Doubled Haploid Production in Crop Plants - Springer

Doubled Haploid Production in Crop Plants

Doubled Haploid Production in Crop Plants A Manual Edited by

M. Maluszynski Joint FAOflAEA Division of Nuclear Techniques in Food and Agriculture, Vienna, Austria

K.J. Kasha University of Guelph, Department of Plant Agriculture, Guelph, Canada

B.P. Forster Scottish Crop Research Institute, Dundee, U.K.

and

I. Szarejko University of Silesia, Department of Genetics, Katowice, Poland

SPRINGER SCIENCE+BUSINESS MEDIA, LLC

A C.I.P. Catalogue record for this book is available from the Library of Congress.

ISBN 978-90-481-6393-9 ISBN 978-94-017-1293-4 (eBook) DOI 10.1007/978-94-017-1293-4

Printed on acid-free paper

AII Rights Reserved © 2003 Springer Science+Business Media New York Originally published by Kluwer Academic Publishers in 2003 Softcover reprint of the hardcover 1st edition 2003 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work.

Editorial The views expressed in the papers, nomenclature used and way of presentation remain the responsibility of the authors concerned, who are also responsible for any reproduction of copyright material. All published papers were reviewed by the following group of 'COST 851' Programme experts: S.B. Andersen, B. Bohanec, P. Devaux, M. Hansen and M. Wedzony. The excellent assistance of Mrs. Katayon Allaf and Kathleen Weindl, Joint FAOIIAEA Division, Plant Breeding and Genetics Section for editorial help, retyping some manuscripts and making corrections in English, is highly appreciated.

Contents Preface ................................................................................................................................................. vii Abbreviations ....................................................................................................................................... ix Colour Figures ...................................................................................................................................... xi 1. Production of doubled haploids in crop plants. An introduction K.J. Kasha and M Maluszynski................................................................................... 1 2. Protocols for major crops Barley 2.1. Doubled haploid production in barley using the Hordeum bulbosum (L.) technique P. Hayes, A. Corey and J. DeNoma ............................................................................. 5 The Hordeum bulbosum (L.) method 2.2. P. Devaux................................................................................................................... 15 Anther culture in barley 2.3 C. Jacquard, G. Wojnarowiez and C. Clement.......................................................... 21 Barley anther culture 2.4. L. Cistue, MP. Valles, B. Echavarri, J.M SanzandA. Castillo ............................... 29 Anther culture for doubled haploid production in barley (Hordeum vulgare L.) 2.5. I. Szarejko .................................................................................................................. 35 Barley isolated microspore culture protocol 2.6. K.J. Kasha, E. Simian, R. Oro and Y.S. Shim ............................................................ 43 Barley isolated microspore culture (IMC) method 2.7. P.A. Davies ................................................................................................................ 49 Wheat Doubled haploid production in wheat through wide hybridization 2.8. MN. Inagaki .............................................................................................................. 53 Protocol of wheat (Triticum eastivum L.) anther culture 2.9. J. Pauk, R. Mihaly and M Puolimatka ...................................................................... 59 Protocol for producing doubled haploid plants from anther culture of wheat 2.10. (Triticum aestivum L.) B. Barnabas ............................................................................................................... 65 Wheat anther culture 2.11. S. Tuvesson, R. von Post and A. Ljungberg ............................................................... 71 Haploid wheat isolated microspore culture protocol 2.12. K.J. Kasha, E. Simian, M Miner, J. Letarte and T. C. Hu ......................................... 77 Production of doubled haploids in wheat (Triticum aestivum L.) through 2.13. microspore embryogenesis triggered by inducer chemicals MY. Zheng, W: Liu, Y. Weng, E. Polle and C.F. Konzak. ......................................... 83 Maize Isolated microspore culture in maize (Zea mays L.), production of doubled-haploid 2.14. via induced androgenesis MY. Zheng, Y. Weng, R. Sahibzada and C.F. Konzak .............................................. 95 Anther culture of maize (Zea mays L.) 2.15. B. Barnabas ............................................................................................................. 103 Rice indica/japanica 2.16. Laboratory protocol for anther culture technique in rice F.J. Zapata-Arias ..................................................................................................... 109 Triticale Triticale anther culture 2.17. S. Tuvesson, R. von Post and A. Ljungberg ............................................................. 117 Protocol for anther culture in hexaploid triticale (x Triticosecale Wittm.) 2.18. M Wt;?dzony ............................................................................................................. 123

111

iv 2.19. 2.20. Rye 2.21. 2.22.

