Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

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

Item Metadata

Download

Media
831-UBC_1991_A1 M48.pdf [ 11.1MB ]
Metadata
JSON: 831-1.0100367.json
JSON-LD: 831-1.0100367-ld.json
RDF/XML (Pretty): 831-1.0100367-rdf.xml
RDF/JSON: 831-1.0100367-rdf.json
Turtle: 831-1.0100367-turtle.txt
N-Triples: 831-1.0100367-rdf-ntriples.txt
Original Record: 831-1.0100367-source.json
Full Text
831-1.0100367-fulltext.txt
Citation
831-1.0100367.ris

Full Text

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. S c , Universidad Nacional Autonoma de Mexico, 1984 M.Sc, Universidad Nacional Autonoma de Mexico, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1991 ©Alicia Mazari 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. ^ . . , BOTANY Department of The University of British Columbia Vancouver, Canada D a t e AUGUST 2 , 1991 DE-6 (2/88) ABSTRACT The c y t o k i n i n s , b e n z y l a d e n i n e and 2 - i s o p e n t e n y l aden ine , promoted the i n v i t r o f o r m a t i o n o f m e r i s t e m a t i c c e n t e r s which l e d to bud and shoot p r o d u c t i o n on e x c i s e d c o t y l e d o n s o f P i n u s ponderosa D o u g l . The organogenic response was dependent on the t ime o f exposure to these growth r e g u l a t o r s . D i f f e r e n c e s between m e r i s t e m a t i c , n o n - m e r i s t e m a t i c r e g i o n s , and 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 medium, were observed by day 5 i n c u l t u r e . P l a s t i d s i n newly m e r i s t e m a t i c c e l l s showed p o o r l y deve loped t h y l a k o i d membranes and some g r a n a , whereas c e l l s i n n o n -m e r i s t e m a t i c t i s s u e s 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 had w e l l d e v e l o p e d i n n e r membranes, more t h y l a k o i d membranes and grana than p l a s t i d s o f m e r i s t e m a t i c c e l l s . A d e c l i n e i n t o t a l p r o t e i n content was d e t e c t e d d u r i n g the f i r s t days i n c u l t u r e . The d e c l i n e was more r a p i d on growth r e g u l a t o r f r e e than on 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 i s t r e n d c o n t r a s t e d w i t h c h l o r o p l a s t markers ( 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 a s s o c i a t e d w i t h p h o t o s y n t h e s i s ) , which were p r e s e n t i n lower c o n c e n t r a t i o n s i n 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 than i n those c u l t u r e d i n growth r e g u l a t o r f r e e medium. Both benzyladenine and 2-isopentenyl 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 of at least two photosynthetic polypeptides i n cotyledons i n the f i r s t 24 hours i n culture. The a b i l i t y of cotyledons to respond i n t h i s way to cytokinins was l o s t after only three days i n culture i n growth regulator free medium. RESUMEN Las c i t o c i n i n a s , benciladenina y 2-isopentenil adenina, promovieron l a formaci6n de centros meristematicos i n v i t r o , los cuales a su vez produjeron brotes y yemas en cotiledones de Pinus ponderosa Dougl. La respuesta organogenica es dependiente del tiempo de exposici6n a estos reguladores de crecimiento. Se observaron diferencias entre las regiones meristematica, no meristematica y t e j i d o de cotiledones cultivados en ausencia de reguladores de crecimiento, despues de 5 dias en c u l t i v o . Los p l a s t i d i o s de l a regi6n meristematica mostraron membranas t i l a c o i d e s poco desarrolladas y algunos grana, mientras que e l t e j i d o no meristematico y e l t e j i d o cultivado s in ci t o c i n i n a s , mostr6 membranas internas bien desarrolladas con mas membranas t i l a c o i d e s y grana que los p l a s t i d i o s de celulas meristematicas. Se observ6 una disminuci6n del contenido de proteina t o t a l durante los primeros dias en c u l t i v o , siendo mas rapido en cotiledones cultivados en ausencia de reguladores de crecimiento que en presencia de ci t o c i n i n a s . Estas observaciones contrastaron con los marcadores de cloroplastos ( c l o r o f i l a y 5 polipeptidos i v asociados con f o t o s i n t e s i s ) , que mostraron una concentraci6n menor en cotiledones tratados con cit o c i n i n a s respecto a aquellos cultivados en medio s i n reguladores de crecimiento. Ambas cit o c i n i n a s , benciladenina y 2-isopentenil adenina, inhibieron l a acumulaci6n de a l menos dos polipeptidos f o t o s i n t e t i c o s en' cotiledones durante las primeras 24 horas en c u l t i v o . La habilidad de los cotiledones para responder de esta forma a las ci t o c i n i n a s se perdi6 en tan solo tres dias en cu l t i v o en medio sin reguladores de crecimiento. v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v i LIST OF TABLES v i i i LIST OF FIGURES ix ABBREVIATIONS x i i ACKNOWLEDGEMENTS x i i i 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 20 1.2. CULTURE METHODS 21 1.3. CULTURE CONDITIONS 23 2. HISTOLOGICAL AND ULTRASTRUCTURAL ANALYSES 2.1. LIGHT MICROSCOPY 24 2.2. TRANSMISSION ELECTRON MICROSCOPY 24 3. PROTEIN AND CHLOROPHYLL ANALYSES 3.1. PROTEIN EXTRACTIONS 25 3.2. TOTAL PROTEIN ANALYSIS 25 3.3. SDS POLYACRYLAMIDE GEL ELECTROPHORESIS . 2 6 3.4. IMMUNOBLOTTING 27 3.5. CHLOROPHYLL DETERMINATION . ... 28 v i 4. PROTEIN AND CHLOROPHYLL AS ORGANOGENIC MARKERS . 30 5. STATISTICAL ANALYSIS 30 RESULTS 1. TISSUE CULTURE 31 1.1. CULTURE MEDIA 31 1.2. GROWTH REGULATOR CONCENTRATIONS 34 1.3. TIME OF EXPOSURE 37 2. HISTOLOGICAL AND ULTRASTRUCTURAL ANALYSES 40 3. PROTEIN AND CHLOROPHYLL ANALYSES 57 3.1. TOTAL PROTEIN DETERMINATION 57 3.2. SDS POLYACRYLAMIDE GEL ELECTROPHORESIS . 60 3.3. IMMUNOBLOTTING 63 3.4. CHLOROPHYLL DETERMINATION 65 4. PROTEIN AND CHLOROPHYLL AS ORGANOGENIC MARKERS . 68 4.1. CULTURE ON GROWTH REGULATOR FREE MEDIUM FOLLOWED BY TRANSFER TO CYTOKININ-CONTAINING MEDIUM 68 4.2. CULTURE ON CYTOKININ-CONTAINING MEDIUM FOLLOWED BY TRANSFER TO GROWTH REGULATOR FREE MEDIUM 73 DISCUSSION 78 1. TISSUE CULTURE 80 2. HISTOLOGY AND ULTRASTRUCTURE 85 3. MOBILIZATION OF RESERVES 8 8 4. PHOTOSYNTHETIC PROTEINS AND CHLOROPHYLL 91 5. PROTEIN AND CHLOROPHYLL AS ORGANOGENIC MARKERS . 96 CONCLUSIONS 102 SUGGESTIONS 104 BIBLIOGRAPHY 105 v i i LIST OF TABLES Page 1. Culture media composition 22 2. Culture conditions 41 3. Responses of Pinus ponderosa cotyledons cultured 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 v i i i L I S T OF FIGURES Page 1. M u l t i p l e b u d s a n d s h o o t s 32 2. 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 m e d i a 33 3. 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 on LP medium 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 , on LP o r SH medium f o r 10 d a y s 36 5. 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 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 BA o r 2 i P f o r 10 a n d 42 d a y s 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 on LP o r SH medium a f t e r d i f f e r e n t t i m e o f e x p o s u r e t o c y t o k i n i n s 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 f r o m g e r m i n a t e d e m b ryos 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 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 (day 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 day 3 i n c u l t u r e i n t h e p r e s e n c e o r a b s e n c e o f c y t o k i n i n s 45 10. 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 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 11. 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 a n d n o n -m 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 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 4 6 i x 12. Micrographs of cotyledons at 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. Micrographs of cotyledons at day 5 i n c u l t u r e on GRF medium 50 14. Micrographs of cotyledons at day 10 i n c u l t u r e i n the presence of c y t o k i n i n s 52 15. Transmission e l e c t r o n micrographs of p l a s t i d s of cotyledons at day 10 i n c u l t u r e i n the presence of c y t o k i n i n s 55 16. Micrographs of cotyledons at day 10 i n c u l t u r e i n GRF medium 56 17. L i g h t micrograph of a cotyledon at day 21, c u l t u r e d i n the presence of 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 to a growth r e g u l a t o r f r e e (GRF) medium 58 18. T o t a l p r o t e i n concentration of cotyledons c u l t u r e d on GRF medium, or i n the presence of BA or 2iP 59 19. SDS-PAGE of cotyledons c u l t u r e d on GRF or BA co n t a i n i n g media from days 0 t o day 10 61 20. Immunoblots of photosynthetic p r o t e i n s , CF1, LSU-RUBP, 33EP, CP2 9, LHCII and LHCI, of cotyledons 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, with 15 uM benzyladenine (BA), or 2-isopentenyl adenine ( 2 i P ) , and c o n t r o l (C) 64 21. LSU-RUBP concentration of c y t o k i n i n (CK) versus growth r e g u l a t o r f r e e (GRF) t r e a t e d cotyledons c u l t u r e d i n v i t r o 66 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 concentrations, of BA or 2iP t r e a t e d cotyledons and those c u l t u r e d on GRF medium 67 23. T o t a l p r o t e i n concentration of cotyledons c u l t u r e d on GRF medium f o r 1, 3, or 5 days and then t r a n s f e r r e d to BA or 2iP c o n t a i n i n g media, compared with GRF t r e a t e d cotyledons .. 69 x 24. Immunoblots of LSU-RUBP and CP29, of cotyledons 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 to BA or 2iP co 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 C h l o r o p h y l l b concentrations of cotyledons c u l t u r e d i n the absence of c y t o k i n i n s f o r 1, 3 and 5 days and then t r a n s f e r r e d to BA or 2iP 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 concentration of cotyledons c u l t u r e d on BA or 2iP c o n t a i n i n g media f o r 1, 3 or 5 days and then t r a n s f e r r e d t o GRF medium, compared with BA or 2iP t r e a t e d cotyledons 74 27. Immunoblots of LSU-RUBP and CP29, of cotyledons c u l t u r e d on BA or 2iP c o n t a i n i n g media and then t r a n s f e r r e d to 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 C h l o r o p h y l l b concentrations of cotyledons c u l t u r e d i n the presence of BA or 2iP f o r 1, 3 or 5 days and then t r a n s f e r r e d to GRF medium 7 6 x i ABBREVIATIONS APP M - apparent molecular mass BA = N^-Benzyladenine or 6-Benzylaminopurine CF1 = coupling 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 = antennal component of photosystem I I DMSO = dimethyl s u l f o x i d e 33EP = 33kD e x t r i n s i c polypeptide FW = f r e s h weight GRF = growth r e g u l a t o r f r e e 2iP = N 6-[2-isopentenyl]adenine or 6-(5,S-Dimethyl a l l y l a m i n o purine) LSU-RUBP = l a r g e subunit 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 = polyacrylamide g e l e l e c t r o p h o r e s i s RUBP = Ribulose-1,5-bisphosphate carboxylase SDS = sodium dodecyl sulphate SE = standard e r r o r of the mean TRIS = T r i s [hydroxymethyl] aminomethane x i i ACKNOWLEDGEMENT S I would l i k e t o acknowledge r e c e i p t o f a gradu a t e s c h o l a r s h i p from my c o u n t r y t h r o u g h t h e U n i v e r s i d a d N a c i o n a l Aut6noma de Mexico. To Dr. 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 Dr. E d i t h L. Camm f o r a l l h er h e l p and a d v i c e w i t h t h e p r o t e i n a n a l y s i s . To M i c h a e l Weis f o r h i s h e l p w i t h t h e m i c r o s c o p i c a l work. To Dr. Dane R o b e r t s , Dr. Denis Lavender, Dr. Anthony G l a s s , Dr. N e i l Towers and Dr. D a v i d Webb f o r t h e i r s u p p o r t and i n t e r e s t i n my academic 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 and s u g g e s t i o n s . To Dr. K a r l D. H a u f f e f o r h i s s u g g e s t i o n s . To E v e l y n L e a f and Susan McDougall 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 who somehow h e l p e d and s u p p o r t e d me d u r i n g a l l t h e s e y e a r s . To t h e Montana Department o f S t a t e Lands f o r t h e g i f t o f ponderosa p i n e seed. x i i i A MIS PAPAS x i v 1 INTRODUCTION The mechanisms of c y t o k i n i n a c t i o n during growth and organ d i f f e r e n t i a t i o n of i n t a c t p l a n t s are not w e l l understood (Tanimoto and Harada, 1982) but i n the l a s t 40 years t i s s u e c u l t u r e techniques have emerged as a t o o l to study growth r e g u l a t o r e f f e c t s . In order to study the r e g u l a t i o n of organized 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, 1982). Cy t o k i n i n s are r e q u i r e d to induce bud and shoot formation i n c o n i f e r s i n v i t r o . L i t t l e i s understood about the b i o l o g i c a l e f f e c t s of these compounds upon t h e i r t a r g e t c e l l s . However, many authors have noted an i n t e r a c t i o n w ith a b i n d i n g receptor (Napier and Venis, 1990) and changes i n genomic expression (e.g. Akoyunoglou and Argyroudi-Akoyunoglou, 1985; Chen and Le 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 biochemical approaches have been used to e l u c i d a t e some metabolic events during d i f f e r e n t i a t i o n i n c u l t u r e d c o n i f e r t i s s u e s . Only i n Pinus r a d i a t a D. Don has the experimental approach been focused towards a systematic s t r u c t u r a l , biochemical and p h y s i o l o g i c a l study of s e v e r a l aspects of d i f f e r e n t i a t i o n (e.g. Kumar et a l . , 1988, 1987; V i l l a l o b o s et a l . , 1985, 2 1984; P a t e l and Thorpe, 1984a; Douglas et a l . , 1982; A i t k e n et a l . , 1981; Yeung et a l . , 1981). They showed that the i n i t i a t i o n of organized development i n v i t r o i n c l u d e d changes at the 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 . However, no p a r t i c u l a r emphasis has been d i r e c t e d towards the development of the p l a s t i d . L i t t l e work, other than t h a t of Thorpe and coworkers, has been done to r e l a t e changes i n metabolism to the a c q u i s i t i o n of developmental determination or the l o s s of competence to form shoots i n c o n i f e r t i s s u e c u l t u r e . Reports of the e f f e c t s of growth r e g u l a t o r s on the development of the photosynthetic apparatus are fragmentary and c o n t r a d i c t o r y when comparing responses of angiosperm and gymnosperm species. Zimmermann and coworkers (1987) reported t h a t treatment of Cucumis  s a t i v u s L. cotyledons with benzyladenine (BA) increased the content and a c t i v i t y of RUBP whereas Stabel and coworkers (1991) reported a suppressed accumulation of the same enzyme i n P i c e a abies (L.) Karst embryos by BA treatment. C h l o r o p h y l l accumulation due to the a c t i o n of c y t o k i n i n s has been reported i n s e v e r a l angiosperms (Mikulovich et a l . , 1981) 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 treatment of Pinus p i n a s t e r A i t . cotyledons 3 i n h i b i t e d c h l o r o p h y l l synthesis (Tranvan et a l . , 1988). These observations i n c o n i f e r s have not been r e l a t e d to the morphological development of the p l a s t i d or the photosynthetic apparatus. The t h e s i s of the research presented i n t h i s document i s that 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 e x c i s e d cotyledons of Pinus ponderosa Dougl. c u l t u r e d i n v i t r o w i l l c o n t r i b u t e to the understanding 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 understanding of c o n i f e r micropropagation. The r e l a t i o n s h i p s among the i n d u c t i o n of organogenic events, s u b c e l l u l a r changes and the development of the photosynthetic apparatus i n c y t o k i n i n t r e a t e d c o n i f e r t i s s u e s have not been e x t e n s i v e l y i n v e s t i g a t e d . I addressed these issues by i n v e s t i g a t i n g the e f f e c t s of the c y t o k i n i n s benzyladenine (BA) and 2-isopentenyl 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 during i n v i t r o organogenesis. The u l t r a s t r u c t u r a l observations of p l a s t i d development were r e l a t e d at the biochemical l e v e l to s e l e c t e d photosynthetic p r o t e i n s and to c h l o r o p h y l l . The photosynthetic p r o t e i n s and c h l o r o p h y l l appeared to have p o t e n t i a l use as markers of c y t o k i n i n i n d u c t i o n f o r the t i m i n g of organogenic events. 4 LITERATURE REVIEW 1. CONTROL OF ORGANIZED DEVELOPMENT IN TISSUE CULTURE Knowledge of c o n i f e r b i o l o g y and f o r e s t biotechnology has increased during the l a s t few years through the use of t i s s u e c u l t u r e techniques. Tissue c u l t u r e has been proposed as a method with 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 propagation i n v i t r o can occur v i a somatic embryogenesis or organogenesis (Thorpe, 1982). The common procedure used f o r c l o n a l propagation i n many c o n i f e r species remains the i n d u c t i o n of ad v e n t i t i o u s shoots (e.g. Von Arnold 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 et a l . , 1984). Shoot organogenesis occurs when u n i p o l a r bud primordia from t i s s u e explants are i n i t i a t e d . These develop i n t o shoots but must be induced to form roots (Thorpe and P a t e l , 1984). The process of somatic embryogenesis r e q u i r e s the presence of both auxins and c y t o k i n i n s but the i n d u c t i o n of shoot organogenesis occurs i n the presence of c y t o k i n i n s only. This need f o r a s i n g l e exogenous growth r e g u l a t o r permits the study of a c y t o k i n i n i n d u c t i o n process without i n t e r a c t i o n with other growth r e g u l a t o r s . The organogenic process can be d i v i d e d i n t o three p h y s i o l o g i c a l stages: 1. the a b i l i t y of a c e l l or t i s s u e 5 to respond to an inducer; 2. the process of i n d u c t i o n per  se which occurs under the i n f l u e n c e of exogenous growth r e g u l a t o r s ; 3. the morphological 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 ( C h r i s t i a n s o n and Warnick, 1987, 1988). A c e l l i s i n a 'permissive' or 'target' c o n d i t i o n when i t can recognize s i g n a l s that i n i t i a t e d i f f e r e n t i a t i o n (Osborne, 1984). Other than the r e a c t i o n to s p e c i f i c s i g n a l s , there are at l e a s t two requirements f o r de novo organized development: c e l l d i f f e r e n t i a t i o n , and c e l l i n t e r a c t i o n . The i n i t i a t i o n of organized development i s a complex morphogenic phenomenon (Thorpe, 1982). Both e x t r i n s i c and i n t r i n s i c f a c t o r s play a r o l e . Organized development i n v i t r o can be regulated by manipulation of the c u l t u r e medium composition, the c u l t u r e environment, and by s e l e c t i o n of an explant that i s i n an appropriate responsive s t a t e (Thorpe, 1980; Thorpe and B i o n d i , 1981) . This l a s t f a c t o r has a s i g n i f i c a n t bearing on shoot p r o d u c t i v i t y (Aitken et a l . , 1981). For example, the response of the embryos cannot be equated to the response observed i n e x c i s e d explants such as cotyledons. In s p i t e of recent major advances, the most s u i t a b l e j u v e n i l e explant i n c o n i f e r s must s t i l l be determined e m p i r i c a l l y , as there are no p r e d i c t i v e methods f o r explant s e l e c t i o n (Thorpe and P a t e l , 1986). 