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Cell suspension culture studies of the Coffea arabica L. Buckland, Elizabeth J. 1972

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CELL SUSPENSION CULTURE STUDIES OF THE COFFEA ARABICA L. BY ELIZABETH J . BUCKLAND B.Sc. ( A g r ) . , U n i v e r s i t y o f B r i t i s h Columbia 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department o f Food S c i e n c e 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 BRITISH COLUMBIA December, 1972. In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Food Science The University of British Columbia Vancouver 8, Canada Date February 20, 1973 i i i ABSTRACT C u l t u r e d t i s s u e s d e r i v e d from the c o f f e e p l a n t , C o f f e a a r a b i c a L., were grown i n v i t r o i n the form of both c a l l u s and suspension c u l t u r e s . The suspension c u l t u r e s grew r a p i d l y and appeared healthy. M i c r o s c o p i c examination showed th a t the c e l l s c h a r a c t e r i s t i c a l l y grew i n long filamentous chains. Suspension c u l t u r e s were examined f o r the presence of thr e e components - f r e e amino a c i d s , c a f f e i n e and chl o r o g e n i c a c i d . By examining these components the species s p e c i f i c i t y c o uld be determined. The f r e e amino acids of the cof f e e bean are thought t o be one of the major precursors of c o f f e e aroma on r o a s t i n g . The c o f f e e suspension c u l t u r e s were shown t o c o n t a i n a s i m i l a r p a t t e r n of f r e e amino acids although the t o t a l content was much higher i n the c u l t u r e s than i n the i n t a c t green c o f f e e bean. A s p a r t i c a c i d , glutamic a c i d , phenylalanine, a l a n i n e , v a l i n e , t h r e o n i n e , s e r i n e , and g l y c i n e were the predominant amino acids present i n the c o f f e e suspension c u l t u r e . Threonine, s e r i n e , g l y c i n e , a l a n i n e and phenylalanine were the major f r e e amino acid s i n the green c o f f e e bean. The f r e e amino a c i d content i n the suspension c u l t u r e e x h i b i t e d an i n i t i a l r i s e , decreased duri n g a c t i v e growth, then incr e a s e d r a p i d l y t o the maximum l e v e l d u r i n g the d e c l i n e of the c u l t u r e . Roasted c o f f e e bean e x t r a c t s were i n v e s t i g a t e d t o a s c e r t a i n whether one solvent could i n preference e x t r a c t some of the major precursors of c o f f e e aroma. Methanol was found t o e x t r a c t m a t e r i a l from green c o f f e e beans which on r o a s t i n g produced c o f f e e aroma. C a f f e i n e was detected i n the c e l l suspension c u l t u r e s . However, problems w i t h the a n a l y t i c a l methods gave r i s e t o question a b l e r e s u l t s . The suspension c u l t u r e s , at maximum i v c a f f e i n e y i e l d , contained 0.03% c a f f e i n e (dry weight) whereas the green c o f f e e bean contained c o n s i d e r a b l y more c a f f e i n e (1.15%, dry weight). The c a f f e i n e content of the t i s s u e s i n c r e a s e d d u r i n g the l a g phase, decreased d u r i n g the r a p i d phase and then i n c r e a s e d again i n the s t a t i o n a r y phase and u l t i m a t e l y p r o d u c t i o n l e v e l l e d o f f d u r i n g the d e l i n e phase of growth. The c e l l c u l t u r e s produced c h l o r o g e n i c ac i d i n low conc e n t r a t i o n s at the maximum 0.14% dry weight i n co n t r a s t t o the green coffee bean which contains 6.5% dry weight. The production or accumulation of chl o r o g e n i c a c i d f o l l o w e d a s i m i l a r p a t t e r n t o tha t of the c e l l c a f f e i n e production over the growth curve. C a f f e i c a c i d was a l s o detected. The c e l l suspension c u l t u r e s of Coffea a r a b i c a L. were shown t o be species s p e c i f i c i n t h e i r b i o c h e m i c a l c a p a b i l i t i e s . V ACKNOWLEDGEMENTS . The author, wishes t o express s i n c e r e g r a t i t u d e t o Dr. P.M. Townsley f o r h i s patien c e , guidance, d i r e c t i o n , and u n f a i l i n g encouragement dur i n g the course of t h i s study. The author a l s o wishes t o extend thanks t o Drs. V/.D. Powrie, J.F. Richards, T.L. Coulthard, and C.A. Hornby f o r t h e i r generous support. Thanks i s a l s o extended t o Drs. K.G. Johnson and D. Rose ( N a t i o n a l Research C o u n c i l Canada) f o r t h e i r time, help, and guidance i n the p r e p a r a t i o n of t h i s paper. The author a l s o wishes t o express g r a t i t u d e t o Miss A. Gelder f o r her t e c h n i c a l a s s i s t a n c e i n the a n a l y s i s of the amino acids and f o r her patie n c e ; snd t o Mr. T. Beveridge and Dr. S.A. Nakai f o r t h e i r t e c h n i c a l a s s i s t a n c e . The author acknowledges the f i n a n c i a l support of the N a t i o n a l Research C o u n c i l Canada and the U n i v e r s i t y of B r i t i s h Columbia which made t h i s study p o s s i b l e . G r a t i t u d e i s extended t o the author's parents, Dr. and Mrs. B.G. G r i f f i t h , and s i s t e r , Dorothy, f o r t h e i r support, encouragement and understanding during the course of t h i s study. v i TABLE OF CONTENTS Abst r a c t i i i Acknowledgements y L i s t of Tables x L i s t of Figures x i INTRODUCTION . . . 1 TERMINOLOGY 3 LITERATURE REVIEW 4 1 . H i s t o r y of plant t i s s u e c u l t u r e 4 2 . The p o t e n t i a l of c e l l s 5 3 . The c o f f e e p l a n t 6 4. Free amino acids i n p l a n t t i s s u e c u l t u r e s and i n green c o f f e e beans 7 5 . Roasting of green c o f f e e beans 12 6 . C a f f e i n e 1 6 7 . Chlorogenic a c i d 1 9 MATERIALS AND METHODS . . . . 2 1 1 . P r e p a r a t i o n of the c u l t u r e s 2 1 2 . Maintenance of the c u l t u r e s 2 1 3 - Growth and sampling of c u l t u r e s f o r analyses 2 3 4. Media 24 5 . Coffee bean p r e p a r a t i o n 2 7 6 . Amino a c i d a n a l y s i s 28 a. P r e p a r a t i o n of the samples f o r a n a l y s i s 2 8 i . E x t r a c t i o n of the f r e e amino acids from the samples . 2 8 i i . H y d r o l y s i s of the f i l t r a t e 2 9 i i i . D e s a l t i n g of the f i l t r a t e s 2 9 i v . Ammonia removal 3 0 v. A n a l y s i s on the amino a c i d a n a l y z e r 3 1 b. Dry weight of samples 3 1 c. C a l c u l a t i o n s 3 1 d. P r o t e i n content of the samples 3 2 e. T h i n l a y e r chromatography of the f r e e amino acids 3 2 v i i 7. Roasting of the green coffee bean, ground coffee and coffee bean extracts 33 a. Roasting of the green coffee bean 33 b. Roasting ground green coffee 33 c. Roasting coffee bean extracts ... 3^ i . Preparation of the samples 3^ i i . Roasting of coffee bean extracts 3^ 8. Caffeine i n the green coffee bean and the coffee c e l l s 35 a. Determination of caffeine 35 b. Paper chromatography of caffeine 36 9. Chlorogenic acid i n the green coffee bean and the coffee suspension culture 36 a. Determination of chlorogenic acid content 36 i . Preparation of the sample s o l u t i o n 37 i i . Determination of chlorogenic acid ............ 38 (a) , untreated determination . 38 (b) . lead treated determination 38 i i i . Preparation of the standard curve 39 v i . C a l c u l a t i o n of the concentration of chlorogenic acid i n the sample s o l u t i o n 39 b. Paper chromatography of the chlorogenic acid 39 RESULTS AND DISCUSSION 41 1. The growth of the culture 41 a. Callus cultures 41 b. Suspension cultures 41 c. Growth of the suspension culture 48 2. Free amino acids i n Coffea arabica L. c e l l suspension cultures and green coffee beans 52 a. Determination of free amino acids - methods 52 b. Free amino acids i n Coffea. arabica L. suspension cultures 53 c. Free amino acids i n green coffee beans 59 d. Protein content of samples used f o r analysis of free amino acids 60 e. Comparison of the free amino acid content of coffee bean and coffee suspension cultures 60 • • * V l l l 3. Roasting of the green coffee "bean and i t s extracts .... 6 l a. Roasting of green coffee 6 l i . Roasting of the green coffee bean i n i t s e n t i r e t y 6 1 i i . Roasting of ground green coffee beans 6 2 i i i . Roasting the green bean p e l l e t 6 3 i v . Discussion on roasting green coffee 6 4 b. Roasting of green coffee bean extracts 6 5 i . Water extracts of green coffee beans ......... 6 5 i i . Methanol extracts of green coffee beans 6 5 i i i . Ethanol extracts of green coffee beans 6 ? i v . Roasting of combined extracts of green coffee.. 6 ? v. Miscellaneous 6 7 v i . Discussion of the roasting of green coffee bean extracts 6 7 4 . Caffeine determination i n Coffea arabica L. c e l l suspension cultures and green coffee beans 6 8 a. Caffeine determination - methods 6 8 b. Caffeine content i n c e l l suspension cultures of Coffea arabica L. 6 8 c. Caffeine content i n green coffee beans ............. 7 3 d. Paper chromatography of caffeine 7 3 e. Discussion 7 3 5 . Chlorogenic acid i n the Coffea a^ab_i_ca L. c e l l suspension cultures and the green bean...... 7 3 a. Chlorogenic acid determination - methods 7 4 b. Chlorogenic acid i n the culture medium .. 7 4 c. Chlorogenic acid i n Coffea arabica L . suspension cultures 7 7 d. Chlorogenic acid i n green coffee beans 7 7 e. Paper chromatography f o r chlorogenic acid i n extracts of green coffee beans and t i s s u e culture samples 7 7 f. Discussion 8 0 SUMMARY ... 8 1 BIBLIOGRAPHY 8 2 i x LIST OF TABLES 1. Comparison of the f r e e amino acids i n i n t a c t t i s s u e s and c a l l u s c u l t u r e s of potato, c a r r o t , and "brittleweed p l a n t s • 8 2 . The fr e e amino acids present i n green c o f f e e beans (var. Columbia) 11 3 . The composition of the PRL - 4 - C - CM medium, l i q u i d and s o l i d 2 3 4. The composition of the B5 medium, l i q u i d and s o l i d .... 2 4 5 . The measurement of growth i n C o f f e a a r a b i c a L. c e l l suspension c u l t u r e s 5 0 6. Free amino acids present i n C o f f e a a r a b i c a L. c e l l suspension c u l t u r e s and green c o f f e e beans 5 6 7. The aroma and c o l o u r produced upon r o a s t i n g at 220°C of c o f f e e e x t r a c t s w i t h v a r i o u s a d d i t i v e s ( i n the presence of water) 6 6 8 . C a f f e i n e content of C o f f e a a r a b i c a L. c e l l suspension c u l t u r e s 6 9 9. The chlorogenic a c i d content of Coffea a r a b i c a L. suspension c u l t u r e s ? 6 X LIST OF FIGURES 1. The pathways proposed f o r the s y n t h e s i s of c a f f e i n e . . . . 18 2. The h o r i z o n t a l shaker used f o r a e r a t i o n of the suspension c u l t u r e s ... 22 3. A C o f f e a a r a b i c a L. c a l l u s c u l t u r e on B5 medium 42 4. A f i l a m e n t from a Cof f e a a r a b i c a L. suspension c u l t u r e showing s p i r a l l i n g f i l a m e n t 44 5« C e l l suspension from a Coffea. a r a b i c a L. suspension c u l t u r e showing two f i l a m e n t s s p i r a l l i n g t o g e t h e r and the v a r y i n g c e l l s i z e 44 6. Healthy C o f f e a a r a b i c a L. c e l l s showing nucleus, n u c l e o l u s , and cytoplasmic strands 46 7. A health y C o f f ea a r a b i c a L* c e l l , w i t h i n a fi l a m e n t s p i r a l 46 8. Coffee c e l l s c o n t a i n i n g unknwon bodies throughout the c e l l 47 9. Dead Cof f e a a r a b i c a L. c e l l s c o n t a i n i n g unknown bodies w i t h i n the c e l l w a l l 47 10. Growth of the Coffea a r a b i c a L. c e l l suspension c u l t u r e s 49 11. Chromatographic s e p a r a t i o n of the f r e e amino acids i n Coff e a a r a b i c a L. c e l l suspensions ( e i g h t e e n day old) c u l t u r e s and green c o f f e e beans a. S e p a r a t i o n of the b a s i c amino acids 54 b. S e p a r a t i o n of the a c i d i c and n e u t r a l amino acids . . 5 5 x l 12. Free amo.no a c i d content of Goffea a r a b i c a L. c e l l suspension c u l t u r e s . 57 13* C a f f e i n e content of C o f f e a a r a b i c a L. c e l l suspension c u l t u r e s 70 14. Paper chromatogram showing presence of c a f f e i n e i n c o f f e e beans, c e l l and medium e x t r a c t s 7 2 15. Standard curves f o r c h l o r o g e n i c a c i d a. Determination of wavelength best s u i t e d f o r c h l o r o g e n i c a c i d d e t e c t i o n 75 b. Standard curve f o r conversion of the absorbance value t o a c o n c e n t r a t i o n value 75 16. Chlorogenic a c i d p r o d u c t i o n by Coffea a r a b i c a L. c e l l suspension c u l t u r e s 78 17. Paper chromatogram showing the presence of c h l o r o g e n i c a c i d i n the e x t r a c t s of Lhe c e i l suspension c u l t u r e s and c o f f e e beans 79 INTRODUCTION The concept of c u l t i v a t i n g i s o l a t e d plant c e l l s was conceived by Haberlandt i n 1902 (White, 1963; N i c k e l l , 1962). Since then, the f i e l d of plant t i s s u e c u l t u r e s has been g r a d u a l l y expanding although i t i s s t i l l at the fundamental stages of development. Pl a n t c e l l suspension c u l t u r e s can be grown i n l i q u i d medium i n a manner s i m i l a r t o m i c r o b i a l c u l t u r e s . U n l i k e the m i c r o b i a l c u l t u r e s , however, the c e l l suspensions are composed g e n e r a l l y of a heterogenous p o p u l a t i o n of c e l l types (Willmer, 1963) and as a consequence growth and subsequent development tend t o be somewhat d i f f i c u l t t o c o n t r o l . The i s o l a t e d p l a n t c e l l i s t h e o r e t i c a l l y b i o c h e m i c a l l y t o t i p o t e n t ; t h a t i s , the c e l l has the f u l l b iochemical p o t e n t i a l found i n the i n t a c t parent plant ( S r i k o r i a n , 1 9&5)• C u l t u r e s have been shown t o produce carbohydrates, amino acids and p r o t e i n s r e a d i l y ( K l e i n , I 9 6 0 ; Staba, 1 9 6 3 ) . Secondary metabolites such as a l k a l o i d s have been detected i n very low nunn-fci-fcies (Puhan and M a r t i n , 1 9 7 1 )» The extent t o which the plant c e l l ' s b iochemical p o t e n t i a l can be c o n t r o l l e d i s s t i l l unknown. At the present time i n most cases the products produced by the p l a n t t i s s u e c u l t u r e s can be obtained and/or produced more economically from the i n t a c t p l a n t . T i s s u e c u l t u r e s d e r i v e d from a Coffea a r a b i c a L. plant were used i n t h i s study t o determine i f they could serve as a p o s s i b l e p o t e n t i a l f o r producing ' c o f f e e ' . Coffee, as the consumer recognizes i t , i s the roasted product of Co f f e a species beans. The r o a s t i n g process through a s e r i e s of 'browning r e a c t i o n s ' transforms the bean from a green c o l o u r w i t h a disagreeable odour t o a chocolate brown product w i t h the c h a r a c t e r i s t i c c o f f e e f l a v o u r and aroma. Numerous authors have suggested t h a t the major precursors of co f f e e f l a v o u r and aroma e x i s t s i n the amino a c i d s , p r o t e i n s and carbohydrate c l a s s e s of compounds (Feldman et a l , I 9 6 9 ; M e r r i t t and Angeline, 1971). I f t h i s theory i s correct and the isola t e d plant c e l l i s species biochemically totipotent, then i t may be possible to produce a coffee flavour and aroma on roasting of the coffee t i s s u e cultures (Townsley and Buckland, 1972). Several components, free amino acids, caffeine and chlorogenic acid, found normally i n the green coffee bean were investigated i n the Coffea arabica L. suspension cultures. The free amino acids of green coffee bean are thought to be one of the major precursors of roasted coffee aroma. Caffeine and chlorogenic acid, while they are not thought to contribute s i g n i f i c a n t l y to roasted flavour and aroma, are normally secondary metabolites found i n the coffee bean. These three components were examined to ascertain whether a coffee suspension culture i s s t i l l species s p e c i f i c i n i t s biochemical p o t e n t i a l . Roasting of green coffee bean extracts was also investigated. The aim was to f i n d i f one of the solvents used would extract enough of the major precursors of coffee aroma. m 1 — ' — ~ X* -i_ 1. * _ L. . . J _ _ . . . X- _ _ - - - - _ . r » _t_ •» .1 • j . n c a x i u <JJ . u j i j . o o o u u j w<3.to u u U Ul i l j j f c i i . ' t ; fc>U:ue U i O i l e i/eu ugni & eu chemical constituents of the green coffee bean with those found i n coffee c e l l suspension cultures. 3 TERMINOLOGY A r t i c l e s dealing with plant t i s s u e culture use varying terminology which often, unfortunately, makes i t d i f f i c u l t to determine exactly what type of t i s s u e culture has been used. The s p e c i f i c terminology to be used i n the present study to describe the various types of plant t i s s u e cultures i s : I*. undifferentiated growth - an embryonic type of c e l l growth i n which a l l or most of the c e l l s present are the same or c l o s e l y related i n structure and function, so are not s p e c i a l i z e d f o r organized functions such as the formation of leaves and roots (Routien et a l . 1956)• 2. (plant) t i s s u e cultures - a general term encompassing a l l forms of plant tissues cultured i n v i t r o such as c a l l u s and c e l l suspension cultures. 3. c a l l u s cultures - homogenous masses of undifferentiated c e l l s 'to a l l outward appearances' grown on s o l i d medium (Tulecke, 1961). 