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Studies on polyacetylene production in normal and transformed tissue cultures of Bidens alba Norton, Robert Allen 1984

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STUDIES ON POLYACETYLENE PRODUCTION IN NORMAL AND TRANSFORMED TISSUE CULTURES OF BIDENS ALBA By ROBERT ALLEN NORTON B. A., The U n i v e r s i t y o f M i s s o u r i , K a n s a s C i t y , Mo. 1970 M.Sc., The U n i v e r s i t y o f M i s s o u r i , K a n s a s C i t y , Mo. 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES ( D e p a r t m e n t o f B o t a n y ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA June 1984 <§> R o b e r t A l l e n N o r t o n , 1984 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . BOTANY D e p a r t m e n t o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3 Date tf Tcj/v-e /Zt' I E - 6 (3/81) i i ABSTRACT The organs of B idens alb a each have a d i f f e r e n t compo-s i t i o n of p o l y a c e t y l e n e s (PAs). F a c t o r s a f f e c t i n g p r o d u c t i o n of these compounds i n t i s s u e and organ c u l t u r e s , and c h a r a c t e r -i s t i c s of c u l t u r e s showing s u s t a i n e d s y n t h e s i s of PAs were - -i n v e s t i g a t e d . A d d i t i o n a l s t u d i e s evaluated t r a n s m i s s i o n of tumour markers and l e a f PAs i n s e x u a l l y produced o f f s p r i n g . P o l y a c e t y l e n e s were separated and q u a n t i t a t e d by high pressure l i q u i d chromatography. Twenty-one compounds, r e p r e -s e n t i n g s i x chromophores, were i d e n t i f i e d or i n d i c a t e d . The p r i n c i p a l compounds were p h e n y l h e p t a t r i y n e (PHT), found i n leaves and stems; phenyldiynene (PDE), found i n stems; PDE-OAc, found i n r o o t s ; and entetraynene acetate (ETE-OAc), found i n r o o t s . Production of PAs past the t h i r d passage could not be achieved i n c a l l u s from normal p l a n t s , d e s p i t e v a r i a t i o n s i n medium for m u l a t i o n s and environmental parameters. C a l l u s e s from crown g a l l s induced on B_. alba and B_. p i lo sa by two s t r a i n s of Agrobacterium tumefaciens, however, continued to produce PAs a f t e r three years i n c u l t u r e . Transformed c a l l u s l i n e s showed wide f l u c t u a t i o n s i n l e v e l s and a d e c l i n i n g p r o p o r t i o n of PDE-OAc over time. Leaf and stem PAs were not found i n tumour c a l l u s s e v e r a l months o l d . C a l l u s contained PAs not found i n the p l a n t . Root c u l t u r e s were used to determine the e f f e c t of e n v i r o n -mental, hormonal, and n u t r i t i o n a l parameters on growth, PA l e v e l and PA composition. Low temperatures, darkness, high k i n e t i n l e v e l s , and in c r e a s e d s u c r o s e / n i t r a t e r a t i o s i n c r e a s e d p r o d u c t i o n i i i of PAs by r o o t s . The p r o p o r t i o n of PDE-OAc i n c r e a s e d i n l i g h t , high k i n e t i n medium, and l a t e l o g phase of growth. Leaf and stem PAs could not be induced i n root c u l t u r e s . P l a n t s regenerated from nopaline g a l l s rooted and set f e r t i l e seed. S e l f s and crosses with normal p l a n t s demonstrated i n h e r i t a n c e of tumour c h a r a c t e r i s t i c s to the f o u r t h generation. However, and succeeding generations had s h a r p l y reduced l e v e l s of n o p a l i n e , and showed d i f f e r e n t i a l l o s s of t r a n s f o r m a t i o n markers. Transformed p l a n t s could form octopine g a l l s and double transformant t i s s u e was obtained. Transformed p l a n t s were m o r p h o l o g i c a l l y d i f f e r e n t from normal p l a n t s . Crosses between 15. alba and B_. p i l o s a , which d i d not con-tain_;PAs i n leaves or stems, y i e l d e d which segregated f o r PHT s y n t h e s i s , but not i n Mendelian r a t i o s . Synthesis of PHT was dominant but showed depressed l e v e l s i n the which was incompatible with a gene dosage e f f e c t . i v TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v LIST OF FIGURES v i i ACKNOWLEDGEMENTS i x PREFACE x CHAPTER I. INTRODUCTION . 1 CHAPTER I I . IDENTIFICATION OF ACETYLENES AND THEIR QUANTIFICATION 10 CHAPTER I I I . INHERITANCE OF TUMOUR CHARACTERISTICS IN BIDENS ALBA 46 CHAPTER IV. STUDIES OF NORMAL CALLUS OF BIDENS ALBA 74 CHAPTER V. STUDIES ON TRANSFORMED CALLUS OF BIDENS ALBA 94 CHAPTER VI. STUDIES ON ROOT CULTURES OF BIDENS ALBA 133 CHAPTER V I I . CHARACTERISTICS OF HYBRIDS BETWEEN BIDENS ALBA AND BIDENS PILOSA 17 3 AFTERWORD 195 APPENDIX 197 LIST OF TABLES Table 1. HPLC r e t e n t i o n times f o r p o l y a c e t y l e n e s i s o -l a t e d from p l a n t s and c u l t u r e s of 13. alba  Table 2. P o l y a c e t y l e n e composition of Bidens alba var. r a d i a t a s e e d l i n g organs and flowers Table 3. Nopaline s y n t h e s i s i n s u c c e s s i v e generations o transformed Bidens a l b a Table 4. Nopaline s y n t h e s i s and phytohormone autotrophy i n cu. l i n e s l t u r e d stem explants from combined F and R I Table 5. R e l a t i v e growth of B. alba explants on d i f f e r e n t media Table 6. Medium i n g r e d i e n induce p o l y a c e t y l e n e syn Table 7. Comparison of po s i t i o n of normal p l a n t o and g a l l s of B. alba and ts evaluated f o r a b i l i t y to thes i s l y a c e t y l e n e l e v e l and compo-rgans, transformed c u l t u r e s , B. p i l o s a  Table 8. Comparison of p o l y a c e t y l e n e s i n leaves of rooted and unrooted transformed p l a n t l e t s Table 9. Comparison of p o l y a c e t y l e n e s i n l i g h t s or dark-grown transformed p l a n t l e t s Table 10. P o l y a c e t y l e n e composition of s e e d l i n g and c u l t u r e d roots of 15, a l b a grown under standard c o n d i t i o n s Table 11. E f f e c t of changes i n c a r b o h y d r a t e / n i t r a t e r a t i o s on p o l y a c e t y l e n e l e v e l s of 15. alba root c u l t u r e s Table 12. E f f e c t of v a r y i n g k i n e t i n c o n c e n t r a t i o n of medium on p o l y a c e t y l e n e composition i n B. alba r o o t s . Table 13. E f f e c t of l i g h t and hormones on root growth and p o l y a c e t y l e n e composition. Table 14. E f f e c t of d e d i f f e r e n t i a t i o n on c u l t u r e s of transformed roots Table 15. S t a b i l i t y of composition, p o l y a c e t y l e n e l e v e l , and growth over time of 15. alba root c u l t u r e s v i Table 16. Comparison of c h a r a c t e r s of 15. alb a, 15. p i l o sa and F hybrids 182 Appendix Table 1. V a r i a t i o n s i n medium hormones used f o r c u l t u r i n g Bidens alb a c a l l u s 198 Appendix Table 2. V a r i a t i o n s i n sugars and vitamins used f o r c u l t u r i n g 15. alba c a l l u s 198 Appendix Table 3. V a r i a t i o n s i n SH medium supplemented with primary m e t a b o l i t e s 199 Appendix Table 4. V a r i a t i o n s i n SH medium macronutrients used f o r c u l t u r i n g 15. alba c a l l u s 200 Appendix Table 5. V a r i a t i o n s i n SH medium supplemented with mixed f a t t y a c i d s 201 v i i LIST OF FIGURES Figure ." 1. Proposed pathways of b i o g e n e s i s of the crep-enynic a c i d f a m i l y of a c e t y l e n i c f a t t y a c i d s Figure 2. P r i n c i p a l p o l y a c e t y l e n e s i n organs of Bidens  a l b a . Figure 3. Compounds d i s c u s s e d : s t r u c t u r e , systematic names, and abbreviated names. Figure '4. HPLC standard curves showing the r e l a t i o n - ". ship between amount of compound and peak area Figure 5. HPLC t r a c e of c a l i b r a t i o n mixture of l e a f and root p o l y a c e t y l e n e s of Bidens alba  Figure 6. UV s p e c t r a of entetrayene compounds Figure .7. Mass s p e c t r a of entetraynene. hydrocarbons. Figure 8. Mass s p e c t r a of entetraynene a l c o h o l s Figure 9. Mass s p e c t r a of entetraynene acetates Figure 10. UV s p e c t r a of entetraynene aldehyde i n d i f f e r e n t s o l v e n t s F i g u r e 11. UV and mass- s p e c t r a of phenyIdiynene hydrocarbon , Figu r e 12. Mass p e c t r a of phenyIdiynene a l c o h o l s . F i g u r e 13. UV and -mass s p e c t r a of phenyIdiynene acetate F i g u r e 14. UV and mass- s p e c t r a of p h e n y l h e p t a t r i y n e hydrocarbon Fig u r e 15. Mass spectrum of p h e n y l h e p t a t r i y n e a c e t a t e . Figure 16. UV spectrum of endiynene acetate .•. Figure 17. UV spectrum of d i e n t r i y n e n e a c e t a t e Figure 18. Paper electropherogram of s i n g l e and double transformant c a l l u s F igure 19. Mo r p h o l o g i c a l d i f f e r e n c e s i n transformed and normal R. a lb a plants-v i i i F i g u r e 20. D i a g r a m o f e v a l u a t i o n p r o c e d u r e f o r p o l y - — a c e t y l e n e p r o d u c t i o n i n c a l l u s c u l t u r e s o f B. a l B a . .. 81 F i g u r e 21. T o t a l p o l y a c e t y l e n e s f o r t r a n s f o r m e d c a l l u s e s g r a d e d by amount o f p i g m e n t a t i o n 103 F i g u r e 22. T o t a l p o l y a c e t y l e n e s i n h i g h and l o w p i g m e n t t a t i o n c a l l u s g r o u p s o v e r t i m e 105 F i g u r e 23. P e r c e n t a g e change i n p h e n y l d i y n e n e a c e t a t e o v e r l o n g - t e r m c u l t u r e o f t r a n s f o r m e d c a l l u s , 107 F i g u r e 24. T o t a l p o l y a c e t y l e n e s o v e r t i m e o f d i f f e r e n t c a l l u s l i n e s o f t r a n s f o r m e d B. a l b a and B. p i l o s a . 109 F i g u r e 25. B i o s y n t h e t i c p a t h w a y s l e a d i n g t o p o l y a c e t y l -enes o c c u r r i n g i n B i d e n s a l b a 115 F i g u r e 26. E f f e c t o f k i n e t i n on p o l y a c e t y l e n e s i n s h o o t s and c a l l u s o f t r a n s f o r m e d p l a n t l e t s 122 F i g u r e 27. E f f e c t o f g r o w t h t e m p e r a t u r e on c u l t u r e d j3. a l b a r o o t s 143 F i g u r e 28. E f f e c t o f pH on g r o w t h and p o l y a c e t y l e n e c o n t e n t of c u l t u r e d j3. a l b a r o o t s 146 F i g u r e 29. E f f e c t of c u l t u r e age on g r o w t h and p o l y -a c e t y l e n e s o f B-. a l b a r o o t c u l t u r e s 148 F i g u r e 30. E f f e c t of v a r y i n g k i n e t i n c o n c e n t r a t i o n on t o t a l ' p o l y a c e t y l e n e s and g r o w t h of c u l t u r e d 13. a l b a r o o t s 154 F i g u r e 31. E f f e c t o f NAA c o n c e n t r a t i o n o n . c u l t u r e d r o o t s o f 13. a l b a 1 5 7 F i g u r e 32. T y p i c a l l e a v e s f r o m p a r e n t a l and F^ h y b r i d B i dens p l a n t s 181 F i g u r e 33. T y p i c a l f l o w e r head o f 13. a l b a , F^, and and 13. p i l o s a 183 F i g u r e 34. Ray f l o w e r s f r o m 13. p i l o s a , F^, and B: a l b a . . 183 F i g u r e 35. P o l l e n m o t h e r c e l l s q u a s h e s of t e l o p h a s e I n u c l e i 185 F i g u r e 36. R e p r e s e n t a t i v e UV s p e c t r a o f p e t r o l e u m e t h e r e x t r a c t s o f 13. a l b a X 13. p i l o s a F^ p l a n t s 1 0 0 F i g u r e 37. F r e q u e n c y d i s t r i b u t i o n o f PHT l e v e l / i n 65 F p r o g e n y o f 13. a l b a X 13. p i l o s a ^ 9 i x AKNOWLEDGEMENTS I would l i k e to express my a p p r e c i a t i o n and thanks to the members of my research committee; Drs. F. R. Ganders, A. D. M. Glass, and P. M. Townsley f o r t h e i r c r i t i c i s m s and h e l p f u l suggestions. I would a l s o l i k e to thank the f o l l o w i n g people for p r o v i d i n g t e c h n i c a l a s s i s t a n c e or m a t e r i a l s : Dr. C h i - K i t Wat and Mr. F e l i p e B a l z a f o r GC/MS analyses; Mr. C h r i s K e l l e r f o r a s s i s t a n c e with root c u l t u r e s ; Dr. Thor Arnason f o r k i n d l y s u p p l y i n g seeds of Bidens p i l o s a ; Dr. Gerald S t r a l e y f o r advice on chromosome p r e p a r a t i o n and s t a i n i n g ; and Dr. M. P. Gordon fo r p r o v i d i n g Agrobacterium s t r a i n s and f o r i n t r o d u c i n g me to crown g a l l r e s e a r c h . S p e c i a l thanks to my research s u p e r v i s o r , Dr. G. H. N. Towers, f o r advice and suggestions too numerous to mention. I would l i k e to thank my wi f e , C a r o l Norton, f o r her patience i n enduring many n i g h t s as a graduate student widow, and f o r her t e n a c i t y i n f e r r e t i n g out s t y l i s t i c and t y p g r a p h i c a l e r r o r s . X PREFACE P o l y a c e t y l e n e s a r e w i d e s p r e a d i n two l a r g e d i c o t f a m i l i e s ( U m b e l l i f e r a e and C o m p o s i t a e ) as w e l l as s e v e r a l s m a l l e r f a m i -l i e s and i n c e r t a i n g r o u p s of f u n g i . Over t h e p a s t t h i r t y y e a r s t h e s e compounds have been e x t e n s i v e l y i n v e s t i g a t e d p r i -m a r i l y by c h e m i s t s and much i s now known a b o u t t h e b i o s y n t h e t i c p a t h w a y s l e a d i n g t o s p e c i f i c compounds. I n r e c e n t y e a r s s t u d i e s on t h e b i o a c t i v i t y o f many o f t h e s e compounds have shown t h a t a number of them have p o w e r f u l p h o t o t o x i c a n t i b i o t i c a c t i v i t i e s . T hese two a s p e c t s , h o w e v e r , s c a r c e l y e x h a u s t t h e p o i n t s o f i n t e r e s t o f t h i s c l a s s o f compounds. Many p l a n t s s y n t h e s i z e a d i f f e r e n t s e t of p o l y a c e t y l e n e s i n e a c h o r g a n o r appendage t y p e , s o m e t i m e s w i t h l i t t l e o v e r l a p f r o m one t o t h e o t h e r . The s t r i n g e n t c o n t r o l e x e r c i s e d o v e r t h e b i o s y n t h e s i s o f a g i v e n p l a n t ' s complement of p o l y a c e t y l e n e s p r e s e n t a number of i n t e r -e s t i n g q u e s t i o n s ; h o w e v e r , v e r y l i t t l e r e s e a r c h i n t h e area o f e i t h e r p h y s i o l o g i c a l o r b i o c h e m i c a l a s p e c t s o f t h e c o n t r o l o f polyacetylene.°synthesis had b e e n r e p o r t e d when t h i s t h e s i s was b egun. Over t h e p a s t few y e a r s t i s s u e c u l t u r e s of p l a n t s h a v e b e e n u s e d e x t e n s i v e l y i n s e v e r a l c a s e s f o r i n v e s t i g a t i n g n o t o n l y t h e b i o s y n t h e t i c s t e p s o f s e c o n d a r y p r o d u c t s b u t a l s o c o n t r o l o f t h e p a t h w a y s ' e.g., t h e work on t h e c r u c i a l i n v o l -vement of p h e n y l a l a n i n e ammonia l y a s e i n p h e n y l p r o p a n o i d m e t a b o l i s m . The s t u d i e s r e p o r t e d i n t h i s t h e s i s were u n d e r -t a k e n w i t h t h e p u r p o s e o f d e v e l o p i n g a t i s s u e c u l t u r e s y s t e m f o r i n v e s t i g a t i n g b i o s y n t h e t i c and r e g u l a t o r y a s p e c t s of p o l y -x i a c e t y l e n e s y n t h e s i s i n B i d e n s a l b a . However, i n i t i a l r e s u l t s w i t h c a l l u s c u l t u r e s were n o t p r o m i s i n g and o t h e r a p p r o a c h e s were t r i e d w h i c h l e d t o tumour and o r g a n c u l t u r e s . R o o t c u l -t u r e s p r o v e d t o be a s e n s i t i v e s y s t e m f o r s t u d y i n g p h y s i o l o g i c a l c o n t r o l o f p o l y a c e t y l e n e s y n t h e s i s , and some c h a r a c t e r i s t i c s o f t h e s e c u l t u r e s a r e p r e s e n t e d h e r e . I n a d d i t i o n , p r e l i m i n a r y s t u d i e s were made of t h e i n h e r i t a n c e o f a m a j o r p o l y a c e t y l e n e o c c u r r i n g i n J3. a l b a . A g a i n , v e r y l i t t l e r e s e a r c h h a s a p p e a r e d on t h e g e n e t i c s o f p o l y a c e t y l e n e s a l t h o u g h t h e p o t e n t i a l f o r i n v o l v e m e n t o f t h i s g r o u p o f compounds i n e c o l o g i c a l a d a p t a t i o n w o u l d a p p e a r t o be h i g h . A d d i t i o n a l s t u d i e s n o t d i r e c t l y r e l a t e d t o p o l y a c e t y l e n e s were c a r r i e d o u t when r e s u l t s o f work on crown g a l l c u l t u r e s i n d i c a t e d t h a t 15. a l b a m i g h t b e h a v e d i f f e r e n t l y f r o m p l a n t s p r e v i o u s l y i n v e s t i g a t e d f o r m a i n t e n a n c e o f t r a n s f o r m a t i o n t h r o u g h m e i o s i s . T h i s t h e s i s i s , t h e r e f o r e , n o t a c o m p l e t e d and d e t a i l e d e x a m i n a t i o n o f any one a s p e c t o f p o l y a c e t y l e n e s b u t , r a t h e r , a s u r v e y o f b o t h c h a r a c t e r i s t i c s o f d i f f e r e n t t i s s u e c u l t u r e s y s t e m s and a l s o some g e n e t i c f e a t u r e s o f 15. a l b a r e l a t e d t o e i t h e r p o l y a c e t y l e n e s y n t h e s i s o r c u l t u r e s o f t h e p l a n t . 1 CHAPTER I INTRODUCTION 2 INTRODUCTION An a c t i v e area of cu r r e n t r e s e a r c h i n p l a n t Biochemistry i s the p r o d u c t i o n of secondary compounds i n t i s s u e c u l t u r e s . One phase of the work reported i n t h i s t h e s i s attempts to apply some of the techniques of t i s s u e c u l t u r e to the problem of producing a s p e c i f i c class- of secondary compounds, p o l y a c e t y l e n e s , i n c u l t u r e d m a t e r i a l . P o l y a c e t y l e n e s , a l s o termed poly-ynes or acetylenes., are found i n 19. higher p l a n t f a m i l i e s and i n Basidiomycete f u n g i (9). They are most c h a r a c t e r i s t i c of members of the U m b e l l i f -erae, Compositae, Santalaceae, and r e l a t e d f a m i l i e s Q5). Higher plants- account f o r 85% of the known compounds with, the remainder i s o l a t e d from f u n g i . Over 725 known acetylenes have been de s c r i b e d C9_) • This group would seem, then, to be one of the most abundantly produced classes- of secondary m e t a b o l i t e s i n p l a n t s , and thus warrant res e a r c h i n t o the f a c t o r s a f f e c t i n g its:., '..synthesis. Polyacetylenes- are considered to be a bio — g e n e t i c a l l y uniform group having a common d e r i v a t i o n from o l e i c a c i d and c o n t a i n i n g one or more ace t y l e n e bonds, u s u a l l y con-jugated- C3 , 5).. The common b i o s y n t h e t i c o r i g i n of p o l y a c e t y l e n e s i s shown i n F i g . 1, which a l s o shows- the proposed r e l a t i o n s h i p s between p r e c u r s o r f a t t y a c ids and r e l a t e d epoxy and a c e t y l e n i c compounds (4). The p l a n t chosen f o r i n v e s t i g a t i n g these compounds was Bidens a l b a var. r a d i a t a , a wi d e l y d i s t r i b u t e d member of the Compositae. This species has been segregated from the more commonly known B. p i l o s a By B a l l a r d 0 . ) • Bidens alfra c o ntains 3 18:1 (9c) » X O l e i c a c i d 18 : 2 ( 9 c l 2 t ) >18:2(9cl2c) — L i n o l e i c a c i d 18 : 2 (9cl2a) .Crepenynic a c i d 9h-18:2(10tl2a) H e l e n y n o l i c a c i d ->-12,13-epoxy-18:l(9c) V e r n o l i c a c i d 9,10-epoxy-18:l(12c) Coronaric a c i d 1 18:3(9cl2al4c) 9,10-epoxy-18:l(12a) I P o l y a c e t y l e n e s F i g . 1. Proposed pathways of b i o g e n e s i s of the crepenynic fa m i l y of a c e t y l e n i c f a t t y a c i d s . A s t e r i s k denotes p o s t u l a t e d enzyme-bound i n t e r m e d i a t e s . Source: Hitchcock and N i c h o l s ; Plant L i p i d Bi ochemi s t r y , 1971. three e a s i l y detected p o l y a c e t y l e n e s : p h e n y l h e p t a t r i y n e (PHT), phenyldiynene CPDE), entetraynene (ETE), and a c e t y l a t e d and alcohol forms of these. These compounds have characteristic d i s t r i b u -t i o n s i n d i f f e r e n t p a r t s of the p l a n t and thus o f f e r a r e l a -t i v e l y simple but i n t e r e s t i n g system f o r i n v e s t i g a t i n g produc-t i o n i n c u l t u r e d m a t e r i a l . F i g u r e 2 shows the p r i n c i p a l a c e t ylenes found i n B. alba and t h e i r d i s t r i b u t i o n i n the p l a n t . Much i s now known about the b i o s y n t h e t i c pathways of p o l y -a c e t y l e n e s as a r e s u l t of work i n the labs of Bohlmann, Bu'Lock, Jones, the Sorensens, and many others . Bohlmann et a l . summar-i z e d t h i s research i n 1973 i n what i s s t i l l the standard r e f -erence f o r work i n p o l y a c e t y l e n e s (3). N e v e r t h e l e s s , much remains to be done; as emphasized by T h a l l e r (10) who noted that "Although the general p r i n c i p l e s of p o l y a c e t y l e n e b i o g e n e s i s are thought to be understood, many d e t a i l s s t i l l wait to be <^^-CsC-CsC-CsC-CH 3 P H E N Y L H E P T A T R I Y N E <^^>-esC-CsC-CH=CH-C H 2 R P H E N Y L D I Y N E N E CH5CH - (C3C) ? CH=CH-CH 2 0Ac E N T E T R A Y N E N E F i g . 2. P r i n c i p a l p o l y a c e t y l e n e s i n organs of Bidens alba, 5 c o r i f i r m e d , n o t . l e a s t t h e b i o g e n e s i s o f t h e t r i p l e b o n d i t s e l f . " A s i m i l a r v i e w was sfatetfc b y B o h l m a n n i n 1 9 7 3 (7). T i s s u e c u l t u r e s h a v e B e e n u s e d e x t e n s i v e l y f o r s t u d y i n g m e t a b o l i c p a t h w a y s i n a v a r i e t y o f h i g h e r p l a n t s . C u l t u r e s t h a t s y n t h e s i z e c o m p o u n d s o f i n t e r e s t o f f e r s e v e r a l a d v a n t a g e s f o r t h e s t u d y o f t h e i r b i o s y n t h e s i s a n d c o n t r o l . 1) A c t i v e c e l l - f r e e s y s t e m s a n d p u r i f i e d e n z y m e p r e p a r a t i o n s c a n b e p r e p a r e d m o r e e a s i l y d u e t o t h e a b s e n c e o f c o m p o u n d s c a u s i n g d e a c t i v a t i o n o f e n z y m e s , n o t a b l y p h e n o l s a n d q u i n o n e s Q8). 2) T i s s u e , c u l t u r e s o f t e n s h o w h i g h e r i n c o r p o r a t i o n r a t e s o f l a b e l l e d p r e c u r s o r s t h a n I n t a c t p l a n t s o r t i s s u e s l i c e s ; a l l o w -i n g g r e a t e r f l e x i b i l i t y i n i d e n t i f i c a t i o n t e c h n i q u e s . As a n e x a m p l e , s u s p e n s i o n c u l t u r e s o f C a t h . a r a n t h . u s r o s e u s h a d d e u t e r i u m i n c o r p o r a t i o n l e v e l s - t w o o r d e r s o f m a g n i t u d e h i g h e r t h a n i n t a c t p l a n t s , a l l o w i n g t h e u s e o f m a s s s p e c t r o m e t r y f o r d e t e c t i o n o f l a b e l l e d c o m p o u n d s C2)'. • 3 ) U n d e r s u i t a b l e c o n - . , d i t i o n s a r e l a t i v e l y h o m o g e n e o u s c e l l p o p u l a t i o n c a n b e o b t a i n e d a n d e n v i r o n m e n t a l a n d n u t r i t i o n a l p a r a m e t e r s c l o s e l y c o n t r o l l e d , a l l o w i n g r a p i d a n d p r e c i s e e v a l u a t i o n o f c h a n g e d c o n d i t i o n s . A n e x a m p l e o f how- c e l l c u l t u r e s - may c o n t r i b u t e i n a u n i q u e way t o u n d e r s t a n d i n g t h e e n z y m o l o g y o f s e c o n d a r y p r o - r d u c t f o r m a t i o n i s Z e n k ' s s u m m a r y o f w o r k o n th.e s y n t h e s i s o f a l k a l o i d s - o f C a t h a r a n t h u s r o s e u s 0L3)'.. E x c e l l e n t r e v i e w s o f t h e u s e o f t i s s u e a n d o r g a n c u l t u r e s f o r t h e s o l u t i o n o f p r o b l e m s i n s e c o n d a r y - p l a n t m e t a b o l i s m h a v e B e e n p u b l i s h e d r e c e n t l y by-O v e r t o n a n d P i c k e n (8 ] a n d K u r z a n d C o n s t a b e l C7.1_, O b t a i n i n g s y n t h e s i s o f s e c o n d a r y p r o d u c t s , h o w e v e r , has p r e s e n t e d a c o n s i d e r a b l e c h a l l e n g e t o t i s s u e c u l t u r l s t s . 6 C a l l u s and suspension c u l t u r e s are most o f t e n accompanied by l o s s or g r e a t l y reduced p r o d u c t i o n of secondary compounds. The b a s i c problem f a c i n g r e s e a r c h e r s attempting to o b t a i n secondary products from t i s s u e c u l t u r e s has been s t a t e d s u c c i n c t l y by K r l k o r i a n and Steward ( 6 ) : Is i t p o s s i b l e to reproduce at w i l l the p a r t i c u l a r Biochemistry- of any p a r t i c u l a r organ, t i s s u e or c e l l . Can one Induce p o t e n t i a l l y t o t i -potent c e l l s to express the Biochemistry that they normally achieve i n a given m o r p h o l o g i c a l s e t t i n g , without the n e c e s s i t y of reproducing that s e t t i n g i n t a c t by the growth of the whole organism? In recent years the e f f o r t s of many workers has r e s u l t e d i n a c h i e v i n g secondary product l e v e l s equal to or exceeding those of the i n t a c t p l a n t s f o r m e t a b o l i t e s i n s e v e r a l product c a t e g o r i e s C 1 2 ) , none of which.were f a t t y a c i d d e r i v a t i v e s . Thus, one phase of research, may Be s a i d to Be r e a l i z i n g part of the goal aBove; a c h i e v i n g l e v e l s of compounds comparable'to those of the p l a n t . The other p a r t , s e l e c t i v e a c t i v a t i o n of path-ways, remains a c h a l l e n g i n g problem i n nearly- a l l systems, In order to study the p o l y a c e t y l e n e s i n B_. alba f o r t h i s t h e s i s , i t was f i r s t necessary to develop a n a l y t i c a l techniques f o r i d e n t i f y i n g and q u a n t i f y i n g them and these r e s u l t s are presented i n Chapter r r . The primary- o b j e c t i v e of the r e s e a r c h re p o r t e d here was to t r y to develop a t i s s u e c u l t u r e .system which could be used f o r B i o s y n t h e t i c , and p o s s i b l y enzymological, s t u d i e s . In attempting to f i n d , c u l t u r e s which, would produce a c e t y l e n e s , v a r i a t i o n s i n media composition and other f a c t o r s were t r i e d . However, no unambiguously p o s i t i v e r e s u l t s could be obtained By these methods, which, are summarized i n Chapter 7 IV. F o r t u i t o u s circumstances l e d to i n v e s t i g a t i n g transformed, crown g a l l , c u l t u r e s f o r p o l y a c e t y l e n e s and they were found to be c o n s i s t e n t producers. These r e s u l t s are reported i n Chap-ter V. An a l t e r n a t i v e approach to the problem of o b t a i n i n g c u l -tures which would produce p o l y a c e t y l e n e s was to use root c u l -t u r e s ; they o f f e r e d advantages f o r i n v e s t i g a t i n g f a c t o r s a f f e c t i n g the s p e c i f i c c o m p o s i t i o n a l p a t t e r n s i n the p l a n t . Chapter VI presents r e s u l t s showing c h a r a c t e r i s t i c s of these c u l t u r e s and the degree to which p o l y a c e t y l e n e composition could be a l t e r e d i n them. The work with crown g a l l c u l t u r e s l e d to the f i n d i n g that 13. alba was unusually s u i t a b l e f o r r e g e n e r a t i o n of p l a n t s from one type of tumour c a l l u s and these p l a n t s showed an unexpected a b i l i t y to s t a b l y transmit the tumourous s t a t e from one gener-a t i o n to another. These r e s u l t s are presented i n Chapter I I I . A f i n a l area of t h i s t h e s i s concentrates on the i n h e r i -tance of p h e n y l h e p t a t r i y n e (PHT) p r o d u c t i o n . T h i s i s the main p o l y a c e t y l e n e of leaves of B_. a l b a . There has been only one r e p o r t of work on the i n h e r i t a n c e of p o l y a c e t y l e n e s i n higher p l a n t s ( 1 1 ) . The a v a i l a b i l i t y of two i n t e r f e r t i l e s p e c i e s ( B . alba and B_. p i l o s a ) , which d i f f e r e d i n t h e i r l e a f compounds presented an o p p o r t u n i t y to determine the number of l o c i i n v o l v e d i n PHT i n h e r i t a n c e and a l s o to l e a r n something about the depen-dence of compound l e v e l on gene dosage. These r e s u l t s are found i n Chapter VII. LITERATURE CITED B a l l a r d , R. E. 19 75. A Biosys tenia t i c and chemosyste-matic study of tHe. Bidens- p j l o s a complex i n North and C e n t r a l America. Ph. D. t h e s i s . U n i v e r s i t y of Iowa, Iowa C i t y , Iowa. B a l s e v i c h , J . , F. ConstaBel, and W. G. W. Kurz. 1982. E f f i c i e n t i n c o r p o r a t i o n of 10-hydroxygeraniol and 10-hydroxynero1 i n t o i n d o l e a l k a l o i d s By a c e l l suspension c u l t u r e of Catharanthus roseus. P l a n t a Medica ^4:231-233. Bohlmann, F., T. Burkhardt, and C. Zdero. 1973. N a t u r a l l y o c c u r r i n g a c e t y l e n e s . Academic Press, Ldn. , N.Y'. Hitchcock, C., and B. W. N i c h o l s . 1971. P l a n t l i p i d B i o c h e m i s t r y . Academic Press, Ldn, N.Y. Jones, E. R. H. , and V. T h a l l e r . 19.78. N a t u r a l a c e t y l -enes. Pages 621-633 lrr\S. P a t a i , ed. The chemistry of the carBon-carBon t r i p l e Bond. John Wiley & Go;. ,N;Y K r i k o r i a n , A. D., and F. C. Steward. 1969. Biochemical d i f f e r e n t i a t i o n : the B i o s y n t h e t i c p o t e n t i a l i t i e s of growing and quiescent t i s s u e . Pages 227^-326 i n F. C. Steward, ed. P l a n t p h y s i o l o g y : a t r e a t i s e , v o l . VB. Academic Press, N.Y., Ldn. Kurz, W. G. W., and F. ConstaBel. 1979. P l a n t c e l l c u l t u r e s , a p o t e n t i a l source of pharmaceuticals. Advances i n A p p l i e d M i c r o B i olp:gy 25:20.9-240.. Overton, K. H., and D. J . Picken. 1977. Studies i n secondary metabolism with, p l a n t t i s s u e c u l t u r e s . F o r t s c h r i t t e : d. Chem. Org. Nat u r s t . 34:249-29.8. Sorensen, "N-. A. 19.77, P o l y a c e t y l e n e s and conservation'.of ,' chemical c h a r a c t e r s i n the Composltate. Pages 385-433 i n V. ff. Heywood, J . B". HarBorne, and B. L. Turner. The B i o l o g y and chemistry of the Compositae, V o l . I. Academic Press^ N.Y., Ldn. T h a l l e r , V. 1979. N a t u r a l a c e t y l e n i c and o l e f i n i c compounds e x c l u d i n g marine n a t u r a l products. Pages 1-19 in. F. D. Gunstone, ed. A l i p h a t i c and r e l a t e d n a t u r a l product chemistry, V o l . I. The Chemical S o c i e t y , Ldn. Van F l e e t , D. S. 1970. Enzyme l o c a l i z a t i o n and the g e n e t i c s of polyenes and p o l y a c e t y l e n e s i n the 9 endodermis. Advancing F r o n t i e r s of Plant Science 26:109-143. 12. Zenk, M. H. 1978. The impact of p l a n t c e l l c u l t u r e on i n d u s t r y . Pages 1-13 In T. A. Thorpe, ed. F r o n t i e r s of p l a n t t i s s u e c u l t u r e 1978. I n t e r n a t i o n a l A s s o c i -a t i o n of Pl a n t T i s s u e C u l t u r e , Calgary, A l f a . 13. Zenk, M. H. 1980. Enzymatic s y n t h e s i s of a j m a l i c i n e and r e l a t e d i n d o l e a l k a l o i d s . L l o y d i a 43:438-451. 10 CHAPTER II IDENTIFICATION OF ACETYLENES AND THEIR QUANTITATION 11 INTRODUCTION I d e n t i f i c a t i o n o f t h e m a j o r a c e t y l e n i c compounds i n J3. a l b a p l a n t s and c u l t u r e s and t h e i r q u a n t i t a t i o n were c e n t r a l t o t h e s t u d i e s f o r t h i s t h e s i s . The p r o c e d u r e s f o r compound i d e n t i -f i c a t i o n and r e s u l t s a r e r e p o r t e d i n t h i s c h a p t e r . I d e n t i f i c a t i o n o f t h e UV a b s o r b i n g m o i e t y , t h e c h r o m o p h o r e , i n a c e t y l e n e i c compounds i s o f t e n s t r a i g h t f o r w a r d s i n c e c o n j u -g a t e d p o l y - y n e n e s y s t e m s t y p i c a l l y e x h i b i t s h a r p a b s o r p t i o n b ands w h i c h a r e d i a g n o s t i c f o r t h e number and p o s i t i o n o f d o u b l e and t r i p l e b onds i n t h e cIl.romoph.ore. P u b l i s h e d t a b l e s c o r r e l a t e UV maxima and i n t e n s i t y w i t h s t r u c t u r e ( 5 , 6 ) . C o n j u g a t e d t r i p l e bonds have a s e t o f a b s o r p t i o n p e a k s w i t h , v e r y h i g h m o l a r e x t i n c t i o n c o e f f i c i e n t s C£. v a l u e s ) i n t h e UV r e g i o n f r o m 210-290 nm and q u i t e s m a l l amounts o f compounds can be d e t e c t e d (6 100, 000. f o r t h r e e o r more, t r i p l e b o n d s ) . C o n j u g a t i o n o f t r i p l e and d o u b l e bonds r e s u l t s i n a s e c o n d s e t of l o n g e r wave-l e n g t h p e a k s , w i t h l o w e r £ v a l u e s , e x t e n d i n g i n t o t h e r e g i o n 290-410 nm. The r e l a t i v e a b s o r p t i o n shown by t h e two s e t s of p e a k s d e p e n d s on t h e r a t i o o f the. number o f d o u b l e and t r i p l e bonds and t h u s c a n y i e l d some i n f o r m a t i o n a b o u t t h e c h r o m o p h o r e ( 5 ) . A d d i t i o n a l i n f o r m a t i o n on t h e g e n e r a l n a t u r e o f t h e t e r -m i n a l g r o u p s o r o t h e r s u b s t i t u t i o n s c a n be i n f e r r e d f r o m t h e o r d e r i n w h i c h compounds, e s p e c i a l l y d i f f e r e n t f o r m s o f t h e same c h r o m o p h o r e , s e p a r a t e , by c h r o m o t o g r a p h y (.9). M e t h o d s o f s e p a r a t i n g a c e t y l e n e s r e l y c h i e f l y on l i q u i d c h r o m a t o g r a p h y , both, c o l u m n and t h i n - l a y e r . S o l i d p h a s e s u s e d 12 . i n c l u d e a l u m i n a ( 7 ) , s i l i c a g e l ( 9 ) , and Sephadex LH-20 ( 1 ) . R e v e r s e p h a s e s i l i c a g e l was u s e d by B u ' L o c k and S m i t h (8) t o s e p a r a t e a c e t y l e n i c f a t t y a c i d s . H i g h p r e s s u r e l i q u i d c hroma-t o g r a p h y (HPLC) was u s e d f o r p r e p a r a t i v e s e p a r a t i o n o f c i s / t r a n s i s o m e r s by Rose e t a l . ( l b ) . Use of l i q u i d c o l u m n (CC) and t h i n - l a y e r c h r o m a t o g r a p h y CTLC) a r e u n d o u b t e d l y t h e methods of c h o i c e f o r i s o l a t i n g m i n o r c omponents o f c o m p l e x m i x t u r e s a t t h e l e v e l s r e q u i r e d f o r c h a r a c t e r i z a t i o n o f new compounds, how-e v e r r e c o v e r i e s a r e s e l d o m q u a n t i t a t i v e a c c o r d i n g t o J o n e s and T h a l l e r ( 1 1 ) . F o r a q u a n t i t a t i v e s t u d y a d i f f e r e n t method seemed a d v i s a b l e . I n t h e p r e s e n t s t u d y s e p a r a t i o n was by HPLC. T h i s method o f f e r s s e v e r a l a d v a n t a g e s : 1) many a c e t y l e n e s a r e u n s t a b l e i n p u r i f i e d f o r m , e s p e c i a l l y i n l i g h t , and HPLC u s e s a c l o s e d s y s t e m w h i c h m i n i m i z e s e x p o s u r e t o l i g h t d u r i n g s e p a r -a t i o n ; 2) Q u a n t i t a t i o n o f compounds i s e a s i l y c a r r i e d o u t a f t e r s t a n d a r d c u r v e s a r e o b t a i n e d ; 3 ) " A n a l y s i s o f s a m p l e s i s r e l a -t i v e l y f a s t — t h e t u r n a r o u n d t i m e was 3 0 m i n u t e s f o r a n a l y t i c a l s e p a r a t i o n s i n t h i s s t u d y ; 4) Th.e a v a i l a b i l i t y o f a v a r i a b l e -w a v e l e n g t h d e t e c t o r a l l o w s s e l e c t i o n o f a w a v e l e n g t h t h a t m i n i -m i z e s d e t e c t i o n o f p e a k s f r o m n o n - a c e t y l e n i c \ compounds and i n c r e a s e s t h e r e l i a b i l i t y o f peak i d e n t i f i c a t i o n and q u a n t i t a t i o n , I n o r d e r t o s i m p l i f y d i s c u s s i o n o f t h e r e s u l t s w h i c h f o l l o w , t h e s t r u c t u r e s and names o f a l l t h e compounds i s o l a t e d o r r e p o r -t e d t o o c c u r i n B. a l b a 4 n 4 B. p i l o s a ' , a r e shown i n F i g . 3 . 13 R-CH=CH-C=C~C=C-C=C- C=C~ CH=CH2 A b b r e v i a t i o n I R=CH3 T r i d e c a - l : l l - d i e n e - 3 : 5 : 7 : 9 - t e t r a y n ETE I I R=CHO T r i d e c a - l : l l - d i e n e - 3 : 5 : 7 : 9 - t e t r a y n - 1 3 - a l E T E - a l III R=CH20H T r i d e c a - l : l l - d i e n e - 3 : 5 : 7 : 9 - t e t r a y n - 1 3 - o l E T E - o l I V R=CH20Ac T r i d e c a - l : l l - d i e n e - 3 : 5 : 7 : 9 - t e t r a y n - 1 3 - a c e t a t e ETE-OAc R-CH=CH-CsC-C=C-V: l-phenylhepta-l : 3-diyn - 5-ene PDE V R=CH3 V i R ^ C H O V a : l-pbenylhepta-l : 3-diyn - 5-ene - 7-al PDE-al VI R = C H 2 0 H l-phenylhepta-l : 3-diyn - 5-ene - 7-ol PDE-ol VI I R=CH2OAc l-phenylhepta-l : 3-diyn - 5-ene - 7-acetate PDE-OAc R - C s C - C = C - C H C - ( ^ ) VII I R=CH 3 l - p l i e n y l h e p t a - l : 3 : 5 ^ t r i y n PHT I X R=CH20H l - p h e n y l h e p t a - l : 3 : 5 - t f i y n - 7 - o l PHT-ol X R=CH2OAc l - p h e n y l h e p t a - l : 3 : 5 - t r i y n - 7 - a c e t a t e PHT-OAc DTE R - C H = C H - C = C - C = C - C S C - C H = C H - C H = C H 2 X I R = C H 3 T r i d e c a - l : 3 : l l - t r i e n e - 5 : 7 : 9 - t r i y n XI I R = C H 2 0 H T r i d e c a - l : 3 : l l - t r i e n e - 5 : 7 : 9 - t r i y n - 1 3 - o l DTE-ol XI I I R = C H 2 O A c T r i d e c a - l : 3 : l l - t r i e n e - 5 : 7 : 9 - t r i y n - 1 3 - a c e t a t e DTE-OAc R - C H = C H - C S C - C H C - C H = C H - C H ? C H 2 - C H 2 - C H 3 X IV R s C H ^ H T r i d e c a - 5 : l l - d i e n e - 7 : 9 - d i y n - 1 3 - o l EDE-ol X V R= C H ^ A C Trideca-5:ll-diene-7:9-diyn-13-acetate EDE-OAc C H j C H = C H - C s C - C = C - C H = C H - C H = C H - C H = C H 2 X V I . T r i d e c a - l : 3 : 5 : l l - t e t r a e n e - 7 : 9 - d i y n TDE C H j C = C - C = C - C = C - C = C - C = C - C H = C H 2 X V I I Trideca-l-ene-3:5:7:9:11-pentayn Pentaynene F i g . 