Protocol of triticale (x Triticosecale Wittmack) microspore culture J. Pauk, R. Mihaly, T. Monostori and M Puolimatka............................................. Protocol for doubled haploid production in hexaploid triticale (x Triticosecale Wittm.) by crosses with maize M W!i!dzony ............................................................................................................. Protocol for rye anther culture S. lmmonen and T. Tenhola-Roininen ..................................................................... Microspore culture of rye S. Pul/i and Y.-D. Guo.............................................................................................

129 135 141 151

Oat 2.23.

Oat haploids from wide hybridization H. W. Rines............................................................................................................... Durumwheat 2.24. Haploid and doubled haploid production in durum wheat by wide hybridization P.P. Jauhar.............................................................................................................. 2.25. Haploid and doubled haploid production in durum wheat by anther culture P.P. Jauhar.............................................................................................................. Timothy Anther culture and isolated microspore culture in timothy 2.26. S. Pul/i and Y.-D. Guo............................................................................................. Ryegrass and other grasses 2.27. Doubled haploid induction in ryegrass and other grasses S.B. Andersen........................................................................................................... Rapeseed 2.28. Microspore culture in rapeseed (Brassica napus L.) J.B.M Custers ........................................................................................................ . Broccoli 2.29. Protocol for broccoli microspore culture J. C. da Silva Dias ................................................................................................... . Other Brassicas 2.30. Microspore culture of Brassica species A. Ferrie................................................................................................................... 2.31. Protocol for microspore culture in Brassica M Hansen................................................................................................................ Tobacco 2.32. Anther and microspore culture in tobacco A. Touraev and E. Heber/e-Bors ............................................................................. Potato 2.33. Haploid production of potatoes by anther culture G.C.C. Tai andXY. Xiong....................................................................................... 2.34. Anther culture through direct embryogenesis in a genetically diverse range of potato (Solanum) species and their interspecific and intergeneric hybrids V.-M Rokka ............................................................................................................. 2.35. Potato haploid technologies MJ. De,Maine .........................................................................................................

155 161 167 173 179 185 195 205 217 223 229

235 241

Linseed/flax 2.36.

Anther culture of linseed (Linum usitatissimum L.) K. Nichterlein. ..... ... ..... ... .... ..... ... ..... ..... ..... ... .... .... ...... ... .... ..... .... .... .... .... .... ... .... .... ...

249

v Sugar beet 2.37. Doubled haploid production of sugar beet (Beta vulgaris L.) E. Wremerth Weich and M W. Leva!! ...................................................................... Asparagus 2.38. Asparagus microspore and anther culture D.J. Wolyn and B. Nichols....................................................................................... Onion 2.39. In vitro gynogenesis induction and doubled haploid production in onion (Allium cepa L.) L. Martinez............................................................................................................... 2.40. Haploid induction in onion via gynogenesis M JakSe and B. Bohanec ................... ..................................................................... Apple 2.41. In vitro androgenesis in apple M Hofer ................................................................................................................... Aspen 2.42. Doubled haploid production in poplar S.B. Andersen........................................................................................................... Cork oak 2.43. Oak anther culture MA. Bueno and JA. Manzanera ....................... ...................................................... Citrus 2.44. Haploids and doubled haploids in Citrus spp. MA. Germano ........ ......................................... ........................................................

255 265

275 281

287

293 297 303

3. Published protocols for other crop plant species M Maluszynski, K.J. Kasha and I. Szarejko............................................................ 309 4. Application of doubled haploid production techniques 4.1. Doubled haploids in breeding W.T.B. Thomas, B.P. Forster and B. Gertsson ........................................................ 4.2. Doubled haploid mutant production I. Szarejko ................................................................................................................ 4.3. Barley microspore transformation protocol by biolistic gun Y.S. Shim and K.J. Kasha ...................................................................................... .. 4.4. Doubled haploids in genetic mapping and genomics B.P. Forster and W. T.B. Thomas.............................................................................