4 . (single c e l l ) suspension cultures - a submerged culture containing single c e l l s and very small c e l l aggregates. The c e l l s have no secondary wall thickening (Vv'illmer, 1963)• 4 LITERATURE REVIEW 1. History of plant t i s s u e culture. As early as 1 9 0 2 , Haberlandt envisaged c u l t u r i n g i s o l a t e d plant c e l l s on a r t i f i c a l medium. He based his 'outrageous' ideas upon Schwann's ( 1 8 3 9 ) concept of c e l l u l a r totipotency. Haberlandt ( 1 9 0 2 ) proposed that studies could be done more re a d i l y on organization and c e l l u l a r r e l a t i o n s h i p s within the plant i f i s o l a t e d plant c e l l s and t i s s u e s could be c u l t i v a t e d . He assumed that there was no l i m i t to c e l l d i v i s i b i l i t y . He, however, was unsuccessful i n his attempts to c u l t i v a t e single c e l l s or tissues mainly as his choice of experimental material was wrong rather than his techniques ( N i c k e l l , 1 9 6 2 ; White, I 9 6 3 ) . Later he stated (translated by White, I 9 6 3 ) "at any rate, the c u l t i v a t i o n of i s o l a t e d c e l l s i n nutrient s o l u t i o n would make possible an experimental approach to many important problems from a new point of view". It was not u n t i l the early 1 9 3 0 ' s that continuous growth i n v i t r o of plant segments was achieved. White ( 1 9 3 ^ ) and Gautheret ( 1 9 3 ^ ) were both successful i n obtaining elongation of i s o l a t e d root t i p s i n v i t r o . In 19391 the f i r s t p u b l i c a t i o n appeared describing the successful c u l t i b a t i o n of both carrot and tobacco cambial c a l l u s cultures over prolonged periods of time (Gautheret, 1939; Nobecourt, 1939; White, 1939). Since then the f i e l d of t i s s u e culture has greatly expanded. In 1 9 5 4 , Muir et a l produced c e l l suspensions from Tagetes erocta and Nicotiana tabacurn calluses. These l i q u i d suspensions contained both single c e l l s and small clumps of c e l l s . Since t h i s f i r s t successful attempt at single c e l l suspensions, singl e c e l l suspensions have been obtained from most dicotyledonous 5 and a few monocotyledonous plants. K l e i n (i960) has stated that almost a l l tissues can be grown although he questioned the actual value of doing so. 2. The p o t e n t i a l of c e l l s . " I f the zygote receives a l l the genetic information which i s e s s e n t i a l f o r the whole organism, and i f i t s subsequent d i v i s i o n s are as consistently equational as they seem to be, then the o r i g i n a l totipotency of the zygote should p e r s i s t i n the array of derivative parenchyma c e l l s despite t h e i r v a r i a t i o n i n form and metabolism" (Blakely and Steward, 1964). Thus meristematic (undifferentiated) c e l l s i s o l a t e d from the parent plant and cultures should have the biochemical p o t e n t i a l of the parent plant (Krikorian, 1965)• Plant c e l l s , however, cultured i n v i t r o tend towards a common c e l l type which i s simple and undifferentiated i n nature. The c e l l s have a narrow range of detectable enzymes and a narrow range of metabolic a c t i v i t y (Tulecke and N i c k e l l , i960). V.'einstein et a l (1962) stated that the ti s s u e cultures were c y t o l o g i c a l l y , p h y s i o l o g i c a l l y , and biochemically d i f f e r e n t from the parent plant. Staba (I963) concluded that single c e l l cultures were s i m i l a r biochemically to lower forms of plant l i f e . He summarized the commercial p o t e n t i a l of plant c e l l cultures as follows: "carbohydrates, amino acids and proteins can be produced by plant suspension cultures. To what extent i t i s economically p r a c t i c a l to grow and regulate such cultures remains to be seen". Staba (I969) l a t e r , however, stated that just because t h i s c e l l type appeared to be 'geared up* f o r active growth and protein production, i t should not be concluded that the c e l l i s not p o t e n t i a l l y able to produce the desired products. Numerous plant products have been i s o l a t e d from c a l l u s and single c e l l suspension cultures. These include a l k a l o i d s , pigments, proteins, enzymes, organic acids, phenolics, saponins, steroids, and terpenoids (Puhan and Martin, 1971). It has 6 been found, however, i n t i s s u e c u l t u r e s the l e v e l of the products detected was below the l e v e l i n the parent p l a n t (Puhan and M a r t i n , 1 9 7 1 ; Tomita, 1 9 7 1 ) . 3• The coffee p l a n t . The coffee found on the r e t a i l s h e l f i s a h i g h l y r e f i n e d product of the C o f f e a s p e c i e s . I t i s w e l l known f o r i t s p l e a s i n g f l a v o u r and aroma and f o r i t s s t i m u l a t i n g e f f e c t s on animal metabolism ( S i v e t z , I963). The Coffea species i s a t r o p i c a l evergreen which o r i g i n a t e d i n the A f r i c a n highlands of Kenya and A b y s s i n i a (Thompson, 1971). Coffee i s grown commercially today i n most of the t r o p i c a l c o u n t r i e s of A f r i c a , A s i a , Oceania, and South and C e n t r a l America. I n the 1971-1972 season the c o f f e e world produced 9,398.4 m i l l i o n pounds of green c o f f e e and exported 6,930 m i l l i o n pounds t o non-coffee producing c o u n t r i e s . For the . 1972-1973 season i t i s f o r e c a s t that 9.603 m i l l i o n pounds of green c o f f e e w i l l be produced and 7,035.6 m i l l i o n pounds w i l l be exported. B r a z i l i s the top cof f e e producing and ex p o r t i n g country w i t h Columbia f o l l o w i n g ( F o r e i g n A g r i c u l t u r a l S e r v i c e , 1971 and 1972). The Cof fea species i s a widely v a r i a b l e p l a n t . Cof fea.  a r a b i c a L. and i t s v a r i e t i e s account f o r n e a r l y a l l of the commercial market w i t h C o f f e a l i b e r i c a and C o f f e a robust a accounting f o r the r e s t (Thompson, 1971). Three species of the C o f f e a f a m i l y (C. a r a b i c a L., C. canephora Robusta, and C. l i b e r i c a B u l l , ex Hiern) have been grown i n v i t r o . The c a l l u s c u l t u r e s obtained were of two types. The f i r s t was white and spongy i n appearance and the second was more compact. Only the Robusta v a r i e t y c a l l u s showed embryoidal development i n the c a l l u s t i s s u e s ( S t a r i t s k y , 1970). 7 4. Free amino acids i n plant t i s s u e c u l t u r e s and green coffee beans. . I n t a c t p l a n t s and plant t i s s u e c u l t u r e s have been shown t o c o n t a i n the usual spectrum of f r e e amino acids and amides (Synge, 1 9 5 5 ; Steward et a l , 1 9 5 8 ; Weinstein et a l , 1 9 5 9 ) • The plant t i s s u e c u l t u r e s , however, have a much lower content of t o t a l f r e e amino acids and amides (Steward et a l , 1 9 5 8 ? Weinstein et a l , 1 9 5 9 ) (Table 1 ) . For example, i n t a c t potato t i s s u e s and potato c a l l u s t i s s u e s c o n t a i n 1 , 2 8 1 and 4 5 2 micrograms of n i t r o g e n per gram f r e s h weight r e s p e c t i v e l y (Steward et a l , 1 9 5 8 ) ; c a r r o t p l a n t s and c a r r o t c a l l u s c u l t u r e s c o n t a i n 8 0 1 and 124 micrograms of n i t r o g e n per gram f r e s h weight r e s p e c t i v e l y (Steward et a l , 1 9 5 8 ) ; and b r i t t l e w e e d (Happlopappus g r a c i l i s ) p l a n t s b r i t t l e w e e d c a l l u s c u l t u r e s c o n t a i n 468 . 8 and 1 2 2 . 6 micrograms n i t r o g e n per gram f r e s h weight r e s p e c t i v e l y ( K r i k o r i a n , 1 9 6 5 ) . Steward et _al ( 1 9 5 8 ) concluded t h a t s e v e r a l amino acid s and amides, e s p e c i a l l y asparagine, glutamine, and a r g i n i n e , were lower i n content i n the plant t i s s u e c u l t u r e s than i n the parent pl a n t w h i l e Y-aminobutyric a c i d v/as higher i n content. K r i k o r i a n ( 1 9 6 5 ) noted th a t g e n e r a l l y t h e r e was l i t t l e d i f f e r e n c e between the c u l t u r e s and the parent plant i n the f r e e amino a c i d content except f o r s p e c i a l i z e d amino compounds which the c u l t u r e d t i s s u e s tended t o be l a c k i n g . I n tobacco suspension c u l t u r e s , Koiwai et a l ( 1 9 7 1 ) found th a t the t o t a l f r e e amino a c i d content tended t o decrease a l i t t l e d u r i n g the l a g phase and then t o inc r e a s e r a p i d l y i n the l o g phase of growth. The p r i n c i p a l amino acids were g l u t amine, asparagine, and Y - a m i n o f r u " t y r i c a c i d , which v a r i e d i n content w i t h the age of the c u l t u r e . The glutamine content was low durin g the l a g phase of growth ajid 8 Table 1 . Comparison of the free amino acids i n intact t i s s u e and c a l l u s cultures of potato, carrot and brittleweed plants. pot at oV carrot V brittleweed* Amino acids intact c a l l u s intact c a l l u s intact c a l l u s aspartic acid 1 1 . 2 2 * 1.64 2 5 . 9 0 1 . 2 0 5 . 2 0 3 . 2 0 glutamic acid 1 6 . 9 5 2 . 7 3 4 2 . 0 0 4 . 5 0 1.40 7 . ^ 0 serine 8 . 8 1 7.28 1 7 . 0 0 1 . 5 0 3 . 5 0 2 . 5 0 glycine 5 . 1 5 8 . 0 0 1 . 7 0 0.40 1 . 3 0 5 . 7 0 asparagine 2 9 1 . 5 0 0 . 0 0 1 5 3 . 0 0 0 . 0 0 present threonine 1 1 . 8 3 4 . 5 0 11.80 0 . 7 0 3 . 2 0 trace alanine 2 0 . 1 5 2 1 . 5 0 9 3 . 0 0 10.40 3.40 2 1 . 2 0 glutamine 5 7 9 . 0 0 1 5 . 7 2 77.80 0 . 0 0 present h i s t i d i n e - - - - 3 . 3 0 2 . 5 l y s i n e 1 2 . 1 0 2 . 6 2 0 . 5 0 0 . 0 0 1 1 . 9 0 2 0 . 1 0 arginine 114 . 5 0 15.46 3 5 . 2 0 0 . 0 0 -methionine 0 . 0 0 2 . 6 1 0 . 0 0 trace pr o l i n e 0 . 0 0 0 . 0 0 4 . 2 0 0 . 1 0 2 . 3 0 5 - 7 0 v a l i n e 2 9 . 1 0 6 . 3 1 8 . 6 0 0 . 9 0 6.40 2 . 7 0 leucines 1 1 . 1 5 8 . 0 5 7 .80 0 . 7 0 9 . 6 0 4 . 7 0 phenylalanine 1 1 . 7 1 6 . 1 8 1 3 . 3 0 2 . 0 0 trace y-aminobutyric acid 4 0 . 7 5 8 8 . 3 0 18.40 2 . 3 0 6 . 3 0 4 . 6 0 g-alanine - - - - 0 . 5 0 0 . 0 0 p i p e c o l i c acid - - - - 3 . 2 0 trace t o t a l 1 2 8 1 . 0 4 5 2 . 0 8 0 1 . 0 124 .0 468 . 8 1 2 2 . 9 V Steward et a l , 1 9 5 8 ; * K r i k o r i a n , 1 9 6 5 . " a l l values expressed as micrograms nitrogen per gram fresh weight - no values given f o r that i n d i v i d u a l amino acid. " then decreased s l i g h t l y a f t e r the peak of growth (day 6 ) . The asparagine content increased during the lag phase, decreased very s l i g h t l y during the log phase and then rapi d l y increased again during the stationary phase of growth, y-aminobutyric acid was found to increase markedly during, the lag phase, and then decrease gradually a f t e r rapid grov/th began (Koiwai et a l , 1 9 7 1 ) • The content of glutamic acid, glycine and l y s i n e decreased during the lag phase of growth and then increased during the log phase of growth. The content of alanine, serine, leucines, and proline increased during the lag phase and then decreased i n content during the log phase. The other amino acids were found to have no recognizable pattern although most of them were elevated during the stationary phase of growth (Koiwai et' a l , 1 9 7 1 ) • y-aminobutyric acid was the only amino acid found i n plant t i s s u e cultures' consistently i n a higher content than i n the parent plant, although other amino acids have been shown to be higher i n the plant culture than i n the intact plant (Stewart et a l , 1 9 5 8 ; Bove et a l , 1 9 5 7 ) . Koiwai et a l ( 1 9 7 1 ) found thaty-aminobutyric acid increased i n content i n the plant t i s s u e cultures only during the lag phase of grov/th and ther e a f t e r gradually decreased. Wickremasinghe et a l ( 1 9 & 3 ) showed thaty-aminobutyric acid accumulated i n rose, bean, sycamore, and brittleweed cultures when the a i r supply was limi t e d , y-aminobutyric acid i s thought to be an intermediate i n the oxidation of glutamic acid to organic acids of the c i t r i c acid cycle. A suppression of the oxidation as a result of a. l i m i t e d supply of a i r would cause an accumulation ofy-aminobutyric acid (Koiwai et a l , 1 9 7 1 ) . 10 The free amino acids and proteins were both found to vary i n content and composition with the age of the culture (Steward et a l , 1 9 5 8 ) . A Paul's s c a r l e t rose suspension culture was found to contain 96 micrograms of nonprotein nitrogen per gram fresh weight and 9 1 5 micrograms of protein nitrogen per gram fresh weight a f t e r four days of c u l t u r i n g . A f t e r twelve days of c u l t i v a t i o n the cultures were found to contain 8k micrograms of nonprotein nitrogen per gram fresh weight and 2 1 9 micrograms of protein nitrogen per gram fresh weight (Fletcher and Beavers, 1 9 7 0 ) . The r e l a t i v e composition of the c e l l protein did not r e f l e c t the r e l a t i v e composition of the free amino acids present. This seems to indicate that more than just simple condensation of the free amino acids occurs i n protein synthesis (Steward et a l , 1 9 5 8 ) . Changes i n the environmental conditions caused changes i n the soluble and insoluble (protein) amino acid content and composition. "When potassium was added to the medium the amount of free amino acids present i n the c a l l u s cultures was strongly influenced (Tulecke, 1 9 6 1 ) . Light caused a s l i g h t increase i n t o t a l amino acid content of peanut c a l l u s cultures. The l i g h t favoured increases i n aspartic acid, serine, threonine, and valine content, while cultures grown i n dark favoured higher l e v e l s of proline and glycine (Krikorian, 1 9 6 5 s K r i k o r i a n and Steward, 1 9 6 9 ) • Green coffee beans contain appreciable amounts of nonprotein amino containing compounds (Underwood and Deatherage, 1 9 5 2 ) . The free amino acids of green coffee beans (var. Columbia) were analysed q u a n t i t a t i v e l y (Table 2 ) . Wolfrom et a l ( i 9 6 0 ) found ten free amino acids plus Y" a mi n°kutyric acid and Walter et a l ( 1 9 7 0 ) found f i v e a d d i t i o n a l free amino acids on further analysis. Table 2. The free amino acids present i n green coffee beans (var. Columbia) (Walter et a l , 1970). amino acid percent concentration aspartic acid 0.33 serine 0.12 asparagine 0.30 glutamic acid 0.49 proline 0.14 glycine 0.02 alanine 0.24 val i n e 0.02 isoleucine 0.03 leucine 0.03 tyrosine 0.04 phenylalanine 0.08 Y-aminobutyric acid 0.30 l y s i n e 0.04 h i s t i d i n e 0.04 arginine 0.04 12 5. Roasting of green coffee beans. The flavour and aroma of the green coffee bean i s not very appealing and i t i s only with roasting that the c h a r a c t e r i s t i c flavour and aroma develop. During the roasting process there occurs a mild p y r o l y s i s of the bean's constituents accompanied by the gradual formation of v o l a t i l e substances (Gianturco, 1 9 6 7 ; Gianturco, 1 9 6 5 ; Gianturco et a l , 1 9 6 4 ) . P y r o l y s i s i s a chemical change occurring at elevated temperatures which r e s u l t s i n the degradation and synthesis of products (Sivetz, 1 9 6 3 ) . The coffee beans are generally roasted at approximately 220°C (Feldman et a l , 1 9 6 9 ) . The bean i n i t i a l l y loses i t s free water (70%) and then, as the i n t e r n a l temperature of the bean begins to r i s e , the bound water i s lost u n t i l the water content i s reduced to one to two percent. The i n t e r n a l temperature of the coffee bean has then reached approximately 400°F and the absorption of heat by the bean i s supplemented by the l i b e r a t i o n of heat from i n t e r n a l p y r o l y t i c reactions. This strong exothermic reaction i s accompanied by the sudden expansion or pu f f i n g of the bean as well as i n t e r n a l rupturing of the c e l l layers. The d r a s t i c hydrolysis of proteins and other plant constituents allows the development of v o l a t i l e s and the release of carbon dioxide. The reactions occurring i n the bean must be stopped abruptly by cooling r a p i d l y at the desired end point as the reactions once i n i t i a t e d occur i n a few seconds. The colour of the bean changes r a p i d l y from green to dark brown during the f i n a l minutes of roasting (Sivetz, 1 9 6 3 ; Feldman et a l , 1 9 6 9 ; Keable, 1 9 1 0 ; Furia, 1 9 7 1 ) . The intact bean has been likened to a small autoclave i n which the chemical constituents react and interact under r e s t r i c t e d conditions (Keable, 1 9 1 0 ) . The p o s s i b i l i t i e s of reactions among the d i f f e r e n t chemical classes present i n the green coffee bean under' p y r o l y t i c conditions are v i r t u a l l y 13 unlimited but there i s some s e l e c t i v i t y evident as expressed by the greatly varying number of compounds formed (Gautschi et a l , 1967). The nature of the react ants and t h e i r analysis before and a f t e r roasting indicate that the M a i l l a r d reaction, Strecker degradation, base catalyzed sugar reaction, etc. occur perhaps along uncommon pathways governed by low water content, l o c a l i z e d buffer systems, and a fluccuating balance of reaction products. The M a i l l a r d type reactions, i f permitted to go to completion, produce both v o l a t i l e s and browning of the product (Gautschi et a l , 19&7)• This type of reaction involves the i n t e r a c t i o n of reducing sugars and amines. The free amino acids of the product would be expected to react more rapidl y than the protein amino acids with the carbohydrates (Feldman et a l , 1969)• There are excellent reviews written on the mechanisms of browning reactions i n foods (Hodge, 19&3 a n^ L I967? Danehy and Pigman, 1951; Reynolds, I963, I965 and I969). The aroma of coffee i s extremely complex i n nature and as a re s u l t much work has been done i n t h i s f i e l d . There are several review a r t i c l e s which give a f a i r l y comprehensive coverage of the f i e l d (Winter et a l , 1967; F r i e d e l et a l , 1971)• The number of compounds i s o l a t e d from the aroma sample i s affected by the method of i t s a c q u i s i t i o n . Using a head space sample, Watanabe (I969) i s o l a t e d and i d e n t i f i e d 313 compounds but concluded that his l i s t was not complete. Weidmann and'Mohr (1970) i s o l a t e d 363 compounds from coffee aroma. They i d e n t i f i e d 131 a c y c l i c compounds, 73 i s o c y c l i c compounds, and 159 heterocyclic compounds. Gautschi et a l (I967) and Weidmann and Mohr (1970) concluded that aroma arises mainly from a large number of v o l a t i l e compounds blended together rather than from an i n d i v i d u a l compound. Reymond et a l , (1963) concluded that the aroma was also influenced by Ik nonvolatile components present. The aroma produced on roasting i s dependent upon numerous factors, f o r example the roasting method, temperature of the roast, the water content of the beans, and the f i n a l grind (Furia, 1 9 7 1 ) • The difference between l i g h t (mild) and dark (French) roast coffee i s r e f l e c t e d by differences i n the v o l a t i l e s present and t h e i r concentrations. The p o s s i b i l i t y of s p e c i f i c v o l a t i l e s having two or more precursors of d i f f e r e n t thermal s t a b i l i t i e s has been demonstrated. Moreover, the formation of s p e c i f i c v o l a t i l e s can occur along two or more pathways which have d i f f e r e n t energy requirements (Self, 19&3! Gianturco, I 9 6 5 and I 9 6 9 ) • Thus the length of the roast i s very important to the production of v o l a t i l e s . The p y r o l y s i s of amino, acids, proteins and peptides has been related to the flavour and aroma of coffee a f t e r roasting (Merritt and Angeline, 1 9 7 1 ) • The degradation and i n t e r a c t i o n products of amino compounds are thought to be the main source of v o l a t i l e s i n roasting coffee (Gianturco et a l , 1 9 6 7 ) . The study, however, of the precursors of coffee flavour and aroma has been l a r g e l y ignored (Gianturco _et a l , I 9 6 7 ) . Russwurm ( 1 9 6 9 ) found that extracts obtained from coffee beans would not produce coffee aroma on roasting unless the frac t i o n s containing sugars and amino acids were combined. Erdman ( 1 9 0 2 ) demonstrated coffee aroma caused by nitrogenous compounds. Although he roasted a mixture of coffee tannic acid and raw sugar only to obtain a. burnt smell, he was able to obtain coffee aroma on roasting when caffeine was added to the mixture before roasting. The primary aroma, of cocoa i s produced by the i n t e r a c t i o n of flavonoids, sugars and amino acids. By roasting a methanolic extract of fermented cocoa beans, cocoa aroma was obtained. 