UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

IN VITRO culture of red clover (TRIFOLIUM PRATENSE L.) and evaluation of regenerated plants Wang, Hong 1985

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1985_A6_7 W35.pdf [ 10.65MB ]
Metadata
JSON: 831-1.0096266.json
JSON-LD: 831-1.0096266-ld.json
RDF/XML (Pretty): 831-1.0096266-rdf.xml
RDF/JSON: 831-1.0096266-rdf.json
Turtle: 831-1.0096266-turtle.txt
N-Triples: 831-1.0096266-rdf-ntriples.txt
Original Record: 831-1.0096266-source.json
Full Text
831-1.0096266-fulltext.txt
Citation
831-1.0096266.ris

Full Text

IN VITRO CULTURE OF RED CLOVER (TRIFOLIUM PRATENSE L. ) AND A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Plan t Science We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1985 © HONG WANG, 1985 EVALUATION OF REGENERATED PLANTS by HONG WANG In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) A b s t r a c t Red c l o v e r ( T r i f o l i u m p r a t e n s e L.) cvs 'Altaswede' (2n=2X=14) and 'Norseman' (2n=4X=28) were used i n the present study to i n v e s t i g a t e t i s s u e c u l t u r e i n i t i a t i o n , p l a n t r e g e n e r a t i o n and the o c c u r r e n c e of somaclonal v a r i a t i o n . Hypocotyl e x p l a n t s of a s e p t i c s e e d l i n g s were i n o c u l a t e d i n t o L2 medium c o n t a i n i n g 0.06 mg/1 P i c l o r a m and 0.1 mg/1 benzyladenine f o r c a l l u s i n d u c t i o n . C a l l i were u s u a l l y induced a f t e r two weeks of c u l t u r e . C a l l u s i n d u c t i o n frequency was 60% to 85% of the e x p l a n t s c u l t u r e d with 'Altaswede' showing a s l i g h t l y higher frequency than 'Norseman'. S a t i s f a c t o r y r e s u l t s were obtained under dark or l i g h t c o n d i t i o n s using e i t h e r t e s t tubes or p e t r i p l a t e s , as c u l t u r e v e s s e l s . A f t e r c a l l u s i n d u c t i o n , an experiment was conducted to r e g u l a t e shoot i n d u c t i o n by s u b c u l t u r i n g the c a l l i on L2 medium c o n t a i n i n g 0.01 mg/1 2,4-dichlorophenoxy a c e t i c a c i d and 2 mg/1 adenine (LSE) and on B 5 medium c o n t a i n i n g 2 mg/1 naphthalene a c e t i c a c i d and 2 mg/1 adenine, media which have been r e p o r t e d to be s h o o t - s u p p o r t i v e . However, both media f a i l e d to i n i t i a t e shoots under the present experimental c o n d i t i o n s . F u r t h e r t e s t s c o n f i r m e d that LSE medium d i d not induce shoots from these c a l l i and that c a l l u s growth on LSE medium s t e a d i l y d e t e r i o r a t e d over s e v e r a l s u b c u l t u r e s . Subsequently, v a r i o u s media were t e s t e d with an emphasis on d i f f e r e n t combinations of growth r e g u l a t o r s . Root d i f f e r e n t i a t i o n from these c a l l i was f r e q u e n t l y observed. i i Shoots were i n i t i a t e d from some c a l l i when they were t r a n s f e r r e d from SCP medium to media c o n t a i n i n g naphthalene a c e t i c a c i d and k i n e t i n . Embryogenic c a l l u s of one genotype was s e l e c t e d and maintained on LSP medium, l e a d i n g to the re g e n e r a t i o n of numerous p l a n t s . Supplementation with a r g i n i n e , glutamic a c i d and c a s e i n h y d r o l y s a t e d i d not show a s i g n i f i c a n t e f f e c t on c a l l u s growth and d i f f e r e n t i a t i o n . The source of c a l l u s i n f l u e n c e d r a t e s of growth and the occurrence of d i f f e r e n t i a t i o n . U s u a l l y 'Norseman' c a l l i grew f a s t e r and produced more r o o t s than 'Altaswede' c a l l i , while shoots were induced only from 'Altaswede' c a l l i . Although 'Norseman' had more shoot t i p s induced to produce m u l t i p l e shoots, the m u l t i p l e shoot number per c u l t u r e of 'Altaswede' was higher than that of 'Norseman'. Shoot t i p c u l t u r e s were a l s o e s t a b l i s h e d t o induce m u l t i p l e shoots and to regenerate p l a n t s v i a root organogenesis. Regenerants from i n i t i a l m u l t i p l e shoots (RG1), m u l t i p l e shoots a f t e r two s u b c u l t u r e s (RG2), three-month c a l l i (RG3) and one-year c a l l i (RG4) were e v a l u a t e d f o r chromosome number s t a b i l i t y , morphology and s e v e r a l b i o c h e m i c a l t r a i t s . When 'Altaswede' p l a n t s were analysed f o r chromosome number, RG1 and RG3 p l a n t s were normal, while one RG2 p l a n t and 23% of 119 RG4 p l a n t s had t e t r a p l o i d chromosome numbers. Regenerated p l a n t s were q u i t e s t a b l e r e g a r d i n g t h e i r isozyme p a t t e r n s of malate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucose isomerase, phosphoglucomutase and shikimate dehydrogenase and t h e i r nodule leghaemoglobin p r o f i l e s . M o r p h o l o g i c a l l y , the l e a f l e t l e n g t h to width r a t i o of RG1, RG2 and RG3 p l a n t s of 'Altaswede' showed s i g n i f i c a n t l y more v a r i a t i o n than c o n t r o l p l a n t s (P<0.01), while RG4 p l a n t s of 'Altaswede' and RG1 and RG2 p l a n t s of 'Norseman' were not d i f f e r e n t from c o n t r o l p l a n t s . I t i s suggested that the absence of d e t e c t a b l e d i f f e r e n c e s i n the RG4 'Altaswede' p l a n t s was a consequence of t h e i r o r i g i n from one o r i g i n a l genotype. V a r i a b i l i t y and s t a b i l i t y of regenerated p l a n t s are d i s c u s s e d . i v A b b r e v i a t i o n s f o r Growth Regulators and Media Auxins 2,4-D: 2,4-dichlorophenoxy a c e t i c a c i d IAA: i n d o l e - 3 - a c e t i c a c i d NAA: naphthalene a c e t i c a c i d PIC: P i c l o r a m ( 4 - a m i n o - 3 , 5 , 6 - t r i c h l o r o p i c l i n i c a c i d ) C y t o k i n i n s ADE: adenine (6-amino pu r i n e ) BA: benzyladenine 2 i P : ( 2 - i s o p e n t e n y l ) adenine KIN: k i n e t i n Media L2: P h i l l i p s and C o l l i n s (1979a) LSE: L2 medium c o n t a i n i n g 0.01 mg/1 2,4-D and 2 mg/1 ADE LSP: L2 meidum c o n t a i n i n g 0.002 mg/1 PIC and 0.2 mg/1 BA SCP: L2 medium c o n t a i n i n g 2 mg/1 2,4-D, 2 mg/1 BA and 2 mg/1 ADE. v Table of Contents Chapter Page A b s t r a c t i i A b b r e v i a t i o n s f o r Growth Regulators and Media i i i Table of Contents v i L i s t of Tables x L i s t of F i g u r e s x i i Aknowledgement x i v 1. INTRODUCTION 1 2. LITERATURE REVIEW 4 2.1 T i s s u e C u l t u r e i n Red C l o v e r 4 2.2 Somaclonal V a r i a t i o n .7 2.2.1 V a r i a t i o n among Regenerated P l a n t s of Se v e r a l Crop Species .8 2.2.1.1 Sugar Cane 8 2.2.1.2 Potato .9 2.2.1.3 Tobacco 11 2.2.1.4 Rice and Wheat 12 2.2.2 O r i g i n of and F a c t o r s A f f e c t i n g Somaclonal V a r i a t i o n 13 2.2.3 Genetic B a s i s 17 3. MATERIALS AND METHODS 22 3.1 General C u l t u r e Methodology 22 3.1.1 Media 22 3.1.2 Explan t s 24 3.1.3 C u l t u r e C o n d i t i o n s 25 3.2 S p e c i f i c C u l t u r e Methodology 26 3.2.1 C a l l u s Induction 26 v i 3.2.2 C a l l u s Maintenance and P l a n t Regeneration : 3.2.2.1 Primary Test of Two Media : 3.2.2.2 C a l l u s Growth on LSE Medium : 3.2.2.3 2,4-D and BA : 3.2.2.4 PIC and BA : 3.2.2.5 NAA and KIN : 3.2.2.6 NAA and BA i n Presence of ADE ... 3.2.2.7 C a l l u s D i f f e r e n t i a t i o n on Four Test Media 3.2.2.8 C a l l u s Growth at High or Low Auxin to C y t o k i n i n R a t i o s 3.2.2.9 C a l l u s Growth on SCP Medium 3.2.2.10 L i g h t E f f e c t on C a l l i Growing on SCP Medium i 3.2.2.11 S e l e c t i o n and Maintenance of Embryogenic C a l l u s 36 3.2.2.12 Growth of Embryogenic and Non-embryogenic C a l l i 3.2.3 Shoot T i p C u l t u r e 3.2.4 Rooting of Shoots ' 38 3.2.5 Scanning E l e c t r o n Microscopy 3.3 Growth and A n a l y s i s of Regenerated P l a n t s 3.3.1 Pl a n t T r a n s f e r and Growth i n Greenhouse .. 3.3.2 Chromosome A n a l y s i s 3.3.3 Leghaemoglobin A n a l y s i s 3.3.4 Isozyme A n a l y s i s 3.3.5 M o r p h o l o g i c a l A n a l y s i s RESULTS 4.1 C a l l u s Induction v i i 4.1.1 C a l l u s I n d u c t i o n Frequency 47 4.1.2 Genotypic E f f e c t on C a l l u s I n d u c t i o n 49 4.1.3 Observations on C a l l u s Induction 49 4.2 C a l l u s Maintenance and P l a n t Regeneration 52 4.2.1 Primary Test of Two Media 52 4.2.2 C a l l u s Growth on LSE Medium 53 4.2.3 2,4-D and BA 61 4.2.4 PIC and BA 61 4.2.5 NAA and KIN 64 4.2.6 NAA and BA i n Presence of ADE 73 4.2.7 C a l l u s D i f f e r e n t i a t i o n on Four Media 75 4.2.8 C a l l u s Growth and Auxin to C y t o k i n i n R a t i o 75 4.2.9 C a l l u s Growth on SCP Medium 79 4.2.10 L i g h t E f f e c t on C a l l i Growing on SCP Medium 84 4.2.11 Observations on Embryogenesis 84 4.2.12 S e l e c t i o n and Maintenance of Embryogenic C a l l i 87 4.2.13 Growth of Embryogenic and Non-embryogenic C a l l i 91 4.2.14 Summary of Experiments on C a l l u s Growth and D i f f e r e n t i a t i o n 94 4.2.15 P l a n t Regeneration from C a l l u s 95 4.3 Shoot T i p C u l t u r e 96 4.3.1 M u l t i p l e Shoot In d u c t i o n 96 4.3.2 M u l t i p l e Shoot Propagation 96 4.4 Growth and A n a l y s i s of Regenerated P l a n t s ......100 4.4.1 P l a n t T r a n s f e r and S u r v i v a l 101 v i i i 4.4.2 Chromosome Number S t a b i l i t y 103 4.4.3 Isozyme A n a l y s i s 106 4.4.4 Nodule Formation and Leghaemoglobin P r o f i l e 114 4.4.5 Plant Morphology 114 5. DISCUSSION 121 5.1 C a l l u s Induction 121 5.2 C a l l u s Maintenance and P l a n t Regeneration 122 5.2.1 Primary Test of Two Media ...122 5.2.2 C a l l u s Growth on LSE Medium 123 5.2.3 C a l l u s Growth on SCP Medium 125 5.2.4 Plant Regeneration: Organogenesis and Embryogenesis 126 . 5.2.5 Genotype E f f e c t ' 133 5.3 Shoot T i p C u l t u r e 136 5.4 Regenerated P l a n t s 136 6. Summary 143 LITERATURE CITED 146 • APPENDIX 1 158 ix L i s t o f T a b l e s Table Page 1. Treatment combinations used to t e s t the e f f e c t of PIC and BA on p l a n t r e g e n e r a t i o n . 31 2. C a l l u s i n d u c t i o n frequency f o r two c u l t i v a r s of red c l o v e r on L2 medium. 48 3. C a l l u s i n d u c t i o n from t h r e e e x p l a n t s d e r i v e d from the same s e e d l i n g . 50 4. C a l l u s growth on LSE medium. 53 5. Response of c a l l i on LSE medium: i n i t i a l and s u b c u l t u r e of Test 1 recorded a f t e r four and f i v e weeks of c u l t u r e , r e s p e c t i v e l y . 55 6. Response of c a l l i from LSE medium s u b c u l t u r e d onto four media. 59 7. Response of c a l l i on L2(D0.01A5) medium. 60 8. Response of c a l l i on media c o n t a i n i n g 2,4-D and BA recorded a f t e r t hree weeks of c u l t u r e . 62 9. Response of 'Altaswede' c a l l i on media c o n t a i n i n g PIC and BA i n presence and absence of a r g i n i n e . 63 10. Response of c a l l i on media with added c a s e i n h y d r o l y s a t e and glutamic a c i d . 65 11. Response of c a l l i of two c u l t i v a r s on media with v a r i o u s combinations of PIC and BA. 67 12. Response of 'Norseman' c a l l i from LSE medium to v a r i o u s combinations of NAA and KIN. 68 13. Response of c a l l i from SCP medium to combinations of NAA and KIN. 70 14. Response of 'Norseman' c a l l i to combinations of NAA and KIN. 72 15. Response of 'Norseman' c a l l i on media c o n t a i n i n g v a r i o u s l e v e l s of NAA i n combination with BA and ADE. 74 x 16. D i f f e r e n t i a t i o n of 'Norseman' c a l l i from L2(D2B2A5) medium on four media. 76 17. Response of 'Norseman' c a l l i at high (11) or low (0.1) auxin to c y t o k i n i n r a t i o . 77 18. Recovery frequency of senescing c a l l i a f t e r t r a n s f e r to SCP medium. 80 19. L i g h t e f f e c t on growth and d i f f e r e n t i a t i o n of 'Altaswede' c a l l i on SCP medium. 85 20. Embryo development and p l a n t l e t formation from embryogenic c a l l i s u b c u l t u r e d onto LSP medium. 89 21. M u l t i p l e shoot i n d u c t i o n from shoot t i p s of red c l o v e r on L2(P0.003B2) mediium. 97 22. Comparison of two c u l t i v a r s of red c l o v e r f o r m u l t i p l e shoot i n d u c t i o n . 97 23. M u l t i p l e shoot p r o d u c t i o n of two c u l t i v a r s of red c l o v e r . 99 24. S u r v i v a l r a t e of p l a n t s t r a n s f e r r e d to greenhouse a f t e r d i f f e r e n t treatments. 102 25. Chromosome numbers of 'Altaswede' p l a n t s regenerated through d i f f e r e n t ways. 104 26. L e a f l e t number i n c o n t r o l (seed-derived) and regenerated p l a n t s of red c l o v e r . 116 27. Means and v a r i a n c e s of l e a f l e t length/width r a t i o s f o r d i f f e r e n t p o p u l a t i o n s of red c l o v e r . 118 28. Comparisons of regenerated p l a n t s and c o n t r o l p l a n t s f o r means of l e a f l e t l e ngth/width r a t i o . 119 29. Comparisons of regenerated and c o n t r o l p l a n t s f o r the v a r i a n c e s of l e a f l e t l e n g t h / width r a t i o . 119 x i L i s t of F i g u r e s F i g u r e Page 1. C a l l u s i n d u c t i o n and genotype e f f e c t . 'Norseman' h y p o c o t y l e x p l a n t s on L2 medium. 51 2. Response of 'Altaswede' c a l l i on LSE medium a f t e r two weeks of c u l t u r e . 56 3. Response of 'Altaswede' c a l l i on LSE medium a f t e r f i v e weeks of c u l t u r e . 57 4. Response of 'Altaswede' c a l l i on L2 medium c o n t a i n i n g 0.005 mg/1 PIC, 1 mg/1 BA and 1 g/1 c a s e i n h y d r o l y s a t e a f t e r f i v e weeks of c u l t u r e . 66 5. Shoot formation from 'Altaswede' c a l l u s on L2(N0.05K2) medium. 71 6. Response of 'Norseman' c a l l i on media with d i f f e r e n t auxin to c y t o k i n i n r a t i o s a f t e r f i v e weeks of c u l t u r e . 78 7. Recovery of senescing c a l l i on SCP medium. 81 8. C a l l u s maintenance on SCP medium. 82 9. Embryogenic c a l l u s of 'Altaswede'. 86 10. Somatic embryo development from embryogenic c a l l u s of 'Altaswede'. 88 11. S e l e c t i o n of c a l l u s capable of embryogenisis. 90 12. Embryogenic c a l l u s a r i s i n g from regenerated p l a n t l e t at the p o i n t of root o r i g i n . 92 13. Embryogenic and non-embryogenic c a l l i of 'Altaswede' 93 14. M u l t i p l e shoot p r o d u c t i o n i n red c l o v e r . 98 15. Karyotype of normal root t i p of red c l o v e r cv. 'Altaswede' (2n=2X=14). 105 16. Root t i p chromosome numbers of a d i p l o i d / t e t r a p l o i d RG4 p l a n t of 'Altaswede'. 107 x i i 17. Zymogram of phosphoglucose isomerase from RG3 p l a n t s of red c l o v e r cv. 'Altaswede'. 110 18. Zymogram of phosphoglucomutase from c a l l i of red c l o v e r cv. 'Altaswede'. 111 19. Zymogram of malate dehydrogenase from RG3 p l a n t s of red c l o v e r cv. 'Altaswede'. 112 20. Zymogram of 6-phospholguconate dehydrogenase from RG3 p l a n t s of red c l o v e r cv. 'Altaswede'. 113 21. C e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s of leghaemoglobin of red c l o v e r cv. 'Altaswede'. 115 x i i i Aknowledgement I am most g r a t e f u l to Dr. F . B . H o l l , my s u p e r v i s o r , f o r h i s d i r e c t i o n , encouragement and h e l p throughout the p e r i o d of my s t u d i e s , and to the members of my t h e s i s committee, Drs. A.D.M.Glass, C.R.Norton and V.C.Runeckles f o r t h e i r reviewing the d r a f t and very u s e f u l comments. S i n c e r e thanks are extended to Ms. Mei Sun f o r her help i n the isozyme a n a l y s i s and to Mr. L.J.Veto f o r h i s guidance and a s s i s t a n c e with the SEM photos of c u l t u r e d m a t e r i a l s . Thanks a l s o go to Drs. R.R.Smith, E.Rosenberg and K.Cole f o r t h e i r a d v i c e with respect to methods for c y t o g e n e t i c a n a l y s i s . I want to express my a p p r e c i a t i o n to the f a c u l t y , s t a f f and students of the Department of P l a n t Science who p r o v i d e d h e l p and f r i e n d s h i p d u r i n g my s t u d i e s at UBC. I am a l s o g r a t e f u l f o r the f i n a n c i a l support of the M i n i s t r y of Education of the People's Republic of China and the c o n t i n u i n g encouragement of my f a m i l y and f r i e n d s i n China. I thank them f o r t h e i r commitments. x i v 1. I N T R O D U C T I O N Legume seed and forage s p e c i e s are the second most important food and feed crops a f t e r c e r e a l s i n world a g r i c u l t u r e . The present and f u t u r e u t i l i t y of p l a n t c e l l and t i s s u e c u l t u r e f o r the improvement of these crops depends upon e f f i c i e n t p l a n t r e g e n e r a t i o n from the c u l t u r e d m a t e r i a l s . U n f o r t u n a t e l y legume s p e c i e s are o f t e n d i f f i c u l t to regenerate from c e l l and c a l l u s c u l t u r e s . I t has been suggested that these s p e c i e s , e s p e c i a l l y the seed legumes, are e q u a l l y or perhaps even more d i f f i c u l t to manipulate in  v i t r o than grasses (Conger, 1981). However, much progress has been made r e c e n t l y i n the achievement of p l a n t r e g e n e r a t i o n i n forage legumes such as a l f a l f a (Medicaqo  s a t i v a L.) (McCoy and Walker, 1984) and red c l o v e r ( T r i f o l i u m pratense L.) ( P h i l l i p s and C o l l i n s , 1983). Red c l o v e r i s an important hay crop w i t h good feed value ( T a y l o r , 1973; T a y l o r and Smith, 1979). I t i s a l s o e x c e l l e n t f o r use as a r o t a t i o n crop due to- i t s s o i l improving p r o p e r t i e s . A red c l o v e r crop may r e t u r n to the s o i l between 125 and 200 kg of n i t r o g e n per ha. a n n u a l l y , thus g r e a t l y r e d u c i n g the need f o r and c o s t of n i t r o g e n f e r t i l i z e r f o r subsequent cr o p s . P l a n t r e g e n e r a t i o n from suspension and c a l l u s c u l t u r e s of non-meristematic t i s s u e s (e.g. h ypocotyl) of red c l o v e r was r e p o r t e d p r e v i o u s l y by P h i l l i p s and C o l l i n s (1979a; 1980). However, r e g e n e r a t i o n was dependent on the genotype used and the frequency of r e g e n e r a t i o n from h y p o c o t y l c a l l u s 1 2 was low ( P h i l l i p s and C o l l i n s , 1979a). T h e r e f o r e , p l a n t r e g e n e r a t i o n c o u l d be a problem when using another c u l t i v a r , or a l a r g e r number of regenerated p l a n t s are r e q u i r e d f o r study. I t i s b e l i e v e d that somaclonal v a r i a t i o n may r e p r e s e n t a novel source of v a r i a t i o n that can be e x p l o i t e d i n p l a n t b r e e d i n g programs ( L a r k i n and Scowcroft, 1981). P r e v i o u s work on the t i s s u e c u l t u r e of red c l o v e r focussed on the development of ijn v i t r o methods. Regenerated p l a n t s were observed to be normal both m o r p h o l o g i c a l l y and c y t o g e n e t i c a l l y (Beach and Smith, 1979; P h i l l i p s and C o l l i n s , 1979a). Information i s l a c k i n g as to whether a b n o r m a l i t i e s are a common phenomenon i n regenerated red c l o v e r p l a n t s . Most r e p o r t s concerned with the changes induced v i a t i s s u e c u l t u r e have been c e n t e r e d on q u a n t i t a t i v e c h a r a c t e r s . Because these c h a r a c t e r s are v a r i a b l e and e a s i l y a f f e c t e d by environmental f a c t o r s , somaclonal v a r i a t i o n i s confounded by environmental v a r i a t i o n . I t i s a l s o more d i f f i c u l t to assess the frequency of v a r i a t i o n f o r q u a n t i t a t i v e c h a r a c t e r s . I r v i n e (1984), using f i v e sugar cane (Saccharum o f f i c i n a r u m ) c l o n e s with d i s t i n c t i v e markers, observed o n l y a low frequency of s t a b l e v a r i a t i o n . The experiments conducted here, u s i n g red c l o v e r as the e x p e r i m e n t a l m a t e r i a l , were designed f o r two main purposes: (1) to i n v e s t i g a t e the r e g u l a t i o n of c a l l u s c u l t u r e d e r i v e d from h y p o c o t y l segments and p l a n t r e g e n e r a t i o n from the c u l t u r e s and (2) to e v a l u a t e the regenerated p l a n t s f o r somaclonal v a r i a t i o n i n the c h a r a c t e r s a n a l y s e d . 2. LITERATURE REVIEW 2.1 T i s s u e C u l t u r e i n Red C l o v e r N i i z e k and K i t a (1973) i n i t i a t e d the t i s s u e c u l t u r e of red c l o v e r u sing anthers as e x p l a n t s to induce c a l l u s . The b a s a l media of M i l l e r (1961) and Bourgin and N i t s c h (1967) m o d i f i e d with v a r i o u s combinations of IAA, NAA, BA and GA were e v a l u a t e d f o r c a l l u s i n d u c t i o n . Only on medium with 17 M M IAA and 0.6 M M BA added was c a l l u s induced. No morphogenesis o c c u r r e d . F o l l o w i n g t h i s r e p o r t o t h e r i n v e s t i g a t o r s (Ahloowalia, 1976; Ranga Rao, 1976) d e s c r i b e d c a l l u s c u l t u r e s i n i t i a t e d from d i f f e r e n t t i s s u e s . These r e p o r t s d e s c r i b e d l i t t l e evidence f o r morphogenesis and no p l a n t s were regenerated. Beach and Smith (1979) c u l t u r e d s e e d l i n g h y p o c o t y l s and o v a r i e s . C a l l u s formed when 2,4-D was present i n combination with auxin and KIN. In the absence of 2,4-D l i t t l e or no c a l l u s r e s u l t e d . Hypocotyl e x p l a n t s produced more c a l l u s than ovary t i s s u e . C a l l u s formation was most s u c c e s s f u l on B 5 medium (Gamborg et a l . , 1968) or a m o d i f i e d B 5 b a s a l medium c o n t a i n i n g 10 mg/1 thiamine, 11 uM NAA and 10 uM each of 2,4-D and KIN. C a l l u s p r o d u c t i o n was poor when B 5 was s u b s t i t u t e d by the mineral s a l t s of Blaydes (1966) or Murashige and Skoog (1962). A f t e r four weeks c a l l i were t r a n s f e r r e d t o another medium f o r shoot-bud d i f f e r e n t i a t i o n . Gamborg's B 5 with 20 mg/1 thiamine, 10 uM NAA and 15 M M ADE was the most e f f e c t i v e combination. Beach 4 5 and Smith (1979) a l s o observed that i t was the white f r i a b l e c a l l u s with i s o l a t e d green segments, r a t h e r than u n i f o r m l y green c a l l u s , that d i f f e r e n t i a t e d i n t o p l a n t s . KIN, 2iP and BA f a i l e d t o induce shoot d i f f e r e n t i a t i o n and the a d d i t i o n of coconut milk d i d not enhance c a l l u s f o rmation. More i n t e n s i v e i n v e s t i g a t i o n s have been conducted by P h i l l i p s and C o l l i n s (1979a; 1980; 1981). They (1979a) f i r s t e v a l u a t e d the b a s a l n u t r i e n t requirements f o r c a l l u s c u l t u r e s induced from h y p o c o t y l segments. V i s u a l e v a l u a t i o n of c u l t u r e s produced u s i n g more than 125 media l e d to the development of a m o d i f i e d medium, r e f e r r e d to as L2. T h i s medium was shown to be more s u p p o r t i v e of red c l o v e r genotypes i n c a l l u s c u l t u r e than other media t e s t e d . PIC was used as the auxin source, as i t was shown to be more e f f i c i e n t i n a c t i o n than 2,4-D ( C o l l i n s et a l . , 1978). C a l l u s was induced on L2 medium c o n t a i n i n g 0.06 mg/1 PIC and 0.1 mg/1 BA, a combination which had r e s u l t e d from the e v a l u a t i o n of s e v e r a l r e g u l a t o r s i n c l u d i n g IAA, NAA, PIC, 2,4-D, BA and KIN ( P h i l l i p s and C o l l i n s , 1979a). P l a n t r e g e n e r a t i o n was achieved when c a l l i were s u b c u l t u r e d onto other media. G e n e r a l l y meristem-derived c a l l i were more capable of r e g e n e r a t i n g p l a n t s than non-meristem d e r i v e d c a l l i . The frequency of genotypes that gave r e g e n e r a t i o n f o r the former was 30% to 80% but f o r the l a t t e r i t was only 1% ( P h i l l i p s and C o l l i n s , 1979a). For r e g e n e r a t i o n from non-meristem d e r i v e d c a l l i , a 2,4-D-containing medium ( i n combination with other auxins) was more e f f e c t i v e than the 6 PIC + BA combination. Pl a n t r e g e n e r a t i o n was a l s o a c h ieved from c e l l suspension c u l t u r e ( P h i l l i p s and C o l l i n s , 1980). E x p l a n t s of h y p o c o t y l and e p i c o t y l s e c t i o n s of s e e d l i n g s were put i n t o l i q u i d L2 medium c o n t a i n i n g 0.06 mg/1 PIC and 0.1 mg/1 BA to i n i t i a t e c e l l suspension c u l t u r e s . C a l l u s recovery and bud i n i t i a t i o n r e s u l t e d a f t e r a small amount of suspension c u l t u r e was i n o c u l a t e d on agar media. Both the k i n d and c o n c e n t r a t i o n of auxin were c r i t i c a l f o r bud i n i t i a t i o n and the medium (de s i g n a t e d LSE) c o n t a i n i n g 0.01 mg/1 2,4-D and 2.0 mg/1 ADE was most e f f e c t i v e . A second medium (LSP), which c o n t a i n s 0.001 mg/1 PIC and 0.2 mg/1 BA was found to be e f f e c t i v e f o r shoot development from buds. Shoot t i p s c o u l d be p r o l i f e r a t e d on a medium c o n t a i n i n g 0.003 mg/1 PIC and 0.5 mg/1 BA ( P a r r o t and C o l l i n s , 1983). In other experiments by Campbell and Tomes (1983) BA was s u p e r i o r t o 2iP and KIN, with maximum shoot number at the c o n c e n t r a t i o n of 2.0 mg/1 BA. Other ijn v i t r o experiments have been conducted to develop methods of s p e c i a l use. P h i l l i p s and C o l l i n s (1979b) e s t a b l i s h e d the procedures f o r meristem c u l t u r e t o o b t a i n virus-symptom f r e e p l a n t s . Embryo c u l t u r e techniques were employed to rescue immature h y b r i d embryos (Evans, 1962; C o l l i n s et a l . , 1981; P h i l l i p s et a l . , 1982) and methods f o r long-term c o l d storage of shoot c u l t u r e s have been developed by Cheyne and Dale (1980). 7 2.2 Somaclonal V a r i a t i o n The phenomenon of v a r i a b i l i t y among regenerated p l a n t s from c e l l and t i s s u e c u l t u r e s was r e p o r t e d more than a decade ago ( S a c r i s t a n and Melchers, 1969; Morel, 1971). However, i t i s only i n the l a s t few years that much a t t e n t i o n has been p a i d to i t . In a review a r t i c l e L a r k i n and Scowcroft (1981) used the term "somaclonal v a r i a t i o n " to d e s c r i b e the v a r i a t i o n observed among regenerated p l a n t s as a consequence of t i s s u e c u l t u r e . Since the r e c o g n i t i o n of the importance of t h i s phenomenon i n the a p p l i c a t i o n of t i s s u e c u l t u r e to p l a n t b r eeding, numerous papers have been p u b l i s h e d a d d r e s s i n g the problem. Somaclonal v a r i a t i o n has been r e p o r t e d f o r many s p e c i e s i n v e s t i g a t e d , although s t a b i l i t y of c h a r a c t e r e x p r e s s i o n i n regenerated p l a n t s has been observed i n other experiments ( L a r k i n and Scowcroft, 1981; 1983a; L o r z , 1983; Scowcroft and L a r k i n , 1983; R e i s c h , 1983; Evans et a l . , 1984). Wherever the o b j e c t i v e i s to o b t a i n p l a n t s i d e n t i c a l t o p a r e n t a l type f o r propagation, somaclonal v a r i a t i o n i s a hazard. When the o b j e c t i v e i s to s e l e c t v a r i a n t s as source m a t e r i a l f o r p l a n t improvement, somaclonal v a r i a t i o n r e p r e s e n t s a novel source of v a r i a t i o n , at l e a s t i n terms of the method by which v a r i a t i o n i s generated. A t i s s u e c u l t u r e c y c l e r e f e r s to the procedure which i n v o l v e s the e s t a b l i s h m e n t of a more or l e s s d e d i f f e r e n t i a t e d c e l l or t i s s u e c u l t u r e from d i v e r s e sources ( m u l t i c e l l u l a r e x p l a n t s , i s o l a t e d s i n g l e c e l l s or 8 p r o t o p l a s t s ) , on d e f i n e d m e d i a , p r o l i f e r a t i o n f o r a number of c e l l g e n e r a t i o n s and t h e f i n a l r e g e n e r a t i o n o f p l a n t s . The r e g e n e r a t e d p l a n t s may e x h i b i t v a r i a t i o n i n s u c h a s p e c t s as m o r p h o l o g y , y i e l d , d i s e a s e r e s i s t a n c e and b i o c h e m i c a l c h a r a c t e r s . A l t h o u g h t h e r e a r e numerous p r o p o s a l s c o n c e r n i n g g e n e t i c and c y t o g e n e t i c a b n o r m a l i t i e s w h i c h c o u l d be r e s p o n s i b l e f o r t h e o b s e r v e d p h e n o t y p i c v a r i a t i o n o f r e g e n e r a n t s , t h e o r i g i n o f t h e s e a b n o r m a l i t i e s i s not y e t c l e a r l y u n d e r s t o o d . In some c a s e s t h e o b s e r v e d v a r i a t i o n i s t r a n s m i s s a b l e t h r o u g h s e x u a l r e p r o d u c t i o n ( R e i s c h , 1983). In t h i s t h e s i s r e p r e s e n t a t i v e e x a m p l e s o f some s p e c i e s and t h e g e n e r a l i t i e s o f t h i s phenomenon w i l l be r e v i e w e d . 