6 In the angiosperm C i c e r arietinum L. (Pino et a l . , 1991) embryos and exc i s e d cotyledons were not equivalent 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 : the absence of the embryonic a x i s markedly reduced the degradation of p r o t e i n s . In general growth r e g u l a t o r s are synthesized w i t h i n the embryonic a x i s and t h e r e a f t e r pass to the storage t i s s u e (Ashton, 1976) . Wounding may increase the endogenous l e v e l of a c t i v e c y t o k i n i n s (Crane and Ross, 1986) . Manipulation of the 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 normally quiescent c e l l s which become organogenic, and i t i s assumed that 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 of m a t e r i a l s by or from the medium i n t o the t i s s u e have been i m p l i c a t e d i n determining the l o c a t i o n of primordia i n i t i a t i o n (Thorpe, 1 9 8 2 ) . However, mechanistic explanations are s p e c u l a t i v e . During i n v i t r o organogenesis, c e l l s have become competent to express 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 of s e l e c t i v e gene a c t i v i t y that manifests 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 the c u l t u r e d t i s s u e s which can be described 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, 1980, 1982; Thorpe and B i o n d i , 1981) . Tissue 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 complicated by the 7 unknown content and p h y s i o l o g i c a l a c t i v i t y of endogenous growth r e g u l a t o r s ( J e l i c and Bogdanovic, 1990). The exact r o l e s of c y t o k i n i n s w i l l only become c l e a r when t h e i r metabolic pathway, t h e i r a c t i o n s i t e ( s ) and the molecular mechanisms i n v o l v e d i n t h e i r a c t i v i t y are c h a r a c t e r i z e d (McGaw and Horgan, 1985) . Even without knowledge of p r e c i s e endogenous l e v e l s of p l a n t growth r e g u l a t o r s , c o n t r o l of development can be achieved by exogenous a p p l i c a t i o n s which regulate macromolecular s y n t h e s i s , carbohydrate 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 that can be c o r r e l a t e d with 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 of the developmental sequence from seed explants to buds and shoots i n v i t r o has been described f o r s e v e r a l c o n i f e r s . Among the species that have been described h i s t o l o g i c a l l y are Pinus  r a d i a t a (Yeung et a l . , 1981; V i l l a l o b o s et a l . , 1985), Pinus strobus L. ( F l i n n , 1987; F l i n n et a l . , 1988), Pinus  p i n a s t e r (Tranvan et a l . , 1988), Pinus contorta Dougl. ex Loud, Pinus r i g i d a M i l l . , P i c e a engelmannii Parry (Thorpe and P a t e l , 1986), Pinus e l d a r i c a (Wagley et a l . , 1987), P i c e a abies (Von Arnold and Gronroos, 1986), P i c e a mariana 8 B.S.P. and Pic e a glauca (Moench) Voss (Rumary et a l . , 1986). In a l l these species the formation of adventitous shoots i n v o l v e s the development of meristemoids. The meristemoids are formed by s p e c i f i c planes of c e l l d i v i s i o n which leads to the formation of 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 ith prominent n u c l e i . Densely s t a i n i n g cytoplasm, and microvacuolation are al s o c h a r a c t e r i s t i c features of meristemoid c e l l s (Thorpe, 1980). In a l l species examined, the meristemoids and meristematic regions o r i g i n a t e d from e i t h e r epidermal and/or immediately subepidermal l a y e r s of the explant. The meristemoids developed i n t o bud primordia and f i n a l l y to a d v e n t i t i o u s shoots with a p i c a l domes and needle primordia. The developmental sequence occurred i n the absence of c a l l u s formation on the c y t o k i n i n (CK)-co 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 occurred at d i f f e r e n t s i t e s i n various explants, or at d i f f e r e n t times and s i t e s w i t h i n the same explant. The reasons behind the d i f f e r e n t i a l behaviour of c e l l s i n contact with the medium i n the embryonic and cotyledonary explant are not c l e a r (Thorpe and P a t e l , 1986). C e l l s i n shoot-forming regions have been examined at the u l t r a s t r u c t u r a l l e v e l . Few u l t r a s t r u c t u r a l s t u d i e s have de a l t with c o n i f e r s c u l t u r e d i n v i t r o . P l a s t i d 9 morphology has not been examined by e l e c t r o n m i c r o s c o p y (Fowke, 1986) i n any c o n i f e r d u r i n g o r g a n o g e n i c e v e n t s . In angiosperms, t h e most s t u d i e d genera where p l a s t i d morphology has been examined a r e Hordeum, PhaseoluS/ Avena, Cucumis, V i c i a , Zea, T r i t i c u m and Pisum. These s t u d i e s have been f o c u s e d on g r e e n i n g o f e t i o l a t e d s e e d l i n g s , where t h e u l t r a s t r u c t u r a l changes i n most s p e c i e s a r e s i m i l a r . The main d i f f e r e n c e s between s p e c i e s o c c u r i n t h e r a t e o f p l a s t i d morphogenesis. E t i o p l a s t s c o n t a i n i n g h i g h l y c r y s t a l l i n e p r o l a m e l l a r b o d i e s and few u n p e r f o r a t e d s t r o m a l l a m e l l a e are r a p i d l y p h o t o t r a n s f o r m e d . The l a m e l l a e d e v e l o p i n t o g r a n a l i n i t i a l s which s u b s e q u e n t l y form r e c o g n i z a b l e g r a n a l s t a c k s ( W e l l b u r n , 1 9 8 2 ) . R e b e i z and R e b e i z (1985) have demonstrated 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 i s an i n t e g r a l p a r t o f 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 p h o t o p e r i o d i c growth c o n d i t i o n s . The e t i o p l a s t s o f angiosperms a l r e a d y c o n t a i n s u b s t a n t i a l amounts of most of t h e enzymes and o t h e r c o n s t i t u e n t s o f t h e c h l o r o p l a s t . C h l o r o p l a s t s d e v e l o p not o n l y from e t i o p l a s t s but a l s o from p r o p l a s t i d s (Whatley, 1974) . These two d e v e l o p m e n t a l pathways cannot be c o n s i d e r e d e q u i v a l e n t . The r e g u l a t i o n o f p l a s t i d and n u c l e a r gene e x p r e s s i o n may change d u r i n g t h y l a k o i d membrane b i o g e n e s i s ( E s k i n s e t a l . , 1 9 8 9 ) . 10 3. CYTOKININS Cy t o k i n i n s , compounds f i r s t recognized by t h e i r a b i l i t y to 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 , are now known to induce a d i v e r s i t y of responses i n p l a n t s . The various 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 or storage forms, d e t o x i f i c a t i o n products, d e a c t i v a t i o n products (formed to reduce c y t o k i n i n a c t i v i t y l e v e l s ) or i n a c t i v a t i o n products (formed as a r e s u l t of t h e i r u t i l i z a t i o n ) (Letham and P a l n i , 1983). Transient changes i n c y t o k i n i n l e v e l s occur during both the -breaking of dormancy and the process of germination i n seeds (e.g. J e l i c and Bogdanovic, 1990; Pino et a l . , 1991). I t i s d i f f i c u l t to p i n p o i n t the exact f u n c t i o n of these growth r e g u l a t o r s i n the germination process (Van Staden, 1983) . This problem a l s o e x i s t s during the organogenic process i n c o n i f e r s . C y t o k i n i n s t r u c t u r e has not been r e l a t e d to the molecular a c t i o n ( s ) of these growth r e g u l a t o r s ( P a r t h i e r , 1979). They are d e r i v a t i v e s of the n u c l e i c a c i d purine adenine and t h e i r a c t i o n may be connected with RNA and p r o t e i n 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 species of angiosperms have been reported. For example, i n Lemna gibba L. p l a n t s grown i n darkness, c y t o k i n i n s r e g u l a t e the expression of RNA at 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 of RNA (Flores and Tobin, 1986), or i n h i b i t i n g the degradation of s p e c i f i c mRNAs (Tobin and Turkaly, 1982). Petunia hybrida M i t c h e l l c u l t u r e s respond to c y t o k i n i n treatment by accumulating RNA (Funckes-Shippy & Levine, 1985), which r e s u l t s from the t r a n s c r i p t i o n of nuclear-encoded genes. These genes code f o r polypeptides that f u n c t i o n w i t h i n the 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 are 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 order to describe the e f f e c t of 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 necessary to describe some aspects of the formation of c h l o r o p l a s t s , a complex developmental process r e s u l t i n g from an i n t e r p l a y between the nuclear and p l a s t i d genomes. Some c h l o r o p l a s t p r o t e i n s are synthesized i n s i d e the developing o r g a n e l l e , many others are synthesized i n the cytoplasm ( E l l i s , 1981; 1984). For example, the 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 the c h l o r o p l a s t and polypeptide components encoded by nuclear DNA and synthesized i n the cytoplasm (Bennett et a l . , 1984). The expression of genes coding p l a s t i d p r o t e i n s may be regulated at many l e v e l s , from t r a n s c r i p t i o n to the assembly and i n t e g r a t i o n of 12 t h e i r p r o t e i n products (Buetow, 1985). In order to understand c h l o r o p l a s t development i t i s important to study both nuclear 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 i n v o l v e s synthesis of c h l o r o p h y l l s and t h e i r precursors 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 to p r o t e i n and l i p i d content. Carotenes and other substances are al s o necessary f o r the f u n c t i o n of the mature c h l o r o p l a s t (Sundqvist et a l . , 1980) . Growth r e g u l a t o r s may exert two types of e f f e c t s on the greening of higher p l a n t s , i n d i r e c t l y v i a the p l a s t i d membranes or d i r e c t l y on some of the 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 Rebeiz, 1985) . Ch l o r o p l a s t development i n va s c u l a r p l a n t s can al 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 (Mullet, 1988) and water s t r e s s (Sundqvist et a l . , 1980). Very l i t t l e i s known about the fundamental endogenous re g u l a t o r s of 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 APPARATUS The hormonal r e g u l a t i o n of c h l o r o p l a s t development i s a l s o complex. At l e a s t four exogenously s u p p l i e d c l a s s e s of 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 inc 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 detected inside the chloroplast (Sundqvist et. a l . , 1980). Cytokinins seem to control the operation of s p e c i f i c genes i n both chloroplast and nuclear genomes i n plant c e l l s (Mikulovich et a l . , 1981) . Some species, for example Secale cereale L. (Feierabend and De Boer, 1978), seem to require more cytokinin for p l a s t i d rather than for cytoplasmic synthesis of ch l o r o p l a s t - s p e c i f i c proteins. For example, RUBP, the large subunit of which i s formed i n the p l a s t i d , showed a greater response to changed cytokinin lev e l s than did chloroplast enzymes of cytoplasmic o r i g i n . The formation of the small subunit of RUBP seemed to be less sensitive to a cytokinin deficiency than was the formation of the complete enzyme (Feierabend and De Boer, 1978). Differences observed i n p l a s t i d formation i n di f f e r e n t species could be due to differences i n the genetic basis of the plant, the physical conditions, 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 into account for a l l studies of chloroplast formation (Sestak, 1985). The requirement of cytokinins for chloroplast 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 subcellular effects (Peaud-Lenoel and Axelos, 1981). They may control s p e c i f i c events, either inside the organelles or i n the cytoplasmic compartment (Lescure and Seyer, 1981). They 14 increase 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; Feierabend and De Boer, 1978) . C y t o k i n i n e f f e c t s have been reported 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 processes (Akoyunoglou and Argyroudi-Akoyunoglou, 1985; P a r t h i e r et a l . , 1985; F l o r e s and Tobin, 1988). They can enhance, induce or suppress the expression of c e r t a i n p r o t e i n s (Chen and L e i s n e r , 1985). In tobacco c e l l suspension c u l t u r e s under continuous l i g h t , the b i o s y n t h e s i s of some nuclear DNA-encoded p l a s t i d p r o t e i n s was found to be regulated by c y t o k i n i n s (Teyssendier de l a Serve et a l . , 1985a). I t has been reported that c y t o k i n i n s s t i m u l a t e grana formation i n the cotyledon c h l o r o p l a s t s of s e v e r a l species of angiosperms, i n c l u d i n g Cucumis s a t i v u s (Harvey et a l . , 1974), Cucurbita pepo L. (Mikulovich et a l . , 1981; Lerbs et a l . , 1984), N i c o t i a n a tabacum L. (Teyssendier 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 (Bracale et a l . , 1988). In dark grown suspension c u l t u r e s of Petunia hybrida, a combination of l i g h t and BA a l s o s t i m u l a t e d greening and the formation of t h y l a k o i d s ( C o l i j n et al _ . , 1982) . The 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 suspension c u l t u r e s was s t r o n g l y reduced i n the absence of c y t o k i n i n s . The d i f f e r e n t i a t i o n of mature c h l o r o p l a s t s under continuous l i g h t occurred 15 only i n c y t o k i n i n supplemented medium (Lescure and Seyer, 1981). In c y t o k i n i n - d e p l e t e d leaves of Secale cereale the 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 smaller than 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 to 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 considered to be developmentally equivalent to other t i s s u e s of the same species ( 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 to d i f f e r from organ to organ (Haru et a l . , 1 9 8 2 ) . 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 anelles and i n t a c t c e l l s cannot be c o r r e l a t e d to c u l t u r e d c e l l responses. For example, BA enhanced the i n organello p r o t e i n synthesis a c t i v i t y of e t i o p l a s t s of 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 (Ohya and Suzuki, 1990). There are numerous examples of the e f f e c t of 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. In Cucurbita pepo cotyledons ( P a r t h i e r et a l , 1985) both BA and l i g h t r e g u l a t e d RUBP gene expression. In Lemna gibba c y t o k i n i n s s t i m u l a t e d the accumulation of mRNA coding f o r the small subunit of RUBP and LHCII i n the dark (Flores and Tobin, 1987, 1988). In 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 to be st 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 c e l l s synthesized mRNA encoding the small subunit of RUBP much e a r l i e r than i n growth r e g u l a t o r - s t a r v e d c e l l s . Larger amounts of RUBP carboxylase were produced i n the cytokinin-supplemented c u l t u r e (Axelos, et a l . , 1987). I t has been shown that c y t o k i n i n s not only a f f e c t angiosperm p l a s t i d s t r u c t u r e but a l s o increase c h l o r o p h y l l accumulation (Mikulovich et a l . , 1981). In Raphanus  s a t i v u s ( L i c h t e n t h a l e r and Buschmann, 1978) k i n e t i n not only enhanced c h l o r o p h y l l accumulation but 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 ) formation i n e t i o l a t e d s e e d l i n g s . 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 of photosynthetic enzymes i n s e v e r a l species of angiosperms. In Cucumis s a t i v u s cotyledons, BA increased s p e c i f i c a c t i v i t y and content of RUBP i n both dark (Harvey et a l . , 1974) and l i g h t (Zimmermann et a l . , 1987). In the same species, BA i n darkness s t i m u l a t e d the synthesis of a 39 kD and s e v e r a l membrane polypeptides lower than 34 kD (Ohya and Suzuki, 1990). In exc i s e d 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 synthesis (Lerbs et a l . , 1984), increased i t s content and st i m u l a t e d a c t i v i t y of the enzyme ( P a r t h i e r et a l . , 1985). In e t i o l a t e d Secale cereale 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 increased a f t e r k i n e t i n treatment 17 (Feierabend and De Boer, 1978). This increase may have been due to a number of factors including stimulation of synthesis, reduction i n the rate 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 stimulated the synthesis of some polypeptides i n dark grown cultures, as well as emulating or enhancing many of the l i g h t - t r i g g e r e d processes (Ohya et a l . , 1986; Ohya and Suzuki, 1990). However, in C i t r u l l u s vulgaris cotyledons (Marziani-Longo et. aJ., 1990), cytokinins could only p a r t i a l l y replace the l i g h t requirement for LHC synthesis. LHC-mRNA, as well as the protein, appeared i n the dark i n the presence of cytokinins, but apparently were not associated with thylakoids. It i s l i k e l y that the f a i l u r e of LHC to integrate into the thylakoids i n excised C i t r u l l u s  vulgaris cotyledons was due to the lack of chlorophyll. Chlorophyll i s known to s t a b i l i z e the association of these proteins with the membranes (Apel and Kloppstech, 1980). Light increased the content and a c t i v i t y of RUBP in excised cotyledons of Cucumis sativus. Its effectiveness decreased with increasing cytokinin concentration. Saturation of BA was reached at lower concentrations i n the l i g h t than i n the darkness, indicating an interaction between photocontrol and cytokinin regulation (Zimmermann et a l . , 1987) . Cytokinins and phytochrome did not act 18 through the same mechanism (Flores and Tobin, 1 9 8 6 ) . The combination of light-stimulated mRNA synthesis and i n h i b i t i o n of mRNA degradation by cytokinins could account for the synergism often observed between l i g h t and cytokinin (Tobin and Turkaly, 1982) . The close c o r r e l a t i o n between the physiological state of the tissue or c e l l and the eff e c t of cytokinins on the greening process would support an in d i r e c t mode of action on the synthesis and accumulation of chlorophyll by the growth regulator (Parthier, 1979) . 