15 The methanol s o l u t i o n extracts flavonoids, sugars and amino acids from the cocoa bean (Rohan and Stewart, 1 9 6 5 ; Rohan, 1 9 6 4 ) . The free amino acids present have been shown to vary greatly i n composition and content between cocoa plant v a r i e t i e s and thus i t i s not s u r p r i s i n g to obtain differences between v a r i e t i e s i n the roasted aroma. Also d i f f e r e n t amino" acids react at d i f f e r e n t rates and to d i f f e r e n t extents under the roasting conditions (Rohan and Stewart, I 9 6 6 ) . The cocoa bean i s fermented before roasting to achieve the desirable aroma and flavour. Roasting an unfermented bean re s u l t s i n an aroma resembling ,roasted broad beans (Rohan, 1 9 6 4 ) . Coffee substitutes have been produced from non coffee plant o r i g i n s . General Foods Corporation ( 1 9 6 3 and 1 9 6 6 ) produced a 'coffee' beverage by heating an aqueous (10%) mixture of proteinaceous material such as peanuts, and a reducing sugar and then d i l u t i n g the roasted product. Calcium carbonate was added to the mixture to maintain the pH between concluded that a synthetic instant coffee with f u l l fresh flavour and aroma was p r a c t i c a l l y impossible to synthesize. 6. Caffeine. Another nonprotein source of nitrogen i n green coffee beans and roasted coffee beans i s c a f f e i n e . It i s well known f o r i t s stimulatory effect of animal metabolism. It i s a xanthine a l k a l o i d with the formulation of: jajTOucrlCft. v ±yOvj 0 C H . 1 * 16 (Clarke, 1 9 6 9 ? Sivetz, I 9 6 3 ) . Caffeine i s present i n most coffee v a r i e t i e s i n varying amounts ( 1 . 2 % i n Coffea arabica; 2.0% i n C. robust a; and none i n wild v a r i e t i e s ) (Lehmajn, 1 9 7 1 ; Feldman et a l , 1 9 6 9 ) . Roasting of the bean causes a gradual decrease i n concentration of caffeine mainly as a res u l t of sublimation (Sharka and Telepcak, 1 9 6 4 ) . Plant alkaloids are generally produced during high metabolic a c t i v i t y (Hamerslag, 1 9 5 0 ) , and decrease i n content with increasing age of the t i s s u e (Hamerslag, 1 9 5 0 ; Beauden-Dufour and Mueller, 1 9 7 1 ; Wanner and Kalberer, 1 9 6 6 ; Kaeber, I 9 6 5 ) • Ogutuga and Northcote ( 1 9 ? 0 a and 1 9 ? 0 b ) studied caffeine biosynthesis i n tea c a l l u s cultures. The calluses a f t e r t h i r t y days growth contained 400 micrograms caffeine per gram dry weight (Ogutuga and Northcote, 1 9 7 0 a ) whereas the intact tea plant contains approximately 3 - 5 % caffeine (Fecak and Struhar, 1 9 7 0 ) . In the c a l l u s medium caffeine was also found to increase rapi d l y near the end of the rapid growth phase of the culture (Ogutuga and Northcote, 1 9 7 0 a ) . The r e l a t i v e proportion of caffeine i n the c a l l u s and i n the medium varied with the age of the culture. Caffeine production i s thought t o be followed eit h e r by i r r e v e r s i b l e discharge from the c e l l or release as a result of c e l l u l a r autolysis (Ogutuga and Northcote, 1 9 7 0 a ) . Caffeine determination has also been determined on sycamore and bean c a l l u s cultures (Ogutuga and Northcote, 1 9 7 0 a ) . The r e s u l t s showed that these two cultures do not synthesize c a f f e i n e . The lack of caffeine i n the sycamore and the bean c a l l u s cultures and the presence of caffeine i n the tea c a l l u s cultures demonstrate the genus s p e c i f i c i t y of the products produced by the c a l l u s cultures (Ogutuga and Northcote, 1 9 7 0 a ) . Caffeine i s synthesized i n intact plants along the pathways f o r normal purine synthesis, followed by methylation of the 17 purine r i n g (Proiser and Serenkov, 1963? Anderson and Gibbs, 1962). Ogutuga and Northcote (1970a and 1970b) proposed two pathways f o r the synthesis of caffeine (Figure 1 ) . Pathway II i s thought to be the major pathway as an increase i n caffeine content occurs during tea l e a f fermentation probably caused by a catabolic breakdown of nucleic acids. When ribonucleic acid was added to the c a l l u s t i s s u e medium an increase i n caffeine content followed (Ogutuga and Northcote, 1970a). Light was found to enhance purine formation and thus caffeine synthesis (Anderson and Gibbs, 1962). Ogutuga and Northcote (1970b), however, reported l i g h t had an i n h i b i t o r y effect upon caffeine production of the tea c a l l u s . The highest y i e l d s were obtained i n calluses grown i n complete darkness (1 ,500 micrograms caffeine per gram dry weight). Precursors of the methylated purine r i n g such as ammonium formate were also found to cause marked increases i n caffeine production (Ogutuga and Northcote, 1970a). 18 Figure 1. The pathways proposed f o r the synthesis of caffeine (Ogutuga and Northcote, 1970a). Pathway II ribonuclease ^Mej* P nucleic acids F 7-methylquanylic • ' deoxyoribonuclease 1 o^iri >5 a 11 i 9 purine pool I i xanthine ! 'LMe] \ / i 3-methylxanthine 1.3-3 imethylxanthine (theophylline) i / 1» 3i 7-trimethylxanthine (caffeine) ac d 7-methylquanosine i deammasej IMH 2 I 7-methylxa.nthine .'( ... v ribose [Me] ribose 7-methyl-' xanthine QvieJ + |Me] 3,7-dimethylxanthine theobromine * [Me] - indicates a methyl group. 19 7. Chlorogenic acid. Chlorogenic acid i s an a c i d i c phenol found normally i n green coffee beans and t h e i r roasted products. Chlorogenic acid i s composed of two simple acids, quinic acid and c a f f e i c acid. The formulation f o r chlorogenic acid i s : (Sivetz, 1 9 6 3 ) . Several isomers of chlorogenic acid have also been i s o l a t e d from green coffee i n small amounts. These isomers are isochlorogenic acid and neochlorogenic acid (Chassevant, 1 9 6 9 ; Smith, I 9 6 3 ) . In green coffee beans the content of chlorogenic acid varies between v a r i e t i e s ( 6 . 5 % dry weight i n Coffea arabica and 7 . 7 % i n C. robust a.) (Lehman, 1 9 7 1 ) and between reports ( 7 « 3 % dry weight i n C. arabica) (Feldman et a l , I 9 6 9 ) . Lehman et a l ( 1 9 6 7 ) reported that chlorogenic acid was present i n green coffee beans from 5 ' 5 % to 7 . 5 % dry weight. Chlorogenic acid and i t s hydrolysis products, quinic acid and c a f f e i c acid, do not contribute to coffee aroma but only to the flavour (Lee, 1 9 6 2 ) . Roasting of the coffee bean causes a decrease i n the content of chlorogenic acid probably as a res u l t of the hydrolysis of the molecule. Lehman et a l ( I 9 6 7 ) reported a f t e r roasting of coffee beans that chlorogenic acid 2 0 content had dropped to 3 . 6 % and 4 . 5 % of the dry weight. Feldman et a l ( 1 9 6 9 ) , however, reported only 0 . 3 % chlorogenic acid was present a f t e r roasting of coffee beans. The content of chlorogenic acid varied greatly with the extent of roasting (Sivetz, 1 9 6 3 ) . Chlorogenic acid has been detected i n t i s s u e cultures of potato (Gamborg, 1 9 6 7 ) and tobacco (Bergman, 1 9 6 3 ) * In the tobacco t i s s u e cultures the l e v e l was found to be increased with the addition of 1 0 % k i n e t i n to the medium. This also increased the content, of other alkaloids present (Bergman, 1 9 6 3 ) • Light also had a stimulatory effect on the production of chlorogenic acid by the ti s s u e culture with continuous l i g h t i n g producing the highest l e v e l . Higher phenolic contents were also obtained i n c a l l u s cultures when a-NAA was added to the medium (Leonova et a l , 1 9 7 0 ) . Two possible pathways f o r the biosynthesis of chlorogenic acid have been demonstrated. The major pathway i s : phenylalanine ¥ cinnamic acid v p-coumarate ^ p p-coumarolylquinic acid — c h l o r o g e n i c acid (Zucker, 1 9 6 3 ; Steck, 1 9 6 8 ) . The secondary pathway which has been shown to exist i s : phenylalanine 5> cinnamic acid p-coumarate -—l»-caffeine - — • c h l o r o g e n i c acid (Zucker, 1 9 ' 6 3 ; Steck, 1 9 6 8 ) . According to Zucker ( 1 9 6 3 ) cinnamic acid i s not only an intermediate i n chlorogenic acid biosynthesis but also stimulates chlorogenic acid synthesis. Colonna and Boudet ( 1 9 ? 1 ) concluded that chlorogenic acid i s not an inert product which accumulates but that i t plays an active metabolic role i n the plant c e l l . 2 1 METHODS AND MATERIALS 1. Preparation of the cultures. Healthy vigorously growing young shoots were taken from a Coffea arabica L. plant growing i n the University H o r t i c u l t u r a l greenhouses. The shoots were stripped of leaves, cut into two inch lengths to f a c i l i t a t e easier handling and s t e r i l i z e d by submersion i n 5 % sodium hypochlorite s o l u t i o n f o r approximately twenty minutes. A l l procedures thereafter were performed a s e p t i c a l l y to avoid contamination. Following s t e r i l i z a t i o n , the two inch lengths were rinsed i n d i s t i l l e d water, cut into pieces or explants approximately three millimeters long and placed on s o l i d medium (Table 3 ) • The implanted f l a s k s of medium were incubated i n darkness at 2 5°C. When the explants were growing well the Callus t i s s u e clumps were broken into fragments which were placed into f l a s k s of l i q u i d medium (T a b l e 3 ) • These f l a s k s were incubated at 25°C i n darkness on a horizontal rotary shaker (New Brunswick Gyrotory Shaker Model G 1 0 ) at approximately 1 6 0 rpm (Figure 2 ) . 2 . Maintenance of the cultures. The c a l l u s cultures on s o l i d medium were transferred to fresh medium (Tables 3 and 4 ) every four to eight weeks to maintain s u f f i c i e n t moisture and nutrients f o r continuous growth of the cultures. The t r a n s f e r was effected by breaking the growing c a l l u s clumps into pieces and placing these pieces i n d i v i d u a l l y onto fresh medium. The l i q u i d cultures were transferred to fresh l i q u i d medium (Tables 3 and 4 ) every two weeks u n t i l s a t i s f a c t o r y growth was well established and thereafter t r a n s f e r was repeated every eight to ten days to maintain the vigour of the cultures. 2 2 a F i g u r e 2. The h o r i z o n t a l shaker used f o r a e r a t i o n of the suspension c u l t u r e s - c o n t a i n i n g c u l t u r e s of d i f f e r e n t ages. 23 Table 3. The composition of the PRL-4-C-CM medium, l i q u i d and s o l i d * (Gamborg et a l , 1 9 6 8 ) . NaHgPO^.HgG 90 m i l l i g r a m s / l i t e r NagHPO^ 30 KC1 200 (NH^SO^ 200 MgS04.?H20 250 KNOj 1 , 0 0 0 Ga.Cl2.2H20 150 KI 0 . 7 5 2,4-D 2 . 0 sucrose 2 0 , 0 0 0 N-Z amine type A** 500 coconut milk 10 m i l l i l i t e r s / l i t e r vitamin stock s o l u t i o n 10 i r o n stock s o l u t i o n 5 micronutrient stock s o l u t i o n 1 * f o r s o l i d medium - 10 grams per l i t e r of agar-agar was added, The solu t i o n was heated u n t i l the agar-agar had melted and then the medium was dispensed. ** enzymatically hydrolyzed casamine. Vitamin stock s o l u t i o n (stored i n a p l a s t i c bottle) n i c o t i n i c acid 10 milligrams/100 m i l l i l i t e r s thiamine KC1 100 pyridoxine HC1 10 myo-inositol 1,000 Iron stock s o l u t i o n (kept frozen) FeS0^.7H20 278 milligrams/100 m i l l i l i t e r s Na2EDTA 372 Micronutrient stock s o l u t i o n (kept frozen) MnSO^.HgO 1,000 milligrams/100 m i l l i l i t e r s H^BO^ 300 * ZnS0^.?H20 300 ..Ka2Mo0^.2H20 25 CuSO^ 25 C o C l 2 . 6 H 2 o 25 24 Table 4. The composition of the B5 medium, l i q u i d and s o l i d * (Gamborg et a l , 1968). NaH 2P0^.H 20 150 m i l l i g r a m s / l i t e r KNO^ 2,500 (NH^) 2 S0^ 13^ MgSO^HgO 250 C a C l 2 . 2 H 2 0 , 150 KI 0.75 sucrose 20,000 2,4-D 0.2 vitamin stock s o l u t i o n * * 10 m i l l i l i t e r s / l i t e r i r o n stock s o l u t i o n * * 5 micronutrient stock s o l u t i o n * * 1 * f o r s o l i d medium - 10 grams agar-agar per l i t e r of medium. The s o l u t i o n was heated u n t i l the agar-agar had melted and then the medium was dispensed. f o r stock s o l u t i o n compositions see Table }, 25 The t r a n s f e r of the l i q u i d cultures was accomplished by placing a ten m i l l i l i t e r of c e l l suspension aliquot into one hundred m i l l i l i t e r s of fresh medium. 3. Growth and sampling of cultures f o r analysis. In preparing the c e l l suspension cultures f o r analysis, seven to ten day old growing suspension cultures were f i l t e r e d a s e p t i c a l l y through miracloth (supplied by Galbiochem) and allowed to drain f o r approximately f i f t e e n minutes. Flasks containing one hundred m i l l i l i t e r s and f i v e hundred m i l l i l i t e r s of fresh medium were then inoculated, with 0.5 grams and 2.5 grams of these f i l t e r e d c e l l s , respectively. For each analysis, s u f f i c i e n t f l a s k s of each size were inoculated to allow removal of one to three fl a s k s each sampling day over a period of twenty-five days. Twenty-five days was chosen f o r the length of the sampling period as the growth, measured by dry weight, had peaked and was beginning to decline by the twenty - f i f t h day of growth* On sampling days, the required number of flas k s were randomly removed from the shaker, and the contents f i l t e r e d through miracloth. The residue of c e l l s was washed with approximately twenty-five m i l l i l i t e r s of d i s t i l l e d water and allowed to drain f o r an add i t i o n a l f i f t e e n minutes. The c e l l s were weighed to est a b l i s h the fresh weight of the c e l l s and then freeze dried using a V i r t i s freeze d r i e r at approximately f i v e microns vaccuum. When dry, the c e l l s were reweighed to est a b l i s h t h e i r dry weight. The dried c e l l s were stored i n a desicator over T e l - t a l e (W.R. Grace and Gomp., Davison Chemical Division) u n t i l use to present spoilage and maintain a constant moisture c ont ent. A l l c e l l analyses were ca r r i e d out using the dried c e l l s as the moisture content was more uniform throughout thus making the dry weight more r e l i a b l e than the fresh weight. 26 The pH of the medium was measured on samples of the f i l t r a t e obtained a f t e r removing the c e l l s from the growing culture. This f i l t r a t e was l a t e r discarded. k. Media. Plant t i s s u e cultures, l i k e microorganisms, require c e r t a i n substances to promote and maintain growth i n v i t r o . E s s e n t i a l requirements may vary depending upon the type of the plant and culture desired. In general, a carbohydrate s o l u t i o n f o r t i f i e d with minerals and vitamins i s required. For i n i t i a t i o n and promotion of rapid grov/th, a growth stimulant may be added i n low concentrations. The s o l i d nutrient medium i n i t i a l l y used to stimulate c a l l u s growth and to maintain the cultures was s o l i d i f i e d PRL-4-C-CM (Gamborg .et a l , 1 9 6 8 ; Table 3 ) . A f t e r several months grov/th, the cultures were transferred to s o l i d B 5 medium (Gamborg .et a l , 1 9 6 8 : Table 4 ) . Both media were dispensed into 2 5 0 m i l i i l i x e r erlenmeyer flasks i n iOO m i l l i l i t e r amounts. The c a l l u s cultures were maintained throughout the study to ensure a ready supply of c a l l u s material i n case of contamination or loss of c e l l vigour i n the suspension cultures. The l i q u i d nutrient medium i n i t i a l l y used f o r the c e l l suspension cultures was l i q u i d PRL-4-C-CM (Gamborg et a l , 1 9 6 8 5 Table 3 ) • A f t e r the cultures became well established on t h i s medium, the medium was then changed to l i q u i d B5 medium (Gamborg et a l , 1 9 6 8 ; Table 4 ) . The l i q u i d media, both the PRL-4-C-CM and the 3 5 , were dispensed i n one hundred m i l l i l i t e r aliquots into 2 5 0 m i l l i l i t e r and 1 0 0 0 m i l l i l i t e r erlenmeyer flasks respectively. The amount used depended upon the dry weight required f o r the planned analyses of the c e l l s . 