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

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IN VITRO  CULTURE OF RED CLOVER (TRIFOLIUM  PRATENSE  EVALUATION OF REGENERATED PLANTS  by HONG WANG  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE  FACULTY OF GRADUATE STUDIES Plant  We a c c e p t to  THE  this  Science  t h e s i s as c o n f o r m i n g  the required  standard  UNIVERSITY OF BRITISH COLUMBIA O c t o b e r , 1985  ©  HONG WANG, 1985  L. ) AND  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 department  or  by  his or  her  representatives.  be  granted by the head of  It is understood  that  copying  publication of this thesis for financial gain shall not be allowed without my permission.  Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6(3/81)  my or  written  Abstract Red  clover  (2n=2X=14) and present plant  study  mg/1  'Altaswede'  'Norseman'. or  light  two  was  callus  shoot  2 mg/1  induction  adenine  naphthalene  results  acetic  to i n i t i a t e  conditions. induce shoots  mg/1  P i c l o r a m and  Callus  of the e x p l a n t s  were  cultured  higher frequency  than  were o b t a i n e d u n d e r test  tubes  acid  from  an  experiment  subculturing  mg/1  or p e t r i  dark plates,  on  B  5  2 mg/1  s h o o t s under  deteriorated  over  of g r o w t h  these c a l l i  i i  L2  mg/1  adenine,  have  media w h i c h  However, b o t h  the present  and  on  medium c o n t a i n i n g 2  confirmed  these c a l l i  from  the c a l l i  to  acid  that  that  media  experimental  LSE medium d i d not  callus  g r o w t h on  LSE  several subcultures.  v a r i o u s media were t e s t e d  differentiation  conducted  acetic  shoot-supportive.  combinations  was  2,4-dichlorophenoxy  and  Further tests  medium s t e a d i l y Subsequently,  by  (LSE) and  been r e p o r t e d t o be  different  s e e d l i n g s were  culture.  t o 85%  induction,  medium c o n t a i n i n g 0.01  failed  somaclonal  Calli  showing a s l i g h t l y  Satisfactory  initiation,  induction.  weeks o f  60%  i n the  vessels.  After  and  culture  aseptic  for callus  conditions using either  as c u l t u r e  regulate  tissue  i n t o L2 medium c o n t a i n i n g 0.06  frequency  'Altaswede'  (2n=4X=28) were u s e d  explants of  induced a f t e r  induction  cvs  the o c c u r r e n c e of  benzyladenine  usually  p r a t e n s e L.)  to i n v e s t i g a t e  Hypocotyl  inoculated  with  'Norseman'  r e g e n e r a t i o n and  variation.  0.1  (Trifolium  w i t h an  regulators. was  emphasis  Root  frequently  observed.  on  S h o o t s were  initiated  transferred  from SCP medium  acetic  and k i n e t i n .  was  acid  from  some c a l l i  t o media c o n t a i n i n g  Embryogenic  callus  naphthalene  of one  s e l e c t e d and m a i n t a i n e d on LSP medium, l e a d i n g  regeneration arginine,  o f numerous p l a n t s .  glutamic acid  a significant The  effect  source of c a l l u s  occurrence grew  on c a l l u s  and p r o d u c e d  s h o o t s were  more  Although  'Norseman' had more  multiple  shoots, the m u l t i p l e  'Altaswede' cultures  was h i g h e r t h a n  calli  from  that  number  biochemical  traits.  for  chromosome  one  RG2  plant  chromosome regarding  When  number,  RG1  their  induced to produce per c u l t u r e of  o f 'Norseman'.  Shoot t i p s h o o t s and  shoots (RG2),  (RG1), three-month  (RG4) were e v a l u a t e d f o r  morphology  'Altaswede' and RG3  Regenerated  isozyme  calli,  calli.  t o induce m u l t i p l e  and 23% o f 119 RG4  numbers.  tips  calli  'Altaswede'  'Altaswede'  multiple  calli  stability,  and t h e  'Norseman'  than  two s u b c u l t u r e s  (RG3) and o n e - y e a r  chromosome  of g r o w t h  organogenesis.  initial  shoots a f t e r  from  differentiation.  s h o o t number  regenerate plants v i a root Regenerants  and  Usually  shoot  were a l s o e s t a b l i s h e d  multiple  rates  roots  induced only  with  h y d r o l y s a t e d i d n o t show  growth  influenced  genotype to the  Supplementation  and c a s e i n  of d i f f e r e n t i a t i o n .  faster  while  to  when t h e y were  patterns  and  several  p l a n t s were a n a l y s e d  p l a n t s were n o r m a l ,  p l a n t s had  tetraploid  p l a n t s were q u i t e of malate  while  stable  dehydrogenase,  6-phosphogluconate  dehydrogenase,  phosphoglucose  phosphoglucomutase  and s h i k i m a t e d e h y d r o g e n a s e  isomerase,  and  their  nodule  leghaemoglobin  leaflet  length  'Altaswede' plants RG2  to width r a t i o  plants  stability  o f RG1,  RG2  more v a r i a t i o n  plants  I t i s suggested that  of 'Altaswede'  i n t h e RG4  origin  the absence  'Altaswede'  f r o m one o r i g i n a l  of r e g e n e r a t e d p l a n t s  iv  plants  the  a n d RG3 p l a n t s o f  o f 'Norseman' were n o t d i f f e r e n t  differences their  Morphologically,  showed s i g n i f i c a n t l y  (P<0.01), w h i l e RG4  plants.  of  profiles.  of  and RG1  and  from c o n t r o l detectable  was a  genotype.  than c o n t r o l  consequence  Variability  are discussed.  and  Abbreviations  f o r Growth R e g u l a t o r s  and M e d i a  Auxins 2,4-D: 2 , 4 - d i c h l o r o p h e n o x y a c e t i c IAA:  indole-3-acetic  NAA:  naphthalene a c e t i c  PIC:  Picloram  acid  acid acid  (4-amino-3,5,6-trichloropiclinic  acid)  Cytokinins ADE: a d e n i n e BA:  (6-amino  purine)  benzyladenine  2iP:  (2-isopentenyl)  KIN:  kinetin  adenine  Media L2:  Phillips  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  LSP:  L2 meidum c o n t a i n i n g  0.002 mg/1  SCP:  L2 medium c o n t a i n i n g  2 mg/1  ADE.  v  2,4-D and 2 mg/1 PIC and 0.2 mg/1  2,4-D, 2 mg/1  ADE BA  BA and 2 mg/1  Table  of Contents  Chapter  Page  Abstract  i i  Abbreviations Table  f o r Growth R e g u l a t o r s  and M e d i a  of C o n t e n t s  List  of T a b l e s  List  of F i g u r e s  i i i vi x x i i  Aknowledgement  xiv  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 S o m a c l o n a l V a r i a t i o n 2.2.1  2.2.2 2.2.3 3.  .7  V a r i a t i o n among R e g e n e r a t e d P l a n t s o f S e v e r a l Crop S p e c i e s  .8  2.2.1.1  Sugar Cane  2.2.1.2  Potato  .9  2.2.1.3  Tobacco  11  2.2.1.4  Rice  12  a n d Wheat  O r i g i n o f and F a c t o r s Somaclonal V a r i a t i o n Genetic  8  Basis  Affecting 13 17  MATERIALS AND METHODS  22  3.1 G e n e r a l  22  C u l t u r e Methodology  3.1.1  Media  22  3.1.2  Explants  24  3.1.3  Culture Conditions  25  3.2 S p e c i f i c 3.2.1  C u l t u r e Methodology  Callus  Induction vi  26 26  3.2.2  C a l l u s M a i n t e n a n c e and P l a n t Regeneration  :  3.2.2.1  Primary Test  :  3.2.2.2  C a l l u s Growth  3.2.2.3  2,4-D  3.2.2.4  PIC and BA  :  3.2.2.5  NAA  a n d KIN  :  3.2.2.6  NAA  a n d BA i n P r e s e n c e o f ADE  3.2.2.7  Callus Differentiation T e s t Media  3.2.2.8  C a l l u s Growth a t H i g h o r Low Auxin to Cytokinin Ratios  3.2.2.9  C a l l u s Growth  o f Two M e d i a on L S E Medium  and BA  :  3.2.3  Shoot T i p C u l t u r e  3.2.4  R o o t i n g of Shoots  3.2.5  Scanning E l e c t r o n Microscopy and A n a l y s i s  Growing  36  and  '  38  of Regenerated Plants  3.3.1  Plant  Transfer  and Growth  3.3.2  Chromosome A n a l y s i s  3.3.3  Leghaemoglobin  3.3.4  Isozyme  3.3.5  Morphological  Analysis  Analysis Analysis  RESULTS 4.1  on F o u r  S e l e c t i o n and M a i n t e n a n c e o f Embryogenic C a l l u s  3.2.2.12 Growth o f E m b r y o g e n i c Non-embryogenic C a l l i  3.3 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 on SCP Medium i 3.2.2.11  :  C a l l u s Induction vii  i n Greenhouse  ..  4.1.1  Callus  Induction  Frequency  4.1.2  Genotypic E f f e c t  on C a l l u s  4.1.3  O b s e r v a t i o n s on C a l l u s  4.2 C a l l u s M a i n t e n a n c e a n d P l a n t  47 Induction  49  Induction  49  Regeneration  52  4.2.1  Primary Test  4.2.2  C a l l u s Growth  4.2.3  2,4-D  4.2.4  PIC and BA  61  4.2.5  NAA  and KIN  64  4.2.6  NAA  and BA i n P r e s e n c e o f ADE  73  4.2.7  Callus Differentiation  4.2.8  C a l l u s Growth Ratio  and A u x i n t o C y t o k i n i n  C a l l u s Growth  on SCP Medium  4.2.9  52  on L S E Medium  53  and BA  4.2.10 L i g h t E f f e c t Medium 4.2.11  o f Two M e d i a  61  on F o u r M e d i a  75  on C a l l i  Growing  79 on SCP 84  O b s e r v a t i o n s on E m b r y o g e n e s i s  4.2.12 S e l e c t i o n Calli  84  and Maintenance of Embryogenic 87  4.2.13 Growth o f E m b r y o g e n i c Non-embryogenic C a l l i  and  4.2.14 Summary o f E x p e r i m e n t s on C a l l u s  91 Growth  and D i f f e r e n t i a t i o n 4.2.15 P l a n t  R e g e n e r a t i o n from C a l l u s  4.3 S h o o t T i p C u l t u r e Multiple  Shoot  4.3.2  Multiple  Shoot P r o p a g a t i o n  4.4.1  and A n a l y s i s Plant  94 95 96  4.3.1  4.4 G r o w t h  75  Induction  of Regenerated P l a n t s  Transfer  and S u r v i v a l  viii  96 96 ......100 101  4.4.2  Chromosome Number  4.4.3  Isozyme  4.4.4  Nodule F o r m a t i o n and Leghaemoglobin Profile  114  P l a n t Morphology  114  4.4.5 5.  Stability  Analysis  106  DISCUSSION  121  5.1  121  Callus Induction  5.2 C a l l u s M a i n t e n a n c e and P l a n t  Regeneration  122  5.2.1  Primary Test  5.2.2  C a l l u s Growth  on L S E Medium  123  5.2.3  C a l l u s Growth  on SCP Medium  125  5.2.4  P l a n t R e g e n e r a t i o n : O r g a n o g e n e s i s and Embryogenesis  126 .  Genotype  133  5.2.5  6.  103  o f Two M e d i a  Effect  ...122  '  5.3 S h o o t T i p C u l t u r e  136  5.4 R e g e n e r a t e d P l a n t s  136  Summary  143  LITERATURE CITED  146 •  APPENDIX  158  1  ix  List  of  Tables  Table 1.  Page Treatment combinations used t o t e s t the e f f e c t o f PIC and BA on p l a n t r e g e n e r a t i o n .  31  C a l l u s i n d u c t i o n f r e q u e n c y f o r two c u l t i v a r s o f r e d c l o v e r on L2 medium.  48  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 t h e same s e e d l i n g .  50  4.  Callus  53  5.  R e s p o n s e o f 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 o f T e s t 1 r e c o r d e d a f t e r f o u r and f i v e weeks o f c u l t u r e , respectively.  55  R e s p o n s e of c a l l i f r o m L S E medium s u b c u l t u r e d onto four media.  59  2. 3.  6.  g r o w t h on LSE medium.  7.  Response  8.  R e s p o n s e of c a l l i on m e d i a c o n t a i n i n g and BA r e c o r d e d a f t e r t h r e e weeks of culture.  9.  10. 11. 12.  13. 14. 15.  of c a l l i  on L2(D0.01A5)  medium.  60  2,4-D 62  R e s p o n s e of ' A l t a s w e d e ' c a l l i on media c o n t a i n i n g PIC and BA i n p r e s e n c e and a b s e n c e of a r g i n i n e .  63  R e s p o n s e o f c a l l i on m e d i a w i t h added c a s e i n h y d r o l y s a t e and g l u t a m i c a c i d .  65  R e s p o n s e o f c a l l i o f two c u l t i v a r s on media w i t h v a r i o u s c o m b i n a t i o n s o f PIC and BA.  67  R e s p o n s e o f 'Norseman' c a l l i from LSE medium t o v a r i o u s c o m b i n a t i o n s of NAA and KIN.  68  R e s p o n s e o f c a l l i from SCP medium t o c o m b i n a t i o n s o f NAA and KIN.  70  R e s p o n s e o f 'Norseman' c a l l i t o c o m b i n a t i o n s o f NAA and KIN.  72  R e s p o n s e o f '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 o f NAA i n c o m b i n a t i o n w i t h BA and ADE.  74  x  16. 17. 18. 19. 20.  21. 22. 23. 24. 25. 26. 27.  28.  29.  D i f f e r e n t i a t i o n of 'Norseman' c a l l i L2(D2B2A5) medium on f o u r m e d i a .  from 76  R e s p o n s e of 'Norseman' c a l l i a t h i g h (11) or low (0.1) a u x i n t o c y t o k i n i n r a t i o .  77  Recovery transfer  80  f r e q u e n c y of s e n e s c i n g c a l l i t o SCP medium.  after  L i g h t e f f e c t on g r o w t h and d i f f e r e n t i a t i o n of ' A l t a s w e d e ' c a l l i on SCP medium.  85  Embryo d e v e l o p m e n t and from e m b r y o g e n i c c a l l i medium.  89  p l a n t l e t formation s u b c u l t u r e d o n t o LSP  M u l t i p l e s h o o t i n d u c t i o n from s h o o t t i p s r e d c l o v e r on L 2 ( P 0 . 0 0 3 B 2 ) mediium. C o m p a r i s o n of two c u l t i v a r s of for m u l t i p l e shoot i n d u c t i o n . M u l t i p l e shoot p r o d u c t i o n of of r e d c l o v e r .  red  two  of 97  clover 97  cultivars 99  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 t o greenhouse a f t e r d i f f e r e n t treatments.  102  Chromosome numbers of ' A l t a s w e d e ' p l a n t s r e g e n e r a t e d t h r o u g h d i f f e r e n t ways.  104  L e a f l e t number i n c o n t r o l and r e g e n e r a t e d p l a n t s of  116  (seed-derived) red c l o v e r .  Means and v a r i a n c e s o f l e a f l e t l e n g t h / w i d t h 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 r e d clover.  118  C o m p a r i s o n s o f r e g e n e r a t e d 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 length/width r a t i o .  119  C o m p a r i s 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 f o r t h e v a r i a n c e s of l e a f l e t length/width r a t i o .  119  xi  List  of  Figures  Figure 1. 2. 3. 4.  5. 6.  Page C a l l u s i n d u c t i o n and g e n o t y p e 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  Response of 'Altaswede' c a l l i a f t e r two weeks o f c u l t u r e .  on L S E medium 56  Response o f ' A l t a s w e d e ' c a l l i a f t e r f i v e weeks o f c u l t u r e .  on L S E medium 57  Response o f ' A l t a s w e d e ' c a l l i on L2 medium c o n t a i n i n g 0.005 mg/1 P I C , 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 o f culture.  66  Shoot f o r m a t i o n from L2(N0.05K2) medium.  71  'Altaswede' c a l l u s  R e s p o n s e o f 'Norseman' c a l l i d i f f e r e n t auxin to c y t o k i n i n five  on  on media w i t h ratios after  weeks o f c u l t u r e .  78  7.  Recovery  8.  C a l l u s maintenance  9. 10.  Embryogenic c a l l u s of 'Altaswede'. S o m a t i c embryo d e v e l o p m e n t from e m b r y o g e n i c c a l l u s of 'Altaswede'.  11. 12. 13.  of s e n e s c i n g c a l l i  S e l e c t i o n of c a l l u s embryogenisis.  on SCP medium.  on SCP medium.  81 82 86 88  c a p a b l e of 90  E m b r y o g e n i c c a l l u s a r i s i n g from r e g e n e r a t e d p l a n t l e t a t the point of root o r i g i n .  92  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 'Altaswede'  93  of  14.  Multiple  15.  K a r y o t y p e of normal r o o t t i p of r e d c l o v e r cv. ' A l t a s w e d e ' (2n=2X=14).  105  Root t i p chromosome numbers o f a d i p l o i d / t e t r a p l o i d RG4 p l a n t o f 'Altaswede'.  107  16.  shoot p r o d u c t i o n  calli  xii  i n red clover.  98  17. 18. 19. 20.  21.  Zymogram o f p h o s p h o g l u c o s e i s o m e r a s e from RG3 p l a n t s o f r e d c l o v e r c v . 'Altaswede'.  110  Zymogram o f p h o s p h o g l u c o m u t a s e from of r e d c l o v e r c v . 'Altaswede'.  111  calli  Zymogram o f m a l a t e d e h y d r o g e n a s e f r o m RG3 p l a n t s of red c l o v e r c v . 'Altaswede'.  112  Zymogram o f 6 - p h o s p h o l g u c o n a t e d e h y d r o g e n a s e from RG3 p l a n t s o f r e d c l o v e r cv. 'Altaswede'.  113  Cellulose acetate electrophoresis leghaemoglobin of r e d c l o v e r c v . 'Altaswede'.  115  xi i i  of  Aknowledgement I am  most g r a t e f u l t o Dr.  his direction,  F.B.Holl,  encouragement and  of my  s t u d i e s , and  Drs.  A.D.M.Glass, C . R . N o r t o n and  reviewing  the  Sincere help  i n the  g u i d a n c e and materials. and  to the  help  d r a f t and thanks are  help  students and  t o Mr.  L.J.Veto  assistance  with  Ministry the  to Drs.  advice  with  Sun  p h o t o s of  respect  for  her  for  his  cultured  R.R.Smith,  of  the  appreciation  Department my  of  E.Rosenberg  t o methods f o r  Education  of  the  encouragement  to the  Plant  financial  I thank them f o r t h e i r  support  f a m i l y and  commitments.  xi v  who  staff  provided  UBC.  People's Republic of my  faculty,  Science  s t u d i e s at  a l s o g r a t e f u l f o r the of  SEM  their  analysis.  continuing  China.  the  period  comments.  isozyme a n a l y s i s and  friendship during  I am  for  Mei  I want t o e x p r e s s my and  V.C.Runeckles  useful  for  committee,  t o Ms.  for their  cytogenetic  thesis  the  extended  Thanks a l s o go  K.Cole  supervisor,  throughout  members of my  very  my  of  of  the  China  friends in  and  1.  Legume s e e d and important  food  agriculture. and  and The  forage  feed  and  f o r the  d e p e n d s upon e f f i c i e n t  to  are  future  the  from c e l l  of  regeneration  legume s p e c i e s  and  p e r h a p s even more d i f f i c u l t  has  than g r a s s e s  species,  (Conger,  been made r e c e n t l y  regeneration sativa  L.)  (Trifolium Red value  clover  (Taylor,  i s an 1973;  f o r use  improving  properties.  soil  between  thus g r e a t l y fertilizer Plant of was  125  as  u s e d and  200  reducing  hay  and  kg  the  regeneration  of  Smith,  frequency  of  of  progress  plant  red  (Medicaqo  clover  Collins,  1979).  1983).  It  feed is also  to- i t s s o i l  per cost  from s u s p e n s i o n and  by  in  return ha. of  to  the  annually, nitrogen  crops.  tissues  previously  to manipulate  c r o p may  f o r and  been  legumes,  c r o p w i t h good  nitrogen  need  (e.g.  was  and  red  clover  C o l l i n s (1979a;  dependent  regeneration  1  callus cultures  h y p o c o t y l ) of  Phillips  However, r e g e n e r a t i o n the  and  A red c l o v e r  f o r subsequent  reported  1980).  important  and  difficult  I t has seed  cell  cultured  often  alfalfa  a r o t a t i o n c r o p due  and  non-meristematic  1984)  (Phillips  Taylor  excellent  achievement  Walker,  p r a t e n s e L.)  crops  However, much  legumes s u c h as  (McCoy and  these  e s p e c i a l l y the  1981).  i n the  i n forage  plant  callus cultures.  are  vitro  of  are  these  or  in world  from the  suggested that equally  s e c o n d most  utility  improvement  plant  Unfortunately  regenerate  species  crops a f t e r c e r e a l s  present  tissue culture  materials.  INTRODUCTION  on  the  genotype  from h y p o c o t y l  callus  2 was  low  (Phillips  regeneration or a  larger  and  c o u l d be number of  Collins,  1979a).  Therefore, plant  a p r o b l e m when u s i n g a n o t h e r regenerated  p l a n t s are  cultivar,  required for  study. It a  i s b e l i e v e d that somaclonal  novel  source  breeding work on  of v a r i a t i o n  programs the  development observed  of  t o be  ijn v i t r o  1979a).  be  represent  exploited  in plant  Scowcroft,  1981).  of  red c l o v e r  f o c u s s e d on  methods.  normal both (Beach  t h a t can  may  and  tissue culture  cytogenetically Collins,  (Larkin  variation  Smith,  Information  the  R e g e n e r a t e d p l a n t s were  morphologically  and  Previous  1979;  Phillips  is lacking  a b n o r m a l i t i e s a r e a common phenomenon  and  as  and  t o whether  in regenerated  red  clover plants. Most tissue  reports concerned  culture  characters. easily  variation  by  environmental  a l s o more d i f f i c u l t  cane  The  experiments  experimental (1) from  factors,  the  Irvine  (1984),  to  o n l y a low  conducted  frequency  here,  investigate  hypocotyl  the  regulation  segments and  variation.  It is  of v a r i a t i o n  using f i v e  for  sugar  distinctive  of s t a b l e  using red clover  m a t e r i a l , were d e s i g n e d  and  somaclonal  frequency  (Saccharum o f f i c i n a r u m ) c l o n e s w i t h  markers, observed  induced v i a  quantitative  environmental  to assess  characters.  on  changes  characters are v a r i a b l e  i s c o n f o u n d e d by  quantitative  the  have been c e n t e r e d  Because these  affected  with  f o r two  of c a l l u s  main  variation. as  the  purposes:  culture  p l a n t r e g e n e r a t i o n from  derived the  c u l t u r e s and  (2) t o e v a l u a t e  somaclonal v a r i a t i o n  the  regenerated  i n the c h a r a c t e r s  plants for  analysed.  2.  2.1 T i s s u e C u l t u r e Niizek red  clover  and  i n Red  Kita  Clover  (1973) i n i t i a t e d  (1961) and  IAA  and  M M BA  0.6  investigators  cultures  initiated  reports described l i t t l e plants  were  successful 5  10  1976;  uM  each 5  was  formed when 2,4-D  callus than  on  B  5  KIN.  resulted.  ovary  (1967) and  GA  on medium w i t h  17  No  report other 1976)  described  tissues.  These  o f 2,4-D  and  substituted  differentiation. 15 M M ADE  no  was  hypocotyls  present  Hypocotyl Callus  explants  KIN. by  the m i n e r a l s a l t s (1962).  to another  After  medium f o r  w i t h 20 mg/1  5  t h e most e f f e c t i v e  4  produced most  or a m o d i f i e d  11  uM  C a l l u s p r o d u c t i o n was  Skoog  2,4-D  f o r m a t i o n was  thiamine,  and  in  (Gamborg e t a l . , 1968)  Gamborg's B was  seedling  In t h e a b s e n c e o f  tissue.  medium  were t r a n s f e r r e d  and  of The  f o r m o r p h o g e n e s i s and  (1979) c u l t u r e d  (1966) o r M u r a s h i g e and calli  BA  induced.  different  evidence  Nitsch  NAA,  Ranga Rao,  b a s a l medium c o n t a i n i n g 10 mg/1  when B  NAA  callus  from  w i t h a u x i n and  o r no  more c a l l u s  B  Smith  Callus  combination little  culture  regenerated.  B e a c h and ovaries.  Only  Following this  (Ahloowalia,  and  o f IAA,  induction.  a d d e d was  morphogenesis o c c u r r e d .  callus  Bourgin  with v a r i o u s combinations  were e v a l u a t e d f o r c a l l u s MM  the t i s s u e  u s i n g a n t h e r s as e x p l a n t s t o induce c a l l u s .  b a s a l media of M i l l e r modified  LITERATURE REVIEW  of  NAA  and  poor  Blaydes  f o u r weeks shoot-bud thiamine,  combination.  10  uM  Beach  5 and  Smith  callus green  (1979) a l s o  with  callus,  BA f a i l e d of  Phillips first  that  green  that  shoot  differentiation  intensive  investigations (1979a;  induced  cultures  development  formation.  have been c o n d u c t e d They  u s i n g more t h a n  o f a m o d i f i e d medium,  for callus  Visual  125 media  referred  by  (1979a)  requirements  from h y p o c o t y l segments.  produced  KIN, 2 i P and  and t h e a d d i t i o n  1980; 1981).  e v a l u a t e d the b a s a l n u t r i e n t  friable  than u n i f o r m l y  into plants.  m i l k d i d not enhance c a l l u s  and C o l l i n s  cultures  i t was t h e w h i t e  segments, r a t h e r  differentiated  t o induce  coconut More  of  isolated  observed  evaluation  l e d to the  t o as L2.  This  medium was shown t o be more s u p p o r t i v e o f r e d c l o v e r genotypes was u s e d  in callus culture as the auxin  efficient  i n action  Callus PIC  was  PIC,  than  2,4-D  BA, a c o m b i n a t i o n  of s e v e r a l  calli.  The f r e q u e n c y  1% ( P h i l l i p s  and C o l l i n s ,  IAA,  from  NAA,  1979a).  Plant  were s u b c u l t u r e d o n t o calli  were more  that  gave r e g e n e r a t i o n  was 30% t o 80% b u t f o r t h e l a t t e r  and C o l l i n s ,  mg/1  than non-meristem d e r i v e d  of genotypes  1979a).  non-meristem d e r i v e d c a l l i , combination  including  Generally meristem-derived  the former  e t a l . , 1978).  which had r e s u l t e d  regulators  capable of r e g e n e r a t i n g p l a n t s  for  (Collins  was a c h i e v e d when c a l l i  other media.  PIC  s o u r c e , a s i t was shown t o be more  2,4-D, BA and KIN ( P h i l l i p s  regeneration  o t h e r media t e s t e d .  i n d u c e d on L2 medium c o n t a i n i n g 0.06  a n d 0.1 mg/1  the e v a l u a t i o n  than  i t was o n l y  F o r r e g e n e r a t i o n from  a 2,4-D-containing  medium ( i n  w i t h o t h e r a u x i n s ) was more e f f e c t i v e  than the  6 PIC  + BA  combination.  Plant  r e g e n e r a t i o n was  suspension c u l t u r e of  h y p o c o t y l and  into BA and  liquid  (Phillips  epicotyl  cell  initiation  was  mg/1  and  2.0  2,4-D  found  0.003 mg/1  mg/1  tips  ADE  PIC  c o u l d be and  0.5  other experiments t o 2 i P and  concentration Other  was  of  by KIN,  2.0  ijn v i t r o  (1979b) e s t a b l i s h e d  Callus  mg/1  recovery  Both  the  bud  PIC  and  A  0.01  second  0.2  mg/1  from  BA  buds.  on a medium c o n t a i n i n g and  Collins,  1983).  Tomes (1983) BA  was  number a t  the  BA. have been c o n d u c t e d  use.  free plants.  Phillips  cold  and  embryos  (1980).  