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UBC Theses and Dissertations

Ascorbic acid production in the cultured tissue of the briar rose, rosa rugosa Wegg, Susan Melanie 1972

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ASCORBIC ACID PRODUCTION IN THE CULTURED TISSUE OF THE BRIAR ROSE, ROSA RUGQSA by SUSAN MELANIE WEGG B.H.Sc, University of Guelph, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Food Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1972 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by h i s representatives.. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of PQ-Q-Q^  S CAJL/VVC5L The University of B r i t i s h Columbia Vancouver 8 , Canada i i ABSTRACT The fleshy hips of the briar rose, Rosa rugosa were cultured on a modified medium developed by Gamborg (1963) to produce callus and suspension cultures. These cells were very light, almost white. Reduced ascorbic acid, determined by the 2,6-dichlorophenolindophenol ti t r a t i o n method, was found in the suspension cultures. This finding prompted an investigation into culture techniques for obtaining an optimum concentration of ascorbic acid. During the growth of the ce l l s , the pH rose and ascorbic acid concentration increased after the sixth day. Agecof culture was also an important factor as., cultures older than three: or four weeks contained virtually no ascorbic acid. Possible precursors of ascorbic acid in plants were added to cultures and their effect on the ascorbic acid level was determined over a period of twenty-four hours. D-glucose, D-(-)-levulose, D-galactose and D-glucurono-Y-lactone caused nb increase. L-gulono-V-lactone brought about a slight increase and a comparatively large increase was obtained with L-galactono-Y-lactone. Confirmation for the latter was obtained using the 2,4-dinitrophenylhydrazine method to rule out the possibility o r microbial contamination and to be sure t h a t a n o t h e r m e t a b o l i t e was n o t r e s p o n s i b l e f o r t h e i n c r e a s e d r e d u c i n g c a p a c i t y o f t h e s y s t e m . O m i s s i o n o f m y o i n o s i t o l f r o m t h e v i t a m i n s o l u t i o n o f t h e medium d e c r e a s e d t o t a l c e l l y i e l d b u t h a d no e f f e c t on t h e l e v e l o f a s c o r b i c a c i d . D e c r e a s i n g t h e s u c r o s e c o n t e n t o f t h e medium by o n e - h a l f c a u s e d a s h a r p d e c r e a s e i n a s c o r b i c a c i d c o n c e n t r a t i o n , i n d i c a t i n g t h a t an a d e q u a t e s u p p l y o f s u g a r i s a p r e r e q u i s i t e f o r o p t i m u m p r o d u c t i o n o f a s c o r b i c a c i d . G r o w i n g l i q u i d s u s p e n s i o n s u n d e r c o n t r o l l e d i l l u m i n a t i o n r e s u l t e d i n i n c r e a s e d a s c o r b i c a c i d v a l u e s w i t h e a c h s u c c e s s i v e t r a n s f e r ; h o w e v e r , t o t a l c e l l y i e l d s d e c r e a s e d e a c h t i m e . I n a d d i t i o n , t h e r e was no e v i d e n c e o f c h l o r o p h y l l p r o d u c t i o n i n t h e c u l t u r e s . T h i s e x p l o r a t o r y w o r k shows t h a t a s c o r b i c a c i d i s p r e s e n t i n t h e t i s s u e c u l t u r e o f R o s a r u g o s a . T h e r e f o r e , t i s s u e c u l t u r e may be u s e f u l f o r e l u c i d a t i n g t h e c o m p l e t e p a t h w a y f o r a s c o r b i c a c i d p r o d u c t i o n w h i c h i s a s y e t s t i l l u n c l e a r . T h i s i n f o r m a t i o n c o u p l e d w i t h t h e e f f e c t s o f a l t e r i n g t h e p h y s i c a l e n v i r o n m e n t o f t h e c e l l s may d i r e c t c u l t u r e s o f R o s a r u g o s a t o p r o d u c e a s c o r b i c a c i d i n q u a n t i t i e s t h a t w o u l d make i t s r e c o v e r y i n d u s t r i a l l y a t t r a c t i v e . iv TABLE OF CONTENTS page INTRODUCTION 1 LITERATURE REVIEW 4 MATERIALS AND METHODS-1. Plant