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The single cell suspension culture of the licorice plant, Glycyrrhiza glabra Wu, Chiu Hui 1970

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THE SINGLE CELL SUSPENSION CULTURE OF THE LICORICE PLANT, GLYCYRRHIZA GLABRA by CHIU HUI WU B. S. A., National Taiwan University A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Food Science We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1970 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my Depar tment o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depa r tment The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8 , Canada i i ABSTRACT The c e l l s of the l i c o r i c e plant, Glycyrrhiza glabra, were cultured as a "single c e l l " suspension. Their growth behaviour, y i e l d and metabolic products were studied. The suspension cultures of the l i c o r i c e plant were established from the f r i a b l e calluses obtained from the ra d i c l e , cotyledon and hypocotyl of the germinated seeds. The single c e l l s , regardless of t h e i r o r i g i n showed l i t t l e difference i n c e l l size and morphology. After an apparent adjustment to the medium, the c e l l s required 11-13 days of incubation to reach the maximum c e l l y i e l d of 1.2 gm/100 ml medium, dry weight. During the growth period, the pH of the growth medium decreased from pH 5.6 to pH 4.7 i n the f i r s t few days and then increased to about pH 6. A l e v e l of 10% coconut milk i n PRL-4-CM medium was found to support good c e l l growth; the lower the coconut milk l e v e l , the longer the growth period required to reach the maximum c e l l y i e l d . I t was also found that 0.57o yeast extract could be used to replace the coconut milk i n the PRL-4-CCM medium. The metabolites detected and examined i n the l i c o r i c e single c e l l suspension culture included a v o l a t i l e apple aroma, a polysaccharide p e c t i n - l i k e material, steroids and t r i t e r -penoids. The analyses of the l i c o r i c e c e l l v o l a t i l e apple aroma found under anaerobic conditions indicated the presence i i i of ethanol and some related esters. The monosaccharides found i n the p e c t i n - l i k e poly-saccharide hydrolysate were glucose, fructose, galactose, arabinose, xylose, galacturonic acid and glucuronic acid. The p e c t i n - l i k e material i n the c e l l preparations reached a maximum y i e l d of 1.1 mg/ml af t e r one month of growth. G l y c y r r h i z i n i c acid, the common l i c o r i c e constituent found i n the root, could not be detected i n the suspension cultures. However, several other related compounds which gave t y p i c a l s t e r o i d and t r i t e r p e n o i d reactions were found. S o r b i t o l and fructose were found to be the two major sugars which accumulated i n free form i n the l i c o r i c e c e l l medium. i v ACKNOWLEDGEMENT I would l i k e to express my sincere gratitude to Dr. P. M. Townsley fo r his encouragement, u n f a i l i n g i n t e r e s t and constructive c r i t i c i s m during the course of t h i s study. I also wish to acknowledge my appreciation to Dr. N. R. Bulley, Dr. V/. D. Powrie, Dr. A. J . Renney, Dr. J . F. Richards and professor E. L. Watson f o r t h e i r valuable suggestions and assistence. Thanks are also extended to my husband, whose under-standing and support has made t h i s study possible. TABLE OF CONTENTS page INTRODUCTION 1 LITERATURE REVIEW 4 I. Plant t i s s u e culture 4 I I . L i c o r i c e 11 MATERIALS AND METHODS 21 I. Plant o r i g i n , c u l t u r a l conditions and preparation of single c e l l suspension 21 A. Origin of the plant ...21 B. Medium 21 i . PRL-4-C medium 21 i i . PRL-4-C-CM medium 22 i i i . PRL-4-DM medium 22 i v . PRL-4-1W-GM medium 23 v. PRL-4-YM medium 23 v i . PRL-4-CHM medium 23 C. Callus formation 23 D. Preparation of single c e l l suspension 24 II . Measurement of the c e l l growth ...24 I I I . Quantitative measurement of the pectin-l i k e material 25 IV. Isolation and hydrolysis of the pectin-l i k e material 25 A. Isolation 25 B. Hydrolysis 26 V. Qualitative carbohydrate analysis; paper chromatography 26 v i , page A. Solvents 26 B. Location reagents 27 VI. Isolation of steroids 27 VII. Extraction and p u r i f i c a t i o n of g l y c y r r h i z i n i c acid from l i c o r i c e root s t i c k or dried c e l l s 27 VIII. Hydrolysis of g l y c y r r h i z i n i c acid 29 IX. Reduction of g l y c y r r h e t i n i c acid 29 X. Qua l i t a t i v e s t e r o i d and t r i t e r p e n o i d analyses 29 XI. Methods used f o r lo c a t i n g the steroids and triterpenoids 30 A. Observation under u l t r a - v i o l e t .30 B. Spray with coloration reagent 30 XII. Measurement of oxygen consumption 31 XIII. Qualitative apple aroma analyses 31 XIV. Enzymatic determination of alcohol 31 EXPERIMENTAL RESULTS 32 Part I. GROWTH EXPERIMENTS 32 I. Callus formation and the preparation of single c e l l suspensions 32 I I . Growth curve 34 I I I . E f f e c t of coconut milk l e v e l and growth factors on the growth of l i c o r i c e c e l l s 41 IV. Yeast extract medium 47 V. Ef f e c t of ascorbic acid and 1-gulonolactone on the growth of l i c o r i c e c e l l s 49 Part I I . PRODUCTS FROM LICORICE SINGLE CELL SUSPENSION CULTURE 52 I. F r u i t smell 52 I I . The p e c t i n - l i k e material 54 v i i page A. Y i e l d 54 B. The components of the p e c t i n - l i k e material 57 I I I . Steroids, triterpenoids and g l y c y r r h i z i n i c acid 62 A. Steroids 62 B. G l y c y r r h i z i n i c a c i d 67 C. Triterpenoids 68 IV. The reducing substances of the l i c o r i c e plant c e l l growth medium 70 DISCUSSION 82 I. L i c o r i c e c e l l culture 82 I I . The products from l i c o r i c e single c e l l suspension culture 85 SUMMARY 91 BIBLIOGRAPHY 93 x i i i LIST OF FIGURES - Figure page 1. Procedure f o r i s o l a t i o n and p u r i f i c a t i o n of g l y c y r r h i z i n i c a c i d from l i c o r i c e root s t i c k or d r i e d plant c e l l s 28 2. Callus formed from cotyledon section; begining stage 35 3. Callus formed from root section; begining stage 35 4. A c a l l u s , white at one end and brown at the other end with cle a r d i v i d i n g l i n e 36 5. Large seaweed-like c e l l s (400X) 36 6(a). Kidney-like c e l l s (400X) 37 6(b). Kidney-like c e l l s (1200X) 37 7(a). C o i l - l i k e c e l l s (400X) 38 7(b). C o i l - l i k e c e l l s (1200X) 38 8. C e l l s i n d i f f e r e n t sizes and morphology (400X) ......39 9. Dried c e l l s grown i n PRL-4-CCM medium and i n PRL-4-17£M-GM medium 39 10. Dry weight and pH changes during the growth period of l i c o r i c e c e l l s 40 11. Dry weight of l i c o r i c e c e l l s i n PRL-4-C medium with d i f f e r e n t coconut milk l e v e l s 44 12. Oxygen uptake by 3.0 ml of 7 days l i c o r i c e c e l l suspension 50 13. Graph of gas chromatography f o r apple aroma from l i c o r i c e growth medium ..53 14. Paper chromatograms f o r p e c t i n - l i k e material hydrolysates (1) 59 15. Paper chromatograms for p e c t i n - l i k e material hydrolysates (2) 60 i x 16. Paper chromatograms f o r p e c t i n - l i k e material hydrclysates (3) 60 17. Paper chromatograms f o r p e c t i n - l i k e material hydrolysates (4) 61 18. TLC f o r chloroform extracts of l i c o r i c e c e l l s grown i n d i f f e r e n t media (1) 63 19. TLC f o r chloroform extracts of l i c o r i c e c e l l s grown i n d i f f e r e n t media (2) 64 20. TLC f o r chloroform extracts of l i c o r i c e c e l l s grown i n d i f f e r e n t media (3) 65 21. TLC f o r chloroform extracts of l i c o r i c e c e l l s grown i n d i f f e r e n t media (4) 66 22. TLC f o r " c e l l g l y c y r r h i z i n i c a c i d " (1) 71 23. TLC f o r " c e l l g l y c y r r h i z i n i c a c i d " (2) 72 24. TLC f o r " c e l l l i c o r i c e " hydrolysate (1) 73 25. TLC f o r " c e l l l i c o r i c e " hydrolysate (2) 74 26. Paper chromatograms f o r reducing substances in l i c o r i c e c e l l growth medium (1) 77 27. Paper chromatograms f o r reducing substances i n l i c o r i c e c e l l growth medium (2) 78 28. Paper chromatograms f o r reducing substances i n l i c o r i c e c e l l growth medium (3) 79 29. Paper chromatograms f o r reducing substances in l i c o r i c e c e l l growth medium (4) 80 30. Paper chromatograms f o r reducing substances i n l i c o r i c e c e l l growth medium (5) 81 31. Paper chromatograms f o r reducing substances i n l i c o r i c e c e l l growth medium (6) 82 X LIST OF TABLES Table page 1. Concentrations of vitamins and amino acids used i n the chemically defined medium 23 2. Growth rate of l i c o r i c e c e l l s 34 3. Y i e l d of l i c o r i c e c e l l s i n PRL-4-DM medium 42 4. Y i e l d of l i c o r i c e c e l l s i n PRL-4-C medium with d i f f e r e n t l e v e l of coconut milk 43 5. Y i e l d of l i c o r i c e c e l l s ( v i s i b l e growth response) i n d i f f e r e n t PRL-4-17.CM-GM medium following 2-3 weeks inoculation 46 6. Y i e l d of l i c o r i c e c e l l s i n yeast extract and casein hydrolysate media 48 7. Y i e l d of l i c o r i c e c e l l s i n PRL-4-CCM with 2,4-D 1-gulonolactone, ascorbic acid or absence of any growth regulator 51 8. Ethanol content i n the l i c o r i c e c e l l aroma as measured by alcohol dehydrogenase ...54 9. Pe c t i n - l i k e material contained i n the cold growth medium f i l t r a t e and hot water c e l l extract of c e l l cultures a f t e r 20 days incubation 55 10. Total p e c t i n - l i k e material from yeast extract media at d i f f e r e n t ages (days) a f t e r inoculation 56 11. The results of g l y c y r r h i z i n i c a c i d assay 69 INTRODUCTION In 1902, Haberlandt formulated the theory of cultu-r i n g the i s o l a t e d vegetative c e l l s of higher plants i n - v i t r o as a method of studying the problems of c e l l d i f f e r e n t i a t i o n and c e l l u l a r i n t e r r e l a t i o n s h i p s . Although he f a i l e d to estab-l i s h a growing tissue culture, his concepts were pursued by several of his students and other investigators. From the time that K o t t e 6 9 ' 7 0 and R o b b i n s 9 5 ' 9 5 f i r s t i n i t i a t e d studies on the growth of excised root t i p s i n s t e r i l e culture solu-tions i n 1922, remarkable culture success has been reached. Callus and c e l l suspension culture from many d i f f e r e n t plant o r i g i n have been successfully grown. The induced d i f f e r e n -t i a t i o n of c a l l u s and single i s o l a t e d c e l l s into vascular elements, stem, leaf and roots and ultimately into whole p l a n t s 2 4 ' 3 3 ' 6 1 ' 1 0 7 ' 1 1 3 ' 1 1 5 ' 1 2 6 has been shown to be possible. As i t has been demonstrated that whole plants can be generated from i s o l a t e d single c e l l s , i t can be assumed that each c e l l possesses the f u l l genetic potential of the whole plant. S i m i l a r l y i t may also be assumed that, any com-pound synthesized by plants i s a potential by-product of c e l l s grown i n solution. For many years, plant c e l l culture was considered too exacting and uncertain f o r general use. More recently, however, techniques have been developed employing r e l a t i v e l y simple media which produced good c e l l y i e l d s . During the past 2 decade much of the research has been directed toward the growth of plant c e l l s i n suspension culture. The culture of plant c e l l s i n l i q u i d media analogus to the culture of microorganisms i s a convenient method for the production of c e l l s of higher plants. Plant c e l l culture i s now approaching the point where one can look upon the c u l t i v a t i o n of i s o l a t e d plant c e l l s as one would the c u l t i v a t i o n of bacteria, asking the same type of questions and employing the same techniques. Thus one may think of these c e l l s as a new class of microorganisms. In f a c t , suspension c e l l cultures derived from plants which synthesize p a r t i c u l a r secondary metabolites such as alka-l o i d s , steroids and terpenoids, have i n many instances been shown to synthesize those same compounds. The amino ac i d composition of the soluble proteins of wheat c o l e o p t i l e and soybean hypocotyl resembled that of soluble proteins from cultured c e l l s of these plants. A l l these observation have stimulated an increasing interest i n plant tissue culture. The development of techniques which allow the c u l t i -vation of plant c e l l s on a large scale and on a routine basis such as the carboy system^-^' 120,loO a n c j ^ n e continuous system 19 4 R 77 199 ' ' ' have been described f o r the submerged c u l t i v a t i o n of a number of c e l l l i n e s . From an i n d u s t r i a l viewpoint any plant metabolite of interest to industry i s a potential product of plant c e l l culture. In the studies to be presented, single c e l l suspen-sion cultures of the commercial l i c o r i c e plant, Glycyrrhiza 3 glabra were studied. The growth conditions, the y i e l d and the potential of producing various products were investigated. 