BIBLIOTECA, UU OYUN 10 AThe Practical Approach Series SERIES EDITORS ». RIcKWOOD Department of Biology, University of Essex Wivenhoe Park, Colchester, Essex CO4 38Q, UK B.D. HAMES Deparoment of Biochemisry and Molecular Biology University of Leeds, Leeds LS2 9IT, UK Affinity Chromatography Anaerobic Microbiology Animal Cel] Culture (2nd Edition) Animal Virus Pathogenesis Antibodies I and Il Basic Cell Culture Behavioural Nouroscience Biochemical Toxicology Biological Data Analysis Biological Membranes Biomechanics—Materials Biomechanics—Structures and Systems Biosensors Carbohydrate Analysis (2nd Edition) Cell—Cell Interactions The Cell Cycle Cell Growth and Division Cellular Calcium Cellular Interactions in Development Cellular Neurobiology Centrifugation (2nd Edition) Clinical Immunology Computers in Microbiology Crystallization of Nucleic Acids and Proteins Cytokines ‘The Cytoskeleton Diagnostic Molecular Pathology Tand Il Directed Mutagenesis DNA Cloning I, 1, and Ill Drosophila Electron Microscopy in Biology Electron Microscopy in Molecular Biology Electrophysiology Enzyme Assays Essential Developmental Biology Essential Molecular Biology I and Experimental Neuroanatomy Extracellular Matrix Fermentation Flow Cytometry (2nd Edition) Gas Chromatography Gel Electrophoresis of Nucleic Acids (2nd Edition) Gel Electrophoresis of Proteins (2nd Edition) Gone Targeting Gene Transcription Genome Analysis Glycobiology Growth Factors Haemopoiesis Histocompatibility Testing HPLC of Macromolecules HPLG of Small Molecules Human Cytogenetics I and II (2nd Edition) Human Genetic Disease Analysis Immobilised Cells and. Enzymes Immunocytochemistry In Situ Hybridization lodinated Density Gradient Media Light Microscopy in Biology Lipid Analysis Lipid Modification of Proteins Lipoprotein Analysis Liposomes Lymphocytes Mammalian Cell Biotechnology Mammalian Development Medical Bacteriology Medical Mycology Medical Virology Microcomputers in Biochemistry Microcomputers in Biology Microcomputers in Physiology Mitochondria Molecular Genetic Analysis of Populations, Molecular Genetics of Yeast Molecular Imaging in Neuroscience Molecular Neurobiology Molecular Plant Pathology Vand I Molecular Virology Monitoring Neuronal Activity Mutagenicity Testing Neural Transplantation Neurochemistry Neuronal Cell Lines NMR of Biological Macromolecules Nucleic Acid Hybridisation Nucleic Acid and Protein Sequence Analysis Nucleic Acids Sequencing Oligonucleotides and Analogues Oligonucleotide Synthesis PCR PCR—Volume 2 Peptide Antigens Peptide Hormone Action Peptide Hormone Secretion Photosynthesis: Energy Transduction Plant Gell Biology Plant Cell Culture Plant Molecular BiologyPlasmids (2nd Edition) Pollination Ecology Postimplantation Mammalian Embryos Preparative Centrifugation Prostaglandins and Related Substances Protein Architecture Protein Blotting Protein Engineering Protein Function Protein Phosphorylation Protein Purification Applications Protein Purification Methods Protein Sequencing Protein Structure Protein Targeting Proteolytic Enzymes Radioisotopes in Biology Receptor Biochemistry Receptor-Bffector Coupling Receptor—Ligand Interactions Ribosomes and Protein Synthesis RNA Processing I and Il Signal Transduction Solid Phase Peptide Synthesis Spectrophotometry and Spectrofluorimetry Steroid Hormones Teratocarcinomas and Embryonic Stem Cells ‘Transcription Factors Transcription and Translation ‘Tumour Immunobiology Virology Yeast Plant Cell Culture A Practical Approach Second Edition Edited by RICHARD A. DIXON and ROBERT A. GONZALES Plant Biology Division, ‘Tho Samuel Roberts Noble Foundation, P.O. Box 2180, Ardmore, Oklahoma 73402, USA ONFORD UNIVERSIFY PRESS Oxtord New York TokyoFes P52 © PS is toot nis tec prinet dls ee proce a suerte 1999 OXFORD (Great Clarendon Stet, Oxford OX2 6DP Onfond University Pressis a deparaien ofthe Universi of Oxon Ie furthers the Unisersy"soljestive of excolene ia researeh scholarship, ‘nd education by publishing worldwide (Ostnd New York Auckland Cape Town Dares Selusm Hong Kong. Karachi Kuala Lumpur Maid Melboume Mewicn City Naito ‘New Delhi ShasghaTaipe! Toren ‘witha it Arssrina Austra Srull Chile Caech Repulic Frans Greece Gustinals Hungary Ialy Japan South Korea Polind. Portal ‘Singapore Switzerland Thailnd Turkey Ukraine Vietnam (Oxf is a episted ade nak f Ostend Universi Inthe UK andi cota her couatios Published inthe United Sats by Oxford University Pres Ine, New York © Oxfond Universiy ress, 1988 Not ta be reprinted hour pessbsion The snort righ ofthe wor have been ser Datahose ight Ost Livesity Press imal) Reprinted 2006 All ght eservod. No arto this publication rmy be epreduced, ‘sore ina retrieval ster of tase, in ay form or by any men witout the prior permission in wring of Oxf Lnveraty Press ‘ras expresly ported by er user terns agree With he appeopot rspropraphies nights gonaation. Engines concerning raadlon bse ne scape of he abuve ahd Be sent he Rigs Departs ‘Oxford University ross he aes ahve ‘You smst mt isi this boo in anyother binding arcover ‘And you mie! impose ths sme conition onan Ae QUiEE ISBN 97S-0-19.86802-6 Preface The second edition of this book incorporates a number of significant changes, to the content and scope of the first edition (published in 1985). These primarily reflect the impact of molecular biology on the plant sciences, Re- generation of plants from cell culture is a key step in many genetic transfor- ‘mation strategies, and protoplasts have increasingly been used as recipient cells for DNA delivery for both transient gene expression and stable transfor- ‘mation studies. The last few years have scen rapid progress in understanding plant stress physiology at the molecular level, hence the inclusion of new sections on cell selection for cold, salinity, and toxin resistance. Whereas in 1985 the manipulation of secondary metabolite levels in cell cultures could only be performed by varying the chemical and physical parameters of the culture environment, today both levels and composition of secondary meta- bolites can be manipulated by gene transfer. We hope the new sections will be of value to plant molecular biologists, particularly by providing specific examples of the different tissue culture systems and approaches that can be used. We have, however, omitted details of recombinant DNA techniques from this volume, as these are readily available from other volumes in this Asin the first edition, this volume describes methods for the establishment ‘and manipulation of well-characterized cell culture systems, as well as more general advice that should enable rescarchers to develop their own protocols with their specific species of interest. Its still true to say that development of tissue culture protocols is in part an empirical science (or perhaps even an ‘art?), but the increasing body of information available makes it ever easier to make generalizations that will have some utility. We hope