 39 years. Seeds of cultivated plants, especially cereal grains and legume seeds, perished quickly in the soil, while seeds of wild plants, especially persistent weeds like docks, lambsquarters, plantains, daisies, poke, purslane and Jimson weed, retained their vitality well. Such weeds therefore cannot be controlled by ploughing the seeds under because the seeds outlive any crop rotation. We should note here that many persistent weed species do not possess hard coats. The poor survival of seeds of many species (and particularly the cultivated species) might not be related to their loss of viability, in the conventional sense. For survival in moist soil a seed not only has to maintain viability, but also has to possess a dormancy mechanism, otherwise it will germinate, degrade, and hence not be accounted for in the early viability tests. In these classical burial experiments it has not been possible to distinguish clearly between how many seeds were lost through germination in situ, and how many lost viability. The longest controlled burial experiment to date is that ofW.J. Beal who, in 1879, selected seeds of 23 different species of plants common in the vicinity of Michigan Agricultural College in East Lansing, mixed 50 seeds of each species with moist sand in unstoppered bottles and buried them in a sandy knoll. At regular intervals since, bottles have been unearthed and viability of the seeds tested. The spring of 1970 marked the 90-year period of this seed viability experiment, and the results of this, and previous tests are presented in Table 1.3. Under the germination conditions used (which may not have always been ideal) only Verbascum blattaria seeds remained viable after 90 years, although many species did persist for 25-30 years, and a few seeds of Oenothera biennis and Rumex crispus for 80 years. Thus the values of longevity for buried seeds of known age and maintained under controlled conditions are far shorter than for seeds dated by indirect, circumstantial evidence.

1.2. Viability of Seeds in Storage Over the past few decades there have been hundreds, if not thousands, of research papers published on this subject, and we have consulted but a fraction of these in the preparation of this account. Instead, we have relied heavily on comprehensive summaries incorporated into books [4, 22, 32] and recent reviews [3,5, 13, 15, 17, 20,21,23-31] - the reader should consult these for more detailed information and for further references. This account is therefore a synopsis which, we trust, includes most of the points essential for the reader to gain some feel for the subject.

+

+

+

+

+ +

+

+

0 ?

+ + + +

0 0

0 0

+ + +

0

+ + + + +

0

0 0

+

+ +

0

+

0

+

+

0

+ +

0 0

+

0

+ +

0 0

+ + + +

+

0 0

0 0

+

0

+

+ +

0 0

+ +

+

0 0 ? 0

0 0

+

0

+

+

0

+

+

0

+

0 0 0 0 0 0 0

0 0

+

0

+

0 0 0 0 19 (38) 9 (18)

+

+

0

+

0

0 0 0 0

+

0

+

0 0 0 0 0 19 (38) 26 (52) 31 (62)

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 12 (24) 2 (4) 34 (68)

60th year 1940

80th year 1960 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 (10) 1 (2) 35 (70)

70th year 1950 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 (14) 4 (8) 37 (74)

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10 (20)

90th C year 1970

a The + signs following each species, both in the upper and in the lower parts of the table, indicate that one or more seeds of that species germinated for the year shown. The number indicates the number of seeds germinating, while the number in parentheses indicates the percent germinated b Perennial C There is some question concerning the identification of Verbascum plants in the early period (1884--1920) as V. thapsus rather than V. blattaria d In previous years incorrect germination conditions might have been used; for further comment see [55] e For 100 years see: Kivilaan and Bandurski, Am. J. Bot. 68, 1290 (1981)

+

+

+ + + + + +

0

0 0

0

+

+

0

+ +d 0 0 0 0 0 0 0

+

0

0 0

0 0

+

+

+ +

0

+ +

0

+

0

+

+

+

0

+

0 0 0 0

+

0 0 0 0 0

0

0

0

0

1 1 1 1 1 1 1 1 1 1 1 or 2 per. b 1 1 1 1 per. b 2 2 2

Agrostemma githago Amaranthus retroflexus Ambrosia artemisifolia Anthemis cotula Brassica nigra Bromus secalinus Capsella bursa-pastoris Erechtites hieracifolia Euphorbia maculata Lepidium virginicum Malva rotundifolia Plantago major Polygonum hydropiper Portulaca oleracea Setaria glauca Stellaria media Ti·ifolium repens Verbascum thapsus C Oenothera biennis Rumex crispus Verbascum blattaria C

0

50th year 1930

5th 10th 15th 20th 25th 30th 35th 40th year year year year year year year year 1884 1889 1894 1899 1904 1909 1914 1920

Duration of life-cycle (years)

Name of species tested a

Table 1.3. Viability of buried seeds in W.J. Beal's buried-seed experiment, 1879-1970

Q

ｾＮ＠

I)Q

=

0

t""

0-

'= "

'


:;::

u

o (J)

OJ ｾ＠

0

1.L.

0

ｾ＠

-0 >-

..c

Ul

o.?:-

1.2

c (J)

01 0 ｾ＠

.2 (J)

..c

0

1.0

-0 ｾ＠ >--

ｾ＠

0

-0

.2 o

(J)

ｾ＠

08 •

01 0

§i 0.6

..c

o.u ｾ＠

Ul

0

(J)

"§.

0.

I

I.D

0

I

(J)

)g

u

:::J

(3

O.L.

Ul

0 -0

0.2

(J)

Ul Ul

0

I

."..----.

0

I

•

0

(J) ｾ＠

0.

x

(J)

00

L.

/•

•

8

12

16

20

2L.

28

Water content (%)

cells could lead to depletion of essential substrates, that would then not be available for germination - but there is no evidence for this. Whether activities of certain enzymes lead to loss of viability during storage (e.g. through depletion of substrates, or production of toxic compounds), or whether lack of enzyme activity (e.g. of membrane repair enzymes) is lethal remain matters for speculation.

1.9. Changes in Food Reserves Since germination is completed before mobilization of the major reserves commences, we should expect their deterioration to affect seedling vigour rather than seed viability. On the other hand, since food mobilization is under the control of the growing embryo, or axis (Chap. 7, Vol. 1), we should not expect this process to occur in non-viable seeds. It has been demonstrated in many laboratory experiments that excised cereal embryos can germinate and establish themselves with no requirement for the endosperm reserves, and that legumes can germinate and grow with a large part of their cotyledons removed. Under less favourable field conditions, however, there undoubtedly is more dependence of the embryo/axis on the stored reserves, particularly before the aerial parts force themselves up out of the soil and commence photosynthesis. As an example, we might note that 25% fewer seedlings emerge from lightweight grains of perennial ryegrass sown at 25 mm soil depth than from heavier grains [47]. The difference in grain weight is probably to a large extent related to stored reserve content - the less the reserve content, the less growth that can be supported during passage through the soil.

Changes in Food Reserves

47

100 >,

ClI

-;:

'0

0 .D

75

2

ClI

>.:0 .:; c0

3

:;::U 'E « ClI L

*'

rn

t.

?;
A 2, Bl , C l ). In some cases, however, germinability is retained at lower temperatures and dormancy is manifest only at higher temperatures (Fig. 2.1C 2), i.e. the seeds are said to show relative dormancy [19, 45]. An example of relative dormancy (wheat) - C 2 typeis shown in Fig. 2.2. together with a contrasting case of complete dormancy ( Avena Jatua). In freshly harvested wheat, dormancy is manifest only at temperatures

62

Dormancy

100

......

80

"-"

-0 Q)

0

c

-

""---\

0

0

60

E Q;

t9

\-

40

?F. 20

•

0 0

5

•

• 10

15

""----•

• • 20

25

30

Temperature (OC)

Fig. 2.2. Relative and complete dormancy exemplified by wheat [Triticum aestivum (_) and wild oat (Avenafatua) (e)]. Germination at different temperatures was determined 16 days after the start of imbibition. To show that Avena fatua is viable, naked caryopses were stimulated to germinate by 0.33 mM gibberellic acid (GA3) at two temperatures (0). Avena fatua: "Montana" strain. Triticum aestivum: cv. Capelle Desprez. By the authors

above about 18° C whereas in A.fatua it is present at all temperatures. Relative dormancy (B2 type) is exemplified by Amaranthus retroflexus [222a]. We should note, in cases of relative dormancy, that though seeds may appear non-dormant at some temperatures they may nevertheless have a slower rate of germination - described by some as "resistance to germination" [108] [see Fig. 3.35 (inset) for an example]. Thus, it seems that the dormancy completely prevents germination at some temperatures but only slows it down at others. It is important not to confuse the failure to germinate due to unsuitable conditions for germination itself with the inability to germinate because of relative dormancy. A state of relative dormancy can be recognized by the beneficial effect of a priming treatment (perhaps chilling, light) or by the increased germinability after the seed has passed some time in the dry state. Priming treatments do not enable non-dormant seeds to germinate outside their "correct" temperature range, nor do such seeds show shifts in temperature tolerance with time. Relative dormancy, particularly the shifts in depth of dormancy during the lifetime of a seed, has been explored by Vegis [19, 245, 246] in connection with the onset of dormancy (see Sect. 2.8).

2.1.1. Categories of Dormancy

Different categories of seed dormancy can be recognized according to their manner of origin. Unfortunately, different terminologies are employed by various authorities, which can give rise to some confusion. Several authorities distinguish only between dormancy and secondary dormancy, the former being the state of the seed as shed from the mother plant, the latter being dormancy induced in a mature, imbibed seed by certain environmental conditions which are unfavourable to germination. The descriptions primary and induced are also applied to these two categories of dormancy. Other authorities divide primary dormancy (or dormancy) in-

What is Dormancy?

63

to innate dormancy and enforced dormancy: both are inherent in the seed at the end of its development on the mother plant, but the second is a dormancy manifest only under certain environmental conditions. Some authorities would consider the latter to be a case of relative dormancy. The variety of terms, some of which are equivalent in their meaning and which the reader will meet in his incursions into the literature, are set out in Table 2.1. Examples are given which, it is hoped, will clarify the situation. In this book we will use the terms dormancy (or primary dormancy), relative dormancy, and secondary dormancy.

2.1.2. Biological Significance of Seed Dormancy

Dormancy is a device for optimizing the distribution of germination in time or space and its importance is therefore best seen in an ecological context. Distribution in time can be achieved by spreading gelJIlination over an extended period. This happens because seeds of many species show variability in depth of dormancy; the population consequently exhibits sporadic release from dormancy and hence irregular germination. This behaviour is of considerable biological value since the temporal dispersal enhances the spread and survival of the species. Termination of dormancy is also brought about by physical factors, such as light and temperature, and their action and interaction can regulate germination to fit in with seasonal changes in the environment. Thus, dispersal of germination can be controlled so as to limit the appearance of new plants to times that are least hazardous for their successful establishment. It may be advantageous, for example, for seedling emergence to be delayed until after the low temperatures of winter have passed. If, to be released from dormancy, the seed requires chilling temperatures for several weeks, germination thus occurs only after but not before the winter temperatures have been experienced. Salisbury [220, 221] recognized four basic patterns with respect to the temporal distribution of germination: (a) Quasi-simultaneous, when germination of all the seeds occurs over a relatively brief period; (b) Continuous, in which members of the population germinate over an extended time period, with no clear peaks; (c) Intermittent, irregular germination over long time periods, showing essentially a multimodal distribution; (d) Periodic germination, which is again multimodal but shows more regular periodicity. These patterns result from the dormancy characteristics as discussed above and also from an interplay between these and the control of germination itself by various environmental factors, such as temperature. The ecological significance of these patterns will be considered more fully in Chapter 6. The dormancy mechanism can also operate to secure a suitable place for germination. For example, those seeds whose dormancy is broken by light are clearly unable to germinate when buried deeply in the soil. Germination is thus limited to the top few millimetres of soil. This can be an advantage to a small seedling carrying food reserves which support growth only for a relatively short time, since it can quickly establish itself as a photosynthesizing autotrophic plant. Spatial distribution of germination can also result from the effect of light quality on the termination of dormancy. As we shall see in Chapter 6, dormancy of light-sensitive

Dormancy

Primary Dormancy dormancy

Enforced Dormancy dormancy

Dormancy Primary dormancy

Secondary Secondary Secondary Secondary Secondary Secondary Secondary Induced dormancy or dormancy dormancy dormancy dormancy dormancy or induced induced dormancy dormancy

2. A seed which in darkness is dormant at temperatures above a certain value. At the higher temperature dormancy can be broken and germination secured by exposing seeds to a short period of light (e.g. Lactuca sativa cvs.)

3. A seed which germinates in darkness but is inhibited by light. After photoinhibition, transfer to darkness does not allow of germination. The seeds have become dormant. This dormancy is terminated by moving the seed to a lower temperature (e.g. Nemophila ins ignis at 23° C)

Dormancy or relative dormancy

Dormancy or primary dormancy Dormancy

Primary Dormancy dormancy

Innate Dormancy dormancy

Bewley and Black

Dormancy Primary dormancy

ViIIiers [20]

1. A seed which, from the time of dispersal from the mother plant is dormant over a range of normal temperatures, but germinates after several weeks prechilling (e.g. for CoryJus ave/lana)

Mayer and PoljakoffMayber [13]

Koller et al. [162]

Crocker [61]

Harper [10] Barton [2] Roberts [15]

Terminology Crocker and Barton [7]

Example of dormancy

Table 2.1. Terminology used to describe categories of dormancy

J

§

o

.j:>.

""

What is Dormancy?

65

seeds is not broken by light filtered through green leaves; thus germination is restricted to places receiving full sunlight, which is clearly of benefit to a newly produced seedling. Dormancy can also operate to regulate the geographical distribution of plants. The coupling of dormancy release to an environmental signal such as chilling temperatures, for example, would clearly limit certain species to climatic zones which experience cold winters and thus prevent spread to warmer regions. Dormancy is often assumed to be important in relation to the prevention of vivipary and precocious germination, i.e. germination while still on the mother plant. This may indeed be so in certain species, e.g. cereals, but it is doubtful that it is of general significance in all those species which produce dormant seeds. In Section 2.8.1 we shall see that dormancy in many cases only develops towards the end of seed maturation, so it cannot be responsible for restraining germination at earlier times. Other factors presumably are operative, such as various controls exerted by the mother plant itself. It has sometimes been implied that the viability of a seed is connected with its dormancy, but as Roberts has pointed out [15] there is scant evidence for this. Indeed, rice grains display no relationship between the degrees of dormancy and viability [15]. Evidence has emerged, however, suggesting that the retention ofviability by certain species is enhanced in imbibed seeds as compared with dry seeds (Chap. I). If dormancy allows such a seed to remain imbibed without germinating, it is therefore also instrumental in prolonging the period of viability beyond that which would occur in the dry state. To this extent then, dormancy and viability might indeed prove to be related.

2.1.3. Dormancy in Cultivated Plants During the domestication of plants, selection has usually been exercised for seeds which germinate relatively promptly and uniformly, so the dormancy characteristics of many cultivated plants differ from those of their wild progenitors. Crop species of Phaseolus, for example, possess little or no dormancy, germination occurring fairly readily upon wetting the seed at a suitable temperature, etc. In the wild Phaseolus, however, (e.g. P.heterophyllus, P.polystachios, P.polyanthus) dormancy is imposed by extremely hard seed coats [142] and in P. acutifolius and P. vulgaris ssp. aborgineus a post-harvest maturation period (after-ripening) is needed to break dormancy [228]. Seeds of the Shirley poppy, a cultivar of Papaver rhoeas, have no dormancy, unlike those of the wild P. rhoeas [23]. Cultivation has clearly selected against seed dormancy in Anemone coronaria. Here, the achenes ofthe wild species are dormant (they require after-ripening) but those of the cultivated De Caen type are not [129]. This, it has been suggested, is how germination of the wild type in its Mediterranean habitat is delayed from the spring until the rainy autumn, unnecessary for the cultivated type, and indeed selection has been practised against it. Although seed dormancy has been minimized in crop plants, it is not the case, as is sometimes implied, that they all lack dormancy. The cereals, wheat, rice, oats and barley are all dormant to some degree, though generally for only a few weeks or months. Seeds of many vegetables (e.g. lettuce, celery, carrot) also show various

66

Dormancy

types of dormancy, and beet is notorious in this respect. In all these cases the dormancy period can be a considerable nuisance to growers because the timing and extent of germination is unpredictable. Dormancy can be a serious inconvenience in the malting industry where the time of germination and associated processes in the barley grain have to be carefully regulated, and also in seed-testing laboratories where the germination levels and viability of a seed crop (such as wheat) have to be defined within a short time after harvest in time for distribution to farmers. On the other hand, a degree of dormancy may be desirable and selection by Man has sometimes favoured species or varieties which have this. For example, in wheat and barley, the agriculturally disastrous phenomenon of pre-harvest sprouting can occur, in which germination takes place on the mother plant. When grown in parts of the world subject to wet, cool weather during the time of seed maturation, these cereals are particularly prone to such premature germination, which results from insufficient dormancy. For this reason, the red-grained, more dormant wheat varieties have been selected for cultivation in areas whose climatic conditions provoke sprouting on the ear (see Sect. 2.4.3 for further discussion on grain colour). Wheat illustrates several other interesting points with regard to seed dormancy and agriculture. Firstly, Man makes conflicting demands upon the seed. We would like sufficient dormancy to prevent pre-harvest sprouting but none to interfere with seed testing and rapid establishment of a new crop. Ideally, up to the time of harvest, there should be a fairly deep dormancy which then rapidly disappears! It could be argued that some of the problems concerning dormancy in wheat are connected with the transfer of a species from its centre of origin into regions to which it is poorly adapted. For example, the absence of appreciable dormancy in maturing grain is not likely to lead to premature germination (with its unfavourable biological and agricultural consequences) in the Middle East where wheat originates, because there it is generally hot and dry during the time of grain ripening. There are cases where deliberate attempts have been made to introduce dormancy. The "dormoat", for example, is a cross between the cultivated oat, Avena sativa, which has little dormancy, and the wild oat, A.fatua, which can have very deep dormancy. The cross is made to introduce dormancy into the cultivated oat so that it can be sown in autumn, when the fields are easily workable, overwinter in the dormant state, and then germinate in the spring as soon as conditions allow. The possible advantage of this is that in certain countries (e.g. Canada) benefit can be obtained from the early spring growth and a consequently modified shoot/root ratio, both of which give rise to a greater grain yield.

2.1.4. Polymorphism and Heteroblasty

Many species produce seeds which differ individually in their degree of dormancy. This partly accounts for the temporal distribution of germination which, as discussed in Section 2.1.2, is an important biological role of seed dormancy. On genetic grounds we expect variability in the offspring of a plant, and there is no reason why such variability should not apply to the control of germination as well as to other physiological and anatomical features. An interesting aspect of the vari-

What is Dormancy?

67

ation in dormancy and germinability is that the seeds of many species fall into two or more discontinuous populations. Frequently, this physiological variability is matched by differences in seed morphology. The term polymorphism originally was used with reference to these morphological differences but now it is applied to physiological variation in dormancy, even if there are no accompanying morphological dissimilarities [52]. Other terms signifying the same phenomenon are also employed, such as heteromorphy, heteroblasty [65, 93] and physiological heterogeneity [160]. For simplicity's sake we will use only one term in this account - polymorphism. Polymorphism is widespread in the Compositae, Cruciferae and Chenopodiaceae, and occurs in other families including the Gramineae. It is manifest by differences in dormancy between seeds of different plants and also by variability in the dormancy characteristics of seeds from the same mother. A good example of the former is provided by Rumex. Cavers and Harper [52] point to the confused data regarding the dormancy and germination of R. crispus and R.obtusifolius. Germination values ranging from 0%-60% can be found for seed of the same age, produced by plants under apparently identical environments and tested under standard conditions. When examined in detail it becomes clear that these differences are due partially to dissimilarities between seed from different plants. For example, it was found that seeds from two individual plants of R. crispus, growing on the same site, are dormant in darkness. Dormancy of seeds from both plants could be terminated by treatment with light together with alternating temperature, but seed from one plant showed a significantly greater response to alternating temperature even in darkness (70% germinated as against 57%). Different plants of Eschscholtzia also produce seed with variable levels of dormancy. In this species individual plants have seeds which are either completely dormant in darkness (no germination) or are only partially dormant (up to 18% germinate) [57]. Polymorphic seeds are also commonly formed by the same parent plant. A wellknown example is Xanthium pennsylvanicum, in which two seeds are contained in the dispersal unit; an upper, deeply dormant one, and a lower, less dormant seed (Fig. 2.3). As we shall see later, the upper, dormant seed has been used extensively for investigations into the mechanism of dormancy. Many other composites produce different seeds in their ray and disc florets. The capitula of Bidens bipinnata has two kinds of cypselas; the outer ones are short, brown, rugose and deeply dormant, while the inner ones are longer, black, smooth and less dormant [64]. In Synedrella nodiflora, the cypselas of the ray florets are also more dormant than those from the disc [211]. Cases are known in which dissimilar seeds are formed by different inflorescences. Individual panicles of Rumex crispus yield seeds with different degrees of dormancy [52] as do the umbels of celery ( Apium graveolens) [239 a]. The depth of dormancy in Avena Jatua also seems to be partially determined by the panicle on which the grains are borne [136a] and in this species, the earlier ripening grains are also the least dormant. Another graminaceous species, Aegilops ovata, provides a further, interesting example of how the location of the grain on the mother plant influences its dormancy. Here, up to six grains are dispersed together in a group of from two to four spikelets. The grains differ greatly in their dormancy and especially in their response to light and temperature. Generally, only one grain in the intact unit germinates; this is the heaviest one from the lower-

68

Dormancy Fig. 2.3. The burr of Xanthium pennsylvanicum

Upper seed

Lower ＭＺｾﾭ seed

Table 2.2. Polymorphism in Portulaca oleracea Plant No.

Percent germinated Capsule 1

Capsule 2

1 2

100

10 94

3

24

4

4

o

o

13

Capsule 3 6

15 98 41

After Egley, 1974 [82J

most spikelet [65]. Finally, we might note an example - Portulaca oleracea (purslane) - where seeds of different fruits (capsules) have different dormancy properties, and where individual plants also differ (Table 2.2). Reference has been made above to morphological differences which can accompany dissimilarities in dormancy (e.g. in Bidens bipinnata). In other species too, the level of dormancy can be predicted from the physical appearance of the seed. Salsola volkensii and Aellenia antrani produce green (chlorophyllous) and non-green seed, the former being almost completely non-dormant, and the latter dormant [196]. Four morphological types of seeds can occur on one plant of Chenopodium album. Seeds may be brown or black, with smooth or reticulate coats. The smooth, black seeds have the deepest dormancy and require a period of chilling to release them [119, 261]. Production of the various seed types in Chenopodium can be regulated by the daylength, a point to which we will return in Section 2.8.4.

Embryo Dormancy

69

Little is known about the control of polymorphism. Clearly, the regulation is both genetic and positional; in the Compositae, for example, polymorphism is maternally determined [119]. As mentioned above, the biological importance of polymorphism is connected with the temporal distribution of germination which results from the variability in dormancy. It is interesting that in weed species, whose seed dormancy is one feature contributing to survival, polymorphism is widespread. In fact, weedy members of a family (e.g. the Compositae) commonly have a higher incidence of polymorphism than the non-weedy members [119].

2.2. Dormancy Mechanisms When we enquire into the mechanism of dormancy we are asking these questions: What is the nature of the blocks within the seed which prevent germination under apparently favourable conditions, and how do they operate? This is clearly not the same as asking what environmental factors cause dormancy or what is required to break dormancy, although a full appreciation of the mechanism of dormancy demands eventual attention to these questions too. There are basically two types of dormancy which involve different mechanisms: (a) Embryo dormancy, where the control of dormancy resides within the embryo itself and (b) Coat-imposed dormancy, in which dormancy is maintained by the structures enclosing the embryo, i.e. the seed coat. Nikolaeva [14] has treated each of these in several sub-divisions, but the basic dichotomy of embryo and coat-imposed dormancy is still retained. In certain species, both types may exist simultaneously or consecutively. Some authors employ the term embryo dormancy in a somewhat looser sense than our usage of it [2], paying attention to where the release mechanism occurs rather than to where the maintenance mechanism is located. Thus, many cases which we would consider as coat-imposed dormancy are described as embryo dormancy, because the growth efficiency of the embryo is enhanced when dormancy is terminated. Confusion should be avoided by giving emphasis to where the imposition of dormancy resides, not to where the release is actuated.

2.3. Embryo Dormancy The most clear-cut, authentic case of embryo dormancy is recognized by the failure of the viable, mature embryo to germinate even when it is isolated from the seed or dispersal unit. Such naked embryos, when placed on a wet substratum, remain dormant even though the conditions are suitable for germination itself. If the intact seed has previously been subjected to a dormancy-breaking treatment, the embryo does germinate, showing that the conditions really are favourable. Dormancy is

70

Dormancy

Table 2.3. Some species showing embryo dormancy Species

Reference

Species

Reference

Azorella selago Acer platanoides Acer pseudoplatanus Acer saccharum Ambrosia artemisifolia Ambrosia trifida Avena Jatua "Montana" Chaenomeles spp. Corylus avellana Crataegus moWs Cydonia japonica Elaeagnus angllstifolia E laeagnus umbellata Euon ymus europaeus Fagus s ylvatica Fraxinus americana Fraxinus excelsior

[72] [205] [239] [253] [260] [66] [39] [149] [136] [68] [157] [118] [117] [48] [101] [229] [247]

Helipterum craspedioides Hordeum spp. (barley) Malus sylvestris Polygonum spp. Pnmus armeniaca Prunus persica Pyrus communis Rosa spp. Rhodotypos ken·ioides Sorbus aucuparia Syringa rejlexa Taxus baccata Taxus baccata var. Jastigiata Triticum aestivum

[191] [109] [99] [141] [43] [99] [149] [134] [99] [101] [138] [169] [168] [155]

Many of the references above are not the first citations of embryo dormancy in the particular species but are fairly recent publications reporting experimental work

marked not only by an inability of the isolated embryo to germinate, which is clearly due to a deficiency in the axis, but also by metabolic blocks within the cotyledons. In Xanthium pennsylvanicum, for example, where embryos of freshly harvested seeds are dormant, excised cotyledons cannot form chlorophyll or expand when exposed to light, while greening and expansion both occur in cotyledons excised from l-year-old seeds which have lost their embryo dormancy [89]. This indicates that metabolic deficiencies exist in the cotyledons as well as in the axes of dormant embryos. Another facet of embryo dormancy is seen in those species whose isolated embryos can germinate, but do so very sluggishly to produce slowly growing dwarf seedlings (physiological dwarfism). Table 2.3 lists several species in which embryo dormancy has been reported. A wide range of families is represented but it may be noted that woody members of the Rosaceae are prominent. In anyone species, variability may be encountered, which probably explains the differences in response sometimes reported in the literature. In apple ( Malus sylvestris) for example, the intensity and extent of embryo dormancy depends upon variety, provenance, year of harvest, and other factors [238]. Embryo dormancy may vary in time. In some species it may be short-lived, its transience being independent of any easily recognizable, dormancy-breaking experience. The embryos of Acer pseudoplatanus (sycamore) pass through a period of dormancy while still on the mother plant when, late in their maturation, they cannot germinate even after removal from the enclosing covers [239]. The converse of this situation holds in Corylus avellana (hazel) where a long-lasting embryo dormancy develops after a short, coat-imposed dormancy. These seeds show no em-

Embryo Dormancy

71

bryo dormancy when freshly harvested, but the dormancy slowly develops in dry storage and is complete after approximately 45 days, e.g. [136]. It can be argued, however, that the case of hazel is really an example of secondary embryo dormancy, induced by the coat. Other cases of induced embryo dormancy are well documented. One of the best-known is Xanthium, where isolated, germinable embryos become dormant when held in damp clay or under other conditions of low oxygen tension [67]. Low oxygen concentrations also induce embryo dormancy in nondormant apple seeds [56]. We have so far considered cases where the whole embryo fails to germinate and grow because of its dormancy. But another, narrower aspect is seen in cases where only part of the embryonic axis is dormant. In several species including Paeonia (e.g. P.suffruticosa, the tree peony), Lilium spp., Viburnum spp., Asarum canadense, and others, radicle growth readily occurs but the epicotyl of the embryonic axis remains dormant - so-called epicotyl dormancy. Such seedlings require a dormancy-breaking treatment, usually chilling, to promote epicotyl growth [2, 7, 233] (see Chap. 3). In other species, both the radicle and epicotyl may be dormant but with different intensities. Thus in Trillium spp., Caulophyllum thalictroides and Smiladna racemosa radicle dormancy is less deep and is overcome by one dormancybreaking experience (chilling) whereas the epicotyl needs a second treatment in order to begin normal growth [27]. These are examples of double dormancy. There are still other instances (Convallaria majalis, Sanguinaria canadensis) where dormancy of the radicle is incomplete and where only the percentage of seeds showing such growth and the extent of the growth are improved by chilling [233]. It is not clear how far, in some of these cases, the radicle dormancy is coat-imposed; the epicotyl dormancy seems, however, to reside within the organ itself. As already mentioned, species are included in Table 2.3 whose embryos, when isolated from dormant seeds, are capable only of very sluggish germination, giving rise to dwarf plants (physiological dwarfism). Such plants have short stems, bear abnormal leaves and show a rosette type of growth habit. In extreme cases, forced plants from excised dormant embryos exhibit no root growth. Physiological dwarfism is shown by varieties of peach (Prunus persica) , apricot (P. armeniaca) , plum (P.domestica), apple (Malus sylvestris), pear (Pyrus communis), and by hawthorn (Crataegus mollis) , Japanese rose tree (Rhodotypos kerrioides) , and Helipterum craspedioides [28, 43, 98-100, 105, 183, 191]. Interestingly, treatments which break dormancy in intact seeds of these species are in some cases effective when applied to a forced, dwarf plant. Thus, chilling (Chap. 3) or gibberellins may completely break embryo dormancy and also convert physiological dwarfs into plants of normal growth habit.

2.3.1. Control Mechanisms in Embryo Dormancy

In those cases where detailed studies have been made the evidence suggests that the control of embryo dormancy involves (a) the cotyledons, and (b) germination inhibitors. (We will not yet consider those morphologically immature embryos in which the control of "dormancy" obviously has an anatomical basis; these instances are discussed in Sect. 2.3.4).