4 • LITERATURE REVIEW I. Plant tissue culture Many attempts were made following Haberlandt's studies during the years of the f i r s t two decades of twentieth century to culture single c e l l s of the palisade tissue of leaves, of medullary parenchyma of epidermis and of various plant h a i r s . However the experimental results of these investigations were discouraging and i n no case could the i s o l a t e d c e l l s be induced to generate tissue masses by c e l l d i v i s i o n . K o t t e 6 9 ' 7 0 and R o b b i n s 9 5 ' 9 6 reported independently i n 1922 the experiments on the growth of root t i p s i n s t e r i l e l i q u i d medium. In 1932, White commenced his work on excised root culture, using seedling root t i p s of Triticum as experi-mental material. In attempts to obtain continuous growth of his excised wheat roots, White systematically varied such c u l -t u r a l factors as l i g h t , temperature, aeration, volume, pH and the inorganic composition of the medium 1 3 3' 1 3 4. Under favour- 1 able conditions, the roots grew rapidly i n culture for about 14 days and then rapidly declined to zero. This cessation of growth was found to be hastened by attempts to subculture the root apices. 1 o c In 1934, White , using a s i m i l a r culture medium but with sucrose instead of dextrose as the source of carbon, succeeded in repeatedly subculturing excised tomato roots with-out diminution of the growth rate. . In addition, the author found that by subculturing the l a t e r a l root t i p s which develop-5 ed during culture, a clone of excised roots i n continuous culture was established from a single r a d i c l e . By 1936^ 8, these roots had passed through 160 passages, each of 7 days duration, and the mean l i n e a r growth rate of the roots had continued at approximately 5 mm per day. At about the same time, reports by Ganthert i n d i c a t -ed that pieces of cambium removed under aseptic conditions from S a l i x capraea, Populus nigra and other trees and placed on a s o l i d i f i e d medium containing Knop's solution, dextrose and cystein hydrochloride would continue to p r o l i f e r a t e f o r some months, givi n g r i s e to a l g a l - l i k e outgrowth^'^. Influenced by contemporary work with root cultures, Ganthert added thiamine and indole a c e t i c acid to his medium and report-ed greatly enhanced growth i n culture of the Sali x capraea c a m b i u m ^ ' A t the same time, Nobecourt reported some suc-cess in cu l t u r i n g explants of cambium from carrots and potato-es using a medium containing Knop's solution supplemented with Berthelet's mixture of accessory salts^,86,87 m Ganthert re-examined the behaviour of carrot explants on Nobecourt's medium modified by incorporating into the medium thiamine, cystein and a low concentration of indole acetic acid. The carrot explants gave r i s e to an "undifferentiated" mass of tissue capable of repeated s u b c u l t u r e ^ , A f e w days e a r l i e r . White had independently reported the continuous c u l t i v a t i o n of a similar undifferentiated c a l l u s derived from the procambial tissue of young stems of a hybrid Nicotiana (N. glauca X N.  l a n g s d o r f f i i ) cultured i n the medium he had developed for exci-6 sed tomato roots containing 0.5% a g a r 1 3 9 . These c a l l u s c u l -tures of Ganthert and White were the forerunners of s i m i l a r cultures derived from the tissues of many dicotyledonous plants. In 1954, Muir, Hildebrandt and Riker reported the growth of l i q u i d cultures containing single c e l l s and small 80 clumps of c e l l s of Tagetes erecta and of Nicotiana tabacum . These cultures arose when fragments of call u s cultures of these species were transferred to a l i q u i d medium agitated on a recip r o c a l shaker. S i m i l a r l y , i n 1955, Steward and Shantz reported that they were studying the growth of carrot phloem explants i n l i g u i d medium i n s p e c i a l l y constructed r o t a t i n g culture vessels, the supernatant f l u i d became turbid owing to the development and growth of free f l o a t i n g c e l l s 1 1 1 . In 1956, Reinert obtained evidence f o r the occurrence of c e l l d i v i s i o n s i n a similar suspension of single c e l l s and c e l l clumps from Picea g l a u c a 9 4 . In the same year, N i c k e l l reported that he had continuously subcultured f o r four years a suspension r i c h i n single c e l l s of hypocotyl of Phaseolus v u l g a r i s ^ 2 . In 1957, Torrey and Shigemura i n t h e i r paper t i t l e d "Growth and controlled morphogensis i n pea root ca l l u s tissue grown i n l i g u i d media" 1 1' stated that under certain n u t r i t i o n a l conditions the s o l i d and woody ca l l u s became f r i a b l e , and when transferred to the fresh medium i n a flas k , mild agitation produced a dense suspension of i s o l a t e d viable c e l l s . The reason for the observed morphogensis was supposed to be due to an appropriate balance between auxin and yeast extract. Also 7 papers from Bergmann 1 4, Torrey and Reinert 1 , Lamport and 71 ^9 Northcote , Earle and T o r r e y o z mentioned the release of c e l l s and tissue fragments from f r i a b l e c a l l u s masses and that the maintainance of a good degree of c e l l separation may often be promoted eith e r by the presence i n the l i q u i d medium of a high auxin concentration, a balance between auxin and k i n e t i n , or a d e f f i c i e n c y of certain vitamins. Suspension cultures have also been successfully established by introducing tissue fragments into the l i q u i d medium obtained from s t e r i l e roots by mechanical disruption with a s t e r i l e homogenizer 2 0. In such cases the tissue frag-* ments f i r s t gave r i s e to small c a l l u s masses from which free c e l l s and small c e l l aggregates were l a t e r released. Es p e c i a l l y designed culture tubes, shakers and methods of agitation have been recommended for single c e l l preparation during the past y e a r s 1 9 ' 4 1 ' 7 2 ' 8 0 ' 1 1 2 ' 1 1 9 ' 1 2 0 . Recently i t has become evident that the rate of c e l l growth and c e l l com-position measured in terms of free c e l l s and c e l l aggregates (the l a t t e r were always present) of a suspension culture were not primarily dependent upon agitation of the cultures but upon the o r i g i n of the plant tissue and the composition of nutrient medium. According to Lamport 7 2, c e l l suspensions can be de-fined as "cultures consisting of single c e l l s and small clumps which can be pipetted accurately". Thus we may think of these c e l l s as a new class of microorganism, and apply the microbio-l o g i c a l techniques to higher plants v i a the c e l l suspension 8 technique. A plant culture suspension composed e n t i r e l y of free growing c e l l s has not, as yet been achieved. However i n recent years a number of plant c e l l strains have been developed from plants with great success, e. g. soybean 3 7' 3**, waxbean 3 7, mung bean 3 7, horse radish p e t i o l e 3 7 , red kidney b e a n 1 2 9 , r o s e 3 ' 7 ' 1 4 1 , sugar cane^ 3, wheat b a r l e y 3 ^ , bush bean, spear-m i n t 1 3 0 , t o b a c c o 1 0 9 , Ammi v i s n a g i 6 6 , maple and gi n k g o 7 2 . In recent years, the induced d i f f e r e n t i a t i o n from undifferentiated callus and single c e l l s has been extensively studied. D i f f e r e n t i a t i o n has been demonstrated i n ca l l u s tissues of t o b a c c o 1 0 7 , endive and p a r s l e y 1 2 6 * 1 2 7 , c a r r o t 6 1 * 1 1 3 , asparagus 1 4 4, p o t a t o 1 1 6 , geranium 2 4* 9 2, convolvulus 3 3 and chrysanthemum 5 5. In addition, i t has been shown that whole plants can be generated from single carrot and tobacco c e l l s grown i n suspension c u l t u r e 6 0 * 1 1 4 . I t , therefore, has been assumed that each c e l l possesses the f u l l genetic potential of the whole plant and that a compound synthesized by adult plants would be a poten t i a l by-product of c e l l s grown i n i s o l a t i o n . The application of microbiological techniques to the culture of c e l l suspensions of higher plants i s p o t e n t i a l l y useful in studying many aspects of plant metabolism, and i n p a r t i c u l a r primary and secondary metabolites from the plant c e l l suspen-sion . A number of papers have been published recently which describe the relationships between plant c e l l cultures and respective the plant organ from which the c e l l s 9 were derived. It has been found that the cultured c e l l s d i f f e r e d biochemically from the parent plant presumably becau-se the cultured c e l l s were primarily meristematic, undifferen-t i a t e d c e l l s , while the int a c t tissue from a plant organ were d i f f e r e n t i a t e d c e l l s . The difference between cultured c e l l s and c e l l s of plant organ was r e f l e c t e d i n the chemical composi-121 tion of t h e i r proteins. Tulecke et a l . observed a notable difference between the protein amino acid composition of Gink-go pollen and c a l l u s cultures derived from po l l e n . A compari-son between the protein amino acids from stems and leaves of the rose and from the c a l l u s culture of rose stem also indica-ted a difference i n the proteins i n these t i s s u e s 1 3 1 , as might be expected. However, very recently, Gamborg and F i n l a y s o n 4 0 analysed the amino acids of the soluble and t o t a l protein from plant c e l l s grown i n suspension culture, the c e l l cultures originated from twelve d i f f e r e n t plant species, and the expl-ants were taken from d i f f e r e n t organs of the plants. The l a t t e r authors found r e l a t i v e l y small differences i n the amino acid composition between c e l l s o r i g i n a t i n g from d i f f e r e n t organs of the same species, and between the same cultures grown in d i f f e r e n t media under the same environmental condi-tions. However some var i a t i o n was found i n the ess e n t i a l amino acid composition p a r t i c u l a r l y in ly s i n e , arginine and methio-nine. The l a t t e r amino acids were found to be proportionally higher i n concentration i n the c e l l proteins than those reported f o r seed proteins. In addition, a number of investigations claim that 10 suspension c e l l cultures derived from plants which synthesize p a r t i c u l a r secondary metabolites, such as a l k a l o i d s , steroids and terpenoids have been shown to synthesize these same compounds i n culture. In 1957, French and G i b s o n 3 6 f i r s t reported the e f f e c t of glutamic a c i d on the biosynthesis of alkaloids (hyoscyamine and hyoscine) i n the three-week o l d cultures.of 109 Datura t a t u l a L. In 1964, Speake et a l . reported that n i -cotine could be successfully i s o l a t e d and i d e n t i f i e d from se-veral l i n e s of c a l l u s tissue culture of Nicotiana tabacum. In 1965, Carew 2 2 reported that the c a l l u s of Alstonia  constricta has the c a p a b i l i t y to biosynthesize reserpine. In 1967, Kaul and Staba ^ demonstrated the biosynthesis of visnagin from A. visnaga c e l l suspension with the y i e l d at 0.31%. In 1965, Williams and Goodwin 1 4 1 examined the t e r -penoids of tissue cultures of Paul's scarlet rose, they found the carotenoids present in tissue cultures are zeaxanthin, violaxanthin, auroxanthin and neoxanthin. (3-carotene, l u t i n and neoxanthin were the main pigments i n stem and leaf from the intact rose. The t o t a l carotenoids in stem and leaf from the intact plant was four and t h i r t y times greater,respective-l y , than the same tissue grown i n culture. The triterpenoids i s o l a t e d and i d e n t i f i e d from tissue culture and from stem and leaf were i d e n t i c a l , ( i - s i t o s t e r o l was the main t r i t e r p e n o i d component, with traces of r - s i t o s t e r o l , lanosterol, ^-amyrin 52 and squalene. In 1968, Heble et a l . i s o l a t e d diosgenin and 11 - s i t o s t e r o l from Solarium xanthocarpum tissue culture with a reported y i e l d higher than that found i n the plant b e r r i e s . A n t i b i o t i c substances have been shown to be produced by many plant tissue culture, e. g. lettuce, cauliflower, aspen and a v o c a d o 4 7 ' 6 5 ' 7 5 . A number of enzymes such as glucanases 3 9, d i f f e r e n t ? f i ? Q *}f) Sft dehydrogenases and transaminases ' ' ' ' have also been demonstrated to be present i n the f i l t r a t e s of plant tissue cultures. In conclusion, although the development of plant tissue culture i s of r e l a t i v e l y recent o r i g i n , the technique promises great p o t e n t i a l . For example, i n plant biology, tissue culture has been used to study d i f f e r e n t i a t i o n , c e l l u -l a r i n t e r r e l a t i o n s h i p s , metabolic pathways and genetic control. In the microbial industry, plant cultures have been suggested as a source of food, food additives, medicines and i n d u s t r i a l materials. I I . L i c o r i c e Several review a r t i c l e s concerning l i c o r i c e have been written within the l a s t f i f t y y e a r s 1 ' 1 1 ' 2 5 ' 7 3 ' 7 8 ' 8 4 ' 1 0 4 . In order to obtain a better insight on the importance of single c e l l suspension cultures of l i c o r i c e c e l l s , i t i s necessary to outline the history, pharmacology, and important chemical components of the l i c o r i c e plant. L i c o r i c e i s a leguminous shrub that may reach a 12 height of