2.2.1 V a r i a t i o n among Regenerated P l a n t s of S e v e r a l Crop S p e c i e s 2.2.1.1 Sugar Cane One i n t e r e s t i n g and i m p o r t a n t a s p e c t o f s o m a c l o n a l v a r i a t i o n i n s u g a r cane has been t h e a p p e a r a n c e o f d i s e a s e - r e s i s t a n t r e g e n e r a n t s f r o m t i s s u e c u l t u r e s o f s u s c e p t i b l e c u l t i v a r s . F o l l o w i n g r e p o r t e d v a r i a t i o n i n m o r p h o l o g i c a l , c y t o g e n e t i c and i sozyme t r a i t s ( H e i n z and Mee, 1969; H e i n z , 1973), e f f o r t s were made t o seek r e s i s t a n c e t o F i j i d i s e a s e (a l e a f h o p p e r t r a n s m i t t e d v i r u s ) and downy mildew ( S c l e r o s p o r a s a c c h a r i ) ( c f . H e i n z e t a l . , 1977). A p r e d o m i n a n t s h i f t t o w a r d i n c r e a s e d r e s i s t a n c e was o b s e r v e d f o r b o t h d i s e a s e s e v e n t h o u g h t h e v a r i e t y u s e d f o r i n i t i a t i o n o f t i s s u e 9 c u l t u r e was a p p a r e n t l y r e s i s t a n t . E x t e n s i v e v a r i a t i o n among sugar cane somaclones to s e v e r a l d i s e a s e s was confirmed (e.g. Heinz et a l . , 1977; L a r k i n and Scowcroft, 1981; 1983b). I n t e r e s t i n g l y , p l a n t s regenerated from c u l t i v a r Q101, which was s u s c e p t i b l e to eyespot d i s e a s e (Helminthosporium s p . ) , s h i f t e d s i g n i f i c a n t l y to the r e s i s t a n c e s i d e of the Q101 r e a c t i o n , whereas a l l 52 somaclones from r e s i s t a n t c u l t i v a r Q47 remained r e s i s t a n t ( L a r k i n and Scowcroft, 1981). Subsequently, L a r k i n and Scowcroft (1983b) i n i t i a t e d c u l t u r e s from a sugar cane which was h i g h l y s u s c e p t i b l e to eyespot d i s e a s e and from which they were able to i s o l a t e somaclones w i t h v a r y i n g degrees of t o l e r a n c e . E v a l u a t i o n of subsequent v e g e t a t i v e g e n e r a t i o n s i n d i c a t e d t h a t a t l e a s t some of the v a r i a t i o n c o u l d be r e t a i n e d through v e g e t a t i v e p r o p a g a t i o n . 2.2.1.2 Potato Potato " i s another s p e c i e s where somaclonal v a r i a t i o n has been e x t e n s i v e l y demonstrated. Shepard et a l . (1980) regenerated p l a n t s from l e a f p r o t o p l a s t s of 'Russet Burbank'. Among 10,000 somaclones screened s i g n i f i c a n t and s t a b l e v a r i a t i o n was found f o r tuber morphology, p l a n t growth h a b i t , p h o t o period response, m a t u r i t y date and y i e l d . In another study (Secor and Shepard, 1981) 65 s e l e c t e d c l o n e s of 'Russet Burbank' from l e a f p r o t o p l a s t s were an a l y s e d f o r 35 c h a r a c t e r s . 10 S i g n i f i c a n t v a r i a t i o n was found f o r 22 of the c h a r a c t e r s and a l l somaclones d i f f e r e d from 'Russet Burbank' i n one or more c h a r a c t e r s . V a r i a t i o n ranged from a minimum of one c h a r a c t e r i n three somaclones to a maximum of 17 c h a r a c t e r s f o r one somaclone. A s i m i l a r r e s u l t was o b t a i n e d by Thomas et a l . (1982); a l l the 23 p r o t o p l a s t - d e r i v e d p l a n t s d i f f e r e d from each other i n one or more of the 10 m o r p h o l o g i c a l c h a r a c t e r s a n a l y s e d . Only one somaclone of the p a r e n t a l type was recovered. M o r p h o l o g i c a l v a r i a t i o n and chromosome i n s t a b i l i t y have a l s o been r e p o r t e d i n other s t u d i e s (Karp et a l . , 1982; Ramulu et a l . , 1983; C r e i s s e n and Karp, 1985). Regenerated p l a n t s were screened f o r r e s i s t a n c e to e a r l y b l i g h t ( A l t e r n a r i a s o l a n i ) and l a t e b l i g h t (Phytophthora i n f e s t a n s ) (Bidney and Shepard, 1981). F i v e out of 500 somaclones were more r e s i s t a n t to e a r l y b l i g h t than the parent; 20 out of 800 were more r e s i s t a n t to l a t e b l i g h t . T h i s a l t e r e d e x p r e s s i o n was r e t a i n e d through s e v e r a l v e g e t a t i v e g e n e r a t i o n s . In c o n t r a s t , Wenzel et a l . (1979) observed remarkable u n i f o r m i t y among the regenerated p l a n t s from potato p r o t o p l a s t c u l t u r e s . The d i s c r e p a n c y between the r e s u l t s of Shepard et a l . (1980) and Wenzel et a l . (1979) may be a t t r i b u t e d to genotypic d i f f e r e n c e , p l o i d y l e v e l (Shepard: 4X; Wenzel: 2X); sample s i z e (Shepard: 10,000; Wenzel: 211) and t i s s u e source of p r o t o p l a s t s (Shepard: l e a f mesophyll; Wenzel: stem t i p 11 c u l t u r e ) . Thomas et a l . (1982) u s i n g the same technique as Wenzel et a l . (1979) observed v a r i a t i o n among the p r o t o p l a s t - d e r i v e d regenerated p l a n t s of a t e t r a p l o i d c u l t i v a r of potato. 2.2.1.3 Tobacco In tobacco a number of r e p o r t s have d e s c r i b e d v a r i a b i l i t y i n p l a n t s d e r i v e d from i_n v i t r o c u l t u r e . Evidence f o r somaclonal v a r i a t i o n was r e p o r t e d i n work by Burk and Matzinger (1976). These authors s e l f - f e r t i l i z e d an i n b r e d v a r i e t y 'Coker' f o r 15 g e n e r a t i o n s f o l l o w i n g i t s r e l e a s e as a pure l i n e , t o ensure a h i g h degree of i n b r e e d i n g . S e l f e d progeny of t i s s u e c u l t u r e - d e r i v e d l i n e s were analysed with re s p e c t to c h a r a c t e r s such as y i e l d , grade index, days to f l o w e r i n g and t o t a l a l k a l o i d s . S i g n i f i c a n t v a r i a b i l i t y was shown f o r a l l ten c h a r a c t e r s i n v e s t i g a t e d . A more d e t a i l e d a n a l y s i s of regenerants was c a r r i e d out by Burk and C h a p l i n (1980) using c u l t u r e s i n i t i a t e d from a h y b r i d , heterozygous f o r two l o c i a f f e c t i n g c h l o r o p h y l l s y n t h e s i s and l e a f c o l o r . Genetic a n a l y s i s of progeny of somaclonal v a r i a n t s i n d i c a t e d t h a t the frequency of mutation was 3.5% and 3.6% at each l o c u s r e s p e c t i v e l y among 1,666 p l a n t s . In a recent study on p l a n t s regenerated from mesophyll p r o t o p l a s t s heterozygous for the s u l f u r l o c u s (Su/su), 79 out of 2,156 morphogenic c o l o n i e s were i d e n t i f i e d to be non-parental and about 75% of the 79 c o l o n i e s were claimed to be somaclonal 12 v a r i a n t s because they produced regenerated p l a n t s of d i f f e r e n t genotypes (Lorz and Scowcroft, 1983). Among the s e l f e d regenerated p l a n t s 37% (33/96) had s e g r e g a t i o n r a t i o s which d e v i a t e d s i g n i f i c a n t l y from the expected 1:2:1 r a t i o . 2.2.1.4 Rice and Wheat Oono (1978) and Kucherenko (1979) s t u d i e d the i n h e r i t a n c e and v a r i a t i o n s of t r a i t s of r i c e (Oryza  s a t i v a L.) somaclones. R e s u l t s showed that r a t h e r high f r e q u e n c i e s of v a r i a t i o n o c c u r r e d f o r t r a i t s l i k e p l a n t h e i g h t and t i l l e r i n g i n progeny of somaclones. An i n t e n s i v e study of v a r i a t i o n i n r i c e somaclones and t h e i r progenies has been c a r r i e d out (Sun et a l . , 1981 c i t e d i n Sun et a l . , 1983; Zhao et a l . , 1982). When the i n h e r i t a n c e of and v a r i a t i o n s f o r some t r a i t s of more than 2,000 p l a n t s were i n v e s t i g a t e d i n the second and t h i r d g enerations of regenerated p l a n t s , only 24% of the r i c e l i n e s were normal f o r a l l the t r a i t s s t u d i e d (Sun et a l . , 1983). The frequency of v a r i a t i o n f o r d i f f e r e n t t r a i t s v a r i e d from 11.5 to 39.5%. A dwarf mutant which was c o n t r o l l e d by a r e c e s s i v e gene was i d e n t i f i e d . Trends were a l s o observed f o r p l a n t s to be s h o r t e r , to produce more p r o d u c t i v e t i l l e r s and f o r 1000-grain weight to be reduced (Sun et a l . , 1983). More recent experiments d e s c r i b e d the i d e n t i f i c a t i o n of promising breeding l i n e s i n y i e l d , m a t u r i t y and height (Zhao et a l . , 1984). V a r i a t i o n i n g r a i n p r o t e i n 13 content has a l s o been d e s c r i b e d among progenies of d i h a p l o i d s regenerated from anther c a l l u s c u l t u r e s ( S c h a e f f e r et a l . , 1984). In wheat ( T r i t i c u m aestivum L.) u s i n g immature embryos as e x p l a n t s improved e f f i c i e n c y of p l a n t r e g e n e r a t i o n (Bhojwani and Hayward, 1977; O'Hara and S t r e e t , 1978; Karp and Maddock, 1984; L a r k i n et a l . , 1984). Somaclonal v a r i a t i o n has been r e p o r t e d f o r wheat i n s e v e r a l cases, but the a n a l y s i s was mainly on regenerated p l a n t s (e.g. L u p i , 1981; Yurkova et a l . , 1982; Karp and Maddock, 1984). Recently more i n t e n s i v e work by L a r k i n et a l . (1984) r e p o r t e d v a r i a n t c h a r a c t e r s among 142 regenerants of 'Yaqui 50E' which i n c l u d e d h e i g h t , awns, t i l l e r number, g r a i n c o l o r , heading date, waxiness, glume c o l o r , g l i a d i n p r o t e i n and a-amylase r e g u l a t i o n and the occurrence of both dominant and r e c e s s i v e mutations. They claimed that chromosome l o s s or a d d i t i o n were not evident as the primary source of v a r i a t i o n . 2.2.2 O r i g i n of and F a c t o r s A f f e c t i n g Somaclonal V a r i a t i o n I t i s important both f o r academic i n t e r e s t s and f o r p r a c t i c a l use to determine whether the observed g e n e t i c v a r i a t i o n simply r e f l e c t s the v a r i a b i l i t y p r e - e x i s t e n t i n somatic c e l l s before the c u l t u r e c y c l e or i s a consequence of the c u l t u r e p r o c e s s . Lorz and Scowcroft (1983) regenerated p l a n t s from p r o t o p l a s t s of Su/su heterozygotes 14 of N i c o t i a n a tobaccum and, using l e a f c o l o r a n a l y s i s , estimated that the m a j o r i t y (92.2-98.4%) of c o l o n i e s were p a r e n t a l type, but 1.4-6.0% were c u l t u r e - i n d u c e d v a r i a n t s and only 0.1-1.8% p r e - e x i s t i n g v a r i a n t s . Thomas et a l . (1982) found that c o n s i d e r a b l e v a r i a t i o n e x i s t e d f o r ten m o rphological c h a r a c t e r s among regenerated p l a n t s of potato from the same c a l l u s o r i g i n a t e d from a s i n g l e c e l l of a t e t r a p l o i d c u l t i v a r , demonstrating c l e a r l y t h a t the v a r i a t i o n was c e l l - c u l t u r e induced. However the occurrence of somaclonal v a r i a t i o n may depend on the genotype and explant used, l e n g t h of c u l t u r e p e r i o d or other iji v i t r o c o n d i t i o n s . I t has been w e l l documented that changes i n k a r y o t y p i c s t r u c t u r e are i n c r e a s e d with the l e n g t h of c u l t u r e p e r i o d ( B a y l i s s , 1980; R e i s c h , 1983). A c c o r d i n g l y , p l a n t s regenerated from long-term c u l t u r e s tend to have more a b n o r m a l i t i e s . Flower and l e a f a b n o r m a l i t i e s were commonly found among tobacco p l a n t s regenerated from long-term (up to 28 months) c u l t u r e s , while they were not observed among the p l a n t s from short-term (1-5 months) c u l t u r e s (Syono and Furuya, 1972). McCoy et a l . (1982) observed that p l a n t s showing c y t o g e n e t i c a b n o r m a l i t i e s d e r i v e d from t i s s u e c u l t u r e s of oat i n c r e a s e d from 12% to 48% and 49% to 88% i n two l i n e s , r e s p e c t i v e l y , when p l a n t s were regenerated a f t e r four and twelve months i n c e l l c u l t u r e . Chrysanthemum  m o r i f o l i u m p l a n t s , regenerated from l e a f c a l l u s , maintained f o r nine years showed a l t e r e d l e a f shape, s m a l l e r and 15 abnormally shaped flowers and e x c e s s i v e l a t e r a l shoot growth as compared with p l a n t s regenerated from c a l l u s i n c u l t u r e f o r only one year ( S u t t e r and Langhans, 1981). One or more components i n the medium under c u l t u r e c o n d i t i o n s may a c t as mutagens to induce v a r i a t i o n i n c u l t u r e d c e l l s . Phytohormones, e s p e c i a l l y s y n t h e t i c auxins, may be the most probable candidate (Lorz, 1983). Shepard (1981) observed d i f f e r e n c e s i n the frequency of v a r i a n t s a f t e r r e g e n e r a t i n g p l a n t s from potato p r o t o p l a s t s using d i f f e r e n t c o n c e n t r a t i o n s of NAA and 2,4-D i n the medium. Few r e p o r t s are a v a i l a b l e on t h i s t o p i c , p a r t l y because there i s no good system with which to work. Deambrogio and Dale (1980) regenerated p l a n t s from t i s s u e c u l t u r e s of b a r l e y (Hordeum vu l g a r e ) using 1, 2, 3 and 4 mg/1 2,4-D. V a r i a t i o n i n such t r a i t s as a l b i n i s m , l e a f shape, and t i l l e r f e r t i l i t y happened only at 4 mg/1 2,4-D. P l a n t s were regenerated on two d i f f e r e n t media (NK and I) from c u l t u r e s which o r i g i n a t e d from a s i n g l e stock c a l l u s of Haworthia  s e t a t a and e i g h t c h a r a c t e r s were analysed (Ogihara, 1981). Chromosome a n a l y s i s r e v e a l e d that NK medium tended to regenerate more t e t r a p l o i d p l a n t s , but fewer p l a n t s with t r a n s l o c a t i o n s than the I medium. The r e l a t i o n s h i p between occurrence of v a r i a n t s and g e n e t i c background of m a t e r i a l was i n d i c a t e d i n some p u b l i c a t i o n s . F i r s t , there i s an obvious d i f f e r e n c e among s p e c i e s concerning the l e v e l of v a r i a b i l i t y . McCoy et a l . (1982) r e p o r t e d a high frequency of chromosome a b n o r m a l i t i e s 16 i n oat (Avena s a t i v a L.) i n one study, while McCoy and P h i l l i p s (1982) observed a high degree of chromosome s t a b i l i t y , although the techniques employed were s i m i l a r i n both cases. Second, d i f f e r e n c e s among c u l t i v a r s of the same s p e c i e s have been c l e a r l y demonstrated. In sugar cane, v a r i a n t s were found among a t o t a l of 4,600 regenerated p l a n t s ( L i u and Chen, 1976). For c l o n e F156 the frequency of m o r p h o l o g i c a l change was 34.0% while f o r clone 146 i t was 1.8%. In potato c o n s i d e r a b l e v a r i a t i o n was observed among the p r o t o p l a s t - d e r i v e d p l a n t s of t e t r a p l o i d c u l t i v a r s while no v a r i a t i o n was observed among the regenerated p l a n t s of a d i p l o i d c u l t i v a r e v a l u a t e d u s i n g s i m i l a r procedures (Wenzel, 1979; Shepard et al.. , 1 980; Thomas et a l . , 1982). I t was suggested that t h i s d i f f e r e n c e was due to d i f f e r e n c e s i n g e n e t i c background r a t h e r than p l o i d y l e v e l of the s t a r t i n g m a t e r i a l s (Lorz, 1983). In an a n a l y s i s of c e l l - d e r i v e d p l a n t s of Oryza s a t i v a L. more than 2,000 p l a n t s were i n v e s t i g a t e d (Sun et a l . , 1983). The percentages of p o l y p l o i d s f o r nine v a r i e t i e s of the 'Hsien' ( i n d i c a ) group were 0-13.3%, but no p o l y p l o i d was d e t e c t e d i n the nine v a r i e t i e s of the 'Keng' (jap o n i c a ) group. D i f f e r e n c e s were a l s o found i n p r o t o p l a s t - d e r i v e d p o t a t o p l a n t s of two t e t r a p l o i d c u l t i v a r s (Karp et a l , 1982). The c y t o g e n e t i c a n a l y s i s of p l a n t s from t i s s u e c u l t u r e s of two Avena s a t i v a l i n e s r e v e a l e d that the l i n e 'Tippecanoe' had twice as many c y t o l o g i c a l a l t e r a t i o n s as the l i n e ' L o d i ' (McCoy et a l . , 1982). 17 Explant source c o u l d a l s o have a s i g n i f i c a n t e f f e c t on somaclonal v a r i a t i o n . P l a n t s regenerated from c a l l u s of four types of e x p l a n t s of pineapple (Ananas comosus L.) were a l t e r e d a c c o r d i n g to the explant source (Wakasa, 1979). P l a n t s from the s l i p , crown and a x i l l a r y buds showed changes only i n spine c h a r a c t e r s , but p l a n t s from syncarp i n c l u d e d v a r i a n t s i n l e a f c o l o r , s p i n e , wax and f o l i a g e d e n s i t y . Van Harten and B r o e r t j e s (1981) showed that m orphological v a r i a t i o n s were observed i n 12.3% of p l a n t s from l e a f l e t d i s k s , but i n 50.3% of p l a n t s from r a c h i s - and p e t i o l e - d e r i v e d c a l l u s . Although i t i s g e n e r a l l y b e l i e v e d that p l a n t s regenerated v i a embryogenesis produce fewer v a r i a n t s than those v i a organogenesis, d i r e c t comparisons between the two r e g e n e r a t i o n pathways are very r a r e . Somatic embryogenesis has been achieved i n many c e r e a l s p e c i e s and obvious phenotypic and c y t o g e n e t i c v a r i a n t s have not been found among the regenerated p l a n t s ( V a s i l and V a s i l , 1981; V a s i l et a l . , 1982). The s t a b i l i t y of regenerated p l a n t s was reconfirmed i n a recent study (Swedlund and V a s i l , 1985). However, chromosome number and s t r u c t u r e changes were observed i n p l a n t s regenerated v i a embryogenesis from T r i t i c u m aestivum L. c u l t u r e s (Karp and Maddock, 1984). 2.2.3 Genetic B a s i s Understanding the g e n e t i c b a s i s of somaclonal v a r i a t i o n i s important to f a c i l i t a t e i t s p r a c t i c a l use f o r p l a n t 18 improvement. Based upon e x i s t i n g experimental knowledge somaclonal v a r i a t i o n may be a t t r i b u t e d to (1) chromosome changes (number and s t r u c t u r e ) ; (2) gene a l t e r a t i o n s ; (3) c y t o p l a s m i c DNA changes and (4) other kinds of h e r i t a b l e change such as t r a n s p o s i b l e elements. Chromosome v a r i a b i l i t y of regenerated p l a n t s has been w e l l documented (D'Amato, 1977; 1978; Sunderland, 1977; B a y l i s s , 1980; L a r k i n and Scowcroft, 1981; K r i k o r i a n et a l . , 1983) . P l o i d y changes are the most f r e q u e n t l y observed chromosomal ab n o r m a l i t y . Prat (1983) regenerated 172 p l a n t s from mesophyll p r o t o p l a s t c u l t u r e of two l i n e s of N i c o t i a n a  s y l v e s t r i s . Seventy two regenerated p l a n t s were d i p l o i d l i k e the o r i g i n a l l i n e s , while the remainder were p o l y p l o i d ; no a n e u p l o i d s were recovered. From h a p l o i d p r o t o p l a s t s of N. s y l v e s t r i s F a c c i o t i and P i l e t (1979) regenerated p l a n t s which were e s s e n t i a l l y d i p l o i d s . P l o i d y changes were a l s o r e a d i l y observed i n potato and tobacco (Karp et a l . , 1982; 1984) . Aneuploids have been recovered among regenerated p l a n t s i n many cases where chromosome abnormality o c c u r s , although the frequency i s u s u a l l y lower than e u p l o i d s ( F a c c i o t i and P i l e t , 1979; Karp et a l . , 1984). A n a l y s i s of p l a n t s d e r i v e d from c u l t u r e d immature embryos of four wheat c u l t i v a r s ( T r i t i c u m aestivum 2n=6X=42) showed that 29% of the 192 p l a n t s examined were an e u p l o i d s ranging i n chromosome number from 38 to 45 (Karp and Maddock, 1984). 19 In a d d i t i o n to chromosome number changes chromosomal rearrangements such as d e l e t i o n s , t r a n s l o c a t i o n s and i n v e r s i o n s may a l s o be expected. T r a n s l o c a t i o n s have been observed among regenerated p l a n t s of ryegrass (Lolium sp.) (Ahloowalia, 1976), potato (Shepard, 1982), oat (McCoy et a l . , 1982), Haworthia s e t a t a (Ogihara, 1981) and wheat X rye h y b r i d s (X T r i t i c o s e c a l e Wittmack) ( L a p i t a n et a l . , 1984). In work with l a r g e chromosomes of Haworthia s e t a t a , Ogihara (1981) was able to demonstrate t r a n s l o c a t i o n s and d e l e t i o n s . More new karyotypes were d e t e c t e d i n regenerated p l a n t s than i n c l o n e d c a l l i . Rearrangements were confirmed to be r e c i p r o c a l t r a n s l o c a t i o n s by the o b s e r v a t i o n of q u a d r i v a l e n t s i n metaphase I. Chromosome b r i d g e s and segments, or segments alone, were i n t e r p r e t e d as evidence of p a r a c e n t r i c i n v e r s i o n s and sub-chromatid a b e r r a t i o n s . P l a n t s regenerated from c a l l u s c u l t u r e s of h y b r i d s of Hordeum vulqareX H. jubatum showed enhanced freqency of b i v a l e n t s and m u l t i v a l e n t s and they were presumed to be the r e s u l t of e i t h e r t r a n s l o c a t i o n or the d e l e t i o n of a p a i r i n g suppressor (Orton, 1980). Increases in recombination r a t e have been observed on tomato ( S i b i et a l . , 1984). P h e n o t y p i c a l l y d i s t i n g u i s h a b l e r e c e s s i v e marker genes were s e l e c t e d i n the 'Monalbo' homozygous background. Cotyledons of i n d i v i d u a l F 2 h y b r i d s e e d l i n g s were used as ir\ v i t r o t i s s u e c u l t u r e m a t e r i a l , while the r e s t of the p l a n t was grown as a c o n t r o l . Recombination r a t e s of the s e l f e d progenies from regenerated 20 p l a n t s and matched c o n t r o l s were compared. For two l o c i 'bs-ms32' on chromosome I i n c r e a s e s of 6.07 to 6.91 map u n i t s were observed and f o r the 'aa-d' region of chromosome I I , the i n c r e a s e s ranged from 4.92 to 6.04 map u n i t s . Cytoplasmic g e n e t i c changes may a l s o be r e s p o n s i b l e f o r observed v a r i a t i o n . The r e s i s t a n c e of corn to l e a f b l i g h t and c u l t i v a r s e n s i t i v i t y to a host t o x i n of Helminthosporium  maydis race T i s t i g h t l y a s s o c i a t e d with Texas male s t e r i l e (cms-T) c y t o p l a s m i c genotypes in seed-derived p l a n t s . Gengenbach et a l . (1977) s e l e c t e d f o r r e s i s t a n c e to t o x i n i n v i t r o and r e g e n e r a t e d r e s i s t a n t p l a n t s with the aim of r e c o v e r i n g r e s i s t a n t , cms-T breeding l i n e s . However, r e s i s t a n c e was a s s o c i a t e d with a concomitant r e v e r s i o n to male f e r t i l i t y . Gengenbach et a l . (1981) observed m i t o c h o n d r i a l DNA v a r i a t i o n i n T - c y t o p l a s m i c male s t e r i l e maize p l a n t s regenerated from t i s s u e c u l t u r e and s e l e c t e d f o r r e s i s t a n c e to Helminthosporium maydis race T. R e s t r i c t i o n enzyme a n a l y s i s of m i t o c h o n d r i a l DNA showed d i f f e r e n c e s between d i f f e r e n t r e g e n e r a t e d p l a n t s and a l s o d i f f e r e n c e s when compared to T or N cytoplasm maize p l a n t s . A s p e c i f i c type of t r a n s p o s i t i o n event i n maize m i t o c h o n d r i a l DNA was shown to be c o r r e l a t e d t o a change in c a l l u s morphology (Chourey and Kemble, 1982). The observed DNA v a r i a t i o n was l i m i t e d to two p l a s m i d - l i k e DNA components c a l l e d SI and S2. When DNA a n a l y s i s was performed on doubled h a p l o i d p l a n t s of N i c o t i a n a s y l v e s t r i s produced from p o l l e n c u l t u r e , h e r i t a b l e 21 q u a n t i t a t i v e and q u a l i t a t i v e changes were observed i n the nucle a r DNA (De Paepe et a l . , 1983). Doubled h a p l o i d p l a n t s c o n t a i n e d , on average, i n c r e a s i n g amounts of t o t a l DNA and i n c r e a s i n g amounts of h i g h l y repeated sequences. More re s e a r c h at the molecular l e v e l w i l l be e s s e n t i a l f o r a b e t t e r understanding of the g e n e t i c b a s i s of somaclonal v a r i a t i o n . 3. MATERIALS AND METHODS 3.1 General C u l t u r e Methodology 3.1.1 Media In t h i s study an e s t a b l i s h e d medium L2 ( P h i l l i p s and C o l l i n s , 1979a) was used except i n one t e s t i n which B 5 (Gamborg et a l . , 1968) (Appendix 1) was used. Composition of b a s a l L2 medium i s as f o l l o w s (mg/1): NH 4N0 3, 1,000; KN0 3, 2,100; KH 2PO„, 325; N a H 2 P 0 „ - H 2 0 , 85; C a C l 2 . 2 H 2 0 , 600; MgS0«-7H 20, 435; FeSO««7H 20 (EDTA), 25; H 3B0 3, 5; KI, 1; MnSO„'H 20, 15; ZnSO«-7H 20, 5; CuSO«'5H 20, 0.1; N a 2 M o O „ 2 H 2 0 , 0.4; CoCl 2-6H 20, 0.1; Thiamine, 2.0; P y r i d o x i n e - H C l , 0.5; M y o - i n o s i t o l , 250; sucrose, 25,000. D i f f e r e n t growth r e g u l a t o r s and c o n c e n t r a t i o n s were added i n v a r i o u s t e s t s depending on the c u l t u r e o b j e c t i v e s . Growth r e g u l a t o r s used were: c y t o k i n i n s : BA, ADE and KIN; auxins: 2,4-D, IAA, NAA and PIC. Stock s o l u t i o n s were made f o r (1) macro-nutrients (NH„N0 3, K N O 3 , KH 2PO f t, N a H 2 P O „ - H 2 0 and MgS0 f l-7H 20) X10; (2) C a C l 2 - H 2 0 , X20; (3) FeSO«-7H 20 (EDTA) X200; (4) m i c r o - n u t r i e n t s (H 3B0 3, MnSO f t«H 20, ZnS0i,'7H20, CuS0««5H 20, Na2MoO(,-2H20 and CoCl 2.6H 20) X400; (5) KI X1000; and (6) thiamine and p y r i d o x i n e X1000. In p r e p a r i n g FeSO a*7H 20 stock s o l u t i o n , 5.00 g F e S O a « 7 H 2 0 were f i r s t d i s s o l v e d i n hot d i s t i l l e d water and then 7.45 g Na 2EDTA were added. The volume was brought up to 500 ml, r e s u l t i n g i n a s o l u t i o n of 22 23 5 mg/1 F e S O a « 7 H 2 0 . The C a C l 2 « 2 H 2 0 stock s o l u t i o n was made s e p a r a t e l y to prevent p r e c i p i t a t i o n i n the stock b a s a l s a l t s . Thiamine and p y r i d o x i n e were prepared as aqueous s o l u t i o n s . Stock s o l u t i o n s were a l s o prepared f o r each of the growth r e g u l a t o r s used: BA, 1 mg/ml; ADE, 1 or 2 mg/ml; KIN, 1 mg/ml; IAA, 1 mg/ml, NAA, 0.2 mg/ml; 2,4-D, 0.5 mg/ml; and PIC, 0.06 mg/ml. BA and ADE were f i r s t d i s s o l v e d i n a smal l amount of 1N KOH and d i s t i l l e d water was added to the a p p r o p r i a t e volume. KIN was f i r s t d i s s o l v e d i n 1N HC1 and subsequently d i l u t e d with water. The auxins 2,4-D, NAA and PIC were d i s s o l v e d i n a small amount of "ethanol p r i o r to the a d d i t i o n of water. Stock s o l u t i o n s were s t o r e d i n c o l o r e d b o t t l e s or b o t t l e s wrapped with t i n f o i l at 4°C except f o r the IAA stock s o l u t i o n which was s t o r e d f r o z e n . Media were prepared by adding a p p r o p r i a t e volumes of stock s o l u t i o n s of s a l t s , v i t a m i n s and growth r e g u l a t o r s t o a l a r g e volume of d i s t i l l e d water. Sugar was then added and the media were s o l i d i f i e d with 8 g/1 agar ( D i f c o Bacto) u n l e s s otherwise s p e c i f i e d . The pH of media was a d j u s t e d to 5.8 with IN KOH bef o r e adding agar and media were a u t o c l a v e d at 121°C, 1.0 kg/cm 2 f o r 15 min. (20 min when a c o n t a i n e r h e l d more than 1,000 ml of medium). Media i n these experiments are s p e c i f i e d by the a b b r e v i a t i o n s of b a s a l media and the i n i t i a l s of growth r e g u l a t o r s . For example, L2(P0.06B0.2) r e f e r s to L2 medium c o n t a i n i n g 0.06 mg/1 PIC and 0.2 mg/1 BA. The l e t t e r b e f o r e 24 the b r a c k e t s r e p r e s e n t s a b a s a l medium. L2 ( P h i l l i p s and C o l l i n s , 1979a) and B 5 (Gamborg et a l . , 1968) r e f e r to two b a s a l media r e s p e c t i v e l y . W i t h i n the b r a c k e t s l e t t e r s symbolize the growth r e g u l a t o r s , u s u a l l y an auxin and a c y t o k i n i n . Symbols f o r growth r e g u l a t o r s were: 2,4-D (D); IAA ( I ) ; NAA (N); PIC (P); ADE (A); BA (B); and KIN (K). Each r e g u l a t o r symbol i s f o l l o w e d by a number which i n d i c a t e s the c o n c e n t r a t i o n used i n mg/1. S e v e r a l media which were used f r e q u e n t l y have been a s s i g n e d s p e c i a l a b b r e v i a t i o n s . A l l these media c o n t a i n L2 b a s a l s a l t s . Growth r e g u l a t o r s used i n these media were as f o l l o w s : (1) L2: c o n t a i n i n g 0.06 mg/1 PIC and 0.1 mg/1 BA; (2) LSE: c o n t a i n i n g 0.01 mg/1 2,4-D and 2 mg/1 ADE; (3) LSP: c o n t a i n i n g 0.002 mg/1 PIC and 0.2 mg/1 BA and (4) SCP: c o n t a i n i n g 2 mg/1 2,4-D, 2 mg/1 BA and 2 mg/1 ADE. L2, LSE and LSP have been s p e c i f i e d by P h i l l i p s and C o l l i n s (1979a; 1980). 3.1.2 Explants T r i f o l i u m pratense L. cv 'Altaswede' and cv 'Norseman' were used. The former i s a d i p l o i d c u l t i v a r and the l a t t e r a t e t r a p l o i d c u l t i v a r . 