to  techniques  (Evans,  e t a l . , 1982)  s t o r a g e of shoot  Cheyne and D a l e  culture  Embryo c u l t u r e  Phillips  to  Collins  f o r meristem  immature h y b r i d  e t a l . , 1981;  by  0.1  for  development  and  the procedures  methods f o r l o n g - t e r m  put  containing  w i t h maximum s h o o t  experiments  were e m p l o y e d t o r e s c u e  been d e v e l o p e d  mg/1  (Parrot  Campbell  mg/1  virus-symptom  Collins  BA  and  agar media.  proliferated  mg/1  Explants  most e f f e c t i v e .  f o r shoot  d e v e l o p methods o f s p e c i a l  1962;  PIC  ( d e s i g n a t e d LSE)  t o be e f f e c t i v e  Shoot  obtain  on  ( L S P ) , w h i c h c o n t a i n s 0.001  superior  mg/1  c o n c e n t r a t i o n o f a u x i n were c r i t i c a l t h e medium  In  1980).  a s m a l l amount o f  inoculated  and  was  after  cell  o f s e e d l i n g s were  suspension c u l t u r e s .  initiation  medium  Collins,  sections  resulted  suspension c u l t u r e k i n d and  and  L2 medium c o n t a i n i n g 0.06  to i n i t i a t e bud  a l s o a c h i e v e d from  and  cultures  have  7  2.2  Somaclonal The  from  phenomenon o f v a r i a b i l i t y  cell  and  d e c a d e ago However,  c u l t u r e s was  (Sacristan  has  Scowcroft  describe  tissue  i t i s only  attention and  Variation  and  to i t .  (1981) u s e d  the v a r i a t i o n  importance  tissue  culture  published been  of t h i s  years that  "somaclonal  Since the  phenomenon  Larkin  variation"  recognition  i n the a p p l i c a t i o n  b r e e d i n g , numerous p a p e r s  in other experiments  1981;  1983a; L o r z ,  1983;  E v a n s e t a l . , 1984). plants  somaclonal  somaclonal  1983;  identical  variation  v a r i a n t s as  least  much  Somaclonal  Scowcroft  and  Wherever  i s a hazard.  have  been has  although  and  has  Scowcroft, 1983;  the o b j e c t i v e  Reisch,  i s to  for propagation,  When t h e o b j e c t i v e for plant  i s to  improvement,  r e p r e s e n t s a n o v e l s o u r c e of  i n terms of t h e method by  as  of  variation  Larkin,  t o p a r e n t a l type  source m a t e r i a l  variation  (Larkin  to  of  of c h a r a c t e r e x p r e s s i o n i n r e g e n e r a t e d p l a n t s  been o b s e r v e d  select  1971).  article  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 ,  obtain  a  among r e g e n e r a t e d p l a n t s  a d d r e s s i n g the problem.  stability  at  Morel,  In a r e v i e w  culture.  to p l a n t  1969;  few  the term  observed  a c o n s e q u e n c e of t i s s u e the  last  plants  r e p o r t e d more t h a n  Melchers,  i n the  been p a i d  among r e g e n e r a t e d  variation,  which v a r i a t i o n  is  generated. A tissue involves  culture cycle  refers  t o the procedure  t h e e s t a b l i s h m e n t o f a more o r  dedifferentiated (multicellular  cell  or t i s s u e  culture  explants, isolated  single  which  less from  diverse  cells  or  sources  8  protoplasts), of The as  cell  regenerated  concerning  genetic  responsible  this  for  the  may  proliferation  final exhibit  are  numerous  the  observed  In  sexual  this  of  in  such  reproduction  phenomenon  yet  variation  some be  could  of  i s not  (Reisch,  of  will  which  variation  observed  examples  aspects  biochemical  abnormalities the  number  plants.  abnormalities  some c a s e s  a  proposals  phenotypic  these  representative of  variation  r e s i s t a n c e and  cytogenetic  of  for  regeneration  and  origin  generalities  2.2.1  the  disease  through  thesis  media,  there  understood.  transmissable  the  yield,  Although  regenerants,  In  and  plants  morphology,  clearly  defined  generations  characters.  be  on  is  1983).  species  and  reviewed.  V a r i a t i o n among R e g e n e r a t e d P l a n t s o f S e v e r a l C r o p Species 2.2.1.1  Sugar  One  Cane  interesting  variation  in  sugar  disease-resistant susceptible  and  cane  important has  regenerants  cultivars.  cytogenetic  and  Heinz,  1969;  resistance  to  virus)  downy  Heinz  and et  increased even  Fiji  disease  mildew  a l . , 1977).  A  r e s i s t a n c e was  though  the  and  1973),  variety  (a  the  from  Following  morphological, Mee,  been  aspect  tissue  efforts  w e r e made  (Sclerospora  used  for  shift  to  seek  (cf.  toward  both  initiation  diseases of  in  (Heinz  transmitted  sacchari)  for  of  variation  traits  leafhopper  of  cultures  reported  predominant  somaclonal  appearance  isozyme  observed  of  tissue  9 culture  was  apparently r e s i s t a n t .  among s u g a r confirmed  cane  (e.g.  Scowcroft,  to eyespot  1981).  Q47  Subsequently, cultures  Evaluation  generations  indicated  could  be  side  somaclones  Larkin  and  of  shifted  of the  Q101  resistant and  Scowcroft  d i s e a s e and  and  susceptible  sp.),  (Larkin  was  plants  was  from  a s u g a r cane  somaclones  tolerance.  Larkin  which  resistant  from  to eyespot  to i s o l a t e  variation  Q101,  t o the r e s i s t a n c e  remained  diseases  Interestingly,  disease (Helminthosporium  susceptible able  1983b).  whereas a l l 52  initiated  to several  e t a l . , 1977;  from c u l t i v a r  significantly reaction,  Heinz  1981;  regenerated  cultivar  somaclones  Extensive variation  which  Scowcroft, (1983b) was  from w h i c h  highly t h e y were  with varying degrees  of  subsequent v e g e t a t i v e  that  at  retained  least  some o f  the  through v e g e t a t i v e  propagation.  2.2.1.2 P o t a t o Potato "is another variation et a l . of  has  s p e c i e s where  been e x t e n s i v e l y  Burbank'.  significant  and  stable  Among  variation  growth h a b i t ,  maturity  d a t e and  yield.  Shepard,  1981)  selected  leaf  65  from  leaf  Shepard protoplasts  10,000 s o m a c l o n e s  morphology, p l a n t  from  demonstrated.  (1980) r e g e n e r a t e d p l a n t s  'Russet  somaclonal  was  found  for tuber  photoperiod  In a n o t h e r c l o n e s of  study  response, (Secor  'Russet  p r o t o p l a s t s were a n a l y s e d f o r 35  screened  and  Burbank'  characters.  10 Significant  v a r i a t i o n was  characters Burbank'  and  or more c h a r a c t e r s .  f r o m a minimum o f 17  similar  result  all  23  the  other  one  character  characters  analysed.  was  obtained  or more of  Only  one  the  Morphological  instability  have a l s o been  ( K a r p e t a l . , 1982; 1985).  resistance blight 1981).  out  of than  more r e s i s t a n t t o retained  through  uniformity  among t h e  protoplast  cultures.  results  Shepard  (1979) may ploidy  be  level  (Shepard:  each  characters type  was  chromosome  i n other  studies  Creissen  were s c r e e n e d  and  for  ( A l t e r n a r i a s o l a n i ) and  parent;  blight.  20  out  This  and  late  Shepard,  et a l .  plants  discrepancy (1980) and  a t t r i b u t e d to genotypic ( S h e p a r d : 4X;  (Shepard: l e a f  800  expression  remarkable  from  potato  between  the  Wenzel e t a l . difference,  W e n z e l : 2X); and  were  generations.  (1979) o b s e r v e d  regenerated The  of  altered  several vegetative  10,000; W e n z e l : 211)  protoplasts  from  s o m a c l o n e s were more r e s i s t a n t  the  late  (1982);  parental  (Bidney  to  A  10 m o r p h o l o g i c a l  reported  In c o n t r a s t , W e n z e l e t a l .  of  somaclone.  the  ranged  somaclones  plants differed  infestans) 500  'Russet  Variation  Ramulu e t a l . , 1983;  (Phytophthora Five  from  v a r i a t i o n and  to e a r l y b l i g h t  the  Thomas e t a l .  Regenerated p l a n t s  to e a r l y b l i g h t  was  by  s o m a c l o n e of  recovered.  Karp,  of  in three  f o r one  protoplast-derived  i n one  f o r 22  a l l somaclones d i f f e r e d  i n one  a maximum of  found  sample  size  t i s s u e source  of  m e s o p h y l l ; W e n z e l : stem t i p  11  culture).  Thomas e t  t e c h n i q u e as among t h e  al.  Wenzel e t  (1982) u s i n g  al.  cultivar  of  same  (1979) o b s e r v e d v a r i a t i o n  protoplast-derived  tetraploid  the  regenerated plants  of  a  potato.  2.2.1.3 T o b a c c o In  t o b a c c o a number of  variability Evidence by  in plants  for  Burk and  derived  Matzinger an  (1976).  inbred  following  ensure a high  degree of  to c h a r a c t e r s  s u c h as  flowering  total  and  a n a l y s i s of  Burk and  Chaplin  hybrid,  of  and  among  was  1,666  regenerated the  f o r two  of  and  plants.  In  were i d e n t i f i e d  the  loci  79  colonies  out  A more  c a r r i e d out initiated  a n a l y s i s of the  at each locus  t o be  to  by from a  affecting chlorophyll  Genetic  79  of  respect  investigated.  cultures  a recent  (Su/su),  with  index, days  indicated that 3.6%  to  S e l f e d progeny  study  from m e s o p h y l l p r o t o p l a s t s  s u l f u r locus  colonies 75%  (1980) u s i n g  15  a pure l i n e ,  r e g e n e r a n t s was  leaf color.  3.5%  as  for  Significant variability  characters  somaclonal v a r i a n t s  mutation  'Coker'  grade  i n work  authors  were a n a l y s e d  alkaloids.  heterozygous  synthesis  lines  culture.  reported  inbreeding.  yield,  shown f o r a l l t e n  detailed  variety  described  i_n v i t r o  These  i t s release  tissue culture-derived  was  from  have  s o m a c l o n a l v a r i a t i o n was  self-fertilized generations  reports  of  on  frequency  plants  heterozygous  2,156  t o be  of  respectively  for  morphogenic  n o n - p a r e n t a l and  were c l a i m e d  progeny  about  somaclonal  12 variants  because  different the  genotypes  selfed  ( L o r z and  regenerated plants  segregation the  they produced  ratios  r e g e n e r a t e d p l a n t s of S c o w c r o f t , 1983). 37%  (33/96)  which d e v i a t e d  e x p e c t e d 1:2:1  Among  had  significantly  from  ratio.  2.2.1.4 R i c e and Wheat Oono  (1978) and K u c h e r e n k o  i n h e r i t a n c e and v a r i a t i o n s sativa  L.)  somaclones.  height  and  intensive  tillering  s t u d y of v a r i a t i o n  p r o g e n i e s has been  cited  i n Sun  et a l . ,  inheritance  of r i c e  of  third  for  in rice  was to for  mutant  traits  varied  from  11.5  1984).  in yield,  Variation  of  only  24%  traits variation A  by a r e c e s s i v e  gene  observed f o r plants  reduced  experiments d e s c r i b e d  (Zhao e t a l . ,  When  t o 39.5%.  t o p r o d u c e more p r o d u c t i v e  breeding l i n e s  1981  i n the second  f r e q u e n c y of  controlled  t o be  and  f o r some t r a i t s  T r e n d s were a l s o  1000-grain weight  promising  The  w h i c h was  shorter,  More r e c e n t  1983).  An  1982).  f o r a l l the  high plant  (Sun e t a l . ,  Zhao e t a l . ,  were n o r m a l  (Sun e t a l . ,  identified. be  like  somaclones  out  of and v a r i a t i o n s  lines  different  dwarf  rather  g e n e r a t i o n s of r e g e n e r a t e d p l a n t s ,  the r i c e  studied  (Oryza  for traits  more t h a n 2,000 p l a n t s were i n v e s t i g a t e d and  the  of somaclones.  carried  1983;  studied  showed t h a t  occurred  i n progeny  their  the  of t r a i t s  Results  f r e q u e n c i e s of v a r i a t i o n  (1979)  tillers  (Sun e t a l . ,  and 1983).  the i d e n t i f i c a t i o n maturity  in grain  and  protein  of  height  13 content  has  also  dihaploids  regenerated  (Schaeffer  e t a l . , 1984).  In wheat embryos a s  1978;  1984).  Somaclonal  on  al.,  1982;  variant which  K a r p and  Maddock,  1984;  has  142  and  date,  d o m i n a n t and  that  chromosome l o s s  the primary  Origin  source  o f and  i s important  practical  use  variation  simply  been r e p o r t e d f o r  Lupi,  cells  the c u l t u r e  regenerated  or a d d i t i o n  of  both  whether the  more  (1984) r e p o r t e d  and  of  'Yaqui  number,  50E'  grain gliadin  the o c c u r r e n c e They  were n o t  of  claimed  evident  Somaclonal  as  L o r z and  cycle  Variation  i n t e r e s t s and observed  the v a r i a b i l i t y  b e f o r e the c u l t u r e  p l a n t s from  Recently  variation.  f o r academic  to determine  mainly  Yurkova et  regenerants  Factors Affecting  process.  was  1981;  1984).  regulation  and  L a r k i n et a l . ,  r e c e s s i v e mutations.  reflects  O'Hara  w a x i n e s s , glume c o l o r ,  a-amylase  both  immature  of p l a n t  the a n a l y s i s  work by L a r k i n e t a l .  heading  protein  somatic  but  (e.g.  c h a r a c t e r s among  using  1977;  i n c l u d e d h e i g h t , awns, t i l l e r  color,  of  plants  L.)  Hayward,  variation  of  callus cultures  efficiency  Maddock,  in several cases,  intensive  It  aestivum  and  K a r p and  regenerated  anther  improved  (Bhojwani  Street,  wheat  from  (Triticum  explants  regeneration  2.2.2  been d e s c r i b e d among p r o g e n i e s  or  for  genetic  pre-existent in is a  Scowcroft  p r o t o p l a s t s of Su/su  consequence  (1983) heterozygotes  14 of  Nicotiana  estimated  tobaccum and,  using  leaf color  analysis,  that  the  majority  parental  type,  but  1.4-6.0% were c u l t u r e - i n d u c e d  and  0.1-1.8% p r e - e x i s t i n g v a r i a n t s .  only  (1982) f o u n d t h a t morphological from t h e  considerable  characters  variation  cultivar, was  cell-culture  used,  length  Thomas e t a l . for  of  clearly  induced. depend  that  the  culture period  of  a  occurrence  genotype  or  potato  the  However t h e on  ten  of  from a s i n g l e c e l l  demonstrating  were  variants  among r e g e n e r a t e d p l a n t s  s o m a c l o n a l v a r i a t i o n may  explant  colonies  variation existed  same c a l l u s o r i g i n a t e d  tetraploid  of  (92.2-98.4%) of  other  and  iji v i t r o  conditions. It  has  been w e l l  structure  are  (Bayliss,  1980;  regenerated  documented  increased  that  with the  Reisch,  1983).  length  F l o w e r and  of  tend  regenerated  28  t h e y were not  months) c u l t u r e s , w h i l e from  Furuya,  short-term  1972).  McCoy e t  showing c y t o g e n e t i c cultures two four  of  lines, and  al.  increased  from  derived  12%  to  r e s p e c t i v e l y , when p l a n t s  plants,  from  regenerated  were commonly  long-term  48%  (Syono  from  n i n e y e a r s showed a l t e r e d l e a f  from and  that  to  the  and plants  tissue 49%  t o 88%  were r e g e n e r a t e d  culture.  (up  o b s e r v e d among  (1982) o b s e r v e d  abnormalities  period  t o have more  months) c u l t u r e s  t w e l v e months i n c e l l  morifolium for  oat  (1-5  karyotypic  plants  leaf abnormalities  f o u n d among t o b a c c o p l a n t s  plants  in  culture  Accordingly,  from l o n g - t e r m c u l t u r e s  abnormalities.  changes  in  after  Chrysanthemum  leaf callus, shape, s m a l l e r  maintained and  15 abnormally as  shaped  compared w i t h  for  only  one  One  cells.  Few  i s no  good  of NAA  system with  (1980) r e g e n e r a t e d  barley  i n such t r a i t s  fertility  happened o n l y a t on  two  as  and  regenerate  more t e t r a p l o i d  translocations The genetic  than  the  relationship  First,  species concerning (1982) r e p o r t e d  the  a high  partly  medium. because  Deambrogio  1,  4 mg/1  2,  3 and  2,4-D.  media  (NK  stock  t h a t NK  Plants  and  I) of  and  2,4-D.  s h a p e , and  callus  tiller  were  from c u l t u r e s Haworthia  (Ogihara,  medium t e n d e d  p l a n t s , but  1981). to  fewer p l a n t s  with  I medium.  between o c c u r r e n c e  b a c k g r o u n d o f m a t e r i a l was  publications.  i n the  using  c u l t u r e s of  e i g h t c h a r a c t e r s were a n a l y s e d  Chromosome a n a l y s i s r e v e a l e d  variants  from t i s s u e  4 mg/1  which o r i g i n a t e d from a s i n g l e setata  topic,  auxins,  Shepard  protoplasts  albinism, leaf  different  of  w h i c h t o work.  using  Variation  1983).  2,4-D  in  synthetic  frequency  and  this  plants  (Hordeum v u l g a r e )  regenerated  variation  (Lorz,  from p o t a t o  r e p o r t s a r e a v a i l a b l e on  there Dale  concentrations  in culture  1981).  induce  candidate  plants  growth  i n t h e medium u n d e r c u l t u r e  d i f f e r e n c e s i n the  regenerating  shoot  from c a l l u s  Phytohormones, e s p e c i a l l y  t h e most p r o b a b l e  different  lateral  Langhans,  a c t a s mutagens t o  (1981) o b s e r v e d after  excessive  ( S u t t e r and  or more components  cultured be  and  plants regenerated  year  c o n d i t i o n s may  may  flowers  there level  i s an  of v a r i a n t s  i n d i c a t e d i n some obvious  d i f f e r e n c e among  of v a r i a b i l i t y .  frequency  and  of chromosome  McCoy e t a l . abnormalities  16 in  oat  (Avena  Phillips  although  cases.  species  the  study,  were f o u n d  techniques  of m o r p h o l o g i c a l  e m p l o y e d were s i m i l a r  demonstrated.  1976).  change was  of  4,600  while  variation  diploid 1979;  Shepard  genetic  the  was  (Lorz,  of O r y z a  polyploids  found  tetraploid analysis lines  'Keng'  The  was  procedures  level of  of t h e  revealed that  'Hsien'  alterations  cultures  line  It  was in  starting  of two  'Lodi'  of group  nine  D i f f e r e n c e s were  The  'Tippecanoe'  as the  (Wenzel,  (indica)  d e t e c t e d i n the  ( K a r p e t a l , 1982).  the l i n e  while  cell-derived  percentages  ( j a p o n i c a ) group.  tissue  among  cultivars  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 cultivars  was  2,000 p l a n t s were  of t h e  no p o l y p l o i d  of p l a n t s f r o m  cytological 1982).  L. more t h a n  e t a l . , 1983).  it  to d i f f e r e n c e s  In an a n a l y s i s  for nine v a r i e t i e s  of t h e  due  r a t h e r than p l o i d y  sativa  (Sun  was  146  observed  Thomas e t a l . , 1982).  difference  1983).  0-13.3%, but  varieties also  this  frequency  r e g e n e r a t e d p l a n t s of a  evaluated using similar  background  investigated  among t h e  e t al.. , 1 980;  that  materials plants  observed  cultivar  suggested  were  was  same  cane,  for clone  In p o t a t o c o n s i d e r a b l e v a r i a t i o n  the  in  regenerated  t h e 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 no  of  In s u g a r  F o r c l o n e F156 34.0%  and  o f chromosome  among c u l t i v a r s  among a t o t a l  Chen,  w h i l e McCoy  a h i g h degree  Second, d i f f e r e n c e s  ( L i u and  1.8%.  i n one  have been c l e a r l y  variants plants  L.)  (1982) o b s e r v e d  stability, both  sativa  had  two  cytogenetic Avena  sativa  t w i c e a s many  (McCoy e t a l . ,  17 Explant somaclonal four  s o u r c e c o u l d a l s o have a s i g n i f i c a n t  variation.  Plants regenerated  t y p e s of e x p l a n t s of p i n e a p p l e  altered Plants only  according from  the  crown and  Harten  in leaf  and  color,  Broertjes  were o b s e r v e d  disks,  i n 50.3%  but  petiole-derived Although regenerated  i t i s generally  et  reconfirmed  produce  Van  leaflet  and  The  (Vasil  stability  i n a recent study  in plants  Triticum  aestivum  that fewer  of  plants variants  have not and  been  found  1981;  Vasil,  ( K a r p and  Vasil was 1985).  c h a n g e s were  regenerated v i a embryogenesis L. c u l t u r e s  two  obvious  regenerated plants  structure  the  embryogenesis  Vasil,  (Swedlund and  than  between  Somatic  s p e c i e s and  cytogenetic variants  observed  from  Maddock,  1984).  Genetic Basis Understanding  is  included  density.  from  comparisons  i n many c e r e a l  However, chromosome number and  2.2.3  rachis-  believed  direct  regenerated plants  a l . , 1982).  changes  morphological  of p l a n t s  pathways a r e v e r y r a r e .  and  1979).  syncarp  foliage  were  callus.  been a c h i e v e d  among t h e  and  from  v i a embryogenesis  regeneration  phenotypic  i n 12.3%  of p l a n t s  those v i a organogenesis,  has  s p i n e , wax  on  of  buds showed  from  (1981) showed t h a t  variations  (Wakasa,  axillary  i n s p i n e c h a r a c t e r s , but p l a n t s  variants  callus  (Ananas comosus L.)  to the e x p l a n t source  slip,  from  effect  important  the g e n e t i c b a s i s of  to f a c i l i t a t e  its practical  somaclonal use  variation  for plant  18 improvement. somaclonal changes  variation  (number and  cytoplasmic change  B a s e d upon e x i s t i n g  DNA  such as  may  be  (4) o t h e r  transposible  elements.  1983) .  1980;  (D'Amato, Larkin  Ploidy  of  t h e most  chromosomal a b n o r m a l i t y .  Prat  from  culture  mesophyll  sylvestris like  protoplast  . Seventy  the o r i g i n a l  two  while  no  a n e u p l o i d s were r e c o v e r e d .  N.  sylvestris Faccioti  readily  observed  and  Sunderland, 1981;  Krikorian  et a l . ,  f r e q u e n t l y observed  of two  the  been  1977;  (1983) r e g e n e r a t e d lines  remainder  172  plants  of N i c o t i a n a diploid  were  polyploid;  From h a p l o i d p r o t o p l a s t s of  Pilet  (1979) r e g e n e r a t e d p l a n t s  diploids.  i n p o t a t o and  (3)  heritable  r e g e n e r a t e d p l a n t s were  lines,  w h i c h were e s s e n t i a l l y  k i n d s of  1978;  Scowcroft,  changes are  (1) chromosome  r e g e n e r a t e d p l a n t s has  1977;  and  to  knowledge  (2) gene a l t e r a t i o n s ;  c h a n g e s and  documented  Bayliss,  attributed  structure);  Chromosome v a r i a b i l i t y well  experimental  Ploidy  tobacco  c h a n g e s were  also  ( K a r p e t a l . , 1982;  1984) . Aneuploids  have been  r e c o v e r e d among r e g e n e r a t e d p l a n t s  i n many c a s e s where chromosome a b n o r m a l i t y the  frequency  Pilet, from  1979;  cultured  (Triticum plants from  38  i s usually  lower  than  K a r p e t a l . , 1984).  t o 45  euploids (Faccioti  f o u r wheat  2n=6X=42) showed t h a t  e x a m i n e d were a n e u p l o i d s ( K a r p and  Maddock,  although and  A n a l y s i s of p l a n t s d e r i v e d  immature embryos o f  aestivum  occurs,  ranging 1984).  29%  cultivars of t h e  192  i n chromosome number  19 In a d d i t i o n  t o chromosome number c h a n g e s chromosomal  rearrangements such as  deletions,  translocations  inversions  expected.  Translocations  may  a l s o be  o b s e r v e d among r e g e n e r a t e d  plants  (Ahloowalia,  (Shepard,  al.,  1982), H a w o r t h i a  hybrids In  work w i t h  More new  large  able  paracentric  inversions  bivalents  and  result  either  recessive  I.  and  cultures  and  plants to  or  the  than  be  of  as  and evidence  of  aberrations. of  hybrids  of  freqency  of  t h e y were presumed t o be deletion  of  the  a pairing  1980).  in recombination  e t a l . , 1984).  rate  seedlings  were u s e d as  while  r e s t of  the  rates  of  C o t y l e d o n s of ir\ v i t r o plant the  have been o b s e r v e d  Phenotypically  m a r k e r genes were s e l e c t e d  Recombination  Ogihara  deletions.  Chromosome b r i d g e s  sub-chromatid  rye  a l . , 1984).  setata,  observation  j u b a t u m showed e n h a n c e d  homozygous b a c k g r o u n d .  the  et  et  wheat X  in regenerated  the  translocation  (Orton,  (Sibi  and  multivalents  Increases tomato  by  from c a l l u s  Hordeum v u l q a r e X H.  suppressor  (Lapitan  and  segments a l o n e , were i n t e r p r e t e d  regenerated  of  1981)  sp.)  (McCoy  R e a r r a n g e m e n t s were c o n f i r m e d  i n metaphase  segments, or  1982), o a t  chromosomes of H a w o r t h i a  translocations  quadrivalents  have been  (Lolium  to demonstrate t r a n s l o c a t i o n s  calli.  reciprocal  ryegrass  (Ogihara,  k a r y o t y p e s were d e t e c t e d  cloned  Plants  setata  (X T r i t i c o s e c a l e W i t t m a c k )  (1981) was  in  1976), p o t a t o  of  and  was  i n the  distinguishable 'Monalbo'  individual F  tissue culture grown as  a  selfed progenies  on  2  hybrid  material,  control. from  regenerated  20 plants  and  matched c o n t r o l s  'bs-ms32' on units II,  chromosome I  were o b s e r v e d  the  increases  Cytoplasmic  genetic  Gengenbach e t in  vitro  and  al.  to  c h a n g e s may  The  to a host  was  variation  to  of  for  leaf blight  Helminthosporium sterile  plants. to  w i t h the  lines.  toxin  aim  of  However,  with a concomitant  al.  reversion  (1981) o b s e r v e d m i t o c h o n d r i a l  i n T - c y t o p l a s m i c male s t e r i l e  regenerated  from  tissue culture  and  t o H e l m i n t h o s p o r i u m m a y d i s r a c e T. analysis different compared  of  mitochondrial  to T or  correlated  Kemble, two  N  analysis  was  selected  to  also  A  when  specific DNA  was  i n c a l l u s morphology  o b s e r v e d DNA  DNA  components c a l l e d SI  s y l v e s t r i s produced  between  differences  The  p e r f o r m e d on  resistance  R e s t r i c t i o n enzyme  showed d i f f e r e n c e s and  DNA  plants for  i n maize m i t o c h o n d r i a l  t o a change  1982).  