that the scope of this second edition will adequately encompass the broad range of areas, both fundamental and applied, in which plant cell culture technology is a central ‘component Ardmore R. November 1993 RA 9Contents List of contributors vi List of abbreviations 1. Initiation and maintenance of callus and cell suspension cultures 1 CL. Franklin and R.A. Dixon 1. Introduction 1 2. Culture media 2 Composition of commonly used culture media 2 Plant growth regulators 9 Preparation of eultre media 8 Proparation and sterilization of explants 1s 4. Initiation of callus and suspension cultures 16 Non-embryogenie cell cultures 6 Embryogenic cll cultures 18 5. Meesurement of growth parameters 2 Fresh and dry weight measurements 21 Tnerease in cell umber 2 Packed cell volume 23 Cel viability 23 Roferences 2 2, Isolation, culture, and regeneration of protoplasts n VW. Blackhall, M. R. Davey, and J, B. Power 1, Introduction a 2, Protoplast isolation wB Enzyme teatment 8 Protoplast purification 2% Visualization of cell walls 0 Determination of protoplast viability 0 Counting protoplasts 30Contents 3. Protoplast culture Media Culture procedures 4, Concluding remarks Referonces Applications of protoplast technology 3A. Fusion and selection of somatic hybrids N.W. Blackhall, M. R. Davey, and. B. Power Introduction Protoplast fusion ‘Chemical fusion Electrical fusion Selection of heterokaryons and somatic hybrid tissues “Manual selection Complementation Flow cytometry 4. Characterization of somatic hybrid plants 5. Concluding remarks References ‘3B. Transiont gene expression and stable transformation R.A, Dixon 1. Introduction 2, Transient gene expression 3. Stable transformation References 3C. Studies with viruses James C. Register Itt 4, Introduction 2. Appropriate uses/limitations of protoplasts 3, Protoplast preparation 4, Inoculation Electroporation 2 2 33 38 9 49 cy 3 55 56 56 37 st 37 Contents 5. Assessing virus accumulation Removal of cross-reacting antibodies Protein dot-blot assay Nucleic acid replication 6. Applications to resistance Resistance at the single cel level Blockage of ouclec acid replication Blockage of views uncoating 7. Concluding remarks Acknowledgements References Selection of plant cells for desirable characteristics 4A. Inhibitor resistance Robert A. Gonzales 1. Introduction 2. Plant cell culture 3. Dose-response curves 4. Mutant selection 5. Notes on mutant selection Acknowledgements References 4B, Cold tolerance D.C. W. Brown and J. Singh 4. Introduction 2. Target systems and selection strategies 3. Assessment of plant survival References 4G. In vitro selection for salt tolerance 1. Winicov Introduction 2, Cellular salt tolerance Somaclonal variation ar Isolation of stable variants salt xi n 8 3 22885. Contents 3. Characteristics of salt tolerance at the regenerated plant level 4. Generation of salt tolerant alfalfa plants through tissue culture Tissue culture Selection for cellular salt tolerance Regeneration of alfalfa plants from salt tolerant cell culture ‘Testing regenerated plants for improved salt tolerance. References 4D. In vitro selection for disease/toxin resistance Jan Brazotot, Kang Fu Yu, and K, Peter Pauls 1. Introduction 2, Toxic preparations from sease-causing organisms 3. Plant tissue cultures for in vitro selection 4, Selection strategies 5. Regenerating and testing plant material for disease resistance Acknowledgements, References Plant regeneration via embryogenic suspension cultures John J, Finer 1. Introduction 2. Production of embryogenic suspension cultures Tnitaton of embryogenic suspension cultures Proliferation Methods for establishing an embryogenic suspension culture Development and maturation of somatic embryos Specie protocols fr plant regeneration via somatic embryogenesis 3. Uses of embryogenic suspension cultures Desiccation and artificial seeds ‘Transformation Cloning of elite germplasm Physiological studies Cryopreservation Acknowledgements References xii aL 8 8 8 a 7 90 2 98 7 Contents 6. Applied aspects of plant regeneration GA. Micropropagation Stefan P. O. Werbrouck and Pierre C. Debergh 1. Introduction 2, Stage 0: the preparative stage Hygienic conditions Physiologica! conditions 3. Stage 1: initiation of culture Choice of explant Environmental conditions 4, Stage 2: shoot multiplication Media ‘Adventitious versus axillary bud formation Number of subcultures 5. Stage 9: shoot elongation and root induction or development Elongation Root induction 6. Stage 4: transfer to greenhouse conditions Acknowledgement References 6B. Virus-free plants Jolin L. Sherwood 1. Introduction 2. Overview of methods Facilites and equipment Heat therapy Selection of tissue and meristem culture Detection of viruses in cultured tissue Detection by infectivity Deteetion of virus oF virus inclusions Detection of virus components 4. Conclusions Acknowledgement Roferences xiii a7 i 17 18 128 128 18 128 19 129 129 130 bo 1B 1 132 132 132 133 134 134 135 13s 13s 135 136 137 137 137 137 138 Bs 138Contents BC. Artificial seeds D.C. W. Brown 1. Introduction, 2, Dried somatic embryos of Medicago sativa 3. Dried microspore-derived embryos of Brassica napus 4, Dried immature embryos of Triticum aestivum Roferences Cryopreservation Erica E. Benson 1. Introduction 2. Theory and terminology Choice of plant material and pre-cuture Pre-growth Cryoprotective treatments Freezing and storage Thawing Recovery 3. Cryopreservation equipment 4. A cryopreservation strategy 5. Protocols for method development re-growth Chemical exyoprotection Cayoprotective dehydration treatments Encapsulation and dehydration Vitafication Freezing ‘Thawing and recovery 6. Protocols for specific applications ‘Chemical eryoprotetion and controled cooling Vitsfcation methods Cayoprotective dehydration methods Encapsvlation and dehydration 7. Post-thaw viability, recovery, and stability Fluorescein diacetate vital stain The tiphenyl tetrazolium chloride (TTC) assay 8. Conclusions Acknowledgements References 138 138 140 144 14s “7 147 147 17 147 148 139 149 v9 150 150 131 151 153 154 155 136 187 1s7 157 159 162 163 168 165 165 166 Contents Secondary products from cultured cells and organs: I. Molecular and cellular approaches Richard J. Robins 1 2 a Introduction Strategies for inducing secondary product formation in suspension cultures Improvement of quinoline alkaloid production in Cinchiona Tedgeriana cutores Bicitation of acridone epoxide alkaloids in Rute graveotens suspension cultures Strategies for ostablishing excised root-organ cultures ‘competent in secondary product formation Establishment of tropane alkaloid producing cultures of Hyoscyamus niger and H. afbus Establishment of pyrrolziine alkaloid producing cultures of Senecio species Strategies for establishing transformed root-organ cultures competent in secondary product formation Establishment of pyridine alkaloid producing cultures of ‘Nicotiana rustica Establishment of tropane alkaloid producing