72

Dormancy

2.3.2. The Role of the Cotyledons

There is good evidence that in many cases the cotyledons are responsible for inhibiting the growth of the axis in dormant embryos. Part of this evidence comes from experiments in which one or both cotyledons have been removed from the isolated, dormant embryo. For example, dormancy is broken by cutting off just one cotyledon of Euonymus europaeus [48], an operation which is also partially effective in hazel [136]. Amputation of both cotyledons is needed to cause germination of Fraxinus excelsior embryos [49]. Embryonic axes of barley cultivars with embryo dormancy can be stimulated to germinate by excision of the scutellum (which is regarded as a modified cotyledon) [109]. These findings strongly suggest that dormancy of the axis in the intact embryo is maintained by some action of the cotyledon(s). The effect has been studied further in apple embryos where the degree of dormancy appears to be a function of the amount of cotyledon which is left attached (Fig. 2.4). In both epicotyl dormancy and physiological dwarfism the cotyledons have been shown to exert a similar inhibitory influence over the axis. Restoration of axial growth is achieved by excising the cotyledons of Viburnum trilobum, a species normally showing epicotyl dormancy [156]. Similarly, removal of the cotyledons from peach embryos taken from dormant seeds leads to normal, nondwarfed growth, though if cotyledon excision is delayed beyond a certain time period, reversion to the normal growth habit does not occur. Thus, the cotyledons seem able to induce changes in the axis from which it cannot recover. Cotyledons are also active in secondary (induced) dormancy. When embryos are isolated from non-dormant apple seeds and held under unfavourable conditions for germination, a secondary dormancy is normally induced. This does not occur, however, in embryos from which portions of the cotyledons have been excised [237]. The physiological and biochemical basis for the action of the cotyledons in dormancy is unknown. Inhibitors, not unexpectedly, have been invoked and, indeed, there is a little evidence, for example in Corylus avellana, that abscisic acid derived from the testa is present in the cotyledons [136]. Further work on this problem is needed. A phenomenon which has commonly been observed in isolated dormant embryos is unequal physiological activity of the two cotyledons. In particular, en60 _ _ _ _0

50 ｾ＠

c:iI

o

1.0 o c

·E 30 Q;

t:)

w

\ ｾ Ｎ＠

::>

20 ./

•

\

\

0

\

\. 0

0.5

1.0

1.5

2.0

2.5

3.0

h

Fig. 2.7. Gas uptake by Beta vulgaris (sugar beet) dispersal units. Changes in gas volume in Warburg flasks were measured after adding water (arrowed) to parts of dispersal units, viz. opercula (e), pericarps (_), testas (D) and naked seeds (0). There is initially a rapid gas evolution (invariably observed when water is added to dry seeds or fruits), followed by gas (oxygen) uptake. Weights of tissue: Testa, 10 g. All others, 100 g. After Coumans et aI., 1976 [59]

abundant mucilage which, especially when the seed is exposed to excessive amounts of water, swells greatly to occupy completely the spaces between multicellular hairs protruding from the coat's surface. This is thought to form such an effective barrier against oxygen that the germination of the seed is much delayed. That interference with oxygen entry is in fact likely to be the action of the imbibed mucilage is supported by the finding that an atmosphere of oxygen overcomes the inhibitory effect of the overwetted mucilage, and germination proceeds [116, 264]. There is good evidence that the mucilage of the coats of Hirschfeldia [197] and Spinacia [125] acts in a similar manner. In both red beet and sugar beet (Beta vulgaris), germination is inhibited by the ovary cap (operculum) covering each seed of the cluster. When the cap is removed, or when the external oxygen concentration around the intact fruits is raised, germination follows [59, 126]. This inhibitory effect in red beet has been attributed partly to the poorly permeable mucilage found around the rim of the cap and partly to the occurrence of inhibitors, possible phenolic compounds. For sugar beet, however, a somewhat different explanation has been offered, based on measurements of oxygen uptake by the dispersal unit [59]. Although composed of dead tissue, the operculum has a comparatively high level of gas consumption, presumably oxygen, (Fig. 2.7) which, it is thought, might thereby deprive the seed and inhibit its germination. It is interesting that Heydecker et al. [126] observed a greatly stimulated oxygen uptake by beet seed clusters supplied with aqueous beet fruit extracts. This

84

Dormancy

was tentatively attributed to an uncoupling effect, but they may have been adding enzymically oxidizable compounds from the operculum. Oxygen consumption by the structures enclosing the embryo or the seed has been observed in several other species. Clearly, when such uptake does occur the diffusion of oxygen through these covering tissues will be strongly reduced. This has been suggested by Come and his colleagues [6, 55] to be the action of the coats in Pyrus malus. Most of their investigations were carried out, in fact, on seeds whose dormancy had been broken (by chilling) but in which germination at certain temperatures nevertheless was controlled by the coat. Oxygen consumption by the coat in this species is attributed to the oxidation of various phenolics such as phloridzin, chlorogenic acid and para-coumarylquinic acid, known to be present in the testa. The relevance of this phenomenon to primary dormancy in apple is doubtful (this species shows embryo dormancy) but it may be important in connection with the induction of secondary dormancy (see Sect. 2.8.7). This is brought about at certain temperatures when, it has been suggested, oxygen consumption by coat phenolics is increased and deprivation of the embryo consequently worsened. Further evidence to connect coat-imposed dormancy with oxygen consumption by the seed coverings is hard to find in the research literature. Phenolics in the hull have been suggested to be involved in barley [208] but firm evidence is lacking. Also in barley, peroxidase, cytochrome oxidase and phosphoglycerate dehydrogenase have been found in the testa [179]. These enzymes could account for some oxygen consumption, but their significance is not yet clear. The case of rice (Oryza sativa), however, provides plausible support for a relationship between dormancy and oxygen consumption by extra-embryonic tissues. Here, dormancy is imposed by the hull; the naked caryopsis germinates readily (see Table 2.7). Germination can also be secured by subjecting intact rice grains (i.e. the hull is present) to an atmosphere of oxygen [217], or by puncturing the glumes over the embryo [227]. The hull, when imbibed, is capable of oxygen uptake, apparently because of the action of a peroxidase which presumably is carried over in the dehydrated tissues. Interestingly, the consumption of oxygen by hulls of dormant grains is about double that of hulls from non-dormant dispersal units, a disparity which coincides with the twofold activity of the peroxidase in dormant hulls. Further, oxygen uptake by the intact, dormant grain is rather more than 50% higher than by the non-dormant grain, and since this difference is not exhibited by the naked caryopses it is apparently due to the effect of the hull. It is worth noting that a similar elevated uptake of oxygen by imbibed, dormant grains has been observed in other cereals, for example, barley [178]; it is not yet clear, however, if this too can be attributed to oxygen-consuming enzymic reactions in the outer covers. Based on these findings, it may be suggested that coat-imposed dormancy in rice is caused by, or at least closely connected with the consumption of oxygen by peroxidases in the hull, which action restricts the amount of oxygen reaching the embryo. Fully consistent with this concept is the observation that peroxidase activity declines during the natural loss of dormancy (after-ripening). Indeed, the correlation between the activity of the enzyme and the degree of dormancy provides further persuasive support for the hypothesis (Fig. 2.8). Accepting that the seed coverings in some species, for one reason or another, limit the amount of oxygen available to the embryo, we may now enquire as to the

Coat-imposed Dormancy

85

c Ｎｾ＠

Cl

'- 1.0 Q)

c.

J:: 0.9

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o 0.8 \ • ., Q)

Ul

o ] 0.7 o ｾ＠

c. Q)

0.6

>

:; Qj

a::

O.

o

20

40

60

80

100

% Germinated

Fig. 2.8. Relationship between dormancy levels and peroxidase activity in hulls of Oryza sativa. Grains of four rice cultivars were after-ripened in dry storage and tested intermittently for dormancy and peroxidase activity. Note that as germinability increases (due to increased time of after-ripening) peroxidase activity declines. Cultivars: .: H6 (after-ripened at 25°-27°C); 0: Mayang Ebos 80 (after ripened at 25°-27°C); T: Seraup 27 (after-ripened at 25°-27°C); 'i7 : H4 (after-ripened at 28°-30°C). After Navasero et al., 1975 [193]

consequences of this. Perhaps the simplest possibility to envisage is that there would be insufficient oxygen to support the level of respiration needed for germination, and the embryo therefore remains dormant. Observations on several species, however, militate against this explanation. It has been found in, for example, Betula pubescens [41], wheat [76], Sinapis arvensis [8] and Stachys alpina [206] that isolated embryos are capable of germinating under extremely low oxygen tensions and even under nitrogen. Moreover, in both Sinapis arvensis and Xanthium pennsylvanicum the amount of oxygen reaching the embryo through the intact seed coat is quite adequate to supply the respiratory requirements of the embryo during its germination [8, 209]. These points are discussed more fully in the case histories considered below (Sects. 2.5.1 and 2.5.2) but it is worth noting now that, at least in these species, the deleterious effects of low oxygen levels (because of coat impedance) must clearly be attributed to factors other than reduced respiration. We shall see that inhibitor production, or the failure to oxidize inhibitors which are already present, have both been invoked as possibilities. There is very little evidence to link the dormancy-imposing action of the seed coat with the accumulation of carbon dioxide inside the enclosing covers. Several reports can be found of germination being inhibited by high concentrations of carbon dioxide, of the order of 20%--40% (see e.g. [4]). Secondary dormancy can be induced by carbon dioxide in isolated embryos of Brassica (=Sinapis) alba [151] and other species. But if the retention of carbon dioxide within the seed coat is indeed to play any part in the imposition and maintenance of dormancy the gas would apparently have to accumulate to a high level. In Cucurbita pepo the inner membrane of the coat has a higher permeability to carbon dioxide than to oxygen; retention of high concentrations of carbon dioxide would not be a likely occur-

86

Dormancy

rence. This is a non-dormant seed, however, and it may be worthwhile to extend these investigations to species with a clear coat-imposed dormancy.

2.4.3. Inhibitors in the Coat

We have already seen (Sect. 2.3.3) that seeds of many species contain germination inhibitors. Some examples in which inhibitors are known to occur in the coats are shown in Table 2.8. In several of these cases it has been considered that the inhibitor(s) in the covering tissues might account for the coat's imposition and maintenance of dormancy, but evidence to support this contention is, in fact, very limited and much of the experimental work is open to criticism. Frequently, the inhibitor has been detected by its action on wheat or oat coleoptile growth, or by its effect on germination of a different species, such as lettuce or cress. Rigorous testing on the species from which the inhibitor has been extracted has often been omitted. Ideally, non-dormant, intact seeds or embryos of the same species should be used for bioassay and the inhibitor should be supplied at a concentration similar to the one occurring in the dormant seed (see Sect. 2.9). Furthermore, the proposition that the coat inhibitor is truly involved in dormancy is strengthened if a correlation can be found between depth of dormancy and level of inhibitor. Absence of such a correlation does not necessarily mean, however, that the inhibitor is not an important component of the dormancy-regulating system (see Sect. 2.8.6). We should be aware also that because of chemical modifications during extraction an inhibitory compound might arise which, in fact, is not active in situ. The kind of finding which has led workers to invoke inhibitors in the mechanism of coat-imposed dormancy can be illustrated by reference to Comptonia peregrina (the sweet fern) [69]. Removal of the pericarp and testa, but not of the pericarp alone, relieves dormancy and elicits germination. But in the intact seed the embryo nevertheless swells sufficiently to rupture the testa without radicle protrusion being accomplished. Here, then, the mechanical barrier, as well as the possible barrier to gaseous exchange, appears to have been considerably weakened, yet the testa remains inhibitory. This suggests that the testa could inhibit by a chemical action and indeed an inhibitor, perhaps ABA, occurs in this tissue. We should note, however, that a possible interference by the testa with leaching of inhibitor from the embryo has not been excluded. The outer coats of two cereals - rice and wheat - have inhibitors which have been reported to decrease as dormancy is lost during the after-ripening process [121,188]. Among the inhibitors in the testa of wheat are catechins and catechin tannins which themselves are precursors of the red pigment, phlobaphene [187]. Thus, the red-coated wheats are thought to have high levels of inhibitory catechins and their derivatives, and the white wheats, low levels. Interestingly, the whitegrained wheats have a high susceptibility to pre-harvest sprouting, whereas the redgrained wheats tend to be much more resistant [95, 199]. It appears, therefore, that the red-grained cultivars have a deeper dormancy than those cultivars lacking pigment (or, more relevantly, its germination-inhibitory precursors) (see Table 2.9). That red pigmentation invariably accompanies deeper dormancy is not, of course, proof of a causal relationship, for the association might reflect either an extremely

Coat-imposed Dormancy

87

Table 2.8. Some seeds known to have inhibitors in the coat" Species

Location of inhibitor

Nature of inhibitorb

Reference

Aegilops ovata AceI' negundo Avena sativa Beta vulgaris

Hull Pericarp Hull Pericarp

[166] [132] [84] [31, 140, 185]

Betula pubescens Bouteloua curtipendula Comptonia peregrina Corylus avellana Elaeagnus angustifolia Elaeagnus umbellata Fraxinus americana Fraxinus ornus Hordeum vulgare

Peri carp Glumes, lemma, paleas Testa Testa Peri carp, testa Pericarp, testa Pericarp Pericarp Hull

Iris spp. Oryza sativa Prunus persica Rosa can ina Rosa arvensis Sinapis arvensis Triticum spp.

Endosperm Hull Testa Peri carp, testa Peri carp, testa Testa Pericarp, testa

Zilla macroptera

Pericarp

A monoepoxylignanolide Possibly ABA Unknown Various phenolic acids, possibly ABA, cis-4cyclohexene-1,2dicarboxirnide, high inorganic ions concentration Unknown Possibly coumarin and derivatives Possibly ABA ABA Possibly coumarin Possibly coumarin ABA ABA Coumarin, phenolic acids, scopoletin Unknown Probably ABA ABA ABA ABA Unknown Catechin, catechin tannins, several unknowns Unknown

[41] [177] [69] [262] [118] [117] [230] [230] [244] [213] [121,122] [71] [134] [134] [263] [187, 188] [38]

" Not all of the named chemicals shown here were rigorously characterized b Also see Table 2.14

Table 2.9. Coat colour and dormancy in wheat Hybrid population

1. Genesee x Turkey 2. Genesee x Seneca 3. Genesee x Red Rock 4. Genesee x Dual 5. Genesee x Redcoat 6. Genesee x Monon 7. Genesee

Number of Grain colour and % dormancy" coat colour genes White Light red Medium red 3 3 3 2 2 2 0

15.0 16.1 10.5 11.1 13.1 1.2 0

77.6 60.0 59.1 53.5 36.7 30.9

76.9 71.0 57.2 60.5 57.6 40.0

Dark red 77.8 61.9 53.8 57.5 63.0 44.6

" Dormancy is given as the percentage of dormant grains after 7 days in a germination test After Freed et aI., 1976 [103]

88

Dormancy 60

"0 ClI

i.0

o

c

'E Q; l:>

;,R. o

20

10

20

30

Time (days)

Fig. 2.9. Effect of coats on germination of Rosa arvensis embryos. Embryos were placed on the wet substratum together with pieces of the coat (pericarp and testa). D.-D. naked embryos; T - T naked embryos in the presence of pericarps and testas (not touching); .a.-.a. naked embryo covered with half-pericarps; e-e intact seeds (i.e. embryo with testa). After Jackson, 1968 [134]

close linkage between the genes for coat colour and those for dormancy, or a pleiotropic phenomenon (i.e. multiple phenotypic effects due to a single gene). N otwithstanding this, it has never been possible, by crossing techniques, to separate coat colour and dormancy. Table 2.9 shows the dormancy found in the progeny of several crosses. The parents in these crosses contribute different numbers of genes to coat colour (there are three genes for red-coatedness). Besides demonstrating the association between testa colour and dormancy, the results also show that the different genes for red pigmentation are associated with different levels of dormancy (compare, for example, crosses 4 and 6). These findings form persuasive evidence linking the control of dormancy in certain wheat cultivars with inhibitors in the coat. In some cases it has been possible to show directly that the influence of the enclosing structures in dormancy is connected with the presence of inhibitors. Embryos of Iris, for example, germinate readily when isolated, though the intact seed exhibits dormancy. The tissue responsible is the endosperm. That inhibitors are involved is strongly suggested by the observation that the germination, even of excised embryos, is considerably retarded if they are in contact with pieces of endosperm [213]. Chemical inhibitors can be isolated from the endosperm of these seeds [145]. A rather similar approach has been taken with dormant achenes of Rosa arvensis [134]. When removed from the enclosing coat (pericarp and testa) these embryos germinate. Their germination behaviour reverts to that of the intact achene, however, if half of a pericarp is replaced over each embryo. A substantial amount of dormancy is reimposed just by setting pericarps and testas on the same substratum as the naked embryo (Fig. 2.9). In some species of Rosa germination can

Coat-imposed Dormancy

89

be provoked by washing the achenes in excess water, a treatment which is presumed to leach out inhibitor. Susceptibility to leaching is often taken as a criterion that inhibitors are involved (Sect. 2.9). Finally, we should note the situation where inhibitors in the coat contribute to the imposition and maintenance of dormancy but are not solely responsible. Seeds (achenes) of birch (Betula pubescens) illustrate this. The pericarp (and possibly the endosperm) contains inhibitors which prevent the germination of excised birch embryos. Furthermore, repeated washing of the seeds (i.e. leaching) partially relieves the dormancy, presumably because inhibitors are removed. Dormancy can be substantially broken, however, simply by slitting the pericarp and endosperm. The result of this operation suggests that gaseous exchange is a factor which must also be considered and that the enclosing tissues might act by impeding the entry of oxygen. The role of the coat therefore is not a simple one, and only part of its effect may be due to inhibitors which it contains. It has been suggested that inhibitors in the pericarp impose a high oxygen requirement upon the embryo which cannot be satisfied because of the coat's relative impermeability to the gas [41]. The relationship between inhibitors and gaseous exchange will be explored further in the case histories discussed later (Sects. 2.5.1 and 2.5.2).

2.4.4. Prevention of the Escape of Inhibitors

Germination inhibitors occur within the inner tissues as well as in the outer coverings of the seed. It is conceivable that the coat impairs the escape of these inhibitors by acting either as a completely impermeable barrier or to reduce the rate at which outward diffusion can take place. The embryo, under these conditions, retains a high level of inhibitor and thus dormancy is maintained. Introduction of the isolated embryo to water encourages the loss of inhibitor, thus eliciting germination. The following criteria, when considered together, can be used to assess whether the enclosing structures might act to impede the exit of inhibitors: (a) After removing all other possible constraints such as interference with gaseous exchange, water uptake and mechanical restriction is dormancy still maintained? (b) Do the inner tissues of the seed (embryo, endosperm) contain germination inhibitors? (c) Does inhibitor diffuse out of the naked embryo on to a wet substratum (or out of the seed or caryopsis if the pericarp or glumes, etc. are the inhibitory tissues )? (d) Is outward diffusion prevented or the rate reduced by the enclosing coat? (e) Are leaching treatments more effective when the coats are removed? (t) If the naked embryo (seed or caryopsis) is allowed to take up water under conditions where outward diffusion of solutes cannot occur, or is severely limited, is dormancy still maintained? Let us now examine a few cases where all or some of these criteria have been applied. Dormancy in most strains of Avenafatua (wild oats) is generally imposed and maintained by the hull (lemma and paleae). When naked caryopses are held on a wet substratum dormancy is relieved and rapid germination ensues. If, however, naked caryopses are held under conditions of high humidity, when they achieve the same total water uptake (although at a slower rate than when in contact with liquid water) dormancy is maintained [39]. Under these latter conditions, since the hull

90

Dormancy

has been removed, constraints due to either mechanical or gaseous exchange effects cannot operate. A hypothesis which is consistent with these findings is that inhibitor is present in the caryopsis which cannot diffuse out (or not at a high enough rate) through the hull. When outward diffusion from naked caryopses is prevented by other means, i.e. by the absence of surrounding liquid water, a similar effect is obtained. The caryopsis indeed contains inhibitors which diffuse out on to a wet substratum but significantly less is lost from an intact dispersal unit than from a naked caryopsis. It appears, therefore, that the hull is a barrier against the escape of inhibitor. There is good evidence that the coat of the deeply dormant upper seed of Xanthium pennsylvanicum (see Fig. 2.3) acts, at least partially, by preventing the escape of inhibitors from the embryo [252]. The embryo contains two water-soluble inhibitors which diffuse out from the naked embryo but not from the intact seed when these are held on a wet substratum. The testa, therefore, acts as an effective barrier against the outward movement of the inhibitors. Moreover, in naked embryos, where the constraints of mechanical restriction or interference with gaseous exchange are circumvented, dormancy is still maintained, provided the imbibed embryo is not allowed contact with liquid water. Thus in these cases dormancy continues when the retention of inhibitor is secured, either naturally by the presence of the testa or experimentally by withholding liquid water from an already imbibed, isolated embryo. In this species, treatment of intact seeds with high oxygen tensions also relieves dormancy. Evidence suggests that this effect may operate by nullifying the inhibitor, a point which is discussed more fully in the case history of dormancy in this species (Sect. 2.5.2).

2.4.5. The Coat as a Light Filter

The great majority of light-sensitive seeds have coat-imposed dormancy (i.e. the naked embryo germinates almost irrespectively of the light/dark condition) and only a few cases are known which show true embryo dormancy (Chap. 3). For reasons which include the effects discussed above, as well as mechanical restraint, the embryo therefore remains dormant under the influence of the coat. As we shall see in Chapter 3, intact light-requiring seeds are stimulated to germinate (i.e. dormancy is broken) when a certain ratio of the active (Prr) and inactive (Pr) forms of phytochrome (P) is established within the embryo by the combined action of the red and far-red components of white light. The ratio required is dependent upon the species. Since the light has to pass through the structures enclosing the embryo it is conceivable that these could act as a filter, altering the proportion of red and far-red radiation reaching the sensitive embryo. This embryo, therefore, would not only bear the burden of coat-imposed dormancy but perhaps also suffer from an ineffectual light environment. Seed dormancy of many species is influenced by the conditions experienced while the seeds are developing on the mother plant (Sect. 2.8.4). One of the effects of the environment is on the production of seeds of different coat thickness and pigmentation. The case of Chenopodium album, in which this occurs, suggests that these two characteristics of the coat can later modify the effectiveness of the light

Coat-imposed Dormancy

91

Table 2.10. Seed-coat thickness in Chenopodium album and sensitivity to light Thickness of coat (Il) 24-28 34-39 44-49 49-53

% Germinated 15 min light

48 h light

62 61 47 27

100 80 91 80

Seeds were incubated in darkness for 24 h then exposed to red light for 15 min or 48 h. It is clear that dormancy of seeds with thicker coats is poorly broken by 15 min light Based on data in Karssen, 1970 [144]

received by the embryo [144]. Seeds with thick, dark coats are less responsive to light than their thin, light-coated fellows (Table 2.10). To secure germination, irradiation for longer time periods is needed; moreover, the efficacy of certain red/farred ratios appears to be modified. This example illustrates that while the possible filtering action of the seed coat does not actually impose dormancy upon the embryo it is a factor which can interfere with the breaking of dormancy. It is likely that this effect could operate in other species and the phenomenon seems worthy of further study. 2.4.6. Mechanical Restraint

There are a large number of instances in which none of the above effects can adequately explain the action of the coat in imposing and maintaining dormancy. These are cases where the isolated embryo germinates, where germination is provoked by various operations on the coat (such as an incision, puncture or removal of a portion), and yet where there seems to be no involvement of gaseous exchange and water uptake effects, or inhibitors in either coat or embryo. It is unnecessary to discuss a range of such examples in detail but the reader can refer to a few recent reports which illustrate the point [29,81,206]. In many cases of these kinds it has been concluded, although usually tentatively, that the coat must act by exerting a mechanical restraint. The coats of many seeds and indehiscent fruits (e.g. nuts) are hard, tough tissues, clearly offering some resistance to the growth of the embryo. Obviously, if the embryo cannot generate enough force to surmount the mechanical constraint it must remain ungerminated. Surprisingly few studies have been made of the mechanical resistance of seed coats or of the force which embryos must create to overcome this. Early investigations are those of Crocker and his colleagues on a range of nuts and hard-coated seeds [61, 63]. Ikuma and Thimann [131] concluded that the endosperm of lettuce imposes dormancy by acting as a mechanical barrier; it is indeed a tissue composed of thick-

92

Dormancy

Fig. 2.10. Toughness of lettuce endosperms. Seeds of Lactuca sativa cv. New York were washed with 5.25% sodium hypochlorite and placed on filter paper wetted with sodium dichloro-isocyanurate (0.25%-0.5%). They were held in 16 h photoperiods at 21 °118°C for 3 days, after which time embryo buckling was apparent. Photograph by courtesy of A. Pavlista

walled cells [l37]. They claimed that the endosperm is normally too resistant for the embryo to penetrate it but that in dormancy breakage cellulolytic and pectolytic enzymes are induced which weaken it, thus permitting the radicle to burst through. While there are certain objections to some of the experiments on which the conclusions are based (the most serious being that the endosperm cell walls are richer in mannans than in cellulose [116 a]), it nevertheless does seem tha t the lettuce endosperm is indeed a tough, resistant coat which an embryo could have difficulty in rupturing. Figure 2.10 shows seeds oflettuce from which the pericarp and testa have been removed. The cultivar is New York which under the conditions used in these experiments is not dormant. When treated with compounds which release chlorine (hypochlorite or dichloro-isocyanurate) the embryo, although still able to germinate, is incapable of breaking the endosperm. The experimenters' interpretation of this result is that the chlorine interferes with the chemical weakening of the endosperm (normally brought about, presumably, by the action of the radicle) but does not prevent radicle growth itself [203]. Whether this suggestion is correct remains to be confirmed, but the experiment impressively demonstrates that the endosperm can indeed offer considerable mechanical resistance, which u·nder certain

Coat-imposed Dormancy

93

circumstances even a growing embryo cannot always overcome. During germination of lettuce, changes in the resistance of the endosperm do occur, but these do not appear to be consistently related to the emergence of the radicle. The force required to puncture the endosperm falls from 0.6 newton for dark-imbibed seeds to 0.42 newton for seeds exposed to light or gibberellin, yet the radicles of seeds treated with the growth regulator are much slower to emerge [235]. It has already been mentioned that the mechanical restriction by the coat is of significance in dormancy only in relation to the force or thrust which the embryo can generate. Hence, measurement of the mechanical strength of a seed coat, taken on its own, does not inform us fully about the relationships in the seed. With this qualification in mind we should note some interesting work on Syringa which suggests that dormancy is associated with mechanical resistance of the coat [138]. Seeds of certain species of this genus (e.g. S. reflexa) are dormant at temperatures below 18° C; dormancy is imposed by the endosperm. Other species (e.g. S.josikaea) are non-dormant. Determinations of the force needed to pierce isolated, imbibed endosperms with a fine needle show that the endosperm from the dormant species is significantly more resistant than that of the non-dormant S.josikaea. Although the thrust generated by the embryos was not measured, it was found that the growth potential of embryos from non-dormant seeds was higher, as judged by the ability to grow in osmotica. Both the thrust of the radicle and the resistance of the testa have been investigated in Xanthium pennsylvanicum by Esashi and Leopold [85]. We should recall that this species produces dimorphic seeds, a smaller, upper seed which retains its dormancy and a larger, lower one which, although dormant when freshly harvested, loses its dormancy on after-ripening. The weights needed to force a sharply pointed perforator through the testa, and to equalize the thrust of the radicle of an isolated embryo were determined for both types of seed. The results are shown in Fig. 2.11. They indicate that the testa of the smaller, dormant seed is, in fact, less resistant than the testa of the non-dormant seed, but that the embryo from the dormant seed cannot generate sufficient thrust to rupture the coat. The conclusion which may be drawn from these findings is that dormancy cannot be attributed only to the mechanical constraint imposed by the testa but it still may play an important role. We have, however, already referred to Xanthium in connection with oxygen, inhibitor and leaching effects. We will shortly go on to consider all these aspects together as one of the case histories which illustrate the complexities of coat effects in dormancy. Finally, further experiments which offer information relevant to the matter of resistance and embryonic thrust should be noted. It has been observed in several species, including lettuce, that embryos isolated from dormant seeds can be prevented from germinating by the application of osmotic restraints [192]. Thus, excised embryos remain dormant when held in solutions above a certain osmolarity. In lettuce, the germination of such embryos can be secured by irradiation with light Gust as in the intact dormant seed). The growth potential of the radicle/hypocotyl axis is thus increased. It has been inferred that this experimental situation is analogous to the condition in the intact seed - that of a constraint external to the embryo which can be overcome by increasing the growth potential or thrust. This will be discussed further in Chapter 4, Section 4.4.5.

94

Dormancy A

B

Rubber stop Embryo

Manometer

Mercury

Water Piston Red ink -

Simulated axis

__

Mercury

Fig. 2.11A, B. Embryonic thrust and testa resistance of the upper (dormant) and lower (non-dormant) seed of Xanthium pennsylvanicum. (A) Imbibed embryos of X. pennsylvanicum were positioned as shown. The thrust was determined by reference to a previous calibration of the manometer against columns of mercury of known weight. (B) For determination of testa resistance coats were used from seeds 20 h after moistening. After Esashi and Leopold, 1968 [85]

A

B

Total embryonic thrust* Testa resistance* Dormant 41 56 Non-dormant 84 67 * The figures are grams weight required to pierce the testa and to equalize the thrust generated by the embryo. Note that the total embryonic thrust is comprised of a passive (imbibitional) expansion and an active (growth) expansion Seed

2.5. Two Case Histories These examples have been chosen as cases that bring together some of the possible mechanisms of coat-imposed dormancy which have been discussed above.

2.5.1. Sinapis arvensis (Charlock)

These seeds, which are roughly spherical with a diameter of about 2 mm, retain their dormancy for many years. The coat, which imposes dormancy, consists of a mucilaginous epidermis, a lignified, pigmented palisade layer and the one-cellthick living remnant of the endosperm (the aleurone layer) [77]. Complete removal of the coat terminates dormancy but dormancy persists (in air) when the coat is only punctured or slit [77, 80]. This suggests that the coat's mechanical resistance is not normally the cause of dormancy for one would expect this to be eliminated by such surgical treatments. Neither is water uptake by the embryo a contributory factor since this proceeds in a completely unimpeded manner in intact seeds, and

Two Case Histories

95

Table 2.11. Oxygen relationships and germination in Sinapis arvensis

Isolated embryos

Intact seeds

External oxygen conc. (atm.)