2? The change i n media composition was made mainly f o r chemical reasons. Once the cultures were well established, a lower concentration of growth stimulator (just 2,4-D) maintained the cultures i n an active growing state. The PRL-4-C-CM medium, s o l i d or l i q u i d contained coconut milk and casein hydrolysate, both of which have an unknown chemical composition. On the other hand, the l i q u i d or s o l i d B5 medium i s completely synthetic i n composition. Thus a l l constituents added are known and also i t i s cheaper to prepare. Chemical analyses f o r t h i s study were s i m p l i f i e d and f a c i l i t a t e d by the use of the a r t i f i c a l medium. The switch to the a r t i f i c a l medium did not noticeably affect the c e l l growth. The flasks containing both s o l i d and l i q u i d media were stoppered with non absorbent cotton wool plugs wrapped i n several layers of cheesecloth to allow t r a n s f e r of gases between the atmosphere and f l a s k , and to minimize the p o s s i b i l i t y of c o^"^" aminat ^  on. 5. Coffee bean preparation. Green coffee beans were prepared f o r analyses and used as a control standard i n each set of t e s t s performed. The beans were imported from San Salvador and obtained through the courtesy of Nabob Foods Limited, Vancouver, B.C. To prepare the beans f o r analyses, the beans were ground fine enough to pass through a f o r t y mesh seive using a. Wiley m i l l (intermediate model, Arthur H. Thomas Company). Dry ice was added to keep the green coffee from becoming pasty during grinding and to c o n t r o l heat generation i n the machine and the coffee during grinding (Honvitz (AOAC), 1970; Schall e r , 1972). To reduce the rate of d e t e r i o r a t i o n and to avoid pastiness of the powder during storage, the ground coffee was stored i n the freezer at-4°C u n t i l used. 28 6. Amino acid analysis. Generally, free amino acid determination was c a r r i e d out by extracting the t i s s u e culture or medium with 7 0 to 8 0 $ ethanol, f i l t e r i n g the extract, drying the extract i n vacuo and then passing the r e s u l t i n g residue through a cation exchange r e s i n . The r e s u l t i n g product was then analyzed on an amino acid analyzer (Simpkins and Street, 1 9 7 0 ) . With.the coffee samples, e s p e c i a l l y the bean, chlorogenic acid interference occurred to such an extent that i t became necessary to eliminate the i n t e r f e r r i n g f a c t o r ( s ) . Samples of both ground coffee and dry c e l l s were analyzed s i m i l a r l y f o r t h e i r free amino acids. a. Preparation of the samples f o r analyses. The samples were prepared using the following steps. The f i l t e r paper used i n t h i s analysis was Whatman number.one which had been well washed v/ith 95% alcohol, then dried under a gentle stream cf a i r t c remove re s i d u a l alcohol. This alcohol treatment was used to remove any free amino acids present i n the f i l t e r paper. i . E x t raction of the free amino acids from the samples (Simpkins and Street, 1 9 7 0 ; Brenner et a l , 1 9 6 3 ) . One gram of each sample to be analyzed was extracted by homogenizing with one hundred m i l l i l i t e r s of b o i l i n g 80/6 ethanol three to f i v e minutes at high speed on an O s t e r i z e r (Galaxie VIII) and then l e f t overnight to ensure complete extraction. The samples were then f i l t e r e d through ethanol washed f i l t e r paper and the residue washed with 80% ethanol. The residue was then discarded. 2 9 The f i l t r a t e was dried i n vacuo using a Rotovat (Model R-Buchi) at 4 5 °C and approximately f i f t e e n millimeters vacuum to remove the ethanol. i i . Hydrolysis of the f i l t r a t e ( F i t z p a t r i c k and Porter, 1 9 6 6 ) • The f i l t r a t e s were hydrolyzed under mild conditions to lower the chlorogenic acid interference. The dry residue was taken up i n one hundred m i l l i l i t e r s of 4 N hydrochloric acid and heated at 120°C i n an o i l bath f o r three hours. A f t e r hydrolysis, the solutions were cooled and f i l t e r e d through prepared f i l t e r paper to remove any p r e c i p i t a t e that had formed during hydrolysis. The f i l t r a t e s were evaporated to dryness i n vacuo at 65°C to remove hydrochloric acid. This was repeated three times by the addition of d i s t i l l e d water to the dry residue. i i i . Desalting of the f i l t r a t e s The samples were desalted to remove any sugars and minerals present. A cation exchange column (Biorad AG 5 0 - 8 x ; 1 0 0 - 2 0 0 mesh; H form), one by twenty centimeters i n dimensions, was used f o r the desalting procedure.. The column was regenerated a f t e r use and maintained at approximately pH 3 . 2 5 with a c i t r a t e buffer ( 0 . 1 5 molar) of that pH. The residue l e f t a f t e r removal of the hydrochloric acid was taken up i n approximately ten m i l l i l i t e r s of a c i t r a t e buffer pH 2 . . 2 and then applied to the prepared column. The flow rate was two to three m i l l i l i t e r s per minute. Following the. sample approximately f i f t e e n m i l l i l i t e r s of c i t r a t e buffer pH 2 . 2 was applied to wash out a l l extraneous material. The amino acids were then eluted from the column with twenty to t h i r t y m i l l i l i t e r s of 2 N ammonium hydroxide. The eluent 30 was c o l l e c t e d and dried i n vacuo at 4 5°C. To remove some of the excessive ammonia, the residue was dried at least three times a f t e r each addition of ten m i l l i l i t e r s of d i s t i l l e d water. i v . Ammonia removal (Beveridge, 1 9 7 2 ) . As the ammonia content was s t i l l to high f o r good separations on the amino acid analyzer, further ammonia removal was necessary. Using solutions of 0.5M ammonium sulphate adjusted over a range of pH 8 to pH 12 with 1M sodium hydroxide, i t was found that the lowest a l k a l i n e pH at which ammonia r e a d i l y v o l a t i l i z e d was pH 10. Ammonia v o l a t i l i z e s more r e a d i l y at higher pHs but the a l k a l i n i t y was kept as low as possible to avoid possible a l k a l i n e hydrolysis. To determine whether t h i s treatment would have any adverse effects upon the amino acid patterns cr resolution, samples of coffee c e l l s and of ground coffee beans were used. These samples were s p l i t i n two and one h a l f was treated. The other h a l f was l e f t untreated. The samples were run on the amino acid analyzer and the results, compared. No differences were noted between the treated and untreated samples except that i n the treated samples, the ammonia concentration was lower and h i s t i d i n e was more r e a d i l y calculated. The dried desalted residue r e s u l t i n g from the protocal of the previous section was then transferred q u a n t i t a t i v e l y to large ( 1 5 0 m i l l i l i t e r ) beakers, using twenty-five to f i f t y m i l l i l i t e r s of d i s t i l l e d water. The pH of these solutions was adjusted to approximately pH 1 0 using 1M sodium hydroxide. The beakers were placed i n a vacuum desiccator containing 31 concentrated sulphuric acid. The desiccator was then pumped down to approximately 0.2 microns vacuum with care being taken to avoid 'bumping' of the samples. A f t e r one and a h a l f hours under vacuum, the samples were removed and t h e i r pH adjusted to approximately pH 6. The samples were frozen to prevent spoilage, when the samples were to be analyzed, the samples were defrosted and dried i n vacuo at 60 °C. v. Analysis on the amino acid analyzer (Moore and Stein, 195*0 *  The dried samples were taken up q u a n t i t a t i v e l y i n f i v e m i l l i l i t e r s of c i t r a t e buffer pH 2 . 2 1 , f i l t e r e d , i f required, and then stored i n the freezer u n t i l needed. The samples were run on a dual column arrangement on a Pheonix amino acid analyzer. b. Dry weights of the samples (Beveridge, l y / 2 ) . The free amino acids were calculated i n terms of dry weight of the ground coffee bean and the dry c e l l s . The dry weight was determined by weighing a sample of 2 5 0 milligrams into an aluminium f o i l cup, placing i t i n an oven at 104°C, and reweighing a f t e r f i v e hours i n the oven and every hour a f t e r u n t i l no further change i n weight was detected. This f i n a l weight was used as the dry weight of the sample. c. Calculations (Phoenix Instrument Book). The amount of free amino acids i n each sample was calculated on the dry weight of that sample. The cal c u l a t i o n s f o r each amino acid involved the measuring of the area enclosed by i t s corresponding peak on the chromatogram using the height 32 times width (HW) method of i n t e g r a t i o n . d. P r o t e i n content of the samples ( G o r n a l l et a l , 1949). The a c i d h y d r o l y s i s i n v o l v e d i n the amino a c i d sample p r e p a r a t i o n could a l s o hydrolyze t o a l i m i t e d extent any p r o t e i n present a f t e r the e t h a n o l i c e x t r a c t i o n . A b i u r e t r e a c t i o n was t h e r e f o r e performed on part of some sample s o l u t i o n s of ground c o f f e e beans and c o f f e e c e l l s before and a f t e r h y d r o l y s i s t o ensure t h a t the p r o t e i n content was minimal. The samples were d r i e d i n vacuo, each was then mixed w i t h t e n m i l l i l i t e r s of d i s t i l l e d water and f i v e m i l l i l i t e r s of the s o l u t i o n was mixed w e l l w i t h one m i l l i l i t e r of b i u r e t reagent. The absorbance of the s o l u t i o n s was then determined at 583 m i l l i m i c r o n s on a Beckman spectrophotometer. As no n o t i c e a b l e r e a c t i o n occurred, the p r o t e i n content of the sample s o l u t i o n s was l e s s than one percent. e. T h i n l a y e r chromatography of the f r e e amino acid s (Jones and Heathcote, i 9 6 0 ) . • • • S e v e r a l of the samples run on the amino a c i d a n a l y z e r were a l s o chromatographed on C e l l u l o s e Mn 300 p l a t e s f o r f u r t h e r i d e n t i f i c a t i o n . The samples were a p p l i e d t o the p l a t e s i n the lower l e f t hand corner and d r i e d . The p l a t e s were then run f i r s t i n i s o p r o p a n o l : water : formic a c i d (40 i 10 : 2) , d r i e d , turned and then run i n t e r t i a r y butanol : methyl e t h y l ketone : ammonia (8876 by volume) J water (25 : 15 : 6.25 « 3.75) i n the second dimension. The p l a t e s were v i s u a l i z e d u s i n g n i n h y d r i n spray (0.3 grams n i n h y d r i n , 20 m i l l i l i t e r s g l a c i a l a c e t i c a c i d , and 5 m i l l i l i t e r s c o l l i d i n e i n 75 m i l l i l i t e r s absolute e t h a n o l ) . The Rf values and the c o l o u r s of v i s i b l e spots were noted. 33 ?. Roasting of the green coffee bean, ground coffee, and coffee bean extracts.  The green coffee bean produces the f a m i l i a r aroma, flavour and colour on roasting. It should thus be possible to roast coffee extracts to obtain coffee aroma. a. Roasting of the green coffee bean. The green coffee bean was placed i n a Coors porcelain f l a t dish and roasted at 220°C i n a muffle furnace (Thermolyte) f o r f i f t e e n minutes and then r a p i d l y cooled i n an ice bath. The beans were then crushed to obtain a coffee aroma. b. Roasting ground green coffee. The green beans were ground i n a Wiley m i l l to pass through a twenty mesh sieve. One gram portions were placed into Coor's porcelain c r u c i b l e dishes and roasted i n a muffle furnace at 220°C f o r varying lengths of time. A dish of water was placed i n the oven i n some cases to rais e the oven's humidity and thus slow down the. evaporation of the sample's moisture. Upon removal from the oven, the samples were covered with a watch glass to r e t a i n any aroma escaping and cooled r a p i d l y i n an ice bath. E f f o r t s to obtain coffee aroma from the ground bean p e l l e t s were made using a Paar's p e l l e t machine. The p e l l e t s were made i n 0.5. 1.0, and 1.5 gram portions. These were roasted at 220°G i n the presence of a dish of water u n t i l the p e l l e t s were dark brown externally. The p e l l e t s were then cooled r a p i d l y and disrupted. The i n t e r n a l colour and aroma were noted. c. Roasting coffee bean extracts,  i . Preparation of the samples. Ten grams of ground coffee (40 mesh) were extracted with one hundred m i l l i l i t e r s of b o i l i n g solvent of each of d i s t i l l e d 3^ water, methanol, and ethanol by homogenization at high speed f o r three to f i v e minutes using an Osterizer. The solutions were l e f t overnight then f i l t e r e d through Whatman f i l t e r paper and the residue washed with the appropriate solvent (approximately twenty m i l l i l i t e r s ) . The methanol and ethanol solutions were reduced i n volume at 45°C i n vacuo to remove excesses of the solvent. The solutions were then d i l u t e d s l i g h t l y by the addition of water. This raised the freezing point c l o s e r to the freezing point of water. A l l the solutions were then freeze dried and stored i n a desiccator u n t i l use. i i . Roasting of coffee bean extracts. The extracts (0 . 5 grams) and one m i l l i l i t e r of water per sample were mixed together and roasted u n t i l brown i n Coor's por c e l a i n c r u c i b l e dishes i n a muffle furnace at 220°C, When brown, coffee extracts were removed from the oven, a watch glass placed over top, and then the dishes were cooled r a p i d l y i n i c e . A dish of water was present i n the oven f o r most of the t e s t mixtures. Various additions were made to the samples. These additives were calcium carbonate, degtrose, caffeine, tannic acid and chlorogenic acid. They were added at f i f t y milligrams each per sample. In some cases 0 .5 N hydrochloric acid or 0 .5 N sodium hydroxide were used i n place of water. The colour and aroma of the samples were noted a f t e r roasting. 8. Caffeine i n the green coffee bean and the coffee c e l l s . ;  The caffeine content of green coffee and coffee c e l l s was determined using a modification of the micro Bailey - Andrew method (Horwitz (AOAC), 1970). a. Determination of caffeine (Korwitz (ACAO), 1970). One gram of ground coffee or of coffee c e l l s was mixed with f i v e grams of powdered magnesium oxide and approximately 35 100 m i l l i l i t e r s of water. The so l u t i o n was heated to b o i l i n g and boiled f o r t y - f i v e minutes, s w i r l i n g occasionally. The s o l u t i o n was then cooled and f i l t e r e d through a Whatman number one f i l t e r paper into a 125 m i l l i l i t e r separatory funnel. Four m i l l i l i t e r s of sulphuric acid ( i n a r a t i o of one m i l l i l i t e r of acid to nine m i l l i l i t e r s of water) was added and the s o l u t i o n was mixed well. The s o l u t i o n was then extracted f i v e times with ten m i l l i l i t e r portions of chloroform. Each extraction was shaken vigorously f o r one minute and then l e f t to s i t u n t i l the emulsion broke. The chloroform layer (the bottom layer) was drained into a second 125 m i l l i l i t e r separatory funnel, when the extraction was completed, f i v e m i l l i l i t e r s of \% potassium hydroxide were added to the chloroform extract. The s o l u t i o n was shaken vigorously f o r one minute and then l e f t u n t i l the emulsion broke. The chloroform layer was then drained o f f through a cotton plug into a k j e l d a h l f l a s k . The chloroform extract was digested on a digestion rack a f t e r the addition of 1.30 grams of potassium sulphate, ~0 milligrams of mercuric oxide, and two m i l l i l i t e r s of concentrated sulphuric acid. The samples were allow to digest f o r one hour a f t e r the solutions became clear, and then were cooled. Ammonium content of the samples was determined by Nessler's reaction. Aliquots of 0.05, 0.1, and 0.2 m i l l i l i t e r s of digest were mixed well with f i v e m i l l i l i t e r s of Messier*s reagent (Fisher) i n duplicate. The solutions were read at 500 m i l l i -microns on a Beckman spectrophotometer. Standards containing twenty, t h i r t y , and f i f t y micrograms of nitrogen were also run (Fitzsimmons and Mason, 1972). 36 C a l c u l a t i o n of the caffeine content was performed using the following equation* ^ 1 0 \ / o p t i c a l density ) (volume factor) average O.D. of/ ' H °f sample » standard / l 0 micrograms V nitrogen The volume factors were as follows: f o r 0 . 2 m i l l i l i t e r sample the volume f a c t o r was 1; f o r 0.1 m i l l i l i t e r sample - 2 ; and f o r the 0 . 0 5 m i l l i l i t e r sample - k. b. Paper chromatography of caffeine (Ogutuva and Northcote, 1970).  Several samples of medium coffee c e l l s and ground coffee beans were run on paper chromatography. The chloroform extract cf these samples was spotted on paper cliromatograriis sno. &xj.oweci to dry. A standard of caffeine (Fisher) was run with the samples. The papers were developed by descending chromatography f o r twenty hours i n the upper phase of n-butanol : water (86 : 14 v/v). Caffeine was detected by u l t r a v i o l e t l i g h t . 9 . Chlorogenic acid i n the green coffee bean and the coffee c e l l s .  