'Altaswede' was o b t a i n e d from Richardson Seed Company L t d and 'Norseman' from C h a r l e s Sharpe & Company L t d . Seeds were germinated i n h a l f - s t r e n g t h L2 b a s a l medium with 5 g/1 sucrose and 8 g/1 agar. The medium was dispensed i n t o 25 X 150 mm t i s s u e c u l t u r e tubes, each r e c e i v i n g 20 ml 25 volume. Seeds were s t e r i l i z e d i n 75% ethanol f o r 5 min., then i n 20% commercial bleach (1.0% sodium h y p o c h l o r i t e i n f i n a l c o n c e n t r a t i o n ) f o r 20 min., and r i n s e d i n s t e r i l e d i s t i l l e d water at l e a s t three times. Six to e i g h t s t e r i l i z e d seeds were put i n t o t e s t tubes to germinate and were incubated f o r 8-10 days i n a growth chamber at 25°C with a photoperiod of 16 hours (200 izEm" 2 s e c " 1 ) . S e e d l i n g h y p o c o t y l s were cut a s e p t i c a l l y w i t h a s c a l p e l i n t o 3-4 mm segments f o r c a l l u s i n d u c t i o n and 3-4 mm shoot t i p s were u t i l i z e d f o r shoot t i p c u l t u r e s . A l l experimental m a n i p u l a t i o n s of e x p l a n t s were c a r r i e d out under a s e p t i c c o n d i t i o n s . T r a n s f e r s were f a c i l i t a t e d u s ing a laminar a i r flow c a b i n e t .(Environmental A i r C o n t r o l Co.). 3.1.3 C u l t u r e C o n d i t i o n s Two kinds of c u l t u r e v e s s e l s were used i n these experiments. When t e s t tubes (25 X 150 mm) were used, bulk medium with a l l components added and pH a d j u s t e d to.5.8 was put i n t o a microwave oven to melt the agar. Once the agar had melted the medium was dispensed i n t o the t e s t tubes. Tubes were capped with p l a s t i c c l o s u r e s ( B e l l c o B i o l o g i c a l Glassware & Equipment). The s u b c u l t u r e i n t e r v a l was u s u a l l y four weeks. When p e t r i p l a t e s were used, the bulk medium was poured i n t o the p l a t e s (30 ml / p l a t e ) i n the laminar a i r flow c a b i n e t when medium temperature had coo l e d to approximately 45°C a f t e r a u t o c l a v i n g and allowed to s e t . F o l l o w i n g 26 explant t r a n s f e r the p l a t e s were wrapped with p a r a f i l m to prevent contamination and reduce moisture l o s s . The s u b c u l t u r e i n t e r v a l f o r p l a t e c u l t u r e s was a l s o four weeks. Six growth chambers were used duri n g the experiments. T h e r e f o r e , d i f f e r e n t experiments may have d i f f e r e n t l i g h t i n t e n s i t i e s . They w i l l be s p e c i f i e d i n d e s c r i p t i o n s of methods f o r i n d i v i d u a l experiments. L i g h t was p r o v i d e d by c ool-white f l u o r e s c e n t tubes. The l i g h t / d a r k p e r i o d was 16/8 hours unless otherwise i n d i c a t e d . The temperature in these chambers was 25±1°C. 3.2 S p e c i f i c C u l t u r e Methodology 3.2.1 C a l l u s Induction Hypocotyl segments 3-4 mm i n l e n g t h were t r a n s f e r r e d onto L2(PO.06BO.1) medium f o r c a l l u s i n d u c t i o n . C u l t u r e s were maintained at 25°C i n growth chambers. C a l l u s i n d u c t i o n was evaluated v i s u a l l y two weeks l a t e r . In batch one, t e s t tubes were used as c u l t u r e v e s s e l s , C u l t u r e s were incubated i n a growth chamber with l i g h t i n t e n s i t y of 500 yEm" 2sec" 1. In batch two, s t e r i l e p e t r i d i s h e s were used c o n t a i n i n g e x p l a n t s from two s e e d l i n g s with each having three to f i v e e x p l a n t s . These p e t r i d i s h e s were put i n t o a growth chamber at a l i g h t i n t e n s i t y of 50 nEm~2sec"1. F i v e batches of c u l t u r e s were e s t a b l i s h e d to induce c a l l u s from hypocotyl e x p l a n t s . Test tubes were used i n 27 three batches and p e t r i p l a t e s i n the other two. Each t e s t tube c o n t a i n e d three e x p l a n t s from one s e e d l i n g . O bservations were made i n two of these three batchess to determine the genotype e f f e c t on c a l l u s i n d u c t i o n i . e . whether any r e l a t i o n e x s i t s between the t h r e e e x p l a n t s for c a l l u s i n d u c t i o n . 3.2.2 C a l l u s Maintenance and P l a n t Regeneration The f o l l o w i n g o b s e r v a t i o n s were made t o e v a l u a t e c a l l u s growth and d i f f e r e n t i a t i o n on v a r i o u s media: (1) green-spotted c a l l u s (GSC): c a l l u s with i s o l a t e d green regions c o n t a i n i n g m e r i s t e m - l i k e s t r u c t u r e s d i s t r i b u t e d on the c a l l u s . I t has been r e p o r t e d t h a t these s t r u c t u r e s c o u l d be an i n d i c a t o r of r e g e n e r a t i o n (Kim and Jang, 1984). C a l l i were scored u s i n g t h i s s t r u c t u r e to p r e d i c t t h e i r p o t e n t i a l f o r d i f f e r e n t i a t i o n . (2) shoot c a l l u s (SC): c a l l u s with one or more shoots, i n d i c a t i n g shoot d i f f e r e n t i a t i o n . (3) root c a l l u s (RC): c a l l u s with one or more r o o t s . (4) brown c a l l u s : c a l l u s with brown c o l o r . T h i s kind of c a l l u s d i d not grow w e l l . (5) dead or n e c r o t i c c a l l u s : c a l l u s which was dark brown and sometimes hard i n t e x t u r e . (6) growth: In some experiments c a l l u s was graded f o r t h e i r r e l a t i v e growth r a t e i n d i c a t e d by p l u s s i g n s (more p l u s s i g n s meaning f a s t e r growth). 28 3.2.2.1 Primary Test of Two Media A f t e r s u c c e s s f u l c a l l u s i n d u c t i o n , attempts were made to achieve p l a n t r e g e n e r a t i o n from c a l l u s c u l t u r e s . Two media were f i r s t t r i e d : (1) B 5 medium c o n t a i n i n g 20 mg/1 thiamine, 2 mg/1 NAA and 2 mg/1 ADE (=B 5(N2A2)) and (2) L2 medium c o n t a i n i n g 0.01 mg/1 2,4-D and 2 mg/1 ADE (=LSE). These media have been repo r t e d to be capable of i n d u c i n g p l a n t r e g e n e r a t i o n (Beach and Smith, 1979; P h i l l i p s and C o l l i n s , 1980). C a l l i were induced on L2 medium and t r a n s f e r r e d to p l a t e s of one of the above media. Each p l a t e was i n o c u l a t e d with three p i e c e s of c a l l u s . C u l t u r e s were maintained i n a chamber with l i g h t i n t e n s i t y of 900 uEm _ 2sec" 1. The c u l t u r e s were eva l u a t e d two weeks l a t e r f o r d i f f e r e n t i a t i o n and growth. Some c a l l i were t r a n s f e r r e d onto f r e s h media of both types and incubated f o r four more weeks before e v a l u a t i o n . F i f t y c a l l i of each c u l t i v a r which had been c u l t u r e d on LSE f o r seven weeks were f u r t h e r s u b c u l t u r e d on LSE medium to determine the e f f e c t of subsequent s u b c u l t u r e s . 3.2.2.2 C a l l u s Growth on LSE Medium Test 1: The LSE medium f a i l e d to s t i m u l a t e shoot i n d u c t i o n i n the p r e v i o u s experiment ( S e c t i o n 3.2.2.1). It was p o s s i b l e that t h i s absence of success c o u l d be a t t r i b u t e d to (1) a small c a l l u s sample s i z e and/or (2) some minor environmental c o n d i t i o n which d i d not meet the requirements f o r shoot i n d u c t i o n . In another 29 experiment, 1452 c a l l i of 'Altaswede' and 1556 c a l l i of 'Norseman' were t r a n s f e r r e d from L2 to LSE medium on August 3 and 4, 1984, and c u l t u r e d f o r four weeks. Subsequently, 984 'Altaswede' c a l l i and 568 'Norseman' c a l l i were s u b c u l t u r e d onto f r e s h LSE medium. Vari o u s l i g h t treatments were a p p l i e d to the c u l t u r e s : (1) L i g h t i n t e n s i t y v a r i e d from 70 to 900 juEirr 2 sec ~ 1 . T h i s g r a d a t i o n was accomplished by s t a c k i n g ten c u l t u r e p l a t e s i n one column i n two chambers with maximum l i g h t i n t e n s i t i e s of 500 or 900 /uEirr 2 sec ~ 1 ; (2) Photoperiods of 16 and 24 hours were used f o r 'Altaswede' c a l l i and 16 hours f o r 'Norseman' c a l l i . T est 2: I t was a l s o p o s s i b l e that c a l l i growing on LSE medium r e q u i r e d t r a n f e r t o another medium f o r shoot emergence because of the absence of some f a c t o r i n LSE medium necessary f o r shoot development. To t e s t t h i s h y p o t h e s i s , c a l l i were f i r s t i n i t i a t e d from h y p o c o t y l e x p l a n t s . They were then t r a n s f e r r e d onto LSE medium f o r two or three weeks. These c a l l i were s u b c u l t u r e d onto the f o l l o w i n g four media: (1) LSP medium, which has been claimed to be s u p p o r t i v e of shoot development ( P h i l l i p s and C o l l i n s , 1980); (2) L2(P0.002B0.2 and h a l f s t r e n g t h of s a l t s ) medium, to ev a l u a t e the e f f e c t of reduced c o n c e n t r a t i o n s of b a s a l s a l t s on c a l l u s d i f f e r e n t i a t i o n ; (3) L2(NO.05KO.5) medium and (4) medium 4, which had only L2 b a s a l medium without growth r e g u l a t o r s added. C a l l i were c u l t u r e d i n p l a t e s with 30 24 hour photoperiod and 100 MEm _ 2sec" 1 f o r four weeks. Test 3: In t h i s t e s t , d i f f e r e n t c a l l u s sources were used. They had been c u l t u r e d on L2(D2B2) medium. C a l l u s from primary c u l t u r e s was t r a n s f e r r e d onto a medium having the same composition as LSE except c o n t a i n i n g 5 mg/1 ADE i n s t e a d of 2 mg/1 ADE. C a l l i were c u l t u r e d i n t e s t tubes at 400 AtEm~ 2sec~ 1 and 16-hour photoperiod f o r three weeks. Because LSE medium f a i l e d to support shoot i n d u c t i o n i n any experiment, f u r t h e r work was c a r r i e d out to determine the medium composition which would be s u p p o r t i v e of shoot i n d u c t i o n . 3.2.2.3 2,4-D and BA In t h i s t e s t d i f f e r e n t l e v e l s of 2,4-D and BA were e v a l u a t e d . Two batches of c u l t u r e were used. The treatments f o r the f i r s t batch were: 2 and 4 mg/1 BA combined with 2 mg/1 2,4-D. The four treatments f o r the second batch were 0.01, 0.1, 2.0 and 10.0 mg/1 2,4-D combined with 2 mg/1 BA and a d d i t i o n a l 5 mg/1 ADE was i n c o r p o r a t e d i n t o the medium c o n t a i n i n g 2 mg/1 2,4-D and 2 mg/1 BA. 3.2.2.4 PIC and BA Three t e s t s were c a r r i e d out. The treatments i n the three t e s t s are l i s t e d i n Table 1. For embryogenesis of c a l l u s , reduced n i t r o g e n , e.g. amino a c i d s , has been shown to be a major f a c t o r (Raghavan, 31 Table 1. Treatment combinations used t o t e s t the e f f e c t of PIC and BA on p l a n t r e g e n e r a t i o n ( c o n c e n t r a t i o n i n mg/1). Other Test No Treatment No PIC BA A d d i t i v e s 1 1 0.001 0.2 1740 Arg 2 0.0005 1 1740 Arg 3 0.001 1 1740 Arg 4 0.005 1 1740 Arg 5 0.01 1 1740 Arg 6 0.05 1 1740 Arg 7 0.0005 1 8 0.001 1 9 0.005 1 10 0.01 1 1 1 0.05 1 1 0.005 1 100 CH 2 0.005 1 500 CH 3 0.005 1 1000 CH 4 0.005 1 5000 CH 5 0.005 1 147 GA 1 0.002 0.2 2 0.005 0.2 3 0.01 0.2 4 0.002 1 5 0.005 1 6 0.01 1 ^ r g = A r g i n i n e ; CH = Casein H y d r o l y s a t e ; GA = Glutamic A c i d 1740 mg/1 a r g i n i n e = lOrnM. 32 1976; W e t h e r a l l and D o u g a l l , 1976; Kohlenbach, 1978). In a l f a l f a both q u a l i t y and number of embryos were s t i m u l a t e d by adding amino a c i d s ( S t u a r t and S t r i c k l a n d , 1984a). In t h i s experiment a r g i n i n e , glutamic a c i d and c a s e i n h y d r o l y s a t e were added to some of the media t e s t e d . Test 1: T h i s t e s t was e s t a b l i s h e d to i n v e s t i g a t e the e f f e c t of v a r i o u s c o n c e n t r a t i o n s of PIC and BA on c a l l u s growth i n presence or absence of a r g i n i n e . Only 'Altaswede' c a l l i were used. C a l l i were p r e v i o u s l y c u l t u r e d on L2(D0.01A2) medium. They were t r a n s f e r r e d onto t e s t media i n p l a t e s and incubated at 400 i i E m ~ 2 s e c ~ 1 with a 16 photoperiod at 25°C. C u l t u r e s were eva l u a t e d three weeks l a t e r . Test 2: Another t e s t was e s t a b l i s h e d to determine the e f f e c t of c a s e i n h y d r o l y s a t e and glutamic a c i d i n c l u s i o n i n medium on c a l l u s growth. Aqueous s o l u t i o n s were prepared u s i n g these substances and t h e i r pH a d j u s t e d to 5.8. A f t e r f i l t e r s t e r i l i z a t i o n , s o l u t i o n s were added to the medium which was c o o l i n g down a f t e r a u t o c l a v i n g to g i v e the d e s i r e d f i n a l c o n c e n t r a t i o n . T r a n s f e r r e d c a l l i from LSE medium were incubated with a 16-hour ph o t o p e r i o d at 280 juEm^sec" 1 i and 25°C for four weeks. Test 3: In t h i s t e s t 'Altaswede' c a l l i were p r e v i o u s l y c u l t u r e d on L2(D2B2N2) medium. For 'Norseman' c a l l i of three sources were used i n the 33 t e s t : (1) L2(D2B2N2) medium, (2) L2(D2B0.5A2) medium and (3) L2(PO.002B0.2) medium. Each p l a t e had two c a l l i incubated with a 24-hour photoperiod at 200 AiEm" 2sec _ 1 and 25°C. C u l t u r e s were evaluated four weeks a f t e r the t r a n s f e r . 3.2.2.5 NAA and KIN Test 1: In t h i s t e s t 'Norseman' c a l l i which had been c u l t u r e d on LSE medium f o r one month were t r a n s f e r r e d onto media with v a r i o u s c o n c e n t r a t i o n s of NAA (0.001, 0.01, 0.1, 1.0 and 5.0 mg/1) combined with 1 mg/1 KIN. P l a t e s c o n t a i n i n g the c u l t u r e s were incubated with a 16-hour photoperiod at 440 juEirr 2 sec ~ 1 and 25°C f o r three weeks. Test 2: Yellow-green c a l l i of cv 'Altaswede' growing on SCP medium were used f o r t h i s t e s t . NAA was t e s t e d at the f o l l o w i n g c o n c e n t r a t i o n s (mg/1): 0.01, 0.05 and 0.1. C o n c e n t r a t i o n s of KIN were 0.5 or 2.0 mg/1. C a l l i were t r a n s f e r r e d onto v a r i o u s medium combinations and c u l t u r e d with a 24-hour photoperiod at 250 MEm~ 2sec" 1 and 25°C f o r three weeks. Test 3: 'Norseman' c a l l i i n t h i s t e s t were d e r i v e d from three sources f o r each treatment: (1) 14 c a l l i from L2(D2A2) medium, (2) 14-20 c a l l i from B 5(D2N2K2)medium and (3) 16 c a l l i from L2(D2B0.5A2) medium. C u l t u r e s were incubated with a 24-hour pho t o p e r i o d at 250 MEm~ 2sec~ 1 and 25°C f o r four weeks. 34 3.2.2.6 NAA and BA i n Presence of ADE The t e s t was designed to determine the e f f e c t of v a r i o u s c o n c e n t r a t i o n s of NAA i n combination with BA and ADE on c a l l u s d i f f e r e n t i a t i o n . 'Norseman' c a l l i from LSE medium were used. D i f f e r e n t l e v e l s of NAA (0, 0.001, 0.01 and o.1 mg/1) and BA (0 and 0.2 mg/1) were used with 2 mg/1 ADE i n a l l t e s t s . C a l l i i n t e s t tubes were incubated with a 16-hour photoperiod at 250 /uEm~2sec"1 and 25°C f o r four weeks. 3.2.2.7 C a l l u s D i f f e r e n t i a t i o n on Four Test Media 'Norseman' c a l l i , p r e v i o u s l y c u l t u r e d on L2(D2B2A5) medium, were t r a n s f e r r e d onto s e v e r a l media: (1) L2(B0.2A2), (2) L2(DO.01B0.2A2), (3) (NO.01B0.2A2) and (4) L2 medium with h a l f s t r e g t h of b a s a l s a l t s and v i t a m i n s c o n t a i n i n g 0.2 mg/1 IAA and 2 mg/1 n i c o t i n i c a c i d . Each l a r g e c a l l u s was d i v i d e d i n t o four p i e c e s with each p i e c e put i n t o one of four media c o n t a i n e d i n t e s t tubes. A l l the c u l t u r e s were incubated with a 16-hour photoperiod at 500 MEm~ 2sec" 1 and 25°C. Response was recorded a f t e r three weeks of c u l t u r e . 3.2.2.8 C a l l u s Growth at High or Low Auxin to C y t o k i n i n R a t i o s 'Norseman' c a l l i from LSE medium were t r a n s f e r r e d onto the f o l l o w i n g two media (1) L2(D11K1) and (2) L2(D1K10). The f i r s t medium had a high auxin to c y t o k i n i n r a t i o (11), while the second one had a very 35 low r a t i o (0.1). C a l l i were c u l t u r e d with a 24-hour ph o t o p e r i o d at 200 MEm" 2sec" 1 and 25°C f o r f i v e weeks. 3.2.2.9 C a l l u s Growth on SCP Medium In experiments when c a l l i were t r a n s f e r r e d onto a medium which d i d not promote e f f i c i e n t c a l l u s growth (e.g. on LSE medium), i t would be u s e f u l to r e s t o r e such c a l l i to a normal v i g o r o u s growth p a t t e r n . T h i s t e s t was e s t a b l i s h e d t o determine the e f f e c t of SCP medium as a v e h i c l e f o r r e s t o r i n g senescing c a l l i . C a l l i were p r e v i o u s l y c u l t u r e d on LSE medium f o r v a r i o u s time p e r i o d s : (1) 'Altaswede' c a l l i and (2) 'Norseman' c a l l i , both of which had been c u l t u r e d on LSE medium f o r 55 days (At t h i s time the c a l l i had stopped growing and were showing brown c o l o r . Some c a l l i were dead); (3) 'Altaswede' c a l l i which had been c u l t u r e d on LSE medium f o r 42 days (Some of c a l l i had senesced, but the c u l t u r e s were g e n e r a l l y b e t t e r than those of (1) and ( 2 ) ) ; (4) 'Norseman' c a l l i which had been c u l t u r e d on LSE medium f o r 37 days and showed s l i g h t browning (Some c a l l i showed a l i t t l e growth); (5) 'Altaswede' c a l l i induced on L2 medium were f i r s t t r a n s f e r r e d onto LSE medium f o r 23 days and then s u b c u l t u r e d t o L2(P0.01B0.2) medium (At t h i s time these c a l l i turned yellow brown or dark brown). C a l l i from the above sources were t r a n s f e r r e d onto SCP medium (1 mg/1 2,4-D) i n p l a t e s . C u l t u r e s were incubated at 25°C and under 16-hour p h o t o p e r i o d with l i g h t i n t e n s i t i e s of 36 250 luEm" 2 s e c " 1 f o r c a l l u s sources (1), (4) and (5)and 700 MEm" 2sec" 1 f o r c a l l u s sources (2) and ( 3 ) . Growth and appearance were recorded a f t e r four weeks of c u l t u r e . C a l l i on SCP medium were c o n t i n u o u s l y s u b c u l t u r e d on f r e s h SCP medium f o r one year. 3.2.2.10 L i g h t E f f e c t on C a l l i Growing on SCP Medium T h i s t e s t was e s t a b l i s h e d to i n v e s t i g a t e the e f f e c t of l i g h t i n t e n s i t y on c a l l u s d i f f e r e n t i a t i o n on SCP medium i n attempts to achieve p l a n t r e g e n e r a t i o n . 'Altaswede' c a l l i , which had been c u l t u r e d on LSE medium f o r one month , were t r a n s f e r r e d onto SCP medium i n p l a t e s and incubated with a 16-hour photoperiod and at 25°C f o r 40 days. D i f f e r e n t l i g h t i n t e n s i t i e s (70-450 MEm~ 2sec" 1) were o b t a i n e d by s t a c k i n g p l a t e s as 14 columns, each having e i g h t t o ten p l a t e s . Each p l a t e c o n t a i n e d t h r e e to four c a l l i . 3.2.2.11 S e l e c t i o n and Maintenance of Embryogenic C a l l u s A f t e r p l a n t l e t r e g e n e r a t i o n was achieved on two media c o n t a i n i n g NAA and KIN, embryogenic c a l l i of these two treatments were t r a n s f e r r e d and then s u b c u l t u r e d on LSP medium. Three c a l l i from these c u l t u r e s were f u r t h e r s u b c u l t u r e d on LSP medium f o r embryo p r o l i f e r a t i o n and p l a n t r e g e n e r a t i o n . E v e n t u a l l y c a l l i from one c a l l u s l i n e (one genotype) 37 was r e t a i n e d . C a l l i which were capable of embryogenesis were thus ob t a i n e d . These embryogenic c a l l i were s u b c u l t u r e d on LSP medium. Test, tubes c o n t a i n i n g these c a l l i were incubated with a 16-hour photoperiod at 500 MEm" 2sec" 1 and 25°C. They were evaluated a f t e r four weeks of c u l t u r e , f o r embryo development and p l a n t l e t p r o d u c t i o n . 3.2.2.12 Growth of Embryogenic and Non-embryogenic C a l l i C a l l i may be i d e n t i f i e d on the b a s i s of the occurrence of embryogenesis, i . e . embryogenic and non-embryogenic. T h i s experiment attempted to determine the d i f f e r e n c e between the two kinds of c a l l i when they were c u l t u r e d on d i f f e r e n t media. Embryogenic c a l l i of 'Altaswede' were obtained from the c u l t u r e s i n which embryogenesis had been observed. Non-embryogenic c a l l i were from another c a l l u s c u l t u r e of 'Altaswede'. A l l c a l l i were c u l t u r e d on the f o l l o w i n g ten media: (1) L2(NO.01 BO.1), (2) L2(N0.05B0.1), (3) L2(NO.1 BO.1), (4) L2(NO.01 BO.5), (5) L2(N0.05B0.5), (6) L2(PO.001 BO.2), (7) L2(N2A2), (8) L2 ba s a l c o n t a i n i n g 2 g/1 yeast e x t r a c t and 10 g/1 agar, (9) L2 b a s a l medium c o n t a i n i n g 600 mg/1 glutamine and 3% sucrose and (10) L2(N0.5K0.5). Non-embryogenic c a l l i had 19 tubes f o r each treatment and embryogenic c a l l i , two tubes per treatment. C u l t u r e s were incubated at 480 MEm" 2sec" 1. 38 3.2.3 Shoot T i p C u l t u r e Shoot t i p s 3-4 mm i n l e n g t h were used as e x p l a n t s f o r m u l t i p l e shoot i n d u c t i o n . Shoot t i p s were f i r s t c u l t u r e d on L2 medium. Three weeks l a t e r they were t r a n s f e r r e d onto L2(PO.003B2) medium 'for m u l t i p l e shoot i n d u c t i o n . Each t e s t tube c o n t a i n e d one shoot t i p . T h i r t y tubes i n one rack were t r e a t e d as a group and groups were randomly a s s i g n e d . C u l t u r e s were eva l u a t e d f o u r weeks l a t e r f o r m u l t i p l e shoot i n d u c t i o n frequency and the number of shoots produced per shoot t i p based on group. M u l t i p l e shoots were s u b c u l t u r e d twice onto L2 medium c o n t a i n i n g 0.002-0.003 mg/1 PIC and 1 mg/1 BA f o r propagation at a s u b c u l t u r e i n t e r v a l of one month. Shoots were i s o l a t e d with a s c a l p e l from the m u l t i p l e shoot r o s e t t e . Only well-formed shoots were u t i l i z e d . These c u l t u r e s were i n c u b a t e d with a 16-hour photoperiod at 200 MEnr 2sec~ 1 and 25°C. 3.2.4 Rooting of Shoots Shoots obtained from shoot t i p c u l t u r e s or from embryogenic c a l l i were t r a n s f e r r e d to r o o t i n g medium c o n s i s t i n g of h a l f s t r e n g t h s a l t s and v i t a m i n s of L2 medium and c o n t a i n i n g 1 mg/1 n i c o t i n i c a c i d , 2.5 mg/1 3-aminopyridine, 0.2 mg/1 IAA, 10 g/1 sucrose and 8 g/1 agar ( P h i l l i p s and C o l l i n s , 1979a). C u l t u r e s were maintained under a 16-hour p h o t o p e r i o d and 250 /zEnr 2 sec ~ 1 . They were ready to be t r a n s f e r r e d t o pots a f t e r one month of growth. 39 3.2.5 Scanning E l e c t r o n Microscopy Samples from embryogenic c u l t u r e s were i n i t i a l l y f i x e d i n 4% g l u t a r a l d e h y d e (primary f i x a t i v e ) f o l l o w e d by 1% osmium t e t r o x i d e i n 0.1 M sodium c a c o d y l a t e b u f f e r f o r at l e a s t one hour (secondary f i x a t i v e ) . F o l l o w i n g f i x a t i o n , samples were dehydrated through a graded s e r i e s of e t h a n o l 30%, 50%, 70%, 85% and 95% f o r 5-10 minutes each and f i n a l l y to a b s o l u t e ethanol with two changes of 30 minutes each. The dehydrated specimens were t r a n s f e r r e d to a c r i t i c a l p o i n t d r y i n g apparatus (CPD; Parr 4770). Specimens were p l a c e d i n the d r y i n g chamber with s u f f i c i e n t a l c o h o l to prevent a i r - d r y i n g p r i o r to c r i t i c a l p o i n t d r y i n g . The cover of the specimen chamber was secured and l i q u i d carbon d i o x i d e admitted to the chamber from an e x t e r n a l tank. The i n l e t v a l v e was kept open and chamber pressure i n c r e a s e d to 820 p . s . i . Three to four minutes l a t e r the exhaust v a l v e was opened f o r t h r e e to four minutes. A f t e r three minutes the exhaust v a l v e was c l o s e d . The exhaust v a l v e was a l t e r n a t e l y opened and c l o s e d every three minutes f o r a t o t a l of 14 times. Then the exhaust valve and the l i q u i d carbon d i o x i d e i n l e t v a l v e s were c l o s e d . Hot water was poured around the specimen chamber, he a t i n g i t to 44°C, s u f f i c i e n t to achieve the c r i t i c a l p o i n t of carbon d i o x i d e i . e . 31°C. During the h e a t i n g process the p r e s s u r e i n the chamber rose above 1,600 p . s . i . (pound per square i n c h ) . A f t e r a steady p r e s s u r e of 1,400 p . s . i . at 44°C had been 40 s t a b i l i z e d , the exhaust valve was slowly opened and the pressure was released to 0 p . s . i . while maintaining the specimen chamber at 44-45°C. The dried specimens were mounted on specimen stubs, transferred to a vacuum evaporator and coated using a "Nanotech Semprep 2" sputter coater. Coated specimens were viewed under a scanning electron microscope (Cambridge Stereoscan 250, Cambridge Instrument Co.) and pictures were taken on Polaroid Type 55 f i l m . 3.3 Growth and A n a l y s i s of Regenerated P l a n t s 3.3.1 P l a n t T r a n s f e r and Growth i n Greenhouse A mixture of s o i l , sand and vermiculite (1:1:1 by volume) was used to f i l l 6 X 6 cm pots and also 50 X 32 cm f l a t s into which the pots were placed. Each f l a t held 28 pots. The f l a t s were wrapped with a piece of brown paper and autoclaved at 121°C, 1 kg/cm2 for 20 minutes and allowed to cool for at least one day p r i o r to receiving plants. S o i l in individual pots was inoculated with commercial Rhizobium t r i f o l i i (Nitragin Co. Ltd) prior to plant transfer. Plants to be transferred were usually cultured on rooting medium for one month. They were removed from the test tubes using forceps and rinsed free of agar in lukewarm s t e r i l i z e d water. Plants were transferred into pots with the root t i p in a downward pos i t i o n . 41 Tr a n s p l a n t e d p l a n t s were t r a n s f e r r e d to the greenhouse. High humidity was pro v i d e d f o r the f i r s t two weeks by c o v e r i n g f l a t s with p i e c e s of Saran Wrap (or t r a n s p a r e n t p l a s t i c f i l m ) . During t h i s p e r i o d they were watered f r e q u e n t l y . The p l a n t s were exposed to normal greenhouse c o n d i t i o n s a f t e r two weeks. One week a f t e r p l a n t s were t r a n s p l a n t e d they were watered with s o l u b l e f e r t i l i z e r (15-15-18). A l l subsequent f e r t i l i z a t i o n was c a r r i e d out using a N - l e s s n u t r i e n t s o l u t i o n a p p l i e d approximately once a week to f a c i l i t a t e root nodule development. An experiment was i n i t i a t e d to determine the e f f e c t of d i f f e r e n t treatments on the s u r v i v a l of regenerated p l a n t l e t s . Shoots of 'Altaswede' induced from c u l t u r e s were rooted in v i t r o f o r s i x weeks. P l a n t s t r a n s f e r r e d were t r e a t e d as f o l l o w s : (1) T r a n s f e r r e d p l a n t s were l e f t under ambient l a b o r a t o r y c o n d i t i o n s f o r the f i r s t week and then t r a n s f e r r e d to the greenhouse ( P l a n t s were covered with Saran Wrap f o r the f i r s t two weeks); (2) P l a n t s were t r e a t e d as i n (1) except they were p l a c e d i n greenhouse f o r the e n t i r e p e r i o d ; (3) P l a n t s were moved d i r e c t l y t o the greenhouse, but were not covered with Saran Wrap. S u f f i c i e n t water f o r growth was s u p p l i e d d u r i n g t h i s p e r i o d . E v a l u a t i o n of s u r v i v a l was made four weeks l a t e r i n a l l treatments. 42 3.3.2 Chromosome A n a l y s i s Root t i p s of regenerated and c o n t r o l p l a n t s (seed-derived) were c o l l e c t e d i n d i v i d u a l l y from f r e s h , t h i c k r o o t s . P r e p a r a t i o n and s t a i n i n g methods were p r o v i d e d by R.R.Smith (Univ. of Wisconsin, Madison, p e r s o n a l communication) with minor m o d i f i c a t i o n s . Root t i p s were p r e t r e a t e d i n 0.003 M 8-hydroxyquinoline at 4°C f o r 4-6 hours. They were then r i n s e d with d i s t i l l e d water and f i x e d i n ethanol - a c e t i c a c i d (3:1 v/v) at room temperature (25°C) o v e r n i g h t . U s u a l l y f i x a t i o n was fo l l o w e d immediately by h y d r o l y s i s , but the m a t e r i a l c o u l d be s t o r e d i n 70% etha n o l a f t e r t h i s s t e p . Root t i p s were h y d r o l y s e d i n concen t r a t e d HCl (ethanol : h y d r o c h l o r i c a c i d = 1:1 v/v) f o r 45-60 sec. and then r i n s e d with 70% ethanol before s t a i n i n g w i t h m o d i f i e d c a r b o l f u c h s i n (Kao, 1976). Root t i p s were squashed and o b s e r v a t i o n of c e l l s made on a Z e i s s l i g h t microscope. For each p l a n t , two root t i p s were squashed and the chromosome number of at l e a s t two c e l l s were examined. When abnormal chromosome numbers were observed a d d i t i o n a l root t i p s and c e l l s were examined. 