several feet, occurring c h i e f l y i n subtropical regions where i t can be found growing wild on r i v e r banks. The roots of the plant are used commercially for the production of l i c o r i c e extract. The l i c o r i c e root; i s c o l l e c t e d i n the f a l l , leaving parts of the root stump to assure the propagation of a new crop. The main licorice-growing countries are found i n the subtropical Eurasian zone which includes Spain, the south of France, I t a l y , Greece, Syria, Iran, Iraq, Russia (Volga and Black sea area) and China. Most commercial l i c o r i c e plants belong to the species Glycyrrhiza glabra L. The products of the l i c o r i c e root, Glycyrrhiza glaba, have been treasured f o r over 40 centuries. Their value has been stated i n many of man's e a r l i e s t medical records. As early as 500 B.C. i n documents of the Greek physician Hippo-crates and i n the essay of the Greek philosopher Thaophrastus, l i c o r i c e was mentioned as sweet Scythian root. In the f i r s t Chinese herbal "Shen nung Pen tsa'o King", l i c o r i c e i s c l a s s i -f i e d amongst the 120 "drugs of the f i r s t c l a s s " which are supposed, to exert godly influence on the body and to lengthen l i f e . L i c o r i c e i s also mentioned i n the e a r l i e s t medical record of the Emperors of Rome, Greek, Egypt i n the l i s t of drugs as a panacea, a cosmetic and a part of the e l i x i e r . In ancient time, l i c o r i c e was used as a remedy for human ailments, p a r t i c u l a r l y as medication f o r dry coughs, inflammation of the throat and other respiratory i n f e c t i o n s . Today l i c o r i c e i s used i n various potions and medi-13 caments, a number of which are included i n the pharmacopoeias of most countries. Sometimes l i c o r i c e extract i s added to correct the b i t t e r a c r i d taste of other drugs; i n other cases, however l i c o r i c e i s considered useful against a va r i e t y of a i l -ments, mainly affections or inflammations of respiratory t r a c t , including throat, bronchia and chest. B e r g e f o r s 1 3 found ground l i c o r i c e root to be an excellent p i l l base. Investigations f o r the physiological and pharmacolo-g i c a l properties of l i c o r i c e have been c a r r i e d out, however, with the complex juice, i t i s seldom possible to a t t r i b u t e some ef f e c t to a s p e c i f i c component of the extract. No i l l e f f e c t s of o r a l administratiin of reasonable quantities have been recorded. The metabolism of the characte-r i s t i c sweet-tasting glycosides i n l i c o r i c e , g l y c y r r h i z i n i c 611 62 63 acid, was investigated by Van Katwijk and Huis i n ' t Veld ' ' and Hudson et a l . According to the l a t t e r authors, glycy-r r h i z i n i c acid i s not metabolized to i d e n t i f i a b l e s t e r o i d substances. The decrease of a r t e r i a l blood pressure by g l y c y r r h i z i n i c acid upon intravenous .injection (Clere et a l . , 8 4 ) i s coupled with a reduction of the surface tension. In 1922, K o f l e r 8 4 found a hemolytic ef f e c t of g l y c y r r h i z i n i c acid i n concentration as low as 0.05%, and Busacca 8 4 confirmed the hemolytic a c t i v i t y , which was considered to be i n accordance with the saponin nature of the compound. However results obtained with the l i c o r i c e extract or with i n s u f f i c i e n t l y p u r i f i e d g l y c y r r h i z i n i c acid preparations are to be judged c a r e f u l l y , as other p h y s i o l o g i c a l l y active constituents are 14 present i n the root extract. A stimulus i n the medicinal status of l i c o r i c e extract started i n Holland. In 1946, Revers reported that the extract had a therapeutic e f f e c t on stomach u l c e r s . Verheyen (1948) suggested that the favorable influence of l i c o r i c e extract on the g a s t r i c ulcer might be related to the spasmoly-t i c (cramp abolishing) action of the succus, which was subse-quently demonstrated i n rat s . The therapeutic e f f e c t of l i c o -r i c e on stomach ulcers was further confirmed by Schulze and Franke (1951), Standner (1952), Lange (1952), B o i l e r (1952), I r l e (1952), Schulze et al.(1954) although Revers (1948) discovered that i n about 20% of the cases i n which stomach ulcer was treated with l i b e r a l doses of pure l i c o r i c e , a side e f f e c t occurred r e s u l t i n g i n edema of the face and extremities, usually accompanied by headaches and sometimes by dizziness. The mechanism by which l i c o r i c e cures ulcers i s yet unknown. According to Czok and Kreienberg (1954) the drug could regulate acid formation i n the stomach, or at least i s considered to be in some way i n t e r r e l a t e d with acid formation (Kreienberg and Harth, 1955). Studying the cause of edema as a result of overdose of l i c o r i c e extract, Borst et a l . (1950) made the most interes-t i n g discovery that l i c o r i c e influenced the water and electro-ly t e ( s a l t ) metabolism of the organism. Daily administration of 20 to 45 grams of l i c o r i c e extract to hos p i t a l i z e d patients resulted i n retention of water, sodium and chloride, whereas potassium output was much increased. This group of investiga-15 7 Q tors came to the conclusion that the o r a l administration of succus l i q u i r i t i a e i s i n almost a l l respects s i m i l a r to that of large doses of intravenously administered deoxycortone. 91 Groen et a l . and Pelser et a l . (1953) considered that glycy-r r h i z i n i c acid was the responsible component f o r the deoxycor-ticosterone- l i k e a c t i v i t y since the ammonium s a l t of g l y c y r r h i -z i n i c acid proved to be active. 21 Card et a l . (1953) showed that g l y c y r r h e t i n i c acid, the aglycon of g l y c y r r h i z i n i c acid had the same ef f e c t on the water and s a l t metabolism as deoxycortone, but the acid, unlike the hormone, did not prolonged the survival time of adrenalectomized ra t s . Hassan et a l . (1954)^ confirmed the el e c t r o l y t e regulating a c t i v i t y of l i c o r i c e . Several derivatives of g l y c y r r h e t i n i c acid and some compounds with analogous structure were also tested c l i n i c a l l y ; o l eanolic acid and u r s o l i c acid proved i n a c t i v e . The use of l i c o r i c e extract for the sole purpose of fla v o r i n g confectionery products started during the eighteenth century when an English chemist, George Dunhill, blended the extract with sugar, molasses, f l o u r and other ingredients to produce so c a l l e d Poute-fract Cakes. This gradually led to the manufacture of other l i c o r i c e confections which up to the present day have been very popular i n Holland, England, France and other European countries where the consumption of these confections i s many times that i n the United States. In some type of chewing gum, l i c o r i c e extract i s incorporated to ensure a f l e x i b l e texture; the antioxidant properties of l i c o -16 r i c e are said to improve keeping q u a l i t y and freshness of the product. In certa i n chocolate candies, l i c o r i c e i s also added to s t a b i l i z e the f a t dispersion, therefore preventing bloom formation. Many l i c o r i c e confectionery l i n e s , e s p e c i a l l y i n Holland, combine the f l a v o r i n g properties of l i c o r i c e with i t s soothing q u a l i t i e s . In cough and throat lozenges, p a s t i l l e s , and gums, l i c o r i c e i s d e f i n i t e l y recognized as a valuable health ingredient. Between d e l i c i o u s l i c o r i c e sweet and phar-maceutical preparations containing l i c o r i c e , there i s a wide f i e l d of l i c o r i c e products having energy-yielding, f l a v o r i n g , and medicinal q u a l i t i e s . The tobacco industry, e s p e c i a l l y i n the United States, i s by f a r the largest user of l i c o r i c e , incorporating the juice into cigarettes, cigars, smoking mixtures, and even snuff tobacco. In tobacco, l i c o r i c e serves several purposes: i t imparts a sweet taste and c h a r a c t e r i s t i c f l a v o r and at the same time enhance the mildness of a tobacco mixture, i t also acts as a moisture c o n t r o l l i n g agent. Another i n t e r e s t i n g use of l i c o r i c e extract i s i n beer, where by virtue of i t s surface-active properties the foaming q u a l i t i e s of the extract increase the head-forming character of the beverage. Like many plants, the l i c o r i c e root or Radix l i q u i -r i t i a e (the name of the drug form) contains glycosides, compounds b u i l t up from an aglycon joined to a sugar residue. In most cases the sugar-free moiety, the aglycon, has a 17 complicated structure containing one or more hydroxylic groups. In the l i c o r i c e root, g l y c y r r h i z i n i c acid, the c h a r a c t e r i s t i c sweet-tasting compound i s one of the most studied of the aglycons. The amount' of t h i s glycoside i n the root may vary markedly, 7-10% i s usually accepted as a r e l i a b l e average figure. The sugar moiety of g l y c y r r h i z i n i c a c i d according to Lythgoe and Trippett (1950) 7 4 i s : I t i s consist of 2-glucopyruronic a c i d residues joined by a @>-1': 2-link. The aglycon of g l y c y r r h i z i n i c acid, g l y c y r r h e t i n i c COOH acid, according to Ruzicka and Jeger (1942) 102 i s : C % ~ ^ COOH CH 3 HO CH3 CH 3 or c a l l e d : A -2-oxy-ll-oxo-oleanene 30 acid . 18 A second glycoside found i n l i c o r i c e root which has been examined closely, i s l i q u i r e t i n . According to Shinoda and. Ueeda (1934) t h i s i s a true glucoside, the aglycon being linked to one molecule of glucose. Pure l i q u i r e t i n consists of c o l o r l e s s c r y s t a l s of melting point 212° C. The aglycon, l i q u i r e t i g e n i n , (m.p. 207"C) has been identified, by Shinoda and Ueeda 1 0 6 as 4',7 dihydroxyflavanone. The structure of the glucoside, 7-hydroxy-4'-glucosidoxy-flavanone i s as follows: 0 X \ / V.0-C6Hn05 0 I s o l i q u i r t i n (Puri and Seshadri, 1954) and Rhamno-1?5 l i q u i r i t i n (Van Hulle, 1968) also can be obtained from l i c o r i c e root. The aglycon of i s o l i q u i r i t i n , i s o l i q u i r i t i g e n i n , shows a remarkable spasmolytic action. The q u a l i t a t i v e detection of l i c o r i c e extract i n pharmaceutical preparations by means of u l t r a v i o l e t l i g h t , according to Steiner (1946) i s apparently based upon the strong fluorescent capacity of these flavanone. A neutral saponin related to the glycosides was cry reported i n l i c o r i c e root by Kobert i n 1915 . This glycoside occurs on i s o l a t i o n as an ether-insoluble resin consisting of glucuronic acid and hemolytic sapogenin. Houseman and Swift i s o l a t e d flora Chinese l i c o r i c e root (1929) a compound of 19 gross formula C20 H21^9» probably related to lapachol. Glabric acid, a t r i t e r p e n o i d constituent i n l i c o r i c e root, was f i r s t reported i n 1956 by Beaton and S p r i n g 1 2 and 35 the structure was determined i n 1968 by Elgamal and Fayez . Liguoric acid, another t r i t e r p e n o i d constituent of g l y c y r r h i z a 34 was reported by Elgamal, Fayez and Snatzk i n 1965 • Recently, the Japanese investigators, Shibata and Saitoh i d e n t i f i e d some additional new constituents i n the l i c o r i c e root, these constituents are reported as l i c o r i c i d i n 1 0 ^ 103 g l y c y r o l , 5-o-methyl g l y c y r o l and i s o g l y c y r o l . L i c o r i c i d i n can be represented as 3',6-diisopentenyl-2',4'5-trihydroxy-7-methoxyisoflavan. The glycyrols have the structures as: Me^ Me Me Me7 (glycyrol, R=H) (isoglycyrol) (5-o-methylglycyrol, R=CH3) Other triterpene compounds meristotropic acid, macedonic acid and enathic acid have been found also i n the roots and rhizomes of G. glabra, G. korsninsJcyi and G. uralensis as reported by a Russian worker" . The purpose of t h i s study i s to apply tissue culture techniques, v i a single c e l l suspension culture to the study 2 0 the growth of the l i c o r i c e and the potential of producing g l y c y r r h i z i n i c acid or other c e l l byproducts of academic and i n d u s t r i a l i n t e r e s t . 21 MATERIALS AND METHODS I. Plant o r i g i n , c u l t u r a l conditions and preparation of  single c e l l suspension. A. Origin of the plant The plant used throughout the studies was Glycyrrhiza  glabra ( l i c o r i c e ) . The seeds of the plant were supplied by a number of botanical and seed suppliers throughout the world. B. Medium The main basal medium used throughout these studies was a modified PRL-4-C medium of Gamborg 3 7, This basal medium, the composition of which i s l i s t e d below, was found suitable for a l l cultures. i . PRL-4-C medium Component NaH2P04H20 Na 2HP0 4 KC1 (NH 4) 2S0 4 Mg304-7H20 KN03 CaCl 2-2H 20 KI iron-EDTA i f mi c ronut r i ent s ** vitamins - •* sucrose N-Z amino type A 2,4-D or NAA mg/liter 90 30 300 200 250 1000 150 0.75 5.0 ml 1.0 ml 10.0 ml 20.0 gm 2.0 gm 2.0 mg mM 0.65 0.21 4.0 1.5 1.0 10.0 1.0 22 *Stock solution. Dissolve i n 100 ml of water: 278 mg FeS04«H20 and 372 mg Na2EDTA. Kept frozen. **Stock solution. Dissolve i n 100 ml of water: 1000 mg MnS04«H20, 300 mg H3BO3, 300 mg ZnSO^B^O, 25 mg Na2Mo04"2H20, 25 mg CUSO4, 25 mg CoCl2'6H20. Kept frozen. ***Stock solution. Dissolve i n 100 ml of water: 10 mg of n i c o t i n i c acid, 100 mg thiamine HC1, 10 mg pyridoxine HC1, and 1000 mg myo-inositol. Kept frozen. In the studies, N-Z amino type A was replaced by casein hydrolysate, and the quantity was decreased to 0.50 gm per l i t e r . i i . PRL-4-C-CM PRL-4-C medium containing 10% coconut milk. The coconut milk was obtained from mature coconuts from the l o c a l market. The milk was drained, f i l t e r e d through Whatman No.l paper and stored frozen i n p l a s t i c b o t t l e s . i i i . PRL-4-DM This i s a chemically defined medium, containing the same mineral s a l t s , sucrose, and growth regulators as the general basal' medium, but the vitamin solution was more complex and the protein hydrolysate was replaced by a mixture of amino acids. 