maize  c y t o p l a s m maize p l a n t s .  event  plasmid-like  Nicotiana  DNA  regenerated plants  transposition  t o be  DNA  units.  fertility. Gengenbach e t  to  chromosome  for resistance  resistance  and  map  responsible  in seed-derived  r e s i s t a n t , cms-T b r e e d i n g  of  loci  6.91 of  map  corn  two  w i t h T e x a s male  recovering  male  6.04  resistant plants  associated  to  region  toxin  (1977) s e l e c t e d  regenerated  6.07  of  associated  genotypes  For  a l s o be  resistance  is tightly  (cms-T) c y t o p l a s m i c  of  'aa-d'  from 4.92  cultivar sensitivity  maydis race T  increases  f o r the  ranged  observed v a r i a t i o n . and  and  were c o m p a r e d .  doubled haploid from p o l l e n  shown  (Chourey  v a r i a t i o n was and  type  limited  S2.  plants  culture,  When of  heritable  21  quantitative nuclear  DNA  a n d q u a l i t a t i v e c h a n g e s were o b s e r v e d (De Paepe e t a l . , 1983).  contained,  on a v e r a g e ,  increasing  amounts o f h i g h l y  research better  variation.  haploid  i n c r e a s i n g amounts o f t o t a l  at the molecular  understanding  Doubled  repeated  level  will  of the g e n e t i c  i n the  sequences.  plants  DNA More  be e s s e n t i a l f o r a b a s i s of somaclonal  and  3.  3.1  General Culture  3.1.1  MATERIALS AND  METHODS  Methodology  Media In t h i s  Collins,  s t u d y an e s t a b l i s h e d  1979a) was  (Gamborg e t a l . , of b a s a l KN0 ,  1968)  (Appendix  435;  2  MnSO„'H 0,  CoCl -6H 0, 2  2  Myo-inositol,  0.1;  250;  sucrose,  25,000.  the c u l t u r e  were: c y t o k i n i n s : BA, and  and KIN;  5  1,000;  5; K I ,  1;  Na MoO„2H 0, 2  2  0.5;  growth  in various  Growth  600;  2  Pyridoxine-HCl, Different  objectives.  ADE  3  0.1;  2  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 d e p e n d i n g on  3  2  3  T h i a m i n e , 2.0;  B  85; C a C l . 2 H 0 ,  2  5; C u S O « ' 5 H 0 ,  2  and  Composition  (EDTA), 25; H B 0 ,  2  15; Z n S O « - 7 H 0 ,  2  used. 4  2  FeSO««7H 0  i n which  (mg/1): N H N 0 ,  325; N a H P 0 „ - H 0 ,  2  (Phillips  test  1) was  L2 medium i s as f o l l o w s  MgS0«-7H 0,  0.4;  u s e d e x c e p t i n one  2,100; K H P O „ ,  3  medium L2  tests  regulators  a u x i n s : 2,4-D, IAA,  used NAA  PIC. Stock  s o l u t i o n s were made f o r (1) m a c r o - n u t r i e n t s  (NH„N0 , KNO3,  KH PO , N a H P O „ - H 0  3  CaCl -H 0, 2  2  X20;  2  micro-nutrients  ft  2  2  (3) F e S O « - 7 H 0 2  3  f t  Na MoO(,-2H 0 and C o C l . 6 H 0 ) 2  2  2  2  X400;  2  t h i a m i n e and p y r i d o x i n e X1000. stock  solution,  hot d i s t i l l e d volume  was  5.00  2  water and t h e n 7.45  b r o u g h t up t o 500 m l ,  22  (2)  2  (4)  ZnS0i,'7H 0, 2  CuS0««5H 0, 2  (5) KI X1000; and  In p r e p a r i n g  g FeSO «7H 0 a  fl  (EDTA) X200;  (H B0 , MnSO «H 0, 3  and MgS0 -7H 0) X10;  were f i r s t  (6)  FeSO *7H 0 a  2  dissolved in  g Na EDTA were a d d e d . 2  resulting  in a solution  The of  23 5 mg/1  FeSO «7H 0. a  separately salts.  The C a C l « 2 H 0  2  2  t o prevent  Thiamine  stock s o l u t i o n  2  precipitation  was made  i n the stock basal  a n d p y r i d o x i n e were p r e p a r e d  as aqueous  solut ions. Stock  solutions  were a l s o p r e p a r e d  growth r e g u l a t o r s used:  0.06 mg/ml.  0.2 mg/ml; 2,4-D, 0.5 mg/ml; a n d  BA a n d ADE were f i r s t  amount of 1N KOH and d i s t i l l e d appropriate  volume.  subsequently PIC  were d i s s o l v e d  addition bottles the  with water.  IAA s t o c k  Stock  solution  a  solutions  The a u x i n s  solutions  wrapped w i t h  5.8 w i t h at  of s a l t s ,  water.  specified.  more t h a n  1.0 kg/cm  a p p r o p r i a t e volumes o f  Sugar was t h e n (Difco  added and  Bacto)  The pH o f media was a d j u s t e d t o agar  f o r 15 m i n .  and media were a u t o c l a v e d (20 min when a c o n t a i n e r h e l d  1,000 ml o f medium).  Media  i n these  abbreviations regulators. containing  2  to the  v i t a m i n s and g r o w t h r e g u l a t o r s t o  IN KOH b e f o r e a d d i n g  121°C,  2,4-D, NAA a n d  a t 4°C e x c e p t f o r  t h e media were s o l i d i f i e d w i t h 8 g/1 a g a r otherwise  i n 1N HC1 a n d  were s t o r e d i n c o l o r e d  tinfoil  by a d d i n g  l a r g e volume o f d i s t i l l e d  unless  to the  w h i c h was s t o r e d f r o z e n .  M e d i a were p r e p a r e d stock  dissolved  i n a small  i n a s m a l l amount o f " e t h a n o l p r i o r  of water. or b o t t l e s  dissolved  w a t e r was added  KIN was f i r s t  diluted  of the  BA, 1 mg/ml; ADE, 1 o r 2 mg/ml; KIN,  1 mg/ml; IAA, 1 mg/ml, NAA, PIC,  f o r each  experiments  are specified  o f b a s a l media and t h e i n i t i a l s F o r e x a m p l e , L2(P0.06B0.2) r e f e r s  0.06 mg/1  P I C and 0.2 mg/1  BA.  by t h e of growth t o L2 medium  The l e t t e r  before  24 the  brackets  Collins, basal  represents  1979a) and  media  symbolize  the  Each  (N);  regulator  indicates  Within  the  the  f o r growth r e g u l a t o r s  PIC  ( P ) ; ADE  symbol  ( A ) ; BA  i s followed  concentration  used  by  A l l these media c o n t a i n  containing  used  0.06  containing  0.01  containing  0.002 mg/1  containing  2 mg/1  and  LSP  mg/1  2,4-D PIC  and  and and  L2  mg/1  2 mg/1 0.2  2,4-D, 2 mg/1  have been s p e c i f i e d  0.1  BA  by  and  Phillips  (K).  media  salts.  follows:  BA;  BA  (D);  special  basal  ADE;  mg/1  KIN  Several  have been a s s i g n e d  PIC  a  which  i n t h e s e m e d i a were as  mg/1  and  were: 2,4-D  i n mg/1.  abbreviations.  Growth r e g u l a t o r s  auxin  a number  two  letters  ( B ) ; and  frequently  and  r e f e r to  brackets  w h i c h were u s e d  L2:  (Phillips  g r o w t h r e g u l a t o r s , u s u a l l y an  Symbols  ( I ) ; NAA  L2  (Gamborg e t a l . , 1968)  5  respectively.  cytokinin. IAA  B  a b a s a l medium.  (2)  (3) and  (1)  LSE:  LSP: (4)  SCP:  2 mg/1  ADE.  L2,  and  Collins  LSE  (1979a;  1980).  3.1.2  Explants Trifolium  were u s e d . a  The  tetraploid  Richardson  p r a t e n s e L. former  cultivar.  into  25  X  mm  and  cv and  'Norseman' the  obtained  'Norseman' f r o m  latter  from Charles  Ltd.  sucrose 150  cultivar  ' A l t a s w e d e ' was  S e e d s were g e r m i n a t e d 5 g/1  ' A l t a s w e d e ' and  is a diploid  Seed Company L t d  S h a r p e & Company  with  cv  and  i n h a l f - s t r e n g t h L2  8 g/1  agar.  The  basal  medium was  medium dispensed  t i s s u e c u l t u r e t u b e s , e a c h r e c e i v i n g 20  ml  25 volume. then  Seeds were s t e r i l i z e d  i n 20% c o m m e r c i a l  final  concentration)  distilled  bleach  three  f o r 8-10 d a y s  with a photoperiod hypocotyls  test  utilized  f o r shoot  manipulations conditions.  Six to eight  tubes  2  t i pcultures.  mm  shoot  into  3-4  tips  were  out under a s e p t i c  T r a n s f e r s were f a c i l i t a t e d  using a laminar a i r  A i rControl Co.).  Culture Conditions  experiments.  When t e s t  v e s s e l s were u s e d  tubes  (25 X 150 mm)  i n these were u s e d ,  medium w i t h a l l components added a n d pH a d j u s t e d put  i n t o a m i c r o w a v e oven  had  melted  Glassware  t o melt  the agar.  t h e medium was d i s p e n s e d  T u b e s were c a p p e d  with p l a s t i c  & Equipment).  into  bulk  t o . 5 . 8 was  Once t h e a g a r  the test  closures (Bellco  The s u b c u l t u r e i n t e r v a l  tubes.  Biological was u s u a l l y  weeks. When p e t r i  into  mm  A l l experimental  o f e x p l a n t s were c a r r i e d  Two k i n d s of c u l t u r e  four  and  Seedling  1  with a scalpel  i n d u c t i o n a n d 3-4  flow c a b i n e t .(Environmental  t o germinate  (200 izEm" s e c " ) .  were c u t a s e p t i c a l l y  forcallus  in sterile  i n a g r o w t h chamber a t 25°C  o f 16 h o u r s  segments  3.1.3  times.  s e e d s were p u t i n t o  were i n c u b a t e d  f o r 5 min.,  ( 1 . 0 % sodium h y p o c h l o r i t e i n  f o r 20 m i n . , a n d r i n s e d  water a t l e a s t  sterilized  i n 75% e t h a n o l  p l a t e s were u s e d ,  t h e p l a t e s (30 m l / p l a t e )  cabinet  when medium t e m p e r a t u r e  45°C a f t e r  t h e b u l k medium was  i n the laminar  poured  a i r flow  had cooled t o approximately  a u t o c l a v i n g and a l l o w e d  to set.  Following  26 explant  transfer  the  plates  prevent contamination subculture Six  and  were wrapped w i t h p a r a f i l m  reduce moisture  i n t e r v a l for plate  cultures  intensities. methods f o r cool-white 16/8  They w i l l  be  3.2  Callus  the  experiments.  tubes.  The  Light  was  light/dark  indicated.  The  period  dishes  was  mm  in length  of  was  temperature  were  500  i n g r o w t h chambers. v i s u a l l y two  yEm" sec" . 2  to  five  In  1  b a t c h two,  explants  explants.  i n t o a g r o w t h chamber a t  Cultures  Callus later. culture  a light  vessels,  light  sterile  f r o m two  petri  seedlings  These p e t r i i n t e n s i t y of  dishes 50  nEm~ sec" . 2  1  Five callus  in  transferred  i n a g r o w t h chamber w i t h  were u s e d c o n t a i n i n g three  weeks  t e s t t u b e s were u s e d as  were i n c u b a t e d  each having put  25°C  evaluated  b a t c h one,  intensity  by  Methodology  segments 3-4  were m a i n t a i n e d a t  Cultures  of  provided  o n t o L2(PO.06BO.1) medium f o r c a l l u s i n d u c t i o n .  In  light  Induction  Hypocotyl  induction  weeks.  25±1°C.  S p e c i f i c Culture  3.2.1  four  s p e c i f i e d in descriptions  hours unless otherwise  t h e s e chambers was  also  have d i f f e r e n t  i n d i v i d u a l experiments. fluorescent  The  was  g r o w t h chambers were u s e d d u r i n g  T h e r e f o r e , d i f f e r e n t e x p e r i m e n t s may  loss.  to  b a t c h e s of c u l t u r e s  were e s t a b l i s h e d  from h y p o c o t y l e x p l a n t s .  Test  to  induce  t u b e s were u s e d  in  with were  27  t h r e e b a t c h e s and tube c o n t a i n e d  petri  plates  three explants  the  w h e t h e r any callus  3.2.2  two.  Each  seedling.  two  of  these  three  genotype e f f e c t  on  callus  relation  exsits  batchess  induction  between t h e  three  test  to  i.e.  explants  for  induction.  Callus The  M a i n t e n a n c e and  following  g r o w t h and (1)  other  f r o m one  O b s e r v a t i o n s were made i n determine  i n the  Plant  Regeneration  o b s e r v a t i o n s were made t o  differentiation  green-spotted c a l l u s  on  various  (GSC): c a l l u s  regions containing meristem-like on  the  callus.  I t has  s t r u c t u r e s c o u l d be and  Jang,  1984).  structure  to  indicator  Calli  predict  with  isolated  structures  of  were s c o r e d  their  callus  media:  been r e p o r t e d  an  evaluate  that  green  distributed  these  regeneration using  potential  (Kim  this  for  differentiation. (2)  shoot c a l l u s indicating callus  (SC):  (RC):  callus  (4)  brown c a l l u s :  (5)  dead or  callus  not  necrotic  sometimes h a r d (6)  g r o w t h : In relative  one  differentiation.  root  did  with  shoot  (3)  callus  callus  grow  or  more  more  w i t h brown c o l o r .  shoots,  roots.  This  kind  of  well.  callus: in  w i t h one  or  callus  w h i c h was  dark  brown  and  texture.  some e x p e r i m e n t s c a l l u s growth r a t e  indicated  s i g n s meaning f a s t e r  growth).  by  was plus  graded signs  for  their  (more  plus  28 3.2.2.1  Primary Test  After  cultures.  Two  containing  20  plant  mg/1  be  Smith,  1979;  induced of  ADE  capable  the  three  on  L2  of  incubated  to  of  (1)  NAA  light  and  Collins,  1980).  E a c h p l a t e was Cultures  intensity  and  two  the  of  900  of  Test  1: The  LSE  induction  i n the  previous  p o s s i b l e that to  reported (Beach  Calli  _ 2  LSE  (1) a s m a l l  with in a The  1  for were  types  and  evaluation.  Fifty  on  LSE  medium  subcultures.  to  stimulate  experiment  (Section  a b s e n c e of  success  callus  sample  shoot  3.2.2.1). could  s i z e and/or  some m i n o r e n v i r o n m e n t a l c o n d i t i o n w h i c h d i d not the  requirements  f o r shoot  LSE  Medium  medium f a i l e d  this  one  been c u l t u r e d on  subsequent  and  were  uEm sec" .  Some c a l l i  w h i c h had  effect  2,4-D  were m a i n t a i n e d  weeks l a t e r  growth.  each c u l t i v a r  ADE  mg/1  inoculated  f o r f o u r more weeks b e f o r e  attributed  2 mg/1  0.01  s e v e n weeks were f u r t h e r s u b c u l t u r e d  was  medium  5  regeneration  o n t o f r e s h media of b o t h  determine  B  t r a n s f e r r e d t o p l a t e s of  3.2.2.2 C a l l u s Growth on  It  2 mg/1  plant  and  medium and  differentiation  for  inducing  were e v a l u a t e d  transferred  tried:  were  callus  medium c o n t a i n i n g  of c a l l u s .  chamber w i t h  from  ( = L S E ) . T h e s e media have been  Phillips  pieces  calli  thiamine,  above m e d i a .  cultures  regeneration  (2) L2  5  2 mg/1  i n d u c t i o n , attempts  media were f i r s t  ( = B ( N 2 A 2 ) ) and  to  Media  successful callus  made t o a c h i e v e  and  of Two  induction.  In  another  be (2)  meet  29 experiment,  1452 c a l l i  of ' A l t a s w e d e '  'Norseman' were t r a n s f e r r e d August  3 and 4,  Subsequently,  f r o m L2 t o L S E medium  1984, and c u l t u r e d  984  'Altaswede'  calli  were s u b c u l t u r e d  light  t r e a t m e n t s were a p p l i e d  Light  intensity  gradation plates  i n one c o l u m n  emergence b e c a u s e  2  explants.  has  f o r 'Altaswede'  also possible tranfer  that  calli  initiated  They were t h e n t r a n s f e r r e d These  four media:  half  and C o l l i n s ,  and  growing  calli  1980);  on  f o r shoot  o f some f a c t o r  i n LSE  To t e s t  this  from h y p o c o t y l onto LSE  were  medium  subcultured  (1) LSP medium,  been c l a i m e d t o be s u p p o r t i v e o f s h o o t  (Phillips  calli  t o a n o t h e r medium  were f i r s t  the f o l l o w i n g  light  calli.  two o r t h r e e weeks.  onto  This  1  (2) P h o t o p e r i o d s  1  of the absence  calli  (1)  ten culture  n e c e s s a r y f o r shoot development.  hypothesis,  Various  70 t o 900 juEirr sec ~ .  2  required  'Norseman'  to the c u l t u r e s :  o f 500 o r 900 /uEirr s e c ~ ;  2: I t was  LSE medium  for  a n d 568  on  i n two chambers w i t h maximum  f o r 'Norseman'  Test  medium  calli  of  weeks.  f r e s h L S E medium.  16 and 24 h o u r s were u s e d  16 h o u r s  of  from  f o r four  was a c c o m p l i s h e d by s t a c k i n g  intensities of  varied  onto  and 1556 c a l l i  which  development  (2) L2(P0.002B0.2 a n d  s t r e n g t h o f s a l t s ) medium, t o e v a l u a t e t h e e f f e c t  reduced c o n c e n t r a t i o n s of b a s a l  differentiation; medium  4, w h i c h  (3) L2(NO.05KO.5)  salts medium  h a d o n l y L2 b a s a l medium  r e g u l a t o r s added.  Calli  were c u l t u r e d  on  callus  a n d (4) without  in plates  growth with  30 24  h o u r p h o t o p e r i o d and Test  3:  In t h i s  were u s e d .  They had  Callus  primary  from  medium h a v i n g containing  the  test,  out  i n any  In t h i s evaluated. treatments  2,4-D  batches  2 mg/1  3.2.2.4 PIC Three  and tests  2 mg/1  2 mg/1  400  a  except  ADE.  Calli  AtEm~ sec~ 2  1  and  to support  shoot  work was  carried  w h i c h would  levels  of 2,4-D  0.1, BA  were u s e d .  were: 2 and  The  and  be  BA  were  The  4 mg/1  BA  four treatments  for  2.0  and  and  10.0  additional  mg/1 5 mg/1  t h e medium c o n t a i n i n g 2  ADE  mg/1  BA.  BA were c a r r i e d listed  embryogenesis of c a l l u s , has  as LSE  onto  weeks.  batch  were 0.01,  the t h r e e t e s t s a r e  acids,  transferred  further  2,4-D.  incorporated into and  sources  on L 2 ( D 2 B 2 ) medium.  of c u l t u r e  first  combined w i t h  2,4-D  callus  BA  2 mg/1  batch  f o r f o u r weeks.  induction.  different  f o r the  the second  1  t h e medium c o m p o s i t i o n  test  combined w i t h  was  experiment,  and  Two  at  for three  shoot  2,4-D  tubes  of  medium f a i l e d  to determine  3.2.2.3  was  instead  in test  s u p p o r t i v e of  different  cultures  16-hour p h o t o p e r i o d  induction  _2  been c u l t u r e d  ADE  B e c a u s e LSE  MEm sec"  same c o m p o s i t i o n  5 mg/1  were c u l t u r e d  100  out.  in Table  reduced  been shown t o be  The  treatments  1.  For  n i t r o g e n , e.g.  a major  factor  in  amino  (Raghavan,  31  Table  Test  ^rg 1740  1. T r e a t m e n t c o m b i n a t i o n s u s e d t o t e s t t h e e f f e c t o f 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). No  Treatment  No  PIC  BA  1 2 3 4 5 6 7 8 9 10 1 1  0.001 0.0005 0.001 0.005 0.01 0.05 0.0005 0.001 0.005 0.01 0.05  0.2 1 1 1 1 1 1 1 1 1  1 2 3 4 5  0.005 0.005 0.005 0.005 0.005  1 1 1 1  1 2 3 4 5 6  0.002 0.005 0.01 0.002 0.005 0.01  0.2 0.2 0.2 1 1 1  = A r g i n i n e ; CH = C a s e i n mg/1 a r g i n i n e = lOrnM.  Other Additives  1  1740 1740 1740 1740 1740 1740  Arg Arg Arg Arg Arg Arg  100 500 1000 5000 147  CH CH CH CH GA  1  1  Hydrolysate;  GA = G l u t a m i c  Acid  32  1976; In  Wetherall  alfalfa  by  Strickland,  of  the  the  1984a).  media  of  onto t e s t iiEm~ sec~ 2  the  2:  effect  inclusion  test  was  pH  a  16  three  Another  of  casein  adjusted  weeks test  and  with  previously  3:  some  to  a  In  was  callus using  5.8.  PIC  and  BA  arginine.  were  incubated at  at  Cultures  e s t a b l i s h e d to and  these  substances  desired f r o m LSE at  and  sterilization,  medium w h i c h was  calli  acid  Aqueous  filter  the  determine  glutamic  growth.  After  to give  test  of  280  'Altaswede' c a l l i  L2(D2B2N2) medium.  three  Only  400  cooling final  medium were juEm^sec"  weeks.  this  on  previously  25°C.  16-hour p h o t o p e r i o d  c u l t u r e d on  'Norseman' c a l l i  of  investigate  later.  Transferred  25°C f o r f o u r Test  to  They were t r a n s f e r r e d  hydrolysate  autoclaving  concentration.  i  Calli  photoperiod  s o l u t i o n s were a d d e d t o t h e  incubated  arginine,  a b s e n c e of  were u s e d .  i n medium on  down a f t e r  were  were a d d e d  concentrations  s o l u t i o n s were p r e p a r e d their  1978).  and  e s t a b l i s h e d to  i n p l a t e s and  with  were e v a l u a t e d Test  (Stuart  hydrolysate  L2(D0.01A2) medium.  media 1  embryos  experiment  i n p r e s e n c e or  'Altaswede' c a l l i on  number of  this  casein  various  growth  cultured  In  Kohlenbach,  tested.  1: T h i s  effect  callus  1976;  a d d i n g amino a c i d s  a c i d and  Test  Dougall,  b o t h q u a l i t y and  stimulated  glutamic  and  s o u r c e s were u s e d  were For in  the  1  33 test: and  (1) L2(D2B2N2) medium,  (3) L2(PO.002B0.2) medium.  calli  Each p l a t e  i n c u b a t e d w i t h a 24-hour p h o t o p e r i o d  AiEm" sec 2  and 2 5 ° C .  _ 1  the t r a n s f e r .  3.2.2.5 NAA  and KIN  Test  1: In t h i s  test  'Norseman' c a l l i  been c u l t u r e d on L S E medium transferred  onto  KIN.  0.1,  1.0 and 5.0 mg/1)  25°C f o r t h r e e Test  growing tested  on SCP medium  were  combined w i t h  a t 440 juEirr sec ~ 2  1  calli  were u s e d  of cv  'Altaswede'  for this  test.  NAA  was  a t t h e 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,  Calli  combinations  Concentrations were t r a n s f e r r e d  2  Test  3:  1  onto  and 25°C f o r t h r e e  'Norseman' c a l l i  from  three sources  from  L2(D2A2) medium,  B (D2N2K2)medium 5  o f KIN were 0.5 various  o r 2.0  medium  and c u l t u r e d w i t h a 24-hour p h o t o p e r i o d a t  250 MEm~ sec"  medium.  w h i c h had  weeks.  2: Y e l l o w - g r e e n  0.05 a n d 0.1. mg/1.  four  P l a t e s c o n t a i n i n g t h e c u l t u r e s were  i n c u b a t e d w i t h a 16-hour p h o t o p e r i o d and  a t 200  media w i t h v a r i o u s c o n c e n t r a t i o n s of  (0.001, 0.01,  1 mg/1  f o r one month  medium  had two  C u l t u r e s were e v a l u a t e d  weeks a f t e r  NAA  (2) L2(D2B0.5A2)  weeks.  in this  f o r each treatment:  and  (2) 14-20 c a l l i (3) 16 c a l l i  from  test  were d e r i v e d  (1) 14  calli  from L2(D2B0.5A2)  C u l t u r e s were i n c u b a t e d w i t h a 24-hour  photoperiod  a t 250 MEm~ sec~ 2  1  and 25°C f o r f o u r  weeks.  34 3.2.2.6 NAA The  and  test  BA  was  i n Presence designed  of  to determine  v a r i o u s c o n c e n t r a t i o n s of NAA and  ADE  from  on  LSE  callus  used  with  and 2 mg/1  o.1  mg/1)  ADE  /uEm~ sec" 2  and  1  and  BA  (0 and  on Four  medium w i t h h a l f  tubes.  tubes  250  Media  (3)  s e v e r a l media: (NO.01B0.2A2)  IAA  was  and  2 mg/1  divided  i n t o one  of  into  at  recorded  500  four pieces  f o u r media c o n t a i n e d  MEm~ sec" 2  after  and  1  t h r e e weeks o f  3.2.2.8 C a l l u s Growth a t H i g h  and  nicotinic  A l l t h e c u l t u r e s were i n c u b a t e d w i t h  16-hour p h o t o p e r i o d R e s p o n s e was  were  in test at  (0,  s t r e g t h of b a s a l s a l t s  mg/1  Each l a r g e c a l l u s  w i t h each p i e c e put test  of NAA mg/1)  Test  onto  (2) L2(DO.01B0.2A2),  v i t a m i n s c o n t a i n i n g 0.2 acid.  calli  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  (4) L2  BA  25°C f o r f o u r weeks.  'Norseman' c a l l i ,  and  0.2  Calli  of  with  levels  16-hour p h o t o p e r i o d  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  (1) L 2 ( B 0 . 2 A 2 ) ,  effect  'Norseman'  Different  in a l l tests.  were i n c u b a t e d w i t h a  the  i n combination  differentiation.  medium were u s e d .  0.001, 0.01  ADE  o r Low  Auxin  in  a  25°C. culture.  to Cytokinin  Ratios 'Norseman' c a l l i onto  the  f o l l o w i n g two  L2(D1K10). cytokinin  The ratio  first  from  LSE  media  medium were  transferred  (1) L2(D11K1) and  medium had  (11), while the  a high auxin second  one  had  (2) to a  very  35 low  ratio  (0.1).  Calli  were c u l t u r e d  p h o t o p e r i o d a t 200 M E m " s e c " 2  3.2.2.9  (e.g.  e x p e r i m e n t s when c a l l i w h i c h d i d n o t promote  on LSE medium),  such c a l l i test  Calli  for five  weeks.  were t r a n s f e r r e d  onto a  as a v e h i c l e  i t would  be u s e f u l  LSE medium  cultured  restore  (At t h i s  a n d (2)  cultured  brown c o l o r .  f o r 42 d a y s  s e n e s c e d , but the c u l t u r e s  slight (5)  on L S E medium  browning  (Some c a l l i  'Altaswede' c a l l i  better  i n d u c e d on L2 medium  t o L 2 ( P 0 . 0 1 B 0 . 2 ) medium  mg/1 and  above  under  s o u r c e s were t r a n s f e r r e d  16-hour  than  growth); were  first  f o r 23 d a y s a n d t h e n (At t h i s  brown o r d a r k b r o w n ) .  2,4-D) i n p l a t e s .  had  which had  showed a l i t t l e  subcultured  the  Some  f o r 37 d a y s a n d showed  o n t o L S E medium  turned yellow  had  (Some o f c a l l i  were g e n e r a l l y  transferred  calli  on  w h i c h h a d been  t h o s e o f (1) and ( 2 ) ) ; (4) 'Norseman' c a l l i cultured  o f SCP  time the c a l l i  (3) ' A l t a s w e d e ' c a l l i  on L S E medium  This  calli.  (1) ' A l t a s w e d e ' c a l l i  b o t h o f w h i c h h a d been  f o r 55 d a y s  were d e a d ) ;  been  growth  on L S E medium f o r  s t o p p e d g r o w i n g a n d were s h o w i n g  cultured  to  f o r r e s t o r i n g senescing  time p e r i o d s :  'Norseman' c a l l i ,  calli  callus  t o determine the e f f e c t  were p r e v i o u s l y  various  efficient  t o a normal v i g o r o u s growth p a t t e r n .  was e s t a b l i s h e d  medium  a n d 25°C  1  24-hour  C a l l u s G r o w t h on SCP Medium  In medium  with a  Cultures  photoperiod  time these  Calli  from  o n t o SCP medium  (1  were i n c u b a t e d a t 25°C  with  light  i n t e n s i t i e s of  36 250 luEm" s e c " 2  f o r c a l l u s sources  1  700 M E m " s e c " 2  and  1  ( 1 ) , (4) and  f o r c a l l u s sources  a p p e a r a n c e were r e c o r d e d  (5)and  (2) a n d ( 3 ) . Growth  after  four  weeks o f  culture. Calli on  on SCP medium were c o n t i n u o u s l y  f r e s h SCP medium  3.2.2.10 L i g h t This effect  G r o w i n g on SCP Medium  t e s t was e s t a b l i s h e d  SCP medium  t o i n v e s t i g a t e the  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  i n attempts  'Altaswede' c a l l i medium  f o r one y e a r .  E f f e c t on C a l l i  of l i g h t  to achieve  in  plates  at  2 5 ° C f o r 40 d a y s .  , were t r a n s f e r r e d  and i n c u b a t e d  Different  3.2.2.11  three  Selection  on L S E  o n t o SCP medium  light intensities  1  14 c o l u m n s , e a c h h a v i n g contained  regeneration.  