origin, cultural conditions and prepara-tion of a single c e l l suspension 14 2. Measurement of c e l l growth 16 3. pH measurement 17 4. Ascorbic acid measurement 17 5. Comparison with l i v i n g tissue 20 6. Location of ascorbic acid 20 7. Comparison between age and colour of cultures and ascorbic acid content 21 3. Possible methods of varying ascorbic acid content of cultures 21 9. Confirmation of precursor studies 23 10. Determination of total ascorbic acid, dehydro-ascorbic acid and diketogulonic acid 24 RESULTS AND DISCUSSION 1. Growth of cultures 26 2. Growth curve 30 3. Location of ascorbic acid 33 V RESULTS AND DISCUSSION (cont.) page 4. Comparison between age and colour of cultures and ascorbic acid content 33 5. Comparison with other l i v i n g tissue 36 6 . Addition of precursors to cultures 36 7 . Omission of myoinositol from the medium 4 5 £. Cells grown under controlled illumination 4 6 9. Effect of sucrose 4# SUMMARY 50 LITERATURE CITED 51 v i LIST OF FIGURES page 1. Possible pathways of ascorbic acid synthesis in plants 5 2. Mechanisms of determining total and reduced ascorbic acid 9 3. Composition of PRL-4 medium 15 4. Typical callus culture of Rosa rugosa cultured on PRL-4-C-CM medium 27 5. Typical flask of cells of Rosa rugosa cultured on PRL-4-C-CM medium 23 6. Microscopic examination of typical c e l l popula-tions of Rosa rugosa from the suspension culture flasks 29 7. Results of growth curve showing pH, fresh weight and ascorbic acid content 31 8. Results of growth curve showing pH, dry weight and ascorbic acid content 32 9. Effect of the addition of selected precursors on the reducing power calculated as ascorbic acid on rose cells 37 10. Effect of the addition of selected precursors on the reducing power calculated as ascorbic acid on rose cells 38 11. Thin layer chromatographs showing the results of the DNPH method of detecting ascorbic acid 41 12. Paper chromatographs showing the disappearance of L-galactono-J*-lactone over time 42 v i i LIST OF TABLES page 1. Comparison of ascorbic acid values of l i v i n g tissue and plant cells 34 2. Average ascorbic acid content of fruits 34 3. Comparison between age and colour of cultures and ascorbic acid content 35 4. Titration values of precursors and precursors dissolved in media 35 5. Comparison of dry weights of dark and light grown cells . 44 6. Effect of altering the environment on f i n a l weight of tissue and ascorbic acid content (12th day) ... 44 7. Spectrophotometric readings for "tota l " ascorbic acid and dehydroascorbic + diketogulonic acids ... 47 3. Summary of results obtained from attempts to vary the ascorbic acid content of rose tissue culture 47 INTRODUCTION The propagation of whole plants from isolated fragments has been carried out for centuries. The majority of these techniques were concerned with organ propagation. A rela-tively new technique for the production of cells per se i s called plant tissue culture. From quite primitive beginnings, techniques which permitted the culture of an increasing number of plant types were gradually established. Cultures were started by excising plant tissue from i t s natural environment and growing them under sterile conditions in a vessel on an a r t i f i c i a l medium. The resulting cells were capable of division with a resulting increase in the c e l l plasma mass (3$). The growth of the tissue was thought to take place as a consequence following wounding of a plant organ. Living parenchymatous cells adjacent to the wound frequently became meristematic forming masses of "undiffer-entiated" cells (44). Such a proliferating tissue was called a callus. Culture of cells i s a combination of an art and a science. After reaching a c r i t i c a l size, the callus may be transferred to a liquid medium where i t w i l l grow as a suspension culture. Growth in agitated liquid media i s generally faster than that of static callus cultures, 2 presumably because the nutrients are more