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Effect of cytokinins on tissue structure, plastid development and photosynthetic proteins in tissue culture… Mazari Hiriart, Alicia 1991

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EFFECT OF CYTOKININS ON TISSUE STRUCTURE, PLASTID DEVELOPMENT AND PHOTOSYNTHETIC PROTEINS IN TISSUE CULTURE OF PINUS PONDEROSA DOUGL. COTYLEDONS DURING ORGANOGENESIS by ALICIA MAZARI HIRIART B.Sc, M.Sc,  U n i v e r s i d a d N a c i o n a l Autonoma de Mexico, 1984 U n i v e r s i d a d N a c i o n a l Autonoma de Mexico, 1986  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Botany)  We accept t h i s t h e s i s as  conforming  to t h e r e q u i r e d s t a n d a r d  THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1991 © A l i c i a M a z a r i H i r i a r t , 1991  In presenting this thesis in partial fulfilment of the  requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by his  or  her  representatives.  It  is  understood  that  copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  ^  .  .  ,  Department of  BOTANY  The University of British Columbia Vancouver, Canada  D  a  t  e  DE-6 (2/88)  AUGUST  2,  1991  ABSTRACT  The c y t o k i n i n s , adenine, centers  promoted the  in vitro  response  2-isopentenyl  formation of  of Pinus ponderosa Dougl.  was d e p e n d e n t  meristematic  on t h e  time  The  of  excised  organogenic  exposure  to  these  regulators. Differences  regions, medium,  between m e r i s t e m a t i c ,  and c o t y l e d o n s were  c u l t u r e d on g r o w t h r e g u l a t o r  cells  tissues  developed  i n n e r membranes,  A decline  days  markers  detected was more  t h a n on c y t o k i n i n  ( c h l o r o p h y l l and 5 p o l y p e p t i d e s  concentrations  was  The d e c l i n e  This trend contrasted with  photosynthesis),  cells.  p r o t e i n content  free  had  more t h y l a k o i d membranes  in culture.  r a p i d on g r o w t h r e g u l a t o r cotyledons.  thylakoid  i n non-  of meristematic  in total  first  cells  in  and n o n - c y t o k i n i n t r e a t e d t i s s u e s  and g r a n a t h a n p l a s t i d s  during the  whereas  free  Plastids  showed p o o r l y d e v e l o p e d  membranes and some g r a n a , meristematic  non-meristematic  o b s e r v e d by day 5 i n c u l t u r e .  newly m e r i s t e m a t i c  well  and  w h i c h l e d t o b u d and s h o o t p r o d u c t i o n on  cotyledons  growth  benzyladenine  w h i c h were p r e s e n t in cytokinin treated  those c u l t u r e d i n growth r e g u l a t o r  in  chloroplast associated  with  lower  cotyledons free  treated  medium.  than  in  Both  benzyladenine  and 2 - i s o p e n t e n y l adenine were e f f e c t i v e i n  i n h i b i t i n g the accumulation p o l y p e p t i d e s i n cotyledons culture.  The  of at l e a s t two i n the f i r s t  photosynthetic  24 hours i n  a b i l i t y of cotyledons to respond i n t h i s  to c y t o k i n i n s was  way  l o s t a f t e r only t h r e e days i n c u l t u r e i n  growth r e g u l a t o r f r e e medium.  RESUMEN  Las c i t o c i n i n a s , b e n c i l a d e n i n a y 2 - i s o p e n t e n i l adenina,  promovieron l a formaci6n  meristematicos  i n vitro,  de centros  l o s cuales a su vez produjeron  b r o t e s y yemas en c o t i l e d o n e s de Pinus ponderosa Dougl. La respuesta organogenica  es dependiente d e l tiempo de  e x p o s i c i 6 n a estos reguladores Se observaron meristematica,  de c r e c i m i e n t o .  d i f e r e n c i a s entre l a s regiones  no meristematica  y t e j i d o de c o t i l e d o n e s  c u l t i v a d o s en ausencia de reguladores despues de 5 d i a s en c u l t i v o . meristematica  de c r e c i m i e n t o ,  Los p l a s t i d i o s de l a r e g i 6 n  mostraron membranas t i l a c o i d e s poco  d e s a r r o l l a d a s y algunos grana, mientras meristematico  que e l t e j i d o no  y e l tejido cultivado s i n citocininas,  mostr6 membranas i n t e r n a s b i e n d e s a r r o l l a d a s con mas membranas t i l a c o i d e s y grana que l o s p l a s t i d i o s de c e l u l a s meristematicas. Se observ6 una disminuci6n t o t a l durante l o s primeros  d e l contenido  de p r o t e i n a  d i a s en c u l t i v o , siendo mas  rapido en c o t i l e d o n e s c u l t i v a d o s en ausencia de reguladores citocininas.  de c r e c i m i e n t o que en p r e s e n c i a de E s t a s observaciones  c o n t r a s t a r o n con l o s  marcadores de c l o r o p l a s t o s ( c l o r o f i l a y 5 p o l i p e p t i d o s  iv  asociados  con f o t o s i n t e s i s ) ,  concentraci6n  que mostraron una  menor en c o t i l e d o n e s t r a t a d o s con  c i t o c i n i n a s respecto a a q u e l l o s c u l t i v a d o s en medio s i n reguladores  de c r e c i m i e n t o .  Ambas c i t o c i n i n a s ,  b e n c i l a d e n i n a y 2 - i s o p e n t e n i l adenina, i n h i b i e r o n l a acumulaci6n de a l menos dos p o l i p e p t i d o s f o t o s i n t e t i c o s en' c o t i l e d o n e s durante l a s primeras 24 horas en c u l t i v o .  La  h a b i l i d a d de l o s c o t i l e d o n e s para responder de e s t a forma a l a s c i t o c i n i n a s se p e r d i 6 en t a n s o l o t r e s d i a s en c u l t i v o en medio s i n reguladores  v  de c r e c i m i e n t o .  TABLE OF CONTENTS Page ABSTRACT  i i  TABLE OF CONTENTS  vi  LIST OF TABLES  viii  LIST OF FIGURES  ix  ABBREVIATIONS  xii  ACKNOWLEDGEMENTS  xiii  DEDICATION  xiv  INTRODUCTION  1  LITERATURE REVIEW 1. CONTROL OF ORGANIZED DEVELOPMENT IN TISSUE CULTURE 4 2. HISTOLOGICAL AND ULTRASTRUCTURAL STUDIES OF ORGANIZED DEVELOPMENT 7 3. CYTOKININS 10 4. THE ROLE OF CYTOKININS IN PLASTID DEVELOPMENT 4.1. NORMAL PLASTID DEVELOPMENT 11 4.2. BIOCHEMICAL AND MORPHOLOGICAL EFFECTS OF CYTOKININS ON THE PHOTOSYNTHETIC APPARATUS 12 5. INTERACTIVE EFFECTS OF CYTOKININS AND LIGHT 18 MATERIALS AND METHODS 1. TISSUE CULTURE 1.1. PLANT MATERIAL 1.2. CULTURE METHODS 1.3. CULTURE CONDITIONS 2. HISTOLOGICAL AND ULTRASTRUCTURAL ANALYSES 2.1. LIGHT MICROSCOPY 2.2. TRANSMISSION ELECTRON MICROSCOPY 3. PROTEIN AND CHLOROPHYLL ANALYSES 3.1. PROTEIN EXTRACTIONS 3.2. TOTAL PROTEIN ANALYSIS 3.3. SDS POLYACRYLAMIDE GEL ELECTROPHORESIS . 3.4. IMMUNOBLOTTING 3.5. CHLOROPHYLL DETERMINATION . ...  vi  20 21 23 24 24 25 25 2 6 27 28  4. P R O T E I N AND C H L O R O P H Y L L 5. S T A T I S T I C A L A N A L Y S I S  A S ORGANOGENIC MARKERS  . 30 30  RESULTS 1. T I S S U E C U L T U R E 1.1. C U L T U R E M E D I A 1.2. GROWTH R E G U L A T O R C O N C E N T R A T I O N S 1.3. T I M E OF E X P O S U R E 2. H I S T O L O G I C A L AND U L T R A S T R U C T U R A L A N A L Y S E S 3. P R O T E I N AND C H L O R O P H Y L L A N A L Y S E S 3.1. TOTAL PROTEIN DETERMINATION 3.2. SDS P O L Y A C R Y L A M I D E G E L E L E C T R O P H O R E S I S . 3 . 3 . IMMUNOBLOTTING 3.4. C H L O R O P H Y L L D E T E R M I N A T I O N 4. P R O T E I N AND C H L O R O P H Y L L A S ORGANOGENIC MARKERS . 4 . 1 . C U L T U R E ON GROWTH R E G U L A T O R F R E E MEDIUM FOLLOWED BY T R A N S F E R TO C Y T O K I N I N C O N T A I N I N G MEDIUM 4.2. C U L T U R E ON C Y T O K I N I N - C O N T A I N I N G MEDIUM FOLLOWED BY T R A N S F E R TO GROWTH R E G U L A T O R F R E E MEDIUM DISCUSSION 1. T I S S U E C U L T U R E 2. H I S T O L O G Y AND U L T R A S T R U C T U R E 3. M O B I L I Z A T I O N OF R E S E R V E S 4. P H O T O S Y N T H E T I C P R O T E I N S AND C H L O R O P H Y L L 5. P R O T E I N AND C H L O R O P H Y L L A S ORGANOGENIC MARKERS  31 31 34 37 40 57 57 60 63 65 68  68  73  78 80 85 88 91 . 96  CONCLUSIONS  102  SUGGESTIONS  104  BIBLIOGRAPHY  105  vii  LIST OF TABLES 1. C u l t u r e media composition  Page 22  2. C u l t u r e c o n d i t i o n s  41  3. Responses of Pinus ponderosa cotyledons c u l t u r e d i n v i t r o a f t e r 10 days of exposure to 15 uM BA or 15 uM 2iP  41  viii  L I S T OF  FIGURES Page  1.  M u l t i p l e buds and shoots  32  2.  T o t a l response, organogenic response and mean b u d s a n d s h o o t s , o f c o t y l e d o n s c u l t u r e d on d i f f e r e n t m e d i a  33  T o t a l response, organogenic response, and mean b u d s a n d s h o o t s , o f c o t y l e d o n s c u l t u r e d on d i f f e r e n t c o n c e n t r a t i o n s o f BA o r 2 i P o n L P m e d i u m f o r 42 d a y s  35  4. T o t a l r e s p o n s e , o r g a n o g e n i c r e s p o n s e , a n d mean b u d s a n d s h o o t s , o f c o t y l e d o n s c u l t u r e d on d i f f e r e n t c o n c e n t r a t i o n s o f BA o r 2 i P , o n L P o r SH m e d i u m f o r 10 d a y s  36  3.  5.  T o t a l response, organogenic response, and mean b u d s a n d s h o o t s , o f c o t y l e d o n s c u l t u r e d on LP medium s u p p l e m e n t e d w i t h d i f f e r e n t c o n c e n t r a t i o n s o f B A o r 2 i P f o r 10 a n d 42 days  38  6. T o t a l r e s p o n s e , o r g a n o g e n i c r e s p o n s e , a n d mean b u d s a n d s h o o t s , o f BA o r 2 i P t r e a t e d c o t y l e d o n s c u l t u r e d o n L P o r SH m e d i u m a f t e r d i f f e r e n t time o f exposure t o cytokinins  39  7. T o t a l r e s p o n s e , o r g a n o g e n i c r e s p o n s e , a n d mean b u d s a n d s h o o t s o f c o t y l e d o n s e x c i s e d from g e r m i n a t e d embryos c u l t u r e d i nt h e p r e s e n c e o f B A o r 2 i P f o r 10 d a y s  42  8. M i c r o g r a p h s o f c o t y l e d o n s a t t h e t i m e o f e x c i s i o n ( d a y 0)  44  9. L i g h t m i c r o g r a p h s o f c o t y l e d o n s a t d a y 3 i n c u l t u r e i n t h e presence o r absence o f cytokinins  45  10. 11.  L i g h t micrograph o f a c o t y l e d o n a t day 5 i n c u l t u r e i n t h e p r e s e n c e o f c y t o k i n i n s ... ,  46  L i g h t micrograph o f t h e m e r i s t e m a t i c and nonm e r i s t e m a t i c r e g i o n s o f a c o t y l e d o n a t day 5 in culture i n the presence of cytokinins  46  ix  12. M i c r o g r a p h s o f c o t y l e d o n s a t day 5 i n c u l t u r e i n the presence of c y t o k i n i n s  47  13. M i c r o g r a p h s o f c o t y l e d o n s a t day 5 i n c u l t u r e on GRF medium  50  14. M i c r o g r a p h s o f c o t y l e d o n s a t day 10 i n c u l t u r e i n t h e presence o f c y t o k i n i n s  52  15. T r a n s m i s s i o n e l e c t r o n micrographs o f p l a s t i d s of c o t y l e d o n s a t day 10 i n c u l t u r e i n t h e presence of c y t o k i n i n s  55  16. M i c r o g r a p h s o f c o t y l e d o n s a t day 10 i n c u l t u r e i n GRF medium  56  17. L i g h t m i c r o g r a p h o f a c o t y l e d o n a t day 21, c u l t u r e d i n t h e p r e s e n c e o f c y t o k i n i n s f o r 10 days and t r a n s f e r r e d t o a growth r e g u l a t o r f r e e (GRF) medium  58  18.  Total p r o t e i n concentration of cotyledons c u l t u r e d on GRF medium, o r i n t h e presence of BA o r 2 i P  59  19.  SDS-PAGE o f c o t y l e d o n s c u l t u r e d on GRF o r BA c o n t a i n i n g media from days 0 t o day 10  61  20.  Immunoblots o f p h o t o s y n t h e t i c p r o t e i n s , CF1, LSU-RUBP, 33EP, CP2 9, LHCII and LHCI, o f c o t y l e d o n s c u l t u r e d i n growth r e g u l a t o r f r e e (GRF) medium, w i t h 15 uM b e n z y l a d e n i n e (BA), or 2 - i s o p e n t e n y l adenine ( 2 i P ) , and c o n t r o l (C)  64  21. LSU-RUBP c o n c e n t r a t i o n o f c y t o k i n i n (CK) v e r s u s growth r e g u l a t o r f r e e (GRF) t r e a t e d cotyledons cultured i n v i t r o 22.  T o t a l C h l o r o p h y l l , C h l o r o p h y l l a and C h l o r o p h y l l b c o n c e n t r a t i o n s , o f BA o r 2 i P t r e a t e d c o t y l e d o n s and t h o s e c u l t u r e d on GRF medium  66  67  23. T o t a l p r o t e i n c o n c e n t r a t i o n o f c o t y l e d o n s c u l t u r e d on GRF medium f o r 1, 3, o r 5 days and t h e n t r a n s f e r r e d t o BA o r 2 i P c o n t a i n i n g media, compared w i t h GRF t r e a t e d c o t y l e d o n s .. 69  x  24. Immunoblots o f LSU-RUBP and CP29, o f c o t y l e d o n s c u l t u r e d on growth r e g u l a t o r f r e e (GRF) media and t h e n t r a n s f e r r e d t o BA o r 2 i P c o n t a i n i n g media 71 25. T o t a l C h l o r o p h y l l , C h l o r o p h y l l a, and Chlorophyll b concentrations of cotyledons c u l t u r e d i n t h e absence o f c y t o k i n i n s f o r 1, 3 and 5 days and t h e n t r a n s f e r r e d t o BA o r 2 i P c o n t a i n i n g media  72  26. T o t a l p r o t e i n c o n c e n t r a t i o n o f c o t y l e d o n s c u l t u r e d on BA o r 2 i P c o n t a i n i n g media f o r 1, 3 o r 5 days and t h e n t r a n s f e r r e d t o GRF medium, compared w i t h BA o r 2 i P t r e a t e d cotyledons  74  27. Immunoblots o f LSU-RUBP and CP29, o f c o t y l e d o n s c u l t u r e d on BA o r 2 i P c o n t a i n i n g media and then t r a n s f e r r e d t o growth r e g u l a t o r (GRF) media 75 28. T o t a l C h l o r o p h y l l , C h l o r o p h y l l a, and Chlorophyll b concentrations of cotyledons c u l t u r e d i n t h e p r e s e n c e o f BA o r 2 i P f o r 1, 3 o r 5 days and t h e n t r a n s f e r r e d t o GRF medium  xi  76  ABBREVIATIONS APP M - apparent  m o l e c u l a r mass  BA = N^-Benzyladenine o r 6-Benzylaminopurine CF1 = c o u p l i n g f a c t o r Chi = c h l o r o p h y l l CK = c y t o k i n i n CP29 = a n t e n n a l component of photosystem I I DMSO = d i m e t h y l s u l f o x i d e 33EP = 33kD e x t r i n s i c p o l y p e p t i d e FW = f r e s h weight GRF  = growth r e g u l a t o r f r e e  2iP = N -[2-isopentenyl]adenine 6  or 6-(5,S-Dimethyl  a l l y l a m i n o purine) LSU-RUBP = l a r g e s u b u n i t of  Ribulose-1,5-bisphosphate  carboxylase LHCII = l i g h t h a r v e s t i n g complex of photosystem I I LHCI = l i g h t h a r v e s t i n g complex of photosystem I PAGE = p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s RUBP = R i b u l o s e - 1 , 5 - b i s p h o s p h a t e SDS  = sodium d o d e c y l  carboxylase  sulphate  SE = s t a n d a r d e r r o r of the mean TRIS = T r i s  [hydroxymethyl]  aminomethane  xii  ACKNOWLEDGEMENT S I would l i k e t o acknowledge r e c e i p t o f a s c h o l a r s h i p f r o m my c o u n t r y t h r o u g h  graduate  the Universidad  N a c i o n a l Aut6noma de M e x i c o . To D r . I a i n E.P. T a y l o r f o r h i s s u p e r v i s i o n .  To D r .  E d i t h L. Camm f o r a l l h e r h e l p a n d a d v i c e w i t h t h e p r o t e i n analysis.  To M i c h a e l W e i s f o r h i s h e l p w i t h t h e  microscopical  work.  To D r . Dane R o b e r t s ,  Dr. Denis  Lavender, Dr. Anthony  G l a s s , D r . N e i l Towers a n d D r . D a v i d Webb f o r t h e i r support  and i n t e r e s t  i n my a c a d e m i c p r e p a r a t i o n .  To B a r r y F l i n n f o r h i s h e l p a n d s u g g e s t i o n s . K a r l D. H a u f f e  f o r h i s suggestions.  To D r .  To E v e l y n L e a f a n d  Susan M c D o u g a l l f o r t e c h n i c a l a s s i s t a n c e . To a l l t h e p e o p l e me d u r i n g a l l t h e s e  who somehow h e l p e d a n d s u p p o r t e d  years.  To t h e M o n t a n a D e p a r t m e n t o f S t a t e L a n d s f o r t h e gift  o f ponderosa pine  seed.  xiii  A MIS  xiv  PAPAS  1  INTRODUCTION  The mechanisms o f c y t o k i n i n a c t i o n d u r i n g growth and organ d i f f e r e n t i a t i o n o f i n t a c t p l a n t s a r e not w e l l understood  (Tanimoto and Harada, 1982) b u t i n t h e l a s t 40  y e a r s t i s s u e c u l t u r e t e c h n i q u e s have emerged as a t o o l t o study growth r e g u l a t o r e f f e c t s .  I n o r d e r t o study t h e  r e g u l a t i o n o f o r g a n i z e d development i n t i s s u e c u l t u r e , a r e l i a b l e t i s s u e c u l t u r e system i s r e q u i r e d (Thorpe, C y t o k i n i n s a r e r e q u i r e d t o induce bud and shoot in conifers i n vitro.  L i t t l e i s understood  1982).  formation  about t h e  b i o l o g i c a l e f f e c t s o f t h e s e compounds upon t h e i r t a r g e t cells.  However, many a u t h o r s have noted an i n t e r a c t i o n  w i t h a b i n d i n g r e c e p t o r (Napier and V e n i s , 1990) and changes i n genomic e x p r e s s i o n Argyroudi-Akoyunoglou,  (e.g. Akoyunoglou and  1985; Chen and L e i s n e r , 1985;  F l o r e s and Tobin, 1987, 1988) . H i s t o l o g i c a l , c y t o l o g i c a l and b i o c h e m i c a l approaches have been used t o e l u c i d a t e some m e t a b o l i c events differentiation i n cultured conifer tissues.  during  Only i n  P i n u s r a d i a t a D. Don has t h e e x p e r i m e n t a l approach been f o c u s e d towards a s y s t e m a t i c s t r u c t u r a l , b i o c h e m i c a l and p h y s i o l o g i c a l study o f s e v e r a l a s p e c t s o f d i f f e r e n t i a t i o n (e.g. Kumar e t a l . ,  1988, 1987; V i l l a l o b o s e t a l . , 1985,  2  1984; P a t e l and Thorpe, A i t k e n et a l . ,  1984a; Douglas e t a l . ,  1981; Yeung e t a l . ,  1981).  1982;  They showed  t h a t t h e i n i t i a t i o n of o r g a n i z e d development i n v i t r o i n c l u d e d changes at t h e h i s t o l o g i c a l and u l t r a s t r u c t u r a l level.  However, no p a r t i c u l a r emphasis has been d i r e c t e d  towards t h e development of t h e p l a s t i d .  L i t t l e work,  o t h e r t h a n t h a t of Thorpe and coworkers, has been done t o r e l a t e changes i n metabolism t o t h e a c q u i s i t i o n o f developmental d e t e r m i n a t i o n or t h e l o s s o f competence t o form shoots i n c o n i f e r t i s s u e  culture.  R e p o r t s of t h e e f f e c t s of growth r e g u l a t o r s on t h e development o f t h e p h o t o s y n t h e t i c apparatus are fragmentary and c o n t r a d i c t o r y when comparing responses o f angiosperm coworkers  and gymnosperm s p e c i e s .  Zimmermann and  (1987) r e p o r t e d t h a t t r e a t m e n t of Cucumis  s a t i v u s L. c o t y l e d o n s w i t h b e n z y l a d e n i n e  (BA) i n c r e a s e d  the c o n t e n t and a c t i v i t y of RUBP whereas S t a b e l and coworkers  (1991) r e p o r t e d a s u p p r e s s e d a c c u m u l a t i o n of t h e  same enzyme i n P i c e a a b i e s (L.) K a r s t embryos by  BA  treatment. C h l o r o p h y l l a c c u m u l a t i o n due t o t h e a c t i o n of c y t o k i n i n s has been r e p o r t e d i n s e v e r a l (Mikulovich et a l . ,  1981)  angiosperms  i n c l u d i n g Raphanus s a t i v u s L.  ( L i c h t e n t h a l e r and Buschmann, 1978).  