Oxygen conc. (atm.) at parts of embryo: Growth (% increase 0.24 mm deepa in length)b Surface

1.000 0.200 0.100 0.050 0.035 0.Q18 0.010 1.000 0.200

1.000 0.200 0.100 0.050

0.Q35 0.Q18 0.010 0.038 c 0.020 c

0.908 0.108 0.059 0.029 0.021 0.011 0.006 0.023 0.012

314 195 84 38 0 0

a Position estimated to be at meristems b % increases in length over 24 hat 25° C, i.e. growth following completion of germination. Calculated from data in Edwards [8] assuming a starting length of 1.5 mm [80] c Calculated from oxygen uptake data Adapted from Edwards, 1973 [8]

the embryo becomes fully imbibed (phase I of water uptake, Chap. 4, Vol. I) within approximately 2 h. Interference with oxygen uptake does seem to be of importance, however, because placing punctured seeds in an atmosphere of oxygen overcomes dormancy. Pretreatment of the seeds for short periods of time (5-20 min) with concentrated sulphuric acid also stimulates subsequent germination, an effect which can be ascribed to damage caused by the chemical to the seed coat. Edwards has carried out a series of investigations [8, 77-80] which answer the following questions: (a) What oxygen concentration, at its surface and at the growing regions, does the isolated embryo require to support its germination and growth? (b) What is the oxygen concentration at the surface of an embryo in the intact seed? (c) Why, if oxygen effects are involved, does the embryo in the intact seed fail to germinate? Oxygen uptake by naked embryos during the fIrst 4 h after isolation is linear at external oxygen concentrations ranging from 0 to 0.1 atm and then flattens off up to 1.0 atm oxygen. Using the linear relationship, Edwards estimated the oxygen concentration which must obtain at the embryo's surface in an intact seed to allow the measured level of uptake by the whole seed to occur (this assumes, of course, that the embryo is the only oxygen consumer in the seed). Further, assuming that the seed and the embryo are spherical, calculations were made of the internaloxygen concentrations at various positions in the embryo. The germination and growth of isolated embryos at various oxygen concentrations were also determined (Table 2.11). The results permit two important conclusions to be made. Firstly, low levels of oxygen at the surface of isolated embryos are sufficient to support their germination and growth (e.g. 0.01-0.05 atm). Secondly, the estimated concentrations of oxygen at the embryo's surface when the intact seed is held in air or oxygen are higher than those needed for appreciable growth of excised embryos. It would seem therefore that the low internal oxygen concentration caused by the

96

Dormancy

seed coat has an inhibitory effect which cannot be explained on the basis of the unsatisfied respiratory requirements of the embryo. Sinapis seeds produce chemically unidentified inhibitors which are active upon Sinapis embryos and wheat coleoptiles [79]. Those found by Edwards occur in the embryo, from which they can be collected by extraction or diffusion. Interestingly, significantly greater amounts of inhibitor are produced by isolated embryos held at low oxygen concentrations than by those in air. Much of the inhibitor diffuses from isolated embryos but relatively little passes from intact seeds [80]. These findings therefore suggest an explanation for the role of the seed coat of Sinapis in the imposition of dormancy. The low oxygen concentration at the surface of the embryo, resulting from the relatively low permeability of the testa, is conducive to the production of inhibitor. This is the primary action of the testa but a secondary effect is that it may prevent the escape of the inhibitor from the inside of the seed. This is an interesting hypothesis which still awaits support from other species.

2.5.2. Xanthium pennsylvanicum (Cocklebur) This genus has long been considered to provide a classic example of dormancy imposed by a coat which has insufficient permeability to oxygen. Early work was done on several species, but most investigators have used x.pennsylvanicum. Dormancy in Xanthium was investigated by Crocker and his colleagues over a period of about 30 years. In 1906, Crocker [60] showed that the smaller seed produced by the upper floret possesses a longer, deeper dormancy period than larger seed of the lower floret (upper and lower seed respectively - see Fig. 2.3); when freshly harvested, both seeds exhibit dormancy, however [252]. The difference in ability to germinate was found by Crocker to be eliminated by the removal of the coats. It seemed clear, moreover, that an oxygen uptake phenomenon was involved when he showed that dormancy could also be relieved by exposing the intact seed to an atmosphere of oxygen. (This work was matched at the same time by Correns [58] studying another Composite, Dimorphotheca, which also has dimorphic seeds in the ray and disc florets). Isolation stimulates oxygen uptake by embryos of both upper and lower seed but more so by the former. Later, it was demonstrated that at 21 0 C the embryo excised from the after-ripened, lower (now non-dormant) seed germinates under approximately 0.006 atm oxygen, whereas that from the dormant, upper seed requires approximately 0.011 atm. Although the oxygen requirements of the isolated embryos from the two seeds differed by a factor of nearly 2, the requirements of the intact seeds differed by a factor of nearly 10 [225, 240]. It became accepted, therefore, that the relative impermeability to oxygen of the testa of the upper seed causes its dormancy. This conclusion is not entirely warranted, however, and it is a curious one to have been drawn in view of the fact that Crocker had already found that the permeability of the two testas seemed to differ very little. As was later pointed out by Porter and Wareing [209], it is important to determine the permeability of the testa and to relate the rate of oxygen entry into the seed with the embryo's requirement for oxygen, i.e. its consumption. There are, in fact, no differences between upper and lower seeds in the permeability of the imbibed testas to oxygen, either just after harvesting (when both upper and lower

Two Case Histories

97

20 A

c .2

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Fig. 2.12A, B. Coat-permeability to oxygen and oxygen consumption by X anthium pennsylvanicum seeds. (A) Freshly harvested upper and lower seed which are dormant. (B) After-ripened seed: the lower one will germinate fully, but the upper one remains dormant. Oxygen consumption by upper seed (_), lower seed (.). Diffusion through testa to lower seed (-), upper seed (---). In (B) time of testa splitting (start of radicle growth) is marked by the arrow. After Porter and Wareing, 1974 [209]

seeds are dormant) or some months later (when only the upper ones are dormant). In all cases, diffusion rates of oxygen of approximately 17 III em - 2 h - 1 are found (Fig. 2.12A and B). Also, up to the time of radicle protrusion (of the non-dormant lower seed) both non-dormant and dormant seeds exhibit very similar oxygen consumptions at a rate about one third of that which the permeability of the testa would actually allow (Fig. 2.12B). Thus, not only are the permeabilities of the two testas (i.e. of dormant and non-dormant seeds) virtually identical, but embryos inside both consume oxygen at below the maximum permissible capacities of the testas. Now the observations of the earlier workers on Xanthium were quite correct; intact, upper seeds do need much higher oxygen levels to elicit germination. But since it cannot be because the testa of the upper seed is a barrier to gaseous diffusion, it must instead be because the embryo itself requires a high oxygen concentration in order to overcome its dormancy. This conclusion seems to contradict the aforementioned finding [225] that isolated embryos of the two kinds of seed differ in their oxygen requirement only by a factor of about 2. According to this, and if

98

Dormancy 16

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Fig. 2. 13A-E. Influence of the coat on leaching of inhibitors from Xanthium pennsylvanicum embryos. Upper seeds used were freshly harvested and deeply dormant. Histograms represent assays (elongation of wheat coleoptile sections, starting length 10 mm) of chromatograms of aqueous extracts or diffusates. Dashed line shows the coleoptile growth in the water control. Chromatograms were run in isopropanol/ammonia developing solvent. (A) Extract of intact dry seed; (B) Diffusate from 5 g intact seeds in 150 cm 3 water for 48 h; (C) Diffusate from 5 g excised embryos in 150cm 3 water for 48 h; (D) Extract of embryos from washed seeds (treated as in B); (E) Extract of washed excised embryos (treated as in C). Note the inhibitory activities (i.e. coleoptile growth less than water control) on chromatograms A, C, D and E at Rfs 0.1-0.3 and 0.4-0.5. After Wareing and Foda, 1957 [252]

the testas have the same permeability, it does not seem possible that the oxygen requirement of the upper seed should increase about fivefold just by being retained in the intact seed. How can these discrepancies be resolved? Two water-soluble inhibitors occur in the embryos of Xanthium seeds (Fig. 2.13A at Rfs 0.1- 0.3 and 0.4-0.5). When intact, upper seeds are placed on a wet substratum the inhibitors do not diffuse out (Fig. 2.13B); however, diffusion does

Two Case Histories

99

Fig. 2.14. Effect of high oxygen 16 tension on extractable inhibitor of Xanthium pennsylvanicum seeds. Hydrated upper seeds were 14 held in 100% oxygen or air for 30h after which they were extracted. Details of chromato12 graphy, etc. as in Fig. 2.13. Note E .§ tile lower levels of inhibitor .J::. (Rfs 0.1-0.3, 0.4-0.5) after treat- rn 10 ment with oxygen. After Wareing c ｾ＠ and Foda, 1957 [252] ｾ＠

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occur from naked embryos, and the inhibitors can be collected from the external liquid (Fig. 2. 13 C). The testa is evidently a barrier to the exit of the inhibitor. Indeed, subjection of intact seeds to a washing treatment (leaching) does not provoke any reduction in the extractable inhibitor content of the embryos (Fig. 2.13D), whereas the extractable inhibitor level does drop in isolated embryos given the same treatment (Fig. 2.13E). Furthermore, it seems likely that the loss of inhibitor from dormant embryos held on a wet substratum is what in fact permits their germination, for if embryos are allowed fully to imbibe in the intact seed and are then excised and transferred to a humid atmosphere in air (non-leaching conditions), they do not germinate. It would appear therefore that the isolation per se does not relieve the dormancy imposed upon the embryo, but instead it is the isolation followed by leaching out of the inhibitors which is responsible. (It is important to note that in the earliest experiments with Xanthium the isolated embryos were always held on a wet medium). But why does exposure of intact, dormant seed to a high oxygen tension also relieve dormancy? The answer to this is that elevated oxygen concentrations seem to cause a reduction in the extractable inhibitor content of the embryo, presumably by enzymic oxidative reactions (Fig. 2.14). Interestingly, those embryos which, though fully imbibed, retain their dormancy when held under non-leaching conditions (see above) are stimulated to germinate by treatment with pure oxygen. In summary, then, this work suggests that in Xanthium the action of the coat in dormancy is primarily a barrier to the loss of inhibitor from the embryo. Transfer of the naked embryo to a wet substratum encourages germination by permitting the

100

Dormancy

escape of inhibitor, not by allowing easier access to oxygen in the air. The fact that treatment with pure oxygen stimulates intact seeds to germinate is, in a sense, a diversion, which led to the apparently fallacious concept that the basis of coat-imposed dormancy rested in the impermeability of the testa to oxygen. Although the inhibitor hypothesis as outlined above provides an attractive interpretation of coat-imposed dormancy in Xanthium, it nevertheless leaves several observations unexplained (see, e.g. [90]). Thornton [240] noted that exposure of intact, upper (dormant) Xanthium seed to high concentrations of oxygen in fact does not promote normal germination, but atypical germination, in which the cotyledons grow, split the testa, and emerge first. Only later does radicle growth commence. (This has been confirmed more recently [86]). A simple explanation of this might be that the oxidative destruction ofthe inhibitor takes place more readily in the cotyledons, but this has not been demonstrated. Another observation which the inhibitor hypothesis does not readily explain is that intact upper seeds can be induced to germinate even at 10% oxygen as long as high (i.e. 10%-40%) concentrations of carbon dioxide are also supplied [240]. Further, it is not clear why the lower seed requires less oxygen, since it appears to contain as much inhibitor [252]. Esashi and his colleagues prefer to explain the action of oxygen on the basis of its interacting effects with ethylene upon the cotyledons [91] (see Chap.4). A mechanical interpretation of the role of the coat has been offered by Esashi and Leopold [85] who showed that the thrust developed by the embryo of the upper seed is inadequate to rupture the testa (see Fig. 2.11). Dormancy is therefore seen as the inability of the embryo to develop sufficient thrust, a capability whose development is thought to involve the production and action of ethylene [87, 146] (see Chap.4). This concept is not necessarily incompatible with the inhibitor hypothesis since it could be argued that the inhibitor is also involved in a system of interacting growth-regulating chemicals. How far the hypotheses can be reconciled, however, remains to be seen.

2.6. Coat-imposed Dormancy - a Retrospective View We have seen the evidence for the possible actions of the seed coat in dormancy. In relatively few cases can we attribute dormancy to the operation of just one of these effects, i.e. the impedance of water entry which prevents germination in hardcoated seeds. In all other cases it is difficult to isolate a single mechanism by which the coat imposes and maintains dormancy. Even in those seeds where interference with oxygen uptake is evidently involved other factors must also operate, such as in Sinapis and Betula. It is possible that in most cases the enclosing structures have more than one effect, perhaps for example, to present a mechanical resistance as well as to create conditions which render it difficult for the embryo to overcome this constraint. Of course, the combination of effects is likely to vary from species to species. It seems clear that many more detailed studies are needed, of the kind carried out by several workers on Xanthium, in which various parameters are thoroughly investigated (e.g. [85-91]). Even here, however, a completely satisfactory explanation is still not available.

The Onset of Dormancy

101

2.7. Relationships Between Coat-imposed and Embryo Dormancy Seeds of some species have components of both coat-imposed dormancy and embryo dormancy. This is found in several woody species. For example, Crataegus mollis embryos need different periods of chilling to break their dormancy depending on the amount of coat that is present. The dormancy of the naked embryo can be overcome by chilling for 3-4 weeks. On the other hand, with the testa left intact, 3-4 months at low temperature are required. Finally, if the pericarp is also present, the seed may require up to 12 months chilling to terminate its dormancy [68]. Clearly, then, this species possesses a basic embryo dormancy, superimposed upon which is dormancy contributed by the enclosing structures. Similar cases are discussed in Chapter 3 (see Fig. 3.32) and are quoted by Crocker and Barton [7]. These are cases where degrees of embryo dormancy and coat-imposed dormancy occur simultaneously. Several species, however, exhibit changing patterns of dormancy, where one type may succeed another. Freshly harvested, upper seeds of Xanthium pennsyivanicum, for example, possess embryo dormancy. After-ripening (Chap. 3) removes this embryo dormancy but coat-imposed dormancy remains. Several grasses show a similar phenomenon, although in these cases we are dealing with dormancy of the caryopsis and dormancy ofthe intact dispersal unit (i.e. with glumes, etc. present). Both Aristida contorta and Bouteloua curtipendula are fully dormant for 3-4 months after harvesting, i.e. the intact dispersal units fail to germinate, and removal of the floral parts (the hull) has no promotive effect on germination. After several months have p3;ssed (after-ripening), the intact dispersal units are still dormant but when the floral parts are removed germination occurs [177, 190]. There are therefore, in time, two components of seed dormancy in these species. The first, short-term dormancy, is a condition within the caryopsis itself (possibly even within the embryo) whereas the second, long-term dormancy, is imposed by the hull. Towards the end of seed maturation, embryos of Acer pseudoplatanus appear to be dormant, for when removed from the enclosing structures they do not germinate. Embryos excised from older, fully matured seed, on the other hand, are not dormant, but the intact dispersal units are - hence dormancy is now imposed by the coat. Thus, there seems to be a succession of the two types of dormancy [239]. A succession also occurs in Corylus, but in the reverse order. At first, the seeds possess coat-imposed dormancy; removal of the testa allows the embryo to germinate. The dormancy characteristics of the seeds change, however, since after a period of storage testa removal is no longer beneficial and the embryos themselves have become dormant [136]. As pointed out earlier (Sect. 2.3), this phenomenon might be analogous to secondary dormancy, induced in this case by the testa.

2.S. The Onset of Dormancy How do seeds become dormant and what controlling factors operate in this process? Although much is known about the maintenance of dormancy and about some of the mechanisms operating in its termination, we have a very incomplete

102

Dormancy

picture of the inception of dormancy. In this section we will, nevertheless, attempt to give some answers to these questions. Firstly, we will consider the matter of timing- when dormancy is initiated. Following this, we will look into some of the controlling factors involved in the onset of dormancy, including secondary dormancy. Finally, we will discuss some details concerning the development of coat-hardness.

2.8.1. Timing

As we have seen in the previous section, seeds of certain species (e.g. Xanthium, Acer pseudoplatanus) show an early embryo dormancy, followed later by a coatimposed dormancy. In the case of Acer, embryo dormancy appears to develop before the end of seed maturation since embryos isolated from maturing seeds fail to germinate. The embryos of an inbred, deeply-dormant line (Montana) of Avena fatua also become dormant at an early stage in their development. When isolated from the caryopsis 10 days after fertilization, and placed in a liquid medium, the embryo is already dormant, but it can be stimulated to germinate by application of gibberellic acid [22]. At this lO-day stage the embryo is still only about half of its mature size. It is interesting that although obviously still capable of further growth (largely by cell division) it is nevertheless unable to make that great increase in axial length (i.e. radicle and coleoptile elongation by cell extension) which is characteristic of seedling emergence from the grain. Nothing is known about the control mechanisms operating here, i.e. why embryogenesis can continue while the next phase of embryo growth cannot occur, but presumably we are confronted here with a mechanism controlling alternative patterns of development. The case of the deeply dormant A.fatua is unusual, however, as young embryos frequently show precocious germination such that even when isolated at a very early stage they shift from their normal embryogenetic development into germination. In these cases, the controls which normally direct embryogenesis must be imposed by the intact, developing seed or by the mother plant. It has long been known that intact, immature grains of cereals do not germinate when transferred from the mother plant directly to a wet substratum (see [12] and [152] for accounts of early findings). This applies for those species which later do not normally show significant dormancy (e.g. Zea mays) as well as for those which do (e.g. barley). At some stage during development the grains become able to germinate. This ability is acquired in wheat at a time which coincides with the beginning of the fall in fresh weight as the grains start to dry (Fig. 2.15). Indeed, it seems likely that partial dehydration is actually responsible since non-germinable grains of wheat, barley and maize at the milky stage can be rendered germinable by drying them down for a day or so [12, 155, 184]. How far this inability of un dried grains to germinate represents true dormancy is at present unclear. In wheat, however, it appears to have many of the characteristics of coat-imposed dormancy; here, fully swollen grains, taken directly from the ear, germinate if the pericarp is removed or pricked, or if placed under high oxygen tensions [184, 212, 256, 257]. It is important to note that wheat and barley frequently show dormancy, perhaps lasting for some weeks or months, even after seed maturation. This dormancy, encountered in the mature, dry grain appears to develop later in the maturation pro-

103

The Onset of Dormancy

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Fig. 2.15. Germinability of maturing wheat grains (cv. W42). Grains were taken from the ears of field-grown wheat from 4 days after anthesis until near harvest-ripeness and their germinability tested (.-.). From 18 days after anthesis grains were collected and dried at room temperature down to 10%-15% water content (this took up to 7 days) and then tested for gerrninability (0-0). Germination tests were done on both types of grain at 20 °C for 7 days. The water content during natural drying is also shown ＨｾＭＩＮ＠ After Mitchell et al. 1980 [184)

cess. Figure 2.15 shows the germinability of wheat (cv. W42) followed from 4 days after anthesis. No germination is shown up to 26 days, then at 28 days there is a sharp increase to 100% germination, coinciding with the natural decrease in water content. Germination is elicited at earlier stages by enforced dehydration. But this increase in germinability prompted by drying is short-lived, for there appears a second phase of non-germinability (possibly true dormancy) which begins when the grain is losing moisture, and which reaches a trough at 38 days when the grain has almost finished drying. The entry into this phase is accelerated some 8-10 days by enforced drying to about 15% moisture content. Thereafter, germinability is regained (i.e. dormancy is lost) probably as the grain rapidly after-ripens. Thus there are two phases of low germinability, the first because undried grains are unable to germinate, the second, later phase occurring when dormancy sets in as drying proceeds. This dormancy quickly disappears, which shows its very ephemeral character in this particular cultivar. The onset of coat-imposed dormancy appears to be a later event in the maturation process of other species too. Seeds of two dicots, Portulaca oleracea (common purslane) and Sida spinosa (prickly sida) can germinate even when immature, beginning in the former species about 5 days after anthesis [82], and in the second, 12-16 days after anthesis. High germinability is retained in both species until natural drying commences, when seeds become dormant. The loss of moisture seems to be a factor responsible for the initiation of the coat-imposed dormancy since artificial drying, at least in Sida, converts an immature, germinable seed into a dormant one. This contrasts markedly with the situation in cereals where drying of immature grain has beneficial effects upon germination. The explanation for this difference is unknown. It seems clear, however, that in the cereals (wheat, for ex-

104

Dormancy

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1l. 28 1.2 56 70 81. 98 112 126 Time after the start of Imbibition (days)

11.0

Fig. 2.18. Effect of temperature during grain development on subsequent dormancy in wild oats. Plants of an inbred line of Avena Jatua were held, after grain set, at 28 °e and 20 °e. Grains were harvested just after the dough stage, dried, and assayed for germination at 4 0c. Note the long-lasting dormancy of grain which developed at 20 °C. After Sawhney and Naylor, 1979 [222] and there is some indication that this tissue is, in fact, mechanically tougher when maturation proceeds under the higher temperatures. The thermoperiod (i.e. the day and night temperature) also influences dormancy. For example, seeds of Anagallis arvenis have a very low dormancy when produced on a regime of300/25° C (day/night), fairly high dormancy at 25°/20° C, and extreme dormancy at 20°/ 15° C [110]. We can see again, incidentally, how low temperatures favour the development of dormancy. The thermo periodic regime determines dormancy in Aegi/ops kotschyi, in which more deeply dormant seeds are produced by mother plants exposed to cycles of 17°/ 12° C than by those at 27°/22° C [266]. In Aegi/ops kotschyi, the photoperiodic regime also influences the development of seed dormancy, long days being more inductive than short days. The role of the photoperiod in the initiation of dormancy in several species is now, in fact, well documented. Perhaps the first study which clearly indicated this was by Lona on Chenopodium amaranticolor [176], who showed that plants held under 16-18 h photoperiods produce thick-coated, small, dormant seeds, whereas those in 6-8 h daily photoperiods give larger, thinner-coated, less dormant seeds. Chenopodium album [143] and C.polyspermum [135] (Table 2.12) behave similarly. In both these species, the photoperiod at the time immediately following flowering is important (compare c and d in Table 2.12 for instance). Long days at this time are considered, for C. album, to be responsible for the induction of a short-term dormancy (Fig. 2.19) but in this instance it is not connected with coat thickness. A longer-lasting dormancy, probably associated with the thicker coats, apparently arises in proportion to the total number of long days received. This effect of an increasing number oflong days, at least in C. polyspermum (note increased thickness of coats from a- d in Table 2.12), is a truly photoperiodic one and is not caused by a greater total amount of photosynthesis [210]. Coat thickness and colour are also determined by the photoperiodic experience during seed development of Ononis sicula [94, Ill, 113, 114]. After long days, the seeds of this species are relatively large, have thick,

109

The Onset of Dormancy

Table 2.12. Capacity for germination, weight and thickness of the coat of seeds of Chenopodium polyspermum, as a result of four different photoperiodic treatments undergone by mother plants from the time of flowering (F) until harvest (H) Treatments

a) F 'IfJ!lfJJ!!!!& H b) v..J c) ｾ＠ d) ｾ＠

% Germinated

To

T60

67.2±6.4 a 75.4±2.5 67.2±5.8 36.2±3.0

65.4±5.0 62.6±3.6 68.2±6.6 41.0±5.4

Weight of seeds (mg)

Thickness of seed coat ＨｾＩ＠

58.7±2.1 60.7± 1.1 55.0±0.4 44.9±0.4

23±0.5 28±0.7 34± 1.4 40±1.3

081624 Days To, tests done on the day of harvest ; T 60, tests done 60 days after harvest; a± standard error of the mean; Wl!llllJ/!lJ SD; ｾ＠ LD After Pourrat and Jacques, 1975 [210]

100

ＮＭＺｾ

/

•/

80

ｯﾰ ＭＺ］ｾ＠

./ /

/

,/

,.. ,..

,/

,/

0/

"2

0

0

c 60

E Q;

II> CII

['0

c

to

-0

Ｎ ｾ＠

20

.2 o c

- Lh

·E

- Ao

Q;

Sde Ah

. Ol

Q;

c:

w

a

I

11.0

If)

•I

50

100

6 >. Ol

60 20

:c .£ 710 nm) at 2.9 Wm - 2 for 4 min were used. Seeds returned to darkness for 36 h before counting. Temperature throughout: 24±OS C By the authors

far-red (a few minutes of each) before transference to darkness. The germination, counted later, revealed that breaking of dormancy occurred only if the last irradiation were red light and, no matter how many red doses had been given, dormancy was retained (i.e. the promotive effect of red was nullified) if the last irradiation in the sequence were far-red, even when as many as 99 or 100 irradiations were giv-

The Phytochrome System Fig. 3.3 Reversal by elevated temperature of red light action in the breaking of dormancy of lettuce (cv. Grand Rapids). Seeds were given a saturating red irradiation at 20° C then kept in darkness at 30° C for different times before transfer, still in darkness, to 20° C. The number of germinated seeds was later determined. After Borthwick et a!., 1954 [43]

100

131

ｏ ｾ｜＠

0

75 "U 0.05 >0.02 >0.45 (50%) 0.16 (50%) 0.1--0.15 0.001 (50%)

[109] [55] [55] [70] [58,238]

[69]

[215]

[169] [107] [130] [195]

The tP values are those effective in promoting germination of all seeds in the population. Where 50% follows the stated tP value, the latter promotes germination of 50% of the seeds a P tot = concentration ofP, plus P f , b Dark germinators (see Chap. 6)

from dormancy when cjJ reaches 0.3 (Fig. 3.6). Of course, in both species irradiation with sunlight would be adequate. In sharp contrast are those seeds whose dormancy is terminated by PfrfPtot ratios as low as 0.001-0.02 (Table 3.4) (e.g. Wittrockia superba, Bilbergia pyramidalis and Amaranthus retroflexus [195]) and thus even farred light (e.g. 720-730 nm) can promote germination. In other species, such as Sinapis arvensis [69] and Eragrostis curvula [215], most seeds in a population require relatively high cjJ values but a few are satisfied by photoequilibria as low as 0.02 - these are the ones which germinate in response to far-red light. Because many seeds of E. curvula are sensitive to this low Prr ratio (Table 3.4) it is impossible to obtain complete photoreversion with far-red at, say, 730 nm which would leave 2%-3% Prr. This is why seeds of this species [215], like those of Picea glehnii and Artemisia monosperma [120], still show a promotion of germination over the dark control when subjected to red/far-red reversal operations (Fig. 3.7). It should now be clear that through the photostationary equilibrium of phytochrome the seed is able to detect the light quality of its environment, in particular the red/far-red ratio. Here lies one important feature of phytochrome for, by the operation of this pigment, the germination of the seed can be restricted to environments having a particular light quality. For example, seeds with a requirement for a relatively high cjJ value will not germinate in light filtered through green leaves which is rich in far-red wavelengths. Germination is therefore limited to more open situations which are more favourable for seedling establishment. The ecological aspects of control of germination by phytochrome will be discussed in Chapter 6. Why is the effectiveness ofPrr dependent upon its photostationary equilibrium? One possibility is that there is some kind of competition between the active Prr and

The Phytochrome System

... . . /

100 "0

80

/' )

0

c 60

...

C!) 0ｾ＠

1.0

20 0

100

_-.l----0

QJ

Ｎｾ＠

II

/ •

-.-r--,0 / e

Darkness Lactuca

"6 ----- Oarkness Chenapodiurr o 0.2 0.1. 0.6 0.8

?fl. 20 ｏｾＭ＠

equilibrium ( value of 0.0012. Note that the effect of this increases with the time before irradiation. After Taylorson and Hendricks, 1971 [195]

3.2.5. Chemistry of Phytochrome The similarity between the absorption spectra ofP r and allo-phycocyanin led Hendricks and co-workers [81 a] to postulate that phytochrome, like the phycocyanin, is a bilitriene. Analysis confirmed this prediction - the pigment is indeed a bilitriene (i.e. an open-chain tetrapyrrole) chromophore linked to a protein moiety. The proposed structure of the chromophore is shown in Figure 3.9 (see [Ill] for further details). The molecular weight of phytochrome is 120,000 but extracts of various plant tissues might also contain a species of 60,000 produced by proteolytic degradation, and sometimes very large molecules (e.g. M.W. 240-800 x 10 3 ) formed by aggregation. The 120,000 M .W. phytochrome probably contains just one chromophore which would explain why a 60,000 M.W. non-photoreversible protein is produced by degradation.

(

------,-1Ｍｲ

Protein

) ｾ＠

S

o

o

Fig. 3.9. Proposed structure of the phytochrome chromophore (P r form). After Kendrick and Spruit, 1977 [111]

The Phytochrome System

139

There is uncertainty about the molecular changes taking place on photoconversion from P r to Pfr. Recent evidence stands against conformational changes in the chromophore or substantial alteration in the conformation of the protein [111]. One change that might occur on photoconversion from P r to Pfr is an aggregation into very large (ca. 800,000 M.W.) phytochrome [76].