w i t h a 16-hour p h o t o p e r i o d a n d  (70-450 M E m ~ s e c " ) were o b t a i n e d  plate  plant  , w h i c h had been c u l t u r e d  f o r one month  2  subcultured  eight  to four  by s t a c k i n g  plates  to ten plates.  Each  calli.  and M a i n t e n a n c e o f E m b r y o g e n i c  Callus After  p l a n t l e t regeneration  media c o n t a i n i n g these  NAA  was a c h i e v e d  and KIN, e m b r y o g e n i c  on two  calli  of  two t r e a t m e n t s were t r a n s f e r r e d a n d t h e n  subcultured cultures  on LSP medium.  were f u r t h e r  Three c a l l i  subcultured  embryo p r o l i f e r a t i o n and p l a n t Eventually  calli  from  these  on LSP medium f o r  regeneration.  f r o m one c a l l u s l i n e  (one g e n o t y p e )  as  37  was  retained.  Calli  w h i c h were c a p a b l e o f  e m b r y o g e n e s i s were t h u s o b t a i n e d . calli  were s u b c u l t u r e d  containing  on LSP medium.  these c a l l i  photoperiod  a t 500  evaluated a f t e r development  2  with a  25°C.  weeks o f c u l t u r e ,  and p l a n t l e t  3.2.2.12 G r o w t h  and  1  embryogenic  Test, t u b e s  were i n c u b a t e d  MEm" sec"  four  These  16-hour  They  were  f o r embryo  production.  of Embryogenic  and  Non-embryogenic  Calli Calli  may  be  identified  occurrence of embryogenesis, non-embryogenic.  when t h e y were c u l t u r e d  cultures  calli  of  i . e . embryogenic  of  between  following  ten media:  were c u l t u r e d  (3) L2(NO.1 BO.1),  L2(N0.05B0.5),  (6) L2(PO.001 BO.2), yeast  calli calli,  had two  on t h e (2)  and  10 g/1  MEm" sec" . 2  1  (5)  (8) L2 agar,  g l u t a m i n e and  Non-embryogenic  19 t u b e s f o r e a c h t r e a t m e n t and tubes per treatment.  culture  (7) L 2 ( N 2 A 2 ) ,  600 mg/1  (10) L 2 ( N 0 . 5 K 0 . 5 ) .  i n c u b a t e d a t 480  from the  (4) L2(NO.01 BO.5),  extract  L2 b a s a l medium c o n t a i n i n g  3% s u c r o s e a n d  media.  (1) L2(NO.01 BO.1),  2 g/1  calli  had been o b s e r v e d .  L2(N0.05B0.1),  (9)  kinds of  were from a n o t h e r c a l l u s  A l l calli  basal containing  and  ' A l t a s w e d e ' were o b t a i n e d  calli  'Altaswede'.  t h e two  on d i f f e r e n t  i n which embryogenesis  Non-embryogenic  of the  This experiment attempted to  determine the d i f f e r e n c e  Embryogenic  on t h e b a s i s  Cultures  embryogenic were  38  3.2.3  Shoot Shoot  multiple  Tip Culture tips  shoot  L2 medium.  3-4 mm  i n l e n g t h were u s e d  induction.  Three  weeks  Shoot  later  tips  c o n t a i n e d one s h o o t  treated  as a group  tip.  and g r o u p s  C u l t u r e s were e v a l u a t e d f o u r induction shoot  frequency  t i p based Multiple  containing  Only  3.2.4  s h o o t s were  Shoots embryogenic consisting and  under a ready  mg/1  were  later  onto test  i n one r a c k  were  assigned.  for multiple  of s h o o t s p r o d u c e d  P I C and 1 mg/1  from  shoot per  1  and  medium  BA f o r  the m u l t i p l e  Shoots  shoot  utilized.  incubated with a  2  L2  o f one month.  s h o o t s were  a t 200 M E n r s e c ~  16-hour  25°C.  Shoots  o b t a i n e d from  shoot  t i p cultures  calli  were t r a n s f e r r e d  of h a l f  strength salts  c o n t a i n i n g 1 mg/1  (Phillips  tubes  on  Each  s u b c u l t u r e d twice onto  well-formed  3-aminopyridine,  induction.  were randomly weeks  with a s c a l p e l  R o o t i n g of  cultured  group.  These c u l t u r e s photoperiod  Thirty  at a subculture i n t e r v a l  isolated  rosette.  shoot  and t h e number  0.002-0.003  propagation were  on  were f i r s t  t h e y were t r a n s f e r r e d  L2(PO.003B2) medium 'for m u l t i p l e tube  as e x p l a n t s f o r  nicotinic  0.2 mg/1  and C o l l i n s ,  IAA,  1979a).  or  to rooting  medium  and v i t a m i n s o f L2  acid, 10 g/1  2.5  s u c r o s e and 8 g/1  C u l t u r e s were 2  to pots after  medium  mg/1  16-hour p h o t o p e r i o d a n d 250 /zEnr s e c ~ .  t o be t r a n s f e r r e d  from  1  agar  maintained They  were  one month o f g r o w t h .  39 3.2.5  Scanning E l e c t r o n  Microscopy  Samples f r o m e m b r y o g e n i c in  4%  glutaraldehyde  (primary  osmium t e t r o x i d e i n 0.1 least  one  hour  graded  series  M  fixative)  fixation,  30%,  m i n u t e s e a c h and  finally  to absolute  The point  i n the  prevent of  dioxide inlet  dehydrated  the  admitted  t o the kept  Three to  was  opened  for three  the  exhaust  to  v a l v e was  times.  During  chamber  rose  After  steady  a  inlet the  above  chamber  95%  with  for  a 5-10  two  from an  and  the  1,600  pressure  heating p.s.i. of  1,400  carbon The  increased  exhaust  valve  to  valve minutes  was  three minutes f o r a v a l v e and Hot  s p e c i m e n chamber, h e a t i n g  the  liquid  A f t e r three  exhaust  exhaust  critical  The  external tank.  v a l v e s were c l o s e d .  the  a l c o h o l to  chamber p r e s s u r e  c l o s e d every  were  point drying.  secured  The  critical  Specimens  sufficient  four minutes.  Then t h e  to achieve  31°C.  4770).  to c r i t i c a l  closed.  total  poured around  and  ethanol  four minutes l a t e r  opened and  i.e.  Parr  open and  alternately  sufficient  (CPD;  prior  p.s.i.  carbon d i o x i d e  85%  through  s p e c i m e n s were t r a n s f e r r e d t o a  820  14  70%,  s p e c i m e n chamber was  v a l v e was  of  50%,  d r y i n g chamber w i t h  air-drying  f o r at  each.  drying apparatus  placed  cover  minutes  1%  buffer  s a m p l e s were d e h y d r a t e d  ethanol  30  by  fixed  fixative).  of  c h a n g e s of  followed  sodium c a c o d y l a t e  (secondary  Following  c u l t u r e s were i n i t i a l l y  the  liquid  water  was  i t to  44°C,  p o i n t of carbon  process  the  (pound p e r p.s.i.  at  pressure  dioxide in  the  square  inch).  44°C had  been  40 s t a b i l i z e d , the exhaust valve was pressure  was  slowly opened and  the  r e l e a s e d t o 0 p . s . i . while m a i n t a i n i n g  the  specimen chamber at 44-45°C. The  d r i e d specimens were mounted on specimen  t r a n s f e r r e d to a vacuum evaporator and "Nanotech Semprep 2" s p u t t e r c o a t e r .  coated u s i n g a Coated specimens were  viewed under a scanning e l e c t r o n microscope Stereoscan 250,  Cambridge Instrument Co.)  taken on P o l a r o i d Type 55  3.3  Growth and  3.3.1  Plant  Analysis  Transfer  A mixture of s o i l , volume) was flats pots. and  used to f i l l  autoclaved  Soil  at  p i c t u r e s were  Plants  Growth  i n Greenhouse  sand and  vermiculite  6 X 6 cm pots and  121°C, 1 kg/cm day  (1:1:1  by  a l s o 50 X 32  Each f l a t  held  cm 28  2  f o r 20 minutes and  allowed  p r i o r to r e c e i v i n g p l a n t s .  i n i n d i v i d u a l pots was  Rhizobium t r i f o l i i transfer.  and  f l a t s were wrapped with a p i e c e of brown paper  to c o o l f o r at l e a s t one  ( N i t r a g i n Co.  i n o c u l a t e d with commercial Ltd) p r i o r to p l a n t  P l a n t s to be t r a n s f e r r e d were u s u a l l y c u l t u r e d  r o o t i n g medium f o r one t e s t tubes using s t e r i l i z e d water. the  Regenerated  i n t o which the pots were p l a c e d . The  (Cambridge  film.  of  and  stubs,  month.  f o r c e p s and  They were removed from  on  the  r i n s e d f r e e of agar i n lukewarm  P l a n t s were t r a n s f e r r e d i n t o pots with  root t i p i n a downward p o s i t i o n .  41 Transplanted  p l a n t s were t r a n s f e r r e d t o t h e g r e e n h o u s e .  H i g h h u m i d i t y was p r o v i d e d  f o r the f i r s t  covering  o f S a r a n Wrap ( o r t r a n s p a r e n t  plastic  flats  with  film).  frequently. conditions  pieces  During  this period  The p l a n t s  t h e y were w a t e r e d  were e x p o s e d  a f t e r two weeks.  t h e y were w a t e r e d w i t h  (15-15-18).  A l l subsequent  a N-less  nutrient  a week t o f a c i l i t a t e An e x p e r i m e n t different  rooted  in  vitro  ambient  laboratory  transferred  initiated  to determine  Plants  conditions  t r a n s f e r r e d were  f o r g r o w t h was  (2) P l a n t s  i n greenhouse  were moved  Sufficient  water  Evaluation  o f s u r v i v a l was made f o u r  directly Saran  under  week and t h e n  ( P l a n t s were c o v e r e d  g r e e n h o u s e , b u t were n o t c o v e r e d w i t h  treatments.  f r o m c u l t u r e s were  f o r the f i r s t  t h e y were p l a c e d  (3) P l a n t s  the e f f e c t of  p l a n t s were l e f t  two w e e k s ) ;  once  regenerated  induced  (1) T r a n s f e r r e d  S a r a n Wrap f o r t h e f i r s t  period;  was c a r r i e d o u t  development.  t o the greenhouse  i n (1) e x c e p t  were  fertilizer  nodule  f o r s i x weeks.  as f o l l o w s :  entire  fertilization  Shoots of 'Altaswede'  treated  as  soluble  t r e a t m e n t s on t h e s u r v i v a l o f  plantlets.  greenhouse  solution applied approximately  root  was  t o normal  One week a f t e r p l a n t s  transplanted  using  two weeks by  with  were  treated  f o r the  to the  Wrap.  supplied during weeks l a t e r  this  period.  ina l l  42 3.3.2  Chromosome Root  tips  Analysis  of regenerated  and c o n t r o l  plants  ( s e e d - d e r i v e d ) were c o l l e c t e d i n d i v i d u a l l y f r o m roots.  Preparation  R.R.Smith  (Univ.  a n d s t a i n i n g methods were p r o v i d e d  of Wisconsin, Madison,  hours. in  They were t h e n  ethanol  step.  s e c . and then  microscope.  on  t i p s and c e l l s  extractions  i c e a t 0-4°C.  of  before Root  tips  for  staining  tips  made on a Z e i s s  two r o o t  in  were  light  were s q u a s h e d a n d were  examined.  numbers were o b s e r v e d a d d i t i o n a l  Analysis a n d sample p r e p a r a t i o n  Nodules  pestle  extraction buffer  i n 70%  a c i d = 1:1 v / v )  ( K a o , 1976).  (about  into a microcentrifuge  with a p l a s t i c  immediately  were h y d r o l y s e d  70% e t h a n o l  of c e l l s  temperature  were e x a m i n e d .  Leghaemoglobin  collected  tips  number o f a t l e a s t two c e l l s  When a b n o r m a l chromosome  All  were  water and f i x e d  be s t o r e d  : hydrochloric  F o r each p l a n t ,  chromosome  3.3.3  Root  fuchsin  s q u a s h e d and o b s e r v a t i o n  root  tips  was f o l l o w e d  could  rinsed with  carbol  distilled  fixation  but the m a t e r i a l  HCl (ethanol  with modified  the  Root  (3:1 v / v ) a t room  Usually  after this  concentrated 45-60  rinsed with  - acetic acid  hydrolysis,  ethanol  by  i n 0.003 M 8 - h y d r o x y q u i n o l i n e a t 4°C f o r 4-6  (25°C) o v e r n i g h t . by  thick  personal  communication) w i t h minor m o d i f i c a t i o n s . pretreated  fresh,  were c a r r i e d o u t  20) from e a c h p l a n t tube.  They were  were  ground  i n the tube a f t e r adding a small containing  drop  0.1 M Na/K p h o s p h a t e a n d 1  43 mM EDTA a t pH 7.4. microcentrifuge  The t u b e s w e r e c e n t r i f u g e d  f o r three  m i n u t e s a t 15,600 X g.  L e g h a e m o g l o b i n c o m p o n e n t s were a n a l y z e d acetate  electrophoresis  electrophoresis  using  the c e l l u l o s e  (CAE) m e t h o d o f H o l l e t a l .  (Gelman, Sepra-Tek e l e c t r o p h o r e s i s acetate  in a  strips  system).  (1983)  Cellulose  ( S e p r a p h o r e I I I ) were  pre-soaked i n buffer containing  (g/1):  Tris  ( 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 ) : 2 0 . 7 2 ; EDTA: 0.15; glycine:  3.53 a n d s u c r o s e :  application  After electrophoresis  forprotein  acetic acid.  f o r t e n minutes and c l e a r e d  f o r haem p r o t e i n s  o - d i a n i s i d i n e method d e s c r i b e d  Isozyme  i n 5%  be f u r t h e r  using the  by H o l l e t a l .  (1983).  Analysis  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  evaluated  t h e 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 isomerase  (PGI),  deyhdrogenase of C l a y t o n was  t h e s t r i p was  S t r i p s from s e v e r a l runs c o u l d  stained s p e c i f i c a l l y  for  sample  i n P o n c e a u S ( 1 0 0 mg/ml i n 7.5% a q u e o u s  t r i c h l o r o a c e t i c acid)  3.3.4  Following  t h e s t r i p was r u n a t 300 V f o r 40-50 m i n u t e s a t  room t e m p e r a t u r e . stained  15.0.  (6-PGDH), p h o s p h o g l u c o s e  phosphoglucomutase  (SKDH).  and T r e t i a k  (PGM) a n d  The m o r p h o l i n e - c i t r a t e ( 1 9 7 2 ) was u s e d .  as f o l l o w s : e l e c t r o d e  shikimate buffer  Buffer  b u f f e r : 0.04 M c i t r i c  ( a n h y d r o u s ) w i t h pH a d j u s t e d  t o 6.1  system  composition acid  with  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  citric  44 acid lot  (anhydrous) w i t h the No.  18  ml  of  the  gel  while oven to  392)  and  buffer  buffer the  sucrose  (35  and  ml  332  s o l u t i o n was  remainder  to b o i l i n g .  the  pH  of  The  the  The  formers,  s o l u t i o n was  shaken  g)  slightly  water  buffer  the  i n one  flask  was  4°C  in  then  added  poured onto  room  an  even  a smooth s u r f a c e . kept a t  1/3  mixture  heated  an  g, and  About  s o l u t i o n was  to achieve  down t o  (49  starch/sugar  d e g a s s e d and  to give  Starch  s o l u t i o n was  shaking  They were c o o l e d  6.4.  into another.  added to  were wrapped w i t h S a r a n Wrap and overnight.  to  were p l a c e d  b o i l i n g buffer  s t a r c h mixture with  mixture.  adjusted  gel  Gels  temperature  prior  to  electrophoresis. Two of  cold  small  young l e a f l e t s  extraction  each w e l l  of  buffer  pestle.  filter  of  the  The  gel.  inserted  gel  side  2 cm  protected  gel  sponges.  was  i n t o 30 dye  drops  were added on  i c e to  t a k e n up  to prevent  directly  3 mm  s l o t s at (red  two  homogenized with  to approximately  wide.  the  food  a  into Wicks  cathodal  dye)  of  wrapped w i t h S a r a n Wrap b o t h ends were e x p o s e d .  from d e s s i c a t i o n  g e l were j o i n e d w i t h t h e  cellulose 3-4°C.  the  was  and  end  was  slot.  still  approximately  1983) placed  material  A wick c o n t a i n i n g  was  pad,  crude e x t r a c t  sequentially  i n one  The  ends of  The  paper wicks precut  were i n s e r t e d  (Helenurm,  a sample g r i n d i n g  isozyme d e n a t u r a t i o n . glass  from e a c h p l a n t  The  Enzyme p r o t e i n s  g e l was  and  at  moved a n o d a l l y  This  wrapping  contamination.  electrode run  but  300 at a  buffer V,  via  50-60 mA  rate  The  at  determined  45  by  their  size,  The  staining  receipes  Staining  into  fixed  resulting  a t 37°C.  i n 4%  methanol  Normally composed  were The  evaluated  of  met  when  three  were  trait  in a  from  from  t o 32 mm  chosen  other  was  standard  from  variance  Changes  ratio  of  in  at  of  were  chosen  two  further  taken:  as  number  obtained  Leaflets  Mean,  f o r each  and c o n t r o l  population  and  were  To  conditions the  middle  measured  30 mm  were  25  mm  ranging  randomly  Ratios variance of  plants.  another  length; approximately  data.  clover  plants.  (2) l e a f l e t s  f o r l e n g t h and width.  regenerated  red  (1) O n l y  f o r 'Altaswede'  the observed  of  seed-derived  i n regenerated  f o r 'Norseman'.  of each  leaf  in leaflet  and  was  factors,  range  d e v i a t i o n were  "populations"  incubated  the f i r s t  measured and  t o 27 mm  and measured  calculated  and  23  introduced  acid.  regenerated  to look  restricted  ranging 28  except  measurements were  leaflets  and  staining  w e l l - s t a i n e d , they  10% a c e t i c  to width  by  were  slices.  individual  Boxes were  gels  5-6  separated  leaflets.  f o r both  variation  were  to the  electrophoretic  into  into  according  Analysis leaf  length  morphological limit  and  of t h r e e  leaflet  were  each  sliced  stain.  After  prepared  Following  poured  slices  3 . 3 . 5 Morphological  is  (1983).  were  the appropriate  darkness  were  s t a r c h g e l s were  solutions  and  charge.  solutions  of V a l l i e j o s  separation,  boxes  shape and  were and  different  plants.  compared w i t h  The  mean  those  of  46 the proper  control.  For the comparison  v a l u e s were c a l c u l a t e d were t e s t e d from  for significance.  regenerated  performed.  by f o l l o w i n g  and c o n t r o l  <7_ _ x! -x  of v a r i a n c e , F  e q u a t i o n : F = o\/o\ and  For the comparison plants,  the u test  c a n be o b t a i n e d by f o l l o w i n g  o f means  was equation  2  1978): o  = /a /n,  +  2  X  1  X  and  then  u =  (x,-x )/a  2  2  x  The  al/n  2  1  significance  testable.  x  2  of d i f f e r e n c e  between means i s t h u s  (Ma,  4.  4.1 C a l l u s  4.1.1  Induction  Callus The  Table callus  Induction  data  2.  induction  the  frequency  the  second  grown  intensity  total  of f i v e  did  calli  'Norseman'  the f i r s t  f o r 'Altaswede'  that  'Altaswede'  intensity  o r when  was a l s o o b s e r v e d  i n another  of c u l t u r e s  were  when e x a m i n e d a f t e r  be s l i g h t l y  initiated  was a l s o a p p a r e n t  of 'Norseman' was  layer  o f w a t e r on t h e a g a r  tubes  wrapped w i t h p a r a f i l m  47  30 d a y s  induction  culture.  greatly  while  which  i f measurements a r e made  the c a l l u s  medium  two  two weeks o f c u l t u r e  Therefore, callus  that  A  for callus  50 t u b e s  when r e - e x a m i n e d  higher  when  batch of c u l t u r e .  In one b a t c h o f c u l t u r e s ,  transfer.  to  increase in c a l l u s  a longer p e r i o d of i n i t i a l  frequency  For  showed an i n c r e a s e d  a t low l i g h t  i n four c u l t u r e s  the explant  It  showed a h i g h e r  from  suggest  An  a r e shown i n  I n d u c t i o n f r e q u e n c i e s were r e c o r d e d a f t e r  f r e q u e n c i e s may after  constant  'Altaswede'  better  batches  not have c a l l u s  after  batch  'Norseman'.  The d a t a  decreased  incubation.  produced  was  dish culture.  light  weeks  2.  frequency  induction.  than  However,  perform  in petri  frequency  i n the second  of i n d u c t i o n  i n Batch  induction  induction  frequency  batch.  e x p l a n t s may  Frequency  for callus  'Altaswede'  frequency  RESULTS  induction  affected  when a  d i d not evaporate 'Altaswede'  was  thin in test  less  48  Table  2. C a l l u s clover  i n d u c t i o n f r e q u e n c y f o r two c u l t i v a r s on L2 medium.  B a t c h No  1  Cultivar  of r e d  2  A l t a s w e d e Norseman A l t a s w e d e  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 p r o d u c i n g callus  192  1 12  236  816  Frequency  (%) 2 X 2  66.7 Contingency  x  64.4  2  86. 7  65.5  Analysis  Batch 1  Altaswede  v s Norseman  X  2  1 = 0 .023 ns  Batch 2  Altaswede  v s Norseman  X  2  = 7 .954 **  Combined data  Altaswede  v s Norseman  X  2  = 4 .030 * (P<0.05)  ns: not signi f icant. 1  significant;  . *:significant;  (P<0.01)  **:  highly  49 affected  4.1.2  Genotypic  Effect  on C a l l u s  Tubes w i t h  callus  induced  grouped number  into  If  a r e shown  one e x p l a n t  in Table  produced  phenotypically  similar.  among t h r e e  a response  3.  depending  the other  response.  two  explants  to the uniform  from  this  from one i n d i v i d u a l environment  explants  produced c a l l i  genotype  originating  that  experiments,  from a n o t h e r  i n t h e same  were  t u b e was e l i m i n a t e d by l a t e r f r o m one i n d i v i d u a l  similarly.  (e.g. i n color)  f r o m one g e n o t y p e The p o s s i b i l i t y  The  from t h e data  Calli  look d i f f e r e n t  were  upon t h e  was e v i d e n t .  It i s clear  callus  genotypes c o u l d  whereas t h e t h r e e c a l l i  simply  i n t h e two b a t c h e s  f r o m one g e n o t y p e b e h a v e d  t u b e a l s o showed a s i m i l a r  similarity  Induction  f o r which c a l l u s  the three e x p l a n t s  different  conditions.  1, 2 o r 3 - c a l l u s t u b e s ,  of e x p l a n t s  results that  by t h e same e x p e r i m e n t a l  was  i n the t e s t  w h i c h showed while  that  explants  i n t h e same c u l t u r e v e s s e l d i d n o t  (Figure 1).  4.1.3  Observations After  on C a l l u s  the i n i t i a l  Induction  transfer,  some e x p l a n t s  f r e s h a p p e a r a n c e and d i d n o t s h r i v e l , 3-4 d a y s s u c h  explants  the  swollen  portion.  the  initial  explant  commenced  o r t u r n brown.  s w e l l e d and c a l l u s During  t h i s process  disappeared.  at the periphery  retained a  could develop the green  The g r e e n  of the explant,  After  color  from  c o l o r of  loss  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 t h r e e e x p l a n t s c o m i n g from t h e same s e e d l i n g i n two b a t c h e s o f c u l t u r e . D a t a for two c u l t u r e b a t c h e s have been c o m b i n e d .  Culture  Cultivar Altaswede  No.  of tubes  No. of callus  cultured  tubes  with  (%)  Norseman  (%)  167  266 197  (100)  114  (100)  (90.4)  109  (95.6)  3-callus  tube  180  2-callus  tube  12  (6.1)  3  (2.6)  1-callus  tube  5  (2.5)  2  (1.8)  51  Figure  1.  C a l l u s i n d u c t i o n and g e n o t y p e 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 individual (upper half of p l a t e ) produced c a l l i , w h i l e e x p l a n t s 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 e x p l a n t s showed e a r l y  callus  development, f o l l o w e d by c o n t i n u i n g d i v i s i o n and c a l l u s formation a f t e r two weeks. did  not produce  E x p l a n t s , which withered a f t e r callus.  initial  Other e x p l a n t s showed s l i g h t  transfer,  browning  with some s w e l l i n g ; these e x p l a n t s c o u l d develop f u r t h e r but seldom produced  vigorous c a l l u s growth.  Calli  formed  under  dark c o n d i t i o n were white while those induced i n the l i g h t u s u a l l y showed some green  4.2 C a l l u s Maintenance  4.2.1  Primary  coloration.  and P l a n t  T e s t o f Two  Approximately  Regeneration  Media  85 c a l l i  on each of the two media.  of each c u l t i v a r were c u l t u r e d In n e i t h e r case was shoot  i n d u c t i o n or bud i n i t i a t i o n observed when recorded a f t e r two weeks of c u l t u r e .  Although c a l l i  medium, more green-spotted c a l l i at  t h i s time.  medium. After  Calli  on B  5  grew f a s t e r on B (N2A2) 5  were observed on LSE medium  were whiter than those on LSE  Some root formation was observed on B  17 days some c a l l i  f r e s h media.  5  medium.  i n both cases were t r a n s f e r r e d  onto  In these s u b c u l t u r e s many of the g r e e n i s h  r e g i o n s became brown and the number of green-spotted c a l l i decreased. C a l l u s response on LSE medium More green-spotted c a l l i 'Norseman' than Then c a l l i  i s shown i n Table 4.  were observed i n c u l t u r e s  'Altaswede'  from  a f t e r two weeks of c u l t u r e .  turned darker and a f t e r the s u b c u l t u r e the number  53 Table  4. C a l l u s g r o w t h on L S E medium. D a t a f o r t h e initial c u l t u r e a n d s u b c u l t u r e a r e shown. The f i r s t c u l t u r e was measured a f t e r two weeks o f c u l t u r e and t h e subculture was measured four weeks after the transfer.  Cultivar  NC  GSC  1  Altaswede  86  21  (24.4) 0  0  1  Norseman  88  40  (45.5) 0  0  2  Altaswede  56  2 ( 3.6)  2  Norseman  59  10  Culture  NC=Number of Calli;  Calli;  RC=Root  Calli  (%)  SC  RC  0  2  (16.9) 0  0  GSC=Green - S p o t t e d  Calli;  SC=Shoot  54 of  green-spotted c a l l i  cultivar weeks. was  50 c a l l i During  callus  of  of t h e  f o r about  callus  onto  LSE  months.  presented  had  promotional  transferred was  callus  in Table  roots.  initiate  three calli  one  Upon  been g r o w i n g  Continuous  on  LSE  subculturing bud  or  of  shoot  with  poorer  culture  from  Some c a l l i 2).  5.  initial  t o show an  senesced;  no  fresh  t u r n e d brown  on  shoot  the  induction.  for a short period. yellow-green  subculture  produced  (Figure  3).  of c a l l i  T h e r e f o r e , the  i n the p r e s e n t case  i s less  were  good of  or  calli  stopped initial  were s u b c u l t u r e d , t h e y  growth  At  calli  Then t h e c o l o r  r o o t s d u r i n g the  the c a l l i  unhealthy  After  t o brown  s p o t s d i s a p p e a r e d and  95%  first  medium, g r o w t h and  r o o t s were p r o d u c e d  approximately or dead.  and  None o f t h e s e a p p l i e d c o n d i t i o n s  After  continued  senesced  the  fresh  Green  (Figure  Medium  L2 medium t o LSE  maintained  changed  growing.  