23 Table 1. Concentrations of vitamins and amino acids used i n the chemically defined medium (PRL-4-DM) Amount per Amount per Vitamin liter(tfg) Amino acids liter(mg) Biotin 0.25 L-asparagine 40 Riboflavin 15 L-glutamine 60 F o l i c a c i d 15 glycine 20 p-aminobenzoic acid 200 L-tryptophan 40 Choline 200 L-phenylalanine 20 Ascorbic acid 400 L-arginine 40 N i c o t i n i c a c i d 500 L-methionine 30 Thiamine HC1 500 Pyridoxine HC1 500 Calcium pantothenate 400 myo-inositol. 100 mg i v . PRL-4-l%CM-GM This medium was PRL-4-C containing 1% coconut milk and d i f f e r e n t growth factors as described i n the text. v. PRL-4-YM This medium was PRL-4-C containing various amount of yeast extract as described i n the text. v i . PRL-4-CHM This medium was PRL-4-C containing various amount of casein hydrolysate as described i n the text. C. Callus formation Seeds of Glycyrrhiza glabra were s t e r i l i z e d by 0.5% 24 NaOCl solution and 70% ethyl alcohol and then placed a s e p t i c a l l y on the surface of a s t e r i l e 1% agar-water medium i n the s t e r i l e p e t r i dishes. The seeds were germinated in the dark at 25 C. The r a d i c l e , cotyledons or hypocotyl of the germina-ted seeds were sectioned and four to s i x of these sections were transferred to the surface of 10 ml of PRL-4-CCM agar medium located i n a 100 ml milk d i l u t i o n b o t t l e . The cultures were incubated i n the dark at 25 C. Callus t i s s u e formed within two weeks to a month. D. Preparation of single c e l l suspension. When the calluses were b i g enough to transfer (about 4 weeks), several were transferred to 100 ml of PRL-4-CCM l i q u i d medium located i n a 250 ml erlenmeyer f l a s k . The culture was incubated on a rotatory shaker (Gyrotory shaker) at a speed of 160 revolutions per minute at 28 C. The c e l l s were grown i n the dark with short periodic exposure to i n d i r e c t overhead fluorescent l i g h t during observation. The c e l l s were transferred to new medium at regular time i n t e r v a l s i n order to maintain culture vigour. I I . Measurement of the c e l l growth The growth of the c e l l s was measured by following c e l l dry weight. The c e l l s from duplicate or t r i p l i c a t e cultures were c o l l e c t e d by f i l t r a t i o n through Whatman No.l paper, d r i e d to a constant weight i n a V i r t i s freeze d r i e r . 25 I I I . Quantitative measurement of the p e c t i n - l i k e material The p e c t i n - l i k e material found i n the growth medium was measured as follows: The cold or hot culture medium with the c e l l s (as described i n text) was vacuum f i l t e r e d through Whatman No. 1 f i l t e r paper. A constant volume of f i l t r a t e was placed into a £" diameter cellophane d i a l y s i s tubing and dialyzed against running cold tap water fo r 16 hours. The dialyzed medium was measured fo r t o t a l high molecular weight carbohydrate by the phenol-sulphuric me t h o d 3 1 . The p r i n c i p l e of t h i s method i s that phenol i n the presence of sulphuric acid can be used for the colormetric micro-determination of sugars and t h e i r methyl derivatives, oligasaccharides, and polysaccharides. A known concentration of glucose solution was used as a reference standard. IV. Isolation and hydrolysis of the p e c t i n - l i k e material A. Isolation The p e c t i n - l i k e material present i n the growth medium was extracted as follows: The cold or b o i l i n g medium (as described i n text) was f i l t e r e d through a whatman No.l paper. Two volumes of 95% ethanol was added to the f i l t r a t e 8 9 . The c o l l o i d a l p r e c i p i t a t e which formed was c o l l e c t e d by centrifugation and washed twice with ethanol, once with acetone and once 26 with ether; a f t e r removal of the ether by a i r drying, the f i n a l p r e c i p i t a t e was freeze dried. B. Hydrolysis The i s o l a t e d p e c t i n - l i k e material was hydrolysed for 3 hours or 18 h o u r s 9 * 1 ^ * 1 1 5 depending on the experiment in 3% (v/v) n i t r i c a c i d containing 0.05% (w/v) urea. The hydrolysates were neutralized with NaOH solut i o n . V. Q u a l i t a t i v e carbohydrate analysis; Paper chromatography The sugars present i n the hydrolysate and growth medium were analyzed by descending paper chromatography on No. 1 f i l t e r paper at room temperature. A. Solvents Solvent A: ethyl acetate-pyridine-water 8:2:1 (v/v) Solvent B: n-butanol-ethanol-water 5:1:4 ( v / v ) 9 Solvent C: ethyl acetate-acetic acid-pyridine-water 5:1:5:3 (v/v) i n a tank eq u i l i b r a t e d with ethyl acetate-pyridine-water 40:11:6 by volume 9. Solvent D: ethyl acetate-acetic acid-formic acid-water 18:3:1:4 ( v / v ) 9 Solvent E: the organic phase from a mixture of phenol, 100 ml of water and 1 ml of formic a c i d 9 . Solvent F: n-propanol-ethyl acetate-water 7:1:2 by volume 1 5. 27 Solvent G: 80% p h e n o l 1 5 . Solvent H: n-butanol-pyridine-water 6:4:3 ( v / v ) 1 5 B. Location reagent The following reagents were used f o r the detection of compounds resolved by paper chromatography. i . For reducing sugars and reducing substances: s i l v e r n i t r a t e acetone solution and sodium hydroxide alcohol solution dip followed by treatment with sodium t h i o s u l f a t e to clear the background 1 0 2. i i . For a l l sugars: a n i l i n e phosphate solution dip followed by heating i n a oven at 105°C f o r 10 m i n u t e s 1 4 5 . VI. Is o l a t i o n of steroids The d r i e d plant c e l l s were ground to f i n e powder and CO the steroids extracted with CHCI3 f o r 24 hours i n a s o x h l e t u o apparatus. The extracts were concentrated under vacuum by using a water aspirator. VII. Extraction and p u r i f i c a t i o n of g l y c y r r h i z i n i c a c i d from  l i c o r i c e root s t i c k or dried c e l l s . The procedure l i s t e d i n figure 1 was designed f o r i s o l a t i o n of ammonium g l y c y r r h i z i n a t e 1 7 ' 28 Dried material (washed with 100% ethanol) residue supernatant(discard) (extracted three times with 50% ethanol) residue (discard) residue (discard) (fraction C) supernatant(fraction A) (concentrated under vacuum) residue(fraction B) (added 1 drop of 12 N. HC1 followed by a suitable volume of 95% ethanol, f i l t e r e d ) supernatant(fraction D) (added 1 drop of 14 N. NH4OH, allowed to stand at 0°C for several hrs and then centrifugated) supernatant (discard) p r e c i p i t a t e s ( f r a c t i o n E) (dried) ammonium glycyrrhezinate F i g . 1 Procedure for i s o l a t i o n and p u r i f i c a t i o n of g l y c y r r h i z i n i c a c i d from l i c o r i c e root s t i c k or dried plant c e l l s 29 VIII. Hydrolysis of g l y c y r r h i z i n i c a c i d G l y c y r r h i z i n i c a c i d was hydrolyzed f o r 4 hrs with 6 N. sulphuric a c i d i n a re f l u x a p p a r a t u s 1 7 ' 7 6 . The product was extracted with chloroform or ether. The reaction can be expressed by equation: , H g l y c y r r h i z i n i c a c i d ^ g l y c y r r h e t i n i c a c i d 2 glucuronic a c i d IX. Reduction of g l y c y r r h e t i n i c acid An amount of LiAlH4 equal to the g l y c y r r h e t i n i c a c i d by weight was refluxed f o r s i x hours i n g l y c y r r h e t i n i c a c i d ether s o l u t i o n 1 7 . The r e f l u x i n g apparatus was assembled with a dry-tube at the top to prevent the entrance of moisture. The product of the reaction was poured c a r e f u l l y onto i c e , and shaken with an equal volume of ether. The ether solution was allowed to stand f o r a period of time and then the ether phase was removed from the water phase with the a i d of a separatory funnel. The substance i n the ether portion was the reduced g l y c y r r h e t i n i c a c i d . X. Qualitative steroids and t r i t e r p e n o i d analysis; thin layer  chromatography (TLC) A thin layer of s i l i c a gel G (£. Merek AG, Darmstadt, Germany) was prepared on glass plates as described by E. Stahl ( 1 9 6 5 ) 1 1 0 . Solvents used were: 3 0 i . chloroform-methanol 2 : 1 by v o l . i i . chloroform-95% ethanol 2 : 1 by v o l . i i i . isopropanol-methanol 2 : 1 by v o l . i v . heptane-benzene-absolute ethanol 5 0 : 5 0 : 1 by v o l . 5 7 53 v. n-hexane-acetone 2 : 1 by v o l . Solvents i , i i , i i i , were used mainly for steroids. Solvents i i i , i v , v, were used mainly for t r i -terpenoids. XI. Methods used for lo c a t i n g the steroids and triterpenoids A. Observation under u l t r a - v i o l e t l i g h t Fluorescent spots of d i f f e r e n t colour and absorbence could be distiguished under u l t r a - v i o l e t l i g h t . Standard samples of g l y c y r r h e t i n i c acid (K & K Laboratories Inc. Plain-view, N. Y. Holywood, C a l i f . ) showed three absorbent spots under UV. B. Spray with coloration reagent i . antimony t r i c h l o r i d e , 2 0 % i n chloroform . i i . antimony pentachloride, 2 0 % in c h l o r o f o r m 5 3 ' 5 7 . i i i . a c e t i c anhydride (107o) and sulphuric acid (10%) i n absolute alcohol. Liebermann-Burchard r e a g e n t 5 7 ' 1 1 0 . After spraying, the plates were heated at 120°C for 5 minutes. XII. Measurement of oxygen consuption Oxygen uptake of the c e l l s was measured by a Gilson d i f f e r e n t i a l respirometer. Each Warburg flask contained 3.0 ml of a 7 days c e l l suspension (approx. 50 mg dry weight of c e l l s ) . The center well contained a f l u t e d f i l t e r paper satu-rated with 0.15 ml of 20% KOH. XIII. Q u a l i t a t i v e apple aroma analyses A Becker research dual column gas chromatograph. type 3810, equipped with dual flame i o n i z a t i o n detectors was used for the analyses of the apple aroma from the l i c o r i c e c e l l growth media. The s t a i n l e s s s t e e l columns contained chromosorb W coated with diethylene g l y c o l succinate polyester; mesh size 80/100; 12 feet long; outside diameter 1/8". XIV. Enzymatic determination of alcohol The enzyme, alcohol dehydrogenase, was used to measure the amount of ethanol contained i n the supernatant growth medium following the method of Vallee and H o c h 1 2 3 ' 1 2 4 . C e l l supernatant (0.5 ml) was added to 0.3 ml of nicotinamide adenine dinucleotide (NAD) solution and 0.2 ml enzyme solution. The amount of ethanol contained i n the growth medium was c a l -culated by comparing the rate of increase in absorbancy at 340 m/^of the unknown solution with standard ethanol sol u t i o n . 32 EXPERIMENTAL RESULTS  PART I. Growth experiments I. Callus formation and the preparation of single c e l l  suspensions The glycyrrhizan seeds sprouted on the agar medium within a week; even though they were incubated i n the dark, the plumules were a dark green colour and remained the same colour for more than one month. The 1/16 inch sections of the hypocotyl, cotyledons and roots a l l formed calluses on PRL-4-CCM agar medium. The time needed for ca l l u s formation varied from ten days to month. The cotyledon sections took a longer time to convert to undifferentiated c e l l masses or ca l l u s than d i d the other tis s u e s . Some of the calluses formed from cotyledons and roots contained two d i f f e r e n t colours within the one c a l l u s , white at one end of the c a l l u s and brown at the other end. The white section of the tissue grew slower than the brown coloured area, as a result the whole call u s gradually became predominantly brown i n colour. The calluses had d i f f e r e n t f r i a b i l i t i e s , some were smooth on the surface with l i t t l e f r i a b i l i t y and others were coarse i n appearance and were very f r i a b l e . When the f r i a b l e calluses were of s u f f i c i e n t s i z e , approximately 1/2 inch in diameter, to provide a large number 33 of fragments, they were transferred to PRL-4-CCM l i q u i d medium containing 2 mg/1 2,4-D (2,4 dichlorophenoxy-acetic a c i d ) . The cultures were incubated i n a rotatory shaker at 28° C i n the dark. The time required f o r the di s i n t e g r e t i o n of the c a l l u s to form fragments consisting one to several c e l l s varied from callus to c a l l u s . Generally speaking at the i n i t i a t i o n the cultures grew and divided rather slowly. The c e l l s of d i f f e -rent o r i g i n (root, hypocotyl, cotyledons) did not display a difference i n morphology. However at least three d i f f e r e n t types of c e l l s could be recognized i n the culture under micro-scope. One type of c e l l was c o i l - l i k e i n shape and a l i g h t yellow colour. The second type of c e l l occurred i n a kidney-l i k e shape and was also l i g h t yellow i n colour, the t h i r d type of c e l l was a very large brown coloured c e l l . C e l l aggregates containing at least ten or more c e l l s were found i n the culture for the f i r s t several transfers. The percentage of single c e l l s vs. clumps increased with every culture transfer, and divided at a faster rate from one ? transfer to another. Following a growth period which included about s i x transfers, the c e l l s appeared to grow i n an almost constant rate. The c o i l - l i k e c e l l s decreased i n quantity either due to t h e i r apparent slow growth or to a change i n th e i r morphology during the growing period. The large yellow-ed c e l l s could be found occasionally i n a l l cultures. L i c o r i c e c e l l s reached the maximum t o t a l dry weight in about 11-13 days. Following inoculation into fresh medium. 