In c o n t r a s t ,  c y t o k i n i n t r e a t m e n t of P i n u s p i n a s t e r A i t . c o t y l e d o n s  3  i n h i b i t e d chlorophyll synthesis  (Tranvan et a l . , 1988).  These o b s e r v a t i o n s i n c o n i f e r s have not been r e l a t e d the m o r p h o l o g i c a l development of the p l a s t i d or  to  the  photosynthetic apparatus.  The  t h e s i s of the r e s e a r c h p r e s e n t e d i n t h i s  document i s t h a t a study of the e f f e c t s of exogenous c y t o k i n i n on a d v e n t i t i o u s  shoot development i n  excised  c o t y l e d o n s of P i n u s ponderosa Dougl. c u l t u r e d i n v i t r o w i l l contribute  t o the u n d e r s t a n d i n g of mechanisms of  c y t o k i n i n a c t i o n as w e l l as the  improvement of  our  u n d e r s t a n d i n g of c o n i f e r m i c r o p r o p a g a t i o n . The  r e l a t i o n s h i p s among the  e v e n t s , s u b c e l l u l a r changes and  induction  of organogenic  the development of  p h o t o s y n t h e t i c apparatus i n c y t o k i n i n t r e a t e d t i s s u e s have not been e x t e n s i v e l y  the  conifer  investigated.  I  addressed t h e s e i s s u e s by i n v e s t i g a t i n g the e f f e c t s of cytokinins benzyladenine  (BA)  and  the  2 - i s o p e n t e n y l adenine  ( 2 i P ) , on p l a s t i d u l t r a s t r u c t u r e d u r i n g i n v i t r o organogenesis.  The  u l t r a s t r u c t u r a l o b s e r v a t i o n s of  p l a s t i d development were r e l a t e d at the b i o c h e m i c a l to selected photosynthetic proteins The  photosynthetic proteins  have p o t e n t i a l use the t i m i n g  and  and  to  level  chlorophyll.  c h l o r o p h y l l appeared t o  as markers of c y t o k i n i n i n d u c t i o n  of organogenic e v e n t s .  for  4  LITERATURE REVIEW  1. CONTROL OF ORGANIZED DEVELOPMENT IN TISSUE CULTURE Knowledge o f c o n i f e r b i o l o g y and f o r e s t b i o t e c h n o l o g y has i n c r e a s e d d u r i n g t h e l a s t few y e a r s t h r o u g h t h e use o f t i s s u e c u l t u r e t e c h n i q u e s .  Tissue  c u l t u r e has been proposed as a method w i t h a p p l i c a t i o n i n f o r e s t t r e e improvement.  C l o n a l p r o p a g a t i o n i n v i t r o can  occur v i a somatic embryogenesis o r organogenesis 1982).  (Thorpe,  The common procedure used f o r c l o n a l p r o p a g a t i o n  i n many c o n i f e r s p e c i e s remains t h e i n d u c t i o n o f a d v e n t i t i o u s shoots  (e.g. Von A r n o l d and E r i k s s o n , 1981;  F l i n n et a l . ,  1986; V i l l a l o b o s e t a l . ,  organogenesis  o c c u r s when u n i p o l a r bud p r i m o r d i a from  t i s s u e explants are i n i t i a t e d .  1984).  Shoot  These develop i n t o  shoots  but must be i n d u c e d t o form r o o t s (Thorpe and P a t e l , 1984).  The p r o c e s s o f somatic embryogenesis r e q u i r e s t h e  presence o f b o t h a u x i n s and c y t o k i n i n s b u t t h e i n d u c t i o n of shoot organogenesis cytokinins only.  o c c u r s i n t h e presence o f  T h i s need f o r a s i n g l e exogenous  r e g u l a t o r p e r m i t s t h e study o f a c y t o k i n i n  growth  induction  p r o c e s s w i t h o u t i n t e r a c t i o n w i t h o t h e r growth  regulators.  The organogenic p r o c e s s can be d i v i d e d i n t o t h r e e p h y s i o l o g i c a l s t a g e s : 1. t h e a b i l i t y o f a c e l l o r t i s s u e  5  to  respond t o an i n d u c e r ;  2. t h e p r o c e s s of i n d u c t i o n p e r  se which o c c u r s under t h e i n f l u e n c e of exogenous growth regulators;  3. t h e m o r p h o l o g i c a l d i f f e r e n t i a t i o n and  growth of a c e l l or group of c e l l s Warnick, 1987, 1988).  ( C h r i s t i a n s o n and  A c e l l i s i n a ' p e r m i s s i v e ' or  ' t a r g e t ' c o n d i t i o n when i t can r e c o g n i z e s i g n a l s t h a t initiate differentiation  (Osborne, 1984).  Other t h a n t h e  r e a c t i o n t o s p e c i f i c s i g n a l s , t h e r e are a t l e a s t r e q u i r e m e n t s f o r de novo o r g a n i z e d development: d i f f e r e n t i a t i o n , and c e l l  two cell  interaction.  The i n i t i a t i o n of o r g a n i z e d development morphogenic phenomenon (Thorpe, 1982). i n t r i n s i c factors play a role.  i s a complex  Both e x t r i n s i c  and  O r g a n i z e d development i n  v i t r o can be r e g u l a t e d by m a n i p u l a t i o n of t h e c u l t u r e medium c o m p o s i t i o n , t h e c u l t u r e environment, and by s e l e c t i o n o f an e x p l a n t t h a t i s i n an a p p r o p r i a t e r e s p o n s i v e s t a t e (Thorpe, 1980; Thorpe and B i o n d i , 1981) . T h i s l a s t f a c t o r has a s i g n i f i c a n t b e a r i n g on shoot productivity  ( A i t k e n et a l . , 1981).  F o r example, t h e  response o f t h e embryos cannot be equated t o the response observed i n e x c i s e d e x p l a n t s such as c o t y l e d o n s . of r e c e n t major advances, t h e most s u i t a b l e  In s p i t e  juvenile  e x p l a n t i n c o n i f e r s must s t i l l be d e t e r m i n e d e m p i r i c a l l y , as t h e r e are no p r e d i c t i v e methods f o r e x p l a n t (Thorpe and P a t e l ,  1986).  selection  6  In t h e angiosperm C i c e r a r i e t i n u m L. (Pino e t a l . , 1991)  embryos and e x c i s e d c o t y l e d o n s were not e q u i v a l e n t  when t r e a t e d under s i m i l a r c o n d i t i o n s : t h e absence o f t h e embryonic a x i s markedly reduced t h e d e g r a d a t i o n o f proteins.  In g e n e r a l  growth r e g u l a t o r s a r e s y n t h e s i z e d  w i t h i n t h e embryonic a x i s and t h e r e a f t e r pass t o t h e s t o r a g e t i s s u e (Ashton, 1976) .  Wounding may i n c r e a s e t h e  endogenous l e v e l o f a c t i v e c y t o k i n i n s  (Crane and Ross,  1986) .  Manipulation  o f t h e c u l t u r e c o n d i t i o n s may r e s u l t i n  n o r m a l l y q u i e s c e n t c e l l s which become o r g a n o g e n i c , and i t i s assumed t h a t t h i s i n v o l v e s s e l e c t i v e gene a c t i v a t i o n . Concentration  and d i f f u s i o n o f m a t e r i a l s by o r from t h e  medium i n t o t h e t i s s u e have been i m p l i c a t e d i n d e t e r m i n i n g the l o c a t i o n o f p r i m o r d i a  initiation  However, m e c h a n i s t i c e x p l a n a t i o n s  (Thorpe,  1982).  are speculative.  D u r i n g i n v i t r o o r g a n o g e n e s i s , c e l l s have become competent t o e x p r e s s t h e i r i n t r i n s i c c a p a c i t y f o r organized  development.  .This c a p a c i t y u l t i m a t e l y i s a  r e f l e c t i o n o f s e l e c t i v e gene a c t i v i t y t h a t m a n i f e s t s i t s e l f through biochemical,  b i o p h y s i c a l , and s t r u c t u r a l  changes i n t h e c u l t u r e d t i s s u e s which can be h i s t o l o g i c a l l y and u l t r a s t r u c t u r a l l y Thorpe and B i o n d i , 1981) .  Tissue  described  (Thorpe, 1980,  1982;  response t o e x t e r n a l l y  added growth r e g u l a t o r s i s u s u a l l y c o m p l i c a t e d by t h e  7  unknown c o n t e n t and p h y s i o l o g i c a l a c t i v i t y o f endogenous growth r e g u l a t o r s  ( J e l i c and Bogdanovic, 1990).  r o l e s of c y t o k i n i n s w i l l  The e x a c t  o n l y become c l e a r when t h e i r  m e t a b o l i c pathway, t h e i r a c t i o n s i t e ( s ) and t h e m o l e c u l a r mechanisms i n v o l v e d i n t h e i r a c t i v i t y a r e c h a r a c t e r i z e d (McGaw and Horgan,  1985) .  Even w i t h o u t knowledge o f  p r e c i s e endogenous l e v e l s o f p l a n t growth r e g u l a t o r s , c o n t r o l o f development  can be a c h i e v e d by exogenous  a p p l i c a t i o n s which r e g u l a t e macromolecular  synthesis,  c a r b o h y d r a t e metabolism, n u t r i e n t uptake k i n e t i c s , and c y t o l o g i c a l responses t h a t can be c o r r e l a t e d w i t h s p e c i f i c developmental changes  (Bornman, 1985).  2. HISTOLOGICAL AND ULTRASTRUCTURAL  STUDIES OF ORGANIZED  DEVELOPMENT The anatomy and h i s t o l o g y o f t h e d e v e l o p m e n t a l sequence from seed e x p l a n t s t o buds and s h o o t s i n v i t r o has been d e s c r i b e d f o r s e v e r a l c o n i f e r s .  Among t h e  s p e c i e s t h a t have been d e s c r i b e d h i s t o l o g i c a l l y a r e P i n u s radiata  (Yeung e t a l . ,  1981; V i l l a l o b o s e t a l . ,  P i n u s s t r o b u s L. ( F l i n n , 1987; F l i n n e t a l . , pinaster  (Tranvan e t a l . ,  Loud, P i n u s r i g i d a M i l l . ,  1985),  1988), P i n u s  1988), P i n u s c o n t o r t a Dougl. ex Picea engelmannii Parry  and P a t e l , 1986), P i n u s e l d a r i c a  (Wagley e t a l . ,  (Thorpe 1987),  P i c e a a b i e s (Von A r n o l d and Gronroos, 1986), P i c e a mariana  8  B.S.P. and P i c e a g l a u c a 1986).  (Moench) Voss (Rumary e t a l . ,  In a l l these species the formation of adventitous  shoots i n v o l v e s t h e development o f m e r i s t e m o i d s . meristemoids  The  a r e formed by s p e c i f i c p l a n e s o f c e l l  d i v i s i o n which l e a d s t o t h e f o r m a t i o n o f s m a l l , u s u a l l y i s o d i a m e t r i c c e l l s w i t h prominent n u c l e i . s t a i n i n g cytoplasm,  and m i c r o v a c u o l a t i o n a r e a l s o  c h a r a c t e r i s t i c features of meristemoid 1980).  Densely  cells  (Thorpe,  I n a l l s p e c i e s examined, t h e meristemoids  m e r i s t e m a t i c r e g i o n s o r i g i n a t e d from e i t h e r and/or immediately The meristemoids  subepidermal  developed  and  epidermal  l a y e r s of the explant.  i n t o bud p r i m o r d i a and f i n a l l y  t o a d v e n t i t i o u s shoots w i t h a p i c a l domes and needle primordia.  The developmental  sequence o c c u r r e d i n t h e  absence o f c a l l u s f o r m a t i o n on t h e c y t o k i n i n (CK)c o n t a i n i n g medium (Thorpe and P a t e l , 1986).  The i n d u c t i o n  of a d v e n t i t i o u s shoots o c c u r r e d a t d i f f e r e n t s i t e s i n v a r i o u s e x p l a n t s , o r a t d i f f e r e n t t i m e s and s i t e s w i t h i n the same e x p l a n t .  The reasons b e h i n d t h e d i f f e r e n t i a l  b e h a v i o u r o f c e l l s i n c o n t a c t w i t h t h e medium i n t h e embryonic and c o t y l e d o n a r y e x p l a n t a r e not c l e a r and P a t e l ,  (Thorpe  1986).  C e l l s i n shoot-forming the u l t r a s t r u c t u r a l l e v e l .  r e g i o n s have been examined a t Few u l t r a s t r u c t u r a l s t u d i e s  have d e a l t w i t h c o n i f e r s c u l t u r e d i n v i t r o .  Plastid  9  m o r p h o l o g y has (Fowke, 1 9 8 6 )  not  b e e n e x a m i n e d by  i n any  e l e c t r o n microscopy  conifer during  organogenic  I n a n g i o s p e r m s , t h e most s t u d i e d g e n e r a where m o r p h o l o g y has  Zea,  s t u d i e s have been f o c u s e d  species occur  T r i t i c u m and  on  greening  where t h e u l t r a s t r u c t u r a l  are  similar.  i n the  plastid  b e e n e x a m i n e d a r e Hordeum, P h a s e o l u S /  Avena, Cucumis, V i c i a ,  seedlings,  events.  The  Pisum.  of  These  etiolated  c h a n g e s i n most  main d i f f e r e n c e s between  r a t e of p l a s t i d morphogenesis.  Etioplasts  containing h i g h l y c r y s t a l l i n e prolamellar bodies unperforated  stromal  phototransformed. initials stacks  and  few  l a m e l l a e are r a p i d l y  The  lamellae develop i n t o  which subsequently  (Wellburn,  species  1982).  granal  form r e c o g n i z a b l e R e b e i z and  Rebeiz  granal  (1985)  d e m o n s t r a t e d t h a t p r o l a m e l l a r body f o r m a t i o n  is  have  an  i n t e g r a l p a r t of c h l o r o p l a s t d i f f e r e n t i a t i o n under n a t u r a l photoperiodic  growth c o n d i t i o n s .  angiosperms a l r e a d y the  enzymes and  only  from e t i o p l a s t s but  f r o m p r o p l a s t i d s (Whatley, 1974) .  and  of  considered  nuclear  of  c o n s t i t u e n t s of the c h l o r o p l a s t .  C h l o r o p l a s t s develop not  of p l a s t i d  etioplasts  c o n t a i n s u b s t a n t i a l amounts o f most  other  p a t h w a y s c a n n o t be  The  T h e s e two  equivalent.  gene e x p r e s s i o n  t h y l a k o i d membrane b i o g e n e s i s  may  also  developmental  The  regulation  change  during  (Eskins et a l . , 1989).  10  3. CYTOKININS Cytokinins,  compounds f i r s t r e c o g n i z e d by t h e i r  a b i l i t y t o induce c e l l d i v i s i o n i n c e r t a i n t i s s u e c u l t u r e s , a r e now known t o induce a d i v e r s i t y o f responses in plants.  The v a r i o u s c y t o k i n i n s may be a c t i v e ,  t r a n s l o c a t i o n o r s t o r a g e forms, d e t o x i f i c a t i o n p r o d u c t s , deactivation  products  (formed t o reduce c y t o k i n i n  l e v e l s ) or i n a c t i v a t i o n products their utilization)  activity  (formed as a r e s u l t o f  (Letham and P a l n i , 1983).  T r a n s i e n t changes i n c y t o k i n i n l e v e l s o c c u r d u r i n g b o t h t h e -breaking o f dormancy and t h e p r o c e s s o f g e r m i n a t i o n i n seeds (e.g. J e l i c and Bogdanovic, 1990; P i n o e t a l . , 1991).  I t i s d i f f i c u l t to pinpoint  f u n c t i o n o f t h e s e growth r e g u l a t o r s process  (Van Staden, 1983) .  the exact  i n the germination  T h i s problem a l s o  exists  d u r i n g t h e organogenic p r o c e s s i n c o n i f e r s . Cytokinin  s t r u c t u r e has not been r e l a t e d t o t h e  molecular action(s) 1979).  of t h e s e growth r e g u l a t o r s  (Parthier,  They a r e d e r i v a t i v e s o f t h e n u c l e i c a c i d p u r i n e  adenine and t h e i r a c t i o n may be connected w i t h RNA and protein synthesis  (Leshem, 1973).  At the molecular l e v e l  c y t o k i n i n e f f e c t s on d i f f e r e n t s p e c i e s o f angiosperms have been r e p o r t e d .  F o r example, i n Lemna g i b b a L. p l a n t s  grown i n d a r k n e s s , c y t o k i n i n s  regulate  the expression of  RNA a t a p o s t - t r a n s c r i p t i o n a l l e v e l , p o s s i b l y by a f f e c t i n g  11  the s t a b i l i t y o f RNA ( F l o r e s and Tobin, 1986), o r i n h i b i t i n g t h e d e g r a d a t i o n o f s p e c i f i c mRNAs (Tobin and Turkaly,  1982).  Petunia hybrida M i t c h e l l cultures  respond t o c y t o k i n i n t r e a t m e n t by a c c u m u l a t i n g RNA (Funckes-Shippy & L e v i n e , 1985), which r e s u l t s from t h e t r a n s c r i p t i o n o f n u c l e a r - e n c o d e d genes.  These genes code  f o r p o l y p e p t i d e s t h a t f u n c t i o n w i t h i n t h e c h l o r o p l a s t and t h e i r i n d u c t i o n l e v e l s a r e f u r t h e r modulated by l i g h t .  4. THE ROLE OF CYTOKININS IN PLASTID DEVELOPMENT 4.1.  NORMAL PLASTID DEVELOPMENT In o r d e r t o d e s c r i b e  t h e e f f e c t o f c y t o k i n i n s on  p l a s t i d development i n t i s s u e c u l t u r e i t i s n e c e s s a r y t o describe  some a s p e c t s o f t h e f o r m a t i o n o f c h l o r o p l a s t s , a  complex d e v e l o p m e n t a l p r o c e s s r e s u l t i n g from an i n t e r p l a y between t h e n u c l e a r and p l a s t i d genomes. proteins  are synthesized  inside the developing  many o t h e r s a r e s y n t h e s i z e d 1984).  Some c h l o r o p l a s t organelle,  i n t h e c y t o p l a s m ( E l l i s , 1981;  F o r example, t h e l i g h t - h a r v e s t i n g c h l o r o p h y l l a/b  complex i s a u n i t assembled from pigment components synthesized  w i t h i n t h e c h l o r o p l a s t and p o l y p e p t i d e  components encoded by n u c l e a r DNA and s y n t h e s i z e d cytoplasm  (Bennett e t a l . , 1984).  i n the  The e x p r e s s i o n o f genes  c o d i n g p l a s t i d p r o t e i n s may be r e g u l a t e d  a t many l e v e l s ,  from t r a n s c r i p t i o n t o t h e assembly and i n t e g r a t i o n o f  12  t h e i r p r o t e i n products  (Buetow, 1985).  In o r d e r t o  u n d e r s t a n d c h l o r o p l a s t development i t i s i m p o r t a n t t o study b o t h n u c l e a r and p l a s t i d coded p r o t e i n s . D i f f e r e n t i a t i o n of p l a s t i d s involves synthesis of c h l o r o p h y l l s and t h e i r p r e c u r s o r s as w e l l as u l t r a s t r u c t u r a l changes l i n k e d t o p r o t e i n and l i p i d content.  Carotenes and o t h e r substances a r e a l s o  n e c e s s a r y f o r t h e f u n c t i o n o f t h e mature c h l o r o p l a s t (Sundqvist e t a l . , 1980) .  Growth r e g u l a t o r s may e x e r t two  t y p e s o f e f f e c t s on t h e g r e e n i n g o f h i g h e r p l a n t s , i n d i r e c t l y v i a t h e p l a s t i d membranes o r d i r e c t l y on some of t h e c h l o r o p h y l l b i o s y n t h e t i c r e a c t i o n s  ( D a n i e l l and  R e b e i z , 1985) . Chloroplast  development i n v a s c u l a r p l a n t s can a l s o  be i n f l u e n c e d by n u t r i e n t s t a t u s , i l l u m i n a t i o n ( M u l l e t , 1988)  and water s t r e s s  (Sundqvist e t a l . , 1980).  Very  l i t t l e i s known about t h e fundamental endogenous r e g u l a t o r s o f p l a s t i d d i f f e r e n t i a t i o n i n angiosperms.  4.2. BIOCHEMICAL AND MORPHOLOGICAL EFFECTS OF CYTOKININS ON THE PHOTOSYNTHETIC The  APPARATUS  hormonal r e g u l a t i o n o f c h l o r o p l a s t development  i s a l s o complex.  A t l e a s t f o u r exogenously  supplied  c l a s s e s o f growth r e g u l a t o r s have a d i r e c t i n f l u e n c e on the c h l o r o p l a s t .  These i n c l u d e c y t o k i n i n s , g i b b e r e l l i n s ,  13  auxins and ABA.  Some of these have been d e t e c t e d  the c h l o r o p l a s t (Sundqvist et. a l . , 1980).  inside  C y t o k i n i n s seem  to c o n t r o l the o p e r a t i o n of s p e c i f i c genes i n both c h l o r o p l a s t and n u c l e a r genomes i n p l a n t c e l l s et a l . , 1981) . L.  (Mikulovich  Some s p e c i e s , f o r example Secale c e r e a l e  (Feierabend and De Boer, 1978), seem t o r e q u i r e more  c y t o k i n i n f o r p l a s t i d r a t h e r than f o r cytoplasmic synthesis of c h l o r o p l a s t - s p e c i f i c proteins.  