effect  from  yellow-brown.  shoots  further  g r o w t h of t h e  as b e i n g a s s o c i a t e d  LSE  1: R e s u l t s of  are  period  had  each  growth.  Test  roots  for a  some new  medium d i d not  C a l l u s G r o w t h on  color  LSE  From  s p o t s d i s a p p e a r e d and  calli  as w e l l  on  obvious  produced  test,  two  differentiation,  4.2.2  no  A l l the green  medium  callus  period  'Altaswede'  termination  sharply decreased.  were c u l t u r e d  this  observed.  was  response; and  t h e end  more  calli  previously of the  formed  culture  of both c u l t i v a r s  were  failure  induce  likely  o f LSE related  to to  light  55  Table  5. R e s p o n s e o f c a l l i on L S E medium: initial culture and subculture o f T e s t 1 r e c o r d e d a f t e r f o u r and f i v e weeks o f c u l t u r e , r e s p e c t i v e l y .  Culture  Cultivar  Light  NC  SC  RC  intensity MEm" s e c " 2  1  Initial  Altaswede  100-900  1452  0  1 3  Subculture  Altaswede  100-900  984  0  0  Initial  Norseman  70-450  1 556  0  1 5  Subculture  Norseman  70-450  568  0  0  NC=Number o f C a l l i ;  SC= Shoot  C a l l i ; RC=Root C a l l i  56  Figure  2.  R e s p o n s e of ' A l t a s w e d e ' 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 d i s a p p e a r and r o o t s were i n d u c e d during this culture period.  Figure  3 . R e s p o n s e o f ' A l t a s w e d e ' c a l l i on L S E medium after five weeks o f c u l t u r e . C a l l i showed an u n h e a l t y growth response. (a) C a l l i became brown and stopped growing ( u p p e r ) , (b) No f r e s h r o o t s were p r o d u c e d and p r e v i o u s l y formed r o o t s t u r n e d brown (lower).  58 condition  or  Test (Table and  sample  2: No  6).  other  As  shoot  The  failure  suggested  these media.  basal  salts  solutions; only  shoots  to induce  medium, o t h e r w i s e on  from  they On  fewer  regulators.  on  these  tests,  w i t h NAA  and  than  KIN.  t h e media u s e d been  formed  on  into  in LSE  shoots  on  full  strength  t h o s e on  half  strength  showed brown c o l o r .  Roots  added  regulators  in Table  as  7,  formed  growth  tended  results  previous tests. t h e y had  to  keep  were s i m i l a r  Calli  of  been b e f o r e t h e i r  these  t e s t s demonstrated  to  'Norseman' transfer  that  LSE  i t a good medium f o r c a l l u s  browning,  senescence  and  response  maintenance. throughout  ultimately  p a t t e r n s t r e n g t h e n e d w i t h the time  medium.  medium  p r o d u c t i o n under c u r r e n t e x p e r i m e n t a l  t h i s medium showed a . s i m i l a r  response  this  L2(NO.05K0.5)  longer.  shoot  nor was  Calli  on  from  induce  conditions  This  embryos had  (1980)  medium i s  In a d d i t i o n tests  media  medium.  Results not  four  Collins  s t u d y , LSP  t h e medium w i t h o u t  shown  were n o t a s w a t e r y  and  f o r m a t i o n on  healthier  these o b t a i n e d through  did  Phillips  the  s h o u l d be a b l e t o d e v e l o p  calli on  no  on  L2(P0.002B0.2), c a l l i  calli  3: As  this  formed  i n the  However, a d d i n g  green-spotted  onto  by  shoot  that  appeared  calli  Test  ever  o f embryo d e v e l o p m e n t .  shown t o i n d u c e  test  was  callus.  of the p r e s e n t  was  this  of  demonstrated  results  supportive  size  death. of  callus  59  Table  6.  Response o f c a l l i from L S E medium subcultured o n t o f o u r media ( c o n c e n t r a t i o n i n mg/1).  Medium  1  2  PIC  0.002  0.002  no  BA  0.2  0.2  regulators  4  3  NAA  0.05  KIN  0.5  growth  full  half  full  full  NC  42  36  39  33  RC  0  0  0  2  GSC  2  1  0  0  BC  3  6  5  6  SALTS Results  NC=Number Calli;  of  Calli;  BC=Blackened  RC=Root Calli  Calli;  GSC=Green-Spotted  60  Table  7. R e s p o n s e  of  calli  on L2(D0.01A5) medium,  Cultivar  Altaswede  Norseman  NC  99  99  GSC  3  9  RC  1  0  Color  brown o r  Rate of G r o w t h  ++  NC=Number o f Calli. ++, +++: growth.  Calli;  More p l u s  black  slightly  brown  +++  GSC=Green-Spotted signs  indicate  Calli;  faster  RC=Root  relative . callus  61  4.2.3  2,4-D Results  on  and BA from B a t c h  t h e s e two media were v e r y s i m i l a r  Obvious d i f f e r e n c e s cultivars. calli  than  structures shoot  existed  'Altaswede' calli.  and  I t was  some c a l l i  t h e number leaf-like remained For  of both c u l t i v a r s  two, 20 t o 25 c a l l i  f o r each  treatment.  Calli  on medium  differentiation.  Data  of c u l t u r e  than  sign f o r  f o u r weeks o f  had t u r n e d brown and One  o f t h e two  brown, w h i l e  one  No  of ' A l t a s w e d e '  s h o o t s were  on t h i s medium  w i t h 2 mg/1  recorded after  were  i n d u c e d on any were  grew  2,4-D  had  observed  faster. root  t h r e e weeks a n d  four  showed t h e same t r e n d .  P I C and BA Test  1: R e s u l t s i n T a b l e  concentrations or  faster  leaf-like  However, g r e e n - s p o t t e d c a l l i  one c a l l u s  4.2.4  two  decreased.  become  cultivar.  green.  on L2(D2B2A5) medium.  weeks  grew  When r e c o r d e d a f t e r  s t r u c t u r e s had a l s o  these media.  Only  that  Calli  green-spotted  'Norseman' c a l l i observed  8.  o f t h e two  had more  of g r e e n - s p o t t e d c a l l i  Batch  cultured  for a single  f o r m e d on L2(D2B2) medium, a b e t t e r  differentiation.  culture,  i n Table  between c a l l i  'Norseman' g e n e r a l l y  'Altaswede'  of  one a r e p r e s e n t e d  roots,  effect  10 mM  on s h o o t  arginine,  tested  9 indicated  d i d not induce c a l l i  that  t o produce  a r g i n i n e a d d e d t o t h e medium or root  high levels  PIC a t t h e  also  h a d no  i n d u c t i o n and i n t h e p r e s e n c e  o f PIC i n d u c e d more  shoots  of  green-spotted  (52  Table  8. R e s p o n s e o f c a l l i on m e d i a recorded after three ( c o n c e n t r a t i o n i n mg/1).  c o n t a i n i n g 2,4-D and BA weeks of culture  Medium  Growth regulator  1  2  3  4  2,4-D  2  2  2  2  BA  2  2  4  4  Cultivar  Altaswede  Norseman  Altaswede  Norseman  NC  50  50  50  36  GSC  3  16  4  17  Morphogenesi s  0  two leaf-like structures  0  0 '  +++  ++  +++  Growth  R a t e ++  NC=Number o f C a l l i ; G S C = G r e e n - S p o t t e d C a l l i . ++, +++: More plus signs indicate faster relative growth.  callus  63  Table  9.  Treatment  Response o f ' A l t a s w e d e ' c a l l i on m e d i a c o n t a i n i n g PIC and BA i n p r e s e n c e and a b s e n c e o f a r g i n i n e (1740 mg/1) (growth regulator concentration in mg/1).  PIC  BA  Arg  NC  RC  SC  GSC  +  14  0  0  0  1  0.001  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  0.2  NC=Number of C a l l i ; RC=Root C a l l i ; GSC=Green-Spotted C a l l i  SC=Shoot  Calli;  64  calli. Test adding had  2: The r e s u l t s  glutamic  no e f f e c t  induction. of  acid  on s h o o t  mg/1  4).  induced  those  Those c a l l i  contrast  factor calli  indicating  i n determining and r o o t s  concentrations  became  Some c a l l i  from  11.  r o o t s were  4.2.5  NAA Test  induced  0.005 mg/1 i.e.  rooted  were In  More  important  green-spotted  PIC a t b o t h  BA  'Altaswede' c a l l i  calli  and  on  green-spotted  'Norseman'.  of ' A l t a s w e d e '  f r o m L2 were a l s o t r a n s f e r r e d Neither  shoots  induced.  and KIN 1: R e s u l t s  when c a l l i  containing  shoots  i s an  onto L2(P0.002B0.2) and L2(P0.02B0.2) media. nor  o f 5,000  formed a t a l l  source  formation.  More  No  1, r o o t s  one e x c e p t i o n  were p r o d u c e d  additives  o f PIC and BA.  that c a l l u s  formed w i t h with  these  media  black.  in Table  of T e s t  root levels  g r o w i n g on t h e s e  o f 'Norseman' on medium  root  L2(P0.005BO.2) medium. calli  Calli  various combinations  to the r e s u l t s  combinations  t o t h e medium  sample s i z e p r o h i b i t e d  on m e d i a w i t h o u t  r e s u l t s were shown using  that  a p p e a r e d a t low  but the s m a l l  casein hydrolysate The  calli  interpretation.  were b r o w n e r t h a n  10 showed  i n d u c t i o n , nor d i d i t i n c r e a s e  More g r e e n - s p o t t e d  a conclusive  i n Table  and c a s e i n h y d r o l y s a t e  casein hydrolysate,  (Figure  presented  NAA  (Table  12) showed t h a t no s h o o t s  f r o m L S E were s u b c u l t u r e d  and KIN.  were  o n t o media  Roots and g r e e n - s p o t t e d  calli  were  65  Table  10. R e s p o n s e of calli on media with h y d r o l y s a t e and g l u t a m i c a c i d .  added  casein  1  Cultivar  NC  RC  SC  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  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  CH o r GA  Altaswede  Norseman  .  (mg/1)  NC=Number of RC=Root C a l l i ; SC=Shoot Calli ; C H = C a s e i n H y t r o l y s a t e ; GA=Glutamic A c i d . 1  A11  media c o n t a i n e d  0.005mg/l PIC and  1 mg/1  Calli; BA.  66  Figure  4.  Response of 'Altaswede' calli on L2 medium c o n t a i n i n g 0.005 mg/1 P I C , 1 mg/1 BA and 1 g/1 casein hydrolysate after f i v e weeks o f 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  Table  11.  Response o f c a l l i o f two c u l t i v a r s on media w i t h v a r i o u s c o m b i n a t i o n s o f PIC a n d BA ( c o n c e n t r a t i o n i n mg/1).  Cultivar  PIC  BA  NC  RC  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)  0..002  0..2  46  18 (39)  33 (72)  0..005  0.,2  44  21  36  0..01  0..2  44  8 (18)  36 (82)  0,.002  1  42  6 (14)  27  0..005  1  42  7 (17)  33 (79)  0..01  1  46  6 (13)  34  Norseman  NC=Number o f C a l l i ;  RC=Root C a l l i ;  (%)  (48)  GSC  GSC=Green-Spotted  (%)  (82)  (64)  (74) Calli  68  Table  12. R e s p o n s e o f 'Norseman' c a l l i from LSE various combinations of NAA ( c o n c e n t r a t i o n i n mg/1).  medium to and KIN  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 o f C a l l i ;  RC=Root C a l l i ;  GSC=Green-Spotted  Calli  69 p r o d u c e d when t h e NAA c o n c e n t r a t i o n was l o w (0.001 a n d 0.01 mg/1). Test  2: S h o o t s were i n d u c e d  experiment  (Table  of c a l l i .  Several  shoots they  shoots  were f r o m e m b r y o s shoots  rooted c a l l i low  1 3 ) . They o r i g i n a t e d f r o m t h e u p p e r  had w e l l - f o r m e d  induced  (Figure 5). 0.05 mg/1  Both treatments NAA.  interface.  A l l these  I n c o n t r a s t , more  concentrations  mg/1. KIN  M o s t o f them  calli  were  14) t h a t r o o t  formation  a n d minimum when t h e a u x i n r c y t o k i n i n r a t i o  (1.0).  The maximum number o f  green-spotted  concentrations The t h r e e  root only  calli  r e s u l t e d when b o t h  NAA a n d  were l o w .  t e s t s above were aimed a t s t u d y i n g  o f NAA a n d K I N on c a l l u s  induced  was  b u t a t 1.0 mg/1 NAA when K I N c o n c e n t r a t i o n was 2.0  Fewer g r e e n - s p o t t e d  Although  was  a t both KIN  a p p e a r e d a t 0.1 mg/1 NAA when K I N c o n c e n t r a t i o n  0.5 mg/1,  was  Few r o o t s o r i g i n a t e d  maximum when NAA c o n c e n t r a t i o n was 0.1-0.2 mg/1  calli  which  in color.  3: I t was shown ( T a b l e  was h i g h e s t  that  Many r o o t s o r i g i n a t e d f r o m one c a l l u s , t h e  from t h e t o p p a r t of c a l l u s .  yellow-green  Some  and r o o t s , s u g g e s t i n g  from s e v e r a l t o s e v e r a l dozens.  from c a l l u s - m e d i u m  part  from a s i n g l e c a l l u s .  w e r e p r o d u c e d when t h e NAA c o n c e n t r a t i o n  number r a n g i n g  Test  arose  cotyledons  contained  (0.01 mg/1).  originated  on two m e d i a i n t h i s  formation  the e f f e c t  growth and d i f f e r e n t i a t i o n . occurred  i n one c a s e .  in a l l tests,  This suggests  shoots  were  t h a t : (1)  e m b r y o g e n e s i s h a d been t r i g g e r e d on SCP medium b e f o r e  calli  70  Table  1 3 . Response of c a l l i of  NAA  f r o m SCP medium t o c o m b i n a t i o n s  and KIN ( c o n c e n t r a t i o n  i n mg/1)  NAA  KIN  NC  sc  RC  0.01  0.5  20  0  15  0.05  0.5  20  2  0.1  0.5  22  0  13  0.05  2.0  20  1 (25)  8  Numbers callus. 1  in  the  NC=Number o f C a l l i ;  (6,4)  1  1 1  b r a c k e t a r e t h e number o f s h o o t s SC=Shoot C a l l i ;  RC=Root  Calli  f o r each  71  Figure  5.  Shoot formation from 'Altaswede' callus on L 2 ( N 0 . 0 5 K 2 ) medium. C a l l u s h a d b e e n c u l t u r e d on SCP medium. S h o o t s o r i g i n a t e d f r o m t h e u p p e r part of the c a l l u s , with some h a v i n g c o t y l e d o n s a n d roots.  72  Table  NAA  14. R e s p o n s e o f 'Norseman' calli t o combinations of NAA a n d KIN ( c o n c e n t r a t i o n i n mg/1).  (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  0.5  0.5  46  6  0.2  2.0  50  11  (22.0)  20  (40.0)  1 .0  2.0  48  10  (20.8)  25  (52.1)  NC=Number o f  Calli;  RC =Root  (23.9) (13.0)  10 7  C a l l i ; GSC=Green-Spotted  1  (21.7) (15.2)  Calli  73  were t r a n s f e r r e d so t h a t  shoots  only  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 a n d (2) c o n c e n t r a t i o n of  NAA  is critical  containing  0.05 mg/1  In T e s t mg/1,  0.001  0.1-0.2  shoots  were i n d u c e d  1 more r o o t s were p r o d u c e d when NAA  mg/1  NAA  important  a t both  KIN c o n c e n t r a t i o n s .  factor  is callus  source.  3 had been c u l t u r e d on medium w i t h compared w i t h  higher  those  used  in Test  high  1.  s u b c u l t u r e more r o o t e d  light  d i f f e r e n c e , the  level  used of  f o r Test  2,4-D  Thus, d u r i n g t h e  calli  3 than  were p r o d u c e d a t  of auxin  i n Test  a l s o apparent  t h a t NAA  was more c r i t i c a l  i n Test  1.  I t was  to root  formation  KIN.  4.2.6 NAA  and BA i n P r e s e n c e o f ADE  After the  was a t  Although  Calli  levels  than  on media  3 more r o o t s were r e s u l t e d a t  c o u l d c o n t r i b u t e to the observed  subsequent  only  NAA.  but i n T e s t  conditions most  because  one month o f c u l t u r e no s h o o t s  f o u r media t e s t e d  (Table  15).  Two  p r o d u c e d on medium w i t h o u t  NAA  This  the accepted  result  formation Slightly levels  c o n t r a s t s with  is facilitated  of  NAA.  rooted  calli  calli  auxin  view t h a t  BA.  root  to cytokinin  were o b s e r v e d  on  were  b u t c o n t a i n i n g 0.2 mg/1  by h i g h  more g r e e n - s p o t t e d  were i n d u c e d  ratios.  at high  74  Table  15. R e s p o n s e o f 'Norseman' c a l l i on media containing various levels o f NAA i n c o m b i n a t i o n w i t h BA a n d ADE ( c o n c e n t r a t i o n i n mg/1). 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  A l l t h e media  containing  1  NC=Number of  Calli;  2  mg/1 ADE.  RC=Root C a l l i ; GSC=Green- S p o t t e d  Calli  75 4.2.7  Callus  Differentiation  As shown to  induce  cultured  shoots  root  was  formation  Although  with  t h e t r e n d was o b v i o u s  related  the r e s u l t  BA a n d ADE p r e s e n t  h a d more  per rooted c a l l u s ,  from  efficient  was  less  that  root  This observation on NAA  any added a u x i n  rooted c a l l i  similar  were  of a u x i n s , the  i n the t e s t s  without  (medium 4) w h i c h was  able  t o a u x i n c o n c e n t r a t i o n and  obtained  Medium  r o o t number  shoots  only ten c a l l i  by t h e p r e s e n c e  was  f o u r m e d i a was  was e n h a n c e d by BA a n d ADE.  ( S e c t i o n 4.2.6).  average  calli.  inhibited  of i n h i b i t i o n  coincides BA  from  Media  16, none o f t h e s e  f o r each treatment,  formation extent  i n Table  on F o u r  but w i t h  and t h e maximum  while  t o t h a t used f o r root  and  t h e medium  f o r root  induction  formation from  callus.  4.2.8  Callus Results  on and  Growth and A u x i n of t h i s  L2(D11K1) medium  test  are presented  i n Table  17.  were g r e y  to dark-grey  in color,  showed no d i f f e r e n t i a t i o n  (Figure 6).  On  medium  t h e r e were b a s i c a l l y  callus  was  was  to Cytokinin Ratio  fast  growing and g r e e n .  brown-yellow  were g r e e n  uniformly medium.  in color.  calli.  green-spotted  calli  green  two c a l l u s  Among  These green  190 c a l l i  calli,  which had i s o l a t e d  One  form  two m e d i a p r o d u c e d  a different  callus  u n l i k e normal green  to cytokinin callus  of  form o f  c u l t u r e d , 134  r e g i o n s , were  b e c a u s e o f t h e h i g h KIN c o n t e n t  The c o n t r a s t i n g a u x i n  friable  L2(D1K10)  forms.  Another  Calli  i n the  ratios  morphology.  of the  76  Table  16. Differentiation of L2(D2B2A5) medium on f o u r mg/1)  Medium 1 composition full  L2 SALTS  'Norseman' c a l l i from media ( c o n c e n t r a t i o n  2  3  4  full  full  half  1  0.01  2,4-D  0.1  NAA BA  0.2  0.2  0.2  ADE  2  2  2  IAA  0.2  Nicotinic ac i d  1.0  Result NC  1 0  10  10  10  RC  7  3  3  2  4  4  3  1  Average Root N o .  2  1  L2 medium w i t h h a l f  strength  2  B a s e d on t h e number  of root  NC=Number o f C a l l i ;  RC=Root  o f s a l t s and calli.  Calli.  vitamins.  77  Table  17.  Response of 'Norseman' c a l l i grown on media o f h i g h (11) o r low (0.1) a u x i n t o 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 ) g r e e n - y e l l o w  RC  none  less  NC=Number o f C a l l i ;  RC=Root  Calli  than  10  78  Figure  6.  Response of 'Norseman' calli on media with different auxin to cytokinin ratios after five weeks o f 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 g r e y i n c o l o r ( u p p e r ) , (b) C a l l i on L2(D1K10) medium ( l o w e r ) . Two forms of calli resulted. The majority of calli were the green-type ( l e f t p e t r i p l a t e ) .  79 R o o t s were o n l y cytokinin NAA  and  BA  (Section  4.2.9  ratio.  f o r m e d on  T h i s phenomenon, o b s e r v e d  ( S e c t i o n 4.2.6) and  4 . 2 . 7 ) , was  Recovery of Senescing  green-spotted calli  SCP  of  were most commonly  It is clear  efficient  in restoring  frequency  decreased  medium. source  SCP  from  here.  calli 18.  isolated  medium  these  the  also  to large These  green  (so-called  results  t h a t SCP  and  regenerated  regions.  i n diameter  senescing c a l l i  on  SCP  (Figure  efficient  7)  SCP medium  t h a t the  was  recovery  l e n g t h of c u l t u r e p e r i o d on when a d i f f e r e n t  medium grew v e r y  large c a l l i  with diameter  one  period  culture  green-spotted  and  (3-4  g r e a t e r than  weeks)  many o f  fast.  LSE  callus  centimeter  (Figure 8).  They were  them had  L 2 ( P 0 . 0 0 2 B 2 ) were c o m p a r e d . similar  t o SCP,  media and  calli  On  grew  Many became  one  nodular  within  structures.  of t h r e e m e d i a , L 2 ( D 2 N 2 K 2 ) , L2(D2B0.5A2)  two  f o u r media  Callus  Most c a l l i  other  with  used.  M a i n t e n a n c e of  was  had  centimeter  on  with  R e c o v e r y was was  one  seen  callus).  test  senescing  i s shown i n T a b l e  than  of  to  Medium  were f a s t - g r o w i n g and  They were b i g g e r  auxin  Callus  frequency calli  confirmed  low  in tests  the comparison  once a g a i n  C a l l u s Growth on  Recovery  t h e medium w i t h  In a  and  L2(D2B0.5A2) medium, w h i c h faster  more g r e e n - s p o t t e d  than  calli  calli  on  the  were p r o d u c e d .  80  Table  18.  Recovery transfer  frequency of t o SCP medium.  senescing  Callus  NC No. of calli Frequency SCP c a l l i  SCP of (%)  NC=Number o f  calli  after  source  1  2  3  4  5  40  48  120  72  144  5  12  42  48  61  12.5  25.0  35.0  66.7  43.3  Calli  81  Figure  7. R e c o v e r y o f s e n e s c i n g c a l l i on SCP medium. Before transfer, t h e s e c a l l i were d a r k e n e d a n d brown i n color and had stopped growing. The calli i l l u s t r a t e d a r e a f t e r f i v e weeks c u l t u r e .  82  Figure  8. C a l l u s m a i n t e n a n c e on SCP medium. R a p i d g r o w t h on t h i s medium p r o d u c e d c a l l i w i t h d i a m e t e r s greater than one centimeter w i t h i n t h r e e t o f o u r weeks, (a) 'Altaswede' calli (upper); (b) 'Norseman' calli (lower).  83  Calli  were e a s i l y  subculturing. LSE  medium  Some c a l l i  on SCP t o J u l y  onto  Calli  from  each  less  were g r e e n - y e l l o w  subculture for  o f two m e d i a : SCP a n d onto  i n c u b a t e d i n a g r o w t h chamber. Although  v i g o r o u s and not f r e s h  them o n l y  9, 1985)  on SCP w i t h o u t  L2(P0.001B0.1) were t r a n s f e r r e d  t h e y were v e r y l a r g e .  7, 1984 and  10, 1985 by s u b c u l t u r i n g  c o u l d be m a i n t a i n e d  many months.  c u l t u r e d on  SCP on O c t o b e r  (December 9, 1984 a n d F e b r u a r y Calli  and  on SCP medium by  w h i c h were o r i g i n a l l y  were t r a n s f e r r e d  maintained twice  maintained  SCP on F e b r u a r y When c h e c k e d  and a c t i v e l y  on J u l y 10,  p a r t s of the c a l l u s  in color,  9, 1985  were  the p e r i p h e r a l  regions  growing.  Embryogenesis Embryogenesis medium.  from  calli  cultured  C a l l u s on t h i s medium  contained c e l l  i n some c a s e s embryos a t t h e g l o b e a n d h e a r t  Although  t h e s e embryos d i d n o t d e v e l o p  increased  i n size,  structures. concentration complete medium leading  on SCP  T h i s o b s e r v a t i o n was c o n f i r m e d by m i c r o s c o p i c  examination. and  can occur  forming  This fact  large  suggests  i n t h i s medium  development.  Calli  c o u l d be t r a n s f e r r e d  further,  clusters  stages. they  d i s o r g a n i z e d embryo-like that  t h e hormone  was t o o h i g h f o r embryos t o w i t h embryos i n d u c e d on SCP t o another  to plantlet regeneration.  medium,  eventually  84  4.2.10 L i g h t E f f e c t on C a l l i Growing on SCP Medium The  results  Because c a l l i transfer SCP  had  and  callus  increased with  these c a l l i . in  this  SCP  are on  low,  intensity.  light  LSE  medium p r i o r  frequency  but  19.  this  I t was  to  of t h e  typical  frequency  a l s o obvious  from  promoted r o o t d i f f e r e n t i a t i o n  from  However, l i g h t  experiment  shown i n T a b l e  v i g o r o u s , the  medium was  light  that  test  been c u l t u r e d  were not  on  the r e s u l t s  of t h i s  quality  w h i c h was  not  c o u l d a l s o have some e f f e c t  on  measured the  results.  4.2.11 O b s e r v a t i o n s on Embryogenesis Embryogenesis sequence: s i n g l e stage  in culture  cell(?)  > cell  > heart stage  microscope  many c e l l  were s m a l l and easily  dense  i n cytoplasm from  the  in cultures.  cluster  was  well  On  SCP  medium some c a l l i  larger  although  nodular  structures.  These  light  cells  They were usually  stage, the  cell  became smooth a t p e r i p h e r y . were f o u n d  well-shaped  early and  t h e y c o u l d grow l a r g e r  Upon t r a n s f e r  seen.  cells  globe  Under a  (Figure 9).  a t t h e g l o b e and  T h e s e embryos were n o t  >  stage.  At t h e g l o b e  s h a p e d and  further  cluster  > torpedo  encountered  mainly  occurs v i a the  c l u s t e r s c o u l d be  distinguishable  developed,  likely  of these embryogenic  media some of t h e embryos d e v e l o p e d  t o have  embryos  heart  stages.  d i d not  develop  in size  calli  normally  t o become  onto to the  other torpedo  85  Table  19. L i g h t e f f e c t on 'Altaswede' c a l l i  growth and differentiation on SCP medium.  Intensity of l i g h t (/uEirr s e c " )  No. o f calli  Typical SCP c a l l i  Rooted calli  70  37  2  0  80  49  1 1  0  90  53  18  0  1 00  46  7  0  1 20  51  12  0  150  52  15  1  190  51  15  1  230  48  8  1  280  50  22  5  450  48  23  8  2  1  of  86  Figure  9.  Embryogenic callus of ' A l t a s w e d e ' . C a l l u s was c u l t u r e d on L2(P0.001B0.1) medium. Cell clusters contain cells which are small and dense in c y t o p l a s m . C a l l u s shown was u n s t a i n e d (mag. X 3 8 ) .  87  s t a g e and e v e n t u l l y formed  to plantlets  v i a embryogenesis  s e e d l i n g s d e r i v e d from observable, consisting  developed seeds.  4.2.12 Selection  Cotyledons  embryogenic  calli  were o b s e r v e d  from  were  first  of the f i r s t  leaf  leaflet.  and M a i n t e n a n c e  R e s u l t s a r e shown  Plantlets  morphologically like  f o l l o w e d by d e v e l o p m e n t of a s i n g l e  10).  (Figure  i n Table  of Embryogenic  20 f o r one s u b c u l t u r e o f  LSP medium  at d i f f e r e n t  Calli  t o LSP medium.  developmental  Embryos  stages.  