34 the c e l l s divided vigorously f o r three to seven days forming many small clumps and the small single c e l l s . A f t e r t h i s i n i t i a l growth most c e l l s enlarged and elongated showing d i f f e -rences i n sizes and morphology. The active movement of small mitochondrial p a r t i c l e s from the region of nucleus to the places around the c e l l wall could be seen c l e a r l y under 400x phase contrast microscope i n healthy c e l l s . I I . Growth curve The growth of l i c o r i c e c e l l s i n PRL-4-CCM medium i s shown i n table 2 and figure 10. Table 2. Growth rate of l i c o r i c e c e l l s Growth period Dry weight PH (days) (grams)* 0 0.0800 5.60 3 0.1600 5.21 5 0.2533 4.97 6 0.3942 4.80 7 0.6127 4.60 8 0.6590 4.55 10 0.8348 4.50 11 0.9699 4.70 12 0.9708 4.75 13 1.0004 4.70 14 1.0108 4.96 17 1.0133 4.88 18 1.0734 5.15 20 0.9621 5.60 24 0.8926 5.90 28 0.8482 6.10 * average of t r i p l i c a t e , grams per f l a s k (100 ml medium). Fig. 3 Callus formed from root section; begining stage. 3 6 F i g . 5 Large seaweed - l i k e c e l l s (400X) F i g . 6(a) Kidney-like c e l l s (1200X) 38 Fig. 7(b) C o i l - l i k e c e l l (1200X) F i g . 9 Dried c e l l s grown in PRL-4-CCM (with 2 mg/1 2,4-D) medium and in PRL-4-l%CM-GM medium 40 ft 6 12 18 24 30 days Figure 10. Dry weight and pH changes during the growth period of l i c o r i c e c e l l s symbols: c i r c l e s , dry weight; t r i a n g l e s , pH. 41 The growth medium of l i c o r i c e c e l l s indicated a strong reducing a b i l i t y when tested with Fehling s o l u t i o n . Two major s i l v e r n i t r a t e reducing compounds were observed following paper chromatography and w i l l be discussed i n a l a t e r section. The protein content of the plant c e l l s and the surrounding medium a f t e r ten days growth was not detectable by the biuret method. This would suggest that the protein l e v e l of the c e l l s and the growth medium was below 0.1 mg/ml. I I I . E f f e c t of coconut milk l e v e l and growth factors on the  growth of l i c o r i c e c e l l s High concentrations of coconut milk have been found necessary f o r the growth of many plant calluses and suspension cultures. However, the addition of t h i s chemically undefined material i n such high l e v e l s l i m i t s the usefulness of c e l l suspension for metabolic studies. As a result an attempt was made to replace coconut milk with synthetic medium. Defined synthetic medium (PRL-4-DM) was selected f o r the experiment. This medium was reported to support the growth of several plant c e l l cultures although the growth rate was considered to be considerably lower than that obtained with the complex media. The growth rate of l i c o r i c e c e l l s on PRL-4-DM medium was compared with PRL-4-CCM medium containing 2,4-D and coconut milk. Results are shown i n 42 table 3. The synthetic medium PRL-4-DM with or without 2,4-D both could not support active sustained growth. Coconut milk was a required ingredient i n the synthetic medium f o r l i c o r i c e c e l l growth. Among the fl a s k s exposed to the same treatment, a va r i a t i o n i n the colour of the c e l l s and the medium was apparent. A deep brown colour consistently indicated unfavor-able growth. In PRLH4*CCM medium the c e l l s and the medium were a pale yellow colour which turned to a yellow-brown color as the c e l l s approach the l a t e stationary phase. Table 3. Yields of l i c o r i c e c e l l s i n PRL-4-DM medium •X- 1st transfer 2nd transfer 3rd t ransfer PRL-4-DM without 2,4-D 0.6832 0.2720 0.0351 PRL-4-DM with 2,4-D 0.7082 0.3482 0.2809 PRL-4-CCM 1.1064 1.0923 1.0983 average of t r i p l i c a t e , grams per fl a s k (100 ml medium). In a subsquent experiment, 2,4-dichlorophenoxyacetic acid was found to be important for c e l l growth. Coconut medium without 2,4-D supported slow single c e l l growth, abnormal dark coloured c e l l s and a tendency toward large s o l i d clumps. 43 Table 4. Y i e l d of l i c o r i c e c e l l s (gm/100 ml) i n PRL-4-C medium with d i f f e r e n t l e v e l of coconut milk (%) l e v e l of coconut milk 1st transfer 2nd transfer 12 days 20 days 11 days 18 days dry wt. pH dry wt. pH dry wt. pH dry wt. pH 0.5% 0.9406 4.54 1.0258 4.75 0.3368 1.0% 0.9353 4.40 0.9340 4.60 0.3547 2.0% 0.9074 4.38 1.0736 4.80 0.5003 3.0% 0.9351 4.35 1.0565 4.80 0.5781 5.0% 0.9863 4.45 1.1217 4.65 0.7142 7.0% 0.9898 4.78 1.0891 4.85 1.0178 10.0% 0.9463 5.14 1.1451 4.80 1.1516 15.0% 1.0553 5.17 1.0452 4.95 1.2272 4.60 0.7048 4.80 4.50 0.7376 4.70 4.40 0.8252 4.70 4.50 0.8659 4.70 4.50 0.9349 4.80 4.70 1.1320 5.00 4.80 1.2114 5.20 4.70 1.2565 5.50 average of t r i p l i c a t e . Because of the improved c e l l growth i n the defined medium containing coconut milk, the optimum l e v e l of coconut milk present i n the medium was determined. From the results shown i n table 4 and figure 11, the lower the coconut milk l e v e l i n the medium, the longer the growth period needed to reach the maximum c e l l y i e l d . When the c e l l s were harvested at 12 days a f t e r inoculation, the r e s u l t i n g c e l l y i e l d was found to increase i n a l i n e a r fashion as the coconut milk l e v e l increased from 0.5% to 7%. Only a s l i g h t difference i n c e l l y i e l d was shown i n coconut milk between the 10% and 157o l e v e l . A longer c e l l growth period decreased the difference i n c e l l dry weight observed between coconut milk l e v e l s . 44 1.0 3.0 5.0 7.0 10.0 15.0 coconut milk l e v e l i n medium (%) Figure 11. Dry weight of l i c o r i c e c e l l s (gm/100 ml) i n PRL-4-C medium with d i f f e r e n t coconut milk leve l s s o l i d l i n e : c e l l s harvested at 11 days incubation dotted l i n e : c e i l s harvested at 18 days incubation 45 Generally two or three c e l l transfers are necessary to show the v i s i b l e differences between plant c e l l treatments. The results of second transfer was taken f o r the graph i n figure 1 1 . The reason f o r the long delay i n c e l l response following a s p e c i f i c treatment was due l i k e l y to the large amount of nutrients transferred with the c e l l s and within the c e l l s . In order to observe the e f f e c t s of various plant hormones on l i c o r i c e c e l l s growing on a medium containing l i m i t i n g amount of coconut milk, l i c o r i c e c e l l s were transferred to 1% coconut milk i n PRL-4-C medium with the addition of d i f f e r e n t plant growth fa c t o r s . As observed i n table 5, the hormone treatments did not promote increased c e l l y i e l d . In fact a decrease in c e l l y i e l d was observed for a l l treatments suggesting that these hormones at these concentration l e v e l s under the condition of the experiment i n the presence of l i m i t i n g concentration of coconut milk did not overcome the coconut milk nutrient deficiency. A great difference i n colour and growth rate was noted between treatments and within a p a r t i c u l a r treatment. During t h i r d growth transfer, a l l the c e l l s changed to deep brown colour and appeared to be i n poor condition. In conclusion, the addition of 107o coconut milk to PRL-4-G medium was found to be the optimum concentration : required f o r c e l l growth rate and good c e l l y i e l d . The addi-t i o n of the hormones, k i n e t i n , g i b b e r e l l i c acid, napthalene acetic acid, indole acetic acid, and 2 ,4-dichlorophenoxyacetic ac i d at the l e v e l s recorded did not replace or augment the growth a c t i v i t y contributed by the coconut milk Table 5. Y i e l d of l i c o r i c e c e l l s ( v i s i b l e growth response)* i n d i f f e r e n t PRL-4-l%C-GM following 2-3 week inoculation PRL-4-17JC 1st transfer 2nd tra n s f e r 3rd transfer 0.5mg/l k i n e t i n 2mg/l k i n e t i n 5mg/l ki n e t i n 0.5mg/l G. A. 2mg/l G. A. 5mg/l G. A. 0.5mg/l NAA 2mg/l NAA 5mg/l NAA Q.5mg/1 IAA 2mg/l IAA 5mg/l IAA 2mg/l 2,4-D** •H-H-• t - m +44-H - f -H - f -H 4 0.9522 0.8196 0.4634 * results of t r i p l i c a t e ** weight (grams/100 ml medium) 47 IV. Yeast extract medium Complex nutrients other than coconut milk such as yeast extract or casein hydrolysate were examined to t h e i r a b i l i t y to replace the coconut milk i n the PRL-4-CCM medium. According to the r e s u l t s of these experiments, as summarized in table 6, yeast extract served as a better nutrient than casein hydrolysate. Yeast extract at the 0.3% or the 0.5% l e v e l s i n PEL-4-C medium gave s a t i s f a c t o r y l i c o r i c e c e l l growth through f i v e or more transfers. I t i s of interest to note that when the c e l l s from the stationary phase of growth were used for inoculation, a much longer lag growth phase was needed for the c e l l to recover t h e i r growth a c t i v i t y as the next day following inoculation the c e l l s and the medium a l t e r -ed from yellow to brown. At higher concentration of yeast extract i n the medium (0.8%) growth was i n h i b i t e d ; a longer lag phase, sometimes as long as one month, was noted. In 0.8% yeast extract medium the c e l l s did not change to a brown colour which possibly indicated a c a p a b i l i t y to avoid oxida-t i v e darkening. One of the most i n t e r e s t i n g observation made when using yeast extract as medium component to grow l i c o r i c e c e l l s was that the c e l l s reached t h e i r maximum y i e l d i n only 10 days a f t e r inoculation. This incubation period was comparatively shorter than c e l l s grown i n PRL-4-CCM medium. The PRL-4-YM medium, that i s the PRL-4-C medium containing yeast extract, usually promoted a low pH at the time of c e l l harvest, i n some cases, t h i s pH was as low as 48 pH 2. Generally speaking, the PRL-4-YM medium supported good c e l l growth and could replace the use of coconut milk. In addition the c e l l s growing i n t h i s medium secreted consi-derable polysaccharide. The y i e l d and the composition of the polysaccharide w i l l be discussed i n l a t e r section. Table 6. Y i e l d of l i c o r i c e c e l l s (gm/100 ml medium) i n yeast extract and casein hydrolysate medium PRL-4-C 1st transfer 2nd transfer 3rd transfer 0.8% YE 0.7890* ## 0.5% YE 1.1922 1.1271 1.1356 0.3% YE 1.0733 0.9518 1.0127 0.2% YE 0.9182 0.7192 0.4223 0.5% CH 0.9120 ** 0.2% CH 0.9412 0.7907 0.5686 0.05% CH 0.8450 0.4339 0.25% YE 0.25% CH & 0.9699 **-0.10% YE 0.10% CH & 1.0219 0.7855 0.4160 0.05% YE 0.05% CH & 0.9052 0.7693 0.3007 * average of t r i p l i c a t e ** l o s t experiments due to the delaying of c e l l growth 49 V. E f f e c t of ascorbic a c i d and 1-gulono-lactone on the growth  of l i c o r i c e c e l l s An experiment was c a r r i e d out to study the e f f e c t s of ascorbic a c i d and 1-gulono-lactone on the growth of l i c o r i c e c e l l s . PRL-4-CCM containing eit h e r 2 mg/1 ascorbic a c i d (A) or 2 mg/1 gulono-lactone (B) were used to grow the c e l l s and a comparison was made with c e l l s grown i n the presence (C) and absence (D) of 2mg/l 2,4-D. Medium contain-ing 2,4-dichlorophenoxyacetic acid supported the best growth, followed by ascorbic acid and gulono-lactone. The colour of the c e l l s and the medium varied from l i g h t yellow through to deep brown, the more active 2,4-D stimulated culture was yellow whereas the slower growing cultures were a brown colour. The c e l l response to these treatments can be shown by the oxygen consumption as measured by the Gilson D i f f e r e n t i a l respiro-meter. (Fig. 12) In order to determined whether the concentration of the above compounds i n the medium had an e f f e c t on c e l l growth, the compounds were studied at several d i f f e r e n t concen-t r a t i o n s . A great variation within the treatment was apparent i n the growth rate and y i e l d of the c e l l s , e s p e c i a l l y i n ascor-b i c acid medium. The large growth va r i a t i o n which ex i s t s between flasks within the same treatment i s rather i n t e r e s t i n g and cannot be explained. In the paper of Naguib on Cunninghamella Sp, 8 1 the function of ascorbic acid i s probably related to the respira-300 o C 250 200 -150 100 10 2 0 30 40 50 60 minutes 7 0 80 90 Figure 12. symbols: Oxygen uptake by 3.0 ml of 7 days c e l l suspen-sion (approx. 