3.2.6. The Pathway of Phytochrome Photoconversion

The photoconversions P r ¢ Pfr occur in more than one step in each direction. An awareness of these transformations helps us to appreciate more fully the role of phytochrome in seed physiology, particularly in relation to changes which occur during dehydration and hydration of developing and mature seeds. The basic phenomena will therefore be summarized; a detailed consideration can be found in the review by Kendrick and Spruit [111]. Intermediates in the transformations P r ¢ Pfr have been detected spectrophotometrically in plant tissues and pigment solutions treated in four ways, viz: a) In vitro flash photolysis at 273 K. Measurements of absorbance are rapidly made after a short flash of actinic light at a high fluence rate. b) In vitro and in vivo absorbance measurements at the temperature of liquid nitrogen, 77 K, when complete photoconversion in either direction does not occur but instead phytochrome is trapped as stable intermediates. c) Dehydrated tissue. Certain steps in the conversions take place only in hydrated tissue, but others proceed even in dried tissue. As in the use of very low temperatures some intermediates may therefore be trapped in cells which have a water content below a certain value. d) Kinetics of absorption changes. Upon irradiation with mixed wavelengths both P r and Pfr become excited and are rapidly cycled. Under these conditions intermediates between P r and Pfr reach high enough concentrations to be detected spectrophotometrically. Several intermediates have been detected by the above methods. Of these, photoproducts are designated by the prefix lumi, and products of dark reactions (relaxations) are given the prefix meta, in line with the terminology used for the visual pigment rhodopsin. Further, the products ofP r are identified by R and those ofPfr conversion by F; hence, lumi-R, meta-R, lumi-F and meta-F have been described. The transformations are not understood in detail but an outline is given in Figure 3.10. The first stable product of the photoconversion of Pr is lumi-R (step 1) which differential spectrometry reveals by a maximum at 698 nm, although its peak absorption is likely to be at wavelengths just a few nanometres shorter than this. Photo reversion oflumi-R to P r occurs at 77 K (step 2) but at a higher temperature (203 K), reversion in darkness can also take place (step 3). At physiological temperatures, however, neither reaction occurs but instead lumi-R quickly converts in darkness to the next intermediate, meta-Ra (step 4). This step is somewhat impeded by dehydration and under this condition, therefore, reversion of lumi-R to P r takes place even at physiological temperatures (step 2 or 3). In hydrated tissue lumi-R is converted to Pfr by three dark relaxation steps through the intermediates

140

The Release from Dormancy

(6981 lumi-R

ＭｾＮ＠

(7101 meto-Ro

'H0

P'r

meto-Fa

ｾ

"'::J

1630-6901 __

8

9

ｾ＠

Ｗ ｮ＠

lumi-F

17201

Fig. 3.10. Transformations of phytochrome. Thin arrows are phototransformations and thick arrows are dark (thermal) transformations. Figures in parentheses are difference spectrum maxima. Numbered steps are discussed in the text

meta-Ra and meta-Rb (steps 4,5 and 6) which can be detected by their absorption close to 710 nm and 650 nm respectively; the steps to meta-Rb (step 5) and to Pfr (step 6) do not occur in dehydrated tissue. Turning now to Pfr the first product in its photoconversion by far-red is thought to be lumi-F (step 7) which has peak absorption close to 720 nm; at physiological temperatures it is a transient intermediate. The reactions around lumi-F are complex but it seems likely that its photoconversion and dark relaxations (steps 8 and 9), which can occur in dehydrated tissue, give a mixture of several intermediates - the meta-Fa complex - showing difference spectrum peaks at 690, 675, 650 and 630 nm. Meta-Fa can also slowly revert to P fr in dehydrated tissues in darkness (step 10). Steps 8, 9 and 10 can also occur in hydrated tissue. Step 11 (meta-Fa to meta-Fb) occurs only in hydrated tissue, so in dried tissue some meta-Fa accumulates. In hydrated tissue the meta-Fa complex is not trapped but rapidly proceeds to P r by dark reactions, through the intermediate meta-Fb (steps 11 and 12). These points are summarized in Figure 3.10 which in some details is still only tentative [111]. The molecular changes involved in these transformations are poorly understood. It is worth noting, however, that changes in conformation of the phytochrome apoprotein can occur only in the hydrated state. The reactions via meta-Rb and meta-Fb could therefore involve alterations in the protein moiety of the photoreceptor (see [Ill]). Since a few of the steps can take place under conditions oflow hydration, they can therefore occur in dry seeds. Consequently, dry seeds exhibit a degree of light-sensitivity although the conversions P r ¢ Pfr are limited. We will return to this point in Sections 3.2.7 and 3.2.8.

The Phytochrome System

141

3.2.7. The State of Phytochrome in Seeds

Phytochrome can be detected and measured in vivo by spectrophotometry (see [19] for an outline of the method). Factors which affect the efficacy of spectrophotometry on seeds are (a) the amount of phytochrome present, (b) the colour and thickness of the seed coat, (c) the presence of various pigments in other seed tissues and (d) the size of the embryo - an important consideration when measurements on isolated embryos are required. Seed phytochrome has been measured in several species, some of which are shown in Table 3.5. In many ofthese a substantial proportion of the photoreceptor is already in the active form (Pfr); most of such seeds are not dormant and, provided that the temperature is satisfactory, they germinate readily in darkness. Since the proportion of Prr is affected by illumination conditions all of these seeds can, however, be prevented from germinating by light of a particular spectral composition. The operation of light as a factor inhibiting germination is considered in detail in Chapter 6. Phytochrome has been measured in dry (or rather only slightly hydrated) seeds of several species. The pigment exists as P r in some and as P rr in others (Table 3.5). During water uptake, the concentration of phytochrome increases, the proportion of each form might change and, moreover, the difference spectrum of the new phytochrome may not always be the same as that of the original pigment. Total spectrophotometrically detectable phytochrome increases in, for example, Cucumis sativus [127, 185], Cucurbita maxima [127], Lactuca sativa cv. Reine de Mai [38, 41], Amaranthus caudatus [112], Pinus palustris and P. nigra [149, 210]. Physiological evidence from other seeds (e.g. Amaranthus retroflexus [195]) also points to an increase in total phytochrome. Increasing levels of detectable phytochrome are due

Table 3.5. The state of phytochrome in seeds Species

Condition

State of phytochrome a

Amaranthus caudatus Cucumis melD Cucumis sativus

Imbibed seeds Non-imbibed seeds

Approx 75% P r Mostly Pfr

8 1.2

[112] [125]

Non-imbibed seeds Non-imbibed seeds Imbibed embryos Imbibed embryos

66%-75% Pfr 66% Pfr Mostly P fr Approx 60% P r

6 4.5 6.4-7.6 2.5-5

[185] [127] [127] [41]

Imbibed seeds Non-imbibed embryos Imbibed seeds Imbibed seeds Imbibed seeds

Mostly P fr 100% P r Approx 70% P r 100% P r 50%-100% P r

Cucumber Gherkin Cucurbita maxima Lactuca sativa

Total P concentration Ll (LlA) x 10- 4

Reference

cv. Reine de Mai N emophila insignis Pinus nigra Raphanus sativus Rumex alpinus Sinapis alba

2.7 4.2 10 13 5-10

[125,126] [149] [125] [28] [41, 126]

a Phytochrome is measured in units of absorbance. See Smith 1975 [19J for an explanation of the method

142

The Release from Dormancy

•

120

Fig. 3.11. Changing phytochrome content of seeds of Amaranthus caudatus. Total phytochrome concentration was measured at different times after the start of imbibition and after the completion of germination in darkness at 25° C (-....), in darkness at 0° C (no germination) (- -.--) and in far-red light (no germi(_. -0-· -). After nation) Kendrick et aI., 1969 [112]

Germino tion period 25°C

100

ｾ＠

•

15% water) lettuce seeds, when irradiated with red light and then dried ( < 7% water), can be stored for at least one year and then will germinate when placed on a wet substratum in complete darkness [6, 225]. In the case just quoted, slow loss by thermal reversion of Pfr in the stored seed does not occur because the process is blocked in dehydrated tissue. But even in dry tissue Pfr can be photoconverted to meta-Fa (this happens, as we have discussed, in Cucumis sativus prior to so-called inverse dark reversion) so dry seeds are sensitive to far-red light. In fact, the stimulatory effect of red irradiation on lettuce and Sinapis arvensis is substantially reduced when the subsequently dried seed is exposed to far-red light [69, 110]. The explanation is that upon rehydration of far-red-treated seeds in darkness a substantial proportion of meta-Fa, produced by the far-red light, passes through meta-Fb to P r and thus insufficient Pfr is available to promote germination. The effectiveness offar-red light on dried, red-treated lettuce seeds depends on the duration and timing of the red-light treatment [110]. Seeds given a short exposure to red light are, when later dried, relatively more sensitive to far-red light than are seeds previously exposed, during drying, to continuous red light. The explanation for this is that in the former, 80% of the phytochrome is converted to Pfr by the red light and is therefore sensitive to far-red light. In the latter (i.e. in continuous red light) there is a cycling between P r and Pfr (because even Pfr absorbs red light - see Fig. 3.5) but, as drying proceeds, intermediates progressively become trapped (e.g. meta-Rb) in a form not affected by subsequent irradiation with far-red light (see Fig. 3.10) when the seeds are dry. We should note, also, that when far-red light is effective on dry seeds its effect can be reversed by red light given when the seeds are still dry. This is because the meta-Fa complex produced by the far-red absorbs in the region 630-690 nm (see Fig. 3.10). The sensitivity in dry, partially dry and hydrated seeds is extremely important in nature at three phases in the life of the seed. Firstly, phytochrome conversions occur as the seed on the mother plant matures and dries in the light. Consigned to the mature seed, therefore, is a mixture ofP r, Pfr and various intermediates, their proportions depending upon the spectral quality, the surrounding chlorophyll, the duration and irradiance of the light, and the time taken to dehydrate. Following this development, light is still effective even on dry seeds, when interconversions are brought about between Pfr and the red-absorbing intermediate, meta-Fa. Lastly, the alternating hydration and dehydration, which might take place in seeds on or just beneath the soil surface allows other photoconversions and dark relaxations to occur. Thus, the condition of phytochrome in the seed is determined by a complex set of experiences - of illumination, dehydration, and hydration. 3.2.9. Reversion of PCr in Darkness

In addition to the phenomena discussed above, another important occurrence influences the state of the photoreceptor: this is the thermal reversion of Pfr to P r

147

The Phytochrome System

..

•

.-

. _ _ _ _ _ _e

e-

• 100 ｃｌｾ＠

50

0ｾ＠

20 10

r

2

6

ｾ＠

E ｾ＠

5

e

1

'0

ＱＰｾ＠

QJ

(

0

15 =

.cu 18 h light/day) are needed. This requirement diminishes as the temperature increases and at 20° C even a single pulse oflight terminates dormancy (Fig. 3.22D). With a further rise in temperature (> 25° C) dormancy begins to disappear and germination eventually occurs in total darkness, at least in B. verrucosa [35, 221]. The responses of Betula can be interpreted in the following manner. The seeds mature and are dispersed already charged with a low level of Pfr which, provided the temperature is high enough (e.g. > 25° C), can later act quickly and effectively to

;/0",\

161

Temperature and the Action of Light

100

"

0 C

Ｎｾ＠

ｾ＠

A

80 60

100

\.-.\-00

40

;1!. 20

0 10 15

5

20 25 30

",/ /"0 /0 __ - ,>--",--",--8,

0

" 80 g 60 Ｎｾ＠ 90

(approx.)

(approx.)

25 20

80

25

20

25

[226]

25 10

80

25

10

40

[226]

NR=not recorded

can completely or partially replace light in the breaking of dormancy (e.g. Nicotiana tabacum, Lythrum salicaria, Agrostis tenuis). Secondly, where there is no substitution for light, there is nevertheless an enhancement oflight action so that more germination is achieved in the light at alternating temperatures than at constant temperature (e.g. Fragaria virginiana, Chenopodium album, Lepidium virginicum). Several questions may be raised regarding the role of alternating temperature in modifying the action oflight on seed dormancy, such as: (a) What temperatures are effective? (b) How different should the temperatures be? (c) What durations at each temperature are required? (d) How many cycles are needed? We shall consider these questions in detail in Section 3.8 but some answers can be offered now. Firstly, an indication of effective temperatures is given in Table 3.10. In most of the

166

The Release from Dormancy

cases these were derived empirically, but a detailed analysis of Rumex spp. has revealed that any temperature alternation within the range 1°_35° C is effective to some extent, that the temperature difference must be at least 5° C (optimum = A 15° C), with one temperature above approximately 15° C, and one below approximately 23° C (see, e.g. Fig. 3.28). In the Highland cultivar of Agrostis tenuis, light action is markedly enhanced by alternating temperature combining 8 h at 24°_27° C with 16 h at 11°-15° C, and even more effective are treatments consisting of23 hat 15° C and 1 hat 35°-40° C [216]. Saturation of the response to 8/16 h alternations is achieved by 4-10 cycles in Rumex (Fig. 3.29), although exposure to 1 cycle at 35° C is effective when given after 8 days exposure to 10° C [219]. The length of time spent at each temperature does not appear to be critical for illuminated seeds, at least under certain alternations, so that in Rumex obtusifolius, for example, complete breaking of dormancy in the light occurs in diurnal cycles of 20°/35° C with the period at 20° C ranging from 8-23 h [220]. Where temperature alternations are extreme, however, an optimum period at the lower temperature is clearly definable [219, 220]. It is not clear what is the mechanism ofthe alternating temperature-light interaction in the breaking of dormancy. One possibility is that the temperature changes are effective in connection with the action and preservation of phytochrome. For example, the low temperature could reduce the rate of dark reversion of PCro while the higher temperature might enhance PCr activity. There is, however, no clear evidence for or against this interpretation. On the other hand, evidence is accumulating that a major effect of single temperature shifts concerns the activity of Pfr and it may be justified to extrapolate from our knowledge ofthese shifts to the situation in longer-term diurnal alternations. A single change in temperature often has nearly the same qualitative and quantitative effects as several temperature alternations in enhancing the action of light. Rumex, for example, responds almost as well to one or two 2-h shifts to 35° C, from 20° C, as to several 8/16 h alternations, especially if the shift has been preceded by several days at 20° C [220]. Temperature shifts, then, almost certainly have the same basic action as alternating temperature. Upward or downward shifts are effective, depending on the species. Shifts to a higher temperature, maybe for only 2 h, can break dormancy in darkness of some seeds of several species (e.g. Barbarea, Sisymbrium, Nicotiana, Nigella and Rumex [98, 145,224]). In most cases, however, the shift combines with the action of light to promote extensive termination of dormancy and induction of germination. This was first well-documented by Toole et al. [211-213], who reported work on Lepidium virginicum and other cruciferous species (Table 3.11). Later it was shown that temperatures in the range 35°-45° C (Fig. 3.25A) for 1-2 h (Fig. 3.25B) have the greatest effect, although in Rumex obtusifolius even 1 min at 40° C is effective. In fact in Rumex spp. it seems that the higher the temperature the shorter must be its duration [123]. In Lepidium the 2-h upward shift to 35° C is most effective when it immediately precedes or is 2 h after the light; a shift immediately after the light has less effect [196]. Moreover, in this species and in Potentilla, low energy levels of light become more effective when immediately preceded by a high-temperature period [191, 211]. Clearly, there is some interaction between Pfr and the shift; and even when the shift breaks dormancy in darkness, as in Rumex crispus, it probably does so by interacting with the residual, low level of PCr in the seeds [197].

Temperature and the Action of Light

100 "0 OJ

Ｎｾ＠

cc

80 60

OJ

40

*

20

l!l

0

A

B

100

/---\

-/-/

-

I'"-"-

2 80 c

Ｎｾ＠

c 60 OJ

l!l

*

167

40 20 0

40 20 30 50 Temperature (0 C) during shift

0 2 4 6 Hours at 40°C

Fig. 3.25A, B. Temperature and time requirements for effects of shifts on seeds of Lepidium virginicum. In (A) seeds were held imbibed in darkness for 3 days at 20 C before a temperature shift for 64 min. After the shift, seeds were irradiated with red light and returned to darkness at 20° C to complete their germination. In (B) seeds were left imbibed at 20° C in darkness for 3 days, then given a high temperature (40°C) for different times before irradiation; seeds were then transferred to darkness at 20° C to complete their germination. After Taylorson and Hendricks, 1972 [196] 0

Table 3.11. Response of some light-requiring seeds to temperature shifts Species

Irradiation

Maximum % of seeds germinated At constant temperature

Barbarea vulgaris Camelina microcarpa Capsella bursa-pastoris Lepidium virginicum Sisymbrium altissimum

Red light Dark Red light Dark Red light Dark Red light Dark Red light Dark

After temperature shift a

%

°C

15/25

20/30

20/35/20

73 32 26 41 14 1 33 0 23 6

30 30 15 15 30 15-30 25 15-35 15 20

64 1 42 17 21 1 87

69 11 0 0 64 1

23 4

5 5

93 51 94 31 76 3 98 3 70 11

Temperature regimes: 15/25:24 hat 15° C, followed by constant 25°C; 20/30:24 hat 20° C, followed by constant 30° C; 20/35/20 :24 h at 20° C, 2 h at 35° C, followed by constant 20° C Red light given before the constant temperature After Toole et aI., 1955, 1957 [211,213]

a

The efficacy of downward shifts is seen in the interesting case of Amaranthus retroflexus. Here, seeds fail to respond to red light (i.e. to the PCr so produced) at temperatures above 35° C. But if, after the light, the seeds are transferred to temperatures less than 32° C for 2 h, the PCr becomes effective and dormancy is terminated [83]. We will consider the explanation of these low- and high-temperature shifts when we discuss the mechanism of action of phytochrome in dormancy breakage (Chap. 4).

168

The Release from Dormancy

3.6.3. Chilling An enhanced response to light is exhibited by seeds of many species which have previously experienced low temperatures (ca. 50 C) in the imbibed state (Table 3.12). In many cases, Betula maximowicziana, Capsella bursa-pastoris and Chenopodium polyspermum being examples, light has no apparent action upon dormancy breakage at constant moderate temperature unless the seeds have been chilled previously [139, 153, 227]. The durations of chilling which increase the response to light of these species (i.e. a few days) do not terminate dormancy in darkness; to achieve this, prolonged periods of chilling, extending over some weeks, must be experienced. Other species are so sensitive to chilling, however, that even their dormancy in darkness is terminated and light is not needed (e.g. Betula pubescens, Pinus spp., Senecio vulgaris). But these two kinds of seed probably differ only quantitatively, perhaps simply because they lie on different parts of a chilling sensitivity curve. The effectiveness of chilling on light- and dark-germination is clearly dependent upon the duration of the low-temperature experience. In Pinus strobus low temperatures for at least 32 days are needed for full effect (Fig. 3.26), but a few hours of chilling are adequate in lettuce [222]. Effective chilling temperatures for Amaranthus retroflexus lie below 20 0 C; chilling at 100 C for 144 h can induce high levels of germination in darkness [194]. Prr must evidently be present in the seeds for chilling to be effective, since exposure to far-red light during the low-temperature treatment almost completely nullifies its effect. Clearly, Prr levels which are normally ineffectual are able to intervene in the dormancy of seeds which have previously been chilled. Thus, in lettuce, the Table 3.12. Some examples of pre-chilling/light interaction in the termination of seed dormancy Species

Betula maximowicziana Capsella bursa-pastoris Chenopodium polyspermum Pinus palustris Fraxinus mandshurica Senecio vulgaris Pinus strobus Pinus taeda Amaranthus retrojlexus Betula pubescens Lactuca sativa Sorghum halapense a

Reference

Termination of dormancy Pre-chilled

Unchilled

Light-treated Dark

Light-treated Dark

+++ ++

+++ +++ +++ +++ +++ +++ +++ +++ +++ +

+ +

++

++ ++ ++ ++ ++

++

+

+ + + + +++ +++ +++

[139] [153] [227] [134] [21] [153] [217] [217] [194] [35] [173,222] [192]

- No effect; + Small effect; + + Moderate effect; + + + Strong effect N.B. In this species red light has an inhibitory effect on the termination of dormancy of chilled seeds a

Temperature and the Release from Dormancy Fig. 3.26. The effect of chilling on the response to light of Pinus strobus. Seeds were given red light after different periods of chilling, then transferred to a temperature suitable for the completion of their germination. Irradiated chilled seeds CD). Unirradiated chilled seeds C.). After Toole et aI., 1962 [217]

169

100

80

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60

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32

small amount ofPfr generated by far-red light can cause germination in seeds which have been exposed to low temperatures for a few hours before irradiation. Effective chilling temperatures lie below 13° C when the amount of Pfr is low (i.e. after farred light) (Fig. 4.12B) but, interestingly, are below 18° C if more Pfr has been formed (i.e. after red light). Further, the beneficial effect of the low temperature is reversed by interposing a temperature of 20 C before Pfr is generated. These findings suggest the occurrence of some subtle changes in the seed, brought about by temperatures below a critical value, which sensitize the seed to Pfro A change in the condition of cell membranes has been suggested [222]. This, again, is a point to which we will return when we discuss the mechanism of phytochrome action (Chap. 4). 0

3.7. Temperature and the Release from Dormancy We have already seen how the release from dormancy by light (i.e. phytochrome) can be modified by the temperature. We will now extend the discussion and look in more detail at the role of temperature in dormancy breakage, but not especially at any interaction with light. Temperature-controlled termination of dormancy occurs in fully-hydrated seeds and in dry seeds. In hydrated seeds, alternating temperatures, temperature shifts, low temperatures (chilling) and, rarely, high temperatures break the dor-

170

The Release from Dormancy

mancy. Dry seeds undergo temperature-sensitive changes, called dry after-ripening, which result in the loss of dormancy. We will consider all four in the following sections.

3.8. Termination of Dormancy by Temperature Alternations and Shifts The first report of the stimulatory effects of alternating temperature in breaking seed dormancy is accredited to von Liebenberg who in 1884 published results of his studies of Poa annua [229]. Very many other species are now known whose imbibed seeds react favourably to fluctuations in temperature. For example, 68 of 85 species tested by Steinbauer and Grigsby [187] responded positively to alternating temperature. It is not always clear whether these temperature treatments affect the termination of dormancy or whether other aspects of germination and growth physiology are primarily involved. In some species, such as Tetragona, alternating temperatures seem to act predominantly on germination rates and uniformity [81], but in many there are well-authenticated effects on coat-imposed dormancy, e.g. in freshly harvested Bidens tripartitus [166], Cynodon dactylon, Typha lati/olia, [136], Nicotiana tabacum [212], Lycopus europaeus [205] and Rumex spp. [219, 220]. Of these, Cynodon, Typha and Lycopus seem to have an absolute requirement for alternating temperature. Alternating temperature is a complex factor having at least nine attributes [220]. These are: (a) the value of the upper temperature, (b) the value of the lower temperature, (c) the difference between these two, i.e. the amplitude, (d) the duration ofthe upper temperature, (e) the duration of the lower temperature, (f) the rate of warming, (g) the rate of cooling, (h) the number of cycles and (i) the time, after the start of imbibition, when cycles start. We can begin to see the importance of some of these individual parameters by inspecting the responses of Rumex obtusifolius, studied in great detail by Totterdell and Roberts [219] (Fig. 3.27). The valley running across the diagonal of this graph is given by the germination in darkness at constant temperatures from 1.5-35° C; clearly, under these conditions the seeds remain dormant. But dormancy is terminated when the seeds experience daily tem-

ｾ＠

r::[

#.

0

35 7"(,>0 'tJf,>I"OI

20

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･ｾｯ＠

10 ｜Ｐｾ･＠

20 ｜ｏｾ＠

Fig. 3.27. The effect of alternating temperature on dormancy breakage in 35 c,\ Rumex obtusifolius. Seeds were held at X' ＠ｾ various combinations of temperature '0 (8 hand 16 h) for 28 days in the dark, after which the germinated seeds were counted. After Totterdell and Roberts, 1980 [219]

Ternlination of Dormancy by Temperature Alternations and Shifts

171

Table 3.13. Effective temperature differentials (amplitude) for the termination of dormancy in several species Species

Temperature differential CC) Reference for 50% germination

Agropyron repens Apium graveolens A trip lex hastata Cardamine pratellsis Gnaphalium uliginosum Lycopus europaeus Polygollum persicaria Rorippa islandica Rumex sanguineus Silene dioica Typha latifolia

4 1.5 6.5 7

5 7 8 9

2.5

3-4 1

[203J [206J [203J [203J [203J [206J [203J [203J [203J [206J [203J

perature fluctuations, with the highest germination values attained towards opposite corners of the figure, i.e. where the amplitude of the temperature alternation is greatest (e.g. 8 h at 35° C combined with 16 h at 20° C). That the amplitude, or temperature differential, can determine the efficacy of alternating temperatures has been established for seeds of several species, and in many the magnitude of the fluctuation needs to be only a few degrees (Table 3.13). Temperature combinations giving effective amplitudes to break dormancy of R. obtusifolius can be determined from Figure 3.27. These combinations can be expressed in another graphical form, by a series of isometric contours as shown for R.obtusifolius and Lycopus europaeus in Figure 3.28. Other significant features, in addition to the temperature differential, are revealed by this graphical treatment. In both cases, there is little or no release from dormancy caused by certain temperature combinations even though the differential is satisfactory. These combinations are in the stippled areas of the diagrams; for Lycopus they are areas where the temperature is never above about 17° C or below approximately 27° C, and for Rumex, where the temperatures are never above 15° C and below 23° C. Certain absolute temperatures must be included in the alternation, therefore, if it is to be effective. Now in R.obtusifolius the minimum effective amplitude is about 5° C (Fig. 3.28) and if, to be effective, the alternation must include one temperature above 15° C and one below about 23° C it follows that the temperature pair to give this amplitude must consist of an upper one of 15° C or more and a lower one of about 23 ° C or less [219]. For Lycopus the upper temperature should be 17° C or more and the lower one 27° C or less. A further feature evident from Figure 3.28 is the assymetry of the response, exhibited, for example, by the 80% germination contour in Lycopus. This is due to the lengths of time experienced by the seeds of each temperature in the cycle. Evidently, the lower temperatures are more effective when given for 16 hjday than for 8 hjday; thus, relatively long periods at the lower temperature are more favourable to dormancy release. The time requirements vary with the temperature, however, and the optimum time for seeds to experience the upper temperature seems to de-

172

The Release from Dormancy

u o

A

30 20 / /

QJ

10

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t-

/

/

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/

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Fig. 3.28A, B. Isometric germination contours in relation to alternating temperatures. (A) Lycopus europaeus; (B) Rumex obtusifolius. The contours show equivalent germination percentages (as indicated on each contour) induced by particular temperature combinations, read off from the vertical and horizontal axes. The stippled areas cover temperature combinations which are poorly or totally ineffective in breaking dormancy. In (B) lines are drawn through points of equivalent amplitude. All the seeds received light during the temperature treatments. After Thompson, 1974 [206] (Lycopus) and Totterdell and Roberts, 1980 [219] (Rumex)

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174

The Release from Dormancy

tion with forestry practice and is recorded by Evlyn in 1664 [64]. He recommended that prior to planting seeds of Acer, Fagus and other species in springtime they should be placed in moist sand or soil and kept outdoors during the winter. Seeds so treated germinated better than those planted directly in the spring. An absolute dependence upon low temperature for removing dormancy is displayed by seeds of several species. But more often the low-temperature experience is just one of several environmental factors which can release a particular seed from dormancy; others are light, alternating temperatures and various chemicals (e.g. nitrate); and chilling can interact with them all. Low temperature is an especially important factor in species living in climates where the cold winters are unsuitable for seedling growth and which might prove fatal to an established seedling. A seed which remains dormant until several weeks or months of cold have been experienced usually will not germinate until the winter is over, at a time favourable for seedling establishment. There are two possible controls for this. One is simply that a winter's duration of cold is required for dormancy to be broken and the second is that generally germination of the now non-dormant seed cannot begin until warmer temperatures arrive (there are exceptions, though, and some species do succeed in germinating even at the same low temperatures that break dormancy see e.g. Fig. 3.30).

3.9.1. Response Types Chilling is commonly, if not invariably, required by seeds of woody species which have embryo dormancy, such as Acer saccharum, Corylus avel/ana, Crataegus spp., Pyrus spp. and many genera in the Rosaceae (see Nikolaeva [14]). Herbaceous and woody species with coat-imposed dormancy also respond to chilling, as do seeds with secondary dormancy, such as thermodormant lettuce. So chilling is effective in a range of dormancy types, a point which is exemplified within the genus Acer. Embryo dormancy is present in mature seeds of A. tataricum, A. saccharum [14, 233] and A. platanoides, but in the latter it is not deep and is lost fairly rapidly during warm storage, to leave only a coat-imposed dormancy [152]. Other speciesA.ginnala, A.negundo [14] and A.pseudoplatanus [200] - emerge at maturity with only coat-imposed dormancy. But although there are different patterns of dormancy in these closely related species, low temperature is effective in them all. We shall see below, however, that the differences in depths of dormancy are reflected by the different durations of chilling which are needed to produce germinable seeds. Many of those species whose dormancy is only expressed at above a certain temperature (i.e. relative dormancy) also respond to chilling. Cereals, such as wheat and barley, Delphinium and Lactuca sativa are good examples. In such cases the temperature range over which germination eventually occurs is considerably widened by a previous experience of low temperature (Fig. 3.31). Dormancy in several chilling-sensitive seeds seems to be localized largely in the epicotyl. In these cases of epicotyl dormancy (e.g. Lilium spp., Paeonia suffruticosa and Viburnum spp. [188]) unchilled seeds produce a growing radicle but for emergence of the epicotyl a low-temperature experience is necessary. It could be argued that many of the species which require chilling to some extent have epicotyl dor-

Termination of Dormancy by Low Temperature Fig. 3.31. The effect of chilling on relative dormancy of Delphinium ambiguum. Germination was tested in the dark with (0) and without (e) 2 weeks' previous chilling at 6° C. Note the greater temperature range over which germination occurs after chilling. Adapted from Ezumah, 1980 [65]

175

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Temperature during germination (OC)

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mancy. The dormant embryos of several rosaceous species (certain cultivars of apple, pear and peach, Crataegus spp., Rhodotypos kerrioides) do succeed in germinating, albeit sluggishly, when they are taken out of the seed. The seedlings grow poorly and remain dwarfed, their very stunted stems bearing small, abnormally shaped, dark green leaves or, in some cases, whitish scale leaves. Interestingly, such dwarf seedlings revert to a normal growth habit after they have been chilled (see [188]). In Chapter 2 we saw that dwarfism may be prevented in some species by removing the cotyledons before germination, suggesting that these organs have a long-lasting inhibitory effect but one removable by low temperature. Most chilling-responsive species are satisfied by a single period of cold. But in those which have double dormancy [188] the radicle and shoot appear to have different requirements for low temperature. In Caulophyllum thalictroides and Trillium erectum, for example, radicle emergence occurs after chilling but the shoot does not appear until after a second period of low temperature has been experienced. Radicle dormancy in Convallaria majalis is also terminated by one low-temperature passage, but dormancy of the shoot seems to deepen just after it emerges from the cotyledonary sheath; this organ resumes growth only after being subjected to cold for some months. The consecutive temperature regimes needed for the production of actively growing Convallaria are, therefore: (1) Three months at 5° C to break radicle dormancy in the seed. (2) Two months at warm temperatures (e.g. 21 ° C) when the root continues to grow and the shoot slowly emerges. (3) Between 3 and 5 months at 5° C to break shoot dormancy. (4) Warm temperatures for growth of the first leaf. (5) A third period at 5° C to encourage subsequent growth of the second leaf-bud [188]. 3.9.2. Temperature and Time Requirements Temperatures from 1°-15° C have been found to break dormancy, but species differ as to the most effective range (Table 3.14). Recorded optima for most species

176

The Release from Dormancy

Table 3.14. Temperature and time requirements temperature Species

Range

CC) 1-5

Abies arizonica AceI' pennsylvanicum AceI' platanoides AceI' pseudoplatanus AceI' tataricum Aralia hispida Corylus avellana Crataegus moWs Delphinium ambiguum Gentiana acaulis Picea canadensis Pinus lambertiana Poa annua Pseudotsuga menziesii Prunus persica Rumex crispus Saponaria officinalis Triticum aestivul11 Sorbus aucuparia

100 80 Ｎｾ＠ 60 ill 40

5 2-6 1-5 1-5

5-10 1.5-15 1-5

Apple Testa removed, endosperm intact Naked embryo \-"

,..>-.