5. INTERACTIVE EFFECTS OF CYTOKININS AND LIGHT Considerable progress i s being made i n understanding how l i g h t , an exogenous regulatory factor, modulates p l a s t i d d i f f e r e n t i a t i o n (Bennett et a l . , 1 9 8 4 ) . The mechanism of the light-induced conversion of the e t i o l a t e d p l a s t i d s to chloroplasts requires not only de novo synthesis of lamellar proteins and photosynthetic enzymes, but synthesis of several types of RNA (Wollgiehn and Parthier, 1980) . Cytokinins are required for bud induction i n Pinus  radiata, but the action of both cytokinin and l i g h t are required for subsequent primordium formation (Thorpe, 1 9 8 8 ) . Light affects organogenic events and i t also plays a predominant role i n chloroplast d i f f e r e n t i a t i o n . 19 The t i m i n g and extent of c h l o r o p l a s t development and gene expression vary depending on the developmental str a t e g y of the organism and i t s use of environmental s i g n a l s (Mullet, 1988). L i g h t a f f e c t s c h l o r o p l a s t development by a c t i n g on various photoreceptors which produces responses to various wavelengths of l i g h t . The best c h a r a c t e r i z e d photomorphogenic system i s that 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 to c h l o r o p l a s t s depends not only on an exogenous c y t o k i n i n supply but a l s o on l i g h t (Peaud-Lenoel and Axelos, 1981). Cy t o k i n i n s cannot replace l i g h t i n inducing the f i n a l stages of c h l o r o p l a s t development, pigment accumulation and f u l l d i f f e r e n t i a t i o n of t h y l a k o i d s (Bracale 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 steps i n the s y n t h e s i s , t r a n s p o r t and assembly of a m a j o r i t y of c h l o r o p l a s t p r o t e i n s . Rather i t i s s t i m u l a t o r y at s e v e r a l d i f f e r e n t l e v e l s ( E l l i s , 1981). 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 complex i s one of the few c h l o r o p l a s t components whose accumulation i n t h y l a k o i d s r e q u i r e s continuous i l l u m i n a t i o n . 20 MATERIALS AND METHODS 1. TISSUE CULTURE 1.1. PLANT MATERIAL Pinus ponderosa seeds from western Montana were s u p p l i e d by the Montana Department of State Lands, F o r e s t r y D i v i s i o n , Missoula, MT, USA. Seeds were imbibed i n running tap water f o r 24 h and stored at 4°C f o r three days.. They were surface s t e r i l i z e d i n 20% commercial bleach (approximately 1 % w/v NaOCl) f o r 15 min and r i n s e d three times i n s t e r i l e d i s t i l l e d water. Seed coats were removed, the embryos were separated from the megagametophyte, and exc i s e d cotyledons were placed 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 contained i n p e t r i dishes. Embryos were germinated 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 ex c i s e d from these embryos at d i f f e r e n t stages of 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 or c u l t u r e d on c y t o k i n i n (CK)-containing medium f o r 10 days, then t r a n s f e r r e d to a GRF medium. An advantage of the P. ponderosa system was that each seed had between 7 and 12 cotyledons. Thus, 21 c o t y l e d o n s from t h e same seed c o u l d be used t o st u d y responses by g e n e t i c a l l y u n i f o r m t i s s u e . The 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 as: ( 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 buds and s h o o t s , as a pe r c e n t a g e o f t o t a l c o t y l e d o n s c u l t u r e d . ( i i ) Organogenic response = number o f c o t y l e d o n s t h a t formed buds and shoots as a p e r c e n t a g e o f t o t a l c o t y l e d o n s c u l t u r e d . ( i i i ) Mean buds and shoots = t h e mean number o f buds and shoots formed p e r o r g a n o g e n i c a l l y r e s p o n d i n g c o t y l e d o n . These measurements were made e i t h e r 35 or 42 days a f t e r c u l t u r e i n i t i a t i o n o f e x c i s e d c o t y l e d o n s . A l l r e s u l t s were based upon measurements from a t l e a s t 50 c o t y l e d o n s p e r c u l t u r e t r e a t m e n t . 1.2. CULTURE METHODS The f o u r c u l t u r e media used (Table 1) are i d e n t i f i e d t h r o u g h o u t t h i s t h e s i s as: DCR (Gupta and Durzan, 1985), LP (Von A r n o l d and E r i k s s o n , 1981), MCM (Bornman, 1983) and SH (Schenk and H i l d e b r a n d t , 1972). The l e t t e r b e s i d e each c h e m i c a l (Table 1) c o r r e s p o n d s t o t h e s u p p l i e r : (a) Amachem ( P o r t l a n d , OR), (b) Baker ( P h i l l i p s b u r g , N J ) , (c) Baker and Adamson ( M o r r i s t o w n , N J ) , (d) BDH (Toronto, 22 TABLE 1. Culture media composition (mg/L) MACRONUTRIENTS KNO3 MgS0 4•7H 20 NH 4H 2P0 4 C a C l 2 NH 4N0 3 KH 2P0 4 CH 4N 20 Ca(N0 3) 2'4H 20 NH 4S0 4 KC1 MICRONUTRIENTS MnS0 4 1 MnS04• H3BO3 ZnSO/i 'H20 '4H20 •7H20 7H 20 KI CuS0 4-5H 20 NaMo04•2HoO CoCl 2'6H 2D N i C l 2 IRON FeS0 4 Na2EDTA ZnEDTA EDTA VITAMINS Thiamine HCl N i c o t i n i c A c i d P y r i d o x i n e HCl Myo-Inositol Glycine Pantothenate F o l i c A c i d B i o t i n SUGARS D-Glucose D-Xylose L-Arabinose AMINOACIDS d b d g d a f d b f c c b c e h h g c d b b b h e h d d h h h b d d h d L-Glutamine L-Alanine L-Cysteine HCl J1 L-Arginine L-Leucine L-Phenylalanine e L-Tyrosine e h e SH LP MCM DCR 2500 1900 2000 340 400 370 250 370 300 - - -150.9 135.9 - 6.42 - 1200 - 400 - 340 270 170 - - 150 -- - 500 556 - - 400 -— — 150 — 10 0.17 22.3 - 2.2 - -5 0.63 1.5 6.2 1 - 3 8.6 1 0.75 0.25 0.83 0.2 0.0025 0.025 0.25 0.1 0 . 025 0.25 0 .25 0.1 0.0025 0.025 0.025 - — — 0.025 15 14 15 . 27.8 20 - 20 37.3 - 4.05 - -- 19 - -5 5 1.7 1 5 2 0.6 0.5 0.5 1 1.2 0.5 1000 100 90 200 - 2 2 2 - - 0.5 -- - 1.1 -— — 0.12 — 180 - 150 - -— 150 — — 0.40 - 0.05 - -- 0.02 - -- 0.01 - -- 0.01 - -- 0.01 - -- 0.01 - -23 O n t a r i o ) , (e) Calbiochem (La J o l l a , CA), (f) F i s h e r ( F a i r Lawn, NJ) (g) MCB ( C i n c i n n a t i , OH), (h) Sigma (St. Louis, MO). Each medium contained 3% (w/v) sucrose and 0.8% (w/v) D i f c o bacto-agar, and was adjusted to a f i n a l pH of 5.8 before a u t o c l a v i n g at 16 p s i (120°C) f o r 15 min. D i f f e r e n t concentrations (0, 1, 5, 15 and 25 uM) and d i f f e r e n t times of exposure to c y t o k i n i n s , e i t h e r benzyladenine (BA) or 2-isopentenyl adenine (2iP) were t e s t e d . The t r a n s f e r d e t a i l s f o r each experiment are provided i n the legends of the f i g u r e s and t a b l e s of r e s u l t s . In most experiments p a i r s of cotyledons from the same seed were t r e a t e d i d e n t i c a l l y so that one of each p a i r could be harvested and sto r e d frozen at -80°C while i t s partner was grown on to ensure that the developmental response was observed. The e a r l y harvested member of the p a i r was discarded i f i t s partner d i d not develop, ensuring the a n a l y s i s of developmentally competent explants. 1.3. CULTURE CONDITIONS A l l c u l t u r e s were maintained i n a growth chamber at 27 + 2°C with 16 h photoperiods provided by f l u o r e s c e n t l i g h t s ( P h i l i p s c o o l white 34 W) with an approximate — 9 — 1 photon fluence r a t e of 80 umol'm ^•s ±. 24 2. HISTOLOGICAL AND ULTRASTRUCTURAL ANALYSIS 2.1. LIGHT MICROSCOPY Cotyledons were harvested 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) glutaraldehyde i n 0.1 M sodium cacodylate b u f f e r (pH 7.3) f o r 3 h at room temperature. Specimens were p o s t f i x e d f o r 2 h at room temperature i n 1% (w/v) osmium t e t r o x i d e , dehydrated i n a graduated ethanol 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 to propylene oxide, and embedded i n Spurr 1 s -resin- (Spurr, 1969) . Sections (0.5 um) were cut using g l a s s knives and s t a i n e d with 0.05 % (w/v) t o l u i d i n e blue 0 i n 1 % (w/v) sodium t e t r a b o r a t e . Sections at day 0 were a l s o s t a i n e d f o r p r o t e i n with 1% a n i l i n e blue black i n 7 % (v/v) aqueous a c e t i c a c i d (Jensen and F i s h e r , 1968). Sections were examined and photographed using a L e i t z Dialux compound microscope. 2.2. TRANSMISSION ELECTRON MICROSCOPY Sections from cotyledons c u l t u r e d i n the presence or absence of CKs f o r 0, 5 and 10 days were f i x e d and embedded as described 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 with 2% (w/v) aqueous urany l acetate and Sato's lead c i t r a t e (Hayat, 25 1989), and examined and photographed using a Zeiss EM 10A transmiss i o n e l e c t r o n microscope operating at 60 kV. 3. PROTEIN AND CHLOROPHYLL ANALYSES Cotyledons were harvested and stored at -80°C p r i o r to p r o t e i n and c h l o r o p h y l l analyses. 3.1. 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 cotyledons were weighed and homogenized i n a 0.2 mL micro t i s s u e g r i n d e r with 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 (pH 6.8), 10 % (w/v) g l y c e r o l , 9 % (w/v) SDS, 5% (v/v) 2-JJ-mercaptoethanol and 0.125 % (w/v) bromophenol blue] per 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 at 13, OOOxg f o r 10 min and denatured by heating at 100°C f o r 7 min. 3.2. TOTAL PROTEIN ANALYSIS To t a l p r o t e i n a n a l y s i s was performed using the modified method of Ghosh et a l . (1988), i n which a known volume of supernatant p r o t e i n e x t r a c t was a p p l i e d to 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 blue R, destained i n 40% methanol:10% a c e t i c a c i d , and d r i e d . Each spot was cut out, the 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 of the e x t r a c t s o l u t i o n was determined at 595 nm. A l l p r o t e i n concentrations were determined by reference to a c a l i b r a t i o n curve prepared with bovine serum albumin s o l u t i o n s and were expressed as ug protein/mg f r e s h weight. 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 per lane] were separated by SDS-PAGE i n 10-15 % polyacrylamide gradient gels c o n t a i n i n g 424 mM T r i s - H C l (pH 9.8), with 5% s t a c k i n g g e l c o n t a i n i n g 125 mM T r i s - H C l (pH 6.8) (Chua 1980). The gels were run i n a Protean I I slab g e l apparatus (Bio-Rad, Richmond, CA, USA) at 25 mA f o r 1.5 h and at 35 mA f o r another 4.5 h. The running b u f f e r contained 15 g/L T r i s , 72 g/L g l y c i n e and 5 g/L SDS. Fol l o w i n g f i x a t i o n i n 40% methanol:10% a c e t i c a c i d , the gel s were s t a i n e d with 0.25 % (w/v) Coomassie b r i l l i a n t blue R, destained i n 40% methanol:10% a c e t i c a c i d , photographed using an Ektachrome tungsten f i l m (Kodak) and scanned at 560 nm using a Beckman DU-64 spectrophotometer. Apparent molecular weights were c a l c u l a t e d by reference to the standard p r o t e i n mixture, SDS-7 (Sigma Chemical Co., St. Louis, MO, USA). 27 3.4. IMMUNOBLOTTING Pr 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 gels to n i t r o c e l l u l o s e (Bio-Rad) by e l e c t r o b l o t t i n g f o r 45 min at 2.5 mA/cmz i n a P o l y B l o t apparatus (American B i o n e t i c s , Hayward, CA, USA). Transfer u n i t s were assembled i n the f o l l o w i n g sequence (Kyhse-Andersen, 1984): a f i l t e r paper soaked i n anode b u f f e r # 1 (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); the 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; the g e l ; and three f i l t e r papers soaked i n cathode s o l u t i o n (25 mM T r i s , 40 mM 6-aminohexanoic a c i d , 20 % methanol, pH 9.4). Immunoblotting was performed using antigen-antibody complexes which were v i s u a l i z e d using g o a t - a n t i r a b b i t l i n k e d to a l k a l i n e phosphatase (White and Green 1987a). The b l o t s were coated with 3% (v/v) f i s h s k i n 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 Na 2HP0 4, 15 mM KH 2P0 4) f o r 1 h. B l o t s were incubated i n antiserum i n the same s o l u t i o n f o r 1 h, then washed twice i n phosphate b u f f e r e d s a l i n e plus 0.05 % (v/v) Tween and once i n phosphate b u f f e r e d s a l i n e , each wash t a k i n g 10 min. This was fol l o w e d by inc u b a t i o n f o r 1 h with a l k a l i n e phosphatase conjugated to goat a n t i - r a b b i t antibody i n b l o c k e r and another two washes i n phosphate-bu f f e r e d s a l i n e f o r 10 minutes each. B l o t s were then 28 washed i n 50 mM T r i s - H C l pH 8.0 followed by a d d i t i o n of substrate [0.1 % (w/v) disodium naphthol AS-MX phosphate, 0.2 % (w/v) f a s t red TR s a l t (both from Sigma Co., St. Louis, 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 the 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 towels (White, 1987) . Six photosynthetic polypeptides were i d e n t i f i e d using r a b b i t p o l y c l o n a l antibodies at d i f f e r e n t d i l u t i o n s . These in c l u d e d the <* and ft subunits of the c h l o r o p l a s t coupling f a c t o r (CF1) (1:400) which appear as a s i n g l e band; the larg e subunit of ribulose-1,5-bisphosphate carboxylase (LSU-RUBP) (1:400); the 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 with the oxygen-evolving complex (33EP) (1:200) (Camm et a l . , 1987); an antennal component of 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 the l i g h t - h a r v e s t i n g complex of 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 the l i g h t h a r v e s t i n g complex of photosystem I (LHCI) (1:400) (White and Green, 1987a). 3.5. CHLOROPHYLL DETERMINATION The c h l o r o p h y l l concentration was determined by the method reported by Hiscox and Israelstam (1979). 29 Cotyledons were weighed (2 to 3 mg), placed i n t o 100 uL DMSO, p r e v i o u s l y warmed to 65°C, f o r 10 min, incubated f u r t h e r f o r 60 min at 65°C, and then removed from the sol v e n t . The c h l o r o p h y l l i n the r e s u l t i n g s o l u t i o n s was determined by measuring absorbance at 663 and 645 nm against a DMSO blank. 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 to t i s s u e f r e s h weight (Arnon, 1949). i ) T o t a l c h l o r o p h y l l concentration = [20.2 D 6 4 5 + 8.02 D 6 6 3] X V/1000 X w i n ug/mg f r e s h weight i i ) C h l o r o p h y l l 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 i i i ) C h l o r o p h y l l 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 at the wavelength s t a t e d V = t o t a l volume of the c h l o r o p h y l l s o l u t i o n W = weight of the 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 used to study the organogenic e f f e c t of c y t o k i n i n s 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 repeated at l e a s t 3 times. An a n a l y s i s of variance was performed on r e s u l t s obtained from the t i s s u e c u l t u r e , the t o t a l p r o t e i n , LSU-RUBP and c h l o r o p h y l l concentrations analyses. A l l experimental data were compared f o r s t a t i s t i c a l d i f f e r e n c e at p r o b a b i l i t y (P) ^ 0 . 0 5 . 31 RESULTS 1. TISSUE CULTURE Both BA and 2iP induced m u l t i p l e bud and shoot formation on e x c i s e d P. ponderosa cotyledons c u l t u r e d i n v i t r o (Figure 1A). Outgrowth was observed 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 I B ) . A f t e r the cotyledons were t r a n s f e r r e d to a GRF medium, l e a f primordia developed (Figure IC) and elongated (Figure ID) a f t e r s e v e r a l weeks. Excised cotyledons 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 elongated. Results i n t h i s s e c t i o n are from experiments designed to s e l e c t the most appropriate c u l t u r e medium, optimal concentrations of exogenous BA or 2iP, and optimal exposure time to these growth r e g u l a t o r s . 1.1. CULTURE MEDIA Figure 2 shows the t o t a l response (percentage of cotyledons that responded e i t h e r by greening or forming buds and shoots), the organogenic response (percentage of cotyledons that formed buds and shoots), and the mean buds and shoots formed per cotyledon 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 2iP 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 primordia (C) formed i n a cotyledon a f t e r c u l t u r e on CK-containing medium f o r 10 days and t r a n s f e r r e d to a GRF medium. Elongated l e a f primordia (D) a f t e r 8 weeks of 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 SH CULTURE MEDIA FIGURE 2. T o t a l response + standard e r r o r of the mean (SE), organogenic response + SE, and mean buds and shoots + SE, of cotyledons c u l t u r e d on d i f f e r e n t media i n the presence of 25 uM BA or 2iP 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 the e f f e c t s of the c y t o k i n i n s , although the t o t a l and the organogenic responses were always higher f o r 2 i P - t r e a t e d cotyledons c u l t u r e d on DCR, LP and MCM media. MCM medium with BA or 2iP was the only one that s i g n i f i c a n t l y i n f l u e n c e d the cotyledons responses and t h i s decreased the mean buds and shoots formed per cotyledon. However, the buds and shoots formed on cotyledons c u l t u r e d on LP medium appeared more robust. This medium was s e l e c t e d t h e r e f o r e f o r the microscopic and biochemical analyses. 