50 mg dry weight) c irc les , ce l ls grown in PEL-4-COM with 2 mg/1 2,4-D; squares, cel ls grown in PPL-4-CCM with 2 mg/1 ascorbic acid; triangles, cel ls grown in PEL-4-COM with 2 mg/1 gulono-lactone; cross, control. 51 ti o n and carbohydrate metabolism. Ascorbic a c i d induces rapid sucrose consumption accompanied by increased keto acid content and re s p i r a t i o n rate. Ascorbic acid also e f f e c t the glucosan and galactosan accumulation and nucleoprotein content. The metabolic pathway of ascorbic acid and the function of 2,4-D i n plant tissue i s unknown. From these experiments, i t i s evident that ascorbic acid and gulono-lac-tone stimulated c e l l growth although at the concentration tested the stimulation i s not as marked as 2,4-D. Table 7. Y i e l d of l i c o r i c e c e l l s (dry wt., gm/100 ml medium) in PRL-4-CCM with 2,4-D, 1-gulono-lactone, ascorbic acid or absence of any growth regulator PRL-4-CCM Y i e l d (dry wt., gm/100 ml medium) Exp. No 1 Exp. No 2 Exp. No 3 2,4-D 2mg/l G. L. 2mg/l G. L. 5mg/l G. L. 10mg/l A. A. 2mg/l A. A. 5mg/l A. A. 10mg/l control 1.3744 0.6670 0.5280 0.6953 0.1219 0.1703 0.2079 0.0239 1.2988 0.6835 0.5939 0.7032 0.2132 0.3015 0.1890 0.1253 1.3957 0.5964 0.5142 0.5838 0.9019 1.4107 0.8693 0.6096 52 PART I I . Products from l i c o r i c e single c e l l suspension  culture I. F r u i t smell L i c o r i c e c e l l cultures removed from the shaker and permitted to stand without shaking developed a pleasant apple odour. The aromatic odour was present i n cultures of c e l l s grown i n the media PRL-4-CCM, PRL-4-l%CM-GM and the medium with gulono-lactone, but d i d not occur i n PRL-4-YM and PRL-4-CHM. The components of the apple aroma present i n the head space above the culture were compared with the compounds found i n a sample of natural apple essence by gas chromatography, Several of the constituents had the same retention time on the column as some of the components found on the l i c o r i c e c e l l aroma (Fig. 13). One of the major peaks was ethanol, which was confirmed by enzymatic assay with alcohol dehydrogenase (table 8). The l i c o r i c e c e l l s a f t e r standing i n t h e i r growth medium were found to contain 0.24% ethanol. 5'55" 5'5 vapour from PEL-4-l%CM-NAA medium, 1 ml injected. 3'30 retention time (minutes) 5'42" 6*], 5" 4'55" 3/31 8 ' 4 0 11'25;'10'5" / vapour of apple essence, 0.4 ml injected. 2'55' 2'30' retention time (minutes) Figure 13. Graph of gas chromatography for apple aroma from l i c o r i c e growth medium speed 4, temp. 100°C, attenu. IX, detect temp. 140CC, i n j e c t temp. 120°C. 54 Table 8. Ethanol content i n the l i c o r i c e c e l l aroma as measured by alcohol dehydrogenase (RCH20H-+-NAD^=i RCH0-*-NADH2), use ethanol solution as standard Reaction medium Ethanol content PRL-4-l%CM-0.5mg/l IAA 0.24% PRL-4-l%CM-0.5mg/l ki n e t i n 0.15% PRL-4-l%CM-2mg/l G. A. 0.12% PRL-4-l%CM-2mg/l 2,4-D 0.20% I I . The p e c t i n - l i k e material The yeast extract medium not only supplied a good medium for c e l l growth, but also stimulated the production of large amounts of polysaccharide from the c e l l s . The polymer could pass through Whatman No.l f i l t e r paper; was p o s i t i v e i n the carbohydrate Molisch reaction; formed a gel which disappeared on heating and could be pr e c i p i t a t e d by an equal volume of ethanol. A. Y i e l d The growth medium from the l i c o r i c e c e l l culture was f i l t e r e d through the No.l Whatman paper, and then dialyzed against running cold tap water for 16 hours. The y i e l d of the p e c t i n - l i k e material was measured by the phenol-sulfuric method. The results of the analyses are shown i n table 9 and table 10. Following the removal of the growth medium, the 55 c e l l s were extracted with 30 ml of b o i l i n g water to obtain the hot water soluble c e l l pectin f r a c t i o n . This f r a c t i o n was dialyzed as described above, the y i e l d of the pectin i n t h i s f r a c t i o n and i n the medium are compared i n table 9. The results indicated that the p e c t i n - l i k e material was present not only in the growth medium of the culture but also i n the c e l l extract. The t o t a l pectin, i . e . medium pectin plus c e l l pectin, found i n c e l l cultures at various culture ages and at various yeast extract concentrations are l i s t e d in table 10. Table 9. P e c t i n - l i k e material contained i n the cold growth medium f i l t r a t e and hot water c e l l extract of c e l l cultures a f t e r 20 days incubation Culture PRL-4-C PRL-4-C PRL-4-C PRL-4-C PRL-4-C :0.8%YE 0.5%YE 0.3%YE 0.2%YE 107<CM medium 500 mg/1 760 mg/1 600 mg/1 600 mg/1 680 mg/1 c e l l 275 280 280 170 190 The amount of p e c t i n - l i k e material extractable from the cultures would appear to depend on the length of the culture incubation. The maximum t o t a l hot water extractable pectin from the cultures occurred a f t e r approximately one 56 Table 10. Total p e c t i n - l i k e material from yeast extract media at d i f f e r e n t aces (days) aft e r inoculation" y i e l d represent by mg/1000 ml medium age (days) 8g/lYE 5g/lYE PRL-3g/lYE 4-C 2g/lYE 5g/lCH 2g/lCH 10%CM 13 675 550 510 480 17 690 550 500 20 690 625 500 24 613 720 680 600 27 980 840 754 612 550 800 30 775 1050 780 32 1100 880 770 770 36 950 650 650 * t o t a l media pectin +• hot water soluble c e l l pectin. month of culture incubation, table 10. The culture of t h i s stage was not i n active growth, figure 2. The maximum pectin concentration was produced i n the medium containing yeast ex-tra c t i n place of coconut milk. The y i e l d represent 10% of the dry weight of the c e l l s . Growth media from other plant tissue cultures, such as bush bean and tobacco, also contained p e c t i n - l i k e material. Therefore the p e c t i n - l i k e material i n plant suspension cultures could be a universal phenomena. The middle lamella, the area located between two adjacent plant c e l l s i s composed of calcium and magnesium pectates. The formation of single c e l l s i n the l i q u i d medium may be due to the solution of the material of the middle lame-l l a and thus possibly account f o r the accummulation of the 57 polysaccharide i n the growth medium. As the c e l l s aged, that i s following active growth i n culture, many of the c e l l s appeared to be i n various stages of c e l l degradation as viewed under the phase contrast microscope. This release of c e l l material may account f o r the increase i n soluble polysaccha-ride. On the other hand i t i s e n t i r e l y possible that t h i s material may be synthesized from simple sugars by enzymes active at a l a t e stage in the c e l l growth cycle. B. The components of the p e c t i n - l i k e material P u r i f i e d p e c t i n - l i k e material was hydrolyzed with 3% (v/v) n i t r i c a cid containing 0.05% (w/v) urea for 3 hrs or 18 hrs at 100°C. The hydrolysate was neutralized by d i l u t e d NaOH solution. The sugar components were i d e n t i f i e d by descending paper chromatography. The hydrosate was developed i n solvent A, B, and C respectively, using glucose, galactose, fructose, arabinose, rhamnose, cellubiose, xylose, galacturonic acid, glucuronic a c i d as known standards. Hydrolysis f o r a period of 18 hours d i d not v i s i b l y increase the number or the amount of free sugars over that found a f t e r 3 hours hydrolysis as shown i n figure 13. Folloxtfing development with the ethyl acetate-pyridine-water solvent, the s i l v e r n i t r a t e reagent indicated four major and one minor zone with a p o s i t i v e reaction. In order to examine the composition of these l a t t e r sugar zones, the pectin hydrolysate was applied as a streak 58 on one or more chromatogram sheets. The chromatograms were developed with solvent A f o r 16 hours. A reference s t r i p re-moved from the side of each of the chromatogram sheets was sprayed with s i l v e r n i t r a t e reagent to locate the zones. On the remaining unsprayed portion of the chromatogram, s t r i p s were removed corresponding to the four major and one minor zones (Fig. 14). The sugars were i s o l a t e d from the s t r i p s by eluated with water. Eluates of zones A-E were chromatographed i n two or three d i f f e r e n t solvent systems, the results are shown from F i g . 15 to F i g . 17. The following conclusion could be drawn fo r the possible components contained i n the p e c t i n - l i k e material hydrolysate. Zone A: galactose Zone B: glucose, fructose and arabinose Zone C: arabinose and fructose Zone D: glucose, fructose and xylose Zone E: galacturonic a c i d and glucuronic acid Thus i t would appear that the p e c t i n - l i k e material from l i c o r i c e c e l l s contained glucose, fructose, galactose, arabinose, xylose, galacturonic a c i d and glucuronic a c i d . 59 Eluates F i g . 14 Paper chromatogram run i n solvent A (ethyl acetate-pyridine-water 8:2:1) for 16 hours. S]_, S 2: P e c t i n - l i k e material hydrolysates Sugar standards: 1. glucose 2. galactose 3. fructose 4. arabinose 5. rhamnose 6. cellobiose 7. xylose 8. galacturonic acid 9. glucuronic acid (The results shown that the hydrolysates do not contain rhamnose or cellobiose) 60 Fig. 15. Paper chromatograms for eluate zone A I. run i n solvent C; I I . run i n solvent A; I I I . run in solvent D. Standards: 1.glucose 2.galactose 3.fructose 4.arabinose 5.xylose. Fig. 16. Paper chromatograms for eluate zone E I. run in solvent A; I I . run i n solvent C. II I . run in solvent D. Standards same as i n f i g . 15 61 F i g . 17. Paper chromatograms for eluates B, G and D. l e f t , run i n solvent C; middle l e f t , run i n solvent H; righ t , run i n solvent A; middle right, run i n solvent A. (The results shown: B contain arabinose, fructose, glucose. C contain arabinose. D contain glucose, fructose, xylose.) 62 I I I . Steroids, triterpenoids and g l y c y r r h i z i n i c acid A. Steroids The c e l l steroids were extracted with chloroform and the extracts were examined by thin-layer chromatography on s i l i c a g e l . The chloroform extracts of the c e l l s grown i n d i f f e r e n t media were applied to the s i l i c a gel and developed i n the desired solvent systems. The plates were i . observed under u l t r a - v i o l e t l i g h t ; i i . sprayed with antimony penta-chloride or i i i . sprayed with Liebermann-Burchard reagent. The results are shown i n figure 18 to figure 21. The extracts contained many components which could be located with UV l i g h t and with indicator reagents. In addition, the c e l l extracts obtained from c e l l s grown on d i f f e r e n t medium indicated mainly the presence of same steroids although there were some minor differences. Five to s i x fluorecent spots and one or two absorbent spots were located under UV l i g h t of 258 miAm Five to s i x colour spots were indicated with Liebermann-Burchard reagent, and about four colour spots with antimony pentachloride reagent. As shown by TLC, at least t e n t a t i v e l y , there were fewer components contained i n the CHCI3 extract of l i c o r i c e stick,than i n s i m i l a r extracts of plant c e l l culture. In !• addition, the components contained i n the plant c e l l extract reacted quite d i f f e r e n t from those found i n l i c o r i c e root extract. 63 L 1 2 3 4 5 6 7 L Figure 18. TLC run i n solvent i i i (isopropanol-methanol 2:1) observation under UV, a l l the spots are f l u o r e -scent. L: l i c o r i c e s t i c k chloroform extract 1-7: chloroform extract of c e l l s grown i n d i f f e r -ent media 64 L I 2 3 4 5 6 7 . L Figure 19. TLC run i n solvent i i i , a f t e r sprayed with Liebermann-Burchard reagent and heated at 105 C for 5 min. L: l i c o r i c e s t i c k chloroform extract 1-7: chloroform extract of c e l l s grown i n d i f f e -rent media A: pink purple C: green E: brown B: purple D: f a i n t purple F: deep brown 65 L: l i c o r i c e s t i c k chloroform extract 1-7: chloroform extract of c e l l s grown i n d i f f e r -ent media. 66 Figure 21. TLC run i n solvent i , observation a f t e r sprayed with antimony penta-chloride chloroform solution and heated under 105°C for 5 min. L: l i c o r i c e s t i c k chloroform extract 1-7: chloroform extracts of c e l l s grown i n d i f f e r -ent media. A: brown C: deep brown B: pale brown D: yellowish brown 67 B. G l y c y r r h i z i n i c acid G l y c y r r h i z i n i c a c i d i s the most important commercial component contained i n the l i c o r i c e plant. The assay methods developed within the l i t e r a t u r e f o r g l y c y r r h i z i n i c acid can be divided into three fundamental groups. i . A measure of the weight of the pr e c i p i t a t e s formed by adding ac i d or certa i n s a l t s into the crude l i c o r i c e extracts. i i . A measure of the sugar moiety. i i i . A determination of the quantity of the aglycon, g l y c y r r h e t i n i c acid, by colorimetric or polargraphic method. The f i r s t two methods are very unspecific, as other carbohydrate constituents of the extract may be simultaneously determined or may in t e r f e r e with the assay. The t h i r d method, i s much more s p e c i f i c than the other two, although i t does not d i f f e r e n t i a t e d between s t r u c t u r a l l y s i m i l a r compounds which might be present i n the c e l l extract. The following procedure for the analysis of glycy-r r h i z i n i c acid was developed to avoid as many a n a l y t i c a l ambiguities as possible. i . The taste of the p u r i f i e d material; true glycy-r r h i z i n i c acid gives a very pronounced sweet taste, reported to be 50 times as sweet as sucrose. In th i s study, p u r i f i e d g l y c y r r h i z i n i c acid was prepared from l i c o r i c e root s t i c k of commerce and was used as standard B i i . The hydrolysis of g l y c y r r h i z i n i c acid to 68 g l y c y r r h e t i n i c a c i d with 6 N. H 2S0 4 f o r s i x hours, followed by extraction the mixture with CHCI3. The free g l y c y r r h e t i n i c a c i d can be i d e n t i f i e d by (a) the Rf on TLC (b) the colour reaction with antimony penta-chloride (c) the colour reaction with Liebermann-Burchard reagent Glycyrrhetinic a c i d gives p o s i t i v e colour reaction (brown) with antimony pentachloride but no reaction with Liebermann Burchard reagent. 