337 351 363 367

5. Protocols for chromosome counting 5.1. Cytogenetic tests for ploidy level analyses- chromosome counting J Maluszynska ......................................................................................................... 391 5.2. Ploidy determination using flow cytometry B. Bohanec .... ................................................. ........ ............. ............................... ...... 397 6. Major media composition and basic equipment for DH laboratory 6.1. Major media composition compiled by I. Szarejko ............................................................................................ 405 6.2. Basic equipment for maize microspore culture laboratory compiled by MY. Zheng, Y. Weng, R. Sahibzada and C.F. Konzak ....................... 415 List of contributors .. ..... ..... ..... .... ..... .... .... ..... .... ..... .... ..... ..... .... ..... .... .... ..... .... ..... ..... .... ... ... .... ..... .... .. 41 7

vii

Preface The production of doubled haploids has become a necessary tool in advanced plant breeding institutes and commercial companies for breeding many crop species. However, the development of new, more efficient and cheaper large scale production protocols has meant that doubled haploids are also recently being applied in less advanced breeding programmes. This Manual was prepared to stimulate the wider use of this technology for speeding and opening up new breeding possibilities for many crops including some woody tree species. Since the construction of genetic maps using molecular markers requires the development of segregating doubled haploid populations in numerous crop species, we hope that this Manual will also help molecular biologists in establishing such mapping populations. For many years, both the Food and Agriculture Organization of the United Nations (FAO) and the International Atomic Energy Agency (IAEA) have supported and coordinated research that focuses on development of more efficient doubled haploid production methods and their applications in breeding of new varieties and basic research through their Plant Breeding and Genetics Section of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. The first FAO/IAEA scientific network (Coordinated Research Programme - CRP) dealing with doubled haploids was initiated by the Plant Breeding and Genetics Section in 1986. This was followed by a CRP on doubled haploids in cereals involving countries in Latin America and by other CRPs connecting doubled haploids with molecular marker techniques or with their application for developing stress tolerant mutant germplasm in crop species. Also, numerous FAOIIAEA training courses were organized by the Joint Division in association with CRP's and Technical Cooperation Projects. Requests from researchers and trainees for systematised protocols dealing with the production of doubled haploids in various species were instrumental in initiating work on this Manual, and the idea to prepare it was strongly supported by European scientists working under the EU large scale program 'COST 851' on 'Gametic cells and molecular breeding for crop improvement'. Many of the authors who provided protocols for the manual participated in these activities, thereby helping to transfer doubled haploid technology in countries and over continents. Also, five leading experts from the EU-COST 851 were invited to review the suitability of the manuscripts presented for publication in the Manual. The Manual focuses on efficient protocols - laboratory recipes - for producing doubled haploids in major crop species. Additionally, references on about 200 doubled haploid protocols for other plant/crop species together with chapters illustrating the current status of the use of doubled haploids in plant breeding and basic research are included. However, the Manual does not include reviews on doubled haploids in particular crops or groups of crops as these were recently the subject of another series of books.

James D. Dargie Director, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture Vienna, Austria

IX

Abbreviations 2,4,5-T

2,4,5-Trichlorophenoxyacetic acid

2,4-D

2,4-Dichlorophenoxyacetic acid

2-HNA

2-Hydroxynicotinic acid

2iP

6-( y ,y-Dimethyloamino)purine

ABA

Abscisic acid

AFLP

Amplified fragment length polymorphism

Ancymidol

a-Cyclopropyl-a-(4-methoxyphenyl)-5-pyrimidinemethanol

BA(BAP)

6-Benzylmaminopurine

BaYMV

Barley yellow mosaic virus

BaYDV

Barley yellow dwarf virus

BSA

Bulk segregant analysis

CPA

p-Chlorophenoxyacetic acid

DAPI

4'6-Diamidino-2-phenylindole dihydrochloride

DH

Doubled haploid(s); doubled haploidy

Dicamba

3,6-Dichloro-o-anisic acid (or 3,6-Dichloro-2-methoxybenzoic acid)