1.2. GROWTH REGULATOR CONCENTRATIONS The t o t a l response of cotyledons to 2iP was s i g n i f i c a n t l y higher than to BA a f t e r 42 days of exposure to these growth r e g u l a t o r s (Figure 3). Neither the organogenic response nor the 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 concentration. D i f f e r e n t concentrations of 2iP s i g n i f i c a n t l y i n f l u e n c e d the organogenic response and the mean buds and shoots, the l a s t one being the most v a r i a b l e measure of growth. A f t e r 10 days of exposure (Figure 4) there were no s i g n i f i c a n t c oncentration e f f e c t s between BA and 2iP, except f o r the mean buds and shoots produced during growth with 15 uM on LP medium (Figure 4C). Of the two c u l t u r e media t e s t e d , the mean buds and shoots on SH medium 35 55 50 S 45 g 40 o </) 35 < i— o on o o o z < o or o C/1 I— o o X (/) a z < (/) a < Ul 3 30 25 20 15 55 1 • BA T 2iP Ul £ 45 o CL 40 35 30 h 25 20 15 • BA T 2iP 5 10 15 20 CONCENTRATION (uM) FIGURE 3. T o t a l response + SE, organogenic response + SE, and mean buds and shoots + SE, of cotyledons c u l t u r e d on d i f f e r e n t concentrations of BA or 2iP on LP medium f o r 42 days. 36 • BA T 2iP 0 U 1 1 1 | L_l I I I I I I i 0 5 10 15 20 25 0 5 10 15 20 25 CONCENTRATION (uM) CONCENTRATION (uM) FIGURE 4. T o t a l response, organogenic response, and mean buds and shoots + SE, of cotyledons c u l t u r e d on d i f f e r e n t concentrations of BA or 2iP, on LP (A-C) or SH (D-F) medium f o r 10 days. 37 (Figure 4F) was l e s s than those formed on LP medium (Figure 4C). This lower response on SH medium was al s o detected on Figure 2. Figure 5 i s a composite of Figures 3 and 4. The t o t a l response, the organogenic response and the 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 cotyledons were c u l t u r e d f o r 10 and 42 days on 1-25 uM BA (Figure 5A-C). However the time of exposure to 2iP only i n f l u e n c e d the mean buds and shoots formed (Figure 5F), and the organogenic response at 1 and 5 uM (Figure 5E). These data.showed th a t cotyledons were more s e n s i t i v e t o the time of exposure to BA than to 2 i P . The optimum concentration of both c y t o k i n i n s i n LP medium f o r mean buds and shoots production was 15 uM (Figures 5C and 5F). This concentration was chosen f o r c u l t u r e d cotyledons to be used i n microscopic and biochemical analyses. 1.3. TIME OF EXPOSURE A l l measures of developmental response showed t h a t , cotyledons were more s e n s i t i v e to the time of exposure to BA than to 2iP (Figures 6A-6F) i n both LP and SH medium. However, a f t e r growth on SH medium f o r 21 days, responses to both growth r e g u l a t o r s were s i m i l a r (Figures 6D-6F). 38 60 •u 50 in z o o. (/7 < I— o 40 30 20 I 1 D ^ 1 0 B 4 2 Ld z o CL t/1 Ul cc o z Ul o o z < o O o O X (/) Q Z < Q m < ui 60 50 40 30 20 10 25 20 15 10 I B -T i T -1 5 15 25 CONCENTRATION (uM) 1 1 5 15 25 CONCENTRATION (uM) FIGURE 5. T o t a l response, organogenic response, and mean buds and shoots, of cotyledons c u l t u r e d on LP medium supplemented with d i f f e r e n t concentrations of BA (A-C) or 2iP (D-F) f o r 10 and 42 days. (Composite of 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 or 2iP t r e a t e d cotyledons c u l t u r e d on LP (A-C) or SH (D-F) medium a f t e r d i f f e r e n t days of exposure to c y t o k i n i n s , and then t r a n s f e r r e d to GRF medium. 40 The g e n e r a l o b s e r v a t i o n o f a maximum response a f t e r 5-10 days of c u l t u r e i n t h e p r e s e n c e o f 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 days exposure t i m e f o r growth o f c u l t u r e s t o be used f o r m i c r o s c o p i c and b i o c h e m i c a l a n a l y s e s . T a b l e 2 summarizes t h e 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 t i s s u e s t o be used 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 a n a l y s e s , and T a b l e 3 summarizes t h e o b s e r v e d n u m e r i c a l r e s p o n s e s o f t h e c o t y l e d o n s under t h e s e c o n d i t i o n s . C o t y l e d o n s which were not e x c i s e d and exposed t o c y t o k i n i n s u n t i l 3 days a f t e r g e r m i n a t i o n , showed t o t a l and o r g a n o g e n i c responses t h a t were s i g n i f i c a n t l y l o wer 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 from day 0 ( F i g u r e s 7A and 7B) d e m o n s t r a t i n g a l o s s o f competence. The d i f f e r e n c e between t h e mean buds and s h o o t s was s i g n i f i c a n t . C o t y l e d o n s 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 response t o 2 i P . 2. HISTOLOGICAL AND ULTRASTRUCTURAL ANALYSES 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 o f c u l t u r e d P. ponderosa c o t y l e d o n s showed t h a t c e l l s from i m b i b e d embryos (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 l o c a t e d n u c l e i , as w e l l as l i p i d d r o p l e t s which o c c u p i e d a l a r g e TABLE 2. Culture c o n d i t i o n s s e l e c t e d f o r Pinus ponderosa cotyledons to be used i n the microscopic and biochemical analyses MEDIUM LP (VON ARNOLD & ERIKSSON, 1981) GROWTH REGULATORS BA (BENZYLADENINE) 2iP (2-ISOPENTENYL ADENINE) CONCENTRATION 15 uM TIME OF EXPOSURE 10 DAYS PHYSICAL CONDITIONS 27 + 2°C 16 h PHOTOPERIOD 80 uE/m2/s PHOTON FLUENCE TABLE 3. Responses (see page 21) + standard e r r o r of the mean of Pinus ponderosa cotyledons c u l t u r e d on GRF or 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 of exposure t o 15 uM BA or 15 uM 2iP TOTAL RESPONSE (%) ORGANOGENIC RESPONSE (%)• MEAN BUDS AND SHOOTS GRF BA 2iP BA 2iP BA 2iP 93+1.3 92+1.5 93+1.3 42+5.6 47+4.8 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 of cotyledons e x c i s e d from germinated embryos c u l t u r e d i n the presence of BA or 2iP 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 of the cytoplasm, and p r o t e i n s bodies which were i d e n t i f i e d h i s t o c h e m i c a l l y (Figures 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 the presence of c y t o k i n i n s (Figure 9A) showed a densely s t a i n e d cytoplasm which contained many l i p i d d r o p l e t s , l a r g e prominent n u c l e i , and l i t t l e to no v a c u o l a t i o n , whereas c e l l s c u l t u r e d on GRF medium contained l a r g e r vacuoles (Figure 9B). M i t o t i c f i g u r e s were observed i n some subepidermal c e l l s below the surface of the cotyledons i n contact with the medium, regardless of growth r e g u l a t o r s supply (Figure 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 not observed at 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 the absence of c y t o k i n i n . A f t e r 5 days i n c u l t u r e , the c y t o k i n i n - t r e a t e d cotyledons contained d i s t i n c t meristematic regions i n the t i s s u e i n contact with the medium (Figures 10 and 11) . The epidermal and subepidermal l a y e r s on t h i s s ide were more compact than those i n the upper region of the cotyledons (Figure 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 meristematic c e l l s contained c e n t r a l l y l o c a t e d n u c l e i (Figure 12A), small vacuoles and some l i p i d d r o p l e t s i n the cytoplasm (Figure 12B). The p l a s t i d s present contained few, sm a l l , s i n g l e t h y l a k o i d s and no grana (Figure 12C). 44 FIGURE 8. Pinus ponderosa cotyledons at the time of e x c i s i o n (day 0). A. L i g h t micrograph 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 bodies (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. Transmission e l e c t r o n micrograph of a c e l l w i t h l i p i d d r o p l e t s (L) occupying almost a l l the cytoplasm. (N=nucleus, PB=protein bodies, R=ribosomes, W=cell w a l l ) . FIGURE 9. Pinus ponderosa cotyledons at day 3 i n c u l t u r e . A. L i g h t micrograph of a cotyledon 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 with l i p i d d r o p l e t s (L) occupying almost a l l the cytoplasm, 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 micrograph of a cotyledon 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 the subepidermal l a y e r of c e l l s . Large vacuoles (V) occupy almost 75 % of the c e l l volume. 46 FIGURE 10. L i g h t micrograph of a Pinus ponderosa cotyledon at 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 showing the meristematic (M) and non-meristematic (NM) regions. FIGURE 11. L i g h t micrograph of a Pinus ponderosa cotyledon at 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 showing the meristematic region (M) wi t h c e l l s t i g h t l y packed with c e n t r a l l y l o c a t e d n u c l e i (N) and the non-meristematic region (NM) with c e l l s c o n t a i n i n g l a r g e vacuoles (V). 47 FIGURE 12. Pinus ponderosa cotyledons at 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 . A. L i g h t micrograph of the meristematic region 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) with one or more n u c l e o l i (Nu), and small vacuoles (V). B. Transmission e l e c t r o n micrograph of a meristematic c e l l c o n t a i n i n g small vacuoles (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 the cytoplasm (N=nucleus). C. Transmission e l e c t r o n micrograph of a p l a s t i d (P) of a meristematic c e l l . Thylakoid membranes (T) occur as s i n g l e v e s i c l e s . D. L i g h t micrograph of the non-meristematic r e g i o n . Large vacuoles (V) occupy almost a l l the c e l l volume. L i p i d d r o p l e t s (L) are s t i l l present i n the cytoplasm. (N=nucleus). E. Transmission e l e c t r o n micrograph of a non-meristematic c e l l w ith la r g e vacuoles (V), larg 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) confined to the periphery of the c e l l s . Note some p l a s t i d d i v i s i o n (*). (pl=plasmodesmata). F. Transmission e l e c t r o n micrograph of a p l a s t i d (P) of a non-meristematic c e l l . Thylakoid membranes (T) and grana (G) are c l e a r . The cytoplasm contained l i p i d d r o p l e t s (L) . 49 In the non-meristematic region, c e l l s had lar g e vacuoles which occupied much of the c e l l volume (Figure 12D), and the or g a n e l l e s and l i p i d d r o p l e t s were confined to the c e l l periphery (Figure 12E). The p l a s t i d s (Figure 12F) of these non-meristematic c e l l s were l a r g e r than those i n meristematic c e l l s and showed d i s t i n c t t h y l a k o i d membranes and grana. A f t e r 5 days i n c u l t u r e on GRF medium, cotyledons were elongated (Figure 13A). Large i n t e r c e l l u l a r spaces and l a r g e vacuoles were observed i n these c e l l s (Figures 13B and 13C), and small l i p i d d r o p l e t s were present (Figure 13D). The p l a s t i d s showed d i s t i n c t t h y l a k o i d membranes and grana (Figure 13E) that were s i m i l a r to those from the non-meristematic 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 (Figure 14A), the meristematic region of the c y t o k i n i n t r e a t e d cotyledons had developed to meristematic domes r e s u l t i n g from the r a p i d c e l l p r o l i f e r a t i o n w i t h i n the epidermal and the subepidermal l a y e r s . Meristematic regions occupied 7-10% of the l o n g i t u d i n a l sectioned area. Each meristematic c e l l contained o r g a n e l l e s such as p l a s t i d s , small vacuoles, and a c e n t r a l nucleus with one or more n u c l e o l i (Figures 14B and 14C). There were no i n t e r c e l l u l a r spaces. 50 FIGURE 13. Pinus ponderosa cotyledons at day 5 of c u l t u r e on GRF medium. A. Li g h t micrograph of an elongated cotyledon. B. L i g h t micrograph showing the epidermal (E) and subepidermal l a y e r s of c e l l s with l a r g e vacuoles (V) and i n t e r c e l l u l a r spaces (IS). C. L i g h t micrograph of 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 (IS), la r g e vacuoles (V) and the cytoplasm confined to the periphery of the c e l l s . (P= p l a s t i d s , N=nucleus). D. Transmission e l e c t r o n micrograph of a c e l l w i t h vacuoles (V), nucleus (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. Transmission e l e c t r o n micrograph of a p l a s t i d (P). Thylakoid membranes (T) and grana (G) are c l e a r . (V=vacuole) . 52 FIGURE 14. Pinus ponderosa cotyledons at day 10 of c u l t u r e i n the presence of c y t o k i n i n s . A. L i g h t micrograph showing the meristematic domes (M) i n the region of the cotyledon i n contact with the medium and the non-meristematic (NM) region. B. L i g h t micrograph of the meristematic region 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) with one or more n u c l e o l i (Nu), small vacuoles (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 the subepidermal l a y e r of c e l l s . C. Transmission e l e c t r o n micrograph of a meristematic c e l l w i th small vacuoles (V), and p l a s t i d s (P) i n the cytoplasm (N=nucleus, Nu=nucleolus, W=cell w a l l ) . D. L i g h t micrograph of the non-meristematic region w i t h a w e l l developed epidermis (E). Note lar g e i n t e r c e l l u l a r spaces (IS), l a r g e vacuoles (V) which occupy almost a l l the 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) confined to the periphery of the c e l l s . E. Transmission e l e c t r o n micrograph of non-meristematic c e l l s with l a r g e vacuoles (V) occupying almost a l l the c e l l volume and the cytoplasm confined to the periphery. C h l o r o p l a s t s (C) with s t a r c h granules were present. (W=cell w a l l ) . 54 The non-meristematic c e l l s of these cotyledons (Figure 14D) were l a r g e r , contained vacuoles which occupied most of the c e l l volume, and t h e i r cytoplasm formed a t h i n p e r i p h e r a l l a y e r (Figure 14E). There were many i n t e r c e l l u l a r spaces. The p l a s t i d s of meristematic c e l l s were s p h e r i c a l or i r r e g u l a r (Figure 15A) with 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 the stroma. These contrasted with the developed c h l o r o p l a s t s observed i n non-meristematic c e l l s (Figure 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. There were some l i p i d granules i n the stroma. The c h l o r o p l a s t s i n these 10 day c u l t u r e s were l a r g e r than those i n both meristematic and non-meristematic c e l l s a f t e r 5 days i n c u l t u r e (compare Figures 12C and 12F w i t h Figures 15A and 15B) . Cotyledons c u l t u r e d i n the absence of c y t o k i n i n s , elongated, and showed a w e l l d i f f e r e n t i a t e d epidermis by day 10 i n c u l t u r e (Figure 16A). There were l a r g e i n t e r c e l l u l a r spaces, and the c e l l s contained la r g e vacuoles which, l i k e those from non-meristematic regions of the c y t o k i n i n t r e a t e d cotyledons, occupied almost a l l the c e l l volume (Figures 16B and 16C). The c h l o r o p l a s t s i n the p e r i p h e r a l cytoplasm contained extensive inner membranes and some s t a r c h g r a i n s (Figure 16D). The 55 FIGURE 15. Transmission e l e c t r o n micrographs of Pinus  ponderosa cotyledons at day 10 of c u l t u r e i n the presence of c y t o k i n i n s . A. P l a s t i d (P) of a meristematic 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) are present. (V=vacuole). B. Mature c h l o r o p l a s t (C) i n a non-meristematic c e l l showing w e l l developed grana (G). (V=vacuole, W=cell wall) . FIGURE 16. Pinus ponderosa cotyledons at day 10 i n c u l t u r e i n GRF medium. A. Li g h t micrograph showing the epidermal (E) and subepidermal l a y e r s of c e l l s w e l l d i f f e r e n t i a t e d , and large vacuoles (V) and i n t e r c e l l u l a r spaces (IS) are present. B. L i g h t micrograph of 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 (IS), l a r g e vacuoles (V) and c h l o r o p l a s t s (C) confined to the periphery of the c e l l s . C. Transmission e l e c t r o n micrograph of c e l l s showing lar g e vacuoles (V) , and c h l o r o p l a s t s (C) confined to the periphery of the c e l l s . D. Transmission e l e c t r o n micrograph of 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 the 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 chloroplasts from GRF treated cotyledons appeared similar to those i n c e l l s of the non-meristematic regions of cytokinin treated cotyledons (compare Figure 15B with Figure 16D). By day 21 i n culture, after 10 days of exposure to cytokinins and 11 days on GRF medium, the meristematic regions had developed to leaf primordia (Figure 17), and some mitotic figures were observed i n the meristematic region. The c e l l s in the non-meristematic region were similar i n appearance to those of 10 day-old cotyledons. 3. PROTEIN AND CHLOROPHYLL ANALYSES In order to characterize biochemically the development of the photosynthetic apparatus during cotyledon culture, protein and chlorophyll analyses were performed on cotyledons cultured i n the presence or absence of cytokinins. 3.1. TOTAL PROTEIN DETERMINATION Figure 18 shows that the t o t a l protein concentration in a l l the cotyledons cultured on LP medium decreased rapidly during the f i r s t days. These results suggest that the most concentrated proteins were storage proteins and FIGURE 17. Li g h t micrograph of a cotyledon at day 21, a f t e r c u l t u r e i n the presence of 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 to a growth r e g u l a t o r f r e e (GRF) medium. Leaf primordia (F) have developed. Note the meristematic region (M) i s s t i l l present with 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 concentration + SE of cotyledons c u l t u r e d on GRF medium, or i n the presence of BA or 2 i P . 60 that they were degraded during the f i r s t days i n c u l t u r e e i t h e r i n the presence or absence of 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 cotyledons which were c u l t u r e d with BA or those grown with 2iP, however a tr e n d towards a f a s t e r decrease of t o t a l p r o t e i n was observed on 2iP t r e a t e d cotyledons. Comparison between cotyledons c u l t u r e d with 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 higher i n BA or 2iP t r e a t e d cotyledons. However, a f t e r 7 days i n c u l t u r e a tr e n d towards a constant p r o t e i n concentration was observed. These r e s u l t s suggest that newly synthesized p r o t e i n s were l e s s concentrated than storage p r o t e i n s . 