effectively absorbed and toxic exudates are dispersed by virtue of the greater contact area and development of convection currents (45). To obtain actively growing suspension cultures, constant agitation either by shaking culture flasks, forced aeration or magnetic st i r r i n g i s practised (24). Production of cells can be stepped up to an industrial scale as are microbial fermentations with relative ease. One major difference i s that maximum plant c e l l yield requires a longer growth period, one to ten weeks, as compared to bacteria which generally attain maximum yield under ideal conditions within one to two days. The particular possibility of large scale plant c e l l production has caused speculation as to the possibility of using plant cells as an alternative in helping to alleviate the world protein shortage. The economic f e a s i b i l i t y of such an operation would be more attractive i f other metabolites could also be recovered. For example, there has been interest in using plant cells for secondary product biosynthesis, biogenesis or biotransformation. Specific examples s t i l l at the research stage are the production of antibiotics in lettuce and cauliflower tissues (6) and the biotransformation of pro-gesterone and pregnenolone by plant suspension cultures (11). To this point, however, the practical use of tissue cultures in industry i s highly speculative (23). 3 Potentially, the techniques of plant tissue culture are well suited for studying environmental and nutritive control and chemical pathways involved in the synthesis of particular metabolites (45). It i s a well established fact, however, that the metabolic pathways present in the parent plant are not necessarily operative in the tissue culture of that plant (46, 50). Therefore, in selecting a particular metabolite for study, i t would seem reasonable to choose one that i s present in appreciable quantity in the parent to increase the proba-b i l i t y of detection in culture. Rose hips have long been known as an excellent source of ascorbic acid. In this investigation these organs have been cultured quite satisfactorily to form both calluses and suspension cultures. A very light coloured, almost white, c e l l suspension (which rapidly darkens as the cells age) results. The present study was undertaken to explore the possible metabolic pathway for ascorbic acid in rose tissue culture and to investigate methods that might increase i t s production. 4 LITERATURE REVIEW Vitamin C has the distinction of being the f i r s t nutri-tional adjunct whose deficiency in the human diet was recognized as a cause of disease. Probably as early as 1700, i t was observed that a lack of fresh fruits and vegetables resulted in scurvy and that this disease could be prevented and cured by the proper diet. Gradual recognition of the "antiscorbutic principle" followed. In 1928, Szent-Gyorgyi (47) isolated from cabbage and later from paprika a substance which he called hexuronic acid. It was later shown to be identical to the antiscorbutic factor. The proof that ascorbic acid was indeed the anti-scorbutic factor was provided by Reichstein, Griissner and Oppenauer (7) who synthesized L-ascorbic acid and showed that their product was physiologically active. More recently, ascorbic acid has been linked to the reduction of incidence of the common cold and heart disease and has thus increasingly come to the attention of the public. Ascorbic acid i s an essential factor in the normal growth, development and nutrition of the human (49). Owing to i t s vitamin activity and since i t i s readily oxidized and reduced, i t i s thought to play a role in a large number of metabolic processes. It i s a prerequisite for the formation 5 0 H-C-OH I HO-C-?H I H-C-OH I H-C I CH20H D-glucose -7 I H-C-OH I 0 — C - H i I H-C-OH 0 I H-C Cr 180° 0-C— I C-H 0 0 -C=0 HO-C-H H-C-I HO-C-H ! HO' H D-glucurono-y-lactone 0=C I HO-C-H I 0 HO-C-H I H-C— I HO-C-H I CH2OH o=c HO :n H c .0-0 H-C—1 I HO-C-H I CH20H L-gulono- L-ascbrbic y-lactone acid H-C-OH I HO-C-H ' —> HO-C-H I H-C-OH I CH2OH H /OH NC-H-C-OH I HO-C-H I HO-C-H 1 H-C 0 COOR G* o 180° COOR I C-H I o=c I HO-C-H I o H-C-OH H-C-OH I — \ I H-C-OH ' H-C — I I HO-C-H HO-C-H 0-C-I HO-C II HO-C H-C-I 0 — Cy HO H CH20H HO-C-H I CH20H D-galactose D-galacturonic acid methyl ester L-galactono- L-ascorbic y-lactone acid Figure 1. Possible pathways of ascorbic acid synthesis in plants. From Loewus, F.A. Tracer studies on ascorbic acid formation in plants. Phytochem. 