-0

./

/. "./

ｾ＠

/

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the breaking of dormancy by low

Time (days) Optimum temperature Cc) 30 90--120 50-60 80 90--120 90-120 30-60 180 14 60-90 30-60 56-112 7 49 60-90 42 7 3-5 60--120

1 5 5 5 5 5 5 5 6 1 1 3-5 4 3-5 5 1.5 5 5 1

Reference [50] [14] [151] [234] [14] [50] [74] [50] [65] [50] [50] [190] [164] [190] [50] [218] [187] [46] [50]

Acer pseudoplatanus

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Time at 5°C (days)

Fig. 3.32. The removal of embryonic and coat-imposed dormancy by low temperature. Seeds or fruits were chilled at 5° C and periodically removed for testing. For the test, part or all of the tissues covering the embryo were removed from some dispersal units. Apple: germination tested at 25° C. After Visser, 1954 [228]; AceI': germination tested at 20 C. After Webb et aI., 1973 [234] 0

are close to 5° C which, for greatest effect, needs to be experienced for periods from a few days (e.g. Triticum) to several months (e.g. Crataegus). The time-dependency of three species is shown in Figure 3.30. The required period of cold is related to the depth of dormancy. This is illustrated by species of AceI' which cover a range of cold requirements from 50 to 120 days (Table 3.14). Those species which require longer exposure times to cold (e.g. A. pennsylvanicum and A. saccharum) have a deep embryo dormancy whereas those which are satisfied by a shorter time (e.g. A.pseudoplatanus) have a less deep, coatimposed dormancy. From the two cases shown in Figure 3.32 we can see how the

Termination of Dormancy by Low Temperature

177

two kinds of dormancy (coat-imposed and embryo) inflict different requirements for cold. In apple, the purely embryonic component of dormancy is removed by about 50 days of chilling, the presence of the endosperm increases the requirement by approximately another 20 days, and the additional influence of the testa raises the requirement by more than another 30 days. On the other hand, the embryo of A.pseudoplatanus itself has no dormancy but the testa and pericarp together impose a need for more than 80 days chilling. Chilling is beneficial only in hydrated seeds. When seeds are placed under stratification conditions, the low temperature only begins to take effect when imbibition by the embryo is completed. The seed coats can exert an influence here since they may impede water entry. When this happens, part of the stratification time is simply spent in reaching the necessary level of hydration of the embryo. In Acer saccharum fruits, for example, removal of the pericarp and rupture of the testa before chilling reduced the necessary stratification time from about 60 to 20-40 days; this is considered to be because these enclosing tissues restrict water entry [233]. Before leaving the time requirement for chilling we should note that this is by no means an absolute for any particular species. The period of low temperature needed to break dormancy varies, for example, with the provenance of the seed. Fraxinus excelsior from latitudes having longer, colder winters produces seed which requires an extended period of cold to break dormancy, compared with seed from regions with less severe winters [223]. On the other hand, a detailed investigation of the stratification requirements of Pinus strobus seeds from 11 provenances suggests that seed from southern regions (Georgia and Tennessee) need longer periods of chilling than those from further north (e.g. Ontario). This is interpreted as a means of securing germination only after the months of alternating warmth and cold have passed (characteristic of these southern regions) when it becomes safe for a seed to germinate and the seedling to emerge [71]. The cold requirement of Polygonum coccineum is also known to vary with the provenance [103]. These examples indicate that the cold requirements of a seed are likely to be an adaptation to the climatic factors which it meets. Precise determinations of the temperature optimum for stratification have rarely been made. One of the most detailed seems to be that by Schander (cited by Stokes [188]) who showed an optimum of 2°_5° C for 85 days' chilling of apple seeds. The optimum appears, however, to be time-dependent and moves to these low temperatures with increasing time of exposure. Considerable caution should be exercised when considering temperature optima since an apparent optimum may result from combined effects of two temperature-dependent but antagonistic processes - breaking of dormancy and induction of secondary dormancy - found to occur, for example in Capsel/a bursa-pastoris and Rumex obtusifolius [164, 218]. For relatively short treatment times (e.g. 3 weeks) in darkness, the temperatures 1.5° C and 10° C are about equally effective in terminating dormancy in Rumex obtusifolius, but with longer times 10° C becomes less and less effectual (Fig. 3.33A). This fall is thought to be due to secondary dormancy which sets in more rapidly at 10° C than at IS C. In the light, however, secondary dormancy is apparently deferred, when even 15° C becomes an efficacious chilling temperature, at least over a period of 2 weeks (Fig. 3.33B). The apparent temperature optimum clearly

178

The Release from Dormancy

100 80 -u

g 60 Ｎｾ＠

t3

40 20

Q

a

2

t..

6

8 10 12 0 2 t.. Duration of chilling (weeks I

6

8

10

12

Fig. 3.33A, B. Temperature-time relations in stratification of Rumex obtusifolius. Seeds were treated at three temperatures for different times in the dark (A) or the light (B). They were then transferred to 25° C for 4 weeks when germinated seeds were counted. After Totterdell and Roberts, 1979 [218]

depends upon the treatment time, and over a short time period temperatures over a wide range (IS-ISOC) have almost equal effects on dormancy. While such an analysis has apparently not been made for other species, it is quite possible that a similar situation is widespread. The development of secondary dormancy may account for the reduction in the effectiveness of a chilling treatment when it is interrupted by a period of higher temperature. For example, transfer of Euonymus europaea seeds to a warm temperature before the completion of the requisite stratification period completely nullifies the effect of the previous chilling and the seeds then have to recapitulate the low-temperature experience to be released from dormancy (Fig. 3.34). This phenomenon presumably occurs during the winter in temperate climates when there are intermittent, relatively warm days.

3.10. Termination of Dormancy by High Temperature

For chilling to be effective, it must in some cases be preceded by a period at warm temperatures. Seeds of several woody species, discussed in detail by Nikolaeva [14] have this requirement. The optimum temperature for the warm period is 20°-25° C for Crataegus spp., 20° C for Fraxinus spp., and 15°-20° C for Euonymus spp.; treatment times range from 1 to 3 months. Following the warm incubation, cold treatments at temperatures 1°_7° C for 4-6 months finally release the seeds from dormancy. In Crataegus and Euonymus the warm treatment accelerates certain changes in the seed coat which are conducive to germination; in Crataegus the endocarp is softened and in Euonymus the coverings open [14]. Many species of Fraxinus (e.g. F. syriaca, F. oxycarpa, F. excelsior), exhibit this requirement for warmth preceding cold stratification [14]. During the period at the higher temperature there occurs growth of the immature embryo which can then benefit from the low-temperature treatment.

Loss of Dormancy in Dry Seeds - After-ripening

179

100

"0 C1>

o Ｎｾ＠

c

50

C1> D

"#

o o

2

l.

6

8

10

12

1l.

Time (weeks)

1. ｢ Ｕ［ｾＲｑｴｃｬ＠

2. (1$;10,;c1

Fig. 3.34. The effect of interpolated high temperature during stratification of Euonymus europaea seeds. Seeds were held at 3° C throughout, apart from interpolated periods at 15°-20° C. Note that in treatment 2 the first 3.5 weeks of chilling is cancelled by the high temperature interruption: the seeds then have to receive another 5 weeks approx. at 3° C. After Nikolaeva, 1969 [14]

Some herbaceous species also require a warm treatment for the release from dormancy. Seeds of Narcissus bulbicoidium, for example, are dormant at constant temperatures 3°-38° C, but an experience of 3-4 weeks at 26° C prior to transfer to lOo-15° C serves to promote germination [207]. Similarly, the bluebell (Hyacinthoides non-scripta) requires a conditioning treatment for several weeks at 26°31 ° C optimum, followed by a germination phase at 11 ° C [208]. This device secures germination in late autumn (in western Europe), overwintering as slow-growing seedlings, and establishment before the appearance of the leaf canopy of the deciduous woodland, which is the species' characteristic habitat.

3.11. Loss of Dormancy in Dry Seeds - After-ripening During our considerations of dormancy we have occasiot;lally touched upon the point that the type and depth of dormancy can change with time. For examples, we can remind ourselves of the situation in Acer pseudoplatanus in which the developing seed seems to pass through a period of embryo dormancy [200] and of that in Corylus avellana and certain cultivars of Prunus persica where embryo dormancy deepens after development and dispersal [41 a, 44a]. But the most widespread change displayed by seeds is the gradual reduction in dormancy - even until it is completely lost. This process, called after-ripening, takes place only in seeds which

180

The Release from Dormancy

Table 3.15. Removal of dormancY by dry after-ripening Species and type of dormancy

Dormancy also terminated by

Ambrosia trifida (embryo)

Chilling

Acer negundo (coat-imposed) Avenafatua (embryo) Betula pubescens (coat-imposed) Bromus secalinus (coat-imposed) Hordeum spp. (coat-imposed) Hyptis suaveolens (coat-imposed) Lactuca sativa (coat-imposed) Oryza sativa (coat-imposed) Rumex crispus (coat-imposed) Sporobolus cryptandrus (coat-imposed) Triticum aestivum (coat-imposed)

Alternative period of dry storage

12 months reduces required cold period by 70% 7-8 months Chilling 30 months Chilling 12 months Chilling, light Chilling 1 months Chilling 0.5-9 months Light 8 months 12-18 months Light, chilling 2-3 months Alt. temp, 60 months (approx. light, chilling 50% of seeds released) Light >48 months Chilling 3-7 months

Reference [52] [14] [140] [32] [187] [4a] [236] [32] [162] [48] [214] [32]

have a low water content; it is common, therefore, in seeds in storage but it presumably also occurs in nature when seeds experience long periods in the dry condition. We find that seeds which, for some time after harvest or dispersal, need light, chilling or alternating temperature to break their dormancy, slowly become partially or completely independent of such requirements and gain the ability to germinate under conditions which were previously unfavourable (Table 3.15). Dry after-ripening does not have an all-or-nothing action but rather has a graded effect. Dormancy in Ambrosia trifida [52], Acer semenovii and Fraxinus excelsior [14], for example, is not totally abolished by dry after-ripening but instead the requirement for chilling is much reduced. Similarly in Rumex crispus 5 years of after-ripening removes dormancy in only about 50% ofthe seeds in a popUlation but, nevertheless, the remainder display enhanced sensitivity to alternating temperature and light; even after 2 years' after-ripening the seeds gain greater responsiveness to these factors [48]. There are interesting cases in which after-ripening removes one component of dormancy (e.g. embryo dormancy) while another (coatimposed) remains. Some of these were considered in Chapter 2, Section 2.7. Many of the best-known effects of after-ripening are in cereals (barley, rice, wheat) and other grasses (e.g. Avenafatua) which lose their dormancy during storage (see [16]). Graminaceous dispersal units commonly exhibit relative dormancy, manifest by the ability to germinate only over a limited temperature range. We have already seen that this kind of dormancy is terminable by a few days of chilling but it is also lost over a few months by the dry grain. After-ripening therefore widens the temperature range over which grains can subsequently germinate, and it also expresses its effect at the lower temperatures of this range by enhancing the rate at which germination occurs (Fig. 3.35). The temperature range for germination of other seeds with relative dormancy, such as the Grand Rapids cultivar of lettuce, is also affected by after-ripening. After-ripened lettuce seeds are no longer

Loss of Dormancy in Dry Seeds - After-ripening Barley

Lelluce cv. Grand Ropids

100 11=11011=11-0- 0----0- -0

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35

Fig. 3.35. The effect of dry after-ripening on relative dormancy. Barley: Germinated grains counted after 10 days. Inset: note that after-ripening increases the rate of germination at 1.so C even though the final percentage is unaffected (After Roberts and Smith, 1977 [16]). Lettuce: Seeds after-ripened for 18 months at 18° C approx. By the authors

dormant in darkness at moderately high temperatures, i.e. they no longer require light to enable them subsequently to germinate at such temperatures (Fig. 3.35). Factors important in after-ripening are: (a) seed moisture content, (b) temperature, and (c) oxygen. The time for after-ripening, referred to in Table 3.15, is influenced by these factors, especially (a) and (b) which, in nature, are more variable than (c). 3.11.1. Moisture Content The moisture content of a seed in storage or on dry earth is established by the surrounding atmosphere and is in equilibrium with the ambient relative humidity. Dry-seed water contents generally fall in the range 5%-15%, according to the species and external conditions. There is little known about the effect of variations within this range on the efficacy of after-ripening but it has been suggested that the rate of the process in Avena fatua and rice is enhanced at the upper values [155, 165]. Complications set in at moisture contents higher than the norm for dry seeds when substantial loss of viability may ensue (see Chap. 1) and, as moisture contents become higher still, secondary dormancy develops. Thus, effective after-ripening can only occur at moisture contents within the limits determined by the retention of viability and the onset of secondary dormancy. 3.11.2. Temperature The rate of after-ripening is temperature-dependent. Indeed, one of the earliest reports on after-ripening in cereals showed that the process could be greatly acceler-

182

The Release from Dormancy -;;;

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Fig. 3.36. The effect of temperature on dry after-ripening in rice. Grains of Oryza sativa cv. Lead 35 were tested for germinability (7 days at 32° C) at time intervals during their afterripening in storage at different temperatures. From these results the mean dormancy period at each after-ripening temperature can be calculated. After Roberts, 1965 [163]

ated by subjecting dry grains to temperatures of 30°-40° C for a few days [22]. Low temperatures prevent or greatly retard the process. Storage of Rumex crispus seeds for 5 years at 2°-4° C, for example, results in no appreciable change in dormancy [48]; on the other hand, R.obtusifolius is somewhat affected by storage for 9 months at IS C [218]. Although higher temperatures have long been known to hasten loss of dormancy, temperature/time relationships have been examined in detail for only a few species, notably rice and barley [16, 162, 163]. This relationship, in one of the cultivars of rice investigated by Roberts, is shown in the two graphs of Figure 3.36. The linear relationship between the logarithm of the mean dormancy period (d) and the temperature (t) conforms to the equation log d = Kd - Cdt, where Cd and Kd are respectively the slope of the regression line, and the intercept. The temperature coefficient (Ql0) for the loss of dormancy (i.e. the reciprocal of the mean dormancy period) may be calculated from these data, and is found to be 3.1 [163]. This compares with 3.9 computed for barley [16]. These values imply that in rice, for example, the rate of after-ripening at, say, 5° C would be expected to be 0.1 of that at 25° C, if the same Ql0 applies over a wide temperature range.

3.11.3. Oxygen After-ripening is accelerated by oxygen-enriched atmospheres and delayed in oxygen-depleted ones, for example in rice and wild oats [162, 178]. In the former species, exposure to 100% oxygen approximately doubled the rate of after-ripening over that occurring in seeds intermittently gassed with nitrogen [162]. Aerobic metabolism apparently is essential to the after-ripening process.

Finale - Replacements and Interactions

183

3.12. Finale - Replacements and Interactions It should now be clear that dormancy in anyone species may be terminated not just by one factor (such as light) but by alternative experiences--chilling, fluctuating temperatures, or after-ripening. For example, dormancy in Betula pubescens and lettuce is broken by light or by chilling; in Nicotiana tabacum by light or by alternating temperatures; in numerous other species, dry after-ripening, or various chemicals, bypass any need for light, chilling, or alternating temperatures. Thus, there are many paths leading to the ending of dormancy and, as we shall see later, one of the most challenging problems is to understand how these routes have the same terminus. But besides seeing these replacement phenomena we can also recognize that one factor might reinforce another's effect. For example, seeds which at first seem insensitive to light turn out to be extremely sensitive when they are chilled for a few days or weeks prior to illumination. Woody species such as Betula maximowicziana [139] and herbaceous species (e.g. Capsel/a bursa-pastoris [153]) fall into this category; and of seven weeds studied by Vincent and Roberts [227] six appeared unaffected by light treatments until after they were chilled. Similarly, chilled seed of Chenopodium polyspermum, Papaver rhoeas and Rumex crispus respond much better to alternating temperatures than do unchilled ones [226]. Seeds of some species do not respond to light at constant temperature but do so at alternating temperature (see Table 3.10). Sensitivity of Amaranthus retroflexus seeds to ethylene is greatly enhanced by prior exposure to alternating temperatures [176a]. In fact, it is commonly found that two or three factors interact to release seeds from dormancy. Phytolacca americana seeds, for example, respond only marginally to light, to alternating temperatures, or to nitrate, but a combination of all three succeeds in breaking dormancy [187]. Similar interactive situations have been widely investigated by Roberts and his colleagues in seeds of eleven weed species [153, 164, 218, 226,227]. An important point to emerge is that the release from dormancy in many species (perhaps the majority in the field) is seldom provoked by one factor alone but is usually a consequence of positive interactions between any two of light, alternating temperature and nitrate (first-order interactions) or among all three (second-order interactions); additionally, chilled seeds may display different interactions from those shown by unchilled seeds. The examples shown in Figure 3.37 are in the form of Richards diagrams, a device developed by F.J.Richards [161] as a means of expressing interactions among factors affecting plant activity; the modus operandi is explained in the figure legend. We can see cases where single factors fail to remove dormancy, e.g. alternating temperatures of 15°/25° C, or light on Chenopodium album. First-order interactions (i.e. greater than additive effects) may occur between only limited pairs, e.g. between alternating temperature and nitrate in P.persicaria, not-between alternating temperature and light. In some, first-order interactions are displayed by two pairs, such as 1°/25° C and light or nitrate in C. album, or three pairs e.g., all pairs from 15°/25° C, light and nitrate in C. album. Second-order interactions are shown by P. persicaria and C. album. Finally, factors may interact only in seed which previously has been chilled (c. polyspermum).

184

The Release from Dormancy

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Fig. 3.37. Release of dormancy by combined action of different factors. Each factor is represented by a line: alternating temperature; - - - - light; -' - . - . - nitrate. The germination percentage when none of these factors is operating (i.e. on water, in darkness, at constant temperature) is given above the 0 factor position. To find the effect of one factor follow the appropriate line (i.e. light, alternating temperature or nitrate) which terminates above the one-factor position. For example, in P. persicaria the basal germination - in darkness, on water, at constant temperature - is about 15%; nitrate has almost no effect, light raises germination to about 25%, and alternating temperature to approx. 50%. Addition of a second factor is indicated by a line extending to the two-factor position, e.g. light and nitrate together end dormancy in about 25% of P. persicaria. Clearly, there are two routes for reaching the two-factor position: one follows, for example, line a then line b', the other line b, then line a', so forming a quadrilateral. When the factors have only additive effects the quadrilateral is a parallelogram (e.g. light and nitrate in P. persicaria): when the interaction is greater than additive the quadrilateral is skewed (e.g. nitrate with alternating temperature (15°/25 0c) in C. album). The addition of a third factor is shown by a line to the three-factor position. Additive or greater than additive effects can again be discerned. After Vincent and Roberts, 1977 [226]

The many interactions shown by these species emphasize the fact that the release from dormancy is not necessarily the prerogative of a single factor, but that the combined action of several may be required; indeed in nature this may be the more common occurrence. This, of course, applies to the population as a whole and it is not yet clear how individual seeds react to single or multiple factors. Obviously, some seeds may respond either to alternating temperature or to light, for example, and some seeds could be affected by both factors acting cooperatively. The response patterns shown in Figure 3.37 include multiple effects upon the same seed, as well as single factor actions on individuals which, in a population, summate to raise the total number whose dormancy is terminated. If seed populations

Hard-coated Seeds

185

do contain individuals some of which require one factor and some another this would be another facet of the polymorphism which has been discussed in Chapter 2. It is feasible to explore this possibility by appropriate selection and genetic techniques, but as far as we are aware this has not been done. Finally, we should note that when in the imbibed condition seeds may change in their sensitivity to different factors, acting separately or in combination. Germination of Amaranthus retroflexus, for example, is stimulated by light, ethylene or high temperature when these are applied separately during the first 24 h after the start of imbibition. But after 48 h, seeds are unaffected by each separate factor and dormancy is broken only by all three applied together [176 b].

3.13. Hard-coated Seeds Several artificial methods are used to soften the hard, impermeable seed coats of the Leguminosae, Cannaceae, Malvaceae, Convolvulaceae and other families and so release them from dormancy. These include treatment with concentrated sulphuric acid or ethanol, mechanical scarification, freezing, heating, radiation, percussion and pressure [17]. But although we know of all these ways to soften seeds in the laboratory, how the softening is accomplished in nature is rather poorly understood. The use of various tools and machinery during cultivation of the land probably causes some mechanical abrasion; this might also be achieved when seeds are rubbed against soil particles by wind and rain action. Chemical abrasion occurs to some extent during passage of seeds through the alimentary canal of animals. Softening by microbial action has been suggested but, as pointed out by Rolston [17], there is little quantitative evidence to support this. The best-known effects in nature seem to be associated with the action of temperature - high temperatures and temperature fluctuations [157]. Softening of the coat by temperature fluctuations occurs in Trifolium subterraneum, Stylosanthes humilies and Lupinus varius but, interestingly, only when the seed coats are relatively dry -less than 8.5% water content in Lupinus varius [156]. In Lupinus these fluctuations cause the strophiole of the coat to crack and water is thus allowed to enter [156]. High temperatures can also act on this region of the hard testa. It has long been known that immersion in boiling water for a few minutes leads to a permanent increase in the subsequent permeability of the coat to water [17]. This treatment has been shown, in Albizzia lophantha, to loosen a mound of cells at the strophiole - the strophiolar plug - which comes away from the coat, to reveal the underlying, relatively thin-walled parenchymatous cells through which water can pass [53J (Fig. 3.38). That the strophiole is indeed the permeable region of heat-treated seeds is demonstrated by sealing the strophiole of heated seeds with a resin; these seeds fail to take up water. Heat-treated, unsealed seeds rapidly imbibe and reach a water content sufficient to allow germination to proceed. The strophiole of several species becomes permeable when the seed is heated [24]. Other areas of the hard coat may be affected by heating. Hot water disrupts the chalazal plug of cotton, and dry heat cracks the micropylar region of Rhus

186

The Release from Dormancy

Fig. 3.38a, b. Effect of heat on the strophiole of Albizzia lophantha seeds. (a) S.E.M. of dry seed showing strophiolar plug (s). (b) S.E.M. of seed exposed to boiling water for 10 s. Note that the strophiolar plug has been ejected. Seed treatment and electron microscopy by 1. Pacy

ovata and the seed coat of various legumes [17]. These effects of high temperature are thought to account for the stimulation of germination by fire . Severalleguminous species in forests break dormancy after being subjected to fire [154]; the flush of Albizzia lophantha, for example, can be correlated with the incidence of forest fires [230]. Presumably dry heat of fires acts through the same mechanism as does boiling water.

3.14. Removal of Dormancy by Chemicals Many different kinds of chemicals when applied to dormant seeds will cause them to germinate (Table 3.16). Some of these chemicals have potential value in agriculture and horticulture to accelerate germination or break the dormancy of seeds [87]. Numerous species of seed respond to these substances, supplied either singly or in combination, although it is difficult to find a seed which is sensitive to all of the compounds. Barley, however, is one example of a seed on which a wide variety of dormancy-breaking agents has been tested (Table 4.10) and it is largely on the basis of the efficacy of these chemicals that a seed-dormancy hypothesis has been constructed by Roberts and his colleagues (see, e.g. [16]); this hypothesis will be discussed in Chapter 4.

Removal of Dormancy by Chemicals Table 3.16. Chemicals which break dormancy Class Growth regulators Gibberellins Cytokinins Ethylene Plant products Fusicoccin Cotylenol Cotylenin Strigol Respiratory inhibitors Azide Cyanide Malonate Hydrogen sulphide Carbon monoxide Sodium fluoride Iodoacetate Dinitrophenol L- and D-Chloramphenicol Hydroxylamine Oxidants Hypochlorite Oxygen Nitrogenous compounds Nitrate Nitrite Hydroxylamine Thiourea Sulphydryl compounds Dithiothreitol 2-Mercaptoethanol 2,3-Dimercaptopropanol Various Acetone Ethanol Methanol Ethyl ether Chloroform Methylene blue Carbon dioxide Phenols Hydroxyquinoline Dimethylglyoxime

Reference· [54,87, 102] [114,201] [113, 193] [115, 121, 122] [115] [115] [49]

[16] [16] [16] [16] [16] [16] [16] [16] [16] [16,82] [88,89] [16] [16,82] [16,82] [16,82] [61]

[16] [16] [16] [199] [199] [199] [199] [199] [16] [16] [16] [16] [16]

• The references given here are to recent publications dealing with the effects of these chemicals

187

188

The Release from Dormancy

It is not possible, or appropriate, in this text, to cover the voluminous literature dealing with seed responses to applied chemicals. We propose only to highlight those features which seem to us to contribute to an understanding of dormancy mechanisms. Some details, and an inroad into the literature, can be found in the references quoted in Table 3.16.

3.14.1. Growth Regulators The ability of these substances, when applied to seeds, to release them from dormancy and promote germination is particularly interesting because, it is thought, it gives us a clue to possible dormancy mechanisms in the seed. All of the active substances - gibberellins, cytokinins and ethylene - are found in seeds; the argument is that if similar chemicals are active when supplied to the seed, why should not those already in or produced by the seed act in a similar manner? Prompted by this argument, adherents of the hormonal theory of dormancy have suggested that seed dormancy is regulated by the balance of promoters and inhibitors (see, e.g. [1, 8, 232]), a concept which we shall turn our attention to in Chapter 4. But before then, let us consider briefly some aspects of the effects of these growth regulators.

3.14.2. Gibberellins These are successful in breaking dormancy in numerous species of seed and also in accelerating germination of non-dormant seeds. Among dormant seeds, both those with coat-imposed dormancy (e.g. barley, lettuce) and those with embryo dormancy (e.g. some strains of Avenafatua - wild oats) are promoted. Effective concentrations generally lie within the range 1O-5-1O- 3M. Of the gibberellins which have been widely tested, GA4 and GA7 mixtures are effective at lower concentrations than those of GA3 (gibberellic acid). In some cases where gibberellin is poorly effective on the intact seed, an effect can be induced by various treatments of the seed coat such as mild scarification, pricking or scratching, e.g. [88]. This suggests that gibberellin might not readily penetrate the coats of some seeds, but, surprisingly, this possibility does not appear to have been investigated thoroughly.

3.14.3. Cytokinins One of the earliest reports on cytokinins in seed dormancy concerned the action of kinetin on lettuce seeds [135]; it was reported that dormancy of such seeds can be overcome by supplying this substance in darkness. It later became clear that kinetin is effective only in the presence of low levels of light, which themselves are inadequate in promoting germination. In complete darkness kinetin, as well as other cytokinins, break the dormancy of only a small percentage of lettuce seeds and even then the emergence of the embryo is usually abnormal, the cotyledons

Removal of Dormancy by Chemicals Fig. 3.39. Effect of applied kinetin, abscisic acid and giberellic acid on germination of lettuce (cv. Grand Rapids) in darkness at 25° C. a Gibberellic acid. b Gibberellic acid + abscisic acid (0.04 mM) + cytokinin (0.05 mM). c Gibberellic acid+abscisic acid (0.04 mM). Based on data of A. A. Khan, after Thomas, 1977 [201]

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growing first [93]. The effect of exogenous cytokinin on seed dormancy remains equivocal. Some cases are known in which applied cytokinin improves germination [201] but rarely, if at all, are these substances as effective as the gibberellins. The action of cytokinins is best seen in their capacity to counteract the effect of inhibitors, especially abscisic acid (ABA), when applied simultaneously. Numerous cases have been elaborated and documented by Khan [114], and discussed by Thomas [201]; an example is shown in Figure 3.39. In this experiment, the dormancy-breaking effect of various concentrations of gibberellic acid (curve a) is prevented by abscisic acid (curve c), whose inhibition is overcome by kinetin (curve b). Hence cytokinins are regarded as haVing a "permissive" role, allowing a second promotor (e.g. gibberellin [GA]) to act. Cytokinins also reverse the action of ABA on isolated lettuce embryos but to do so no gibberellin is required; the latter is required only by the intact seed [27]. 3.14.4. Ethylene Dormancy in seeds of many species is broken by ethylene, supplied either directly as the gas or by means of an ethylene-generating chemical such as Ethrel [1l3]. Effective concentrations are in the range 0.1-200 Jll/1. Germination of seeds of several weed species such as Spergula arvensis [101, 146], Striga lutea [59], Amaranthus retroflexus, A. albus, A. spinosus, Chenopodium album and Ambrosia artemisifolia [193] is promoted by ethylene; but of 43 weed species examined by Taylorson [193], seeds of 32 species showed no response. Of those that did respond, several demonstrated interactions between light and ethylene (see also [146]). Because of the presence of ethylene in soil, the known effect of the gas on dormant seeds is of great interest and may be of considerable ecological importance. Cocklebur (Xanthium pennsylvanicum) seeds are affected by ethylene, but the more dormant upper seeds are less sensitive and only 20% of them break dormancy in response to a concentration which stimulates 80% of the lower seeds [108]. We will return to the role of ethylene in these seeds in Chapter 4 when we discuss the evidence that the substance participates in the regulation of dormancy.