cultures of Datura, siramonium Stratogies for establishing shoot-organ cultures ‘competent in secondary product formation Induction and propagation of shoot cultures of C. ledgeriana Establishment of transformed shoot-orgaa cultures of Mentha citrata and M.piperita Use of cultures for studies of the biochemistry of secondary production formation Feeding of precursors [Extraction and assay of enzymes Influence of morphology on secondary product form: Produetion stability in eultures—effcct of inducing morphology Production stability in eultures—effect of disrupting morphology Chromosomal stability in cultures Genetic manipulation of secondary product formation ‘Over-expression of a yeast ornithine decarboxylase gene in NN. rustica root eultores ‘Over-expression of a tryptophan decarboxylase gene in Peganumt armala Over-expression ofa lysine decarboxylase gene in N tabacum Acknowledgements References 169 169 170 17 im 174 15 176 im 178 119 381 181 12 184 tsa 187 189 190 11 wh wm 194 9s 196 197 197Contents Secondary products from cuulured cells and organs: Il. Large scale culture A. H, Scragg 1. Introdu Production of natural or secondary pxoteiis Biotransformations Enzyme production Production of human proteins Micropropagation 2. Properties of plant coll suspensioxsnsand organized cultures Culture rheology Shear sensitivity of plant cell cultures 3. Bioreactor design Bioreactors for suspension cultures Bioreactors for organized cultures Bioreactors for transformed cultures Biorcactors for immobilized cultures 4, Bioreactor use 5. Concluding remarks References Appendix. Addresses of suppliers Index xvi 199 199 201 201 201 201 om 203 205 208 216 217 219 m 2m 28 2 Contributors ERICA E, BENSON Department of Molecular and Life Sciences, ‘The University of Abertay Dundee, Bell Street, Dundee DD1 1HG, Scotland, UK. NW. BLACKHALL Department of Life Science, University of Nottingham, University Park, Nottingham NG7 2RD, UK. JAN BRAZOLOT Department of Crop Science, Ontario Agricultural College, University of Guelph, Guelph, Ontario NIG 2W1, Canada. D.C. W, BROWN Plant Research Center, Central Experimental Farm, Agriculture Canada, Ottawa, Ontario K1A 0C5, Canada. M. R. DAVEY Department of Life Science, University of Nottingham, University Park, Nottingham NG7 2RD, UK. PUERRE C. DEBERGH Laboratory of Horticulture, State University Gent, Faculty of Agricultural Sciences, Coupure Links 653, B-9000 Gent, Belgium R.A. DIXON Plant Biology Division, Samuel Roberts Noble Foundation, Inc., P.O. Box 2180, Ardmore, Oklahoma 73402, USA. JOHN J. FINER Department of Agronomy, Ohio State University, 1680 Madison Avenue, Wooster, Ohio 44691, USA. 1. FRANKLIN Plant Biology Division, Samuel Roberts Noble Foundation, Inc., PO Box 2180, Ardmore, Oklahoma 73402, USA. ROBERT A. GONZALES Plant Biology Division, Samuel Roberts Noble Foundation, Inc., PO Box 2180, Ardmore, Oklahoma 73402, USA. K, PETER PAULS Department of Crop Science, Ontario Agricultural College, University of i elph, Ontario NIG 2W1, Canada,Contributors 1. B. POWER Department of Life Science, University of Nottingham, University Park, Nottingham NG7 2RD, UK. JAMES C. REGISTER It Pioneer Hi-Bred International, Inc., Department of Analytical Biochemistry, 7300 NW 62nd Ave., PO Box 1004, Johnston, Iowa 50131, USA. RICHARD J. ROBINS Genetics and Microbiology Department, Institute of Food Research (Norwich Laboratory), Norwich Research Park, Colney, Norwich NR47UA, UK. AH. SCRAGG Department of Molecular Biology, University of the West of England, Cold- harbour Lane, Frenchay, Bristol BSI6 1OY, UK. JOHN L. SHERWOOD Department of Plant Pathology, Oklahoma State University, Stillwater, Oklahoma 74078, USA. 4. SINGH Plant Research Center, Central Experimental Farm, Agriculture Canada, Ottawa, Ontario KIA OCS, Canada. STEPAAN P. 0. WERBROUCK Laboratory of Horticulture, State University Gent, Faculty of Agricultural Sciences, Coupure Links 653, B-9000 Gent, Belgium. 1, wINtcov. Departments of Microbiology and Biochemistry, University of Nevada, Reno, Nevada 89557, USA. KANG FU YU Department of Crop Science, Ontario Agricultural College, University of Guelph, Guelph, Ontario NIG 2W1, Canada. xviii ABA ‘ADC bs BAP BSA camv cat or CPM 24D D¢ DFMA DFMO DM DMso EDTA FDA nD iM GA ac dus Hopes APLC Lay 1AA tA we « Upc Meoit Mis MIC MS tedium NAA NMR NDA ap hs / Abbreviations abscisic acid arginine decarboxylase Gamborg’s BS medium 6-benzylaminopurine bovine serum albumi ‘cauliflower mosaic virus chloramphenicol acetyltransferase coat protein coat protein-mediated protection 2,4-dichlorophenoxyacetic acid direct current e-difluoromethyl-DL-arginine ‘cdifluoromethyl-Dt-ornithine dry mass dimethylsulfoxide ethylenediamine tetraacetic acid fluorescein diacetate flame ionization detector fresh mass, gibberellic acid ‘gas chromatography B-glucuronidase N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid high performance liquid chromatography ‘concentration resulting in 50% inhibition indole acetic acid indole-3-butyric acid isopentenyladenine kinetin (furfurylaminopurine) lysine decarboxylase methanol 2-(N-morpholino)-cthanesulfonic acid minimum concentration resulting in 100% inhibition Murashige and Skoog medium I-naphthaleneacetic acid nuclear magnetic resonance naphthoxyacetic acid hine decarboxylase phosphate-buffered salinepcPA PCR PCV PDA PEG PGR PND PVDF SH medium TDC DZ. T™V Zea ZR Abbreviations p-chlorophenoxyacetic acid polymerase chain reaction packed cell volume potato dextrose agar polyethylene glycol plant growth regulator putresceine N-methyltransferase phosphorus/nitrogen detector polyvinylidene difluoride relative humidity Schenk and Hildebrandt medium stirred-tank bioreactor tryptophan decarboxylase hidiazuron tobacco mosaic virus triphenyl tetrazolium chloride zeatin riboside Initiation and maintenance of callus and cell suspension cultures C. 1 PRANKLIN and R, A. DIXON 1. Introduction Establishing dedifferentiated cultures from organized plant tissues was a ‘major goal of early plant cell culture studies. Today, this isa relatively routine procedure which is an essential prerequisite for a range of subsequent approaches including regeneration, embryogenesis, growth of large scale cultures, and selection strategies. The key to establishing callus and cell suspension cultures is the choice of the optimum culture medium com- ponents, proper explant source, and plant growth regulator concentrations. ‘Thisis still largely empirical. However, a large number of plant species from a wide-range of families have now been successfully grown in culture, and ‘access to this information allows the researcher to make a good guess at conditions which may be suitable for the growth of species whose culture requirements have not been determined. In the fitst edition of this book (1), we discussed in some detail the basic ‘equipment and facilities necessary for media preparation and the initiation and growth of plant cell cultures. These have also been described in other more comprehensive reference books (2, 3). The following list outlines the major items you will require: «© chemical balance © pH meter + volumetric flasks and beakers for media preparation ‘# culture vessels (Petri dishes and conical flasks) ‘# autoclave ‘+ laminar flow hood for aseptic transfers ‘* open platform orbital shakers (such as those sold by L. H. Engineering, UK, or New Brunswick Scientific, USA) ‘* constant temperature room or incubator(s) which can be set at 25 + 2°C ‘© fluorescent lighting controlled by a timerC. 