1 7 (d) the reduction of the g l y c y r r h e t i n i c a c i d with LiAlH4 i n ether s o l u t i o n . The reduced g l y c y r r h e t i n i c acid gives a pink colour to Liebermann-Burchard r e a g e n t 1 7 . The r e s u l t s of the analyses f o r l i c o r i c e i n the c e l l plant cultures are l i s t e d i n table 11. From these re s u l t s i t can be seen that g l y c y r r h i z i n i c a c i d d i d not occur i n the l i c o r i c e plant c e l l culture. Either there was no g l y c y r r h i z i n i c a c i d produced by the c e l l s or the assay method was not s e n s i t i v e enough to indicate the possi-ble presence of small amount of g l y c y r r h i z i n i c a c i d . C. Triterpenoids When the freeze-dried l i c o r i c e plant c e l l s were treated according to the procedure fo r p u r i f y i n g g l y c y r r h i -z i n i c acid, a product (CGZ) was obtained which appeared v i s i -b l y to be quite s i m i l a r to the p u r i f i e d g l y c y r r h i z i n i c a c i d Table 11. The r e s u l t s of g l y c y r r h i z i n i c acid assay 69 g l y c y r r h e t i n i c l i c o r i c e root l i c o r i c e c e l l s a c i d s t i c k from suspension a. colour of f r a c t i o n A, f i g . 1 b. colour of fr a c t i o n B f i g . 1 c. colour and v i s c o s i t y of f r a c t i o n C, •• f i g . 1 d. colour of fr a c t i o n D, f i g . 1 e. colour of f r a c t i o n E, f i g . 1 f. taste of the f i n a l product i n f i g . 1 g. Rf of the abs. spots of the CHC1 3 extract from hydrolysa-te on TLC, under UV h. colour reaction with S b C l 5 i n CHCI3 i . colour reaction with AC2O/H2SO4 j.colour reaction of the reduced material with Ac 20/H 2S04 brown deep brown yellow deep brown deep brown deep brown high v i s c o s i t y high v i s c o s i t y reddish brown reddish brown not sweet yellow powdery strong l i c o r i c e sweet yellow powdery s a l t y 0.65,0.78,0.92 0.92 (n-hexane- (n-hexane-acetone) acetone) po s i t i v e brown negative Dositive p o s i t i v e brown,yellow negative p o s i t i v e p o s i t i v e brown 5 p o s i t i v e spots 70 from l i c o r i c e s t i c k s (LGZ). However g l y c y r r h i z i n i c a c i d could not be found i n the preparation when tested by taste and by TLC. After CGZ was hydrolyzed (CGZH) and fractionated on TLC, two brown coloured and one yellow coloured compounds were produced when sprayed with SbCls chloroform solution, and f i v e colour compounds (brown and purple) were produced when sprayed with Liebermann-Burchard reagent. None of the compounds corresponded eith e r to g l y c y r r h i z i n i c acid or to gl y c y r r h e t i n i c acid and therefore were considered to be some other trite r p e n o i d s . IV. The reducing substances of the l i c o r i c e plant c e l l  growth medium Ascorbic acid i s found u n i v e r s a l l y i n the d i f f e r e n -t i a t e d tissues of a l l plants. I t i s also found i n some guantity i n certain plant cultures . The high reducing a b i l i t y of the medium of l i c o r i c e c e l l s as indicated by the reduction of Fehling solution suggested the presence of reducing substances. The reducing substance d i d not appear to be ascorbic acid as t h i s acid could not be found by paper chromatographic analysis. In an attempt to determine whether ascorbic acid can accumulate i n the growth medium, ascorbic acid, 1-gulonolactone, were added to the medium. Both ascorbic acid and 1-gulonolactone remain stable i n the medium for at least several days. 1-gulonolactone was not converted to ascorbic a c i d 71 C I '\ Fluorescent absorbent spots 7-O blue white fluorescent fluorescent fluorescent o 0 Gly.t. LGZ CGZ1 CGZ2 LGZH CGZH Gly.t. Figure 22. TLC run i n isopropanol-95% ethanol 2:1 observation under UV Gly.t.: g l y c y r r h i t i n i c acid LGZ: i s o l a t e d g l y c y r r h i z i n i c acid from l i c o r i c e s t i c k CGZ1,2: is o l a t e d " g l y c y r r h i z i n i c a c i d " from c e l l s LGZH: s t i c k l i c o r i c e hydrolysate CGZH: c e l l l i c o r i c e hydrolysate 72 brown brown cp yellow • Hp brown / brown •W o d • Gly.t. LGZ CGZ1 CGZ2 LGZH CGZH Gly.t. Figure 23. TLC run i n isopropanol -957o ethanol 2:1 observation a f t e r sprayed with antimony penta-chloride chloroform solution and heated under 105 C for min. Gly.t.: g l y c y r r h e t i n i c a c i d LGZ: i s o l a t e d g l y c y r r h i z i n i c acid ( l i c o r i c e ) from l i c o r i c e s t i c k CGZ1,2: i s o l a t e d " l i c o r i c e " from c e l l s LGZH: s t i c k l i c o r i c e hydrolysate CGZH: c e l l l i c o r i c e hydrolysate 73 brown 6 purple before heat brown af t e r heat — ^ v j h i t e luo r e s cen t ^ — ^ b l u e fluorescent CGZH 2nd dimension Figure 24. Two dimension TLC f o r c e l l l i c o r i c e hydrolysate 1st dimension was run i n n-hexane-acetone 2:1 and then observed under UV 2nd dimension was run i n isopropanol-95% ethanol 2:1, and then sprayed with antimony pentochloride chloroform solution and heated under 105 C f o r 5 min. 74 O O brown C J o r purple O o white fluorescent blue fluorescent yellow LGZH CGZH / brown LGZH CGZH (a) (b) Figure 25. TLC run in solvent v. (n-hexane-acetone 2:1) (a) observation under UV (b) observation a f t e r sprayed with Lieberxnann-Burchard reagent LGZH: st i c k l i c o r i c e hydrolysate CGZH: c e l l l i c o r i c e hydrolysate 75 as indicated by paper chromatographic analyses. 0=C — I HO-C-H i HO-C-H 0 -2H o=c — I I HO-C-H 0 H HO-C-H I CH2OH 1-gulonolactone H-C I HO-C-H I CH2OH 2-keto-gulono-lactone 0=C I HO-C II HO-C ' I H-C 0 HO-C-H I CH2OH 1-ascorbic a c i d As forementioned, the growth medium of l i c o r i c e c e l l s gave two major and one or two minor s i l v e r n i t r a t e reducing spots when i t was analyzed by the paper chromatogra-phic method. I t was found the two major s i l v e r reducing spots showed only one spot when the chromatogram was developed with a n i l i n e phosphate (Fig. 26). The spot that could not be shown with a n i l i n e phos-phate was te n t a t i v e l y i d e n t i f i e d as s o r b i t o l . As shown i n f i g . 27 and f i g . 28, i t did not appear to be mannose, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, nor was i t o r i g i n a l l y contained i n PRL-4-CCM medium. I t was not d u l c i t o l , i n o s i t o l , glucosamine, gluconolactone, but appeared to be s o r b i t o l or mannitol. A paper chromatogram (Fig. 29) which was run i n solvent G for 50 hours and then was developed with s i l v e r n i t r a t e reagent resulted i n good resolution of the compound. By comparing the three spots shown i n M-+-GM (mannitol+growth medium) and the two spots shown i n S-hGM (sorbitol-f-growth medium) coluum, i t would 76 appear that the unknown sugar was s o r b i t o l . The other major spot on the paper chromatogram which was developed with a n i l i n e phosphate reagent was i d e n t i f i e d as fructose as shown i n f i g . 30. The sucrose i n the medium disappeared long before the c e l l maximum y i e l d was reached (Fig. 271, 2811). on further incubation, fructose was found to be completely absent leaving r e l a t i v e l y small quantities of s o r b i t o l (Fig. 31). F i n a l l y on continued incubation, s o r b i t o l also disappeared. 77 Figure 26. Paper chromatograms run i n solvent A (ethyl acetate-pyridine-water 8:2:1) M: l i c o r i c e growth medium Standards: glucose, galactose, fructose, arabinose, xylose. l e f t : developed i n s i l v e r n i t r a t e reagent r i g h t : developed i n a n i l i n e phosphate reagent 78 (I) ( I D Figure 27. Paper chromatograms run i n solvent A (ethyl acetate-pyridine-water 8:2:1) CM: PRL-4-CCM medium before inoculation GM: l i c o r i c e growth medium Standards: galactose, glucose, fructose, arabinose, xylose. l e f t sheet of I and I I : developed in s i l v e r n i t r a t e reagent right sheet of I and I I : developed i n a n i l i n e phos-phate reagent 79 (I) ( I D Figure 28. Paper chromatograms run i n solvent A (ethyl acetate-pyridine-water 8:2:1) In f i g . ( I ) , material applied: d u l c i t o l , s o r b i t o l , PRL-4-CCM medium, l i c o r i c e growth medium, mannitol, i n o s i t o l , glucose, developed i n s i l v e r n i t r a t e reagent In f i g . ( I I ) , materials applied: s o r b i t o l , mannitol, PRL-4-CCM, medium, l i c o r i c e growth medium, glucose, galactose, fructose. l e f t sheet, developed i n s i l v e r n i t r a t e reagent right sheet, developed i n a n i l i n e phosphate reagent. 80 Figure 29. Paper chromatograms I. run i n solvent F (807.. phenol) I I . run i n solvent G (isopropanol-ethyl acetate-water 7:1:2) f o r 50 hours. materials applied: mannitol s o r b i t o l , mannitol l i c o r i c e growth medium, mannitol, growth medium, s o r b i t o l , s o r b i t o l growth medium, fructose. developed with s i l v e r n i t r a t e reagent 81 Figure 30. Paper chromatograms I. run i n solvent G I I . run in solvent A I I I . run i n solvent F. materials applied: sucrose, sorbose, CM, GM, glucose, fructose, galactose. Figure 31. Paper chromatograms run i n solvent A materials applied: No. 1-19 l i c o r i c e growth medium, (PRL-4-CCM) 1. medium with 2,4-D, 13 days old; 13,17,18,19, medium with 2,4-D, older c e l l s ; 2,3,4, medium with gulonic lactone; 5-10, medium with ascorbic acid, 11,12, medium without growth factors; 5-7,11, c e l l s and medium changed to brown> developed with s i l v e r n i t r a t e reagent. 82 DISCUSSION I. L i c o r i c e c e l l culture L i c o r i c e c e l l s were cultured successfully i n PRL-4-CCM and PRL-4-YM media. In both cases, a high l e v e l of a complex nutrient, coconut milk or yeast extract, was needed. The l i q u i d endosperm, coconut milk, has been found to be required by many normal cal l u s and suspension t i s s u e s 1 4 Considerable inte r e s t has been shown i n the i d e n t i f i c a t i o n of the active growth constituents present i n coconut milk. Pollard, Shantz and Steward (1961) 9 3 have divided the active components of coconut milk into three f r a c t i o n s , namely, a nitrogenous component (consisting of reduced nitrogen com-pounds p a r t i c u l a r l y amino acids and t h e i r amides), a neutral component (in which the most prominent constituents are m-i n o s i t o l , s c y l l o - i n o s i t o l and s o r b i t o l ) and an active compo-nent which has r e s i s t e d analysis but i s regarded as including auxin or auxin precursors. In l a t e r evidence i t was found that the effects of the active components of coconut milk cannot be replaced by k i n e t i n or i t s a nalogues 1 4 3. There i s also evidence that the active component contains b i o l o g i c a l l y active substances possibly leucoanthyanin-like compounds, chemically d i s t i n c t from the known ki n i n s . The culture medium, successfully used by W h i t e 1 3 5 .(1934) f o r the continuous culture of excised tomato roots. 