2, 109-123, 1963. 6 of collagen, intercellular cement, dentine, cartilage, callus, osteoid tissue of bones, blood vessel walls and connective tissue ( 9 ) . It i s also indispensable in the healing of wounds and the uniting of fractures. Ascorbic acid must be supplied to the body as man has lost the a b i l i t y to synthesize i t himself. It i s produced biologically to a greater or lesser degree in a l l plants. Ascorbic acid may then be passed to man when plant material is ingested. The greatest concentrations of this compound are found in areas directly concerned with plant c e l l growth ( 2 9 ) . The complete biosynthetic pathway of ascorbic acid in plants i s s t i l l not clear ( 3 0 ) . From the evidence to date, i t can be represented by one or both of the following sequences (34, 2 9 , 26) as also illustrated in Fig. 1. (1) D-glucose-> D-glucurono-y-lactone —^ L-gulono-y-lactone —^ L-ascorbic acid (2) D-galactoseD-galacturonic acid methyl ester—} L-galactono-y-lactone r-^L-ascorbic acid In general, the evidence for the formation of ascorbic acid from hexose sugars i s suggestive rather than conclusive. Sugars are interconvertible and once incorporated into the metabolic cycle, give rise to a variety of compounds, among which substances acting as precursors might be found ( 3 1 ) . The ascorbic acid of plants i s formed continually in the green organs of the plant.,. A number of workers have 7 established a close relationship between photosynthesis and ascorbic acid formation (8). Numerous attempts have been made to identify chloroplasts as the site and chlorophyll as the necessary agent for synthesis of the vitamin. Asselbergs (2), for example, found a correlation between the ascorbic acid content of apple leaves and their surface area. Sur, Roy and Guha (7) investigated synthesis of the vitamin by germinating seeds of Phaseolus radiatus and found that in sunlight about 60% more ascorbic acid was produced than by growth in the dark. Giroud, Ratsimamanga and Leblond (13), by extraction methods, found a direct relationship between the concentration of ascorbic acid and the presence of chlorophyll. Plant cells should be a good means to study this phenomenon as they are generally cultured in darkness so that l i t t l e pigmentation is observed. Therefore, a comparison between light and dark grown cultures should provide some information in. this-regard.. Certain problems may arise, however, in that when exposed to light, many cultures become only pale green or may have no pigmentation at a l l (45). Ai supposed parallelism between growth rate and ascorbic acid content of higher plants has led to the suggestion that i t might function as a growth factor or even as an indis-pensable phytohormone, especially for young growing organs (1). The concept of a vitamin i s s t i l l inseparable from that of a 8 growth factor in the mind of many biologists although the action of some vitamins has l i t t l e to do with c e l l enlarge-ment or c e l l division (39). Apart from the indirect evidence afforded by high concentrations of ascorbic acid in rapidly growing tissues and organs of embryos, young plants, animals and man u t i l i z e more ascorbic acid than old animals. There is l i t t l e evidence either in vivo or in culture for a direct effect of ascorbic acid on any biological phenomena one associates with the complex process of growth. Also, evidence in favour