1 Franklin and R. A. Dixon applied. However, ‘microbial contamination of cultures is difficult to avoi ‘completely if a transfer hood is not used. 2. Culture media first decision to be made when initiating a plant callus culture is the media are described in the Titerature, but of these, only a few have foun widespread usage fora range of plant species (c.g. MS,SH,and BS, see Table). In culture medium containing 20 ot more components, there is an infin possibility for variation of composition. However, there is sufficient informa: wr in the literature 10 indicate which features of the culture medium are portant, requiting optimization, and which, within certain prescribed limits, are of less importance. Table 2 presents a very selective overview of plant species which have been successfully grown in callus or suspension Piiture, and provides details of the basic medium and growth regulator concentrations. For an extensive list of media formulations for various plant species refer to George et al. (12), Ifyou wish to work with a species listed in Table 2, it would be wise £0 start with the culture conditions referred (0 therein, However, remember that there may be significant differences be- tween the behaviour of individual cultivars of a species with respect to growth jn culture, and that some optimization of culture conditions may be necessary (Gee Section 2.2) It your species isnot listed in Table 2, or George etal. (12), Gecide on your initial test medium by comparing media used for closely related species. 2.1 Composition of commonly used culture media ‘This and the following section outline the components found in plant cell aa ai dia, Methods for preparing culture media are discussed in Section Se probably the most commonly used plant culture medium is that of Murashige and Skoog (4) (Table 1). As with nearly all plant culture media its components can be divided into six groups. ‘These are: «¢ major inorganic nutrients « trace elements « iron source «vitamins carbon source «plant growth reeut 1: Initiation and maintenance of callus and cell suspension cultures ‘These divisions are both functional and practical (i. they reflect the way in which the various stock solutions are made up). Note that plant growth regulator concentrations are omitted from the basic medium formulation. ‘This is because, by alteration of growth regulator concentrations, itis possible to greatly affect growth and differentiation in culture. Growth regulator concentrations required for maintenance of a dedifferentiated callus line of one species may induce organogenesis in another species. The variations in growth regulator levels required for callus growth of different species are lear from a brief inspection of Table 2. It is important to remember that reference to a culture medium by its abbreviation (c.g. MS, SH) generally implies the culture medium minus growth regulators. These are listed in full after the medium name, e.g. MS medium supplemented with 10°* M 2,4-D and 5 x 10"? M kinetin. More detailed advice on choice of plant growth regulators is given in Section 2.2. ‘Components of a few selected tissue culture media are compared in Table 1. One of the important components of the basal medium is nitrogen. MS medium has high levels of inorganic nitrogen while other formulations, such as NN, have very low levels. Some species may require or tolerate higher levels of nitrogen in the medium than others. Certain explants of some species do not require or can not tolerate higher levels of nitrogen in the medium, e.g. immature ovules of Impatiens platypetala (81). With some species, e.g. rice, the ratio between NH and NO; is very critical for cell culture and plant regeneration (42). In general, there is a tendency to use lower levels of NILE than NO; in plant tissue culture media, The amount of NHj in MS medium is almost half that of NOz, while other formulations, such as SH, BS, and GD, contain even lower levels of Nz. here are several reports inthe literature describing the influence of organic nitrogen in the tissue culture medium. Organic nitrogen has been shown to enhance somatic embryogenesis in some species, e.g. orchard grass (56), Agrostis (57), and organogenesis in others, ¢.g. green bean (11). Most com- ‘monly, inorganic nitrogen in the medium is supplemented with organic nitro- igen in the form of proline or glutamine (57-59). In a few tissue culture systems the organic nitrogen (e.g. glutamine) serves as the sole nitrogen source in the medium (11). The components of the SI medium used for ijenerating shoots from bean cotyledons (11) are identical to those of MS medium except for the nitrogen source and sucrose concentration (Table 1). The inorganic nitrogen is compictely replaced by 10 mM glutamine (1462 mg/ lite) which is filter sterilized and added to the autoclaved and cooled imedium, Deletion of KNO, lowers the K* concentration in the medium by IK.8 mM, but this can be compensated for by adding 18.8 mM (1400 mg/litre) KC! to the medium. We have not observed any harmful effects of elevated CL levels in our bean tissue culture system. The LM medium (9) listed in Table 1 is idea! for initiation and maintenance of rapidly growing loblolly pine cell cultures (C. 1. Franklin, unpublished a‘Table 1. A comparison of components in commonly used plant tissue culture media or media modified for a particular species or for 8 ‘specific purpose (see text) Constituent Cancion ner mau (ngte Me ene ce an ot catnoy, a ‘KNOS 2 1900 2500 2500 1000 1900 1900 950, wt, beet ow tok wah a0 twas, Ps mes 7,0 mmm watt cecisan8 a eto =o "2 ma xe sm ‘sco hsro, 0 wm awe Noro 1 Mosoe,0 woot E wins na = : e tis) © E30 Polo Sok anna om 80, Gyo kg occurs | nh cam a te 2S0.740 te Pte a Saal an Ss . us ee cus0.sH.0 0.025 02 0.025, 0.025 0.025 0.50 0.025 0.025 NapMo0,24.0 025, on 025 0.026 025 125 0.25 025 CoCiz6H;0 0.025 on 0.025, 025 0.025 0125, 0.028 FeS047H,0 28 150 28 28 28 278 278 Sequestrene 330 Fe a NarEDTA 33 200 a3 373 313 23 m3 Nicotinic acid os 50 10 on os 5 os: Pyridoxine-Hc 0s 05 19 on 1 on os 08: Thiamine Hct on 50 10.0 10 1 on os: on PBiotin 02 oor 0.05 Folie acid ot os. ‘mye-Inositol 100 1000 100 10 100 100 100 100 Giycine 20 4 on, 2 20 Glutamine 1asz Sucrose 30000, 30000 20000, 20000 20000 30000 20000 10000 Additional components of KM mesium (mili: leotinamige 1: easlum e-pentothenst t folic aid 