3.3.3 Leghaemoglobin A n a l y s i s A l l e x t r a c t i o n s and sample p r e p a r a t i o n were c a r r i e d out on i c e at 0-4°C. Nodules (about 20) from each p l a n t were c o l l e c t e d i n t o a m i c r o c e n t r i f u g e tube. They were ground wi t h a p l a s t i c p e s t l e i n the tube a f t e r adding a small drop of e x t r a c t i o n b u f f e r c o n t a i n i n g 0.1 M Na/K phosphate and 1 43 mM EDTA a t pH 7.4. The tubes were c e n t r i f u g e d i n a m i c r o c e n t r i f u g e f o r t h r e e minutes a t 15,600 X g. Leghaemoglobin components were a n a l y z e d u s i n g the c e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s (CAE) method of H o l l e t a l . (1983) (Gelman, Sepra-Tek e l e c t r o p h o r e s i s s y s t e m ) . C e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s s t r i p s (Sepraphore I I I ) were pr e - s o a k e d i n b u f f e r c o n t a i n i n g ( g / 1 ) : T r i s ( T r i s - ( h y d r o x y m e t h y l ) a m i n o m e t h a n e ) : 20.72; EDTA: 0.15; g l y c i n e : 3.53 and s u c r o s e : 15.0. F o l l o w i n g sample a p p l i c a t i o n the s t r i p was run a t 300 V f o r 40-50 minutes a t room t e m p e r a t u r e . A f t e r e l e c t r o p h o r e s i s t h e s t r i p was s t a i n e d f o r p r o t e i n i n Ponceau S (100 mg/ml i n 7.5% aqueous t r i c h l o r o a c e t i c a c i d ) f o r t e n mi n u t e s and c l e a r e d i n 5% a c e t i c a c i d . S t r i p s from s e v e r a l runs c o u l d be f u r t h e r s t a i n e d s p e c i f i c a l l y f o r haem p r o t e i n s u s i n g t h e o - d i a n i s i d i n e method d e s c r i b e d by H o l l e t a l . (1983). 3.3.4 Isozyme A n a l y s i s In t h e s e e x p e r i m e n t s q u a l i t a t i v e changes were e v a l u a t e d f o r t he f o l l o w i n g f i v e enzymes: m a l a t e dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (6-PGDH), phosphoglucose isomerase ( P G I ) , phosphoglucomutase (PGM) and s h i k i m a t e deyhdrogenase (SKDH). The m o r p h o l i n e - c i t r a t e b u f f e r system of C l a y t o n and T r e t i a k (1972) was used. B u f f e r c o m p o s i t i o n was as f o l l o w s : e l e c t r o d e b u f f e r : 0.04 M c i t r i c a c i d (anhydrous) w i t h pH a d j u s t e d t o 6.1 w i t h N - ( 3 - a m i n o p r o p y l ) - m o r p h o l i n e ; g e l b u f f e r : 0.002 M c i t r i c 44 a c i d (anhydrous) with the pH a d j u s t e d to 6.4. S t a r c h (49 g, l o t No. 392) and sucrose (35 g) were pl a c e d i n one f l a s k and 18 ml g e l b u f f e r and 332 ml water i n t o another. About 1/3 of the b u f f e r s o l u t i o n was added to the s t a r c h / s u g a r mixture while the remainder of the b u f f e r s o l u t i o n was heated i n an oven to b o i l i n g . The b o i l i n g b u f f e r s o l u t i o n was then added to the s t a r c h mixture with shaking to achieve an even mixture. The s o l u t i o n was degassed and was poured onto g e l formers, shaken s l i g h t l y to give a smooth s u r f a c e . Gels were wrapped with Saran Wrap and kept at room temperature o v e r n i g h t . They were c o o l e d down to 4°C p r i o r to e l e c t r o p h o r e s i s . Two s m a l l young l e a f l e t s from each p l a n t and two drops of c o l d e x t r a c t i o n b u f f e r (Helenurm, 1983) were added to each w e l l of a sample g r i n d i n g pad, p l a c e d on i c e to prevent isozyme d e n a t u r a t i o n . The m a t e r i a l was homogenized with a g l a s s p e s t l e . The crude e x t r a c t was taken up d i r e c t l y i n t o f i l t e r paper wicks precut to approximately 3 mm wide. Wicks were i n s e r t e d s e q u e n t i a l l y i n t o 30 s l o t s at the cathodal end of the g e l . A wick c o n t a i n i n g dye (red food dye) was i n s e r t e d i n one s i d e s l o t . The g e l was s t i l l wrapped with Saran Wrap but approximately 2 cm of both ends were exposed. T h i s wrapping p r o t e c t e d the g e l from d e s s i c a t i o n and c ontamination. The ends of g e l were j o i n e d with the e l e c t r o d e b u f f e r v i a c e l l u l o s e sponges. The g e l was run at 300 V, 50-60 mA at 3-4°C. Enzyme p r o t e i n s moved a n o d a l l y at a r a t e determined 45 by t h e i r s i z e , shape and c h a r g e . The s t a i n i n g s o l u t i o n s were p r e p a r e d a c c o r d i n g t o t h e r e c e i p e s o f V a l l i e j o s ( 1 9 8 3 ) . F o l l o w i n g e l e c t r o p h o r e t i c s e p a r a t i o n , s t a r c h g e l s were s l i c e d i n t o 5-6 s l i c e s . S t a i n i n g s o l u t i o n s were p o u r e d i n t o i n d i v i d u a l s t a i n i n g b o x e s and r e s u l t i n g s l i c e s were s e p a r a t e d and i n t r o d u c e d i n t o t h e a p p r o p r i a t e s t a i n . Boxes were i n c u b a t e d i n d a r k n e s s a t 37°C. A f t e r g e l s were w e l l - s t a i n e d , t h e y were f i x e d i n 4% m e t h a n o l and 10% a c e t i c a c i d . 3 . 3 . 5 M o r p h o l o g i c a l A n a l y s i s N o r m a l l y e a c h l e a f e x c e p t t h e f i r s t l e a f o f r e d c l o v e r i s composed of t h r e e l e a f l e t s . Changes i n l e a f l e t number were e v a l u a t e d f o r b o t h r e g e n e r a t e d and s e e d - d e r i v e d p l a n t s . The l e a f l e t l e n g t h t o w i d t h r a t i o was c h o s e n a s a n o t h e r m o r p h o l o g i c a l t r a i t t o l o o k a t i n r e g e n e r a t e d p l a n t s . To l i m i t v a r i a t i o n by o t h e r f a c t o r s , two f u r t h e r c o n d i t i o n s were met when measurements were t a k e n : (1) O n l y t h e m i d d l e o f t h r e e l e a f l e t s was measured and (2) l e a f l e t s m e a sured were i n a r e s t r i c t e d r a n g e o f l e n g t h ; a p p r o x i m a t e l y 25 mm r a n g i n g f r o m 23 t o 27 mm f o r ' A l t a s w e d e ' and 30 mm r a n g i n g f r o m 28 t o 32 mm f o r 'Norseman'. L e a f l e t s were r a n d o m l y c h o s e n and measured f o r l e n g t h and w i d t h . R a t i o s were c a l c u l a t e d f r o m t h e o b s e r v e d d a t a . Mean, v a r i a n c e and s t a n d a r d d e v i a t i o n were o b t a i n e d f o r e a c h o f d i f f e r e n t " p o p u l a t i o n s " o f r e g e n e r a t e d and c o n t r o l p l a n t s . The mean and v a r i a n c e o f e a c h p o p u l a t i o n were compared w i t h t h o s e o f 46 the proper c o n t r o l . For the comparison of v a r i a n c e , F va l u e s were c a l c u l a t e d by f o l l o w i n g e q uation: F = o\/o\ and were t e s t e d f o r s i g n i f i c a n c e . For the comparison of means from regenerated and c o n t r o l p l a n t s , the u t e s t was performed. <7_ _ can be ob t a i n e d by f o l l o w i n g e q u a t i o n (Ma, x! -x 2 1978): o = / a 2 / n , + a l / n 2 X 1 X 2 and then u = ( x , - x 2 ) / a x 1 x 2 The s i g n i f i c a n c e of d i f f e r e n c e between means i s thus t e s t a b l e . 4. RESULTS 4.1 C a l l u s Induction 4.1.1 C a l l u s Induction Frequency The data f o r c a l l u s i n d u c t i o n frequency are shown i n Table 2. 'Altaswede' i n the second batch showed a higher c a l l u s i n d u c t i o n frequency than 'Norseman'. For 'Norseman' the frequency of i n d u c t i o n was constant from the f i r s t to the second batch. However, 'Altaswede' showed an i n c r e a s e d frequency i n Batch 2. The data suggest that 'Altaswede' e x p l a n t s may perform b e t t e r at low l i g h t i n t e n s i t y or when grown i n p e t r i d i s h c u l t u r e . An i n c r e a s e i n c a l l u s i n d u c t i o n frequency f o r 'Altaswede' was a l s o observed when l i g h t i n t e n s i t y decreased i n another batch of c u l t u r e . A t o t a l of f i v e batches of c u l t u r e s were i n i t i a t e d f o r c a l l u s i n d u c t i o n . I n d u c t i o n f r e q u e n c i e s were recorded a f t e r two weeks i n c u b a t i o n . In one batch of c u l t u r e s , 50 tubes which d i d not have c a l l u s when examined a f t e r two weeks of c u l t u r e produced c a l l i i n four c u l t u r e s when re-examined 30 days a f t e r the explant t r a n s f e r . T h e r e f o r e , c a l l u s i n d u c t i o n f r e q u e n c i e s may be s l i g h t l y h igher i f measurements are made a f t e r a longer p e r i o d of i n i t i a l c u l t u r e . It was a l s o apparent that the c a l l u s i n d u c t i o n frequency of 'Norseman' was g r e a t l y a f f e c t e d when a t h i n l a y e r of water on the agar medium d i d not evaporate i n t e s t tubes wrapped with p a r a f i l m while 'Altaswede' was l e s s 47 48 Table 2. C a l l u s i n d u c t i o n frequency f o r two c u l t i v a r s of red c l o v e r on L2 medium. Batch No 1 2 C u l t i v a r Altaswede Norseman Altaswede Norseman No e x p l a n t s c u l t u r e d 288 174 272 1246 No e x p l a n t s producing 192 c a l l u s 1 12 236 816 Frequency (%) 66.7 64.4 86. 7 65.5 2 X 2 Contingency x 2 A n a l y s i s Batch 1 Altaswede vs Norseman X 2 = 0 .023 ns 1 Batch 2 Altaswede vs Norseman X 2 = 7 .954 ** (P<0.01) Combined data Altaswede vs Norseman X 2 = 4 .030 * (P<0.05) 1 ns: not s i g n i f i c a n t ; . * : s i g n i f i c a n t ; **: h i g h l y s i g n i f i c a n t . 49 a f f e c t e d by the same ex p e r i m e n t a l c o n d i t i o n s . 4.1.2 Genotypic E f f e c t on C a l l u s Induction Tubes with c a l l u s induced i n the two batches were grouped i n t o 1, 2 or 3 - c a l l u s tubes, depending upon the number of e x p l a n t s f o r which c a l l u s was e v i d e n t . The r e s u l t s are shown i n T a b l e 3. I t i s c l e a r from the data that the three e x p l a n t s from one genotype behaved s i m i l a r l y . If one explant produced c a l l u s the other two i n the same tube a l s o showed a s i m i l a r response. C a l l i o r i g i n a t i n g from d i f f e r e n t genotypes c o u l d look d i f f e r e n t (e.g. i n c o l o r ) whereas the three c a l l i from one genotype were p h e n o t y p i c a l l y s i m i l a r . The p o s s i b i l i t y t h a t t h i s s i m i l a r i t y among three e x p l a n t s from one i n d i v i d u a l was simply a response to the uniform environment i n the t e s t tube was e l i m i n a t e d by l a t e r experiments, which showed that e x p l a n t s from one i n d i v i d u a l produced c a l l i while e x p l a n t s from another genotype i n the same c u l t u r e v e s s e l d i d not (F i g u r e 1). 4.1.3 Observations on C a l l u s Induction A f t e r the i n i t i a l t r a n s f e r , some e x p l a n t s r e t a i n e d a f r e s h appearance and d i d not s h r i v e l , or t u r n brown. A f t e r 3-4 days such e x p l a n t s s w e l l e d and c a l l u s c o u l d develop from the swollen p o r t i o n . D u r i n g t h i s process the green c o l o r of the i n i t i a l explant d i s a p p e a r e d . The green c o l o r l o s s commenced at the p e r i p h e r y of the ex p l a n t , l e a v i n g the 50 Table 3. C a l l u s i n d u c t i o n f o r three explants coming from the same s e e d l i n g i n two batches of c u l t u r e . Data f o r two c u l t u r e batches have been combined. C u l t u r e C u l t i v a r  Altaswede (%) Norseman (%) No. of tubes c u l t u r e d No. of tubes with c a l l u s 3 - c a l l u s tube 2 - c a l l u s tube 1 - c a l l u s tube 266 197 (100) 180 (90.4) 12 (6.1) 5 (2.5) 167 114 (100) 109 (95.6) 3 (2.6) 2 (1.8) 51 F i g u r e 1. C a l l u s i n d u c t i o n and genotype e f f e c t . 'Norseman' h y p o c o t y l e x p l a n t s on L2 medium. E x p l a n t s from one i n d i v i d u a l (upper h a l f of p l a t e ) produced c a l l i , w h ile explants from a second genotype d i d not a f t e r two weeks c u l t u r e . 52 center green. At day 7-8 some explants showed early c a l l u s development, followed by continuing d i v i s i o n and c a l l u s formation after two weeks. Explants, which withered after i n i t i a l transfer, did not produce c a l l u s . Other explants showed s l i g h t browning with some swelling; these explants could develop further but seldom produced vigorous c a l l u s growth. C a l l i formed under dark condition were white while those induced in the l i g h t usually showed some green coloration. 4.2 Callus Maintenance and Plant Regeneration 4.2.1 Primary Test of Two Media Approximately 85 c a l l i of each c u l t i v a r were cultured on each of the two media. In neither case was shoot induction or bud i n i t i a t i o n observed when recorded after two weeks of culture. Although c a l l i grew faster on B5(N2A2) medium, more green-spotted c a l l i were observed on LSE medium at t h i s time. C a l l i on B 5 were whiter than those on LSE medium. Some root formation was observed on B 5 medium. After 17 days some c a l l i in both cases were transferred onto fresh media. In these subcultures many of the greenish regions became brown and the number of green-spotted c a l l i decreased. Callus response on LSE medium i s shown in Table 4. More green-spotted c a l l i were observed in cultures from 'Norseman' than 'Altaswede' after two weeks of culture. Then c a l l i turned darker and after the subculture the number 53 Table 4. C a l l u s growth on LSE medium. Data f o r the i n i t i a l c u l t u r e and s u b c u l t u r e are shown. The f i r s t c u l t u r e was measured a f t e r two weeks of c u l t u r e and the s u b c u l t u r e was measured four weeks a f t e r the t r a n s f e r . C u l t u r e C u l t i v a r NC GSC (%) SC RC 1 Altaswede 86 21 (24.4) 0 0 1 Norseman 88 40 (45.5) 0 0 2 Altaswede 56 2 ( 3.6) 0 2 2 Norseman 59 10 (16.9) 0 0 NC=Number of C a l l i ; GSC=Green -Spotted C a l l i ; SC=Shoot C a l l i ; RC=Root C a l l i 54 of green-spotted c a l l i was s h a r p l y decreased. From each c u l t i v a r 50 c a l l i were c u l t u r e d on LSE f o r a f u r t h e r three weeks. During t h i s p e r i o d no obvious growth of the c a l l i was observed. A l l the green spots disappeared and one c a l l u s of 'Altaswede' produced some new r o o t s . Upon t e r m i n a t i o n of the t e s t , c a l l i had been growing on LSE medium f o r about two months. Continuous s u b c u l t u r i n g of c a l l u s onto LSE medium d i d not i n i t i a t e bud or shoot d i f f e r e n t i a t i o n , as w e l l as being a s s o c i a t e d with poorer c a l l u s growth. 4 . 2 . 2 C a l l u s Growth on LSE Medium Test 1: R e s u l t s of the i n i t i a l and the f i r s t s u b c u l t u r e are p resented i n Table 5. None of these a p p l i e d c o n d i t i o n s had promotional e f f e c t on shoot i n d u c t i o n . A f t e r c a l l i were t r a n s f e r r e d from L2 medium to LSE medium, growth and good c o l o r was maintained f o r a short p e r i o d . Then the c o l o r of c a l l u s changed from f r e s h yellow-green to brown or yellow-brown. Green spots disappeared and c a l l i stopped growing. Some c a l l i produced r o o t s d u r i n g the i n i t i a l c u l t u r e (Figure 2 ) . A f t e r the c a l l i were s u b c u l t u r e d , they c o n t i n u e d to show an unhealthy growth response; more c a l l i senesced; no f r e s h r o o t s were produced and p r e v i o u s l y formed r o o t s turned brown ( F i g u r e 3 ) . At the end of the c u l t u r e p e r i o d approximately 95% of c a l l i of both c u l t i v a r s were senesced or dead. T h e r e f o r e , the f a i l u r e of LSE to induce shoots i n the present case i s l e s s l i k e l y r e l a t e d to l i g h t 55 Table 5. Response of c a l l i on LSE medium: i n i t i a l c u l t u r e and s u b c u l t u r e of Test 1 recorded a f t e r four and f i v e weeks of c u l t u r e , r e s p e c t i v e l y . C u l t u r e C u l t i v a r L i g h t NC SC RC i n t e n s i t y MEm"2 s e c " 1 I n i t i a l Altaswede 100-900 1452 0 1 3 Sub c u l t u r e Altaswede 100-900 984 0 0 I n i t i a l Norseman 70-450 1 556 0 1 5 Sub c u l t u r e Norseman 70-450 568 0 0 NC=Number of C a l l i ; SC= Shoot C a l l i ; RC=Root C a l l i 56 F i g u r e 2. Response of 'Altaswede' c a l l i on LSE medium a f t e r two weeks of c u l t u r e . C a l l i were browning, green spots began to disappear and r o o t s were induced d u r i n g t h i s c u l t u r e p e r i o d . F i g u r e 3 . Response of 'Altaswede' c a l l i on LSE medium a f t e r f i v e weeks of c u l t u r e . C a l l i showed an unhealty growth response. (a) C a l l i became brown and stopped growing (upper), (b) No f r e s h r o o t s were produced and p r e v i o u s l y formed r o o t s t u r n e d brown (lo w e r ) . 58 c o n d i t i o n or sample s i z e of c a l l u s . T est 2: No shoot was ever formed on the four media (Table 6 ) . As demonstrated by P h i l l i p s and C o l l i n s (1980) and other r e s u l t s of the present study, LSP medium i s s u p p o r t i v e of embryo development. In a d d i t i o n L2(NO.05K0.5) was shown to induce shoots i n the t e s t s with NAA and KIN. The f a i l u r e to induce shoot formation on the media used i n t h i s t e s t suggested that no embryos had been formed on LSE medium, otherwise they should be a b l e to develop i n t o shoots on these media. On L2(P0.002B0.2), c a l l i on f u l l s t r e n g t h b a s a l s a l t s appeared h e a l t h i e r than those on h a l f s t r e n g t h s o l u t i o n s ; fewer c a l l i showed brown c o l o r . Roots formed only from c a l l i on the medium without added growth r e g u l a t o r s . However, adding r e g u l a t o r s tended to keep gree n - s p o t t e d c a l l i l o n g e r . Test 3: As shown in Table 7, r e s u l t s were s i m i l a r to these o b t a i n e d through p r e v i o u s t e s t s . C a l l i of 'Norseman' were not as watery as they had been before t h e i r t r a n s f e r onto t h i s medium. R e s u l t s from these t e s t s demonstrated that LSE medium d i d not induce shoot p r o d u c t i o n under c u r r e n t experimental c o n d i t i o n s nor was i t a good medium f o r c a l l u s maintenance. C a l l i on t h i s medium showed a . s i m i l a r response throughout these t e s t s , browning, senescence and u l t i m a t e l y death. T h i s response p a t t e r n strengthened with the time of c a l l u s on t h i s medium. 59 Table 6. Response of c a l l i from LSE medium s u b c u l t u r e d onto four media ( c o n c e n t r a t i o n i n mg/1). Medium 1 2 3 4 PIC 0.002 0.002 no growth BA 0.2 0.2 r e g u l a t o r s NAA 0.05 KIN 0.5 SALTS f u l l h a l f f u l l f u l l R e s u l t s NC 42 36 39 33 RC 0 0 0 2 GSC 2 1 0 0 BC 3 6 5 6 NC=Number of C a l l i ; RC=Root C a l l i ; GSC=Green-Spotted C a l l i ; BC=Blackened C a l l i 60 Table 7. Response of c a l l i on L2(D0.01A5) medium, C u l t i v a r Altaswede Norseman NC GSC RC Color Rate of Growth 99 3 1 brown or black ++ 99 9 0 s l i g h t l y brown +++ NC=Number of C a l l i ; GSC=Green-Spotted C a l l i ; RC=Root C a l l i . ++, +++: More p l u s s i g n s i n d i c a t e f a s t e r r e l a t i v e . c a l l u s growth. 6 1 4.2.3 2,4-D and BA R e s u l t s from Batch one are presented i n Table 8. C a l l i on these two media were very s i m i l a r f o r a s i n g l e c u l t i v a r . Obvious d i f f e r e n c e s e x i s t e d between c a l l i of the two c u l t i v a r s . 'Norseman' g e n e r a l l y had more gre e n - s p o t t e d c a l l i than 'Altaswede' and 'Norseman' c a l l i grew f a s t e r than 'Altaswede' c a l l i . I t was observed that two l e a f - l i k e s t r u c t u r e s formed on L2(D2B2) medium, a b e t t e r s i g n f o r shoot d i f f e r e n t i a t i o n . When recorded a f t e r four weeks of c u l t u r e , some c a l l i of both c u l t i v a r s had turned brown and the number of green-spotted c a l l i decreased. One of the two l e a f - l i k e s t r u c t u r e s had a l s o become brown, while one remained green. For Batch two, 20 to 25 c a l l i of 'Altaswede' were c u l t u r e d f o r each treatment. No shoots were induced on any of these media. However, green-spotted c a l l i were observed on L2(D2B2A5) medium. C a l l i on t h i s medium grew f a s t e r . Only one c a l l u s on medium with 2 mg/1 2,4-D had roo t d i f f e r e n t i a t i o n . Data recorded a f t e r three weeks and four weeks of c u l t u r e showed the same t r e n d . 4.2.4 PIC and BA T e s t 1: R e s u l t s i n Table 9 i n d i c a t e d t hat PIC a t the c o n c e n t r a t i o n s t e s t e d d i d not induce c a l l i to produce shoots or r o o t s , 10 mM a r g i n i n e added to the medium a l s o had no e f f e c t on shoot or root i n d u c t i o n and i n the presence of a r g i n i n e , h i g h l e v e l s of PIC induced more gre e n - s p o t t e d (52 Table 8. Response of c a l l i on media c o n t a i n i n g 2,4-D and BA recorded a f t e r three weeks of c u l t u r e ( c o n c e n t r a t i o n i n mg/1). Growth r e g u l a t o r 1 Medium 2 3 4 2,4-D 2 2 2 2 BA 2 2 4 4 C u l t i v a r Altaswede Norseman Altaswede Norseman NC 50 50 50 36 GSC 3 16 4 17 Morphoge-n e s i s 0 two l e a f - l i k e s t r u c t u r e s 0 0 ' Growth Rate ++ +++ ++ +++ NC=Number of C a l l i ; GSC=Green-Spotted C a l l i . ++, +++: More p l u s s i g n s i n d i c a t e f a s t e r r e l a t i v e c a l l u s growth. 63 Table 9. Response of 'Altaswede' c a l l i on media c o n t a i n i n g PIC and BA i n presence and absence of a r g i n i n e (1740 mg/1) (growth r e g u l a t o r c o n c e n t r a t i o n i n mg/1). Treatment PIC BA Arg NC RC SC GSC 1 0.001 0.2 + 14 0 0 0 2 0.0005 1 + 16 0 0 0 3 0.001 1 + 14 0 0 0 4 0.005 1 + 14 0 0 0 5 0.01 1 + 14 0 0 2 6 0.05 1 + 18 0 0 7 7 0.0005 1 - 20 0 0 1 8 0.001 1 - 21 0 0 1 9 0.005 1 - 20 0 0 2 10 0.01 1 - 22 0 0 1 1 1 0.05 1 - 20 0 0 2 NC=Number of C a l l i ; RC=Root C a l l i ; SC=Shoot C a l l i ; GSC=Green-Spotted C a l l i 64 c a l l i . T e st 2: The r e s u l t s presented i n Table 10 showed that adding glutamic a c i d and c a s e i n h y d r o l y s a t e t o the medium had no e f f e c t on shoot i n d u c t i o n , nor d i d i t i n c r e a s e root i n d u c t i o n . More green-spotted c a l l i appeared at low l e v e l s of c a s e i n h y d r o l y s a t e , but the s m a l l sample s i z e p r o h i b i t e d a c o n c l u s i v e i n t e r p r e t a t i o n . C a l l i growing on these media were browner than those on media without these a d d i t i v e s (Figure 4 ) . Those c a l l i of 'Norseman' on medium of 5,000 mg/1 c a s e i n h y d r o l y s a t e became b l a c k . The r e s u l t s were shown i n Tab l e 11. No shoots were induced u s i n g v a r i o u s combinations of PIC and BA. In c o n t r a s t to the r e s u l t s of Test 1, r o o t s formed at a l l combinations i n d i c a t i n g that c a l l u s source i s an important f a c t o r i n determining root f o r m a t i o n . More green-spotted c a l l i and r o o t s formed with 0.005 mg/1 PIC at both BA c o n c e n t r a t i o n s with one e x c e p t i o n i . e . 'Altaswede' c a l l i on L2(P0.005BO.2) medium. More ro o t e d c a l l i and green-spotted c a l l i were produced from 'Norseman'. Some c a l l i of 'Altaswede' from L2 were a l s o t r a n s f e r r e d onto L2(P0.002B0.2) and L2(P0.02B0.2) media. N e i t h e r shoots nor r o o t s were induced. 4.2.5 NAA and KIN Test 1: R e s u l t s (Table 12) showed that no shoots were induced when c a l l i from LSE were s u b c u l t u r e d onto media c o n t a i n i n g NAA and KIN. Roots and green-spotted c a l l i were 65 T a b l e 10. Response of c a l l i on media with added c a s e i n h y d r o l y s a t e and glutamic a c i d . 1 C u l t i v a r CH or GA (mg/1) NC RC SC Altaswede 100 CH 10 0 0 500 CH 1 1 0 0 1000 CH 10 0 0 5000 CH 10 0 0 147 GA 10 0 0 Norseman 100 CH 12 0 0 500 CH 12 0 0 . 1000 CH 12 0 0 5000 CH 10 0 0 147 GA 12 1 0 NC=Number of C a l l i ; RC=Root C a l l i ; SC=Shoot C a l l i ; CH=Casein H y t r o l y s a t e ; GA=Glutamic A c i d . 1A11 media contained 0.005mg/l PIC and 1 mg/1 BA. 66 F i g u r e 4. Response of 'Altaswede' c a l l i on L2 medium c o n t a i n i n g 0.005 mg/1 PIC, 1 mg/1 BA and 1 g/1 c a s e i n h y d r o l y s a t e a f t e r f i v e weeks of c u l t u r e . C a l l i showed s l i g h t l y brown c o l o r a t i o n . 67 T a b l e 11. Response of c a l l i of two c u l t i v a r s on media with v a r i o u s combinations of PIC and BA ( c o n c e n t r a t i o n i n mg/1). C u l t i v a r PIC BA NC RC ( % ) GSC (%) Altaswede 0. ,002 0. .2 1 4 4 (29) 8 (57) 0. .005 0. .2 14 2 (24) 9 (64) 0. ,01 0. .2 13 0 3 (20) 0. .002' 1 1 4 0 5 (36) 0. .005 1 14 4 (29) 1 1 (79) 0. .01 1 14 4 (29) 7 (50) Norseman 0. .002 0. .2 46 18 (39) 33 (72) 0. .005 0. ,2 44 21 (48) 36 (82) 0. .01 0. .2 44 8 (18) 36 (82) 0, .002 1 42 6 (14) 27 (64) 0. .005 1 42 7 (17) 33 (79) 0. .01 1 46 6 (13) 34 (74) NC=Number of C a l l i ; RC=Root C a l l i ; GSC=Green-Spotted C a l l i 68 Table 12. Response of 'Norseman' c a l l i from LSE medium to v a r i o u s combinations of NAA and KIN ( c o n c e n t r a t i o n i n mg/1). NAA KIN NC RC GSC 0.001 1 16 3 6 0.01 1 20 1 3 0.1 1 22 0 1 1.0 1 20 0 2 5.0 1 20 0 3 NC=Number of C a l l i ; RC=Root C a l l i ; GSC=Green-Spotted C a l l i 69 produced when t h e NAA c o n c e n t r a t i o n was low (0.001 and 0.01 mg/1). Test 2: Shoots were induced on two media i n t h i s e xperiment ( T a b l e 13). They o r i g i n a t e d from the upper p a r t of c a l l i . S e v e r a l s h o o t s a r o s e from a s i n g l e c a l l u s . Some sho o t s had w e l l - f o r m e d c o t y l e d o n s and r o o t s , s u g g e s t i n g t h a t they were from embryos ( F i g u r e 5 ) . Both t r e a t m e n t s which induced shoots c o n t a i n e d 0.05 mg/1 NAA. In c o n t r a s t , more r o o t e d c a l l i were produced when the NAA c o n c e n t r a t i o n was low (0.01 mg/1). Many r o o t s o r i g i n a t e d from one c a l l u s , the number r a n g i n g from s e v e r a l t o s e v e r a l dozens. Most of them o r i g i n a t e d from the t o p p a r t of c a l l u s . Few r o o t s o r i g i n a t e d from c a l l u s - m e d i u m i n t e r f a c e . A l l these c a l l i were y e l l o w - g r e e n i n c o l o r . T e s t 3: I t was shown (T a b l e 14) t h a t r o o t f o r m a t i o n was maximum when NAA c o n c e n t r a t i o n was 0.1-0.2 mg/1 a t bo t h KIN c o n c e n t r a t i o n s and minimum when the a u x i n r c y t o k i n i n r a t i o was h i g h e s t ( 1 . 0 ) . The maximum number of g r e e n - s p o t t e d c a l l i appeared a t 0.1 mg/1 NAA when KIN c o n c e n t r a t i o n was 0.5 mg/1, but a t 1.0 mg/1 NAA when KIN c o n c e n t r a t i o n was 2.0 mg/1. Fewer g r e e n - s p o t t e d c a l l i r e s u l t e d when bo t h NAA and KIN c o n c e n t r a t i o n s were low. The t h r e e t e s t s above were aimed a t s t u d y i n g the e f f e c t of NAA and KIN on c a l l u s growth and d i f f e r e n t i a t i o n . A l t h o u g h r o o t f o r m a t i o n o c c u r r e d i n a l l t e s t s , s h o o t s were in d u c e d o n l y i n one c a s e . T h i s s u g g e s t s t h a t : (1) embryogenesis had been t r i g g e r e d on SCP medium b e f o r e c a l l i 7 0 Table 1 3 . Response of c a l l i from SCP medium to combinations of NAA and KIN ( c o n c e n t r a t i o n i n mg/1) NAA KIN NC s c RC 0 . 0 1 0 . 5 2 0 0 15 0 . 0 5 0 . 5 2 0 2 ( 6 , 4 ) 1 1 1 0 . 1 0 . 5 2 2 0 13 0 . 0 5 2 . 0 2 0 1 ( 2 5 ) 8 1Numbers i n the bracket are the number of shoots f o r each c a l l u s . NC=Number of C a l l i ; SC=Shoot C a l l i ; RC=Root C a l l i 71 F i g u r e 5. Shoot f o r m a t i o n from 'Altaswede' c a l l u s on L2(N0.05K2) medium. C a l l u s had been c u l t u r e d on SCP medium. Shoots o r i g i n a t e d from t h e upper p a r t of the c a l l u s , w i t h some h a v i n g c o t y l e d o n s and r o o t s . 