F o r example,  RUBP, the l a r g e subunit of which i s formed i n the p l a s t i d , showed a g r e a t e r response  t o changed c y t o k i n i n l e v e l s  d i d c h l o r o p l a s t enzymes o f cytoplasmic o r i g i n .  than  The  formation of the small subunit of RUBP seemed t o be l e s s s e n s i t i v e t o a c y t o k i n i n d e f i c i e n c y than was the formation of the complete enzyme D i f f e r e n c e s observed  (Feierabend and De Boer, 1978).  i n p l a s t i d formation i n d i f f e r e n t  s p e c i e s c o u l d be due t o d i f f e r e n c e s i n the g e n e t i c b a s i s of the p l a n t , the p h y s i c a l c o n d i t i o n s , and the age of the plant.  A l l these c h a r a c t e r i s t i c s should be taken  account  f o r a l l s t u d i e s of c h l o r o p l a s t formation  into (Sestak,  1985). The  requirement  of c y t o k i n i n s f o r c h l o r o p l a s t  d i f f e r e n t i a t i o n i s one of t h e i r most s t r i k i n g effects  (Peaud-Lenoel and Axelos,  1981).  subcellular  They may c o n t r o l  s p e c i f i c events, e i t h e r i n s i d e the o r g a n e l l e s or i n the cytoplasmic compartment  (Lescure and Seyer,  1981).  They  14  i n c r e a s e the a c t i v i t y of enzymes by de novo i n d u c t i o n ( P a r t h i e r , 1979;  F e i e r a b e n d and De Boer, 1978) .  e f f e c t s have been r e p o r t e d  Cytokinin  on gene a c t i v a t i o n ,  t r a n s c r i p t i o n , t r a n s l a t i o n and p o s t - t r a n s l a t i o n p r o c e s s e s (Akoyunoglou and A r g y r o u d i - A k o y u n o g l o u , 1985;  P a r t h i e r et  al.,  enhance,  1985;  F l o r e s and  Tobin, 1988).  i n d u c e or suppress the e x p r e s s i o n (Chen and L e i s n e r ,  1985).  They can  of c e r t a i n p r o t e i n s  In t o b a c c o c e l l  suspension  c u l t u r e s under c o n t i n u o u s l i g h t , the b i o s y n t h e s i s  of some  nuclear  be  DNA-encoded p l a s t i d p r o t e i n s was  r e g u l a t e d by c y t o k i n i n s  found t o  ( T e y s s e n d i e r de l a Serve et a l . ,  1985a). I t has been r e p o r t e d formation  that cytokinins stimulate  i n the c o t y l e d o n  c h l o r o p l a s t s of s e v e r a l  of angiosperms, i n c l u d i n g Cucumis s a t i v u s 1974), C u c u r b i t a  pepo L.  species  (Harvey et a l . ,  ( M i k u l o v i c h e t a l . , 1981;  et a l . , 1984), N i c o t i a n a tabacum L.  grana  Lerbs  ( T e y s s e n d i e r de l a  Serve et a l . , 1985b), and C i t r u l l u s v u l g a r i s Schrad ( B r a c a l e et a l . , 1988). In dark grown s u s p e n s i o n c u l t u r e s of P e t u n i a  hybrida,  a c o m b i n a t i o n of l i g h t and BA  s t i m u l a t e d g r e e n i n g and the f o r m a t i o n ( C o l i j n et a l _ . , 1982) .  The  of  also  thylakoids  l a m e l l a r system development of  N i c o t i a n a tabacum c e l l s u s p e n s i o n c u l t u r e s was reduced i n the absence of c y t o k i n i n s .  The  strongly  differentiation  of mature c h l o r o p l a s t s under c o n t i n u o u s l i g h t  occurred  15  o n l y i n c y t o k i n i n supplemented medium (Lescure and Seyer, In c y t o k i n i n - d e p l e t e d leaves of Secale cereale t h e  1981).  p l a s t i d s i z e was s i g n i f i c a n t l y s m a l l e r t h a n i n k i n e t i n t r e a t e d leaves  (Feierabend and De Boer, 1978) .  In a d d i t i o n t o the growth r e g u l a t o r e f f e c t s , t i s s u e c u l t u r e systems are i n f l u e n c e d by the n u t r i t i o n a l medium. The  c u l t u r e d c e l l s cannot be c o n s i d e r e d t o be  developmentally species  e q u i v a l e n t t o o t h e r t i s s u e s o f the same  ( P a r t h i e r , 1979) because s p e c i f i c c y t o k i n i n  a c t i o n s are known t o d i f f e r from organ t o organ (Haru e t al.,  1982).  S i m i l a r e f f e c t s on i s o l a t e d o r g a n e l l e s and  i n t a c t c e l l s cannot be c o r r e l a t e d t o c u l t u r e d c e l l responses.  For example, BA enhanced the i n o r g a n e l l o  p r o t e i n s y n t h e s i s a c t i v i t y o f e t i o p l a s t s o f Cucumis s a t i v u s but had no e f f e c t on i s o l a t e d p l a s t i d s Suzuki,  (Ohya and  1990).  There are numerous examples o f the e f f e c t o f c y t o k i n i n s on mRNA and gene a c t i v i t y i n angiosperms. C u c u r b i t a pepo c o t y l e d o n s  In  ( P a r t h i e r e t a l , 1985) b o t h BA  and l i g h t r e g u l a t e d RUBP gene e x p r e s s i o n . c y t o k i n i n s s t i m u l a t e d the a c c u m u l a t i o n  I n Lemna g i b b a  o f mRNA c o d i n g f o r  the s m a l l s u b u n i t o f RUBP and LHCII i n the dark ( F l o r e s and Tobin,  1987, 1988).  I n N i c o t i a n a tabacum c e l l  suspension  c u l t u r e s LHCP-mRNA t r a n s l a t i n g a c t i v i t y was  found t o be s t i m u l a t e d by k i n e t i n  (Teyssendier  de l a Serve  16  et a l . , 1985a).  Cytokinin-supplemented tobacco  cells  s y n t h e s i z e d mRNA encoding t h e s m a l l s u b u n i t o f RUBP much e a r l i e r t h a n i n growth r e g u l a t o r - s t a r v e d c e l l s . amounts o f RUBP c a r b o x y l a s e cytokinin-supplemented  Larger  were produced i n t h e  c u l t u r e (Axelos, e t a l . , 1987).  I t has been shown t h a t c y t o k i n i n s not o n l y  affect  angiosperm p l a s t i d s t r u c t u r e b u t a l s o i n c r e a s e c h l o r o p h y l l accumulation sativus  ( M i k u l o v i c h e t a l . , 1981).  I n Raphanus  ( L i c h t e n t h a l e r and Buschmann, 1978) k i n e t i n not  o n l y enhanced c h l o r o p h y l l a c c u m u l a t i o n b u t a l s o had a marked e f f e c t on p r o t o c h l o r o p h y l l ( i d e ) f o r m a t i o n i n etiolated  seedlings.  These growth r e g u l a t o r s a l s o enhanced a c t i v i t i e s o f photosynthetic  enzymes i n s e v e r a l s p e c i e s o f angiosperms.  In Cucumis s a t i v u s c o t y l e d o n s , a c t i v i t y and content 1974)  and l i g h t  BA i n c r e a s e d  specific  o f RUBP i n b o t h dark (Harvey e t a l . ,  (Zimmermann e t a l . , 1987).  I n t h e same  s p e c i e s , BA i n darkness s t i m u l a t e d t h e s y n t h e s i s o f a 39 kD and s e v e r a l membrane p o l y p e p t i d e s (Ohya and S u z u k i , 1990).  lower t h a n 34 kD  In excised Cucurbita  cotyledons,  c y t o k i n i n s s t i m u l a t e d de novo RUBP oxygenase s y n t h e s i s (Lerbs e t a l . , 1984), i n c r e a s e d i t s content  and s t i m u l a t e d  a c t i v i t y o f t h e enzyme ( P a r t h i e r e t a l . , 1985).  In  e t i o l a t e d Secale c e r e a l e leaves, the a c t i v i t y of s e v e r a l c h l o r o p l a s t enzymes was i n c r e a s e d a f t e r k i n e t i n t r e a t m e n t  17  (Feierabend been due  and  De Boer, 1978).  T h i s i n c r e a s e may  have  to a number of f a c t o r s i n c l u d i n g s t i m u l a t i o n of  synthesis,  reduction  i n the r a t e of t h e i r degradation,  or  by a c t i v a t i o n (Harvey et a l . , 1974). Cytokinins polypeptides  stimulated  the s y n t h e s i s  of some  i n dark grown c u l t u r e s , as w e l l as emulating  or enhancing many of the l i g h t - t r i g g e r e d processes et a l . , 1986;  Ohya and  Suzuki,  C i t r u l l u s v u l g a r i s cotyledons  1990).  (Ohya  However, i n  (Marziani-Longo et. aJ.,  1990), c y t o k i n i n s c o u l d only p a r t i a l l y r e p l a c e the requirement f o r LHC  synthesis.  light  LHC-mRNA, as w e l l as  the  p r o t e i n , appeared i n the dark i n the presence of c y t o k i n i n s , but thylakoids.  apparently  were not  associated  with  I t i s l i k e l y that the f a i l u r e of LHC  i n t e g r a t e i n t o the t h y l a k o i d s v u l g a r i s cotyledons  was  due  i n excised  to  Citrullus  to the l a c k of c h l o r o p h y l l .  C h l o r o p h y l l i s known to s t a b i l i z e the a s s o c i a t i o n of these p r o t e i n s with the membranes  (Apel and Kloppstech,  L i g h t i n c r e a s e d the content and e x c i s e d cotyledons  a c t i v i t y of RUBP i n  of Cucumis s a t i v u s .  decreased with i n c r e a s i n g c y t o k i n i n S a t u r a t i o n of BA was  1980).  Its effectiveness  concentration.  reached at lower c o n c e n t r a t i o n s  in  the l i g h t than i n the darkness, i n d i c a t i n g an i n t e r a c t i o n between p h o t o c o n t r o l et a l . , 1987) .  and  cytokinin regulation  Cytokinins  (Zimmermann  and phytochrome d i d not  act  18  through the same mechanism  (Flores and Tobin, 1 9 8 6 ) .  The  combination o f l i g h t - s t i m u l a t e d mRNA s y n t h e s i s and i n h i b i t i o n o f mRNA degradation by c y t o k i n i n s  could  account  f o r the synergism o f t e n observed between l i g h t and cytokinin  (Tobin  The  and Turkaly, 1982) .  c l o s e c o r r e l a t i o n between the p h y s i o l o g i c a l  s t a t e o f the t i s s u e or c e l l and the e f f e c t o f c y t o k i n i n s on the greening process would support an i n d i r e c t mode o f a c t i o n on the s y n t h e s i s the  growth r e g u l a t o r  and accumulation o f c h l o r o p h y l l by  ( P a r t h i e r , 1979) .  5. INTERACTIVE EFFECTS OF CYTOKININS AND LIGHT C o n s i d e r a b l e progress i s being made i n understanding how l i g h t ,  an exogenous r e g u l a t o r y  f a c t o r , modulates  p l a s t i d d i f f e r e n t i a t i o n (Bennett e t a l . , 1 9 8 4 ) . mechanism o f t h e l i g h t - i n d u c e d plastids to chloroplasts synthesis but  synthesis  Parthier,  c o n v e r s i o n o f the e t i o l a t e d  requires  of lamellar proteins  The  not only de novo  and p h o t o s y n t h e t i c enzymes,  o f s e v e r a l types o f RNA (Wollgiehn and  1980) .  Cytokinins  are r e q u i r e d  f o r bud i n d u c t i o n  i n Pinus  r a d i a t a , but the a c t i o n o f both c y t o k i n i n and l i g h t are required 1988).  f o r subsequent primordium formation  (Thorpe,  L i g h t a f f e c t s organogenic events and i t a l s o  a predominant r o l e i n c h l o r o p l a s t d i f f e r e n t i a t i o n .  plays  19  The  t i m i n g and  gene e x p r e s s i o n  e x t e n t of c h l o r o p l a s t development  v a r y depending on the  s t r a t e g y of the organism and signals  ( M u l l e t , 1988).  i t s use  and  developmental of e n v i r o n m e n t a l  Light affects chloroplast  development by a c t i n g on v a r i o u s p h o t o r e c e p t o r s which produces responses t o v a r i o u s wavelengths of l i g h t .  The  b e s t c h a r a c t e r i z e d photomorphogenic system i s t h a t i n v o l v i n g phytochrome (Buetow, 1985). The  transformation  of p r o p l a s t i d s t o  chloroplasts  depends not o n l y on an exogenous c y t o k i n i n s u p p l y but on l i g h t  (Peaud-Lenoel and A x e l o s ,  cannot r e p l a c e  1981).  also  Cytokinins  l i g h t i n i n d u c i n g the f i n a l s t a g e s of  c h l o r o p l a s t development, pigment a c c u m u l a t i o n and differentiation  of t h y l a k o i d s  full  ( B r a c a l e et a l . , 1988).  However, l i g h t i s not e s s e n t i a l f o r a l l s t e p s i n the synthesis, transport chloroplast proteins. different levels  and  assembly of a m a j o r i t y  R a t h e r i t i s s t i m u l a t o r y at  (Ellis,  1981).  b i n d i n g p r o t e i n complex i s one  The  chlorophyll  of the few  illumination.  several a/b  chloroplast  components whose a c c u m u l a t i o n i n t h y l a k o i d s continuous  of  requires  20  MATERIALS AND METHODS  1.  TISSUE CULTURE  1.1.  PLANT MATERIAL P i n u s ponderosa seeds from western Montana were  s u p p l i e d by t h e Montana Department o f S t a t e Lands, F o r e s t r y D i v i s i o n , M i s s o u l a , MT, USA.  Seeds were i m b i b e d  i n r u n n i n g t a p water f o r 24 h and s t o r e d a t 4°C f o r t h r e e days..  They were s u r f a c e s t e r i l i z e d i n 20% commercial  bleach  ( a p p r o x i m a t e l y 1 % w/v NaOCl) f o r 15 min and r i n s e d  t h r e e t i m e s i n s t e r i l e d i s t i l l e d water.  Seed c o a t s were  removed, t h e embryos were s e p a r a t e d from t h e megagametophyte, and e x c i s e d c o t y l e d o n s were p l a c e d l o n g i t u d i n a l l y on t i s s u e c u l t u r e medium c o n t a i n e d i n p e t r i dishes. Embryos were g e r m i n a t e d i n v i t r o i n t e s t tubes c o n t a i n i n g growth r e g u l a t o r f r e e  (GRF) medium.  Cotyledons  were e x c i s e d from t h e s e embryos a t d i f f e r e n t s t a g e s o f growth and e i t h e r used d i r e c t l y f o r a n a l y s i s o r c u l t u r e d on c y t o k i n i n  ( C K ) - c o n t a i n i n g medium f o r 10 days, t h e n  t r a n s f e r r e d t o a GRF medium. An advantage o f t h e P. ponderosa system was t h a t each seed had between 7 and 12 c o t y l e d o n s . Thus,  21  c o t y l e d o n s f r o m t h e same s e e d c o u l d be u s e d t o s t u d y responses  by g e n e t i c a l l y u n i f o r m  The  tissue.  morphogenic c a p a c i t y o f t h e c o t y l e d o n s  was  measured a s : (i) T o t a l response  = number o f c o t y l e d o n s t h a t  responded,  by e i t h e r g r e e n i n g o r p r o d u c t i o n o f b u d s a n d s h o o t s , a s a percentage (ii)  of t o t a l  cotyledons  Organogenic response  formed buds and s h o o t s  cultured.  = number o f c o t y l e d o n s t h a t  as a p e r c e n t a g e  of t o t a l  cotyledons  cultured. (iii)  Mean b u d s a n d s h o o t s = t h e mean number o f b u d s a n d  shoots  formed p e r o r g a n o g e n i c a l l y responding  cotyledon.  T h e s e m e a s u r e m e n t s were made e i t h e r 35 o r 42 d a y s culture i n i t i a t i o n  of excised cotyledons.  after  A l l results  were b a s e d upon m e a s u r e m e n t s f r o m a t l e a s t 50 c o t y l e d o n s per c u l t u r e  treatment.  1.2. CULTURE METHODS The  f o u r c u l t u r e media used  throughout LP and  t h i s t h e s i s a s : DCR  (Gupta  (Von A r n o l d a n d E r i k s s o n , 1 9 8 1 ) , SH  ( T a b l e 1) c o r r e s p o n d s  ( P o r t l a n d , OR), (b) B a k e r  B a k e r a n d Adamson  and Durzan,  MCM  (Schenk a n d H i l d e b r a n d t , 1 9 7 2 ) .  each chemical Amachem  ( T a b l e 1) a r e i d e n t i f i e d 1985),  (Bornman, 1983) The l e t t e r  beside  to the supplier:  (a)  ( P h i l l i p s b u r g , N J ) , (c)  ( M o r r i s t o w n , N J ) , (d) BDH  (Toronto,  22  TABLE 1. C u l t u r e media c o m p o s i t i o n SH  MACRONUTRIENTS d KNO3 b MgS0 •7H 0 d NH H P0 g CaCl d NH N0 a KH P0 f CH N 0 d Ca(N0 ) '4H 0 b NH S0 f KC1 4  4  2500 400 300 150.9  2  2  4  2  4  2  4  -  3  4  2  3  4  2  4  MICRONUTRIENTS MnS0 'H 0 MnS0 •'4H 0 H3BO3 ZnSO/i •7H 0 KI  —  CoCl '6H D NiCl  c c b c e h h g c  IRON F e S 0 7H 0 Na EDTA ZnEDTA EDTA  d b b b  VITAMINS Thiamine HCl Nicotinic Acid P y r i d o x i n e HCl Myo-Inositol Glycine Pantothenate Folic Acid Biotin  h e h d d h h h  SUGARS D-Glucose D-Xylose L-Arabinose  b d d  1  4  2  4  2  2  CuS0 -5H 0 NaMo0 •2HoO 4  2  4  2  2  2  4  2  2  -  5 1 1 0.2 0.1 0.1  -  5 5 0.5 1000  —  —  e  e  135.9 1200 340  MCM  --  -  -  DCR  2000 250  340 370  --  -  6.42 400 170  270 150 500 400 150  10  --  1  -  —  15 20  AMINOACIDS h L-Glutamine d L-Alanine L-Cysteine HCl J h L-Arginine e L-Leucine L-Phenylalanine L-Tyrosine  LP 1900 370  --  -  2  (mg/L)  -  556 —  0.17  2.2 0.63  -1.5  0.0025 0 . 025 0.0025  3 0.25 0.025 0.25 0.025  —  —  14  15 20  -0.75  -  4.05 19  5 2 1 100 2  —  180 150 150  0.40 0.05 0.02 0.01 0.01 0.01 0.01  .  --  22.3  -  6.2 8.6 0.83 0.25 0 .25 0.025 0.025  27.8 37.3  -  -  1.7 0.6 1.2 90 2 0.5 1.1 0.12  1 0.5 0.5 200 2  --  —  -  -  —  —  -  -  --  -  --  ---  23  Ontario),  (e) Calbiochem (La J o l l a , CA), (f) F i s h e r  Lawn, NJ) (g) MCB  (Fair  ( C i n c i n n a t i , OH), (h) Sigma (St. L o u i s ,  MO).  Each medium c o n t a i n e d  (w/v)  D i f c o b a c t o - a g a r , and was a d j u s t e d t o a f i n a l pH o f  5.8 b e f o r e  3% (w/v) s u c r o s e and 0.8%  a u t o c l a v i n g a t 16 p s i (120°C) f o r 15 min.  Different concentrations  (0, 1, 5, 15 and 25 uM) and  d i f f e r e n t t i m e s o f exposure t o c y t o k i n i n s , e i t h e r benzyladenine  (BA) o r 2 - i s o p e n t e n y l  adenine (2iP) were  tested.  The t r a n s f e r d e t a i l s f o r each experiment a r e  provided  i n t h e legends o f t h e f i g u r e s and t a b l e s o f  results. In most experiments p a i r s o f c o t y l e d o n s  from t h e  same seed were t r e a t e d i d e n t i c a l l y so t h a t one o f each p a i r c o u l d be h a r v e s t e d  and s t o r e d f r o z e n a t -80°C w h i l e  i t s p a r t n e r was grown on t o ensure t h a t t h e developmental response was observed. p a i r was d i s c a r d e d ensuring  The e a r l y h a r v e s t e d  member o f t h e  i f i t s p a r t n e r d i d not d e v e l o p ,  t h e a n a l y s i s o f d e v e l o p m e n t a l l y competent  explants.  1.3.  CULTURE CONDITIONS A l l c u l t u r e s were m a i n t a i n e d i n a growth chamber a t  27 + 2°C w i t h 16 h p h o t o p e r i o d s p r o v i d e d by f l u o r e s c e n t lights  ( P h i l i p s c o o l w h i t e 34 W) w i t h an approximate —9  —1  photon f l u e n c e r a t e o f 80 umol'm ^•s . ±  24  2. HISTOLOGICAL AND ULTRASTRUCTURAL ANALYSIS 2.1. LIGHT MICROSCOPY Cotyledons  were h a r v e s t e d a f t e r 0, 3, 5, 10 and 21  days i n c u l t u r e and f i x e d i n 2.5 % (v/v) g l u t a r a l d e h y d e i n 0.1 M sodium c a c o d y l a t e b u f f e r (pH 7.3) f o r 3 h a t room temperature.  Specimens were p o s t f i x e d f o r 2 h a t room  t e m p e r a t u r e i n 1% (w/v) osmium t e t r o x i d e , dehydrated graduated  in a  e t h a n o l s e r i e s (30, 50, 70, 85, 95, 100 and  100%), t r a n s f e r r e d t o p r o p y l e n e S p u r r s -resin- (Spurr, 1969) . 1  o x i d e , and embedded i n  Sections  (0.5 um) were c u t  u s i n g g l a s s k n i v e s and s t a i n e d w i t h 0.05 % (w/v) t o l u i d i n e b l u e 0 i n 1 % (w/v) sodium t e t r a b o r a t e . S e c t i o n s a t day 0 were a l s o s t a i n e d f o r p r o t e i n w i t h 1% a n i l i n e b l u e b l a c k i n 7 % (v/v) aqueous a c e t i c a c i d 1968).  (Jensen and F i s h e r ,  S e c t i o n s were examined and photographed u s i n g a  L e i t z D i a l u x compound  microscope.  2.2. TRANSMISSION ELECTRON MICROSCOPY S e c t i o n s from c o t y l e d o n s c u l t u r e d i n t h e presence o r absence o f CKs f o r 0, 5 and 10 days were f i x e d and embedded as d e s c r i b e d f o r l i g h t microscopy.  