04; p-eminobenzoie sla 0.02: choline chloride 1; siboflevin 02; aacorbs exis 2 itemin A Ot vitamin By O01. viternin Bye 002 secur pyruvate 2: ete abe 4 mate acid a0; hanaie sed US acess, Fiboae, xylose, mannose, shamnose. cellobose, sorb), and mannitol, 260 sesh adaninn equanin, trying urscll argorea ane ana ne 1 21 caning mids 01 each except glataming 86 laine O6, glutamic ad 06 ane cyssing OF IMS = Murashigo and Skoog (), SH = Sehenk and Hidebranct 65 = Garnborg sta (3), GD ~ Gresshft and Doy (1, KM = Kao and Michayick 6), LM = ‘vay era), NN Nth end Nise (0), and St Franklin ta [¥),“oul 2 Mod and growth regulator lees ued for cals and cal clues ol eeced epces Vor an etensve Ie of ter iar eons evel 12) spaces splat Man’ Growth egos (8) neterence Allure sum s aaia 1s ‘raids thatine Send, or ot s 2avaee+K2s ® freon hypoges Ccnyedon ws heat ” Aopraoue oft voce ts papas+K18 ® ‘vena save ioe or ier be we BaD 9 + BAP Ad» CM 10% ® a $ paps ken bo wagore s Nass? = a? a atari ine es pape 2 Caneye s NAADS + Ko9-+ CM 15% B icone idorione ts baba + Kop % Cotes saea $ babs. Keo = Sten ws ea 1082 for 256.4 ae ws Rast s aPon 2 Gomera =p. Shoot ae wn Naa 260 + EM 10% = Estee Fone pene we 24 as + cM 10% » Euphroatrcolt stom us a4bas © NAA TO = Gite bicbe Pollen : aaDananie ron ote Bin er Eaydon hs bao ane 2 Goss rst myposan § Naa ig? EAPO +AIS22 38 wort noe or is paves kar x Fordeum vulgare wet *s nepea s Renn Lemna gibbe Frond s 24-0453 + 2 49 36 Lithops leslie’ Leat ws 24D 45+ K 465 3 Mammilaria elonge Tubercle s 240274 +K47-83 (on IP 49-98 39 Mangitera indica Nucellue s 24D9 33 Medicago sativa Leaf, ypocorv!, or catyleden 24045 + K O99 + Ad 74 40 Nicotiana tabacum Leat MS (ori 24045 a Onze sativa Immature embryo, s 2408 2 Pennisetum americanum Inflorescence s 240113 e Petunia hybrid eat s 26094K12 “4 Phoenix dactyiters Shoottips or embryo s 24-0 440 + 2iP 145 6 Pinus taeda Stem iw 240226 3 Pisum sativum Root s 24D 68+NAAOS+IAAST+K12 46 resis virata izome 8 24:09 + CM 10% 0 Saccharum sp. Leat Ms 24023-1386 @ Sorghum bicolor Leat Ms. 2409+ Kos 4 Theobroma eacao Cotyledon 85 24065+K09 50 Triticum aestivum Immature embryo s 2409 st Vitis vinifera ‘Stem, leat, or fruit 3s NAA OS + KO9 + CM 18% 82 Yucea schidigera Hypocoty! Ms 24045 5 Zamia pumila Embryo 85 24023 +k 47-186 4 Zea mays Immature embryo 8 2.4045-08 6 “MS = Murashige and Skoog medium (4; LS = Linger and Skoog meciur {1 85 ‘meiom formulation or modted forthe study eeporas: 28:0 ~ 26 ds lorphmnovyaete BAP = -bonzvlaminepurne: K= town; Ad = sdanine, dS > edenina ou ambos B5 medium (6): Wh = White's madium (14) S = apes Std; NAA = T-naphinaleneacatc sig, IAA ~ indole aca sae P= Nisopestenylarinopurin’ CM = covgnat mi: Pes peerC.1. Franklin and R. A. Dixon results) and may be suitable for other conifers as well. The GD and NN media are relatively ‘low-salt’ media. The GD medium (7) bas been very useful for recovering loblolly pine plantlets from regenerated shoots (C. I, Franklin, ‘unpublished results). The NN medium (10) is useful for regenerating haploid plants from pollen grains. The KM medium listed in Table 1 (medium No. 6, in ref. 8) is very complex with several additives such as organic acids, amino acids, nucleic avid bases, etc., yet it is a defined medium, and it has been reported to support the growth of Vicia hajastana cells at low initial popula- tion density (25-50 cells/ml) (8). However, addition of undefined media components such as casein hydrolysate and coconut water (see below) was ceessaty for culturing V. hajastana cells at a much lower density of 1-2 cells! ml (medium No. 8, in ref. 8). ‘A comparison between the media in Table 1 features: « all the media are fully defined (i.e. no added biological extracts) ‘ a chelated iron source (Fe-EDTA) is preferred (less easily depleted) ‘® MS and SH are ‘high salt’ media ‘¢ MS and SH use both ammonium and nitrate ions as source of nitrogen ights several important SH medium contains a very high level of myo-inositol sucrose is the preferred carbon source Generally, use of fully-defined media is recommended, as the addition of biological supplements (casein hydrolysate, yeast extract, or cocomut milk) introduces possible variation due to differences in the composition of differ- ent batches of additive. However, these additives may be beneficial and may supplement or replace the vitamins in certain cases, e.g. White’s medium (14). Yeast extract and casein hydrolysate are readily available from most microbiological suppliers (e.g. Oxoid Ltd. in the UK, Sigma Chemical Com- pany, or Difeo Laboratories in the USA). You can purchase coconut milk from the suppliers listed above or prepare it according to Protocol 1. Protocol 1. Preparation of coconut milk 1. Drain the milk from a large number of coconuts.” 2. Deproteinize by boiling for 10 min. . Fier through Whatman No. 1 filter paper. Autoclave in small batches (< 100 mi) 5. Store at ~70°C until required . Thaw and add to culture medium at a final level of ~10% (v/v). + Use of lage oumbor of coconuts minimizes variation. Check the mith fom each coconut Invilly before pooling, ond discard any which appoors tas | eee 1: Initiation and maintenance of callus and cell suspension cultures 2.2 Plant growth regulators Most plant culture media contain an auxin and a cytokinin, These two classes of growth regulators, at concentrations generally around 10 wM, hetp main- tain dedifferentiated cell growth and promote cell division respectively. ‘The most commonly used plant growth regulators are listed in Table 3. 2,4-D is the most widely used synthetic auxin, especially for gramineous species. 2,4- D and NAA have largely replaced the naturally occurring auxin, TAA, in cell culture media, as the latter is readily oxidized by plant cells. Often, callus growth may require lower levels of auxin than needed for callus induction. Very low levels of auxin, or complete omission, may often induce organo- enesis. TRAP and kinetin are the most commonly used cytokinins, Very few culture media employ GA, for initiation or growth of callus cultures. However, this, growth regulator has been shown to be beneficial for the growth of potato cells in callus and suspension culture (61). New or less eommonty used plant ‘growth regulators and their properties are listed in Table 4. These compounds are mainly either synthetic analogues of native growth regulators such as [AA or zeatin, or were originally developed as herbicides. Some of these com- pounds are more potent than the native growth regulators Usually, manipulation of auxin and eytokinin levels will be successful in fining a growth regulator balance necessary for the required behaviour in culture. An experimental approach for such a manipulation is outlined in Protocol 2. If desired results are not obtained using commonly used growth regulators, it is worth trying some of the novel growth regulators listed in Table 4. Most of the aurxins and cytokinins commonly used in plant