of a relationship between ascorbic acid and growth must be weighed against the fact that some non-growing tissues in plants, such as citrus f r u i t s and rose hips, have a relatively high ascorbic acid content (39). Actual quantitative determination of ascorbic acid in plant extracts i s a problem in that absolute certainty of results i s not possible. The methods available are generally of two types—bioassays and chemical analyses. The former, however, are time-consuming, expensive, and leave much to be desired insofar as precision i s concerned. However, they do have the advantage of measuring the summation of chemical entities that possess Vitamin C activity but exclude materials devoid of Vitamin C activity. The situation regarding chemical analyses remains dynamic. The complex biological relationship between the compounds possessing Vitamin C activity, as well as the chemical 9 Dye (pink) L-ascorbic , Dye Dehydro-acid (colourless) ascorbic acid (A) Reduced ascorbic acid by 2,6-dichlorophenolindophenol 0-G I HO-C !i HO-C J H-C i) o -2H o-=c-I o=c 0 H20 f2H 0=C I HO-C-H I CH2OH L-ascorbic acid H-C — 1 HO-C-H I CH20H Dehydro-ascorbic acid O-C-OH O-G-OH I I 0=C .DNPH RHNN-C I > I Q-C ' RHNN^ C I I H-C-OH H-G-OH I - I HO-C-H HO-C-H I i CH2OH GH2OH Diketo-L- bis - 2 , 4-dinitro-gulonic phenylnydrazone acid (B) Total ascorbic acid by 2,4-dinitrophenylhydrazine Figure 2. Mechanisms of determining total and reduced ascorbic acid. 10 similarity of these compounds to others which are inactive, has made the existence of a single, simple and specific method as yet impossible. Chemical analyses can be divided into two groups: 1) determination of reduced form; 2) determination of "total" Vitamin C content. The former i s based on the oxidizing-reducing properties of ascorbic acid or i t s a b i l i t y to couple with diazotized aniline derivatives to form coloured hydrazides, while the latter is based on oxidation to diketogulonic acid and subsequent formation of a highly coloured hydrazone (3). The mechanisms are shown in Fig. 2. A l l of these methods suffer from lack of specificity to one extent or another. For this work, the oxidation-reduction method using 2,6-dichlorophenolindophenol as indicator was chosen as oxidation methods measure substances other than reduced ascorbic acid, in particular dehydroascorbic acid (DHA) and diketogulonic acid (Fig. 2). The relatively labile nature of DHA (35) suggests that once ascorbic acid in a food has been oxidized to this compound, the value of the product as a source of Vitamin C has been impaired (3). Also DHA i s usually found in plant tissues In low concentrations and i s always associated with ascorbic acid in much higher concentra-tions (23). Studies indicate that the indophenol,method i s reliable for the estimation of ascorbic acid in most plant tissues (41)* 11 The visual t i t r a t i o n method is based upon the reduction of the dye by an acid solution of ascorbic acid. In the absence of interfering substances, the capacity of an extract of the sample to reduce a standard solution of the dye, as determined by t i t r a t i o n , i s directly proportional to the ascorbic acid content. A serious error might be expected in the analysis of preparations that contain iron or copper as these two metals are efficient catalysts in hastening the oxidation of the ascorbic acid (14). The most effective acid in preventing this latter oxidation i s metaphosphoric (3). It has been found that the s t a b i l i t y of ascorbic acid solutions is not merely a function of pH but depends also on the nature of the acid (37). In addition to acting to prevent ascorbic acid oxidation, metaphosphoric acid i s also a protein precipitant and thereby aids in the removal of enzymatic oxidases and f a c i l i t a t e s c l a r i f i c a t i o n of the plant extracts. A small amount of ethylenediaminetetra-acetic acid (EDTA) added to the metaphosphoric acid-plant extract solution had