The chlorogenic acid content of ground coffee and dry coffee c e l l s was determined following the A0AC method (Horwitz, 1 9 7 0 ) . a. Determination of chlorogenic acid. i . Preparation of the sample so l u t i o n . Samples of 7 0 0 milligrams of both ground coffee bean and dry. coffee c e l l s were placed i n centrifuge tubes and twenty-five 37 m i l l i l i t e r s of petroleum ether was added and mixed i n thoroughly. The samples were then centrifuged at 5|000 rpm f o r ten minutes i n a S e r v a l l angle centrifuge (Ivan S o r v a l l Inc) and the supernatant decanted o f f and discarded. This extraction with petroleum ether was repeated two more times to ensure complete removal of any l i p i d s present. The remaining residue was placed on a watch glass and l e f t to dry u n t i l the odour of petroleum ether was no longer detectable (approximately h a l f an hour). The samples were then transferred with small amounts of water to erlenmeyer fla s k s (300 m i l l i l i t e r s i z e f o r the dry c e l l s and one l i t e r size f o r the coffee bean). Because of the low concentration of chlorogenic acid present i n the dry c e l l s , and because of the shortage of experimental material, i t was necessary to reduce the t o t a l volume of the sample solu t i o n of the dry c e l l s to increase the chlorogenic acid concentration s u f f i c i e n t l y f o r detection. B o i l i n g d i s t i l l e d water was Then added to the dry residues i n the erlenmeyer flasks - 4-00 m i l l i l i t e r s t o the ground coffee bean and 90 m i l l i l i t e r s to the dry coffee c e l l s . The solutions were then rapi d l y brought to a b o i l and boiled gently f o r f i f t e e n minutes. The flasks were swirled frequently to keep the sample submerged. Flasks were cooled r a p i d l y to room temperature. The cooled solutions were trans f e r r e d to volumetric fl a s k s and made up to volume - 500 m i l l i l i t e r s f o r the ground coffee and 125 m i l l i l i t e r s f o r the dry c e l l s . The solutions were then f i l t e r e d through Whatman number one f i l t e r paper discarding the f i r s t twenty-five to t h i r t y m i l l i l i t e r s of f i l t r a t e . The remaining f i l t r a t e was used f o r the sample s o l u t i o n . i i . Determination of the chlorogenic acid. (a). Untreated determination. Ten m i l l i l i t e r s of the sample s o l u t i o n was transfered to a one hundred m i l l i l i t e r volumetric flask and d i l u t e d to volume 38 w i t h d i s t i l l e d water. The a b s o r p t i o n of the s o l u t i o n was measured at 324 m i l l i m i c r o n s against d i s t i l l e d water u s i n g a D.B. spectrophotometer (Beckman). ( b ) . Lead t r e a t e d determination. F i f t y m i l l i l i t e r s of sample s o l u t i o n were t r a n s f e r r e d t o a one hundred m i l l i l i t e r v o l u m e t r i c f l a s k . One m i l l i l i t e r of a s a t u r a t e d potassium acetate s o l u t i o n and f i v e m i l l i l i t e r s of a b a s i c lead acetate s o l u t i o n (PbfoAclg w i t h a s p e c i f i c g r a v i t y of 1 . 2 5 ) were added t o the sample s o l u t i o n w h i l e s w i r l i n g the f l a s k . The r e s u l t i n g s o l u t i o n was placed i n a b o i l i n g water bath f o r f i v e minutes and then cooled r a p i d l y t o room temperature u s i n g tap water. The s o l u t i o n was then p l a c e d i n an i c e bath and mechanically s t i r r e d . A f t e r one hour the f l a s k was removed from the i c e bath, and the s t i r r e r washed down w i t h d i s t i l l e d water. The s o l u t i o n was warmed to. room temperature u s i n g tap water, d i l u t e d t o volume w i t h d i s t i l l e d water and Then f i l t e r e d through Whatman number one f i l t e r paper d i s c a r d i n g the. f i r s t twenty t o t h i r t y m i l l i l i t e r s of f i l t r a t e . The absorbance was read on the remaining f i l t r a t e immediately at 324 m i l l i m i c r o n s on the D.B. spectrophotometer. i i i . P r e p a r a t i o n of the standard curves. A standard s o l u t i o n of c h l o r o g e n i c a c i d ( s u p p l i e d by J.T. Baker Co.) was made (40 m i l l i g r a m s per l i t e r ) and from t h i s s o l u t i o n a s e r i e s of standards were d e r i v e d . The s e r i e s was made by t r a n s f e r r i n g a given a l i q u o t (one m i l l i l i t e r t o 20 m i l l i l i t e r s ) t o a one hundred m i l l i l i t e r v o l u m e t r i c f l a s k and d i l u t i n g the s o l u t i o n t o volume using d i s t i l l e d water. The a b s o r p t i o n of t h i s s e r i e s was read at 324 m i l l i m i c r o n s on a D.B. spectrophotometer against d i s t i l l e d water.. 39 i v . C a l c u l a t i o n of the concentration of chlorogenic acid i n the sample solutions.  The o p t i c a l readings obtained f o r the untreated and the lead treated sample solutions were converted to apparent concentrations using the. standard curve f o r chlorogenic acid. The corrected concentrations f o r the samples were then calculated using the following formula: corrected concentration = Co - (Ci - 0.00045)/5 Co - apparent concentration of chlorogenic acid i n s o l u t i o n taken f o r absorbance measurement without lead treatment. C i - apparent concentration of chlorogenic acid i n the f i l t r a t e a f t e r lead treatment. 0.00045 - corre c t i o n f a c t o r f o r the s o l u b i l i t y of lead chlorogenate a f t e r the lead acetate treatment i n milligrams per m i l l i l i t e r s . b. Paper chromatography of the chlorogenic acid ( F i t e l s o n . 1 9 6 9 ) •  Paper chromatograms were run on the extracts of dry coffee c e l l s and the coffee beans. One gram of sample was extracted with 8 O/0 b o i l i n g ethanol, homogenized at high speed on an O s t e r i z e r and then allov i to stand overnight to ensure complete extraction. The sample solutions were then f i l t e r e d through Whatman number one f i l t e r paper and the f i l t r a t e evaporated to dryness i n vacuo at 45°C. The residue was taken up i n a minimal amount of water and applied to a paper chromatogram (Whatman number one). A reference s o l u t i o n containing chlorogenic acid, c a f f e i c acid, malic acid, t a r t a r i c acid, oxalic acid and c i t r i c acid was run with the sample solutions. The chromatogram was developed i n the upper phase of n-butanol : water t acetic acid (4 : 5 * 1 ) f o r fourteen hours. The paper was then dried and examined under ultra, v i o l e t l i g h t . The paper was then sprayed with an a n i l i n e f u r f u r a l spray reagent ( 0 . 3 m i l l i l i t e r s of a n i l i n e , and 0 . 3 m i l l i l i t e r s of f u r f u r a l , i n one hundred m i l l i l i t e r s of methanol). 40 RESULTS AND DISCUSSION 1. The growth of the cultures. a. Callus cultures. Undifferentiated growth became v i s i b l e on one or both ends of the coffee stem explant a f t e r approximately one month of incubation. The i n i t i a l growth was slow and brownish i n colour. The morphology of the c a l l u s was firm, smooth and compact. A f t e r several months of incubation, the growth pattern and morphology of the c a l l u s cultures had changed markedly. The calluses were pale grey i n colour and grew very r a p i d l y . The morphology of the calluses was i r r e g u l a r l y lobed and very f r i a b l e (Figure 3). This type of growth was maintained during the study. These c a l l u s cultures appeared to be very s i m i l a r to the f r i a b l e coffee cultures obtained by S t a r i t s k y (19?0). b. Suspension cultures. Single c e l l growth was apparent approximately three weeks a f t e r inoculating the l i q u i d medium with c a l l u s fragments. The growth of the cultures was observed by a gradual increase i n the medium's opaqueness and by the accumulation of debris and c e l l s at the high l i q u i d l i n e (Figure 2). Growth could also be observed by microscopic observation. 41 I n i t i a l l y , growth of the suspension cultures was slow and e r r a t i c with obvious v a r i a t i o n between cultures inoculated from the same f l a s k . Small calluses were formed during the early stages of the suspension cultures. These were gradually eliminated over time by t r a n s f e r r i n g only the c e l l suspension (that i s only the material which remained i n suspension a f t e r allowing the contents to s e t t l e f o r a few minutes). 42b Figure 3« A Coffea arabica L. c a l l u s culture grown on B5 medium. 43 The growth rate of the c e l l suspension cultures increased with continual t r a n s f e r and uniformity became more apparent between the cultures and the growth rate from the same source of inoculum. Once uniform growth was obtained the cultures were grown f o r study. The c e l l suspension cultures were composed mainly of long f i l a m e n t - l i k e strand of c e l l s with minor amounts of single c e l l s and small c e l l aggregates (clumps) present. Generally plant c e l l suspension cultures are composed mainly of single c e l l s and small c e l l aggregates (Nishi and Sugano, 1970s Townsley and Buckland, 1972; Rose, 1972). The long filamentous type of growth observed i s very s i m i l a r to algae filaments (Figures 4 and 5)< S p i r a l l i n g of the filaments e i t h e r singly (Figure 4) or i n pairs (Figure 5) was frequently observed. Occasionally v a r i a t i o n of c e l l size within the chains was observed (Figure 5)• This v a r i a t i o n i n si z e may be related to the stage of elongation or d i v i s i o n of -t- V, ^  i ; ^,,^1 nv, r~-f 4-V, ~ ~ .~ 1 ~1 ~ ~ "I "I - -unidimensional and d i v i s i o n occurred only i n a plane perpendicular to the long axis of the c e l l , thus producing long filaments. Ni s h i and Sugano (1970) reported the occurrence of long filamentous growth i n carrot suspension cultures grown on l i q u i d medium containing IAA. Carrot cultures grown on the same l i q u i d but containing d i f f e r e n t growth hormones d i f f e r r e d i n c e l l u l a r morphology and i n the mode of c e l l u l a r d i v i s i o n . Differences i n morphology and c e l l d i v i s i o n have been attributed previously mainly to n u t r i t i o n a l differences although differences between c e l l cultures on the same medium can be caused by c e l l clone ( l i n e ) s e l e c t i o n (Martin, 1972; Townsley and Buckland, 1972). In b a c t e r i a l cultures i t has been demonstrated that l i m i t i n g l e v e l s of c e r t a i n nutrients such as divalent cations, 44a 44b Figure 4. A filament from a Coffea arabica L. suspension culture showing filament s p i r a l l i n g (lOOx magnification) from a twelve day old culture. Figure 5« C e l l suspension from Coffea arabica L. culture showing filaments s p i r a l l i n g together and varying c e l l s i z e within filaments (lOOx magnification) from a twelve day old culture. k5 and glutamate can cause filament formation (Macdonald, 1 9 7 1 ) ' The l i m i t i n g l e v e l s of these nutrients does not allow normal separation of the c e l l s a f t e r d i v i s i o n . Separation i s normally caused by the action of e x t r a c e l l u l a r a u t o l y t i c enzymes (Macdonald, 1 9 7 1 ) . This l a t t e r b a c t e r i a l mechanism i s not known to operate i n plant c e l l suspension cultures. However, i t i s possible that the separation of the plant c e l l s i n v i t r o i s mediated by a s i m i l a r mechanism dependent upon the growth hormone present and the l e v e l of c e r t a i n nutrients. During the 'log phase' of growth the i n d i v i d u a l c e l l appeared to be healthy and quite active metabolically when observed under the microscope. The nucleus, n u c l e o l i and cytoplasmic strands were generally distinguishable (Figures 6 and 7 ) • Movement along the cytoplasmic strands was also observed frequently. D i v i s i o n of the c e l l s within a chain was occasionally noted. In the older c e l l s , small bodies of unknown composition began to appear i n large numbers (Figures 8 and 9 ) . These bodies were s t i l l v i s i b l e a f t e r the c e l l had died (Figure 9 ) ' Possibly, these bodies were some type of starch granule. Some layering within these structures can be observed i n Figure 9« Layering i s commonly observed i n most types of starch granules. These bodies did not react to the a p p l i c a t i o n of iodine which turns blue - black on contact with amylose starch. Amylopectin starch turns a f a i n t red brown with the iodine test and i s sometimes d i f f i c u l t t o detect. These bodies could, therefore, be starch granules composed mainly of amylopectin. Starch granules have been previously observed i n suspension cultures of potato (Gamborg, 1 9 ^ 7 ) and i n other plant suspension cultures (Rose, 1 9 7 2 ) . Rose ( 1 9 7 2 ) has observed starch granules i n several plant suspension cultures s i m i l a r to the ones observed i n the coffee suspension culture (Figures 8 and 9 ) . 4 6 a 46b Figure 6. Healthy Coffea arabica L. c e l l s showing nucleus, nucleolus, and cytoplasmic strands (magnification - 400x) from a ten day old culture. Figure 7. A healthy Coffea arabica L. c e l l within a filament s p i r a l (magnification -400x) from a fourteen day old culture. 4?b Figure 8. Coffea arabica L. c e l l s containing unknown bodies throughout the c e l l (magnification - 400x) from a fourteen day old culture. Figure 9 . Dead c e l l s of Coffea arabica L. cultures containing unknown bodies within the c e l l wall (magnification - kOOx) from a twenty-five day old culture. 48 c. Growth of the suspension culture. Growth of the suspension cultures was measured i n grams fresh weight and grams dry weight of the c e l l s . The pH value of the medium was determined on the f i l t r a t e following the removal of c e l l s . The fresh weight of the c e l l s i s not a r e l i a b l e value as a c t i v e l y d i v i d i n g c e l l s show an increase i n water uptake (Figure 10). Thus, the value obtained may vary greatly from actual c e l l weight. The dry weight of the c e l l s i s a more accurate representation of actual c e l l growth (Figure 10). The pH of the medium indicates the u t i l i z a t i o n of n i t r a t e and possibly the release of c e l l metabolites into the medium. The growth curves obtained were s i m i l a r to plant c e l l growth curves obtained by other authors (Simpson and Street, 1970; Townsley, 1972). The i n i t i a l l ag phase of the culture averaged about four days and was followed by a period of rapid growth. Rapid growth (generally termed log phase of growth) started at approximately day eight and continued f o r six days. Growth continued f a i r l y r a p i d l y peaking at day eighteen. Exponential growth was not obtained during the rapid phase of growth. Several authors have interpreted a1 log phase of growth i n plant suspension cultures on an arithmetic plot only (Gamborg, 1967; Simpson and Street, 1970; N i s h i and Sugano, 1971). Rose (1972), however, does not accept the concept of exponential growth as being applicable to the rapid growth phase i n plant c e l l cultures. The stationary phase of growth exists while the rate of anabolic growth of the c e l l population i s approximately equivalent to the rate of catabolic growth. This phase averaged only four days i n these cultures. The stationary phase was then followed by a decline i n both fresh and dry F r e s h w e i g h t - g r a m s K> 10 to C J t > <5 — H> Figure 10. Growth of the Coffea arabica L. c e l l suspension cultures. Points are average of eight to ten samples (values given i n Table 5 ) • symbols: D — D pH of medium o o dry weight of f i l t e r e d c e l l s • v fresh weight of f i l t e r e d c e l l s T able 5. The measurement of growth i n Coffea arabica L. c e l l suspension cultures time fresh weight (days) of c e l l s * (grams) 0 0.50 2 0.75 4 1.12 6 2.23 8 4 . 0 5 10 7.32 12 9.26 1 4 1 4 . 8 8 16 22.42 1 6 25•90 20 26.40 22 26.29 25 25.67 2 8 25.77 32 20.40 *values given are average dry weight pH of of c e l l s * medium* (grams) 4.30 0.025 ^.39 0.037 ^.53 0.078 4.76 0.113 4.99 0.262 5.51 0.431 5.90 0.595 5.99 0.698 6.37 0. { 6 4 6.55 0.758 6.49 0.743 6.80 0.668 '7..02 0 . 6 6 4 7.50 0.585 7.93 eight to ten samples. 51 weight of the c e l l s . This decline occurs when the i n t e r n a l rate of catabolism exceeds the rate of anabolism (Figure 10 and Table 5)• The stationary phase i s generally more extended (tobacco -Nich i and Sugano, 1971; soya bean - Gamborg, 196?) than was observed i n these coffee cultures. The rapid decline soon a f t e r the rapid c e l l growth would seem to indicate the presence of material(s) which cause the i n h i b i t i o n of c e l l growth or death of the c e l l s . This material i s possibly a secreted c e l l metabolite or a l i m i t i n g medium constituent. Dead c e l l s , observed early i n the culture's growth, increased i n number with the increase i n age of the culture (Figure 9) e s p e c i a l l y a f t e r rapid growth had ceased. The pH of the medium rose with the increase i n growth (Figure 10 and Table 5)• Generally the pH of the medium (both PRL-4-C-CM and B5) decreases or remains at a constant T _ ^ t r o i n n r » o r ^ n i H ^"^ovt^ ceasedj "^or example, i n rose (Wegg, 1972) and bean (Buckland, 1972) cultures. The rapid increase i n the pH value of the medium a f t e r growth had ceased indicates release of c e l l metabolites into the medium ei t h e r by secretion or by c e l l l y s i s . In eithe r case, i t i s probably the dead c e l l s which release the compound(s) which cause the increase i n pH with the decline i n growth. The c e l l y i e l d of the cultures was f a i r l y good although higher y i e l d s have been obtained by other authors using d i f f e r e n t suspension cultures (Nishi and Sugano, 1971; Buckland, 1972). The c e l l growth was quite healthy and uniform throughout the study. No contamination by mold or other microorganisms was observed. 52 2. Feee amino acids i n Coffea arabica L. c e l l suspension cultures green coffee beans.  Free amino acids were detected and analyzed i n suspension culture and green beans of Coffea arabica L. The most noteable difference was i n t o t a l content of the free amino acids. The coffee suspension culture contained J01.7 micromoles of amino acids per gram dry weight of t i s s u e at the maximum whereas the coffee bean contained 2 5 . 3 micromoles of amino acids per gram dry weight of t i s s u e . There were also differences i n the composition of amino acids present. a. Determination of free amino acids - methods. The procedure followed i n preparing the samples f o r analysis involved a mild hydrolysis of the samples. This hydrolysis was e s s e n t i a l to r i d coffee bean samples of i n t e r f e r i n g substances. The same procedure was ca r r i e d out with the suspension culture samples to allow direct comparison of the r e s u l t s . The hydrolysis was very e f f e c t i v e since the abberant behavior of coffee bean samples on amino acid analysis was eliminated. The mild acid hydrolysis also hydrolyzed the amides to t h e i r respective amino acids (glutamine to glutamic acid and asparagine to aspartic a c i d ) . This was concluded to be the reason no evidence of amides present was found on the r e s u l t i n g chromatogram (Figure 11) whereas there were indications of the presence of amides when the samples were run on t h i n layer chromatograms. Y-aminobutyric acid was not detectable on the amino acid analyzer used f o r analysis. A standard run containing a f a i r l y high concentration of Y- a mi n°butyric acid f a i l e d to show where t h i s amino acid would elute. 