3.2. SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS Figure 19 shows Coomassie blue s t a i n e d SDS-polyacrylamide gels of 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 cotyledons from days 0 to 10 i n c u l t u r e . The most concentrated polypeptides i n day 0 cotyledons had apparent molecular masses of 48, 37, 31, 22 and 21 kD which corresponded to storage p r o t e i n s ( E l l i s and Judd, 1987) and decreased during the f i r s t days i n c u l t u r e , f o l l o w i n g the same trend as the t o t a l p r o t e i n concentrations (Figure 18). Some of these storage p r o t e i n s are s i m i l a r to those 61 FIGURE 19. SDS-pdlyacrylamide gels of cotyledons 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 to day 10. (S=molecular weight standards). 63 found i n s e v e r a l Pinus species ( G i f f o r d , 1988) . Cotyledons which d i d not respond to the d i f f e r e n t treatments t e s t e d showed no d e c l i n e of t h e i r storage p r o t e i n s through time i n c u l t u r e , and there were no newly synthesized p r o t e i n s . Besides the storage p r o t e i n s , the most prominent band observed had an apparent molecular mass of 48-51 kD and was i d e n t i f i e d by immunoblotting as LSU-RUBP. Three low molecular mass polypeptides of 18, 16 and 14 kD were observed as reported by G i f f o r d (1988) and were present from day 0 u n t i l day 10 of c u l t u r e . 3.3. IMMUNOBLOTTING To i n v e s t i g a t e the e f f e c t of c y t o k i n i n s on the development of the photosynthetic apparatus the accumulation of s i x photosynthetic polypeptides was examined by immunoblotting (Figure 20). In a l l cases, there 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 cotyledons t r e a t e d with c y t o k i n i n than i n those c u l t u r e d on GRF medium or i n the cotyledons from germinated embryos (C). However, BA was a greater i n h i b i t o r of accumulation of these photosynthetic p r o t e i n s than 2 i P . The antibody f o r CP29 cross - r e a c t e d with the l i g h t h a r v e s t i n g complex of photosystem I I (LHCII) (White and Green, 1987b; White and Green, 1988). LHCI was not detected i n cotyledons t r e a t e d 64 TREATMENT GRF BA 2iP C DAYS IN CULTURE 4 5 6 7 4 5 6 7 4 5 6 7 4 5 6 7 APP M (kD) • — , . . LHCI 24 — 23 — FIGURE 20. Immunoblots of photosynthetic p r o t e i n s : the coupling f a c t o r (CF1), the la r g e subunit of r i b u l o s e - 1 , 5 -bisphosphate carboxylase (LSU-RUBP), the 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 with the oxygen-evolving complex (33EP), an antennal component of 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 the l i g h t - h a r v e s t i n g complex of 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 the l i g h t h a r v e s t i n g complex of photosystem I (LHCI), of cotyledons 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, wit h 15 uM benzyladenine (BA), or 15 uM 2-isopentenyl adenine ( 2 i P ) , and c o n t r o l (C) from germinated embryos, r e s p e c t i v e l y . 65 with BA or 2iP, but i t was present i n the 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 of LSU-RUBP showed a r a p i d increase during the f i r s t days i n c u l t u r e on GRF medium and a somewhat slower accumulation i n c y t o k i n i n - t r e a t e d cotyledons (Figure 21). By day 10 there was a s i g n i f i c a n t d i f f e r e n c e i n the concentration between cotyledons c u l t u r e d on GRF medium compared with those c u l t u r e d i n the presence of e i t h e r c y t o k i n i n . 3.4. CHLOROPHYLL DETERMINATION The e f f e c t of c y t o k i n i n treatment extended t o c h l o r o p h y l l content. Consistent with the reduced accumulation of photosynthetic polypeptides, the t o t a l c h l o r o p h y l l (Figure 22 A,D) and c h l o r o p h y l l a (Figure 22 B,E) concentrations 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 cotyledons than i n those 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 (Figure 22 C,F) was not apparent u n t i l the seventh day. FIGURE 21. The lar g e subunit of ribulose-1,5-bisphosphate carboxylase (LSU-RUBP) concentration + SE of c y t o k i n i n (CK) versus growth r e g u l a t o r f r e e (GRF) t r e a t e d cotyledons c u l t u r e d i n v i t r o . 67 u. cn £ CD 3 o < I— o cn £ cn 3 o 15 10 5 -i 1 1 r 12 10 8 6 4 2 0 i 1 1 r B i i I I i i cn £ cn 3 0 2 4 6 8 10 TIME IN CULTURE (DAYS) i i i r D t . T I L T 2IP a GRF n 1 1 r E i r / / Ki r a ' T T 2IP • GRF _L i r F i r a r / v - ^  ••-cr T 2IP • GRF J L J L 0 2 4 6 8 10 TIME IN CULTURE (DAYS) FIGURE 22. T o t a l C h l o r o p h y l l SE (B,E) and C h l o r o p h y l l b + BA or 2iP t r e a t e d cotyledons (GRF) t r e a t e d cotyledons. + SE (A,D), C h l o r o p h y l l a + SE (C,F) concentrations, of versus growth r e g u l a t o r f r e e 68 4. PROTEIN AND CHLOROPHYLL AS MARKERS OF ORGANOGENESIS P r o t e i n and c h l o r o p h y l l were used as markers of the pperiod i n which c y t o k i n i n s could a l t e r the development of the photosynthetic apparatus. In these experiments, the cotyledons were c u l t u r e d i n the absence or presence of c y t o k i n i n s f o r 1, 3, and 5 days and then t r a n s f e r r e d to CK-containing or GRF medium, r e s p e c t i v e l y . 4.1. CULTURE ON GRF AND TRANSFERRED TO CK CONTAINING MEDIUM. Figure 23 shows the t o t a l p r o t e i n concentration i n cotyledons c u l t u r e d f o r 1, 3 or 5 days on GRF medium and then t r a n s f e r r e d to BA (Figure 23A) or 2iP (Figure 23B) c o n t a i n i n g medium. Cotyledons c u l t u r e d f o r 5 days 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 y t o k i n i n - c o n t a i n i n g medium showed l e s s t o t a l p r o t e i n concentration, as GRF c u l t u r e d cotyledons, than cotyledons exposed to the exogenous c y t o k i n i n a f t e r only 1 or 3 days i n c u l t u r e on GRF medium. A t r e n d towards a higher p r o t e i n concentration was observed when the cotyledons were c u l t u r e d i n BA a f t e r 1 or 3 days i n GRF compared with 2 i P -t r e a t e d cotyledons. These r e s u l t s can be compared with r e s u l t s i n Figure 18, where the cotyledons 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 p r o t e i n contents than cotyledons 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 concentration at days 5, 7 and 10, of cotyledons c u l t u r e d on GRF medium f o r 1, 3, or 5 days and then t r a n s f e r r e d t o BA (A) or 2iP (B) c o n t a i n i n g media, compared wi t h GRF t r e a t e d cotyledons. 70 c y t o k i n i n s . The longer the cotyledon c u l t u r e on GRF medium p r i o r to t h e i r t r a n s f e r t o CK-containing medium, suggests that the cotyledons tend to respond as i f they were c u l t u r e d countinuously on GRF medium. Results of immunoblotting (Figure 24) f o r LSU-RUBP and CP2 9 showed that the accumulation of these photosynthetic p r o t e i n s was slowed i n cotyledons that were t r a n s f e r r e d to 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 than 4 days on GRF medium whereas, i n cotyledons that were not t r a n s f e r r e d u n t i l 4-5 days, these polypeptides accumulated to l e v e l s s i m i l a r to those c u l t u r e d only on GRF medium f o r the same length of time (Figure 24). In t h i s case, the p r o t e i n l e v e l s seemed to be lower i n 2iP than i n BA-t r e a t e d cotyledons. S i m i l a r patterns were observed f o r 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 concentrations (Figure 25). Cotyledons t r a n s f e r r e d to BA-co 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 the 8th and 9th day i n c u l t u r e (Figure 25A-25C). Although i t i s tempting to 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 followed by t r a n s f e r to 2iP, trends were not s t a t i s t i c a l l y d i f f e r e n t (Figures 25D-25F). DAYS/ IN GRF IN CULTURE APP M (kD) A LSU-RUBP 4 8 - 5 1 — 5 7 10 5 7 10 i i i 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 but ^ ~ CP29 29-30--. LHCII 2 8 — m » 1 • LSU-RUBP 4 8 - 5 1 — • -CP29 29-30^. LHCII 2 8 — 2 5 X m FIGURE 2 4 . Immunoblots of the larg e subunit of r i b u l o s e -1,5-bisphosphate carboxylase (LSU-RUBP), and an antennal component of photosystem I I (CP29), of cotyledons 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 to benzyladenine (BA) (A) or 2iP (B) co 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. To 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 concentrations (C,F) at days 5-10 of cotyledons c u l t u r e d i n the absence of c y t o k i n i n s f o r 1, 3 or 5 days and then t r a n s f e r r e d to BA (A,B,C) or 2iP (D,E,F) cont 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 p r o t e i n concentrations i n cotyledons c u l t u r e d f o r 1, 3 or 5 days on BA (Figure 26A) or 2iP (Figure 26B) co n t a i n i n g media fol l o w e d by t r a n s f e r to GRF medium, showed a higher p r o t e i n concentration i n cotyledons which were i n contact w i t h CK f o r a longer p e r i o d of time. These p r o t e i n l e v e l s were higher when the cotyledons were c u l t u r e d on CK c o n t a i n i n g medium through a l l the time i n c u l t u r e (Figures 26A and 26B). The cotyledons t h a t had a longer c y t o k i n i n exposure tended to have higher p r o t e i n l e v e l s than the ones c u l t u r e d i n the presence of c y t o k i n i n s f o r only 1 or 3 days. These r e s u l t s can be compared with Figure 18, where cotyledons c u l t u r e d i n the presence of c y t o k i n i n s showed higher t o t a l p r o t e i n contents than cotyledons c u l t u r e d i n the absence of these growth r e g u l a t o r s . Cotyledons c u l t u r e d i n the presence of BA or 2iP f o r 1-5 days contained the same l e v e l s of LSU-RUBP or CP29 as cotyledons c u l t u r e d continuously i n the presence of c y t o k i n i n s (Figure 27). 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- (Figure 28A-28C) or 2 i P - (Figure 28D-28F) t r e a t e d cotyledons f o r 1-5 days, 74 FIGURE 26. T o t a l p r o t e i n concentration at days 5, 7 and 10, of cotyledons c u l t u r e d on BA (A) or 2iP (B) c o n t a i n i n g media f o r 1, 3 or 5 days and then t r a n s f e r r e d t o GRF medium, compared wi t h BA (A) or 2iP (B) t r e a t e d cotyledons. 75 DAYS/ IN CK IN CULTURE APP M (kD) A LSU-RUBP 4 8 - 5 1 — 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 CP29 LHCII 29-30 — 2 8 — 2 7 — 2 5 — B LSU-RUBP 48-51— CP29 LHCII 2 9 - 3 0 — 28-21 ~ 25" — • • FIGURE 27. Immunoblots of the larg e subunit of r i b u l o s e -1,5-bisphosphate carboxylase (LSU-RUBP), and an antennal component of photosystem I I (CP29), of cotyledons c u l t u r e d on benzyladenine (BA) (A) or 2iP (B) co n t a i n i n g media and then t r a n s f e r r e d to 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) concentrations at days 5-10 of cotyledons c u l t u r e d i n the presence of BA (A,B,C) or 2iP (D,E,F) f o r 1, 3 or 5 days and then t r a n s f e r r e d t o GRF media. although a f t e r longer c y t o k i n i n exposure, c h l o r o p h y l l l e v e l s seemed to f a l l . DISCUSSION P. ponderosa cotyledons c u l t u r e d i n v i t r o were used as a model to study some of the c y t o k i n i n r e l a t e d changes to the photosynthetic apparatus during shoot organogenesis. The developmental process which leads to organogenesis 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 biochemical 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 are a requirement f o r i n  v i t r o bud and shoot formation. The r o l e s of endogenous r e g u l a t o r s are poorly understood and were not addressed i n t h i s study. D i f f e r e n t stages i n t h i s developmental process have been described from other species. Exogenous c y t o k i n i n s induce explants to give r i s e t o buds and shoots. Induction i s l i m i t e d by the explant competence and i s presumed to be achieved when the explant i s seen to contain c e l l s or groups of c e l l s determined f o r bud and shoot development ( C h r i s t i a n s o n and Warnick, 1988). Once i n d u c t i o n has occurred, the inducer or the i n d u c t i v e c o n d i t i o n s are no longer needed. Competence has been defined as the c a p a c i t y to respond to the i n d u c t i v e e f f e c t s of the medium 79 ( C h r i s t i a n s o n and Warnick, 1988). In the case of P. ponderosa cotyledons, t h i s s t a t e could be maintained up to 3 days on GRF medium a f t e r which cotyledons which were c u l t u r e d on GRF medium had l o s t t h e i r competence to form buds and shoots and there i s no e f f e c t of the c y t o k i n i n at the biochemical l e v e l (see Figures 23, 24 and 25). The observed changes during 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 to d i s r u p t or a l t e r the photosynthetic apparatus that i s c o i n c i d e n t a l with expression of organogenic competence. The a l t e r a t i o n of competence was s t u d i e d i n cotyledons c u l t u r e d w i t h c y t o k i n i n s f o r d i f f e r e n t times (see Figures 26, 27 and 28). With as l i t t l e as 24 hours of exposure, the c y t o k i n i n induced a response seen as the lowering of photosynthetic 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 organogenesis. However, a longer exposure to c y t o k i n i n s increased the organogenic response (see Figure 6). The r e s u l t s of my work i n d i c a t e that the f i r s t days of 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 process, suggesting that the c y t o k i n i n may i n t e r a c t with c e l l u l a r t a r g e t s that become u n a v a i l a b l e or are degraded soon a f t e r cotyledon e x c i s i o n . C y t o k i n i n s have been reported to enter c e l l s p a s s i v e l y (Van Staden et a l . , 1986). 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 Brinegar et a l . , 1985) which seem to have the dual f u n c t i o n of storage p r o t e i n s and r e g u l a t o r s of c y t o k i n i n a v a i l a b i l i t y . C y t o k i n i n s are a l s o known to i n t e r a c t with RNA at t r a n s c r i p t i o n a l and/or post-t r a n s c r i p t i o n a l l e v e l s . The i n d u c t i o n probably occurs through the c o n t r o l of the t r a n s c r i p t i o n of s p e c i f i c genes, as evidenced by numerous repor t s that c y t o k i n i n s enhance, induce or suppress the expression of c e r t a i n p r o t e i n s (Chen and L e i s n e r , 1985). In the case of P. ponderosa, the reduced accumulation of photosynthetic p r o t e i n s seems to be a marker that i n d u c t i o n has occurred. In t h i s case, both the nuclear and c h l o r o p l a s t coded photosynthetic p r o t e i n s that were s t u d i e d showed the same p a t t e r n of response, suggesting that the c y t o k i n i n e f f e c t was at the nuclear, cytoplasmic and o r g a n e l l e l e v e l s . The experiments performed could not r e s o l v e the mechanism f u r t h e r . The foregoing paragraphs are the 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 aspects of c y t o k i n i n e f f e c t s during organogenesis reported 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 lead to organ or embryo formation (Thorpe, 1982) . D i f f e r e n t 81 species and d i f f e r e n t t i s s u e s of one species 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 experience with Pinus' ponderosa i s no exception. Both the c y t o k i n i n s t e s t e d induced m u l t i p l e bud and shoot formation on e x c i s e d P. ponderosa cotyledons. These r e s u l t s d i f f e r e d from those reported by E l l i s and Bild e r b a c k (1984) who found that only BA produced r e l i a b l e m u l t i p l e bud formation and that cotyledons d i d not respond t o 2iP ( E l l i s , 1986). I could not repeat E l l i s ' s r e s u l t s and thus i t was necessary to optimize the 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, using both c y t o k i n i n s , BA and 2 i P . P. ponderosa provided one major advantage, namely that cotyledons e x c i s e d from the same seed responded i n the same way. I could thus study up t o 8 r e p l i c a t e s using cotyledons from one seed, or could subject cotyledons from the same seed t o c u l t u r e under d i f f e r e n t treatments i n the c e r t a i n t y t h a t they were g e n e t i c a l l y uniform. I observed t h a t the cotyledons were more s e n s i t i v e to the time of exposure to c y t o k i n i n s than t o a p a r t i c u l a r n u t r i e n t medium or to the c y t o k i n i n c o n c e n t r a t i o n . These observations c o i n c i d e d with those from other species such as Pinus c o n t o r t a (Patel and Thorpe, 1984b), where d i f f e r e n t i a t i o n of shoot primordia 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 times, and Pinus strobus where a longer exposure to BA 82 produced stunted shoots, which elongated p o o r l y a f t e r subculture ( F l i n n et a l . , 1986). C y t o k i n i n s c l e a r l y i n h i b i t e d cotyledon elon g a t i o n , the stunted buds and shoots observed i n the cotyledons could had al s o been formed a f t e r a long time of exposure to these growth r e g u l a t o r s . The composition of the c u l t u r e medium can pl a y an important r o l e i n organogenesis (Thorpe, 1980), however, i n t h i s research 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 the four media t e s t e d . Low t o t a l and organogenic responses were observed a f t e r a long exposure (42 days) and a high concentration of c y t o k i n i n s (25 uM) (see Figure 2). Lowered concentrations and the reduced times of exposure to these growth r e g u l a t o r s l e d to an increased response. However, of the four media t e s t e d , cotyledons c u l t u r e d on LP medium (Von Arnold and Er i k s s o n , 1981) showed more robust buds and shoots. This could be due t o the a d d i t i o n a l amino acids and sugars which were present only i n t h i s medium (see Table 1). Although concentration was not a s i g n i f i c a n t f a c t o r on bud and shoot production i n the presence of 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. These were c o r r e l a t e d t o the time of exposure (see Figure 5), which was al s o the only v a r i a b l e t o which the cotyledons 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 2 i P . The cotyledons were more s e n s i t i v e to the time of exposure to BA than to 2 i P . This was a l s o t r u e f o r Pinus  c o n t o r t a where the exposure of whole embryos to BA f o r more than 3 weeks caused a gradual d e c l i n e i n the number of shoots formed (Patel and Thorpe, 1984b). In Pinus  strobus embryos c u l t u r e d i n v i t r o , both c y t o k i n i n s , BA and 2iP, were e q u a l l y caulogenic at optimal concentrations, 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 species BA was 10 t o 20 times more potent than 2iP a f t e r 4 