190

The Release from Dormancy

3.14.5. Plant and Fungal Products Fusicoccin and Cotylenins. Fusicoccin was isolated from the fungus Fusicoccum amygdali and shown by Lado et al. to promote the germination of lettuce seeds inhibited by abscisic acid (in this sense it acts rather like cytokinin, except that it does not require a second promoter) and to accelerate germination of maize and radish [121, 122]. Similar compounds, cotylenol and cotylenin E, (Fig. 3.40) isolated from Cladosporium spp., also have some dormancy-breaking action on lettuce [115]. The effect offusicoccin has excited particular interest as information is available about its possible mode of action. We will return to this point in Chapter 4. Strigol. Seeds of the parasite, witch weed (Striga lutea), do not germinate unless stimulated by the root exudation of a potential host plant. One factor, strigol (Fig. 3.40), isolated from rootlets of cotton, is a highly potent stimulant and, at a concentration of 10- 11 M, causes 50% germination [49]. Roots of many other plants also induce adjacent Striga seeds to germinate but it is not yet clear whether they all produce strigol. It is not known, also, if strigol affects seeds of many other species.

Fusicoccin

Cotylenol

:: CH 2 0CH 3

OH Cotylenin E

Fig. 3.40. Some plant and fungal products which promote germination

Removal of Dormancy by Chemicals

191

3.14.6. Respiratory Inhibitors As far as the breaking of dormancy is concerned the most potent respiratory inhibitors are those which inhibit cytochrome oxidase - cyanide, azide, hydroxylamine, hydrogen sulphide and carbon monoxide; other inhibitors are less effective, at least on dormant barley seeds [16]. Cyanide is active on seeds with coat-imposed dormancy such as barley [16] and lettuce [198] and those with embryo dormancy, such as apple [57], where it stimulates isolated embryos. D-threo-chloramphenicol, found to break dormancy in lettuce seeds [33] was at first thought to act through an effect on protein synthesis. It is now believed to act upon respiration (see Chap. 4). The action of respiratory inhibitors in breaking seed dormancy plays a prominent part in the hypothesis developed by Roberts [16] which will be considered in detail in Chapter 4, Section 4.5.5.

3.14.7. Oxidants Oxygen. The effect of high concentrations of oxygen on the relief of dormancy is widely reported; several species of seed which respond are listed in Table 2.7. One interpretation which is placed on this effect is that the barrier presented by the seed coat to the entry of oxygen from air is overcome. As we saw in Chapter 2, the evidence for this is equivocal; in any case, many seed species whose dormancy is terminated by removal of the coat are unaffected by exposure to high tensions of oxygen. Hypochlorite. Sodium hypochlorite breaks seed dormancy in several species including Avenafatua, Polygonum convolvulus and Saponaria vaccaria (see, e.g. [88, 89]). Treatment times must be short (up to 8 h) since prolonged exposure is injurious. The action of this compound may be to modify the seed coat, to allow enhanced entry of water or promotive chemicals, to oxidize inhibitory chemicals in the coat, or to reduce its mechanical rigidity.

3.14.8. Nitrogenous Compounds Nitrate and nitrite have long been known to stimulate germination and to break dormancy in many species. For example, 58 responsive species of graminaceous seeds are listed by Roberts and Smith [16], and many dicotyledonous weed species are also known to be sensitive to treatment with these substances (e.g. [226]). In recent years the action of nitrate, nitrite and hydroxylamine has been intensively examined [16, 82]. The effects ofthese substances have prompted the formulation of hypotheses concerning dormancy mechanisms, but there is no general agreement as to their mechanism of action (see Chap. 4, Sect. 4.5.5). One of the first chemicals shown to break seed dormancy (of lettuce) was thiourea [209]. Seeds of several other species respond similarly (e.g. [180]). Cocklebur(X. pennsylvanicum) seeds are stimulated by thiourea and hydroxylamine but

192

The Release from Dormancy

not by nitrate and nitrite. There is evidence, however, that the two promotive chemicals may act at different sites since the growth behaviour of the embryo differs in response to each one [61]. For example, hydroxylamine appears to induce hypocotyl extension and thiourea promotes a high degree of cotyledon expansion.

3.14.9. Sulfhydryl Compounds Several sulfhydryl-containing chemicals (listed in Table 3.16) have been shown to break dormancy. There is no clear view as to why they are effective, whether by virtue of their reducing action or because of some other property [16].

3.14.10. Various Other Chemicals Including Anaesthetics Many different chemical compounds can break dormancy. We will not deal with all of them here but only with some which seem important because they might affect seeds in the natural environment or because they can tell us something about the possible mechanism of dormancy. Carbon dioxide has long been known to reduce dormancy. Concentrations above 40%, which do not occur in nature, are effective on lettuce [209 a]. But lower concentrations (e.g. 0.5%-5%), which might possibly be in the soil atmosphere in certain situations, terminate dormancy in several species such as Avenafatua and Trifolium subterraneum [23 a, 78 a]. The alcohols, ethanol and methanol, have recently been shown to break seed dormancy of several species including Panicum capillare, Digitaria sanguinalis, D. ischaemum, Echinochloa crus-galli and Setaria faberi; concentrations of about 0.5 M are effective, in some cases (e.g. D.sanguinalis) when combined with a light treatment [199]. Small effects on lettuce seed dormancy are also known [150]. Chloroform, ethyl ether, and acetone reduce dormancy in Panicum capillare seeds [199]. Interestingly, all the aforementioned substances have been considered as anaesthetics, on the basis of their action upon cell membranes (see [199]). We will discuss the importance of the state of membranes in relation to seed dormancy in Chapter 4. Among the other compounds listed in Table 3.16 under "various", methylene blue deserves mention. This well-known electron acceptor reduces dormancy in barley seeds [16]. Its oxidizing action has been fitted into the hypothesis of Roberts, to be discussed in the next chapter.

References

Some Works of General Interest 1. Amen, R.D.: A model of seed dormancy. Botan. Rev. 34, 1-31 (1968) 2. Barton, L.V.: Seed dormancy: general survey. In: Encyclopedia of Plant Physiology. Ruhland, W. (ed.). Berlin: Springer, 1965, Vo!' 15/2, pp. 699-720 3. Borthwick, H.A.: History of phytochrome. In: Phytochrome. Mitrakos, K., Shropshire, W. (eds.). London, New York: Academic Press, 1972, pp. 3-23 4. Briggs, W.R., Rice, H.V.: Ann. Rev. Plant Physio!. 23, 293-334 (1972) 4a. Crocker, W., Barton, L.V.: Physiology of seeds. Waltham, Mass.: Chronica Botanica, 1957 5. Evenari, M.: Seed germination. In: Radiation Biology, Vo!' III. Hollaender, A. (ed.). New York: McGrawHill, 1956, pp. 519-549 6. Evenari, M.: Light and seed dormancy. In: Encyclopedia of Plant Physiology. Ruhland, W. (ed.). Berlin: Springer, 1965, Vo!. 15/2, pp. 804-807 7. Heydecker, W. (ed.).: Seed ecology. London: Butterworths, 1972 8. Jann, R.C., Amen, R.D.: What is germination? In: The Physiology and Biochemistry of Seed Dormancy and Germination. Khan, A.A. (ed.). Amsterdam: North Holland Pub!. Co., 1977, pp. 7-28 9. Kendrick, R.E.: Photocontrol of seed germination. Sci. Prog. (London) 63, 347-367 (1976) 10. Kendrick, R.E., Frankland, B.: Phytochrome and plant growth. Inst. BioI. Stud. BioI., No. 68. London: Arnold, 1976 11. Kendrick, R.E., Smith, H.: The assay and isolation of phytochrome. In: Chemistry and Biochemistry of Plant Pigments. Goodwin, T.W. (ed.). London, New York: Academic Press, 1976, Vo!' 2, 2nd. edn. pp. 334-364 12. Khan, A.A. (ed.): The Physiology and Biochemistry of Seed Dormancy and Germination. Amsterdam: North Holland Pub!. Co., 1977 12a. Mitrakos, K., Shropshire, W. (eds.): Phytochrome. London, New York: Academic Press, 1972 13. Mohr, H.: Lectures on photomorphogenesis. Berlin, Heidelberg, New York: Springer, 1972

193

14. Nikolaeva, M.G.: Physiology of deep dormancy in seeds. Leningrad: Izdakel'stvo 'Nauka'. Isr. Prog. Sci. Trans!., Jerusalem, 1969 14a. Quail, P.H.: Phytochrome. In: Plant Biochemistry. Bonner, J., Varner, J.E. (eds). New York, London: Academic Press, 3rd edn., 1976, pp. 683-711 15. Rollin, P.: Phytochrome control of seed germination. In: Phytochrome. Mitrakos, K., Shropshire, W. (eds.). London, New York: Academic Press, 1972, pp. 229-256 16. Roberts, E.H., Smith, R.D.: In: The Physiology and Biochemistry of Seed Dormancy and Germination. Khan, A.A. (ed.). Amsterdam: North Holland Pub!. Co., 1977, pp. 385-411 17. Rolston, M.P.: Water impermeable seed dormancy. Botan. Rev. 44, 365396 (1978) 18. Satter, R.L., Galston, A.W.: The physiological functions of phytochrome. In: Chemistry and Biochemistry of Plant Pigments. Goodwin, T.W. (ed.). London, New York: Academic Press, 1976, Vo!' I, 2nd edn. pp. 681-735 19. Smith, H.: Phytochrome and photomorphogenesis. London, New York: McGraw-Hill, 1975 20. Smith, H., Kendrick, R.E.: The structure and properties of phytochrome. In: Chemistry and Biochemistry of Plant Pigments. Goodwin, T.W. (ed.). London, New York: Academic Press, 1976 Vol. I, 2nd edn., pp. 378-434 20a. Toole, V.K.: Effects of light, temperature and their interactions on the germination of seeds. Seed Sci. Techno!. 1, 339-396 (1973)

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120a. Koller, D., Sachs, M., Negbi, M.: Plant Cell PhysioI. 5, 85-100 (1965) 12L Lado, P., Rasi-Caldogno, F., Colombo, R.: PhysioI. Plant. 31, 149-152 (1974) 122. Lado, P., Rasi-Caldogno, F., Colombo, R.: PhysioI. Plant. 34, 359-364 (1975) 123. Le Deunff, Y.: C. R. Acad. Sci. Ser. D 279, 1583-1586 (1974) 124. Maier, W.: Jahrb. Wiss. Bot. 78, 1-42 (1933) 125. Malcoste, R.: C. R. Acad. Sci. Ser. D 269, 701-703 (1969) 126. Ma1coste, R.: C. R. Acad. Sci. Ser. D 269, 1415-1418 (1969) 127. Malcoste, R., Boisard, J., Spruit, C.J.P., Rollin, P.: Meded. Landbouwhogesch. Wageningen 70--16, 1-16 (1970) 128. Ma1coste, R., Tzanni, H., Jacques, R., Rollin, P.: Planta 103, 24-34 (1972) 129. Mancinelli, A.L., Borthwick, H.A., Hendricks, S.B.: Bot. Gaz. 127, 1-5 (1966) 130. Mancinelli, A.L., Tolkowsky, A.: Plant PhysioI. 43, 489-494 (1968) 131. Mancinelli, A.L., Yaniv, Z., Smith, P.: Plant PhysioI. 42, 333-337 (1967) 132. McArthur, J.A., Briggs, W.R.: Planta 91, 146-154 (1970) 133. McDonough, W.T.: Nature (London) 214, 1147-1148 (1967) 134. McLemore, B.F., Hansbrough, T.: PhysioI. Plant. 23, I-tO (1970) 135. Miller, C.O.: Plant PhysioI. 31, 318319 (1956) . 136. Morinaga, T.: Am. J. Bot. 13, 141-158 (1926) 137. Mott, J.J., McComb, A.J.: Ann. Bot. (London) 39, 1071-1075 (1975) 138. Nagao, M., Esashi, Y., Tanaka, T., Kumagai, T., Fukumoto, S.: Plant Cell Physiol. 1,39-47 (1959) 139. Nagata, H., Black, M.: J. Jpn. For. Soc. 59, 368-371 (1977) 140. Naylor, J.M., Simpson, G.M.: Can. J. Bot. 39, 281-295 (1961) 141. Negbi, M., Black, M., Bewley, J.D.: Plant PhysioI. 43, 35-40 (1968) 142. Nordstrom, L.: K. Skoghogsk. Skr. 17,91-108 (1953) 143. Nyman, B.: Nature (London) 191, 1219-1220 (1961) 144. Nyman, B.: Studia Forestalia Suecica. No.2, Stockholm: Skogshogsk,1963

145. Ogawara, K., Ono, K.: Proc. Jpn. Acad. 30, 504-509 (1956) 146. Olatoye, S.T., Hall, M.A.: In: Seed Ecology. Heydecker, W. (ed.). London: Butterworths, 1972, pp. 233-240 147. Orlandini, M., Bulard, c.: BioI. Plant. 14, 260--265 (1975) 148. Orlandini, M., Bulard, c.: Trav. Sci. Parco Nat. Vanoise VI, 95-114 (1975) 149. Orlandini, M., Ma1coste, R.: Planta 105,310--316 (1972) 150. Pecket, R.C., Al-Charchapchi, F.: J. Exp. Bot. 58, 7-11 (1978) 151. Pinfield, N.J., Davies, H.V.: Z. PtlanzenphysioI. 90, 171-181 (1978) 152. Pinfield, N.J., Davies, H.V., Stobart, A.K.: Physiol. Plant. 32, 268-272 (1974) 153. Popay, A.I., Roberts, E.H., J. Ecol. 58, 103-122 (1970) 154. Purdie, R.W.: Austr. J. Bot. 25, 35-46 (1977) 155. Quail, P.H.: Ph. D. thesis, Univ. Sydney, Australia (1968) (cited in Simpson [179]) 156. Quinlivan, BJ.: Austr. J. Agric. Res. 19,507-515 (1968) 157. Quinlivan, BJ.: J. Austr. Inst. Agric. Sci. 19, 507-515 (1971) 158. Remer, W.: Ber. Dtsch. Bot. Ges. 22, 328-339 (1904) 159. Resiihr, B.: Planta 30, 471-506 (1939) 160. Reynolds, T., Thompson, P.A.: Physiol. Plant. 28, 516-522 (1973) 161. Richards, F.J.: Ann. Bot. (London) 5, 249-261 (1941) 162. Roberts, E.H.: J. Exp. Bot. 13, 75-94 (1962) 163. Roberts, E.H.: J. Exp. Bot. 16, 341349 (1965) 164. Roberts. E.H., Benjamin, S.K.: Seed Sci. Technol. 7, 379-392 (1979) 165. Roberts, E.H., Totterdell, S.: Plant Cell Environ. 4, 97-106 (1981) 166. Rollin, P.: Rev. Gen. Bot. 63, 461-477 (1956) 167. Rollin, P.: C. R. Acad. Sci. 247, 14841487 (1958) 168. Rollin, P.: Can. J. Bot. 42, 463-471 (1964) 169. Rollin, P.: Bull. Soc. Fr. PhysioI. Veg. 14, 47-63 (1968) 170. Rollin, P., Bidault, Y.: Photochem. PhotobioI. 2, 59-71 (1963) 171. Rollin, P., Maignan, G.: C. R. Acad. Sci. Ser. D 263, 756-759 (1966)

References 172. Rollin, P., Ma1coste, R., Eude, D.: Planta 91, 227-234 (1970) 173. Roth-Bejerano, N., Koller, D., Negbi, M.: Plant Physiol. 41, 962-964 (1966) 174. Roth-Bejerano, N., Koller, D., Negbi, M.: Isr. J. Bot. 20, 28-40 (1971) 175. Scheibe, J., Lang, A.: Plant Physiol. 40, 485-492 (1965) 176. Scheibe, J., Lang, A.: Photochem. Photobiol. 9, 143-150 (1969) 176a. Schonbeck, M.W., Egley, G.H.: Plant Cell Environ. 4, 237-242 (1981) 176b. Schonbeck, M.W., Egley, G.H.: Plant Physiol. 68, 175-179 (1981) 177. Senebier, J.: Mem. Phys. Chym. Vol. III, Geneva 1782 178. Simmonds, J.A., Simpson, G.M.: Can. J. Bot. 49, 1833-1839 (1971) 179. Simpson, G.M.: In: Dormancy and Development Arrest. Clutter, M. (ed.). London, New York: Academic Press, 1978, pp. 168-220 180. Singh, C., Bhan, A.K., Kaul, B.L.: Seed Sci. Technol. 2, 421-425 (1974) 181. Small, J.G.C., Spruit, C.J.P., BlaauwJensen, G., Blaauw, O.H.: Planta 144, 125-131 (1979) 182. Small, J.G.c., Spruit, C.J.P., BlaauwJensen, G., Blaauw, O.H.: Planta 144, 133-136 (1979) 183. Smith, H., Holmes, M.G.: Photochem. Photobiol. 25, 547-550 (1977) 184. Soriano, A.: Rev. Invest. Agric. 7, 315-340 (1953) 185. Spruit, C.J.P., Mancinelli, A.L.: Planta 88, 303-310 (1969) 186. Steams, P., Olson, J.: Am. J. Bot. 45, 53-58 (1958) 187. Steinbauer, G.P., Grigsby, B.: Weeds 5,175-182 (1957) 188. Stokes, P.: In: Encyclopedia of Plant Physiology. Ruhland, W. (ed.). Berlin: Springer, 1965, Vol. 15/2, pp. 746-803 189. Tadmor, N.H., Koller, D., Rawitz, E.: Ktavim (Engl. Ed.) 9, 177-205 (1958) (cited by Evenari, 1965 [6]) 190. Taylor, J.S., Wareing, P.P.: Plant Cell Environ. 2, 165-171 (1979) 191. Taylorson, R.B.: Weed Sci. 17, 144148 (1969) 192. Taylorson, R.B.: Plant Physiol. 55, 1093-1097 (1975) 193. Taylorson, R.B.: Weed Sci. 27, 7-10 (1979) 194. Taylorson, R.B., Hendricks, S.B.: Plant Physiol. 44, 821-825 (1969)

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195. Taylorson, R.B., Hendricks, S.B.: Plant Physiol. 47, 619-622 (1971) 196. Taylorson, R.B., Hendricks, S.B.: Plant Physiol. 49, 127-130 (1972) 197. Taylorson, R.B., Hendricks, S.B.: Plant Physiol. 50, 645-648 (1972) 197a. Taylorson, R.B., Hendricks, S.B.: Plant Physiol. 49, 663-665 (1972) 198. Taylorson, R.B., Hendricks, S.B.: Plant Physiol. 52, 23-27 (1973) 199. Taylorson, R.B., Hendricks, S.B.: Planta 145, 507-510 (1979) 200. Thomas, H., Webb, D.P., Wareing, P.P.: J. Exp. Bot. 24, 958-967 (1973) 201. Thomas, T.H.: In: The Physiology and Biochemistry of Seed Dormancy and Germination. Khan, A.A. (ed.). Amsterdam: North Holland Publ. Co., 1977, pp. 111-144 202. Thomas, T.H., Palevitch, D., Biddington, N.L., Austin, R.B.: Physiol. Plant. 35, 101-106 (1975) 203. Thompson, K., Grime, J.P., Mason, G.: Nature (London) 267, 148-149 (1977) 204. Thompson, P.A.: Physiol. Plant. 21, 833-841 (1968) 205. Thompson, P.A.: J. Exp. Bot. 20, 1-11 (1969) 206. Thompson, P.A.: J. Exp. Bot. 25, 164175 (1974) 207. Thompson, P.A.: New Phytol. 79, 287-290 (1977) 208. Thompson, P.A., Cox, S.A.: Ann. Bot. (London) 42, 51-62 (1978) 209. Thompson, R.C., Kosar, W.P.: Plant Physiol. 14, 567-573 (1939) 209a. Thornton, N.C.: Contrib. Boyce Thompson Inst. 8, 25-40 (1936) 210. Tobin, E.M., Briggs, W.R.: Plant Physiol. 44, 148-150 (1969) 211. Toole, RH., Toole, V.K., Borthwick, H.A., Hendricks, S.B.: Plant Physiol. 30, 15-21 (1955) 212. Toole, E.H., Toole, V.K., Borthwick, H.A., Hendricks, S.B.: Plant Physiol. 30, 473-478 (1955) 213. Toole, E.H., Toole, V.K., Hendricks, S.B., Borthwick, H.A.: Proc. Int. Seed Test. Assoc. 22, 1-9 (1957) 214. Toole, V.K.: J. Agric. Res. 62, 691-715 (1941) 215. Toole, V.K., Borthwick, H.A.: Plant Cell Physiol. 9, 125-136 (1968) 216. Toole, V.K., Koch, E.J.: Crop Sci. 17, 806-811 (1977)

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217. Toole, V.K., Toole, E.H., Borthwick, H.A., Snow, A.G.: Plant Physio!. 37, 228-233 (1962) 218. Totterdell, S., Roberts, E.H.: Plant Cell Environ. 2, 131-137 (1979) 219. Totterdell, S., Roberts, E.H.: Plant Cell Environ. 3, 3-12 (1980) 220. Totterdell, S., Roberts, E.H.: Plant Cell Environ. 4, 75-80 (1981) 221. Vaartaja, 0.: Can. J. Bot. 34, 377-388 (1956) 222. Van Der Woude, W.J., Toole, V.K.: Plant Physio!. 66, 220--224 (1980) 223. Varasova, N.N.: Acta Inst. Bot. Acad. Sci. URSS Ser. IV. Bot. Exp. 11, 370-387 (1956) (cited in Stokes, 1956 [188]) 224. Vicente, M., Noronha, K., Silberschmidt, K., Meneghini, M.: Phyton (Buenos Aires) 25, 11-13 (1968) 225. Vidaver, W., Hsiao, A.I.: Can. J. Bot. 50, 687-689 (1972) 226. Vincent, E.M., Roberts, E.H.: Seed Sci. Techno!. 5, 659-670 (1977) 227. Vincent, E.M., Roberts, E.H.: Seed Sci. Techno!. 7, 3-14 (1979)

228. Visser, T.: Proc. K. Ned. Akad. Wet. C. 57, 175-185 (1954) 229. von Liebenberg, A.: Bot. Zentralb!.18, 21-26 (1884) 230. Wallace, W.R.: J. R. Soc. West. Austr. 49, 33-44 (1965) (cited by Dell, 1980 [53]) 231. Wareing, P.F., Black, M.: Nature (London) 181, 1420--1421 (1958) 232. Wareing, P.F., Saunders, P.F.: Ann. Rev. Plant Physio!. 22, 261-288 (1971) 233. Webb, D.P., Dumbroff, E.B.: Can. J. Bot. 47, 1555-1561 (1969) 234. Webb, D.P., Van Staden, J., Wareing, P.F.: J. Exp. Bot. 81, 741-750 (1973) 235. Wulff, R., Medina, E.: Plant Cell Physio!. 10, 503-511 (1969) 236. Wulff, R., Medina, E.: Plant Cell Physio!. 12, 567-579 (1971) 237. Yaniv, Z., Mancinelli, A.L.: Plant Physio!. 42, 1479-1482 (1967) 238. Yaniv, Z., Mancinelli, A.: Plant Physio!. 43, 117-120 (1968) 239. Yokohama, Y: Bot. Mag. 78, 452-460 (1965)

Chapter 4. The Control of Dormancy

4.1. Introduction Two questions which concern us in this chapter are: (a) What is the mechanism of dormancy? (b) What is the mechanism of the release from dormancy? An answer to one of these questions would obviously contribute towards an answer to the other, since if we understood its mechanism we would also begin to appreciate the changes which must occur in order to end dormancy. To some extent we have already considered the first of these two questions when we discussed dormancy inherent in the embryo and that which is imposed by the tissues surrounding it (Chap. 2). But this division is rather a superficial description of dormancy which tells us little or nothing about its fundamental causes. The basic cause of dormancy is the inability of the embryonic axis to overcome the constraints against growth which are acting upon it; constraints which reside within the embryo itself (embryo dormancy) or belong to the enclosing structures (coat-imposed dormancy). Hence, both forms of dormancy should be seen as reflections of the inadequacy or insufficiency of the embryonic axis, albeit one which may be exposed only by the presence of the coat or, perhaps, by inhibitory influences at work within the embryo itself. We recognize this inadequacy by the failure of the radicle to emerge from the seed, i.e. to complete the germination process. But many events occur during germination (Fig. 4.1) and it seems possible that a block on anyone could account for Hydration - - - - - - Germination - - - - - - - - - Cell expansion- Radicle (Imbibition)

emergence Hormone action?

Enzyme activation Gene activation

Respiratory activity A TP synthesis

Enzyme synthesis

Energy charge increase

Wall loosening Water uptake Turgor changes

Synthesis of cellular components Inter-and intracellular transport

Fig. 4.l. Some cellular events occurring during seed germination. Germination includes those events from initial imbibition until radicle emergence (Chap. 1, Vol. 1). Arrest of any of these could cause dormancy. (see text)

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the failure of the embryonic axis to grow. The problem is to discover where the block(s) lie, but this is a very difficult task with present techniques and the current state of knowledge. What is evident, however, is that dormancy cannot be equated with overall metabolic inactivity, as we shall see. Our approach to the questions posed at the start of this chapter will be as follows: Firstly, we will consider the possible causes of and events associated with dormancy, covering the metabolism of dormant seeds, chemical inhibition and the evidence suggesting that the state of cellular membranes may be of especial importance. Our discussion of the mechanisms involved in the termination of dormancy will be of events occurring at two levels. The first level consists of the perception of the effective factors and the events closely following this, i.e. the primary events. The second level includes all those processes which cannot be directly affected by the releasing factors (e.g. light and cold) but which result from the primary events. To amplify briefly: the production of P rr and its initial action in the dormant seed are primary events which, in themselves, cannot break dormancy. To achieve this, P rr and its action must be "transduced" into processes along the path of germination (Fig. 4.1), which culminates in radicle emergence; these, then are the secondary events.

4.2. Dormancy - Events and Causes 4.2.1. Metabolism of Dormant and After-ripened Seeds

The metabolism of non-dormant seeds during germination has been discussed in considerable detail in Volume 1 of this book. In this section we will concentrate on the metabolism of imbibed, dormant seeds and, where possible, comparisons will be made with that of after-ripened seeds (i.e. those which have lost their dormancy during prolonged storage). The metabolism of seeds whose dormancy has been released by hormones and other chemicals, and by environmental factors (light and cold temperatures) will be discussed in later sections of this chapter. Studies on the respiration of dormant seeds have established that they are capable of gaseous exchange with the surrounding atmosphere. For example, lettuce seeds maintained in the dormant state by incubation on water at 25° C in darkness Table 4.1. Respiratory quotients of dormant and non-dormant excised embryos of Avena Jatua during the period 0-10 h after the start of imbibition on water CO 2 Evolution (pljh per 10 embryos±S.E.)

R.Q.