53 Sampling f o r amino acid analyses was started s i x days a f t e r inoculation, but three samples f o r neutral and a c i d i c amino acids were lo s t (Table 6 ) . b. Free amino acids i n Coffea arabica L. suspension cultures.  The t o t a l free amino acid content increased with the age of the culture from 3 2 . 0 micromoles of amino acids per gram dry weight (day 6 ) to 3 0 1 ' 7 micromoles of amino acids per gram dry weight (day 2 5 ) (Table 6 and Figure 1 2 ) . The concentration of free amino acids increased r a p i d l y during active growth and again during culture decline. The t o t a l a c i d i c and neutral amino acids followed the same pattern as t o t a l amino acids and the basic amino acids remained r e l a t i v e l y stable i n concentration, elevating s l i g h t l y i n the stationary and decline phase (Figure 1 2 ) . There, was. a marked drop i n t o t a l amino acids and t o t a l a c i d i c and neutral amino acid content at day twenty-two. It was less marked i n the basic amino acids but s t i l l present. This drop occurred just a f t e r the maximum c e l l growth had been attained (day 2 0 ) . The free amino acids were rapidly synthesized during active growth and with completion of active growth the synthesis decreased. The r i s e at day twenty-five i n the content of amino acids may be a result of c e l l u l a r breakdown accompanying c e l l and culture death (Figure 1 2 ) . The content of i n d i v i d u a l amino acids varied with the age of the cultures. A t y p i c a l chromatogram of an eighteen day culture can be seen i n Figure 1 1 . The free amino acids, leucine, t y r o s i n e , p r o l i n e , arginine, and unknown one and two increased gradually i n content during rapid growth of the culture, and then l e v e l l e d o f f or decreased s l i g h t l y during the decline i n growth of the culture. These amino acids were present i n the culture i n r e l a t i v e l y low concentrations (Table 6 ) . 54 a Figure 11. Chromatographic separation on the amino acid analyzer of the free amino acids i n Coffea arabica L. green beans and c e l l suspension culture. a. Separation of basic amino acids i . Green coffee bean - sample a p p l i c a t i o n contained 0.075 gram dry weight of sample.  i i . Coffee c e l l suspension culture (18 days old) sample ap p l i c a t i o n contained 0.0375 grams dry weight of sample. '  — - j j - — 1. a c i d i c and neutral amino acids 2. unknown 1 - possibly tryptophan 3• unknown a* 4. l y s i n e 5' h i s t i d i n e 6. ammonia 7. unknown b* 8. arginine •unknown a and b were not calculated, i n d i v i d u a l l y i n t a b ulation of amino acids (Table 6). 5 5 b Figure 11. Chromatographic separation on the amino acid analyzer of the free amino acids i n Coffea arabica green beans and c e l l suspension cultures. b. Separation of neutral and a c i d i c amino acids. i . Green coffee bean - sample a p p l i c a t i o n contained 0 . 0 6 2 5 grams dry weight of sample.  i i . Coffee c e l l suspension culture (18 days old) -sample a p p l i c a t i o n contained 0 . 0 1 7 5 grams dry weight of sample.  Identity of peaks 1. unknown c* 2. unknown d* 3 . unknown e* 4. aspartic acid 5 . threonine 6 . serine 12. methionine . 7. glutamic acid 1 3 . isoleucine 8. proline 14. leucine 9 . glycine 1 5 . tyrosine 10. alanine 16. phenylalanine 11. valine 17. unknown 2 ^unknowns c, d, and e were not calculated i n d i v i d u a l l y i n t a b u l a t i o n of amino acids (Table 6 ) . Table 6. Free amino acids present i n Cof f e a a r a b i c a L. suspension c u l t u r e s and green c o f f e e beans. Coffea a r a b i c a L.. suspension c u l t u r e s (days a f t e r i n o c u l a t i o n ) green c o f f e e beans amino acid s 6 8 10 12 14 16 18 20 22 25 a s p a r t i c a c i d 0.512 _•»> 0.470 7.265 32.496 20.451 91.998 0.321 threonine - - - 1.133 3.502 6.957 9.260 12.418 7.420 14.575 5-764 s e r i n e - - - 1.373 7.^65 11.888 7.757 9.306 7.653 19.866 4.500 glutamic a c i d - - - 6.372 5.972 13.962 20.825 24.832 15.292 36.242 0.474 p r o l i n e - - - 0.056 0.101 0.178 0.155 0.424 0.175 0.568 0.178 g l y c i n e - - - 1.889 4.867 4.836 5.175 6. 349 5-173 11.352 I .058 a l a n i n e - - - 6.569 9.453 5.758 11.858 17.164 11.014 27.266 3.209 c y s t e i c a c i d - - - _•«• + + 0.854 4.097 _# v a l i n e - - - 1.478 2.079 3.152 6.139 12.828 7.?4l 20.933 0.888 methi nine - - - 0.330 + 0.633 1.376 2.571 1.411 3.639 _# i s o l e u c i n e - - • - 0.611 0.937 2.348 6.670 10.87? 5-783 10.585 0.517 l e u c i n e ' - - - 0.761 l.?22 2.056 3.233 3.409 1.610 2.664 O.656 t y r o s i n e - - - 0.342 0.680 1.458 I .926 2.315 1.405 2.364 0.559 phenylalanine - - - 3.168 5.^23 19.574 17.317 25-779 13.455 31.559 1.147 l y s i n e 1.204 1.120 1.534 0.933 1.129 0.882 2.710 4.676 7.646 8.743 0.410 h i s t i d i n e 1.043 0.628 0.697 0.439 0.625 2.421 1.624 3.912 3.142 6.457 0.199 a r g i n i n e 0.662 0.700 1.014 1.796 0.486 1.181 1.466 0.943 0.593 3.350 0.117 unknown 1 1.331 1.672 2.188 1.251 0.858 O.960 0.824 3.535 1.358 I .163 0.212 unknown 2 - - - 0.216 0.273 0.566 0.458 0.320 0.640 t o t a l b a s i c s 0 4.220 4.441 5.433 4.605 3.097 5.444 6.625 13.065 12.73? 19.692 1.436 t o t a l a c i d l c s and n e u t r a l s 0 - - - 27.419 46.508 80.326 99.414 161.852 101.285 281.966 23.86? t o t a l 0 - - - 32.024 49.605 85.770 106.039 174.917 114.022 301.658 25.303 - not analyzed; -* none detected; + t r a c e present only; amino acids not l i s t e d . ' t o t a l s i n c l u d e minor unknown values given are the average of two samples - expressed i n micromoles amino a c i d per gram dry weight of sample. ON D r y _ w e i g h t in g r a m s A m i n o a c i d c o n t e n t - u m o l e s / g d r y wt 57b Figure 12. Free amino acid content of Coffea arabica L. suspension culture. points are averages of two samples (values given i n Table 6) symbols: v ™ — v - dry weight of f i l t e r e d c e l l s (values from Table 5) o———o t o t a l free amino acid content of cultures © ;—« t o t a l free a c i d i c and neutral amino acid content of cultures n ™ « ^ ~ a t o t a l free basic amino acid content of cultures 58 Cysteic acid was not detectable i n the culture u n t i l the stationary phase and then only i n low concentrations. The concentration of cysteic acid was elevated during the decline phase of growth. It would appear the apparent synthesis of c y s t e i c acid i s depressed during the rapid phase of growth although i t may a c t u a l l y be u t i l i z e d f a s t e r than i t i s produced. Alanine, threonine, serine, h i s t i d i n e and phenylalanine increased i n concentration during the rapid phase and continued to increase through the stationary and decline phases of growth. The rest of the amino acids increased r a p i d l y during the rapid growth and stationary phases of the culture and remained elevated through the decline phase of growth (Table 6). The major free amino acids present were aspartic acid, glutamic acid, phenylalanine, alanine, and v a l i n e . The predominant amino acid was aspartic acid. Asparagine has been shown to be' the predominant amino acid i n potato and carrot c a l l u s t i s s u e cultures grown i n darkness while glutamine predominates i n t i s s u e cultures grown i n l i g h t (Steward e_t a l , 1958) . Asparagine has also been shown to increase r a p i d l y during the. stationary phase of growth i n tobacco suspension cultures (Koiwai et a l , 1971) . A comparison between the free amino acids detected i n coffee suspension cultures and tobacco suspension cultures (Koiwai et a l , 1971) showed that the free amino acid patterns were not the same. In the coffee suspension cultures, aspartic acid, glutamic acid, glanine, valine and phenylalanine predominated while i n the tobacco suspension cultures glutamine, asparagine, threonine, glutamic acid, phenylalanine, and Y-aminobutyric acid predominated. This demonstrates a species difference between the cultures. 59 c. Free amino acids i n green coffee. . Although the free amino acids detected i n the green coffee bean were i n higher concentrations (Table 6 ) than had previously been reported (Walter et a_l, 1 9 7 0 ; Table 2 ) the same amino acids were detected. Threonine was an exception. Threonine was not only found to be present i n the sample used f o r t h i s study but was also considered to be one of the major amino acids ( 5 « 8 micromoles per gram dry weight) present. There was no y-aminobutyric acid detected i n the samples analyzed. The differences i n composition of the amino acid p r o f i l e from those obtained by Walter et a l ( 1 9 7 0 ) and by Buckland ( 1 9 7 2 ) may be a result of numerous factors such as differences between v a r i e t i e s , c u l t i v a t i o n , harvesting and post harvesting p r a c t i s e s . Threonine, serine, alanine, phenylalanine and glycine were the major amino acids present i n the sample analyzed. There was no trace of methionine or cysteic acid. P r o l i n e , h i s t i d i n e and arginine were present i n low but detectable l e v e l s (Table 6 and Figure 1 1 ) . d. Protein content of samples used f o r analysis of free amino acids.  A biuret reaction was performed on samples of the ethanol extract of both green bean and coffee suspension cultures to determine the protein content. No protein was detectable. Therefore less than one percent protein was present i n the samples before and a f t e r hydrolysis (Beveridge, 1 9 7 2 ) . This check was done to ensure that as l i t t l e protein as possible was being hydrolyzed during preparation of samples. 60 e. Comparison of the free amino acid content of coffee bean and coffee suspension cultures.  Where were much higher concentrations of t o t a l and i n d i v i d u a l amino acids i n the suspension culture samples, e s p e c i a l l y near the end of the growth cycle, than i n the coffee bean. Cysteic acid and methionine were both found i n the suspension culture but were not detectable i n the green bean. Cysteic acid was very low i n concentration and appeared only l a t e i n the growth cycle while methionine was present at low concentrations throughout the growth of the c e l l s . The major amino acids present i n the suspension cultures were aspartic acid, glutamic acid, alanine, v a l i n e , and phenylalanine while the predominant free amino acids i n the coffee bean were threonine, serine, glycine, alanine and phenylalanine (Table 6). The suspension culture contained f a i r l y high concentrations of threonine, serine, glycine and isoleucine while the coffee bean contained v a l i n e . One would expect differences e s p e c i a l l y i n the asparagine (aspartic acid) and glut amine (Glutamic acid) l e v e l s . Without including aspartic acid and glutamic acid content the major free amino acids were s i m i l a r (Table 6) . Intact plant t i s s u e has been shown to contain higher amounts of free amino acids than i t s c a l l u s cultures (Steward et a l , 1958; Krikorian, 1965; Table 1). The content of free amino acids detected i n coffee suspension cultures was i n the expected range. The coffee bean, however, was very low i n free amino acids. This may be a result of the non active state of the bean - that i s the bean had been k i l l e d by drying 61 a f t e r harvest. Thus, the content found may not be representative of fresh green beans. The content, however, r e f l e c t s the concentration of free amino acids i n the coffee beans before roasting. The major i d e n t i f i e d free amino acids present i n the bean are present as major i d e n t i f i e d free amino acids i n the c e l l cultures. 3. Roasting of the green coffee bean and i t s extracts,  a. Roasting of green coffee. i . Roasting of the green coffee bean i n i t s e n t i r e t y . Before roasting the coffee bean i s grey green i n colour, very hard and has a very disagreeable odour. A f t e r roasting the bean was dark brown i n colour externally, beaded with o i l droplets, and had no distinguishable odour. The aroma was released o n c r u s h i n g the bean. The roasted bean crushed quite e a s i l y i n d i c a t i n g a change i n the bean possibly due to gas release and water evaporation as well as component reactions. The bean was dark brov/n i n colour i n t e r n a l l y . Longer roasting of the coffee bean caused a rapid change i n colour to black, the o i l droplets on the surface increased and the product smelt burnt before and a f t e r crushing. The important phase of the reaction which occurs on roasting i s very rapid and thus the reaction must be stopped immediately at the right point of roasting to produce a s a t i s f a c t o r y product. i i . Roasting of ground green coffee beans. Grinding the coffee bean before roasting was found to have a profound effect upon the c a p i b i l i t y of the grounds to 62 roast. The grounds, on roasting f i f t e e n t o twenty minutes at 220°C, turned a deep brown colour. No o i l droplets were observed on the surfaces of the grounds and no coffee aroma was detected even a f t e r attempting to crush the roasted grounds. The grounds were very hard even a f t e r roasting longer than twenty minutes. No aroma was detectable a f t e r having f r e s h l y roasted grounds i n a small covered container overnight. Roasting the grounds i n the presence of water was attempted. It was hoped that the presence of a moister atmosphere i n the oven would slow down water evaporation from the green coffee grounds and thus enable 'coffee roasting reactions' to occur. Although the roasting procedure was s l i g h t l y slower the r e s u l t s were the same as roasting without water present. When the roasted grounds were ground the i n t e r n a l areas of i n d i v i d u a l grounds were found to be of a l i g h t roast colour or s t i l l green bean colour. No roasted coffee aroma could be detected upon grinding. Tne grounds were very d i f f i c u l t to roast evenly e s p e c i a l l y when roasted i n large quantities as-the i n d i v i d u a l grounds tended to s t i c k together, and heat t r a n s f e r was not even throughout the mass. i i i . , R o a s t i n g of the green bean p e l l e t . Ground green coffee was pelle t e d and then roasted to determine i f a lar g e r sized object would roast more na t u r a l l y . The r e s u l t s were s i m i l a r to those obtained f o r the ground green coffee. A f t e r f i f t e e n minutes of roasting at 220°C the external surfaces of the p e l l e t were deep brown i n colour. No o i l droplets were present and no detectable aroma was released other than burnt or green bean aroma. The i n t e r n a l grounds were externally paler i n colour than were the external grounds and no o i l was observed on these grounds eit h e r . 63 The smaller p e l l e t s roasted somewhat f a s t e r and were darker i n t e r n a l l y than the larger p e l l e t s but otherwise the r e s u l t s were the same. In the presence of water roasting of a l l the p e l l e t s was slower but s t i l l produced s i m i l a r r e s u l t s . The p e l l e t s , on roasting, expanded mainly upward - that i s , along the perpendicular axis. There was more expansion of the p e l l e t s expanded, cracking across the axis causing layering of the p e l l e t . The apparent expansion of the p e l l e t s could be attributed to several factors, f o r example evaporation of i n t e r n a l water and/or release of gases causing i n t e r n a l pressure which was r e l i e v e d by expansion of the p e l l e t . This i s s i m i l a r to the expansion which occurs on roasting coffee beans. The roasted p e l l e t s e a s i l y fragmented into grounds without the release of 'coffee' aroma. The i n d i v i d u a l grounds whether i n t e r n a l l y or externally located within the p e l l e t were s t i l l very hard and on crushing did not release the 'coffee' aroma. i v . Discussion on roasting of green coffee. The green coffee bean roasted i n the manner expected from the l i t e r a t u r e . The ground green coffee bean, however, did not produce coffee aroma and did not brown well. The coffee bean has been likened to a miniature autoclave i n which the reactions occur i n a controlled environment (Keable, 1910). It has also been suggested that the s e l e c t i v i t y of the reactions that occur i s also governed by low water content, l o c a l i z e d buffer systems and a f l u c t u a t i n g balance of reaction products. By grinding the coffee bean before roasting the r e s t r i c t e d environment i s destroyed and the balance that exists within the bean i s upset. Roasting, however, should s t i l l produce a browning of the ground bean 64 although even browning throughout the samples was not obtained probably as a re s u l t of uneven thermal conductivity. I n t e r n a l l y the coffee bean i s almost completely anaerobic r e s t r i c t i n g the reactions that occur, allowing interactions by non-volatile compounds and r e s t r a i n i n g the v o l a t i l e s formed within. On roasting, the covering of the intact bean becomes impermeable to gases or v o l a t i l e s t r y i n g to pass outward. Thus the v o l a t i l e s are retained. Grinding of the coffee bean allows i n t e r n a l surfaces to be exposed to aerobic conditions and w i l l , thus, upset the course of the roasting reactions. The rate of water evaporation i s very important as the browning reactions only occur over a narrow range of t o t a l moisture content. The grounds of green coffee beans cannot r e t a i n moisture and the water l e v e l possibly decreased too r a p i d l y f o r complete browning to occur. In summary, i t would appear that while the intact bean i s caT}ab3.e of producing a coffee colour, aroma, and flavour, the ground bean has l o s t t h i s a b i l i t y at least under the conditions studied. b. Roasting of green coffee bean extracts. Extracts obtained from green coffee were roasted i n various mixtures i n an attempt to i s o l a t e an extract which when roasted would produce a coffee aroma. The extract would then contain some of the major precursors of coffee aroma. The solvents used were water, methanol, and ethanol. i . Water extracts of green coffee beans. The water extract of the green coffee bean was a cloudy grey green before freeze drying and when l y o p h i l i z e d was a d u l l green colour. It contains water soluble proteins, free 65 amino acids, simple carbohydrates, some chlorophyll and other compounds. Water extracts of the coffee bean on the whole did not produce a fresh coffee aroma on roasting (Table ?a) although roasting of the extract alone or with calcium carbonate produced a f a i n t s t a l e coffee aroma and nice coffee brown colour. Otherwise no coffee aroma could be detected on roasting the water extract with other additives. The colour of the end products of water extracts with other additives was dark brown to black. i i . Methanol extract of green coffee beans. The methanol extract of green coffee was c l e a r with no colour before freeze drying. The l y o p h i l i z e d product was white and f l a k y . The extract contains free amino acids, some peptides, and other compounds. On roasting the extract produced a f a i r l y good coffee aroma although with not quite the f u l l body obtained from f r e s h l y roasted coffee beans (Table 7b). The additives did not increase the coffee aroma produced on roasting but tended to mask i t s l i g h t l y . i i i . Ethanol extract of green coffee beans. The ethanol extract of green coffee was c l e a r and yellow i n colour before freeze drying and a f t e r was white and flaky. The extract contained free amino acids, some carbohydrates and other compounds. This extract on roasting alone or with additives tended to produce an aroma reminiscent of roasted o i l seeds (such as peanuts; Table 7c). 