cell culture work are synthetic. IAA, Gay, zeatin, and ABA are naturally occurring plant growth regulators. In the last several years, a number of new naturally plant growth regulating compounds have been discovered. These have not yet found use in cell culture formulations, but the most interesting are listed below since they may prove useful in the future. It is worth noting that a number of them are phenolic compounds chydrodiconifery! alcohol glycosides (cytokinin-like) (70, 71) chlorogenic acid (protects TAA from oxidation) (72) i avonoids (inhibit auxin transport) (74) rious ethylene inhibitors arious Gas biosynthesis inhibitors (62) A wide-range of other plant secondary products have been listed in the cearlier literature as potential naturally occurring growth regulators (75). Much of the older plant cell culture literature listed concentrations of 9 monates (promote leaf senescence) (73)‘Table 3. Commonly used plant growth regulators Class Name ‘Abbreviation Comments? Auxin Indole-S-acetic acid i Use for callus induction at 10-30 uM. Lowering to 1~10 nM organogenesis. Is inactivated by light and readily oxidized by plant calls. The synthetic auxins below have largely superceded lAA for tissue culture studies Indole-s-buryrie seid 3a Use for rooting shoots regenerated vie organogensis. Either ‘maintain at low concentration (1=50 uM) throughout rooting process, or expose to high concentration (100-250 uN) for 2-10 days and then transfer to hormone-tree medium. Can also use as a dip for in vitro or ax vitro rooting of shoots. 24Diehlorophenoxyscetic cid 2.4 ‘Most commonly used synthetic auxin for inducing callus land maintaining callus and suspension cells in dedifferentiated state. Usually used ac sole auxin source (1-0 uM), or in combination with NAA. p-Chlorophenoxyacetic acid CPA Similar to 24-D, but less commonly used. ‘-Naphthaleneacetic acid Naa ‘Synthetic analague of IAA. Commaniy used, either as sole ‘auxin source (2-20 uM for ealus induction and growth of callus and suspension cultures; 0.2-2 wM for root induction), or in combination with 24°. SS Cytokinin 6-Furfurylaminopurine {kinein)—K ‘Often inckuded in culture mecia tor callus induction, growth Of callus and cell suspensions, end induction of ‘morphogenesis [1~20 uM). Higher concentrations (20-50 uM) can be used to induce the rapld munipleation of shoots, axllary/advertitious buds, oF meristems. 6-Benzylaminopurine BAP Includes in culture madia for callus induction, and growin of callus and call suspensions (0.55.0 uM), and for induction of morphogenesis (1-10 uM. More commonly ‘sed than kinetin for inducing rapid mutiplication of shoots, ‘buds, or meristems (5-50 uM} ‘Nsopentenylaminopurine 2P Less commonly used than X oF BAP for callus induction and ‘groweh (2-10 pM), induction of morphogenesis {10-25 MI, (oF multiplication of shoots, buds, or meristems (30-50 nM) Zeatin 2ea Seldom used in callus or suspension media. Can be used for induction of morphogenesis (0.08~10 pM). Zea is ‘thermoiabile and must not be autoclaved. Gibberellin Gibberolin Ay A; Seldom used in callus or suspension medium (one ‘exception being potato (58). Can promate shoot growth when added to shoot induction medium st 0.03-14 .M. ‘Also used to enhance development in embryo/ovule ‘cultures (0.348 ,MI. GAy is thermolabile and must not be autoclaved. Used at concentrations of 0.0410 uM to prevent precocious ‘germination, and promote normal development of somatic ‘embryos (60). Abscisic acid Abscisic acid ABA “The concentrations givanrecrasent vals takon frm the tersture fra rang of lant species. Speci applications fr spaces not desert in the taratre lest check Tobie 2 should be determined experimentalC.1. Franklin and R. A, Dixon Fable & Novel and/or lose commonly used plant growth regulators for plant tissue culture [Abbrevietion/ M,_--Function/ectivity Referenco compound Choreehoin chaside?™ OCC 1581 Inititorof GAy 82 Thomyrnose machors pid. — ecPU 2427 Gratin tke 8) herve wee eroaisic aid icamba «2210 Renin Pa 2Noprnonpoosicid NOR moe? Ann e Prenvacat oc Pan foo2Auwn & eammnoase. oe eo tremoropletne sid ‘watesuron? +02 2202 owatninike BusTicNorophenonyocote BAT ‘sa Nx = a eatin iboside* zn 3884 Ofoknin © “iar starieation recommended Peete Gee We REE ee ee Protocol 2. Experimental approach to optimize growth regulator levels 41. Select one or more commonly used media formulations (e.g. MS, SH, or BS). 2. Optimize the growth regulator(s) requirements by setting up experi. ments usiag concentrations and ratios of auxin and cytokinin listed below. 3. Select the combination of explant source, medium, and growth regular tor levels giving best results. 4. Further optimization (if necessary} can be done by investigating the ‘offects of growth regulator levels in between the concentrations listed here or by using other auxins or cytokinins. 24D Kinetin (NM) wean bo asa a ee | as) an ns am ts deo zsns 25" 2525 280 ee” ins an tas 8 3 eo os wh toasts Bo zs 2s warts per million). The modern convention is ws the conversion from. growth regulators in meg/litre : to express all concentrations as molarity. Table 5 sho -¢ to 4M for the commonly used plant growth regulators 2 1: Initiation and maintenance of callus and cell suspension cultures ‘Table 5. Conversion of mglltea to uM concentiations for commonly used plant growth regulators om NA aD WA A BARK ro 662 2210782002282 2152 mMB?_—-2192 2.0005 0.0004 0.0005 0.0005 a.oso# 10005 0.6008 0.0005, 2008 0.00% 0008 ans onad 0.005 0.005 doz? 0023, 0028 © cc25 0020280028 bas 00459057 0045 ek 0K 027 02% 0285 ©0748-0222 a2a6 dst 04s? 057d oase «ane tes 0a 4a name tz zou 228288 2a Sar 452571 age aaa yeas 2282 ask) 2220332 carl a2 570897) aa ana aa. 13s ete 26912962 oY tH817 12303 2a 22624-26838 74606 22207273234 4808 NAA = anaphialeneacstie acid; 240 = 24-ichlrophenoxyacatic acid: IAA = indole acetic ai TBa~ indole butyre a8: BAP = 6banayiaminoputing;K= kon; 21 = sopentenylamingpurinw, Zan ~ Dentin. 