72 Table 14. Response of 'Norseman' c a l l i t o combinations of NAA and KIN ( c o n c e n t r a t i o n i n mg/1). NAA (mg/1) KIN (mg/1) NC RC (%) GSC (%) 0.01 0.5 48 8 (16.7) 6 (12.5) 0.05 0.5 44 8 (18.2) 8 (18.2) 0.1 0.5 46 11 (23.9) 10 (21.7) 0.5 0.5 46 6 (13.0) 7 (15.2) 0.2 2.0 50 11 (22.0) 20 (40.0) 1 .0 2.0 48 10 (20.8) 25 (52.1) NC=Number of C a l l i ; RC =Root C a l l i ; GSC=Green-Spotted C a l l i 1 73 were t r a n s f e r r e d so that shoots o n l y come from c a l l i which were p r e v i o u s l y c u l t u r e d on SCP medium and (2) c o n c e n t r a t i o n of NAA i s c r i t i c a l because shoots were induced only on media c o n t a i n i n g 0.05 mg/1 NAA. In Test 1 more roo t s were produced when NAA was at 0.001 mg /1 , but i n Test 3 more r o o t s were r e s u l t e d at 0.1-0.2 mg/1 NAA at both KIN c o n c e n t r a t i o n s . Although l i g h t c o n d i t i o n s c o u l d c o n t r i b u t e to the observed d i f f e r e n c e , the most important f a c t o r i s c a l l u s source. C a l l i used f o r Test 3 had been c u l t u r e d on medium with high l e v e l of 2,4-D compared with those used i n Test 1. Thus, d u r i n g the subsequent s u b c u l t u r e more rooted c a l l i were produced at higher l e v e l s of auxin i n Test 3 than i n Test 1. I t was a l s o apparent that NAA was more c r i t i c a l to root formation than KIN. 4.2.6 NAA and BA i n Presence of ADE A f t e r one month of c u l t u r e no shoots were induced on the four media t e s t e d (Table 15). Two rooted c a l l i were produced on medium without NAA but c o n t a i n i n g 0.2 mg/1 BA. T h i s r e s u l t c o n t r a s t s with the accepted view that root formation i s f a c i l i t a t e d by hi g h auxin to c y t o k i n i n r a t i o s . S l i g h t l y more green-spotted c a l l i were observed at hig h l e v e l s of NAA. 74 Table 15. Response of 'Norseman' c a l l i on media c o n t a i n i n g v a r i o u s l e v e l s of NAA i n combination with BA and ADE 1 ( c o n c e n t r a t i o n i n mg/1). NAA BA NC RC GSC 0 0.2 22 2 3 0.001 0.2 22 0 5 0.01 0.2 22 0 5 0. 1 0.2 22 0 8 1 A l l the media c o n t a i n i n g 2 mg/1 ADE. NC=Number of C a l l i ; RC=Root C a l l i ; GSC=Green -Spotted C a l l i 75 4.2.7 C a l l u s D i f f e r e n t i a t i o n on Four Media As shown i n Table 16, none of these four media was ab l e to induce shoots from c a l l i . Although only ten c a l l i were c u l t u r e d f o r each treatment, the t r e n d was obvious that root formation was i n h i b i t e d by the presence of auxins, the extent of i n h i b i t i o n was r e l a t e d to auxin c o n c e n t r a t i o n and root formation was enhanced by BA and ADE. T h i s o b s e r v a t i o n c o i n c i d e s with the r e s u l t o b t a i n e d i n the t e s t s on NAA and BA ( S e c t i o n 4.2.6). Medium without any added auxin but with BA and ADE present had more ro o t e d c a l l i and the maximum average root number per rooted c a l l u s , while the medium (medium 4) which was s i m i l a r t o t h a t used f o r root formation from shoots was l e s s e f f i c i e n t f o r root i n d u c t i o n from c a l l u s . 4.2.8 C a l l u s Growth and Auxin t o C y t o k i n i n R a t i o R e s u l t s of t h i s t e s t are presented i n Table 17. C a l l i on L2(D11K1) medium were grey t o dark-grey i n c o l o r , f r i a b l e and showed no d i f f e r e n t i a t i o n ( F i g u r e 6). On L2(D1K10) medium there were b a s i c a l l y two c a l l u s forms. One form of c a l l u s was f a s t growing and green. Another form of c a l l u s was brown-yellow i n c o l o r . Among 190 c a l l i c u l t u r e d , 134 were green c a l l i . These green c a l l i , u n l i k e normal g r e e n - s p o t t e d c a l l i which had i s o l a t e d green r e g i o n s , were u n i f o r m l y green because of the hi g h KIN content i n the medium. The c o n t r a s t i n g auxin to c y t o k i n i n r a t i o s of the two media produced a d i f f e r e n t c a l l u s morphology. 76 Table 16. D i f f e r e n t i a t i o n of 'Norseman' c a l l i from L2(D2B2A5) mg/1) medium on four media ( c o n c e n t r a t i o n Medium 1 composition 2 3 4 1 L2 SALTS f u l l f u l l f u l l h a l f 2,4-D 0.01 NAA 0.1 BA 0.2 0.2 0.2 ADE 2 2 2 IAA 0.2 N i c o t i n i c ac i d 1.0 Re s u l t NC 1 0 10 10 10 RC 7 3 3 2 Average Root No. 2 4 4 3 1 1 L2 medium with h a l f s t r e n g t h of s a l t s and v i t a m i n s . 2 Based on the number of root c a l l i . NC=Number of C a l l i ; RC=Root C a l l i . 77 Table 17. Response of 'Norseman' c a l l i grown on media of h i g h (11) or low (0.1) auxin to c y t o k i n i n r a t i o . L2(D11K1) L2(D1K10) NC 190 190 Color white to grey (1)brown-yellow (2 )green-yellow RC none l e s s than 10 NC=Number of C a l l i ; RC=Root C a l l i 78 F i g u r e 6. Response of 'Norseman' c a l l i on media with d i f f e r e n t auxin to c y t o k i n i n r a t i o s a f t e r f i v e weeks of c u l t u r e , (a) C a l l i on L2(D11K1) medium; very f r i a b l e and grey i n c o l o r (upper), (b) C a l l i on L2(D1K10) medium (l o w e r ) . Two forms of c a l l i r e s u l t e d . The m a j o r i t y of c a l l i were the green-type ( l e f t p e t r i p l a t e ) . 79 Roots were on l y formed on the medium with low auxin to c y t o k i n i n r a t i o . T h i s phenomenon, observed i n t e s t s with NAA and BA ( S e c t i o n 4.2.6) and the comparison of four media ( S e c t i o n 4.2.7), was once again confirmed here. 4.2.9 C a l l u s Growth on SCP Medium Recovery of Senescing C a l l u s Recovery frequency of senescing c a l l i to l a r g e green-spotted c a l l i i s shown i n Table 18. These regenerated c a l l i were fast-g r o w i n g and had i s o l a t e d green r e g i o n s . They were bigger than one centimeter i n diameter ( F i g u r e 7) were most commonly seen on SCP medium ( s o - c a l l e d SCP c a l l u s ) . I t i s c l e a r from these r e s u l t s that SCP medium was e f f i c i e n t i n r e s t o r i n g s e n e s c i n g c a l l i and that the recovery frequency decreased w i t h the l e n g t h of c u l t u r e p e r i o d on LSE medium. Recovery was a l s o e f f i c i e n t when a d i f f e r e n t c a l l u s source was used. Maintenance of C a l l u s Most c a l l i on SCP medium grew very f a s t . Many became l a r g e c a l l i with diameter g r e a t e r than one centimeter w i t h i n one c u l t u r e p e r i o d (3-4 weeks) ( F i g u r e 8). They were green-spotted and many of them had nodular s t r u c t u r e s . In a t e s t of three media, L2(D2N2K2), L2(D2B0.5A2) and L2(P0.002B2) were compared. On L2(D2B0.5A2) medium, which was s i m i l a r to SCP, c a l l i grew f a s t e r than c a l l i on the other two media and more green-spotted c a l l i were produced. 80 Table 18. Recovery frequency of senescing c a l l i a f t e r t r a n s f e r to SCP medium. C a l l u s source 1 2 3 4 5 NC 40 48 120 72 144 No. of SCP c a l l i 5 12 42 48 61 Frequency of SCP c a l l i (%) 12.5 25.0 35.0 66.7 43.3 NC=Number of C a l l i 81 F i g u r e 7. Recovery of senescing c a l l i on SCP medium. Before t r a n s f e r , these c a l l i were darkened and brown i n c o l o r and had stopped growing. The c a l l i i l l u s t r a t e d are a f t e r f i v e weeks c u l t u r e . 82 F i g u r e 8. C a l l u s maintenance on SCP medium. Rapid growth on t h i s medium produced c a l l i with diameters g r e a t e r than one centimeter w i t h i n three t o fo u r weeks, (a) 'Altaswede' c a l l i (upper); (b) 'Norseman' c a l l i ( l o wer). 83 C a l l i were e a s i l y maintained on SCP medium by s u b c u l t u r i n g . Some c a l l i which were o r i g i n a l l y c u l t u r e d on LSE medium were t r a n s f e r r e d onto SCP on October 7, 1984 and maintained on SCP to J u l y 10, 1985 by s u b c u l t u r i n g them only twice (December 9, 1984 and February 9, 1985) C a l l i c o u l d be maintained on SCP without s u b c u l t u r e f o r many months. C a l l i from each of two media: SCP and L2(P0.001B0.1) were t r a n s f e r r e d onto SCP on February 9, 1985 and incubated i n a growth chamber. When checked on J u l y 10, they were very l a r g e . Although p a r t s of the c a l l u s were l e s s v igorous and not f r e s h i n c o l o r , the p e r i p h e r a l r e g i o n s were green-yellow and a c t i v e l y growing. Embryogenesis Embryogenesis can occur from c a l l i c u l t u r e d on SCP medium. T h i s o b s e r v a t i o n was confirmed by m i c r o s c o p i c examination. C a l l u s on t h i s medium c o n t a i n e d c e l l c l u s t e r s and i n some cases embryos at the globe and heart s t a g e s . Although these embryos d i d not develop f u r t h e r , they i n c r e a s e d i n s i z e , forming l a r g e d i s o r g a n i z e d embryo-like s t r u c t u r e s . T h i s f a c t suggests t h a t the hormone c o n c e n t r a t i o n i n t h i s medium was too hig h f o r embryos to complete development. C a l l i w i t h embryos induced on SCP medium c o u l d be t r a n s f e r r e d to another medium, e v e n t u a l l y l e a d i n g to p l a n t l e t r e g e n e r a t i o n . 84 4.2.10 Light E f f e c t on C a l l i Growing on SCP Medium The r e s u l t s of t h i s t e s t are shown i n Table 19. Because c a l l i had been c u l t u r e d on LSE medium p r i o r to t r a n s f e r and were not v i g o r o u s , the frequency of the t y p i c a l SCP c a l l u s on SCP medium was low, but t h i s frequency i n c r e a s e d with l i g h t i n t e n s i t y . I t was a l s o obvious from the r e s u l t s that l i g h t promoted root d i f f e r e n t i a t i o n from these c a l l i . However, l i g h t q u a l i t y which was not measured in t h i s experiment c o u l d a l s o have some e f f e c t on the r e s u l t s . 4.2.11 Observations on Embryogenesis Embryogenesis i n c u l t u r e l i k e l y occurs v i a the sequence: s i n g l e c e l l ( ? ) > c e l l c l u s t e r > globe stage > heart stage > torpedo stage. Under a l i g h t microscope many c e l l c l u s t e r s c o u l d be seen. These c e l l s were small and dense i n cytoplasm ( F i g u r e 9). They were e a s i l y d i s t i n g u i s h a b l e from the l a r g e r c e l l s u s u a l l y encountered i n c u l t u r e s . At the globe stage, the c e l l c l u s t e r was w e l l shaped and became smooth at p e r i p h e r y . On SCP medium some c a l l i were found to have embryos developed, mainly at the globe and e a r l y heart stages. These embryos were not well-shaped and d i d not develop f u r t h e r although they c o u l d grow l a r g e r i n s i z e t o become nodular s t r u c t u r e s . Upon t r a n s f e r of these embryogenic c a l l i onto other media some of the embryos developed normally to the torpedo 85 Table 19. L i g h t e f f e c t on growth and d i f f e r e n t i a t i o n of 'Altaswede' c a l l i on SCP medium. I n t e n s i t y No. of T y p i c a l Rooted of l i g h t c a l l i SCP c a l l i c a l l i (/uEirr 2 s e c " 1 ) 70 37 2 0 80 49 1 1 0 90 53 18 0 1 00 46 7 0 1 20 51 1 2 0 150 52 15 1 190 51 1 5 1 230 48 8 1 280 50 22 5 450 48 23 8 86 F i g u r e 9. Embryogenic c a l l u s of 'Altaswede'. C a l l u s was c u l t u r e d on L2(P0.001B0.1) medium. C e l l c l u s t e r s c o n t a i n c e l l s which are smal l and dense i n cytoplasm. C a l l u s shown was u n s t a i n e d (mag. X38). 87 stage and e v e n t u l l y to p l a n t l e t s ( Figure 1 0 ) . P l a n t l e t s formed v i a embryogenesis developed m o r p h o l o g i c a l l y l i k e s e e d l i n g s d e r i v e d from seeds. Cotyledons were f i r s t o b s e r v a b l e , f o l l o w e d by development of the f i r s t l e a f c o n s i s t i n g of a s i n g l e l e a f l e t . 4 .2 .12 S e l e c t i o n and Maintenance of Embryogenic C a l l i R e s u l t s are shown i n Table 20 f o r one s u b c u l t u r e of embryogenic c a l l i from LSP medium to LSP medium. Embryos were observed at d i f f e r e n t developmental s t a g e s . Those embryos which had been d e v e l o p i n g normally d u r i n g the c u l t u r e p e r i o d s developed i n t o p l a n t l e t s . F o r t y to f i f t y p e rcent of c u l t u r e s showed p l a n t s developed, with 20% of tubes having more than two p l a n t l e t s ( Figure 1 1 ) . In some c u l t u r e s many embryos a t the torpedo stage were observed growing at the s u r f a c e of c a l l u s . There were a few tubes where no p l a n t l e t or torpedo embryos were observed. However embryos at e a r l i e r stages were observed which c o u l d develop normally a f t e r subsequent t r a n s f e r . In a d d i t i o n , there were a few tubes (approximately 10%) i n which c a l l i were d i s o r g a n i z e d or had an unhealthy c o l o r . Embryogenic c a l l i had been maintained i n c u l t u r e f o r over one year without l o s s i n g the c a p a c i t y of r e g e n e r a t i n g p l a n t s . When shoots o r i g i n a t e d from such c a l l i were c u l t u r e on rooting-medium, p l a n t l e t s formed and i n a few cases embryogenic c a l l u s (with obvious embryos) was produced near 88 F i g u r e 10. Somatic embryo development from embryogenic c a l l u s of 'Altaswede'. C a l l i were c u l t u r e d on L2(PO.001 BO.1) medium. Embryos were observed by scanning e l e c t r o n mcroscopy. (a) An embryo present i n the c e n t e r . Two c o t y l e d o n s are observable (upper, mag. x43). (b) F u r t h e r development of an embryo (lower, mag. X52) 89 Table 20. Embryo development and p l a n t l e t formation a f t e r s u b c u l t u r e of embryogenic c a l l i onto LSP medium. C u l t u r e No 1 1 2 No of tubes 84 99 Tubes with p l a n t l e t development 35 51 1 - p l a n t l e t tube 12 15 2 - p l a n t l e t tube 5 1 6 3-10 p l a n t l e t tube 12 17 >10 p l a n t l e t tube 5 3 Tubes with embryos at torpedo Stage 41 41 a few embryos 24 20 many embryos ^5 17 21 Tubes with c a l l u s only (embryos e a r l i e r than torpedo stage) 8 7 yel l o w green c a l l u s 2 4 d i s o r g a n i z e d embryos 6 2 brown or dark c a l l u s 0 1 1 These were two batches of c u l t u r e s from the embryogenic c a l l u s . 90 F i g u r e 11. S e l e c t i o n of c a l l u s c a p a b l e of em b r y o g e n e s i s . Embryogenic c a l l i of 'Altaswede' s u b c u l t u r e d on L2 medium c o n t a i n i n g 0.001-0.002 mg/1 PIC and 0.2 mg/1 BA produced embryos c o n t i n u o u s l y and showed p l a n t l e t development. 91 the p l a c e where r o o t s were i n t i a t e d ( F igure 12). 4.2.13 Growth of Embryogenic and Non-embryogenic C a l l i Embryognic c a l l i and non-embryogenic c a l l i were m o r p h o l o g i c a l l y d i f f e r e n t . Embryogenic c a l l i had f i n e g r a i n - l i k e s t r u c t u r e s (embryos). These s t r u c t u r e s were very easy to separate from each o t h e r . Non-embryogenic c a l l i , a lthough they v a r i e d i n t e x t u r e from f r i a b l e to compact, do not have these s t r u c t u r e s . Under a l i g h t microscope, embryogenic c a l l i had many v i s i b l e embryos, mostly at the globe and heart stages. When measured a f t e r one month of c u l t u r e , embryogenic c a l l i , compared with non-embyrogenic c a l l i , grew f a s t e r , had f r e s h yellow-green c o l o r and remained a l i v e f o r a longer time ( F i g u r e 13). A l l treatments showed the same tendency. Non-embryogenic c a l l i turned brown and showed l i t t l e or no growth. Embryo-like s t r u c t u r e s were present i n embryogenic c u l t u r e s , although they became l a r g e i n s i z e i n treatments (6) and ( 8 ) . At t h i s time, r o o t s formed on embryogenic c a l l i i n treatments (2) and ( 8 ) . Cotyledons were a l s o observed on the embryogenic c a l l i of treatment ( 8 ) . A f t e r three months without s u b c u l t u r e a l l non-embryogenic c a l l i were dead, whereas most of the embryogenic c a l l i were s t i l l a l i v e . 92 F i g u r e 12. Embryogenic c a l l u s a r i s i n g from regenerated p l a n t l e t at the p o i n t of root o r i g i n . 93 F i g u r e 13. E m b r y o g e n i c and n o n - e m b r y o g e n i c c a l l i o f ' A l t a s w e d e ' . From l e f t t o r i g h t , T u b e s 1 and 2 c o n t a i n L2 medium w i t h 0.05 mg/1 NAA and 0.1 mg/1 BA. Tubes 3 and 4 c o n t a i n L2 medium w i t h 2 g/1 y e a s t e x t r a c t and 10 g/1 a g a r . T u b e s 1 and 3 had e m b r y o g e n i c c a l l i w h i l e T u b e s 2 and 4, n o n - e m b r y o g e n i c c a l l i . C a l l i had been c u l t u r e d on t h e s e media f o r f o r t y d a y s . 94 4.2.14 Summary of Experiments on Callus Growth and D i f f e r e n t i a t i o n In t h i s s e r i e s of experiments c a l l u s d i f f e r e n t i a t i o n was e v a l u a t e d on v a r i o u s media. Some c o n s i s t e n t c o n c l u s i o n s may be made from these r e s u l t s although the responses v a r i e d with i n d i v i d u a l experiments. (1) There were d i f f e r e n c e s i n c a l l u s growth and d i f f e r e n t i a t i o n due to c u l t i v a r . U s u a l l y 'Norseman' c a l l i grew f a s t e r than 'Altaswede' c a l l i and a l s o produced more green-spotted c a l l i . Shoots were induced only from 'Altaswede' c a l l i , but more c a l l i with r o o t s were u s u a l l y formed from 'Norseman'. ( 2 ) More r o o t s were formed from c a l l i c u l t u r e d at a low auxin t o c y t o k i n i n r a t i o or i n the absence of a u x i n s . T h i s r e l a t i o n s h i p was apparent i n experiments u s i n g PIC and BA (PIC at 0.0005 and 0.001 mg/1) and on NAA and BA (without NAA) and i n the experiment s p e c i f i c a l l y t e s t i n g the auxin to c y t o k i n i n r a t i o (more r o o t s at r a t i o equal to 0.1). A t y p i c a l example of t h i s r e l a t i o n s h i p was observed i n the experiment i n which four media were compared. More c a l l i produced r o o t s and g r e a t e r numbers of r o o t s per c a l l u s on the medium without auxins than on any one of the other three media with low or moderate l e v e l s of 2,4-D, NAA or IAA. (3) C a l l u s source i s an important f a c t e r d etermining the r e s u l t . When non-vigorous c a l l i were used as the source, l e s s growth and d i f f e r e n t i a t i o n was observed. 95 In the experiment on PIC and BA, Test 1 showed no root d i f f e r e n t i a t i o n and no obvious d i f f e r e n c e between d i f f e r e n t PIC and BA combinations. When other c a l l u s sources were used i n Test 3 root formation occurred i n a l l treatments and d i f f e r e n c e s between treatments were e v i d e n t . (4) Shoots were induced a f t e r c a l l i of 'Altaswede' c u l t u r e d on SCP medium were t r a n s f e r r e d onto medium c o n t a i n i n g NAA (0.05 mg/1) and KIN with NAA. (5) NAA c o n c e n t r a t i o n was more important than that of KIN i n root or shoot i n d u c t i o n , as demonstrated i n the experiment on NAA and KIN. (6) (6) A d d i t i o n s of amino a c i d s (glutamic a c i d and a r g i n i n e ) and c a s e i n h y d r o l y s a t e d i d not show any obvious promotive e f f e c t on c a l l u s growth compared with c a l l i on media without these a d d i t i v e s . 4.2.15 P l a n t Regeneration from C a l l u s P l a n t r e g e n e r a t i o n from c a l l u s was achieved i n s e v e r a l cases. In a study of c a l l u s c u l t u r e on the f o l l o w i n g media: (1) L2(P0.002B0.2), (2) L 2 ( h a l f s t r e n g t h s a l t s , P0.002B0.2) and (3) L2(NO.05K0.5), one shoot was formed on the L2(N0.05K0.5) medium. T h i s o b s e r v a t i o n was the f i r s t i n d i c a t i o n of any p l a n t r e g e n e r a t i o n from c a l l u s i n t h i s study. Another experiment using NAA and KIN was t h e r e f o r e i n i t i a t e d , l e a d i n g to r e g e n e r a t i o n of some p l a n t s from c a l l i (see S e c t i o n 4.2.5). C a l l i capable of embryogenesis were 96 s u b c u l t u r e d onto LSP medium. Embryos were produced c o n t i n u o u s l y , g i v i n g r i s e t o a number of p l a n t s (see S e c t i o n 4.2.13). 4.3 Shoot T i p C u l t u r e 4.3.1 M u l t i p l e Shoot Induction R e s u l t s of m u l t i p l e shoot i n d u c t i o n and s t a t i s t i c a l a n a l y s i s are presented i n Table 21 and 22. The average number of m u l t i p l e shoots was based on the number of shoot t i p s producing m u l t i p l e shoots. The m u l t i p l e shoot i n d u c t i o n r a t e of 'Norseman' was higher than that of 'Altaswede'. However, when the shoot t i p c o u l d be induced to produce any m u l t i p l e shoots, 'Altaswede' produced more m u l t i p l e shoots per shoot t i p than 'Norseman' ( F i g u r e 14). 4.3.2 M u l t i p l e Shoot Propagation As shown i n Table 23 'Altaswede' not o n l y had more shoots i n the i n d u c t i o n c u l t u r e , but a l s o i n the two s u b c u l t u r e s . M u l t i p l e shoots u s u a l l y arose from a shoot r o s e t t e with c a l l u s at i t s base and green hard t i s s u e at the c e n t e r . Once the m u l t i p l e shoot r o s e t t e was e s t a b l i s h e d a d d i t i o n a l m u l t i p l e shoots were e a s i l y produced by d i v i d i n g the r o s e t t e i n t o s e v e r a l p i e c e s and s u b c u l t u r i n g each p i e c e . Rapid propagation of m u l t i p l e shoots can be accomplished t h i s way i n a short time p e r i o d . 97 Table 21. M u l t i p l e shoot i n d u c t i o n from shoot t i p s of red c l o v e r on L2(PO.003B2) medium. C u l t i v a r R e p l i c a t e No of shoot t i p s c u l t u r e d No of with t i p s MS Average Ni of MS1 Altaswede 1 30 9 4.8 2 30 1 2 4.8 3 30 1 1 4.5 4 30 9 4.7 5 30 9 5.0 T o t a l 1 50 50 Norseman 1 30 17 3.9 2 30 18 3.6 3 30 18 3.9 4 30 15 3.9 T o t a l 120 68 1 MS = M u l t i p l e Shoots Table 22. Comparison of two c u l t i v a r s of red c l o v e r f o r m u l t i p l e shoot i n d u c t i o n . C u l t i v a r Altaswede Norseman No of tubes With MS1 10 B 2 17 A Average No of shoots 4.8 A 3.8 B 1 MS = M u l t i p l e Shoots t r e a t m e n t s followed by d i f f e r e n t c a p i t a l l e t t e r s are s i g n i f i c a n t l y d i f f e r e n t at P^0.01. 98 e 14. M u l t i p l e shoot production i n red c l o v e r . A s i n g l e i s o l a t e d shoot was c u l t u r e d on L2(P0.003B1) medium and showed m u l t i p l e shoot p r o l i f e r a t i o n . 'Altaswede' ( r i g h t ) produced more, but sma l l e r shoots than 'Norseman' ( l e f t ) . 99 Table 23. M u l t i p l e shoot p r o d u c t i o n by two. c u l t i v a r s of red c l o v e r . M u l t i p l e shoot were induced on L2(PO.003B2) medium. They were s u b c u l t u r e d on media c o n t a i n i n g 0.002-0.003 mg/1 PIC and 1 mg/1 BA f o r propagation at a s u b c u l t u r e of one month. C u l t u r e M u l t i p l e shoot No / tube Altaswede Norseman Induction c u l t u r e 4.8 A 1 3.8 B F i r s t s u b c u l t u r e 10.8 a 6.1 b Second s u b c u l t u r e 15.2 A 8.1 B t r e a t m e n t s are s i g n i f i c a n t l y d i f f e r e n t at P^0.01 when followed by d i f f e r e n t c a p i t a l l e t t e r s or at P£0.05 when followed by d i f f e r e n t small l e t t e r s . 100 4.4 Growth and A n a l y s i s of Regenerated P l a n t s During t h i s study, p l a n t s were regenerated through s e v e r a l d i f f e r e n t t i s s u e c u l t u r e pathways. To s i m p l i f y the d i s c u s s i o n , the types of regenerants are d e s c r i b e d as f o l l o w s and given an a p p r o p r i a t e a b b r e v i a t e d d e s i g n a t i o n : (1) RG1 p l a n t s : those regenerated from m u l t i p l e shoots induced on m u l t i p l e shoot i n d u c t i o n medium (L2(PO.003B2)) without s u b c u l t u r e of the m u l t i p l e shoots; (2) RG2 p l a n t s : these regenerated from m u l t i p l e shoots which were d e r i v e d by s u b c u l t u r i n g the o r i g i n a l m u l t i p l e shoots twice on L2 medium c o n t a i n i n g 0.002-0.003 mg/1 PIC and 1 mg/1 BA. (3) RG3 p l a n t s : p l a n t s regenerated from m u l t i p l i c a t i o n of c a l l u s - d e r i v e d shoots. Shoots were f i r s t regenerated from 'Altaswede' c a l l i on L2(N0.05K0.5) and and L2(N0.05K2) media. C a l l i had been i n c u l t u r e f o r three months by that time. These shoots were then m u l t i p l i e d twice by shoot t i p c u l t u r e ; (4) RG4 p l a n t s : p l a n t s regenerated d i r e c t l y from embryogenic c a l l i , which were d e r i v e d by s e l e c t i n g one 'Altaswede' c a l l u s l i n e and s u b c u l t u r i n g . Before p l a n t r e g e n e r a t i o n , c a l l i had been maintained i n c u l t u r e f o r n e a r l y one year. P l a n t s of a l l types were d e r i v e d from 'Altaswede' while regenerants of 'Norseman' were only a v a i l a b l e as RG1 and RG2 p l a n t s . 101 4.4.1 P l a n t T r a n s f e r and S u r v i v a l The ultimate objective of the tissue culture cycle must be to produce vigorous regenerated plants. It i s important, therefore, to ensure a high rate of survival when pl a n t l e t s are transferred out of i_n v i t r o conditions. The t r a n s i t i o n from the test tube to greenhouse i s a s i g n i f i c a n t , environmental change for the p l a n t l e t s . Appropriate conditions are required to f a c i l i t a t e plant adaptation to the new environment and that conditions be changed gradually. In the experiment testing the effect of d i f f e r e n t treatments on plant s u r v i v a l , plants incubated in the laboratory for the f i r s t week were exposed to a r e l a t i v e l y low l i g h t intensity (120 i u E m~ 2sec~ 1) and high humidity. Plants were weak and many died in the subsequent transfer to the greenhouse. As a consequence, the sur v i v a l rate was low. When plants were transferred to the greenhouse without Saran wrap protection some plants died only a few days after the transfer. Dessication in the lower humidity environment was a contributing factor in the low survival rate (39%) (Table 24). The plants which were covered with Saran wrap for two weeks in the greenhouse had the highest survival rate (94%). In the present study five batches of regenerated plants were transferred. During these transfers two other observations were made regarding the conditions which supported successful plant transfer. (1) Maintaining shoots 1 02 Table 24. S u r v i v a l r a t e of p l a n t s t r a n s f e r r e d to greenhouse a f t e r d i f f e r e n t treatments Treatment L a b o r a t o r y / Green house Green house green house +Saran wrap -Saran wrap P l a n t s 112 224 28 t r a n s f e r r e d P l a n t s 60 211 11 s u r v i v e d S u r v i v a l r a t e (%) 54 94 39 103 in r o o t i n g medium f o r more than one month p r i o r to t r a n s p l a n t a t i o n appeared to c o n t r i b u t e to b e t t e r s u r v i v a l . It might be a n t i c i p a t e d t h a t d u r i n g t h i s extended p e r i o d , humidity w i t h i n the c u l t u r e tubes was somewhat lowered and p l a n t s were much b i g g e r . Such p l a n t s were more r e s i s t a n t to w i l t i n g . (2) In February, n a t u r a l l i g h t i n t e n s i t y i s moderate and p l a n t s can be t r a n s f e r r e d to the greenhouse l i g h t c o n d i t i o n s d i r e c t l y . When l i g h t i n t e n s i t y i s very high ( i . e . d u r i n g summer days) a p e r i o d of maintenance at reduced l i g h t i n t e n s i t y should precede exposure to the f u l l s t r e n g t h of s u n l i g h t . During the t r a n s f e r of RG4 p l a n t s of 'Altaswede' on June 17, both l i g h t i n t e n s i t y and temperature in the greenhouse were h i g h . L i g h t i n t e n s i t y was reduced using a screen of black p o l y e t h y l e n e f i l m on a frame above the p l a n t s . Of the 130 p l a n t s t r a n s f e r r e d more than 95% s u r v i v e d . 4.4.2 Chromosome Number S t a b i l i t y RG1, RG2, RG3 and RG4 p l a n t s of 'Altaswede' were examined f o r t h e i r chromosome number. The normal karyotype of 'Altaswede' i s 2n=2X=14. Root t i p squashes of seed-derived p l a n t s were u t i l i z e d as c o n t r o l s . The data are summarized i n Table 25. A l l the c o n t r o l p l a n t s showed normal chromosome numbers (F i g u r e 15). Of 45 RG1 p l a n t s examined no abnormal chromosome number was observed while one d i p l o i d / t e t r a p l o i d mosaic p l a n t was d e t e c t e d among 28 RG2 p l a n t s . For a l l the 170 RG3 p l a n t s no v a r i a t i o n was 104 Table 25. Chromosome numbers of 'Altaswede' p l a n t s regenerated v i a d i f f e r e n t t i s s u e c u l t u r e pathways. P o p u l a t i o n Number of p l a n t s with abnormal t e t r a p l o i d surveyed chromosome t e t r a p l o i d / d i p l o i d number mosaic RG1 45 0 0 0 RG2 28 1 0 1 RG3 170 0 0 0 RG4 119 27 23 4 C o n t r o l 26 0 0 0 F i g u r e 15. Karyotype of normal root t i p of red c l o v e r cv. 'Altaswede' (2n=2X=14) (mag. X1570). 106 found. In c o n t r a s t 27 out of 119 RG4 'Altaswede' p l a n t s had t e t r a p l o i d chromosome numbers ( 2 8 ) . Most (23) of t h e s e 27 p l a n t s w i t h a l t e r e d chromosome number were t e t r a p l o i d s , which were l i k e l y produced t h r o u g h chromosome d o u b l i n g d u r i n g the c a l l u s c u l t u r e s t a g e . Only f o u r p l a n t s were d i p l o i d / t e t r a p l o i d mosaics ( F i g u r e 16). These r e s u l t s s u p p o r t e d the s i n g l e c e l l o r i g i n of most embryos, from which t h e s e p l a n t s were r e g e n e r a t e d . The f o u r mosaic p l a n t s c o u l d come e i t h e r from embryos which had o r i g i n a t e d from more than one c e l l o r i g i n or from embryos which had some c e l l ( s ) undergo changes i n chromosome number d u r i n g embryogenesis. I t was a l s o demonstrated t h a t p l a n t s r e g e n e r a t e d from s h o r t - t e r m c u l t u r e s ( e i t h e r m u l t i p l e shoot c u l t u r e s or c a l l u s c u l t u r e s ) tended t o have a normal chromosome number, w h i l e chromosome a b n o r m a l i t i e s c o u l d happen t o the r e g e n e r a t e d p l a n t s from c u l t u r e s w i t h a p r o l o n g e d c u l t u r e p e r i o d . Another i n t e r e s t i n g a s p e c t of the o b s e r v a t i o n i s t h a t no a n e u p l o i d chromosome numbers were o b s e r v e d . 