3. Compounds discussed: s t r u c t u r e s , systematic names, and abbreviated names . 14 MATERIALS AND METHODS E x t r a c t i o n and P r e p a r a t i o n o f Samples f o r HPLC A n a l y s i s A l l w o rk was c a r r i e d o u t w i t h room l i g h t o f f and c r u d e e x t r a c t s were k e p t i n p e t r o l e u m e t h e r (30-60°C, J . T. B a k e r Chem. Co., P h i l l i p s b u r g , N. J . " R e s i - A n a l y z e d " , o r e q u i v a l e n t ) u n t i l i m m e d i a t e l y p r i o r t o a n a l y s i s t o m i n i m i z e d e g r a d a t i o n of compounds by l i g h t . F o r a n a l y t i c a l work 1-2 g s a m p l e s of t i s s u e were u s e d . B e f o r e f r e s h w e i g h t s were d e t e r m i n e d l i q u i d c u l t u r e d o r g a n s were p a t t e d d r y w i t h p a p e r t o w e l s , s u s p e n s i o n c u l t u r e s were f i l t e r e d o n t o M i r a c l o t h ( C a l B i o c h e m ) , and a g a r c u l t u r e s had a d h e r i n g a g a r s c r a p e d o f f . D u p l i c a t e s a m p l e s w e re t a k e n and d r i e d f o r 24 h o u r s a t 100 , ' c , c o o l e d t o room t e m p e r a t u r e and d r y w e i g h t s d e t e r m i n e d . A f t e r w e i g h i n g , s a m p l e s were homogen-i z e d w i t h 15 ml o f r e d i s t i l l e d m e t h a n o l i n 20 x 15Q mm g l a s s c u l t u r e t u b e s by b r e a k i n g and s h e a r i n g t h e t i s s u e w i t h a g l a s s p e s t l e made i n t h e l a b o r a t o r y w h i c h c l o s e l y f i t t e d t h e c u l t u r e t u b e s . A f t e r h o m o g e n i z a t i . o n , 5 ml o f d i s t i l l e d w a t e r was added f o l l o w e d by 5 ml o f p e t r o l e u m e t h e r ( 3 Q - 6 0 " C ) . The c o n t e n t s were t h e n s h a k e n v i g o r o u s l y f o r 2Q s e c o n d s and c e n t r i f u g e d t o c l a r i f y t h e o r g a n i c p h a s e w h i c h was t h e n drawn o f f by p i p e t t e . The b r e i was e x t r a c t e d t h r e e t i m e s w i t h p e t r o l e u m e t h e r , a l i -q u o t s c o m b i n e d , made up t o v o l u m e , and a UV s p e c t r u m made of t h e c r u d e e x t r a c t . F o r l i q u i d medium e x t r a c t i o n s 25 ml of medium was e x t r a c t e d f o u r t i m e s w i t h p e t r o l e u m e t h e r . S e v e r a l s e r i e s o f p r e l i m i n a r y e x t r a c t i o n s o f l e a f t i s s u e and c u l t u r e d 15 r o o t s were e x t r a c t e d up t o f i v e t i m e s w i t h , p e t r o l e u m e t h e r and two t i m e s w i t h , m e t h a n o l . The r e s u l t s showed t h a t t h e a v e r a g e r a t i o o f a c e t y l e n e s f o r t h e n + l / n e x t r a c t i o n was e q u a l o r l e s s t h a n 0.33. T h i s i n d i c a t e d t h a t t h r e e e x t r a c t i o n s w o u l d r e c o v e r a p p r o x i m a t e l y 9.6% o f t h e compounds e x t r a c t a B l e . I f a n a l y s i s was t o be i m m e d i a t e t h e n t h e c r u d e e x t r a c t was e v a p o r a t e d t o d r y n e s s w i t h n i t r o g e n gas i n dim l i g h t and t h e r e m a i n i n g o i l d i s s o l v e d I n HPLC g r a d e m e t h a n o l . I f a n a l y s i s was t o be d e l a y e d t h e n t h e s a m p l e was s t o r e d a t -70 *C i n p e t r o -l e u m e t h e r . The UV s p e c t r a of s a m p l e s s t o r e d as l o n g as s i x months were i d e n t i c a l t o i n i t i a l s p e c t r a . I n o r d e r t o m a x i m i z e t h e a c c u r a c y of t h e HPLC a n a l y s i s , s a m p l e s were made up i n metha-n o l a t a l e v e l s u c h t h a t t h e a v e r a g e HPLC peak w o u l d have an o p t i c a l d e n s i t y (OD) of a b o u t 0,2, A c c o r d i n g l y , t h e OD o f HPLC p e a k s r e s u l t i n g f r o m i n j e c t i o n o f d i f f e r e n t amounts o f a c r u d e e x t r a c t of c o m b i n e d l e a f and r o o t m a t e r i a l was c o r r e l a t e d w i t h , a b s o r b a n c e a t 287 nm. The f o l l o w i n g r e l a t i o n g e n e r a l l y gave the. v o l u m e r e q u i r e d , g i v e n a 20 u l s a m p l e i n j e c t i o n : V =0D x V x 0.035 s p w here V i s t h e v o l u m e o f m e t h a n o l ( m l ) , and V i s t h e v o l u m e s p o f p e t r o l e u m e t h e r ( m l ) . F o r p r e p a r a t i v e work t h e c o n s t a n t i s 0 . 002 , i n s t e a d o f 0.035. S p e c t r a were r u n on a P y e - U n i c a m SP8-100 UV/VIS s p e c t r o -p h o t o m e t e r s e t a t b a n d w i d t h of 0.5 nm i n p e t r o l e u m e t h e r ( 3 0 -60°C) u n l e s s o t h e r w i s e n o t e d . H i g h P r e s s u r e L i q u i d C h r o m a t o g r a p h y S e p a r a t i o n and q u a n t i t a t i o n o f t h e p o l y a c e t y l e n e s i n t h e 16 o i l r e s u l t i n g f r o m e x t r a c t i o n was a c c o m p l i s h e d by u s e o f a V a r i a n A e r o g r a p h m o d e l 5000 l i q u i d c h r o m a t o g r a p h w i t h a S e r i e s 634 U V / v i s a b l e s p e c t r o p h o t o m e t e r as t h e d e t e c t o r u n i t . D e t e c t -i n g w a v e l e n g t h was 287 nm f o r a l l q u a n t i t a t i v e w o r k . V a r i a n M i c r o p a k MCH-10 r e v e r s e - p h a s e a n a l y t i c a l (30 cm x 4 mm) and p r e p a r a t i v e (50'cm x 8 mm) c o l u m n s were u s e d . The s o l v e n t s were HPLC g r a d e a c e t o n i t r i l e and d i s t i l l e d w a t e r . A t t h e t i m e t h i s w ork was s t a r t e d no p r e v i o u s r e p o r t s o f HPLC use f o r a c e t y l e n e a n a l y s i s c o u l d be f o u n d so i t was n e c e s s a r y t o d e v e l o p a p r o g r a m of c h a n g e s i n s o l v e n t c o m p o s i t i o n and f l o w r a t e t h a t w o u l d o p t i m i z e t h e f o l l o w i n g c o n s i d e r a t i o n s : 1) m a x i m i z e r e s o -l u t i o n o f c o mponents i n a m i x t u r e c o n t a i n i n g a l l t h e p o l y a c e t y l -enes o c c u r r i n g i n E. a l b a p l a n t s ; 2) M i n i m i z e t h e amount o f t i m e r e q u i r e d f o r t h e p r o g r a m t o c y c l e ; 3) F l u s h o u t n o n - p o l a r compounds t e n d i n g t o be r e t a i n e d on t h e c o l u m n and r e - e s t a b l i s h s t a r t i n g c o n d i t i o n s . Two s o l v e n t s y s t e m s were t r i e d ; a c e t o -n i t r i l e / w a t e r and m e t h a n o l / w a t e r ; t h e f o r m e r seemed t o g i v e t h e b e s t r e s u l t s o v e r a l l and was u s e d t h r o u g h o u t t h e w o r k . S o l v e n t c o m p o s i t i o n and f l o w r a t e were programmed t o t h e f o l l o w i n g , c y c l e : ( t i m e i n m i n u t e s : % a c e t o n i t r i l e ) 0:61, 1 0 : 6 1 , 20:100, 24:100, 2 7 : 6 1 ; ( t i m e : f l o w r a t e i n m l / m i n u t e ) 0:1, 19.: 1, 20:2, 24:2, 2 7 : 1 . A s i m i l a r p r o g r a m w a s - d e v e l o p e d f o r / p r e p a r a t i v e w ork: ( t i m e : % a c e t o n i t r i l e ) 0:55 , 16:60, 23:65, 28:10.0, 32:55. F l o w was 5 m l / m i n u t e a t a l l t i m e s . Changes i n f l o w and compo-s i t i o n w e r e l i n e a r o v e r t i m e . I n g e n e r a l t h e e l u t i n g p e a k s w ere homogeneous s i n c e t h e p r o g r a m s were d e v e l o p e d u s i n g a c o m p l e x m i x t u r e o f compounds f r o m b o t h r o o t s and l e a v e s . E l u t i n g p e a k s were c o l l e c t e d and 17 a UV s p e c t r u m o b t a i n e d f o r e a c h p e a k when d i f f e r e n t t y p e s o f s a m p l e s w e r e r u n . T h i s was d o n e f o r t h e f o l l o w i n g r e a s o n s : t o i n s u r e ' t h a t c o e l u t i o n o f m a j o r c o m p o u n d s d i d n o t o c c u r ; t o 1" _--!. c h e c k t h a t c o e l u t i n g v e r y m i n o r c o m p o u n d s d i d n o t c h a n g e i n a m o u n t ; a n d t o d e t e c t new c o m p o u n d s w h i c h m i g h t e l u t e a t a s s i g n e d i r e t e n t i o n t i m e s . To i n s u r e h o m o g e n e i t y , m a j o r p e a k s w e r e s p l i t i n t o two h a l v e s a n d t h e two s p e c t r a c o m p a r e d . A s d e s c r i b e d b e l o w , i n t h o s e i n s t a n c e s i n w h i c h two c o m p o u n d s w i t h d i f f e r e n t c h r o m o p h o r e s e l u t e d a t a s i n g l e p e a k , i t was p o s s i b l e t o d e t e r -m i n e t h e c o m p o s i t i o n b y c o l l e c t i n g t h e p e a k a n d o b t a i n i n g t h e c o m p o s i t e s p e c t r u m . T h e a d v i s a b i l i t y o f f o l l o w i n g t h i s t y p e o f p r o c e d u r e s h o u l d be e m p h a s i z e d , e s p e c i a l l y when c u l t u r a l c o n d i t -i o n s c h a n g e s i g n i f i c a n t l y o r t h e m a t e r i a l b e i n g e v a l u a t e d i s s u s - .: p e c t e d o f b e i n g v a r i a b l e i n c o m p o s i t i o n . A t t h e b e g i n n i n g a n d e n d o f e a c h s e t o f a n a l y s e s a s t a n d a r d m i x t u r e o f l e a f a n d r o o t c o m p o u n d s was r u n t o c a l i b r a t e t h e s y s t e m . HPLC Q u a n t i t a t i o n o f M a j o r C o m p o u n d s The HPLC t r a c e was q u a n t i t a t e d u s i n g s t a n d a r d c u r v e s f o r t h e t h r e e p r i n c i p l e c o m p o u n d s : p h e n y l h e p t a t r i y n e ( P H T ) , p h e n l y d i y n e n e - O A c ( P D E - O A c ) , a n d e n t e t r a y n e n e - O A c ( E T E - O A c . C o m p o u n d s w e r e i s o l a t e d o n a p r e p a r a t i v e c o l u m n a n d made up t o s p e c i f i c c o n c e n t r a t i o n s u s i n g p u b l i s h e d e x t i n c t i o n c o e f f i c i e n t s . Known a m o u n t s w e r e t h e n r e p e a t e d l y r u n o n a n a n a l y t i c a l c o l u m n a n d t h e r e s u l t i n g t r a c e s m e a s u r e d f o r p e a k h e i g h t a n d a r e a . F o u r t o s i x l e v e l s w e r e e a c h r u n s i x o r m o r e t i m e s f o r e a c h c o m p o u n d . T h e l e a s t s q u a r e s l i n e a r r e g r e s s i o n f o r t h e d a t a p o i n t s was o b t a i n e d w i t h t h e l i n e a r r e g r e s s i o n f u n c t i o n o f a T e x a s I n s t r u m e n t s SR-51 c a l c u l a t o r . T h e s e r e l a t i o n s o f 1 8 q u a n t i t y v e r s u s peak h e i g h t and a r e a were u s e d t o q u a n t i t a t e a l l p e a k s of t h e same c h r o m o p h o r e . Compounds w i t h t h e same chromo-p h o r e b u t a d i f f e r e n t t e r m i n a l g r o u p , e.g. E T E - o l i n s t e a d o f ETE-OAc, were c o n v e r t e d as t h e r a t i o o f t h e m o l e c u l a r w e i g h t s o f t h e v a r i a n t and t h e s t a n d a r d . The s t a n d a r d c u r v e s of t h e t h r e e b a s e compounds a r e shown i n F i g . 4. The d e t a i l e d p r o c e d u r e f o r c o v e r t i n g a peak t r a c e t o amount of compound p e r gram of s a m p l e f o l l o w s . An o u t p u t f r o m t h e s p e c t r o p h o t o m e t e r a c t i v a t e s a r e c o r d e r pen a s s e m b l y s u c h t h a t an o u t p u t of 2Q0 mv d r i v e s t h e pen f u l l s c a l e and c o r r e s p o n d s to an OD of 2.00, and o u t p u t o f 50 mv t o an OD of 0.5, e t c . The o u t p u t can be v a r i e d f r o m I mv (OD 0.01) t o 200 mv. I f i s t h e number o f mv f o r f u l l s c a l e t h e n Q.01 x R =0D. F u l l s c a l e v d e f l e c t i o n c o v e r s a d i s t a n c e o f 240 mm so t h a t 0D=P, x K x R , h v where P is'.peak h e i g h t (mm) and K = l / 2 4 0 x 0.01 = 4.167 x 1 0 ~ 5 . A r e a i s c a l c u l a t e d by m u l t i p l y i n g OD by peak w i d t h , i n m i n u t e s ( t ) , a t h peak h e i g h t ( % t ) . M u l t i p l y i n g by t h e r a t i o o f t o t a l s a m p l e C v ). t o i n j e c t i o n amount (V\ ) , t h e v o l u m e o f t h e i n j e c t i o n l o o p , and d i v i d i n g by s a m p l e w e i g h t (w) g i v e s t h e t o t a l a r e a (A ) p e r gram s a m p l e : A =-h t P, x % t x R x K x V w x V . X A r e a i s t h e n c o n v e r t e d t o ug compound p e r gram s a m p l e w i t h t h e i n v e r s e e q u a t i o n s of t h e s t a n d a r d c u r v e s : ETE-OAc ug/g=2.805m-0.0; PDE-OAc ug/g=24.15m-0.016; PHT ug/g=30.26m-Q.121. I n a few i n s t a n c e s r e l a t i v e l y m i n o r compounds c o e l u t e t o g e t h e r ; i n t h e s e c a s e s t h e c o m p o s i t e peak was c o l l e c t e d , m e a s u r e d , and a UV s p e c t r u m r u n . K n o w i n g t h e e x t i n c t i o n c o e f f i -19 A. area (ad. x t) 1 2 3 4 AMOUNT (M§) T — i r i | — i I i i | i 1 3 5 7 9 11 A M T. (yg) F i g . 4. HPLC standard curves showing the r e l a t i o n s h i p Between amount of compound and peak area. A) PDE-OAc. B) ETE-OAc. C): PHT. V e r t i c a l bars are + 1 SD. 20 c i e n t s and assuming n o n i n t e r a c t i o n of the components i t i s p o s s i b l e to c a l c u l a t e the c o n c e n t r a t i o n s of each compound, s i n c e a b s o r p t i o n (or o p t i c a l d e n s i t y ) i s a d d i t i v e , by s o l v i n g a p a i r of simultaneous equations of the form OD>I=61>2 c 1 + 6 ^ 1 c 2 where and \^ are the two wavelengths of o b s e r v a t i o n , £ i s the e x t i n c t i o n c o e f f i c i e n t and c i s the c o n c e n t r a t i o n (13, p. 240). Although the method, i n p r i n c i p l e , i s a p p l i c a b l e to any number of s p e c t r a l l y d i s t i n c t components by adding an a d d i t i o n a l equation and wavelength f o r each a d d i t i o n , i t was only very o c c a s i o n a l l y used i n t h i s study f o r peaks with three components. The r e s u l t s were checked i n s e v e r a l i n s t a n c e s a g a i n s t a known mixture and agreement was w i t h i n 10%. A refinement of t h i s approach using more p o i n t s and a computer program to r e c o n s t r u c t the component s p e c t r a arid c a l c u l a t e c o n c e n t r a t i o n s has r e c e n t l y been p u b l i s h e d (15). Workers have sometimes estimated the amounts and composition of mixtures d i r e c t l y from the crude l i p i d e x t r a c t ( . 8 ) . In t h i s study q u a n t i t a t i o n from the crude e x t r a c t was only used i n one study i n v o l v i n g l e a f e x t r a c t s , i n which one compound predominated, and as a check on r e s u l t s using standard curves. Q u a n t i t a t i o n of Minor Compounds In a few cases minor compounds were t e n t a t i v e l y i n d e n t i f i e d and i t was not p r a c t i c a l to e s t a b l i s h a standard curve d i r e c t l y . Q u a n t i t a t i o n of the HPLC peaks of these compounds was done by e s t i m a t i n g the standard curve slope from the f o l l o w i n g c o n s i d -2 1 e r a t i o n s . F r o m a s p e c t r u m o f t h e p u r i f i e d m i n o r c o m p o u n d t h e e x t i n c t i o n c o e f f i c i e n t a t t h e d e t e c t i n g w a v e l e n g t h ( £ ^ ) w a s d e t e r m i n e d b y m e a s u r i n g t h e p e a k h e i g h t a t t h a t w a v e l e n g t h ( H ^ ) a n d t h e n e a r e s t p e a k ( H ^ ) , a n d t a k i n g t h e r a t i o t i m e s t h e k n o w n £ o f t h a t p e a k . T h u s £.A J=CH J/H ) X £ A . T h e r a t i o o f £ A J f , a d p~ w m a x ' d t h e e x t i n c t i o n c o e f f i c i e n t o f a c o m p o u n d w i t h a k n o w n s t a n d a r d c u r v e , t o £ ^ t i m e s t h e s l o p e o f t h e r e g r e s s i o n l i n e o f t h e k n o w n c o m p o u n d (m') y i e l d s t h e e s t i m a t e d s l o p e f o r t h e m i n o r c o m p o u n d ( m ) : m=C£Aj i / E A ^ x m ' x ( m w / m w ' ) . T h e f i n a l t e r m (mw/mw') c o r r e c t s f o r t h e d i f f e r e n c e i n t h e m o l e c u l a r w e i g h t s o f t h e m i n o r c o m p o u n d (mw) a n d t h e k n o w n (mw') c o m p o u n d s , The a c c u r a c y o f t h e m e t h o d was c h e c k e d b y c a l c u l a t i n g t h e s l o p e s f o r PHT a n d PDE-OAc f r o m t h e d a t a f o r E T E - O A c . I n t h e f o r m e r c a s e t h e c a l c u l a t i o n y i e l d s a s l o p e o f 2 9 . 1 v s . 3 0 . 3 d e r i v e d f r o m a c t u a l d e t e r m i n a t i o n ; f o r t h e l a t t e r c o m p o u n d t h e c a l c u -l a t e d s l o p e i s 2 3 . 5 v s . 2 4 . 1 . A g r e e m e n t was w i t h i n a b o u t 3% f o r t h e c a l c u l a t e d a n d d e t e r m i n e d s l o p e s . A l l s l o p e s d e t e r -m i n e d i n t h i s way w e r e a s s u m e d t o i n t e r s e c t t h e o r i g i n . T h u s , o n e c a r e f u l l y d e t e r m i n e d s t a n d a r d c u r v e c a n f o r m t h e b a s i s f o r c a l c u l a t i n g t h e c u r v e s f o r r e l a t e d c o m p o u n d s i f s m a l l a m o u n t s o f t h e p u r i f i e d c o m p o u n d s a r e a v a i l a b l e f o r r e f e r e n c e s p e c t r a a n d t h e £ v a l u e s a r e k n o w n . T h e f o l l o w i n g c o m p o u n d s w e r e q u a n t i t a t e d by- t h i s - m e t h o d : EDE-OAc (mw 2 3 0 ) s l o p e = Q . 0 3 s p e c t r u m r e f e r e n c e Cla) D T E - o l (mw 1 8 2 ) 11 0 . 0 0 3 1 " " Q5) DTE-OAc (mw 2 2 4 ) " 0 . 0 0 3 8 " " ( 5 ) E T E - a l (mw 1 7 8 ) ' 11 0 . 0 0 3 4 " " C3) 22 Gas Chromatography/Mass Spectrometry (GC/MS) P r e p a r a t i v e HPLC was used to ob t a i n s u f f i c i e n t compound for GC/MS a n a l y s i s : e l u t i n g peaks were c o l l e c t e d , e x t r a c t e d with petroleum ether, d r i e d with sodium s u l f a t e , evaporated with n i t r o g e n gas and d i s s o l v e d i n s p e c t r a l grade n-hexane. Samples were then i n j e c t e d i n t o a Finnegan model 1020 automated GC/MS with a 30 m x 0.25 mm, WE-554 fused s i l i c a c a p i l l a r y column. The i n i t i a l oven temperature of 15Q °C was hel d one minute with a 10 'c/min ramp to 25Q *C which was held f o r 10 minutes. . I n j e c t i o n temperature was 25Q*C. Ion source temperature f o r EIMS was 9_5 °C and the e l e c t r o n energy 70 eV. P u r i f i c a t i o n of PHT Sev e r a l hundred mg of PHT was i s o l a t e d using the f o l l o w i n g procedure: 1 kg of f r e s h B-. alb a leaves was e x t r a c t e d four times with. 4 1 of co l d -methanol, e x t r a c t s combined and evap-orated under reduced pressure to 750 ml and an equal volume of water added and followed by e x t r a c t i o n seven times with 500 ml of heptane. Heptane e x t r a c t s were combined, evaporated to 250 ml, d r i e d w i t h sodium s u l f a t e and a p p l i e d to 4 s i l i c a g e l 60 columns (70-230 mesh .ASTM, E. Merck, Darmstadt) 70. mm x 80 mm made up i n heptane. E l u t i o n with, heptane and c o l l e c t i n g 10.0 ml f r a c t i o n s - gave PHT i n f r a c t i o n s 7-1Q. These f r a c t i o n s were combined and evaporated under reduced pr e s s u r e . Formation of l i g h t yellow- c r y s t a l s - occurred a f t e r most of the so l v e n t was l o s t . These were r e d i s s o l v e d i n heptane and r e c r y s t a l i z e d twice at 4 *C to y i e l d white, elongated c r y s t a l s which were 9_5% pure by CG/MS. The major impurity was PDE-*0Ac (J3.5%I. 23 Pl a n t s Bidens alba s e e d l i n g s were harvested 55 days a f t e r sowing. Pl a n t s were grown i n an incubator at 25 °C i n s o i l . I l l u m i - . : n a t i o n was 12 hours at 6,000 lux. Flower p a r t s were from mature p l a n t s grown under s i m i l a r c o n d i t i o n s . 24 RESULTS Separation An HPLC t r a c e of the mixture of l e a f and root e x t r a c t s used to c a l i b r a t e runs i s shown i n F i g . 5. Not a l l the com-ponents which were e v e n t u a l l y i d e n t i f i e d are shown: some compounds only occurred under c e r t a i n c o n d i t i o n s or i n c e l l c u l t u r e s . A l i s t , i n e l u t i o n order, of compounds that were p o s i t i v e l y or t e n t a t i v e l y i d e n t i f i e d i n p l a n t s and c u l t u r e s of B. a l b a i s shown i n Table 1. Compounds i d e n t i f i e d as "a" or "b" i n the compound number column are i d e n t i c a l i n molecular weight and n e a r l y i d e n t i c a l i n UV and mass s p e c t r a . They are presumed to be c i s / t r a n s isomers. The normal order of e l u t i o n from s i l i c a g el columns, i . e . , i n order of i n c r e a s i n g p o l a r i t y , i s reversed f o r reversed phase columns. Although d i f f e r e n t p o l a r i t y groups mainly separate together there i s some o v e r l a p ; some hydrocarbons e l u t e before a c e t a t e s , e.g (V) before (IVa,b). The correspondence i n e l u t i o n between a n a l y t i -c a l and p r e p a r a t i v e columns d i f f e r e d i n only one i n s t a n c e : (II) el u t e d a f t e r CXVI) and PHT on the former and before these com-pounds from the l a t t e r column. I d e n t i f i c a t i o n : Enetetraynene Compounds Three sets of compounds were i s o l a t e d which had very s i m i -l a r UV s p e c t r a ( F i g . 6) d i f f e r i n g by only % to 1 nm i n the d e t e c t i n g r e g i o n . The s p e c t r a agreed with p u b l i s h e d data f o r the ETE chromophore (2,3,17) and the order of HPLC e l u t i o n suggested q u i t e d i f f e r e n t p o l a r i t i e s f o r the compounds. The 25 L 1 1 1 1 — r 15 20 I I I | I • • < ! • • 0 TIME 5' 10' F i g . 5. HPLC t r a c e of c a l i b r a t i o n m i x t u r e of l e a f and r o o t p o l y a c e t y l e n e s of B i d e n s a l b a . (~la,b) P D E - o l ; (2a,b) DTE; C3) PDE-OAc; C4) E T E - o l ; C5) PDE; (6) PHT; (7a,b) ETE-OAc; (8a,b) ETE. 26 T a b l e 1. HPLC r e t e n t i o n t i m e s f o r p o l y a c e t y l e n e s i s o l a t e d f r o m p l a n t s a n d c u l t u r e s o f B. a l b a . C o mpound no . Compound C o l u m n r e t e n t i o n t i m e ( m i n . ) V i a VIb I X XIV X l l a I l i a X l l b V I I XV X I l l b X H I a V x n i b x v i V I I I I I I V a IVb l a l b a n a l y t i c p r e p a r a t i v e P D E - o l P D E - o l P H T - o l E D E - o l D T E - o l E T E - o l D T E - o l P D E-OAc E T E - O A c PHT-OAc E T E - o l D T E - O A c PDE DTE-OAc' * TDE PHT E T E - a l E T E - O A c E T E - O A c E T E E T E 5 . 1 5.6 6 . 0 6.4 7 . 3 7 . 8 8 . 0 8.7 SL. 1 9.5 10 . 2 11. 2 11. 8 12.6 12 . 8 12 . 8 13 . 7 14.1 16 . 7 19. 7 20.1 9.0 - 9 . 6 10 . 5 11. 3 13 .1 14.0 14. 3 16 . 6 18.4 17 . 1 17 . 7 20.0 20.5 22 .1 23 . 3 23. 3 22 . 6 24 . 8 27.6 29.2 29.9 T e n t a t i v e i d e n t i f i c a t i o n - - b a s e d o n s p e c t r u m a n d r e t e n t i o n t i m e o n l y . 27 F i g . 6. TJT s p e c t r a of e n t e t r a y n e n e compounds. A) ,-entetraynene h y d r o c a r b o n . B) e n t e t r a t y e n e a c e t a t e . 28 74496. 10. 22784. 10. F i g . 7, Mass- s p e c t r a of e n t e t r a y n e n e h y d r o c a r b o n s . A) i s o m e r " a " . B) i s o m e r "B". 29 A 188 245S. 18. 126 118 151 86 74 44 51 M/E 48 63 r 68 88 188 122 138 134 146 128 148 163 • I • 168 188 F i g . 8 . M a s s B, i s o m e r " b " s p e c t r a o f e n t e t r a y n e n e a l c o h o l s , A) i s o m e r " a " . F i g . 9 . Mass s p e c t r a of entetraynene a c e t a t e s . A) isomer B) isomer "b". 31 A F i g . 10. UV s p e c t r a of entetraynene aldehyde i n different., solvents-. AJ spectrum i n petroleum ether (30-60 C). B) p a r t i a l spectrum i n petroleum ether ( s o l i d l i n e ) and i n metha-nol (dotted l i n e ) . Peak at 3 9:0 nm due to contaminating ETE-OAc. F i g . 11, U'V and mass s p e c t r a of pheny Idiynene hydrocarbon. A) UV spectrum. B) mass spectrum. 33 M/E 58 108 150 288 250 100.0- B 153 24576. 18. 50.0- 182 M/E 13 51 63 76 87 5S A 63, .81 38 50 108 110 ik 126 138 163 150 132 205 ' - I ' 223 246 250 F i g 12 M a s s s p e c t r a o f p h e n y l d i y n e n e a l c o h o l s . A) i s o m e r a . B) i s o m e r " b " . F i g . 13. UV and mass s p e c t r a of ph.enyldiynene a c e t a t e . A) UV spectrum. B) mass spectrum. 3 5 A F i g , 14. try and mass- s p e c t r a of p h e n y l h e p t a t r i y n e hydrocarbon, A) UV spectrum. B) -mass: spectrum, 36 5 0 . 8 -162 5769. 10. 43 180 152 126 87 98 U l 51 T <a 7 T ulLil ,iii,liili.,iii,i,Jili IVE 68 108 122 120 134 194 140 160 188 200 F i g . 15. Mass spectrum of p h e n y l h e p t a t r i y n e a c e t a t e . 37 o.d. 450 F i g . 16 ( t o p ) . UV spectrum of endiynene acetate . F i g . 17 Cbtm). UV spectrum of d i e n t r i y n e n e a c e t a t e . 38 ETE chromophore i s d i s t i n g u i s h a b l e from the t e t r a y n d i e n e isomer by UV spectrum d i f f e r e n c e s (10,17). The mass spectrum of the hydrocarbons shows a parent i o n of 164 ( F i g . 7), corresponding to the fomula C-^Hg; the a l c o h o l s a parent ion of 180 ( F i g . 8) which corresponds to C 1_H o0; the ace t a t e s have a parent ion of 222 13 o ( F i g . 9) which corresponds to C i 5 H i o ° 2 a n d h a v e c h a r a c t e r i s t i c base peaks at m/e 43 (CH^CO). The i d e n t i f i c a t i o n s of the compounds i n t h i s group are based on the UV s p e c t r a , e l u t i o n o r d e r s , mass s p e c t r a , and reported occurrences of these compounds i n _B. p i l o s a . As shown i n Table 1, two forms of each compound are found, almost c e r t a i n l y c i s / t r a n s isomers. The conjugated aldehyde of the ETE chromophore was a l s o i s o l a t e d and t e n t a t i v e l y i d e n t i f i e d on the b a s i s of i t s UV spec-trum ( F i g . 10a) as reported (2,3) and r e t e n t i o n time. In a d d i -t i o n the aldehyde f u n c t i o n was i n d i c a t e d by the l o s s of the long wavelength peaks when the compound was d i s s o l v e d i n metha-nol ( F i g . 10b) (12). The a d d i t i o n a l peaks i n t h i s f i g u r e are due to contaminating ETE-OAc. Repeated attempts to o b t a i n a mass spectrum with p u r i f i e d compound using the GC/MS were u n s u c c e s s f u l — a p p a r e n t l y because of i n s t a b i l i t y of the compound under GC c o n d i t i o n s . PhenyIdiynene Compounds The presence of the a l c o h o l and acetate of phenyldiynene i n roots of _B. p i l o s a has been reported (4) but not the hydro-carbon. Three i s o l a t e s with a UV spectrum i n agreement with t h i s chromophore ( F i g s . 11,13) (3,7) were found and HPLC r e t e n -t i o n times suggested the above compounds. The mass spectrum of the hydrocarbon ( F i g . 11) shows the l i k e l y parent ion to be 166 with a base peak at 165 (-H); the a l c o h o l s ( F i g . 12) 39 have the expected parent i on of 182 and the acetate ( F i g . 13) a parent i o n of 224 which corresponds to the formulas ^ ^ 2 ^ 1 0 ' C ^ ^ H ^ Q O , and ^ ^ ^ 1 2 ^ 2 R E S P E C F C 1 v e l y • The mass s p e c t r a of known standards of a l l three compounds were i d e n t i c a l to those of the i s o l a t e s . Only the a l c o h o l occurred as two isomers. P h e n y l h e p t a t r i y n e This aromatic p o l y a c e t y l e n e occurs p r i m a r i l y as the hydro-carbon i n leaves and stems of 13. al b a and i s e a s i l y i d e n t i f i e d by i t s c h a r a c t e r i s t i c UV spectrum (Fig.14a) which has been reported (4). Minute amounts of what appeared to be the a l c o -hol were i s o l a t e d but i n i n s u f f i c i e n t q u a n t i t y f o r f u r t h e r ana-l y s i s . The mass spectrum of PHT has the expected parent i on at 164 f o r C-^Hg (.Fig- 14b) and was i d e n t i c a l with the spectrum of known compounds. The acetate has a parent i on of 222 ( f i g . 15) corresponding to C^^R^^O^ and a prominent peak at m/e 43 (CH 3CO). Other P o l y a c e t y l e n e s A f i n a l group of compounds occurred at l e v e l s too low to allow f u r t h e r c h a r a c t e r i z a t i o n ^ or l o s s occurred under GC c o n d i t i o n s . The only data o b t a i n a b l e were UV s p e c t r a and HPLC r e t e n t i o n times. Compounds i n d i c a t e d on t h i s b a s i s were an endiynene a l c o h o l and acetate (EDE-ol, EDE-OAc) ( F i g . 16); two d i e n t r i y n e n e a l c o h o l s and acetates (DTE-ol, DTE-OAc) ( F i g . 17) and an e n d i y n t r i e n e hydrocarbon (EDT). The a b s o r p t i o n maxima and r e l a t i v e i n t e n s i t i e s agree with p u b l i s h e d values (5,10,16). 40 P o l y a c e t y l e n e s In B. alb a Table 2 shows the composition of B. alba s e e d l i n g s and flowers of mature p l a n t s . B.._ p l l o s u s L. C=B.- p i l o s a L.) was examined by Bohlmann et a l . C 4 ) . Roots of- t h e i r m a t e r i a l con-tai n e d PDE-OAc CVITl, ETE ( T.) , ETE-al ( J l ' l , ETE-ol C U T ) , and ETE-OAc (TV); leaves contained PHT ( V I I I ) , PHT-ol (IX), PHT-OAc (X), and t r a c e pentaynene (XVII). I t i s not known whether the m a t e r i a l i n v e s t i g a t e d here i s the same taxpnt 'r as the one that Bohlmann et a l . examined, however the compositon was s i m i l a r . The two r e p o r t s d i f f e r i n three r e s p e c t s : R, alba lacked the ETE-al i n roots and the a l c o h o l and acetate of PHT i n l e a v e s , although the compounds occurred i n flowers and seeds. B. alba contained EDE-OAc which was not reported "for fr. p i l o s u s . The absence of the aldehyde was i n t e r e s t i n g because t h i s compound was found l a t e r i n crown g a l l c u l t u r e s . No t r a c e of the penta^ ynene was ever observed i n any J3. alba m a t e r i a l . UV s p e c t r a f o r s e v e r a l very minor compounds- not reported by Bohlmann et a l . (4) were obtained from tumour c u l t u r e s (XI-XV). Although only three chromophores occur at s i g n i f i c a n t l e v e l s i n B. alba each may have up to four d i f f e r e n t t e r m i n a l groups Cas i n I-IV) and each may occur i n c i s or trans forms. Table 2 • Polyacetylene composition of Bidens a l b a var. r a d i a t a s e e d l i n g organs and flowers. Polyacetylene composition (wt % of t o t a l ) T o t a l Organ PHT-ol PHT-OAc PHT PDE-OAc PDE ETE-ol ETE-OAc ETE EDE-OAc product A B (mg/g d.w.) Root - - 5.7 58.3 2.9 0.4 4.9 19.3 0.8 7.7 8.7 Stem - - 54.2. 2.2 43.5 - t t - - 2.3 P e t i o l e - - 94.1 - 5.9 - t t - - 1.5 Leaf - - 98.3 - 1.7 - - 4.6 F l o w e r 3 - - 97.3 - 2.7 - - 11.9 head P e t a l b - 6.0 92.7 - 1.2 0.1 - 4.2 Seed 0.8 19.8 78.2 - 1.2 - - 6.9 P o l l e n 0 - - 100.0 - - - - 0.1 detect minor compounds, weight. Minus l i g u l a t e ' p e t a l s ^ L i g u l a t e p e t a l s . Small amounts of m a t e r i a l made i t impossible to Symbols: -, not d e t e c t a b l e ; t, t r a c e ; d.w., dry 42 DISCUSSION There are advantages and disadvantages of using HPLC f o r the studies reported l a t e r . Obtaining enough m a t e r i a l f o r f u r t h e r a n a l y s i s was r e l a t i v e l y f a s t and e f f i c i e n t ; a few or, sometimes, only one run of sample on a p r e p a r a t i v e column usu-a l l y y i e l d s enough m a t e r i a l f o r GC/MS a n a l y s i s (approximately 10 ug). The .loading c a p a c i t y , however, i s r a t h e r low and minor compounds r e q u i r e d a number of runs and sometimes r e - e x t r a c t i o n and p u r i f i c a t i o n with a second run. C i s / t r a n s isomers are e a s i l y separated as shown i n this study and others (14). Q u a n t i t a t i o n i s simple once the r e q u i s i t e standard curves have been determined. T h i s can be coordinated with the part above and the compounds i s o l a t e d used to e s t a b l i s h , the curves. The standard curves can a l s o be c a l c u l a t e d with reasonable p r e c i s i o n . For e x t e n s i v e work with the same group of compounds standard curves are e f f i c i e n t and i f a programmable i n t e g r a t o r i s brought i n t o the system then r o u t i n e peak i d e n t i f i c a t i o n and q u a n t i t a t i o n can be made automatic. However i n other s i t u a ^ , t i o n s TLC or CC may be f a s t e r and i n some cases e s t i m a t i o n d i r e c t l y from a crude e x t r a c t may be p o s s i b l e . D e t e c t i o n of the e l u t i n g peaks i s c o n t r o l l e d by the set wavelength, and s e l e c -t i n g t h i s value is- n e c e s s a r i l y - a compromise f o r complex mix-t u r e s . I f the wavelength is- too short peaks of no i n t e r e s t may be picked up and obscure minor peaks- of i n t e r e s t . If the value i s too long then s e n s i t i v i t y of d e t e c t i o n of some compounds may be q u i t e low. The d e t e c t i n g wavelength may- c o i n — 43 c i d e with a v a l l e y i n the r e g i o n of the a b s o r p t i o n spectrum with r e l a t i v e l y low £ v a l u e s . The set value should be i n a re g i o n i n which as many as p o s s i b l e of the compounds expected would have high absorbance, even f o r -valleys. For t h i s study 28 7 nm was chosen s i n c e i t was at a maximum f o r one of the p r i n c i p a l chromophores, entetraynene, and was s t i l l f a i r l y s e n s i t i v e f o r phenyldiynene, P h e n y l h e p t a t r i y n e had the poorest s e n s i t i v i t y ( F i g . 4). If a p a r t i c u l a r chromophore present i n only small amounts i s to be monitored i t may be p o s s i b l e to s e l e c t a wavelength n e a r l y unique to i t , e.g. } pentaynene (XVIT) was reported to be present i n t r a c e amount i n B. p i l o s a by Bohlmann et a l . CAl' There was no i n d i c a t i o n of i t i n the r e g u l a r runs but i t could e a s i l y be missed i f i t was at low c o n c e n t r a t i o n . It has a peak at 410 nm wh.ich i s not overlapped by any of the other expected a c e t y l e n e s . Samples were run at t h i s wavelength at high s e n s i t i v i t y but there was s t i l l no i n d i c a t i o n of the compound, suggesting that i t was absent or present at very low l e v e l s . 44 L I T E R A T U R E C I T E D 1. B i n d e r , R. G., a n d A. C. C a r t h a m u s R. E. L u n d i n , S. K i n t , J . M. K l i s i e v i c z , W a i s s , J r . 1 9 7 8 . P o l y a c e t y l e n e s f r o m t i n c t o r i u s . P h y t o c h e m i s t r y 1 7 : 3 1 5 - 3 1 7 . l a . B o h l m a n n , F., M. A h m e d , M. G r e n z , R. M. K i n g , a n d H. R o b i n s o n . 1 9 8 3 . B i s a b o l e n e d e r i v a t i v e s a n d o t h e r c o n s t i t u e n t s f r o m C o r e o p s i s s p e c i e s . P h y t o c h e m i s t r y 2 2 : 2 8 5 8 - 2 8 5 9 . 2. B o h l m a n n , F., C. A r n d t , H. B o r n o w s k i , a n d K.-M. K l e i n e . 1 9 6 2 . U b e r d i e p o l y i n e d e r g a t t u n g B i d e n s L. Chem. B e r . 9 5 : 1 3 1 5 - 1 3 1 9 . 3. B o h l m a n n , F., H. B o r n o w s k i , a n d C. A r n d t . 1 9 6 2 . N a t u r -l i c h v o r k o m m e n d e a c e t y l e n v e r b i n d u n g e n . F o r t s c h r . Chem. F o r s c h . 4 : 1 3 8 - 2 7 2 . B o h l m a n n , F ., H. B o r n o w s k i , a n d K.-M. K l e i n e , n e u e p o l y i n e a u s dem t r i b u s H e l i a n t h e a e , 9 7 : 2 1 3 5 - 2 1 3 8 . 1 9 6 4 . U b e r Chem. B e r . B o h l m a n n , F., T. B u r k h a r d t , a n d C. Z d e r o . 1 9 7 3 . N a t u r -a l l y o c c u r r i n g a c e t y l e n e s . A c a d e m i c P r e s s , L d n . , N.Y B o h l m a n n , F., a n d H. J . M a n n h a r d t . 1 9 5 7 . A c e t y l e n v e r b i n -d u n g e n i m p f l a n z e n r e i c h . F o r t s c h r . d. C h e m i e N a t u r -s t o f f e 1 4 : 1 - 7 0 . 7. B r a u n , T., L. S k a t t e b o l , a n d N. A. S o r e n s e n , S t u d i e s r e l a t e d t o n a t u r a l l y o c c u r r i n g p o u n d s . X V I T I . T h e s y n t h e s i s o f some e n e s r e l a t e d t o C o m p o s i t a e c o m p o u n d s . S c a n d . 8 : 1 7 5 7 - 1 7 6 2 . 1 9 5 4 . a c e t y l e n e com-p h e n y l a c e t y l -A c t a Chem. 8. B u ' L o c k , J . D., a n d G. N. S m i t h . 1 9 6 3 . A c e t y l e n i c f a t t y a c i d s i n s e e d s a n d s e e d l i n g s o f s w e e t q u a n d o n g . P h y t o c h e m i s t r y 2 : 2 8 9 - 2 9 6 . 9. C h i n , C , M. C. C u t l e r , E. R. H. J o n e s , J . L e e , S. S a f e , a n d V. T h a l l e r . 1 9 7 0 . N a t u r a l a c e t y l e n e s . P a r t X X X I . C 1 4 - t e t r a h y d r o p y r a n y l a n d o t h e r p o l y a c e t y l e n e s f r o m t h e C o m p o s i t a e D a h l i a c o c c i n e a C a v . v a r . c o c c i n e a , J . Chem. S o c . (C):314-322. 1 0 . I c h i h a r a , K . - I . , a n d M. N o d a . 1 9 7 5 . P o l y a c e t y l e n e s f r o m i m m a t u r e s e e d s o f s a f f l o w e r ( C a r t h a m u s t i n c t o r i u s L . ) A g r . B i o l . Chem. 3 9 : 1 1 0 3 - 1 1 0 8 . 1 1 . J o n e s , E. R. H. c o m p o u n d s . a n d V. T h a l l e r . 1 9 7 4 . P o l y a c e t y l e n e M e t h o d i c u m C h i m i c u m 1 1 : 1 7 5 - 1 7 9 . . 1 2 . Lam, J o r g e n . P e r s o n a l c o m m u n i c a t i o n , 45 Montgomery, R., and C. A. Swenson. 1969. Q u a n t i t a t i v e problems i n the bio c h e m i c a l s c i e n c e s . W. H. Freeman and Co., San F r a n c i s c o . Rose, A. F., B. A. Butt, and T. Jermy. 1980. P o l y a c e t y l -enes from the r a b b i t b r u s h , Chrysothamnus nauseosus. Phytochemistry 19:563-566. Saguy, I., I. J . Kopelman, and S. M i z r a h i . 1978. Computer aided d e t e r m i n a t i o n of beet pigments. J . Food S c i . 43 : 124-12.7 . Skattebo'l, L. and N. A. Sorensen. 1959. Studies r e l a t e d to n a t u r a l l y o c c u r r i n g a c e t y l e n e compounds. XXVII. The synthesis- of a mixture of t r i d e c a - 1 : 3 : 1 1 - t r i e n e -5:7:9-triyne (IT) and t r a n s - l - p h e n y l h e p t a - 1 : 3 - d i y n -5-ene (V). A novel c y c l i s a t i o n r e a c t i o n . Acta Chem. Scand." 13:2101-2106. Sorensen, J . and N. A. Sorensen. 