further ascorbic acid stabilizing a b i l i t y (10). Titrations must be carried out as rapidly as possible to minimize interference from other reducing substances such as cysteine, glutathione, sodium sulfide and reductones i f they are present. To obtain information about total ascorbic acid as a 12 comparison, another approach i s required. Methods of reducing DHA to ascorbic acid so that i t may be determined by tit r a t i o n include reducing the sample with H2S or Escherichia c o l i . There are problems with both methods, however. Tewari and Krishman (48) have shown that part of the DHA i s irreversibly destroyed by H2S treatment. King (21) also found that many aldehydes, ketones and quinones give rise to interfering reactions when reduced by H2S. Mapson and Ingram (33) have found that the reducing a b i l i t y produced using E. c o l i i s in part due to reduction of nitrate to n i t r i t e . On acidification, the nitrous acid formed rapidly oxidizes the ascorbic acid. For these reasons, to estimate "t o t a l " ascorbic acid, the 2,4-dinitrophenylhydrazine (DNPH) was employed (3). The total ascorbic acid i s determined by oxidation with bromine. To determine the amount contributed by DHA and diketogulonic acid, stannous chloride i s ground with sample to preferentially protect the reduced ascorbic acid present. Currently, ascorbic, acid i s made commercially by the following process (18). D-glucose D-sorbitol I L-sorbose i diacetone-L-sorbose i diacetone-2-keto-L-gulonic acid i 2-keto-L-gulonic acid i L-ascorbic acid 13 This process must occur by a number of steps. Simplification of the procedure to one operation may eventually represent an advantage economically. Plant cells offer potential in this respect. Methods of increasing the quantity of ascorbic acid formed in rose cells were investigated. Many of the pre-cursors found effective for plants were administered to the cultures. These included D-glucose, D-(-)-levulose, D-galactose, D-glucurono'-J'-lactone, L-gulono-tf-lactone and L-galactono-Y-lactone. Other possible methods explored were growing the cells under controlled illumination and by decreasing the myoinositol and sucrose concentrations in the medium. From these investigations, i t i s hoped that information regarding regulation of ascorbic acid production under the conditions of tissue culture can be obtained. 14 MATERIALS AND METHODS 1. Plant origin, cultural conditions and preparation of a single c e l l suspension  A. Origin of the plant The plant used throughout the studies was a briar rose, Rosa rugosa (36). Samples were obtained from a plant actively growing on the U.B.C. campus. B. Medium The basal medium used throughout the studies was a medium developed by Gamborg (PRL-4) (12), the composition of which is l i s t e d in Fig. 3» In the studies, N-Z amine type A was replaced by casamine hydrolysate (PRL-4-C) and the quantity was decreased to 0.5 gm./l. Ten per cent coconut milk was also added to the medium (PRL-4-C-CM). The coconut milk was obtained from mature coco-nuts purchased at a local market. The milk was drained, f i l t e r e d through Whatman No. 1 paper and stored frozen in plastic bottles. The complete media was st e r i l i z e d at 15 psi for 20 minutes before use. For solid media, 1$ Bacto-Difco agar was added. 15 Ingredient mg./l. NaH2P0/..H20 90 Na2HPOi. 30 KC1 300 {NH4)2S04 200 MgS0WH20 250 KNO3 1000 CaCl2'2H20 150 KI ^ .75 Iron* 28 (5 ml.) Micronutrients** 1.0 ml. Vitamins*** 10.0 ml. Sucrose 20 gm. N-Z Amine Type A 2.0 mg. 2,4-D 2.0 mg. Final pH 6.2 Fe EDTA stock solution Dissolved in 100 ml.: FeS04.7H20 278 mg. Na2EDTA 372 mg. Keep frozen jkjfc .. ....... Stock solution: Dissolved in 100 ml. H2O: 1 gm. MnSOA^O, 300 mg. H0BO0, 300 mg. ZnSO. .7H20, 25 mg. Na2MoO. .2H20, 25 mg. GuS04, 25 mg. CaCl 2»oH 20 * Stock solution: Dissolved in 100 ml. H2O: 10 mg. nicotinic acid, 100 mg. thiamine, 10 mg. pyridoxine, 1 gm. myoinositol Figure 3. Composition of PRL-4 medium-From Gamborg, O.L. and Eveleigh, D.E. Culture methods and detection of glucanases in suspension cultures of wheat and barley. Can. J. Biochem. 