Those  embryos w h i c h had been d e v e l o p i n g n o r m a l l y d u r i n g t h e culture  periods developed  percent  of c u l t u r e s  t u b e s h a v i n g more  into plantlets.  showed p l a n t s d e v e l o p e d ,  than  two p l a n t l e t s  c u l t u r e s many embryos a t t h e t o r p e d o growing  at the s u r f a c e of c a l l u s .  where no p l a n t l e t  a  few t u b e s  disorganized  over  11).  s t a g e were  There  s t a g e s were o b s e r v e d  subsequent  transfer. 10%)  (approximately  o r h a d an u n h e a l t h y  Embryogenic  (Figure  calli  one y e a r w i t h o u t  In some  observed  were a few  tubes However  which c o u l d d e v e l o p  In a d d i t i o n ,  i n which c a l l i  t h e r e were  were  color.  had been m a i n t a i n e d lossing  fifty  w i t h 20% o f  o r t o r p e d o embryos were o b s e r v e d .  embryos a t e a r l i e r normally a f t e r  Forty to  the capacity  in culture for of regenerating  plants. When s h o o t s o r i g i n a t e d rooting-medium, embryogenic  plantlets  callus  from  formed  such c a l l i  were c u l t u r e  on  and i n a few c a s e s  ( w i t h o b v i o u s embryos) was p r o d u c e d  near  88  Figure  10. Somatic embryo development from embryogenic callus 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 s c a n n i n g e l e c t r o n m c r o s c o p y . ( a ) An embryo p r e s e n t in the center. Two cotyledons are observable (upper, mag. x 4 3 ) . (b) F u r t h e r d e v e l o p m e n t o f an embryo ( l o w e r , mag. X52)  89  Table  20.  Embryo d e v e l o p m e n t and p l a n t l e t f o r m a t i o n a f t e r s u b c u l t u r e o f e m b r y o g e n i c c a l l i o n t o LSP medium. Culture  No  1  1  2  84  99  Tubes with p l a n t l e t development  35  51  1-plantlet  tube  12  15  2-plantlet  tube  5  1 6  12  17  5  3  T u b e s w i t h embryos a t torpedo Stage  41  41  a  24  20  17  21  8  7  callus  2  4  embryos  6  2  callus  0  1  No  of t u b e s  3-10 >10  plantlet plantlet  few  tube tube  embryos  many embryos  ^5  Tubes with callus (embryos e a r l i e r torpedo stage) yellow  green  disorganized brown  or dark  These callus. 1  only than  were two b a t c h e s o f c u l t u r e s  from  the  embryogenic  90  Figure  11. S e l e c t i o n o f c a l l u s c a p a b l e of embryogenesis. 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 ' s u b c u l t u r e d on L 2 medium c o n t a i n i n g 0.001-0.002 mg/1 PIC and 0.2 mg/1 BA p r o d u c e d e m b r y o s c o n t i n u o u s l y a n d 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  (Figure  12).  4.2.13 Growth of Embryogenic and Non-embryogenic Embryognic  calli  morphologically grain-like easy  different.  structures  to separate  they v a r i e d  not  these  embryogenic  calli  fresh time  compared  growth.  calli  after  calli,  t o compact,  do  a l i g h t microscope, embryos,  mostly at the  and  calli,  calli  At t h i s  grew f a s t e r ,  remained a l i v e  turned  for a  had  longer  brown and showed l i t t l e  became l a r g e  time, roots (8).  on t h e e m b r y o g e n i c c a l l i  were d e a d , whereas  embryogenic  A l l t r e a t m e n t s showed t h e same t e n d e n c y .  t h r e e months w i t h o u t  alive.  were v e r y  Non-embryogenic  w i t h non-embyrogenic  although they  (8).  fine  structures  from f r i a b l e  Under  were  had  one month o f c u l t u r e ,  i n t r e a t m e n t s (2) and  observed  calli  E m b r y o - l i k e s t r u c t u r e s were p r e s e n t  cultures, and  calli  stages.  13).  Non-embryogenic  (6)  in texture  yellow-green color (Figure  These  had many v i s i b l e  When m e a s u r e d calli,  (embryos).  structures.  g l o b e and h e a r t  Embryogenic  from each o t h e r .  although have  and n o n - e m b r y o g e n i c  Calli  in size  f o r m e d on  i n embryogenic i n treatments embryogenic  C o t y l e d o n s were  also  of t r e a t m e n t ( 8 ) .  s u b c u l t u r e a l l non-embryogenic  most  o r no  of the embryogenic  calli  After calli  were  still  92  Figure  12. Embryogenic callus arising from p l a n t l e t at the p o i n t of root o r i g i n .  regenerated  93  Figure  13. Embryogenic and non-embryogenic calli of ' A l t a s w e d e ' . From l e f t t o r i g h t , Tubes 1 and 2 contain L 2 m e d i u m w i t h 0.05 mg/1 NAA a n d 0.1 mg/1 BA. T u b e s 3 a n d 4 c o n t a i n L 2 medium with 2 g/1 yeast extract a n d 10 g/1 a g a r . T u b e s 1 a n d 3 h a d embryogenic calli while Tubes 2 and 4, non-embryogenic c a l l i . C a l l i h a d b e e n c u l t u r e d on these media f o r f o r t y days.  94  4 . 2 . 1 4 Summary of Experiments on C a l l u s Growth and Differentiation In  this  series  of e x p e r i m e n t s  was  e v a l u a t e d on v a r i o u s m e d i a .  may  be made from  with  individual  these r e s u l t s  due  grew f a s t e r  a l t h o u g h the responses  in callus  to c u l t i v a r .  than  'Altaswede'  'Altaswede'  were u s u a l l y (2)  from  This  to cytokinin relationship  BA  ( w i t h o u t NAA)  ratio  ratio  calli  was  and  apparent 0.001  A typical  observed  greater  without with  low  a u x i n s t h a n on any  (3) C a l l u s the  source  result.  source,  cultured  less  levels  i s an  mg/1)  ratio  and  More c a l l i  one  and  low  of a u x i n s . using  on NAA  PIC  and  BA  specifically (more r o o t s a t  example o f  this i n which  produced  roots  on t h e medium  o f t h e o t h e r t h r e e media  o f 2 , 4 - D , NAA  important  or  IAA.  facter determining  When n o n - v i g o r o u s c a l l i growth  roots  at a  i n experiments  numbers o f r o o t s p e r c a l l u s  or moderate  induced  with  i n the experiment  f o u r media were c o m p a r e d . and  also  were  i n the absence  the a u x i n t o c y t o k i n i n  r e l a t i o n s h i p was  and  b u t more c a l l i  i n the experiment  equal to 0.1).  and  'Norseman'  Shoots  from c a l l i  or  (PIC a t 0.0005 and  testing  varied  'Norseman'.  More r o o t s were f o r m e d  auxin  and  formed  calli,  growth  Usually  p r o d u c e d more g r e e n - s p o t t e d c a l l i . from  conclusions  experiments.  differentiation  only  differentiation  Some c o n s i s t e n t  (1) T h e r e were d i f f e r e n c e s  calli  callus  were u s e d a s  differentiation  was  the  observed.  95  In  the  experiment  differentiation different  PIC  on  and  and  no  BA  s o u r c e s were u s e d all  PIC  and  Test  1 showed no  root  o b v i o u s d i f f e r e n c e between  combinations.  in Test  t r e a t m e n t s and  BA,  3 root  When o t h e r  formation  callus  occurred  in  d i f f e r e n c e s between t r e a t m e n t s were  evident. (4) S h o o t s were i n d u c e d cultured  on  containing  SCP NAA  NAA  KIN  i n root  the  experiment  (6)  Additions  mg/1)  concentration  arginine)  or  shoot on of  and  calli  4.2.15 P l a n t Plant cases.  In  and  (3)  and  Regeneration  regeneration  L2(N0.05K0.5) medium.  study.  leading  Section  on  from  plant  that  of  in  and  show  growth  any  compared  these a d d i t i v e s .  was  achieved  c u l t u r e on  shoot  the  strength was  using  NAA  in  salts,  was  several  following  formed on  observation  regeneration  Calli  acid  d i d not  callus  to regeneration  4.2.5).  than  Callus  L2(half  This  NAA.  demonstrated  (glutamic  from c a l l u s  Another experiment  initiated, (see  o f any  with  (6)  hydrolysate  a s t u d y of c a l l u s (2)  KIN  KIN.  amino a c i d s  L2(NO.05K0.5), one  indication  'Altaswede'  more i m p o r t a n t  media w i t h o u t  (1) L 2 ( P 0 . 0 0 2 B 0 . 2 ) ,  of  i n d u c t i o n , as  NAA  casein  on  and  was  obvious promotive e f f e c t with  calli  medium were t r a n s f e r r e d o n t o medium (0.05  (5)  after  media:  P0.002B0.2) the  the  first  from c a l l u s  in  and  therefore  of  c a p a b l e of  KIN  was  some p l a n t s  this  from  calli  e m b r y o g e n e s i s were  96  subcultured  o n t o LSP medium.  continuously,  giving rise  Embryos were  t o a number  produced  of p l a n t s  (see Section  4.2.13).  4.3  Shoot  T i p Culture  4.3.1 M u l t i p l e Results analysis number tips  Shoot  Induction  of m u l t i p l e  are presented  of m u l t i p l e  rate  'Altaswede'. to produce multiple  s h o o t s was b a s e d  shoots  shoots.  shoots,  shoots per shoot  Shoot  The m u l t i p l e  i n Table  Multiple  23  the  rosette  way  induced more  ( F i g u r e 14).  shoot  time  a  shoot  t i s s u e a t the  established  produced  and s u b c u l t u r i n g  s h o o t s c a n be  period.  from  hard  r o s e t t e was  s h o o t s were e a s i l y  h a d more  i n t h e two  shoots u s u a l l y arose  into several pieces  i n a short  be  produced  not o n l y  c u l t u r e , but a l s o  R a p i d p r o p a g a t i o n of m u l t i p l e this  t h a t of  'Norseman'  'Altaswede'  Once t h e m u l t i p l e  additional multiple  of shoot  shoot  t i pcould  'Altaswede'  t i p than  average  on t h e number  r o s e t t e w i t h c a l l u s a t i t s b a s e and g r e e n center.  The  Propagation  i n the induction  subcultures.  statistical  of 'Norseman' was h i g h e r t h a n  any m u l t i p l e  shown  and  21 and 22.  However, when t h e s h o o t  4.3.2 M u l t i p l e As  induction  in Table  producing multiple  induction  shoot  by  dividing  each  piece.  accomplished  97  Table  21.  Multiple red clover  Cultivar  Altaswede  Norseman  1  MS  Table  shoot induction from shoot on L2(PO.003B2) medium.  tips  of  No of s h o o t tips cultured  No o f t i p s w i t h MS  A v e r a g e Ni of MS  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  Total  1 50  50  1  30  17  3.9  2  30  18  3.6  3  30  18  3.9  4  30  15  3.9  Total  120  68  Replicate  = Multiple  1  Shoots  22. C o m p a r i s o n o f two cultivars m u l t i p l e shoot i n d u c t i o n .  of r e d  clover  for  Cult ivar Altaswede  Norseman  10 B  17 A  No of t u b e s W i t h MS  1  2  A v e r a g e No of s h o o t s 4.8 A MS = M u l t i p l e S h o o t s  3.8 B  1  treatments followed by different s i g n i f i c a n t l y d i f f e r e n t a t P^0.01.  capital  letters  are  98  e  14. M u l t i p l e s h o o t p r o d u c t i o n i n r e d c l o v e r . A s i n g l e i s o l a t e d s h o o t was c u l t u r e d on L 2 ( P 0 . 0 0 3 B 1 ) medium and showed multiple shoot proliferation. 'Altaswede' (right) produced more, but s m a l l e r s h o o t s t h a n 'Norseman' ( l e f t ) .  99  Table  23. M u l t i p l e s h o o t p r o d u c t i o n by two. c u l t i v a r s o f r e d c l o v e r . M u l t i p l e s h o o t were i n d u c e d on L2(PO.003B2) medium. They were s u b c u l t u r e d on media containing 0.002-0.003 mg/1 PIC a n d 1 mg/1 BA f o r propagation a t a s u b c u l t u r e o f one month.  Culture  Multiple  s h o o t No / t u b e  Altaswede  Norseman  Induction culture  4.8  3.8 B  First subculture  10.8 a  6.1  Second subculture  15.2 A  8.1 B  A  1  b  treatments are s i g n i f i c a n t l y different f o l l o w e d by d i f f e r e n t c a p i t a l letters or f o l l o w e d by d i f f e r e n t s m a l l l e t t e r s .  at at  P^0.01 P£0.05  when when  100 4.4 G r o w t h and A n a l y s i s During through  this  of Regenerated  study,  several different  simplify  plants  Plants  were  regenerated  t i s s u e c u l t u r e pathways.  To  the d i s c u s s i o n , the types of regenerants are  described  as f o l l o w s  and g i v e n  an a p p r o p r i a t e  abbreviated  designation: (1)  RG1  plants:  those  induced  on m u l t i p l e  without  subculture  (2)  p l a n t s : these  RG2  were d e r i v e d twice mg/1 (3)  regenerated  shoot  from m u l t i p l e  i n d u c t i o n medium  of the m u l t i p l e regenerated  by s u b c u l t u r i n g  on L2 medium c o n t a i n i n g  shoots  (L2(PO.003B2))  shoots; from m u l t i p l e  the o r i g i n a l 0.002-0.003  shoots  multiple mg/1  which  shoots  PIC a n d 1  BA. RG3  plants: plants  callus-derived  shoots.  'Altaswede' c a l l i media.  Calli  time.  These  regenerated  from m u l t i p l i c a t i o n of  S h o o t s were  first  regenerated  from  on L2(N0.05K0.5) a n d a n d L2(N0.05K2)  h a d been  i n culture f o r three  months by  s h o o t s were t h e n m u l t i p l i e d t w i c e  that  by s h o o t t i p  culture; (4)  RG4  plants: plants  regenerated  calli,  w h i c h were d e r i v e d  callus  line  calli Plants  by s e l e c t i n g one  and s u b c u l t u r i n g .  had been m a i n t a i n e d  directly  Before  o f a l l t y p e s were d e r i v e d  from  embryogenic  'Altaswede' regeneration,  in culture for nearly  r e g e n e r a n t s of 'Norseman' were o n l y plants.  plant  from  one  'Altaswede'  year.  while  a v a i l a b l e a s RG1  and  RG2  101 4.4.1  Plant Transfer  The be  and S u r v i v a l  u l t i m a t e o b j e c t i v e of the t i s s u e c u l t u r e c y c l e must  t o produce vigorous  regenerated p l a n t s .  t h e r e f o r e , to ensure a high  I t i s important,  r a t e of s u r v i v a l when p l a n t l e t s  are t r a n s f e r r e d out of i_n v i t r o c o n d i t i o n s .  The t r a n s i t i o n  from the t e s t tube to greenhouse i s a s i g n i f i c a n t , environmental change f o r the p l a n t l e t s .  Appropriate  c o n d i t i o n s a r e r e q u i r e d to f a c i l i t a t e p l a n t a d a p t a t i o n t o the new environment and that c o n d i t i o n s be changed gradually. In the experiment t e s t i n g the e f f e c t treatments on p l a n t s u r v i v a l , p l a n t s laboratory low  light  f o r the f i r s t  of d i f f e r e n t  incubated  i n the  week were exposed t o a r e l a t i v e l y  i n t e n s i t y (120 i u E m ~ s e c ~ ) and high 2  1  humidity.  P l a n t s were weak and many d i e d i n the subsequent t r a n s f e r t o the greenhouse. low.  As a consequence, the s u r v i v a l r a t e was  When p l a n t s were t r a n s f e r r e d t o the greenhouse without  Saran wrap p r o t e c t i o n some p l a n t s d i e d only a few days a f t e r the t r a n s f e r .  Dessication  i n the lower humidity environment  was a c o n t r i b u t i n g f a c t o r i n the low s u r v i v a l r a t e (39%) (Table  24).  The p l a n t s which were covered with Saran wrap  f o r two weeks i n the greenhouse had the h i g h e s t rate  survival  (94%). In the present  were t r a n s f e r r e d . observations  study f i v e batches of regenerated  plants  During these t r a n s f e r s two other  were made regarding  the c o n d i t i o n s which  supported s u c c e s s f u l p l a n t t r a n s f e r .  (1) M a i n t a i n i n g  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 a f t e r d i f f e r e n t treatments  to  greenhouse  Treatment Laboratory/ g r e e n house Plants transferred Plants survived Survival  rate  (%)  G r e e n house +Saran wrap  Green house - S a r a n wrap  112  224  28  60  211  11  54  94  39  103 in  r o o t i n g medium  transplantation It  might  appeared  to contribute  be a n t i c i p a t e d t h a t  humidity plants  f o r more t h a n one month p r i o r t o  within  this  Such p l a n t s  (2) In F e b r u a r y ,  natural  survival.  extended  t h e c u l t u r e t u b e s was somewhat  were much b i g g e r .  wilting.  during  to better  period,  l o w e r e d and  were more r e s i s t a n t t o  light  intensity is  m o d e r a t e a n d p l a n t s c a n be t r a n s f e r r e d t o t h e g r e e n h o u s e light high  conditions ( i . e . during  reduced  light  strength  i n t e n s i t y should  on June  During  17, b o t h  a screen  plants.  of b l a c k  precede  i n t e n s i t y i s very of maintenance a t exposure  to the f u l l  t h e t r a n s f e r o f RG4 light  t h e g r e e n h o u s e were h i g h .  using the  When l i g h t  summer d a y s ) a p e r i o d  of s u n l i g h t .  'Altaswede' in  directly.  i n t e n s i t y and temperature  Light  i n t e n s i t y was  polyethylene  Of t h e 130 p l a n t s  p l a n t s of  film  reduced  on a frame  above  t r a n s f e r r e d more t h a n  95%  survived.  4.4.2  Chromosome Number S t a b i l i t y RG1,  examined of  RG2, RG3 and RG4 for their  'Altaswede'  seed-derived summarized  plants  chromosome number.  i s 2n=2X=14.  Root  p l a n t s were u t i l i z e d i n Table  o f ' A l t a s w e d e ' were  25.  (Figure  as c o n t r o l s .  15).  e x a m i n e d no a b n o r m a l chromosome number diploid/tetraploid  RG2 p l a n t s .  karyotype  t i p squashes of  A l lthe c o n t r o l p l a n t s  n o r m a l chromosome numbers  one  The n o r m a l  mosaic p l a n t  The d a t a a r e showed  Of 45 RG1  plants  was o b s e r v e d  was d e t e c t e d  while  among  28  F o r a l l t h e 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' regenerated v i a d i f f e r e n t tissue culture  Population surveyed  Number o f p l a n t s w i t h abnormal chromosome tetraploid number  plants pathways.  tetraploid /diploid mosaic  RG1  45  0  0  0  RG2  28  1  0  1  RG3  170  0  0  0  RG4  119  27  23  4  Control  26  0  0  0  Figure  15. K a r y o t y p e o f n o r m a l r o o t t i p o f red ' A l t a s w e d e ' (2n=2X=14) (mag. X 1 5 7 0 ) .  clover  cv.  106 found.  In c o n t r a s t  27 o u t o f 119 RG4  t e t r a p l o i d chromosome  numbers  ( 2 8 ) . M o s t ( 2 3 ) o f t h e s e 27  p l a n t s w i t h a l t e r e d chromosome w h i c h were l i k e l y  produced  during the c a l l u s c u l t u r e  supported the s i n g l e c e l l  one c e l l undergo  tetraploids,  t h r o u g h chromosome d o u b l i n g Only  (Figure origin  t h e s e p l a n t s were r e g e n e r a t e d . come e i t h e r  number were  stage.  d i p l o i d / t e t r a p l o i d mosaics  ' A l t a s w e d e ' p l a n t s had  f o u r p l a n t s were  16).  These  results  o f most e m b r y o s , f r o m  which  The f o u r m o s a i c p l a n t s  could  from embryos w h i c h had o r i g i n a t e d  f r o m more  o r i g i n o r f r o m e m b r y o s w h i c h h a d some c e l l ( s ) changes  i n chromosome  I t was a l s o d e m o n s t r a t e d short-term cultures  number d u r i n g  embryogenesis.  t h a t p l a n t s r e g e n e r a t e d from  (either multiple  shoot c u l t u r e s or  c a l l u s c u l t u r e s ) t e n d e d t o h a v e a n o r m a l chromosome w h i l e chromosome  Another  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  i n t e r e s t i n g aspect 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  4.4.3  Isozyme  Isozymes  number,  a b n o r m a l i t i e s c o u l d happen t o t h e  regenerated plants period.  than  numbers were o b s e r v e d .  Analysis  are a t t r a c t i v e  f o r direct genetic  study  because  they represent, the primary products of s t r u c t u r a l  genes.  Changes i n c o d i n g sequences  to  will  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 many c a s e s  i n isozyme  lead  patterns.  RG3 a n d RG4 p l a n t s o f ' A l t a s w e d e ' were a n a l y s e d f o r their  isozyme p a t t e r n s o f f i v e enzymes.  Comparisons  were  made t o t h e i s o z y m e p a t t e r n s f r o m s e e d - d e r i v e d p l a n t s .  107  Figure  16. Root t i p chromosome numbers o f two RG4 p l a n t s o f ' A l t a s w e d e ' (mag. X 1 5 7 0 ) . ( a ) 2n=2x=14 (upper); (b) 2n=4x=28 ( l o w e r ) .  108  Results  showed  patterns This  observation order  regenerated isozyme  patterns  were  calli  should  indicate  not exceed  Four  o r more  the occurrence  the  overall  not  exceed  isozyme three.  i n isozyme  starting genotypes  the p l a n t s  always  enzymes  differ  i n t h e two  period.  In t h i s  used the from  could  be d e t e r m i n e d  in culture. original one  should whereas  F o r RG4  genotype.  genotype,  show any  the o r i g i n a l  plants  Because  the majority  the isozyme deviation  pattern  from  from  each  these  two  patterns  the c u l t u r e and  possible  one  easier  genotype i s  to  a l l the plants  determine  were  of the regenerated of the o r i g i n a l  the pattern  should  as the  isozyme  than  i t was  enzymes  from  genotypes  when m o r e  Similarly,  differ  regenerated  is  will  a r e used  no v a r i a t i o n h a d h a p p e n e d d u r i n g  variation  callus  type.  provided  way  isozyme  i f each  which  induced  plants  f o r one enzyme  genotypes  i n the  were  a l l t h e RG3  f o r a l l the five  o f two  species.  original  calli  of a non-parental  patterns  should  even  patterns  I f two  the  of expected  three  plants.  out-crossing  which  Because  patterns  materials,  in seed-derived  t o know  from  t h e number  isozyme  variation occurred  i t i s important  of the p l a n t s  i n the  f o r an  whether  regenerated.  three  different.  other  to determine  patterns  from  b u t one  i s not unusual  plants  plants  were  variation existed  o f a l lt h e enzymes  In  and  that  should  be  derived plants  genotype, i n low  frequency. Two five  hundred  enzymes.  No  a n d t e n RG3  plants  were  investigated for  variation attributable to tissue  culture  109 was  observed  f o r phosphoglucose  isozyme p a t t e r n s T h e s e two  genotype with the  another the  (PGM)  was  band and  two  may  from  vary  since  found  isozymes.  five  had  loci  to  four  bands  For  represented  by  only  consequence  of  the  (Figure  enzyme.  had  two  bands.  Sixty the  five  RG4  observed variation  one  i n RG3 had  enzyme.  three The  f o r the  were o b t a i n e d .  one  sample  b a n d s , one  (SKDH) two t o two  pattern  had  of  the  three  induced  that  (6-PGDH)  pattern,  (Figure  170),  could  20).  For  isozyme p a t t e r n s the  band and  be  a  were  three  another  calli one  f o r isozyme p a t t e r n s  of  had  for  the  same p a t t e r n  g e n o t y p e s w h i c h had  from the  For  regenerated  determined  genotypes of  A l l these plants  bands  enzyme b e c a u s e i t  One  one  dense  plants.  and  ( i . e . plant  c u l t u r e process  than  No  number of  I t was  plants, demonstrating  been  The  more bands  a l l seed-derived 19).  17).  phosphoglucomutase  18).  p l a n t s were s c r e e n e d  e a c h enzyme as  two  genotypes.  6-phosphogluconate dehydrogenase  One  enzymes.  (Figure  had  for  Two  one  among s e e d - d e r i v e d  (Figure  o b s e r v e d , w h i c h were due this  with  i s a dimeric  five  dehydrogenase  in  locus  locus  responsible  isozyme p a t t e r n s  shikimate  one  plants  (MDH)  likely  i s monomeric.  PGI  bands  three  are  original  A l l t h e s e p l a n t s had  faint  five  f o r the  heterozygous  i n RG3  malate dehydrogenase plants  one  at  heterozygous  homozygous one  variation  (PGI).  were d e s i g n a t e d  genotypes d i f f e r e d  homozygous and  isomerase  that  culture  no  been  qualitative  cycle.  110  Figure  17. Zymogram o f p h o s p h o g l u c o s e i s o m e r a s e f r o m RG3 p l a n t s o f r e d c l o v e r c v . 'Altaswede'. Lane 3 from left represents a n o t h e r g e n o t y p e f r o m w h i c h some p l a n t s were r e g e n e r a t e d .  111  +  Figure  18. Zymogram of p h o s p h o g l o c o m u t a s e f r o m c a l l i o f r e d clover c v . ' A l t a s w e d e ' . From l e f t t o r i g h t , l a n e s 1-19: embryogenic calli and lanes 20-29 non-embryogenic c a l l i .  112  Figure  19. Zymogram o f m a l a t e d e h y d r o g e n a s e f r o m RG3 p l a n t s o f r e d c l o v e r c v . ' A l t a s w e d e ' . A l l p l a n t s had f i v e bands.  1 13  Figure  20. Zymogram o f 6 - p h o s p h o g l u c o n a t e d e h y d r o g e n a s e f r o m RG3 p l a n t s o f r e d c l o v e r c v . ' A l t a s w e d e ' . Lane 3 from left r e p r e s e n t s a n o t h e r g e n o t y p e from w h i c h some p l a n t s were r e g e n e r a t e d . Lane 11 f r o m l e f t i s an abnormal p a t t e r n o b s e r v e d , w h i c h h a d an e x t r a band.  1 14  4.4.4 Nodule Formation and Leghaemoglobin All  plants analyzed  leghaemoglobin p r o f i l e The  normal  f o r chromosome number  formed normal  and  study.  two m i n o r  The m a j o r  'Altaswede' No  appearing  21, has t h r e e components,  t h e CAE  and RG2  variation  conditions  and  33 RG3  (i.e.  band  o r a l t e r e d m o b i l i t y ) was  quantitative  differences  were p r e s e n t , individuals,  t h e s e may sample  size  root nodules.  of r e d c l o v e r  new  detected.  (intensity  and r a t i o  be a r e f l e c t i o n and/or  For  p l a n t s were  missing,  one  used i n t h i s  band m i g r a t e s most s l o w l y .  91 RG1  qualitative  under  and  symbiotic  leghaemoglobin p r o f i l e  n o d u l e s , a s shown i n F i g u r e major  Profile  analysed. band  Although between  bands)  of d i f f e r e n c e s  infecting  Rhizobium  among  strain.  4.4.5 P l a n t Morphology  Leaflet  Number  Among t h e r e g e n e r a t e d p l a n t s from  four  26).  