Thin s e c t i o n s  were mounted on copper g r i d s , s t a i n e d w i t h 2% (w/v) aqueous u r a n y l a c e t a t e and Sato's  lead c i t r a t e  (Hayat,  25  1989), and examined and photographed u s i n g a Z e i s s EM 10A transmission  e l e c t r o n microscope o p e r a t i n g  a t 60 kV.  3. PROTEIN AND CHLOROPHYLL ANALYSES C o t y l e d o n s were h a r v e s t e d and s t o r e d a t -80°C p r i o r t o p r o t e i n and c h l o r o p h y l l  3.1.  analyses.  PROTEIN EXTRACTION A l l p r o t e i n e x t r a c t i o n s were performed on i c e . The  c o t y l e d o n s were weighed and homogenized i n a 0.2 mL m i c r o t i s s u e g r i n d e r w i t h 10-30 uL S D S • s o l u b i l i z i n g b u f f e r [62.5 mM T r i s - H C l 5%  (pH 6.8),  10 % (w/v) g l y c e r o l , 9 % (w/v) SDS,  (v/v) 2-JJ-mercaptoethanol and 0.125 % (w/v) bromophenol  b l u e ] p e r mg f r e s h weight t i s s u e .  Samples were  c e n t r i f u g e d a t 13, OOOxg f o r 10 min and d e n a t u r e d by heating  3.2.  a t 100°C f o r 7 min.  TOTAL PROTEIN ANALYSIS T o t a l p r o t e i n a n a l y s i s was performed u s i n g t h e  modified  method o f Ghosh e t a l . (1988), i n which a known  volume o f s u p e r n a t a n t p r o t e i n e x t r a c t was a p p l i e d t o Whatman f i l t e r paper, a i r d r i e d , s t a i n e d f o r 10 min i n 0.25 % (w/v) Coomassie b r i l l i a n t b l u e R, d e s t a i n e d methanol:10% a c e t i c a c i d , and d r i e d .  i n 40%  Each spot was c u t  out, t h e s t a i n e d p r o t e i n was e x t r a c t e d i n 2.0 mL 0.1%  26  (w/v) SDS f o r 1 h, and absorbance was d e t e r m i n e d a t 595 nm.  of the extract  solution  A l l p r o t e i n c o n c e n t r a t i o n s were  d e t e r m i n e d by r e f e r e n c e t o a c a l i b r a t i o n curve p r e p a r e d w i t h b o v i n e serum albumin s o l u t i o n s and were e x p r e s s e d as ug p r o t e i n / m g f r e s h w e i g h t .  3.3. SDS POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) Denatured p r o t e i n s  [40 ug p r o t e i n p e r l a n e ] were  s e p a r a t e d by SDS-PAGE i n 10-15 % p o l y a c r y l a m i d e g r a d i e n t g e l s c o n t a i n i n g 424 mM T r i s - H C l g e l c o n t a i n i n g 125 mM T r i s - H C l  (pH 9.8), w i t h 5% s t a c k i n g (pH 6.8) (Chua 1980).  g e l s were r u n i n a P r o t e a n I I s l a b g e l apparatus  The  (Bio-Rad,  Richmond, CA, USA) a t 25 mA f o r 1.5 h and a t 35 mA f o r another 4.5 h. The r u n n i n g b u f f e r c o n t a i n e d 15 g/L T r i s , 72 g/L g l y c i n e and 5 g/L SDS. F o l l o w i n g f i x a t i o n i n 40% methanol:10%  acetic acid,  the g e l s were s t a i n e d w i t h 0.25 % (w/v) Coomassie b r i l l i a n t b l u e R, d e s t a i n e d i n 40% methanol:10% a c i d , photographed  acetic  u s i n g an Ektachrome t u n g s t e n f i l m  (Kodak) and scanned a t 560 nm u s i n g a Beckman DU-64 s p e c t r o p h o t o m e t e r . Apparent m o l e c u l a r w e i g h t s were c a l c u l a t e d by r e f e r e n c e t o t h e s t a n d a r d p r o t e i n m i x t u r e , SDS-7 (Sigma Chemical Co., S t . L o u i s , MO, USA).  27  3.4.  IMMUNOBLOTTING P r o t e i n s were t r a n s f e r r e d from a n a l y t i c a l g e l s t o  nitrocellulose  (Bio-Rad) by e l e c t r o b l o t t i n g f o r 45 min a t  2.5 mA/cm i n a P o l y B l o t apparatus (American B i o n e t i c s , z  Hayward, CA, USA).  T r a n s f e r u n i t s were assembled i n t h e  f o l l o w i n g sequence (Kyhse-Andersen, 1984): a f i l t e r soaked i n anode b u f f e r # 1  paper  (0.3 M T r i s , 20 % methanol, pH  10.4); two f i l t e r papers soaked i n anode b u f f e r # 2 (25 mM T r i s , 20% methanol pH 10.4); t h e n i t r o c e l l u l o s e paper soaked i n d i s t i l l e d water; t h e g e l ; and t h r e e papers soaked i n cathode s o l u t i o n  filter  (25 mM T r i s , 40 mM 6-  aminohexanoic a c i d , 20 % methanol, pH 9.4). Immunoblotting was performed u s i n g a n t i g e n - a n t i b o d y complexes which were v i s u a l i z e d u s i n g g o a t - a n t i r a b b i t l i n k e d t o a l k a l i n e phosphatase  (White and Green 1987a).  The b l o t s were c o a t e d w i t h 3% (v/v) f i s h g e l a t i n i n phosphate-buffered s a l i n e  (1.37 M NaCl, 27 mM  KC1, 81 mM N a H P 0 , 15 mM K H P 0 ) f o r 1 h. 2  4  2  4  skin  B l o t s were  i n c u b a t e d i n a n t i s e r u m i n t h e same s o l u t i o n f o r 1 h, t h e n washed t w i c e i n phosphate b u f f e r e d s a l i n e p l u s 0.05 % (v/v)  Tween and once i n phosphate b u f f e r e d s a l i n e ,  wash t a k i n g 10 min.  each  T h i s was f o l l o w e d by i n c u b a t i o n f o r 1  h w i t h a l k a l i n e phosphatase c o n j u g a t e d t o goat a n t i - r a b b i t a n t i b o d y i n b l o c k e r and another two washes i n phosphateb u f f e r e d s a l i n e f o r 10 minutes each.  B l o t s were t h e n  28  washed i n 50 mM T r i s - H C l pH 8.0 f o l l o w e d by a d d i t i o n o f substrate  [0.1 % (w/v) d i s o d i u m n a p h t h o l AS-MX phosphate,  0.2 % (w/v) f a s t r e d TR s a l t  (both from Sigma Co., S t .  L o u i s , MO, USA)] d i s s o l v e d i n 50 mM T r i s - H C l pH 8.0. B l o t s were developed u n t i l t h e optimum c o l o r i n t e n s i t y was obtained  (0.25-1 hr) and d r i e d on paper t o w e l s  (White,  1987) . Six photosynthetic  polypeptides  using rabbit polyclonal antibodies These i n c l u d e d t h e <* and ft s u b u n i t s coupling  were i d e n t i f i e d  at d i f f e r e n t d i l u t i o n s . of the chloroplast  f a c t o r (CF1) (1:400) which appear as a s i n g l e  band; t h e l a r g e s u b u n i t carboxylase  of ribulose-1,5-bisphosphate  (LSU-RUBP) (1:400); t h e e x t r i n s i c 33kDa  p r o t e i n a s s o c i a t e d w i t h t h e o x y g e n - e v o l v i n g complex (33EP) (1:200) (Camm e t a l . , 1987); an a n t e n n a l component o f photosystem I I (CP29) (1:200) (White and Green, 1987b); a c h l o r o p h y l l - b i n d i n g p r o t e i n from t h e l i g h t - h a r v e s t i n g complex o f photosystem I I (LHCII);  and a second  c h l o r o p h y l l - b i n d i n g p r o t e i n from t h e l i g h t  harvesting  complex o f photosystem I (LHCI) (1:400) (White and Green, 1987a).  3.5.  CHLOROPHYLL DETERMINATION The  chlorophyll concentration  was d e t e r m i n e d by t h e  method r e p o r t e d by H i s c o x and I s r a e l s t a m  (1979).  29  C o t y l e d o n s were weighed (2 t o 3 mg), p l a c e d i n t o 100 uL DMSO, p r e v i o u s l y  warmed t o 65°C, f o r 10 min, i n c u b a t e d  f u r t h e r f o r 60 min a t 65°C, and t h e n removed from t h e solvent.  The c h l o r o p h y l l i n t h e r e s u l t i n g s o l u t i o n s was  d e t e r m i n e d by measuring absorbance  a t 663 and 645 nm  a g a i n s t a DMSO b l a n k . The t o t a l c h l o r o p h y l l , c h l o r o p h y l l a and c h l o r o p h y l l b were c a l c u l a t e d and r e l a t e d t o t i s s u e f r e s h (Arnon,  weight  1949).  i) Total chlorophyll = [20.2 D  concentration  + 8.02 D  6 4 5  6 6 3  ] X V/1000 X w i n ug/mg f r e s h  weight i i ) Chlorophyll a = [12.7 D  6 6 3  - 2.69 D  6 4 5  ] X V/1000 X w i n ug/mg f r e s h  weight iii)  Chlorophyll b = [22.9 D  6 4 5  - 4.68 D  g 6 3  ] X V/1000 X W i n ug/mg f r e s h  weight  where  D = absorbance  a t t h e wavelength  stated  V = t o t a l volume o f t h e c h l o r o p h y l l s o l u t i o n W = weight o f t h e f r e s h t i s s u e e x t r a c t e d  30  4. PROTEIN AND CHLOROPHYLL AS ORGANOGENIC MARKERS Cotyledons cytokinins  used t o study t h e organogenic  e f f e c t of  were used for. p r o t e i n and c h l o r o p h y l l  analyses.  5. STATISTICAL ANALYSIS A l l experiments  were r e p e a t e d a t l e a s t 3 t i m e s .  An a n a l y s i s o f v a r i a n c e was performed on r e s u l t s o b t a i n e d from t h e t i s s u e c u l t u r e , t h e t o t a l p r o t e i n , LSURUBP and c h l o r o p h y l l c o n c e n t r a t i o n s a n a l y s e s . A l l e x p e r i m e n t a l d a t a were compared f o r s t a t i s t i c a l at p r o b a b i l i t y  (P) ^ 0 . 0 5 .  difference  31  RESULTS  1. TISSUE CULTURE Both BA and 2 i P i n d u c e d m u l t i p l e bud formation vitro  on e x c i s e d P. ponderosa c o t y l e d o n s  ( F i g u r e 1A).  Outgrowth was  shoot  cultured in  o b s e r v e d a f t e r 10 days  i n c u l t u r e with c y t o k i n i n (Figure IB). cotyledons  and  were t r a n s f e r r e d t o a GRF  A f t e r the  medium, l e a f  p r i m o r d i a developed ( F i g u r e IC) and e l o n g a t e d  (Figure  ID)  a f t e r s e v e r a l weeks. Excised cotyledons (GRF)  medium  c u l t u r e d on growth r e g u l a t o r f r e e  elongated.  R e s u l t s i n t h i s s e c t i o n are from e x p e r i m e n t s d e s i g n e d t o s e l e c t the most a p p r o p r i a t e c u l t u r e medium, optimal concentrations  of exogenous BA or 2 i P , and  optimal  exposure time t o t h e s e growth r e g u l a t o r s .  1.1.  CULTURE MEDIA F i g u r e 2 shows the t o t a l response (percentage of  cotyledons  t h a t responded e i t h e r by g r e e n i n g  or  forming  buds and s h o o t s ) , the organogenic response (percentage of cotyledons  t h a t formed buds and s h o o t s ) , and the mean buds  and shoots formed per c o t y l e d o n  a f t e r c u l t u r e on d i f f e r e n t  media i n the presence of 25 uM BA or 2 i P f o r 42 days.  32  FIGURE 1. M u l t i p l e buds and shoots ( A ) , outgrowths ( B ) , and l e a f p r i m o r d i a (C) formed i n a c o t y l e d o n a f t e r c u l t u r e on C K - c o n t a i n i n g medium f o r 10 days and t r a n s f e r r e d t o a GRF medium. E l o n g a t e d l e a f p r i m o r d i a (D) a f t e r 8 weeks o f c u l t u r e on GRF medium.  V —  Ld  (/> z o Q.  1/1  < I—  o  >—' UJ  (/)  z o  CL  in ui Q:  o o o z < o  DC  o  o o X  </) Q  z < in o m  DCR  LP MCM CULTURE MEDIA  SH  FIGURE 2. T o t a l response + s t a n d a r d e r r o r o f t h e mean (SE), organogenic response + SE, and mean buds and shoots + SE, o f c o t y l e d o n s c u l t u r e d on d i f f e r e n t media i n t h e presence o f 25 uM BA o r 2 i P f o r 42 days.  34  There were no s i g n i f i c a n t  d i f f e r e n c e s between t h e e f f e c t s  of t h e c y t o k i n i n s , a l t h o u g h t h e t o t a l and t h e organogenic responses were always h i g h e r f o r 2 i P - t r e a t e d c o t y l e d o n s c u l t u r e d on DCR, LP and MCM media.  MCM medium w i t h BA o r  2iP was t h e o n l y one t h a t s i g n i f i c a n t l y i n f l u e n c e d t h e c o t y l e d o n s responses and t h i s decreased shoots  formed p e r c o t y l e d o n .  t h e mean buds and  However, t h e buds and shoots  formed on c o t y l e d o n s c u l t u r e d on LP medium appeared more robust.  T h i s medium was s e l e c t e d t h e r e f o r e f o r t h e  m i c r o s c o p i c and b i o c h e m i c a l  analyses.  1.2. GROWTH REGULATOR CONCENTRATIONS The t o t a l response o f c o t y l e d o n s t o 2 i P was s i g n i f i c a n t l y h i g h e r than t o BA a f t e r 42 days o f exposure t o t h e s e growth r e g u l a t o r s ( F i g u r e 3 ) . N e i t h e r t h e organogenic  response n o r t h e mean buds and shoots produced  were s i g n i f i c a n t l y r e l a t e d t o BA c o n c e n t r a t i o n .  Different  concentrations of 2iP s i g n i f i c a n t l y influenced the organogenic  response and t h e mean buds and s h o o t s , t h e  l a s t one b e i n g t h e most v a r i a b l e measure o f growth. A f t e r 10 days o f exposure ( F i g u r e 4) t h e r e were no significant except  c o n c e n t r a t i o n e f f e c t s between BA and 2 i P ,  f o r t h e mean buds and shoots produced d u r i n g growth  w i t h 15 uM on LP medium ( F i g u r e 4C). Of t h e two c u l t u r e media t e s t e d , t h e mean buds and shoots on SH medium  35  55 50  S g o </) < i—  o  45 40 35 30  1 • BA  25  T  20  2iP  15 55 • BA 2iP  T Ul  £ o CL on  45 40  o  35  o o z < o or o  30  h  25 20 15  C/1 I—  o o X  (/) a  z <  (/) a < Ul  3  5  10  15  20  CONCENTRATION (uM)  FIGURE 3. T o t a l response + SE, organogenic response + SE, and mean buds and shoots + SE, o f c o t y l e d o n s c u l t u r e d on d i f f e r e n t c o n c e n t r a t i o n s o f BA o r 2 i P on LP medium f o r 42 days.  36 • BA T  0 U 0  1  1  1  5  10  15  | 20  CONCENTRATION (uM)  L_l I I 25 0  I  5  I  I  I  i  10  15  20  25  CONCENTRATION (uM)  FIGURE 4. T o t a l response, organogenic response, and mean buds and shoots + SE, o f c o t y l e d o n s c u l t u r e d on d i f f e r e n t c o n c e n t r a t i o n s o f BA o r 2 i P , on LP (A-C) o r SH (D-F) medium f o r 10 days.  2iP  37  ( F i g u r e 4F) was l e s s t h a n t h o s e formed on LP medium ( F i g u r e 4C). T h i s lower response on SH medium was a l s o d e t e c t e d on F i g u r e 2. F i g u r e 5 i s a composite o f F i g u r e s 3 and 4.  The  t o t a l response, t h e organogenic response and t h e mean buds and shoots were s i g n i f i c a n t l y d i f f e r e n t when c o t y l e d o n s were c u l t u r e d f o r 10 and 42 days on 1-25 uM BA ( F i g u r e 5AC).  However t h e time o f exposure t o 2 i P o n l y i n f l u e n c e d  t h e mean buds and shoots formed  ( F i g u r e 5 F ) , and t h e  organogenic response a t 1 and 5 uM ( F i g u r e 5 E ) . These data.showed t h a t c o t y l e d o n s were more s e n s i t i v e t o t h e time o f exposure t o BA t h a n t o 2 i P . The optimum c o n c e n t r a t i o n o f b o t h c y t o k i n i n s i n LP medium f o r mean buds and shoots p r o d u c t i o n was 15 uM ( F i g u r e s 5C and 5 F ) .  T h i s c o n c e n t r a t i o n was chosen f o r  c u l t u r e d c o t y l e d o n s t o be used i n m i c r o s c o p i c and biochemical analyses.  1.3. TIME OF EXPOSURE A l l measures o f developmental response showed t h a t , c o t y l e d o n s were more s e n s i t i v e t o t h e time o f exposure t o BA t h a n t o 2 i P ( F i g u r e s 6A-6F) i n b o t h LP and SH medium. However, a f t e r growth on SH medium f o r 21 days, t o b o t h growth r e g u l a t o r s were s i m i l a r  responses  ( F i g u r e s 6D-6F).  38  D  60  ^10 B 4 2  1  •u 50 in z o o. (/7 40 < I—  o  30  I  20  B  60 Ld  z o  -  1  50  CL  t/1 Ul  cc  o z Ul o o z < o O  40 30 20 10 25  o O X  (/)  Q Z  <  20 15  Q  m  <  ui  10  I 1  T i  T -  5 15 25 CONCENTRATION (uM)  1  5 15 25 CONCENTRATION (uM)  FIGURE 5. T o t a l response, organogenic response, and mean buds and s h o o t s , o f c o t y l e d o n s c u l t u r e d on LP medium supplemented w i t h d i f f e r e n t c o n c e n t r a t i o n s o f BA (A-C) o r 2 i P (D-F) f o r 10 and 42 days. (Composite o f f i g u r e s 3 and 4) .  39  FIGURE 6. T o t a l response + SE, organogenic response + SE, and mean buds and shoots + SE, of' BA o r 2 i P t r e a t e d c o t y l e d o n s c u l t u r e d on LP (A-C) o r SH (D-F) medium a f t e r d i f f e r e n t days o f exposure t o c y t o k i n i n s , and t h e n t r a n s f e r r e d t o GRF medium.  40  The 5-10  g e n e r a l o b s e r v a t i o n o f a maximum r e s p o n s e  days of c u l t u r e i n t h e presence  of e i t h e r  growth  r e g u l a t o r l e d t o t h e s e l e c t i o n o f 10 d a y s e x p o s u r e f o r growth  after  time  o f c u l t u r e s t o be u s e d f o r m i c r o s c o p i c and  biochemical analyses. Table 2 summarizes t h e c u l t u r e c o n d i t i o n s  selected  f o r t i s s u e s t o be u s e d i n m i c r o s c o p i c and b i o c h e m i c a l analyses,  and T a b l e 3 s u m m a r i z e s t h e o b s e r v e d n u m e r i c a l  responses  of the c o t y l e d o n s under these  conditions.  C o t y l e d o n s w h i c h were n o t e x c i s e d and e x p o s e d cytokinins until and o r g a n o g e n i c  3 d a y s a f t e r g e r m i n a t i o n , showed  total  r e s p o n s e s t h a t were s i g n i f i c a n t l y  lower  t h a n t h o s e c u l t u r e d w i t h c y t o k i n i n f r o m day and 7B)  0  d e m o n s t r a t i n g a l o s s of competence.  2. HISTOLOGICAL AND Histological  embryos  was  Cotyledons e x c i s e d from 7 d a y - o l d s e e d l i n g s  showed no o r g a n o g e n i c  P. p o n d e r o s a  ( F i g u r e s 7A The  d i f f e r e n c e b e t w e e n t h e mean b u d s and s h o o t s significant.  to  response t o 2 i P .  ULTRASTRUCTURAL ANALYSES  and u l t r a s t r u c t u r a l  analyses of  c o t y l e d o n s showed t h a t c e l l s  (day 0) c o n t a i n e d l a r g e c e n t r a l l y  as w e l l as l i p i d d r o p l e t s w h i c h o c c u p i e d a  from  cultured  imbibed  located large  nuclei,  TABLE 2. C u l t u r e c o n d i t i o n s s e l e c t e d f o r P i n u s ponderosa c o t y l e d o n s t o be used i n t h e m i c r o s c o p i c and b i o c h e m i c a l analyses MEDIUM  LP  GROWTH REGULATORS  BA (BENZYLADENINE) 2 i P (2-ISOPENTENYL ADENINE)  CONCENTRATION  15 uM  TIME OF EXPOSURE  10 DAYS  PHYSICAL CONDITIONS  27 + 2°C 16 h PHOTOPERIOD 80 uE/m /s PHOTON FLUENCE  (VON ARNOLD & ERIKSSON, 1981)  2  TABLE 3. Responses (see page 21) + s t a n d a r d e r r o r o f t h e mean o f P i n u s ponderosa c o t y l e d o n s c u l t u r e d on GRF o r c y t o k i n i n - c o n t a i n i n g medium a f t e r 10 days o f exposure t o 15 uM BA o r 15 uM 2 i P TOTAL RESPONSE (%)  ORGANOGENIC RESPONSE (%)•  GRF  BA  2iP  BA  93+1.3  92+1.5  93+1.3  42+5.6  2iP 47+4.8  MEAN BUDS AND SHOOTS BA  2iP  9 + 0.5 8.4+0.6  42  0  2  4  6  8  COTYLEDON AGE (DAYS)  FIGURE 7. T o t a l response, organogenic response, and mean buds and shoots + SE o f c o t y l e d o n s e x c i s e d from g e r m i n a t e d embryos c u l t u r e d i n t h e presence o f BA o r 2 i P f o r 10 days and then t r a n s f e r r e d t o GRF medium.  43  p o r t i o n o f t h e c y t o p l a s m , and p r o t e i n s b o d i e s which were i d e n t i f i e d histochemically  ( F i g u r e s 8A and 8B).  