4.4.3 Isozyme A n a l y s i s Isozymes a r e a t t r a c t i v e f o r d i r e c t g e n e t i c study because they r e p r e s e n t , t h e p r i m a r y p r o d u c t s of s t r u c t u r a l genes. Changes i n c o d i n g sequences w i l l i n many ca s e s l e a d t o q u a n t i t a t i v e or q u a l i t a t i v e changes i n isozyme p a t t e r n s . RG3 and RG4 p l a n t s of 'Altaswede' were a n a l y s e d f o r t h e i r isozyme p a t t e r n s of f i v e enzymes. Comparisons were made t o the isozyme p a t t e r n s from s e e d - d e r i v e d p l a n t s . 107 F i g u r e 16. Root t i p chromosome numbers of two RG4 p l a n t s of 'Altaswede' (mag. X1570). (a) 2n=2x=14 (upper); (b) 2n=4x=28 ( l o w e r ) . 108 R e s u l t s showed t h a t v a r i a t i o n e x i s t e d i n t h e i s o z y m e p a t t e r n s o f a l l t h e enzymes b u t one i n s e e d - d e r i v e d p l a n t s . T h i s o b s e r v a t i o n i s not u n u s u a l f o r an o u t - c r o s s i n g s p e c i e s . I n o r d e r t o d e t e r m i n e w h e t h e r v a r i a t i o n o c c u r r e d i n t h e r e g e n e r a t e d p l a n t s i t i s i m p o r t a n t t o know t h e o r i g i n a l i s o z y m e p a t t e r n s o f t h e p l a n t s f r o m w h i c h c a l l i were i n d u c e d and p l a n t s were r e g e n e r a t e d . B e c a u s e a l l t h e RG3 p l a n t s were f r o m t h r e e c a l l i t h e number of e x p e c t e d i s o z y m e p a t t e r n s s h o u l d n o t e x c e e d t h r e e even i f e a c h c a l l u s i s d i f f e r e n t . F o u r o r more p a t t e r n s f o r one enzyme w i l l i n d i c a t e t h e o c c u r r e n c e o f a n o n - p a r e n t a l t y p e . S i m i l a r l y , t h e o v e r a l l i s ozyme p a t t e r n s f o r a l l t h e f i v e enzymes s h o u l d n o t e x c e e d t h r e e . I f two g e n o t y p e s w h i c h d i f f e r f r o m e a c h o t h e r i n isozyme p a t t e r n s o f two enzymes a r e u s e d a s t h e s t a r t i n g m a t e r i a l s , t h e p l a n t s r e g e n e r a t e d f r o m t h e s e two g e n o t y p e s s h o u l d a l w a y s d i f f e r i n t h e two i s o z y m e p a t t e r n s p r o v i d e d no v a r i a t i o n had h a p p e n e d d u r i n g t h e c u l t u r e p e r i o d . In t h i s way t h e o r i g i n a l g e n o t y p e s a n d p o s s i b l e v a r i a t i o n c o u l d be d e t e r m i n e d when more t h a n one g e n o t y p e i s u s e d i n c u l t u r e . F o r RG4 p l a n t s i t was e a s i e r t o d e t e r m i n e t h e o r i g i n a l g e n o t y p e . B e c a u s e a l l t h e p l a n t s were d e r i v e d f r o m one g e n o t y p e , t h e m a j o r i t y o f t h e r e g e n e r a t e d p l a n t s s h o u l d show t h e isozyme p a t t e r n o f t h e o r i g i n a l g e n o t y p e , whereas any d e v i a t i o n f r o m t h e p a t t e r n s h o u l d be i n low f r e q u e n c y . Two h u n d r e d and t e n RG3 p l a n t s were i n v e s t i g a t e d f o r f i v e enzymes. No v a r i a t i o n a t t r i b u t a b l e t o t i s s u e c u l t u r e 109 was observed f o r phosphoglucose isomerase (PGI). Two isozyme p a t t e r n s were de s i g n a t e d f o r the o r i g i n a l genotypes. These two genotypes d i f f e r e d at one l o c u s with one homozygous and another one heterozygous ( F i g u r e 17). The genotype with the heterozygous l o c u s had two more bands than the homozygous one s i n c e PGI i s a d i m e r i c enzyme. No v a r i a t i o n was found in RG3 p l a n t s f o r phosphoglucomutase (PGM) isozymes. A l l these p l a n t s had three bands, one dense band and two f a i n t bands (F i g u r e 18). The number of bands may vary from three to f i v e among see d - d e r i v e d p l a n t s . For malate dehydrogenase (MDH) a l l seed-derived and regenerated p l a n t s had f i v e bands ( F i g u r e 19). I t was determined that f i v e l o c i are l i k e l y r e s p o n s i b l e f o r the enzyme because i t i s monomeric. For 6-phosphogluconate dehydrogenase (6-PGDH) four isozyme p a t t e r n s were ob t a i n e d . One p a t t e r n , represented by only one sample ( i . e . p l a n t 170), c o u l d be a consequence of the c u l t u r e process ( F i g u r e 20). For shikimate dehydrogenase (SKDH) two isozyme p a t t e r n s were observed, which were due to two genotypes of the three c a l l i i n t h i s enzyme. One p a t t e r n had one band and another one had two bands. S i x t y RG4 p l a n t s were screened f o r isozyme p a t t e r n s of the f i v e enzymes. A l l these p l a n t s had the same p a t t e r n f o r each enzyme as one of the three genotypes which had been observed i n RG3 p l a n t s , demonstrating t h a t no q u a l i t a t i v e v a r i a t i o n had been induced from the c u l t u r e c y c l e . 110 F i g u r e 17. Zymogram of phosphoglucose isomerase from RG3 p l a n t s of red c l o v e r cv. 'Altaswede'. Lane 3 from l e f t r e p r e s e n t s another genotype from which some p l a n t s were regenerated. 111 + F i g u r e 18. Zymogram of phosphoglocomutase from c a l l i of r e d c l o v e r cv. 'Altaswede'. From l e f t to r i g h t , l a n e s 1-19: embryogenic c a l l i and la n e s 20-29 non-embryogenic c a l l i . 112 F i g u r e 19. Zymogram of malate dehydrogenase from RG3 p l a n t s of red c l o v e r cv. 'Altaswede'. A l l p l a n t s had f i v e bands. 1 13 F i g u r e 20. Zymogram of 6-phosphogluconate dehydrogenase from RG3 p l a n t s of red c l o v e r cv. 'Altaswede'. Lane 3 from l e f t r e p r e s e n t s another genotype from which some p l a n t s were regenerated. Lane 11 from l e f t i s an abnormal p a t t e r n observed, which had an e x t r a band. 1 14 4.4.4 Nodule Formation and Leghaemoglobin P r o f i l e A l l p l a n t s analyzed f o r chromosome number and leghaemoglobin p r o f i l e formed normal symbiotic root nodules. The normal leghaemoglobin p r o f i l e of red c l o v e r nodules, as shown i n F i g u r e 21, has three components, one major and two minor under the CAE c o n d i t i o n s used i n t h i s study. The major band migrates most s l o w l y . For 'Altaswede' 91 RG1 and RG2 and 33 RG3 p l a n t s were analysed. No q u a l i t a t i v e v a r i a t i o n ( i . e . band m i s s i n g , new band appearing or a l t e r e d m o b i l i t y ) was d e t e c t e d . Although q u a n t i t a t i v e d i f f e r e n c e s ( i n t e n s i t y and r a t i o between bands) were p r e s e n t , these may be a r e f l e c t i o n of d i f f e r e n c e s among i n d i v i d u a l s , sample s i z e and/or i n f e c t i n g Rhizobium s t r a i n . 4.4.5 Plant Morphology L e a f l e t Number Among the regenerated p l a n t s l e a f l e t number changes from four to seven were observed with high frequency (Table 26). However t h i s v a r i a t i o n was a l s o observed on 'Altaswede' c o n t r o l p l a n t s . The absence of such v a r i a t i o n i n 'Norseman' c o u l d , i n p a r t , be a consequence of the small sample s i z e . A l l the p l a n t s with changed l e a f l e t number were mosaic and had some le a v e s of normal l e a f l e t number. I t would appear that the t i s s u e c u l t u r e regime may have a s t i m u l a t o r y e f f e c t on the occurrence of a l t e r e d l e a f l e t 115 + F i g u r e 21. C e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s of leghaemoglobin of red c l o v e r cv. 'Altaswede'. There are three components, one major and two minor. The major band migrates most s l o w l y . 116 Table 26. L e a f l e t number i n c o n t r o l (seed-derived) and regenerated p l a n t s of red c l o v e r . P o p u l a t i o n C u l t i v a r No of P l a n t s with changed p l a n t s l e a f l e t number (%) RG 1 Altaswede 69 6 (8.7) RG2 Altaswede 100 8 (8.0) RG3 Altaswede 263 5 (1.9) RG4 Altaswede 124 3 (2.4) RG 1 Norseman 93 23 (24.7) C o n t r o l Altaswede 84 5 (6.0) C o n t r o l Norseman 20 0 1 17 number, but such a c o n c l u s i o n would r e q u i r e c a r e f u l comparison and a n a l y s i s of v a r i a t i o n i n seed-derived and regenerated p l a n t s . Length to Width R a t i o of L e a f l e t The mean, variance, and standard d e v i a t i o n of r a t i o s of d i f f e r e n t " p o p u l a t i o n s " are pre s e n t e d i n Table 27. RG1, RG2, RG3 and RG4 p l a n t s were ana l y s e d f o r 'Altaswede'. Because RG4 p l a n t s were grown d u r i n g the summer months an a d d i t i o n a l group of c o n t r o l p l a n t s (CK2) were grown along with them. The mean of CK2 p l a n t s i s a l i t t l e h i g h e r than t h a t of CK1 probably because at that time both temperature and l i g h t i n t e n s i t y were h i g h and p l a n t s were growing more q u i c k l y . S t a t i s t i c a l a n a l y s i s of these data i s shown i n Tables 28 and 29. Means of r a t i o s f o r d i f f e r e n t p o p u l a t i o n s of regenerated p l a n t s of the two c u l t i v a r s are not s i g n i f i c a n t l y d i f f e r e n t from the means of c o n t r o l p l a n t s (P<0.05). That i s , the l e a f l e t l e n g t h to width r a t i o of regenerated p l a n t s d i d not become longer or s h o r t e r as a r e s u l t of iri v i t r o c u l t u r e procedures. However, as shown by the data i n Table 29 the v a r i a n c e s f o r RG1, RG2 and RG3 p l a n t s of 'Altaswede' are a l l s i g n i f i c a n t l y l a r g e r (P^O.01) than that of c o n t r o l p l a n t s . T h i s o b s e r v a t i o n suggests that i n the regenerated p l a n t s g r e a t e r v a r i a t i o n e x i s t e d f o r the r a t i o . N e v e r t h e l e s s the v a r i a n c e of RG4 p l a n t s i s not s i g n i f i c a n t l y d i f f e r e n t from that of the c o n t r o l group. 118 Table 27. Means and v a r i a n c e s of l e a f l e t length/width r a t i o s f o r d i f f e r e n t p o p u l a t i o n s of red c l o v e r . C u l t i v a r P o p u l a t i o n n 1 X s 2 . , n n Altaswede CK1 51 1 . 127 0.074 5.48X10 RG 1 41 1 . 1 30 0. 149 22.0X10 RG2 61 1 .162 0.131 17.2X10 RG3 63 1 .094 0. 125 15.6X10 RG4 1 03 1 .279 0.099 9.80X10 CK2 49 1 .269 0.092 8.46X10 Norseman CK 35 1 . 156 0.086 7.40X10 RG 1 41 1 .130 0.087 7.57X10 RG2 101 1 . f 7 1 0.077 5.93X10 - 3 - 3 - 3 - 3 1 n : sample s i z e 119 Table 28. Comparisons between means of regenerated p l a n t s and c o n t r o l p l a n t s f o r l e a f l e t length/width r a t i o C u l t i v a r Comparison Altaswede RG1/CK1 0.118 n s 1 RG2/CK1 1 .785 ns RG3/CK1 1.761 ns RG4/CK2 0.610 ns Norseman RG1/CK 1.307 ns RG2/CK 0.905 ns 1 ns: not s i g n i f i c a n t . Table 29. Comparisons between v a r i a n c e s of regenerated p l a n t s and c o n t r o l p l a n t s f o r l e a f l e t length/width r a t i o C u l t i v a r Compari son F Altaswede RG1/CK1 4.019 ** 1 RG2/CK1 3.139 ** RG3/CK1 2.853 ** . RG4/CK2 1.158 ns Norseman RG1/CK 1.023 ns RG2/CK 0.801 ns 1 **: s i g n i f i c a n t at PS0.01 l e v e l ; 120 T h i s d i f f e r e n c e i s l i k e l y because RG4 p l a n t s were regenerated from only one genotype so that l e s s v a r i a t i o n might be a n t i c i p a t e d . For 'Norseman', however, the r e s u l t was q u i t e d i f f e r e n t . The v a r i a n c e s of RG1 and RG2 p l a n t s were not d i f f e r e n t from that of c o n t r o l p l a n t s . The d i f f e r e n c e between the two c u l t i v a r s may be r e l a t e d to p l o i d y d i f f e r e n c e , with the t e t r a p l o i d c u l t i v a r 'Norseman' showing more r e s i s t a n c e to change. Morphology of RG4 'Altaswede' P l a n t s S e v e r a l a l b i n o p l a n t s were observed among the RG4 p l a n t l e t s regenerated from the 'Altaswede' c a l l i . Three of them were i s o l a t e d and s u b c u l t u r e d onto LSP medium; they g r a d u a l l y senesced and d i e d d u r i n g the f i r s t and second s u b c u l t u r e s . In g e n e r a l , regenerated p l a n t s grew more q u i c k l y and had greener leaves than c o n t r o l p l a n t s when recorded ten weeks a f t e r the p l a n t t r a n s f e r . These trends were not obvious when recorded only f i v e weeks a f t e r the t r a n s f e r . There were some t e t r a p l o i d p l a n t s among RG4 p l a n t s , t y p i c a l l y r e f l e c t e d i n the p r o d u c t i o n of l a r g e r l e a f l e t s and p e t i o l e s . 5. DISCUSSION 5.1 C a l l u s Induction A f t e r the e x p l a n t s were i n o c u l a t e d on L2(P0.06B0.2) medium c a l l u s p r o d u c t i o n u s u a l l y commenced w i t h i n two weeks. For t h i s study a t o t a l of f i v e batches of c u l t u r e s from two c u l t i v a r s were i n i t i a t e d , and c a l l i were produced r e l a t i v e l y e a s i l y . S i m i l a r l y P h i l l i p s and C o l l i n s (1979a) observed that one t h i r d of 100 genotypes of red c l o v e r t e s t e d showed e x c e l l e n t response while only one f o u r t h gave poor c a l l u s p r o d u c t i o n . Since L2 had been o p t i m i z e d f o r c a l l u s growth by P h i l l i p s and C o l l i n s (1979a), i t i s not s u r p r i s i n g that i t gave good r e s u l t s f o r c a l l u s i n d u c t i o n i n t h i s study. L2 medium uses PIC as the s o l e a u x i n . For c a l l u s i n d u c t i o n only 0.06 mg/1 PIC was r e q u i r e d . Because PIC i s more e f f e c t i v e than 2,4-D i t i s a d e s i r a b l e auxin f o r use i n c a l l u s c u l t u r e . I t a l s o has been used as an auxin i n experiments i n which embryos were formed from l e a f - d e r i v e d c a l l u s of pea (Pisum sativum L.) (Jacobsen and Kysely, 1984). PIC may be a promising growth r e g u l a t o r f o r medium ma n i p u l a t i o n of c e l l c u l t u r e s and p l a n t r e g e n e r a t i o n from legumes. I t was observed that c a l l i formed from e x p l a n t s under v a r i o u s l i g h t c o n d i t i o n s : dark, moderate and high l i g h t i n t e n s i t i e s . 'Altaswede' appeared to show a higher frequency of c a l l u s i n d u c t i o n under low l i g h t or dark c o n d i t i o n s than under high i n t e n s i t y l i g h t . In 'Norseman' 121 122 such an obvious d i f f e r e n c e was not observed. C a l l i formed from both c u l t i v a r s on e i t h e r p l a t e s or i n t e s t tubes. C a l l i of d i f f e r e n t s p e c i e s and even of the same s p e c i e s d i f f e r markedly i n t e x t u r e and c o l o r (Thorpe, 1982). C a l l u s induced i n the dark i s white to p a l e yellow w h i l e t h a t i n i t i a t e d under l i g h t may be p a l e yellow or more s t r o n g l y pigmented. For example red c l o v e r c a l l u s from h y p o c o t y l e x p l a n t s was white with a s l i g h t green c o l o r a t i o n when i n i t i a t e d i n l i g h t . 5.2 Callus Maintenance and Plant Regeneration 5.2.1 Primary Test of Two Media No shoots (or bud i n i t i a l s ) were induced on e i t h e r one of the two media (B 5 and LSE) on which p l a n t r e g e n e r a t i o n i n red c l o v e r has been rep o r t e d (Beach and Smith, 1979; P h i l l i p s and C o l l i n s , 1980). Genotype d i f f e r e n c e s may c o n t r i b u t e i n pa r t to the d i f f e r e n c e between the r e s u l t s i n t h i s study and those of p r e v i o u s r e p o r t s because of d i f f e r e n c e s i n c u l t i v a r s . The c a l l u s source d i f f e r e n c e c o u l d a l s o account f o r the d i f f e r e n t r e s u l t s . Beach and Smith (1979) induced c a l l u s on B 5(N2D2K2) medium, while i n t h i s work c a l l u s i n d u c t i o n o c c u r r e d on L2(PO.06B0.1) medium. The f a i l u r e of LSE medium to induce bud i n i t i a l s w i l l be d i s c u s s e d i n the next s e c t i o n . 1 23 5 . 2 . 2 C a l l u s Growth on LSE Medium In a s e r i e s of t e s t s designed to induce shoots, c a l l i were t r a n s f e r r e d onto LSE medium. A poor response was observed i n a l l these t e s t s . A f t e r t r a n s f e r onto LSE medium, c a l l i showed l i t t l e growth, began to brown a f t e r a s h o r t p e r i o d i n c u l t u r e and the frequency of green-spotted c a l l i decreased. A f t e r continuous s u b c u l t u r e on LSE medium (approximately two months i n c u l t u r e ) c a l l i stopped growing, t u r n e d to brown and black as they d i e d . P h i l l i p s and C o l l i n s (1980) r e p o r t e d the i n d u c t i o n of bud formation with LSE medium on 35-40% of p l a t e s i n o c u l a t e d w i t h c e l l suspension c u l t u r e s of red c l o v e r c u l t i v a r ' A r l i n g t o n ' . In t h e i r experiment suspension c u l t u r e s were e s t a b l i s h e d by i n o c u l a t i n g h y p o c o t y l and e p i c o t y l s e c t i o n s i n t o l i q u i d SL-2 medium ( P h i l l i p s and C o l l i n s , 1980). The suspensions were maintained i n l i q u i d c u l t u r e f o r s i x months u s i n g about 5 ml o l d c u l t u r e to i n o c u l a t e 25 ml f r e s h medium. Approximately one ml of the suspension was i n o c u l a t e d onto s o l i d media to induce buds and shoot i n d u c t i o n was v e r i f i e d by s u b c u l t u r i n g the induced buds onto medium c o n t a i n i n g 0.004 mg/1 PIC and 1 mg/1 BA. In comparing t h e i r experimental c o n d i t i o n s t o those of t h i s study d i f f e r e n c e s i n c u l t i v a r and procedure were e v i d e n t . C u l t i v a r d i f f e r e n c e s i n response have been observed ( P h i l l i p s and C o l l i n s , 1979a). D i f f e r e n c e s between the i n v i t r o behavior of the two c u l t i v a r s used i n the present study were a l s o obvious. C u l t i v a r d i f f e r e n c e s may be 124 exaggerated or masked by the confounding e f f e c t of i n d i v i d u a l genotypes. From the r e s u l t s of c r o s s e s made among regenerated p l a n t s , P h i l l i p s and C o l l i n s (1980) concluded t h a t these p l a n t s were a l l d e r i v e d from e x p l a n t s of a s i n g l e s e e d l i n g . T h i s i n d i v i d u a l s e e d l i n g genotype was l i k e l y more capable of r e g e n e r a t i o n than other genotypes. T h e r e f o r e , i t i s p o s s i b l e that one c u l t i v a r c o n t a i n s a mixture of genotypes with d i f f e r e n t c a p a c i t i e s f o r r e g e n e r a t i o n . The most l i k e l y reason f o r the d i s c r e p a n c y i n observed response was the procedure used to induce p l a n t r e g e n e r a t i o n . P h i l l i p s and C o l l i n s (1980) used a suspension c u l t u r e as the inoculum f o r shoot i n d u c t i o n . I t i s p o s s i b l e t h a t embryos or pre-embryos had a l r e a d y been i n i t i a t e d d u r i n g the c e l l suspension c u l t u r e c y c l e of t h e i r experiment, whereas c a l l i on LSE medium i n t h i s study had not been i n i t i a t e d to undergo embryogenesis. T h i s argument was supported by the r e s u l t s of another experiment ( S e c t i o n 4.2.4), i n which c a l l i from LSE medium d i d not have embryos or shoots developed on the media c o n t a i n i n g 0.0005-0.005 mg/1 PIC and 0.2-1 mg/1 BA. T h i s o b s e r v a t i o n i n d i c a t e d t hat c a l l i grown on LSE i n these experiments had not passed the p o i n t of d e t e r m i n a t i o n (Dodds, 1982). C u l t u r e s of c e l l s pre-determined f o r embryogenesis or embryos may produce embryogenic c a l l i which can grow and d i f f e r e n t i a t e on a medium which would not support d i f f e r e n t i a t i o n or growth of non-embryogenic c a l l i . As demonstrated i n t h i s study, 125 embryogenic and non-embryogenic c a l l i performed d i f f e r e n t l y on a l l the ten media t e s t e d with embryogenic c a l l i more s u c c e s s f u l than non-embryogenic c a l l i . In a d d i t i o n , embryogenic c a l l i r e q u i r e d l e s s exogenous auxin and c y t o k i n i n f o r growth. T h i s o b s e r v a t i o n was r e i n f o r c e d by the f a c t that c a l l i from one genotype capable of embryogenesis produced embryos on LSP medium. Non-embryogenic c a l l i were c u l t u r e d on the same medium, but no response was observed f o r shoot i n d u c t i o n . The f a i l u r e of shoot i n d u c t i o n from c a l l i on LSE medium was probably a consequence of the use of i n d i v i d u a l genotypes with a low c a p a c i t y f o r r e g e n e r a t i o n or a r e f l e c t i o n of the f a i l u r e to t r i g g e r the process l e a d i n g to embryogenesis b e f o r e c a l l i were t r a n s f e r r e d onto LSE medium f o r these genotypes which were capable of r e g e n e r a t i o n . 5.2.3 C a l l u s Growth on SCP Medium SCP medium was shown i n these experiments to be ab l e to r e v i t a l i z e senescing c a l l i . SCP medium may be u s e f u l i n cases where c a l l i of poor q u a l i t y and d e t e r i o r a t i n g performance need to be recovered. C a l l i from d i f f e r e n t sources showed a s i m i l a r response, e s p e c i a l l y those c u l t u r e d on LSE medium. Recovery frequency decreased w i t h the age of c a l l i c u l t u r e d on LSE medium, i n d i c a t i n g that a t h r e s h o l d f o r recovery l i k e l y e x i s t s . C a l l i on SCP medium c o u l d be maintained i n good c o n d i t i o n f o r a time p e r i o d up to four months b e f o r e 126 s u b c u l t u r e . Where c a l l u s l i n e maintenance i s d e s i r a b l e , SCP can be used t o i n c r e a s e the s u b c u l t u r e i n t e r v a l from the t y p i c a l one month to two or three months. Embryogenesis was a l s o observed on t h i s medium (SCP). Embryogenesis i s u s u a l l y i n i t i a t e d when c a l l u s i s t r a n s f e r r e d from media c o n t a i n i n g high l e v e l s of 2,4-D to media c o n t a i n i n g l i t t l e or no 2,4-D (e.g. R e i n e r t et a l . , 1977). I t i s p o s s i b l e to i n i t i a t e the process on media c o n t a i n i n g 2,4-D, but obvious embryos can only be observed on media with low or non-existent l e v e l of 2,4-D. In the present, study embryos at the globe and heart stages were de t e c t e d on SCP medium. P r o g r e s s i v e l y longer c u l t u r e p e r i o d s f o r c a l l i e v e n t u a l l y l e a d to d i s o r g a n i z a t i o n of embryos and embryo-like s t r u c t u r e s . As a r e s u l t , many s t r u c t u r e s which were m o r p h o l o g i c a l l y s i m i l a r to embryos at the globe or heart stages were observed, but o f t e n much l a r g e r i n s i z e . F u r t h e r development of embryos induced on SCP medium r e q u i r e d t r a n s f e r of the c u l t u r e s onto other media. 5.2.4 P l a n t Regeneration: Organogenesis and Embryogenesis P l a n t s can be regenerated from c a l l u s c u l t u r e s through one of two pathways: organogenesis and embryogenesis (e.g. R e i n e r t et a l . , 1977; Narayanswamy, 1977; Bhojwani and Razdan, 1983). A shoot bud from organogenesis and an embryo from embryogenesis are d i s t i n g u i s h a b l e on the b a s i s of r e c o g n i z a b l e morphological d i f f e r e n c e s . The former i s a 1 27 monopolar s t r u c t u r e , while the l a t t e r i s a b i p o l a r one. In some cases, root formation may be incomplete f o r shoots developed from embryos due to r e g u l a t o r s p r e s e n t i n the medium. However, i t i s s t i l l p o s s i b l e to determine the o r i g i n of r e g e n e r a t i o n by other c r i t e r i a . Embryos have a s p e c i f i c morphology and p l a n t l e t s from these embryos have c o t y l e d o n s . For example, red c l o v e r shoots from embryos are r e c o g n i z a b l e by three c r i t e r i a : (1) Embryo s t r u c t u r e s are v i s i b l e on c a l l u s before shoot development; (2) Shoots have two c o t y l e d o n s ; (3) The f i r s t l e a f a f t e r the c o t y l e d o n i s u n i f o l i a t e . In organogenesis c a l l u s c u l t u r e s are f i r s t t r a n s f e r r e d onto a medium which induces shoots from c a l l u s . These shoots are connected a n a t o m i c a l l y with the c a l l u s t i s s u e . The shoots must be t r a n s f e r r e d to another medium f o r root i n d u c t i o n . The most frequent type of d i f f e r e n t i a t i o n from c a l l u s i s root formation ( R e i n e r t et a l . , 1977). The p r i m o r d i a of organs are formed, as a r u l e , from s i n g l e or sma l l groups of parenchymatous c e l l s . In tobacco, formation of buds, r o o t s or c a l l u s can be c o n t r o l l e d at w i l l , depending on the r a t i o between auxin and c y t o k i n i n i n the medium (Skoog and M i l l e r , 1957). High c o n c e n t r a t i o n s of k i n e t i n caused bud i n i t i a t i o n , whereas high l e v e l s of auxin favored r o o t i n g . Other s p e c i e s vary i n t h e i r requirements f o r organogenesis and r e g e n e r a t i o n may be more complicated. P l a n t growth r e g u l a t o r s , other medium components and e x p l a n t (source, s i z e and p h y s i c a l age) as w e l l as temperature, 128 p h o t o p e r i o d , l i g h t i n t e n s i t y , pH and sugar c o n c e n t r a t i o n may a l l p l a y a determining r o l e i n organogenesis (Narayanswamy, 1977; R e i n e r t et a l . , 1977; S t r e e t , 1977; F l i c k et a l . , 1983). The e a r l y phase of shoot formation v i a organogenesis i s s i m i l a r to that of root f o r m a t i o n . T r a n s f e r of t i s s u e to c o n d i t i o n s s u p p o r t i n g organized growth leads to the appearance of meristemoids, which are l o c a t e d on the s u r f a c e or embedded i n the t i s s u e . Continued d i v i s i o n of the s u r f a c e meristems o f t e n leads to formation of small protuberances, g i v i n g t i s s u e a nodular appearance ( c a l l e d nodular c a l l u s , s i m i l a r to green-spotted c a l l i h e r e ) . The meristemoids, i n i t i a l l y a p o l a r , are developmentally p l a s t i c and capable of g i v i n g r i s e to e i t h e r root or shoot p r i m o r d i a (Thorpe, 1982). G e n e r a l l y , r o o t s are endogenously o r i g i n a t e d from c a l l u s , whereas shoots are formed exogenously (Bhojwani and Razdan, 1983). The process of con t i n u e d primordium development t o form organs i s e s s e n t i a l l y s i m i l a r to that i n the i n t a c t p l a n t (Thorpe, 1982) . Often b e f o r e organogenesis, green-spotted c a l l i (or nodular c a l l i ) have been observed ( R e i n e r t et a l . , 1977; Ohyama and Oka, 1982; Thorpe, 1982; Bhojwani and Razdan, 1983) . Such c a l l u s morphology may be an i n d i c a t o r of p o t e n t i a l shoot formation (Kim and Jang, 1984). Green-spotted c a l l u s showed c e l l d i v i s i o n i n the re g i o n s of green c o l o r . Areas of h i g h a c t i v i t y i n c a l l u s can l e a d t o the formation of meristemoids, which are the s i t e s of organ f o r m a t i o n . I n i t i a l l y , when these s t r u c t u r e s 129 were observed in t h i s study i t was thought that they were indicators of potential shoot formation. However, the results showed that these green spots only formed roots, suggesting that either the environmental factors or endogenous regulation (or both) favored d i f f e r e n t i a t i o n to roots instead of shoots. As observed in these experiments, when the green spots grew out of the c a l l u s , they appeared i n i t i a l l y as green bud-like structures (under ill u m i n a t i o n ) . Further elongation occurred and white hairs emerged from the top region as they became more c l e a r l y defined roots. Green spots on c a l l u s in this study were e s s e n t i a l l y an indication of root organogenesis. A s i g n i f i c a n t effect on root formation from c a l l u s was observed with growth regulators, culture sequence, c u l t i v a r and l i g h t i n t e n s i t y . More root formation from c a l l u s generally resulted when cultures were grown on media lacking auxin or with a low auxin to cytokinin r a t i o . This trend was observed throughout the study. In tobacco ('Wisconsin No 38') p i t h culture Skoog and M i l l e r (1957) have demonstrated that the r e l a t i v e r a t i o of auxin (IAA) to cytokinin (KIN) determine the nature of organogenesis. Roots