1954. Studies r e l a t e d to n a t u r a l l y o c c u r r i n g a c e t y l e n e compounds. XVII. Four new p o l y a c e t y l e n e s from garden v a r i e t i e s of Coreops i s . Acta Chem. Scand. 8:1741-1756. 46 CHAPTER I I I INHERITANCE OF TUMOUR CHARACTERISTICS IN BIDENS ALBA i 47 INTRODUCTION The s t u d i e s r e p o r t e d In this- chapter were an o f f s h o o t of the r e s e a r c h on t i s s u e c u l t u r e s f o r p o l y a c e t y l e n e p r o d u c t i o n . They are presented here i n order to keep the s e c t i o n s d e a l i n g with, t i s s u e c u l t u r e s together and to in t r o d u c e the background i n f o r m a t i o n on crown g a l l , Crown g a l l i s a n e o p l a s t i c disease-of p l a n t s induced By ;.. s t r a i n s of AgroBacterjum tumef acjens c a r r y i n g one of a group of plasmids ( T i plasmlds-J e s s e n t i a l f o r tumour i n d u c t i o n (49_, 50)~. A p o r t i o n of the plasmid (T—DNA)' Becomes c o v a l e n t l y l i n k e d to host p l a n t DNA (~46"X txi the nucleus 0.21. £n yiiyQ t r a n s c r i p t i o n of T-DNA has- Been demonstrated 051 and a mi n i -mum of seven polyadenylated RXAs have Been i d e n t i f i e d from octopine tumours C33)~ and ni;ne from nopaline tumours C4Q.I, Three s p e c i f i c mRNAs from T-DNA have Been shown to Be t r a n s -l a t a b l e i n a wheat germ system (42)'. One of the products appeared to Be the enzyme ly-sopl.ne dehydrogenase, which, pro^ duces octopine i n tumour c e l l s - . The presence of T—DNA i s essen-t i a l f o r maintenance of t r a n s f o r m a t i o n (56 , 5.7)1 which, is: gener-a l l y c onsidered to Be confirmed By three c r i t e r i a 09.)_; 1). p r o d u c t i o n of opines By the t i s s u e , 2) growth of t i s s u e explants on a simple medium devoid of phy tohormones , and 3)_ demonstra-t i o n of the presence of T—DNA i n the t i s s u e , A f o u r t h t r a i t i s a l s o used; i n s u s c e p t l B U l t y of tumour—derived p l a n t s t.o i n f e c t i o n By- - v i r u l e n t A. tumef aciens- s t r a i n s (52X.« Opines f a l l i n t o three estaBllsh.ed groups (34X.: IX octopine and 48 r e l a t e d amino a c i d d e r i v a t i v e s , 2) nopaline and r e l a t e d com-pounds, 3) agropine. A f o u r t h type, a g r o c i n o p i n e s , has r e c e n t l y been p a r t i a l l y c h a r a c t e r i z e d and d e s c r i b e d (17). The f i r s t two groups appear to be widely accepted as being s p e c i f i c to crown g a l l t i s s u e (22,32) and there are no r e p o r t s of the l a s t two being found i n normal t i s s u e . A c h a r a c t e r i s t i c f e a t u r e of nopaline tumours i s t h e i r ten-dency to d i f f e r e n t i a t e i n t o r e l a t i v e l y normal organs and t i s s u e s (teratomata) when the tumour e i t h e r grows near the apex of the p l a n t (2,47) or when i t i s c u l t u r e d on a s y n t h e t i c med-ium (7,8,28). However under a p p r o p r i a t e c o n d i t i o n s agropine and octopine tumours may a l s o be induced to d i f f e r e n t i a t e (54). Regeneration, from s i n g l e c e l l s , of f l o w e r i n g p l a n t s which set seed when g r a f t e d to normal p l a n t s has been demonstrated (9,48). Regeneration of complete; rooted tobacco p l a n t s from tumours has been shown by E i n s e t and Cheng (16) and from s i n g l e c e l l s by S a c r i s t a n and Melchers (39). However, t i s s u e from the regenerated p l a n t s showed a l o s s of tumour markers i n both stud-i e s . Loss of markers has a l s o been reported f o r regenerants of A r a b i d o p s i s t h a l i a n a (1). In another case (using Ln v i t r o t r a n s f o r m a t i o n ) regenerants showed a v a r i e t y of combinations of markers (23). In g e n e r a l , then, teratomas u s u a l l y d i f f e r -e n t i a t e to form shoots but not roots (9,26,48,54) and regener-ated p l a n t s may show a l o s s of tumour markers. Braun, using tobacco, was the f i r s t to show complete recovery i n p l a n t s grown from seed set by transformed p l a n t s (9). L a t e r , s t u d i e s of Braun's l i n e (RT37) using DNA s o l u t i o n h y b r i d i z a t i o n (56) and Southern b l o t s (25) confirmed the l o s s 4 9 o f T-DNA; f r o m p l a n t s i n t h e f o r m e r s t u d y a n d f r o m a n d a n t h e r - d e r i v e d h a p l o i d p l a n t s i n t h e l a t t e r s t u d y . Y a n g e t a l . ( 5 8 ) h a v e d e m o n s t r a t e d r e t e n t i o n o f t h e e n d s o f T-DNA (from p T i - T 3 7 ) i n F^ p l a n t s o f t h e same l i n e a l t h o u g h a l l o t h e r m a r k e r s w e r e l o s t . T u r g e o n e t a l , C48) i d e n t i f i e d t h e s t a g e a t w h i c h r e c o v e r y o c c u r r e d b y c h e c k i n g f l o w e r p a r t s a n d h a p -l o i d s d e r i v e d f r o m a n t h e r s . T h e i r r e s u l t s i n d i c a t e d t h a t t h e l o s s o f T-DNA o c c u r r e d a t m e i o s i s . T h i s f i n d i n g w as c o n f i r m e d i n l a t e r s t u d i e s C & , 2 5 , 5 6 ) . The t w o p r o b l e m s i n t h e s e r e p o r t s ( n o n r o o t i n g o f r e g e n -e r a n t s a n d l o s s o f T-DNA a t m e i o s i s ) a r e o f c o n s i d e r a b l e i n t e r -e s t t o m o l e c u l a r b i o l o g i s t s - who a n t i c i p a t e us:e o f t h e T i . p l a s -m i d a s a v e c t o r , w i t h , i n s e r t s i n t h e T-DNA, f o r i n c o r p o r a t i n g new g e n e t i c m a t e r i a l i n t o h.osrt p l a n t g e n o m e s ( 1 0 , 3 0 , 35 , 5 2 ) , T h e f e a s i b i l i t y o f i n s e r t i o n o f e x o g e n o u s DNA i n t o T-DNA, a n d s t a b l e i n c o r p o r a t i o n i n t o frost DNA, h a s b~een s h o w n ("_18,2Q,36)_. T h u s i t i s : s i g n i f i c a n t t h a t t w o g r o u p s r e c e n t l y r e p o r t e d e v i -d e n c e f o r t h e t r a n s - m i s s i o n o f T—DNA i n t o t h e F^ a n d g e n e r -a t i o n s o f t o b a c c o . W i l l i a m s e t a l . C52X u s e d i n v i t r o t r a n s -f o r m a t i o n o f t o b a c c o p r o t o p l a s t s t o o b t a i n a n o p a l i n e — t y p e c l o n e w h i c h was r e g e n e r a t e d i n t o a r o o t l e s s s h o o t a n d , a s a g r a f t , s e t s e e d w h e n c r o s s e d w i t h n o r m a l t o b a c c o y i e l d i n g p l a n t s w h i c h r e t a i n e d t r a n s f o r m a t i o n - m a r k e r s . O t t e n e t a l , ( 3 7 ) o b t a i n e d a t o b a c c o m u t a n t ( G V 2 1 0 0 ) w i t h l y s o p i n e d e h y d r o -g e n a s e a c t i v i t y w h i c h was- a b l e t o r o o t , ..set s e e d , a n d p r o -d u c e a n d F ^ p r o g e n y - r e t a i n i n g m a r k e r s o f t h e i n c i t i n g p l a s -m i d . T h e y . d e m o n s t r a t e d M e n d e l i a n I n h e r i t a n c e o f T-DNA m a r k e r s a n d o b t a i n e d b o t h Homo— a n d fremlzygous S' p l a n t s . T h e p l a s m i d 50 used was p T i B 6 S 3 c o n t a i n i n g a Tn7 i n s e r t i o n w h i c h , however, was d e l e t e d i n p l a n t n u c l e a r DNA a l o n g w i t h a d j a c e n t T-DNA s e q u e n c e s ( 1 3 , 3 7 ) , R e c e n t l y T-DNA w i t h a d e f i n e d gene i n s e r t e d i n t o (and i n a c t i v a t i n g ) a locus, a f f e c t i n g r o o t f o r m a t i o n has been shown to be s t a b l y t r a n s m i t t e d to t h e R^ g e n e r a t i o n ( 4 a ) . These r e s u l t s d e m o n s t r a t e t h e p r a c t i c a l i t y of o b t a i n i n g com-p l e t e p l a n t s w h i c h can r e p r o d u c e s e x u a l l y and t r a n s m i t f o r e i g n T-DNA from g e n e r a t i o n to g e n e r a t i o n i n a p r e d i c t a b l e p a t t e r n . T h e r e i s some e v i d e n c e t h a t e x p r e s s i o n of i n s e r t e d genes may r e q u i r e a d d i t i o n a l g e n e t i c m a n i p u l a t i o n s (1Q,271, R e p o r t e d h e r e a r e t h e r e s u l t s of work, w i t h B i d e n s a l b a showing t h e p r o d u c t i o n of t r a n s f o r m e d whole p l a n t s and t r a n s -m i s s i o n of tumourous- t r a i t s through, f o u r g e n e r a t i o n s - u s i n g b a c -t e r i a c o n t a i n i n g an u n m o d i f i e d n o p a l l n e - t y p e p l a s m i d and i n v i v o t r a n s f o r m a t i o n . These r e s u l t s a g r e e w i t h th.e work of o t h e r s b u t show some i n t e r e s t i n g d i f f e r e n c e s . 51 MATERIAL AND METHODS B a c t e r i a l and Plant C e l l Lines Bidens alba var. r a d i a t a ( S c h u l t z - B i p . ) B a l l a r d as delim-i t e d by B a l l a r d (4,2 9.') was obtained from southern F l o r i d a ji., USA. Voucher specimens are on f i l e at UBC. Agrobacterium  tumefaciens s t r a i n A208 ( c o n t a i n i n g plasmid pTi-T37) and k i l l CpTi-B.6-806) were both obtained from Dr. M. P. Gordon, U. of Washington, S e a t t l e . The origins of the s t r a i n s are d e s c r i b e d i n Sciaky et a l . (43) and C h i l t o n et a l . (_11). G a l l s induced by A208 produce n o p a l i n e and those of A277 , o c t o p i n e . C u l t u r e Methods A l l t i s s u e cultures: were grown at 25 *C i n 100 x 10 mm p l a s t i c c u l t u r e p l a t e s on Schenk and Hildebrandt ( S H )medium (41) with 7 g/1 agar, pff 5,7, Hormones were omitted except as noted. Medium was s t e r i l i z e d by a u t o c l a v i n g . For tumour i n d u c t i o n and c u l t u r e the f o l l o w i n g procedure was used: the p bacterium was grown at 25 C on medium c o n t a i n i n g 0.8% n u t r i e n t b r o t h, 0.1% yeast e x t r a c t , and Q.5% sucrose (17a). A drop of the 48 hour c u l t u r e was placed on a p l a t e of SH medium and 2 spread and p i e c e s of _B. a 1 Ba c a l l u s (approx, 1 cm x 3 mm) were added to the p l a t e . The c a l l u s had p r e v i o u s l y been i s o l a t e d from stem t i s s u e and maintained as- c a l l u s on SH medium plus 4 mg/1 NAA f o r 2% years. The v i s c o u s suspension of b a c t e r i a r e s u l t i n g a f t e r ten days- growth was taken up i n a s y r i n g e and i n o c u l a t e d i n t o a stem internode punctured through with the needle. This method of growing the inoculum was t e s t e d . J 52 v i s - a - v i s l i q u i d nutrient;, medium by i n o c u l a t i n g B. alba and Helianthus annus 'Mammoth Russian', suggested as an assay p l a n t by Gordon (19), with both s t r a i n s . Results (not shown) showed s l i g h t l y l a r g e r g a l l s r e s u l t i n g from the l i q u i d medium but the d i f f e r e n c e was not considered s i g n i f i c a n t . T o t a l c e l l counts 9 9 for l i q u i d medium y i e l d e d 2 x 10 b a c t e r i a / m l and 8.5 x 10 f o r agar c u l t u r e s . Tumours were harvested a f t e r three months of growth, s t e r i l i z e d a ccording to the method of Johnson et a l . (21), and incubated i n darkness f o r 3 weeks on SH medium to which f i l t e r - s t e r i l i z e d s o l u t i o n s of c a r b e n i c i l l i n disodium (Ayerst Labs) and kanamycin s u l f a t e (Sigma), 0.5 g/1 and 0.13 g/1 r e s p e c t i v e l y , had been added. Seeds were s t e r i l i z e d by immersion f o r one minute i n 7Q% ethanol followed by t h i r t y minutes i n 10% commercial bleach with 1% detergent (Sparkleen) and then r i n s e d four times i n s t e r i l e d i s t i l l e d water. G a l l explants were t r a n s f e r r e d to f r e s h medium every three weeks. A f t e r the appearance of organized shoots from A208 t i s s u e (approximately s i x weeks a f t e r h a rvest) c u l t u r e s were t r a n s f e r r e d to a 25 °C i l l u m i n a t e d incubator ( L i f e l i n e f l o u r e s -cent bulbs, S y l v a n i a ) with an i l l u m i n a n c e - of 60QQ lux and a photoperiod of 16 hours. P l a n t s were t r a n s f e r r e d to medium i n f l a s k s as necessary. Rooting i n the c a l l u s surrounding the base of the shoots began about 3^ months a f t e r h a r v e s t . A f t e r the rooted p l a n t s were w e l l e s t a b l i s h e d they were potted i n s t e r i l e p o t t i n g s o i l and grown to maturity i n a growth chamber as above except that i l l u m i n a n c e was 8QQ0 lux and the r e l a t i v e humidity was i n i t i a l l y 70%. 53 Opine Assay Leaf t i s s u e , minus .petiole, was used except as noted, f o r nopaline d e t e r m i n a t i o n . P r e l i m i n a r y work showed that the nop-a l i n e content i n c r e a s e d from the top leaves to the 3rd or 4th l e a f down ( i n young p l a n t s ) . Samples were taken from the 4th l e a f or lower. For g a l l t i s s u e only the outer, nonwoody l a y e r of t i s s u e , which was peeled o f f , was used. About 0.25 gram of f r e s h t i s s u e was weighed and crushed thoroughly with an equal amount of 95% ethanol and c e n t r i f u g e d . The supernatant was taken to dryness on a r o t a r y evaporator and r e d i s s o l v e d i n d i s t i l l e d water. An amount of e x t r a c t e q u i v a l e n t to 0.05 gram of t i s s u e was a p p l i e d to a sheet of Whatman no. 1 chromato-graphy paper. On each sheet three sets of standards were run, namely, a r g i n i n e (Sigma), octopine (Sigma), and nopaline ( C a l -biochem), so that one set contained 2 ug of each component, the second 4, and the t h i r d 6 ug. Samples were e l e c t r o p h o r e s e d f o r two hours by the procedure of Otten et a l . (38), the paper d r i e d and sprayed thoroughly with a s o l u t i o n of phenanthrene-quinone a c c o r d i n g to the method of Yamada and Itano (55). A f t e r d r y i n g the i n t e n s i t y of sample spots was estimated under long wave UV by comparison to the standards. I f a sample was o u t s i d e the standard range i t was rerun at twice or h a l f the amount above. In t h i s way the amount of opine i n a sample spot was graded i n t o the f o l l o w i n g c a t e g o r i e s : absent, present i n t r a c e amount, or present at 2, 3, 4, 5, or 6 ug/spot. M u l t i -p l y i n g by the a p p r o p r i a t e f a c t o r y i e l d s the estimated opine/g f r e s h weight. A s e r i e s of r e p l i c a t e runs were made with sample order randomized from run to run and the grade assignments 5 4 compared; the assessments on average v a r i e d l e s s than one grade (data not pr e s e n t e d ) . In Table 3 when only hemizygous p l a n t s r e s u l t e d the homozygote l e v e l i s assumed to be twice the hemi-zygo te. Crosses Bidens a l b a var. r a d i a t a has smal l flowers organized i n t o heads of 4 0 - 6 0 f l o r e t s . The anthers surround the s t y l e i n a manner t y p i c a l f o r the Compositae making i t t e c h n i c a l l y imprac-t i c a l to remove them i n c r o s s e s . However, the p a r t i c u l a r l i n e of p l a n t s used i n t h i s study i s l a r g e l y s e l f - i n c o m p a t i b l e ; when p l a n t s are i s o l a t e d from p o l l i n a t o r s and allowed to s e l f , seed-set i s only 4 - 8 % of the l e v e l obtained when the p l a n t s are out-crossed (data not presented). To minimize s e l f s i n crosses the heads to be used i n the cross were chosen at a stage i n f l o w e r i n g such that most of the s t y l e s had emerged from the anthers and were covered with, p o l l e n on the non-receptive por-t i o n of the s t y l e t i p . The r e c i p i e n t heads were then blown v i g o r o u s l y to d i s p e r s e as much, of the p o l l e n a,s p o s s i b l e . Remaining unopened f l o r e t s were plucked out and d i s c a r d e d . The cross: was then made by rubbing the heads together. Under these c o n d i t i o n s an estimated background l e v e l of s e l f s of about 5% was expected, assuming equal v i a b i l i t y of p o l l e n from the two p l a n t s . Crosses of transformed to w i l d type p l a n t s were to normal untransformed p l a n t s grown from the same seed pool as the p a r e n t a l p l a n t s . : .-. - . s r . 55 RESULTS AND DISCUSSION In h e r i t a n c e of Nopaline Synthesis One o b j e c t i v e of t h i s study was to see i f Mendelian i n h e r -i t a n c e of a T-DNA marker could be demonstrated f o r B. a l b a i n the absence of s i n g l e - c e l l c l o n e s . Explanted g a l l t i s s u e formed shoots and then roots a f t e r approximately s i x weeks and 3h months r e s p e c t i v e l y . Two d i f f e r e n t sets of g a l l s ( i n o c u -l a t e d at d i f f er en t ;. t imes J showed the same c a p a c i t y f o r regen-e r a t i n g r o o t i n g p l a n t s . These regenerants were the p a r e n t a l g e n e r a t i o n (R). The second generation CR^) was produced by s e l f i n g p a r e n t a l p l a n t s and s e l e c t i n g the seed from one of these with h i g h e s t nopaline content as the l i n e f o r f u r t h e r study. Outcrosses and f u r t h e r s e l f s were made as shown i n Table 3. I n d i v i d u a l R^ p l a n t s sh.ould, theref ore, be equiva-l e n t to s i n g l e — c e l l regenerants but the d i f f e r e n t p l a n t s would not n e c e s s a r i l y be clones of the same l i n e s i n c e the parent p l a n t was d e r i v e d from a m u l t i c e l l u l a r tumour fragment. If i t i s assumed that the parent (_R) f o r the R^ generati o n was homozygous then the crosses which f o l l o w can only f i t the data i f l o s s e s of the t r a i t o c c u r j 6% f o r the R^, about 43% f o r the F^, and 24% f o r the R^. If i t i s assumed that the parent was hemizygous- then the R^ can only be c o r r e c t i f low and high l e v e l s are i n the same category ( i . e., nopaline"* 1) and the depar-ture from a 1:3 r a t i o i s not s i g n i f i c a n t . This i m p l i e s that gene dose i s not r e s o l v e d by nopa l i n e l e v e l . The F^ crosses and the.R, do not d i f f e r s i g n i f i c a n t l y (by chi-square t e s t ) 56 from the expected r a t i o s i n t h i s case. However, the f i n a l gen-e r a t i o n shown, R 2 F 1 _ ^ ' ^ o e s d i f f e r s i g n i f i c a n t l y from an expec-ted 1:3 r a t i o . These p l a n t s were the progeny of a s e l f e d p l a n t which must have been hemizygous, s i n c e the parent was a w i l d - t y p e plant which had r e c e i v e d transformed p o l l e n . However a l l of these p l a n t s were nop a l i n e n e g a t i v e . Thus the r e s u l t s appear to be incompatible with e i t h e r assumption unless the t r a i t i s l o s t at a v a r i a b l e r a t e . Data presented i n the n e x t / s e c t i o n suggests that the f i r s t assumption i s more l i k e l y to be c o r r e c t . An a d d i t i o n a l s e l f , not shown i n Table 3, was a l s o made; an F^-A p l a n t was s e l f e d but d i d not y i e l d enough, p l a n t s f o r s t a t i s t i c a l a n a l y s i s ( s i x nopaline"*', one n o p a l i n e ) but the .. r e s u l t d i d demonstrate i n h e r i t a n c e of n o p a l i n e s y n t h e s i s to the f o u r t h g e n e r a t i o n . Along with the apparent l o s s of n o p a l i n e s y n t h e s i s as a t r a i t , , n o p a l i n e l e v e l s a l s o d e c l i n e d i n the s u c c e s s i v e gener-a t i o n s of F^, , and R2F^-C; d e c l i n i n g from 300 ug/g f.w. (R^) to 20 ug/g f.w. (R 2) and to 0.0 (R 2F -C); to 100 ug/g f.w. (F^-A) and 60 ug/g f.w. (F^-C). Although l e v e l s c o n s i s t e n t l y declined a f t e r the Rp the amount v a r i e d c o n s i d e r a b l y from gener-a t i o n to g e n e r a t i o n . This i s not e a s i l y explained but, c o n s i d -e r i n g the c e n t r a l i t y of a r g i n i n e and c^-ketoglutarate ( s u b s t r a t e s f o r n o p a l i n e synthase) i n metabolism, i t seems u n l i k e l y that these r e s u l t s are due to p h y s i o l o g i c a l f a c t o r s . Comparisons of the n o p a l i n e l e v e l s i n t i s s u e s grown under d i f f e r e n t c o n d i t i o n s ( l i g h t / d a r k , with/without auxin) and d i f f e r e n t t i s s u e types ( l e a f and r o o t , c a l l u s and l e a f ) showed that l e v e l s d i f f e r e d by no more than a f a c t o r of two at most (data not p r e s e n t e d ) . 57 Table 3. Nopaline s y n t h e s i s i n s u c c e s s i v e generations of transformed Bidens a l b a . Generation O r i g i n Nopaline l e v e l N Nopaline (% p l a n t s analyzed) amount N.D. Low High (ug/g f . R Parentali.regenerants . : • Q 54 46 13 260 R l R (high) self 6 31 63 16 300 F 1 " A W.t.b$ X RjCf"(low) 46 54 0 3 9. 100 F 1 " C R $ (low) X W.t.cf 40 60 0 43 60 R2 R x • (low) self 24 45 31 84 20 R 9 F 1 ~ C F,-C (low) self 1Q0 Q 0 21 n.d. w) Estimated n o p a l i n e f o r homozygote. Untransformed, w i l d - t y p e , p l a n t s . A b b r e v i a t i o n s : n.d., not d e t e c t a b l e ; N, t o t a l p l a n t s assayed. 58 I conclude that n o p a l i n e s y n t h e s i s i s t r a n s m i t t e d through meiosis but that v a r i a b l e r a t e s of l o s s of e x p r e s s i o n of the t r a i t prevent determination of the number of l o c i i n v o l v e d i n the nopaline t r a i t . D i f f e r e n t i a l T r a i t E xpression To check f o r l o s s of the tumour marker auxin autotrophy + + — (aut ), approximately equal numbers of nop and nop p l a n t s were randomly s e l e c t e d from the and F^ l i n e s and stem i n t e r -node s e c t i o n s (5 mm s l i c e s ) were explanted to SH medium. Explants were t r a n s f e r r e d as r e q u i r e d and growth evaluated at the end of ten weeks. Seven of the n o p T / a u t + c u l t u r e s were grown on medium supplemented with 5 mM a r g i n i n e (1) f o r an a d d i t i o n a l four weeks. Six remained negative and one c u l t u r e was p o s i t i v e . The r e s u l t s of growth on r e g u l a r medium are shown i n Table 5. Note that the r a t i o of nop to nop has no s i g n i f i c a n c e . R e s u l t s f o r the two l i n e s , F^ and R^, were s i m i -l a r and combined. The data suggests that there was not only a l o s s of nopaline s y n t h e s i s , while au.xin autotrophy was r e t a i n e d (.'10 out of 28), but that there appeared to be a bia s to the d i r e c t i o n of l o s s ; the r e t e n t i o n of n o p a l i n e s y n t h e s i s and l o s s of autotrophy (5 out of 26) o c c u r r i n g l e s s f r e q u e n t l y . This i s a l s o i n d i c a t e d i n the genera t i o n R 2F^-C (Table 4) which had 4 out of 21 p l a n t s showing hypertrophy of the lower stem but a l l 21 lacked n o p a l i n e . I assume that auxin autotrophy i s a r e l i a b l e i n d i c a t o r of t r a n s f o r m a t i o n s i n c e I have not been s u c c e s s f u l i n g e t t i n g h a b i t u a t e d t i s s u e a f t e r repeated attempts. However, the p o s s i b i l i t y that passage through t i s s u e c u l t u r e a f f e c t s t h i s t r a i t i s not r u l e d out. 59 Table 4. Nopaline s y n t h e s i s and phytohormone autotrophy i n stem explants from combined T"^  and l i n e s . Type of plant A u t o t r o p h i c Non-auto t r o p h i c Nopaline p o s i t i v e 21 5 Nopaline negative 1Q 18 C o n t r o l s 3 0. 8 Wild-type, n o p a l i n e n e g a t i v e . S e v e r a l s t u d i e s have shown that d i f f e r e n t i a l l o s s of t r a i t s may occur i n c e l l s transformed i n v i t r o by b a c t e r i a (52,53) or plasmids (14,23). Loss may occur a f t e r d i f f e r e n t i a t i o n (44,51) or be l o s t i n roots and r e t a i n e d i n the parent c a l l u s (36), The l a t t e r has a l s o been ohs-erved i n t h i s study i n octopine negative + roots from an oct A277 callus* o f ' a l b a . The data from these t w x D s e c t i o n s suggest a p r o g r e s s i v e l o s s of T-DNA or non-expression from the R^ genera t i o n onward. Known fe a t u r e s of the o r g a n i z a t i o n of the T37 T-DNA are al s o c o n s i s r tent w i t h this- view; the presence of m u l t i p l e copies- of T-DNA was- suggested by the work of Yang et a 1.. (57) and work by Lemmers et a l . (25) i n d i c a t e d that nopaline tumours can car r y from one to at l e a s t four T—DNA c o p i e s i n tandem; evidence by Zamb'ryski et a l . (5 9) i n d i c a t e d that T-DNA was organized as two to f i v e tandem repeats- i n s e r t e d i n t o r e p e t i t i v e DNA. C h i l t o n et a l . (12J concluded that at l e a s t some T—DNA copies were tan-dem and that there were two or more i n s e r t s ( i n c l u d i n g i n s e r t s i n T—DNAX, Decreases of T—DNA i n d i f f e r e n t i a t e d t i s s u e , r e l a -t i v e to parent c a l l u s , has been observed (561 as w e l l as a l t e r -60 a t i o n i n the p a t t e r n of border fragments produced by r e s t r i c t i o n a n a l y s i s i n c a l l u s and d i f f e r e n t i a t e d t i s s u e s (25). These stud-i e s show that T-DNA may undergo a l t e r a t i o n s as the host c e l l s change s t a t e and these a l t e r a t i o n s are c o n s i s t e n t with the type of changes seen..in J 3 . a l b a . However, Barton et a l . (4a) have reported that l e v e l s of n o p a l i n e v a r i e d g r e a t l y among seed-l i n g s of a cloned nopaline parent and the copy number f o r nopal-ine synthase T-DNA showed l i t t l e c o r r e l a t i o n with product l e v e l . Perhaps i n a c t i v a t i o n and l o s s can both, occur as separate a l t e r -a t i o n s . The data above does not allow a choice to be made between these two a l t e r n a t i v e s - . S u p e r i n f e c t i o n There i s conflicting,.data on whether transformed p l a n t s are s u s c e p t i b l e to s u p e r i n f e c t i o n with, the same or other s t r a i n s of A. tumefaciens. In some cases (48,52,58) i t was found that trans formants were r e s i s t a n t to r e i n f e c t i o n . However, Otten et a l . (37} reported that b~oth. h.omo- and hemizygous S^ p l a n t s formed g a l l s upon i n o c u l a t i o n with three d i f f e r e n t s t r a i n s of b a c t e r i a . Double trans-formants- (producing both, octopine and n o p a l i n e ) were obtained. I i n o c u l a t e d f o r t y n opaline p o s i t i v e R^ p l a n t s with s t r a i n A277 and obtained g a l l formation on 9_2.5% of p l a n t s (each p l a n t was i n o c u l a t e d i n three p l a c e s ) ; 77.5% of the p l a n t s formed g a l l s comparable i n s i z e to those formed on four c o n t r o l p l a n t s ; 15% showed a weaker response and 7.5% showed no i n d i c a t i o n of g a l l formation. There were no c o n s i s t e n t d i f f erences i n the g a l l s induced on low and high n o p a l i n e p l a n t s . Some no p a l i n e p o s i t i v e plants- were i n o c u l a t e d with s t r a i n A20.8, G a l l s formed, but extremely s-lowly; i t took, s e v e r a l months: f o r 61 tumour growth to become ev i d e n t . Tissue from an octopine g a l l on a low nopaline. p l a n t was grown on e i t h e r SH medium or SH medium plus a r g i n i n e (SH-A). The l e v e l s of both opines showed marked changes with, time, however on SH-A medium octopine was c o n s i s t e n t l y present at higher l e v e l s than n o p a l i n e . A r e p r e s e n t a t i v e assay of callus- a f t e r two months c u l t u r e i s shown i n Fig.:.18. C a l l u s from internode t i s s u e of the parent p l a n t shows only nopaline on both media (lanes 1 & 3) whereas A277 g a l l c a l l u s from the same p l a n t shows q u i t e low l e v e l s of both compounds on r e g u l a r medium ("lane 2} and a higher r e l a t i v e l e v e l of octopine on SH-A medium (lane 4). Normal con-t r o l c a l l u s shows, no trace, of eith.er compound (lane 5). M o r p h o l o g i c a l v a r i a t i o n P l a n t s from the R^, F^ and l a t e r g e nerations, which were transformed by at l e a s t one of the; two c r i t e r i a showed s e v e r a l c o n s i s t e n t d i f f e r e n c e s i n morphology- from c o n t r o l p l a n t s : 1) transformed p l a n t s were i n v a r i a b l y smaller than c o n t r o l s (Fig... 19A)_. 2) Transformed p l a n t s had w e l l developed b a s a l l a t e r a l branches r e s u l t i n g i n a " p i t c h f o r k " h a b i t ( F i g . 19A,B). 3) Nearly a l l o l d e r transformed plants- showed i r r e g u l a r and abnormal t h i c k -ening of the stem corte x , i n c r e a s i n g both towards the base of the p l a n t and with, age, i n young p l a n t s t h i s s w e l l i n g was- most pro-nounced i n the h y p o c o t y l and upper r o o t , g i v i n g them a r a d i s h -l i k e appearance ( F i g . 19B) . With, age the lower nodes e s p e c i a l l y became swollen and bulbous ( F i g . 19.C). Frequently the p e t i o l e bases- of lower l e a v e s would also show- extreme hypertrophy ( F i g . 19.D) . This was e s p e c i a l l y evident i f the leaves^ were broken at the p e t i o l e base. 4) Ray flowers f r e q u e n t l y showed growth of a 62 F i g . 1 8 . P a p e r e l e c t r o p h e r o g r a m o f s i n g l e a n d d o u b l e t r a n s -f o r m a n t c a l l u s . L a n e s 1 , 3: s i n g l e t r a n s f o r m a n t o n n o r m a l a n d a r g i n i n e m e d i u m r e s p e c t i v e l y . L a n e s 2,4: d o u b l e t r a n s f o r m a n t on n o r m a l a n d a r g i n i n e m e d i u m . L a n e 5: n o r m a l c a l l u s o n a r g i n i n e m e d i u m . A m o u n t o f t i s s u e p e r s a m p l e : 1 a n d 2, 0.05 g; 3 a n d 4, 0.02 g; 5, 0.03 g. A b b r e v i a t i o n s : s , s t a n d a r d s ; a r g , a r g i n i n e ; ?, u n k n o w n ; o c t , o c t o p i n e ; n o p , n o p a l i n e . 6 3 A C F i g . 19. Morphological d i f f e r e n c e s i n transformed and normal B. alba p l a n t s . A) L e f t , normal p l a n t ; r i g h t , R 2 transformant B) LeftjF-^ transformant showing basal hypertrophy; r i g h t , con-t r o l . C) Old Ri plant showing enlargement of nodes. D) Old R± plant showing enlargement of p e t i o l e base. E) L e f t , normal flower; r i g h t , R 2 flower, note spur development on rays. 64 spur of p e t a l t i s s u e from the inner Cupper) s i d e ( F i g . 19E). S i m i l a r a b e r r a t i o n s have been d e s c r i b e d f o r transformed tobacco p l a n t s (4,22). In contrast, Otten et a l . (15) d e s c r i b e d F^ and S^ p l a n t s as appearing normal, apparently as a r e s u l t of a d e l e t i o n i n the T-DNA of that l i n e . Other d i f f e r e n c e s appeared to be due to passage through t i s s u e c u l t u r e s i n c e they occurred i n both transformed p l a n t s and regenerants from stem segments but at much lower frequency i n normal, s eed^gr own, p l a n t s . S i m i l a r e . f f e c t s o f i n v i t r o c u l t u r e have been shown for a number of other p l a n t s C24,45). Hypertrophic growth on some of the p l a n t s grown from seed resembled g a l l growth, suggesting s o i l - b o r n e contamination of the p l a n t s by b a c t e r i a . A number of t e s t s were performed to exclude t h i s p o s s i b i l i t y . T obtained the f o l l o w i n g r e s u l t s : 1) normal seeds- germinated and grown i n s o i l from h y p e r t r o -phic p l a n t s were normal. 2) Seeds from transformed p l a n t s which were germinated on s t e r i l e medium and t r a n s f e r r e d to s t e r i l e s-oll gave p l a n t s that showed the same combinations of markers: as i n Table 4 as w e l l as hypertrophy. 3) Normal seed-l i n g s wounded and I n o c u l a t e d w i t h s o i l from h y p e r t r o p h i c p l a n t s healed normally. 4) G a l l — l i k e growths from p l a n t s which were nopa l i n e negative i n the leaves were negative i n the g a l l s . 5) Normal internode and " g a l l " ti,ss-ue from h y p e r t r o p h i c p l a n t s s u r f a c e — s t e r i l i z e d and explanted to hormone-free medium showed a u t o t r o p h i c growth: with no s i g n of b a c t e r i a l contamination. These r e s u l t s - i n d i c a t e d that there was no contamination of the s o i l by b a c t e r i a r c a r r y i n g a normal T i plasmid. Some of the m o r p h o l o g i c a l c o r r e l a t e s - of t r a n s f o r m a t i o n i n o f f s p r i n g of B. a l b a shown i n this- study- have been seen i n 65 tobacco. Binns et a l . (6) d e s c r i b e d regenerant p l a n t s with thickened nodes and abnormal shoots a r i s i n g from them. In the p l a n t s here the shoots from swollen nodes were normal. Wullems et a l . (52,54) d e s c r i b e d p l a n t s regenerated from somatic f u s i o n h y brids of crown g a l l and normal c e l l s as being s h o r t e r , t h i c k e r and having decreased a p i c a l dominance as primary g r a f t s . A f t e r two or more g r a f t t r a n s f e r s n e a r l y normal shoots r e s u l -ted. These authors a l s o showed that s p r a y i n g normal p l a n t s with k i n e t i n r e s u l t e d i n p l a n t s s i m i l a r to primary g r a f t p l a n t s . Wullems et a l . (52) a l s o noted the small s i z e and slower development of an F^ n o p a l i n e - p r o d u c i n g p l a n t . These r e s u l t s c o n t r a s t with those of Otten et a l . (37) who des-c r i b e d t h e i r F^ and p l a n t s as being completely normal i n ap p earanc e. Three types of c u l t u r e s have been i s o l a t e d from A208 tumours: a compact, slow-growing c a l l u s t i s s u e with no tendency to form teratomata', a l o o s e , f a s t e r growing c a l l u s which shows no organ formation; and shoot-forming c a l l u s which grows only as r e l -a t i v e l y normal, though dimnutive, c l u s t e r s of shoots. From the l a t t e r , l i n e s have been i s o l a t e d i n which root formation i s suppressed i n about 90% of the explants from a given clump. The tendency to form roots can be almost e l i m i n a t e d and l i n e s can be i s o l a t e d i n which both shoot and root formation i s suppressed. Amasino and M i l l e r (3) d e s c r i b e d three t i s s u e types s i m i l a r to these and each was c h a r a c t e r i z e d by a s p e c i f i c balance of hormones. They showed that normal c a l l u s grown on medium with hormone l e v e l s corresponding to those of the transformed c u l t u r e s assumed 66 the same growth form. Whether each of the growth forms i n B_. alb a has i t s own p a r t i c u l a r hormone balance i s not known al t h o u the work of Amasino and M i l l e r ( 3 ) suggests that t h i s i s the case, i n d i c a t i n g that i s o l a t e s from tumour explants of B_. a l b a are capable of assuming s e v e r a l s t a b l e s t a t e s . 67 SUMMARY Bidens a l b a d i f f e r s from p l a n t s commonly used i n crown g a l l r e s e a r c h i n i t s c a p a c i t y f o r regeneration of whole p l a n t s from g a l l s induced by an unmodified T-37 plasmid and i t s r e t e n t i o n of T-DNA t r a i t s through s e v e r a l sexual g e n e r a t i o n s . However, i t shows a marked decrease i n ex p r e s s i o n of nopaline s y n t h e s i s from gene r a t i o n to generation and l o s s of e i t h e r auxin autotrophy or nopaline s y n t h e s i s can occur suggesting e i t h e r p r o g r e s s i v e l o s s of T-DNA or changes i n i t s e x p r e s s i o n . Nopaline p o s i t i v e p l a n t s are capable of having octopine g a l l s induced on them to y i e l d double transformants, suggesting that i n s u s c e p t i b i l i t y to t r a n s f o r m a t i o n i s not a r e l i a b l e i n d i c a t o r that t i s s u e i s transformed. Transformed p l a n t s were d i s t i n -guished by s e v e r a l m o r p h o l o g i c a l d i f f e r e n c e s - some of which were probably due to an a l t e r e d hormone balance i n such p l a n t s , others p o s s i b l y due to passage through, t i s s u e c u l t u r e . 68 L I T E R A T U R E C I T E D A e r t s , M., M. J a c o b s , J . - P . H e r n a l s t e e n s , a n d J . S c h e l ' l . 1 9 7 9 . I n d u c t i o n a n d _ o f A r a b i d o p s i s t h a l i a n a c r o w n g a l l t u m o r s L e t t . 1 7 : 4 3 - 5 0 . M. V a n M o n t a g u i n v i t r o c u l t u r e P l a n t S c i A g h i o n - P r a t , D. 1 9 6 5 . F l o r a l i n t o b a c c o s t e m s . N a t u r e m e r i s t e m - o r g a n i z i n g g r a d i e n t 2 0 7 : 1 2 1 1 . A m a s i n o , R. M . ( a n d C. 0. M i l l e r . o f t o b a c c o c r o w n g a l l t u m o r P h y s i o l . 6 9 : 3 8 9 - 3 9 2 . 1 9 8 2 . H o r m o n a l c o n t r o l m o p r h o l o g y . P l a n t 4. B a l l a r d , R. E. 19.75. A b i o s y s t e m a t i c a n d c h e m o s y s t e m a t i c s t u d y o f t h e B i d e n s p i l o s a c o m p l e x i n N o r t h a n d C e n -t r a l A m e r i c a . P h . D . t h e s i s . U n i v e r s i t y o f I o w a , I o w a C i t y . 4 a . B a r t o n , K. A., A. N. B i n n s , A. J . M. M a t z k e , a n d J . - D . C h i l t o n . 19.83. R e g e n e r a t i o n o f i n t a c t p l a n t s c o n -t a i n i n g f u l l l e n g t h c o p i e s o f g e n e t i c a l l y e n g i n e e r e d T-DNA a n d t r a n s m i s s i o n o f T-DNA t o R^ p r o g e n y . C e l l 3 2 : 1 0 3 3 - 1 0 4 3 . 5. B e u c k l e e r , M. D e . , M. L e m m e r s , G. De V o s , L. W i l l m i t z e r , M. V a n M o n t a g u , a n d J . S c h e l l . 1 9 8 1 . F u r t h e r i n s i g h t o n t h e t r a n s f e r r e d - D N A o f o c t o p i n e c r o w n g a l l . M o l . G e n . G e n e t . 1 8 3 : 2 8 3 - 2 8 8 . 6, B i n n s , A. N , H. N. Wood, a n d A. C. B r a u n . 