2.3 Preparation of culture media ‘The most efficient way of preparing plant culture media is first to make up stock solutions of major inorganic nutrients, trace elements, iron source, vitamins, and individual plant growth regulators. An example of how to do his for the MS medium is given in Table 6. Store the vitamins frozen at 20°C in small batehes (e.g. enough for one litre of medium), thaw, and mix fully before use. The other stocks can be kept in a refrigerator at 4°C, but should be frequently checked and discarded if precipitation occurs; do not store inorganic stock solutions longer than one month. It is advisable to make up small stocks of growth regulators fresh for each batch of media, as small changes in concentration due to precipitation can seriously affect growth of the cultures. Protocol 3 outlines the preparation of media for growth of cell suspension cultures, For semi-solid media, add agar at a final concentration of 6=10 g/ litre prior to autoclaving. Itis important to use a good quality, bacteriological grade agar for plant cell culture work. Suitable agars for most work are New Zealand Agar (BDH Chemicals, Ltd, UK), Oxoid Bacteriological Agar No. 1 (Oxoid Ltd, UK), BiTek agar (Difco Laboratories, Detroit, MI, USA), or phytagar (GIBCO Laboratories, NY, USA). For pouring Petri dishes of agar medium, itis convenient to autoclave 500 mi batches in 1 litre conical flasks to dissolve the agar, allow the medium to coo! to around 40°C, and then pour. 13G.1 Franklin and R. A. Dixon ‘Table 6. Composition and preparation of Murashige and Skoog medium Constituent _Molarity in. Concentration of ‘Volume of stock Storage of medium stock solution per tra of stock solution mg/l) ‘medium {on [Major inorganic nutrients NH4NO3 208 x 10% 33000 KNO 1.88 x 10° 38000, caC.2H,0 300 10 8800 0 44°C MgSO.7H.0 1.60 x 10% 7400 KPO, 1.25 x 10 3000, ‘Trace elements Kt 5.00 x 10% 166 13805 1100 x 104 7240 MnSO.4H0 999 10 4460 ZnSOeTH.0 2.00 10 1720 5 4c NazMoO42H:0 1.00 x 10° 50 CuS0.8H,0 100x107 5 Coct,gH0 100x107 5 Iron source FeS0,7H,0 100x104 8560 y ‘eget Na,EDTA2H,O 1.00 x 10 7460 Organic supplement ‘myo-inositol 4.80 x 10 20000 Nicotinic acid 465 10 100 Pyridoxine HCl 240 x 10 100 5 20°C Thiamine-HC! 3.00 107 100 (in 5 ml aliquots) Giycine 300 x 10° 400 Cordon source Sucrose agox107 ~ ‘Add 28 solid (20 gilt) Protocol 3. Preparation of plant cell culture media 1, Prepare stock solutions’ of major inorganic nutrients, trace elements, vitamins, and plant growth regulators using analytical reagent grade chemicals and double distilled water. 2. For 1 litre of liquid medium, pipette the required volumes of each stock solution into a 1 litre glass beaker on a magnetic stirrer. 3. Add sucrose as solid. Stir until itis fully dissolved, adding more water if necessary.” 14 4: Initiation and maintenance of callus and cetl suspension cultures 4. Adjust the volume to ~ 950 ml with double dis 5. Adjust the pH to the required value (usually 5.8-5.9) with 0.5 M NaOH. 6. Transfer to a 1 litre measuring cylinder or volumetric flask, and adjust volume to 1 litre with double distilled water. illed water. 7. Transfer back to the flask and stir for complete mixing. 8. Transfer 75 ml batches of medium to clean 250 m! conical flasks, plug with non-absorbent cotton wool or sponge plugs, cover the tops with aluminium foil, and autoclave for 18 min at 120°C (1.06 ka/em). * Seon inorganic stock sobtions #1 4°C for & maximum of ane meth. Oispente vitamins in Or slquots and store at 20°C: Prapare groweh regulator stocks fresh esc te: ausine are Unvoly tested into solution with NGOH, whereas cytokinin are disoived inde NEOM oF ‘squbous ethanol grows regulators ae tharmoltile fe Taber 3 an, et sterlirad “iter the autoclaved medium hos cote * coneantates media mey be stored frozen {-20°0) at this stage. ‘Note that, although the pH of most plant culture media is adjusted to between pH 5.5 and 6.0 prior to autoclaving, the final pH may be stightly lower due to the production of sugar acids during autoclaving. ‘The tength of time required to sterilize a batch of culture medium by autoclaving will depend upon the volume of medium in the flask. For 250 ml batches, 25 minutes at 120°C is adequate. Thermolabite growth regulators (see Tables 3 and 4) must be added to the medium after it has cooled 10 40°C (semi-solid media) or below. 3. Preparation and sterilization of explants A wide-range of plant organs and tissues can be used as a source of explants for the initiation of callus cultures; this can be appreciated from a glance at Table 2. The choice of explant is generally dictated by the aims of the research. Some explant sources are preferable to others if embryogenic cul- lures are to be established for regeneration studies (see below). Likewise, for biochemical studies of secondary product synthesis, explant source may be critical, For example, alfalfa roots appear to maintain much of their secondary product metabolism even when dedifferentiated and grown as a suspension culture (76). Protocols 4 and 5 outline sterilization procedures for seed and tuber tissues. It is often a good idea to use seedlings from sterilized seed as a source of sterile root, shoot, and leaf material. Small seed can be directly plated on callus induction medium; the seed will germinate and then the tissues will callus directly. In such cases, the callus may be of mixed origin with respect to the initiating cell type. With larger seed, itis possible to dissect sterile radicle nd plumule explants directly after sterilization of the seed coat (Protocol 6). 15C.1. Franklin and R.A. Dixon tiation of callus Protocol 4. Sterilization of seeds for 1, Submerge seeds in absolute ethanol for 10 sec, or in 10% commercial bleach solution for 15 min with constant agitation. 2. Rinse in sterile distilled water (soak overnight if large seeds are to be used for dissection of radicles, see Protocol 61 3, Select seeds with intact testas," and submerge in 10% commercial bleach solution, containing 0.05% (v/v) Teepo! detergent, for 20 min? 4. Wash three times in sterile distilled water. '. Prepare seeds for callus initiation by one of the following strategies. {a) For small seeds, plate out whole seeds on agar medium for callus induction or germination. (b) Germinate seeds on agar culture medium lacking growth regula- tors, Use sterile root, shoer, of leaf tissue for callus initiation. {c) Dissect and plate out radicle or plumule tips for callus initiation (see Protocol! 6). * bamage estes are more really apparent shor the seed has soaked in sn aqueous soon, Microbial contaminant can gain entry through damagod testes where they ae subsequeny plotected from tho bleach solton Prone lf clcum hypechlonte or 1% wy) bromine water ar cltrntiv sterling agers, Protocol 5. Sterilization of storage organs (e.g. potato or artichoke tubers) prior to initiation of callus 1. Serub clean under cunning top-water 2, Submerge in 10% commercial bleach solution for 30 min. 3. Wash three t storile distilled water. 4. Cut off an area of the skin with a sterile scalpel 5. Remove small discs of tisoue with a sterile cork Borer (5-10 mm diameter} and scalpel. 