weeks of exposure ( F l i n n et a l . , 1986). The l a c k of P. ponderosa response to 2iP reported by E l l i s (1986), may a l s o r e f l e c t t h i s d i f f e r i n g s e n s i t i v i t y . The organogenic c a p a c i t y to form buds diminishes i n many c o n i f e r s soon a f t e r germination (Bonga, 1982). This observation was confirmed i n t h i s study of P. ponderosa where, only 3 days a f t e r germination, the cotyledons showed a s i g n i f i c a n t l y lower response than those c u l t u r e d with c y t o k i n i n from day 0 (see Figure 7). In the same species, E l l i s and B i l d e r b a c k (1989) reported that cotyledons became incompetent to form buds when embryos were i n i t i a l l y placed on GRF medium f o r 2 days before being 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 reported f o r other species', such as cucumber, where the e f f e c t s of BA on growth and 84 c h l o r o p h y l l synthesis a l s o decreased w i t h cotyledon age (Haru et a l . , 1982) . Cyt o k i n i n s c o n t r o l determination and the organogenic competence of P. ponderosa cotyledons ( E l l i s and Bil d e r b a c k , 1989). Although P . ponderosa cotyledons d i d not have a high organogenic response (number of cotyledons th a t formed buds and shoots) (42-47%), t h i s system could permit the separation of the i n d u c t i o n and determination phases ( C h r i s t i a n s o n and Warnick, 1987, 1988), since the t o t a l response (number of cotyledons that responded by e i t h e r greening or production of 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 biochemical analyses r e q u i r e d s e l e c t i o n of developmentally competent explants before the development was morphologically apparent. I e x p l o i t e d the f a c t t h a t cotyledons from the same embryo behaved i n the same way by s e t t i n g up every treatment w i t h p a i r s of cotyledons from a s i n g l e seed. One of the p a i r was removed from c u l t u r e at the t e s t time and stored frozen at -80°C. The other cotyledon was allowed to develop 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 only on the sampled partner of a cotyledon which expressed an organogenic response. " 85 2. HISTOLOGICAL AND ULTRASTRUCTURAL ANALYSES Several authors have described 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 during development of buds and shoots i n c o n i f e r s induced 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 of p l a s t i d s i s l e s s w e l l understood and has not been reported i n d e t a i l . P. ponderosa, l i k e other c o n i f e r species reported, formed meristematic centers or meristemoids which l e d to a d v e n t i t i o u s bud and shoot formation. When t r a n s f e r r e d to a GRF medium, needle primordia elongated. This developmental sequence occurred without c a l l u s formation on CK-containing medium i n apparently the same way as i n other species (Thorpe and P a t e l , 1986). As i n Pinus r a d i a t a ( V i l l a l o b o s et a l . , 1985), P. ponderosa cotyledons, i n the presence of BA and 2iP during the f i r s t days i n c u l t u r e , developed meristematic regions whose c e l l s were non-vacuolated and t i g h t l y packed (see Figure 12A). Cotyledons grown on GRF 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 maturation (see Figure 13) s i m i l a r to t h a t reported from P. r a d i a t a (Patel and Thorpe, 1984a). However, P. ponderosa cotyledons d i f f e r e d 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 that they d i d not form the promeristemoid-like structure's reported 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 contact with 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 with those i n c e l l s of the non-meristematic region and those i n non-CK t r e a t e d cotyledons. This delay of inner membrane formation i n the 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 meristematic c e l l s . E i t h e r BA or 2iP induced t h i s response. P. r a d i a t a cotyledons c u l t u r e d on GRF medium al s o developed an apparently f u n c t i o n a l photosynthetic apparatus (Kumar et a l . , 1988). The nature and extent 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 s t a t e " of the cotyledons (Longo et a l . , 1979) and perhaps to the p l a s t i d developmental stage (Mikulovich et a l . , 1981). For example, c h l o r o p l a s t s may 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 stages of p l a s t i d s i n higher p l a n t s are r e a d i l y i n t e r c o n v e r t e d (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 spinach showed only 2 p r o t e i n s present i n seeds, presumably i n p r o p l a s t i d s , and 10 present i n e t i o p l a s t s (Paproth and Hauska, 1985). However, some photosynthetic 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 reported to be immunologically i d e n t i c a l (Radunz et a l . , 1985). My research d i d not pursue the 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 the two c o n i f e r systems, P. ponderosa and P. r a d i a t a , may have been because the P. ponderosa cotyledons were ex c i s e d from imbibed seeds while those from P. r a d i a t a were obtained from 5 to 7 days o l d germinated seedlings i n the dark ( V i l l a l o b o s , 1983). Hence the explants were not at the same developmental and p h y s i o l o g i c a l s t a t e . Unfortunately n e i t h e r the q u a n t i f i c a t i o n of p l a s t i d numbers nor t h e i r inner 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 research because p r o p l a s t i d numbers were d i f f i c u l t to determine by l i g h t microscopy and the sec t i o n s r e q u i r e d f o r e l e c t r o n microscopy were too t h i n to allow an accurate assessment. The same problems stopped other microscopic e v a l u a t i o n of the development of inner membranes. S e r i a l s e c t i o n i n g could provide a p a r t i a l s o l u t i o n , but was im p r a c t i c a b l e on a sc a l e l a r g e enough to provide 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 the mature c h l o r o p l a s t the 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 continuously t u r n i n g over (Whatley, 1978, Sundqvist et a l . , 1980). The p l a s t i d and photosynthetic apparatus development have not been p r e v i o u s l y described during i n v i t r o organogenesis i n c o n i f e r s . The greening of e t i o l a t e d seedlings i n angiosperms has been widely 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, 1982; Wellburn et a l . , 1985). The 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 cotyledons that were ex c i s e d from the s e e d l i n g and t r e a t e d with 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 germination. Thus, before i n t r o d u c t i o n to c u l t u r e the p l a s t i d s appeared as comparatively la 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 Besides the e f f e c t of c y t o k i n i n s on the promotion of meristematic development and i n h i b i t i n g development of p l a s t i d s ' inner membranes, breakdown of reserve m a t e r i a l was retarded i n the cotyledons which were c u l t u r e d i n the presence of BA or 2 i P . These observations d i f f e r e d from other rep o r t s i n gymnosperms that 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 reserves i n the seeds during germination ( 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 increased i n P i c e a  s i t c h e n s i s C a r r i e r e and Pinus s y l v e s t r i s L. seeds f o l l o w i n g the i n d u c t i o n of germination (Taylor and Wareing, 1979). However, i n a l l the s t u d i e s done with endogenous c y t o k i n i n s i t has proved d i f f i c u l t to p i n p o i n t the exact f u n c t i o n of the c y t o k i n i n s i n the germination process (Van Staden, 1983). 89 In P. ponderosa, l i p i d s and protein bodies were the p r i n c i p a l cotyledon reserves i n excised cotyledons. After 3 days i n culture with cytokinins, the c e l l s s t i l l contained many l i p i d droplets, but small vacuoles seemed to have replaced protein bodies. C e l l s cultured on GRF medium for 3 days contained vacuoles which occupied almost a l l the c e l l volume (see Figure 9). The disappearance of protein bodies also correlated with the lowered t o t a l protein concentration i n the cotyledons, which also dropped faster on GRF medium than on CK-containing medium (see Figure 18). This suggests that the most of the proteins present were storage proteins and that t h e i r degradation may have proceeded more slowly on CK treated than on non-CK treated cotyledons. The degradation of storage proteins was also observed on the SDS-polyacrylamide gels (see Figure 19). Storage proteins similar to those described for other Pinus species (Gifford, 1988) decreased during the f i r s t days i n culture. These observations suggested that cytokinins retarded the breakdown of l i p i d and protein reserves by c o n t r o l l i n g a s h i f t from normal germination metabolism early i n culture. This was also observed i n Pinus strobus (Flinn et a l . , 1989) during the f i r s t 7 days i n culture, where the l i p i d content also decreased more rapidly'on non-CK than on CK-containing media, and there was a delay 90 i n storage p r o t e i n degradation on BA c o n t a i n i n g medium. In t h i s species a l s o the d i f f e r e n c e i n p r o t e i n degradation between c e l l s c u l t u r e d i n the presence or absence of BA was r e f l e c t e d i n the u l t r a s t r u c t u r e of p r o t e i n bodies. A f t e r 3 days on GRF medium, p r o t e i n bodies 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 bodies were i n t a c t and densely s t a i n e d . Pinus  r a d i a t a cotyledons, from 5 day-old 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 bodies a f t e r one day i n c u l t u r e . They were almost completely devoid of storage metabolites by day 5 (Douglas et a l . , 1982). However, the observations on Pinus species d i f f e r e d from the 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 the degradation of reserve m a t e r i a l (Longo et a l . , 1979), or Raphanus s a t i v u s cotyledons, 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 the appearance of a l a r g e c e n t r a l vacuole during the f i r s t days a f t e r e x c i s i o n (Thomas et a l . , 1980). The low molecular weight polypeptides observed i n P. ponderosa cotyledons have been reported from other gymnosperm species ( G i f f o r d , 1988). An increased synthesis of these low molecular weight polypeptides, a f t e r 2 days i n c u l t u r e , has been reported f o r Pseudotsuga m e n z i e s i i (Mirb.) Franco (Yasuda et a l . , 1980) which 91 r e f l e c t s the r e a l monomeric s t a t e of these molecules and not products of p r o t e o l y t i c cleavage (Hasegawa et a l . , 1979). 4. PHOTOSYNTHETIC PROTEINS AND CHLOROPHYLL Tissues 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 patterns of p r o t e i n synthesis and accumulation (Reynold, 1989). My observations of P. ponderosa cotyledons provides f u r t h e r s p e c i f i c support f o r the developmental assumption t h a t the i n i t i a t i o n of organized development observed at the 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 to change the content and spectrum of both s t r u c t u r a l and enzymatic p r o t e i n s (Thorpe, 1980, 1982) . The formation of c h l o r o p l a s t s r e s u l t s from nuclear 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 cotyledons grown with and without CK,. i t seemed reasonable that photosynthetic p r o t e i n s , both nuclear 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 of c y t o k i n i n s . The observations of CK e f f e c t s upon development of inner membranes i n the p l a s t i d s c o i n c i d e d with a changed . accumulation of the photosynthetic polypeptides which cro s s - r e a c t e d with a n t i b o d i e s r a i s e d against angiosperm 92 photosynthetic p r o t e i n s , the c* and E> subunits of CF1, LSU-RUBP, 33EP, CP29, LHCII and LHCI. These showed lower l e v e l s i n the presence of c y t o k i n i n s (see Figures 20 and 21). Antibody f o r CP29 cross- r e a c t e d with the l i g h t h a r v e s t i n g complex of photosystem I I , because CP29 i s immunologically r e l a t e d to LHCII and LHCI (White and Green, 1987b, Green, 1988). The c h l o r o p h y l l concentration was a l s o lower i n the c y t o k i n i n treatment compared to the GRF-treated cotyledons (see Figure 22). The e f f e c t s of c y t o k i n i n s i n P. ponderosa cotyledons are s i m i l a r t o those i n other species of gymnosperms. For example, i n Pinus  r a d i a t a c h l o r o p l a s t s were al 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 (Patel and Thorpe, 1984a). In P i c e a abies (Stabel et al., 1990, 1991) the l a r g e subunit 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 synthesized 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 c y t o k i n i n pulse of embryos. In P. ponderosa cotyledons a reduction i n the l e v e l of a 52 kD peptide, p o s s i b l y the l a r g e subunit of RUBP, was detected a f t e r only one week ( E l l i s and Judd, 1987). This decrease of enzyme amount i s c o n s i s t e n t with a decrease i n RUBP a c t i v i t y i n cotyledons of Pinus r a d i a t a c u l t u r e d i n the presence of c y t o k i n i n s (Kumar et aJL. 1988) . In Pinus p i n a s t e r c u l t u r e d cotyledons, the continuous presence of growth r e g u l a t o r s 93 al s o i n h i b i t e d the synthesis of c h l o r o p h y l l s (Tranvan et a l . , 1988). However, i n other gymnosperm species l i k e Pinus n i g r a Arnold, l i g h t grown embryos reacted to 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 synthesis ( J e l i c and Bogdanovic, 1990) and, i n the dark, cotyledons d i d not accumulate p r o t o c h l o r o p h y l l (Bogdanovic and J e l i c , 198 9). This s t i m u l a t i o n depended on the type of c y t o k i n i n and on the co 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 inner membranes and the reduced accumulation of photosynthetic p r o t e i n s i n P. ponderosa cotyledons i n the presence of c y t o k i n i n s c ontrasted 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 these growth r e g u l a t o r s i n angiosperms. I t may be that the 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 formation i s concerned, i s that the l a t t e r have the enzymatic machinery to c a r r y the reduction of p r o t o c h l o r o p h y l l i n the dark and t h i s appears to be confined to the cotyledons i n most gymnosperms (Kirk 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, besides the 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 formation i n gymnosperms (Cahay et a l . , 1985). However, even i f they can synthesize c h l o r o p h y l l i n the dark, they do not have photosynthetic c a p a c i t y (Wellburn, 1982, 1984), and c e r t a i n r e a c t i o n s i n the photosynthetic 94 e l e c t r o n t r a n s p o r t chain remain i n a c t i v e during greening i n darkness (Wollgiehn and P a r t h i e r , 1980). The suppression of c h l o r o p l a s t development i n P i c e a abies would be a consequence of the general i n h i b i t i o n of s e e d l i n g maturation that r e s u l t s from the bud-induction treatment (Stabel et a l . , 1990). The extent to which photosynthetic 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 with 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 the "dogma" i s that photosynthesis plays a minor r o l e i n p r o v i d i n g carbon skeletons and energy f o r metabolic events a s s o c i a t e d with shoot and bud i n d u c t i o n (Kumar et a l . , 1988). However, i n P. ponderosa cot y l e d o n s ' s t a r c h grains were detected i n the 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 cotyledons, both on medium co n t a i n i n g 3% sucrose, suggesting t h a t the 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 during i n v i t r o c u l t u r e . In Pinus r a d i a t a cotyledons c u l t u r e d i n v i t r o the c h l o r o p h y l l and carotenoid contents i n the c u l t u r e d cotyledons were lower than i n germinated seedlings (Douglas et a l . , 1982). The net photosynthetic r a t e of 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 co n t a i n i n g sugar (Kozai et a l . , 1988). I t appears that carbohydrates play two r o l e s " i n organ-forming t i s s u e s , as a carbon and energy source and 95 as an osmotic agent (Thorpe, 1980). Sucrose a p p l i e d 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 s ynthesis (Dalton and S t r e e t , 1977), while CO2 enrichment of Cymbidium p l a n t l e t s i n v i t r o promoted photosynthesis (Kozai et a l . , 1990). C0 2 and ethylene, which b u i l t up i n Pinus r a d i a t a cotyledons during the f i r s t 10 t o 15 days i n c u l t u r e , promoted morphogenesis, and excessive accumulation a f t e r bud i n i t i a t i o n caused some degree of d e d i f f e r e n t i a t i o n (Kumar et a l . , 1987). Photosynthesis indeed does occur at a reduced l e v e l i n green m a t e r i a l c u l t u r e d i n the l i g h t (Hughes, 1981). This low l e v e l of photosynthesis 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 other e f f e c t s through the a c t i v a t i o n of pigment receptors other than those a s s o c i a t e d d i r e c t l y with photosynthesis, 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). Li 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 at the same molecular s i t e s or i n the same metabolic pathway ( P a r t h i e r , 1979). I t i s p o s s i b l e to suggest however, that there are some common steps i n a metabolic chain of r e a c t i o n s p r o v i d i n g the a c t i v a t i o n of the p l a s t i d rRNA synthesis by BA and by l i g h t (Mikulovich et a l . , 1981) . 