1 9 8 1 . S u p p r e s -s i o n o f t h e t u m o r o u s s t a t e i n c r o w n g a l l t e r a t o m a s o f t o b a c c o : A c l o n a l a n a l y s i s . D i f f e r e n t i a t i o n 1 9 : 9 7 - 1 0 2 ' • B r a u n , A. R e v . C. 19 5 4 . T h e P l a n t P h y s i o l p h y s i o l o g y 5 : 1 3 3 - 1 6 1 . o f p l a n t t u m o r s A n n , B r a u n , A. C„ 19.58. A p h y s i o l o g i c a l b a s i s f o r a u t o n o m o u s g r o w t h , o f t h e c r o w n - g a l l t u m o r c e l l . P r o c . N a t l . A c a d . S c i . 44:344-349... B r a u n , A. C. 19-59.. A d e m o n s t r a t i o n o f t h e r e c o v e r y o f t h e c r o w n - g a l l t u m o r c e l l w i t h t h e u s e o f c o m p l e x t u m o r s o f s i n g l e - c e l l o r i g i n . P r o c . N a t l . A c a d . S c i . 45:9.32-9_3 8 . 1 0 , C h i l t o n , M.-D., M, W, B e y a n , N. Y a d a v , A. J . M. M a t z k e , M. B y r n e , M. G - r u l a , K, B a r t o n , J , V a n d e r l e y d e n , A. De F r a m o n d , a n d W. M. B a r n e s . 1 9 8 1 . T a i l o r i n g t h e A g r o b a c t e r i u m t u m e f a c i e n s p l a s m i d a s a v e c t o r f o r p l a n t g e n e t i c e n g i n e e r i n g . S t a d l e r Symp. 1 3 : 3 9 - 5 1 . 69 1 1 . C h i l t o n , M.-D., M. H. Drummond, D. J . M e r l o , D. S c i a k y , A. L. M o n t o y a , M. P. G o r d o n , a n d E. W. N e s t e r . 1 9 7 7 . S t a b l e i n c o r p o r a t i o n o f p l a s m i d DNA i n t o h i g h e r p l a n t c e l l s : T h e m o l e c u l a r b a s i s o f c r o w n g a l l t u m o r i g e n e -s i s . C e l l 1 1 : 2 6 3 - 2 7 1 . 1 2 . C h i l t o n , M.-D., R. E. S a i k i , N. Y a d a v , M. P. G o r d o n , a n d F. Q u e t i e r . 1 9 8 0 . T-DNA f r o m A g r o b a c t e r l u m t u m e f a c i e n s p l a s m i d i s i n t h e n u c l e a r DNA f r a c t i o n o f c r o w n g a l l t u m o r c e l l s . P r o c . N a t l . A c a d . S c i . 7 7 : 4 0 6 0 - 4 0 6 4 . 1 3 . De G r e v e , H., H. D e c r a e m e r , J . S e u r i n c k , M. V a n M o n t a g u , a n d J . S c h e l l . 19.81. T h e f u n c t i o n a l o r g a n i z a t i o n o f t h e o c t o p i n e A g r o b a c t e r l u m t u m e f a c i e n s p l a s m i d p T i B 6 S 3 . P l a s m i d 6 : 2 3 5 - 2 4 8 . 1 4 . D r a p e r , J . , M. R. D a v e y , J . P. F r e e m a n , E. G. C o c k i n g , a n d B. J . C o x . 1 9 8 2 . T i p l a s m i d h o m o l o g o u s s e q u e n c e s p r e s e n t i n t i s s u e s f r o m A g r o b a c t e r i u m p l a s m i d - t r a n s -f o r m e d P e t u n i a p r o t o p l a s t s . P l a n t C e l l P h y s i o l . 3 2 : 4 5 1 - 4 5 8 . 1 5 . D r ummond, M. H., M. P. G o r d o n , W. E. N e s t e r , a n d M.-D. C h i l t o n . 19.77. F o r e i g n DNA o f b a c t e r i a l p l a s m i d o r i g i n i s t r a n s c r i b e d i n c r o w n g a l l t u m o r s . N a t u r e 2 6 9 : 5 3 5 - 5 3 6 . 1 6 . E i n s e t , J . W,t a n d ..Cheng. 1 9 7 9 , R e g e n e r a t i o n o f t o b a c c o p l a n t s f r o m c r o w n g a l l t u m o r s . I n V i t r o 1 5 : 7 0 3 - 7 0 8 . 1 7 . E l l i s , J . G. a n d P. J , M u r p h y . 1 9 8 1 . F o u r new o p i n e s f r o m c r o w n g a l l t u m o r s - - t h e i r d e t e c t i o n a n d p r o p e r t i e s . M o l . G e n . G e n e t . 1 8 1 : 3 6 - 4 3 . 1 7 a . F a v u s , S., 0, G o n z a l e z , P. B r o w n , a n d A. G a l s k y , 1 9 7 7 . I n h i b i t i o n o f c r o w n - g a l l t u m o r f o r m a t i o n o n p o t a t o d i s c s b y c y c l i c - A M P a n d p r o t a g l a n d i n s E]_ a n d E 2 . P l a n t C e l l P h y s i o l . 1 8 : 4 6 9 - 4 7 2 . 1 8 . G a r f i n k e l , D. J . , R, B, S i m p s o n , L, W, Ream, F. F, W h i t e , M. P. G o r d o n , a n d E. W, N e s t e r . 19.81. G e n e t i c a n a l y -s i s o f c r o w n g a l l : F i n e s t r u c t u r e map o f t h e T-DNA b y s i t e - d i r e c t e d m u t a g e n e s i s . C e l l 2 7 : 1 4 3 - 1 5 3 . 19... G o r d o n , M. P. 1 9 8 1 . Tumor f o r m a t i o n i n p l a n t s . P a g e s 5 3 1 - 5 7 0 i n A, M a r c u s , e d . T h e b i o c h e m i s t r y o f p l a n t s : A c o m p r e h e n s i v e t r e a t i s e , V o l . 6. A c a d e m i c P r e s s , N.Y, 2 0 . H e r n a l s t e e n s , J ,-P . 1 9 8 0 . T h e A g r o b a c t e r i u m t u m e f a c i e n s T i p l a s m i d a s a h o s t v e c t o r s y s t e m f o r i n t r o d u c i n g f o r e i g n DNA i n p l a n t c e l l s . N a t u r e 2 8 7 : 6 5 4 - 6 5 6 . 2 1 . J o h n s o n , R., R. H. G u d e r l a n , F. E d e n , M.-D. C h i l t o n , M. P. G o r d o n , a n d E. W, N e s t e r . 19.74. D e t e c t i o n a n d q u a n -t i t a t i o n o f o c t o p i n e i n n o r m a l p l a n t t i s s u e a n d i n c r o w n g a l l t u m o r s . P r o c . N a t l . A c a d . S c i . 7 1 : 5 3 6 - 5 3 9 . . 70 2 2 . Kemp, J . D, , E. E a c k , a n d D, W. S u t t o n , 19.80, C r o w n g a l l s p e c i f i c g e n e p r o d u c t s - - o c t o p i n e a n d n o p a l i n e s y n t h a s e . P a g e s 489.-49.6 i n C. J . L e a v e r , e d . Genome o r g a n i -z a t i o n a n d e x p r e s s i o n i n p l a n t s . P l e n u m P r e s s , N.Y., L d n . 2 3 . K r e n s , F. A., L. M o l e n d i j k , G. J . W u l l e m s , a n d R. A. S c h i l p e r o o r t . 1 9 8 2 . I n v i t r o t r a n s f o r m a t i o n o f p l a n t p r o t o p l a s t s w i t h T i - p l a s m i d DNA. N a t u r e 2 9 6 : 7 2 - 7 4 . 2 4 . L a r k i n , P. J . , a n d W. R. S c o w c r o f t . 1 9 8 1 . S o m a c l o n a l v a r i -a t i o n — a n o v e l s o u r c e o f v a r i a b i l i t y f r o m c e l l c u l t u r e s f o r p l a n t i m p r o v e m e n t . T h e o r . A p p l . G e n e t . 6 0 : 1 9 7 - 2 1 4 . 2 5 . L e m m e r s , M., M, D. B e u c k e l e e r , M. H o l s t e r s , P. Z a m b r y s k i , A. D e p i c k e r , J . P. H e r n a l s t S e n s , M, V a n M o n t a g u , a n d J . S c h e l l , 19.80. I n t e r n a l o r g a n i z a t i o n , b o u n d a r i e s a n d i n t e g r a t i o n o f T i - p l a s m i d DNA i n n o p a l i n e c r o w n g a l l t u m o r s . J , M o l , B i o l . 1 4 4 : 3 5 3 - 3 7 6 . . 2 6 . M a r t o n , L , , G, J , W u l l e m s , L , M o l e n d i j k , R. A. S c h i l p e r -o o r t , 1979., In v i t r o t r a n s f o r m a t i o n o f c u l t u r e d c e l l s f r o m N i c o t i a n a t a b a c u m b y A g r o b a c t e r i u m t u m e -f a c i e n s . N a t u r e 2 7 7 : 1 2 9 - 1 3 1 . 2 7 . M a r x , J . L. 19.82. T i p l a s m i d s a s g e n e c a r r i e r s . S c i e n c e 2 1 6 : 1 3 0 6 - 1 3 0 7 . 2 8 . M e i n s , F . , J r , 19.71. R e g u l a t i o n o f p h e n o t y p i c e x p r e s -s i o n i n c r o w n — g a l l t e r a t o m a t i s s u e s o f t o b a c c o . D e v e l o p m e n t a l B i o l o g y 2 4 : 2 9 1 - 3 0 0 . 2 9 . M e l c h e r t , T, E. 19.75. New c o m b i n a t i o n s i n t h e C o r e o p s i -d i n a e , P h y t o l o g i a 32 ; 29JL-29.8 . 3 0 . M e r l o , D, J , 19.7 9., T i p l a s m i d s o f A g r o b a c t e r i u m : P o t e n t i a l f o r g e n e t i c e n g i n e e r i n g . S t a d l e r Symp. 11:69-9.0. 3 1 . M e r l o , D. J . , R. C, N u t t e r , A. L. M o n t o y a , D. J . 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S c h r o d e r , and J , S c h r o d e r . 1982. T r a n s f e r and e x p r e s s i o n o f genes i n p l a n t s . B i o l o g y o f t h e C e l l 45:467. 41. S c h e n k , R. V.,and A. C. H i l d e b r a n d t . 1972. Medium and t e c h n i q u e s f o r i n d u c t i o n and g r o w t h o f m o n o c o t y l e d o n -ous and d i c o t y l e d o n o u s p l a n t c e l l c u l t u r e s . Can. J . B o t . 5 0:199-204. 42. S c h r o d e r , G.,and J . S c h r o d e r . 1982. H y b r i d i z a t i o n s e l e c -t i o n and t r a n s l a t i o n o f T-DNA e n c o d e d mRNAs f r o m o c t o -p i n e t u m o r s . M o l . Gen. G e n e t . 185:51-55. 43. S c i a k y , D., A. L. M o n t o y a , and M.-D. C h i l t o n . 1978. F i n g e r p r i n t s o f A g r o b a c t e r i u m T i p l a s m i d s . P l a s m i d 1:238-253. 44. S c o t t , I . M. 1979. O p i n e c o n t e n t o f u n o r g a n i z e d and t e r a -t o m a t o u s t o b a c c o crown g a l l t i s s u e s . P l a n t S c i . L e t t . 1 6 : 2 3 9-248. 45. S k i r v i n , R. M. 1978. N a t u r a l and i n d u c e d v a r i a t i o n i n t i s s u e c u l t u r e . E u p h y t i c a 27:241-266. 72 4 6 . T h o m a s h o w , M. F., R. N u t t e r , A. L. M o n t o y a , M. P. G o r d o n , a n d E. W. N e s t e r . 19.80. I n t e g r a t i o n a n d o r g a n i z a t i o n o f T i p l a s m i d s e q u e n c e s i n c r o w n g a l l t u m o r s . C e l l 1 9 : 7 2 9 - 7 3 9 . 4 7 . T u r g e o n , R. 1 9 8 1 . S t r u c t u r e o f g r a f t e d c r o w n - g a l l t e r a t o m a s h o o t s o f t o b a c c o : r e g u l a t i o n o f t r a n s f o r m e d c e l l s . P l a n t a 1 5 3 : 4 2 - 4 8 . 4 8 . T u r g e o n , R.,H. N. Wood, a n d A. C. B r a u n . 1 9 7 6 . S t u d i e s o n t h e r e c o v e r y o f c r o w n g a l l t u m o r c e l l s . P r o c . N a t l . A c a d . S c i . 7 3 : 3 5 6 2 - 3 5 6 4 . 4 9 . V a n L a r e b e k e , N., G. E n g l e r , M. H o l s t e r , S. V a n Den E l s a c k e r , I , Z a e n e n , R, S. S c h i l p e r o o r t , a n d J . S c h e l l . 1 9 7 4 . L a r g e p l a s m i d i n A g r o b a c t e r i u m t u m e f a c i e n s e s s e n t i a l f o r c r o w n g a l l - i n d u c i n g a b i l i t y . N a t u r e 2 5 2 : 1 6 9 - 1 7 0 . 5 0 . W a t s o n , B,, T. C. C u r r i e r , M. P. G o r d o n , M.-D. C h i l t o n , a n d E. W. N e s t e r . 1 9 7 5 . P l a s m i d r e q u i r e d f o r v i r u -l e n c e o f A g r o b a c t e r i u r n t u m e f a c i e n s . J . B a c t e r i o l . 1 2 3 : 2 5 5 - 2 6 4 . 5 1 . Wood, H. N., A. N. B i n n s , a n d A. C. B r a u n . 1 9 7 8 . D i f f e r -e n t i a l e x p r e s s i o n o f o n c o g e n i c i t y a n d n o p a l i n e s y n -t h e s i s i n i n t a c t l e a v e s d e r i v e d f r o m c r o w n g a l l t e r a -t o m a s o f t o b a c c o . D i f f e r e n t i a t i o n 1 1 : 1 7 5 - 1 8 0 . 5 2 . W u l l e m s , G. J . , L. M o l e n d i j k , G. Ooms, R. A. S c h i l p e r o o r t . 19_81. R e t e n t i o n o f t u m o u r m a r k e r s i n F^ p r o g e n y p l a n t s f r o m i_n v i t r o i n d u c e d o c t o p i n e a n d n o p a l i n e t u m o r t i s s u e s . C e l l 24:719.0.727. 5 3 . W u l l e m s , G, J , , L , M o l e n d i j k , G. Ooms, a n d R. A. S c h i l -p e r o o r t . 19.81. D i f f e r e n t i a l e x p r e s s i o n o f c r o w n g a l l m a r k e r s i n t r a n s f o r m a n t s o b t a i n e d a f t e r i n v i t r o  A g r o b a c t e r i u m t u m e f a c i e n s - i n d u c e d t r a n s f o r m a t i o n o f c e l l w a l l r e g e n e r a t i n g p r o t o p l a s t s d e r i v e d f r o m N i c o t i a n a t a b a c u m . P r o c . N a t l . A c a d . S c i . 7 8 : 4 3 4 4 -4 3 4 8 . 5 4 . W u l l e m s , G. J , , L , M o l e n d i j k , and R, A- S c h i l p e r o o r t , 1 9 8 0 . T h e e x p r e s s i o n o f t u m o u r m a r k e r s i n i n t r a -s p e c i f i c s o m a t i c H y b r i d s o f n o r m a l a n d c r o w n g a l l c e l l s f r o m N i c o t i a n a t a b a c u m . T h e o r , A p p l . G e n e t 5 6 : 2 0 3 - 2 0 8 . 5 5 . Y a m a d a , S. t a n d H, A. I t a n o . 1 9 6 6 , P h e n a n t h . r e n e q u i n o n e a s a n a n a l y t i c a l r e a g e n t f o r a r g i n i n e a n d o t h e r mono-s u b s t i t u t e d g u a n i d i n e s . B i o c h i m , B i o p h y s , A c t a 1 3 0 : 5 3 8 - 5 4 0 . 5 6 . Y a n g , F., A. L. M o n t o y a , D. J . M e r l o , M. H. Drummond, M.-D. C h i l t o n , E. W, N e s t e r , a n d M. P. G o r d o n . 1 9 8 0 . 73 Fore i g n DNA sequences i n crown g a l l teratomas and t h e i r f a t e during the l o s s of the tumorous t r a i t s . Mol. Gen. Genet. 177:707-714. 57. Yang, F. M., A. L. Montoya, E. W. Nester, and M. P. Gordon. 19.80. Plant tumor r e v e r s a l a s s o c i a t e d with the l o s s of f o r e i g n DNA. In V i t r o 16:87-92. 57. Yang, F a n d R. B, Simpson. 19.81, Revertant s e e d l i n g s from crown g a l l tumors r e t a i n a p o r t i o n of the Bac-t e r i a l T i plasmid sequence. Proc. N a t l . Acad. S c i . 78:4151-4155. 59.. ZamBryski, P., M. H o l s t e r s , K, Kruger, A. Depicker, J . S c h e l l , M. Van Montagu, and H. M. Goodman., 1980. Tumor DNA s t r u c t u r e i n p l a n t c e l l s transformed by A. tumefaciens. Science 209:1385-1391. 7 4 CHAPTER IV STUDIES OF NORMAL CALLUS OF BIDENS ALBA 75 INTRODUCTION This chapter d e s c r i b e s attempts to o b t a i n p r o d u c t i o n of p o l y a c e t y l e n e s i n normal c a l l u s c u l t u r e of J i . a l b a . In :an' effort to uncover c l u e s to the p o s s i b l e requirements of such c u l t u r e s a survey of r e p o r t s of other p l a n t s which produced unusual f a t t y a c i d products was made ( f o r recent reviews see 22 , 27). The most useful of;Such cultures appear to have been those of two species of Malva, which produced l e v e l s of cyclopropane and cyclopropene f a t t y a c i d s ( s t e r c u l i c and m a l v a l i c a c i d s and t h e i r dihydro analogs) comparable to or exceeding those of the seeds (43). C a l l u s c u l t u r e s were used to e s t a b l i s h the b i o s y n t h e t i c pathway f o r these compounds (42). The b i o s y n t h e -s i s of f a t t y a c i d s having a t e r m i n a l c y c l o p e n t e n y l r i n g was' s t u d i e d with suspension c u l t u r e s of Ides i a p o l y c a r p a , which were found to elongate a p r e c u r s o r i n t o the f i n a l product (23,28). However, c u l t u r e s of a r e l a t e d p l a n t , Hydnocarpus  a n t h e l m i n i c a , were u n s u i t a b l e f o r studying these compounds (33). S p e c i a l i z e d f a t t y a c i d s have been found i n c a l l u s of Pinus e l l i o t t i i ( 1 9 1 and Petroselinum crispum (6). In the l a t t e r high l e v e l s of p e t r o s e l e n i c a c i d (18:1,6c) found i n the p l a n t s were absent from c u l t u r e s , but an isomer, v a c c e n i c a c i d (18:1,11c), was present. There seem to be no r e p o r t s of f a t t y a c i d s r e s t r i c t e d to seed t i s s u e being produced i n s i g -n i f i c a n t amount i n c a l l u s , although Jones (17) obtained f o r -mation of e r u c i c a c i d (22:1,13c) i n embryo-like s t r u c t u r e s which d i f f e r e n t i a t e d from Crambe c a l l u s . 7i The r e p o r t s summarized above represent i n s t a n c e s of t i s s u e c u l t u r e s producing more than t r a c e q u a n t i t i e s . o f unusual f a t t y acids.. However, no s p e c i a l m o d i f i c a t i o n s to c u l t u r a l c o n d i t i o n s were made to enhance or induce the p r o d u c t i o n of these com-pounds. These p l a n t s would appear to be c o n s t i t u t i v e producers under the standard c o n d i t i o n s used. Many negative r e p o r t s , however, demonstrate that p r o d u c t i o n of s p e c i a l i z e d f a t t y a c i d d e r i v a t i v e s , as with other groups of secondary compounds, i s e x c e p t i o n a l i n t i s s u e c u l t u r e s . (11,17,20,29,35,36,40). No r e p o r t s could be found of attempts to i n f l u e n c e e x p r e s s i o n of unusual f a t t y a c i d s through n u t r i t i o n a l changes, although i t has been repo r t e d that p l a n t membrane p h o s p h o l i p i d s could be e x p e r i m e n t a l l y a l t e r e d (24). Reports of p o l y a c e t y l e n e s i n normal t i s s u e c u l t u r e s of Compositae have uni f o r m l y been r e s t r i c t e d to short—term c u l -tures ( l e s s than three months). Jente C.16) r e p o r t e d the e a r l i e s t i n s t a n c e of es tablis-hing c a l l u s c u l t u r e s and deter? mining the p o l y a c e t y l e n e s present. She found extremely low l e v e l s of 0.5 ug/g fresh, wt. i n c a l l u s of~ Centaurea r u t h e n i c a versus 5.0 mg/g f .w. i n roots- of the p l a n t . P o l y a c e t y l e n e s were found on l y i n . c u l t u r e s which had been grown i n l i g h t or a l t e r n a t i n g l i g h t and dark. The compounds i d e n t i f i e d i n c a l l u s - were d i f f e r e n t from those o c c u r r i n g i n the i n t a c t p l a n t , although b l o g e n e t i c a l l y r e l a t e d . There was no d i f f e r e n c e i n a c e t y l e n e s between c u l t u r e s e s t a b l i s h e d from roots or l e a f m a t e r i a l C16")*. i c h i h a r a and No da (14). i n v e s t Igate.d Car thamus  t l n c t o r j u s - s e e d l i n g s and c a l l u s c u l t u r e s e s t a b l i s h e d from r o o t s , hypocotyls and cotyledons- of s e e d l i n g s . T i s s u e s were 77 incubated with i l l u m i n a t i o n f o r 14 days before h a r v e s t i n g . Under these c o n d i t i o n s most a c e t y l e n i c compounds i n c r e a s e d i n amount, however no data on c u l t u r e s over two weeks o l d were r e p o r t e d . These authors a l s o demonstrated s y n t h e s i s of p o l y a c e t y l e n e s i n p r o t o p l a s t s and chopped seed p r e p a r a t i o n s . Tagetes erec ta was i n v e s t i g a t e d by S e t i a (31) f o r b i o s y n t h e s i s of both p o l y a c e t y l e n e s and thiophenes. C a l l u s c u l t u r e s f a i l e d to produce s i g n i f i c a n t amounts of e i t h e r type of compound when the medium (B^) contained 1 ppm 2,4-D. However, cultures grown on med-ium e i t h e r l a c k i n g hormones or wtih 2 , 3 , 5 - t r i c h l o r o b e n z o i c or 2 , 3 - d i c h l o r o b e n z o i c a c i d s (1-4 ppm) were able to s y n t h e s i z e thiophenes but not p o l y a c e t y l e n e s i n low but i s o l a t a b l e amounts. S p e c t r a l evidence suggested the presence of a p o l a r t r i y n d i e n e chromophore but only i n minute amounts. C u l t u r e s began to d i e w i t h i n 6 to 7 days and no growth occurred a f t e r 14 days. The highest product y i e l d was found a f t e r 13 to 16 days (.31). The f o l l o w i n g p o i n t s summarize the main f i n d i n g s of these s t u d i e s : 1) l i g h t may be necessary f o r product formation. 2) Types of hormones used i n the medium could be a f a c t o r i n s u s t a i n i n g b i o s y n t h e s i s . 3) Compounds normally not found i n s i g n i f i c a n t amount may accumulate i n c u l t u r e d t i s s u e . The work summarized i n t h i s chapter attempted to improve on these s t u d i e s and e s t a b l i s h c u l t u r e s which would s y n t h e s i z e p o l y a c e t y l e n e s as a normal part of c a l l u s growth. Se v e r a l reviews of f a c t o r s a f f e c t i n g a wide range of secondary prod-ucts s y n t h e s i z e d i n t i s s u e c u l t u r e s served as guides f o r s e l e c t -ing parameters to evaluate f o r p o l y a c e t y l e n e p r o d u c t i o n (1,5,38), 78 MATERIALS AND METHODS Explant s Seeds were washed i n 70% ethanol f o r one minute and then soaked ten minutes i n a 10% commercial bleach (Javex) s o l u t i o n with 0.5% detergent as a wetting agent, gently a g i t a t e d , r i n s e d three or more times i n s t e r i l e d i s t i l l e d water and germinated i n dark at 25*C on moistened and s t e r i l i z e d f i l t e r paper d i s c s . A f t e r germination s e e d l i n g s were t r a n s f e r r e d to the l i g h t and grown to approximately four cm i n length and then d i v i d e d i n t o r o o t s , h y p o c o t y l , and cotyledons and explanted to separate p l a t e s of medium. Media The f o l l o w i n g media f o r m u l a t i o n s were t e s t e d : White (41), N i t s c h (26), H e l l e r Cl2a), ER (7), MS (25), Hild e b r a n d t (13), B,. (9), and SH (30); MS medium was made up with equimolar con-c e n t r a t i o n s of f e r r o u s sulphate and disodium EDTA (32), A l l fo r m u l a t i o n s , except as noted, were made up with 0.7% agar ( D i f c o , Bacto-agar), 3% sucrose ( F i s h e r S c i e n t i f i c ) , 1 mg/1 2,4-dichlorophenoxyacetic a c i d (2,4—D) (Sigma) and pH 5.7, adjusted with potassium hydroxide or h y d r o c h l o r i c a c i d , Media were autoclaved 20 minutes at 15 lb i n - 2 and then poured i n t o 15 x 90 mm p l a s t i c p e t r i ' p l a t e s . The standard i n c u b a t i o n f o r c u l t u r e s was 25*C with no i l l u m i n a t i o n . Supplements Inorganic s a l t s , organic a c i d s (except f a t t y a c i d s ) , 79 glucose, i n o s i t o l , and k i n e t i n were added to the medium p r i o r to a u t o c l a v i n g . A l l other compounds were f i 1 t e r - s t e r i i i z e d with 0.2 micron M i l l i p o r e f i l t e r s and added a f t e r a u t o c l a v i n g . F a t t y a c i d supplements were added to cooled medium a f t e r f i l -t e r - s t e r i l i z i n g , shaken v i g o r o u s l y to form an emulsion and then poured. A number of d i f f e r e n t e m u l s i f i e r s f o r l i p i d s were t r i e d i n c l u d i n g ethylene g l y c o l , g l y c e r i n , T r i t o n X-100, Tween-20, -40, and -80, e t h a n o l , l e c i t h i n , sodium acetate and tragacanth; these i n v a r i o u s combinations. A l l are l i s t e d i n standard r e f e r e n c e s on emulsions (2,37) as s u i t a b l e f o r l i p i d s of the type added. However, most were u n s a t i s f a c t o r y due to the low r a t i o of l i p i d to aqueous phase i n medium. In a d d i t i o n , heated medium enhanced emulsion breakdown and cream-in g . The best combination obtained, which formed a s t a b l e emulsion, was f a t t y a c i d : Tween-8Q: ethanol i n the r a t i o 35:8;7 (v / v / v ) . C o n t r o l s showed l i t t l e e f f e c t on c a l l u s growth from the Tween-80 and ethanol at the hi g h e s t l e v e l s used. O l e i c , l i n o l e i c , and l i n o l e n i c a c i d s were P u r i f i e d grade, c o n t a i n i n g at l e a s t 90% of the f r e e a c i d ( F i s h e r S c i e n t i f i c ) . C a l l u s T e s t i n g C a l l u s f o r use i n t e s t s of medium supplements were l i n e s which had been s u b c u l t u r e d two or more times and showed the presence of smal l amounts of a c e t y l e n e s . Four explants were made to each of three p l a t e s and a t o t a l of s i x l i n e s t e s t e d . A f t e r growth on the medium f o r 2-3 weeks the c a l l u s from each p l a t e was combined, e x t r a c t e d , and a UV spectrum taken and checked f o r any i n d i c a t i o n of p o l y a c e t y l e n e s y n t h e s i s . I f an e f f e c t was to occur i t might only be found with c e r t a i n l i n e s 8 0 so as many l i n e s as p o s s i b l e were t e s t e d . Approximately t h r e e - q u a r t e r s of the c a l l u s from a p e t r i ^ p l a t e was taken f o r e x t r a c t i o n ; the remainder was- l e f t f o r s u b c u l t u r i n g . The petroleum ether e x t r a c t was evaporated with a stream of n i t r o -gen, made up to 5 ml, and a UV spectrum made of i t . This would u s u a l l y represent the e x t r a c t from at l e a s t 5 g of c a l l u s and p o s s i b l y more. Under these c o n d i t i o n s the UV spe~ct;rum~frOm approximately Q. 02 g of r o o t s or ft, 0.1 g of l e a f gave s p e c t r a i n which the main chromophores could b?e recog-n i z e d . Hence, the p o l y a c e t y l e n e s i n c a l l u s c o n t a i n i n g 0,5% of the l e v e l found i n the p l a n t should have been r e a d i l y r e c o g n i z a b l e . The spectrum f o r a l i n e was c l a s s i f i e d as negative or p o s i t i v e f o r product and compared to l a t e r s p e c t r a of the same l i n e to determine the e f f e c t of f u r t h e r s u b c u l t u r i n g . F i g u r e 20 shows the o v e r a l l s t r u c t u r e of the e v a l u a t i o n procedure followed i n attempting to f i n d some def i n e d c o n d i t i o n which would r e s u l t i n product synthesis: i n c a l l u s . Mercuric C h l o r i d e Treatment C o n d i t i o n s f o r mercuric c h l o r i d e e l i c i t a t i o n were adapted from data of Hargreaves 0 - 2 ) . A c o n c e n t r a t i o n of 3 mM mercuric c h l o r i d e and exposure times of 5, IQ, and 15 minutes were used. Treated and c o n t r o l c a l l u s e s were r i n s e d f i v e times i n s t e r i l e d i s t i l l e d water and incubated f o r four days on SH medium w i t h 4.0 mg/1 NAA. D u p l i c a t e p l a t e s of f i v e c a l l u s e s f o r each of four l i n e s were t e s t e d at each treatment combina-t i o n . Two l i n e s had been c u l t u r e d f o r two years and two f o r four years at the time of the experiment, 81 S e e d l i n g e x p l a n t s t o 8 d i f f e r e n t m e d i a . 1 S e l e c t i o n o f 3 m e d i a w i t h b e s t g r o w t h 1 E v a l u a t i o n o f g r o w t h v s . hormone l e v e l s . H a r v e s t and e v a l u a t e "** a l l c a l l u s f o r com-:' pounds . E v a l u a t e o r g a n d i f f e r e n c e s . S t a n d a r d a u x i n l e v e l . E s t a b l i s h m e n t of 220 l i n e s f r o m s e e d l i n g . G r o w t h ' f o r t h r e e m o nths. J H a r v e s t and e v a l u a t e e x t r a c t o f a l l l i n e s . C u l t u r e s s h o w i n g some i n d i c a t i o n o f p r o d u c t , S u b c u l t u r e . R e - e x a m i n e . D i s c a r d l i n e i f s p e c t r u m f e a t u r e l e s s . E v a l u a t i o n o f medium v a r i a t i o n s . \ E v a l u a t i o n o f e a c h v a r i a t i o n w i t h , a t l e a s t 6 l i n e s p e r t r e a t m e n t . F i g . 20. D i a g r a m o f e v a l u a t i o n p r o c e d u r e f o r p o l y a c e t y l e n e p r o d u c t i o n i n c a l l u s c u l t u r e s o f B. a l b a . 82 RESULTS AND DISCUSSION Growth on D i f f e r e n t M e d i a The i n i t i a l s t e p i n c u l t u r i n g IS. a l b a was t o f i n d how g r o w t h was a f f e c t e d by d i f f e r e n t m e d i a . I n i t i a l work showed t h a t c a l l u s f o r m a t i o n o c c u r r e d on B,. medium w i t h 1.0 mg/1 2,4-D (2 , 4 - d i c h l o r o -p h e n o x y a c e t i c a c i d ) and t h i s l e v e l o f a u x i n was u s e d i n a l l m e d i a . E i g h t f o r m u l a t i o n s were t r i e d w i t h f i v e e x p l a n t s of e a c h s e e d l i n g o r g a n e x p l a n t e d p e r p l a t e and t r i p l i c a t e s f o r e a c h o r g a n and medium. G r o w t h was e v a l u a t e d a t t h e end o f t h r e e weeks by v i s u a l l y r a n k i n g t h e amount of new t i s s u e f o r m e d . R e s u l t s a r e shown i n T a b l e 5. T h e r e was l i t t l e d i f f e r e n c e i n g r o w t h r e s p o n s e among t h e t h r e e o r g a n s . M e d i a h a v i n g l o w s a l t l e v e l s ( H i l d e b r a n d t , W h i t e , N i t s c h , and H e l l e r ) p r o d u c e d d i s t i n c t l y l e s s g r o w t h t h a n t h o s e w i t h h i g h e r s a l t l e v e l s . The l o w s a l t g r o u p was e l i m i n a t e d f r o m f u r -t h e r work a f t e r a d d i t i o n a l s u b c u l t u r e s showed no p r o d u c t f o r m a t i o n , and ER medium was e l i m i n a t e d due t o i t s s i m i l a r i t y t o MS and i t s l a c k of p r o d u c t . The r e m a i n i n g m e d i a (MS, SH, B<-) p r o v i d e d t h e b e s t g r o w t h and were u s e d i n s u b s e q u e n t e x p e r i m e n t s . T a b l e 5. R e l a t i v e g r o w t h of _B. a l b a e x p l a n t s on d i f f e r e n t m e d i a . Organ Medium t y p e H i l d e - W h i t e N i t s c h H e l l e r SH MS ER B 5 R o o t + + + + ++ ++ ++ +++ H y p o c o t y l + + + + ++ ++ ++ ++ C o t y l e d o n + + + + + ++ ++ +++ Symb o I s : +, l e a s t i g r o w t h ; +++, most g r o w t h . 83 C a l l u s and P r o d u c t F o r m a t i o n E x p l a n t s c a l l u s e d r e a d i l y on 2,4-D s u p p l e m e n t e d SH, , and MS m e d i a , a l t h o u g h some r o o t f o r m a t i o n occurred w i t h l e a f t i s s u e , and by t h e end o f t h e f i r s t month most o f t h e o r i g i n a l e x p l a n t had b e e n r e p l a c e d by c a l l u s . E x t r a c t i o n o f c a l l u s t i s s u e a t t h e end o f t h e s e c o n d month o f c u l t u r e showed no PHT was p r e s e n t i n l e a f o r s t e m c a l l u s . L e a f c a l l u s had low l e v e l s o f ETE-R (/the p a r t i c u l a r f o r m c o u l d n o t he d e t e r -m i n ed f r o m t h e s p e c t r u m , a l t h o u g h i t was p r o b a b l y t h e a c e t a t e ) due t o r o o t f o r m a t i o n . T h e r e were o n l y t r a c e amounts o f a c e t -y l e n e s i n s t e m and r o o t c a l l u s . By t h e end o f the. t h i r d month p o l y a c e t y l e n e s were n o t d e t e c t a b l e i n most o f t h e c u l t u r e s . I f t r a c e s w e re s t i l l pr-es-ent i t was a l w a y s e i t h e r ETE-R o r P D E - R , ( p r e s u m a b l y t h e a c e t a t e ) . M e r c u r i c C h l o r i d e T r e a t m e n t M e r c u r i c c h l o r i d e i s an a b i o t i c e l i c l t o r w h i c h has been f o u n d t o c a u s e p r o d u c t i o n o f p h y t o a l e . x i n s i n some p l a n t s (10)_. A f t e r o t h e r methods?- had b e e n t r i e d and f o u n d n o t t o r e s u l t i n p r o d u c t s y n t h e s i s , t h i s a p p r o a c h was t r i e d . C a l l u s treated w i t h m e r c u r i c c h l o r i d e t u r n e d brown and showed e x t e n -s i v e , b u t n o t c o m p l e t e , c e l l d e a t h . T h e r e was no i n d i c a t i o n o f e l i c i t a t i o n o f p o l y a c e t y l e n e s . I t i s p o s s i b l e t h a t t h e age o f t h e l i n e s was a f a c t o r i n t h e n e g a t i v e r e s u l t s ; F e t t and Z a c h a r i u s (8) f o u n d t h a t p h y t o a l e x i n p r o d u c t i o n c o u l d be i n d u c e d i n a r e c e n t l y e s t a b l i s h e d pea c e l l s u s p e n s i o n c u l t u r e by b a c t e r i a b u t a s i m i l a r l i n e i n c u l t u r e f o r 15 y e a r s showed no r e s p o n s e . A t t h e t i m e t h i s e x p e r i m e n t was done a l l t h e l i n e s u s e d were a t l e a s t two y e a r s o l d . 84 M i s c e l l a n e o u s T r e a t m e n t s A f e w f a c t o r s w e r e c h e c k e d i n a s e r i e s o f c u r s o r y e x p e r i -m e n t s f o r o b v i o u s e f f e c t s : o n p r o d u c t f o r m a t i o n . T h e e f f e c t o f t e m p e r a t u r e s h o c k was checked b y e x p o s i n g c u l t u r e s t o e i t h e r l o w ( 4 * C ) o r h i g h ( 4 0 ° C ) t e m p e r a t u r e s o v e r n i g h t , r e t u r n i n g t h e m t o s t a n d a r d c o n d i t i o n s , a n d h a r v e s t i n g t wo d a y s l a t e r , C a l l u s e s w e r e a l s o i n c u b a t e d u n d e r a g e r m i c i d a l l a m p f o r t i m e s r a n g i n g f r o m t w o h o u r s t o o v e r n i g h t a n d s i m i l a r l y h a r v e s t e d . C u l t u r e s w e r e h a r v e s t e d a t a l l p h a s e s - o f g r o w t h , i n c l u d i n g v e r y l a t e i n t h e p a s s a g e w h e n m o s t o f t h e m e d i u m was- g o n e a n d t h e t e x t u r e o f t h e c a l l u s b e c a m e q u i t e l o o s e . C a l l u s e s c o n t a m i n a t e d w i t h a v a r i e t y o f unknown;} f u n g i a n d b a c t e r i a w e r e h a r v e s t e d . N o n e o f t h e m a t e r i a l c h e c k e d u n d e r t h e s e c o n d i t i o n s s h o w e d a n y i n d i -c a t i o n o f p r o d u c t . s y n t h e s i s . Summary o f R e s u l t s - f r o m M e d i u m M o d i f i c a t i o n s A s s h o w n i n F i g . 2 0 , t h e n e x t s t e p i n e v a l u a t i o n was t o d e t e r m i n e i f t h e r e was a n y e f f e c t f r o m t h e s o u r c e o f t h e e x p l a n t o n p r o d u c t i n t h e s u h c u l t u r e d c a l l u s . A t t h e . t i m e c a l l u s <f counv; •'„ - t h e t h r e e o r g a n s was h a r v e s t e d ( " f o u r t h p a s s a g e ) n o n e o f t h e c u l t u r e s w e r e p r o d u c i n g a p p r e c i a b l e l e v e l s o f p r o d u c t . I t was c o n c l u d e d t h a t u n d e r t h e s e c o n d i t i o n s * t h e s o u r c e o f t h e e x p l a n t was u n i m p o r t a n t . T h e e x p l a n t e d o r g a n s o r i g i n -a l l y h a d c h a r a c t e r i s t i c p a t t e r n s - a n d l e v e l s o f p o l y a c e t y l e n e s s i m i l a r t o t h o s e i n T a b l e 2. Op i n i o n s - o n t h e i m p o r t a n c e o f t h e s o u r c e o f t h e . e x p l a n t f o r s u b s e q u e n t b i o s y n t h e t i c c a p a c i t y - v a r y w i d e l y ; Bb'hm (3)1 c o n -s i d e r s i t i r r e l e v a n t , w h i l e S t a b a C34) c i t e s e v i d e n c e , s u p p o r t i n g t h e i m p o r t a n c e o f e x p l a n t s o u r c e . I t s i m p o r t a n c e f o r s u b s e -85 quent morphogenesis has Been reviewed by Thorpe (38a) but e f f e c t s on secondary product s y n t h e s i s seem to be v a r i a b l e . Loh et a l . (21) r e c e n t l y reported that no d i f f e r e n c e i n pro-duct could be demonstrated f o r c a l l u s from d i f f e r e n t p a r t s of Cannabis s a t i v a ; cannabinoids .were n o t s y n t h e s i z e d i n any of the c a l l u s e s from d i f f e r e n t p a r t s of the p l a n t . However, the amount and type of hormones- r e q u i r e d f o r growth was s p e c i f i c f o r the o r i g i n of the explant. C a l l u s from the d i f f e r e n t organs of Bidens seedlings', seemed to show about the same amount of growth, as shown i n Table 5. In the next stage of e v a l u a t i o n the l e v e l of auxin f o r best growth was determined f o r both NAA (eC-nap t h a i one a c e t i c a c i d ) and 2,4-D. Best growth f o r 2,4-D was at 1.0 mg/1 and f o r NAA, 4.0 mg/1. There was no d e t e c t a b l e d i f f e r e n c e between NAA and 2,4-D i n product formation, however, so 2,4-D was used f o r the balance of the experiments, except as noted. The next step was to c u l t u r e as many separate s e e d l i n g s as p r a c t i c a l and determine i f there were s i g n i f i c a n t d i f f e r e n c e s i n the c a l l u s d e r i v e d from these, i . e . would a small percent of the s e e d l i n g s be c o n s t i t u t i v e f o r p o l y a c e t y l e n e s y n t h e s i s i n c a l l u s . A l t o g e t h e r c a l l u s from 220 s e e d l i n g s was checked f o r product; some were grown on MS medium, some on SH, and some on . C a l l u s was s u b c u l t u r e d three times and evaluated during the f o u r t h passage. Most of the l i n e s showed no i n d i -c a t i o n of any p o l y a c e t y l e n e s at t h i s stage and were^ t h e r e f o r e , d i s c a r d e d . A few showed low l e v e l s a,nd these were continued and used f o r the experiments with various- media.,'- and e n v i r o n -mental f a c t o r s . 86 T a b l e 6 . Medium i n g r e d i e n t s - e v a l u a t e d f o r a b i l i t y t o i n d u c e p o l y a c e t y l e n e s y n t h e s i s . Hormones 2,4-D NAA NAA x k i n e t i n GA^ x k i n e t i n S u g ars and v i t a m i n s I n o s i t o l S u c r o s e S u c r o s e C+>- l i g b t ) G l u c o s e A s c o r b i c a c i d P r i m a r y m e t a b o l i t e s A c e t a t e (A) M a l a t e (M) C i t r a t e (C) A + M, A + C, + C, A + H + C M a c r o n u t r i ' e n t s KN0 3, MgS0 4, NH' 4ff 2P0 4, C a C l 2 , K t t 2 P 0 4 P r o d u c t p r e c u r s o r s or r e l a t e d p r o d u c t s O l e i c a c i d L i n o l e i c a c i d L i n o l e n i c a c i d Combinations- of a l l t h r e e 8 7 The o b j e c t i v e of the next step was to t e s t as many as p o s s i b l e of the l i n e s that s t i l l showed tr a c e s of product a g a i n s t v a r i a t i o n s i n medium and determine i f product l e v e l continued the sharp d e c l i n e shown by most l i n e s or i f the d e c l i n e i n product could be stopped or reversed. The only e v a l u a t i o n made was whether or not the UV spectrum of a l i n e showed constancy or improvement i n product l e v e l . There was no exact rmeasurement of growth or other c h a r a c t e r i s t i c s except i n one or two cases. Table 6 l i s t s the f a c t o r s examined. The Appendix contains d e t a i l e d t a b l e s showing the medium compon-ents which were v a r i e d , range of l e v e l s t e s t e d , number of L.o. l e v e l s t e s t e d , and some general comments on how growth was a f f e c t e d (see Appendix Tables 1 to 5). The f i n a l r e s u l t was that none of the treatments induced measurable l e v e l s of p o l y a c e t y l e n e s . The product l e v e l i n a l l of the l i n e s which had been used f o r these experiments f e l l below the l i m i t of d e t e c t i o n w i t h i n two f u r t h e r s u b c u l -tures or l e s s . Although changes i n t e x t u r e and growth r a t e v a r i e d c o n s i d e r a b l y w i t h the treatments, none induced product s y n t h e s i s . F a t t y a c i d supplements r e s u l t e d i n hard, dense c a l l u s somewhat l i k e the transformed c a l l u s d e s c r i b e d l a t e r , but there was no t r a c e of product i n any of the e x t r a c t s . Greening of c a l l u s was induced by lowering sucrose l e v e l to l e s s than 1%Z and i l l u m i n a t i n g the c a l l u s , but again the .... r e s u l t s were n e g a t i v e . It ; i s c o n c e i v a b l e that loss- of b i o s y n t h e t i c c a p a c i t y might be i r r e v e r s i b l e and that attempting to regain• synth.esis once l o s t , would net be p r a c t i c a l . However, many non-producing 88 c u l t u r e s have Been shown to Be capable, o f s y n t h e s i z i n g s e c o n -d a r y compounds when e x p o s e d to e l i c i t o r s of v a r i o u s t y p e s , i n c l u d i n g heavy m e t a l s (IT))., S a f y n o l , a p o l y a c e t y l e n i c p h y t o a l e x i n , i s p r o d u c e d By C a r t h amu g t i n c t o r i u s i n r e s p o n s e to i n f e c t i o n ( 1 5 ) . More r e c e n t l y s a f y n o l has Been e l i c t e d i n s u s p e n s i o n c u l t u r e s of C . t i n c t o r i u s By c e l l w a l l p r e p a r a t i o n s f r o m two d i f f e r e n t f u n g a l p a t h o g e n s (3 9:). One o f t h e c u l t u r e l i n e s of B. a l B a e s t a B l i s h e d f o r t h e work i n t h i s c h a p t e r has Been shown to s y n t h e s i z e l o w l e v e l s of PHT i n r e s p o n s e t o a f u n g a l B r o t h p r e p a r a t i o n (4).. These r e p o r t s show t h a t c a l l u s and s u s p e n s i o n c u l t u r e s w h i c h have s t o p p e d m a k i n g p o l y a c e t y l e n e s n e v e r t h e l e s s ) r e t a i n the c a p a c i t y t o make them i f a p p r o p r i a t e l y t r e a t e d . The a p p r o a c h used In t h i s c h a p t e r , however, d i d n o t r e s u l t i n f i n d i n g d e f i n e d c o n d i t i o n s f o r c o n t i n u o u s s y n t h e s i s . 