DMSO

Dimethyl sulphoxide

EDTA

Ethylenediaminetetraacetic acid

EI

Ethyleneimine

ELS

Embryo-like structure

EMS

Ethyl methanesulfonate

ENU

N-nitroso-N-ethylurea

GA

Gibberelic acid

GA3

Gibberelin A3

IAA

Indole-3-acetic acid

IBA

Indole-3-butric acid

X

MES

2-(N-Morpholino)ethanesulphonic acid

MNH(MNU)

N-nitroso-N-methylurea

MNNG

N-methyl-N -nitro-N-nitrosoguanidine

NAA

1-N aphtalenacetic acid

NaN3

Sodium azide

Nr

Fast neutrons

PAA

Phenylacetic acid

Picloram

4-Amino-3,5,6-trichloropicolinic acid

RAPD

Random amplified polymorphic DNA

RFLP

Restriction fragment length polymorphism

RYMV

Rye yellow mosaic virus

SSD

Single seed descent

SSR

Simple sequence repeat (microsatelite)

Thidiazuron

1-Phenyl-3-(1 ,2,3-thidiazol-5-yl) urea

TIBA

2,3,5-Triiodobenzoic acid

Colour Figures

xii

Figure 2.2-1. a) Vernalization of H. bulbosum plants in a cold-frame; b) Emasculation of a barley spike; c) H. bulbosum spike sheading pollen(*); d) Collecting H. bulbosum pollen; e) Pollination of a barley spike; f) Spray a barley spike 1-2 days after pollination. Figures originated from Florimond Desprez and from Crop and Food Research(*), Christchurch, NZ (courtesy of Dr. Paul Johnson).

xiii

Figure 2.2-2. a) Print of a bar coded label; b) Bagged spikes after spraying; c) Embryo excision from seeds(*); d) Embryo culture on medium(*); e) Developing embryos on medium(*); f) Plantlets ready to be transferred into potting mix. Figures originated from Florimond Desprez and from Crop and Food Research(*), Christchurch, NZ (courtesy of Dr. Paul Johnson).

xiv

Figure 2.2-3. a) Transfer ofplantlets into potting mix; b) Plants at 2-3 tiller stage; c) Haploid plants of barley prepared to be treated with colchicine; d) Colchicine treatment of haploid plants of barley; e) Plants in soil after colchicine treatment; f) Doubled-haploid plants covered with a bag; g) Doubled-haploid plants after self-pollination; h) Manual collection of fertile spikes from doubledhaploid plants; i) Gathering the fertile spikes of each doubled-haploid in a bag; j) Identification of each bag with a label. Figures originated from Florimond Desprez.

XV

Figure 2.4-1. Barley anther culture. a) Anthers after four days of pre-treatment in mannitol; b) Embryos after 12-14 days in induction medium; c) Embryos after 18-20 days in induction medium; d) Well-developed embryos before transferring to regeneration medium; e) Green and albino plants in regeneration medium; f) Transfer of plants to soil; g) Doubled haploid plants cultivated in the greenhouse; h) Field trials of doubled haploid lines.

xvi

a

b

e

d

e

f

g

h

Figure. 2.5-1. Stages of microspore development in barley: a) tetrads; b) early uninucleate; c) early-mid uninucleate; d) mid uninucleate; e) midlate uninucleate; t) late uninucleate; g) anaphase of the first microspore division; h) bi-nucleate.

xvii

Fig. 2.5-2. Production of barley doubled haploids through anther culture: a) tillers containing spikes at the proper developmental stage (the arrow indicates a distance between the flag and the penultimate leaf); b) a microspore at the mid uninucleate stage; c) a microspore at the mid-late uninucleate stage; d) spikes selected for cold pre-treatment; e) induction plates 30 days after anther inoculation; t) microsporederived calli/embryos on Ficoll medium; g) an anther with embryo-like structures on induction medium; h) an androgenic embryo 35 days after anther inoculation; i) regenerating calli/embryos 14 days after transfer onto regeneration medium; j) a plantlet developing from callus on regeneration medium; k) regenerants on rooting medium; 1) young plants transferred into soil; m) spontaneously doubled, fertile microspore derived barley plants (DHl); n) field evaluation ofDH2lines.

xviii

Figure 2.7-l. a) Eberbach blender model 8580; b) 100 !1ffi sieves; c) Band of viable microspores at the interface of maltose and mannitol in the neck of the volumetric flask.