(pljh per 10 embryos ± S. E.) 12.07±0.55 12.16±0.41

11.74±0.63 11.59±1.14

0.97 0.95

0 1 Uptake

Dormant Non-dormant

After Simmonds and Simpson, 1971 [187]

Dormancy - Events and Causes

201

A

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Fig. 4.2 A, B. Oxygen consumption by Grand Rapids lettuce seeds incubated (A) in darkness on GA3 (0.29 mM) (e); ABA (0.5 mM) + GA3 (0.29 mM) (A); or (B) with an irradiation of 2 min red light after 2 h (e). Seeds imbibed in darkness on water (0). ± S.E. of five replicates is presented. Emergence of radic1es from the first seeds to complete germination was noticed after 9-10 h from the start of imbibition. (A) After Bewley, 1979 [2]; (B) By J. Krochko and Bewley - previously unpublished

consume about 80 J.LI 02/h/ 100 seeds on completion of imbibition (Fig. 4.2A and B), and may continue to do so for several days. Imbibed dormant seeds of Xanthium pennsylvanicum also consume appreciable quantities of oxygen (Fig. 2.12A): after-ripening does not enhance this level of consumption before gennination is completed (Fig. 2.12B). Excised embryos from dormant and after-ripened wild oats (Avena Jatua) respire at equal rates during the first 10 h after imbibition starts, and have the same R .Q. value (Table 4.1). This observation is in contrast to the claim that intact dormant grains of wild oats consume less oxygen than after-ripened grains during the first 10- 12 h [47] . The differences between these results may be related to variation in the length of after-ripening received by each grain batch [187], to restrictions on oxygen uptake by intact grains [22], or to differential respiration in the endospenns (aleurone layer) rather than in the embryos of intact dormant and after-ripened grains. Embryos excised from wild oat grains at intervals of several months during

12

202

The Control of Dormancy

Table 4.2. ATP content of dormant Grand Rapids lettuce seeds imbibed in darkness at 25° C Time from start of imbibition (h)

o 1 3 5 7 9

ATP content (nmolf300 mg seeds dry wt)

o 71

113 114 123 121

By J. Krochko and Bewley-previously unpublished after-ripening, and then imbibed, consume similar amounts of oxygen [187] - evidence that loss of dormancy need not be accompanied by any substantial changes in respiratory capacity. Oxygen uptake and CO 2 output by non-dormant barley and rice grains have been reported to be less than that by dormant grains during the first 6 h from the start of imbibition [140]. Measurements of respiration by dehusked, dormant, and after-ripened grains, and of excised embryos therefrom, were not made, however. Thus we do not know if the respiratory changes were in the embryo per se. In some species, including certain cereals, oxidation of seed coat components occurs upon imbibition, thus making difficult the measurement of respiration in terms of oxygen consumption alone - see Chapter 2, Section 2.4.2 for further details. From references cited in [140], it appears that differences in respiration between dormant and after-ripened cereal grains are not commonly observed. Endogenous levels of malic acid in grains of wild oats appear to be correlated inversely with dormancy of the population. On the other hand, levels of another citric acid cycle intermediate, succinic acid, are similar in dormant and after-ripened grains [92]. The significance, if any, of this observation to respiration and to dormancy is obscure. Oxygen consumption by dormant lettuce seeds increases over the first 2 h from the start of imbibition, and then levels off (Fig. 4.2A and B). Dormant lettuce seeds synthesize A TP from early times of imbibition and maintain high levels (Table 4.2). Likewise, axes from dormant seeds of maple (Acer saccharum) incubated at 20° C synthesize considerable amounts of ATP, resulting in a high energy charge (Table 4.13). Thus the inability of dormant seeds to germinate is not due to an overall inhibition of respiratory metabolism nor, it appears, to an impairment of energy production in the form of ATP. Incorporation of radioactive precursors into protein and RNA occurs in the dormant seeds of a number of species, including lettuce [35, 77, 78], Agrostemma githago [95], wild oat [47], cocklebur [182], Vaccaria pyramidata [97], Melandrium noctiflorum [96], and Paulownia tomentosa [86]. Failure to detect synthesis of proteins and RNA in other dormant seeds probably can be attributed to inadequate uptake of radioactive precursor to the sites of synthesis within the seed. Polyribosomes are absent from dry lettuce seeds but are formed during and following imbibition in conditions which maintain dormancy (Fig. 4.3A). Incorporation of radioactive leucine into protein accompanies polyribosome formation (Fig. 4.3A). A

Fig. 4.3. (A) Changes in fresh weight (.A.), polyribosome levels (D), and protein synthesis (e) in dormant lettuce seeds (cv. Grand Rapids) imbibed in darkness on water at 25 °C. Treatment of the ribosomal pellet, extracted from dry seeds, with pancreatic ribonuclease does not reduce the area under the polyribosome peak. Hence dry seeds contain no polyribosomes. (The apparent basal level of 15% polysomes is due to absorption by free ribosomes alone.) After Fountain and Bewley, 1973 [76]. (B) Protein synthesis by longdormant lettuce seeds. Seeds were incubated on water in darkness at 25 °C for the times indicated, the embryos then were dissected out and protein synthesis monitored for I h by following incorporation of supplied 3H-Ieucine into a buffer-soluble protein fraction. By Fountain, 1974 [75]

report [144] that polyribosomes are formed only after a germination stimulus (i.e. light) is received by lettuce seeds is incorrect: the techniques used for polyribosome extraction and analysis probably were inadequate. Even after being hydrated for 96 h in darkness, dormant lettuce embryos stiII are capable of protein synthesis, albeit at a reduced rate in comparison to earlier times (Fig. 4.3B). The claims that dormant seeds of Portulaca oleracea (common purslane) conduct very little protein synthesis and no RNA synthesis [172, 173] are questionable - again on the basis of the techniques used. Autoradiographic studies have shown that protein synthesis occurs in the axis, scuteIIum, coleorhiza and aleurone layer of dormant wild oat grains. The amount

204

The Control of Dormancy

of synthesis in the whole embryos, however, is not substantially different from that in the embryos of after-ripened grains [47,52]. While a quantitative analysis of soluble protein synthesis in dormant and after-ripened wild oat grains remains to be carried out, an analysis of membrane proteins has shown that their turnover in dormant grains is greater than in non-dormant (presumably after-ripened) grains, although no new classes of membrane proteins are produced [52]. Phospholipid components of the membrane (and in particular phosphatidyl inositol) also are synthesized and degraded continuously in the dormant wild oat embryo [53]. It has been suggested [52, 53] that this continuous generation and recycling of cellular membranes is necessary to maintain the metabolic integrity of the hydrated cells of the dormant embryo. This might be important in relation to the maintenance of cellular activity during long-term dormancy in the hydrated state (see Sect. 1.7.l). So far, membrane turnover has been demonstrated to occur in wild oats over a 7-day period: whether such a mechanism could remain in operation for at least 6 years, as would be required for dormant Fraxinus excelsior seeds (Sect. 1.7.1) remains to be demonstrated. Some workers have considered that seeds maintain dormancy because of their inability to produce hydrolytic enzymes to mobilize their major stored reserves. This is unlikely because, as emphasized in Volume 1, the mobilization of reserves is almost exclusively a post-germination phenomenon, and is not required for radicle emergence to occur. For similar reasons, dormancy of wild oat grains is probably not due to an inability to convert the products of starch hydrolysis to sucrose [46]. 4.2.2. Dormancy and Maturation Seeds of ash (Fraxinus excelsior) are shed from the mother tree while their embryos are incompletely developed. After imbibition of the seed the embryo matures (a process that takes about 3 months at room temperature) but the mature embryo remains deeply dormant and requires chilling (cold-stratification) for dormancy to be broken (Chap. 3). An ultrastructural study has been made of the developmental changes which the dormant ash embryo undergoes during maturation, when the morphologically complete embryo grows in size from approximately half to nearly the full size of the seed [226]. Its lipid content falls from 20% to 4% of the dry weight, and lipid bodies disappear. Protein content increases 14%-24%. Starch grains (amyloplasts) also form, particularly in the regions of the inner cortex and root cap. Rough endoplasmic reticulum appears to proliferate and dictyosomes become more numerous. These observations illustrate the extensive changes which cells ofthese embryos undergo - their dormancy cannot be equated with metabolic inactivity. 4.2.3. Chemical Inhibition We have already considered the possibility that inhibitors are responsible for the imposition and maintenance of dormancy. The evidence for this is equivocal and there is no proof that such chemicals are the cause of dormancy. For example, it

Dormancy - Events and Causes Stage 0

Stage 1

Stage 2

OOQ

205

Stage 3

Outer

tesla -

Fig. 4.4. Stages of growth in germinated seeds of Chenopodium album. Stage 0 stage before growth; Stage 1 outer testa split near radicle; Stage 2 radicle extends but is retained within inner testa and endosperm layer; Stage 3 radicle penetrates inner layers. After Karssen, 1976 [116]

seemed possible that dormant seeds have higher levels of abscisic acid (ABA) than do the non-dormant kinds; this, indeed, may sometimes be so, e.g. in two species of Fraxinus, F. americana (dormant) and F. ornus (non-dormant) [195], and among several Rosa spp. [108 a]. However, no such difference occurs between the non-dormant Avena sativa and the highly dormant A.fatua [28 a] or among species and cultivars of Pyrus with different degrees of dormancy [58]; and many non-dormant seeds, such as the Great Lakes cultivar of lettuce [142], and Acer saccharinum [181 a] contain very high amounts of ABA - in these particular cases ca. 700 Ilg/kg FW! But because inhibitors have attracted so much attention, and have so often been invoked as causal agents, it is worth spending some time to consider them further. If we attribute dormancy to the action of these inhibitors, we must then enquire as to how they operate; but, unfortunately, there is very little information available to help us to deal with this question. Of the inhibitors commonly present in seeds, most is known about the biochemical and physiological action of ABA. Applied ABA can inhibit nucleic acid and protein synthesis (Sec. 4.5.2), and so it is possible that these processes are held in check by endogenous inhibitor. On this basis, the action of the inhibitor in controlling dormancy would be to prevent these essential metabolic processes which, in a seed proceeding through germination, start very soon after the beginning of imbibition (Chap. 5, Vol. 1). Thus, germination would be inhibited in its early stages and dormancy would then be marked by lowered metabolic activity even in the early period after initial hydration. It is known, however, that even in dormant seeds which contain ABA (e.g. lettuce [142]) considerable synthesis of macromolecules occurs. But an equally compelling argument against the role of ABA is that this inhibitor can be shown to prevent only the very latest phase of germination, that involving cell expansion itself. Studies by Karssen on Chenopodium album demonstrate this particularly well [116]. Radicle growth in this seed can be divided temporally into 3 stages (Fig. 4.4). The radicle splits the outer integument (testa) in stage 1, but the inner testa remains intact; in stage 2, further growth occurs, still inside the intact inner integument, but finally, in stage 3, the radicle bursts through this coat. Application of different concentrations of ABA to germinating seeds does not stop them reaching stages 1 and 2,

206

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The Control of Dormancy Fig. 4.5. The effect of abscisic acid on the three stages of radicle growth in unscarified Chenopodium album seeds irradiated with 1 h red light at 24 h intervals. Vertical bars standard deviation of the mean. The stages are numbered as in Fig. 4.4. After Karssen, 1976 [116]

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but stage 3 is arrested (Fig. 4.5). Studies with 14C-Iabelled ABA demonstrate that substantial amounts of applied inhibitor enter the seeds within 16 h from the start of imbibition - well before seeds have reached stage 1. Work on lettuce [32], Sinapis alba [185] and Haplopappus gracilis [82] also suggests a late action of abscisic acid. Addition of this inhibitor to Sinapis can be delayed for several hours after the start of imbibition, but it nevertheless stops radicle elongation as long as it is applied just before this process is due to start. We should note that in both Sinapis and Haplopappus ABA does not appreciably affect an already elongating radicle, suggesting that the processes for the initiation of elongation are different from those required for its continuation. This point, incidentally, also arises out of studies on osmotic inhibition of germination and radicle growth [98, 99]. As far as the action of ABA is concerned, this conclusion seems reasonable: the inhibitor does not arrest the early events of germination but instead acts upon incipient radicle emergence. Support for this view comes from the observation that ABA prevents the increase in acidification of the medium surrounding Raphanus sativus seeds and Zea mays embryos [131] and inhibits the increase in transmembrance potential in cortical cells of Raphanus axes [23 a]. Acidification and the change in potential are considered to be due to proton extrusion which may be an essential prelude to cell elongation [49]. If we accept that an inhibitor such as ABA is present within the seed and maintains dormancy, but acts only at the time of radicle emergence, it means that dormant seeds complete almost all of the germination processes and are held, poised, with their radicles ready to elongate. This hardly seems likely, however; one telling argument against it is that in many seeds (e.g. apple [136]) endogenous ABA disappears long before dormancy has been terminated by chilling; and it even disappears at temperatures at which dormancy is retained! [23] - see Section 4.5.3. We can find little support, therefore, for the possibility that dormancy generally is imposed by an inhibitor such as ABA; further doubt comes from studies on the fate of ABA during the breaking of dormancy, discussed in Section 4.4.3. Of course, it is arguable that a controlling chemical other than ABA, or one with similar properties, is operative. There is no strong candidate for such a role and so the

Dormancy - Events and Causes

207

matter remains an open question for now. We should nevertheless note the claims that have been presented for the participation of certain short-chain fatty acids in the control of dormancy. These substances, particularly the C 7 , C s and C 9 acids, have attracted interest since the discovery that they can inhibit various processes in plants, including seed germination [28, 199]. In Avena spp. the occurrence of the acids correlates well with dormancy. At harvest, in the virtually non-dormant A. sativa the total C 6-C 10 acids amounts to 172 ng/grain, (31 ng are the inhibitory C 7 , C s and C 9 ), whereas the highly dormant A.fatua contains 4482 ng/grain, of which 2458 ng are C 7 , C s and C 9 acids [28 a]. Such levels could well account for the failure of A.Jatua to germinate, but much needs to be done before we can be sure that these fatty acids are indeed involved in the control of dormancy. One reason why such substances are particularly interesting, however, is that they might participate in the metabolism of membrane constituents; their incorporation into phospholipids, for example, could affect the properties of membranes. As we shall see in later sections, there is some evidence to suggest that the state of cell membranes is important in the maintenance and relief of dormancy.

4.2.4. Membrane Properties and Dormancy Much of the evidence linking dormancy with the state of cell membranes comes from the effects of temperature on germination and upon the termination of dormancy. In this section, we will mainly consider the first of these two but also touch upon the second. In Chapters 2 and 3 we referred to cases of relative dormancy, in which dormancy is expressed only at comparatively high temperatures. When we examine this response to temperature a feature which strikes us is that dormancy is not increasingly displayed as the temperature rises but rather that it develops abruptly when a particular temperature is exceeded. In Figure 4.6, dormancy is expressed as the mean germination period as a function of temperature. It is apparent that seeds germinate readily (i.e. they have little or no dormancy) at all temperatures below approximately 18° C, but at this temperature there is a precipitous decline in germinability when dormancy becomes expressed. This characteristic is well known in numerous species, including many which require light for the termination of their dormancy (see Fig. 3.22). The sharp discontinuity in the response to temperature presumably results from a sudden change in the condition of the cells in the embryo. One explanation of this is that a subtle balance of metabolic reactions proceeding at different rates is achieved at a particular temperature. But another possibility which is attracting much attention is that cell membranes are involved, since they are known to undergo sharp transitions in their physical state at particular temperatures; and consequently, enzymes associated with them undergo abrupt changes in activity too (see, e.g. [170]). These reversible thermotrophic transitions are changes in the lipid bilayers from an ordered quasi-crystalline phase (gel) at low temperatures, to a random, fluid phase at higher temperature [21, 143, 169] (Fig. 4.7A and B).The temperature at which the transition takes place depends upon the class of lipid in the membrane, the length of the non-polar acyl chain, the extent of unsaturation of

208

The Control of Dormancy Fig. 4.6. Temperature-dependent mancy in barley (e) and wheat After Roberts and Smith, 1977 barley; and Gosling and Butler, viously unpublished, wheat

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Dormancy - Events and Causes

209

Increase Temperature

........ Decrease liquid crystalline

Below transition temperatu re (Quasi-crystalline phase)

Downward transition (decrease in temperature)

Upward transition (increase in temperature)

Above transition temperature ( liquid-crystalline phase)

Fig. 4.7 a, b. Changes in the state of membranes with temperature transitions. (a) Changes in the phospholipid bilayer from the crystalline (gel) phase to liquid crystalline phase. (b) Possible changes involving the disposition of membrane proteins (a-i). (b) After Armond and Stahlein, 1972 [21J

at a temperature (32°-35° C) which is similarly effective in seeds of several other species (e.g. Barbarea verna, Fig. 4.8); a membrane transition at this temperature seems to be common to many species [103, 105]. We should note that determinations of leakage have been made on whole seeds and therefore the measured changes represent only an average for the seed. We do not know whether the increased permeability discussed above occurs in all the regions of the seed (axis, cotyledons, endosperm) or only in certain parts. Obviously, if the changes are to have any meaning with regard to dormancy we would expect them to take place at least in the embryonic axis, and particularly the radic1e/hypocotyl. Transitions in membranes isolated from seeds have been followed by means of fluorescent probes. The latter are mostly polycyclic hydrocarbons which fluoresce when excited with ultra-violet light (ca. 360 nm), at wavelengths which depend upon the polarity of the probe's environment and its viscosity. These molecular probes, when associated with an ordered, crystalline membrane, are orientated at the lipid- water interface, but as the membrane becomes more fluid the probes can enter the non-polar environment inside the membrane; the fluorescence peak then shifts to a shorter wavelength. By the use of this technique upon isolated, fairly

210

The Control of Dormancy

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The Control of Dormancy

a more comprehensive list of a wider variety of seed species and chemicals which have been tested, the reader is directed to the references in the footnote to this table. Not all cultivars (or harvests) of the cited cereal grains and dicot seeds respond in a similar quantitative manner to applied inhibitors: in some cultivars they promote little or no germination over the same concentration range that they are strongly promotive in others. The reasons for this are not known: it is possible that penetration of inhibitors into vital tissues is limiting in the unstimulated seeds. The most effective germination promoters in rice (cv. Radin China 4) are inhibitors of terminal oxidation reactions of the mitochondrial electron transport chain. While these are effective in other cereals too, inhibitors of the citric acid cycle (e.g. malonate in wild oats and Rika barley) and of glycolysis (in Rika barley) also promote germination (Table 4.10). In an attempt to explain these and other observations, Roberts has proposed a mechanism for dormancy breaking which depends upon shifts in respiratory metabolism [8, 9]. We do not intend to consider in detail the arguments for and against this proposal- ample discussion of the indirect evidence in its favour has already been published [8-10]. Here we will content ourselves with an overview of the theory, its strengths and its weaknesses. The initial premise inherent in Roberts' proposal is that dormant seeds have certain respiratory deficiencies which are absent from non-dormant seeds. One such deficiency apparently is imposed by the citric acid cycle, which is supraoptimal in its activity and utilizes available oxygen to the exclusion of other oxygenrequiring processes. It is one of these latter processes which must operate successfully for dormancy to be broken. Raising the ambient oxygen for dormant seeds may promote germination by providing an excess of oxygen which can be utilized by the normally oxygen-starved processes. The proposal also assumes that attenuation of the electron transport chain by inhibitors of the citric acid cycle and terminal oxidation reactions reduces their requirement for oxygen, thus freeing more for the alternative processes which can then proceed, resulting in dormancy breaking. Net oxygen uptake by dormant and non-dormant seeds may be expected to be the same, only the pathway through which it is utilized will be different. A diagrammatic representation of the proposed scheme is presented below. Dormont

Dormancy

1

0 2-

. Requir ing processes

Non- dormont

processes Germination

The pentose phosphate pathway has been suggested to be the alternative oxygen-requiring process essential for germination [8, 9,188]. This pathway is cyanide-

Secondary Events in the Release from Dormancy

247

insensitive, but requires regeneration of NADP by oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH 2 ) - it is claimed that this reaction is limiting under conditions of low oxygen availability. Promotion to germination of dormant seeds by hydrogen acceptors (e.g. nitrate, nitrite and methylene blue: Table 4.10) is assumed to be mediated through re-oxidation ofNADPH 2 (nitrate and nitrite presumably induce their appropriate NADPHrdependent reductases), thus stimulating operation of the pentose phosphate pathway. Approximate estimations of the relative activities of the glycolytic and pentose phosphate pathways in dormant seeds have been obtained experimentally by comparing their ability to utilize glucose-6-l 4C and glucose-1-l 4C. The technique involves application of glucose-6-l 4C and glucose-1-l 4C to separate samples of the same tissue followed by collection and estimation of evolved l4C0 2 from each. The results are expressed as a C 6 /C l ratio. The success of the technique depends upon the fact that during glycolysis a glucose molecule is split into two 3C units and the carbons in position I and 6 of the glucose molecule both end up in the same position in pyruvate. Consequently both units are decarboxylated in an identical fashion in the citric acid cycle. Thus if all glucose-6-l 4C and glucose-1-l 4C applied to a tissue is respired solely by glycolysis, then the C 6 /C l ratio will be unity. On the other hand, if the pentose phosphate pathway is operative the carbon in position 1 of glucose becomes removed by 6-phosphogluconate dehydrogenase during the conversion of 6-phosphogluconate to ribulose-5-phosphate; carbon 6 is not removed. Hence the operation of the pentose phosphate pathway in a tissue reduces the C 6 /C l ratio due to the greater release of l4C0 2 from glucose-1-l 4C. Non-dormant grains of Pallas barley show a lower C 6 /C l ratio than do dormant grains ([8] and Table 5.5, Vol. 1), which is indicative of a more active pentose phosphate pathway. Similarly, excised embryos of dormant wild oats have a C 6 /C l ratio higher than that of non-dormant (after-ripened) embryos [187]. The C 6 /C l ratio of sour cherry (Prunus cerasus) seeds declines with increased time of after-ripening [128]. That release from dormancy by chemical agents is associated with ratio changes has been demonstrated for oats and two barley cultivars (Table 4.11), although some changes are small, e.g. the promotive effects ofKCN and NaN0 2 on Proctor barley germination are not striking. The validity of using C 6 /C l ratios to obtain quantitative estimates of the amounts of glucose entering glycolysis or the pentose pathway has been questioned [54]. It has been pointed out, for example, that the rate at which l4C0 2 appears from labelled glucose depends in part upon the pool size of various intermediates. If large pools of citric acid cycle intermediates accumulate within a seed (perhaps due to metabolic changes induced by dormancy-breaking agents) then these would effectively dilute the radioactivity of carbon entering the cycle, giving a low yield of l4C0 2 . Glucose passing through the pentose phosphate pathway would (assuming a small pool of hexose phosphates) release C l with relatively little dilution. This would lead to over-estimation of pentose phosphate pathway activity. On the other hand, under-estimation of the pentose phosphate pathway would result from reversibility of the aldolase reaction (see Sect. 5.1.7, Vol. 1) in association with rapid triose phosphate isomerization, which would tend to equilibrate C 6 + Cl. Thus, although there is an apparent tendency for dormancy-breaking agents to lower the C 6 /C l ratio, the changes might not be as significant as they appear to be.

248

The Control of Dormancy

Table 4.1 1. Changes in the C6/C 1 ratio prior to radicle emergence associated with breaking of cereal grain dormancy by various chemicals Species

Avenafatua (embryos) Barley grains cv. Golden Promise Barley grains cv. Proctor

Chemical treatment

GA3 (50 ppm) Malonate (1O- 2 M) 2-Mercaptoethanol (5x to- 2M) Dithiothreitol (to-1M) GA 3(tO- 3M) NaN0 2(10- 2 M)

KCN(IO- 3M)

Reference

C6/C 1 ratio Treated

Untreated dormant control

0.64-0.65 0.73-0.80 (83) 0.03-0.04 (89)

0.76-0.84 0.83-0.85 (17) 0.36-0.42 (8)

[187] [188] [9]

0.07-0.09 (90)

0.2-0.37 (to)

[9]

0.11 (100) 0.20 (32) 0.16 (26)

0.24 (10) 0.22 (10) 0.19 (10)

[8] [8] [8]

Brackets after the C 6/C 1 ratios for treated and untreated dormant control values show, where known, the final germination percentage promoted by chemical treatment

If we are to accept that the operation of the pentose phosphate pathway is in some way important for the breaking of dormancy, then the answers to two key questions must be sought. Namely, how is the pentose phosphate pathway blocked in dormant seeds and what does this pathway provide which is essential for stimulation of germination? In relation to the first question, direct comparisons between the quantities and activities of two pentose phosphate dehydrogenases - glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase - in dormant and non-dormant barley grains have shown no differences prior to radicle emergence from the latter (unpublished results cited in [9]). Likewise, no significant differences have been found between the levels of oxidized and reduced forms of the co-enzymes involved in glycolysis (NADjNADH 2 ) and the pentose phosphate pathway (NADPj NADPH 2 ) in dormant and non-dormant grains over the same time period (unpublished results cited in [9]). Thus, the relative inactivity of the pentose phosphate pathway in dormant barley would not appear to be due to the absence of these dehydrogenases or co-enzymes, although estimations of enzyme activities in vitro may not reflect their activities in vivo. Levels of extractable glucose-6-phosphate dehydrogenase rise with increasing time after imbibition in non-dormant grains of wild oats, but decline in dormant grains (Fig. 4.27). Levels of 6-phosphogluconate dehydrogenase change similarly, but to a lesser extent (Fig. 4.27). Enzymes of the glycolysis pathway either remain at constant levels (e.g. aldolase) in dormant and non-dormant grains for 48 h after imbibition, or increase equally (e.g. glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase) in both [127]. It has been suggested [127], therefore, that the ability to maintain or increase the initial levels of pentose phosphate pathway enzymes following imbibition is obligatory for germination. This might be so, but the evidence is not conclusive. First, the whole grain was used for these experiments, and changes in enzyme levels in the aleurone layer could be a contribut-

Secondary Events in the Release from Dormancy

249

Fig. 4.27 A, B. Enzyme activities during steeping of dormant (0) and non-dormant (.) wild oat grains. Activities of (A) glucose-6-phosphate dehydrogenase and (B) 6-phosphogluconate dehydrogenase are expressed in Katals (activity effecting the conversion of one mol of substrate per s). Arrow indicates the time of radicle protrusion from the first grains of a population of non-dormant grains. After Kovacs and Simpson, 1976 [127]

ing factor. Second, radicle protrusion from non-dormant grains commences after about 12 h, 50% complete germination by 16 h and most members of a population complete germination by 24 h (Simpson - personal communication). Hence it is difficult to ascribe the block to germination of dormant grains to differences in levels of pentose phosphate pathway enzymes alone, for their levels in non-dormant and dormant grains are similar over the first 12 h, and yet only the former will germinate subsequently. The marked increase in enzyme levels after 12 h, and particularly after 24 h, in non-dormant grains presumably is associated with seedling establishment as more grains complete their germination. For, as indicated in Volume 1, Chapter 5, the pentose phosphate pathway appears to play an increasingly important role in seedling tissues during their development [240]. The decline in extractable pentose phosphate pathway enzyme activity in dormant grains at longer times after imbibition is interesting. It would be of relevance to determine if dormancy-breaking agents, such as GA, applied after 36-48 h, induce germination and a concomitant increase in enzyme activities. Grains whose dormancy has been broken by imbibing and then steeping in GA for 5 days contain higher levels of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase than those steeped continuously in water (Fig. 4.28). Some GA-treated grains will have completed germination by this time, however, and the increases in enzyme levels could reflect changes during seedling development rather than during germination. Non-dormant grains steeped for 5 days in water or GA have (in comparison to dormant grains) higher, but identical, enzyme levels (Fig. 4.28). By 5 days, though, grains subjected to both treatments will have developed into quite large seedlings. Here again we feel that more experiments are required to show that changes in pentose phosphate pathway enzymes are associated with the germination process per se. On a technical note, Gosling and Ross [84] have found that assays of glucose6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase involving crude extracts from cotyledons of hazel do not provide for accurate quantification of their activities. They recommend that these enzymes be partially purified before

250

-'>

The Control of Dormancy Fig. 4.28. Effect of GA (flll) on the levels of 6-phosphogluconate dehydrogenase (6PGDH) and glucose-6-phosphate dehydrogenase (G6PDH) activities in dormant (D) and nondormant (ND) wild oat grains steeped for 5 days. 0: water controls. After Kovacs and Simpson, 1976 [127]

..,

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being assayed. They also note that the above-mentioned studies on these two enzymes from wild oats employed crude extracts: hence the results should be treated with due caution. Gosling and Ross [85] themselves have noted that glucose-6phosphate dehydrogenase and 6-phosphogluconate dehydrogenase increase in hazel cotyledons during cold-stratification at 5° C (using crude and partially purified extracts) compared to warm-stratified controls. The major increase occurs coincidentally with the breaking of dormancy, and prior to the completion of germination. While this is an interesting observation, it is not clear how changes in a respiratory path within the cotyledons might affect the embryonic axis and cause it to change from a dormant to a germinating state. Using the previously-mentioned, and not necessarily ideal, criteria for pentose phosphate pathway activity (C 6 /C 1 ratios; glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities), it appears that this pathway is no more active in non-dormant (i.e. after-ripened) lower cocklebur seeds than it is in dormant lower ones [183]. As mentioned previously, it has been suggested that the rate of reoxidation of NADPH 2 is a limiting reaction in dormant grains. This could be a consequence of oxygen starvation from an active NADPH 2 -oxidase system because of the greater affinity of oxygen for cytochrome oxidase of the electron-transport chain. Alternatively, the limitation could result from the relative inactivity of the NADPH 2 -oxidase system in dormant grains. What is the nature of this oxidase system? A variety of mechanisms has been suggested for the re-oxidation ofNADPH 2 in plants [19] but none of them appears to fit the requirements for the postulated NADPH 2 -oxidase system. The role of the pentose phosphate pathway is to provide NADPH 2 for reductive biosynthesis and, as yet, no other unique function for the pathway has been demonstrated in any plant. Thus, in relation to its putative role in dormancy breaking, the significance of enhanced pentose phosphate pathway activity is enigmatic. It provides no intermediates which have yet been shown to be essential for the completion of germination and, furthermore, reoxidation ofNADPH 2 does not appear directly to involve molecular oxygen which is assumed to be made more available as a consequence of reduced cytochrome oxidase activity. Acceptance, at

Secondary Events in the Release from Dormancy

251

the present time, of the pentose phosphate pathway as the key to dormancy breaking also requires an assumption that the pathway provides some unknown vital intermediate, and/or there is reoxidation ofNADPH z by an undefined oxidase system (but see later). There can be little doubt that for termination of dormancy a critical supply of ATP is required. The precise mechanism by which ATP is synthesized during early imbibition and prior to the completion of germination is not known although there are several possible pathways, including an alternative electron transport system which does not utilize cytochrome oxidase (and, like the pentose phosphate pathway, is cyanide insensitive!) - is this, therefore, a candidate for being the alternative oxygen-requiring process? The effects on ATP synthesis of blocking reactions of the citric acid cycle and electron transport chain have not been measured but, obviously, sufficient ATP still must be produced for germination to occur. At high concentrations of cyanide and azide, though, germination is no longer promoted, but inhibited [177, 211]. It is possible that at low, promotive concentrations of inhibitors reactions of the citric acid cycle and electron transport chain still proceed (as suggested by the relatively high C 6 /C 1 ratios for most seeds in Table 4.11) to the extent that critical ATP levels can be maintained. High inhibitor concentrations might promote germination, but this promotive effect presumably is negated by their inhibition of ATP synthesis. The extent of ATP formation via nucleotide synthesis pathways or via substrate level phosphorylation is not known, and so the ameliorating effects of these alternative methods of synthesis on the ATP pools in the presence of inhibitors must remain a subject for speculation. Much of the indirect evidence in support of Roberts' proposal comes from studies using respiratory inhibitors. The specificity of these inhibitors has almost invariably been assumed, and not tested critically. Such assumptions may, in some instances, have been too bold. Low concentrations of cyanide promote germination of pigweed (Amaranthus albus) and lettuce (Lactuca sativa) seeds [100]. This observation at first was held to be consistent with the notion that a promotive, cyanide-insensitive pathway is favoured due to inhibition of terminal oxidases by this chemical. Unfortunately, promotive low concentrations of cyanide do not reduce oxygen uptake by the seeds - only higher concentrations which inhibit germination do so [211]. Another explanation for this effect has been proposed recently. Cyanide supplied to these seeds reacts with cysteine to yield f3-cyanolalanine and hydrogen sulphide - activity of the enzyme responsible for this reaction must be high because no free cyanide accumulates within the seed. The f3-cyanolalanine then is converted to asparagine and aspartic acid, which may be incorporated into protein. The following scheme outlines the pathway: HCN + L-Cysteine

a

) f3L-Cyanolalanine+H zS

H2 O L-Aspartic Acid ,

I

c

! 1

Protein

bE L-Asparagine

I

H2 O

252

The Control of Dormancy Fig. 4.29. Effects of various concentrations of thiourea on germination of lettuce seeds after 48 h (e) and inhibition of catalase activity (0) after 24h. Catalase activity of controls incubated on water was 8.5±O.1 pmol of H 2 0 2 lost/mg protein/ min. After Hendricks and Taylorson, 1975 [102]