t o s e v e n were o b s e r v e d w i t h h i g h  However t h i s  also  plants.  The  absence of such  size.  were m o s a i c It  would  o b s e r v e d on  be a c o n s e q u e n c e  A l l the p l a n t s w i t h changed and had  appear  stimulatory  in part,  some l e a v e s o f normal  that  effect  the t i s s u e  changes  frequency (Table  was  'Norseman' c o u l d ,  sample  number  variation  'Altaswede' c o n t r o l in  leaflet  culture  variation  of the  small  leaflet  number  leaflet  number.  r e g i m e may  on t h e o c c u r r e n c e o f a l t e r e d  have  leaflet  a  115  + Figure  21. Cellulose acetate electrophoresis of leghaemoglobin of red c l o v e r cv. 'Altaswede'. There a r e three components, one major and two m i n o r . The m a j o r band m i g r a t e s most s l o w l y .  116  Table  26. L e a f l e t number in control (seed-derived) r e g e n e r a t e d p l a n t s of r e d c l o v e r .  Population  Cultivar  No of plants  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  Control  Altaswede  84  5 (6.0)  Control  Norseman  20  0  and  P l a n t s w i t h changed l e a f l e t number (%)  (24.7)  1 17 number,  but such  comparison  and a n a l y s i s  regenerated  Length  require careful  of v a r i a t i o n  i n s e e d - d e r i v e d and  plants.  t o Width R a t i o of L e a f l e t  The  mean, v a r i a n c e , and s t a n d a r d  different RG2,  a c o n c l u s i o n would  RG3  d e v i a t i o n of r a t i o s of  "populations" are presented a n d RG4  B e c a u s e RG4 additional  p l a n t s were a n a l y s e d  i n Table for  p l a n t s were grown d u r i n g t h e summer months an group of c o n t r o l  plants  (CK2) were grown  them.  The mean o f CK2 p l a n t s i s a l i t t l e  that  o f CK1  probably  light  RG1,  'Altaswede'.  with  and  27.  intensity  because a t t h a t time  along  higher  both  than  temperature  were h i g h and p l a n t s were g r o w i n g  more  quickly. Statistical  analysis  of t h e s e  28 a n d 29.  Means  regenerated  p l a n t s o f t h e two c u l t i v a r s  significantly (P<0.05).  result  different  That  regenerated  i n Table  in  the regenerated  ratio.  are not  procedures.  r a t i o of  However, a s shown by  f o r RG1,  are a l l s i g n i f i c a n t l y plants.  plants  longer or s h o r t e r as a  RG2  and  larger  RG3 (P^O.01)  T h i s observation suggests  plants greater variation  Nevertheless  significantly  p o p u l a t i o n s of  l e n g t h to width  29 t h e v a r i a n c e s  that of c o n t r o l  i n Tables  t h e means o f c o n t r o l  p l a n t s d i d n o t become  p l a n t s of 'Altaswede' than  from  culture  i s shown  for different  i s , the l e a f l e t  of iri v i t r o  the data  of r a t i o s  data  existed  f o r the  t h e v a r i a n c e o f RG4 p l a n t s i s n o t  different  from  t h a t of the c o n t r o l  that  group.  118  Table  27. Means a n d v a r i a n c e s o f l e a f l e t l e n g t h / w i d t h for d i f f e r e n t populations of red c l o v e r .  Cultivar  Population n  1  X  s .,  ratios  2  n  n  A l t a s w e d e CK1  51  1 . 127  0.074  5.48X10  RG 1  41  1 . 1 30  0. 149  22.0X10 -  3  RG2  61  1 .162  0.131  17.2X10 -  3  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  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 -  Norseman  1  n:  sample  size  3  3  119 Table  28. C o m p a r i s o n s and c o n t r o l  between means o f plants for leaflet  regenerated length/width  Cultivar  Comparison  Altaswede  RG1/CK1  0.118  RG2/CK1  1 .785 ns  RG3/CK1  1.761  ns  RG4/CK2  0.610  ns  RG1/CK  1.307  ns  RG2/CK  0.905 ns  Norseman  1  ns  1  ns: not s i g n i f i c a n t .  Table  29. C o m p a r i s o n s between variances 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 l e n g t h / w i d t h ratio  Cultivar  Compari son  F  Altaswede  RG1/CK1  4.019  **  RG2/CK1  3.139  **  RG3/CK1  2.853 ** .  RG4/CK2  1.158  ns  RG1/CK  1.023  ns  RG2/CK  0.801  ns  Norseman  1  plants ratio  **: s i g n i f i c a n t  a t PS0.01  level;  1  120 This  difference  regenerated might  is likely  from o n l y  one g e n o t y p e  be a n t i c i p a t e d .  was q u i t e  different.  b e c a u s e RG4  ploidy  so t h a t  were  less variation  F o r 'Norseman', however, t h e r e s u l t The v a r i a n c e s  were n o t d i f f e r e n t from t h a t difference  plants  o f RG1  and RG2  of c o n t r o l p l a n t s .  between t h e two c u l t i v a r s may  resistance  M o r p h o l o g y o f RG4 Several  'Norseman'  to change.  'Altaswede'  albino  The  be r e l a t e d t o  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  s h o w i n g more  plants  plants  Plants  were o b s e r v e d among t h e RG4  plantlets  regenerated  from t h e ' A l t a s w e d e ' c a l l i .  them were  i s o l a t e d and s u b c u l t u r e d  o n t o LSP medium;  gradually  s e n e s c e d and d i e d  the f i r s t  during  Three of they  and second  subcultures. In had  general,  greener  regenerated p l a n t s  leaves  than c o n t r o l p l a n t s  weeks a f t e r t h e p l a n t  transfer.  o b v i o u s when r e c o r d e d  only  five  petioles.  when r e c o r d e d  These trends weeks  T h e r e were some t e t r a p l o i d p l a n t s typically  grew more q u i c k l y and  were n o t  a f t e r the t r a n s f e r .  among RG4  r e f l e c t e d i n the p r o d u c t i o n  ten  plants,  of l a r g e r  leaflets  and  5.  5.1  Callus Induction After  t h e e x p l a n t s were i n o c u l a t e d  medium c a l l u s For  this  production usually  study a t o t a l of f i v e  cultivars  were i n i t i a t e d , and  easily.  Similarly  that  third  one  excellent  of  100  by P h i l l i p s and  Collins  gave g o o d r e s u l t s  0.06  effective callus  mg/1  Collins  t h a n 2,4-D  culture.  experiments  fourth  callus  o f pea  1984).  I t a l s o has  PIC may  manipulation  Because  callus growth  PIC  that  study.  induction i s more  a u x i n f o r use i n  from  leaf-derived  ( J a c o b s e n and K y s e l y ,  be a p r o m i s i n g g r o w t h cultures  showed  been u s e d as an a u x i n i n  s a t i v u m L.)  of c e l l  in this  For c a l l u s  embryos were formed  (Pisum  tested  gave p o o r  induction  required.  two  relatively  i t i s not s u r p r i s i n g  i t is a desirable  i n which  from  been o p t i m i z e d f o r c a l l u s  (1979a),  weeks.  (1979a) o b s e r v e d  of red c l o v e r  one  for callus  PIC was  two  were p r o d u c e d  medium u s e s PIC a s t h e s o l e a u x i n . only  L2(P0.06B0.2)  commenced w i t h i n  calli  genotypes  S i n c e L2 had  on  b a t c h e s of c u l t u r e s  P h i l l i p s and  response while only  production.  it  DISCUSSION  and  regulator  plant  f o r medium  r e g e n e r a t i o n from  legumes. It various  was  observed that  calli  formed  f r o m e x p l a n t s under  l i g h t c o n d i t i o n s : d a r k , m o d e r a t e and  intensities. frequency conditions  'Altaswede'  of c a l l u s  appeared  induction  t h a n under  t o show a h i g h e r  under  high intensity  121  high l i g h t  low  l i g h t or  light.  In  dark 'Norseman'  L2  122  s u c h an o b v i o u s d i f f e r e n c e from  both c u l t i v a r s Calli  differ induced  on e i t h e r  of d i f f e r e n t  markedly  under  pigmented.  Calli  or i n t e s t  formed  tubes.  s p e c i e s a n d even o f t h e same s p e c i e s  i s white  light  (Thorpe,  1982).  to pale yellow while  may be p a l e y e l l o w o r more  F o r example  e x p l a n t s was w h i t e initiated  plates  i n t e x t u r e and c o l o r  i n the dark  initiated  was n o t o b s e r v e d .  red clover  with a s l i g h t  callus  green  from  Callus  that strongly  hypocotyl  coloration  when  in light.  5.2 C a l l u s Maintenance  and P l a n t  Regeneration  5.2.1 Primary Test of Two Media No s h o o t s of  ( o r bud i n i t i a l s )  t h e two media  red c l o v e r Phillips  and L S E ) on w h i c h p l a n t  5  has been r e p o r t e d ( B e a c h  and C o l l i n s ,  contribute  in  results  in  reports  because  callus  (B  part  this  1980). to  study of  results.  5  occurred to  induce  section.  difference  and  those  could also  in this  on L2(PO.06B0.1) medium. bud i n i t i a l s  will  of  in  Beach and S m i t h  B ( N 2 D 2 K 2 ) medium, w h i l e  and S m i t h ,  the  one  regeneration in 1979;  Genotype d i f f e r e n c e s  differences  source d i f f e r e n c e  different  were i n d u c e d on e i t h e r  between  may the  previous  cultivars.  account  The  f o r the  (1979) i n d u c e d c a l l u s on  work c a l l u s  induction  The f a i l u r e  o f L S E medium  be d i s c u s s e d i n t h e n e x t  1 23 5.2.2  In  observed  onto  LSE  i n a l l these  medium, c a l l i period  tests.  in culture  calli  decreased.  (approximately  After  two  and  cell  by  into  SL-2  liquid  5 ml  solid  (Phillips vitro  by  and  and  of the  epicotyl  Collins, culture  PIC  and  1979a).  b e h a v i o r of t h e two  s t u d y were a l s o o b v i o u s .  were  sections  1980).  The  f o r s i x months  25 ml  suspension  subculturing  i n response  Collins,  of  inoculated  suspension cultures  to inoculate  i n c u l t i v a r and  differences  induction  of r e d c l o v e r c u l t i v a r  ml  0.004 mg/1  growing,  died.  fresh was shoot  t h e i n d u c e d buds  1 mg/1  t h e i r experimental conditions  study d i f f e r e n c e s Cultivar  one  medium  stopped  m e d i a t o i n d u c e buds and  verified  medium c o n t a i n i n g comparing  calli  in l i q u i d  old culture  Approximately  was  s u b c u l t u r e on LSE  35-40% o f p l a t e s  (Phillips  using  induction  they  medium on  medium  LSE  of g r e e n - s p o t t e d  frequency  i n o c u l a t i n g h y p o c o t y l and  onto  onto  C o l l i n s (1980) r e p o r t e d t h e  were m a i n t a i n e d  inoculated  transfer  was  the  continuous  suspensions about  response  calli  t o brown a f t e r a  In t h e i r e x p e r i m e n t  established  shoots,  g r o w t h , began  suspension c u l t u r e s  'Arlington'.  medium.  After  b l a c k as  f o r m a t i o n w i t h LSE  with  and  A poor  months i n c u l t u r e )  t o brown and  Phillips  to induce  medium.  showed l i t t l e  short  turned  Medium  a s e r i e s of t e s t s d e s i g n e d  were t r a n s f e r r e d  bud  LSE  C a l l u s Growth on  In  t o those of  procedure have been  Differences  c u l t i v a r s used  BA.  onto  this  were e v i d e n t . observed between t h e i n  i n the p r e s e n t  C u l t i v a r d i f f e r e n c e s may  be  124 exaggerated individual  o r masked by genotypes.  the c o n f o u n d i n g  From t h e r e s u l t s  among r e g e n e r a t e d p l a n t s , concluded of  that  a single  likely  mixture  and  of  o f c r o s s e s made Collins  (1980)  t h e s e p l a n t s were a l l d e r i v e d f r o m  seedling.  more c a p a b l e  Therefore,  Phillips  effect  This individual  seedling  of r e g e n e r a t i o n than  i t is possible  of g e n o t y p e s  that  one  other  cultivar  with d i f f e r e n t  explants  genotype  was  genotypes.  contains a  capacities  for  regeneration. The response  most was  likely  reason  the procedure  f o r the d i s c r e p a n c y i n observed used  to  regeneration.  Phillips  and  culture  inoculum  f o r shoot  that  as t h e  the c e l l  experiment, been  was  supported  4.2.4),  initiated by  PIC  0.2-1  on mg/1  from  BA.  of d e t e r m i n a t i o n (Dodds,  non-embryogenic  initiated their  in this  f o r embryogenesis which can  calli.  study  had  T h i s argument  experiment have  (Section embryos  This observation indicated  i n these experiments  medium w h i c h would not  suspension  t h e media c o n t a i n i n g 0.0005-0.005  point  calli  of  a  It i s possible  LSE medium d i d n o t  grown on LSE  embryogenic  cycle  of another  calli  pre-determined  a l r e a d y been  on LSE medium  the r e s u l t s  i n which c a l l i  and  (1980) u s e d  t o undergo embryogenesis.  shoots developed  mg/1  plant  induction.  suspension c u l t u r e  whereas c a l l i  not  or  Collins  embryos o r p r e - e m b r y o s had  during  induce  1982).  not  passed  C u l t u r e s of  o r embryos may  grow and  support  had  As d e m o n s t r a t e d  in this  the  cells  produce  differentiate  differentiation  that  on  a  or growth of study,  125 embryogenic on  a l l t h e t e n media t e s t e d  successful  than  embryogenic cytokinin the  and n o n - e m b r y o g e n i c  fact  required  f o r growth. that  embryogenesis  calli  Non-embryogenic no  response  of  shoot  calli  less  calli.  trigger  In a d d i t i o n ,  exogenous a u x i n a n d  one g e n o t y p e  were c u l t u r e d  capable of  f o r shoot  from c a l l i  on t h e same medium, b u t induction.  genotypes  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 the process leading  were t r a n s f e r r e d  onto  The  failure  on L S E medium was p r o b a b l y a  c o n s e q u e n c e o f t h e use o f i n d i v i d u a l capacity  more  embryos on LSP medium.  was o b s e r v e d  induction  differently  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  from  produced  performed  w i t h embryogenic c a l l i  non-embryogenic  calli  calli  w i t h a low  of t h e f a i l u r e t o  t o embryogenesis  before  L S E medium f o r t h e s e g e n o t y p e s  calli which  were c a p a b l e o f 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 t h e s e e x p e r i m e n t s  revitalize  senescing c a l l i .  c a s e s where c a l l i performance sources  for  showed a s i m i l a r  cultured  Recovery  condition  quality  response,  and d e t e r i o r a t i n g Calli  from  especially  different those  cultured  f r e q u e n c y d e c r e a s e d w i t h t h e age o f  on L S E medium,  recovery l i k e l y Calli  SCP medium may be u s e f u l i n  need t o be r e c o v e r e d .  on L S E medium. calli  of poor  t o be a b l e t o  indicating  that  a  threshold  exists.  on SCP medium c o u l d be m a i n t a i n e d f o r a time p e r i o d  i n good  up t o f o u r months b e f o r e  126  subculture. can  Where c a l l u s  be u s e d t o i n c r e a s e  typical  one month  transferred  i s desirable,  interval  a l s o o b s e r v e d on t h i s initiated  f r o m media c o n t a i n i n g little  (SCP).  when c a l l u s i s high  o r no 2,4-D  low o r n o n - e x i s t e n t  from t h e  medium  levels  (e.g.  o f 2,4-D  Reinert  the process  level  et a l . ,  be  observed  of 2,4-D.  In t h e  present,  s t u d y embryos a t t h e g l o b e and h e a r t  stages  detected  on SCP medium.  culture  periods  for calli  Progressively  eventually  embryos a n d e m b r y o - l i k e  lead  globe or heart  structures.  larger  in size.  stages  Further  SCP medium r e q u i r e d  were  t o d i s o r g a n i z a t i o n of  s t r u c t u r e s w h i c h were m o r p h o l o g i c a l l y the  longer  to  on media  2,4-D, but o b v i o u s embryos c a n o n l y  media w i t h  SCP  months.  I t i s possible to i n i t i a t e  containing on  the subculture  i s usually  media c o n t a i n i n g 1977).  maintenance  t o two o r t h r e e  E m b r y o g e n e s i s was Embryogenesis  line  As a r e s u l t ,  many  s i m i l a r t o embryos a t  were o b s e r v e d , b u t o f t e n development  o f embryos  much  induced  t r a n s f e r of the c u l t u r e s onto  on  other  media.  5.2.4  Plant Plants  R e g e n e r a t i o n : O r g a n o g e n e s i s and E m b r y o g e n e s i s c a n be r e g e n e r a t e d  from c a l l u s  cultures  through  one o f two p a t h w a y s : o r g a n o g e n e s i s and e m b r y o g e n e s i s ( e . g . Reinert  e t a l . , 1977; Narayanswamy,  Razdan,  1983).  A shoot  1977; B h o j w a n i  and  bud f r o m o r g a n o g e n e s i s a n d an  embryo  from e m b r y o g e n e s i s a r e d i s t i n g u i s h a b l e on t h e b a s i s o f recognizable  morphological  differences.  The f o r m e r  is a  1 27  monopolar  structure,  some c a s e s , r o o t developed medium. origin  from embryos due  of  For example,  recognizable  by  two  b e f o r e shoot  c o t y l e d o n s ; (3) The  from  red clover  first  one.  i n the the  Embryos have a  t h e s e embryos have  shoots  from  embryos  (1) Embryo s t r u c t u r e s development;  leaf  In  f o r shoots  to determine  other c r i t e r i a .  three c r i t e r i a :  callus  incomplete  possible  plantlets  cotyledons.  is a bipolar  to regulators present  r e g e n e r a t i o n by  on  be  it is still  m o r p h o l o g y and  visible  latter  f o r m a t i o n may  However,  specific  w h i l e the  after  are  are  (2) S h o o t s  have  the c o t y l e d o n i s  unifoliate. In onto  organogenesis  a medium w h i c h  induces  shoots are connected The  s h o o t s must be  induction. callus  The  i s root  primordia  of  medium  on  most  are  the r a t i o  favored  rooting.  bud  organogenesis  Plant  growth  (source,  size  to another  (Reinert formed,  can  tissue.  medium f o r r o o t  as a r u l e ,  be  Other and  from  High  at  single  cytokinin  formation  i n the  c o n c e n t r a t i o n s of  in their  r e g e n e r a t i o n may  of  age)  as w e l l  auxin  requirements  be more c o m p l i c a t e d .  o t h e r medium components and  physical  or  will,  whereas h i g h l e v e l s  species vary  from  The  In t o b a c c o ,  controlled  1957).  initiation,  regulators, and  These  e t a l . , 1977).  between a u x i n and  and M i l l e r ,  caused  transferred  from c a l l u s .  of p a r e n c h y m a t o u s c e l l s .  (Skoog  first  f r e q u e n t type of d i f f e r e n t i a t i o n  formation  kinetin  for  shoots  are  a n a t o m i c a l l y with the c a l l u s  buds, r o o t s or c a l l u s  depending  cultures  transferred  of organs  s m a l l groups  callus  as  explant  temperature,  128 photoperiod, all  play  1977;  intensity,  a determining role  Reinert  1983). is  light  phase of shoot  to that  of r o o t  Transfer  s u p p o r t i n g o r g a n i z e d growth  appearance  of meristemoids,  embedded  i n the t i s s u e .  s u r f a c e meristems protuberances, nodular  and  giving  callus,  meristemoids,  often  initially  capable of g i v i n g  (Thorpe,  1982).  originated  continued  to green-spotted c a l l i  to either  (Bhojwani  similar  and R a z d a n , development  to that  h a v e been o b s e r v e d  Such c a l l u s shoot  plastic  t o form organs i s plant  (Thorpe,  green-spotted c a l l i (or  (Reinert  et a l . ,  1982; B h o j w a n i  f o r m a t i o n (Kim and J a n g ,  r e g i o n s of green  The p r o c e s s o f  1977;  and R a z d a n ,  m o r p h o l o g y may be an i n d i c a t o r o f  Green-spotted c a l l u s color.  showed c e l l  1984).  division  i n the  Areas of h i g h a c t i v i t y  t o the formation of meristemoids,  of organ  The  or shoot p r i m o r d i a  i n the i n t a c t  Ohyama a n d Oka, 1982; T h o r p e ,  sites  root  1983).  calli)  lead  here).  whereas s h o o t s a r e formed  nodular  can  (called  apolar, are developmentally  Often before organogenesis,  potential  of the  a nodular appearance  1982) .  1983) .  on t h e s u r f a c e  l e a d s t o f o r m a t i o n of s m a l l  rise  callus,  primordium  essentially  are located  G e n e r a l l y , r o o t s a r e endogenously  from  exogenously  of t i s s u e t o  leads t o the  Continued d i v i s i o n  tissue  similar  et a l . ,  formation v i a organogenesis  formation.  which  (Narayanswamy,  1977; F l i c k  conditions  or  c o n c e n t r a t i o n may  i n organogenesis  e t a l . , 1977; S t r e e t ,  The e a r l y  similar  pH a n d s u g a r  formation.  Initially,  which  when t h e s e  in callus are the structures  129 were observed i n t h i s study i t was  thought that they were  i n d i c a t o r s of p o t e n t i a l shoot f o r m a t i o n .  However, the  r e s u l t s showed that these green spots only formed  roots,  suggesting that e i t h e r the environmental f a c t o r s or endogenous r e g u l a t i o n  (or both) favored d i f f e r e n t i a t i o n to  r o o t s i n s t e a d of shoots.  As observed i n these  experiments,  when the green spots grew out of the c a l l u s , they i n i t i a l l y as green b u d - l i k e s t r u c t u r e s  (under  appeared  illumination).  F u r t h e r e l o n g a t i o n occurred and white h a i r s emerged from the top r e g i o n as they became more c l e a r l y d e f i n e d r o o t s . spots on c a l l u s i n t h i s study were e s s e n t i a l l y an of root  Green  indication  organogenesis.  A s i g n i f i c a n t e f f e c t on root formation from c a l l u s observed with growth r e g u l a t o r s , c u l t u r e sequence, and l i g h t  intensity.  More root formation from  cultivar  callus  g e n e r a l l y r e s u l t e d when c u l t u r e s were grown on media auxin or with a low auxin to c y t o k i n i n r a t i o . was  observed throughout  the study.  In tobacco  No 38') p i t h c u l t u r e Skoog and M i l l e r demonstrated cytokinin Roots  lacking  This trend ('Wisconsin  (1957) have  that the r e l a t i v e r a t i o of auxin (IAA) to  (KIN) determine the nature of organogenesis.  formed  from the t i s s u e i n the absence  in the presence of 0.18-3.0 mg/1 the presence of 1.0 mg/1  of k i n e t i n  1957).  and  IAA, and shoots formed i n  k i n e t i n , p a r t i c u l a r l y with  c o n c e n t r a t i o n s i n the range of 0.005-0.18 mg/1 Miller,  was  IAA  (Skoog  and  Since then, q u a n t i t a t i v e changes have been  found to be d e c i s i v e i n a number of other p l a n t  tissue  130 systems callus  (Narayanaswamy, c u l t u r e s was  study.  studied, four  systems  low  2,4-D  induced a f t e r a  four  concentrations  Nevertheless, "induction  demonstrated  o r g a n o g e n e s i s has  relatively et  roots  high  culture  and  low  period  kinetin  ratio  of  clover in  this  u n l i k e many been  after  induction  kinetin  a l , 1976).  day  medium" t h a t  i n red  shown t h a t ,  produced  (Walker  i t i s the  formation  i t was  in which  days exposure to  Root  d i f f e r e n t , as  c a l l u s from a l f a l f a  relatively  2,4-D  quite  S i m i l a r l y in a l f a l f a ,  conventional  by  1977).  and  S h o o t s were  in r e l a t i v e l y  high  concentrations.  auxin  to c y t o k i n i n  c o n t r o l l e d root  or  in  the  shoot  organogenesis. Shoot  formation  observed.  However, s h o o t  propagation  of m u l t i p l e  o r g a n o g e n e s i s was media. with  Rooting  shoot  f r o m c a l l u s v i a o r g a n o g e n e s i s was organogenesis d i d occur  shoot  dependent  cultures.  upon h i g h  f r o m s h o o t s was  induction.  A  rooting  shoots t r a n s f e r r e d onto the  and  could  i m p r o v e d by  during  this  case  cytokinin-containing  relatively  the  be  In  easy  compared  f r e q u e n c y of about r o o t i n g medium was  elevating  not  IAA  85%  of  observed  concentration  slightly. Plant  regeneration  embryogenesis. somatic of  with convicing  Somatic  counterparts  also  Although the  e m b r y o g e n e s i s has  reports  1983).  may  be  list  been  of  with appropriate  via  species  for  reported  evidence  embryos s h o u l d  achieved  i s smaller  resemble root,  i s long,  their  s h o o t and  which the  number  (Ammirato, zygotic cotyledon  131  organs with  s h o o t s c a p a b l e of  Somatic pattern: NAA  or  e m b r y o g e n e s i s of most  growth i n i t i a t i o n  IAA),  on  a p p e a r a n c e of  a medium c o n t a i n i n g no  (Raghavan,  a l . , 1978;  Ammirato,  that  successive  changes  Explant  components a r e The of  treatments  encourage the totipotency the  development  does not  differentiation  and  leading cells  to  are  the  with prominent  (Thorpe,  1982).  benefit  to another.  the  1977;  suggested  are  auxin  and  other  medium  of  embryo d e v e l o p m e n t Brawley  e m b r y o g e n e s i s of of  this  that  C a l l u s and  smaller,  cell colonies  contain  cells.  division, isodiametric  stained  cytoplasm  i . e . meristem-like  cells,  i s needed  Sometimes t h e  change  cultured cells or p o l l e n  to  i n one  transfer  cultures  of  from  inconspicuous  cell  usually  should  regeneration,  parenchyma  t y p e of  specific  cells  Although  cell  A  occurrence  a l . , 1984).  cultured  in achieving  and  of  et  polarity.  ensure  i s the  nuclei densely  In a n t h e r  or  e t a l . , 1977).  vacuolated  formation  been  to  f a c t o r s a f f e c t i n g embryogenesis.  occur.  cytoplasm.  