89 SUMMARY' 1) M e d i a c o n t a i n i n g h i g h : l e v e l s o f s a l t s CMS, SH, ER, a n d B,.) g a v e t h e B e s t c a l l u s g r o w t h , f o r B i d e n s ^ a l B a . M a x i m u m g r o w t h was o b t a i n e d w i t h , e i t h e r 1,0. -mg/1 2 , 4—D o r 4,Q mg/1 NAA. C h o i c e o f m e d i u m d i d n o t a f f e c t t h e l o s s - o f p o l y a c e t -y l e n e s y n t h e s i s i n s u c c e s s i v e c u l t u r e s - , 2 ) T h e p a r t o f s e e d l i n g s c u l t u r e d ( r o o t , h y p o c o t y l , o r c o t y l e d o n ) h a d l i t t l e e f f e c t o n t h e g r o w t h , o f c a l l u s a r i d no e f f e c t o n p o l y a c e t y l e n e s y n t h . e s t s . 3 1 I f _B_, a l B a s e e d l i n g s : d i f f e r i n t h e c a p a c i t y t o con-; t i n u e p o l y a c e t y l e n e s y n t h e s i s - i n c a l l u s - , th_en t h e f r a c t i o n o f p l a n t s w h i c h s h o w c o n t i n u e d s y n t h e s i s o f p r o d u c t i s l e s s t h a n c o u l d B e d e t e c t e d i n a s a m p l e o f 2 2 0. s e e d l i n g s . 4) P r o d u c t i o n o f p o l y a c e t y l e n e s : c o u l d n o t B e a c h i e v e d B y v a r y i n g s e l e c t e d n u t r i t i o n a l a n d e n v i r o n m e n t a l p a r a m e t e r s . 90 LITERATURE CITED Alfermann, A. 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Recent advances i n the p r o d u c t i o n of m e d i c i n a l substances by pl a n t c e l l c u l t u r e s . Pages 3-26 In W. Barz, E. Reinhard, and M. H. Zenk, eds. Plant t i s s u e c u l t u r e and i t s b i o - t e c h n o l o g i c a l a p p l i c a t i o n . S p r i n g e r - V e r l a g , H e i d e l b e r g . 38a. Thorpe, T. A. 1980. Organogenesis jLn 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 bi o c h e m i c a l a s p e c t s . I n t l . Rev. C y t o l . , supp. 11A:71-111. 39. T i e t j e n , K., and U. Matern. Mode of a c t i o n of a l t e r n a r i a carthami t o x i n i n s a f f l o w e r (Carthamus t i n c t o r i u s L.) Phytochemical S o c i e t y of North America Newsletter, J u l y , 1981. A b s t r a c t C-23. 40. Van de Voort, F., and P. M. Townsley. 1974. A gas chromatographic comparison of the f a t t y a c i d s of the green c o f f e e bean, Cof f ea a r a b i c a and the sub-merged c o f f e e c e l l c u l t u r e . J. I n s t . Can. S c i . Technol. Aliment. 7:82-85. 41. White, P. R. 1954. The c u l t i v a t i o n of animal and pla n t c e l l s . Ronald Press, N.Y. 42. Yano, I., L. J . M o r r i s , B. W. N i c h o l s , and A. T. James. 1972. The b i o s y n t h e s i s of cyclopropane and c y c l o -propene f a t t y a c i d s i n higher p l a n t s (Malvaceae). L i p i d s 7:35-45. 43. Yano, I., B. W. N i c h o l s , L. J . M o r r i s , and A. T. James. 1972. The d i s t r i b u t i o n of cyclopropane and c y c l o -propene f a t t y a c i d s i n higher p l a n t s (Malvaceae). L i p i d s 7:30-34. 94 CHAPTER V S T U D I E S ON TRANSFORMED C A L L U S OF BIDENS A L B A 95 INTRODUCTION In attempts to o b t a i n c u l t u r e s of Bidens alba which would produce p o l y a c e t y l e n e s , two f u r t h e r approaches were t r i e d : the establishment'' of c u l t u r e s of crown g a l l tumours and root c u l -t u r e s . In t h i s chapter the r e s u l t s of s t u d i e s on tumours, and tumour c u l t u r e s , of B. alb a, B. p i l o s a , • :and B-. c y n a p i i f o l i a are presented. No systematic survey of the secondary products of crown g a l l tumours or of c u l t u r e s prepared from them seems to have been reported i n the more than seventy years- that crown g a l l has been i n t e n s i v e l y s t u d i e d by p l a n t p h y s i o l o g i s t s , biochemists, p a t h o l o g i s t s , and molecular b i o l o g i s t s . The f o l l o w i n g para-graphs summarize the main r e p o r t s that deal p r i m a r i l y with, secondary products i n tumours or c u l t u r e s . Kado (.31) has reviewed and t a b u l a t e d recent l i t e r a t u r e on primary and secondary- m e t a b o l i t e s of crown g a l l tumour c e l l s . Older r e f e r e n c e s may be found i n K r i k o r i a n and Steward (.38) and K l e i n (34). Kado concluded that of the many primary m e t a b o l i t e s and enzymes i n v e s t i g a t e d , most appear to d i f f e r q u a n t i t a t i v e l y r a t h e r tfran q u a l i t a t i v e l y . Often i t "appeared th.at d i f f e r e n c e s were due to higher growth r a t e s f o r tumour c e l l s than f o r nor-mal c e l l s (31). Of secondary m e t a b o l i t e s , a l k a l o i d s have been most i n v e s t i -gated. K l e i n (33V,'. r e f e r r i n g to unpublished data of Mika, s t a t e s that Datura stramonium tumours c o n t a i n almost 500% more hyosclne than roots and up to 300% more tropane a l k a l o i d than 96 the whole p l a n t . Tomatine, a g l y c o s y l a t e d s t e r o i d a l k a l o i d , i s r e p o r t e d l y found i n tomato stem g a l l s (15,35) and sporad-i c a l l y i n normal c a l l u s c u l t u r e s over two years o l d , But not i n suspension c u l t u r e s (55). Catharanthus- roseus tumour c u l t u r e s produce a l k a l o i d s i n c a l l u s and l i q u i d c u l t u r e s s i m i l a r to those found i n the p l a n t But at lower l e v e l s . Sus-pension c u l t u r e s were able to e f f e c t c o n v e r s i o n of v i n d o l i n e to other a l k a l o i d s (4). Non-protein amino acids- are found i n some i n t a c t g a l l s ; Y-hydroxyvaline occurs i n stems, l e a v e s , and tumours of Kalanchoe d a i g r emo n t i an a (50)" at l e v e l s higher than i n non-tumourous t i s s u e (39). Bean leaves i n o c u l a t e d with Agrobac t e r ium tumef aciens- s t r a i n 13333 produce Y-amino b u t y r i c a c i d which promotes tumour growth at l e v e l s as.low as 1 ug per l e a f (48). Ubiquinone i s present i n tumours of Par thenocls sus sp. at l e v e l s s i m i l a r to those of normal t i s s u e s (46), However., sus-pension c u l t u r e s of tobacco tumour c e l l s contained l e s s than h a l f as much ubiquinone, as- normal c e l l s and one - t h i r d as much as much as a normal l i n e s e l e c t e d f o r high y i e l d (28), Brown and Tenniswood (12.)] r e p o r t e d that normal tobacco callus, three weeks a f t e r s u b c u l t u r e , d i f f e r e d i n composition from crown g a l l tumour c a l l u s . The l a t t e r contained fewer of the coumarin.com-pounds found i n normal c e l l s - but at higher l e v e l s . The authors, emphasize that t r a n s f o r m a t i o n leads to marked q u a l i t a t i v e j. _ changes i n the metabolism of p h e n o l i c compounds. In recent years Japanese i n v e s t i g a t o r s have shown i n t e r e s t i n the p r o d u c t i o n of secondary products from crown g a l l , Misawa (43]L has reviewed s e v e r a l patents- f o r p r o d u c t i o n of compounds 97 from tumour c u l t u r e s , I n c l u d i n g b e r b e r i n e from Coptis j aponlca, saponins and sapogenlns from ginseng, betanin from s e v e r a l _ p l a n t s , and s t e v i o s i d e from STtev i a rebaudiana. Ginseng tumour c a l l u s contained about the same l e v e l of crude saponin as normal c a l l u s . M a t r i c a r i a chamomllla appears to be the only Composite f o r which data on secondary products i n normal and transformed t i s s u e s i s a v a i l a b l e . R e i c h l i n g et a l . (53 J determined essen-t i a l o i l s , f l a v o n o i d s , coumarins-, and p h e n o l i c a c i d s i n f l o w e r s , herb, r o o t , and tumours of t h i s p l a n t . They found that stem tumours c o n t a i n approximately 1/10 the l e v e l of e s s e n t i a l o i l s of flowers and about the same amount as herb and root t i s s u e . Among, ; the compounds i d e n t i f i e d were two a c e t y l e n i c compounds, c i s - and t r a n s - e n - y n - d l c y c l o e t h e r s , which, were present i n a l l p a r t s of the p l a n t and i n both, n o p a l i n e and octopine type j tumours. Flavonoids and two coumarins C h e r n i a r i n and u m b e l l i f -eroneN) were present i n shoots and flowers but not i n roots or tumours. The occurrence~of i n u l i n i n Jerusalem a r t i c h o k e was i n v e s t i g a t e d by Kaneko (32) and found not to he present i n either normal or tumour c a l l u s . In summary, there i s o n l y one r e p o r t i n which a p o l y -a c e t y l e n e has been detected i n transformed c e l l s and a compara-t i v e l y few reports- of other secondary products. The l i t e r a -ture on the p h y s i o l o g y and b i o c h e m i s t r y of crown g a l l i s exten-s i v e but i t i s c l e a r that there i s no way to p r e d i c t what .. ; e f f e c t t r a n s f o r m a t i o n wllllhave; onmost aspects of c e l l metabol-. ism, i n c l u d i n g secondary products. Teuscher, however, has suggested (60) that s i n c e crown g a l l tumours c o n t a i n secon-93 dary products, they might r e t a i n t h i s c h a r a c t e r i s t i c i n c u l -t u r e ; p a r a l l e l i n g tumour bus-.- endocrine c e l l s of animals, which can produce hormones while c u l t u r e d on a r t i f i c i a l s u b s t r a t e s . Evidence to date n e i t h e r confirms nor d i s c o n f i r m s t h i s conjec-ture although the evidence presented here shows that i t a p p l i e s to p o l y a c e t y l e n e s i n two species of Bidens. The o b j e c t i v e of the work, d e s c r i b e d i n t h i s chapter was to determine i f tumour cultures- might be u s e f u l i n p o l y a c e t y l -ene r e s e a r c h , and whether p o l y a c e t y l e n e e x p r e s s i o n was, d i f f e r -ent i n transformed c e l l s . I n i t i a l work attempted to i n c r e a s e l e v e l s of product i n transformed c u l t u r e s . 99 MATERIALS AND METHODS B a c t e r i a l and P l a n t Lines O r i g i n s and c u l t u r e /methods f o r Bidens alba v a r . r a d i a t a and Agrobacterium tumef aclens s t r a i n s are given i n Chapter 3. Also found there are the methods f o r transforming p l a n t t i s s u e and c u l t u r i n g both t i s s u e s and b a c t e r i a . Seeds of Bidens p i l o s a var. minor (Blume) Sh e r f f were obtained from B e l i z e C i t y , B e l i z e through Dr. Thor Arnason. Seeds of Bid ens c y n a p j i f o l j a H. B. K. were obtained from Jamaica, W, T, through, the Chem-i s t r y Department of the U n i v e r s i t y of West I n d i e s . C u l t u r e of Teratomas A l l c u l t u r e s were grown i n 15 mm x 9.0. mm p l a s t i c p e t r i . ". plates- on SH medium with. 7 g/1 agar, 3% sucrose, pH 5,7, and no hormones except as noted. I l l u m i n a t i o n was from L i f e l i n e fluorescent; bulbs CSylvanla). with, illuminance: of 6,00.0 lux . Teratogenic c a l l u s from tumours of" A. tumef aclens s t r a i n A2Q8 spontaneously d i f f e r e n t i a t e d shoots and then formed r o o t s i n c a l l u s at the base of the shoots. If organized t i s s u e was r e p e a t e d l y e x c i s e d from c a l l u s and the c a l l u s c u l t u r e d , then d i f f e r e n t i a t i o n eventually- stopped o c c u r r i n g . This: happened whether the t i s s u e was grown i n l i g h t or dark. I f , however ? d i f f e r e n t i a t e d shoot t i s s u e s were l e f t attached to the l i g h t -grown c a l l u s then more shoots formed. The c l u s t e r of shoots could be d i v i d e d and grown s e p a r a t e l y . T h i s process was. ;',.>-repeated f o r at l e a s t eighteen s u b c u l t u r e s . C u l t u r e s were subculttired every- three weeks- or harvested, The whole process. 100 i s s i m i l a r to the technique of propagation By means of pre-cocious a x i l l a r y Branches as- descriBed By K r i k o r i a n (37). Some d i v i s i o n s d i d not root and these non-rooting d i v i s i o n s were suBdivided r e p e a t e d l y to oB t a i n a l i n e which had a continuous h i s t o r y of suppressed r o o t s . Some'teratomas were grown i n l i q u i d c u l t u r e i n 125 ml Erlenmever f l a s k s with 75 ml of medium and IncuBated i n dark or l i g h t . These c u l t u r e s were suBcultured once Before h a r v e s t i n g . Transformation of T i s s u e S l i c e s In an attempt to i n c r e a s e the y i e l d of explanted tumours, an a l t e r n a t i v e method of i n o c u l a t i o n to/ that des-criB~ed i n Chap-te r ITT was t r i e d . Stem internode segments- of B. a 1 Ba p l a n t s approximately 4 cm long were s u r f a c e s t e r i l i z e d with. 70% ethanol f o r one" minute followed By ten -minutes i n a-10% commercial Bleach s o l u t i o n f a v e x ) and -rinsed 4X with s t e r i l e d i s t i l l e d water. The end 5 mm were trimmed o f f and tfLe remaining stem cut i n t o 5 mm s l i c e s - and placed on s-olidlfi;ed 1% agar i n pe.tri.„ p l a t e s . S l i c e s ' were incuBate.d overnight i n dark at 25 C and i n o c u l a t e d the next day with; enough. 48—hour B a c t e r i a l suspen^ s i o n to cover the s-urface of each, segment. A f t e r two week.Sj s m a l l outgrowths- 1-2 mm i n diameter were explanted to a n t i b i o t i c medium, Only one of these explants s u r v i v e d to y i e l d c a l l u s , the l i n e designated F277 IV, This- l i n e produced octopine and grew -rapidly as l o o s e , f r i a b l e c a l l u s . A s i m i l a r method of t r a n s f o r m a t i o n has- Been descriBed By Braun C9--I and mo-re r e c e n t l y By Barton and C h i l t o n ( 2 ) . 101 RESULTS AND DISCUSSION P h y s i c a l C h a r a c t e r i s t i c s of C a l l u s T i s s u e C a l l u s d e r i v e d from both types of p l a n t tumours d i f f e r e d from normal c a l l u s i n s e v e r a l c h a r a c t e r i s t i c s : 1) transformed c a l l u s was harder and denser than normal c a l l u s , appearing to have more l i g n i f i e d t i s s u e . Clumps of transformed c a l l u s had to be cut with a s c a l p e l f o r s u b c u l t u r i n g while normal c a l l u s could be s l i c e d e a s i l y with t h i n f o r c e p s . 2) The r a t i o of wet to dry weight f o r tumour c u l t u r e s was always lower than f o r normal t i s s u e , the former was t y p i c a l l y about 12 and the l a t t e r about 18. 3) Growth of normal t i s s u e was g e n e r a l l y f a s t e r than transformed t i s s u e . Doubling time f o r dense transformed t i s s u e was t y p i c a l l y 30 days but only 11 days, f o r normal t i s s u e . 4) Tumour c a l l u s contained s c a t t e r e d c a v i t i e s c o n t a i n i n g anJ amber o i l resembling the m a t e r i a l found i n r e s i n canals of i n t a c t p l a n t organs. These were almost c e r t a i n l y accumulations of a c e t y l e n i c compounds. S i m i l a r s t r u c t u r e s were not found i n normal t i s s u e . An exception ,to t h i s p a t t e r n was- tumour l i n e F277 IV,' which was d e r i v e d from a tumour induced on a stem s e c t i o n ; t h i s l i n e was s i m i l a r to normal t i s s u e i n a l l the p o i n t s above except that i t produced p o l y a c e t y l e n e s . The b a s i s f o r these d i f f e r e n c e s i s not c l e a r , although i t may r e f l e c t d i f f e r e n c e s i n l e v e l s and r a t i o s of hormones which become e s t a b l i s h e d i n c a l l u s (see l a t e r d i s c u s s i o n ) . 102 Pigmentation and Product L e v e l The p o l y a c e t y l e n e s i n v e s t i g a t e d i n t h i s study are c o l o u r -l e s s compounds, but t h e i r d e t e c t i o n i s r e l a t i v e l y easy, i n v o l v -ing a simple e x t r a c t i o n and UV spectrophotometry of the e x t r a c t . However, t h i s method r e q u i r e s from 10 mg to 1 g of m a t e r i a l and the number of such t e s t s that could be run set a severe l i m i t on the d e t e c t i o n of l i n e s with high ..yield. A g r o u p - s p e c i f i c TLC spray reagent f o r most ac e t y l e n e s i s known (44) but i t i s not s e n s i t i v e enough f o r small groups of c e l l s squashed d i r e c t l y onto a TLC p l a t e and developed, as has been s u c c e s s f u l with n i c o t i n e s e l e c t i o n i n tobacco (45). T h e r e f o r e , some other method was necessary i f s e l e c t i o n of i n f r e q u e n t high y i e l d i n g c u l t u r e s was to be p r a c t i c a l . Soon a f t e r a number of transformed (oct-) c a l l u s l i n e s had been e s t a b l i s h e d v a r i a t i o n i n c o l o u r of the c a l l u s e s was apparent. Some had dark brown spots i n v a r y i n g amounts while others were n e a r l y white. Prelminary work suggested a c o r r e l a t i o n between amount of c o l o u r and acetylene content. An experiment was set up to t e s t t h i s i d e a . F i f t e e n d i f f e r e n t c a l l u s e s from one l i n e were graded i n t o f i v e groups on the b a s i s of the amount of pigment present. The average product l e v e l s f o r the groups are shown i n F i g . 21. The c o r r e l a t i o n c o e f f i c i e n t f o r t h i s data was 0.72 and the c r i t i c a l value (P=0.01) was 0.68. The n u l l h ypothesis was r e j e c t e d (P<0.01). Composition (not shown) changed i n a r e g u l a r way a l s o ; c l a s s 1 showed a hig h e r p r o p o r t i o n of PDE-OAc (88%) than c l a s s 5 (75%) and the other c l a s s e s had in t e r m e d i a t e p e r c e n t -ages. The bulk of the remaining product was ETE-OAc. 103 Ii 1 I I L 1 2 3 4 5 COLOUR CLASS F i g . 21 • T o t a l p o l y a c e t y l e n e s f o r t r a n s f o r m e d c a l l u s e s g r a d e d by amount of p i g m e n t a t i o n . C l a s s 1 w h i t e , c l a s s 5 h e a v i l y m o t t l e d . B a r s show - 1 SD. 104 The c o r r e l a t i o n of pigmentation with product l e v e l sug-gested that continued s e l e c t i o n of l i n e s might lead to i n c r e a s e d l e v e l s over time. A c c o r d i n g l y , the l i n e s from the previous experiment were separated i n t o a high group and a low group based on t h e i r c l a s s f i c a t i o n by colour (not on t h e i r product l e v e l ) , with a t o t a l of f o u r t e e n i n each category. At each s u b c u l t u r e the most h i g h l y pigmented c a l l u s was t r a n s f e r r e d i n the high l i n e s and the whitest c a l l u s i n the low l i n e s . At e i g h t months only the hi g h e s t of the pigmented l i n e s and the lowest of the white l i n e s were r e t a i n e d (four each.) and colour s e l e c t i o n continued. Fig.22 shows the r e s u l t s a f t e r n e a r l y 18 months of s e l e c t i o n . The i n i t i a l l i n e was- d e r i v e d from an explant o r i g i n a l l y s t a r t e d i n c u l t u r e 10 months e a r l i e r . There was l i t t l e d i f f e r e n c e over the f i r s t p e r i o d of s e l e c t i o n between the two groups, indeed at one p o i n t the white l i n e exceeded the dark l i n e by a small margin. I t appeared that s e l e c t i o n on the b a s i s of pigmentation had l i t t l e e f f e c t i n d i f f e r e n t i a t -ing the l i n e s over time, or even m a i n t a i n i n g an i n i t i a l d i f f -erence. The t o t a l l e v e l , however, i n c r e a s e d a p p r e c i a b l y f o r both s e r i e s during t h i s i n t e r v a l , A f t e r the s-eidies were s e l e c -ted on the basis- of product l e v e l at eig h t months 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 two groups- began to appear. The p o i n t s at 10% months represent the l e v e l s a f t e r s e l e c t i o n ; i n e f f e c t the s t a r t i n g points- f o r the next s e l e c t i o n s e r i e s . Over the next 10 months the low l i n e d e c l i n e d n e a r l y 5Q% while the high l i n e i n c r e a s e d by 43%. The sharp change i n r e l a t i v e , l e v e l s i s shown i n the decreasing r a t i o <of low to high l i n e s i n F i g , 22. rJ,',. • 105 . . .8 -t I I 1 I I I l I i i I 2 4 6 8 10 12 14 1 6 18 TIME (MONTHS) F i g , 2 2 . T o t a l p o l y a c e t y l e n e s i n h i g h and l o w p i g m e n t a t i o n c a l l u s g r o u p s o v e r t i m e . R a t i o o f l o w l i n e s t o h i g h l i n e s i s i n p a r e n t h e s e s . A r r o w h e a d i n d i c a t e s r e s e l e c t i o n o f g r o u p s by l e v e l o f p r o d u c t . 106 The l a t e r d i v e r g e n t trends: may i n d i c a t e an e f f e c t of s e l e c t i o n on the reduced set of l i n e s , but the absence of a c o n t r o l s e r i e s without s e l e c t i o n makes t h i s u n c e r t a i n . The pigmenta-t i o n , once s e l e c t e d , was s t a b l e f o r the three::.', years the l i n e s were maintained. Segregation of tumour c a l l u s i n t o v i s u a l l y d i f f e r e n t types has been r e p o r t e d by Scott ( 56 ) , Amasi.no and M i l l e r CL)~» and Meins CA2). The l a s t two studies- were able to f i n d n u t r i e n t and hormone conditions- which, could induce formation of the different tumour types-. Differences- i n secondary products were not examined. Other s t u d i e s Have repo r t e d c o r r e l a t i o n s between c a l l u s c o l o u r and the presence of c o l o u r l e s s products ( " 8 , 26 ) . In some cases r e l a t e d compounds were coloured and provided a s e l e c t a b l e c h a r a c t e r . Tt i s poss-ible: that the pigment i n E, alba c a l l u s represented polymerized p o l y a c e t y l e n e s , a common r e a c t i o n of these compounds when exposed to l i g h t or to d r y i n g out i n a i r , but a separate a n a l y s i s : of the pigment was not attempted. The connection between pigment and product l e y e l remains u n c l e a r . F i g u r e 23. shows the combined p o l y a c e t y l e n e composition data :"for the two s e l e c t e d l i n e s , I n c l u d i n g g a l l t i s s u e and the c a l l u s p r i o r to s e l e c t i o n . Composition showed a d i s t i n c t downward trend i n . PDE-OAc percentage over time. Long-term changes i n product r a t i o s have been reported f o r the.baine and p r o t o p i n e type a l k a l o i d s i n Papayer braeteaturn c u l t u r e s (61) but the reason f o r such slow changes is- not c l e a r . On the b a s i s of the r e s u l t s from work with, roots reported, l a t e r , the i n d i c a t i o n would be that levels- of c y t o k i n l n s were d e c l i n i n g 107 i o o r 8 10 12 14 16 TIME MONTHS 18 20 22 24 26 28 30 V 42 F i g . 23 . Percentage change i n phenyldiynene acetate over long-term c u l t u r e of transformed c a l l u s . over time i n JB. alba c a l l u s . Changes i n composition and product l e v e l f o r c a l l u s of octopine l i n e F277 IV, which grew r a p i d l y , were followed at three-day i n t e r v a l s over the course of a 27 day c u l t u r e p e r i o d . No c o n s i s t e n t p a t t e r n was found f o r composition; the PDE-OAc percentage f l u c t u a t e d between 45% and 60% with a mean at 52%. P o l y a c e t y l e n e l e v e l v a r i e d l i t t l e from 0.04 mg/g dry wt. and t h i s l i n e showed no tendency to accumulate product during any phase of the c u l t u r e c y c l e . 108 S t a b i l i t y of P o l y a c e t y l e n e P r o d u c t i o n over Time Several d i f f e r e n t l i n e s of transformed c a l l u s were c u l -tured and harvested f o r a n a l y s i s at v a r i o u s times a f t e r explant-i n g . F i g u r e 24 shows tfr.es-e r e s u l t s . A l l of the l i n e s except that d e r i v e d from B. p11osa showed i n s t a b i l i t y of l e v e l over long-term c u l t u r e . Levels of t o t a l product ranged up to 20% of the l e v e l found i n c u l t u r e d r o o t s but the l e v e l to which the c u l t u r e s seemed to be s t a b i l i z i n g was- less- than 10% of that value. (Table 10 shows tfre values f o r c u l t u r e d r o o t s ) . Wide f l u c t u a t i o n s - i n secondary compound p r o d u c t i o n from t i s s u e c u l t u e s are w e l l known, although s t a b l e l i n e s producing v a r i o u s product have been rep o r t e d (e.g. 3,41,661, The de t e r -minants of s t a b i l i t y - are not known, Tumour c e l l s might be expected to be at l e a s t i n i t i a l l y - u nstable f o r reasons r e l a t e d to t h e i r o r i g i n ; T - D N A , which, maintains the transformed s t a t e , can undergo a l t e r a t i o n s res-ulting i n d e l e t i o n s of n e a r l y a l l or p o r t i o n s of the i n s e r t , l e a d i n g to changes i n expre s s i o n of those T—DNA genes which; a f f e c t c e l l f u n c t i o n s . Evidence that this: occurs i n E , a l b a has been presented i n Chapter 111, Extended c u l t u r e of the transformed lines- might r e s u l t i n more s t a b l e p r o d u c t i o n but more than twenty-five s u b c u l t u r e s have not e f f e c t e d such a change i n the. lines- s-tudl.ed here, However, there i s no evidence that T—DNA a l t e r a t i o n s account f o r the long—term i n s t a b i l i t y . It seems- more, l i k e l y that changes i n the T—DNA would produce a v a r i e t y of d i f f e r e n t c a l l u s types e a r l y i n c u l t u r e and .these would be r e l a t i v e l y s t a b l e to f u r -ther changes-. 1 0 9 1 I I 1 I I I I I I [ l I 1 ' 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 T I M E ( M O N T H S ) F i g . 24. T o t a l p o l y a c e t y l e n e s o v e r t i m e o f d i f f e r e n t c a l l u s l i n e s o f t r a n s f o r m e d B. a l b a and 13. p i l o s a . F208 l i n e s a r e A208 (nop+) t y p e B. a l b a , F277 an A277 ( o c t + ) l i n e , and B208 an A208 l i n e o f J3. p i l o s a . E a c h p o i n t i s t h e v a l u e o b t a i n e d f o r t h e c o m b i n e d c a l l u s f r o m two o r more p l a t e s . 110 D e t a i l e d A n a l y s i s of P o l y a c e t y l e n e s i n Transformed and Normal  Tissue Shown i n Table 7 i s a breakdown of the composition and product l e v e l s f o r the two species of Bidens c u l t u r e d . Results f o r both normal as w e l l as s i n g l y and doubly t r a n s -formed c a l l u s are shown. The data show the unique p a t t e r n of p o l y a c e t y l e n e b i o s y n t h e s i s found i n tumour c e l l s . There i s a c o n s i s t e n t p a t t e r n of r a t i o s and of the p r o d u c t i o n of compounds found only at d e t e c t a b l e l e v e l s i n tumour c e l l s . In s i t u tumours of 13. alba d i f f e r from the surrounding t i s s u e i n having low l e v e l s of PHT and high l e v e l s of PDE-OAc i n s t e a d of PDE. Octopine and nopaline g a l l s were q u a l i t a t i v e l y s i m i l a r but d i f f e r e d markedly with respect to the p r o p o r t i o n of PDE. Growth of tumours as c a l l u s , away from the i n f l u e n c e of the host t i s s u e , r e s u l t e d i n a d i f f e r e n t p a t t e r n of com-pounds. The most i n t e r e s t i n g change was the occurrence of s i g -n i f i c a n t amounts of the ETE aldehyde (ETE-al) i n a l l tumour c a l l u s e s . This compound was not detected i n normal t i s s u e or i n g a l l s . It i s i n t e r e s t i n g that t h i s compound was found by Bohlmann (as noted i n Chapter II) i n the m a t e r i a l he worked with but was not present in B. a 1 b a except under these c o n d i t i o n s . Small percentages of the p u t a t i v e d i e n t r i y n e n e a l c o h o l (DTE-ol) were found consistently in a l l three types of cultured tumour c a l l u s . The t r i e n d i y n e n e a l c o h o l (TDE-ol) was a l s o detected only i n tumour c a l l u s but at l e v e l s too low to estimate. A l l the c a l l u s types showed convergence to a s i m i l a r range of com-pounds and p r o p o r t i o n s suggesting that the d i f f e r e n c e s between g a l l types were a f u n c t i o n of the p l a n t environment. The T a b l e 7 . C o m p a r i s o n of p o l y a c e t y l e n e l e v e l and c o m p o s i t i o n of n o r m a l p l a n t o r g a n s , t r a n s f o r m e d c u l t u r e s , and g a l l s o f 13. a l b a and 13. p i l o s a . V a l u e s a r e t h e a v e r a g e of a t l e a s t f i v e d i f f e r -e n t s a m p l e s . C a l l u s v a l u e s were a v e r a g e d o v e r one y e a r . T i s s u e / T o t a l P o l y a c e t y l e n e C o m p o s i t i o n (wt % o f t o t a l ) S p e c i e s p r o d u c t (mg/g d.w.) PHT PDE-OAc PDE ETE-o l 3 E T E -OAc ETE-A l d ETE EDE-OAc DTE o l E. a l b a L e a f k . 6 98.3 - 1.7 - - - - - -Stem 2 . 3 54. 2 2 . 2 43.5 - t - - - -Root 8.7 5 . 7 58 . 3 2 . 9 0 . 4 24 . 2 - 0.8 7 . 7 t + Oct stem g a l l 0.24 7 . 4 67 . 7 8.9 3.4 4.4 - - 6 . 3 1.9 Nop' s tem 3 a11 0.41 7 . 1 5 5.8 31 . 3 0 . 5 5 . 3 - - t O c t + c a l l u s 0. 30 - 55 . 7 - 16 . 5 10.0 5 . 2 t 9 . 1 3.5 N o p + c a l l u s 0 . 25 - 48.1 - 5.8 15 . 2 14 . 9 t 11. 2 6.8 ' O c t + - n o p + c a l l u s 0.26 - 51.3 - 7.4 14. 7 15 . 3 t 6 . 2 6 . 0 N o r m a l c a l l u s B. p i l o s a Leaf Stem Root Nop + c a l l u s Normal c a l l u s 0.1 3 . 5 0.08 - 43.2 - 50. 5 - 77.8 56.7 0.4 40.1 4.3 11.8 t 0.4 8 . 6 6.1 Includes ETE-OAc (A) and (B). Double transformant. Symbols, a b r e v i a t i o n s : not d e t e c t a b l e ; t, t r a c e ; d.w., dry weight. 113 absence of PDE i n c u l t u r e s of nopaline g a l l s was the most s t r i k i n g change although PHT was a l s o absent from s u b c u l t u r e d c a l l u s . C a l l u s c u l t u r e s c h a r a c t e r i s t i c a l l y had higher per-centages of PDE-OAc, compared to ETE-OAc, than any of the plant organs. No ac e t y l e n e s were detected i n normal c a l l u s of e i t h e r s p e c i e s . Bidens p i l o s a had a simpler, more r e s t r i c t e d range of compounds than 15. a l b a ; PHT and PDE were both absent from p l a n t s and c u l t u r e s . Nopaline t i s s u e was g e n e r a l l y s i m i l a r to r o o t s , however EDE-OAc was absent from transformed c a l l u s and ETE-al was found only i n c a l l u s . As i n the other tumour c u l t u r e s , the r a t i o of PDE-OAc to ETE-OAc was l a r g e r than i n pl a n t organs. Two f e a t u r e s of Table 7 are no t a b l e : 1) the appearance i n tumour c u l t u r e s of compounds not found i n e i t h e r normal pl a n t m a t e r i a l or i n g a l l s . 2) The absence of compounds from c u l t u r e d tumour t i s s u e which were found i n the g a l l s . The accumulation of metabolic i n t e r m e d i a t e s or the occurrence of new compounds i n p l a n t t i s s u e c u l t u r e s i s not unusual according to Staba (58) and there are many r e p o r t s d e s c r i b i n g such r e -s u l t s (e.g.7,20,21,59), i n c l u d i n g one f o r p o l y a c e t y l e n e s i n Centaurea r u t h e n i c a (29) by Jente. Although the compounds found by Jente were present i n very low l e v e l s (approximately 0.1% of the l e v e l s found i n her p l a n t s ) there seemed to be a completely d i f f e r e n t set of compounds i n c a l l u s as compared to the p l a n t s . The compounds were shown to be b i o g e n e t i c a l l y r e l a t e d , however. It i s i n t e r e s t i n g that Jente's c u l t u r e s only produced a c e t y l e n e s under l i g h t , or a l t e r n a t i n g l i g h t and dark, 114 b u t n o t i n d a r k . 13. a l b a c u l t u r e s showed a s i m i l a r p a t t e r n t o t h a t of r o o t s d e s c r i b e d l a t e r ; i l l u m i n a t i o n d e c r e a s e d t h e l e v e l o f a c e t y l e n e s by a t l e a s t 60% ( r e s u l t s n o t s h o w n ) . The q u e s t i o n of what f a c t o r s m i g h t a c c o u n t f o r n o v e l compounds has b een d i s c u s s e d by Bbhm (7) who s u g g e s t s t h a t t h e c h a n g e d l o c a l i z a t i o n o f s u b s t r a t e and enzyme i s i m p o r t a n t and a l s o t h e f o r m a t i o n o f new compounds f r o m a c c u m u l a t e d i n t e r -m e d i a t e s . B u ' L o c k (13) s t a t e s t h a t s e c o n d a r y p r o d u c t enzymes g e n e r a l l y have lo w s p e c i f i c i t i e s and m o d i f y a v a r i e t y of s u b -s t r a t e s ; t h e f i n a l p r o d u c t p r o d u c e d d e p e n d i n g on t h e t y p e of s u b s t r a t e a v a i l a b l e . O t h e r i m p o r t a n t f a c t o r s , a c c o r d i n g t o B u ' L o c k , a r e t h e p h y s i o l o g i c a l h i s t o r y and c o n d i t i o n s o f c u l t u r e . W h i c h of t h e s e f a c t o r s i s r e l e v a n t t o tumour c a l l u s i s n o t c l e a r ; c e r t a i n l y t h e p h y s i o l o g i c a l and n u t r i t i o n a l s t a t u s o f t h e c a l l u s must be d i f f e r e n t . One f a c t o r w o u l d be t h e c h a n g e d h o r m o n a l l e v e l o f tumour t i s s u e as compared t o n o r m a l t i s s u e . Work d e s c r i b e d l a t e r w i t h e x o g e n o u s l y s u p p l i e d hormones s u g g e s t s t h a t a t l e a s t p a r t o f t h e d i f f e r e n c e , n a m e l y t h e r e l a t i v e p r o p o r t i o n s of PDE-OAc and ETE-OAc, c a n be a c c o u n -t e d f o r by i n c r e a s e d c y t o k i n i n l e v e l s i n c a l l u s . T h e r e I s , h o w e v e r , no d a t a on t h e a c t u a l hormone l e v e l s i n B i d e n s c a l l u s t o c o n f i r m t h i s s u g g e s t i o n . The p o s s i b i l i t y t h a t i n t e r m e d i a t e s m i g h t a c c u m u l a t e c a n be e v a l u a t e d i f t h e b i o s y n t h e t i c p a t h w a y s f o r t h e compounds a r e known. A b i o s y n t h e t i c scheme f o r t h e p o l y a c e t y l e n e s w h i c h o c c u r i n B. a l b a i s shown i n F i g . 25. ' T h i s i n f o r m a t i o n was c o m p i l e d f r o m Bohlmann e t a l . ( 5 ) . J e n t e and R i c h t e r (30) have i s o l a t e d l a b e l l e d PHT ( V I I I ) f r o m C o r e o p s i s l a n c e o l a t a f e d w i t h 115 X V I I I X I X XX X X I X X I I X X I I I X X I V XXV X X V I X X V I I I C H 3 < C H 2 ) 7 C H = C H ( C H 2 ) 7 C 0 2 R | - IH] C H 3 (CH 2 ) CH=CH C H 2 C H = C H ( C H ^ C C ^ R \ -IH] CH 3 (CH 2 ) ( ,C=CCH 2 CH=CH ( C H j L C O ^ ) -IH] CH 3 (CH 2 ) 2 CH=C HC=C CH 2 CH=C H (C H 2 ) ? C 0 2 R ( -IH] C H 3(C H 2 ) 2 (CSCL, C H 2 C H=C H C H 2 ) ? C 0 ^ \ -IH] -CH 3 CH=CH ( C = C ) C H 2 C H = C H ( C H 2 ) ? C 0 2 R I 2 x P-oxicfation C H 3 C H = C H ( C = C ) 2 C H 2 C H = C H ( C H 2 ) A O R | 10], H 2 0 CH 3 CH=CH(C=C) 2 (CH=CH) 2 (CH 2 ) 2 CH 2 OH -IQL_^N \ [ 0 ] , H 2 0 . C 0 2 C H 3 C H = C H ( C = C ) 3 C H = C H ( C H 2 ) CHjOH | -IH] C H 3 C H = C H ( C = C ) 4 ( C H 2 ) 2 C H 2 0 H ) 10], H ^ . C 0 2 C H 3 C H = C H ( C 5 C ) A C H = C H 2 •IH] X X I X C H 3 ( C = C ) 3 C H 2 C H = C H ( C H ^ C O j R \ 2 x 0-oxidation XXX C H 3 ( C S C ) 3 C H 2 C H = C H ( C H 2 ) 3 C 0 2 R | 10] xxxi {c^icsayfl^ OH ,[H 20],-I c 02l ' X V I IH20],-[C021 1 X I 10] X X X I I I R C H = C H ( C = R 0 2 C ^ j ^ X X X I I X X X I V R C H ^ H I C ^ O J R 0 2 C OH c H 3 ( c = c r 3 i P 5 l R 0 2 C ^ T OH V I I I C H 3 ( C = -iHjCfclCOpJ - t H 2 0 ] , - K 0 2 ] [ RCH=CH(C=C ) 2T|^J V, V a , V I R = C H 3 , C H 0 , C H 2 0 H , V I I C H 2 O A C X V I CH3CH=CH(C=C ) 2 ( CH=CH)2CH=CH 2 X I C H 3 C H = C H ( C = C ) 3 C H = C H - C H = C H 2 F i g . 25. B i o s y n t h e t i c p a t h w a y s l e a d i n g t o p o l y a c e t y l e n e s o c c u r r i n g i n B i d e n s a l b a . S o u r c e : B o h l m a n n , B u r k h a r d t , and Z d e r o : N a t u r a l l y o c c u r r i n g a c e t y l e n e s , 1973. 116 l a b e l l e d crepenynic a c i d (XX) and o l e i c a c i d (XVIII), confirm-ing t h i s part of the pathway. Assuming the e s s e n t i a l c o r r e c t -ness of F i g . 25, i t i s clear- that n e a r l y a l l of the compounds found i n the s t u d i e s with J 3 . alba were t e r m i n a l products which only undergo m o d i f i c a t i o n at the t e r m i n a l methyl group; oxida-t i o n to the a l c o h o l or aldehyde or a c e t y l a t i o n . An a l t e r n a -t i v e b i o s y n t h e t i c route to the aromatic a c e t y l e n e s has been suggested by Sorensen ( r e f . 