Figure 2.7-2. a) Microspore colonies in liquid medium prior to subculture to solidified medium; b) Regenerants growing on solidified medium; c) Regenerants on rooting medium; d) Regenerant plants in soil; e) Regenerants transferred to greenhouse.

xix

Figure. 2.8-1. Stages in producing wheat doubled haploids through ultra-wide crosses. a) Wheat spikes at ear emergence (/eft), emasculation (center) and seed setting (right) . b) Germination of maize pollen on wheat stigma. Bar 0.1 mm. c) Injection of 2,4-D into wheat spike using a syringe. d) Pearl millet pollen at collection (upper) and after being dried for two hours (lower). Bar 0.1 mm . e) Detached-tiller culture of wheat at emasculation (/eft), pollination (center) and seed setting (right) . t) Wheat seeds obtained from self-pollination, and from crosses with maize, pearl millet and sorghum (from left to right) . Bar 2 mm. g) Apparatus used for embryo rescue on a laminar flow bench.

XX

Figure 2.8-1 (continuation). h) Wheat embryos obtained from self-pollination, and from crosses with maize pearl millet and sorghum (from left to right), showing differences in size and shape among crosses. Bar 2 mm. i) Plant developing from cultured embryos (from left to right; 0, 1, 2 and 3 weeks after incubation). j) Culture room accommodating test tubes and plastic dishes. k) Wheat haploid plants transferred to potted soil for further development. 1) Somatic chromosomes (2n=3x=21) of a wheat haploid plant. Bar 1OJ.Ull. m) Colchicine treatment of wheat haploid plants at tillering. n) Wheat plants transferred to potted soil after colchicine treatment. o) Wheat spikes of colchicine-treated plants, covered with glassine bag (/eft), and producing doubled haploid grains (right). p) Doubled haploid plants grown for seed multiplication. q) Doubled haploid lines grown in the field, showing uniformity within lines.

xxi

Figure 2.9-1. a) Vacuolated, mid uninucleated wheat microspore after two weeks in cold pretreatment. Don't use older stages for wheat anther isolation; b) Float anther culture using P-4mf culture medium with embryo-like-structures (ELS) at the 6th week of culture; c) A responsive anther with five individual ELS; d) Rooting and tillering of androgenic haploids.

Figure 2.9-2. a) Root tip cytology from a wheat haploid plant with 21 chromosomes (2n=3x=21); b) Colchicine treatment of haploids under a well-controlled greenhouse condition; c) Acclimatisation of transplanted colchicine treated plantlets; d) Microspore-derived DH breeding lines in greenhouse propagation.

Figure 2.9-3 . Yield test and propagation of DH lines in Szeged, Hungary. Seed and bread of the first anther culture-derived DH variety : GK Delibab (mirage), released and patented in 1992 (upper right comer).

xxii

a

Figure 2.10-1. a) Triticum aestivum microspore in the late uninuclear stage of development, n nucleus; b) doubled chromosome set in a rnicrospore nucleus (fEM picture).

Figure 2.10-2. Effect of colchicine on microspore androgenesis in vitro; a) C-rnitosis; b) symmetrical division; c) microspore derived embryo.

Figure 2.10-3. Pollen embryos (e) and callus (c) in anther culture.

Figure 2 .10-4. a) Regeneration of pollen plants; b) Doubled haploid plants grown in the growth chamber.

xxiii

Figure 2.13-1. Wheat microspores are induced by an inducer chemical to develop toward embcyoid fonnation. a) Uni-nucleate microspores, naturally programmed for gametophytic pathway toward pollen fonnation; b) Triggered by inducer chemical, microspores are reprogrammed for sporophytic development toward embcyoid fonnation.

Figure 2.13-2. a) Pretreatment apparatus in "flask" system; b) Disinfected spikes.

a Figure 2.13-3. a) Cut florets in blender cup ready to be blended; b) Blender cup assembled to the blender for blending to release rnicrospores.

xxiv

Figure 2.13-4. From left to right: 100 J.Ul1 (a) and 38J.Ull (b) mesh filter in beakers, overlay of 0.3 M mannitol+ microspores over 0.58 M maltose (c), and band with androgenic wheat microspores after gradient centrifugation (d).