83 contained a balanced solution of inorganic s a l t s , sucrose and 0.01% of yeast extract. The extract was e s s e n t i a l for c o n t i -nued root growth. Robbins and Bartley (1937) 9 7 and W h i t e 1 3 7 (1937) showed that the yeast extract could be p a r t i a l l y replaced by c r y s t a l l i n e thiamine hydrochloride. Robbins and Schmidt ( 1 9 3 9 ) 9 8 ' 9 9 ' 1 0 0 then reported that a mixture of t h i a -mine and pyridoxine would permit the i n d e f i n i t e culture of excised tomato roots and therefore concluded that the growth promoting a c t i v i t y of yeast extract was due to i t s content of these vitamins. Further addition of other B vitamins f a i l e d to enhance the growth of tomato roots of the s t r a i n used by Robbins and Schmidt. This was confirmed by Bonner (1943). However, Bonner and Devirian (1939) 1 6 and Robbins ( 1 9 4 1 ) 1 0 1 showed that the growth of other tomato root clones was further enhanced by addition of n i a c i n ( n i c o t i n i c a c i d ) . White (1937) found that, with a clone of tomato roots, the yeast extract could be f u l l y replaced by thiamine plus a mixture of nine amino acids and l a t e r reported that the nine amino acids could be f u l l y replaced by an appropriate concentration of glycine 136,137,139, Yeast extract was reported to be an extremely good source of the B-vitamins (B^2 excepted). These vitamins are n i c o t i n i c acid, pentothenic acid, thiamine, p-amino-benzoic acid, r i b o f l a v i n , f o l i c acid, pyridoxine and b i o t i n . In the experiments,reported i n t h i s investigation, i t was found that the 10% coconut milk i n the PRL-4-CCM medium could be replaced by 0.5% yeast extract. I t would seem 84 possible that the high l e v e l of B-vitamins may play an impor-tant role i n l i c o r i c e c e l l growth and d i v i s i o n . The amount of B-vitamin added from yeast extract to the medium which supported growth was calculated to be about 100-1500 times that contained i n PRL-4-C medium and about 50 times the concentration contained i n White's medium. Due to the comple-x i t y of the yeast extract, one has to be very careful i n drawing any conclusion about the e f f e c t i v e factors present, but i t i s clear that the high l e v e l of B-vitamin as contained in 0.5% yeast extract d i d not show any i n h i b i t i o n to the c e l l groxvth. Because the PRL-4-YM supported s a t i s f a c t o r y c e l l growth, the p o s s i b i l i t y of formulating a new defined medium for l i c o r i c e c e l l s i s suggested. L i c o r i c e c e l l s vary i n growth rate and colour. The c e l l s required a considerable time to adopt to the culture medium; once the c e l l s have adopted to the new medium, the c e l l growth rate and y i e l d increases with the number of culture transfers. During the experiments with c e l l s grown i n the media with the added factors, ascorbic acid and 1-gulonolactone, a number of flasks of c e l l s and growth medium changed to dark brown colour. This colour response appear to be in d i c a t i o n of culture f a i l u r e . On the other hand, i n other l i g h t coloured cultures, both the ascorbic acid and the gulonolactone media supported an much better growth than the c e l l s grown i n the medium without added growth factors. 85 I I . The products from l i c o r i c e single c e l l suspension  culture The products r e a d i l y detected i n l i c o r i c e single c e l l suspension culture included a v o l a t i l e apple aroma, a polysaccharide p e c t i n - l i k e material, steroids and t r i t e r -penoids and reducing substances a l l of which are secreted by the c e l l s into the medium during growth. The v o l a t i l e apple aroma was composed of i n part by ethanol and some esters which were formed by c e l l s under appa-rent anaerobic condition. Most of the components of t h i s v o l a t i l e apple aroma have not been i d e n t i f i e d . The mechanism of ethanol formation i n these c e l l s i s unknown. The monosaccharides found i n the p e c t i n - l i k e mater-i a l hydrolysate include glucose, fructose, galactose, arabi-nose, xylose, galacturonic a c i d and glucuronic acid. The y i e l d of the p e c t i n - l i k e material reached a maximum of 1.1 mg/ml one month a f t e r inoculation. These results are s i m i l a r to the results reported i n a very recent paper of Olson et a l . 8 8 . The l a t t e r investigators found the components of the polysaccaride i n tobacco suspension culture medium to be arabinose, xylose, glucose, galactose, mannose and galacturo-nic acid. The tobacco c e l l cultures have a maximum y i e l d of the polysaccharide of 1.2 mg/ml. The time needed to reach the maximum polysaccharide y i e l d varied with the incubation tempe-rature. Large amounts of pe c t i c substances are known to be involved i n holding plant c e l l s together. Thus, the p e c t i c 86 substances present i n the media from c e l l suspensions suggest-ed that these are l i k e l y derived from the i n t e r c e l l u l a r cements or a precursor thereof. In 1966, Stoddart, Barrett and Northcote reported the analyses of the pectins of sycamore c a l l u s t i s s u e , 1 1 5 they found that the p e c t i c substances were more heterogeneous and complex than those of apple f r u i t . The polysaccharide composition of the c e l l walls of sycamore cambium and sycamore ca l l u s tissues were found to be d i r e c t l y comparable; both contained galactose, glucose, mannose, arabinose, xylose, rhamnose and galacturonic a c i d . They supposed that the d i f f e -rences observed i n the pectins from c a l l u s , cambium and f r u i t appeared not to be that of species difference but a character-i s t i c of the nature of the growth and growth conditions of the c e l l s and therefore related to the problems of the control and the mechanism of plant c e l l growth and d i f f e r e n t i a t i o n . A considerable number of the components of the l i c o r i c e root have been i s o l a t e d and i d e n t i f i e d 3 4 * 3 5 ' 1 0 3 * 1 0 5 . Among these components steroids and triterpenoids occur i n considerable amount. It i s not s u r p r i s i n g therefore that steroids and triterpenoids should occur i n l i c o r i c e c e l l culture suspension. However, the assay method applied i n t h i s investigation d i d not indicate the presence of g l y c y r r h i z i n i c a c i d . The i d e n t i f i c a t i o n of the steroids or triterpenoids present i n the c e l l extract i s important not only because of the commercial i n t e r e s t but also because of the interest i n the genetic r e l a t i o n s h i p between the i n t a c t plant tissue and: 87 single c e l l suspension culture. S o r b i t o l and fructose were found to be the two major sugars present i n the medium of l i c o r i c e c e l l s a f t e r 12 days incubation. Burstrom demonstrated that excised wheat roots hydrolyzed sucrose i n l i q u i d media, 1 8 Tulecke, et a l . 1 2 1 and B a l l 7 gave s i m i l a r i l l u s t r a t i o n s i n t h e i r papers. Wang and Staba analyzed that in carboy I of the dual-carboy system of spearmint, sucrose was completely hydrolyzed to dextrose and fructose by the t h i r d day of culture, both dextrose and fruc-tose were absent i n the culture medium a f t e r 8 d a y s 1 3 0 . This investigation i s the f i r s t record of the occur-rence of s o r b i t o l i n plant c e l l suspension cultures. The role that s o r b i t o l plays i n carbohydrate metabolism and the synthesis of and metabolic pathways of s o r b i t o l present a very i n t e r e s t i n g research subject. A c y l i c polyhydric alcohols such as s o r b i t o l and mannitol occur i n many species of plant, although i n the majority of cases neither quantitative data on the amounts present nor information on t h e i r metabolic role are av a i l a b l e . Donen (1939) suggested that s o r b i t o l i s formed i n plum f r u i t when the concentration of hexoses reaches a c r i t i c a l l e v e l , whereas Kid, West, G r i f f i t h and Potter (1938 ) 6' found that much of the s o r b i t o l i n pears was replaced by hexulose (probably fructose) as the f r u i t ripens. S o r b i t o l could also be u t i l i z e d for polysaccharide synthesis i n leaves q of Rosaceae and Gramineae (Barker 1955°, Altermatt and Neish 1956 2) but nothing was known of the extent of t h i s conversion 88 under natural conditions. Andrew and Hough (1958) i n the experiments with 14CC>2, o r i g i n a l l y designed to y i e l d informa-ti o n on the biosynthesis of leaf polysaccharides, indicated that the s o r b i t o l i n plum leaves has an important role i n the metabolism of these leaves. The incorporation of 1 4 C into s o r b i t o l was considerably greater than into starch. Further investigation of s o r b i t o l metabolism i n plum leaves by Anderson, Andrew and Hough (1961, 1962 ) 3 ' 4 indicated that des-p i t e the large amounts of s o r b i t o l present i n the leaves, s o r b i t o l was r a p i d l y e q u i l i b r a t e d with the primary products of photosynthesis. When D-( 1 1 4 C ) and D-( 6 1 4C)-glucose were metabolized by plum leaves, about 40% of the 1 4 C detected in the leaves a f t e r 5 hours was i n s o r b i t o l . Labelling patterns showed that the conversion of D-glucose into s o r b i t o l probably occurs without rupture of the carbon chain. To a small extent, D-glucuronic a c i d appeared to be converted into s o r b i t o l when fed to plum leaves, only about 20% of the a c t i v i t y of the a c i d was detected i n the leaves a f t e r 5 hours and 80% of t h i s was present i n s o r b i t o l . Whetter and Tapper ( 1 9 6 6 ) 1 3 2 reported s o r b i t o l i n germinating apple seed, and i n a l l organs of the developing seedling including the root. Photosynthesizing cotyledons appeared to be a s i t e of synthesis of s o r b i t o l . S o r b i t o l was considered to be a reserve form of carbohydrates i n seeds and cotyledons of apple. When spur shoots of the apple tree with or without f r u i t were enclosed i n p l a s t i c bags and exposed to 1 4 C 0 2 , Hansen (1967) 5 0 found approximately 90% of the 1 4 C 89 taken up by the leaves was transferred to nearby f r u i t within 5 days. Immediately following application, the leaves contained 58-807° of the added 1 4 C as s o r b i t o l , 7-9% as sucrose and 1-47. i n the form of glucose. Within f i v e days, the amount of s o r b i t o l 1 4 C was greatly reduced while the glucose 1 4 C increased. The s o r b i t o l * 4C and t o t a l s o r b i t o l and glucose were highest i n leaves from shoots without f r u i t . Williams, Martin and Stahly (1967 ) 1 4 2 applied uniformly 1 4 C l a b e l l e d s o r b i t o l or sucrose to the underside of the leaves on young vigorously growing Delicious apple trees i n the green house and studied the rates of absorption and translocation. S o r b i t o l 1 4 C moved re a d i l y i n the tree and 14 C could be found in sugars, amino acids, and other organic compounds i n plant. P h l o r i z i n became radioactive soon a f t e r the application of the l a b e l l e d sorbitol;, S o r b i t o l was absorbed and translocated at a f a s t e r rate than sucrose and continued to move into apple f r u i t s a f t e r watercore began 5to develop. Wieneke ( 1 9 6 9 ) 1 4 0 also reported that a f t e r assimilation of 14CC-2 or absorption of s o r b i t o l 1 4 C from the leaves, s o r b i t o l and also sucrose were transported i n the bark to the apple f r u i t . After one minute, s o r b i t o l was the most l a b e l l e d sugar. The exact role of s o r b i t o l i n carbohydrate metabo-lism i s s t i l l i n confusion, but i t i s clear that s o r b i t o l plays an important role since i t i s e a s i l y transported and incorporated i n t o glucose, fructose, polysaccharides and c e l l material. The enzymes, aldose reductase (R-CH0 + TPNH -+H +^=^ 90 RCH2OH+TPN) and s o r b i t o l dehydrogenase (sorbitol-)- DPN=?=^ fructose + DPNH-+H*) have been demonstrated i n animals and bacteria, but not i n plant t i s s u e s . However, i t i s believed the two enzymes are also present i n plant t i s s u e due to the incorporation of s o r b i t o l into glucose, fructose and other carbohydrates. 91 SUMMARY The c e l l s of the L i c o r i c e plant, Glycyrrhiza glabra, were cultured as a "single c e l l " suspension. Their growth behaviour, y i e l d and metabolic products were studied. The suspension cultures of the l i c o r i c e plant were established from the f r i a b l e calluses obtained from the ra d i c l e , cotyledon and hypotyl of the germinated seeds. The single c e l l s , regardless of t h e i r o r i g i n showed l i t t l e difference i n c e l l size and morphology. After an apparent adjustment to the medium, the c e l l s required 11-13 days of incubation to reach the maximum c e l l y i e l d of 1.2 gm/100 ml medium, dry weight. During the growth period, the pH of the growth medium decreased from pH 5.6 to pH 4.7 i n f i r s t few days and then increased to about pH 6.0. A l e v e l of 10% coconut milk i n PRL-4-CM medium was found to support the best c e l l growth; the lower the coconut milk l e v e l , the longer the growth period required to reach the maximum c e l l y i e l d . I t was also found that 0.5% yeast extract could be used to replace the coconut milk i n the PRL-4-CCM medium. The metabolites detected and examined i n the l i c o r i c e single c e l l suspension culture included a v o l a t i l e apple aroma, a polysaccharide p e c t i n - l i k e material, and some steroids and triterpenoids. The analyses of the l i c o r i c e c e l l v o l a t i l e apple aroma found under anaerobic conditions indicated the presence 92 of ethanol and some related esters. The monosaccharides found i n the p e c t i n - l i k e poly-saccharide hydrolysate were glucose, fructose, galactose, arabinose, xylose, galacturonic acid and glucuronic a c i d . The pe c t i n - l i k e material i n the c e l l preparations reached a maximum y i e l d of 1.1 mg/ml a f t e r one month of growth. G l y c y r r h i z i n i c acid, the common l i c o r i c e constituent found i n the root, could not be detected i n the suspension cultures. However, several other related compounds which gave t y p i c a l s t e r o i d and t r i t e r p e n o i d reactions were found. So r b i t o l and fructose were found to be the two major sugars which accumulated i n free form i n the l i c o r i c e c e l l medium. 93 BIBLIOGRAPHY 1. Adams, A. V. 1953. Pharmacology of l i c o r i c e . Pharm. J . 171, 34. 2. 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