microvacuolation,  could  (Reinert  in i t s e l f  highly  (2,4-D,  et a l . ,  I t has  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  These c e l l s nuclei  will  a similar  a weaker a u x i n  Reinert  e t a l . , 1977;  favoring  follows  i n n u t r i e n t media  i s widely accepted  capacity  various  feature  (Reinert  1976;  nitrogen,  important  critical  bipolarity  These  reduced  levels,  1983).  for embryogenesis  source,  species  plants.  a medium w i t h a u x i n  2,4-D  Evans e t  significant  into  embryos a f t e r c a l l u s t r a n s f e r  reduced  growth r e g u l a t o r s  the  developing  and  of many  factors  from one  state  Nicotiana  132 tobaccum  e m b r y o g e n e s i s and  subsequent p l a n t  were s t u d i e d w i t h e x p o s u r e d u r i n g atmospheric pressure, as  a result  anaerobic  of a d d i t i o n  a d d i t i o n s of a b s c i s i c and e a c h o f e i g h t  o f 0.5  acid,  showed t h a t  suitable  factors  significantly  to reduced  e n v i r o n m e n t s , water  stress  M m a n n i t o l t o t h e medium and  ascorbic  amino a c i d s  Results  culture  regeneration  acid,  ( H a r a d a and  dithiothreitol Imamura,  1983).  treatments with a l l these  enhanced  embryo d e v e l o p m e n t  in  culture. P l a n t s were r e g e n e r a t e d this  study.  containing  Calli  were f i r s t  relatively  high  transferred  PIC and BA.  Of  to i n i t i a t e  and q u a l i t y 1984a;  calli  f a c t o r s which c o n t r o l of reduced n i t r o g e n  o f t h e most  or  c a n t h e n be  1984b).  enhanced  by s i m i l a r  addition  o f amino a c i d s  stimulated  additives  Development  and  required.  induced  i t is  plantlet  medium. iri v i t r o  somatic  supplied  to  i m p o r t a n t ( K o h l e n b a c h , 1978).  embryo f o r m a t i o n was by amino a c i d  be  and have  from t h e s e embryos on a s i n g l e  i s one  alfalfa  and KIN  S u b c u l t u r e may  embryo p r o d u c t i o n  embryogenesis the l e v e l  In  NAA  of e m b r y o g e n e s i s h a s been  s e v e r a l major  cultures  medium  concentrations.  These embryogenic  regeneration.  Once t h e p r o c e s s  development  on SCP  and BA  containing  v i a embryogenesis i n  o n t o LSP medium f o r embryo d e v e l o p m e n t  eventual plant  possible  cultured  2,4-D  Embryos d e v e l o p e d on media containing  from c a l l i  (Stuart  i n b o t h number and  Strickland,  of embryos t o p l a n t l e t s  additives.  In the p r e s e n t  ( a r g i n i n e and g l u t a m i c  was  also  study the a c i d ) d i d not  133 enhance  callus  cultured  differentiation.  previously  t o undergo  surprising  that  embryogenesis  the a d d i t i o n  significantly  alfalfa  (Stuart  and S t r i c k l a n d ,  a  of a h i g h l y  triggered  r e g e n e r a b l e was  should  be p o s s i b l e  acids in  initiated reported  In t h e i r  genotype  ( c a p a b l e of  obtained  from non-meristem ( B e a c h and S m i t h ,  from c a l l u s  Phillips  et a l .  hypocotyl  (1984) o b t a i n e d c a l l i  e x p l a n t s which c a l l i  organogenesis except f o r rare Success  in regeneration  on c u l t u r e  5.2.5  Genotype Genotypic  morphology  for a l f a l f a .  cultures  and  has  been  Collins,  regeneration. from l e a f  d i d not undergo instances  of root  and  any formation.  from t h e s e c u l t u r e s m a i n l y  p r o c e d u r e s and g e n o t y p e  study i t  i n red clover  e x p l a n t s of r e d c l o v e r 1979;  which i s  o f amino  reported  1979a) i t i s n o t a l w a y s e a s y t o a c h i e v e Bhojwani  easily  line  the a d d i t i o n  results  experiment  in this  s t i m u l a t e s embryogenesis  regeneration  be  Since a c a l l u s  whether  comparison with the e a r l i e r Though p l a n t  embryogenesis i n  1884a).  eventually  to test  significantly  t h e same  Embryogenesis c o u l d  by a r o u t i n e method.  highly  while  stimulated  regenerable used.  been  i t i s perhaps not  on embryo i n d u c t i o n  e m b r y o g e n e s i s ) was  were  o f amino a c i d s d i d n o t show any  s u b s t a n c e s have  clone  these c a l l i  on LSE medium a n d had n o t  stimulated  promotional e f f e c t  Because  depends  used.  Effect effects  and p l a n t  on c a l l u s  regeneration  induction,  callus  were o b s e r v e d t h r o u g h o u t  134  this  study.  calli  I t i s obvious  that  performed d i f f e r e n t l y  both  callus  'Norseman*  on t h e v a r i o u s m e d i a t e s t e d f o r  growth and d i f f e r e n t i a t i o n .  grew f a s t e r  than  'Altaswede' c a l l i .  more r o o t e d  calli  and g r e e n - s p o t t e d  induced  only  more s h o o t s while  from  'Norseman'  'Norseman' calli,  'Altaswede' c u l t u r e s .  per m u l t i p l e shoot  shoots  and 'Altaswede'  produced  but shoots  were  'Altaswede' had  r o s e t t e than  o f 'Norseman' were b i g g e r  calli  'Norseman',  than  those of  'Altaswede'. Differences  i n the c a p a c i t y of regeneration  r e p o r t e d among c u l t i v a r s Collins, plant  1979a).  of r e d c l o v e r  Intra-varietal  regeneration  species,  including red clover  Phillips  and C o l l i n s  petiole plants  explants from o n l y  attempting  source  of v a r i b i l i t y  both  callus  1974; and  variation  that genetic  which  f o r most  e t a l . , 1984),  that cotyledon  in callus  variance 5^n v i t r o  root  initiation,  regenerating Studies  significant  growth,  colony  c h l o r o p h y l l p r o d u c t i o n and  (Keyes e t a l . , 1980).  i n d u c t i o n and p l a n t  regeneration  Chen e t a l . , 1978; S h a r p a n d E v a n s , 1984).  o r stem  characters  d i f f e r e n c e s seem t o be a m a j o r  Nitzsche,  while  c u l t u r e s of r e d  was a  included rapid callus  embryogenesis  Genetic  Trifolium.  1% o f t h e g e n o t y p e s c u l t u r e d .  revealed  somatic  (Bhojwani  of r e d c l o v e r y i e l d e d c a l l i  clover  vascularization,  for in vitro  i n three  (1979a) r e p o r t e d  to evaluate  investigated,  ( P h i l l i p s and  variation  was a l s o o b s e r v e d  h a s been  Genetic  factor  limiting  (Green e t a l . , ,  1982; Subba Rao  s t u d i e s on some in v i t r o  1 35  characters, by  relating  to regeneration,  s e v e r a l l a b o r a t o r i e s (Zamir  Bingham,  1980; S k v i r s k y  In a l f a l f a ,  crosses  regeneration 1980). these  line  were made between a h i g h  a n d low r e g e n e r a t o r ratio  suggested  was c o n t r o l l e d  Nesticky If be  some r n v i t r o that they  regenerated  regeneration Selection  and y i e l d  should  callus  characters  of other  ( M e z e n t s e v , 1980;  respond  of only  for friability  regeneration  line  1981).  (Sondahl  of a l f a l f a tissue  plant  12% (Bingham e t a l . , 1975).  and i n t e n s i v e p r o l i f e r a t i o n of 'F71' h a s r e s u l t e d i n t h r e e  callus  s t r u c t u r e and improved  A program aiming  t o improve  growth maize  Zea d i p l o p e r e n n i s a s a  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  e t a l . , 1984).  In r e d c l o v e r , s e l e c t i o n possible.  lines  an i n i t i a l  was r e p o r t e d u s i n g  o f germplasm  and  superior populations f o r Genetic  plants with  from a m a i z e  to breeding  were s e l e c t e d f r o m h y p o c o t y l  frequency  (Bartkowiak,  source  from  c h a r a c t e r s a r e h i g h l y h e r i t a b l e i t may  l i n e s c h a r a c t e r i z e d by u n i f o r m rate  I_n v i t r o  the expression  culture studies.  67% r e g e n e r a t i o n  callus  genes.  i n attempts t o develop  in tissue  culture  among t h e p r o g e n y o f  e t a l . , 1983).  selection  with  of  ( R e i s c h a n d Bingham,  observed  by two d o m i n a n t  such as f e r t i l i t y  expected  use  frequency  t h a t bud d i f f e r e n t i a t i o n  may a l s o be c o r r e l a t e d w i t h characters  e t a l . , 1980; R e i s c h a n d  e t a l . , 1982; M e i n s e t a l . , 1983).  The s e g r e g a t i o n crosses  have been c a r r i e d o u t  Callus lines  f o r r e g e n e r a b i l i t y may a l s o be  or p l a n t  l i n e s may be o b t a i n e d  by  136  selection  techniques  f o r improved  especially  plant  facilitate  tissue culture  5.3  regeneration.  provided Shoot  propagation  Such l i n e s would  t i p cultures  were e s t a b l i s h e d  L 2 ( P 0 . 0 0 4 B 1 ) medium cultures  were a l s o  of r e d c l o v e r (Parrot  established  1983).  In the p r e s e n t  study  media c o n t a i n i n g induction  shoot  Results  and p r o p a g a t i o n .  characters not  using  1983). medium  5  study  Shoot t i p and a f f e c t i n g  (Campell  tips  and  Tomes,  were c u l t u r e d  on L2  for multiple  Procedures described f o r m u l t i p l i c a t i o n of  in plants  Plants  from t h e a n a l y s i s  isozyme p a t t e r n s  characters.  reported  techniques.  Regenerated  regenerated  B  response.  d i f f e r e n t l e v e l s o f PIC a n d BA  these experiments are a v a i l a b l e vitro  was  using  i n another  materials.  from two c u l t i v a r s ,  and C o l l i n s ,  were e v a l u a t e d  in  production  different in their  factors  shoot  shoot  a r a p i d method t o p r o p a g a t e v a l u a b l e  Meristem-tip culture  and  greatly  studies.  by m u l t i p l e  w h i c h were s i g n i f i c a n t l y  5.4  characters,  Shoot T i p C u l t u r e Clonal  by  ir\ v i t r o  of f i v e  of leghaemoglobin  enzymes showed t h a t  f r o m c a l l u s c u l t u r e were q u i t e These d a t a are very  indicated either  stable  that  cycle  plants f o r these  these  g e n e t i c a l l y so t h a t  e a s i l y i n d u c e d by a s i n g l e c u l t u r e  somaclonal v a r i a t i o n  stable  profiles  variation i s  or that  i s n o t common i n t h e s e g e n o t y p e s o f r e d  137 clover.  The a n a l y s i s o f p r o t e i n s  evaluate  gene p r o d u c t s  varition  f o r such q u a l i t a t i v e t r a i t s  species for  offers a direct  f o r genetic  variation.  When 45 RG1 multiple  shoots  multiple  cultures  initially  i n d u c e d , were a n a l y s e d f o r  s h o o t s underwent  v i a embryogenesis  (callus  t o be n o r m a l .  subculture, mosaic.  P l a n t s (170)  from s h o r t - t e r m  had been c u l t u r e d  via  embryogenesis  had  c h a n g e d chromosome numbers,  a very  to length  callus  f o r three  months) d i d n o t  subsequently  (Sunderland,  1977).  (D'Amato,  within  the formation  I n some c a s e s ,  1977).  one y e a r )  variation i s  I t takes place i n which c o n s i s t s of the nuclear  o f a s p i n d l e and t h e  u n d e r g o e s a f u r t h e r DNA  chromosome numbers i n c r e a s e d period  ( nearly  Chromosome d o u b l i n g i s  of endomitosis,  of sister-chromatids  membrance b u t w i t h o u t  regenerated  i n d i c a t i n g that  of c u l t u r e p e r i o d .  by t h e p r o c e s s  separation  nucleus  term c u l t u r e s  commen change f o r c u l t u r e d c e l l s .  some c e l l s a  from l o n g  After  one o f 28 p l a n t s  show any v a r i a t i o n , whereas 23% o f 119 p l a n t s  related  reported  ' A l t a s w e d e ' p l a n t s , w h i c h came from t h e  e x a m i n e d was a d i p l o i d / t e t r a p l o i d regenerated  freqently  characters.  chromosome number t h e y were a l l f o u n d the  Somaclonal  may be d e m o n s t r a b l e f o r  i n w h i c h t h i s v a r i a t i o n h a s been  q u a n t i t a t i v e and m o r p h o l o g i c a l  way t o  cells  replication  with  doubled  a f t e r a prolonged  culture  Accordingly  plants  regenerated  long-term c u l t u r e s tend  t o have more p o l y p l o i d s .  In r e d  clover  showed p l a n t s  from  previous  short-term  reports  callus  c u l t u r e s were n o r m a l  regenerated  i n chromosome  from  number  138 ( B e a c h and S m i t h ,  1979;  Phillips  and C o l l i n s ,  However, when p l a n t s were r e g e n e r a t e d diploid  cultivar  culture  f o r s i x months b e f o r e  regeneration, (Phillips In  w h i c h had  two  americanum  L.  r e g e n e r a t e d p l a n t s was 1982;  1985).  regenerated plants wheat c u l t i v a r s  Panicum  1983).  one was  101  tetraploid  In c o n t r a s t ,  and a l l t h e  from c u l t u r e d  (Triticum  immature  tetraploid  cultures  i n which o r i g i n  o f s h o o t s was  regenerated  discrepancy  192  calli  not  and  four  plants  among t h e r e p o r t s  from the  identified.  chromosome number  result  (1984)  abnormalities  from embrogenic  chromosome a b n o r m a l i t i e s c o u l d  (Swedlund  embryos o f  a e s t i v u m L . ) , 29% o f  study demonstrated that  others  when K a r p and Maddock  t o p l a n t s both from embryogenic  The  americanum  were o b s e r v e d i n t h e c u l t u r e s  happened  clover.  1981;  plants  of Pennisetum  were a n e u p l o i d s and chromosome  occurred plants  maximum and  and V a s i l ,  When  calli  Pennisetum  o f chromosome number i n  examined  present  from  achieved in  a l t h o u g h a h i g h e r p e r c e n t a g e of  and a n e u p l o i d c e l l s and V a s i l ,  hybrid,  observed ( V a s i l  from embryogenic  were d i p l o i d s ,  regeneration  stability  Vasil,  L . were i n v e s t i g a t e d ,  tetraploids  L . , P e n n i s e t u m purpureum,  a e s t i v u m L., h i g h  regenerated  p l a n t s were  1980).  X P_j_ purpureum  et a l . ,  of  v i a e m b r y o g e n e s i s has been  P e n n i s e t u m americanum  Vasil  two  of a  in suspension  the i n i t i a t i o n  c e r e a l s and g r a s s e s p l a n t calli  from c a l l i  cultured  out of twenty  and C o l l i n s ,  embryogenic  Triticum  been  1979a).  calli  The  change of r e d  concerning  from d i f f e r e n c e s i n  139  length  of c u l t u r e  period,  culture  procedure  and m a t e r i a l s  used. Somatic (e.g.  Vasil  plants  from  suggests than  embryos a r e b e l i e v e d e t a l . , 1982). one c a l l u s  that  either  one c e l l  cell(s)  the p l a n t s callus  frequency.  stage of  effect  t h e c h a n g e s may  factors  (Dr.  than  in  more  i n some  from m u l t i p l e  observed  among  s h o o t s o r from  1962).  leaflet  the e x p r e s s i o n of  number  five  by two g e n e s  with  When a p l a n t  shows o n l y  number and t h e  Simon, T e c h n i c a l u n i v e r s i t y In t h i s  remainder  of Munich,  study a l l the p l a n t s  were m o s a i c ,  g e n e t i c changes.  indicating  of phenotypic  that  plasticity  Therefore, the fact  p l a n t s had v a r i e d  leaflet  numbers  that  with  they rather more  than  p l a n t s d o e s n o t n e c e s s a r i l y mean t h a t t h e  culture  plants  from  be a c o n s e q u e n c e o f e n v i r o n m e n t a l  arose as a r e s u l t  seed-derived tissue  U.  leaflet  regenerated  study  embryo o r i g i n a t e d  i s controlled  communication).  from  regenerated  embryogenesis.  (Simon,  normal,  probably  some  s e e d - d e r i v e d p l a n t s a l t h o u g h a t lower  few l e a v e s w i t h a l t e r e d  changed  cells  T h i s phenomenon was, however, a l s o  in red clover  personal  single  i n the present  I t has been r e p o r t e d t h a t  complementary a  the somatic  regenerated either  among  leaflets  were m o s a i c  that  number c h a n g e s were f r e q u e n t l y  cultures.  observed  The f a c t  from  o r t h e chromosome number c h a n g e d  a t an e a r l y  Leaflet  to arise  c y c l e had i n d u c e d h e r i t a b l e  for this  trait.  g e n e t i c changes  140 Interestingly, more v a r i a t i o n those  regenerated  i n the length to width  o f 'Norseman'.  callus  p l a n t s of 'Altaswede' ratio  of l e a v e s  P l a n t s w h i c h were r e g e n e r a t e d  c u l t u r e s of one g e n o t y p e  showed a r e d u c e d  R e g e n e r a t e d p l a n t s o f 'Norseman' d i d n o t d i f f e r seed-derived tetraploid to  such  c o n t r o l s with  nature  o f 'Norseman' may  results  regenerated  variation  than  for qualitative  i s more l i k e l y  quantitative height,  demonstrated  characters  d r y weight  variation. from the The  i t s stability  the r e p o r t s  2.2.1.1.).  were r e p o r t e d . regenerated  However,  Thus  relatively  length,  In sugar  high  plant  cane,  f r e q u e n t l y b u t most  from t h e d o n o r study  frequencies  (1976) r e p o r t e d F156  s t a b l e and v i s i b l e  five  (Section of v a r i a n t s  that  34% of  differed  i n morphological  where  somaclonal  i n work on  to q u a n t i t a t i v e characters  f r o m one c l o n e  i n another  distinctive  such as l e a f l e t  L i u a n d Chen  plants  significantly  t o be r e p o r t e d  among  for quantitative  characters.  h a s been r e p o r t e d  relate  Usually,  variation  t o occur  or g r a i n weight.  somaclonal v a r i a t i o n  subclones  characters. with  m a r k e r s were s e l e c t e d f o r  c u l t u r e , the r a t e of change of t h e s p e c i f i c  qualitative two o r t h r e e missing  from  variability.  increase  that  p l a n t s was more l i k e l y  characters  tissue  to this  than  changes.  These  of  respect  showed  characters,  freckle  p l a n t s per thousand.  internode  detected,  leaf  they  and z i g - z a g  and l e a f  blotch,  F o r two o t h e r  stalk,  were n o t t r a n s m i s s a b l e  although through  was  characters,  c h a n g e s were vegetative  141 propagation Very  (Irvine,  few  1984).  r e p o r t s are  regenerated  p l a n t s from  (Groose  Bingham,  al.,  and  1985),  efficient  callus  in  efforts  regimes.  be  are  or use  The  non-meristem d e r i v e d  approached  by  two  been r e p o r t e d  g e n o t y p e s can  second  culture.  only  research. be  such  To  of  m a t e r i a l s are  solid  o b t a i n e d by  and  lines  t h a t the  effect  are p o s s i b l e to good  screening  f o r and  variation  required.  regenerator  sufficiently r a n g e of  proper  These t r a i t s  s u s c e p t i b l e to existing  traits  s h o u l d be  plants  have one  time looked  to  of at  f o r the  then  i n both  i t will  be  the  experimental r e p o r t s have  are  dealt  inherently quite  environmental  of t h e e x p e r i m e n t .  several single  are a v a i l a b l e  homozygous s t a t e s ,  to estimate  Most of p r e v i o u s  traits.  c o n d i t i o n s at the  traits  evidence  somaclonal  with quantitative variable  regenerative  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.  provide  frequency  r e q u i r e s the  Nevertheless,  l a r g e p l a n t p o p u l a t i o n s t o ensure variability  approach  proper  and  Since a genotypic  i n many c a s e s ,  further  ways.  r e q u i r e d t o d e f i n e the  of genotypes showing h i g h  from j j n v i t r o  facilitate  legumes  i n p a r t b e c a u s e of t h e c o n t i n u i n g d i f f i c u l t y  c o n d i t i o n s i n c l u d i n g medium c o m p o s i t i o n  potential  of  c u l t u r e s of  J o h n s o n e t a l . , 1983;  T h i s p r o b l e m may  development  these  the a n a l y s i s  et  environmental  has  1983;  and  on  Damiani  additional  culture  cell  p l a n t r e g e n e r a t i o n from  cultures. First,  available  Qualitative  s t a t e d purposes.  If  gene-controlled t r a i t s heterozygous  and  and  p o s s i b l e to estimate  the  142  frequencies more c u l t u r e  of dominant cycles.  or r e c e s s i v e  mutants a f t e r  one o r  6. When h y p o c o t y l  Summary  e x p l a n t s of  red  and  'Norseman' were c u l t u r e d  the  explants produced c a l l i  on  induction  (P40.05) i n t h e  combined d a t a  medium, p r e v i o u s l y  red  clover,  under was  the  was  not  two  weeks.  than  the  described  'Altaswede'  medium, 60-85%  frequency of  cvs  batches  in culture  experimental conditions  of  'Altaswede'  'Norseman'  two  s u c c e s s f u l l y used  of  to  tested.  studies  induce  of  shoots  t h i s study,  nor  i t a good medium f o r c a l l u s m a i n t e n a n c e .  On  SCP  could  medium, c a l l i be  initiated the  on  SCP  They  t h i s medium f o r up  to  Embryogenesis  medium, but  s t a g e s were not  was  embryos able  at  to  normally.  were u s u a l l y  auxin  growth r a t e .  subculturing.  e a r l y heart  further  More r o o t s low  a high  i n some c a l l i  g l o b e and  develop  had  s u c c e s s f u l l y grown on  f i v e months w i t h o u t  a  L2  within  showed a h i g h e r  LSE  clover  formed  to c y t o k i n i n  from c a l l i  cultured  ratio  or  i n the  absence  S h o o t s were formed a f t e r c a l l i  of  'Altaswede'  on  o n t o media  at  of  auxins.  NAA KIN  SCP and  medium were t r a n s f e r r e d KIN.  concentration  was  containing  more c r i t i c a l  than  concentration.  Additions and  NAA  cultured  casein  effect  on  of  amino a c i d s  hydrolysate  (arginine  d i d not  and  show any  glutamic  promotive  c a l l u s growth or d i f f e r e n t i a t i o n .  143  acid)  144  By  s e l e c t i n g and s u b c u l t u r i n g  number  of p l a n t s  h a v e been  Callus capability selected  lines  Embryogenic calli  f o r more  calli  than  containing Usually roots  to produce shoot  mg/1  shoot  tips  cultured  shoots  were  them on L2 media  PIC and 1 mg/1  BA.  grew f a s t e r a n d p r o d u c e d  calli,  'Altaswede'  while shoots calli.  'Norseman' had more  multiple  number  from  by s u b c u l t u r i n g  'Altaswede'  shoots c u l t u r e ,  retained in  non-embryogenic  These m u l t i p l e  'Norseman' c a l l i  induced o n l y from  culture.  one y e a r .  induced  0.002-0.003  than  a  used.  s h o o t s were  propagated  calli,  from  was  grew f a s t e r t h a n  on L2(PO.003B2) medium. easily  regenerated  f o r embryogenesis  on t e n media  Multiple  embryogenic  were  In m u l t i p l e shoot  tips  s h o o t s , but the average  f o r 'Altaswede'  more  was h i g h e r t h a n  induced  multiple that f o r  'Norseman'. Plants  were r e g e n e r a t e d  initial twice either  multiple  shoots  from m u l t i p l e  shoots, e i t h e r  (RG1) o r m u l t i p l e  s u b c u l t u r e s (RG2), o r from c a l l u s short-term  long-term  ( t h r e e months) c a l l u s  (one y e a r ) c a l l u s  (RG4).  shoots a f t e r  cultures, (RG3) o r  145  12.  Regenerated p l a n t s of 'Altaswede' chromosome normal.  number.  One  A l l RG1  o f 28 RG2  p l a n t s had a l t e r e d  13.  chromosome  were  p l a n t s were  numbers.  RG4  A l l these  or d i p l o i d / t e t r a p l o i d  mosaics.  recovered.  R e g e n e r a t e d 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  profiles  6-PGDH, PGI, PGM 14.  a n d RG3  p l a n t s a n d 27 o u t o f 119  p l a n t s were t e t r a p l o i d s No a n e u p l o i d s  were a n a l y s e d f o r  For l e a f l e t of  controls.  RG1,  RG2  ratio,  different  p l a n t s were compared w i t h RG2  and RG3  showed more v a r i a t i o n plants,  w h i l e RG4  p l a n t s of  for this  ratio  from  the c o r r e s p o n d i n g  populations those of  'Altaswede' than  p l a n t s of 'Altaswede'  p l a n t s o f 'Norseman' were n o t  different  MDH,  a n d SKDH.  length/width  regenerated  and i s o z y m e p a t t e r n s o f  control  a n d RG1  significantly control plants.  and  LITERATURE  CITED  A h l o o w a l i a , B S (1976) Chromosome c h a n g e s i n p a r a s e x u a l l y produced r y e g r a s s . I n : J o n e s , K a n d Brandham,PE ( e d s ) : C u r r e n t chromosome r e s e a r c h . E l s e v i e r Press: Amsterdam, p p l 1 5 - 1 2 2 . A h l o o w a l i a , B S (1982) P l a n t r e g e n e r a t i o n i n wheat. C r o p S c i . 22,405-410.  from c a l l u s  culture  Ammirato,PV (1983) E m b r y o g e n e s i s . 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Zhao,CZ; Zheng,KL; Q i , X F ; Sun,ZX and Fu,YP (1982) The c h a r a c t e r s o f r i c e s o m a t i c 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 p r o g e n i e s i n paddy f i e l d . Acta Genet. S i n . 9,320-322. Zhao,CZ; Sun,ZX; Zheng,KL; Q i , X F 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. Scientia Agri. S i n . 5,35-40.  Appendix 1  C o m p o s i t i o n o f B5 medium: m i n e r a l s a l t s and o r g a n i c compounds e x c e p t g r o w t h r e g u l a t o r s Macronutrients KNO3 CaCl^^O MgSCv7H O (NH ) S0 NaH P04.H 0  2500 150 250 134 150  z  4  2  4  2  mg/1  2  Micronutrients  mg/1  KI H B0 * MnS0;H 0 ZnS0 *7H O Na Mo0 -2H 0 CuS0 -5H 0 CoCl -6H 0 Na EDTA FeSO^I^O  0.75 3.0 10 2.0 0.25 0.025 0.025 37.3 27.8  Organic  mg/1  3  3  2  4  2  z  4  4  2  2  2  2  a  compounds  Inositol Nicotinic acid Pyridoxine*HCl Thiamine-HCl Sucrose  100 1.0 1.0 10.0 20000  

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