9, Chap. I) i n which compound (XI) i s the p r e c u r s o r f o r c y c l i z a t i o n . Other than t h i s suggestion^ there i s no known or p o s t u l a t e d i n t e r c o n v e r s i o n s of any of these compounds except the compound which was t e n t a t i v e l y d e s i nated EDE-OAc. A l l of the a c e t y l e n e s p o s i t i v e l y i d e n t i f i e d were C^^ compounds or d e r i v a t i v e s , thus when a compound was i s o l a t e d which had the endiynene chromophore spectrum, i t was assumed that i t would have a chain l e n g t h , before any t e r m i n a l m o d i f i c a t i o n s . In f a c t , however, although a C - ^ Q acet ylene with t h i s chromophore i s w e l l known as m a t r i c a r i a e s t e r , I have been unable to f i n d a record of a C ^ a c e t y l e n e with t h i s s t r u c t u r e . A p o s s i b i l i t y i s that the compound i s an i n t e r m e d i a t e between compounds XXIV and XXV. If t h i s scheme i s c o r r e c t then EDE-OAc could be the C ^ p r e c u r s o r f o r ( I ) , (XVI), and (XI). The low l e v e l s found i n t i s s u e s and the apparent i n s t a b i l i t y to GC/MS a n a l y s i s d i d not allow f u r t h e r c h a r a c t e r i z a t i o n of the compound. The presence of PHT and PDE i n stems and stem g a l l s , i n c l u d i n g the b a s a l c a l l u s of p l a n t l e t s , suggests their synthe-s i s i s not t i g h t l y l i n k e d to a s t r u c t u r e or s i g n a l that only occurs i n one type of d i f f e r e n t i a t e d organ; as, f o r example ) 117 seems to be the case with many seed storage l i p i d s . The r e -port by Bohlmann et a l . (5) that PHT can be found i n the roots of s e v e r a l s p e c i e s of Coreopsis (a genus c l o s e l y r e l a t e d to Bidens) and one other species of Bidens , Bidens cernua, sup-ports t h i s view. However, e x t r a c t s of roots of Coreopsis  v e r t i c i l l a t a and 13. cernua growing l o c a l l y showed no i n d i c a -t i o n of PHT, although both are reported to c o n t a i n i t (5). V a r i a b i l i t y i n e x p r e s s i o n of aromatic acetylenes s y n t h e s i z e d by a d i f f e r e n t scheme from that shown i n F i g . 25 has been reported f o r Ar t eme s i a c a p i l l a r i s . P l a n t s growing by the seaside i n Japan c o n t a i n c a p i l l e n and c a p p i l l i n only i n the r o o t s , but p l a n t s growing along r i v e r b a n k s c o n t a i n both com-pounds i n roots and leaves (65). The l a c k of PHT and PDE i n the c u l t u r e d tumour t i s s u e suggests that the f a c t o r s common to stems and leaves are not maintained i n c u l t u r e , although they apparently are t r a n s m i t t e d from stems to the b a s a l c a l l u s of unrooted p l a n t l e t s , as shown i n the experiment d e s c r i b e d on p. 118. Thus the problem of e x p r e s s i o n of these compounds seems not to be s t r o n g l y l i n k e d to morphological d i f f e r e n t i a t i o n . However, i t is d i f f i c u l t to imagine that r e l a t e d p l a n t s would have such d i f f e r e n t hormone d i s t r i b u t i o n s that the root of one would be s i m i l a r to the l e a f and stems of another. The i m p l i -c a t i o n , then, seems to be that a combination of f a c t o r s , or a balance between hormones, environmental, and n u t r i t i o n a l con-d i t i o n s i s r e s p o n s i b l e . E f f e c t of t r a n s f o r m a t i o n on Bidens c y n a p i i f o l i a To t e s t f o r the p o s s i b i l i t y that i n f e c t i o n by A. tume-f a c i e n s might induce p o l y a c e t y l e n e s y n t h e s i s i n normally 118 non-producing t i s s u e s , Bidens c y n a p i i f o l i a p l a n t s were inocu-l a t e d with A208 (nop +) b a c t e r i a . Large g a l l s formed on the stems three months a f t e r i n f e c t i o n and these were devoid of UV-detectable a c e t y l e n e s , as were the stems and leaves of the p l a n t . Although i t i s very u n l i k e l y that genes c a r r i e d by plasmid T-DNA would s p e c i f i c a l l y cause s y n t h e s i s of p o l y a c e t -y l e n e s , as f o r opines, nevertheless t h i s simple experiment could t e s t f o r t h i s p o s s i b i l i t y . The negative r e s u l t supports the i n d i r e c t involvement of t r a n s f o r m a t i o n i n a c e t y l e n e s y n t h e s i s i n tumour t i s s u e . Transformed P l a n t l e t s The a v a i l a b i l i t y of r o o t i n g and non-rooting p l a n t l e t s on the same medium made i t p o s s i b l e to o b t a i n i n f o r m a t i o n on whether roots were a f a c t o r i n p o l y a c e t y l e n e s y n t h e s i s i n l e a v e s , e s p e c i a l l y PHT formation. P l a n t l e t s were di-minutlve but well-formed with small leaves 5-10 mm long and 2-3 mm wide. Several shoots were e a s i l y grown i n a standard p e t r i p l a t e . Table 8 shows the r e s u l t s of a number of sample analyses. Each sample was composed of the leaves from s e v e r a l p l a n t l e t s . Formation of PHT continued i n unrooted p l a n t s , although the composition was somewhat d i f f e r e n t from that of normal l e a v e s ; there was a c h a r a c t e r i s t i c i n c r e a s e i n l e v e l of the PHT a l c o h o l . Unrooted p l a n t s were perhaps most s i m i l a r to seed i n compo-s i t i o n , except that the p r o p o r t i o n s of PHT-ol and PHT-OAc were reversed ( c f . Table 2). Leaves of rooted p l a n t s were most l i k e normal l e a v e s . Rooted p l a n t s had a p p r e c i a b l y more PHT than unrooted p l a n t s suggesting that roots may have pro-119 T a b l e 8. C o m p a r i s o n o f p o l y a c e t y l e n e s i n l e a v e s o f r o o t e d and u n r o o t e d t r a n s f o r m e d p l a n t l e t s . C o e f f i c i e n t o f v a r i a t i o n i n p a r e n t h e s e s . P o l y a c e t y l e n e c o m p o s i t i o n (wt %) a . T o t a l N PHT PHT-OAc P H T - o l PDE p r o d u c t (mg/g d.w. )  N o n - r o o t i n g 78.1 3.9 14.7 3.0 7.8 (3.7) 6 R o o t i n g 91.0 1.0 5.5 2.5 11.8 (16.2) 6 Number of s a m p l e s a n a l y z e d . v i d e d some f a c t o r w h i c h e i t h e r p r o m o t e d an i n c r e a s e i n s y n -t h e s i s o r d e c r e a s e d d e s t r u c t i o n o f PHT. A s h i e l d i n g e f f e c t f r o m i n c r e a s e d c h l o r o p l a s t pigments may have have r e d u c e d d e s -t r u c t i o n by l i g h t ; r o o t e d shoots were m a r k e d l y d a r k e r g r e e n t h a n n o n - r o o t e d s h o o t s . C h l o r o p h y l l , h o w e v e r , i s u n l i k e l y t o be e s s e n t i a l f o r p r o t e c t i o n o f a c e t y l e n e s ; t h e w h i t e p e t a l s o f r a y f l o w e r s c o n t a i n l e v e l s o f PHT a b o u t 40% o f t h o s e o f l e a v e s ( T a b l e 2). R o o t l e s s s h o o t s were grown i n l i q u i d c u l t u r e i n d a r k and i n l i g h t t o d e t e r m i n e i f l i g h t was n e c e s s a r y f o r PHT f o r m a t i o n . These r e s u l t s a r e shown i n T a b l e 9. Due t o t h e l a c k o f e x p a n s i o n o f l e a f l a m i n a grown i n d a r k , i t was n e c e s s a r y t o h a r v e s t stems as w e l l as l e a v e s and p e t i o l e s . T h i s e x p l a i n s t h e s i g n i f i c a n t d i f f e r e n c e s i n c o m p o s i t i o n b e t w e e n t h e s e c u l t u r e s and t h e a g a r - g r o w n s h o o t s . L i g h t - g r o w n c u l t u r e s c o n t a i n e d l e s s t h a n 20% o f t h e amount o f p r o d u c t o f t h o s e grown i n dark.- The main d i f f e r e n c e i n c o m p o s i t i o n was t h e h i g h p r o p o r t i o n o f PDE i n i l l u m i n a t e d c u l t u r e s , w h i c h was n e a r l y t w i c e as h i g h as i n d a r k c u l t u r e s . D a r k c u l t u r e s were 12D Table 9 . Comparison of p o l y a c e t y l e n e s i n l i g h t - or dark-grown transformed p l a n t l e t s . C o e f f i c i e n t of v a r i a t i o n i n parentheses. Light-grown p l a n t s r e c e i v e d 6,000 lux continuous l i g h t . „ , P r o d u c t composition (wt %) C u l t u r e _ . 1 T o t a l type PHT PDE ETE-OAc product . N (mg/g d.w.)  Dark-grown 51.8 40.8 7.4 2.3 (17.2) 7 Light-grown 19.8 79.0 0.6 0.4 (38.6) 5 3 I n c l u d e s PHT, PHT-OAc, and PHT-ol. Number of samples analy z ed. s i m i l a r i n l e v e l and composition to s e e d l i n g stems ( c f . Table 2). Since these were s u b c u l t u r e s of dark-grown shoots, the amount of c a r r y - o v e r of compounds from the green c u l t u r e s used as a source should have been s m a l l . The presence of PHT and PDE i n e t i o l a t e d shoots i n d i c a t e d , then, that c h l o r o p l a s t s and l i g h t are not r e q u i r e d f o r t h e i r s y n t h e s i s . In a general way t h i s agrees with r e s u l t s f o r Crepis rubra; chopped seeds did not r e q u i r e l i g h t f o r formation of crepenynic a c i d (25). In c o n t r a s t , c h l o r o p l a s t involvement i s reported f o r epoxide a c e t y l e n e s i n s a f f l o w e r (27) and i n a c e l l - f r e e homogenate of Chrysanthemum f l o s c u l o s u m f o r p r o d u c t i o n of a b i c y c l i c a c e t y l e n e (6). Hagimori et a l . (24), i n a s i m i l a r type of study, used d i f f e r e n t i a t e d shoots and c a l l u s , e i t h e r of which could be grown to be green or c o l o u r l e s s , . and found that c h l o r o p l a s t s were not e s s e n t i a l f o r the s y n t h e s i s of d i g i t o x i n i n D i g i t a l i s  purpurea. 121 E f f e c t o f K i n e t i n on P l a n t l e t s The e f f e c t o f medium s u p p l e m e n t e d w i t h k i n e t i n on t h e acetylene c o n t e n t o f b o t h l e a v e s and t h e b a s a l c a l l u s o f i l l u m i n a t e d t r a n s f o r m e d p l a n t l e t s i s shown i n F i g . 26. A s u r p r i s i n g r e s u l t was t h a t l e a v e s were much more s e n s i t i v e t o t h e e f f e c t of k i n e t i n , e s p e c i a l l y i n l e v e l o f p r o d u c t , t h a n was c a l l u s . The two t i s s u e s seemed t o be a l m o s t c o m p l e t e l y i n d e p e n -d e n t of e a c h o t h e r i n r e s p e c t t o t h e i r a c e t y l e n e s ; c o m p o s i t i o n d i f f e r e d s h a r p l y and was c o n s i s t e n t f o r e a c h p a r t a c r o s s t h e r a n g e o f k i n e t i n t e s t e d . The r e s u l t s s u g g e s t e d t h a t p o l y a c e t y l -enes a r e n o t t r a n s p o r t e d f r o m l e a f t o c a l l u s . I_n s i t u s y n t h e -s i s o f a c e t y l e n e i c a c i d s has been shown i n sweet quandong (14) and i n o t h e r p l a n t s ( 2 7 ) . I n c r e a s i n g k i n e t i n g e n e r a l l y r e s u l t e d i n an i n c r e a s i n g p r o p o r t i o n o f PDE-OAc i n l e a v e s , a l t h o u g h t h e p r o p o r t i o n was l e s s t h a n o n e - h a l f t h a t o f t h e c a l l u s . S i g n i f i c a n t amounts o f ETE-OAc were o n l y p r e s e n t i n l e a v e s when a u x i n was p r o -v i d e d w i t h k i n e t i n . The l e v e l s o f p r o d u c t i n t h e c a l l u s may have been r e d u c e d by l i g h t d e s t r u c t i o n ; c a l l u s c o n t a i n e d o n l y s m a l l amounts ( v i s u a l e s t i m a t e ) o f c h l o r o p h y l l . The e f f e c t • o f k i n e t i n on t o t a l p r o d u c t i n l e a v e s i s d i f f i c u l t t o r a t i o n -a l i z e ; as shown l a t e r , r o o t c u l t u r e s showed i n c r e a s e d p r o d u c t o v e r t h e same r a n g e o f k i n e t i n t e s t e d i n s h o o t s . When 0.1 mg/1 NAA.was i n c l u d e d a l o n g w i t h k i n e t i n , l e a f p r o d u c t d r o p p e d s h a r p l y b u t i n c a l l u s t h e l e v e l was s t a b l e . The j o i n t e f f e c t of a u x i n and k i n e t i n was n o t f u r t h e r i n v e s t i g a t e d b u t i t a p p e a r e d t h e r e may h a v e been i n t e r a c t i o n b e t w e e n t h e two h o r m o n e s , a f f e c t i n g p r o d u c t l e v e l as w e l l as c o m p o s i t i o n i n 122 1.0 0*8 z o o Q. S o o 0-2 .0 4.QJ 5 3.0-Q PDE-OAC PHT ETE-OAC mn • 0 5 2. OH < a. < t- 1-oH o C L L E A F ( L ) CALLUS (C ) "* 1 / 0 0 1 I 0-033 0-1 KINETIN > 0-33 3.3 (MG/L ) .1NAA 3-0K/N F i g . 26. E f f e c t o f k i n e t i n on p o l y a c e t y l e n e s i n s h o o t s a n d c a l l u s o f t r a n s f o r m e d p l a n t l e t s . P D E - O A c , PHT, a n d E T E - O A c i n c l u d e a l l f o r m s o f t h e s e c h r o m o p h o r e s . C i s c a l l u s , L i s l e a f m a t e r i a l . B a r s r e p r e s e n t + 1 s t a n d a r d d e v i a t i o n . 123 l e a v e s . There i s evidence that f a c t o r s t r a n s f e r r e d from roots to leaves i n the t r a n s p i r a t i o n stream r e g u l a t e the r a t e of growth and p h o t o s y n t h e t i c a c t i v i t y i n leaves (17). Other s t u d i e s i n d i c a t e that c y t o k i n i n s are produced i n the roots and provide a source f o r leaves with the stems p o s s i b l y serv-ing as a r e s e r v o i r (18,57). Exogenous a p p l i c a t i o n of b e n z y l -adenine can compensate f o r c h l o r o p h y l l r e d u c t i o n r e s u l t i n g from p a r t i a l root e x c i s i o n (16). The s t i m u l a t o r y e f f e c t of r o o t i n g on greening and product seemed to agree with a cyto-k i n i n e f f e c t ; however, k i n e t i n i n the medium did not produce the same e f f e c t . The form or type of c y t o k i n i n s u p p l i e d may be c r i t i c a l . Aspects of Crown G a l l Physiology P r o d u c t i o n of secondary compounds i n crown g a l l c u l t u r e s (other than the c h a r a c t e r i s t i c opines) at l e v e l s s i g n i f i c a n t l y higher than f o r normal t i s s u e c u l t u r e s i s u n l i k e l y to be general (e.g., coloured products would c e r t a i n l y not have gone un n o t i c e d ) , but the r e s u l t s i n t h i s chapter suggest that p o l y -acetylene s y n t h e s i s may be p e c u l i a r l y a f f e c t e d by t r a n s f o r -mation i n at l e a s t two c l o s e l y .related species of Bidens. Although the net e f f e c t s of t r a n s f o r m a t i o n on c e l l s are by no means e n t i r e l y c l e a r some of the reported changes may bear on secondary product s y n t h e s i s . Increased i o n - t r a n s p o r t c a p a b i l i t i e s or a l t e r e d membrane p e r m e a b i l i t y , e s p e c i a l l y to K +, has been suggested to e x p l a i n r e s u l t s showing i o n - a c t i v a t i o n by high s a l t medium of b i o s y n -t h e t i c pathways (11,63). Late r work on the uptake of q u i n i -124 c r i n e showed that normal c e l l membrane p e r m e a b i l i t y was unchanged by r a i s i n g the l e v e l of s a l t s (64). Lentz et a l . (40) confirmed the d i f f e r e n c e i n i n t e r n a l l e v e l s of K + but found that r e g u l a t i o n of uptake was d i f f e r e n t i n h a b i t u a t e d normal c e l l s and tumour c e l l s . Normal c e l l s i n c r e a s e d the r a t e of K + uptake when i n t e r n a l l e v e l s were low but tumour c e l l s did not. The l e v e l s of K , Mg , and Ca were a l l found to be higher i n tumour c e l l s of potato d i s c s than i n + ++ normal c e l l s . Levels of K and Mg were u n a f f e c t e d by 2,4-DNP, i n d i c a t i n g that a c t i v e t r a n s p o r t was not i n v o l v e d . Radosevich and Galsky concluded that i n c r e a s e d c o n c e n t r a t i o n s of c e r t a i n cations may be a s p e c i f i c property of crown g a l l t umour s (51). There is evidence that membrane p e r m e a b i l i t y may c o n t r o l the q u a n t i t y of anthocyanins i n rose and red cabbage, and p o s s i b l y the q u a l i t a t i v e composition of l i g n i n s i n poplar stems (52). Krauspe (36) has pointed out that changes i n p e r m e a b i l i t y a l s o occur t e m p o r a r i l y a f t e r wounding and Braun has suggested that transformation r e s u l t s i n a p e r s i s t e n t a c t i v a t i o n of the wound response (10). Studies of membrane l i p i d s were repo r t e d by P h i l l i p s and Butcher (49) who found that the o v e r a l l p a t t e r n of phos-p h o l i p i d s was s i m i l a r f o r normal and tumour c a l l u s c u l t u r e s but tumour c e l l s contained more p h o s p h a t i d y l c h o l i n e and l e s s p h o s p h a t i d i c a c i d and p h o s p h a t i d y l i n o s i t o l than normal c e l l s . Cockerham et a l . (19) evaluated the p o s s i b i l i t y that membrane changes were due to secondary d i f f e r e n c e s a s s o c i a t e d with the r a p i d growth of transformed c e l l s , Working with Vinca rosea they found that f u l l y transformed c e l l s had l e s s than one-half 125 the amount of p h o s p h o l i p i d s of normal c e l l s . P a r t i a l l y t r a n s -formed c e l l s were s i m i l a r to normal c e l l s and when s t i m u l a t e d to grow at a r a t e comparable to f u l l y transformed c e l l s the l e v e l of p h o p h o l i p i d d i d not change a p p r e c i a b l y . The authors concluded that membrane a l t e r a t i o n is a d i r e c t r e s u l t of t r a n s f o r m a t i o n and not a consequence of growth r a t e . Para-f i n i c hydrocarbons of tobacco were compared i n c u l t u r e d t e r a -toma tumour t i s s u e , h a b i t u a t e d c a l l u s and i n t a c t p l a n t s (61). Tumour c e l l s were found to be q u a l i t a t i v e l y i d e n t i c a l to seed-l i n g s i n composition. A t h i r d c h a r a c t e r i s t i c f e a t u r e of most types of crown g a l l t i s s u e i s the c a p a c i t y f o r growth on medium l a c k i n g aux-ins and c y t o k i n i n s . Evidence exists that, f o r at l e a s t some p l a n t s , t r a n s f o r m a t i o n r e s u l t s i n higher than normal auxin l e v e l s i n tumour t i s s u e (1,10,22). However, other s t u d i e s show that t h i s i s not g e n e r a l l y true (47,62). The mechanism fo r a l t e r a t i o n of hormone l e v e l , whether by d i r e c t l y coded T-DNA s y n t h e t i c genes or By modulation of p l a n t hormone b i o -s y n t h e s i s , i s s t i l l not known (54). The connection between these three areas i s not c l e a r at present and more i n f o r m a t i o n i s needed on changes i n p e r m e a b i l i t y , memBrane composition of transformed c e l l s , and hormone l e v e l s i n normal and transformed c e l l s at s i m i l a r growth stages. R e s o l v i n g the ways i n which tumour c e l l s d i f f e r from normal c e l l s would c e r t a i n l y help to understand why transformed _B. alBa c e l l s produce p o l y a c e t y l e n e s and normal c a l l u s c e l l s do not. 126 SUMMARY Crown g a l l s induced on _B. alba by two d i f f e r e n t s t r a i n s of Agrobac t er ium t ume f ac ien s were c u l t u r e d and i n v e s t i g a t e d f o r p o l y a c e t y l e n e p r o d u c t i o n and composition. An e f f o r t to use a v i s u a l d i f f e r e n c e i n some c a l l u s e s as guide f o r s e l -e c t i n g those with higher product l e v e l s r e s u l t e d i n a l i n e with a higher content but an u n c e r t a i n r e l a t i o n to the s e l -e c t i o n c r i t e r i o n . C a l l u s grown over s e v e r a l years showed a c o n t i n u i n g a b i l i t y to s y n t h e s i z e polyacetylenes at l e v e l s up to 20% of that in cultured roots. However, l e v e l s f l u c t u a t e d widely over time,and composition showed a decrease i n the p r o p o r t i o n of phenyldiynene acetate (PDE-OAc). Octopine, n o p a l i n e , and double transformant c a l l u s , a l l showed s i m i -l a r i t i e s i n composition c h a r a c t e r i z e d by high l e v e l s of PDE-OAc arid the presence of minor compounds not found i n plant t i s s u e s . Work with transformed p l a n t l e t s showed that roots and l i g h t were unnecessary f o r s y n t h e s i s of l e a f and stem a c e t y l e n e compounds. Leaves of p l a n t l e t s g e n e r a l l y showed i n h i b i t i o n of p h e n y l h e p t a t r i y n e s y n t h e s i s by k i n e t i n i n the medium, but c a l l u s a s s o c i a t e d with the p l a n t l e t s showed an i n c r e a s e . 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W. , and K. Spanier. 1981. Phytohormones i n the formation of crown g a l l tumors. Planta 153: 326-337. 63. Wood, H. N., and A. C. Braun. 1961. Studies on the reg-u l a t i o n of c e r t a i n e s s e n t i a l b i o s y n t h e t i c systems i n normal and crown-gall tumor c e l l s . Proc. N a t l . Acad. S c i . 47:1907-1913. 64. Wood, H. N., and A. C. Braun. 1965. Studies on the net uptake of s o l u t e s by normal and crown-gall tumor c e l l s . Proc. N a t l . Acad. S c i . 54:1532-1538. 65. Yano, K. 1975. V a r i a t i o n i n acetylene content of d i f f -erent ecotypes of Artemisa c a p i l l a r i s . Phytochemistry 14:1783-1784. 66. Zenk, M. H. 1978. The impact of p l a n t c e l l c u l t u r e on i n d u s t r y . Pages 1-13 i_n T. A. Thorpe, ed. F r o n t i e r s of p l a n t t i s s u e c u l t u r e . I n t e r n a t i o n a l A s s o c i a t i o n f o r P l a n t T i s s u e C u l t u r e , Calgary, A lb. 67. Z i t o , S. W., and E. J . Staba. 1982. Thebaine from root c u l t u r e s of Papaver bracteatum. P l a n t a Medica 45: 53-54. 133 CHAPTER VI STUDIES ON ROOT CULTURES OF BIDENS ALBA 134 INTRODUCTION The v a r i a t i o n s i n medium components, a d d i t i v e s and environ-mental c o n d i t i o n s that were t r i e d i n unsuccessful attempts to o b t a i n c u l t u r e s of Bidens a l b a that produced a p p r e c i a b l e l e v e l s of p o l y a c e t y l e n e s have been d e s c r i b e d i n Chapter I I I . Rather than continue to experiment with aging c a l l u s which produced no acetylenes i t seemed more promising to work with a system which alr e a d y produced acetylenes and to determine how the l e v e l and composition was a f f e c t e d by f a c t o r s known to enhance l e v e l s of secondary products or l i p i d s i n other systems. If s u b s t a n t i a l l y i n c r e a s e d product could be achieved then these c o n d i t i o n s should serve as a b e t t e r s t a r t i n g point f o r o b t a i n -ing p o l y a c e t y l e n e s i n c a l l u s and suspension c u l t u r e s . In a d d i t i o n , i n f o r m a t i o n from root c u l t u r e s could help i n under-standing the f a c t o r s c o n t r o l l i n g b i o s y n t h e t i c p a t t e r n s i n the p l a n t . The relevance of work with excised roots to the phy-s i o l o g y of the i n t a c t plant has been a f f i r m e d by S t r e e t (5). Excised root c u l t u r e s have been used i n secondary product s t u d i e s to determine the s i t e of b i o s y n t h e s i s of compounds, examples being work demonstrating the p r o d u c t i o n of a t r o p i n e i n roots of Atropa belladonna (45), the b i o s y n t h e s i s of n i c o -t i n e i n root t i p s of tobacco, and anabasine i n a separate r e g i o n of the root (17,35). Other s t u d i e s have used root c u l -tures to show an i n c r e a s e i n hyoscyamine when p r o t e i n s y n t h e s i s was i n h i b i t e d by puromycin (11), to determine the e f f e c t of p r e c u r s o r s and i n h i b i t o r s on l e v e l s of tomatine i n excised 135 tomato roots (32), and to i n v e s t i g a t e the b i o s y n t h e s i s of tropane a l k a l o i d s i n Datura metel (26). Root c u l t u r e s of Dioscorea d e l t o i d e a produced only t r a c e amounts of d i o s g e n i n while c a l l u s d e r i v e d from roots produced a p p r e c i a b l e amounts (21). There i s a report of a patent f o r p r o d u c t i o n of saponins from ginseng root c u l t u r e s (20). In g e n e r a l , however, there seems to have been s u r p r i s i n g l y l i t t l e use made of root c u l -tures f o r e i t h e r b i o s y n t h e t i c s t u d i e s or as sources f o r the l a r g e number of u s e f u l compounds which occur i n r o o t s . Excised root c u l t u r e s of 13. alba provided s u i t a b l e e x p e r i -mental m a t e r i a l f o r the f o l l o w i n g reasons. 1) The roots could be s u b c u l t u r e d r e p e a t e d l y on a d e f i n e d medium, thus p r o v i d i n g genetic c o n t i n u i t y . 2) The p h y s i c a l and chemical environment could be e a s i l y c o n t r o l l e d and growth under both l i g h t and dark c o n d i t i o n s could be s t u d i e d . 3) Growth, with an appro-p r i a t e hormone l e v e l , was r a p i d , a l l o w i n g experiments to be conducted on m a t e r i a l which had doubled s e v e r a l times, so that only a small percentage of the f i n a l c u l t u r e was from m a t e r i a l grown under the standard c o n d i t i o n s of the inoculum source. 4) C u l t u r e d roots lacked secondary t h i c k e n i n g , a l l o w i n g much e a s i e r homogenization f o r e x t r a c t i o n and e l i m i n a t i n g a v a r i a b l e which could a f f e c t product s y n t h e s i s . 5) C u l t u r e d roots pro-duced a r e l a t i v e l y s t a b l e p a t t e r n of p o l y a c e t y l e n e s at l e v e l s and composition comparable to i n t a c t r o o t s . The s t u d i e s reported here o u t l i n e the c h a r a c t e r i s t i c s of 13. a lb a root c u l t u r e s and the e f f e c t of v a r i o u s treatments on p o l y a c e t y l e n e l e v e l and composition. The o v e r a l l o b j e c t i v e was to develop a c u l t u r e system which would f a c i l i t a t e work 136 on f a c t o r s which determine the p a t t e r n of compounds found i n the i n t a c t p l a n t . The r e s u l t s show the value of t h i s c u r r e n t l y r a t h e r l i t t l e used technique of t i s s u e c u l t u r e (defined i n the broad sense) f o r i n v e s t i g a t i n g the c o n t r o l of p o l y a c e t y l e n e s y n t h e s i s . 137 MATERIALS AND METHODS Cul t u r e Methods Root c u l t u r e s were e s t a b l i s h e d from s i n g l e roots a r i s i n g from e i t h e r s t e r i l e l e a f or stem explants of 15. alba grown on SH agar medium which contained 4.0 mg/1 NAA. Stock c u l -tures were store d on SH agar p l a t e s without hormones and were kept i n the dark at 25*C. Roots were stored i n t h i s way f o r four to s i x months between t r a n s f e r s . Under these c o n d i t i o n s growth was q u i t e slow and when a t i p segment was placed i n l i q u i d shake c u l t u r e a l a g phase of approximately seven days occurred before vigorous growth resumed. Roots were a l s o s t o r e d i n l i q u i d medium without hormones. A s i n g l e segment was used to s t a r t a l i q u i d c u l t u r e , which was then used to s t a r t both experimental and c o n t i n u i n g ; stock c u l t u r e s . The l a t t e r served as the source f o r the next experimental and stock c u l t u r e , and so on. T r a n s f e r s were made by p u l l i n g out, with f o r c e p s , a t u f t of young roots from the parent clump and i n o c u l a t i n g the experimental or stock f l a s k . E q u a l i t y of explants was determined v i s u a l l y . Although t h i s method was not p r e c i s e , t y p i c a l sets of explants had a f r e s h weight of 0.2 g and a c o e f f i c i e n t of v a r i a t i o n of 15%. The v a r i a t i o n i n weights at time of harvest compared favourably with s t u d i e s using s i n g l e roots (17,33a). If environmental c o n d i t i o n s were being e v a l u a t e d, and the medium was i d e n t i c a l f o r a l l c o n d i t -i o n s , then i n o c u l a t e d f l a s k s were randomized before being assigned to an experimental group. Roots were then grown for 138 11 days i n 125 ml Erlenmeyer f l a s k s c o n t a i n i n g 75 ml of l i q u i d SH medium with 0.5 mg/1 NAA. Medium pH was 5.7, adjusted with KOH or HCl, and f l a s k s were stoppered with foam plugs ( I d e n t i -plugs, Western S c i e n t i f i c ) covered with two l a y e r s of paper towel and sealed at the neck with a rubber band. Incubation was at 25°C i n dark on a r o t a r y shaker at 125 rpm. Medium was autoclaved f o r 20 mintues at 15 l b / s q . i n . These are the standard c o n d i t i o n s and any v a r i a t i o n s are noted. Transformed Roots Transformed roots were c u l t u r e d from both A208 and A277 c a l l u s l i n e s , which rooted as d e s c r i b e d i n Chapter I I I . Both l i n e s were t e s t e d f o r the presence of opines and found to con-t a i n the expected types. However, the k i l l l i n e a f t e r a few months i n c u l t u r e was negative f o r o c t o p i n e . The A208 l i n e remained p o s i t i v e f o r n o p a l i n e . C u l t u r e s were grown as f o r normal roo t s . Harves t i n g C u l t u r e s were harvested by pouring o f f the medium and gently removing the clump of roots with f o r c e p s . The clump was patted dry between paper towels, weighed and a r e p r e s e n t a t i v e one gram sample used f o r e x t r a c t i o n and a n a l y s i s . The remain-ing t i s s u e was d r i e d between paper towels f o r 24 hours at 100°C, allowed to c o o l to room temperature and weighed. Sample and tot a l dry weight (per f l a s k ) were c a l c u l a t e d from the r a t i o of f r e s h to dry weight. "Growth" as used here means the cumu-l a t i v e dry weight per f l a s k versus time. 139 A n a l y s i s Roots were e x t r a c t e d and prepared f o r HPLC a n a l y s i s and q u a n t i t a t i o n as d e s c r i b e d i n Chapter 2 . Average doubling time ( t ^ ) was c a l c u l a t e d with the f o l l o w -ing equation: t ^  = t/["log (w^/w^ ) / log l\ where t i s the time i n t e r v a l f o r growth and w^  and w^  are the s t a r t i n g and ending weights r e s p e c t i v e l y . 140 RESULTS AND DISCUSSION Comparison of P o l y a c e t y l e n e Composition of Cul t u r e d and I n t a c t  Roo t s Table 10 shows a comparison of p o l y a c e t y l e n e l e v e l and composition f o r 13. al b a s e e d l i n g roots and dark-grown c u l t u r e d r o o t s . I n t a c t roots contained small amounts of PHT and PDE which were not found i n c u l t u r e d r o o t s . The. small amounts of both compounds may have been c o n t r i b u t e d by the crown of the r o o t ; p l a n t s were harvested by c u t t i n g o f f the roots at ground l e v e l and the upper p o r t i o n of the root clump i n c l u d e d some stem m a t e r i a l . The p r i n c i p a l d i f f e r e n c e i n composition between the two types of roots was the higher percentage of PDE—OAc i n i n t a c t roots and a corresponding lower percentage of ETE-OAc. Higher p r o p o r t i o n s of PDE-OAc were found i n c u l t u r e d roots when e i t h e r NAA or k i n e t i n l e v e l s were higher than the standard 0.5 mg/1 NAA (see l a t e r ) , suggesting that i n t a c t roots may c o n t a i n l e v e l s of hormones higher than those used f o r the c u l t u r e d r o o t s . The d i f f e r i n g environment and the presence of the shoot must prevent the exce s s i v e c a l l u s i n g found i n c u l t u r e d roots at higher hormone l e v e l s . The o v e r a l l s i m i l a r i t y of the two types of roots seemed to j u s t i f y use of c u l t u r e d roots as a r e p r e s e n t a t i v e system f o r the study of p o l y a c e t y l e n e s . The next step was to see how va r i o u s f a c t o r s a f f e c t e d the l e v e l and composition of a c e t y l -enes i n t h i s m a t e r i a l . T a b l e 10- P o l y a c e t y l e n e c o m p o s i t i o n of s e e d l i n g and c u l t u r e d r o o t s of 15. a l b a grown u n d e r s t a n d a r d c o n d i t i o n s . P o l y a c e t y l e n e c o m p o s i t i o n (wt % o f t o t a l ) T o t a l Root t y p e PHT PDE-OAc PDE EDE-OAc E T E - o l ETE-OAc ETE p r o d u c t A B (mg/g d.w.) S e e d l i n g 5.7 58.3 2.9 7.7 0.4 4.9 19.3 0.8 8.7 C u l t u r e d - 38.7 - 7.6 0.7 3.9 47.1 2.0 3.3 S y m b o l : -, n o t d e t e c t a b l e . 142 E f f e c t of Temperature on Roots Fi g u r e 27A shows the dependence of p o l y a c e t y l e n e l e v e l and f i n a l dry weight per f l a s k on i n c u b a t i o n temperature. With i n c r e a s i n g temperature over the range 18-33 °C product l e v e l decreased at very n e a r l y a constant r a t e . Growth a l s o showed a n e a r l y l i n e a r response to temperature; i n c r e a s i n g up to 30°C but d e c r e a s i n g sharply at 33°C. C u l t u r e s grown at 15°C and o 35 C showed no a p p r e c i a b l e growth at e i t h e r temperature over the c u l t u r e p e r i o d of 11 days and are not shown. Tomato roots are a l s o reported to grow best at 30*C with a sharp d e c l i n e i n growth at higher temperatures, and other s p e c i e s show a s i m i l a r s e n s i t i v i t y (40). Figure 27B shows the e f f e c t of temperature on composition. Only the major component, PDE-OAc i s shown, s i n c e most of the remaining product was ETE-OAc, which changed r e c i p r o c a l l y to PDE-OAc. Low temperatures did not s i g n i f i c a n t l y a f f e c t PDE-OAc and high temperatures (30-33°C) r e s u l t e d i n a maximum decrease of about 10% i n t h i s component. I t might be supposed t h a t f at higher than normal temperatures^ the composition of c u l t u r e s would tend to resemble those grown longer at lower tempera-t u r e s ; however, as F i g . 29B shows, t h i s was not the case. In o l d e r c u l t u r e s grown at 2 5°C the p r o p o r t i o n of PDE-OAc i n c r e a -ses. Thus the small d e c l i n e appears to be caused by other f a c t o r s . The o v e r a l l composition was r e l a t i v e l y s t a b l e over changes i n temperature while product l e v e l s changed s h a r p l y . A d e c l i n e i n a l k a l o i d formation with i n c r e a s i n g temperature has been reported by C o u r t o i s and Guern (8) f o r Catharanthus  roseus suspension c u l t u r e s . Their e x p l a n a t i o n f o r the e f f e c t 143 4 . 0 -3 . 0 -2 . 0 -1 . 0 -T O T A L P R O D U C T ( M G / G D.WJ D R V W T . / F L A S K (G) -0 .8 • 0 . 6 - 0 . 4 -0 .2 18 2 0 2 2 2 4 2 6 2 8 3 0 3 2 T E M P . ( - C ) B ^P D E - O A C W T . % T O T . P R O D . 4 0 -3 0 -2 0 -1 0 -i i , i • i • I i 1 8 i2 26 3 0 T E M P . C C J F i g . 27- E f f e c t o f g r o w t h t e m p e r a t u r e on c u l t u r e d B_. a l b a r o o t s . A) E f f e c t on p o l y a c e t y l e n e l e v e l and g r o w t h . B) E f f e c t on p o l y a c e t y l e n e c o m p o s i t i o n . Each p o i n t i s t h e a v e r a g e f o r t h r e e f l a s k s . B a r s show + 1 SD. 144 was that the r a t i o between b i o s y n t h e s i s and degradation was modified by temperature. Given the small change i n composition over the temperature range t e s t e d , and the l a r g e changes i n product l e v e l , t h i s e x p l a n a t i o n seems u n l i k e l y f o r Ii. alba r o o t s , unless degradation was e i t h e r n o n s e l e c t i v e or n e a r l y e q u a l l y balanced f o r the d i f f e r e n t p o l y a c e t y l e n e s . One p o s s i b l e e x p l a n a t i o n of the i n v e r s e r e l a t i o n between product l e v e l and temperature could be that s u b s t r a t e a v a i l -a b i l i t y i s i n c r e a s e d at lower temperatures. Many p l a n t s show in c r e a s e s i n f a t t y a c i d l e v e l s (30) and u n s a t u r a t i o n as tem-perature decreases (10,18,31). However, there are exceptions to t h i s general r u l e (12,42) and i n some p l a n t s g e n e t i c con-t r o l can o v e r r i d e p h y s i c a l f a c t o r s (19). Ne v e r t h e l e s s , if. c u l t u r e d B. al b a roots are t y p i c a l , then higher l e v e l s of l i n o l e i c a c i d r e s u l t i n g from c o l d treatment, might be d i v e r t e d to product s y n t h e s i s . The e f f e c t of temperature on l i p i d composition i s thought to act e i t h e r d i r e c t l y on the desaturase system or through i n c r e a s e d l e v e l s of d i s s o l v e d oxygen at lower temperatures (15, 16). Since oxygen i s r e q u i r e d f o r f a t t y a c i d d e s a t u r a t i o n (9), the same argument might a l s o apply to the formation of the t r i p l e bond of p o l y a c e t y l e n e s ; there i s evidence that oxygen i s r e q u i r e d f o r t h i s r e a c t i o n as w e l l (4,14). It would be i n t e r e s t i n g to know the e f f e c t of d i f f e r e n t oxygen l e v e l s on p o l y a c e t y l e n e s y n t h e s i s i n c u l t u r e d r o o t s . A l t e r n a t i v e l y , the enzymes f o r t r i p l e bond formation may be i n h i b i t e d or i n a c t i v a t e d at temperatures not i n h i b i t o r y to growth. Haigh et a l . (14) found that the conversion of o l e a t e 145 to crepenynate i n chopped Crepis rubra seeds was completely i n h i b i t e d at 32 *C. However, the e f f e c t of a range of temper-atures on o l e a t e c o n v e r s i o n was not r e p o r t e d . Martin (28) has reported that temperatures below 25 *C reduced n i t r o g e n u t i l i z a t i o n i n Ipomoea to a g r e a t e r extent than sucrose u t i l i z a t i o n . If a s i m i l a r e f f e c t occurs i n Bidens then i t could account, at l e a s t p a r t l y , f o r the l e v e l s of p o l y a c e t y l e n e . A l a t e r experiment (see below) shows that i n c r e a s e d sugar to n i t r a t e r a t i o s favoured i n c r e a s e d product f o rmation. Martin (28) notes that " l i t t l e i f anything seems to have been reported on temperature optima f o r growth versus those f o r m e t a b o l i t e p r o d u c t i o n . " The data reported here are the f i r s t f o r p o l y a c e t y l e n e s . Further work needs to be done to e l u c i d a t e the mechanism causing product l e v e l and growth to show opposite trends. E f f e c t of pH on Roots Resu l t s from growing roots i n unbuffered medium are shown i n F i g . 