Figure 2.13-5. Wheat microspore embryogenesis was triggered by an inducer chemical and nursed by optimal culture condition in vitro; a) Dividing microspores at day 7 in liquid culture medium; b) Developing pre-embryos at day 21; c) Mature embryos at day 30; d) Embryos geminated on solid medium.

Figure 2.13-6. a) Wheat microspore-derived plants in trays; b) Plants in trays with plastic cover.

XXV

Figure 2.13-7. Tube filter, which can be placed inside 30xl0 mrn or larger culture Petri-dishes.

Figure 2.13-8. Beaker filter, which can be placed inside another beaker and/or beaker filter.

Figure 2.13-9. a) Near 100% dividing microspores; b) Mature wheat embryoids from embryogenic microspores.

xxvi

Figure 2.14-1. a) Tassel following sampling; b) Tassels or florets ready for disinfection, c) Pretreated florets ready for microspore isolation; d) Blender and blender cup.

Figure 2.14-2. a) Filtering through 100 IJ.IIlfilter; b) Microspores trapped on 50 IJ.IIl mesh filter; c) Viable microspores in band(s); d) Potentially embl)'ogenic microspore band.

xxvii

Figure 2.14-3. a) Maize microspores ready for induction culture; b) Cell divisions in 3d culture; c) Multicellular structures (lith day); d) Pro-embryoids emerging from the exine.

Figure 2.14-4. a) Some mature embryoids/calli; b) Plantlets on Reg-IT medium; c) Plantlets on Reg-III (for rooting); d) Plantlets ready for transfer to GH.

xxviii

Figure 2.14-5. a) Plants 4 dafter the transplant; b) Plants 10 dafter the transplant; c) Plants 5 weeks after the transplant; d) Mature ears from DH plants.

xxix

Figure 2.15-l. Development of the microspores during cold pre-treatment. a) mid uninuclear microspore; b) late uninuclearmicrospore.

Figure 2.15-2. Androgenic development of maize microspores. a) microspore divided asymmetrically; b) callus; c) embryo formation.

Figure 2.15-3. Androgenic response of the anthers of the DHI05xHMv5405 hybrid induced without (a) and in the presence of colchicine (b).

Figure 2.15-4. Plantlet differentiation (a) and growth (b).

XXX

Figure 2.15-5. DH line (No.109) selected from anther culture of the exotic genotype Chi 592.

Figure 2.15-6. DH line No.IOS selected from anther culture of an SC hybrid SR88xChi 592 of partly exotic origin

xxxi

Figure 2.16-1. a) Selected rice seeds; b) Growing of donor plants; c) Panicles showing different flag leave length; d)Wrapped panicles in moisten paper towel ready for cold shock; e) Florets containing anthers. Anthers containing microspores at the right stage of development should not exceed half of the floret length; f) Microspore at the early uninucleate stage of development; g) Microspore at the late uninucleate stage of development; h) Microspore at the early binucleate stage of development; i) Microspore at the late binucleate stage of development

xxxii

Figure 2.16-2. a) Plating of anthers under sterile conditions; b) Close up of figure a; c) Callus induction; d) Embryogenic globular structures developed from microspores; e) Calli being plated for plant regeneration; t) Shoot development in regeneration medium; g) Regenerated plants; h) Regenerated plants placed in Yoshida's culture solution for a good root development; i) Haploid plant ready to be diploidized by immersing the plant in a colchicine solution.

xxxiii

Figure 2.18-1. Stages of hexaploid triticale anther culture, (a-d) induction phase; a) Longitudinally broken anthers of variety Vero show embryo-like structures at the end of the fourth week of the induction phase. b) 5th week of anther culture of variety Bogo conditioned with ovaries (one can be noticed in the centre), androgenic structures of various shapes are visible; c) and d) Variability of shapes and sizes of androgenic structures produced from the anthers of variety Salvo after 5 weeks of the induction phase; e and t) Regeneration phase; e) Green and albino plants regenerated 3 weeks after placing androgenic structures onto the regeneration medium . t) Green plants were rooted for two weeks in magenta boxes. At this stage they are most sensitive to the colchicine treatment according to the protocol 1.