1 i v . Roasting of combined extracts of green coffee bean. It was found that the combinations resulted i n aroma reminiscent of roasted o i l seeds (Table 7d). Table 7. The aroma and colour produced upon roasting at 220°C of coffee extracts v/ith various additives ( i n the presence of water). a. roasting of water extract. mixture roasted c o n t r o l (v/ater extract only) con t r o l and calcium carbonate (CaCOo) cont r o l and IN sodium hydroxide (NaOH) con t r o l and CaCOj and IN NaOH con t r o l and dextrose con t r o l and IN hydrochloric acid (HG1) control and acid and destrose and GaCO^ b. roasting of methanol extract. control c o n t r o l c o n t r o l c o n t r o l c o n t r o l cont rol. (methanol extract only) and and and and and CaCO 3 NaOH' NaOH and CaCO' dextrose HG1 (IN) con t r o l and dextrose and acid and CaCO-c. roasting of ethanol extract. c o n t r o l (ethanol extract only) c o n t r o l c o n t r o l c o n t r o l c o n t r o l c o n t r o l c o n t r o l and and and and and and CaCO 3 IN NaOH CaCO 3 and dextrose IN HC1 dextrose NaOH (IN) and acid and CaCO' d. roasting of combined extracts water and ethanol extracts water and methanol extracts ethanol and methanol extracts colour produced brown dark brown black brown black brown dark brown black brown l i g h t brown brownish l i g h t brown black brown black brown medium brown medium brown black brown brown dark brown black brown black brown medium brown dark brown black brown dark brown dark brown carmel brown aroma produced very s t a l e coffee very s t a l e coffee non d e s c r i p t i v e * non d e s c r i p t i v e burnt carmel sweet smelling burnt and sweet coffee-cocoa coffee l i k e non descriptive non descriptive sweetened coffee sweet, a c i d i c and coffee l i k e sweet and coffee l i k e roasted peanuts roasted sunflower seeds non descriptive non descriptive cooked vegetable protein sweet and a c i d i c burnt sugar roasted broad beans roasted broad beans bland roasted peanuts * non descriptive - there was a strong aroma present i n each case but i t was d i f f i c u l t to rel a t e i t to anything f o r description. The aroma was not pleasant and was not co f f e e - i s h at a l l . 67 v. Miscellaneous. The addition of caffeine to a mixture of sugar and tannic acid was stated by Erdman (1902) to produce a coffee aroma. When the l a t t e r materials.were roasted alone, i n mixtures, or i n various combinations with water, methanol, and ethanol extracts coffee aroma was not detected, A musky woody aroma was produced i n a l l roasted mixtures containing tannic acid. Caffeine appeared to have no effect on the aroma production. v i . Discussion of the roasting of green coffee bean extracts.  Rohan and Stewart (1965) and Rohan (1965) found that roasting a methanol extract from fermented cacao beans produced a cocoa aroma and thus contained the primary precursors of cocoa aroma. In t h i s study methanol was also found to be the best solvent f o r extracting the precursors of coffee aroma from coffee beans. It would, therefore, appear that the major precursors of coffee aroma couiibe amino acids, sugars, and flavonoids (Rohan, 1965) although other compound classes are involved i n producing a more f u l l bodied coffee aroma (Gianturco, 1967). 4. Caffeine determination i n Coffea. arabica L. c e l l suspension cultures and green coffee beans.  Caffeine was detected i n the c e l l s of a Coffea arabica L. c e l l suspension culture although the results obtained were v a r i a b l e . a. Caffeine determination - methods. The usual method f o r caffeine determination (Horwitz AOAC , 1970) was modified s l i g h t l y f o r microdetermination. The t r i a l k j e l d a h l determinations performed were very low and variable between samples. It was hoped that Nessler's reagent would be more se n s i t i v e but a c o l l o i d a l orange p r e c i p i t a t e which formed i n some cases i n t e r f e r r e d with the o p t i c a l density reading. 68 b. Caffeine content i n c e l l suspension. Cultures of Coffea arabica L.  Caffeine was detected i n coffee suspension cultures (Table 8 ) . Although the apparent values were possibly higher than actual values the trend could be detected. The values were possibly higher than actual values as a result of the presence of c o l l o i d a l matter. The caffeine content decreased during active growth and increased i n content during nonactive growth periods (Figure 1 3 and Table 8 ) . Tea c a l l u s cultures (Ogutuva and Northcote, 1 9 7 0 a and 1 9 7 0 b ) were found also to synthesize caffeine i n a s i m i l a r pattern -only during non active growth did caffeine, accumulate. The concentration produced i n the tea c a l l u s culture grown i n the dark was 1 , 5 0 0 micrograms per gram dry weight at maximum. The coffee suspension culture was not as productive - producing' only 3 6 0 micrograms per gram dry weight. Ogutuva and Northcote ( 1 9 7 0 a ) suggested that the caffeine which appeared i n the l a t t e r portion of the rapid phase of growxh i n t ea ca l l u s cultures was formed by those c e l l s which had fin i s h e d active growth. According to the l a t t e r authors some of the caffeine possibly was formed as a result of the catabolic breakdown of nucleic acids rather than from direct synthesis of purines within the c e l l . The caffeine produced within the c e l l i s eithe r secreted immediately into surrounding medium and i s unable to reenter active c e l l s or i s concentrated within the c e l l i n a membrane bound vacuole removing the caffeine from contact with the l i v i n g c e l l (Ogutuva and Northcote, 1 9 7 0 a 1 9 7 0 b ) . The caffeine i n coffee cultures could be produced as i t i s i n tea. t i s s u e s . A f t e r active growth the c e l l may contain high amounts of nucleic acids and may route t h e i r disposal v i a caffeine which the c e l l by some means can secrete or envelop to remove i t from direct contact with the c e l l . This process 69 Table 8. Caffeine content of Coffea arabica L. c e l l suspension cultures. time (days) dry weight of cultures (grams)* caffeine content of c e l l s * (Yg/g dry wt) pH of medium' 0 0.016 - 4.30 4 0.050 - 4.60 6 0.066 75.0 4.70 8 0.110 37.5 4.70 10 0.263 51.0 5.55 12 0.431 100.0 5.75 14 0.605 150.0 6.20 16 0.677 185_.0 6.70 J.O 0.790 225.0 6.80 20 0.757 301.0 6.20 22 0.733 375.0 6.85 2? O.665 360.0 7.60 coffee bean *values given are averages of two samples (the caffeine i s done i n six r e p l i c a t i o n s of each sample). ?0a J : ! • I I L_ 4 1 0 1 4 1 8 2 2 2 7 T i m e ( d a y s ) 70b Figure 13• Caffeine content of Coffea. arabica L. c e l l suspensions. Values are average of two samples and three r e p l i c a t i o n s of each (Values given i n Table 8). Symbolsi « © dry weight of Coffea arabica L. C e l l s • c caffeine content of c e l l s 71 would predominately occur i n older c e l l s although some breakdown would occur even i n a c t i v e l y d i v i d i n g c e l l s . The c e l l may also synthesize caffeine d i r e c t l y f o r a short space of time u n t i l back feeding of caffeine to the i n i t i a l steps i n purine formation i n h i b i t s the biosynthetic sequence. c. Caffeine content i n green coffee beans. The caffeine content detected i n green coffee beans was 1.15/0 which i s f a i r l y near the values obtained by Lehman (1971) and Feldman et a l (1969). d. Paper chromatography of c a f f e i n e . Caffeine was detected i n c e l l suspension cultures, culture medium and green coffee beans. Theobromine was possibly the other major spot present (Figure 14) . Theobromine did not i n t e r f e r e in-the analysis a s . i t was removed by the addition of a c i d i c water to the chloroform extract. The standard caffeine sample was net pure caffeine according to the paper- chromatographic r e s u l t s . e. Discussion. Caffeine was produced by the coffee suspension culture at approximately 0.03% dry weight i n contrast to the beans' content of 1.15/6 dry weight. It i s obvious that one of the enzyme systems involved i n caffeine production i s s t i l l present. Caffeine production may be a method f o r the c e l l to dispose of non e s s e n t i a l nucleic acids. 5« Chlorogenic acid i n the Coffea arabica L. c e l l suspension culture and the green bean.  Chlorogenic acid was detected i n the c e l l s of a Coffea arabica L. c e l l suspension culture (Figure 17)' As with most secondary 72a 72b Figure 14. Paper chromatogram showing the presence of caffeine i n green coffee bean and Coffea arabica L. c e l l s and medium extracts. A. medium extract B. coffee c e l l extract n „ „ .r1 -O _ • - — i _i oct J. x c n i c Di»aiiuBi'u D. coffee bean extract spot i d e n t i f i c a t i o n 1. caffeine 2. possibly theobromine 73 metabolites produced i n t i s s u e cultures the maximum l e v e l detected i n the cultures (0.14/6 dry weight) was well below the l e v e l normally present i n green coffee beans (6.5% dry weight) (Table 9)* a. Chlorogenic acid determination - methods. Chlorogenic acid analysis was not started u n t i l s i x days a f t e r inoculation as there was i n s u f f i c i e n t material available f o r the determination. The method (Horwitz (AOAC), 1970) used was modified to accomodate the amount of material available f o r analysis. The spectrum scan of a chlorogenic acid standard showed that the. optimum absorption was at 324 millimicrons as suggested i n Horwitz (1970) (Figure 15a). The c a l c u l a t i o n of the chlorogenic acid content of the samples was done on an enlargement of Figure 15b. The difference i n content of chlorogenic acid between the coffee bean .and the culture sample i s obvious. The method used did not d i s t i n g u i s h among the d i f f e r e n t isomers of chlorogenic acid (chlorogenic acid, isochlorogenic acid and neochlorogenic acid) and i t i s possible that i t determined only chlorogenic acid and not i t s isomers. However, no trace of the isomers was detected on paper chromatograms. 74 b. Chlorogenic acid i n the culture medium. The presence of chlorogenic acid was not detected using paper chromatography i n the culture medium. This may have been a result of the presence of only minute quantities or the complete absence of chlorogenic acid i n the medium. The pH of the culture medium rose f a i r l y s t e a d i l y during the growth cycle of the culture i n d i c a t i n g no notable secretion of acid into the medium (Table 9). An a n a l y t i c a l determination on the medium was not attempted as the detection of i t s presence by q u a l i t a t i v e paper chromatography f a i l e d . 75a 75b Figure 15« Standard curves f o r chlorogenic acid. a. Determination of wavelength best suited f o r chlorogenic acid detection. b. Standard curve f o r conversion of the absorbance value to a concentration value. - a sample of green coffee bean and of a c e l l suspension are dotted i n . 76 Table 9. The chlorogenic a c i d content of Coffea a r a b i c a L. suspension c u l t u r e s . time dry weight c h l o r o g e n i c a c i d pH of (days) of c e l l s * content of c e l l s * medium* (grams) (mg/g dry wt) 0 0.020 4.30 2 0.025 - 4.39 4 0.049 - 4.58 6 0.122 0.632 4.93 8 . 0.136 O.929 5.36 10 O.36I O.607 5.70 12 0.421 0.061 5-73 14 O.676 0.000 6.16 16 0.848 0.337 6.15 18 0.760 1.084 6 . 7 O 20 O.702 1.323 6.96 22 0.731 1.147 7.10 25 0.647 1.375 6.99 28 0.666 1.092 7.50 32 0.585 1.125 7.93 co f f e e bean 65.220 *values given are the average of f o u r samples. 77 c. Chlorogenic acid content i n Coffea arabica L. c e l l suspension cultures.  The chlorogenic acid content of the cultures appeared to r i s e and then decrease before rapid growth began. A f t e r the rapid phase of growth of the culture had ceased the content of chlorogenic acid rose r a p i d l y l e v e l l i n g o f f during the decline of the culture. Chlorogenic acid did not appear to be produced by the culture during active growth (Figure 16). It would appear that only the older c e l l s , not a c t i v e l y d i v i d i n g , can synthesize chlorogenic acid as seen by the production of chlorogenic acid only during periods of l i t t l e or no net culture growth (Figure 16 and Table 9)- The maximum l e v e l of chlorogenic acid was obtained just a f t e r the peak of growth (as expressed i n dry weight) had been reached. To determine i f chlorogenic acid was produced normally by other cultures a bean (Buckland, 1972) and a rose (Wegg, 1972) suspension culture were analyzed. There was no chlorogenic acid detected i n e i t h e r sample. d. Chlorogenic acid i n green coffee beans. The content of chlorogenic acid i n the green coffee beans was 6.5% dry weight (Table 9)• The normal range i s 5«5 to 7«5% dry weight (Lehmann et a l , 1967). e. Paper chromatography of chlorogenic acid i n extracts of green coffee bean and coffee t i s s u e cultures.  Paper chromatography of extracts of the green coffee bean and the c e l l suspensions i l l u s t r a t e d the presence of c a f f e i c acid and chlorogenic acid (Figure 17). Samples from twelve, fourteen, and sixteen day old cultures f a i l e d to show the presence of chlorogenic acid although traces of c a f f e i c acid were detected. ?8b Figure 16. Chlorogenic acid production of Coffea arabica L. c e l l suspension culture. Values are the average of four samples (values are given i n Table 9 ) . T~ ~ "I — oy i n u \j j _ o i -o dry weight of Coffea arabica L. c e l l suspension cultures. chlorogenic acid content of Coffea arabica L . c e l l suspension cultures. 79a o A 79b Figure 17• Paper chromatogram showing the presence 'of chlorogenic acid i n extracts from green coffee and a coffee suspension culture. Key: A. organic acid standard 1. oxalic acid 2. t a r t a r i c acid 3 ' c i t r i c acid 4. malic acid 5» chlorogenic acid 6. c a f f e i c acid B. c e l l suspension culture extracts twenty day old culture C . green coffee bean Areas dotted i n were fluorescent under u l t r a v i o l e t l i g h t and the darkened areas were detected using a n i l i n e - f u r f u r a l reagent. The areas outlined with a s o l i d black l i n e appeared white using both detection methods. 80 f. Discussion. Since chlorogenic acid was detected i n Coffea arabica L. cultures an enzyme system responsible f o r chlorogenic acid production i s , therefore, present i n the c e l l s . This synthesizin system would not appear to be active during c e l l d i v i s i o n . However, according to Colonna and Boudet.(1971) chlorogenic acid may play an active metabolic role during c e l l d i v i s i o n and hence only accumulates during non active periods i n the c e l l cycle. The enzyme system(s) f o r chlorogenic acid production was shown to be species s p e c i f i c as no chlorogenic acid was detected i n the rose or bean suspension cultures whose parent tissues do not normally contain chlorogenic acid. 81 SUMMARY Suspension cultures were derived from the Coffea arabica L. plant. These cultures were healthy and grew rapid l y . C e l l s c h a r a c t e r i s t i c a l l y grew i n long filamentous chains and i n many cases contained unknown bodies which possibly could have been amylopectin starch. The cultures were analyzed q u a n t i t a t i v e l y f o r t h e i r content of free aminoacids, chlorogenic acid and c a f f e i n e . The t o t a l free amino acid content was found to be greater i n the t i s s u e culture c e l l s than i n the coffee bean. The major free amino acids present i n the Coffea arabica L. culture c e l l were aspartic acid, glutamic acid, phenylalanine, alanine, valine, threonine, serine, and glycine whereas i n the green coffee bean threonine, serine, glycine, alanine and phenylalanine were the predominate ones. Roasting of green coffee bean indicated that grinding before roasting disrupts the reactions involved i n browning and flavour and aroma development of the coffee bean. The i n a b i l i t y to properly roast ground coffee bean i s probably p a r t l y a result of poor thermal conductivity. The roasting of various green coffee bean extracts indicated that methanol was the best solvent f o r extracting major 'coffee.' precursors. The roasting of the methanol extract produced a mild but s t a l e coffee aroma. Caffeine and chlorogenic acid were both detected i n the c e l l s of Coffea arabica L. The content was, however, well below the content normally detected i n the parent plant. Determination of chlorogenic acid i n other t i s s u e cultures (rose and bean) detected no chlorogenic acid present. In summary, Coffea arabica L. suspension culture c e l l s were shown to be species s p e c i f i c i n t h e i r a b i l i t y to produce s i m i l a r free amino acid patterns, caffeine and chlorogenic acid, the l a t t e r two i n lower qua n t i t i e s . 