28. The pH of the medium a f t e r 10 days growth of a l l except the roots grown at pH 3.3 and 9.0 was 5,5 + 0.1. Roots c u l t u r e d at the lowest and highest pH showed very l i t t l e growth or p o l y a c e t y l e n e content. Composition (not shown) was s i m i l a r f o r a l l pH values except the two extremes, which showed in c r e a s e d PDE-OAc. Both maximum growth and product c o i n c i d e d with the standard medium pH of 5.7. The range f o r growth of B. a l b a r o o t s , as w e l l as the c a p a c i t y f o r a d j u s t i n g pH, was s i m i l a r to r e p o r t s f o r c u l t u r e s of other p l a n t s (6, p.46). Growth and product l e v e l g e n e r a l l y d e c l i n e d as the s t a r t i n g 146 T O T AL P R O D U C T DRY WT./ (MG/G D.W.) F L A S K ( G ) I I I I I I I 3 4 5 6 7 8 9 p H F i g . 28. E f f e c t o f pH on g r o w t h and p o l y a c e t y l e n e c o n t e n t of c u l t u r e d 13. a l b a r o o t s . C u l t u r e s were grown f o r t e n days u n d e r s t a n d a r d c o n d i t i o n s . R e s u l t s a r e a v e r a g e v a l u e s f o r t h r e e f l a s k s f o r e a c h p o i n t . B a r s show + 1 SD; d r y w e i g h t SD v a l u e s were s i m i l a r t o p r o d u c t v a l u e s . 147 pH was lower or higher than 5.7. D i f f e r e n c e s i n the amount of growth may be due to the time r e q u i r e d by the roots to change the pH of the medium to an e q u i l i b r i u m l e v e l p e r m i t t i n g r a p i d growth. E f f e c t of C u l t u r e Age on Roots Fig u r e 29A shows the r e l a t i o n s h i p between age of c u l t u r e and the amount of t i s s u e formed, the p o l y a c e t y l e n e content per gram of t i s s u e and per f l a s k , and average doubling time. Growth fo l l o w e d a p a t t e r n . t y p i c a l of many types of c u l t u r e s and showed the f o l l o w i n g phases: l a g phase (days 0-2), expo-n e n t i a l or l o g phase (days 2-14) and s t a t i o n a r y phase (from day 14). The p o l y a c e t y l e n e l e v e l of root c u l t u r e s , per gram of t i s s u e , decreased during the l a g phase and reached a minimum during the p e r i o d of most r a p i d growth (days 4-6), i n c r e a s e d during m i d - t o - l a t e log phase (days 8-14) and d e c l i n e d again as growth ceased. T o t a l a c e t y l e n e s per f l a s k changed l i t t l e d uring e a r l y log phase growth but r a p i d l y i n c r e a s e d as growth began to slow and decreased as growth began to stop, suggesting that the compounds were being metabolized or excreted i n t o the medium. E x t r a c t i o n of the medium i n s e v e r a l experiments, however, f a i l e d to show s i g n i f i c a n t amounts of a c e t y l e n e s ; l e v e l s were t y p i c a l l y l e s s than 1 ug per ml of medium. Figu r e 29B shows the change i n composition as root c u l t u r e s age; only the PDE-OAc component i s shown. Low l e v e l s of PDE-OAc were c o r r e l a t e d with the p e r i o d of most r a p i d growth and n e a r l y doubled as growth ceased. A much more pronounced change i n composition of tobacco roots has been repo r t e d by S o l t et a l . (35). In the e a r l y part of the c u l t u r e p e r i o d anabasine and 148 4.0« T OT A L PR ODUCT ( M G / F L A S K ) 2.4-2.2- 3.0-2.0-1.8-1.6-1-4 1 .2-1.0-0&-0.6 OA~i 0.2-2.0-1.0-T 0 T A L PRODUCT 0 (MG/GCLVtJ DOUBLING TIME (HRS) I 82 133 ( 8 3 I 92 1121 | > 2 0 0 , 1 2 9 B 6 0 -50-4 0 -30 2 0 -1 0 -> I I 1 I I 4 8 12 CULTURE AGE (DAYS) PDE-OAC WT.% T QT. PROD. 16 T -2 0 24 DRY WT,/ FLASK (G) 1.2 8 •0.6 4 •0.3 2 0.16 0-08 0 .04 -0.0 2 8 12 AGE (DAYS) 16 20 24 F i g . 29. E f f e c t o f c u l t u r e age on g r o w t h and p o l y a c e t y l e n e s °f J*- a l b a r o o t c u l t u r e s . A) T o t a l p r o d u c t p e r gram r o o t and p e r f l a s k , d o u b l i n g t i m e , and g r o w t h . B) C o m p o s i t i o n of p o l y a c e t y l e n e s . E a ch p o i n t i s t h e a v e r a g e f o r t h r e e f l a s k s . B a r s show + 1 SD. Dry w e i g h t s c a l e i s l o g a r i t h m i c . 149 n i c o t i n e were present at a r a t i o of 2:1, but toward the end of the passage the r a t i o changed to 4:1. The authors concluded that the two products were sy n t h e s i z e d i n separate p o r t i o n s of the r o o t . A n a l y s i s of separate regions of Bid ens r o o t s , however, showed l i t t l e d i f f e r e n c e i n composition (see below), but l a r g e d i f f e r e n c e s i n amount of product. L e v e l of secondary product formation i n c e l l c u l t u r e s i s sometimes s t r o n g l y dependent on the growth stage of the c u l t u r e (7) or the r a t e at which the c u l t u r e grows (34). Product syn-t h e s i s i s f r e q u e n t l y a s s o c i a t e d with l a t e l o g and s t a t i o n a r y phase, e s p e c i a l l y i n microorganisms (25,27). However, s t u d i e s of a l k a l o i d p r o d u c t i o n i n roots of tobacco (17) and tomato (32) show a strong c o r r e l a t i o n between growth and amount of product, c h a r a c t e r i s t i c of compounds s y n t h e s i z e d i n young t i s s u e . Other evidence would make the l a t e s y n t h e s i s of a c e t y l e n e s u n l i k e l y i n 13. alba root c u l t u r e s : r e s i n c a n a l s , the s t r u c t u r e s i n which ac e t y l e n e s accumulate i n roots (36, 43), form i n J3. alba root t i p s ahead of the e a r l i e s t v i s i b l e xylem t i s s u e (data not presented) and the p o l y a c e t y l e n e l e v e l i n the young-est growth of a c u l t u r e i s higher than i n the o l d e r core p o r t i o n (see l a t e r ) . The minimum l e v e l of p o l y a c e t y l e n e s occurred during the p e r i o d of most r a p i d growth, suggesting e i t h e r that s y n t h e s i s did not keep pace with growth or that primary m e t a b o l i t e s were u n a v a i l a b l e f o r secondary product s y n t h e s i s . Work with f u n g i has i n d i c a t e d that carbon i s shun-ted to l i p i d p r o d u c t i o n as p r o t e i n and n u c l e i c a c i d syntheses d i m i n i s h (.22) and that a r a p i d i n c r e a s e i n lipids f o l l o w i n g n i t r o g e n l i m i t a t i o n i n f u n g i r e f l e c t s a constant r a t e of l i p i d 150 s y n t h e s i s as c e l l p r o l i f e r a t i o n s l o w s . The r a p i d r i s e i n p o l y a c e t y l e n e s t h a t f o l l o w s t h e p e r i o d o f most r a p i d g r o w t h s u g g e s t s t h a t a s i m i l a r e f f e c t o c c u r s i n r o o t c u l t u r e s . E v i -d e n c e s u p p o r t i n g t h i s e f f e c t was p r o v i d e d i n t h e n e x t e x p e r i -men t . The l a t e d r o p i n a v e r a g e t i s s u e p r o d u c t l e v e l was somewhat u n e x p e c t e d and when c o m b i n e d w i t h t h e d e c l i n e i n t o t a l p r o d u c t p e r f l a s k i n d i c a t e d t h a t b r e a k d o w n o f t h e p o l y a c e t y l e n e s was o c c u r r i n g . S o r e n s e n (37 ) n o t e s t h a t i n t h o s e c a s e s where up-t a k e of l a b e l l e d p o l y a c e t y l e n e s by p l a n t s has b een s t u d i e d , t h e y a r e r a p i d l y m e t a b o l i z e d w i t h a h a l f - l i f e o f one t o two d a y s . E v i d e n t l y r o o t p o l y a c e t y l e n e s a r e n o t i n e r t end p r o d u c t s , p a s s i v e l y a c c u m u l a t e d ; r a t h e r , t h e y seem t o be q u i t e s e n s i t i v e t o t h e c o n t i n u a l c h a n g e s w h i c h o c c u r u n d e r b a t c h c u l t u r e c o n -d i t i o n s . E f f e c t of S u c r o s e / N i t r a t e R a t i o s on R o o t s The e f f e c t of c h a n g e s i n t h e s u c r o s e and p o t a s s i u m n i t r a t e l e v e l s on p o l y a c e t y l e n e p r o d u c t i o n and c o m p o s i t i o n i s shown i n T a b l e 11. The n o r m a l l e v e l o f s u c r o s e and n i t r a t e i n SH medium i s 3% and 0.25% r e s p e c t i v e l y w i t h n i t r a t e a c c o u n t i n g f o r two-t h i r d s o f medium n i t r o g e n . G rowth and p r o d u c t i n c r e a s e d as s u c r o s e i n c r e a s e d f r o m 1%% t o 6% b u t b o t h d e c l i n e d s h a r p l y a t h i g h e r c o n c e n t r a t i o n s of s u c r o s e . C o m p o s i t i o n showed a more c o m p l e x p a t t e r n : PDE-OAc d e c r e a s e d as s u c r o s e i n c r e a s e d and i n c r e a s e d as n i t r a t e l e v e l d e c r e a s e d . D o u b l i n g t h e s u c r o s e l e v e l f r o m 3% t o 6% r e s u l t e d i n a 25% i n c r e a s e I n p r o d u c t ; d e c r e a s i n g n i t r a t e l e v e l by \ and h i n c r e a s e d i t by 37% and 25%. S i m i l a r e f f e c t s on f a t a c c u m u l a t i o n i n f u n g i have been 151 Table 11. E f f e c t of changes in c a r b o h y d r a t e / n i t r a t e r a t i o s on po l y a ce t y l ene l e v e l s of B. a lba root c u l t u r e s . Resu l t s are averages f o r three f lask 's of each medium type grown under s tandard c o n d i t i o n s . Med i urn type Root dry wt/ f l a s k (g) PDE-0Ac b wt. % of t o t a l To ta l p r o d u c t b KNCU (%r Sucrose (%) (mg/g d. w.) (CV) 0 . 2 5 1 . 5 0 . 1 9 3 0 . 9 a b 2 . 8 5 b ( 2 6 ) 0 . 2 5 3 . 0 a 0 . 3 1 3 6 . 1 b c 3 . 1 2 b ( 1 0 ) 0 . 2 5 6 . 0 0 . 3 3 3 0 . 6 a b 3 . 9 1 a ( 1 2 ) 0 . 2 5 1 2 . 0 0 . 2 6 2 8 . 3 a 0 . 5 6 c ( 2 7 ) 0 . 2 5 1 6 . 0 0 . 1 2 2 4 . 2 a 0 . 7 0 c ( 2 1 ) 0 . 1 2 5 3 . 0 0 . 3 4 4 1 . 2 c 3 . 8 9 a ( 1 4 ) 0 . 063 3 . 0 0 . 3 0 3 7 . 9 c 4 . 2 8 a ( A) a S t a n d a r d SH medium. b Means w i t h i n a column f o l l o w e d by the same l e t t e r a r e n o t s i g -n i f i c a n t l y d i f f e r e n t a t t h e 5% l e v e l by Duncan's m u l t i p l e range t e s t . 152 found. The C:N r a t i o i s reported to be the most important n u t r i t i o n a l parameter f o r l i p i d p r o d u c t i o n by f u n g i (44). Although i t al s o appears to be important i n root c u l t u r e s , the e f f e c t of other components should be i n v e s t i g a t e d , e.g., the e f f e c t of d i f f e r e n t l e v e l s of myo-inos i t o 1 and phosphate and ammonium versus n i t r a t e . Hagimori et a l . (.13) found that r e d u c t i o n of b a s i c n i t r o g e n by o n e - t h i r d i n c r e a s e d the y i e l d of d i g i t o x i n from D i g i t a l i s purpurea without suppressing growth. The l e v e l s of sucrose optimal f o r p o l y a c e t y l e n e s y n t h e s i s i n t h i s experiment (6%) were c o n s i d e r a b l y lower than the 40% l e v e l of glucose reported to be optimal f o r many fu n g i (44). Zenk (46) has noted that i n many cases I n c r e a s i n g sucrose l e v e l above the normal 2-3% improves secondary product y i e l d . The c o n c e n t r a t i o n of sucrose was a key f a c t o r i n o b t a i n i n g very high y i e l d s of r o s m a r i n i c a c i d from Coleus c u l t u r e s , 7% sucrose being optimal (47). S i m i l a r r e s u l t s have been reported f o r s h i k o n i n p r o d u c t i o n from ginseng (41), anthraquinones from Cinchona pubescens (29), p h e n o l i c s from Paul's s c a r l e t rose (24) , and a l k a l o i d s and polyphenols from Catharanthus roseus (25) . Although not a l l secondary products are i n c r e a s e d by higher l e v e l s of sucrose (41), the number of such cases suggests that sugar l e v e l i s one of the f a c t o r s that should be evaluated e a r l y i n any attempt to enhance product y i e l d . 153 E f f e c t of K i n e t i n on Roots The r e s u l t s of growing ]S. alba roots i n medium supplemented with k i n e t i n are shown i n F i g . 30 and Table 12. No auxin was added to these c u l t u r e s . Figure 30 shows that low l e v e l s of k i n e t i n i n c r e a s e d growth moderately, but at 0.135 mg/1 growth i n c r e a s e d s h a r p l y and a d d i t i o n a l k i n e t i n had l i t t l e effect on growth^ however, t o t a l product i n c r e a s e d over the expected l e v e l (from F i g . 29) of 2.5 mg/g f o r 11 day c u l t u r e s , to a maximum of over 5.0 mg/1 at the highest l e v e l of k i n e t i n (1.2 mg/1). There was a l s o a r e g u l a r e f f e c t on composition as shown i n Table 12. The weight percent of ETE-ol, ETE and the p u t a t i v e EDE-OAc were changed r e l a t i v e l y l i t t l e by the l e v e l of k i n e t i n . The percentage of PDE-OAc and ETE-OAc, however, showed r e g u l a r and r e c i p r o c a l changes with k i n e t i n l e v e l ; the former i n c r e a -sed by 14.4% from the lowest to the highest k i n e t i n l e v e l while the ETE-OAc d e c l i n e d by 11.5%. The expected percentage of PDE-OAc f o r 11 day c u l t u r e s grown with 0.5 mg/1 NAA would be 36-40% ( F i g . 29) which agrees with the l e v e l of t o t a l p o l y -a c e t y l e n e s i n c u l t u r e s grown at k i n e t i n l e v e l s of 0.045 and 0.135 mg/1, i . e . , at the l e v e l s marking the t r a n s i t i o n to i n c r e a s e d growth and product. Roots grown at the highest l e v e l of k i n e t i n were t h i c k e r and s h o r t e r than those grown at lower l e v e l s . In t h i s respect they resembled roots grown i n l i g h t , transformed r o o t s , and o l d e r r o o t s ; a l l of which had higher l e v e l s of PDE-OAc. These p o i n t s suggest that the l e v e l of c y t o k i n i n s may be one of the f a c t o r s r e s p o n s i b l e f o r both the thickened growth and the i n c r e a s e d PDE—OAc l e v e l . Other, unknown f a c t o r s seem to be i n v o l v e d i n determining the l e v e l 154 -V T DRY W T / T O T A L • F L A S K (G) KJNETIN (MG/L) F i g . 3 0 . E f f e c t o f v a r y i n g k i n e t i n c o n c e n t r a t i o n on t o t a l p o l y a c e t y l e n e s and g r o w t h o f c u l t u r e d B. a l b a r o o t s . Each p o i n t i s t h e a v e r a g e f o r t h r e e f l a s k s . B a r s show + 1 SD. 155 Table 12. E f f e c t of v a r y i n g k i n e t i n c o n c e n t r a t i o n of medium on p o l y a c e t y l e n e composition i n Ji. alba r o o t s . Roots were grown under standard c o n d i t i o n s except f o r omission of NAA. Figures are the average of three f l a s k s f o r each c o n c e n t r a t i o n . C o e f f i -c i e n t of v a r i a t i o n i n parentheses. K i n e t i n (mg/1) P o l y a c e t y l e n e composition (wt % of t o t a l ) PDE-0Ac b EDE-OAc ETE-ol ETE- OAc ^ ab ETE 0.0 29 . 9 14 . 4 1.1 53 . 0a 1.6 (0.4) (0.5) (1.9) (0. 8) (9.2) 0.005 33. l b 12 . 5 1. 1 51. 8 a 1. 5 (0.1) (19.1) (.8.9) (5. 9) (4.4) 0. 015 32.5b 11.3 1.3 52 . 9a 2 . 0 (8.5) (20.6 (18.2) (1. 8) (10.0) 0. 045 36 . 3 12 . 0 0.9 48 . 9 1. 9 (1.9) (0.8) (1.0) (1. 9) (8.3) 0. 135 41. 3 11. 4 0.9 44 . 7 1. 7 (2.3) (6.6) (7.0) (8. 8) (12.4) 0.405 44.2a 12 . 6 1. 0 40. 3b 1. 9 (1.4) (4.1 (4.2) (0. 2) (4.3) 1.22 44.3a 10 . 2 1.0 41. 2b 3.3 (0.8) (2.2) (.5.9) (1. 5) (3.2) 3 T o t a l f o r ETE-OAc (A)+(B). b Means w i t h i n a column followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t at the 5% l e v e l by Duncan's m u l t i p l e range t e s t . 156 of product. Butcher and S t r e e t (5) have d i s c u s s e d s t u d i e s showing an in c r e a s e d percentage of dry weight f o r roots grown with auxin and provided evidence that t h i s was due to i n c r e a s e d sucrose uptake. In a l l the s t u d i e s reported here with J3. alba , however, in c r e a s e s i n k i n e t i n r e s u l t e d i n a decreased f r e s h weight to dry weight r a t i o and auxin i n i n c r e a s e d r a t i o s . Whether k i n e t i n s t i m u l a t e d sucrose uptake i s not known but the e f f e c t s were s i m i l a r to those from i n c r e a s i n g the carbo n / n i t r o g e n r a t i o . E f f e c t of NAA on Roots The r e l a t i o n s h i p between NAA c o n c e n t r a t i o n and p o l y a c e t y l e n e l e v e l and growth i s shown i n F i g . 31A. Growth was n e a r l y a l i n e a r f u n c t i o n of NAA l e v e l except at the highest l e v e l . Product l e v e l showed a more complex r e l a t i o n s h i p with higher l e v e l s of NAA c l e a r l y i n h i b i t i n g product accumulation. Due to the amount of t i s s u e formed at the higher l e v e l s of NAA, n u t r i e n t l i m i t a t i o n could have a f f e c t e d the amount of product, however F i g . 29 shows that a d e c l i n e i n product d i d not take place u n t i l t i s s u e dry weight i n excess of 0.64 g / f l a s k was formed and t h i s l e v e l was only reached at the hi g h e s t NAA l e v e l . The standard l e v e l of 0.5 g/1 was near the p o i n t of maximum product. Changes i n composition are shown i n F i g . 31B. Changes i n the p r o p o r t i o n of PDE-OAc were l e s s pronounced than with k i n -e t i n but showed the same trend. In g e n e r a l , roots showed an i n c r e a s e i n PDE-OAc formation as e i t h e r NAA or k i n e t i n l e v e l s i n c r e a s e d . It i s i n t e r e s t i n g that both k i n e t i n and NAA i n c r e a s e d 157 T O T A L PR ODUCT (MG/G D. W.) 4.0-3.0-2-0-1-0-DRY W T / F L A S K (G) 0.01 0-0 4 NAA (MG/L) 0.16 0.6 4 1.28 5 0-40 -30 20 10 PDE-OAC WT.% T 0 L P R O D , B r i i i 0.01 0-0 4 0.16 0.64 NAA (MG/L) 1.28 F i g . 31. E f f e c t of NAA c o n c e n t r a t i o n on c u l t u r e d r o o t s of J i . a l b a . A) E f f e c t on g r o w t h and p r o d u c t l e v e l . B) E f f e c t on p o l y a c e t y l e n e c o m p o s i t i o n . Each p o i n t i s t h e a v e r a g e f o r t h r e e f l a s k s . B a r s show + 1 SD. 158 growth but only k i n e t i n showed an i n c r e a s e i n product over the l e v e l of roots grown i n medium l a c k i n g hormones. The e f f e c t of hormones on p r o d u c t i o n of secondary products i n t i s s u e c u l t u r e s can be c r i t i c a l i n some cases, e.g. (29), but there seem to be no general r u l e f o r d e c i d i n g which to use f o r any given product. I n h i b i t i o n by high l e v e l s of auxin, however, i s f r e q u e n t l y reported (3,25) and t h i s was found f o r p o l y a c e t y l e n e p r o d u c t i o n at l e v e l s of NAA which promoted the most r a p i d growth i n Bidens. E f f e c t of L i g h t on Roots The e f f e c t of l i g h t on root c u l t u r e s grown with k i n e t i n or NAA i s shown i n Table 13. Two f e a t u r e s were immediately apparent: 1) the product r a t i o s of i l l u m i n a t e d c u l t u r e s were q u i t e d i f f e r e n t from dark-grown r o o t s ; the p r o p o r t i o n of PDE-OAc was over twice that of dark c u l t u r e s . 2) K i n e t i n r e s u l t e d i n 2 to 3 times higher t o t a l product l e v e l s than NAA. T o t a l product l e v e l was h i g h e s t at the higher k i n e t i n l e v e l , confirm-ing the e f f e c t seen with dark-grown c u l t u r e s i n F i g . 30. Com-pa r i s o n of light-grown k i n e t i n c u l t u r e s with s i m i l a r dark-grown c u l t u r e s ( F i g . 30) showed that l i g h t reduced the product l e v e l to about 1/3 of dark c o n d i t i o n s . However, an even sharper decrease was seen i n NAA and hormone'-free c u l t u r e s ; t o t a l pro-duct l e v e l was l e s s than 1/6 and 1/16 of the dark l e v e l s . K i n e t i n markedly enhanced product l e v e l i n light-grown c u l t u r e s r e l a t i v e to NAA and hormone-free media. This r e s u l t was con-t r a r y to the e f f e c t shown on transformed shoots; k i n e t i n up to 3.3 mg/1 caused decreases i n l e a f product. E v i d e n t l y l e a f t i s s u e responds to the same hormonal environment very d i f f e r -T a b l e 13. E f f e c t o f l i g h t and hormones on r o o t g r o w t h and p o l y a c e t y l e n e c o m p o s i t i o n . C o e f f i c i e n t of v a r i a t i o n i n p a r e n t h e s e s . Each c o m b i n a t i o n i s t h e a v e r a g e of f i v e f l a s k s . I l l u m i n a t e d r o o t s r e c e i v e d 1400 l u x c o n t i n u o u s l i g h t ( + ) . Hormone (mg/1) L i g h t Dry wt/ f l a s k (g) P r o d u c t PDE-OAc c ompos i t i o n EDE-OAc (wt %) ETE-OAc 3 p r o d u c t (mg/g d.w.) NAA 0.67 30. 1 9 . 0 60 . 9 3.2 (0.5 ) (12) (19) (35) (4) ( 1 2 ) NAA + 0.36 74 . 3 7 . 0 18. 7 0 . 5 (0 . 5 ) (16) (3) (9) (10) ( 1 0 ) None + 0 . 04 83.5 3 . 2 13 . 3 0.2 (13) (3) (11) (28) (44) K i n e t i n + 0. 16 82.8 4.9 12 . 4 1.2 (0.1 ) (27) (4) (37) (13) (18) K i n e t i n + 0 . 21 70 . 7 8.0 21.1 1. 7 (1.0 ) (26) (3) (5) (9). ( 2 1 ) I n c l u d e s ETE and E T E - o l , t h e t o t a l o f w h i c h amounted t o l e s s t h a n 1% f o r i l l u m i n a t e d c u l t u r e s . 160 e n t l y from r o o t s ; the p r o p o r t i o n of PDE-OAc i n i l l u m i n a t e d leaves i n c r e a s e d s l i g h t l y with i n c r e a s i n g k i n e t i n . Light-grown r o o t s , however, showed decreasing PDE-OAc with i n c r e a s i n g k i n -e t i n . Changes i n composition Between l i g h t and dark-grown Ruta graveolens c u l t u r e s producing Cg and C^ v o l a t i l e o i l com-ponents have a l s o Been reported (7); light-grown c u l t u r e s resembled leaves i n composition and dark-grown c u l t u r e s were s i m i l a r to r o o t s . Changes i n n u t r i e n t c o n d i t i o n s had l i t t l e e f f e c t i n t h i s system; composition of the v o l a t i l e o i l i n c u l -tures was c h i e f l y determined By l i g h t . Bidens roots seem to Be constrained By morphology to produce a p a r t i c u l a r set of of a c e t y l e n e s , and although the r e l a t i v e p r o p o r t i o n s of these may Be a l t e r e d , sharp d i s c o n t i n u i t i e s i n the type of product or the form of the product were not oBserved. E f f e c t of D e d i f f e r e n t i a t i o n on P o l y a c e t y l e n e s of Transformed  Roots In t h i s experiment transformed roots (nop +) were used to examine the e f f e c t of d e d i f f e r e n t i a t i o n on p o l y a c e t y l e n e pro-d u c t i o n . Transformed roots were s i m i l a r to normal roots i n types of p o l y a c e t y l e n e s present and i n the enhancement of growth by NAA; although 0.5 mg/1, as used here, was probably higher than necessary f o r good growth^ i t was used f o r c o n s i s -tency. The primary d i f f e r e n c e s between the two types of roots was the l e v e l of PDE-OAc, which was about 10% higher than i n normal r o o t s . Transformed roots were used because they seemed to be more s e n s i t i v e to k i n e t i n than normal r o o t s , showing a gre a t e r tendency to form c a l l u s i n the o l d e r p o r t i o n of the roots and to shed c e l l s i n t o the medium. K i n e t i n was p r e f e r r e d 161 as the hormone s i n c e e a r l i e r experiments had shown i t to i n c r e a s e product l e v e l at the c o n c e n t r a t i o n necessary f o r d e d i f f e r e n t i a t i o n . The d e t a i l s on s e p a r a t i o n of the roots i n t o d i f f e r e n t regions and promotion of d e d i f f e r e n t i a t i o n are shown i n the legend of Table 14. Two main trends were evident from the data: 1) product l e v e l decreased s h a r p l y as the o r g a n i z a t i o n of the c u l t u r e decreased; the youngest p o r t i o n of the root had the hi g h e s t p o l y a c e t y l e n e l e v e l and the o l d e s t p o r t i o n , the lowest l e v e l . Suspension c u l t u r e l e v e l s were the lowest of the s e r i e s , i n d i c a t i n g that o r g a n i z a t i o n played a c r u c i a l r o l e i n accumu-l a t i o n of p o l y a c e t y l e n e s . 2) The second trend was the change i n composition of the c u l t u r e s . Within the organized root EDE-OAc was s i m i l a r i n p r o p o r t i o n to normal r o o t s , however as o r g a n i z a t i o n was l o s t the p r o p o r t i o n of EDE-OAc i n c r e a s e d between three and s i x times to become the dominant a c e t y l e n e compound present. Both PDE-OAc and ETE-OAc decreased i n per-centage. Another f e a t u r e of composition i n the d i s o r g a n i z e d t i s s u e was the appearance of small amounts of PHT and the p u t a t i v e d i e n t r i y n e n e acetate (DTE-OAc) which were otherwise not found i n c u l t u r e d r o o t s . The change i n r e l a t i v e percent-ages of the two forms of ETE-OAc was a l s o s t r i k i n g ; ETE-OAc(A) showed a steady i n c r e a s e r e l a t i v e to the (B) isomer. No other c u l t u r e c o n d i t i o n s t u d i e d r e s u l t e d i n the near e q u a l i t y of these two isomers. None of the e f f e c t s from the parameters s t u d i e d i n t h i s chapter could reasonably be e x t r a p o l a t e d to give the type of composition found i n the c a l l u s e d roots and suspension c u l t u r e s , T a b l e 14. E f f e c t of d e d i f f e r e n t i a t i o n on c u l t u r e s o f t r a n s f o r m e d r o o t s . C a l l u s i n g was p r o m o t e d by g r o w i n g r o o t s i n l i q u i d medium w i t h 1.5 mg/1 k i n e t i n f o r one p a s s a g e and t r a n s f e r r i n g t h e most h e a v i l y c a l l u s e d r o o t s t o f l a s k s f o r t h e n e x t p a s s a g e . The f r e e c e l l s f rom t h e same p a s s a g e were u s e d as t h e i n o c u l u m f o r s u s p e n s i o n c u l t u r e s . Root t i p was t h e f i r s t 2 cm of r o o t f r o m t h e t i p , m i d d l e r o o t was t h e n e x t 3-4 cm, and t h e r e m a i n d e r o f t h e r o o t was the o l d e s t r o o t . R o o t s and s u s p e n s i o n c u l t u r e s grown u n d e r s t a n d a r d c o n d i t i o n s e x c e p t as n o t e d . T i s s u e s t a g e R e l a t i v e c a l l u s P o l y a c e t y l e n e c o m p o s i t i o n (wt % o f t o t a l ) T o t a l PDE-OAc EDE-OAc DTE-OAc PHT E T E - o l ETE-OAc p r o d u c t A B (mg/g d.w.) Root t i p 47 . 1 9 . 2 0.4 4.3 39.0 2.72 M i d d l e r o o t 43.7 9.0 0.6 5.1 41.6 2.36 O l d e s t r o o t 45 . 8 9 . 5 0.9 6.2 37.7 0.64 C a l l u s e d r o o t ++ 28 . 8 30. 2 1. 9 3.2 1.7 13.8 20.4 0.49 C a l l u s e d r o o t +++ 19 . 9 55.4 1.1 1.4 8.2 11.2 0.06 S u s p e n s i o n c u l t u r e 31. 2 41. 4 1.3 3.0 5.4 9.0 8.7 0.02 Grown w i t h 1.5 mg/1 k i n e t i n . S y m b o l s : 0, no v i s a b l e c a l l u s i n g ; +, ++, +++ i n c r e a s i n g c a l l u s f o r m a t i o n ; -, no d e t e c t a b l e compound. 163 The appearance of PHT e s p e c i a l l y was q u i t e unexpected. The i m p l i c a t i o n i s that the o r g a n i z a t i o n i n roots determines both l e v e l and composition and l o s s of o r g a n i z a t i o n not only changes the r e l a t i v e p r o p o r t i o n s of t y p i c a l compounds but a l s o allows the s y n t h e s i s of compounds e i t h e r normally not present or pre-sent at much lower l e v e l s . Staba (38) has suggested that shoot or root c u l t u r e s could be modified f o r growth i n fermentors by using high l e v e l s of growth r e g u l a t o r s to reduce t h e i r s i z e . An example he c i t e d was degenerated Chrysanthemum shoot c u l t u r e s which were grown with 20 ppm benzyladenine. Although i t was simple to d e r i v e l e s s organized c u l t u r e s from roots i t d i d not look promising to use these f o r f u r t h e r s t u d i e s , due to the low l e v e l s of product. One problem i s that i f p o l y a c e t y l e n e s are s e c r e t e d i n t o the medium i n c e l l suspensions then they are much more l i a b l e to degradation, e s p e c i a l l y i f l i g h t i s present. A p o s s i b l e method f o r i n c r e a s i n g the p r o d u c t i o n of p o l y a c e t y l e n e s under these c o n d i t i o n s i s suggested by the work of Beiderbeck (1) and others (2) who show that a suspension c u l t u r e of M a t r i c a r i a  chamomi11a tumour c e l l s y i e l d a marked i n c r e a s e i n the recovery of l i p o p h i l l i c compounds when the c e l l s are c u l t i v a t e d i n a two phase system c o n s i s t i n g of n u t r i e n t medium and s y n t h e t i c t r i g l y c e r i d e , which accumulates the product. Production of c o n i f e r y l aldehyde, i n the same c u l t u r e , i n c r e a s e d 6 0 - f o l d by adding a c t i v a t e d c h a r c o a l to the medium and r e c o v e r i n g adsorbed product from the c h a r c o a l (23), A s i m i l a r system could work with suspension c u l t u r e s of J3. alba tumour c e l l s ; a l i p o p h i l l i c 164 phase could e f f i c i e n t l y accumulate small amounts of non-polar p o l y a c e t y l e n e s r e l e a s e d i n t o the medium. Charcoal might work i n r e c o v e r i n g a c e t y l e n i c a l c o h o l s and, perhaps, some of the p o s t u l a t e d and known a c i d i c i n t e r m e d i a t e s . Use of the method with root c u l t u r e s or suspension c u l t u r e s might s t a b i l i z e ex-c r e t e d product and y i e l d a b e t t e r estimate of the amount of p o l y a c e t y l e n e s a c t u a l l y produced by c u l t u r e d r o o t s . S t a b i l i t y of Root C u l t u r e s The f i n a l t a b l e of t h i s chapter i s a c o m p i l a t i o n of the data f o r c o n t r o l c u l t u r e s which were run f o r each experiment, i n c l u d i n g c o n t r o l s f o r some experiments not reported here. The t o t a l time that t h i s p a r t i c u l a r i s o l a t e was a c t i v e l y sub-c u l t u r e d was f i v e months, however the l i n e had been maintained on agar p l a t e s as d e s c r i b e d i n M a t e r i a l s and Methods f o r n e a r l y three years p r i o r to being a c t i v e l y c u l t u r e d . The r e s u l t s , shown i n Table 15, g e n e r a l l y showed repro-d u c i b i l i t y from run to run, however an o v e r a l l d r i f t i n the data over time was c l e a r , e s p e c i a l l y between weeks 8 and 13. When the data was grouped i n t o two c l a s s e s , weeks 3-8 and 13-20, and t e s t e d f o r s i g n i f i c a n c e using a t w o - t a i l e d t - t e s t , the means f o r PDE-OAc, ETE-OAc (A+B), t o t a l product, and dry weight were a l l s i g n i f i c a n t l y d i f f e r e n t (P<0.01) using e i t h e r group as the standard. The s i g n i f i c a n c e of the changes i n the minor components was not c a l c u l a t e d . Bidens root c u l t u r e s , then, showed s t a t i s t i c a l l y s i g n i f i c a n t changes over time i n a l l the parameters measured; composition, t o t a l product, and growth. The c u l t u r e s appeared to change between weeks 8 and 13. 165 T a b l e 15. S t a b i l i t y o f c o m p o s i t i o n , p o l y a c e t y l e n e l e v e l , and g r o w t h o v e r t i m e of B. a l b a r o o t c u l t u r e s . F i g u r e s a r e t h e a v e r a g e of N f l a s k s f o r e a c h age . C u l t u r e s grown u n d e r s t a n d a r d ' c o n d i t i o n s , O r i g i n o f r e i s o l a t e d e s c r i b e d i n t e x t . P r o d u c t c o m p o s i t i o n (wt % of t o t a l ) T o t a l Dry wt/ Age N PDE- EDE ETE- ETE- OAc ETE p r o d u c t f l a s k ( w e e k s) OAc OAc o l A B (mg/g d.w •) (g) 3 3 36 . 1 8.6 0. 7 5.5 47.3 1.8 3.4 0 .43 5 4 34 . 3 9 . 1 0.6 2 . 1 52 . 1 1.8 3.4 0.27 6 3 38.8 6 . 9 0.3 4.7 47 . 6 1. 7 4 . 9 0 . 40 8 2 35 . 7 8.9 0. 7 3 . 5 49 . 5 1.7 3.2 0. 35 8 3 36 . 1 9 . 0 0.8 6.6 45 . 3 2 . 2 4 . 5 0.31 13 3 41. 4 6.7 0.8 3.3 45.5 2 . 3 1.9 0 .62 14 3 43 . 4 3. 7 0 . 9 2 . 0 47 . 9 2 . 1 2.9 0.34 16 2 44 . 6 7 . 6 0.6 5 . 7 39 . 8 1.7 2 . 1 0.57 17 2 37.6 8.3 0.9 2.1 48 . 7 2 . 4 3.6 0.29 19 3 44.2 7 . 7 0.8 2 . 3 42 . 6 2 . 4 3.2 0 .60 20 2 44.8 8 . 4 1. 1 2 .1 41. 8 1. 7 3.4 0 . 66 a 3 2 44 . 9 7 . 6 0.8 2 . 5 42.5 1.7 2 . 3 0.60 S t a t i s t i c a l A n a l y s i s (A+B) 3-8 x = 3 6.7 52 . 3 ** 3.9 0 . 3 4 X * N=15 SE= 0 .82 0.76 0.243 0. 021 13- x = 42 . 7 47 . 6 2 . 8 0.51 20 N=15 SE= 0.72 0.76 0.224 0.039 a R e i s o l a t e . " " ( P < 0 . 0 1 ) . 166 During t h i s time they were being maintained i n l i q u i d medium without hormones and without a g i t a t i o n . A c t i v e growth was s t a r t e d by t r a n s f e r r i n g to the standard growth medium with shaking. As a check a new c u l t u r e was s t a r t e d from the same l i n e which had been s t o r e d on agar medium. A f t e r growing up and s u b c u l t u r i n g , the m a t e r i a l was harvested as u s u a l . This r e i s o l a t e i s shown i n Table 15. I n e x p l i c a b l y , i t showed almost i d e n t i c a l c h a r a c t e r i s t i c s to the second group of a c t i v e r o o t s ; d i f f e r i n g only i n t o t a l product l e v e l . A p o s s i b l i t y i s that an unnoticed change occurred i n e i t h e r the e n v i r o n -mental c o n d i t i o n s or i n the medium, r e s u l t i n g i n a s i m i l a r change i n both c u l t u r e s . Note though that transformed c a l l u s showed a d e c l i n e i n PDE-OAc over some 42 months i n c u l t u r e ( F i g . 23). C o n s i d e r i n g the more r a p i d growth of roots r e l a -t i v e to c a l l u s , the p h y s i o l o g i c a l ages of the c u l t u r e s which r e s u l t e d i n s i m i l a r changes i n composition were probably com-par a b l e . Whether the change i n the roots occurred as a r e s u l t of a chance s e l e c t i o n of roots with s l i g h t l y d i f f e r e n t char-a c t e r i s t i c s from the main stock c u l t u r e , from some change due to storage or medium, or from a developmental change of p h y s i o l -ogy i s u n c l e a r . Rucker et a l , (33) have repo r t e d that root c u l t u r e s of D i g i t a l i s purpurea produced c a r d i a c g l y c o s i d e s f o r two passages (two months) but were not detected on the t h i r d passage. Growth of roots a l s o i n c r e a s e d s u b s t a n t i a l l y with each sub-c u l t u r e . Steward and K r i k o r i a n (39) have s t a t e d that ", . . r o o t -t i p c u l t u r e s of many p l a n t s , (both d i c o t s and monocots) even-1 6 7 tually 'peter out'." This does not appear to be the case f o r B. alba root c u l t u r e s . However, i f a very s t a b l e l i n e was r e q u i r e d f o r experimental work, then more i n f o r m a t i o n on the long-term s t a b i l i t y of Bidens root c u l t u r e s would be r e q u i r e d . The ease with which a clone can be maintained on agar allows a l i n e to be e f f e c t i v e l y ' s tored' while i n a c t i v e use. However, the s t a b i l i t y of slow-growing agar c u l t u r e s must s t i l l be demon-s t r a t e d . I do not think that the s m a l l , but s i g i n i f i c a n t , change i n the root c u l t u r e s shown over the course of these experiments i n v a l i d a t e s the r e s u l t s or c o n c l u s i o n s drawn from them. 168 SUMMARY E x c i s e d r o o t c u l t u r e s o f B i d e n s a l b a grown i n d a r k n e s s w i t h 0.5 g/1 NAA i n l i q u i d SH medium were e v a l u a t e d f o r g r o w t h , p o l y a c e t y l e n e l e v e l and p o l y a c e t y l e n e c o m p o s i t i o n as n u t r i t i o n a l o r e n v i r o n m e n t a l c o n d i t o n s were a l t e r e d . Factors w h i c h i n c r e a s e d p o l y a c e t y l e n e s i n r o o t s above t h e s t a n d a r d l e v e l w e r e : l a t e l og. p h a s e g r o w t h , l o w e r e d t e m p e r -a t u r e , h i g h l e v e l s of k i n e t i n , and h i g h s u c r o s e t o n i t r a t e r a t i o s . F a c t o r s w h i c h d e c r e a s e d p o l y a c e t y l e n e l e v e l s w e r e : l o g and s t a t i o n a r y p h a s e g r o w t h , h i g h t e m p e r a t u r e , l o w s u c r o s e to n i t r a t e r a t i o s , h i g h l e v e l s o f NAA, and i l l u m i n a t i o n . C o m p o s i t i o n was s h i f t e d t o i n c r e a s e d s y n t h e s i s o f p h e n y l d i y n -ene a c e t a t e by h i g h l e v e l s o f k i n e t i n and i l l u m i n a t i o n . . T r a n s f o r m e d r o o t s were e v a l u a t e d f o r t h e e f f e c t o f o r g a n i -z a t i o n on p r o d u c t l e v e l and c o m p o s i t i o n . Young r o o t t i p s had t h e h i g h e s t l e v e l o f p r o d u c t and t h e o l d e s t , b a s a l , r e g i o n o f r o o t s , t h e l o w e s t . D i s o r g a n i z a t i o n o f r o o t s , i n d u c e d by h i g h k i n e t i n l e v e l s , r e s u l t e d i n low l e v e l s of p r o d u c t w i t h a q u i t e a t y p i c a l c o m p o s i t i o n . As c u l t u r e s , r o o t s showed a s m a l l b u t s t a t i s t i c a l l y s i g n i f i c a n t c hange i n c h a r a c t e r i s t i c s o v e r t i m e . N e v e r t h e l e s s , j3. a l b a r o o t s a r e a s e n s i t i v e s y s t e m f o r i n v e s t i g a t i n g f a c t o r s a f f e c t i n g p o l y -a c e t y l e n e s y n t h e s i s . 