A f t e r 3 days i n c u l t u r e c e l l s grown i n t h e p r e s e n c e of c y t o k i n i n s  ( F i g u r e 9A) showed a d e n s e l y  c y t o p l a s m which c o n t a i n e d  stained  many l i p i d d r o p l e t s ,  prominent n u c l e i , and l i t t l e t o no v a c u o l a t i o n , c e l l s c u l t u r e d on GRF medium c o n t a i n e d  large whereas  l a r g e r vacuoles  ( F i g u r e 9B). M i t o t i c f i g u r e s were o b s e r v e d i n some s u b e p i d e r m a l c e l l s below t h e s u r f a c e o f t h e c o t y l e d o n s i n contact supply  w i t h t h e medium, r e g a r d l e s s  o f growth  regulators  ( F i g u r e 9B). A c y t o k i n i n e f f e c t on m i t o t i c  a c t i v i t y was n o t observed a t day 3 i n c u l t u r e , but more d i f f e r e n t i a t e d c e l l s were observed i n t h e absence o f cytokinin. A f t e r 5 days i n c u l t u r e , t h e c y t o k i n i n - t r e a t e d cotyledons contained t i s s u e i n contact The  d i s t i n c t meristematic regions  i n the  w i t h t h e medium ( F i g u r e s 10 and 11) .  e p i d e r m a l and s u b e p i d e r m a l l a y e r s on t h i s s i d e were  more compact t h a n t h o s e i n t h e upper r e g i o n o f t h e cotyledons  ( F i g u r e 12A) and s e v e r a l m i t o t i c f i g u r e s were  observed.  The m e r i s t e m a t i c c e l l s c o n t a i n e d  centrally  l o c a t e d n u c l e i ( F i g u r e 12A), s m a l l v a c u o l e s and some droplets i n the cytoplasm present contained  lipid  ( F i g u r e 12B). The p l a s t i d s  few, s m a l l , s i n g l e t h y l a k o i d s and no  grana ( F i g u r e 12C).  44  FIGURE 8. P i n u s ponderosa c o t y l e d o n s at t h e time o f e x c i s i o n (day 0 ) . A. L i g h t m i c r o g r a p h of c e l l s t i g h t l y packed c o n t a i n i n g p r o t e i n b o d i e s (PB) and c e n t r a l l y l o c a t e d n u c l e i (N), B. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h of a c e l l w i t h l i p i d d r o p l e t s (L) o c c u p y i n g almost a l l t h e c y t o p l a s m . (N=nucleus, PB=protein b o d i e s , R=ribosomes, W=cell w a l l ) .  FIGURE 9. P i n u s ponderosa c o t y l e d o n s a t day 3 i n c u l t u r e . A. L i g h t m i c r o g r a p h o f a c o t y l e d o n c u l t u r e d on c y t o k i n i n c o n t a i n i n g medium showing c e l l s w i t h l i p i d d r o p l e t s (L) o c c u p y i n g almost a l l t h e c y t o p l a s m , and c e n t r a l l y l o c a t e d n u c l e i (N). B. L i g h t m i c r o g r a p h o f a c o t y l e d o n c u l t u r e d on GRF medium, showing some m i t o t i c a c t i v i t y (*) on t h e subepidermal l a y e r o f c e l l s . Large v a c u o l e s (V) occupy almost 75 % o f t h e c e l l volume.  46  FIGURE 10. L i g h t m i c r o g r a p h o f a P i n u s ponderosa c o t y l e d o n at day 5 i n c u l t u r e i n t h e p r e s e n c e o f c y t o k i n i n s showing the m e r i s t e m a t i c (M) and n o n - m e r i s t e m a t i c (NM) r e g i o n s .  FIGURE 11. L i g h t m i c r o g r a p h of a P i n u s ponderosa c o t y l e d o n at day 5 i n c u l t u r e i n t h e presence of c y t o k i n i n s showing the m e r i s t e m a t i c r e g i o n (M) w i t h c e l l s t i g h t l y packed w i t h c e n t r a l l y l o c a t e d n u c l e i (N) and t h e n o n - m e r i s t e m a t i c r e g i o n (NM) w i t h c e l l s c o n t a i n i n g l a r g e v a c u o l e s ( V ) .  47  FIGURE 12. P i n u s ponderosa c o t y l e d o n s a t day 5 i n c u l t u r e i n t h e presence o f c y t o k i n i n s . A. L i g h t m i c r o g r a p h o f t h e m e r i s t e m a t i c r e g i o n showing c e l l s t i g h t l y packed, c e n t r a l l y l o c a t e d n u c l e i (N) w i t h one o r more n u c l e o l i (Nu), and s m a l l v a c u o l e s ( V ) . B. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h o f a m e r i s t e m a t i c c e l l c o n t a i n i n g s m a l l v a c u o l e s (V), some l i p i d d r o p l e t s (L) and some p r o p l a s t i d s (P) i n t h e c y t o p l a s m (N=nucleus). C. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h o f a p l a s t i d (P) o f a meristematic c e l l . T h y l a k o i d membranes (T) o c c u r as single vesicles. D. L i g h t m i c r o g r a p h o f t h e n o n - m e r i s t e m a t i c r e g i o n . Large v a c u o l e s (V) occupy almost a l l t h e c e l l volume. Lipid d r o p l e t s (L) a r e s t i l l p r e s e n t i n t h e c y t o p l a s m . (N=nucleus). E. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h o f a n o n - m e r i s t e m a t i c c e l l w i t h l a r g e v a c u o l e s (V), l a r g e i n t e r c e l l u l a r spaces (IS), and p l a s t i d s (P) c o n f i n e d t o t h e p e r i p h e r y o f t h e cells. Note some p l a s t i d d i v i s i o n (*). (pl=plasmodesmata). F. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h o f a p l a s t i d (P) o f a non-meristematic c e l l . T h y l a k o i d membranes (T) and grana (G) a r e c l e a r . The c y t o p l a s m c o n t a i n e d l i p i d d r o p l e t s (L) .  49  In t h e n o n - m e r i s t e m a t i c  r e g i o n , c e l l s had l a r g e  v a c u o l e s which o c c u p i e d much o f t h e c e l l volume ( F i g u r e 12D),  and t h e o r g a n e l l e s and l i p i d d r o p l e t s were c o n f i n e d  t o t h e c e l l p e r i p h e r y ( F i g u r e 12E). The p l a s t i d s 12F) o f t h e s e n o n - m e r i s t e m a t i c  (Figure  c e l l s were l a r g e r than  t h o s e i n m e r i s t e m a t i c c e l l s and showed d i s t i n c t  thylakoid  membranes and grana. A f t e r 5 days i n c u l t u r e on GRF medium, c o t y l e d o n s were e l o n g a t e d  ( F i g u r e 13A). Large i n t e r c e l l u l a r  spaces  and l a r g e v a c u o l e s were observed i n t h e s e c e l l s ( F i g u r e s 13B and 13C), and s m a l l l i p i d d r o p l e t s were p r e s e n t ( F i g u r e 13D).  The p l a s t i d s showed d i s t i n c t  membranes and grana  thylakoid  ( F i g u r e 13E) t h a t were s i m i l a r t o  t h o s e from t h e n o n - m e r i s t e m a t i c  c e l l s of c y t o k i n i n t r e a t e d  cotyledons. By day 10 ( F i g u r e 14A), t h e m e r i s t e m a t i c r e g i o n o f t h e c y t o k i n i n t r e a t e d c o t y l e d o n s had developed t o m e r i s t e m a t i c domes r e s u l t i n g from t h e r a p i d proliferation layers.  cell  w i t h i n t h e e p i d e r m a l and t h e subepidermal  M e r i s t e m a t i c r e g i o n s o c c u p i e d 7-10% o f t h e  l o n g i t u d i n a l sectioned area.  Each m e r i s t e m a t i c  cell  c o n t a i n e d o r g a n e l l e s such as p l a s t i d s , s m a l l v a c u o l e s , and a c e n t r a l n u c l e u s w i t h one o r more n u c l e o l i and 14C). There were no i n t e r c e l l u l a r  ( F i g u r e s 14B  spaces.  50  FIGURE 13. P i n u s ponderosa c o t y l e d o n s a t day 5 o f c u l t u r e on GRF medium. A. L i g h t m i c r o g r a p h o f an e l o n g a t e d c o t y l e d o n . B. L i g h t m i c r o g r a p h showing t h e e p i d e r m a l (E) and subepidermal l a y e r s o f c e l l s w i t h l a r g e v a c u o l e s (V) and i n t e r c e l l u l a r spaces ( I S ) . C. L i g h t m i c r o g r a p h o f c e l l s w i t h l a r g e i n t e r c e l l u l a r spaces ( I S ) , l a r g e v a c u o l e s (V) and t h e c y t o p l a s m c o n f i n e d t o t h e p e r i p h e r y o f t h e c e l l s . (P= p l a s t i d s , N=nucleus). D. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h o f a c e l l w i t h v a c u o l e s (V), n u c l e u s (N) and p l a s t i d s ( P ) . ( I S = i n t e r c e l l u l a r space, L = l i p i d d r o p l e t ) . E. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h o f a p l a s t i d ( P ) . T h y l a k o i d membranes (T) and grana (G) a r e c l e a r . (V=vacuole) .  52  FIGURE 14. P i n u s ponderosa c o t y l e d o n s a t day 10 o f c u l t u r e i n t h e presence o f c y t o k i n i n s . A. L i g h t m i c r o g r a p h showing t h e m e r i s t e m a t i c domes (M) i n t h e r e g i o n o f t h e c o t y l e d o n i n c o n t a c t w i t h t h e medium and t h e n o n - m e r i s t e m a t i c (NM) r e g i o n . B. L i g h t m i c r o g r a p h o f t h e m e r i s t e m a t i c r e g i o n showing c e l l s t i g h t l y packed, c e n t r a l l y l o c a t e d n u c l e i (N) w i t h one o r more n u c l e o l i (Nu), s m a l l v a c u o l e s (V) and p l a s t i d s (P). Note some m i t o t i c a c t i v i t y (*) i n t h e subepidermal layer of c e l l s . C. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h o f a m e r i s t e m a t i c c e l l w i t h s m a l l v a c u o l e s (V), and p l a s t i d s (P) i n t h e c y t o p l a s m (N=nucleus, Nu=nucleolus, W=cell w a l l ) . D. L i g h t m i c r o g r a p h o f t h e n o n - m e r i s t e m a t i c r e g i o n w i t h a w e l l developed e p i d e r m i s ( E ) . Note l a r g e i n t e r c e l l u l a r spaces ( I S ) , l a r g e v a c u o l e s (V) which occupy almost a l l t h e c e l l volume, n u c l e i (N) and c h l o r o p l a s t s (C) c o n f i n e d to the periphery of the c e l l s . E. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h o f n o n - m e r i s t e m a t i c c e l l s w i t h l a r g e v a c u o l e s (V) o c c u p y i n g almost a l l t h e c e l l volume and t h e c y t o p l a s m c o n f i n e d t o t h e p e r i p h e r y . C h l o r o p l a s t s (C) w i t h s t a r c h g r a n u l e s were p r e s e n t . (W=cell w a l l ) .  54  The n o n - m e r i s t e m a t i c c e l l s o f t h e s e c o t y l e d o n s ( F i g u r e 14D)  were l a r g e r , c o n t a i n e d v a c u o l e s which  o c c u p i e d most of t h e c e l l volume, and t h e i r c y t o p l a s m formed a t h i n p e r i p h e r a l l a y e r many i n t e r c e l l u l a r  ( F i g u r e 14E).  There were  spaces.  The p l a s t i d s of m e r i s t e m a t i c c e l l s were s p h e r i c a l or irregular  ( F i g u r e 15A) w i t h s i n g l e t h y l a k o i d membranes,  o c c a s i o n a l l y some grana, and s t a r c h g r a i n s i n t h e stroma. These c o n t r a s t e d w i t h t h e d e v e l o p e d c h l o r o p l a s t s i n non-meristematic c e l l s  observed  ( F i g u r e 15B) which showed more  t h y l a k o i d membranes and more d i s t i n c t grana. some l i p i d g r a n u l e s i n t h e stroma.  There were  The c h l o r o p l a s t s i n  t h e s e 10 day c u l t u r e s were l a r g e r t h a n t h o s e i n b o t h m e r i s t e m a t i c and n o n - m e r i s t e m a t i c c e l l s a f t e r 5 days i n culture  (compare F i g u r e s 12C and 12F w i t h F i g u r e s 15A  and  15B) . C o t y l e d o n s c u l t u r e d i n t h e absence of c y t o k i n i n s , e l o n g a t e d , and showed a w e l l d i f f e r e n t i a t e d e p i d e r m i s by day 10 i n c u l t u r e  ( F i g u r e 16A).  There were l a r g e  i n t e r c e l l u l a r spaces, and t h e c e l l s c o n t a i n e d l a r g e v a c u o l e s which, l i k e t h o s e from n o n - m e r i s t e m a t i c r e g i o n s of t h e c y t o k i n i n t r e a t e d c o t y l e d o n s , o c c u p i e d almost a l l t h e c e l l volume ( F i g u r e s 16B and 16C).  The  chloroplasts  i n the p e r i p h e r a l c y t o p l a s m c o n t a i n e d e x t e n s i v e i n n e r membranes and some s t a r c h g r a i n s ( F i g u r e 16D).  The  55  FIGURE 15. T r a n s m i s s i o n e l e c t r o n micrographs o f P i n u s ponderosa c o t y l e d o n s a t day 10 o f c u l t u r e i n t h e p r e s e n c e of c y t o k i n i n s . A. P l a s t i d (P) o f a m e r i s t e m a t i c c e l l . Some t h y l a k o i d membranes ( T ) , grana (G), and a s t a r c h g r a i n (SG) a r e p r e s e n t . (V=vacuole). B. Mature c h l o r o p l a s t (C) i n a n o n - m e r i s t e m a t i c c e l l showing w e l l d e v e l o p e d grana (G). (V=vacuole, W=cell wall) .  FIGURE 16. P i n u s ponderosa c o t y l e d o n s a t day 10 i n c u l t u r e i n GRF medium. A. L i g h t m i c r o g r a p h showing t h e e p i d e r m a l (E) and subepidermal l a y e r s o f c e l l s w e l l d i f f e r e n t i a t e d , and l a r g e v a c u o l e s (V) and i n t e r c e l l u l a r spaces (IS) a r e present. B. L i g h t m i c r o g r a p h o f c e l l s w i t h l a r g e i n t e r c e l l u l a r spaces ( I S ) , l a r g e v a c u o l e s (V) and c h l o r o p l a s t s (C) confined t o the periphery of the c e l l s . C. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h o f c e l l s showing l a r g e v a c u o l e s (V) , and c h l o r o p l a s t s (C) c o n f i n e d t o t h e periphery of the c e l l s . D. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h o f a developed c h l o r o p l a s t (C) showing w e l l developed grana (G) and a s t a r c h g r a i n (SG) i n t h e stroma. ( I S = i n t e r c e l l u l a r space, V=vacuole, W=cell w a l l ) .  57  c h l o r o p l a s t s from GRF t r e a t e d cotyledons to those  appeared s i m i l a r  i n c e l l s of the non-meristematic regions of  c y t o k i n i n t r e a t e d cotyledons  (compare F i g u r e 15B with  F i g u r e 16D). By day 21 i n c u l t u r e , a f t e r 10 days o f exposure t o c y t o k i n i n s and 11 days on GRF medium, the meristematic regions had developed  to leaf primordia  some m i t o t i c f i g u r e s were observed region.  (Figure 17), and  i n the meristematic  The c e l l s i n the non-meristematic r e g i o n were  s i m i l a r i n appearance t o those of 10 day-old  cotyledons.  3. PROTEIN AND CHLOROPHYLL ANALYSES In order t o c h a r a c t e r i z e b i o c h e m i c a l l y the development of the p h o t o s y n t h e t i c apparatus d u r i n g cotyledon c u l t u r e , p r o t e i n and c h l o r o p h y l l analyses were performed on cotyledons  c u l t u r e d i n the presence or  absence of c y t o k i n i n s .  3.1.  TOTAL PROTEIN  DETERMINATION  F i g u r e 18 shows t h a t the t o t a l p r o t e i n c o n c e n t r a t i o n i n a l l the cotyledons  c u l t u r e d on LP medium  r a p i d l y d u r i n g the f i r s t  days.  decreased  These r e s u l t s suggest  that  the most concentrated p r o t e i n s were storage p r o t e i n s and  FIGURE 17. L i g h t m i c r o g r a p h o f a c o t y l e d o n a t day 21, a f t e r c u l t u r e i n t h e p r e s e n c e o f c y t o k i n i n s f o r 10 days and t r a n s f e r r e d t o a growth r e g u l a t o r f r e e (GRF) medium. Leaf p r i m o r d i a (F) have d e v e l o p e d . Note t h e m e r i s t e m a t i c r e g i o n (M) i s s t i l l p r e s e n t w i t h c e l l s t i g h t l y packed and c e n t r a l l y l o c a t e d n u c l e i . (NM=non-meristematic r e g i o n ) .  FIGURE 18. T o t a l p r o t e i n c o n c e n t r a t i o n + SE o f c o t y l e d o n s c u l t u r e d on GRF medium, o r i n t h e presence o f BA o r 2 i P .  60  t h a t they were degraded d u r i n g t h e f i r s t days i n c u l t u r e e i t h e r i n t h e presence  o r absence o f c y t o k i n i n s .  There were no s i g n i f i c a n t d i f f e r e n c e s between c o t y l e d o n s which were c u l t u r e d w i t h BA o r those grown w i t h 2iP,  however a t r e n d towards a f a s t e r decrease  p r o t e i n was observed  of t o t a l  on 2 i P t r e a t e d c o t y l e d o n s .  Comparison between c o t y l e d o n s c u l t u r e d w i t h c y t o k i n i n and those grown on GRF medium showed t h a t , by day 4 i n c u l t u r e , t o t a l p r o t e i n l e v e l s were s i g n i f i c a n t l y h i g h e r i n BA o r 2 i P t r e a t e d c o t y l e d o n s .  However, a f t e r 7  days i n c u l t u r e a t r e n d towards a c o n s t a n t p r o t e i n c o n c e n t r a t i o n was observed.  These r e s u l t s suggest  newly s y n t h e s i z e d p r o t e i n s were l e s s c o n c e n t r a t e d  that than  storage p r o t e i n s .  3.2. SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS F i g u r e 19 shows Coomassie b l u e s t a i n e d SDSp o l y a c r y l a m i d e g e l s o f p r o t e i n e x t r a c t s from GRF and BA t r e a t e d c o t y l e d o n s from days 0 t o 10 i n c u l t u r e .  The most  c o n c e n t r a t e d p o l y p e p t i d e s i n day 0 c o t y l e d o n s had apparent m o l e c u l a r masses o f 48, 37, 31, 22 and 21 kD which corresponded and d e c r e a s e d  t o s t o r a g e p r o t e i n s ( E l l i s and Judd, 1987) d u r i n g t h e f i r s t days i n c u l t u r e ,  following  the same t r e n d as t h e t o t a l p r o t e i n c o n c e n t r a t i o n s ( F i g u r e 18).  Some o f t h e s e s t o r a g e p r o t e i n s a r e s i m i l a r t o those  61  FIGURE 19. S D S - p d l y a c r y l a m i d e g e l s of c o t y l e d o n s c u l t u r e d on GRF or BA c o n t a i n i n g media from days 0 t o day 10. (S=molecular weight s t a n d a r d s ) .  63  found i n s e v e r a l P i n u s s p e c i e s ( G i f f o r d , 1988) . C o t y l e d o n s which d i d not respond t o t h e d i f f e r e n t t r e a t m e n t s t e s t e d showed no d e c l i n e o f t h e i r  storage  p r o t e i n s t h r o u g h time i n c u l t u r e , and t h e r e were no newly synthesized proteins. B e s i d e s t h e s t o r a g e p r o t e i n s , t h e most  prominent  band observed had an apparent m o l e c u l a r mass o f 48-51 kD and was i d e n t i f i e d by i m m u n o b l o t t i n g  as LSU-RUBP.  Three low m o l e c u l a r mass p o l y p e p t i d e s o f 18, 16 and 14 kD were observed as r e p o r t e d by G i f f o r d  (1988) and were  p r e s e n t from day 0 u n t i l day 10 o f c u l t u r e .  3.3. IMMUNOBLOTTING To i n v e s t i g a t e t h e e f f e c t o f c y t o k i n i n s on t h e development o f t h e p h o t o s y n t h e t i c apparatus t h e a c c u m u l a t i o n o f s i x p h o t o s y n t h e t i c p o l y p e p t i d e s was examined by i m m u n o b l o t t i n g  (Figure 20).  In a l l cases,  t h e r e was l e s s c r o s s - r e a c t i n g p r o t e i n i n c o t y l e d o n s t r e a t e d w i t h c y t o k i n i n than i n t h o s e c u l t u r e d on GRF medium o r i n t h e c o t y l e d o n s from germinated embryos (C). However, BA was a g r e a t e r i n h i b i t o r o f a c c u m u l a t i o n o f t h e s e p h o t o s y n t h e t i c p r o t e i n s than 2 i P .  The a n t i b o d y f o r  CP29 c r o s s - r e a c t e d w i t h t h e l i g h t h a r v e s t i n g complex o f photosystem  I I (LHCII)  Green, 1988).  (White and Green, 1987b; White and  LHCI was not d e t e c t e d i n c o t y l e d o n s t r e a t e d  64  TREATMENT GRF BA DAYS IN CULTURE 4567 4 5 6 7 APP M (kD) • —,  LHCI  .  2iP 4 5 6 7  .  