formed from the tissue in the absence of kinetin and in the presence of 0.18-3.0 mg/1 IAA, and shoots formed in the presence of 1.0 mg/1 kinet i n , p a r t i c u l a r l y with IAA concentrations in the range of 0.005-0.18 mg/1 (Skoog and M i l l e r , 1957). Since then, quantitative changes have been found to be decisive in a number of other plant tissue 130 systems (Narayanaswamy, 1977). Root formation i n red c l o v e r c a l l u s c u l t u r e s was q u i t e d i f f e r e n t , as demonstrated i n t h i s study. S i m i l a r l y i n a l f a l f a , i t was shown t h a t , u n l i k e many co n v e n t i o n a l systems i n which organogenesis has been s t u d i e d , c a l l u s from a l f a l f a produced ro o t s a f t e r i n d u c t i o n by four days exposure to r e l a t i v e l y high k i n e t i n and r e l a t i v e l y low 2,4-D (Walker et a l , 1976). Shoots were induced a f t e r a four day c u l t u r e p e r i o d i n r e l a t i v e l y high 2,4-D c o n c e n t r a t i o n s and low k i n e t i n c o n c e n t r a t i o n s . N e v e r t h e l e s s , i t i s the r a t i o of auxin to c y t o k i n i n i n the " i n d u c t i o n medium" that c o n t r o l l e d root or shoot organogenesis. Shoot formation from c a l l u s v i a organogenesis was not observed. However, shoot organogenesis d i d occur d u r i n g propagation of m u l t i p l e shoot c u l t u r e s . In t h i s case organogenesis was dependent upon hig h c y t o k i n i n - c o n t a i n i n g media. Rooting from shoots was r e l a t i v e l y easy compared with shoot i n d u c t i o n . A r o o t i n g frequency of about 85% of the shoots t r a n s f e r r e d onto the r o o t i n g medium was observed and c o u l d be improved by e l e v a t i n g IAA c o n c e n t r a t i o n s l i g h t l y . P l a n t r e g e n e r a t i o n may a l s o be achieved v i a embryogenesis. Although the l i s t of s p e c i e s f o r which somatic embryogenesis has been r e p o r t e d i s l o n g , the number of r e p o r t s with c o n v i c i n g evidence i s smaller (Ammirato, 1983). Somatic embryos sh o u l d resemble t h e i r z y g o t i c c o u n t e r p a r t s with a p p r o p r i a t e r o o t , shoot and c o t y l e d o n 131 organs with shoots capable of d e v e l o p i n g i n t o p l a n t s . Somatic embryogenesis of most s p e c i e s f o l l o w s a s i m i l a r p a t t e r n : growth i n i t i a t i o n on a medium with auxin (2,4-D, NAA or IAA), appearance of embryos a f t e r c a l l u s t r a n s f e r to a medium c o n t a i n i n g reduced 2,4-D l e v e l s , a weaker auxin or no growth r e g u l a t o r s (Raghavan, 1976; R e i n e r t et a l . , 1977; Evans et a l . , 1978; Ammirato, 1983). I t has been suggested that the s u c c e s s i v e changes i n n u t r i e n t media are s i g n i f i c a n t f o r embryogenesis ( R e i n e r t et a l . , 1977). Explant source, reduced n i t r o g e n , auxin and other medium components are important f a c t o r s a f f e c t i n g embryogenesis. The c r i t i c a l f e a t u r e of embryo development i s the occurrence of b i p o l a r i t y ( R e i n e r t et a l . , 1977; Brawley et a l . , 1984). These treatments f a v o r i n g embryogenesis of c u l t u r e d c e l l s should encourage the development of t h i s p o l a r i t y . Although t o t i p o t e n c y i s widely accepted i n a c h i e v i n g r e g e n e r a t i o n , the c a p a c i t y does not i n i t s e l f ensure that c e l l d i f f e r e n t i a t i o n w i l l occur. C a l l u s and c e l l c o l o n i e s from v a r i o u s t i s s u e s c o n s i s t e s s e n t i a l l y of parenchyma c e l l s . These c e l l s are h i g h l y v a c u o l a t e d and c o n t a i n inconspicuous n u c l e i and cytoplasm. A s p e c i f i c type of c e l l d i v i s i o n , l e a d i n g to the formation of s m a l l e r , u s u a l l y i s o d i a m e t r i c c e l l s with prominent n u c l e i densely s t a i n e d cytoplasm and m i c r o v a c u o l a t i o n , i . e . m e r i s t e m - l i k e c e l l s , i s needed (Thorpe, 1982). Sometimes the change in one of many f a c t o r s c o u l d b e n e f i t the c u l t u r e d c e l l s to t r a n s f e r from one s t a t e to another. In anther or p o l l e n c u l t u r e s of N i c o t i a n a 132 tobaccum embryogenesis and subsequent p l a n t r e g e n e r a t i o n were s t u d i e d with exposure dur i n g c u l t u r e to reduced atmospheric p r e s s u r e , anaerobic environments, water s t r e s s as a r e s u l t of a d d i t i o n of 0.5 M mannitol to the medium and a d d i t i o n s of a b s c i s i c a c i d , a s c o r b i c a c i d , d i t h i o t h r e i t o l and each of e i g h t amino a c i d s (Harada and Imamura, 1983). R e s u l t s showed t h a t s u i t a b l e treatments with a l l these f a c t o r s s i g n i f i c a n t l y enhanced embryo development i n c u l t u r e . P l a n t s were regenerated from c a l l i v i a embryogenesis i n t h i s study. C a l l i were f i r s t c u l t u r e d on SCP medium c o n t a i n i n g r e l a t i v e l y h i g h 2,4-D and BA c o n c e n t r a t i o n s . Embryos developed on media c o n t a i n i n g NAA and KIN or c o n t a i n i n g PIC and BA. These embryogenic c a l l i can then be t r a n s f e r r e d onto LSP medium f o r embryo development and eventual p l a n t r e g e n e r a t i o n . S u b c u l t u r e may be r e q u i r e d . Once the process of embryogenesis has been induced i t i s p o s s i b l e to i n i t i a t e embryo p r o d u c t i o n and have p l a n t l e t development from these embryos on a s i n g l e medium. Of s e v e r a l major f a c t o r s which c o n t r o l iri v i t r o somatic embryogenesis the l e v e l of reduced n i t r o g e n s u p p l i e d to c u l t u r e s i s one of the most important (Kohlenbach, 1978). In a l f a l f a embryo formation was s t i m u l a t e d i n both number and q u a l i t y by amino a c i d a d d i t i v e s ( S t u a r t and S t r i c k l a n d , 1984a; 1984b). Development of embryos to p l a n t l e t s was a l s o enhanced by s i m i l a r a d d i t i v e s . In the present study the a d d i t i o n of amino a c i d s ( a r g i n i n e and glutamic a c i d ) d i d not 133 enhance c a l l u s d i f f e r e n t i a t i o n . Because these c a l l i were c u l t u r e d p r e v i o u s l y on LSE medium and had not been s t i m u l a t e d to undergo embryogenesis i t i s perhaps not s u r p r i s i n g that the a d d i t i o n of amino a c i d s d i d not show any promotional e f f e c t on embryo i n d u c t i o n while the same substances have s i g n i f i c a n t l y s t i m u l a t e d embryogenesis i n a l f a l f a ( S t u a r t and S t r i c k l a n d , 1884a). In t h e i r experiment a clone of a h i g h l y regenerable genotype (capable of embryogenesis) was used. Embryogenesis c o u l d be e a s i l y t r i g g e r e d by a r o u t i n e method. Si n c e a c a l l u s l i n e which i s h i g h l y regenerable was e v e n t u a l l y o b t a i n e d i n t h i s study i t should be p o s s i b l e to t e s t whether the a d d i t i o n of amino a c i d s s i g n i f i c a n t l y s t i m u l a t e s embryogenesis i n red c l o v e r i n comparison with the e a r l i e r r e s u l t s r e p o r t e d f o r a l f a l f a . Though p l a n t r e g e n e r a t i o n from c a l l u s c u l t u r e s i n i t i a t e d from non-meristem e x p l a n t s of red c l o v e r has been r e p o r t e d (Beach and Smith, 1979; P h i l l i p s and C o l l i n s , 1979a) i t i s not always easy to a c h i e v e r e g e n e r a t i o n . Bhojwani et a l . (1984) obtained c a l l i from l e a f and h y p o c o t y l e x p l a n t s which c a l l i d i d not undergo any organogenesis except f o r rare i n s t a n c e s of root formation. Success i n r e g e n e r a t i o n from these c u l t u r e s mainly depends on c u l t u r e procedures and genotype used. 5.2.5 Genotype E f f e c t Genotypic e f f e c t s on c a l l u s i n d u c t i o n , c a l l u s morphology and p l a n t r e g e n e r a t i o n were observed throughout 134 t h i s study. I t i s obvious that 'Norseman* and 'Altaswede' c a l l i performed d i f f e r e n t l y on the v a r i o u s media t e s t e d f o r both c a l l u s growth and d i f f e r e n t i a t i o n . 'Norseman' c a l l i grew f a s t e r than 'Altaswede' c a l l i . 'Norseman' produced more roo t e d c a l l i and green-spotted c a l l i , but shoots were induced only from 'Altaswede' c u l t u r e s . 'Altaswede' had more shoots per m u l t i p l e shoot r o s e t t e than 'Norseman', while shoots of 'Norseman' were bigger than those of 'Altaswede'. D i f f e r e n c e s i n the c a p a c i t y of r e g e n e r a t i o n has been re p o r t e d among c u l t i v a r s of red c l o v e r ( P h i l l i p s and C o l l i n s , 1979a). I n t r a - v a r i e t a l v a r i a t i o n f o r i n v i t r o p l a n t r e g e n e r a t i o n was a l s o observed i n three T r i f o l i u m . s p e c i e s , i n c l u d i n g red c l o v e r (Bhojwani et a l . , 1984), while P h i l l i p s and C o l l i n s (1979a) r e p o r t e d that c o t y l e d o n or stem p e t i o l e e x p l a n t s of red c l o v e r y i e l d e d c a l l i r e g e n e r a t i n g p l a n t s from only 1% of the genotypes c u l t u r e d . S t u d i e s attempting t o e v a l u a t e v a r i a t i o n i n c a l l u s c u l t u r e s of red c l o v e r r e v e a l e d that g e n e t i c v a r i a n c e was a s i g n i f i c a n t source of v a r i b i l i t y f o r most 5^n v i t r o c h a r a c t e r s i n v e s t i g a t e d , which i n c l u d e d r a p i d c a l l u s growth, colony v a s c u l a r i z a t i o n , root i n i t i a t i o n , c h l o r o p h y l l p r o d u c t i o n and somatic embryogenesis (Keyes et a l . , 1980). Ge n e t i c d i f f e r e n c e s seem to be a major f a c t o r l i m i t i n g both c a l l u s i n d u c t i o n and p l a n t r e g e n e r a t i o n (Green et a l . , , 1974; Chen e t a l . , 1978; Sharp and Evans, 1982; Subba Rao and N i t z s c h e , 1984). Genetic s t u d i e s on some in v i t r o 1 35 c h a r a c t e r s , r e l a t i n g to r e g e n e r a t i o n , have been c a r r i e d out by s e v e r a l l a b o r a t o r i e s (Zamir et a l . , 1980; R e i s c h and Bingham, 1980; S k v i r s k y et a l . , 1982; Meins et a l . , 1983). In a l f a l f a , c r o s s e s were made between a high frequency of re g e n e r a t i o n l i n e and low re g e n e r a t o r (Reisch and Bingham, 1980). The se g r e g a t i o n r a t i o observed among the progeny of these c r o s s e s suggested that bud d i f f e r e n t i a t i o n from c a l l u s was c o n t r o l l e d by two dominant genes. I_n v i t r o c h a r a c t e r s may a l s o be c o r r e l a t e d with the e x p r e s s i o n of other c h a r a c t e r s such as f e r t i l i t y and y i e l d (Mezentsev, 1980; Ne s t i c k y et a l . , 1983). I f some rn v i t r o c h a r a c t e r s are h i g h l y h e r i t a b l e i t may be expected that they should respond to breeding and s e l e c t i o n i n attempts to develop s u p e r i o r p o p u l a t i o n s f o r use i n t i s s u e c u l t u r e s t u d i e s . G e n e t i c l i n e s of a l f a l f a with 67% r e g e n e r a t i o n were s e l e c t e d from h y p o c o t y l t i s s u e c u l t u r e regenerated p l a n t s with an i n i t i a l p l a n t r e g e n e r a t i o n frequency of only 12% (Bingham e t a l . , 1975). S e l e c t i o n f o r f r i a b i l i t y and i n t e n s i v e p r o l i f e r a t i o n of c a l l u s from a maize l i n e 'F71' has r e s u l t e d i n th r e e c a l l u s l i n e s c h a r a c t e r i z e d by uniform s t r u c t u r e and improved growth r a t e (Bartkowiak, 1981). A program aiming to improve maize r e g e n e r a t i o n was r e p o r t e d using Zea d i p l o p e r e n n i s as a source of germplasm f o r i t s c a p a c i t y of r e g e n e r a t i o n (Sondahl et a l . , 1984). In red c l o v e r , s e l e c t i o n f o r r e g e n e r a b i l i t y may a l s o be p o s s i b l e . C a l l u s l i n e s or p l a n t l i n e s may be o b t a i n e d by 136 s e l e c t i o n techniques f o r improved ir\ v i t r o c h a r a c t e r s , e s p e c i a l l y p l a n t r e g e n e r a t i o n . Such l i n e s would g r e a t l y f a c i l i t a t e t i s s u e c u l t u r e s t u d i e s . 5 . 3 Shoot T i p C u l t u r e C l o n a l propagation by m u l t i p l e shoot p r o d u c t i o n p r o v i d e d a r a p i d method to propagate v a l u a b l e m a t e r i a l s . Shoot t i p c u l t u r e s were e s t a b l i s h e d from two c u l t i v a r s , which were s i g n i f i c a n t l y d i f f e r e n t i n t h e i r response. M e r i s t e m - t i p c u l t u r e of red c l o v e r was r e p o r t e d using L2(P0.004B1) medium (Pa r r o t and C o l l i n s , 1983). Shoot t i p c u l t u r e s were a l s o e s t a b l i s h e d using B 5 medium and a f f e c t i n g f a c t o r s were eva l u a t e d i n another study (Campell and Tomes, 1983). In the present study shoot t i p s were c u l t u r e d on L2 media c o n t a i n i n g d i f f e r e n t l e v e l s of PIC and BA f o r m u l t i p l e shoot i n d u c t i o n and propagation. Procedures d e s c r i b e d i n these experiments are a v a i l a b l e f o r m u l t i p l i c a t i o n of p l a n t s by in v i t r o t echniques. 5 . 4 Regenerated P l a n t s R e s u l t s from the a n a l y s i s of leghaemoglobin p r o f i l e s and isozyme p a t t e r n s of f i v e enzymes showed that p l a n t s regenerated from c a l l u s c u l t u r e were q u i t e s t a b l e f o r these c h a r a c t e r s . These data i n d i c a t e d e i t h e r t h a t these c h a r a c t e r s are very s t a b l e g e n e t i c a l l y so that v a r i a t i o n i s not e a s i l y induced by a s i n g l e c u l t u r e c y c l e or that somaclonal v a r i a t i o n i s not common i n these genotypes of red 137 c l o v e r . The a n a l y s i s of p r o t e i n s o f f e r s a d i r e c t way t o ev a l u a t e gene products f o r gen e t i c v a r i a t i o n . Somaclonal v a r i t i o n f o r such q u a l i t a t i v e t r a i t s may be demonstrable f o r s p e c i e s i n which t h i s v a r i a t i o n has been f r e q e n t l y r e p o r t e d f o r q u a n t i t a t i v e and morphological c h a r a c t e r s . When 45 RG1 'Altaswede' p l a n t s , which came from the m u l t i p l e shoots i n i t i a l l y induced, were analysed f o r chromosome number they were a l l found to be normal. A f t e r the m u l t i p l e shoots underwent s u b c u l t u r e , one of 28 p l a n t s examined was a d i p l o i d / t e t r a p l o i d mosaic. P l a n t s (170) regenerated v i a embryogenesis from short-term c a l l u s c u l t u r e s ( c a l l u s had been c u l t u r e d f o r three months) d i d not show any v a r i a t i o n , whereas 23% of 119 p l a n t s regenerated v i a embryogenesis from long term c u l t u r e s ( n e a r l y one year) had changed chromosome numbers, i n d i c a t i n g that v a r i a t i o n i s r e l a t e d to l e n g t h of c u l t u r e p e r i o d . Chromosome d o u b l i n g i s a very commen change f o r c u l t u r e d c e l l s . I t takes p l a c e i n some c e l l s by the process of endomitosis, which c o n s i s t s of a s e p a r a t i o n of s i s t e r - c h r o m a t i d s w i t h i n the nucl e a r membrance but without the formation of a s p i n d l e and the nucleus subsequently undergoes a f u r t h e r DNA r e p l i c a t i o n (Sunderland, 1977). In some cases, c e l l s with doubled chromosome numbers i n c r e a s e d a f t e r a prolonged c u l t u r e p e r i o d (D'Amato, 1977). A c c o r d i n g l y p l a n t s regenerated from long-term c u l t u r e s tend to have more p o l y p l o i d s . In r e d c l o v e r p r e v i o u s r e p o r t s showed p l a n t s regenerated from short-term c a l l u s c u l t u r e s were normal i n chromosome number 138 (Beach and Smith, 1979; P h i l l i p s and C o l l i n s , 1979a). However, when p l a n t s were regenerated from c a l l i of a d i p l o i d c u l t i v a r which had been c u l t u r e d i n suspension c u l t u r e f o r s i x months bef o r e the i n i t i a t i o n of r e g e n e r a t i o n , two out of twenty two p l a n t s were t e t r a p l o i d s ( P h i l l i p s and C o l l i n s , 1980). In c e r e a l s and grasses p l a n t r e g e n e r a t i o n from embryogenic c a l l i v i a embryogenesis has been a c h i e v e d i n Pennisetum americanum L., Pennisetum purpureum, Pennisetum  americanum L. X P_j_ purpureum h y b r i d , Panicum maximum and T r i t i c u m aestivum L., h i g h s t a b i l i t y of chromosome number i n regenerated p l a n t s was observed ( V a s i l and V a s i l , 1981; V a s i l et a l . , 1982; V a s i l , 1983). When 101 p l a n t s regenerated from embryogenic c a l l i of Pennisetum americanum L. were i n v e s t i g a t e d , one was t e t r a p l o i d and a l l the others were d i p l o i d s , although a higher percentage of t e t r a p l o i d and a n e u p l o i d c e l l s were observed i n the c u l t u r e s (Swedlund and V a s i l , 1985). In c o n t r a s t , when Karp and Maddock (1984) regenerated p l a n t s from c u l t u r e d immature embryos of four wheat c u l t i v a r s ( T r i t i c u m aestivum L . ) , 29% of 192 p l a n t s examined were aneuploids and chromosome a b n o r m a l i t i e s happened to p l a n t s both from embryogenic c a l l i and from the c u l t u r e s i n which o r i g i n of shoots was not i d e n t i f i e d . The present study demonstrated t h a t chromosome number change o c c u r r e d p l a n t s regenerated from embrogenic c a l l i of red c l o v e r . The d i s c r e p a n c y among the r e p o r t s c o n c e r n i n g chromosome a b n o r m a l i t i e s c o u l d r e s u l t from d i f f e r e n c e s i n 139 l e n g t h of c u l t u r e p e r i o d , c u l t u r e procedure and m a t e r i a l s used. Somatic embryos are b e l i e v e d to a r i s e from s i n g l e c e l l s (e.g. V a s i l et a l . , 1982). The f a c t that some regenerated p l a n t s from one c a l l u s were mosaic i n the present study suggests t h a t e i t h e r the somatic embryo o r i g i n a t e d from more than one c e l l or the chromosome number changed i n some c e l l ( s ) at an e a r l y stage of embryogenesis. L e a f l e t number changes were f r e q u e n t l y observed among the p l a n t s regenerated e i t h e r from m u l t i p l e shoots or from c a l l u s c u l t u r e s . T h i s phenomenon was, however, a l s o observed among seed-derived p l a n t s although at lower frequency. I t has been r e p o r t e d that the e x p r e s s i o n of f i v e l e a f l e t s i n red c l o v e r i s c o n t r o l l e d by two genes with complementary e f f e c t (Simon, 1962). When a p l a n t shows only a few l e a v e s with a l t e r e d l e a f l e t number and the remainder normal, the changes may be a consequence of environmental f a c t o r s (Dr. U. Simon, T e c h n i c a l u n i v e r s i t y of Munich, p e r s o n a l communication). In t h i s study a l l the p l a n t s with changed l e a f l e t number were mosaic, i n d i c a t i n g t h at they probably arose as a r e s u l t of phenotypic p l a s t i c i t y r a t h e r than from g e n e t i c changes. T h e r e f o r e , the f a c t t h at more regenerated p l a n t s had v a r i e d l e a f l e t numbers than s e e d - d e r i v e d p l a n t s does not n e c e s s a r i l y mean th a t the t i s s u e c u l t u r e c y c l e had induced h e r i t a b l e g e n e t i c changes i n p l a n t s f o r t h i s t r a i t . 140 I n t e r e s t i n g l y , regenerated p l a n t s of 'Altaswede' showed more v a r i a t i o n i n the l e n g t h to width r a t i o of leaves than those of 'Norseman'. P l a n t s which were regenerated from c a l l u s c u l t u r e s of one genotype showed a reduced v a r i a t i o n . Regenerated p l a n t s of 'Norseman' d i d not d i f f e r from the seed-derived c o n t r o l s with respect t o t h i s v a r i a b i l i t y . The t e t r a p l o i d nature of 'Norseman' may i n c r e a s e i t s s t a b i l i t y t o such changes. These r e s u l t s demonstrated that v a r i a t i o n among regenerated p l a n t s was more l i k e l y to occur f o r q u a n t i t a t i v e c h a r a c t e r s than f o r q u a l i t a t i v e c h a r a c t e r s . Thus somaclonal v a r i a t i o n i s more l i k e l y to be r e p o r t e d i n work on q u a n t i t a t i v e c h a r a c t e r s such as l e a f l e t l e n g t h , p l a n t h e i g h t , dry weight or g r a i n weight. In sugar cane, somaclonal v a r i a t i o n has been r e p o r t e d f r e q u e n t l y but most of the r e p o r t s r e l a t e to q u a n t i t a t i v e c h a r a c t e r s ( S e c t i o n 2.2.1.1.). U s u a l l y , r e l a t i v e l y h i g h f r e q u e n c i e s of v a r i a n t s were r e p o r t e d . L i u and Chen (1976) re p o r t e d that 34% of regenerated p l a n t s from one cl o n e F156 d i f f e r e d s i g n i f i c a n t l y from the donor i n mor p h o l o g i c a l c h a r a c t e r s . However, i n another study where f i v e subclones with d i s t i n c t i v e s t a b l e and v i s i b l e markers were s e l e c t e d f o r t i s s u e c u l t u r e , the r a t e of change of the s p e c i f i c q u a l i t a t i v e c h a r a c t e r s , l e a f f r e c k l e and l e a f b l o t c h , was two or three p l a n t s per thousand. For two other c h a r a c t e r s , m i s s i n g internode and z i g - z a g s t a l k , although changes were d e t e c t e d , they were not t r a n s m i s s a b l e through v e g e t a t i v e 141 propagation ( I r v i n e , 1984). Very few r e p o r t s are a v a i l a b l e on the a n a l y s i s of regenerated p l a n t s from c e l l and c a l l u s c u l t u r e s of legumes (Groose and Bingham, 1983; Johnson et a l . , 1983; Damiani et a l . , 1985), i n p a r t because of the c o n t i n u i n g d i f f i c u l t y i n e f f i c i e n t p l a n t r e g e n e r a t i o n from non-meristem d e r i v e d c u l t u r e s . T h i s problem may be approached by two ways. F i r s t , a d d i t i o n a l e f f o r t s are r e q u i r e d to d e f i n e the proper c u l t u r e c o n d i t i o n s i n c l u d i n g medium composition and environmental regimes. The second approach r e q u i r e s the development or use of genotypes showing high r e g e n e r a t i v e p o t e n t i a l from j j n v i t r o c u l t u r e . Since a genotypic e f f e c t has been r e p o r t e d i n many cases, such l i n e s are p o s s i b l e to f a c i l i t a t e f u r t h e r r e s e a r c h . N e v e r t h e l e s s , good regenerator genotypes can only be o b t a i n e d by s c r e e n i n g s u f f i c i e n t l y l a r g e p l a n t p o p u l a t i o n s to ensure t h a t the range of v a r i a b i l i t y f o r r e g e n e r a t i o n i s a p p r o p r i a t e l y sampled. To p r o v i d e s o l i d evidence f o r and to estimate the frequency of somaclonal v a r i a t i o n proper experimental m a t e r i a l s are r e q u i r e d . Most of p r e v i o u s r e p o r t s have d e a l t with q u a n t i t a t i v e t r a i t s . These t r a i t s are i n h e r e n t l y q u i t e v a r i a b l e and s u s c e p t i b l e to e x i s t i n g environmental c o n d i t i o n s at the time of of the experiment. Q u a l i t a t i v e t r a i t s should be l o o k e d a t f o r the s t a t e d purposes. I f p l a n t s have one to s e v e r a l s i n g l e g e n e - c o n t r o l l e d t r a i t s and these t r a i t s are a v a i l a b l e i n both heterozygous and homozygous s t a t e s , then i t w i l l be p o s s i b l e to estimate the 142 f r e q u e n c i e s of dominant or r e c e s s i v e mutants a f t e r one or more c u l t u r e c y c l e s . 6. Summary When hypoc o t y l e x p l a n t s of red c l o v e r cvs 'Altaswede' and 'Norseman' were c u l t u r e d on L2 medium, 60-85% of the e x p l a n t s produced c a l l i w i t h i n two weeks. 'Altaswede' showed a higher i n d u c t i o n frequency than 'Norseman' (P40.05) in the combined data of the two batches t e s t e d . LSE medium, p r e v i o u s l y d e s c r i b e d i n c u l t u r e s t u d i e s of red c l o v e r , was not s u c c e s s f u l l y used to induce shoots under the experimental c o n d i t i o n s of t h i s study, nor was i t a good medium f o r c a l l u s maintenance. On SCP medium, c a l l i had a high growth r a t e . They c o u l d be s u c c e s s f u l l y grown on t h i s medium f o r up to f i v e months without s u b c u l t u r i n g . Embryogenesis was i n i t i a t e d i n some c a l l i on SCP medium, but embryos at the globe and e a r l y heart stages were not a b l e to develop f u r t h e r normally. More roo t s were u s u a l l y formed from c a l l i c u l t u r e d a t a low auxin to c y t o k i n i n r a t i o or i n the absence of a u x i n s . Shoots were formed a f t e r c a l l i of 'Altaswede' c u l t u r e d on SCP medium were t r a n s f e r r e d onto media c o n t a i n i n g NAA and KIN. NAA c o n c e n t r a t i o n was more c r i t i c a l than KIN c o n c e n t r a t i o n . A d d i t i o n s of amino a c i d s ( a r g i n i n e and glutamic a c i d ) and c a s e i n h y d r o l y s a t e d i d not show any promotive e f f e c t on c a l l u s growth or d i f f e r e n t i a t i o n . 143 144 By s e l e c t i n g and s u b c u l t u r i n g embryogenic c a l l i , a number of p l a n t s have been regenerated from c u l t u r e . C a l l u s c a p a b i l i t y f o r embryogenesis was r e t a i n e d i n s e l e c t e d l i n e s f o r more than one year. Embryogenic c a l l i grew f a s t e r than non-embryogenic c a l l i on ten media used. M u l t i p l e shoots were induced from shoot t i p s c u l t u r e d on L2(PO.003B2) medium. These m u l t i p l e shoots were e a s i l y propagated by s u b c u l t u r i n g them on L2 media c o n t a i n i n g 0.002-0.003 mg/1 PIC and 1 mg/1 BA. U s u a l l y 'Norseman' c a l l i grew f a s t e r and produced more root s than 'Altaswede' c a l l i , w hile shoots were induced only from 'Altaswede' c a l l i . In m u l t i p l e shoots c u l t u r e , 'Norseman' had more shoot t i p s induced to produce m u l t i p l e shoots, but the average m u l t i p l e shoot number f o r 'Altaswede' was higher than that f o r 'Norseman'. P l a n t s were regenerated from m u l t i p l e shoots, e i t h e r i n i t i a l m u l t i p l e shoots (RG1) or m u l t i p l e shoots a f t e r twice s u b c u l t u r e s (RG2), or from c a l l u s c u l t u r e s , e i t h e r short-term (three months) c a l l u s (RG3) or long-term (one year) c a l l u s (RG4). 145 12. Regenerated p l a n t s of 'Altaswede' were a n a l y s e d f o r chromosome number. A l l RG1 and RG3 p l a n t s were normal. One of 28 RG2 p l a n t s and 27 out of 119 RG4 p l a n t s had a l t e r e d chromosome numbers. A l l these p l a n t s were t e t r a p l o i d s or d i p l o i d / t e t r a p l o i d mosaics. No aneuploids were recovered. 13. Regenerated p l a n t s showed c o n s i d e r a b l e s t a b i l i t y f o r leghaemoglobin p r o f i l e s and isozyme p a t t e r n s of MDH, 6-PGDH, PGI, PGM and SKDH. 14. For l e a f l e t l ength/width r a t i o , d i f f e r e n t p o p u l a t i o n s of regenerated p l a n t s were compared with those of c o n t r o l s . RG1, RG2 and RG3 p l a n t s of 'Altaswede' showed more v a r i a t i o n f o r t h i s r a t i o than c o n t r o l p l a n t s , while RG4 p l a n t s of 'Altaswede' and RG1 and RG2 p l a n t s of 'Norseman' were not s i g n i f i c a n t l y d i f f e r e n t from the c o r r e s p o n d i n g c o n t r o l p l a n t s . L I T E R A T U R E C I T E D Ahloowalia,BS (1976) Chromosome changes i n p a r a s e x u a l l y produced r y e g r a s s . In: Jones,K and Brandham,PE ( e d s ) : Current chromosome r e s e a r c h . E l s e v i e r P r e s s : Amsterdam, ppl15-122. Ahloowalia,BS (1982) P l a n t r e g e n e r a t i o n from c a l l u s c u l t u r e in wheat. Crop S c i . 22,405-410. Ammirato,PV (1983) Embryogenesis. In: Evans,DA; Sharp,WR; Ammirato,PV and Yamada,Y (eds) Handbook of P l a n t C e l l C u l t u r e . Macmillan P u b l i s h i n g Co.: New York. pp82-l23. Bartkowiak,E (1981) T i s s u e c u l t u r e of maize: s e l e c t i o n of f r i a b l e c a l l u s l i n e s . P l a n t C e l l Reports 1,52-55. Bayliss,MW (1980) Chromosome v a r i a t i o n i n p l a n t t i s s u e . In: V a s i l , I K (ed) I n t e r n a t i o n a l Review of Cytology, Suppl. 11A: P e r s p e c t i v e s i n Pl a n t C e l l and T i s s u e C u l t u r e . Academic P r e s s : New York, ppl13-144. Beach,KH and Smith,RR (1979) P l a n t r e g e n e r a t i o n from c a l l u s of red and crimson c l o v e r . P l a n t S c i . L e t t . 