96 5. PROTEIN AND CHLOROPHYLL AS ORGANOGENIC MARKERS. Since the organogenic response was more s e n s i t i v e to the time of exposure to c y t o k i n i n s (see Figures 5 and 6) and to the delay of c y t o k i n i n exposure a f t e r germination (see Figure 7), these two v a r i a b l e s were al s o t e s t e d at the biochemical l e v e l . To c o r r e l a t e the organogenic response of the cotyledons and the a b i l i t y of c y t o k i n i n s t o induce a re d u c t i o n on the accumulation of photosynthetic p r o t e i n s , these were used as markers of two important t i m i n g events: the minimum exposure time t o c y t o k i n i n s needed to induce a response, and the du r a t i o n of the "developmental window" a f t e r which cotyledons lose t h e i r competence to respond t o the 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 the competence of the cotyledons to respond to the c y t o k i n i n i s l o s t a f t e r 5 days germination (see Figure 7) or c u l t u r e on GRF medium. However, the c y t o k i n i n can s t i l l decrease the p r o t e i n concentrations a f t e r 3 days germination (see Figure 24). My r e s u l t s i n d i c a t e d the CK must be introduced on or before the t h i r d day of c u l t u r e on GRF medium to induce a red u c t i o n of photosynthetic p r o t e i n accumulation (see Figure 24). However, the changes i n c h l o r o p h y l l l e v e l s only showed the same p a t t e r n as the p r o t e i n concentration at days 9 and 10 f o r BA but not f o r 2iP treatment (see 97 Figure 25). Organogenic responses t o the growth r e g u l a t o r s d i f f e r e d , the cotyledons were more s e n s i t i v e to the time of exposure to BA than to 2iP (see Figure 6). Changes i n t o t a l p r o t e i n concentration i n cotyledons a l s o c o r r e l a t e d with these observations, being higher when a CK exposure was delayed f o r 1 or 3 days (see Figure 23) than when the delay was f o r 5 days. The t o t a l p r o t e i n concentration i n GRF-treated cotyledons was always lower than i n CK-treated cotyledons (see Figure 18). These biochemical observations c o r r e l a t e d with organogenic responses of e x c i s e d cotyledons c u l t u r e d on BA-containing medium a f t e r 3 days of embryo growth on GRF medium (see Figure 7). These r e s u l t s c o i n c i d e d with observations by E l l i s and Bi l d e r b a c k (1989) w i t h the same species. They reported t h a t an e a r l y exposure t o BA was necessary f o r cotyledons to r e t a i n t h e i r competence f o r response to BA by forming de novo buds. A delayed BA exposure of 24 h s i g n i f i c a n t l y reduced the number of buds and meristemoids produced by the cotyledons, whereas a 48 h delay l e d to a l o s s of cotyledonary competence to form buds and meristemoids. C y t o k i n i n s can a l s o induce a reduction of photosynthetic p r o t e i n accumulation and of 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 of exposure (see Figures 27 and 28). The longer 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 (see Figure 27), although t h i s higher accumulation at day 5 could be due to 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 running the g e l . A t r e n d towards a lower c h l o r o p h y l l c oncentration was observed with 5 days of exposure to e i t h e r BA or 2iP (see Figure 28). The t o t a l cotyledon p r o t e i n concentration was higher i n CK-treated cotyledons compared with only a 1-5 day exposure to CK (see Figure 26). These observations suggest that a longer exposure to CKs r e t a r d s more e f f i c i e n t l y the breakdown of storage p r o t e i n s (see Figure 18). These r e s u l t s c o r r e l a t e d with organogenic responses of the cotyledons i n which a longer exposure to CKs (more than 5 days) induced a higher organogenic response and a higher mean buds and shoots (see Figure 6) than with 3 days of exposure. E l l i s and B i l d e r b a c k (1989) working w i t h the same species observed th a t the i n d u c t i o n of bud and shoot formation occurred soon a f t e r embryo e x c i s i o n , and that t h i s stimulus 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. These observations are c o n s i s t e n t with my observations that photosynthetic p r o t e i n s were a l s o very s u s c e p t i b l e to 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 formation, during the f i r s t 3 days of c u l t u r e . The f i r s t 1-3 days of exposure to CK i seem to be the most c r i t i c a l time p e r i o d frame f o r 99 i n d u c t i o n of biochemical changes to the photosynthetic apparatus. A s i m i l a r response i n angiosperms i s known from Lemna gibba i n which BA must be present f o r more than one hour to s t i m u l a t e the LHC and SSU-RUBP mRNA l e v e l s . However, when BA i s present 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 great as a f t e r 24 h (Flores and Tobin, 1987). In 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 cotyledon 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 synthesis (Haru et a l . , 1982). The r a t e of macromolecule synthesis was greater i n organogenic c u l t u r e s of Solanum ca r o l i n e n s e L., and the "organogenic polypeptides" are produced i n regenerating explants w i t h i n the f i r s t day of c u l t u r e , w e l l before there are any signs of d i f f e r e n t i a t i o n (Reynold, 1989) . For a comprehensive understanding 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 and hormonal r e g u l a t i o n , i t w i l l be necessary to examine the i n t r a c e l l u l a r concentrations of the hormones and t h e i r compartmentalization (Zimmermann et a l . , 1987). In order to understand how the growth r e g u l a t o r stimulus i s transduced i n t o observed p h y s i o l o g i c a l responses, i t i s e s s e n t i a l to 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 that i s shown to bi n d a growth r e g u l a t o r (Napier and Venis, 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 to ask, "what relevance has the study of i n v i t r o c u l t u r e to understanding of t r e e growth e i t h e r i n the f i e l d or during i n v i v o s t u d i e s ? " . A long-standing issue i n p l a n t b i o l o g y 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 reproduction i n n a t u r a l propagation. Some genera, such as Rubus, T r i f o l i u m and Lolium, may r e l y on v e g e t a t i v e propagation f o r much of t h e i r reproduction. Several gymnosperms, i n c l u d i n g e s p e c i a l l y Sequoiadendron, Juniperus and s e v e r a l of the more shrubby taxa, can be propagated s u c c e s s f u l l y by l a y e r i n g . The economically important t r e e forms of Pinus, L a r i x , P i c e a and Abies are not e a s i l y propagated by t h i s or other asexual methods. While a r t i f i c i a l propagation i s easy f o r species t h a t use v e g e t a t i v e reproduction i n v i v o , the attainment of c l o n a l propagation from mature c o n i f e r s remains a goal f o r c o n i f e r biotechnology. The mature seed cotyledon has been adopted as a model f o r the study of the fundamental mechanism of shoot i n i t i a t i o n from non-meristematic v e g e t a t i v e c o n i f e r explants. I have demonstrated that the formation of meristematic regions 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 with lower accumulation of photosynthetic 101 polypeptides 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 of meristematic c e l l s which were on the contact side between the cotyledon and the with CK-c 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 of lowered photosynthetic 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 of p l a s t i d s , and the organogenic competence of the cotyledons e x c i s e d from P. ponderosa seeds. There i s a narrow 'window of competence' during the f i r s t 1-3 days a f t e r germination, i n which shoots can be i n i t i a t e d i n e x c i s e d cotyledons by a p p l i c a t i o n of exogenous c y t o k i n i n . The task remains to extend t h i s window or to f i n d methods of re-opening i t i n explants of developmentally young t i s s u e (e.g. emerging leaves) from mature t r e e s so that c l o n i n g of mature stock can be achieved. CONCLUSIONS Cyt o k i n i n s induce the formation of meristematic regions i n cotyledons of Pinus ponderosa c u l t u r e d i n v i t r o . A delay of inner membrane formation i s observed i n p l a s t i d s of these meristematic regions. A red u c t i o n on the accumulation of s i x photosynthetic polypeptides was observed on the 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 of the organogenic i n d u c t i o n . C y t o k i n i n s induce an i n h i b i t o r y response of the photosynthetic polypeptides a f t e r 24 h of exposure. I f the c y t o k i n i n i s not present during the f i r s t 3 days a f t e r germination, there i s no i n d u c t i o n of the i n h i b i t o r y response. . Since cotyledons can respond or not organogenically, the processes of i n d u c t i o n and competence can be s t u d i e d i n t h i s system. 104 SUGGESTIONS Biochemical and molecular s t u d i e s concerning the e a r l y changes i n the system should concentrate on the f i r s t 24 h of c u l t u r e . Related to 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 cotyledons t r e a t e d with c y t o k i n i n s could be used as a c o n t r o l to separate events c y t o k i n i n versus l i g h t dependent. C h l o r o p l a s t development could be fol l o w e d during the development of l e a f primordia a f t e r t r a n s f e r r i n g the cotyledons from CK-containing medium to GRF medium. The f u n c t i o n of the low molecular weight polypeptides which were present through a l l the time i n c u l t u r e should be s t u d i e d . The c y t o k i n i n receptors could 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 . Future research using photosynthetic polypeptides as markers of organogenesis could be d i r e c t e d towards a b e t t e r o p t i m i z a t i o n of the c u l t u r e , using lower concentrations of c y t o k i n i n s and d i f f e r e n t time of exposure. 105 REFERENCES AITKEN J . , HORGAN K.J. and THORPE T.A. 1981. Influence of explant s e l e c t i o n on the shoot-forming c a p a c i t y of j u v e n i l e t i s s u e of Pinus r a d i a t a . Can. J . For. Res. 1^:112-117 AKOYUNOGLOU G. and ARGYROUDI-AKOYUNOGLOU J . 1985. Post-t r a n s l a t i o n a l r e g u l a t i o n 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 . In Regulation 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 . Akoyunoglou G. and Senger H., eds. Alan R. L i s s Inc., NY, pp 571-582 APEL K. and KLOPPSTECH K. 1980. The e f f e c t of l i g h t on the bi o s y n t h e s i s of the 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 p r o t e i n . Evidence f o r the requirement of c h l o r o p h y l l a f o r the s t a b i l i z a t i o n of the apoprotein. P l a n t a 150:426-430 ARNON D.I. 1949. Copper enzymes i n i s o l a t e d c h l o r o p l a s t s polyphenoloxidase i n Beta v u l g a r i s . P l a n t P h y s i o l . 24:1-15 ASHTON F.M. 1976. M o b i l i z a t i o n of storage p r o t e i n of seeds. Ann. Rev. Plant P h y s i o l . 27:95-117 AXELOS M., TEYSSENDIER DE LA SERVE B. and PEAUD-LENOEL C. 1987. Level of messenger RNA encoding small subunit r i b u l o s e bisphosphate carboxylase i s enhanced by c y t o k i n i n s i n tobacco c e l l suspension c u l t u r e s . Biochimie 69:671-675 BENNETT J . , JENKINS G.I., CUMING A.C, WILLIAMS R.S. and HARTLEY M.R. 1984. Photoregulation of t h y l a k o i d b i o g e n e s i s : the case of the 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. In Ch l o r o p l a s t Biogenesis. E l l i s R.J., ed. Cambridge Univ. Press, Cambridge, pp 167-192 BOGDANOVIC M. and JELIC G. 1989. In vi v o c h l o r o p h y l l forms i n the Pinus n i g r a Am. cotyledons during germination i n the dark and i n the l i g h t . Photosynthetica 23(4):674-677 BONGA J.M. 1982. Vegetative propagation i n r e l a t i o n to j u v e n i l i t y , maturity and rej u v e n a t i o n . In. Tissue Culture i n F o r e s t r y . Bonga J.M. and Durzan D.J., eds. Martinus N i j h o f f , The Hague, pp 387-412 106 BORNMAN CH . 1983. P o s s i b i l i t i e s and c o n s t r a i n t s i n the regeneration of t r e e s from cotyledonary needles of P i c e a abies i n v i t r o . P h y s i o l . P l a n t . 57:5-16 BORNMAN C H . 1985. Hormonal c o n t r o l of growth and d i f f e r e n t i a t i o n i n c o n i f e r t i s s u e s i n v i t r o . B i o l . P l a n t . 27:511-538 BRACALE M., LONGO G.P., ROSSI G. and LONGO CP . . 1988. E a r l y changes i n morphology and polypeptide p a t t e r n of p l a s t i d s from watermelon cotyledons induced by benzyladenine or l i g h t are very s i m i l a r . P h y s i o l . P l a n t . 72:94-100 BRINEGAR A.C, STEVENS A. and FOX J.E. 1985. B i o s y n t h e s i s and degradation of a wheat embryo c y t o k i n i n - b i n d i n g p r o t e i n during embryogenesis and germination. P l a n t P h y s i o l . 79:706-710 BUETOW D.E. 1985. C h l o r o p l a s t development. In Regulation 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 . Akoyunoglou G. and Senger H., eds. Alan R. L i s s Inc., NY, pp 427-432 CAHAY C , MICHEL-WOLWERTZ M.R. and BROUERS M. 1985. C h a r a c t e r i z a t i o n of e t i o c h l o r o p l a s t membrane f r a c t i o n s i s o l a t e d from dark-grown pine (Pinus j e f f r e y i ) . Ln Regulation 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 . Akoyunoglou G. and Senger H., eds. Alan R. L i s s Inc., NY, pp 283-289 CAMM E.L., GREEN B.R., ALLRED D.R. and STAEHELIN L.A. 1987. A s s o c i a t i o n of the 33 kDa e x t r i n s i c polypeptide ( w a t e r - s p l i t t i n g ) w i t h PSII p a r t i c l e s : immunochemical q u a n t i f i c a t i o n of r e s i d u a l polypeptide a f t e r membrane e x t r a c t i o n . Photos. Res. 13:69-80 CHEN CM. and LEISNER S.M. 1985. Cytokinin-modulated gene expression i n e x c i s e d pumpkin cotyledons. P l a n t P h y s i o l . 77:99-103 CHRISTIANSON M.L. and WARNICK D.A. 1987. P h y s i o l o g i c a l genetics of organogenesis i n v i t r o . In Genetic manipulation of woody p l a n t s . Hanover J.W. and Keathley D.E., eds. Plenum Press, NY, pp 101-115 CHRISTIANSON M.L. and WARNICK D.A. 1988. Organogenesis i n  v i t r o as a developmental process. HortScience 23 (3) :515-519 107 CHUA N.H. 1980. E l e c t r o p h o r e t i c a l a n a l y s i s of c h l o r o p l a s t p r o t e i n s . Ln Methods i n Enzymology. P i e t r o A.S., ed. Academic Press, NY 60:434-446 COLIJN CM., SIJMONS P., MOL J.N.M., KOOL A.J. and NIJKAMP H.J.J. 1982. L i g h t and benzylaminopurine induce changes i n u l t r a s t r u c t u r e and gene expression i n p l a s t i d s of Petunia hybrida c e l l c u l t u r e s . Curr. Gen. 6^:129-135 CRANE K.E. and ROSS C J . 1986. E f f e c t s of wounding on c y t o k i n i n a c t i v i t y i n cucumber cotyledons. P l a n t P h y s i o l . 82:1151-1152 DALTON C C and STREET H.E. 1977. Influence of a p p l i e d carbohydrates on the growth and greening of c u l t u r e d spinach (Spinacia oleracea L.) c e l l s . P l a n t S c i . L e t t . 10_:157-164 DANIELL H. and REBEIZ C A . 1985. Ch l o r o p l a s t c u l t u r e XI. Involvement of phytohormones i n the greening of higher p l a n t s . I_n Regulation 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 . Akoyunoglou G. and Senger H., eds. Alan R. L i s s Inc., NY, pp 63-70 DOUGLAS T.J., VILLALOBOS V.M., THOMPSON M.R. and THORPE T.A. 1982. L i p i d and pigment changes during shoot i n i t i a t i o n i n c u l t u r e d explants of Pinus r a d i a t a . P h y s i o l . P l a n t . 55:470-477 ELLIS R.J. 1981. Ch l o r o p l a s t p r o t e i n s : s y n t h e s i s , t r a n s p o r t and assembly. Ann. Rev. Plan t P h y s i o l . 32:111-137 ELLIS R.J. 1984. I n t r o d u c t i o n : p r i n c i p l e s of c h l o r o p l a s t b i o g e n e s i s . In. C h l o r o p l a s t Biogenesis. E l l i s , R.J., ed. Cambridge Univ. Press, Cambridge, pp 1-9 ELLIS D.D. and BILDERBACK D.E. 1984. M u l t i p l e bud formation by c u l t u r e d embryos of Pinus ponderosa. J Plan t P h y s i o l . 115:201-204 ELLIS D.D. 1986. The competency of Pinus ponderosa Laws, cotyledons to respond to benzyladenine i n t i s s u e c u l t u r e . Ph.D. t h e s i s . U n i v e r s i t y of Montana, Montana, USA ELLIS D.D. and BILDERBACK D.E. 1989. Temporal competence of embryonic Pinus ponderosa cotyledons to form m u l t i p l e buds i n v i t r o . Amer. J . Bot. 76(3):348-355 108 ELLIS D.D. and JUDD R.C. 1987. SDS-PAGE a n a l y s i s of bud forming cotyledons of Pinus ponderosa. P l a n t C e l l T i s s . Org. C u l t . 11;57-65 ESKINS K., WESTHOFF P. and BEREMAND P.D. 1989. L i g h t q u a l i t y and i r r a d i a n c e l e v e l i n t e r a c t i o n i n the c o n t r o l of expression of l i g h t - h a r v e s t i n g complex of photosystem I I . P l a n t P h y s i o l . 91:163-169 FEIERABEND J . and DE BOER J . 1978. Comparative a n a l y s i s of the a c t i o n of c y t o k i n i n and l i g h t on the formation of the ribulosebisphosphate carboxylase and p l a s t i d b i o g e n e s i s . P l a n t a 142:75-82 FLINN B.S., WEBB D.T. and GEORGIS W. 1986. In v i t r o c o n t r o l of caulogenesis by growth r e g u l a t o r s and media components i n embryonic explants of eastern white pine (Pinus s t r o b u s ) . Can. J . Bot. 64:1948-1956 FLINN B.S. 1987. Anatomical and u l t r a s t r u c t u r a l changes during caulogenic determination of eastern white pine (Pinus strobus L.) cotyledons i n v i t r o . M. Sc. t h e s i s , Queen's U n i v e r s i t y , Kingston, Ontario, Canada FLINN B.S., WEBB D.T. and NEWCOMB W. 1988. The r o l e of c e l l c l u s t e r s and promeristemoids i n determination and competence f o r caulogenesis by Pinus strobus cotyledons i n v i t r o . Can. J . Bot. 66;1556-1565 FLINN B.S., WEBB D.T. and NEWCOMB W. 1989. Morphometric a n a l y s i s of reserve substances and u l t r a s t r u c t u r a l changes during caulogenic determination and l o s s of competence of Eastern White pine (Pinus strobus) cotyledons i n v i t r o . Can. J . Bot. 67:779-789 FLORES S. and TOBIN E.M. 1986. Benzyladenine modulation of the expression of the two genes f o r nuclear-encoded c h l o r o p l a s t p r o t e i n s i n Lemna gibba: apparent post-t r a n s c r i p t i o n a l r e g u l a t i o n . P l a n t a 168:340-349 FLORES S. and TOBIN E.M. 1987. Benzyladenine r e g u l a t i o n of the expression of two nuclear genes f o r c h l o r o p l a s t p r o t e i n s . In Molecular b i o l o g y of p l a n t growth c o n t r o l . Fox J.E. and Jacobs M., eds. Alan R. L i s s Inc., NY, pp 123-132 FLORES S. and TOBIN E.M. 1988. C y t o k i n i n modulation of LHCPmRNA l e v e l s : the involvement of post-t r a n s c r i p t i o n a l r e g u l a t i o n . P l a n t Mol. B i o l . 