C 4 5 6 7  24 — 23 —  FIGURE 20. Immunoblots o f p h o t o s y n t h e t i c p r o t e i n s : t h e c o u p l i n g f a c t o r (CF1), t h e l a r g e s u b u n i t o f r i b u l o s e - 1 , 5 b i s p h o s p h a t e c a r b o x y l a s e (LSU-RUBP), t h e e x t r i n s i c 33kD p r o t e i n a s s o c i a t e d w i t h t h e o x y g e n - e v o l v i n g complex (33EP), an a n t e n n a l component o f photosystem I I (CP29), a c h l o r o p h y l l - b i n d i n g p r o t e i n from t h e l i g h t - h a r v e s t i n g complex o f photosystem I I (LHCII), and a second c h l o r o p h y l l - b i n d i n g p r o t e i n from t h e l i g h t h a r v e s t i n g complex o f photosystem I (LHCI), o f c o t y l e d o n s c u l t u r e d on growth r e g u l a t o r f r e e (GRF) medium, w i t h 15 uM b e n z y l a d e n i n e (BA), o r 15 uM 2 - i s o p e n t e n y l adenine ( 2 i P ) , and c o n t r o l (C) from g e r m i n a t e d embryos, r e s p e c t i v e l y .  65  w i t h BA o r 2 i P , but i t was p r e s e n t i n t h e c o n t r o l and i n GRF t r e a t e d  cotyledons.  Gel scan q u a n t i f i c a t i o n o f LSU-RUBP showed a r a p i d increase and  d u r i n g t h e f i r s t days i n c u l t u r e on GRF medium  a somewhat s l o w e r a c c u m u l a t i o n i n c y t o k i n i n - t r e a t e d  cotyledons  (Figure  2 1 ) . By day 10 t h e r e was a s i g n i f i c a n t  difference  i n t h e c o n c e n t r a t i o n between c o t y l e d o n s  c u l t u r e d on GRF medium compared w i t h t h o s e c u l t u r e d i n t h e presence o f e i t h e r  3.4. CHLOROPHYLL  cytokinin.  DETERMINATION  The e f f e c t o f c y t o k i n i n t r e a t m e n t extended t o c h l o r o p h y l l content.  C o n s i s t e n t w i t h t h e reduced  accumulation of photosynthetic polypeptides, the t o t a l chlorophyll  (Figure  22 A,D) and c h l o r o p h y l l a (Figure  22  B,E) c o n c e n t r a t i o n s were s i g n i f i c a n t l y lower i n 4 day c y t o k i n i n t r e a t e d c o t y l e d o n s t h a n i n t h o s e grown on GRF medium.  The s i g n i f i c a n t d i f f e r e n c e between c h l o r o p h y l l b  concentrations seventh day.  (Figure  22 C,F) was not apparent u n t i l t h e  FIGURE 21. The l a r g e s u b u n i t o f r i b u l o s e - 1 , 5 - b i s p h o s p h a t e c a r b o x y l a s e (LSU-RUBP) c o n c e n t r a t i o n + SE o f c y t o k i n i n (CK) v e r s u s growth r e g u l a t o r f r e e (GRF) t r e a t e d c o t y l e d o n s cultured i n v i t r o .  67 i  1  1  r  i  i  i  r  D  15 u. cn £ CD 3  10  o  t  <  . T  5  I—  o  -  T 2IP a GRF  i 12  1  1  n  r  1  I  L  1  r  i  E  B  r  10 cn  8  £  /  cn 3  6  r  a  /  Ki  '  4  o  2 0  T i  i  I  i  I  i  T 2IP • GRF  _L  i  r  r  i  F  cn £ cn  a  r  3  / v- ^  T 2IP • GRF  ••-cr 0  2  4  6  8  TIME IN CULTURE (DAYS)  10  0  J  L  2  4  J 6  8  L 10  TIME IN CULTURE (DAYS)  FIGURE 22. T o t a l C h l o r o p h y l l + SE (A,D), C h l o r o p h y l l a + SE (B,E) and C h l o r o p h y l l b + SE (C,F) c o n c e n t r a t i o n s , o f BA o r 2 i P t r e a t e d c o t y l e d o n s v e r s u s growth r e g u l a t o r f r e e (GRF) t r e a t e d c o t y l e d o n s .  68  4. PROTEIN AND  CHLOROPHYLL AS MARKERS OF ORGANOGENESIS  P r o t e i n and pperiod  c h l o r o p h y l l were used as markers of  the  i n which c y t o k i n i n s c o u l d a l t e r the development of  the p h o t o s y n t h e t i c  apparatus.  In t h e s e e x p e r i m e n t s ,  the  c o t y l e d o n s were c u l t u r e d i n the absence or p r e s e n c e of c y t o k i n i n s f o r 1, 3, and C K - c o n t a i n i n g or GRF  4.1.  CULTURE ON GRF  5 days and t h e n t r a n s f e r r e d t o  medium, r e s p e c t i v e l y .  AND  TRANSFERRED TO CK CONTAINING  MEDIUM. Figure  23 shows the t o t a l p r o t e i n c o n c e n t r a t i o n  c o t y l e d o n s c u l t u r e d f o r 1, 3 or 5 days on GRF t h e n t r a n s f e r r e d t o BA c o n t a i n i n g medium.  ( F i g u r e 23A)  or 2 i P  in  medium  (Figure  23B)  C o t y l e d o n s c u l t u r e d f o r 5 days on  medium p r i o r t o t h e i r t r a n s f e r t o  as  c u l t u r e d cotyledons,  the  t h a n c o t y l e d o n s exposed t o  GRF  exogenous c y t o k i n i n a f t e r o n l y 1 or 3 days i n c u l t u r e medium.  concentration  A t r e n d towards a h i g h e r was  r e s u l t s i n Figure  on  protein  o b s e r v e d when the c o t y l e d o n s were  c u l t u r e d i n BA a f t e r 1 or 3 days i n GRF treated cotyledons.  GRF  cytokinin-containing  medium showed l e s s t o t a l p r o t e i n c o n c e n t r a t i o n ,  GRF  and  compared w i t h  2iP-  These r e s u l t s can be compared w i t h 18, where the c o t y l e d o n s c u l t u r e d i n the  absence of c y t o k i n i n s showed a lower t o t a l  protein  c o n t e n t s t h a n c o t y l e d o n s c u l t u r e d i n the presence of  69  FIGURE 23. T o t a l p r o t e i n c o n c e n t r a t i o n a t days 5, 7 and 10, o f c o t y l e d o n s c u l t u r e d on GRF medium f o r 1, 3, o r 5 days and t h e n t r a n s f e r r e d t o BA (A) o r 2 i P (B) c o n t a i n i n g media, compared w i t h GRF t r e a t e d c o t y l e d o n s .  70  cytokinins.  The l o n g e r t h e c o t y l e d o n c u l t u r e on GRF  medium p r i o r t o t h e i r t r a n s f e r t o C K - c o n t a i n i n g medium, suggests t h a t t h e c o t y l e d o n s t e n d t o respond as i f t h e y were c u l t u r e d c o u n t i n u o u s l y on GRF medium. R e s u l t s of immunoblotting  ( F i g u r e 24) f o r LSU-RUBP  and CP2 9 showed t h a t t h e a c c u m u l a t i o n o f t h e s e p h o t o s y n t h e t i c p r o t e i n s was slowed i n c o t y l e d o n s t h a t were t r a n s f e r r e d t o c y t o k i n i n - c o n t a i n i n g medium a f t e r l e s s t h a n 4 days on GRF medium whereas, i n c o t y l e d o n s t h a t were not transferred until to  4-5 days, t h e s e p o l y p e p t i d e s accumulated  l e v e l s s i m i l a r t o t h o s e c u l t u r e d o n l y on GRF medium f o r  t h e same l e n g t h o f time  ( F i g u r e 2 4 ) . I n t h i s case, t h e  p r o t e i n l e v e l s seemed t o be lower i n 2 i P t h a n i n BAtreated cotyledons. S i m i l a r p a t t e r n s were observed f o r t h e t o t a l c h l o r o p h y l l , c h l o r o p h y l l a and c h l o r o p h y l l b concentrations  ( F i g u r e 2 5 ) . C o t y l e d o n s t r a n s f e r r e d t o BA-  c o n t a i n i n g medium a f t e r 5 days on GRF medium accumulated s u b s t a n t i a l l y more c h l o r o p h y l l s between t h e 8 t h and 9 t h day i n c u l t u r e  ( F i g u r e 25A-25C).  Although i t i s tempting  t o note i n c r e a s i n g c h l o r o p h y l l l e v e l s a f t e r growth on GRF f o l l o w e d by t r a n s f e r t o 2 i P , t r e n d s were not s t a t i s t i c a l l y different  ( F i g u r e s 25D-25F).  DAYS/ IN GRF IN CULTURE APP M (kD)  5 7 10 5 7 10  A LSU-RUBP  4 8 - 5 1 — but ^ ~  CP29  29-30--.  LHCII  2 2 2 5 7 10  28—  LSU-RUBP  48-51—  CP29  29-30^.  LHCII  i i i 5 7 10  28— 2 5  3 3 3 5 7 10  5 5 7 10  4 4 4 5 7 10  m »  •  1  •  -  m  X  FIGURE 2 4 . Immunoblots o f t h e l a r g e s u b u n i t o f r i b u l o s e 1,5-bisphosphate c a r b o x y l a s e (LSU-RUBP), and an a n t e n n a l component o f photosystem I I (CP29), o f c o t y l e d o n s c u l t u r e d on growth r e g u l a t o r f r e e (GRF) media and then t r a n s f e r r e d t o b e n z y l a d e n i n e (BA) (A) o r 2 i P (B) c o n t a i n i n g media.  72  Cn  \  B 3  -C  o < h-  o  CO  E CO 3  5 co cn 3  o  5  6  7  8  9  10  TOTAL TIME CULTURE(DAYS)  5  6  7  8  9  10  TOTAL TIME CULTURE(DAYS)  FIGURE 25. T o t a l C h l o r o p h y l l + SE (A,D), C h l o r o p h y l l a + SE (B.E), and C h l o r o p h y l l b + SE c o n c e n t r a t i o n s (C,F) a t days 5-10 o f c o t y l e d o n s c u l t u r e d i n t h e absence o f c y t o k i n i n s f o r 1, 3 o r 5 days and t h e n t r a n s f e r r e d t o BA (A,B,C) o r 2 i P (D,E,F) c o n t a i n i n g media.  73  4.2.  CULTURE ON CK-CONTAINING MEDIUM FOLLOWED BY TRANSFER  TO GRF MEDIUM. The  t o t a l protein concentrations  i n cotyledons  c u l t u r e d f o r 1, 3 o r 5 days on BA ( F i g u r e 26A) o r 2 i P ( F i g u r e 26B) c o n t a i n i n g media f o l l o w e d by t r a n s f e r t o GRF medium, showed a h i g h e r p r o t e i n c o n c e n t r a t i o n i n c o t y l e d o n s which were i n c o n t a c t p e r i o d of time.  w i t h CK f o r a l o n g e r  These p r o t e i n l e v e l s were h i g h e r  when t h e  c o t y l e d o n s were c u l t u r e d on CK c o n t a i n i n g medium t h r o u g h a l l the time i n c u l t u r e  ( F i g u r e s 26A and 26B).  c o t y l e d o n s t h a t had a l o n g e r  The  c y t o k i n i n exposure tended t o  have h i g h e r p r o t e i n l e v e l s t h a n t h e ones c u l t u r e d i n t h e p r e s e n c e o f c y t o k i n i n s f o r o n l y 1 o r 3 days. r e s u l t s can be compared w i t h F i g u r e  These  18, where c o t y l e d o n s  c u l t u r e d i n t h e presence o f c y t o k i n i n s showed h i g h e r  total  p r o t e i n c o n t e n t s t h a n c o t y l e d o n s c u l t u r e d i n t h e absence of t h e s e growth r e g u l a t o r s . C o t y l e d o n s c u l t u r e d i n t h e p r e s e n c e o f BA o r 2 i P f o r 1-5 days c o n t a i n e d  t h e same l e v e l s o f LSU-RUBP o r CP29 as  cotyledons cultured continuously cytokinins  ( F i g u r e 27).  i n t h e presence o f  There were no s i g n i f i c a n t  d i f f e r e n c e s between t o t a l c h l o r o p h y l l , c h l o r o p h y l l a, and c h l o r o p h y l l b l e v e l s i n e i t h e r BA- ( F i g u r e 28A-28C) o r 2iP-  ( F i g u r e 28D-28F) t r e a t e d c o t y l e d o n s f o r 1-5 days,  74  FIGURE 26. T o t a l p r o t e i n c o n c e n t r a t i o n a t days 5, 7 and 10, o f c o t y l e d o n s c u l t u r e d on BA (A) o r 2 i P (B) c o n t a i n i n g media f o r 1, 3 o r 5 days and t h e n t r a n s f e r r e d t o GRF medium, compared w i t h BA (A) o r 2 i P (B) t r e a t e d cotyledons.  75  DAYS/ IN CK IN CULTURE APP M (kD) A LSU-RUBP  48-51—  CP29  29-30 —  LHCII  5 7 10 5 7 10  1 1 1 5 7 10  —  2 2 2 5 7 10  3 3 3 5 7 10  •  4 4 4 5 7 10  5 5 7 10  28— 27— 25—  B LSU-RUBP 4 8 - 5 1 — CP29 LHCII  •  29-30— 2821 ~ 25"  FIGURE 27. Immunoblots o f t h e l a r g e s u b u n i t o f r i b u l o s e 1,5-bisphosphate c a r b o x y l a s e (LSU-RUBP), and an a n t e n n a l component o f photosystem I I (CP29), o f c o t y l e d o n s c u l t u r e d on b e n z y l a d e n i n e (BA) (A) o r 2 i P (B) c o n t a i n i n g media and then t r a n s f e r r e d t o growth r e g u l a t o r (GRF) media.  FIGURE 28. T o t a l C h l o r o p h y l l + SE (A,D), C h l o r o p h y l l a + SE (B, E ) , and C h l o r o p h y l l b + SE (C,F) c o n c e n t r a t i o n s a t days 5-10 o f c o t y l e d o n s c u l t u r e d i n t h e p r e s e n c e o f BA (A,B,C) or 2 i P (D,E,F) f o r 1, 3 o r 5 days and t h e n t r a n s f e r r e d t o GRF media.  a l t h o u g h a f t e r l o n g e r c y t o k i n i n exposure, l e v e l s seemed t o  fall.  chlorophyll  DISCUSSION  P. ponderosa c o t y l e d o n s c u l t u r e d i n v i t r o were used as a model t o s t u d y some of t h e c y t o k i n i n r e l a t e d changes t o t h e p h o t o s y n t h e t i c apparatus d u r i n g shoot organogenesis. The developmental p r o c e s s which l e a d s t o o r g a n o g e n e s i s i n v i t r o i n v o l v e s gene a c t i v a t i o n , which i s manifested through b i o c h e m i c a l then s t r u c t u r a l  changes.  Exogenously a p p l i e d c y t o k i n i n s a r e a requirement f o r i n v i t r o bud and shoot f o r m a t i o n .  The r o l e s o f endogenous  r e g u l a t o r s are p o o r l y u n d e r s t o o d and were not a d d r e s s e d i n t h i s study. D i f f e r e n t s t a g e s i n t h i s developmental p r o c e s s have been d e s c r i b e d from o t h e r s p e c i e s .  Exogenous c y t o k i n i n s  induce e x p l a n t s t o g i v e r i s e t o buds and s h o o t s . I n d u c t i o n i s l i m i t e d by t h e e x p l a n t competence and i s presumed t o be a c h i e v e d when t h e e x p l a n t i s seen t o c o n t a i n c e l l s or groups of c e l l s d e t e r m i n e d f o r bud shoot development  ( C h r i s t i a n s o n and Warnick, 1988).  i n d u c t i o n has o c c u r r e d , t h e i n d u c e r or t h e i n d u c t i v e c o n d i t i o n s are no l o n g e r needed. Competence has been d e f i n e d as t h e c a p a c i t y t o respond t o t h e i n d u c t i v e e f f e c t s of t h e medium  and Once  79  ( C h r i s t i a n s o n and Warnick, 1988).  In t h e case o f P.  ponderosa c o t y l e d o n s , t h i s s t a t e c o u l d be m a i n t a i n e d up t o 3 days on GRF  medium a f t e r which c o t y l e d o n s which were  c u l t u r e d on GRF  medium had l o s t t h e i r competence t o form  buds and shoots and t h e r e i s no e f f e c t o f t h e c y t o k i n i n at the b i o c h e m i c a l l e v e l The  observed  (see F i g u r e s 23, 24 and  25).  changes d u r i n g c u l t u r e w i t h c y t o k i n i n  suggest an a c t i o n t o d i s r u p t or a l t e r the p h o t o s y n t h e t i c apparatus  t h a t i s c o i n c i d e n t a l w i t h expression of  organogenic  competence.  The  a l t e r a t i o n of competence  was  studied i n cotyledons c u l t u r e d with c y t o k i n i n s f o r d i f f e r e n t times  (see F i g u r e s 26, 27 and 28).  W i t h as  l i t t l e as 24 hours o f exposure, the c y t o k i n i n induced  a  response seen as the l o w e r i n g of p h o t o s y n t h e t i c p r o t e i n s and c h l o r o p h y l l f o l l o w e d by some o r g a n o g e n e s i s . a l o n g e r exposure t o c y t o k i n i n s i n c r e a s e d the response The  However, organogenic  (see F i g u r e 6 ) . r e s u l t s o f my work i n d i c a t e t h a t the f i r s t  days  o f c u l t u r e are c r i t i c a l f o r the i n d u c t i o n p r o c e s s , s u g g e s t i n g t h a t t h e c y t o k i n i n may  interact with c e l l u l a r  t a r g e t s t h a t become u n a v a i l a b l e or are degraded soon a f t e r cotyledon  excision.  C y t o k i n i n s have been r e p o r t e d t o e n t e r passively  (Van Staden e t a l . , 1986).  cells  Once i n s i d e they  b i n d t o c y t o k i n i n - b i n d i n g p r o t e i n s (see f o r example work  80  on T r i t i c u m durum by B r i n e g a r  et a l . , 1985)  which seem t o  have the d u a l f u n c t i o n of s t o r a g e p r o t e i n s and of c y t o k i n i n a v a i l a b i l i t y . i n t e r a c t w i t h RNA transcript ional The  regulators  C y t o k i n i n s are a l s o known t o  at t r a n s c r i p t i o n a l and/or p o s t levels.  i n d u c t i o n p r o b a b l y o c c u r s t h r o u g h t h e c o n t r o l of  t h e t r a n s c r i p t i o n of s p e c i f i c genes, as e v i d e n c e d by numerous r e p o r t s t h a t c y t o k i n i n s enhance, i n d u c e or suppress t h e e x p r e s s i o n L e i s n e r , 1985).  of c e r t a i n p r o t e i n s  (Chen  and  In the case of P. ponderosa, t h e  a c c u m u l a t i o n of p h o t o s y n t h e t i c marker t h a t i n d u c t i o n has  reduced  p r o t e i n s seems t o be  occurred.  a  In t h i s case, b o t h  t h e n u c l e a r and c h l o r o p l a s t coded p h o t o s y n t h e t i c  proteins  t h a t were s t u d i e d showed the same p a t t e r n of response, suggesting  t h a t the c y t o k i n i n e f f e c t was  cytoplasmic  and o r g a n e l l e l e v e l s .  The  at t h e  nuclear,  experiments  performed c o u l d not r e s o l v e t h e mechanism f u r t h e r . The  f o r e g o i n g paragraphs are t h e b a s i s f o r the  d e t a i l e d d i s c u s s i o n on a l l a s p e c t s of c y t o k i n i n e f f e c t s d u r i n g organogenesis r e p o r t e d i n t h i s t h e s i s .  1. TISSUE CULTURE Much work i n c o n i f e r t i s s u e c u l t u r e has  been  d i r e c t e d t o o p t i m i z i n g the c u l t u r e c o n d i t i o n s t h a t l e a d t o organ or embryo f o r m a t i o n  (Thorpe, 1982) .  Different  81  s p e c i e s and d i f f e r e n t t i s s u e s o f one s p e c i e s respond d i f f e r e n t l y when c u l t u r e d i n v i t r o and my e x p e r i e n c e w i t h Pinus' ponderosa i s no e x c e p t i o n .  Both t h e c y t o k i n i n s  t e s t e d i n d u c e d m u l t i p l e bud and shoot f o r m a t i o n on e x c i s e d P. ponderosa c o t y l e d o n s .  These r e s u l t s d i f f e r e d from  t h o s e r e p o r t e d by E l l i s and B i l d e r b a c k  (1984) who found  t h a t o n l y BA produced r e l i a b l e m u l t i p l e bud f o r m a t i o n and t h a t c o t y l e d o n s d i d not respond t o 2 i P ( E l l i s , c o u l d not r e p e a t E l l i s ' s to  1986).  I  r e s u l t s and t h u s i t was n e c e s s a r y  o p t i m i z e t h e c u l t u r e c o n d i t i o n s f o r t h i s system, u s i n g  b o t h c y t o k i n i n s , BA and 2 i P . P. ponderosa p r o v i d e d one major advantage, namely t h a t c o t y l e d o n s e x c i s e d from t h e same seed responded i n the  same way.  I c o u l d t h u s s t u d y up t o 8 r e p l i c a t e s u s i n g  c o t y l e d o n s from one seed, o r c o u l d s u b j e c t c o t y l e d o n s from the  same seed t o c u l t u r e under d i f f e r e n t t r e a t m e n t s i n t h e  c e r t a i n t y t h a t t h e y were g e n e t i c a l l y u n i f o r m . I o b s e r v e d t h a t t h e c o t y l e d o n s were more s e n s i t i v e to  t h e t i m e o f exposure t o c y t o k i n i n s t h a n t o a p a r t i c u l a r  n u t r i e n t medium o r t o t h e c y t o k i n i n c o n c e n t r a t i o n .  These  o b s e r v a t i o n s c o i n c i d e d w i t h t h o s e from o t h e r s p e c i e s such as P i n u s c o n t o r t a  ( P a t e l and Thorpe, 1984b), where  d i f f e r e n t i a t i o n o f shoot p r i m o r d i a and t h e i r  subsequent  development was markedly a f f e c t e d by c y t o k i n i n exposure t i m e s , and P i n u s s t r o b u s where a l o n g e r exposure t o BA  82  produced s t u n t e d s h o o t s , which e l o n g a t e d p o o r l y a f t e r subculture  (Flinn et a l . ,  1986).  Cytokinins clearly  i n h i b i t e d c o t y l e d o n e l o n g a t i o n , t h e s t u n t e d buds and shoots observed i n t h e c o t y l e d o n s c o u l d had a l s o been formed a f t e r a l o n g time o f exposure t o t h e s e growth regulators. The c o m p o s i t i o n o f t h e c u l t u r e medium can p l a y an important r o l e i n organogenesis  (Thorpe, 1980), however,  i n t h i s r e s e a r c h no s i g n i f i c a n t d i f f e r e n c e s were observed i n t i s s u e performance on t h e f o u r media t e s t e d .  