16,231-237. Bhojwani,SS and Hayward,C (1977) Some o b s e r v a t i o n s and comments of t i s s u e c u l t u r e of wheat. Z. p f l a n z e n p h y s i o l . 85,341-347. Bhojwani,SS and Razdan,MK (1983) P l a n t T i s s u e C u l t u r e : Theory and P r a c t i c e . E l s e v i e r : Amsterdam. p p 7 l - n 2 . Bhojwani,SS; M u l l i n s , K and Cohen,D (1984) I n t r a - v a r i e t a l v a r i a t i o n f o r in vitro p l a n t r e g e n e r a t i o n i n the genus Trifolium. E u p h y t i c a 33,915-921. Bidney,DL and Shepard,JF (1981) Phenotypic v a r i a t i o n i n p l a n t s regenerated from p r o t o p l a s t s : the potato system. Biotechnology and B i o e n g i n e e r i n g 23,2691-2701. Bingham,FT; Hurley,LV; Kartz,DM and Saunders,JW (1975) Breeding a l f a l f a which regenerates from c a l l u s t i s s u e in c u l t u r e . Crop S c i . 15,719-721. Blaydes,DF (1966) I n t e r a c t i o n of k i n e t i n and v a r i o u s i n h i b i t o r s i n the growth of soybean t i s s u e . P h y s i o l . P l a n t . 19,748-758. Bourgin,JP and N i t s c h , J P ( 1967) Obtention de Ni cot i ana h a p l o i d e s a p a r t i r d'etamines c u l t i v e e s in vitro. Ann. P h y s i o l . Veg. 9,377-382. 146 147 Brawley,SH; Wetherell,DF and Robinson,KR (1984) E l e c t r i c a l p o l a r i t y i n embryos of w i l d c a r r o t precedes co t y l e d o n d i f f e r e n t i a t i o n . Proc. N a t l . Acad. S c i . (USA) 81,6064-6067. Burk,LG and Matzinger,DF (1976) V a r i a t i o n among an t h e r - d e r i v e d double d i h a p l o i d s from an i n b r e d l i n e of tobacco. J . Hered. 67,381-384. Burk,LG and C h a p l i n , J F (1980) V a r i a t i o n among a n t h e r - d e r i v e d h a p l o i d s from a m u l t i p l e d i s e a s e - r e s i s t a n t h y b r i d . Crop S c i . 20,334-338. Campbell,CT and Tomes,DT (1983) Establishment and m u l t i p l i c a t i o n of red c l o v e r p l a n t s by in vitro shoot t i p c u l t u r e . P l a n t C e l l T i s s u e Organ C u l t u r e 3,49-57. Chen,CH; Lo,PF and Carabia,JV (1978) Regeneration of p l a n t l e t e from c a l l u s c u l t u r e s of i n d i a n g r a s s . Crop S c i . 19,117-118. Cheyne,VA and Dale,PJ (1980) Shoot t i p c u l t u r e i n forage legumes. Plant S c i . L e t t . 19,303-309. Chourey,PS and Kemble,RJ (1982) T r a n s p o s i t i o n event i n t i s s u e c u l t u r e d c e l l s of maize. In: Fujiwara,A (ed) Proc. 5th I n t l . Cong. P l a n t T i s s u e C u l t u r e . Jpn. Assoc. Plant T i s s u e C u l t u r e : Tokyo. pp425-426. Clayton,JW and Tretiak,DN (1972) A m i n o - c i t r a t e b u f f e r s f o r pH c o n t r o l i n s t a r c h g e l e l e c t r o p h o r e s i s . J . F i s h e r i e s Res. Board Can. 29,1169-1172. C o l l i n s , G B ; Vian,WE and P h i l l i p s , G C (1978) Use of 4 - a m i n o - 3 , 5 , 6 - t r i c h l o r o p i c o l i n i c a c i d as an auxin source i n p l a n t t i s s u e c u l t u r e . Crop S c i . 18,286-288. C o l l i n s , G B ; Taylor,NL and P h i l l i p s , G C (1981) S u c c e s s f u l h y b r i d i z a t i o n of red c l o v e r with p e r e n n i a l Trifolium s p e c i e s v i a embryo rescue. Proceedings of Fourteenth I n t . Grasslands Congress. Westview Press, Boulder, Colorado. Conger,BV (1981) Agronomic Crops In: Conger,BV (ed) C l o n i n g A g r i c u l t u r a l P l a n t s v i a In V i t r o Techniques. CRC P r e s s : Boca Raton, F l o r i d a . pp165-2l5 Creissen,GP and Karp,A (1985) K a r y o t y p i c changes i n potato p l a n t s regenerated from p r o t o p l a s t s . P l a n t C e l l T i s s u e Organ C u l t u r e 4,171-182. D'Amato,F (1977) C y t o g e n e t i c s of d i f f e r e n t i a t i o n i n t i s s u e and c e l l c u l t u r e s . In: R e i n e r t , J and Bajaj,YPS (eds) A p p l i e d and Fundamental Aspects of P l a n t C e l l , T i s s u e 148 and Organ C u l t u r e . S p r i n g e r - V e r l a g : B e r l i n , pp 343-357. D'Amato,F (1978) Chromosome number v a r i a t i o n i n c u l t u r e d c e l l s and regenerated p l a n t s . In: Thorpe,TA (ed) F r o n t i e r s of P l a n t T i s s u e C u l t u r e 1978. Univ. of Calgary Press, C a l g a r y . pp287-295. Damiani,F; M a r i o t t i , D ; P e z z o t t i , M and A r c i o n i , S (1985) V a r i a t i o n among p l a n t s regenerated from t i s s u e c u l t u r e of Lotus corniculatus L. Z. p f l a n z e n z i i c h t g 94,332-339. Deambrogio,E and Dale,PI (1980) E f f e c t of 2,4-D on the frequency of regenerated p l a n t s i n b a r l e y {Hordeum vulgare) c u l t i v a r 'Akha' and on g e n e t i c v a r i a b i l i t y between them. C e r e a l Res. Commun. 8,417-424. De Paepe,R; Prat,D and Huguet,T (1983) H e r i t a b l e DNA changes i n doubled h a p l o i d p l a n t s o b t a i n e d by p o l l e n c u l t u r e of Nicotiana sylvestris. P l a n t S c i . L e t t . 28,11-28. Dodds,JH (1982) Experiments i n P l a n t T i s s u e C u l t u r e . Cambrige Univ. P r e s s : Cambridge. p66. Evans,AM (1962) Species h y b r i d i z a t i o n i n Trifolium: I. Methods of overcoming i n c o m p a t i b i l i t y . E u p h y t i c a 11,164-176. Evans,DA and Sharp,WR (1983) S i n g l e gene mutations i n tomato p l a n t s regenerated from t i s s u e c u l t u r e . Science 221,949-951. Evans,DA; Sharp,WR; and F l i c k , C E (1978) Growth and behavior of c e l l c u l t u r e s : embryogenesis and organogenesis. In: Thorpe,TA (ed) P l a n t T i s s u e C u l t u r e : Methods and A p p l i c a t i o n s i n A g r i c u l t u r e . Academic P r e s s : New York. pp45-114. Evans,DA; Sharp,WR and Medina-Filho,HP (1984) Somaclonal and gametoclonal v a r i a t i o n . Amer. J . Bot. 71,759-774. F a c c i o t i , D and P i l e t , P E (1979) P l a n t s and embryoids from h a p l o i d Nicotiana sylvestris p r o t o p l a s t s . P l a n t S c i . L e t t . 15,1-6. F l i c k , C E ; Evans,DA and Sharp,WR (1983) Organogenesis. In: Evans,DA; Sharp,WR; Ammirato,PV and Yamada,Y (eds) Handbook of Pl a n t C e l l C u l t u r e V.1. Macmillan P u b l i s h i n g Co.: New York. pp13-8l. Fukui,K (1983) S e q u e n t i a l occurrence of mutations on a growing r i c e c a l l u s . Theor. A p p l . Genet. 65,225-230. Gamborg,OL; M i l l e r , R A and Ojima,K (1968) N u t r i e n t 1 49 requirements of suspension c u l t u r e s of soybean root c e l l s . Exp. C e l l Res. 50,151-158. Gengenbach,BG; Connell,JA; Pring,DR and Conde,MF (1981) M i t o c h o n d r i a l DNA v a r i a t i o n i n maize p l a n t s regenerated d u r i n g t i s s u e c u l t u r e s e l e c t i o n . Theor. Appl. Genet. 59,161-167. Gengenbach,BG; Green,CE and Donovan,CM (1977) I n h e r i t a n c e of s e l e c t e d p a t h o t o x i n r e s i s t a n c e i n maize p l a n t s regenerated from c e l l c u l t u r e s . Proc. N a t l . Acad. S c i . (USA) 74,5113-5117. Green,CE; P h i l l i p s , R L and Kleese,RA (1974) T i s s u e c u l t u r e s of maize (Zea may L . ) : i n i t i a t i o n , maintenance and o r g a n i c growth f a c t o r s . Crop S c i . 4,54-58. Groose,RW and Bingham,ET (1983) Somaclonal v a r i a t i o n among p l a n t s regenerated from a l f a l f a t i s s u e c u l t u r e . 75th Ann. Meet. Amer. Soc. of Agronomy, Washington. p65. Harada,H and Imamura,J (1983) F a c t o r s t h a t s t i m u l a t e p o l l e n embryogenesis. In: C e l l and T i s s u e C u l t u r e Techniques f o r C e r e a l Crop Improvement. S c i . P r e s s : B e i j i n g . PP145-158. Heinz,DJ (1973) Sugar-cane improvement through induced mutations using v e g e t a t i v e propagules and c e l l c u l t u r e t e c h n i q u e s . In: Induced Mutations i n V e g e t a t i v e l y Propagated P l a n t s (Proc. of a P a n e l . 11-15 September, 1972) I n t l . Atomic Agency: Vienna. pp53-59. Heinz,DJ and Mee,GWP (1969) P l a n t d i f f e r e n t i a t i o n from c a l l u s t i s s u e of Saccharum s p e c i e s . Crop S c i . 9,346-348. Heinz,DJ; Krishmamurthi,M; N i c k e l l , L G and Maretzki,A (1977) C e l l , t i s s u e and o r g a n " c u l t u r e i n sugarcane improvement. In: R e i n e r t , J and Bajaj,YPS (eds) A p p l i e d and Fundamental Aspects of P l a n t C e l l T i s s u e and Organ C u l t u r e . S p r i n g e r - V e r l a g : B e r l i n . pp3-17. Helenurm,S (1983) Genetic d i f f e r e n t i a t i o n of Hawaii Bidens. M.Sc. T h e s i s . The Univ. of B r i t i s h Columbia. H o l l , F B ; M i l l i r o n , M L and D e l a f i e l d , S J ( 1 9 8 3 ) Q u a l i t a t i v e v a r i a t i o n i n root nodule leghemoglobins: i n t r a - s p e c i f i c v a r i a t i o n i n component p r o f i l e s . P l a n t S c i . L e t t . 32,321-326. I r v i n e , J E (1984) The frequency of marker changes i n sugercane p l a n t s regenerated from c a l l u s c u l t u r e s . P l a n t C e l l T i s s u e Organ C u l t u r e 3,201-209. 150 Jacobsen,H and Kysely,W (1984) In d u c t i o n of somatic embryos i n pea, Pi sum sativum L. P l a n t C e l l T i s s u e Organ C u l t u r e 3,319-324. Johnson,LB; Schlarbaum,SE and S t u t e v i l l e , D L (1983) Chromosome v a r i a b i l i t y i n a l f a l f a p l a n t s regenerated from p r o t o p l a s t s . 75th Ann. Meet. Soc. of Agronomy, Washington. p68. Kao,KN (1976) A chromosome s t a i n i n g method f o r c u l t u r e d c e l l s . In: Gamborg,OL and Wetter,LR (eds) P l a n t T i s s u e C u l t u r e Method. N a t i o n a l Research C o u n c i l of Canada, P r a i r i e R e g i o n a l Laboratory, Saskatoon. pp63-65. Karp,A and Maddock,SE (1984) Chromosome v a r i a t i o n i n wheat p l a n t s regenerated from c u l t u r e d immature embryos. Theor. A p p l . Genet. 67,249-255. Karp,A; Nelson,RS; Thomas,E and Bright,SWJ (1982) Chromosome v a r i a t i o n i n p r o t o p l a s t - d e r i v e d potato p l a n t s . Theor. Ap p l . Genet. 63,265-272. Karp,A; R i s i o t t , R ; Jones,MGK and Bright,SWJ (1984) Chromosome d o u b l i n g i n monohaploid and d i h a p l o i d potatoes by r e g e n e r a t i o n from c u l t u r e d l e a f e x p l a n t s . P l a n t C e l l T i s s u e Organ C u l t u r e 3,363-373. Keyes,GJ; C o l l i n s , G B and Taylor,NL (1980) Genetic v a r i a t i o n i n t i s s u e c u l t u r e of red c l o v e r . Theor. Appl. Genet. 58,265-271. Kim,SG and Jang,JR (1984) Regeneration of p l a n t s from c a l l u s t i s s u e of cucumber (Cucumis sativus L.) s e e d l i n g c o t y l e d o n s . P l a n t P h y s i o l . 75(1.suppl.),15. Kohlenbach,HW (1978) Comparative somatic embryogenesis. In: Thorpe,TA (ed) F r o n t i e r s of P l a n t T i s s u e C u l t u r e 1978. Univ. of C a l g a r y P r e s s : C a l g a r y . p59. K r i k o r i a n , A D ; O'Connor,SA and F i t t e r , M S (1983) Chromosome number v a r i a t i o n and karyotype s t a b i l i t y i n c u l t u r e s and c u l t u r e - d e r i v e d p l a n t s . Kucherenko,LA (1979) T i s s u e c u l t u r e i n r i c e improvement: experience i n the USSR. In: Innovative approaches to r i c e b r e e d i n g . IRRI, M a n i l a , P h i l i p p i n e s , pp93-102. In: Evans,DA; Sharp,WR; Ammirato,PV and Yamada,Y (eds) Handbook of P l a n t C e l l C u l t u r e V.1. Macmillan P u b l i s h i n g Co.: New York. pp541-58l. Lapitan,NLV; Sears,RG and G i l l , B S (1984) T r a n s l o c a t i o n s and other k a r y o t y p i c s t r u c t u r a l changes i n wheat X rye h y b r i d s regenerated from t i s s u e c u l t u r e . Theor. A p p l . Genet. 68,547-554. 151 L a r k i n , P J and Scowcroft,WR (1981) Somaclonal v a r i a t i o n — a novel source of v a r i a b i l i t y from c e l l c u l t u r e s f o r p l a n t improvement. Theor. A p p l . Genet. 60,197-214. L a r k i n , P J and Scowcroft,WR (1983a) Somaclonal v a r i a t i o n and crop improvement. In Kosuge,T; Meredith,C and Hollaender,A (eds) Genetic E n g i n e e r i n g of P l a n t s . Plenum P r e s s : New York. pp289-314. L a r k i n , P J and Scowcroft,WR (1983b) Somaclonal v a r i a t i o n and eyespot t o x i n t o l e r a n c e i n sugar cane. Pl a n t C e l l T i s s u e Organ C u l t u r e 2,111-121. L a r k i n , P J ; Ryan,SA; B r e t t e l l , R I S and Scowcroft,WR (1984) H e r i t a b l e somaclonal v a r i a t i o n i n wheat. Theor. Appl. Genet. 67,443-455. Linsmaier,EM and Skoog,F (1965) Organic growth f a c t o r requirements of tobacco t i s s u e c u l t u r e s . P h y s i o l . P l a n t . 18,100-127. Liu,MC and Chen,WH (1978) T i s s u e and c e l l c u l t u r e as a i d s to sugar cane breeding I . C r e a t i o n of genetic v a r i a t i o n through c a l l u s c u l t u r e . E u p h y t i c a 25,393-403. L6rz,H (1983) V a r i b i l i t y i n t i s s u e c u l t u r e d e r i v e d p l a n t s . In: Mentell,SH and Smith,H (eds) Pl a n t B i o t e c h n o l o g y . Cambrige U n i v e r s i t y P r e s s : Cambrige. ppl03-114. L6rz,H and Scowcroft,WR (1983) V a r i a b i l i t y among p l a n t s and t h e i r progeny regenerated from p r o t o p l a s t s of Su/su heterozygates of Nicotiana tobaccum. Theor. A p p l . Genet. 66,67-75. Lupi,MC; Bennici,A; B a r o n c e l l i , S ; Gennai,D and D'Amato,F (1981) In v i t r o r e g e n e r a t i o n of durum wheat. 2. D i p l o n t i c s e l e c t i o n i n aneusomatic p l a n t s . Z. pf l a n z e n z u c h t g 87,167-171. Ma,YH (ed) ( 1978) Experiments and S t a t i s t i c s . A g r i c u l t u r e P r e s s : B e i j i n g , chap 4 and 5. McCoy,TJ and P h i l l i p s , R L (1982) Chromosome s t a b i l i t y i n maize (Zea mays) t i s s u e c u l t u r e s and s e c t o r i n g i n some regenerated p l a n t s . Can. J . Genet. C y t o l . 24,559-565. McCoy,TJ and Walker,K (1984) A l f a l f a . In: Ammirato,PV; Evans,DA; Sharp,WR and Yamada,Y (eds) handbook of P l a n t C e l l C u l t u r e V.3. Macmillan P u b l i s h i n g Co.: New York. PP171-192. McCoy,TJ; P h i l l i p s , R L and Rines,HW (1982) Cy t o g e n e t i c v a r i a t i o n i n t i s s u e c u l t u r e regenerated p l a n t s of Avena 152 sativa: High frequency of chromosome l o s s . Can. J . Genet. C y t o l . 24,37-50. McWilliam,AA; Smith,SH and Street,HE (1974) The o r i g i n and development of embryoids i n suspension c u l t u r e of c a r r o t . Ann. Bot. 38,234-250. Me i n s , F . J r ; Foster,R and Lutz,JD (1983) Evidence f o r a Mendelian f a c t o r c o n t r o l l i n g the c y t o k i n i n requirement of c u l t u r e d tobacco c e l l s . Developmental G e n e t i c s 4,129-141 . Mezentsev,AV (1980) Induced c a l l u s formation and morphogenesis i n the t i s s u e c u l t u r e of l u c e r n e and p o s s i b i l i t i e s of r e g u l a t i n g i t . Sel'kokhozyarstvennaya B i o l o g i y a 15,379-383 (Ru). ( c f . P l a n t Breeding A b s t r a c t 1980:10368). M i l l e r , C O (1961) A k i n e t i n - l i k e compound i n maize. Proc. N a t l . Acad. S c i . (USA) 47,170-174. Morel,G (1971) The impact of p l a n t t i s s u e c u l t u r e i n p l a n t b r e e d i n g . In: Lupton,FGH; Jenkins,G and Johnson,R (eds) The Way ahead i n P l a n t Breeding. S i x t h Congress, E u c a r p i a , Cambridge. p p l 8 5 - l 9 4 . Murashige,T and Skoog,F (1962) A r e v i s e d medium f o r r a p i d growth and bioassays with tobacco t i s s u e c u l t u r e s . P h y s i o l . P l a n t . 15,473-497. Narayanaswamy,S (1977) Regeneration from t i s s u e c u l t u r e s . In: R e i n e r t , J and Bajaj,YPS (eds) A p p l i e d and Fundamental Aspects of P l a n t C e l l T i s s u e Organ C u l t u r e . S p r i n g e r - V e r l a g : B e r l i n . pp179-206. Nesticky,M; Movak,FJ; P i o v a r c i , A and Dolezelova,M (1983) G e n e t i c a n a l y s i s of c a l l u s growth of maize (Zea mays L.) in vitro. Z. p f l a n z e n z u c h t g 91,322-329. N i i z e k i , M and K i t a , F (1973) S t u d i e s on p l a n t c e l l and t i s s u e c u l t u r e . I I I . In vitro i n d u c t i o n of c a l l u s from anther c u l t u r e of forage c r o p s . J . Fac. A g r i c . Hokkaido Univ. 57,293-300. Ogihara,Y (1981) T i s s u e c u l t u r e i n Haworthia P a r t 4: Genetic c h a r a c t e r i z a t i o n of p l a n t s regenerated from c a l l u s . Theor. A p p l . Genet. 60,353-363. 0'Hara,JF and Street,HE (1978) Wheat c a l l u s c u l t u r e : the i n i t i a t i o n , growth and organogenesis of c a l l u s d e r i v e d from v a r i o u s explant sources. Ann. Bot. 42,1029-1038. Ohyama,K and Oka,S (1982) M u l t i p l e shoot formation from mulbery {Morus alba L.) h y p o c o t y l s by 153 N - ( 2 - c h l o r o - 4 - p y r i d y l ) - N ' - p h e n y l u r e a . In: Fujiwara,A (ed) Proc. 5th I n t l . Cong. Plant T i s s u e C u l t u r e . Jpn. Assoc. P l a n t T i s s u e Culture:Tokyo. pp149-l50. Oono,K (1978) Test tube breeding of r i c e t i s s u e c u l t u r e . Trop. A g r i c . Res. Ser 11, 109-123. Orton,TJ (1980) Chromosome v a r i a b i l i t y i n t i s s u e c u l t u r e s and regenerated p l a n t s of Hordeum. Theor. A p p l . Genet. 56,101-112. Parrot,WA and C o l l i n s , G B (1983) C a l l u s and s h o o t - t i p c u l t u r e of e i g h t Trifolium s p e c i e s in vitro with r e g e n e r a t i o n v i a somatic embryogenesis of T. rubens. P l a n t S c i . L e t t . 28,189-194. P h i l l i p s , G C and C o l l i n s , G B (1979a) In vitro t i s s u e c u l t u r e of s e l e c t e d legumes and p l a n t r e g e n e r a t i o n from c a l l u s c u l t u r e s of red c l o v e r . Crop S c i . 19,59-64. P h i l l i p s , G C and C o l l i n s , G B (1979b) V i r u s sympton-free p l a n t s of red c l o v e r using meristem c u l t u r e . Crop S c i . 19,213-216. P h i l l i p s , G C and C o l l i n s , G B (1980) Somatic embryogenesis from c e l l suspension c u l t u r e s of red c l o v e r . Crop S c i . 20,323-326. P h i l l i p s , G C and C o l l i n s , G B (1981) Growth and s e l e c t i o n of red c l o v e r (Trifolium pratense L.) c e l l s on low l e v e l s of phosphate. Agronomy A b s t r a c t s p l 8 7 . P h i l l i p s , G C and C o l l i n s , G B (1983) Red c l o v e r and other forage legumes. In. Sharp,WR; Evans,DA; Ammirato,PV and Yamada,Y (eds) Handbook of P l a n t C e l l C u l t u r e V.2. Macmillan P u b l i s h i n g Co.:New York, ppl69-209. P h i l l i p s , G C ; C o l l i n s , G B and Taylor,NL (1982) I n t e r s p e c i f i c h y b r i d i z a t i o n of red c l o v e r (Trifolium pratense L.) with T. s ar os i e ns e Hazsi using in vitro embryo rescue. Theor. Appl. Genet. 62,17-24. Prat,D (1983) Genetic v a r i a b i l i t y induced i n Nicotiana sylvestris by p r o t o p l a s t c u l t u r e . Theor. A p p l . Genet. 64,223-230. Raghavan,V (1976) Experimental Embryogenesis i n V a s c u l a r P l a n t s . Academic Pr e s s , New York. Ramulu,KS; D i j k h u i s , P and Roset,S (1983) Phenotypic v a r i a t i o n and p l o i d y l e v e l of p l a n t s regenerated from p r o t o p l a s t s of t e t r a p l o i d potato (Solanum tuberosum L. cv ' B i n j t e ' ) . Theor. Appl. Genet. 65,329-338. 1 54 Ranga Rao,V (1976) Nitrogenase a c t i v i t y in Rhizobium a s s o c i a t e d with leguminous and non-leguminous t i s s u e c u l t u r e s . P l a n t S c i . L e t t . 6,77-83. R e i n e r t , J ; Bajaj,YPS and Z b e l l , B (1977) Aspects of o r g a n i z a t i o n — organogenesis, embryogenesis , c y t o d i f f e r e n t i a t i o n . In: Street,HE (ed) Pl a n t T i s s u e and C e l l C u l t u r e . Univ,. of C a l i f o r n i a P r e s s: Berkeley and Los Angeles. pp389-427. Reisch,B (1983) Genetic v a r i a b i l i t y i n regenerated p l a n t s . In: Evans,DA; Sharp,WR; Ammirato,PV and Yamada,Y (eds) Handbook of P l a n t C e l l C u l t u r e V.1. Macmillan P u b l i s h i n g Co.: New York. pp748-769. Reisch,B and Bingham,ET (1980) The g e n e t i c c o n t r o l of bud formation from c a l l u s c u l t u r e s of d i p l o i d a l f a l f a . P l a n t S c i . L e t t . 20,71-77. Sacristan,MD and Melchers,G (1969) The c a r y o l o g i c a l a n a l y s i s of p l a n t s regenerated from tumerous and other c a l l u s c u l t u r e s of tobacco. Molec. Gen. Genet. 105,317-333. Schaeffer,GW; Sharpe,FT J r . ; Cregan,PB (1984) V a r i a t i o n f o r improved p r o t e i n and y i e l d from r i c e anther c u l t u r e . Theor. Appl. Genet. 67,383-389. Scowcroft,WR and L a r k i n , P J (1983) Somaclonal v a r i a t i o n and g e n e t i c improvement of crop p l a n t s . In. B e t t e r Crops fo r Food. Pitman Books, London (Ciba Foundation Symposium 9). p177-l93. Secor,GA and Shepard,JF (1981) V a r i a b i l i t y of p r o t o p l a s t - d e r i v e d potato c l o n e s . Crop S c i . 21,102-105. Sharp,WR and Evans,DA (1982) A p p l i c a t i o n of somatic embryogenesis to crop improvement. In: Fujiwara,A (ed) Proc. 5th I n t l . Cong. P l a n t T i s s u e and C e l l C u l t u r e . Jpn. Assoc. P l a n t T i s s u e C u l t u r e : Tokyo. p759-762. Shepard,JF (1981) P r o t o p l a s t s as sources of di s e a s e r e s i s t a n c e i n p l a n t s . Ann. Rev. Phytopath. 19,145-166. Shepard,JF (1982) The r e g e n e r a t i o n of potato p l a n t s from l e a f - c e l l p r o t o p l a s t s . S c i . Amer. 246,154-166. Shepard,JF; Bidney,D and Shahen,E (1980) Potato p r o t o p l a s t s i n c r o p improvement. Science 28,17-24. Shimada,T; Sasakuma,T and Tsunewaki,K (1969) In vitro c u l t u r e of wheat t i s s u e I. C a l l u s formation, organ r e d i f f e r e n t i a t i o n and s i n g l e c e l l c u l t u r e . Can. J . of 1 55 Genet, and Cyto. 11,294-304. Sibi,M; B i g l a r y , M and Demarly,Y (1984) Increases i n the r a t e of recombinations i n tomato (Lycopersicon esculentum L.) Theor. A p p l . Genet. 68,317-321. Simon,U (1962) I n h e r i t a n c e of p o l y p h y l l i n red c l o v e r [Trifolium pratense L.) . Crop S c i . 2,258. Skoog,F and M i l l e r , C (1957) Chemical r e g u l a t i o n of growth and organ formation i n p l a n t t i s s u e s c u l t u r e d in vitro. Symp. Soc. Exp. B i o l . 11,118-131. Skvirsky,RC; Hanson,MR and Ausubel,FM (1982) A g e n e t i c approach f o r s t u d y i n g p l a n t r e g e n e r a t i o n : a n a l y s i s of c y t o k i n i n - c o n t r o l l e d shoot morphogenesis from t i s s u e e x p l a n t s of Petunia hybrida. In: Earle,ED and Demarly,Y V a r i a b i l i t y i n P l a n t s Regenerated from T i s s u e C u l t u r e . Praeger: New York. pplOl-120. Sondahl,MR; Evans,DA; P r i o l i , L M and Silva,WA (1984) T i s s u e c u l t u r e r e g e n e r a t i o n of p l a n t s i n Zea dipioperennis a c l o s e r e l a t i v e of c o r n . Bio/Technology 2,455-458. Street,HE (1977) Embryogenesis and c h e m i c a l l y induced organogenesis. In: Sharp,WR; Larsen,PO and Raghavan,V (eds) P l a n t C e l l and T i s s u e C u l t u r e : P r i n c i p l e s and A p p l i c a t i o n s . ppl23-153. Stuart,DA and S t r i c k l a n d , S G (1984a) Somatic embryogenesis from c e l l c u l t u r e s of Medicago saliva L. I. The r o l e of amino a c i d s a d d i t i o n s to the r e g e n e r a t i o n medium. Pl a n t S c i . L e t t . 34,165-174. Stuart,DA and S t r i c k l a n d , S G (1984b) Somatic embryogenesis from c e l l c u l t u r e s of Medicago sativa L. I I . The i n t e r a c t i o n of amino a c i d s with ammonium. Pla n t S c i . L e t t . 34,175-181. Subba Rao,MV and Nitzsche,W (1984) Genetic d i f f e r e n c e s i n c a l l u s growth and organogenesis of e i g h t p e a r l m i l l e t l i n e s . E u p h y t i c a 33,923-928. Sun,ZX; Zhao,CZ; Zheng,KL; Qi,XF and Fu,YP (1983) Somaclonal g e n e t i c s of r i c e , Oryza sativa L. Theor. Appl. Genet. 67,67-73. Sunderland,N (1977) Nuclear Cytology. In: Street,HE (ed) P l a n t T i s s u e and C e l l C u l t u r e . Univ. of C a l i f o r n i a P r e s s : Berkeley and Los Angeles. pp177-206. S u t t e r , E and Langhans,RW (1981) A b n o r m a l i t i e s i n Chrysant hemum regenerated from long term c u l t u r e s . Ann. Bot. 48,559-568. 1 56 Swedlund,B and V a s i l , I K (1985) Cy t o g e n e t i c c h a r a c t e r i z a t i o n of embryogenic c a l l u s and regenerated p l a n t s of Pennisetum americanum (L. ) K. Schum. Theor. A p p l . Genet. 69,575-581. Syono,K and Furuya,T (1972) Abnormal flower formation of tobacco p l a n t s regenerated from c a l l u s c u l t u r e s . Mag. Tokyo 85,273-284. Taylor,NL (1973) Red c l o v e r and a l s i k e c l o v e r . In: Heath,ME; Metcalfe,DS and Barnes,RF Forages: tbe Science of G r a s s l a n d A g r i c u l t u r e . Iowa S t a t e Univ. P r e s s , Ames, Iowa. pp148-158. Taylor,NL and Smith,RR (1979) Red c l o v e r breeding and g e n e t i c s . Adv. Agron 31,125-154. Thomas,E; Bright,SWJ; F r a n k l i n , J ; Lancaster,V; M i f l i n , B J and Gibson,R (1982) V a r i a t i o n amongst p r o t o p l a s t - d e r i v e d p o t a t o p l a n t s (Sol anum tuberosum cv. 'Maris Bard'). Theor. Appl. Genet. 62,65-68. Thorpe,TA (1982) C a l l u s o r g a n i z a t i o n and de novo formation of shoots, r o o t s and embryos in v i t r o . In: Tomes,DT; E l l i s , B E ; Harney,PM; Kasha,KJ and Peterson,RL (eds) A p p l i c a t i o n of P l a n t C e l l and T i s s u e C u l t u r e to A g r i c u l t u r e and I n d u s t r y . Univ. of Guelph, Guelph ppl15-138. V a l l i e j o s , C E (1983) Enzyme a c t i v i t y s t a i n i n g . In: Tanksley,SD and Orton,TJ Isozymes i n P l a n t G e n e t i c s and Breeding Part A. E l s e v i e r Science P u b l i s h e r s , Amsterdam. pp469-516. Van Harten,AM and B r o e r t j e s , C (1981) In v i t r o a d v e n t i t i o u s bud techniques f o r v e g e t a t i v e propagation and mutation breeding of potato (Sol anum tuberosum L.) I I . S i g n i f i c a n c e f o r mutation b r e e d i n g . E u p h y t i c a 30,1-8. V a s i l , I K (1983) Regeneration of p l a n t s from s i n g l e c e l l s of c e r e a l s and g r a s s e s . In: Lurquin,P and K l e i n h o f s , A (eds) Genetic E n g i n e e r i n g i n E u k a r y t e s . Plenum, New York. pp233-252. V a s i l , V and V a s i l , I K (1981) Somatic embryogenesis and p l a n t r e g e n e r a t i o n from suspension c u l t u r e s of p e a r l m i l l e t (Penni set urn americanum). Ann. Bot. 47,669-678. V a s i l , I K ; V a s i l , V ; Lu,C; Qzias-Akins,P; Haydu,Z and Wang,DY (1982) Somatic embryogenesis i n c e r e a l s and g r a s s e s . In: Earle,DE and Demarly,Y (eds) V a r i a b i l i t y i n P l a n t s Regenerated from T i s s u e C u l t u r e . Praeger, New York. pp3-21. 157 Wakasa,K (1979) V a r i a t i o n i n the p l a n t s d i f f e r e n t i a t e d from t i s s u e c u l t u r e of p i n e a p p l e . Jpn. J . Breeding 29,13-22. Walker,KA; Wendeln,ML and Jaworski,EG (1976) Organogenesis i n c a l l u s c u l t u r e of Medicago sativa L.: The temperal s e p a r a t i o n of i n d u c t i o n p rocess from d i f f e r e n t i a t i o n p r o c e s s . P l a n t S c i . L e t t . 16,23-30. Wenzel,G; Schieder,0; Przewozny,T; Sopory,Sk and Melchers,G (1979) A comparison of s i n g l e c e l l d e r i v e d Solanum tuberosum p l a n t s and a model f o r t h e i r a p p l i c a t i o n i n breeding programs. Theor. Appl. Genet. 55,49-55. Wetherell,DF and Dougall,DK (1976) Sources of n i t r o g e n s u p p o r t i n g growth and embryogenesis i n c u l t u r e d w i l d c a r r o t t i s s u e . P h y s i o l . P l a n t . 37,97-103. Yurkova,GN; Levenko,BA and Novozhilov,PV (1982) P l a n t r e g e n e r a t i o n i n wheat t i s s u e c u l t u r e . Biochem. P h y s i o l . P f l a n z . 177,337-344. Zamir,D; Jones,RA and Kedar,N (1980) Anther c u l t u r e of m a l e - s t e r i l e tomato (Lycopersi con esculentum mi 11 . ) mutants. P l a n t S c i . L e t t . 17,353-361. Zhao,CZ; Zheng,KL; Qi,XF; Sun,ZX and Fu,YP (1982) The c h a r a c t e r s of r i c e somatic t i s s u e d e r i v e d p l a n t s and t h e i r progenies i n paddy f i e l d . A cta Genet. S i n . 9,320-322. Zhao,CZ; Sun,ZX; Zheng,KL; Qi,XF and Fu,YP (1984) A p p l i c a t i o n of somatic c e l l c u l t u r e t o r i c e v a r i e t y improvement. S c i e n t i a A g r i . S i n . 5,35-40. Appendix 1 Composition of B5 medium: m i n e r a l s a l t s and o r g a n i c compounds except growth r e g u l a t o r s M a c r o n u t r i e n t s mg/1 KNO3 2500 C a C l ^ ^ O 150 MgSCv7H zO 250 ( N H 4 ) 2 S 0 4 134 NaH2P04.H20 150 M i c r o n u t r i e n t s mg/1 KI 0.75 H 3B0 3 * 3.0 MnS0;H 20 10 ZnS04*7HzO 2.0 Na 2Mo0 4-2H 20 0.25 CuS0 4-5H 20 0.025 CoCl 2-6H 20 0.025 Na aEDTA 37.3 FeSO^I^O 27.8 Organic compounds mg/1 I n o s i t o l 100 N i c o t i n i c a c i d 1.0 P y r i d o x i n e * H C l 1.0 Thiamine-HCl 10.0 Sucrose 20000 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0096266/manifest

Comment

Related Items