6. Place discs face down on callus induction me 4, Initiation of callus and suspension cultures 4.1 Non-embryogenic cell cultures Initiate callus by gently pressing the explant on to the surface of a suitable agar-solidified medium. Explants are usually placed horizontally, although stem sections may produce more callus if placed vertically with one eut end in 1: Initiation and maintenance of callus and cell suspension cultures he agar. Consult Tables 1 and 2, and Section 2.1, for details of suitable culture media. Remember that inclusion of an auxin and cytokinin will be necessary for callus growth, and that somewhat higher auxin concentrations may be required for callus initiation as compared to callus growth in some cases. After plating out explants, it is important (0 seal Petri plates with Ciingilm or Parafilm in order to prevent desiccation. Plates should then be incubated at approximately 25°C either in the dark or, if beneficial, under low level illumination, Protocol 6. Initiation of callus cultures from radicle tips of French bean (Phaseolus vulgaris) 1, Place seeds in 8.5 cm diameter Petri dishes (15 seeds/disht 2. Fill dishes with 10% commercial bleach solution, ensuring seeds are fully covered. Leave for 20 min. 3. Wash the seeds in the Petri dish with three changes of 20 mi sterile distiled water. Leave seeds In Petri dish to soak overnight in sterile distilled water Discard any seeds with cracked testas {use sterile forceps). Re-sterilize the seeds in 10% bleach solution for 20 min, Wash the seeds with three changes of 20 ml sterile . Transfer the seeds to sterile Petri dishes (5 seeds/dish). 9. Using sterile forceps to hold the seed, make two cuts with a sterile scalpel in order to allow the testa to be pulled back to reveal the radicle, led water. 10. Cut out the radicle tips (2-3 mm} and transfer to Petri dishes contain- ing sterile distilled water (16 tipsfdish. 111, Transfer the radiele tips to SH medium supplemented with 10° M PCPA, 2 x 10° M2,4-D, and 10°” M kinotin (5 tips/Petri dish). Seal the dishes with Parafilm and transfer to a dark cabinet at 25°C. If sufficient callus for subculture has not formed within three to eight weeks, it will be necessary to re-evaluate the culture medium and conditions. Subculture by removing hewly formed callus with a sterile scalpel and trans- {erring to fresh medium. Be careful not (o squash the somewhat fragile callus too much when re-plating, and do not cut the callus into 100 many very small pieces. The optimum inoculum size varies depending on the species, but itis best to err on the large side while the culture is in the early stages of estab- lishment. Once well established, transfer approximately 3-10 mm? of callus clumps from actively growing regions to the fresh medium, Most callus 17C. Franklin and R. A. Dixon cultures will require regular subculture at approximately one month intervals. For newly initiated callus, it may be necessary to transfer the entire callus to the fresh medium for the first two or three subcultures. Fast growing callus ‘cultures may require subculture at two to three week intervals. In general, suspension cultures form readily after transfer of callus to shaken flasks of the culture medium minus agar. A large inoculum may be necessary (0 initiate the suspension, and agitation rates on orbital shakers, should be in the range of 30-15) r.p.m, with an orbital motion stroke of 2-4 om, If the callus culture is non-friable, very litle will break off the callus ‘lumps and the necessary inoculum size of freely suspended cells and small cell clusters may not be attained. In such cases, modifications may have to be ‘made to the callus maintenance or induction media in order to produce a ‘more friable callus; the approaches here are somewhat empirical ‘When the newly established suspension culture has reached a suitable cell density for subculture, remove as much of the remaining unbroken callus ‘material and large clumps as possible, This can be done by either transferring the single cells and small clumps with a sterile syringe (the orifice of which will exclude large clumps), by filtration, by allowing the large material to settle and pipetting off from the top of the culture, or simply by direct pouring Remember, however, that there is a minimum inoculum size below which cell suspensions do not readily resume active growth following transfer, and that in some cultures most of the growth occurs on the surface of small clumps. ‘The minimum density of cells required for cell suspension cultures depends ‘on the rate of growth and the composition of the medium. Generally, ten per cent of initial cell density to the total volume of the culture will be sufficient Addition of medium in which the cell line had previously been growing (con- ditioned medium) can sometimes stimulate growth of cell suspensions at low inoculum density. The growth rate of the culture will often increase if the cells are transferred before the culture reaches stationary phase. For some pur- poses, ¢.g. isolation of protoplasts which retain viability after electroporation (Gee Chapter 3), it is very important to have a vigorously growing culture. 4.2 Embryogenic cell cultures Callus and cell cultures having the potential to produce plantlets via somatic embryogenesis are referred to as embryogenic cultures (see Chapter 5). Embryogenic cultures are widely used for obtaining transgenic plants, soma- clonal variants, and mutants. Transgenic maize, wheat, rice, and oat plants have been regenerated from embryogenic callus or cell cultures transformed via biolistic bombardment-mediated DNA. transfer. Protoplasts, isolated from embryogenic rice cell cultures, have been transformed via electro- poration or polyethylene glycol-mediated DNA transfer for obtaining trat genic plants (see Chapter 3B). In grasses, two different types of embryogenic cali are produced, ‘The fist 18 i: Initiation and maintenance of callus and cell suspension cultures type, referred to as type Lembryogenic callus, is organized, compact, white or pale yellow in colour, and slow growing, The other type, referred to as type TI callus, is friable, soft, somewhat translucent, and rapidly growing. Use of a dissecting microscope will make it easier to identify the non-embryonic callus from the embryogenic callus. Generally, the non-embryogenic callus s friable, {ast growing, and lacking in organized structures. Embryogenic callus, on the other hand, is usually compact (except for type II callus in the case of grasses) nd will show organized structures especially during the later stages of differ- ntiation (see Figure 2), ‘Choosing the right explant is very critical for establishing embryogenic cell cultures. Immature parts of a plant (¢.g. immature embryos/inflorescence, young leaves/petioles, and hypocotyls from young seedlings) have been used as explants for producing embryogenic callus, e.g. immature embryos for wheat (51), maize (55), and soybean (60); young leaves/coleoptiles for cauca- sian bluestem (82). Proioco! 7 describes the initiation and maintenance of nbryogenic cell cultures from seed explants of the forage grass caucasian bluestem (Bothriochloa caucavica), and the regeneration of plants from these cultures. With some species, there is a tendency for the embyrogenic cell cultures t0 Jose embryogenic potential (i.e. ability to regenerate plants) alter a prolonged culture period. Therefore, itis essential periodically to test the regenerability of these cultures. As an alternative, cell clumps from embryogenic cultures showing good embryogenic potential can be cryo- preserved for future use (see Chapter 7) Figure 1. Embryogenic (0) and non-embryogenic (ne) callus of caucasian bluestem (x 7). 19