XXXIV

Figure 2.19-l. In vitro androgenesis of isolated triticale microspores in directly isolated microspore culture; a) Freshly isolated late-stage uninucleated microspores in 0.3 M mannitol; b) Separation of viable (band between 0.58 M maltose and 0.3 M mannitol) and dead (at the bottom of the tube) microspores from blendor macerated crude head suspension on the maltose/mannitol cushion; c) Dividing multicellular (dense cells) and non-responsive (dead) microspores 1-week after isolation; d) Well-growing microspore-derived aggregates 2-week after isolation; e) ELS in the 4th week of culturing while non-divided microspores (little white points) are visible in background; f) Germinated bipolar embryo on regeneration medium with first and second leaf initiatives (up) and at the opposite side (down) little root is visible at 6-7 week after isolation; g) Microspore-derived colchicine treated androgenic triticale spike with sectorial fertility (arrow).

XXXV

Figure 2.20-1. a) At the left, kernel-like structure (enlarged ovary) obtained after pollination of triticale variety Salvo with maize variety Gama and treatment with 100 mg/L dicamba applied one day later. At the right kernel developing after the selfpollination of the same cultivar of the same age, i. e. 18 day after pollination; b-d) Triticale haploid embryos at time of isolation for culture; e and f) Triticale haploid embryos germinating in vitro.

xxxvi

Figure 2.21-1. a,b) Optimal microspore developmental stages for rye anther culture, (a) late uninucleate stage, (b) first pollen mitosis (anaphase); c) Responding rye anthers on induction medium; d,e) Proliferation of ELS and callus on induction medium; t) Green plant regeneration through gametic embryogenesis on induction medium; g) anther culture derived green rye regenerants on regeneration medium; h) Anther culture derived green plants from F 1 cross.

xxxvii

Figure 2.24-L Stages of embryo germination and development of haploid durum plants: a) A small embryo (arrow) in the empty karyopsis (without endosperm), 16 days after pollination and daily post-pollination treatments with 3 mg/L 2, 4-D + 120 mg/L AgNO:J; b) Lower: the embryo shown in A is rescued onto hormonefree MS medium placed in an incubator in the druk Upper: a torpedo-shaped, small embryo rescued from a different karyopsis. Some of these small embryos are also viable; c,d) Stages of embryo sprouting on the medium in the dalk; e) A young plantlet (without chlorophyll) with well developed coleoptile and several primary roots, transferred to a lighted incubator; f) Green plantlet with small primary roots. Note chlorophyll that developed under light.

-.--'" . '

~"

Figure 2.24-2. Somatic and meiotic chromosomes of haploid durum plants: a) Conventionally stained 14 somatic chromosomes from root tips. Note the satellited chromosomes IB and 6 B; I B has a smaller satellite; b) Conventionally stained meiotic chromosomes from pollen mother cells, note 14 unpaired chromosomes (univalents). Pairing between the A-genome and the B-genome chromosomes is suppressed by Phi (see also Fig. d); c) Somatic chromosomes (after fluorescent GISH) from root tips of haploid plantlets. Note 7 A-genome chromosomes (green) and 7 B-genome chromosomes (red). Labeled (biotinylated) total genomic DNA from A· genome donor, T. urartu, was used as a probe; d) Meiotic chromosomes from pollen mother cells of immature anthers of haploid plants. Fluorescent GISH reveals the 7 univalents of the A genome (green) and 7 univalents of the B genome (red). Note Phi-induced suppression of pairing.

xxxviii

Figure 2.25-l. Different stages of durum anther culture, green and albino regenerants, and chromosomes of haploid durum plantlet. a) Mid uninucleate microspore; note the nucleus and germpore; b) Appropriately staged yellowish green anthers, cultured on liquid medium; note that both lobes are in contact with the medium; c) An anther on BAD-3 induction medium, three weeks after culture showing the formation of several calli; d) Callus initiated on BAC-1 medium transferred to BAD-I differentiation medium 4 weeks after initial anther culture; e) Differentiating callus showing embryoid formation with a shoot initial. Note chlorophyll that developed in the darl