Low t o t a l  and organogenic responses were observed a f t e r a l o n g exposure (25 uM)  (42 days) and a h i g h c o n c e n t r a t i o n o f c y t o k i n i n s (see F i g u r e 2 ) . Lowered c o n c e n t r a t i o n s and t h e  reduced t i m e s o f exposure t o t h e s e growth r e g u l a t o r s l e d t o an i n c r e a s e d response.  However, o f t h e f o u r media  t e s t e d , c o t y l e d o n s c u l t u r e d on LP medium (Von A r n o l d and E r i k s s o n , 1981) showed more r o b u s t buds and s h o o t s .  This  c o u l d be due t o t h e a d d i t i o n a l amino a c i d s and sugars which were p r e s e n t o n l y i n t h i s medium (see T a b l e 1 ) . A l t h o u g h c o n c e n t r a t i o n was not a s i g n i f i c a n t  factor  on bud and shoot p r o d u c t i o n i n t h e presence o f e i t h e r growth r e g u l a t o r , some d i f f e r e n c e s were observed. were c o r r e l a t e d t o t h e t i m e o f exposure  These  (see F i g u r e 5 ) ,  which was a l s o t h e o n l y v a r i a b l e t o which t h e c o t y l e d o n s showed a s i g n i f i c a n t response d i f f e r e n c e between BA and  83  2iP.  The  c o t y l e d o n s were more s e n s i t i v e t o t h e time  exposure t o BA than t o 2 i P .  T h i s was  of  a l s o t r u e f o r Pinus  c o n t o r t a where t h e exposure o f whole embryos t o BA f o r more than 3 weeks caused a g r a d u a l d e c l i n e i n t h e number of shoots formed ( P a t e l and Thorpe, 1984b).  In P i n u s  s t r o b u s embryos c u l t u r e d i n v i t r o , b o t h c y t o k i n i n s , BA 2iP,  and  were e q u a l l y c a u l o g e n i c at o p t i m a l c o n c e n t r a t i o n s ,  and shoot  formation occurred without p e r c e p t i b l e c a l l u s  production.  However, i n t h i s s p e c i e s BA was  10 t o 20  t i m e s more p o t e n t than 2 i P a f t e r 4 weeks of exposure ( F l i n n et a l . , to  1986).  The  2 i P r e p o r t e d by E l l i s  differing  l a c k of P. ponderosa  (1986), may  response  also reflect  this  sensitivity.  The organogenic  c a p a c i t y t o form buds d i m i n i s h e s i n  many c o n i f e r s soon a f t e r g e r m i n a t i o n o b s e r v a t i o n was  (Bonga, 1982).  This  c o n f i r m e d i n t h i s study o f P. ponderosa  where, o n l y 3 days a f t e r g e r m i n a t i o n , t h e showed a s i g n i f i c a n t l y lower response w i t h c y t o k i n i n from day  cotyledons  than t h o s e c u l t u r e d  0 (see F i g u r e 7 ) .  In t h e same  s p e c i e s , E l l i s and B i l d e r b a c k (1989) r e p o r t e d t h a t c o t y l e d o n s became incompetent t o form buds when embryos were i n i t i a l l y p l a c e d on GRF  medium f o r 2 days b e f o r e  b e i n g t r a n s f e r r e d t o a BA-supplemented medium.  This  e f f e c t has a l s o been r e p o r t e d f o r o t h e r species', such cucumber, where t h e e f f e c t s of BA on growth  and  as  84  c h l o r o p h y l l s y n t h e s i s a l s o d e c r e a s e d w i t h c o t y l e d o n age (Haru e t a l . ,  1982) .  C y t o k i n i n s c o n t r o l d e t e r m i n a t i o n and t h e organogenic competence o f P. ponderosa c o t y l e d o n s ( E l l i s and B i l d e r b a c k , 1989).  A l t h o u g h P . ponderosa c o t y l e d o n s d i d  not have a h i g h organogenic response t h a t formed buds and shoots)  (number o f c o t y l e d o n s  (42-47%), t h i s system c o u l d  p e r m i t t h e s e p a r a t i o n o f t h e i n d u c t i o n and d e t e r m i n a t i o n phases  ( C h r i s t i a n s o n and Warnick, 1987, 1988), s i n c e t h e  t o t a l response  (number o f c o t y l e d o n s t h a t responded by  e i t h e r g r e e n i n g o r p r o d u c t i o n o f buds and shoots) observed was 92-93% (see Table 3 ) . The h i s t o l o g i c a l , u l t r a s t r u c t u r a l and b i o c h e m i c a l a n a l y s e s r e q u i r e d s e l e c t i o n o f d e v e l o p m e n t a l l y competent e x p l a n t s b e f o r e t h e development apparent.  was m o r p h o l o g i c a l l y  I e x p l o i t e d t h e f a c t t h a t c o t y l e d o n s from t h e  same embryo behaved i n t h e same way by s e t t i n g up e v e r y t r e a t m e n t w i t h p a i r s o f c o t y l e d o n s from a s i n g l e seed. One o f t h e p a i r was removed from c u l t u r e a t t h e t e s t time and s t o r e d f r o z e n a t -80°C.  The o t h e r c o t y l e d o n was  a l l o w e d t o d e v e l o p u n t i l organogenesis was apparent. A n a l y t i c a l s t u d i e s were performed o n l y on t h e sampled p a r t n e r o f a c o t y l e d o n which e x p r e s s e d an organogenic response.  "  85  2. HISTOLOGICAL AND  ULTRASTRUCTURAL ANALYSES  S e v e r a l a u t h o r s have d e s c r i b e d the c e l l anatomy, h i s t o l o g y and u l t r a s t r u c t u r e d u r i n g development o f buds and shoots i n c o n i f e r s i n d u c e d by c y t o k i n i n s i n v i t r o . However, u l t r a s t r u c t u r a l a n a l y s i s o f p l a s t i d s i s l e s s w e l l understood  and has not been r e p o r t e d i n d e t a i l .  P.  ponderosa, l i k e o t h e r c o n i f e r s p e c i e s r e p o r t e d , formed m e r i s t e m a t i c c e n t e r s or meristemoids  which l e d t o  a d v e n t i t i o u s bud and shoot f o r m a t i o n . a GRF  When t r a n s f e r r e d t o  medium, needle p r i m o r d i a e l o n g a t e d .  developmental  This  sequence o c c u r r e d w i t h o u t c a l l u s  formation  on C K - c o n t a i n i n g medium i n a p p a r e n t l y t h e same way o t h e r s p e c i e s (Thorpe and P a t e l , As i n P i n u s r a d i a t a  1986).  ( V i l l a l o b o s e t a l . , 1985), P.  ponderosa c o t y l e d o n s , i n t h e presence  o f BA and 2 i P d u r i n g  t h e f i r s t days i n c u l t u r e , developed m e r i s t e m a t i c whose c e l l s were n o n - v a c u o l a t e d F i g u r e 12A).  Cotyledons  as i n  regions  and t i g h t l y packed  grown on GRF  (see  medium showed l i t t l e  m i t o t i c a c t i v i t y by day 5 i n c u l t u r e and an advance towards c e l l d i f f e r e n t i a t i o n and m a t u r a t i o n  (see F i g u r e  13) s i m i l a r t o t h a t r e p o r t e d from P. r a d i a t a Thorpe, 1984a).  (Patel  However, P. ponderosa c o t y l e d o n s  and  differed  from t h i s w e l l c h a r a c t e r i z e d P. r a d i a t a system i n t h a t they d i d not form the p r o m e r i s t e m o i d - l i k e structure's r e p o r t e d by V i l l a l o b o s and coworkers (1985) .  86  At the u l t r a s t r u c t u r a l l e v e l , the p l a s t i d s i n P. ponderosa c e l l s which were i n c o n t a c t w i t h the  CK-  c o n t a i n i n g medium, showed l i t t l e t h y l a k o i d development compared w i t h those i n c e l l s o f the  non-meristematic  r e g i o n and those i n non-CK t r e a t e d c o t y l e d o n s .  This delay  of i n n e r membrane f o r m a t i o n i n t h e c h l o r o p l a s t s was c h a r a c t e r i s t i c of m e r i s t e m a t i c c e l l s . i n d u c e d t h i s response. GRF  E i t h e r BA or 2 i P  P. r a d i a t a c o t y l e d o n s c u l t u r e d on  medium a l s o developed  p h o t o s y n t h e t i c apparatus  an a p p a r e n t l y f u n c t i o n a l (Kumar e t a l . ,  1988).  The  nature  and e x t e n t of c y t o k i n i n a c t i o n on p l a s t i d s has been a t t r i b u t e d t o the somewhat nebulous " p h y s i o l o g i c a l of the c o t y l e d o n s  (Longo e t a l . ,  p l a s t i d developmental  stage  For example, c h l o r o p l a s t s may  1979)  state"  and perhaps t o the  (Mikulovich et a l . ,  1981).  develop i n d i f f e r e n t ways  from p r o p l a s t i d s and e t i o p l a s t s  (Whatley,  1974),  and  d i f f e r e n t s t a g e s o f p l a s t i d s i n h i g h e r p l a n t s are r e a d i l y interconverted  (Schnepf,  1980).  A comparison of t h y l a k o i d  p r o t e i n s i n seeds and e t i o l a t e d p l a n t s of s p i n a c h showed o n l y 2 p r o t e i n s p r e s e n t i n seeds, presumably i n p r o p l a s t i d s , and 10 p r e s e n t i n e t i o p l a s t s Hauska, 1985).  (Paproth  However, some p h o t o s y n t h e t i c p r o t e i n s of  e t i o p l a s t s and c h l o r o p l a s t s of Phaseolus  v u l g a r i s L. have  been r e p o r t e d t o be i m m u n o l o g i c a l l y i d e n t i c a l al.,  1985).  and  (Radunz e t  My r e s e a r c h d i d not pursue t h e d i f f e r e n c e s  87  between e t i o p l a s t and c h l o r o p l a s t development.  However,  d i f f e r e n c e s observed between t h e two c o n i f e r systems, P. ponderosa and P. r a d i a t a , may have been because t h e P. ponderosa c o t y l e d o n s were e x c i s e d from imbibed seeds w h i l e t h o s e from P. r a d i a t a were o b t a i n e d from 5 t o 7 days o l d g e r m i n a t e d s e e d l i n g s i n t h e dark  (Villalobos,  1983).  Hence t h e e x p l a n t s were not a t t h e same d e v e l o p m e n t a l and physiological state. Unfortunately neither the q u a n t i f i c a t i o n of p l a s t i d numbers n o r t h e i r i n n e r membrane s t r u c t u r e was p o s s i b l e i n t h i s r e s e a r c h because p r o p l a s t i d numbers were d i f f i c u l t t o determine by l i g h t m i c r o s c o p y and t h e s e c t i o n s r e q u i r e d for  e l e c t r o n m i c r o s c o p y were t o o t h i n t o a l l o w an a c c u r a t e  assessment.  The same problems stopped o t h e r  microscopic  e v a l u a t i o n o f t h e development o f i n n e r membranes.  Serial  s e c t i o n i n g c o u l d p r o v i d e a p a r t i a l s o l u t i o n , b u t was i m p r a c t i c a b l e on a s c a l e l a r g e enough t o p r o v i d e s t a t i s t i c a l l y adequate i n f o r m a t i o n .  Besides  these  problems, i n t h e mature c h l o r o p l a s t t h e i n t e r n a l membrane p r o t e i n and l i p i d system i s c o n t i n u o u s l y t u r n i n g over (Whatley, 1978, S u n d q v i s t  e t a l . , 1980).  The p l a s t i d and p h o t o s y n t h e t i c apparatus development have not been p r e v i o u s l y d e s c r i b e d d u r i n g i n v i t r o organogenesis i n c o n i f e r s .  The g r e e n i n g  of e t i o l a t e d  s e e d l i n g s i n angiosperms has been w i d e l y s t u d i e d as a  88  model of c h l o r o p l a s t morphogenesis (Wellburn, W e l l b u r n et a l . , 1985).  The  1982;  e f f e c t s of c y t o k i n i n s  on  o r g a n e l l e development used c o t y l e d o n s t h a t were e x c i s e d from the s e e d l i n g and t r e a t e d w i t h growth r e g u l a t o r s e v e r a l days a f t e r onset of g e r m i n a t i o n .  Thus, b e f o r e  i n t r o d u c t i o n t o c u l t u r e the p l a s t i d s appeared as c o m p a r a t i v e l y l a r g e and w e l l d i f f e r e n t i a t e d e t i o p l a s t s .  3. MOBILIZATION OF RESERVES B e s i d e s the e f f e c t of c y t o k i n i n s on the promotion of m e r i s t e m a t i c development and  i n h i b i t i n g development of  p l a s t i d s ' i n n e r membranes, breakdown of r e s e r v e was  retarded  material  i n the c o t y l e d o n s which were c u l t u r e d i n the  p r e s e n c e of BA or 2 i P .  These o b s e r v a t i o n s  d i f f e r e d from  o t h e r r e p o r t s i n gymnosperms t h a t endogenous c y t o k i n i n s a c c e l e r a t e d the m o b i l i z a t i o n of r e s e r v e s during germination  i n the  ( J e l i c and Bogdanovic, 1988).  Endogenous c y t o k i n i n a c t i v i t y a l s o i n c r e a s e d s i t c h e n s i s C a r r i e r e and P i n u s s y l v e s t r i s L. f o l l o w i n g the i n d u c t i o n of g e r m i n a t i o n Wareing, 1979).  seeds  i n Picea seeds  (Taylor  and  However, i n a l l the s t u d i e s done w i t h  endogenous c y t o k i n i n s i t has p r o v e d d i f f i c u l t t o the e x a c t f u n c t i o n of the c y t o k i n i n s i n the process  (Van  Staden, 1983).  pinpoint  germination  89  In P. ponderosa, l i p i d s and p r o t e i n bodies were the p r i n c i p a l cotyledon r e s e r v e s i n e x c i s e d c o t y l e d o n s . 3 days i n c u l t u r e with c y t o k i n i n s , the c e l l s  still  c o n t a i n e d many l i p i d d r o p l e t s , but small vacuoles to  have r e p l a c e d p r o t e i n b o d i e s .  After  seemed  C e l l s c u l t u r e d on  GRF  medium f o r 3 days c o n t a i n e d vacuoles which occupied a l l the c e l l  volume  p r o t e i n bodies  (see F i g u r e 9).  The  almost  disappearance  of  a l s o c o r r e l a t e d with the lowered t o t a l  p r o t e i n c o n c e n t r a t i o n i n the cotyledons, which a l s o dropped f a s t e r on GRF (see F i g u r e 18).  medium than on CK-containing  T h i s suggests  medium  t h a t the most of the  p r o t e i n s present were storage p r o t e i n s and t h a t t h e i r degradation may  have proceeded more slowly on CK t r e a t e d  than on non-CK t r e a t e d c o t y l e d o n s . storage p r o t e i n s was polyacrylamide  gels  a l s o observed  The degradation on the  (see F i g u r e 19).  SDS-  Storage  s i m i l a r to those d e s c r i b e d f o r other Pinus (Gifford,  1988)  decreased  culture.  These o b s e r v a t i o n s suggested  of  proteins  species  d u r i n g the f i r s t days i n that cytokinins  r e t a r d e d the breakdown of l i p i d and p r o t e i n r e s e r v e s controlling a shift early in culture.  from normal germination This was  ( F l i n n et a l . , 1989)  a l s o observed  d u r i n g the f i r s t  where the l i p i d content  a l s o decreased  non-CK than on CK-containing  by  metabolism  i n Pinus  strobus  7 days i n c u l t u r e , more r a p i d l y ' o n  media, and t h e r e was  a delay  90  i n s t o r a g e p r o t e i n d e g r a d a t i o n on BA c o n t a i n i n g medium. In t h i s s p e c i e s a l s o t h e d i f f e r e n c e i n p r o t e i n d e g r a d a t i o n between c e l l s c u l t u r e d i n t h e presence  o r absence o f BA  was r e f l e c t e d i n t h e u l t r a s t r u c t u r e o f p r o t e i n b o d i e s . A f t e r 3 days on GRF medium, p r o t e i n b o d i e s were f l o c c u l a n t and s t a i n e d l i g h t l y whereas, on BA c o n t a i n i n g medium, p r o t e i n b o d i e s were i n t a c t and d e n s e l y s t a i n e d .  Pinus  r a d i a t a c o t y l e d o n s , from 5 d a y - o l d embryos germinated i n the dark and c u l t u r e d i n v i t r o , a l s o showed a d e c l i n e i n l i p i d and p r o t e i n b o d i e s a f t e r one day i n c u l t u r e .  They  were almost c o m p l e t e l y d e v o i d o f s t o r a g e m e t a b o l i t e s by day 5 (Douglas e t a l . ,  1982).  However, t h e o b s e r v a t i o n s  on P i n u s s p e c i e s d i f f e r e d from t h e angiosperm C i t r u l l u s v u l g a r i s , where BA a c c e l e r a t e d t h e d e g r a d a t i o n o f r e s e r v e material  (Longo e t a l . ,  1979), o r Raphanus s a t i v u s  c o t y l e d o n s , where c y t o k i n i n s a l s o a c c e l e r a t e d p r o t e i n body breakdown and t h e appearance o f a l a r g e c e n t r a l d u r i n g t h e f i r s t days a f t e r e x c i s i o n  vacuole  (Thomas e t a l . ,  1980). The low m o l e c u l a r weight p o l y p e p t i d e s observed  i n P.  ponderosa c o t y l e d o n s have been r e p o r t e d from o t h e r gymnosperm s p e c i e s ( G i f f o r d , 1988).  An i n c r e a s e d  s y n t h e s i s o f t h e s e low m o l e c u l a r weight p o l y p e p t i d e s , a f t e r 2 days i n c u l t u r e , has been r e p o r t e d f o r Pseudotsuga menziesii  (Mirb.) Franco  (Yasuda e t a l . ,  1980) which  91  r e f l e c t s t h e r e a l monomeric s t a t e o f t h e s e m o l e c u l e s and not p r o d u c t s o f p r o t e o l y t i c c l e a v a g e  (Hasegawa e t a l . ,  1979).  4. PHOTOSYNTHETIC PROTEINS AND CHLOROPHYLL T i s s u e s e n t e r i n g d i f f e r e n t developmental pathways i n v i t r o can be d i s t i n g u i s h e d by t h e i r p a t t e r n s o f p r o t e i n s y n t h e s i s and a c c u m u l a t i o n observations  (Reynold,  1989). My  o f P. ponderosa c o t y l e d o n s  provides  further  s p e c i f i c support f o r t h e developmental assumption t h a t t h e i n i t i a t i o n o f o r g a n i z e d development observed a t t h e h i s t o l o g i c a l and u l t r a s t r u c t u r a l l e v e l s , i s preceded by a s h i f t i n metabolism t o change t h e content b o t h s t r u c t u r a l and enzymatic p r o t e i n s  and spectrum o f  (Thorpe, 1980,  1982) . The  formation  o f c h l o r o p l a s t s r e s u l t s from n u c l e a r  and c h l o r o p l a s t coded p r o t e i n s  ( E l l i s , 1981, 1984).  Since  I showed c y t o l o g i c a l d i f f e r e n c e s between c o t y l e d o n s w i t h and w i t h o u t photosynthetic  CK,. i t seemed r e a s o n a b l e  that  p r o t e i n s , b o t h n u c l e a r and c h l o r o p l a s t  coded, may be a f f e c t e d by presence o f c y t o k i n i n s . observations  grown  The  o f CK e f f e c t s upon development o f i n n e r  membranes i n t h e p l a s t i d s c o i n c i d e d w i t h a changed . accumulation of the photosynthetic  polypeptides  cross-reacted with antibodies r a i s e d against  which  angiosperm  92  photosynthetic  p r o t e i n s , the c* and E> s u b u n i t s  RUBP, 33EP, CP29, LHCII and LHCI.  (see F i g u r e s  A n t i b o d y f o r CP29 c r o s s - r e a c t e d  harvesting  w i t h the  20  and  light  complex of photosystem I I , because CP29 i s  i m m u n o l o g i c a l l y r e l a t e d t o LHCII and LHCI (White Green, 1987b, Green, 1988). was  LSU-  These showed lower  l e v e l s i n the p r e s e n c e of c y t o k i n i n s 21).  of CF1,  The  chlorophyll  and  concentration  a l s o lower i n the c y t o k i n i n t r e a t m e n t compared t o  GRF-treated cotyledons  (see F i g u r e 22).  The  the  e f f e c t s of  c y t o k i n i n s i n P. ponderosa c o t y l e d o n s are s i m i l a r t o t h o s e i n other species  of gymnosperms.  For example, i n P i n u s  r a d i a t a c h l o r o p l a s t s were a l s o f u l l y developed a f t e r 3 days i n c u l t u r e i n the absence of c y t o k i n i n s Thorpe, 1984a). 1991)  In P i c e a a b i e s  the l a r g e s u b u n i t  (Patel  ( S t a b e l e t al.,  and  1990,  of RUBP, the c h l o r o p h y l l  a/b  b i n d i n g p r o t e i n and a 23 kD component of photosystem I I were s y n t h e s i z e d c y t o k i n i n pulse reduction  of embryos.  