11:409-415 109 FOWKE L.C. 1986. U l t r a s t r u c t u r a l cytology of c u l t u r e d p l a n t t i s s u e , c e l l s and p r o t o p l a s t s . In C e l l c u l t u r e and somatic c e l l genetics of p l a n t s . V a s i l I . , ed. Academic Press, NY 3:323-342 FUNCKES-SHIPPY C.L. and LEVINE A.D. 1985. C y t o k i n i n regulates the expression of nuclear genes r e q u i r e d f o r photosynthesis. In Molecular Biology of the Photosynthetic Apparatus. Steinback K.E., ed. Cold Spring Harbor Lab., pp 407-411 GHOSH S., GEPSTEIN S., HEIKKILA J . J . and DUMBROFF E.B. 1988. Use of a scanning densitometer or an ELISA p l a t e reader f o r measurement of nanogram amounts of p r o t e i n i n crude e x t r a c t s from b i o l o g i c a l t i s s u e s . Anal. Biochem. 169:227-233 GIFFORD D.J. 1988. An e l e c t r o p h o r e t i c a n a l y s i s of the seed p r o t e i n s from Pinus monticola and eight other species of pine. Can. J . Bot. 66:1808-1812 GREEN B.R. 1988. The c h l o r o p h y l l - p r o t e i n complexes of higher p l a n t photosynthetic membranes or j u s t what green band i s that? Photos. Res. 15:3-32 GUPTA P.K. and DURZAN D.J. 1985. Shoot m u l t i p l i c a t i o n from mature t r e e s of Douglas f i r (Psedotsuga menziesii) and sugar pine (Pinus lambertiana). P l a n t C e l l Rep. 4_:177-179 HARU K., NAITO K. and SUZUKI H. 1982. D i f f e r e n t i a l e f f e c t s of benzyladenine and potassium on DNA, RNA, p r o t e i n and c h l o r o p h y l l contents and on expansion growth of detached cucumber cotyledons i n the dark and l i g h t . P h y s i o l P l a n t . 55:247-254 HARVEY B.M.R., LU B.C. and FLETCHER R.A. 1974. Benzyladenine a c c e l e r a t e s 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 and s t i m u l a t e s photosynthetic enzyme a c t i v i t y i n cucumber cotyledons. Can. J . Bot. 52:2581-2586 HASEGAWA P.M., YASUDA T. and CHENG T.Y. 1979. E f f e c t of auxin and c y t o k i n i n on newly synthesized p r o t e i n s of c u l t u r e d Douglas f i r cotyledons. P h y s i o l P l a n t . 46:211-217 HAYAT M. 1989. P r i n c i p l e s and techniques of e l e c t r o n microscopy. B i o l o g i c a l a p p l i c a t i o n s . CRC, F l o r i d a . , pp 208-327 110 HISCOX J.D. and ISRAELSTAM G.F. 1979. A method f o r the e x t r a c t i o n of c h l o r o p h y l l from l e a f t i s s u e without maceration. Can. J . Bot. 57:1332-1334 HUGHES K.W. 1981. In v i t r o ecology: exogenous f a c t o r s a f f e c t i n g growth and morphogenesis i n p l a n t c u l t u r e systems. Environ. Exp. Bot. 21:281-288 JELIC G./ and BOGDANOVIC M. 1988. Antagonism between a b s c i s i c a c i d and c y t o k i n i n i n c h l o r o p h y l l synthesis i n pine s e e d l i n g s . P l a n t Science 61:197-202 JELIC G., and BOGDANOVIC M.. 1990. The r e l a t i o n s h i p between c h l o r o p h y l l accumulation and endogenous c y t o k i n i n i n the greening cotyledons of Pinus n i g r a Arn. P l a n t Science 71:153-157 JENSEN W.A. and FISHER D.B. 1968. Cotton embryogenesis: the entrance and discharge of the p o l l e n tube i n the embryo sac. P l a n t a 78:158-183 KHYSE-ANDERSEN J . 1984. E l e c t r o b l o t t i n g of m u l t i p l e g e l s : a simple apparatus without b u f f e r tank f o r r a p i d t r a n s f e r of p r o t e i n s from polyacrylamide to n i t r o c e l l u l o s e . J . Biochem. Biophys. Meth. 10:204-209 KIRK J.T.O. and TILNEY-BASSETT R.A.E. 1978. The p l a s t i d s : t h e i r chemistry, s t r u c t u r e , growth and i n h e r i t a n c e . E l s e v i e r , North Holland, pp 720-787 KOZAI T., KOYAMA Y. and WATANABE I. 1988. M u l t i p l i c a t i o n of potato p l a n t l e t s in_ v i t r o w i t h sugar f r e e medium under high photon photosynthetic f l u x . Acta H o r t i c . 230:121-127 KOZAI T., OKI H. and FUJIWARA K. 1990. Photosynthetic c h a r a c t e r i s t i c s of Cymbidium p l a n t l e t i n v i t r o . Plant C e l l T i s s . Org. Cult. 2^:205-211 KUMAR P.P., REID D.M. and THORPE T.A. 1987. The role of ethylene and carbon dioxide i n d i f f e r e n t i a t i o n of shoot buds i n excised cotyledons of Pinus radiata i n v i t r o . Physiol. Plant. 69:244-252 KUMAR P.P., BENDER L. and THORPE T.A. 1988. A c t i v i t i e s of ribulose biphosphate carboxylase and phosphoenolpyruvate carboxylase and 14-C-bicarbonate f i x a t i o n during i n v i t r o culture of Pinus radiata cotyledons. Plant Physiol. 87:675-679 LERBS S., LERBS W., KLYACHKO N.L., ROMANKO E.G., KULAEVA O.N., WOLLGIEHN R. and PARTHIER B. 1984. Gene expression i n c y t o k i n i n - and lig h t - m e d i a t e d p l a s t o g e n e s i s of Cucurbita cotyledons: r i b u l o s e - 1 , 5 -bisphosphate carboxylase/oxygenase. P l a n t a 162:289-298 LESCURE A.M. and SEYER P. 1981. E f f e c t of c y t o k i n i n on p l a s t i d d i f f e r e n t i a t i o n i n tobacco c e l l suspensions. In Metabolism and Molecular A c t i v i t i e s of C y t o k i n i n s . Guern J . and Peaud-Lenoel C , eds. Springer-Verlag, B e r l i n , pp 298-307 LESHEM Y. 1973. The molecular and hormonal b a s i s of p l a n t growth r e g u l a t i o n . Pergammon Press, Oxford, pp 121-128 LETHAM D.S. and PALNI L.M.S. 1983. The b i o s y n t h e s i s and metbolism of c y t o k i n i n s . Ann. Rev. P l a n t P h y s i o l . 34:271-279 LICHTENTHALER H. and BUSCHMANN C. 1978. Co n t r o l of c h l o r o p l a s t development by red l i g h t , blue l i g h t and phytohormones. Ln C h l o r o p l a s t development. Akoyunoglou G. et a l , eds. E l s e v i e r , N. Holland, pp 801-816. LONGO G.P., PEDRETTI M., ROSSI G. and LONGO C P . 1979. E f f e c t of benzyladenine on the development of p l a s t i d s and microbodies i n ex c i s e d watermelon cotyledons. P l a n t a 145:209-217. McGAW B.A. and HORGAN R. 1985. C y t o k i n i n metabolism and the c o n t r o l of c y t o k i n i n a c t i v i t y . B i o l . P l a n t . (Praha) 21_{2-3) : 180-187 MARZIANI LONGO G.P., BRACALE M., ROSSI G. and LONGO C P . 1990. Benzyladenine induces the appearance of LHCP-mRNA and of the re l e v a n t p r o t e i n i n dark-grown e x c i s e d watermelon cotyledons. P l a n t Mol. B i o l . 14:569-573 MIKULOVICH T.P., ROMANKO E.R., YU-SELIVANKINA S., KUKINA I.M. and WOLLGIEHN R. 1981. C y t o k i n i n a c t i o n on RNA synth e s i s i n c h l o r o p l a s t s . In. Metabolism and Molecular a c t i v i t i e s of c y t o k i n i n s . Guern J . and Peaud-Lenoel C , eds. Springer-Verlag, B e r l i n , pp 287-297 MULLET J.E. 1988. C h l o r o p l a s t development and gene expression. Ann. Rev. Pla n t P h y s i o l . 39:475-502 NAPIER R.M. and VENIS M.A. 1990. Receptors f o r p l a n t growth r e g u l a t o r s : Recent advances. J . P l a n t Growth Regul. 9:113-126 112 OHYA T., KOJIMA S., NAITO K. and SUZUKI H. 1986. D i f f e r e n t i a l e f f e c t s of benzyladenine and potassium on ribulose-1,5-bisphosphate carboxylase synthesis i n detached cucumber cotyledons. J...Plant P h y s i o l . 125:115-121 OHYA T. and SUZUKI H. 1990. Benzyladenine- and l i g h t -s t i m u l a t e d p l a s t i d p r o t e i n synthesis i n e x c i s e d cucumber cotyledons. P l a n t P h y s i o l . Biochem. 28(1) : 27-35 OSBORNE D.J. 1984. Concepts of t a r g e t c e l l s i n p l a n t d i f f e r e n t i a t i o n . C e l l D i f f . 14:161-169 PAPROTH B. and HAUSKA G. 1985. Thylakoid 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 spinach. In. Regulation 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 . Akoyunoglou G. and Senger H., eds. Alan R. L i s s Inc., NY pp 193-196 PATEL K.R. and THORPE T.A. 1984a. Histochemical examination of shoot i n i t i a t i o n i n c u l t u r e d cotyledon explants of r a d i a t a pine. Bot. Gaz. 145(3):312-322 PATEL K.R. and THORPE T.A. 1984b. In v i t r o d i f f e r e n t i a t i o n of p l a n t l e t s from embryonic explants of lodgepole pine (Pinus c o n t o r t a Dougl. ex Loud.). P l a n t C e l l T i s s . Org. C u l t . 3:131-142 PARTHIER B. 1979. The r o l e of phytohormones (cytokinins) i n c h l o r o p l a s t development. Biochem. P h y s i o l . Pflanzen 174 :173-214 PARTHIER B., LERBS S., LEHMANN J . and WOLLGIEHN R. 1985. C y t o k i n i n - c o n t r o l l e d r i b u l o s e - 1 , 5 - b i s p h o s p h a t e carboxylase gene e x p r e s s i o n i n pumpkin c o t y l e d o n s . B i o l . P l a n t . 27(2-3): 131-138 PEAUD-LENOEL C. and AXELOS M. 1981. P l a s t i d p r o t e i n s of c y t o p l a s m i c o r i g i n as molecular markers of c y t o k i n i n a c t i v i t y . Ln Metabolism and molecular a c t i v i t i e s of c y t o k i n i n s . Guern J . and Peaud- Lenoel C , eds. S p r i n g e r - V e r l a g , B e r l i n , pp 308-316 PINO E., MARTIN L., GUERRA H., NICOLAS G. and VILLALOBOS N. 1991. E f f e c t of d i h y d r o z e a t i n on the m o b i l i z a t i o n of p r o t e i n r e s e r v e s i n p r o t e i n bodies d u r i n g the germination of chick-pea seeds. J . P l a n t P h y s i o l . 137:425-432 113 RADUNZ A., SCHMID G.H., BERTRAND M. and DUJARDIN E. 1985. Comparative s e r o l o g i c a l s t u d i e s on some 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 var. commodore L. In Regulation 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 . Akoyunogloy G., and Senger H., eds. Alan R. L i s s Inc., NY, pp 197-203 REBEIZ C C . and REBEIZ C A . 1985. C h l o r o p l a s t biogenesis 53: U l t r a s t r u c t u r a l study of c h l o r o p l a s t development during photoperiodic greening. In Regulation 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 . Akoyunogloy G., and Senger H., eds. Alan R. L i s s Inc., NY, pp 389-396 REYNOLD T.L. 1989. Changes i n RNA, p r o t e i n and t r a n s l a t a b l e messenger RNA synthesis and accumulation during adventive organogenesis i n somatic t i s s u e c u l t u r e s of Solanum c a r o l i n e n s e . P l a n t S c i . 65:77-85 RUMARY C , PATEL K.R. and THORPE T.A. 1986. P l a n t l e t formation i n black and white spruce. I I . H i s t o l o g i c a l a n a l y s i s of a d v e n t i t i o u s shoot formation i n v i t r o . Can. J. Bot. 64:997-1002 SCHENK R.U. and HILDEBRANDT A.C. 1972. Medium and techniques f o r i n d u c t i o n and growth of monocotyledonous and dicotyledonous p l a n t c e l l c u l t u r e s . Can. J . Bot. 50 :199-204 SCHNEPF E. 1980. Types of p l a s t i d s : t h e i r development and i n t e r c o n v e r s i o n s . In C h l o r o p l a s t s . R e i n e r t , J . , ed. Springer-Verlag, B e r l i n , pp 1-27 SESTAK Z. 1985. General features i n u l t r a s t r u c t u r e and composition of c h l o r o p l a s t s during t h e i r development. In Regulation 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 . Akoyunoglou G. and Senger H., eds. Alan R. L i s s Inc, NY, pp 397-405 SPURR A. 1969. A low v i s c o s i t y epoxy r e s i n embedding medium f o r e l e c t r o n microscopy. J . U l t r a s t r u c t u r e Res. 26:31-34 STABEL P., ERIKSSON T. and ENGSTROM P. 1990. Changes i n p r o t e i n synthesis upon cytokinin-mediated a d v e n t i t i o u s bud i n d u c t i o n and during s e e d l i n g development i n Norway spruce, P i c e a abies. P l a n t P h y s i o l . 92:1174-1183 STABEL P., SUNDAS A. and ENGSTROM P. 1991. C y t o k i n i n treatment of embryos i n h i b i t s the synthesis of c h l o r o p l a s t p r o t e i n s i n Norway spruce. P l a n t a 183:520-527 114 SUNDQVIST c , BJORN L.O. and VIRGIN H.I. 1980. Factors 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 . In C h l o r o p l a s t s . Reinert J . , ed. Springer-Verlag, B e r l i n , pp 201-224 TANIMOTO S. and HARADA H. 1982. E f f e c t of c y t o k i n i n and a n t i c y t o k i n i n on i n i t i a l stage of a d v e n t i t i o u s bud d i f f e r e n t i a t i o n i n the epidermis of Torenia stem segments. P l a n t C e l l P h y s i o l . 23 (8) -.1371-1376 TAYLOR J.S. and WAREING P.F. 1979. The e f f e c t of l i g h t on the endogenous l e v e l s of c y t o k i n i n s and g i b b e r e l l i n s i n seeds of S i t k a spruce (Picea s i t c h e n s i s C a r r i e r e ) . P l a n t C e l l Environ. 2:173-179 TEYSSENDIER DE LA SERVE B., AXELOS M. and PEAUD-LENOEL C. 1985a. K i n e t i n - i n d u c e d accumulation of mRNA encoding the apoprotein of the 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 p r o t e i n complex i n tobacco c e l l suspension. In Regulation 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 . Akoyunoglou G. and Senger H., eds. Alan R. L i s s Inc., NY, pp 565-570 TEYSSENDIER DE LA SERVE B., AXELOS M. and PEAUD-LENOEL C. 1985b. Cy t o k i n i n s modulate the expression of genes encoding the p r o t e i n of the 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. Pla n t Mol. B i o l . 5_:155-163 THOMAS J . , KUGRENS P. and ROSS C.W. 1980. C y t o l o g i c a l and biochemical aspects of cytokinin-enhanced growth of r a d i s h (Raphanus sativus) cotyledons. Amer. J . Bot. 67 (4) :456-464 THORPE T.A. 1980. Organogenesis i n v i t r o : s t r u c t u r a l , p h y s i o l o g i c a l , and biochemical aspects. I n t . Rev. C y t o l . Suppl. 11A:71-111 THORPE T.A. 1982. Morphogenesis and regeneration i n t i s s u e c u l t u r e . In Genetic Engineering: A p p l i c a t i o n s to a g r i c u l t u r e . Owens L.D., ed. Rowman and A l l a n h e l d , Totowa, pp 285-303 THORPE T.A. 1988. In v i t r o somatic embryogenesis. In ISI A t l a s of science: animal and p l a n t sciences. P h i l a d e l p h i a , PA, pp 81-88 THORPE T.A. and BIONDI S. 1981. Regulation of pl a n t organogenesis. Adv. C e l l C u l t . 1^:213-239 THORPE T.A. and PATEL K.R. 1984. C l o n a l propagation: a d v e n t i t i o u s buds. In C e l l c u l t u r e and somatic c e l l genetics of p l a n t s . V o l . 1. Academic Press, NY, pp 49-60 THORPE T.A. and PATEL K.R. 1986. Comparative morpho-h i s t o l o g i c a l s t u d i e s on the s i t e s of shoot i n i t i a t i o n i n various c o n i f e r explants. N. Z. J . For. Science 1_6 (3) :257-268 TOBIN E.M. and TURKALY E. 1982. K i n e t i n a f f e c t s rates of degradation of mRNAs encoding two major c h l o r o p l a s t p r o t e i n s i n Lemna gibba L. G-3. J . Pla n t Growth Regul. 1_:3-13 TRANVAN H., TROTON D. and CALVAYRAC R. 1988. Morphological, h i s t o l o g i c a l and l i p i d changes during a d v e n t i t i o u s budding i n Pinus p i n a s t e r c u l t u r e d cotyledons. J . Exp. Bot. 39 (204) :907-915 VAN STADEN J . 1983. Seeds and c y t o k i n i n s . P h y s i o l . P l a n t . 58:340-346 VAN STADEN J . , FORSYTH C , BERGMAN L. and VON ARNOLD S. 1986. Metabolism of benzyladenine by e x c i s e d embryos of Pi c e a abies. P h y s i o l . P l a n t . 66:427-434 VILLALOBOS V.M. 1983. The e a r l y events a s s o c i a t e d with organogenesis i n c u l t u r e d r a d i a t a pine cotyledons. Ph.D. t h e s i s , Department of Biology, U n i v e r s i t y of Calgary, A l b e r t a , Canada VILLALOBOS V.M., OLIVER M.J., YEUNG E.C. and THORPE T.A. 1984. C y t o k i n i n induced switch i n development i n exc i s e d cotyledons of r a d i a t a pine c u l t u r e d i n v i t r o . P h y s i o l . P l a n t . 61:483-489 VILLALOBOS V.M., YEUNG E.C. and THORPE T.A. 1985. O r i g i n of a d v e n t i t i o u s shoots i n e x c i s e d r a d i a t a pine cotyledons c u l t u r e d i n v i t r o . Can. J . Bot. 63:2172-2176 VON ARNOLD S. and ERIKSSON T. 1981. In v i t r o s t u d i e s of a d v e n t i t i o u s shoot formation i n Pinus c o n t o r t a . Can. J . Bot. 59:870-874 VON ARNOLD S. and GRONROOS R. 1986. Anatomical changes and peroxidase a c t i v i t y a f t e r c y t o k i n i n treatments inducing a d v e n t i t i o u s bud formation on embryos of P i c e a abies• Bot. Gaz. 147 (4) :425-431 116 WAGLEY L.M./ GLADFELTER H.J. and PHILLIPS G.C. 1987. De  novo shoot organogenesis of Pinus e l d a r i c a Medw. i n v i t r o . I I . Macro and micro-photographic evidence of de  novo regeneration. P l a n t C e l l Rep. _6:167-171 WELLBURN A.R. 1982. Bi o e n e r g e t i c and u l t r a s t r u c t u r a l changes a s s o c i a t e d with c h l o r o p l a s t development. I n t . Rev. C y t o l . 80:133-191 WELLBURN A.R. 1984. U l t r a s t r u c t u r a l , r e s p i r a t o r y and metabolic changes a s s o c i a t e d with c h l o r o p l a s t development. In Topics i n photosynthesis. C h l o r o p l a s t Biogenesis. Baker N.R. and Barber J . , eds. E l s e v i e r S c i . Publ., Amsterdam, 5_:253-303 WELLBURN A.R., GOUNARIS I . , OWEN J.H., LAYBOURN-PARRY J.E.M. and WELLBURN F.A.M. 1985. Development of bi o e n e r g e t i c f u n c t i o n i n light-grown s e e d l i n g s . _In Regulation 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 . Akoyunoglou G. and Senger H., eds. Alan R. L i s s Inc, NY, pp 283-289 WHATLEY J.M. 1974. C h l o r o p l a s t development i n primary leaves of Phaseolus v u l g a r i s . New P h y t o l . 73:1097-1110 WHATLEY J.M. 1978. A suggested c y c l e of p l a s t i d developmental i n t e r r e l a t i o n s h i p s . New P h y t o l . 80:489-502 WHITE M.J. 1987. Developmental and immunological s t u d i e s on c h l o r o p h y l l p r o t e i n s . Ph.D. Thesis. The U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B.C., Canada WHITE M.J. and GREEN B.R. 1987a. Antibodies t o the photosystem I c h l o r o p h y l l a+b antenna c r o s s - r e a c t with polypeptides of CP29 and LHCII. Eur. J . Biochem. 163:545-551 WHITE M.J. and GREEN B.R. 1987b. Polypeptides belonging t o each of the three major c h l o r o p h y l l a+b p r o t e i n complexes are present i n a c h l o r o p h y l l - b - l e s s b a r l e y mutant. Eur. J . Biochem. 165:531-535 WHITE M.J. and GREEN B.R. 1988. Intermittent-light chloroplasts are not developmentally equivalent to chlorina f2 chloroplasts i n barley. Photos. Res. 15:195-203 WOLLGIEHN R. and PARTHIER B. 1980. RNA and protein synthesis i n p l a s t i d d i f f e r e n t i a t i o n . In Chloroplasts. Reinert J . , ed. Springer-Verlag, B e r l i n , pp 95-145 YASUDA T., HASEGAWA P.M. and CHENG T.Y. 1980. A n a l y s i s of newly synthesized p r o t e i n s during d i f f e r e n t i a t i o n of c u l t u r e d Douglas f i r cotyledons. P h y s i o l . P l a n t . 48:83-87 YEUNG E.C., AITKEN J . , BIONDI S. and THORPE T.A. 1981. Shoot h i s t o g e n e s i s i n cotyledon explants of r a d i a t a pine. Bot. Gaz. 142 (4) :494-501 ZIMMERMANN K.H., CHUDY M., PREUSSER E. and GORING H. 1987. R e l a t i o n s h i p s between l i g h t and hormone a c t i o n on ribulose-1,5-bisphosphate carboxylase i n cucumber cotyledons. Biochem. P h y s i o l . Pflanzen 182:407-415 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0100367/manifest

Comment

Related Items