82 BIBLIOGRAPHY Anderson, L. and Gibbs, M. 1962. Biosynthesis of caffeine i n the coffee plant. J . B i o l . Chem. 23?:1941-4. Beauden-Dufour, D., and Mueller, L.E. 1971' E f f e c t of s o l a r r a d i a t i o n and age on the content of caffeine and nitrogen i n leaves and f r u i t s of three species of coffee trees. T u r r i a l b a 21(4):387-392. Chemical Abstracts 76:70258h. Bergman, L. 1963. (published 1965). The effect of k i n e t i n on the metabolism of plant t i s s u e cultures. Proc. Int. Conf. Plant Tissue Culture, Penn State Univ. 171-181. Beveridge, T. 1972. Personal communication. University of B.C. Blakely, L.M., and Steward, F.C. 1964. Grov/th and organized development of cultured c e l l s . V. The grov/th of colonies from free c e l l s on nutrient agar. Amer. J . Bot. 51(7): 780-791. Bove, J . , Bave, C , and Raveux, G. 1957- Extraction, separation et determination, de certains composes hydrosolubles (giucides solubles, acid carboxyliques non v o l a t i l s de U 2 A C 5 et acides amines solubles) dans les plant c e l l s et diverses cultures de t i s s u s de Citrus lemonum. Revue Gen. Bot. 64:572-592. Brenner, M.f Niederwiesser, A., and Pataki, G. 1963. Amino acids and derivatives, pages 391-440. In Stahl, E. ( e d i t o r ) . , Thin Layer Chromatography. Academic Press Inc., Publishers, New York. Broderick, J . J . 1968. Coffee - a new approach. Amer. Perfumer  and Cosmetics 83:37-38. Buckland, E.J. 1972. Unpublished data. Chassevant, F. 1969. P h y s i o l o g i c a l and pharmacological actions of chlorogenic acid . Ann. Nutr. Aliment. 23(1): 1R-14R. Clarke, E.G. ( e d i t o r ) . 1969. I s o l a t i o n and i d e n t i f i c a t i o n of  drugs. Pharmaceutical Press, London, page 234. Colonna, J.P., and Boudet, A . 1971- K i n e t i c study of the incorporation of C-14 l a b e l l e d quinic and cinnamic acids into chlorogenic acid of coffee (Coffea d-awevrei race  excelsa) l e a f t i s s u e s . C.R. Acad. S c i . , Ser. D 272(7): 952-955. Chem. Abstracts 74"« 108212n.' 83 Danehy, J.P., and Pigman, W.W. 1 9 5 1 ' R e a c t i o n s between s u g a r s and n i t r o g e n o u s compounds and t h e i r r e l a t i o n s h i p t o c e r t a i n f o o d p r o d u c t s . Adv. i n Food R e s e a r c h 3 * 2 4 1 - 2 9 0 . Erdmann, E. 1 9 0 2 . B e i t r a g z u r K e n n t n i s s des K a f f e c o l e s . B e r . 3 5:1846. from K i r c h n e r , J.G. 1 9 4 9 . The c h e m i s t r y o f f r u i t and v e g e t a b l e f l a v o u r s . Adv. i n Food R e s e a r c h 2s 2 7 7 - 2 8 3 . Fecak, B., and S t r u h a r , M. 1970. E v a l u a t i o n o f s e v e r a l t y p e s of t e a . Prum. P o t r a v i n 2 1 ( 1 ) 5 7 - 1 8 . Chem A b s t r a c t s ?2: 118457u. Feldman, J.R., Ryder, W.'S.,. and Kung, J.T. 1 9 6 9 . Importance o f non v o l a t i l e comoounds t o t h e f l a v o u r o f c o f f e e . J ' A g r i c . Fd. Chem/ 1?(4) j - 7 3 3 - 7 3 9 • F i t e l s o n , J . 1 9 6 9 . P a p e r c h r o m a t o g r a p h i c d e t e c t i o n o f manor o r g a n i c a c i d s i n f r u i t j u i c e s . J . A. 0 . A. C. 5 2:646-649. F i t z p a t r i c k , T . J . , and P o r t e r , W.L. 1 9 6 6 . Changes i n t h e s u g a r s and amino a c i d s i n c h i p s made from f r e s h , s t o r e d , and r e - c o n d i t i o n e d p o t a t o e s . Amer. Pot at o J . 43:238-248. Fitzsimmons,• R.C., and Mason, D. 1972. p e r s o n a l communication. F l e t c h e r , J.S., and B e e v e r s , K. 1 9 7 0 . A c e t a t e metabolism, i n c e l l s u s p e n s i o n c u l t u r e s . P l a n t P h y s i o l . 4 5 ( 6 ) : 7 6 5 - 7 7 2 . F o r e i g n A g r i c . S e r v i c e s . (May - June) 1971 - 1 9 ? 2 . U.S.D.A. F r i e d e l , P., Kr a m p l , V., R a d f o r d , T., Renner, J.A., Shephard, F.W., and G i a n t u r c o , M.A. 1 9 7 1 . Some c o n s t i t u e n t s o f t h e aroma complex o f c o f f e e . J . A g r i c . Fd. Chem., 1 9 ( 3 ) : 5 3 0 - 5 3 2 . F u r i a , B. 1 9 7 1 . C o f f e e . I n F u r i a , B. ( e d i t o r ) , Handbook o f F l a v o u r I n g r e d i e n t s . C.R.C. Gamborg, O.L. 1 9 6 7 . A r o m a t i c m e t a b o l i s m i n p l a n t s , v. The b i o s y n t h e s i s o f c h l o r o g e n i c a c i d and l i g n i n i n p o t a t o c e l l c u l t u r e s . Can. J . Biochem. 4 5 :1451-1457• Gamborg, O.L., and E v e l e i g h , D.E. 1 9 6 8 . C u l t u r e methods and d e t e c t i o n o f g l u c a n a s e s i n s u s p e n s i o n c u l t u r e s o f wheat and b a r l e y . Can. J . Biochem. 46:417-421. G a u t h e r e t , R.J. 1 9 3 9 ' S u r l a p o s s i b i l i t e de r e a l i s e r l a c u l t u r e i n d e f i n i e des t i s s u s de t u b e r c u l e s de c a r o t t e . C.R. Acad. S c i . ( P a r i s ) 208:118-120. 84 Gautheret,' R. J . 1 9 3 4 . Culture du t i s s u cambial. C.R. Acad. S c i . (Paris) 1 9 8 : 2 1 9 5 - 2 1 9 6 . Gautschi, F. , Winter, M. , Flament, I. , Willhalm, B., and S t o l l , M. I 9 6 7 . The chemistry of coffee aroma - a survey of present knowledge. In T h i r d Int. Colloquium on the Chem. of Coffee. pub. 1 9 6 8 . Assoc. S c i . Int. du Cafe. Gautschi, F., Winter, M., Flament, Y., Willhalm, B., and S t o l l , M. 1 9 6 7 . New developments i n coffee aroma research. J . Agric. ' Fd. Chem. 1 5 ( 1 ) : 1 5 - 2 3 . General Foods Corporation, 1 9 6 6 . Beverage aromatic materials. Ger. 1 , 2 2 6 , 4 0 5 . (patent) General Foods Corporation. 1 9 6 3 * A. method of manufacturing flavours. Belg. 6 3 1 , 1 0 1 . (patent) Gianturco, M.A. 1 9 6 7 . Coffee flavour. In Schultz, H.W. (e d i t o r ) , Symposium on Foods: the chemistry and physiology of flavours. Avi publishing .Comp. , Inc., New York, pages 4 3 1 - 4 4 9 . Gianturco, M.A. 1 9 6 5 * Coffee flavours. In Chemistry and  Physiology of Flavours. pages 4 3 1 - 4 4 9 . r i : „ -u .. r-.fi A r » - ~ ; . A O TI — i ~ j ~ i * n - a -i _. - _ : . - ir u ± m i K U i ^ O | i«i . n . , u j . e u i i i u c i i . m u , .n • >_> • , r j . x c u . c j . , X " . , e u i u i ' X c H i d g , t a i l , v . 1 9 6 4 . The v o l a t i l e constituents of coffee. i v . . Furanic and p y r r o l i c compounds. Tetrahedron 2 0 : 2 9 5 1 - 2 9 6 1 . Gornall, A.G., Bardawell, C.J., and David, M.A. 1 9 4 9 . Determination of serum proteins by means of the biuret reaction. J . B i o l . Chem. 1 7 7 : 7 5 1 - 7 6 6 . Haberlandt, G. 1 9 0 2 . Kulturversuche mit i s o l i e r t e n Pflanzenzellen. S. B. Akad. Wiess. Wien. Math.-Naturw. Kl 1 1 1 : 6 9 . see N i c k e l l , 1 9 6 2 ; White, 1 9 6 3 ; and White, 1 9 5 4 . Hamerslag, W. 1 9 5 0 . The technology and chemistry of al k a l o i d s . D. van Nostrand Comp., Inc. New York. Hodge, J.E. 1 9 6 7 . O r i g i n of flavour i n foods- - nonezymatic browning reactions. In Schultz, H.W. ( e d i t o r ) . Symposium  on Foods: the chemistry and physiology of flavours. Avi Publishing Comp., Inc. New York. pages~ 4 ~ 6 5 - 4 9 1 . Hodge, J.E. 1 9 5 3 - Browning reaction theories integrated i n review - dehydrated foods - chemistry of browning reactions i n model systems. J . Agric. Fd. Chem. 1 ( 1 5 ) « 9 2 8 - 9 4 3 . 85 Horwitz, W. ( e d i t o r ) . 1 9 7 0 - O f f i c i a l methods of analysis. Assoc. of O f f i c i a l A g r i c u l t u r a l Chemists (A. 0 . A. C.) Washington. Jones, K., and Heathcote, J.G. i 9 6 0 . The rapid r e s o l u t i o n of n a t u r a l l y occurring amino acids by t h i n layer chromatography. J . Chromatog. 1 0 6 - 1 1 1 . Kaeber, P. 1 9 6 5 * Breakdown of caffeine on the leaves of Coffea arabica. Nature 2 0 5 ( 4 9 7 1 ) » 5 9 7 - 5 9 8 . Keable, B.B. 1 9 1 0 . Coffee. Pitman and Sons Ltd., London, (revised by Sanderson, H.S.) K l e i n , R.M. i 9 6 0 . Plant t i s s u e cultures, a possible source of plant constituents. Econ. Bot. 14; 2 . 8 6 - 2 8 9 • Koiwai, A., Noguchi, M., and Tomaki, E. 1 9 7 1 * Changes i n the amino acid composition of tobacco c e l l s i n suspension culture. Phytochem. 1 0 : 5 6 1 - 5 6 6 . K r i k o r i a n , A.D. 1 9 6 5 « The synthetic p o t e n t i a l i t i e s of cultured plant c e l l s and t i s s u e s . PhD. C o r n e l l University. K r i k o r i a n , A.D., and Steward, F.C. 1 9 6 9 . Biochemical d i f f e r e n t i a t i o n : the biosynthet 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 . In Steward, F.C. ( e d i t o r ) . Plant Physiol. 11B: 2 2 7 - 3 2 6 . Lee, S. 1 9 6 2 . Role of chlorogenic acid i n coffee. Tea Coffee  Trade J . 1 2 3 ( 1 ) : 1 3 . Lehman, G. 1 9 7 1 . [Physiological important constituents of coffee] . Ernachr.-Umsch. 1 8 ( 2 ) : 4 3 - 4 ? . Lehman, G., Hahns, H.G., and Luzuriaga, 0 . 1 9 6 7 . [Chlorogenic acid content i n raw coffee, roast coffee, and coffee extract powders]. Deut. Lebensm. Reindsch. 6>3( 9 ) : 2 7 3 - 2 7 5 . Leonova, L.A., Gamanets, L.V., and Gamburg, K.Z. 1 9 7 0 . [Effect of auxin on polyphenol content i n c a l l u s t i s s u e of tobacco plants grown i n suspended c u l t u r e ] , F i z i o l . Rast. 1 7 ( 4 ) : 7 3 1 - 7 3 7 . Martin, S. 1 9 7 2 . Personal communications. National Research Council Canada (Ottawa). McDonald, I.J . 1 9 7 - 1 . Filamentous forms of Streptococcus cremoris and Streptococcus l a c t i s . Observations on structure and s u s c e p t i b i l i t y to l y s i s . Can. J . M i c r o b i o l . 1 7 ( 7 ) : 8 9 7 - 9 0 2 . 86 Moore, S., and Stein, W.H. 1 9 5 4 . Procedures f o r the chromato-graphic determination of amino acids on four percent cross linked sulphonated polystyrene resins. J . B i o l . Chem. 2 1 1 : 8 9 3 - 9 0 6 . Muir, W.H., Hildebrandt, A.C., and Riker, A.J. 1 9 5 ^ . Plant t i s s u e cultures produced from single i s o l a t e d c e l l s . S c i . 1 1 9 : 8 7 ? - 8 ? 8 . N i c k e l l , L.G. 1 9 6 2 . Submerged growth of plant c e l l s . Adv. Applied M i c r o b i o l . 4 : 2 1 3 - 2 3 6 . N i s h i , A., and Sugano, N. 1 9 7 0 . Grov/th and d i v i s i o n of carrot c e l l s i n suspension culture. Plant and C e l l Physiol. 1 1 : 7 5 7 - 7 6 5 -Nobecourt, P. 1 9 3 9 - Sur l a perennite et 1 'augmentation de volume des cultures de t i s s u s vegetaux. C.R. Soc. B i o l . (Paris) 1 3 0 : 1 2 7 0 . Ogutuga, D.B.A., and Northcote, D.H. 1 9 7 0 a . Caffeine formation i n tea c a l l u s t i s s u e . J . Exp. Bot. 2 1 ( 6 7 ) : 2 5 8 - 2 7 3 . Ogutuga, D.B.A., and Northcote, D.H. 1 9 7 0 b . Biosynthesis of caffeine i n tea c a l l u s t i s s u e . Biochem. J . 1 1 7 : 7 1 5 - 7 2 0 . Phoenix Pr e c i s i o n instruments Com. Liquid chromatography handbook. Philadelphia, U.S.A. Proiser, E., and Serenkov, G.P. 1 9 6 3 . [ Biosynthesis of caffeine i n t ea leaves]. Biokhimiya 2 8 ( 5 ) » 8 5 7 - 8 6 1 . Chem. Abstracts 6 0 : 3 2 7 7 g . " v. . ' Puhan, Z., and Martin, S.N. 1 9 7 1 . The i n d u s t r i a l p o t e n t i a l of plant c e l l culture. Progress i n Ind. M i c r o b i o l . 9 : 1 4 - 3 9 . Reymond, D., Chavan, P., and E g l i , R.H. 1 9 6 3 . Gas chromatograph analysis of the highly v o l a t i l e constituents of roasted coffee. Recent Adv. i n Fd. Res. 3 : 1 5 1 - 1 5 7 . Reynolds, T.M.. 1 9 6 9 . Nonenzymatic browning - sugar amine inte r a c t i o n s . In Schultz, H.W. ( e d i t o r ) . Symposium.on foods; carbohydrates and t h e i r r o l e s . Avi Publishing Comp., Inc, Weatport, Conn, pages 2 1 9 - 2 5 2 . Reynolds, T.M. I 9 6 5 . Chemistry of nonenzymatic browning I I . Adv. i n Fd. Research 1 4 : 1 6 7 - 2 8 3 . Reynolds, T.M. 1 9 6 3 . Chemistry of nonenzymatic browning. I. the reaction between aldoses and amines. Adv. i n Fd. Research 1 2 : 1 - 5 2 . 87 Rohan, T.A. 1964. The precursors of chocolate aroma: a comparative study of fermented and unfermented cocoa beans. J . Fd. S c i . 2 9 ( 4 ) : 4 5 6 - 4 5 9 * Rohan, T.A., and Stewart, T. 1 9 6 6 . The precursors of chocolate aroma: changes i n the free amino acids during the roasting of cocoa beans. J . Fd. S c i . 31(2)1202-205. Rohan, T.A., and Stewart, T. 1 9 6 5 - The precursors of chocolate aroma: the d i s t r i b u t i o n of free amino acids i n di f f e r e n t commercial v a r i e t i e s of cocoa beans. J . Fd. S c i . 30(3)* 4 1 6 - 4 1 9 . Rose, D. 1 9 7 2 . Personal communication, National Research Council Canada. (Ottawa). Routien, J.B., Tenafly, N.J., and N i c k e l l , L.G. 1 9 5 6 . C u l t i v a t i o n of plant t i s s u e . U.S. (patent) 2 , 7 4 7 , 3 3 ^ . Russwurm, H. 1 9 6 9 . Fractionation and analysis of aroma. precursors i n green coffee. Colloquium Inst. Chim. Cafes  Verts, T o r r e f i e s Leur Deriv. Fourth (pub. 1 9 7 0 ) . pages 1 0 3 -1 0 7 . . S c h a l l e r , D. 1 9 7 2 . Personal communication. Previously of " U n i v e r s i t y of B r i t i s h Columbia.; Schwann, T. I 8 3 9 . Mikroskopische leutersuchungen Uber die Ubereinstimmung i n der Struktur und dem wachstume der T i e r e und Pflanzen. In Engelmann, W. ( e d i t o r ) . 1910. Ostwalds K l a s s i k e r der exa.kten. # 1 ? 6 . Wessinschaften, L e i p z i p . (see White, 1 9 6 3 ; N i c k e l l , 1 9 6 2 ) . S e l f , R., Casey, J . C , and Swain, T. 1 9 6 3 . The low b o i l i n g v o l a t i l e s of cooked foods. Chem. Ind. (London) 863-864. Sharka, B., and Telepcak, M. 1 9 6 4 . Content alternations of components during roasting . Prumsl. Pot ravin. 1 5:188 - 1 8 9 . Chem. Abstracts 6 1 : 6 2 8 3 h . Simpkins, I., and Street, H.E. 1 9 7 0 . Studies on the grov/th i n culture of plant c e l l s . v i i . E f f e c t s of k i n e t i n on the carbohydrate and nitrogen metabolism of Acer pseudoplatanus L. c e l l s grown i n suspension culture. J . Exp. Bot. 2 1 : 1 7 0 - 1 8 5 . • " Sivetz, M. I 9 6 3 . Coffee processing technology. Vol. I I . Avi Publishing Comp., Inc. Smith, R.F. 1 9 6 3 . The chlorogenic acids i n coffee. Cafe, Cacao, The 7 : 2 4 5 - 2 5 2 . " 88 Staba, E.J. 1969* Plant t i s s u e culture as a technique f o r the phytochemist. Recent Adv. i n Phytochem. 2:75-106. Staba, E.J. 1963' The biosynthetic p o t e n t i a l of plant t i s s u e cultures. Development i n Ind. M i c r o b i o l . 4:193-198. S t a r i t s k y , G. 1970. Embryoid formation i n c a l l u s tissues of coffee. Acta Bot. Neerl. 19(4)i509-514. Steck, to'. 1968. Metabolism of cinnamic acid i n plants: chlorogenic acid formation. Phytochem. 7(10)J1711-171?• Steward, F.C., Thompson, J.F., and Poll a r d , J.K. 1958-Contrasts i n the nitrogenous composition of rapidly growing and nongrowing plant t i s s u e s . J.Exp.Bot. 9:1-10. Synge, R.L.M. 1955• Peptides (bound amino acids) and free amino acids. In Modern Methods i n Plant Analysis. Springer Verlag Berline. page 1-22. Thompson, 7*.J. 1971- The story of coffee. Horticulture XLIX (11):20-21, 34-35. Tomita, Y. 1971. (Tissue cultures of higher plants. Chemistry of natural substances). Korvo # 100( 3): 79-90. Townsley. P.M. 1 9 7 ? . Unpublished date. University -of B.C. Townsley, P.M., and Buckland, E.J. 1972. Patent pending. Townsley, P.M., and Buckland, E.J. 1972. Unpublished.data. University of B r i t i s h Columbia. Tulecke., to. 1961. Recent progress and the goals of plant tis s u e culture. Torrey Botanical Club B u l l e t i n 88(5): 350-360. Tulecke, to., and N i c k e l l , L.G. i960. Methods, problems, and results of growing plant c e l l s under submerged conditions. Trans. N.Y. Acad. S c i . Ser. II 22sl96-206. Underwood, G.E., and Deatherage, F.E. 1952. Nitrogen compounds of coffee. Fd.Res. 17:419-424. Walter, W., Grigat, H.G., and Heukeshoven, J . 1970. Uber f r e i e Amino sauren im grunen Kafee. N aturwissensehaft en 57(5):246-247. ~ " Wanner, W., and Kalberer,. P. 1966. (Degradation of caffeine i n Coffea arabica). Abh. Deut. .Akad. Wiss. B e r l i n , Kl Chem., Creol. B i o l . ( 3 ) : 6 0 ? - 6 l 0 . Chem. Abstracts 66: 83124e. 89 Watanabe,.S. I 9 6 9 . (Coffee and cocoa aroma research). Korvo # 9 2 : 5 3 - 6 6 . Chem. Abstract 7 5 : 6 2 1 9 5 z . Wegg, S . 1 9 7 2 . Unpublished t h e s i s . M.Sc. t h e s i s . University of B r i t i s h Columbia. Weidmann, H.L., and Mohr, W. 1 9 7 0 . ( S p e c i f i c i t y of roasted coffee aroma). Lebensm. -Wiss. Technol. 1 3 ( 2 ) : 2 3 - 3 2 . Weinstein, L.H., N i c k e l l , L.G., Laurencot, H.J., and Tulecke, W. 1 9 5 9 » Biochemical and p h y s i o l o g i c a l studies of t i s s u e cultures and the plant parts from which they were derived. I. Agave toumeyana T r e l . Boyce Thompson  Inst. Contributions 2 0 : 2 3 9 - 2 5 0 . Weinstein, L.H., Tulecke, W., N i c k e l l , L.G., and Laurencott, H.G. 1 9 6 2 . Biochemical and ph y s i o l o g i c a l studies of ti s s u e cultures and the plant parts from which they are derived. I I I . Paul's scarlet rose. Contributions Boyce Thompson Inst. 2 1 : 3 7 1 - 3 8 6 . White, P.R. 1 9 6 3 . The c u l t i v a t i o n of animal and plant c e l l s , (second e d i t i o n ) . Ronald Press Comp. N.Y. White, P.R." 1 9 3 9 . P o t e n t i a l l y unlimited growth of excised plant c a l l u s i n an a r t i f i c i a l nutrient. Amer. J . Bot. . 2 6 : 5 9 - 6 4 . White, P.R. 1 9 3 4 . P o t e n t i a l l y unlimited growth of excised tomato root t i p s i n a l i q u i d medium. Plant Physiol. 91 5 8 5 - 6 0 . 0 . Wickremasinghe, R.L., Swain, T., and Goldstein, J.L. I 9 6 3 . Accumulation of amino acids i n plant c e l l t i s s u e cultures. Nature 1 9 9 ( 4 9 0 0 ) : 1 3 0 2 - 1 3 0 3 . Willmer, E.N. 1 9 6 3 . Tissue culture. (fourth edition) Wiley, New York. Winter, M., Gautschi, F., Flament, I., Willhalm, B., and St o i l , M. 1 9 6 7 . ( V o l a t i l e components of coffee aroma). In Solms, J . ( e d i t o r ) . Aroma -Geschmacksstoffe  Lebensm., Fortbildungskur. Forster -Verlag A. -G., Zurich, Switzerland, pages 1 6 5 - 1 9 8 . Chem. Abstract 6 9 : 9 5 H 4 b . Wolfrom, M.L., Plunkett, R.A., and Laver, M.L. i 9 6 0 . Carbohydrates of the green bean. J . Agric. and Fd. Chem. 8 ( l ) : 5 8 - 6 5 . Zucker, M. 1 9 6 3 . Influence of l i g h t on synthesis of protein and chlorogenic acid i n potato tuber t i s s u e . Plant  Physiol. 3 8 ( 5 ) s 5 7 5 - 5 8 0 . 

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