169 LITERATURE CITED L. B e i d e r b e c k , R. 1982. Two-phase c u l t u r e — a method f o r t h e i s o l a t i o n o f l i p o p h i l i c s u b s t a n c e s f r o m p l a n t s u s p e n -s i o n c u l t u r e s ( i n German). Z. P f l a n z e n p h y s i o l . 108: 27-30. 2. B i s s o n , W., R. B e i d e r b e c k , and J . R e i c h l i n g . 1983. 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N a t u r w i s s e n . 64:585-586. 173 CHAPTER VII CHARACTERISTICS OF HYBRIDS BETWEEN BIDENS ALBA AND BIDENS PILOSA 174 INTRODUCTION The g e n e t i c c o n t r o l o f p o l y a c e t y l e n e s y n t h e s i s has r e c e i v e d l i t t l e s t u d y . However, i f c e l l c u l t u r e s a r e t o be u s e d f o r r e s e a r c h i n t o t h e c o n t r o l o f t h e pathway i t w o u l d be o f i n t e r -e s t t o know i f p o l y p l o i d y a f f e c t s s y n t h e s i s , and w h e t h e r t h e genes f o r s y n t h e s i s o f i n d i v i d u a l compounds a r e i n h e r i t e d i n a s i m p l e M e n d e l i a n p a t t e r n . I n t h e c o u r s e o f t h e s t u d i e s p r e s e n t e d e a r l i e r , B i d e n s p i l o s a ( s e n s u l a t o ) p l a n t s f r o m s e v -e r a l p a r t s o f t h e w o r l d were e x a m i n e d f o r PHT i n l e a v e s . One of t h e s e a c c e s s i o n s , T3. p i l o s a v a r . m i n o r ( B l u m e ) S h e r f f , f r o m B e l i z e C i t y , B e l i z e was c h o s e n f o r c r o s s i n g s t u d i e s w i t h B_. a l b a . C h a r a c t e r i s t i c s o f t h i s a c c e s s i o n w h i c h made i t s u i t a b l e f o r t h i s s t u d y were t h e f o l l o w i n g : 1) i t does n o t s y n t h e s i z e d e t e c t a b l e l e v e l s o f PHT ( o r any o t h e r p o l y a c e t y l e n e s ) i n i t s l e a v e s . 2) I t c r o s s e s r e a d i l y w i t h 13. a l b a as t h e f e m a l e p a r e n t . 3) S e v e r a l c h a r a c t e r s a p p e a r t o d i s t i n g u i s h t h e two t a x a s u f f i c i e n t l y t o a l l o w r e a d y i d e n t i f i c a t i o n o f F^ h y b r i d p l a n t s and s e l f s . The f o l l o w i n g p o i n t s were t o be i n v e s t i g a t e d : 1) e v i d e n c e f o r t h e h y b r i d i z a t i o n o f 13. a l b a and 13. p i l o s a ; 2) l e v e l s o f l e a f PHT i n F^ and S^ p l a n t s ; 3) i n h e r i t a n c e p a t t e r n and l e v e l s f o r l e a f p o l y a c e t y l e n e s i n F^ p l a n t s . The o n l y s t u d i e s i n w h i c h h i g h e r p l a n t c r o s s e s have been made and t h e o f f s p r i n g e v a l u a t e d f o r a c e t y l e n e s a r e t h o s e o f Van F l e e t ( 17) who w o r k e d w i t h C o r e o p s i s s a x i c o l a and C. g r a n d -i f l o r a . H i s r e s u l t s a r e d i f f i c u l t t o e v a l u a t e s i n c e no q u a n t i -t a t i v e d a t a were g i v e n f o r e i t h e r p o l y a c e t y l e n e l e v e l s o r f o r 175 numbers o f p l a n t s u s e d i n c r o s s e s , and a l l t h e d a t a a r e a p p a r -e n t l y f o r F^ p l a n t s . However, t h e f o l l o w i n g p o i n t s s u m m a r i z e t h e r e l e v a n t a s p e c t s of Van F l e e t ' s r e s u l t s : 1) r o o t p o l y a c e t -y l e n e s o f p a r e n t s were s i m i l a r and h y b r i d s showed no q u a l i t a -t i v e c h a n g e s . 2) Most of t h e p a r e n t s c o n t a i n e d PHT as t h e p r i n c i p l e a c e t y l e n e i n s t e m s and l e a v e s b u t some C. g r a n d i f l o r a and most o f t h e h y b r i d s had a m i x t u r e o f t r i e n e - t r i y n e and p h e n y l d i y n e n e . 3) H y b r i d s a p p e a r e d t o be a d d i t i v e f o r p o l y -a c e t y l e n e s f r o m b o t h p a r e n t s b u t t h e p r o p o r t i o n s of compounds d i f f e r e d w i d e l y f r o m t h e p a r e n t a l l e v e l s ( 1 7 ) . W i l d and h o r t -i c u l t u r a l f o r m s of D a h l i a s c a p i g e r a (2) and D. c o c c i n e a ( 8 ) have been compared b u t t h e g e n e t i c r e l a t e d n e s s o f t h e t a x a was n o t g i v e n i f known. S t u d i e s on t h e h e r i t a b i l i t y o f a c e t y l e n e l e v e l s i n f u n g i have been r e p o r t e d - by B i s t i s and A n c h e l ( 4 ) and C a r e y e t a l . ( 7 ) . L e v e l s i n d i k a r y o n s a p p e a r t o depend on t h e m a t i n g t y p e s i n v o l v e d as w e l l as on t h e component homo-k a r y o n s t r a i n s . However, t h e r e was no e v i d e n c e of a gene d o s a g e e f f e c t . H y b r i d i z i n g a PHT p r o d u c i n g t a x o n w i t h a c o m p a t i b l e n o n p r o -d u c i n g t a x o n c o u l d i n d i c a t e i f gene dose a f f e c t s PHT l e v e l o r i f PHT s y n t h e s i s i s d o m i n a n t o r r e c e s s i v e . I n my v i e w c r o s s i n g a p r o d u c e r and a n o n - p r o d u c e r may be c o n s i d e r e d t o be t h e i n v e r s e o f p o l y p l o i d i z a t i o n w i t h r e s p e c t t o t h e t r a i t PHT s y n -t h e s i s . L e v i n ( 1 1 ) has r e c e n t l y r e v i e w e d t h e l i t e r a t u r e on p o l y p l o i d y e f f e c t s on c e l l m e t a b o l i s m and i t i s c l e a r t h a t enzyme a c t i v i t i e s of p r i m a r y m e t a b o l i s m v a r y w i d e l y w i t h c h a n g e s i n p l o i d y , s o m e t i m e s s h o w i n g d o s a g e e f f e c t s , some-t i m e s c o m p e n s a t i o n , and s o m e t i m e s d e c r e a s i n g b e l o w c o m p e n s a t i o n 176 l e v e l — d e p e n d i n g on the enzyme and the plant examined. The e f f e c t of p l o i d y changes on secondary products, other than f l a v o n o i d s , seems to be l e s s i n v e s t i g a t e d but Levin (11) c i t e s a number of examples of drug a l k a l o i d s and terpenes showing l a r g e to moderate i n c r e a s e s i n q u a n t i t i e s with i n c r e a s e s i n p l o i d y l e v e l . Q u a l i t a t i v e changes were a l s o found i n one study. Grant (9) d i s c u s s e s gene dosage e f f e c t s observed i n vi t a m i n A content of corn endosperm and flower c o l o u r i n P a h l i a and Anti r r h i n u m . There seems to be no r e g u l a r c o r r e -l a t i o n between p l o i d y and product l e v e l even i n a u t o p o l y p 1 o i d s . The s i t u a t i o n with a l l o p o l y p l o i d s , as i n the present study, might be expected to be even more v a r i a b l e . 177 MATERIALS AND METHODS Crosses Seeds for the p a r e n t a l s t r a i n s of Bidens alba L. var. r a d i a t a (Schz. Bip.) B a l l a r d ex Melchert and B_. p i l o s a var. minor (Blume) Sherff were sown i n f l a t s of s t e r i l i z e d black s o i l , sphagnum, and l e a f mulch (2:1:1) and grown under L i f e l i n e f l u o r e s c e n t bulbs ( S y l v a n i a ) with an i l l u m i n a n c e of 6,000 lux 0 and photoperiod of 18 hours at a temperature of 25 C and 70% r e l a t i v e humidity. P a r e n t a l flower heads were s e l f e d by rubbing together f l o w e r i n g heads of the same plant and crossed by rubbing the other s p e c i e s . Crossed heads were tagged and r e c r o s s e d as new flowers opened. Heads s e t t i n g seed were c o l l e c t e d at maturity and d i v i d e d i n t o the r e s u l t i n g four groups ( i . e . s e l f s of each type and the two r e c i p r o c a l c r o s s e s ) . These seeds were then sown i n separate f l a t s and grown under g l a s s -house c o n d i t i o n s with the n a t u r a l l i g h t f o r Vancouver, B.C. from August to October. and S^ were sown i n 1981 and the T? i n 1982. To check fo r year to year v a r i a t i o n B. alba F2 _  p l a n t s were grown from the same seed source as the p a r e n t a l gen e r a t i o n and sown at the same time as the F^. A f t e r the f i r s t flowers i n a c l u s t e r of head began to open } a p l a s t i c bag, p e r f o r a t e d with approximately 50 needle h o l e s , was placed over the c l u s t e r and c l o s e d at the base by a s t r i p of p l a s t i c . Head diameter of unbagged heads was measured from 178 the t i p of one ray flower to the t i p of the opposite flower i n heads i n which a l l flowers had opened. The ray flowers were counted and plucked from the head and the length of the c o r o l l a measured from the p o i n t of attachment to the ovary to the most d i s t a n t extension of the p e t a l . P i l o s i t y was estimated v i s u a l l y with the p a r e n t a l p l a n t s s e r v i n g as +and ++ standards f o r 73. alba and 13. p i l o s a r e s p e c t i v e l y . Seed awns of F^ and F2 having a comparable length were counted, but s e t a t e l e s s than approximately l / 5 t h the length of the two main awns were not i n c l u d e d . Voucher specimens of the two parents and the F^ hybrids are deposited at UBC. E x t r a c t i o n s P r e l i m i n a r y r e s u l t s showed that PHT c o n c e n t r a t i o n i n leaves v a r i e d with p o s i t i o n on a given p l a n t . Since i t was not p r a c t i c a l to evaluate whole p l a n t s , the 3rd l e a f from the base, not i n c l u d i n g the c o t y l e d o n s , was used f o r q u a n t i t a t i o n of the S^ and F^. For the F^, the 3rd and 6th f u l l y opened leaves from the top of the plant were used. In a p r e l i m i n a r y set of e x t r a c t i o n s of 12 p l a n t s , t h e PHT content of both leaves at the t h i r d node was compared i n order to determine i f there was a p p r e c i a b l e v a r i a t i o n i n l e a f p a i r s . The c o r r e l a t i o n c o e f f i c i e n t was 0.98 and the n u l l hypothesis r e j e c t e d (P<0.01). Therefore only one l e a f was sampled per node. Only the lamina of the l e a f was used fo r e x t r a c t i o n . E x t r a c t i o n s were performed as d e s c r i b e d i n Chapter 2. PHT accounts f o r over 95% of the a c e t y l e n e s i n leaves of J3. alba (see Table 2 ) , and any a d d i t i o n a l acetylene chromo-phores are r e a d i l y detected i n the spectrum, e i t h e r by the 1 7 9 presence of a d d i t i o n a l a b s o r p t i o n peaks or by changes i n the r a t i o s between PHT peaks. Therefore the t o t a l PHT content was estimated from the UV spectrum of the combined petroleum ether e x t r a c t s . The amount was determined from the absorbance at 310 nanometers using the p u b l i s h e d e x t i n c t i o n c o e f f i c i e n t of 33,700 (10). A tangent to the v a l l e y s on e i t h e r side of the peak at 310 nm was drawn and peak height measured from t h i s b a s e l i n e . A s i m i l a r b a s e l i n e was drawn f o r the peak at 290 nm and the r a t i o checked f o r agreement with the r a t i o f o r pure compound. This procedure was adapted from Morton (14, p.60). Chromosome Counts Flower buds were f i x e d i n modified Carnoy's s o l u t i o n con-s i s t i n g of c h l o r o f o r m : absolute e t h a n o l : g l a c i a l a c e t i c a c i d (4:3:1, v/v/v) (1) and anthers s t a i n e d i n a c e t o - o r c e i n or aceto-carmine, mounted i n Hoyer's medium (3) and squashed. Counts were made on m e i o t i c p o l l e n mother c e l l s . S t a t i s t i c s C o r r e l a t i o n c o e f f i c i e n t s , c h i - s q u a r e , and student's t - t e s t s were c a l c u l a t e d according to S t r i c k b e r g e r (16). For t - t e s t s when more than two p o p u l a t i o n s were i n v o l v e d , means were e v a l -uated i n p a i r s . 180 RESULTS AND DISCUSSION F-^  V e g e t a t i v e Characters T y p i c a l leaves from p a r e n t a l and F^ p l a n t s are shown i n F i g . 32. Leaves are from the 6th node or h i g h e r . Bidens  alba t y p i c a l l y develops simple leaves at the f i r s t few nodes, changing to t r i f o l i a t e at about the 5th node. A few upper leaves have 5 l e a f l e t s . Bidens p i l o s a leaves are t y p i c a l l y t r i f o l i a t e at lower nodes and have 5-7 l e a f l e t s at the upper nodes. Leaves of F^ hybrids with 13. alba as the p i s t i l l a t e parent had 5-7 l e a f l e t s i n the upper nodes and 3 l e a f l e t s at the lower nodes. P u t a t i v e F^ p l a n t s with 13. p i l o s a as the p i s t i l l a t e parent were i n d i s t i n g u i s h a b l e from 13. p i l o s a i n a l l c h a r a c t e r s scored (Table 16), apparently the r e s u l t of s e l f i n g . P i l o s i t y of the F^ was i n t e r m e d i a t e to s i m i l a r to the 13. alba parents. The t r a i t i s not e a s i l y evaluated p r e c i s e l y , but i t does appear to be more v a r i a b l e i n the hybrids than i n the parents. About h a l f the F^ were l i k e B. alba and h a l f i n t e r m e d i a t e between the two parents. F l o r a l Characters The number of ray flowers per head were s i m i l a r i n the two parents (Table 16) and the d i f f e r e n c e was not s t a t i s t i c a l l y s i g n i f i c a n t . Both ray flower length and flower diameter were c l o s e l y s i m i l a r , as would be expected, and only the former (which had somewhat l e s s v a r i a n c e ) i s l i s t e d i n Table 16. The average length of ray flower c o r o l l a s i n the F^ was n e a r l y i n t e r m e d i a t e between the p a r e n t a l types, d i f f e r i n g by l e s s than 181 F i g . 32. T y p i c a l leaves from parental and F i h y b r i d Bidens p l a n t s . Leaves were taken from the s i x t h node, or higher, from the base. Column A) 15. alba; B) 15. p i l o s a ; C) B. alba ^ X B. p i l o s a ^ . T a b l e 16. C o m p a r i s o n o f c h a r a c t e r s of B. a l b a , B. p i l o s a , and h y b r i d s B. a l b a , S F , B. a l b a g B. p i l o s a , S. F^, Ti p i l o s a J Ray f l o w e r s no./head (SE) 5.61 (0.048) 5.47 (0.062) 5 . 09 ( 0 . 0 4 5 ) 5.24 (0 . 0 7 6 ) c o r o l l a l e n g t h (mm) (SE) 16 . 58 (0.091) 11.78 (0.13) 5 . 91 ( 0 . 0 3 3 ) 5.97 ( 0 . 0 5 8 ) s t y l e p r e s e n t N 254 151 + 153 50 No. awns/ achene (SE) N P i ' l o s i t y Time of f l o w e r i n i PHT (mg/g d.w.) (SE) 2 . 12 (0 . 0 1 6 ) 500 + mid Aug . -e a r l y Sept 4.8 (0.29) 22 2. 19 (0.025) 250 ± . + e a r 1 y t o l a t e S ept 2.8 (0.35) 15 3.11 (0.025 250 ++ e a r l y Oct e a r l y Nov nd 27 3 . 06 ( 0 . 0 2 2 ) 250 ++ e a r l y Oct e a r l y Nov nd 15 " D i f f e r s s i g n i f i c a n t l y from F , B. a l b a P < 0 . 0 5 ; P < 0 . 0 1 . A b b r e v i a t i o n s : N, number of u n i t s s c o r e d ; SE, s t a n d a r d e r r o r ; nd, not d e t e c t a b l e ; d.w, d r y we i g h t . P u t a t i v e h y b r i d . 183 184 5% from the a r i t h m e t i c mean. Parents and F^ d i f f e r e d s i g n i f i -c a n t l y f o r t h i s c h a r a c t e r . R e p r e s e n t a t i v e flower heads from the p a r e n t a l and F^ p l a n t s are shown i n F i g . 33. In Table 16 " s t y l e p r e s e n t " means that the ray flowers were scored "+" i f they had an evident s t y l e . According to B a l l a r d (1) these flowers are f e r t i l e i n 13. p i l o s a var. minor. The t r a i t i s suppressed i n the F^ as i n the 13. alba parent, suggesting that absence of a s t y l e i s dominant. Figure 34 shows t y p i c a l ray flowers f o r parents and F^; note the l a c k of a s t y l e i n 13. alba and the F^ and the i n t e r m e d i a t e c o r o l l a l e n g t h i n the F ^ D i f f e r e n c e s i n number of achene awns does not q u i t e reach s i g n i f i c a n c e at the 0.05 l e v e l ; the average p r o b a b i l i t y f o r the mean of the F^ and J3. alba compared to the mean of the B. p i l o s a S^ and p u t a t i v e F^ i s 0.08. The d i f f e r e n c e between J3. alba and the F^ i s not s i g n i f i c a n t . Time of f l o w e r i n g i n the F^ i s i n t e r m e d i a t e between the parents (Table 16). A l l seeds were pla n t e d on the same day i n i d e n t i c a l s o i l and grown together i n the same glasshouse. Chromosome Counts B a l l a r d (1) showed that 13. alba i s a t e t r a p l o i d (x=24) and — • p i l o s a a h e x a p l o i d (x=,36). Since these counts would imply that the h y b r i d would be p e n t a p l o i d (x=30), and probably i n f e r -t i l e , counts were made on s e l e c t e d p l a n t s used i n t h i s study. F i g u r e 35 shows a t r a c i n g of r e p r e s e n t a t i v e p r e p a r a t i o n s of telophase p o l l e n mother c e l l s and confirms B a l l a r d ' s r e s u l t s . M e iosis i n the hybrids seemed to be r e g u l a r . The c h a r a c t e r i s t i c s of the F^ p l a n t s may be summarized as 185 F i g . 35. P o l l e n mother c e l l squashes of telophase I n u c l e i . A) B. alba (x=24), B) F hyb r i d (x=30), C) B. p i l o s a (x=36). X 500 1 8 6 f o l l o w s : 1) F^ are i n t e r m e d i a t e between the two parents i n f l o w e r i n g time, ray flower c o r o l l a l e n g t h , and p i l o s i t y . 2) F^ are s i m i l a r to the B. p i l o s a parent i n l e a f morphology and s i m i l a r to the J i . alba parent i n achene awn number and i n lack of f e r t i l e ray f l o w e r s . 3) Hybrids have the expected chromosome number. PHT Content of Hybrids The mean PHT l e v e l i n hybrids was c l o s e to one-half the l e v e l i n the B. a l b a parent (Table 16) suggesting a gene dosage e f f e c t on product l e v e l . However, because of the l i m i -ted number of p l a n t s which were recovered from the c r o s s e s , and the high v a r i a n c e i n 15. alba and the F^ h y b r i d s , the values obtained were not s t a t i s t i c a l l y s i g n i f i c a n t . Nevertheless the data were not incompatible with a dosage e f f e c t and t h i s p o s s i -b i l i t y could be evaluated f u r t h e r i n the F^. PHT Levels i n F^ P l a n t s Progeny from s e l f e d F^ segregated i n t o p l a n t s with or without PHT i n l e a v e s . Figure 36 shows r e p r e s e n t a t i v e s p e c t r a f o r each type. The sharp peak at 250 nm (£l48,000) f o r PHT makes p o s s i b l e d e t e c t i o n of l e v e l s much lower than those found i n 13. a l b a , hence there was l i t t l e ambiguity i n c l a s s i f y i n g the p l a n t s f o r t h i s t r a i t . None of the p l a n t s showed any s i g -n i f i c a n t i n c r e a s e i n other acetylene chromophores. The f r e -quency d i s t r i b u t i o n of PHT l e v e l s f o r 65 F^ p l a n t s i s shown i n F i g . 37. Scores are the average of l e v e l s i n leaves at the 3rd and 6th nodes from the top. The d i s t r i b u t i o n i n both s e r i e s was s i m i l a r , with a c o r r e l a t i o n c o e f f i c i e n t of r=0.88 187 (N=58), the n u l l hypothesis (random c o r r e l a t i o n ) r e j e c t e d (P*0. 01) . A n a l y s i s of the F ^  data i s c o n s i d e r a b l y complicated by the d i f f e r e n c e i n p l o i d y l e v e l i n the two parents, as shown e a r l -i e r . The f e r t i l i t y and apparently r e g u l a r meiosis of the F^ are d i f f i c u l t to account f o r i n view of the presumed penta-p l o i d l e v e l . As F i g . 37 shows, seven of the 65 p l a n t s were PHT n e g a t i v e , i n d i c a t i n g that 13. a l b a chromosomes l i k e l y p a i r with p i l o s a homologues. A p o s s i b i l i t y i s that the base chomo-some number i s s i x i n s t e a d of twelve. This would allow r e g u l a r d i v i s i o n . However, Stuessy has r e c e n t l y reviewed the s y s t e -matics of the Heliantheae (.15) and there i s no i n d i c a t i o n that a base number of s i x occurs i n the s u b t r i b e c o n t a i n i n g Bidens, Coreopsidinae, and only four genera are reported to have a base of s i x . C a l c u l a t i o n of gamete r a t i o s and r a t i o s on the basis of a d e c a p l o i d F^ gives a very low frequency f o r the occurrence of the r e c e s s i v e (PHT n e g a t i v e ) : 1 i n 1190. Even i f doses of one and two B.alba chromosomes (versus a maximum of ei g h t i n the ? 2^ i s grouped with the n u l l c l a s s the expected frequency i s l e s s than h a l f of that found and the p r o b a b i l i t y (by chi-square t e s t ) i s much l e s s than 0.0.1. The r e s u l t s are c l e a r l y not l i k e l y to be e x p l a i n e d by p o s i t i n g a lOx F^. A second a l t e r n a t i v e i s to c a l c u l a t e the expected r a t i o s assuming the pent&ploid F^ produces 2n and 3n gametes with a l l combina-t i o n s e q u a l l y probable. L e t t i n g "A" be the alba a l l e l e and "a" the p i l o s a , the f o l l o w i n g combinations and r a t i o s are expected: 1 AA: 3 AAa: 6 Aaa: 6 Aa: 3 aa: 1 aaa. In the F^ 16 out of 400 p l a n t s should be r e c e s s i v e . The chi-square value of 1 8 8 F i g . 36. R e p r e s e n t a t i v e UV s p e c t r a o f p e t r o l e u m e t h e r e x t r a c t s of _B. a l b a X I i . p i l o s a F^ p l a n t s . A) T y p i c a l s p e c t r u m o f PHT p o s i t i v e l e a v e s ; B) t y p i c a l s p e c t r u m o f PHT n e g a t i v e l e a v e s . 189 no.of plants 0 .1 .2 .4 .6 .8 1.0 1.4 1.8 2.2 2.6 3.0 M 3-8 4-2 PHT (mg/g.d.w.) F i g . 37. F r e q u e n c y d i s t r i b u t i o n of PHT l e v e l s i n 65 F2 p r o g e n y of Ti. a l b a X Ti. p i l o s a . Mean PHT l e v e l f o r s e v e n Ti. a l b a c o n t r o l p l a n t s was 4.9 mg/g d r y wt. 30.4 a g a i n has a p r o b a b i l i t y f a r l e s s t h an 0.01 and t h i s hypo-t h e s i s i s r e j e c t e d . I f o n l y 2n gametes a r e assumed to be formed t h e n t h e f o l l o w i n g g a m e t i c r a t i o s a r e e x p e c t e d : 1 AA: 6 Aa: 3aa and t h e c o r r e s p o n d i n g z y g o t e r a t i o s : 1 AAAA: 12 AAAa: 42 AAaa: 36 Aaaa: 9 a a aa. Under t h i s a s s u m p t i o n the c h i - s q u a r e v a l u e i s 0.845 w h i c h has a p r o b a b i l i t y g r e a t e r t h a n 0.1. T h i s would i m p l y t h a t PHT s y n t h e s i s s e g r e g a t e s as a s i n g l e - f a c t o r M e n d e l i a n t r a i t . However, t h i s e x p l a n a t i o n i s c o u n t e r to the chromosome number f o u n d i n p o l l e n mother c e l l s . A s e c o n d a s p e c t of t h e d a t a i n F i g . 37 i s t h e asymmetry of the d i s t r i b u t i o n . Only one p l a n t had a l e v e l c o m p a r a b l e to t h e c o n t r o l p l a n t s (4.2 mg/g v s . 4.9 mg/g r e s p e c t i v e l y ) w i t h t h e b u l k of t h e s c o r e s c l u s t e r e d a r o u n d 0.6 mg/g. A s e a -s o n a l d i f f e r e n c e seems n o t to be i n v o l v e d s i n c e c o n t r o l p l a n t s grown from seed from the same p o o l as t h e p a r e n t a l p l a n t s i n T a b l e 16, and sown a t the same t i m e , gave a l e v e l v e r y c l o s e 190 to that of the (4.9 mg/g vs. 4.8 mg/g). The r e s u l t s c l e a r l y show that PHT l e v e l Is not p r o p o r t i o n a l to gene dosage. A l l of the a l t e r n a t i v e s d i s c u s s e d r e s u l t i n roughly symmetrical d i s t r i b u t i o n s with maxima at combinations having from two-f i f t h s to one-half of the chromosomes from 13. a l b a . If a simple dosage e f f e c t was present then the d i s t r i b u t i o n i n F i g . 37 would peak at about 2.4 mg/g f.w. Apparently a d d i t i o n a l l o c i which a f f e c t l e v e l but not presence or absence of the t r a i t are s e g r e g a t i n g i n the F2. Future Work Evidence presented by B a l l a r d (1) suggests that the two taxa examined here are d e r i v e d from a common parent, 13. odo-ra t a . Given the dominant c h a r a c t e r of PHT s y n t h e s i s t h i s would suggest that B. p i l o s a var. minor has e i t h e r l o s t the c a p a c i t y to a c t i v a t e a f u n c t i o n a l gene set or to have acquired the a b i l i t y to convert PHT to a d e r i v a t i v e . There i s no sug-g e s t i o n of the l a t t e r e i t h e r i n the l i t e r a t u r e (6, Table VIII) or i n the present r e s u l t s ; PHT appears to be a t e r m i n a l pro-duct. It would be i n t e r e s t i n g , then, to t e s t a range of non-producers l i k e p i l o s a var. minor ( e s p e c i a l l y d i p l o i d l i n e s ) i n crosses with producers to see i f trans complementation i s p o s s i b l e . An F^ with enhanced s y n t h e s i s of PHT could serve as an i n i t i a l i n d i c a t o r f o r l i n e s i n which complementation might be o c c u r r i n g . Levy, i n r e l a t e d s t u d i e s , (12) has i n t e r -preted the gain of f l a v o n o i d compounds i n a u t o p l o i d c u l t i v a r s of Phlox drummond i i as f u n c t i o n a l d e r e p r e s s i o n of p r e v i o u s l y s i l e n t s t r u c t u r a l genes, a f a c t Mears (13) has taken to imply that the genes were a c t i v e i n w i l d p l a n t s but became i n a c t i v e 191 i n the c u l t i v a r s and were r e a c t i v a t e d by chromosome doubling with c o l c h i c i n e . The e x i s t e n c e , according to Bohlmann (5,6) of another s p e c i e s of Bidens and s e v e r a l s p e c i e s of Coreopsis i n which PHT i s s y n t h e s i z e d i n roots and not leaves r a i s e s a qu e s t i o n about d i f f e r e n t i a t i o n : i s the PHT pathway a b i o s y n t h e t i c module the exp r e s s i o n of which depends on a s i g n a l ( s ) t i g h t l y l i n k e d to organ d i f f e r e n t i a t i o n as a morp h o l o g i c a l s t a t e or does e x p r e s s i o n r e f l e c t l e s s s p e c i f i c c o n d i t i o n s such as hor-mone balance, with d i f f e r e n t s i t e s of expre s s i o n normally due to d i s s i m i l a r i t y i n hormonal (or other) c o n d i t i o n s i n the d i f f e r e n t organs to which e x p r e s s i o n i s confined? If the l a t t e r i s true then s y n t h e s i s of root compounds i n leaves could be t r i g g e r e d by a l t e r i n g inducer l e v e l s e i t h e r a r t i f i -c i a l l y or g e n e t i c a l l y . Some evidence that t h i s may be the case with f l a v o n o i d s i s d i s c u s s e d by Mears (13): many changes i n f l a v o n o i d t i s s u e s p e c i f i c i t y occurred during i n d u c t i o n of a u t o t e t r a p l o i d s from d i p l o i d c u l t i v a r s of Phlox drummondii. It would be extemely i n t e r e s t i n g to see what p a t t e r n of expres-s i o n occurs i n crosses between taxa producing PHT i n roots only with taxa producing PHT i n stems and leaves only, e s p e c i a l l y i f t i s s u e hormone l e v e l s were determined as w e l l . SUMMARY Hybrids between t e t r a p l o i d (4x=48) 15. alba and hexaploid (6x=72) 15. p i l o s a y i e l d f e r t i l e (5x=60) e x h i b i t i n g a mix-ture of morphological t r a i t s and a roughly i n t e r m e d i a t e l e v e l of PHT i n the l e a v e s . S e l f e d F^ p l a n t s gave an F^ which segre-gated f o r PHT s y n t h e s i s . Synthesis i s dominant but showed depressed l e v e l s incompatible with a s i n g l e gene dosage e f f e c t . The r a t i o s of segregants do not appear to agree with expected values f o r the p e n t a p l o i d . 193 LITERATURE CITED 1. B a l l a r d , R. E. 1975. A b i o s y s t e m a t i c and chemostematic study of the Bidens p i l o s a complex i n North and Cen-t a l America. Ph. D. t h e s i s . U n i v e r s i t y of Iowa, Iowa C i t y . 2. Bedford, C. T., D. Bhattacharjee, J . R. F. F a i r b r o t h e r , E. R. H. Jones, S. Safe, and V. T h a l l e r . 1976. Na t u r a l a c e t y l e n e s . Part XLIX. P o l y a c e t y l e n e s from D a h l i a s c a p i g e r a (A. D i e t r . ) Link and Otto var. s c a p i g e r a f. s c a p i g e r a and some D a h l i a h y b r i d s . J . Chem. Soc. Pkn. 1:735-741. 3. Beeks, R. M. .1955. Improvements i n the squash technique f o r p l a n t chromosomes. E l A l i s o 3:131-134. 4. B i s t i s , G., and M. Anchel. 1966. Evidence f o r genetic con-t r o l of p o l y a c e t y l e n e p r o d u c t i o n i n a Basidiomycete. Mycologia 58:270-274. 5. Bohlmann, F., M. Ahmed, M. Granz, L. M. King, and H. Robin-son. 1983. Bisabolene d e r i v a t i v e s and other c o n s t i t -uents from Coreopsis s p e c i e s . Phytochemistry 22:2858-2859. 6. Bohlmann, F., T. Burkhardt, and C. Zdero. 1975. N a t u r a l l y o c c u r r i n g a c e t y l e n e s . Academic Press, Ldn, N.Y. 7. Carey, S., M. Anchel, and G. B i s t i s . 1974. P o l y a c e t y l e n e p r o d u c t i o n i n C l i tocybe t r u n c i c o l a : E f f e c t of mating-type combination. Mycologia 66:327-332, 8. Chin, C., M. C. C u t l e r , E. R. H. Jones, J. Lee, S. Safe, and V. T h a l l e r . 1970. N a t u r a l a c e t y l e n e s . Part X X X I . Ci4~tetrahydropyrany1 and other p o l y a c e t y l e n e s from the Composite D a h l i a coccinea Cav. var. co c c i n e a . J . Chem. Soc. (C):314-322. 9. Grant, V. 1975. Genetics of f l o w e r i n g p l a n t s . Columbia U n i v e r s i t y Press, N.Y. 10. I c h i h a r a , K.-I., and M. Noda. 1975. P o l y a c e t y l e n e s from immature seeds of s a f f l o w e r (Carthamus t i n c to r i u s L . ) . Agr. B i o l . Chem. 39:1103-1108. 11. L e v i n , D. A. 1983. P o l y p l o i d y and no v e l t y i n f l o w e r i n g p l a n t s . Amer. N a t u r a l i s t 122:1-25. 12. Levy, M. 1976. A l t e r e d g l y c o f l a v o n e e x p r e s s i o n i n induced a u t o t e t r a p l o i d s of Phlox drummondii. Biochem. Syst. E c o l . 4:249-259. 194 Mears, J . A. 1980. Chemistry of p o l y p l o i d s : a summary with comments on Parthenium (Asteraceae-Ambrosiinae). Pages 77-101 in W. H. Lewis, ed. P o l y p l o i d y : b i o l o g -i c a l r e l e v a n c e . Plenum Press, N. Y., Ldn. Morton, R. A. 1975. Biochemical spectroscopy, V o l . I. John Wiley and Sons, N. Y. Stuessy, T. F. 1977. H e l i a n t h e a e - - s y s t e m a t i c review. Pages 621-761 Ln V. H. Heywood, J . B. Harborne, and B. L. Turner, eds. The b i o l o g y and chemistry of the Compositae, V o l . I I . Academic Press, Ldn., N.Y., and San Franc i s co. S t r i c k b e r g e r , M, N. 1976. Genetics, 2nd edn. Macmillan P u b l i s h i n g Co., Inc., N. Y. Van F l e e t , D. S. 1970. Enzyme l o c a l i z a t i o n and the g e n e t i c s of polyenes and p o l y a c e t y l e n e s i n the endo-dermis. Advancing F r o n t i e r s of Plant Science 26:109-143. 195:.' AFTERWORD The i S t u d i e s presented i n t h i s t h e s i s have shown some of the p o s s i b i l i t i e s f o r using Bidens alba t i s s u e and organ cui-.. tures f o r studying the p h y s i o l o g i c a l f a c t o r s c o n t r o l l i n g p o l y -a c e t y l e n e l e v e l and composition. C a l l u s c u l t u r e s from normal p l a n t s could not be induced to produce d e t e c t a b l e l e v e l s of p o l y a c e t y l e n e s ; however, transformed c a l l u s did produce a char-a c t e r i s t i c set of compounds, some of which were novel, at f r a c t i o n a l l e v e l s of those found i n the p l a n t . Further work needs to be done to determine what f a c t o r s account f o r the d i f f e r e n c e between normal and transformed c e l l s ' , e.g., i s the composition and l e v e l of hormones the c r i t i c a l d i f f e r e n c e or are other p h y s i o l o g i c a l f a c t o r s involved? Root c u l t u r e s proved to be a simple, e f f i c i e n t , and con-s i s t e n t system f o r i n v e s t i g a t i n g po l y ac e ty lene:; synthes i s . F a c t o r s having a s i g n i f i c a n t e f f e c t on composition or l e v e l i n c l u d e d the f o l l o w i n g : temperature, growth phase, carbohydrate/ n i t r a t e r a t i o , hormone type and l e v e l , and presence of absence of l i g h t . Whether these determinants are i n t e g r a t e d i n t o fewer, or perhaps one, b a s i c system c o n t r o l l i n g the l e v e l and compo-s i t i o n of product remains to be determined. I t i s p o s s i b l e that a l l the f a c t o r s above r e s u l t i n changes i n the r a t i o s and l e v e l s of hormones which, i n t u r n , act on b i o s y n t h e s i s . The s t u d i e s presented here have i n d i c a t e d some f a c t o r s which can modify the p o l y a c e t y l e n e r a t i o s i n roots but not how non-root compounds can be induced. Other s i g n a l s are apparently necessary f o r 196 p r o d u c t i o n of l e a f and stem compounds i n r o o t s . D e d i f f e r e n -t l a t i o n of roots r e s u l t e d i n p r o d u c t i o n of some of these com-pounds at low l e v e l s , i n d i c a t i n g that the s p e c i f i c type of t i s s u e o r g a n i z a t i o n i s important fo r s p e c i f i c i t y . The c e n t r a l problem of how the e x p r e s s i o n of organ s p e c i f i c compounds are r e g u l a t e d , however, remains to be s o l v e d . Bidens a l b a , however, has s e v e r a l c h a r a c t e r i s t i c s which make i t s u e f u l f o r s t u d i e s of t h i s type. R e s u l t s from crosses of By a l b a and <B. • p i l o s a i n d i c a t e d that at l e a s t one of the p r i n c i p a l compounds of Ii. a l b a , PHT, was i n h e r i t e d as a u n i t , but other l o c i apparently a f f e c t l e v e l s of product. The a v a i l a b i l i t y of an i n t e r f e r t i l e s p e c i es l a c k i n g the b i o s y n t h e t i c pathway f o r PHT o f f e r a d d i t i o n a l p o s s i b i l i t i e s f o r s tudying the c o n t r o l of p o l y a c e t y l e n e e x p r e s s i o n , although the combination used i n t h i s study r e s u l t e d i n problems of i n t e r p r e t a t i o n due to the d i f f e r e n t p l o i d y l e v e l s i n v o l v e d . Transmission of crown g a l l tumour c h a r a c t e r i s t i c s through sexual r e p r o d u c t i o n presents an o p p o r t u n i t y to see how a d d i t i o n of f o r e i g n DNA to a p l a n t genome i s i n t e g r a t e d i n t o the t o t a l l i f e c y c l e of the p l a n t . In a d d i t i o n , i t suggests that i n s e r t i o n s of other genes would be r e t a i n e d , and B_. alba may be p a r t i c u l a r l y s u i t a b l e f o r g e n e t i c engineering s t u d i e s . In summary, t h i s t h e s i s i s a c o n t r i b u t i o n to understanding the way i n which a f a s c i n a t i n g and important set of compounds, p o l y a c e t y l e n e s , are i n t e g r a t e d i n t o the complex web of pathways which f u n c t i o n so p r e c i s e l y i n t h i s , and many other, r a t h e r o r d i n a r y weeds. APPENDIX 198 Appendix Table 1. V a r i a t i o n s i n medium hormones used f o r c u l t u r i n g Bidens alba c a l l u s . A l l media were made with 3% sucrose. Hormone Range t e s t e d Cmg/1) No. l e v e l s t e s t e d Medium Remarks 2,4-D 0-2 . 0 5 B 5 Maximum growth at 1.0 mg/1. NAA 0.02-10.0 9„ SH: Maximum growth at 4.0 mg/1. NAA x K i n e t i n 0.01-1.2 x 0.06-10.0 11 SH GA 3 x 0.04-0.4 x 91 SH Maximum growth at K i n e t i n 0.04-0.4 0.125 mg/1 each. A b b r e v i a t i o n s : 2, 4-D, 2,4-dichlorophenoxyacetic a c i d ; NAA, O^-napthalene a c e t i c a c i d ; GA^, g i b b e r e l l i c a c i d . Appendix Table 2. V a r i a t i o n s i n fo r c u l t u r i n g B. al b a c a l l u s . sugars and vitamins: used Supple- .' ment :Range te s t e d c g / D No. l e v e l s ' Medium Remarks I n o s i t o l 0-1.6 7 • i . s L i t t l e d i f f e r e n c e i n growth. Sucrose . 2.5-80 6 1SH Maximum growth at 50 g/1. b Sucrose ( + l i g h t ) 2.5^80 6 1SH Maximum growth at 50 g/1. b Sucrose ( + l i g h t ) 2.5 1 0SH No growth, some green c a l l u s . Glucose 15-30 2 1SH Poor growth. A s c o r b i c a c i d 0.01-0.4 5 1 B 5 Best growth on 0.05 g/1, 0.2 and above k i l l e d c e l l s . Concentration of 2,4-D (mg/1) precedes medium abbreviation. I l l u m i n a t i o n 6000 lux, continuous. 199 Appendix Table 3. Variations, i n SH medium supplemented with primary m e t a b o l i t e s . A l l media made with 1 mg/1 2,4-D and 2% sucrose. Supplement Range t e s t e d No. of l e v e l s (molar) t e s t e d Acetate (.A) 1 0 - 1 - 1 0 ~ 4 4 Malate CM) 1 0 _ 2 - 1 0 _ 5 4 C i t r a t e (C) 1 0 _ 2 - 1 0 " 5 4 A + M 3 x IO" 4 (A) + ; 3 x 10" 4 (M) 1 A + C " " 1 M + C 2.2 x 10~ 4(M). + 7.4 x 10~4C.C) 1 A + M+ C 2.2 + 2.2 + 7.4.x 10~ 4 1 200 Appendix Table 4. Varia t i o n s - i n SH medium macronutrients used f o r c u l t u r i n g B. alB"a c a l l u s . Composition i n mg/1 except sucrose C g / l ) - "C" i s the c o n t r o l l e v e l , the standard l e v e l f o r SH medium. A l l media made with 1 mg/1 2,4-D. Sucrose KN0 3 MgS0 4 NH 4H 2P0 4 C a C l 2 KH 2P0 4 a30 a2500 a400 a300 a200 0 C 1250 C C C 0 c 625 C C C 0 c 313 C C C 0 c C C 6 00 C 0. c C C 1200 C 0 c C C 240O C 0. 100 6 25 C 1200 C 0 50 C C 0 C 680 50 1250 C 0 C 6 80. 50 62 5 C 0 C 680 .'050. 1250 C 0 C 1360 C C 8 0.0 C C Q. C C 16 00 C C 0. C C C c 4 00. 0 C C C C 80Q 0 50 1250 C 60 0. C 0 50 1250 C 600 C Q C o n t r o l l e v e l , standard l e v e l f o r SK medium. 201 Appendix Table 5. V a r i a t i o n s i n SH medium supplemented with mixed f a t t y a c i d s . L i p i d s s o l u b i l i z e d i n ethanol and Tween-80. Fig u r e s are the average of s i x c a l l u s e s . A l l media made with 3% sucrose and 1.0 mg/1 2,4-D. Supplement O l e i c a c i d L i n o l e i c a c i d l i n o l e n i c a c i d Fresh wt. Cml/1) (ml/1) (ml/1) ( g / c a l l u s ) a o 0 0 2 . 0 0.31 0 0 2.2 0.31 0.05 0 1.4 0.31 0 0. 05 1.8 0.31 0.05 0 . 05 1.6 0 0. 05 0.05 1.3 1.0 0 0 1.0 0 1.0 0 1.0 0 0 1.0 2 . 0 0.95 0.05 0 1.5 0.95 0 0. 05 0.8 0 0.1 0.1 1.2 0.95 0.05 0.05 1.4 1.0 0.1 0 1.5 1.0 0 0.1 1.2 V 0 0 1.9. cL Ethano1, Tween-80 c o n t r o l . ^ C o n t r o l , no a d d i t i v e s . 

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