100 e

Catalase inhibition

60

,

e ./

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Thiourea conc. (log

Ml

Enzymes: (a) L-cysteine hydrogen sulphide lyase, (b) pL-cyanolalanine lyase and (c) L-asparaginase. p-Cyanoalanine promotes germination of Amaranthus, as to a lesser extent does aspartate, although asparagine does not, perhaps due to restrictions on uptake into the seed [211]. It is claimed that aspartate/asparagine incorporation into proteins influences germination of Amaranthus, lettuce, and also Lepidium virginicum seeds imbibed in cyanide solutions. This implies that proteins essential for germination require the presence of these amino acids for their synthesis, and that these are normally limiting in dormant seeds. As yet, there is no evidence that this is so. We still need to know, for example, if proteins synthesized by seeds stimulated to germinate are qualitatively different from those of dormant seeds, and if proteins in germinating seeds contain higher levels of aspartate/asparagine than those of dormant seeds. More recently, another hypothesis for dormancy-breaking has been put forward, this time to explain the promotive action of thiourea, nitrite and hydroxylamine salts on lettuce and Amaranthus seeds [102]. Promotion of germination by these agents is accompanied by an irreversible inhibition of extractable catalase activity, which is most striking for thiourea-induced lettuce seed germination (Fig. 4.29). Neither respiration nor peroxidase activity are affected at concentrations of these promoters which stimulate germination. These observations have led to the suggestion that inhibition of catalase activity spares H 2 0 2 for peroxidase action which is linked to the pentose phosphate pathway in the following manner: H2 0

Inhibitors

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rt

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Substrates

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ff

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Germination

Secondary Events in the Release from Dormancy

253

Enzymes: (a) o-glucose-6-phosphate: NADP I-oxidoreductase; (b) 6-phospho-ogluconate-5-lactone hydrolase; (c) 6-phospho-o-gluconate: NADP 2-oxidoreductase (decarboxylating); (d) NAD(P)H quinone oxidoreductase; (e) peroxidase; (0 catalase; (g) ATP: NAD 2'-phosphotransferase. Although some enzymes (e.g. d and e) are present in dry and imbibed lettuce and Amaranthus seeds [WI], it has not been shown that they or other enzymes are co-operatively linked together in the manner shown in the above scheme, although it has been suggested that in wheat germ there exists an NADPH 2 -oxidase system which involves two different proteins, one of which is a peroxidase [50]. Other weaknesses in this scheme include the unsubstantiated assumption that the pentose phosphate pathway and reoxidation of NADPH 2 are important for dormancy breaking in lettuce and Amaranthus, and that H 2 0 2 is produced prior to the completion of germination. Hydrogen peroxide evolution occurs during fJ-oxidation of fatty acids in glyoxysomes (Chap. 6, Vo1.I), but mobilization oflipid reserves cannot be detected in lettuce axes, or even whole seeds, until many hours after radicle emergence [89]. Furthermore, isocitrate lyase, a marker enzyme for glyoxysome activity, is not present in the germinating lettuce seed [133]. The possibility that the pentose phosphate pathway is not involved in dormancy breaking oflettuce is suggested by the claim that neither light, GA, nor mercaptoethanol cause shifts in the C 6 /C 1 ratio [10]. But the effects of thiourea on C 6 /C 1 ratios have not been tested nor, on the other hand, is it known iflight- and GA-induced germination oflettuce seed is accompanied by changes in catalase activity. A further weakness in the proposal relates to the time at which catalase was measured. As noted in the legend to Figure 4.29 catalase was only assayed 24 h from the start of imbibition, and it was not stated how many seeds had completed germination at this time. As shown in Figure 4.30, lettuce seeds ofthe same cultivar will complete germination on 1O-1-1O- 3 M thiourea within 12-24 h. Henceitis debatable that thiourea could be affecting catalase activity associated with growth, rather than with germination. Certainly, at 12 h from the start of imbibition there is little or no suppression of catalase activity by thiourea [2]. Finally in this section, a brief word about o-threo- and L-chloramphenicol-induced germination, which has been reported for lettuce [35] and barley [9]. Protein synthesis in chloramphenicol-promoted lettuce seeds is less than in water-imbibed dormant controls, and it has been proposed that some protein synthesis inhibitory to germination is itself inhibited by chloramphenicol, thus allowing dormancy to be broken [35]. There are problems in this interpretation, however. For example, other inhibitors of protein synthesis either inhibit germination, or are ineffective [79]. Also, the specificity ofo-chloramphenicol is for inhibition of protein synthesis on 70S ribosomes (found only in organelles) and not 80S ribosomes, on which the major seed protein synthesis is carried out. L-threo-Chloramphenicol has its maximum inhibitory effect on protein synthesis in all parts oflettuce seeds at 0.5 mg/ml and yet at this concentration it only promotes about 25% of the seeds to germinate (Fig. 4.31). Increasing the concentration of the inhibitor promotes more germination, but has no further inhibitory effect on protein synthesis. This lack of coincidence between germination promotion and inhibition of protein synthesis at these higher concentrations suggests that these two phenomena are not linked. How then does chloramphenicol act? L-threo-Chloramphenicol, for one, is known to be an inhibitorofrespiration [91J, and it might promote germination in this way. Thus the

254

The Control of Dormancy

60

Fig. 4.30. The time course of germination of Grand Rapids lettuce seeds imbibed at 25 DC on 10- 1 M (X), 10- 2 (0) and 10 - 3 M (e) thiourea, or on water (vertical bar). After Bewley, 1979 [2] x

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Fig. 4.31. The relationship between promotion of germination and inhibition of protein synthesis in lettuce seeds treated with L-threo-chloramphenicol. 0- - - -0: % germinated; . - .: Protein synthesis in the endosperm; e--e: Protein synthesis in the cotyledons; 0: Protein synthesis in the radicle/hypocotyl. By Black, previously unpublished 0-

Secondary Events in the Release from Dormancy

255

observed reduction in protein synthesis could be, in part, an indirect consequence of reduced respiration. In our discussion on the role of the pentose phosphate pathway in dormancybreaking we have taken a cautious, perhaps sceptical attitude. Others, however, are more convinced and in an extensive review of the topic Roberts and Smith [10] present a careful and well-argued outline of the evidence in favour of the hypothesis. Certainly, credit is due to Roberts for deriving a working model from the myriad of seemingly unrelated observations. However, we feel that the evidence is not strong enough for the hypothesis to have been accepted to the extent that it has. But, on the other hand, we can offer no comprehensive alternative scheme to explain the published data. Our thesis is that many more rigorous studies need to be carried out to determine the role of the various oxidative pathways, their intermediates, enzymes and cofactors. Much evidence comes from studies using inhibitors and chemicals whose effect on cellular metabolism is undetermined: this needs to be remedied.

4.5.6. Phytochrome-induced Changes in Metabolism Although the dormancy of many species of seeds is known to be broken by irradiation with red light (Chap. 3), metabolic responses to light prior to radicle emergence have been studied to any extent only in three species - lettuce, and two spp. of Pinus. The claim that stimulation of some seeds by light is mediated through phytochrome (Pfr)-induced synthesis of promotive hormones has been dealt with elsewhere (Sect. 4.4.2). Here, in keeping with other parts of this section, we will confine our discussions to the possible influence oflight (viz. phytochrome) on respiration, nucleic acid and protein synthesis. A slight stimulation of oxygen consumption occurs in lettuce seeds (cv. Grand Rapids) irradiated with 5 min red light to break dormancy, compared with unirradiated controls (Fig. 4.2B). This occurs prior to the completion of germination. Unfortunately, the effects of red light, followed by dormancy-maintaining far-red light were not tested. In another study on the same cultivar [238] it was found that red light-irradiated lettuce seeds consumed more oxygen than did far-red light-irradiated ones, but appropriate dark controls were not tested. Using seeds of lettuce cultivar Cannington Forcing, it has been shown that red light slightly promotes oxygen consumption during germination, compared to dormant dark-imbibed, control seeds, and that far-red light reverses the promotive effect of red light [159]. The significance of elevated oxygen consumption in relation to dormancy breaking is not apparent at the present time, and preliminary studies suggest that ATP levels in Grand Rapids lettuce seeds are not modulated by light treatments (J. Krochko and Bewley - unpublished). Seeds of the White Paris cultivar of lettuce germinate in darkness at 25° C, but they can be rendered dormant by exposure to far-red light. The development of mitochondria in intact White Paris lettuce seeds maintained in the dormant state is the same as in non-dormant seeds. For example, during the first 12 h from the start of imbibition, oxygen uptake by mitochondria isolated from dormant and from non-dormant seeds is the same (Fig. 4.32). Also, their respective respiratory control ratios are the same (Y. Morohashi - unpub-

256

The Control of Dormancy 5 A

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36

Fig. 4.32 A, B. Changes in the respiratory rate of mitochondrial fractions isolated from farred-irradiated (.) and dark-imbibed (0) seeds oflettuce cv. White Paris. Oxygen uptake was measured using an oxygen electrode, with (A) a-ketoglutarate or (B) succinate as respiratory substrates. Attempts to repeat these experiments on the more commonly-used cultivar of lettuce, Grand Rapids, were unsuccessful because of the presence in the brown seed (strictly, fruit) coats of contaminating substances (probably phenolics) which are deleterious to the activity of the isolated mitochondrial fractions. White Paris lettuce seeds have a white coat with only a low level of contaminating substances. By Y. Morohashi and Bewley, previously unpublished

lished). Following radicle protrusion from the dark-imbibed seeds (after 11-12 h) mitochondria isolated from the germinated seeds become much more active, while those isolated from the still-dormant seeds undergo only a little further development (Fig. 4.32). Red light-stimulated seeds of Scots pine (Pinus sylvestris) release more CO 2 under aerobic and anaerobic conditions than do dark controls or seeds irradiated with red-far-red [151]. The increase is not large, however, and only becomes substantial after the red light-treated seeds have completed germination. Oxygen consumption during or after germination was not measured. Embryos of lettuce seeds irradiated with red light show no stimulation of 32p_ phosphate incorporation into RNA before germination is completed [78]. Nor does red light enhance or far-red light reduce 3H-uracil incorporation into RNA of Melandrium noctiflorum seeds [96]. These results suggest that dormancy-break-

Secondary Events in the Release from Dormancy

257

ing by phytochrome does not involve synthesis of new RNA molecules. While this may be so, the possibility that RNA synthesis is promoted following red light treatment cannot be ruled out. If synthesis of a few specific messenger RNAs for proteins essential for germination was stimulated by red light, the techniques used would have been inadequate to detect them. Moreover, since the response to light might occur only in the radicle, and maybe within a few cells of this organ, any changes could, again, be too small to detect. This argument can, of course, be applied quite widely when it is not possible to detect any light-, temperature- or hormone-induced response; testing its validity is the problem! Although the effects of red light on lettuce-seed protein synthesis have not been studied directly, there appears to be a detectable increase in polyribosome levels above dark-imbibed controls prior to the completion of germination [125]. Polyribosomes are present in dormant lettuce seeds [76], and are not induced only after irradiation with light (see Sect. 4.2). In imbibed, dormant Pinus thunbergii embryos polyribosomes appear to remain at a very low level for at least a month; within 4 h of a brief exposure to red light (given after 5 or 30 days), however, they begin to increase [239]. This effect of red light is partially reversible by a subsequent far-red irradiation. We should point out, though, that the yield of polyribosomes extracted in these experiments was very low, even in red light-treated seeds, and the only substantial increase was detectable in the dimer peak. In vivo protein synthesis using labelled precursor was not followed: this would have given a better indication of the synthetic capacity of light- and dark-imbibed seeds. Protein synthesis occurs in dormant Melandrium noctiflorum seeds and appears to be unaffected by red or far-red light [96]. Failure of lettuce seeds to germinate when imbibed in darkness on water at supraoptimal (25° C) temperatures is due to the inability of the embryo to overcome the mechanical restraining force of the surrounding endosperm. This is some evidence, which was detailed in Section 4.5.5, that red light (acting through the phytochrome system) induces in the embryo a decrease in water potential equal to that which is required for the radicle to penetrate the endosperm [146]. How then does phytochrome act to reduce water potential? One possibility (among others) is that it induces hydrolysis of storage polymers in the radicle to yield low M.W. compounds, and these would effectively lower the osmotic potential of the radicle cells. Lettuce seeds irradiated with red light, but incubated in 0.5 molal mannitol, do not germinate. Electron micrographs of radicle cells of seeds treated in this manner show digestion of protein and lipid bodies by 24 h, whereas no digestion is apparent in cells of control seeds maintained in a state of dormancy by incubation on mannitol in darkness [145]. Therefore it is claimed that mannitol-treated seeds exposed to red light produce osmotically active substances by protein and lipid degradation, but radicle extension is prevented by the hypertonic solution in which they are incubated. Seeds held in darkness do not germinate because there is no hydrolysis of reserves, so they do not develop the growth potential to break through the endosperm. Biochemical analysis of irradiated seeds imbibed in water has not revealed any decline in lipid content, or any substantial increase in low M.W. sugars (sucrose, raffinose) in the radicles prior to elongation [89], nor do embryos stimulated by red light [42, 133] or GA [75, 77] contain less protein, or more amino acids than do water controls. It is difficult, therefore, to reconcile the observations derived from the ultrastructural study with those obtained by direct

258

The Control of Dormancy

chemical analyses. It could be argued that changes in osmotic potential need only occur in a few cells for a short time just as radicle elongation commences. It was not indicated in the ultrastructural study [145] if the observed changes in the radicles of irradiated seeds were confined to a few cells - nor is it clear whether the cell studies in the radicles of irradiated seeds were located in an identical position to those in the radicles of non-irradiated seeds. Seeds of Scots pine stimulated to germinate by red light undergo no changes in lipolytic or amylolytic activity prior to radicle emergence [152-155]. Some changes in sugar levels occur in embryos of dark- and light-treated seeds; starch levels fluctuate more in irradiated seeds, and glucose and fructose levels are initially lower (0-9 h after imbibition) and then higher (18-24 h) than dark controls [156]. The significance to dormancy breaking of these differences, which are quite small, is not known.

4.5.7. Low-temperature-induced Changes in Metabolism As outlined earlier in Chapter 3, some seeds, in particular those of woody perennials, require exposure to low temperatures in the hydrated state (usually 2°_5° C) before they will break dormancy and successfully germinate at higher temperatures (15°-25° C). In this section we will discuss those metabolic events which occur during a low temperature (chilling or cold-stratification) treatment, and consider their possible importance in the breaking of dormancy. As in previous sections, we must express concern over some of the approaches used by experimenters in determining the relationship between metabolic events during cold-stratification and the breaking of dormancy itself. Such concerns have been elaborated by Nikolaeva [7], who notes that little use has been made of the comparative method of investigation in such studies. She points out, for example, that changes observed in cold-treated dormant seeds are not always compared with those taking place in seeds maintained at warm temperatures (which process we shall call warm-stratification) for similar time periods (see Sect. 4.4.3). Furthermore, little work has been done to compare changes in seeds undergoing cold-stratification with those of non-dormant seeds maintained in the cold. We have omitted reference to the majority of the non-comparative studies because they provide little or no insight into the mechanism of dormancy breaking. Also omitted is reference to the extensive work on the effects of cold temperatures on non-dormant seeds, e.g. most cereal grains (on which vernalization studies have been conducted), soybean and cotton (which are chilling-sensitive during imbibition). We should also remind ourselves at this stage that some seeds are shed from the parent plant when the embryos are still in an immature state and that their maturation, including cell division and cell growth, occurs in the hydrated seed either at cold or at warm temperatures (or both) - see Table 4.12 for examples. The metabolism of seeds undergoing development can be expected to be different from those whose embryos are in the mature state when shed and which undergo insignificant growth prior to termination of dormancy, e.g. seeds of many fruit trees, conifers and maples. It should be noted also that some seeds germinate at low temperatures following their cold-stratification period. Thus, some metabolic changes

Secondary Events in the Release from Dormancy

259

Table 4.12. The growth of immature embryos of some seeds imbibed at cold or at normal temperatures Species

Seeds with immature embryos at shedding Growth at 0-5° C

Growth at 15° C

Notes

Gingseng (Panax spp.) No

Yes

Spindle tree

Yes

After embryo growth a cold period is required to break dormancy Ditto. Development can continue in the cold following 2-3 months warmth Response varies with the geographical location at which seeds are grown. Some will only mature if maintained in warmth After embryo growth a cold period is required to break dormancy Seeds germinate on maturation in the warmth, but the epicotyl remains dormant if not chilled Seeds kept in warmth do not mature completely, nor germinate Ditto

No

(Euonymus verrucosa)

Ash (Fraxinus excelsior)

Yes Yes (more rarely)

Fraxinus nigra

No

Yes

European cranberry bush

No

Yes

Yes

Limited

Yes

Limited

(Viburnum opulus)

Cowparsnip (Heracieum sphondylium)

Sour cherry (Prunus cerasus)

observed after long periods of hydration at cold temperatures may be related to germination or growth of the embryo rather than to the dormancy-breaking process per se. A number of studies have concentrated on the effects of cold-stratification on respiration. In general, the techniques used involve maintenance of seeds at low temperatures (0°_5° C) for several weeks or months before measurement of respiration for a short time period (hours). But usually these measurements are conducted at elevated temperatures of 18°-25 C! Hence results from such experiments do not characterize seed respiration under conditions of chilling, but rather give some indication of the changing nature of, or potential for, respiration brought about by cold treatments. It has generally been observed that over the first few days following imbibition, dormant, cold-requiring seeds maintain a higher level of respiration at 15°-25° C than they do at 0°_5° C. This trend is gradually reversed with time, however, and respiration by seeds kept in the cold remains constant (or rises) whereas that of warm-stratified seeds declines, e.g. box elder, ash, maples and sour cherry [7, 166, 186]. In cockscomb rattleweed (Rhinanthus crista-galli) the respiration rate of seeds maintained at 2° C (but determined at 22° C) is apparently higher than in those kept at 20° C [218]. No substantial qualitative differences in respiration (R.Q.) have been reported between dormant and cold-stratified seeds, and the significance of the differences in respiratory patterns exhibited by seeds kept in the 0

260

The Control of Dormancy

Table 4.13. Effect of stratification at 5° C and 20° C on the level of adenylates in embryonic axes of Acer saccharum Days

Adenylates (nmoljg dry wt) ATP

0 1 5 10 20 40 75

ADP

Stratification period at 5° C 46.9±12.6 180.2±34.6 137.9±13.4 163.3 ± 24.7 238.9±27.9 119.4±14.1 327.2±31.7 98.1±17.0 354.3±63.2 99.8±21.0 416.3±48.8 92.1±11.5 456.3±70.7 91.4±29.3

Energy charge AMP 69Q.8±31.1 194.9±15.0 57.3± 14.1 60.2±14.0 63.6± 18.3 63.2±12.2 56.8±11.8

0.15 0.44 0.72 0.78 0.78 0.80 0.83

690.8±31.1 67.2±16.5 91.9±11.7 118.7±13.3

0.15 0.80 0.78 0.56

Stratification period at 20° C 0 3 10 22

46.9± 12.6 437.4± 40.7 475.7 ± 102.0 172.9± 17.1

180.2±34.6 108.1 ± 13.5 109.4±41.0 9O.9±12.0

After Simmonds and Dumbroff, 1974 [186]

cold or warmth remain unexplained. During both warm- and cold-stratification of sugar maple (Acer saccharum) seeds, ATP levels in the embryonic axes rise while those of ADP and AMP decline (Table 4.13). This is accompanied by an increase in energy charge (E.C.) which expresses the amount of metabolically-available energy within a cell. As pointed out in Volume 1, Chapter 5, when the E.C. is above 0.5, ATP-utilizing systems increase their activities; and above 0.8, cells may actively metabolize and multiply. Since axes of warm-stratified A.saccharum are not impeded in their production of ATP and have a high E.C., the inability of these seeds to break dormancy is unlikely to be a consequence of a block to energy production. Prolonged storage at warm temperatures results in a lowering of ATP levels and E.C., but it is not known if these reductions are the cause or effect of the loss of germination potential. An increase in E.C., but not in ATP levels, has been reported in the embryos of ponderosa pine seeds during stratification at 5° C [48]. A causal relationship between cold-temperature treatment and high-energy charge cannot be established, however, because measurements were not made on warm-stratified seeds. During cold-stratification of sour cherry (Prunus cerasus) an increased rate of respiration (both with time and over warm-stratified controls) occurs in the embryonic axis and leaf primordium [166]. This is accompanied by an accumulation of phosphate in the embryonic axis as organic phosphates, including nucleotide high-energy phosphates [157]. In the axes of warm-stratified seeds, phosphate accumulates also, but principally as inorganic phosphate. Since embryo development can occur at cold, but not warm temperatures (Table 4.12) the implication is that development at warm temperatures (and hence subsequent germination) is arrested due to a block in phosphate metabolism. This possibility has not been fully substantiated and further studies are needed.

Secondary Events in the Release from Dormancy

261

Respiration of seeds at cold-stratification temperatures (2°_5° C) occurs at a low rate but, obviously, at one which is adequate for the realization of those events essential for the breaking of dormancy. In some seeds which require chilling, dormancy is imposed by the outer covering structures, which could restrict oxygen uptake into the embryo (Sect. 2.4.2). Because of the low rate of respiration at cold temperatures, the presence of covers does not appear to be a factor hampering gas exchange, whereas during warm-stratification, when seeds maintain dormancy, oxygen uptake may be limiting. Thus, during cold-stratification a low rate of respiration in the presence of ample oxygen (and with controlled metabolic utilization thereof) could result in cellular events leading to the breaking of dormancy. At warm temperatures, on the other hand, a paucity of oxygen could lead to abnormal or uncontrolled metabolism, which would serve to prolong or even deepen dormancy. The possibility that reduced RNA synthesis in dormant Prunus seeds results from impaired phosphate metabolism (i.e. impaired synthesis of nucleic acid precursors) has been raised, but not strongly supported [157]. Studies on the Bartlett pear (Pyrus communis) suggest an increase in RNA synthesis, including messenger RNA, over 38 days of cold-stratification [124]. This work has a number of technical shortcomings, however, as well as inadequate warm-stratified controls. There is no substantial increase in RNA synthesis induced by cold-stratification in either the cotyledons or axes of Acer platanoides, compared with the appropriate warm controls [55]. A small, but not necessarily significant increase occurs after about 80 days of cold temperatures. But this might be related to the initiation of radicle growth which occurs after this time, rather than to the removal of dormancy. Consequently, no clear-cut relationship has been established between RNA synthesis and the breaking of dormancy. Activities of aminoacyl-tRNA synthetases in coldstratified pear embryos increase over those in control embryos kept at 25° C [203] - the importance of these increases, unfortunately, is not apparent at this time. It is claimed that there is an increase and then a decline in the in vitro translational capacity of polysomes extracted from pear embryos during cold-stratification [18]. But an inadequate number of warm-stratified controls was carried out: changes during cold-stratification were followed over a 41-day period, but for the warm controls only a 10-day period was followed! Even during this 10-day period in vitro polysome activity in the controls increased sixfold. Villiers [227] has shown that during chilling the nucleoli within the cells of the meristem of the radicle of mature embryos of Fraxinus excelsior increase in volume and complexity of structure, but this does not occur if the seeds are kept at 22° C. Other changes in the cytoplasm (including ER proliferation) occur at both cold and warm temperatures. Total cellular RNA increases only in the cold; this observation combined with that of increased nucleolar size would appear to indicate enhanced ribosome synthesis. That this is ultimately important for the relief of dormancy is doubtful [227], since large numbers of ribosomes, both free and membrane-bound (as shown by electron micrographs), are present in the embryo during warm-stratification. The capacity of these ribosomes to become involved in protein synthesis has not been tested, however. Cold-stratification might induce changes in nucleic acid metabolism in cotyledon slices of hazel (Cory Ius avellana) commencing about 10 days after the start of chilling [236]. Whether such changes in the cotyledons are essential for the

262

The Control of Dormancy

Table 4.14. Changes in storage materials and hydrolytic enzymes during cold-stratification of dormant seeds. Examples of experiments lacking adequate warm-stratified controls Species

Claim for changes during cold-stratification

Reference

Sour Cherry (Prunus cerasus) Apple (Pyrus malus) cv. Antonowska

Stored lipids decrease in cotyledons

[129]

Proteinase and phosphatase activities increase

[137]

Protein biosynthesis and soluble protein content increases Chilling provides essential pool of amino acids for germination a Stachyose and raffInose decline. Starch accumulates Hydrolysis of lipids by acid lipase Accumulation of starch

[137]

Phasic changes in soluble nitrogen and amino acid pools Decline in stored fat, increase in sugars

[60]

AmurMaple (Acer ginnala) Pinus morrisonicola a

[137] [167] [241] [57]

[115]

In addition, there is inadequate experimental evidence to support this claim

termination of dormancy and for germination of the axis is unknown. Chilled axes do, apparently, accumulate radioactively labelled adenine into nucleotides, which may indicate increased synthesis of these and of nucleic acids [38]. Again, the importance of these changes remains unestablished. At the present time we are left with the possibility that dormancy of warmstratified seeds could result from a block to nucleic acid metabolism or to the synthesis of certain key metabolic or specifically dormancy-breaking proteins. This possibility has not been substantially supported by experimental evidence, however. There is ample evidence from the literature that during cold-stratification of dormant seeds of some species, decomposition of reserves occurs, accompanied by an increase in hydrolytic enzymes. For example, chilling of Acer platanoides seeds at 5° C is accompanied by an increase in the amounts of free amino acids and sugars in the cotyledons and embryonic axes. There is no increase at 17° C, a temperature which is not conducive to the breaking of dormancy [56]. But the importance of this reserve mobilization to dormancy breaking during cold-stratification is not clearly established, and in some species similar events also occur during warm-stratification. Unfortunately, in a large number of studies (some of which are noted in Table 4.14) mobilization of reserves has been studied under coldstratification conditions but the appropriate warm controls have not been carried out. Hence, despite claims to the contrary (e.g. [136]), there is little compelling evidence that such changes are important for, or even related to, the breaking of dormancy. Other workers have shown that there is more reserve mobilization in seeds placed under germination conditions after cold-stratification than in non-stratified seeds under the same conditions. This is hardly surprising, for the comparison is being made between dormant seeds and those which have had their dormancy broken, and are germinated and growing!

Secondary Events in the Release from Dormancy Fig. 4.33. (A) Dry weights of embryos (0) and endosperms (.(',) of cow parsnip (Heracleum sphondylium) as a percentage of seed dry weight during stratification at 2 °C (-----) and 15°C (- -). (B) Endosperm and embryo nitrogen contents during treatment at 2 °C, expressed as a percentage of total seed nitrogen. A-A: insoluble endosperm nitrogen; 6 - 6 : soluble endosperm nitrogen; e-e: endosperm total nitrogen (soluble and insoluble); 0-0: embryo total nitrogen. No changes were observed in the nitrogen fractions at 15 °C. After Stokes 1952, 1953 [200, 201]

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Fig. 6.17. Germination beneath plants of different ages. Plantago major seeds were exposed to light beneath a Sinapis alba crop at different times after planting. As the S. alba grows the red/far-red ratio beneath them changes as shown, with accompanying changes in the phytochrome photoequilibrium (cfJ) established by the light. After Frankland and Poo, 1980 [77]

ratio which occurs as the leaf canopy thickens. The canopy will also vary with time in many field situations - in deciduous forests as the leaves appear and later drop, and under herbaceous plants as the leaf area index increases and then falls with senescence and death. Thus seeds on the soil surface underneath such plants experience a changing light environment and consequently exhibit a changing pattern of response in their germination (Fig. 6.17). By using both the low-energy mode of phytochrome and the effect of HIR farred light, both activated by leaf-filtered light, seeds are able to detect their proximity to other plants. Germination beneath established plants, which clearly would be an unfavourable situation, can be prevented. Seeds will remain inhibited until such time as a light environment becomes available which is relatively rich in red light, and which can, therefore, support photosynthesis and seedling establishment. This is likely to be the mechanism by which germination in woodlands is arrested until gaps in the canopy appear, perhaps when individual trees die and fall

Temperature

297

or when tree clearance by Man takes place. Many species which are absent from established forests rapidly appear as pioneers when the forests are thinned or felled. Cecropia glaziovi seeds, for example, are dispersed by bats and other small mammals of Brazilian forests, yet seedlings are not found in the forests inhabited by these animals. There is a rich seed bank on the forest soil, but because the species is inhibited by canopy light, germination is arrested until tree clearance occurs [259]. Interestingly, seeds of species which are able to colonize shaded sites (e.g. Centaurium erythrea) are less sensitive to far-red rich canopy light [21S]. Differences in sensitivity among seeds of different species is probably an important factor influencing species distribution.

6.6. Temperature Numerous studies have contributed to our knowledge of the effect of temperature on seed germination (see, e.g. [5, 11, 12, 15,65,151]). In this section we shall consider the major findings. One difficulty which arises in assessing these is to disentangle temperature effects on germination itself from those concerned with dormancy. A few examples should clarify this point. Seeds of many species benefit from exposure to fluctuating temperatures so that their final germination percentages are very much higher than those of the untreated controls. But in many of these cases removal of the tissues enclosing the embryo allows germination to occur over a fairly wide range of constant temperatures [176, 177]. Clearly, such seeds have a coat-imposed dormancy which can be circumvented by alternating temperatures, and it would be incorrect to regard the alternating temperatures as a requirement for germination itself. Seeds whose dormancy is terminated only by cold stratification (in some species chilling for a few days is efficacious) would obviously fail to germinate if maintained at relatively high temperatures. But they may germinate at low temperatures because then their requirement for chilling is satisfied and they can complete the germination process, albeit sluggishly in some cases. Such seeds would thus seem to have a low optimum temperature for germination. That this is not really the case can be shown simply by transferring chilled, now non-dormant seeds, to higher temperatures when they germinate quite adequately. Where seeds have relative dormancy they may increasingly be unable to germinate as the temperature is raised and their optimum for germination will be at low temperatures. However, the temperature optimum can be widened and shifted to higher values by previously exposing seeds to chilling temperatures for a few hours or days. In the following account we will try to avoid the complications introduced by dormancy and, as far as possible, confine our considerations to the germination process per se, in non-dormant seeds.

6.6.1. Temperature Minima, Optima and Maxima When seeds are set to germinate over a temperature range an optimum soon becomes apparent (Fig. 6.1S, curve A). Over the short time period, we can clearly rec-

298

Environmental Control of Germination

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