In P. ponderosa c o t y l e d o n s a  i n the l e v e l of a 52 kD p e p t i d e ,  large subunit ( E l l i s and  at a s i g n i f i c a n t l y lower r a t e a f t e r a  of RUBP, was  Judd, 1987).  detected  possibly  a f t e r o n l y one  the week  T h i s decrease of enzyme amount i s  c o n s i s t e n t w i t h a decrease i n RUBP a c t i v i t y i n c o t y l e d o n s of P i n u s r a d i a t a c u l t u r e d i n the presence of (Kumar et aJL. 1988) . cotyledons,  In P i n u s p i n a s t e r  cytokinins  cultured  the c o n t i n u o u s presence of growth  regulators  93  a l s o i n h i b i t e d t h e s y n t h e s i s of c h l o r o p h y l l s al.,  1988).  (Tranvan e t  However, i n o t h e r gymnosperm s p e c i e s  like  P i n u s n i g r a A r n o l d , l i g h t grown embryos r e a c t e d t o exogenous c y t o k i n i n s by s t i m u l a t i n g c h l o r o p h y l l ( J e l i c and Bogdanovic, 1990)  synthesis  and, i n t h e dark, c o t y l e d o n s  d i d not accumulate p r o t o c h l o r o p h y l l  (Bogdanovic and  Jelic,  198 9 ) . T h i s s t i m u l a t i o n depended on t h e t y p e o f c y t o k i n i n and on t h e c o n c e n t r a t i o n . The a l t e r e d development  of p l a s t i d i n n e r membranes  and t h e reduced a c c u m u l a t i o n o f p h o t o s y n t h e t i c p r o t e i n s i n P. ponderosa c o t y l e d o n s i n t h e presence of c y t o k i n i n s c o n t r a s t e d w i t h r e p o r t s showing a s t i m u l a t o r y e f f e c t of t h e s e growth r e g u l a t o r s i n angiosperms.  I t may be t h a t  t h e e s s e n t i a l d i f f e r e n c e between angiosperms  and  gymnosperms as f a r as c h l o r o p l a s t f o r m a t i o n i s concerned, i s t h a t t h e l a t t e r have t h e e n z y m a t i c machinery t o c a r r y t h e r e d u c t i o n of p r o t o c h l o r o p h y l l i n t h e dark and t h i s appears t o be c o n f i n e d t o t h e c o t y l e d o n s i n most gymnosperms ( K i r k and T i l n e y - B a s s e t t , 1978).  An  a l t e r n a t i v e dark enzymatic pathway, b e s i d e s t h e l i g h t mediated as i n angiosperms, has been suggested f o r c h l o r o p h y l l f o r m a t i o n i n gymnosperms (Cahay e t a l . ,  1985).  However, even i f t h e y can s y n t h e s i z e c h l o r o p h y l l i n t h e dark, they do not have p h o t o s y n t h e t i c c a p a c i t y ( W e l l b u r n , 1982, 1984), and c e r t a i n r e a c t i o n s i n t h e p h o t o s y n t h e t i c  94  e l e c t r o n t r a n s p o r t c h a i n remain i n a c t i v e d u r i n g g r e e n i n g i n darkness  ( W o l l g i e h n and P a r t h i e r , 1980).  The  s u p p r e s s i o n of c h l o r o p l a s t development i n P i c e a a b i e s would be a consequence of t h e g e n e r a l i n h i b i t i o n of s e e d l i n g m a t u r a t i o n t h a t r e s u l t s from t h e b u d - i n d u c t i o n treatment  (Stabel et a l . ,  1990).  The e x t e n t t o which p h o t o s y n t h e t i c s t r u c t u r e s c o n t r i b u t e t o events a s s o c i a t e d w i t h shoot and  bud  i n d u c t i o n i n Pinus r a d i a t a cotyledons c u l t u r e d i n v i t r o i s not known.  C e r t a i n l y t h e "dogma" i s t h a t p h o t o s y n t h e s i s  p l a y s a minor r o l e i n p r o v i d i n g carbon  skeletons  and  energy f o r m e t a b o l i c events a s s o c i a t e d w i t h shoot and induction  (Kumar e t a l . ,  1988).  bud  However, i n P. ponderosa  c o t y l e d o n s ' s t a r c h g r a i n s were d e t e c t e d i n t h e p l a s t i d s of c y t o k i n i n and GRF  c u l t u r e d c o t y l e d o n s , b o t h on medium  c o n t a i n i n g 3% s u c r o s e , s u g g e s t i n g t h a t t h e s t a r c h s y n t h e s i z i n g machinery i s a c t i v e d u r i n g i n v i t r o In P i n u s r a d i a t a c o t y l e d o n s c u l t u r e d i n v i t r o  culture.  the  c h l o r o p h y l l and c a r o t e n o i d c o n t e n t s i n t h e c u l t u r e d c o t y l e d o n s were lower than i n germinated (Douglas  et a l . ,  1982).  seedlings  The net p h o t o s y n t h e t i c r a t e o f  p l a n t l e t s i n v i t r o i s reduced when c u l t u r e d on a medium c o n t a i n i n g sugar  (Kozai et a l . ,  1988).  I t appears t h a t c a r b o h y d r a t e s organ-forming  p l a y two  roles"in  t i s s u e s , as a carbon and energy source  and  95  as an osmotic agent (Thorpe,  1980).  Sucrose  applied to  c u l t u r e d spinach c e l l s d i r e c t l y i n h i b i t e d c h l o r o p h y l l synthesis  (Dalton and S t r e e t , 1977), w h i l e CO2  enrichment  o f Cymbidium p l a n t l e t s i n v i t r o promoted p h o t o s y n t h e s i s (Kozai e t a l . ,  1990).  C0  2  and e t h y l e n e , which b u i l t up i n  P i n u s r a d i a t a c o t y l e d o n s d u r i n g t h e f i r s t 10 t o 15 days i n c u l t u r e , promoted morphogenesis, and e x c e s s i v e accumulation  a f t e r bud i n i t i a t i o n caused some degree o f  dedifferentiation  (Kumar e t a l . ,  i n d e e d does occur a t a reduced cultured i n the l i g h t  1987).  Photosynthesis  l e v e l i n green m a t e r i a l  (Hughes, 1981).  T h i s low l e v e l o f  p h o t o s y n t h e s i s can i n f l u e n c e growth and morphogenesis i n vitro-.  A d d i t i o n a l l y , l i g h t may have o t h e r e f f e c t s  the a c t i v a t i o n o f pigment r e c e p t o r s o t h e r than associated d i r e c t l y with photosynthesis,  through  those  through  photoperiod e f f e c t s or through i n t e n s i t y of l i g h t  (Hughes,  1981). L i g h t and c y t o k i n i n do not i n t e r a c t i n p l a s t i d d i f f e r e n t i a t i o n a t t h e same m o l e c u l a r s i t e s o r i n t h e same m e t a b o l i c pathway ( P a r t h i e r , 1979).  I t i spossible to  suggest however, t h a t t h e r e a r e some common s t e p s i n a metabolic chain of reactions p r o v i d i n g the a c t i v a t i o n of t h e p l a s t i d rRNA s y n t h e s i s by BA and by l i g h t ( M i k u l o v i c h et a l . ,  1981) .  96  5. PROTEIN AND  CHLOROPHYLL AS ORGANOGENIC MARKERS.  S i n c e t h e organogenic response was more s e n s i t i v e t o t h e time of exposure t o c y t o k i n i n s  (see F i g u r e s 5 and 6)  and t o the d e l a y of c y t o k i n i n exposure a f t e r g e r m i n a t i o n (see F i g u r e 7 ) , t h e s e two v a r i a b l e s were a l s o t e s t e d a t the biochemical l e v e l .  To c o r r e l a t e t h e organogenic  response of t h e c o t y l e d o n s and t h e a b i l i t y of c y t o k i n i n s t o induce a r e d u c t i o n on t h e a c c u m u l a t i o n of p h o t o s y n t h e t i c p r o t e i n s , t h e s e were used as markers of two i m p o r t a n t t i m i n g e v e n t s : t h e minimum exposure time t o c y t o k i n i n s needed t o induce a response, and t h e d u r a t i o n of the "developmental window" a f t e r which c o t y l e d o n s l o s e t h e i r competence t o respond t o t h e c y t o k i n i n a f t e r c u l t u r e on growth r e g u l a t o r f r e e  (GRF) medium.  These experiments  demonstrated t h a t t h e competence o f t h e c o t y l e d o n s t o respond t o t h e c y t o k i n i n i s l o s t a f t e r 5 days g e r m i n a t i o n (see F i g u r e 7) or c u l t u r e on GRF medium.  However, t h e  c y t o k i n i n can s t i l l decrease t h e p r o t e i n c o n c e n t r a t i o n s a f t e r 3 days g e r m i n a t i o n (see F i g u r e 2 4 ) . My r e s u l t s i n d i c a t e d t h e CK must be i n t r o d u c e d on or b e f o r e t h e t h i r d day of c u l t u r e on GRF medium t o induce a r e d u c t i o n of p h o t o s y n t h e t i c p r o t e i n a c c u m u l a t i o n (see F i g u r e 24).  However, t h e changes i n c h l o r o p h y l l  levels  o n l y showed t h e same p a t t e r n as t h e p r o t e i n c o n c e n t r a t i o n a t days 9 and 10 f o r BA but not f o r 2 i P t r e a t m e n t (see  97  F i g u r e 2 5 ) . Organogenic responses  t o t h e growth  r e g u l a t o r s d i f f e r e d , t h e c o t y l e d o n s were more s e n s i t i v e t o the time o f exposure t o BA than t o 2 i P (see F i g u r e 6 ) . Changes i n t o t a l p r o t e i n c o n c e n t r a t i o n i n c o t y l e d o n s a l s o c o r r e l a t e d w i t h t h e s e o b s e r v a t i o n s , b e i n g h i g h e r when a CK exposure was d e l a y e d f o r 1 o r 3 days (see F i g u r e 23) than when t h e d e l a y was f o r 5 days. c o n c e n t r a t i o n i n GRF-treated than i n CK-treated cotyledons  The t o t a l p r o t e i n  c o t y l e d o n s was always lower (see F i g u r e 1 8 ) . These  biochemical observations c o r r e l a t e d with responses  organogenic  o f e x c i s e d c o t y l e d o n s c u l t u r e d on B A - c o n t a i n i n g  medium a f t e r 3 days o f embryo growth on GRF medium (see F i g u r e 7 ) . These r e s u l t s c o i n c i d e d w i t h o b s e r v a t i o n s by E l l i s and B i l d e r b a c k (1989) w i t h t h e same s p e c i e s .  They  r e p o r t e d t h a t an e a r l y exposure t o BA was n e c e s s a r y f o r c o t y l e d o n s t o r e t a i n t h e i r competence f o r response by f o r m i n g de novo buds.  t o BA  A d e l a y e d BA exposure o f 24 h  s i g n i f i c a n t l y reduced t h e number o f buds and meristemoids produced by t h e c o t y l e d o n s , whereas a 48 h d e l a y l e d t o a l o s s o f c o t y l e d o n a r y competence t o form buds and meristemoids. C y t o k i n i n s can a l s o induce a r e d u c t i o n o f p h o t o s y n t h e t i c p r o t e i n a c c u m u l a t i o n and o f c h l o r o p h y l l a f t e r as l i t t l e as 24 hours o f exposure (see F i g u r e s 27 and 28).  The l o n g e r exposure t o BA (5 days) r e s u l t e d i n a  98  higher p r o t e i n concentration t h i s higher accumulation  (see F i g u r e 27),  although  at day 5 c o u l d be due t o the  uneven d i s t r i b u t i o n of p r o t e i n bands formed when r u n n i n g the g e l .  A t r e n d towards a lower  c o n c e n t r a t i o n was e i t h e r BA or 2 i P  chlorophyll  observed w i t h 5 days of exposure t o (see F i g u r e 28).  p r o t e i n c o n c e n t r a t i o n was  The t o t a l  cotyledon  higher i n CK-treated  cotyledons  compared w i t h o n l y a 1-5  day exposure t o CK  26).  suggest t h a t a l o n g e r exposure t o  CKs  These o b s e r v a t i o n s  (see F i g u r e  r e t a r d s more e f f i c i e n t l y the breakdown of  proteins  (see F i g u r e 18).  These r e s u l t s c o r r e l a t e d w i t h  organogenic responses of the c o t y l e d o n s exposure t o CKs  storage  i n which a l o n g e r  (more t h a n 5 days) i n d u c e d  a higher  organogenic response and a h i g h e r mean buds and (see F i g u r e 6) t h a n w i t h 3 days of exposure. Bilderback  shoots  Ellis  and  (1989) w o r k i n g w i t h the same s p e c i e s observed  t h a t the i n d u c t i o n of bud and shoot f o r m a t i o n  occurred  soon a f t e r embryo e x c i s i o n , and t h a t t h i s s t i m u l u s became f u l l y o p e r a t i o n a l i n the f i r s t 48 h. are c o n s i s t e n t w i t h my  These  observations that  observations  photosynthetic  p r o t e i n s were a l s o v e r y s u s c e p t i b l e t o r e g u l a t i o n by  CK,  as r e f l e c t e d i n bud and shoot f o r m a t i o n , d u r i n g the  first  3 days of c u l t u r e .  The  first  1-3  days of exposure t o  i seem t o be t h e most c r i t i c a l time p e r i o d frame f o r  CK  99  i n d u c t i o n of b i o c h e m i c a l changes t o the p h o t o s y n t h e t i c apparatus. A s i m i l a r response i n angiosperms i s known from Lemna g i b b a i n which BA must be p r e s e n t f o r more t h a n hour t o s t i m u l a t e the LHC  one  and SSU-RUBP mRNA l e v e l s .  However, when BA i s p r e s e n t f o r 8 h, the s t i m u l a t i o n i s n e a r l y as g r e a t as a f t e r 24 h ( F l o r e s and Tobin, In  1987).  cucumber a f t e r 48 h under i l l u m i n a t i o n , BA produced an  a d d i t i v e e f f e c t on c o t y l e d o n growth but had almost no f u r t h e r e f f e c t on c h l o r o p h y l l s y n t h e s i s (Haru e t a l . , 1982).  The  organogenic  r a t e o f macromolecule s y n t h e s i s was  greater i n  c u l t u r e s of Solanum c a r o l i n e n s e L., and  "organogenic  the  p o l y p e p t i d e s " are produced i n r e g e n e r a t i n g  e x p l a n t s w i t h i n the f i r s t day o f c u l t u r e , w e l l b e f o r e t h e r e are any s i g n s of d i f f e r e n t i a t i o n  (Reynold,  For a comprehensive u n d e r s t a n d i n g  1989) .  of the h i g h l y  complex i n t e r r e l a t i o n s between photomorphogenesis hormonal r e g u l a t i o n , i t w i l l be n e c e s s a r y  and  t o examine t h e  i n t r a c e l l u l a r c o n c e n t r a t i o n s o f the hormones and  their  compartmentalization  In o r d e r  to  understand  transduced  how  (Zimmermann e t a l . ,  1987).  the growth r e g u l a t o r s t i m u l u s i s  i n t o observed  p h y s i o l o g i c a l responses,  i t is  e s s e n t i a l t o e l u c i d a t e the f u n c t i o n of any p r o t e i n t h a t i s shown t o b i n d a growth r e g u l a t o r (Napier and V e n i s ,  1990).  100  I t i s a l e g i t i m a t e concern of f i e l d b i o l o g i s t s t o ask, "what r e l e v a n c e has the study o f i n v i t r o c u l t u r e t o understanding  o f t r e e growth e i t h e r i n the f i e l d o r d u r i n g  in vivo studies?". A long-standing issue i n plant biology i s the s i g n i f i c a n c e of v e g e t a t i v e r e p r o d u c t i o n i n n a t u r a l propagation. L o l i u m , may  Some genera,  such as Rubus, T r i f o l i u m  and  r e l y on v e g e t a t i v e p r o p a g a t i o n f o r much of  t h e i r reproduction.  S e v e r a l gymnosperms, i n c l u d i n g  e s p e c i a l l y Sequoiadendron, J u n i p e r u s and s e v e r a l of the more shrubby t a x a , can be propagated layering.  s u c c e s s f u l l y by  The e c o n o m i c a l l y i m p o r t a n t t r e e forms of P i n u s ,  L a r i x , P i c e a and A b i e s are not e a s i l y propagated  by  this  or o t h e r a s e x u a l methods. While a r t i f i c i a l p r o p a g a t i o n i s easy f o r s p e c i e s t h a t use v e g e t a t i v e r e p r o d u c t i o n i n v i v o , t h e  attainment  of c l o n a l p r o p a g a t i o n from mature c o n i f e r s remains a g o a l for  conifer biotechnology.  The mature seed c o t y l e d o n  has  been adopted as a model f o r t h e study o f t h e fundamental mechanism of shoot i n i t i a t i o n  from  non-meristematic  vegetative conifer explants. I have demonstrated t h a t t h e f o r m a t i o n of m e r i s t e m a t i c r e g i o n s by exogenous c y t o k i n i n i s preceded  or  i s c o i n c i d e n t a l w i t h lower a c c u m u l a t i o n of p h o t o s y n t h e t i c  101  p o l y p e p t i d e s and c h l o r o p h y l l and a l t e r e d t h y l a k o i d membrane development o f m e r i s t e m a t i c c e l l s which were on the c o n t a c t s i d e between t h e c o t y l e d o n and t h e w i t h CKc o n t a i n i n g c u l t u r e medium.  There i s a c o r r e l a t i o n between  the t i m i n g o f lowered p h o t o s y n t h e t i c p r o t e i n  accumulation,  the u l t r a s t r u c t u r a l a l t e r a t i o n o f p l a s t i d s , and t h e organogenic  competence o f t h e c o t y l e d o n s e x c i s e d from P.  ponderosa seeds.  There i s a narrow 'window o f competence'  d u r i n g t h e f i r s t 1-3 days a f t e r g e r m i n a t i o n ,  i n which  shoots can be i n i t i a t e d i n e x c i s e d c o t y l e d o n s by a p p l i c a t i o n o f exogenous c y t o k i n i n .  The t a s k remains t o  extend t h i s window or t o f i n d methods o f r e - o p e n i n g explants of developmentally  young t i s s u e  i t in  (e.g. emerging  l e a v e s ) from mature t r e e s so t h a t c l o n i n g o f mature s t o c k can be  achieved.  CONCLUSIONS  C y t o k i n i n s induce t h e f o r m a t i o n o f m e r i s t e m a t i c r e g i o n s i n c o t y l e d o n s o f P i n u s ponderosa c u l t u r e d i n vitro.  A d e l a y o f i n n e r membrane f o r m a t i o n i s observed i n p l a s t i d s of these meristematic  A r e d u c t i o n on t h e a c c u m u l a t i o n p o l y p e p t i d e s was observed  regions.  of s i x photosynthetic  on t h e c y t o k i n i n t r e a t e d  cotyledons.  CP2 9 and LSU-RUBP can be used as markers o f t h e organogenic  induction.  C y t o k i n i n s induce an i n h i b i t o r y response o f t h e p h o t o s y n t h e t i c p o l y p e p t i d e s a f t e r 24 h o f exposure.  I f t h e c y t o k i n i n i s not p r e s e n t d u r i n g t h e f i r s t 3 days a f t e r g e r m i n a t i o n , t h e r e i s no i n d u c t i o n o f t h e i n h i b i t o r y response.  .  S i n c e c o t y l e d o n s can respond  or not o r g a n o g e n i c a l l y ,  the p r o c e s s e s o f i n d u c t i o n and competence can s t u d i e d i n t h i s system.  be  104  SUGGESTIONS  B i o c h e m i c a l and m o l e c u l a r s t u d i e s c o n c e r n i n g t h e e a r l y changes i n t h e system s h o u l d c o n c e n t r a t e on t h e f i r s t 24 h o f c u l t u r e . Related t o the l i g h t - c y t o k i n i n i n t e r a c t i o n ,  dark-  i  grown c o t y l e d o n s t r e a t e d w i t h c y t o k i n i n s c o u l d be used as a c o n t r o l t o s e p a r a t e events c y t o k i n i n v e r s u s  light  dependent. C h l o r o p l a s t development c o u l d be f o l l o w e d d u r i n g t h e development o f l e a f p r i m o r d i a a f t e r t r a n s f e r r i n g t h e c o t y l e d o n s from C K - c o n t a i n i n g medium t o GRF medium. The  f u n c t i o n o f t h e low m o l e c u l a r  p o l y p e p t i d e s which were p r e s e n t through  weight a l l t h e time i n  c u l t u r e s h o u l d be s t u d i e d . The  c y t o k i n i n r e c e p t o r s c o u l d be a good i n d i c a t i o n  to separate c y t o k i n i n s e f f e c t at the nuclear,  cytoplasmic  or o r g a n e l l e l e v e l . F u t u r e r e s e a r c h u s i n g p h o t o s y n t h e t i c p o l y p e p t i d e s as markers o f organogenesis  c o u l d be d i r e c t e d towards a  better optimization of the culture, using  lower  c o n c e n t r a t i o n s o f c y t o k i n i n s and d i f f e r e n t time o f exposure.  105  REFERENCES AITKEN J . , HORGAN K.J. and THORPE T.A. 1981. 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