46, 417, 1968. 16 C. Callus formation Hips of Rosa rugosa were ster i l i z e d in 5$ sodium hypochlorite for 20 minutes and quickly rinsed in d i s t i l l e d water. They were then dissected with a sterile scalpel in a ste r i l e petrie dish. Small portions of the inner contents were trans-ferred to the surface of 10 ml. of PRL-4-C-CM agar medium in a 100 ml. milk dilution bottle. The cultures were incubated in the dark at 26 °C. Callus tissue formed within two weeks to a month. D. Preparation of a single c e l l suspension When the calluses were large enough to transfer, several were placed in 100 ml. of PRL-4-C-CM liquid medium in a 250 ml. erlenmeyer flask. The culture was incubated on a rotary shaker (Gyrotary shaker) at a speed of 160 revolutions per minute in a 1" circular orbit at 26°C. The cells were grown in the dark with short periodic exposure to indirect over-head fluorescent light during observation. The cells were transferred to a new medium at regular time intervals in order to maintain culture vigour. 2. Measurement of c e l l growth The growth of the cells was measured by following c e l l dry weight every other day for two weeks. The cells 17 from duplicate cultures were collected by f i l t r a t i o n through Mira-cloth and a weighed portion was dried to constant weight in a V i r t i s freeze dryer. The remaining cells were used in ascorbic acid determination. pH measurement The f i l t r a t e from the cells used for dry weight determination was used for this purpose. The pH was measured using a Corning Model 5 pH meter. Ascorbic acid measurement Ascorbic acid was determined by the 2,6-dichloro-phenolindophenol ti t r a t i o n method (3). A* Standardization of the dye (daily) A 5-ml aliquot of the standard ascorbic acid solution (containing 1 mg. ascorbic acid) was diluted with 5 ml. 3$ HPO3. It was titrated with the dye solution to a pink colour which persisted for 15 seconds. Since this volume of dye represented 1 mg. of ascorbic acid, the ascorbic acid equivalent (T) of 1 ml. of the dye solution was equal to 1 divided by the volume in, 1 ml. of the dye solution used in this t i t r a t i o n . 18 B. Procedure ( i) Extraction A sample of cells l e f t after f i l t e r i n g for dry weight determination was rinsed with d i s t i l l e d water to remove any adhering media. This was usually about 5 gm. These cells were quickly placed in a jar and 40 ml. of 6% HPO-j-EDTA was added. The jar was attached to an Osterizer and blended at 1 0 , 4 0 0 rpm. for one minute to mascerate the ce l l s . This slurry was then transferred to a 100 ml. volumetric flask and diluted to volume with 3$ HPO3. The sample was then f i l t e r e d through Whatman No. 1 f i l t e r paper, discarding the f i r s t few ml. of f i l t r a t e . A phase contrast microscope was used to check 1 0 0 $ efficiency of c e l l masceration. ( i i ) Titration A 10 ml. aliquot of the f i l t r a t e from the preceding step was pipetted into a small erlen-meyer flask (50 ml.). The solution was titrated immediately with the standardized solution of 2,6-dichlorophenolindophenol to a faint pink end point which persisted for 15 seconds. The colour of the dye dissolved in the dilute sodium bicarbonate solution was blue, but in 19 an acid medium such as the stabilizing solution used in this determination, the dye assumed a pink colour. Therefore, the colour change in this method, at the end point, was from colour-less to pink. In the titrations of the extracts with the dye, i t was desirable to add the dye quite rapidly to a point where the resulting pink colour did not immediately disappear. As rapidly as possible, the dye was added dropwise with constant mixing of the solution u n t i l the faint pink colour of the solution resulting from the unoxidized dye persisted for 15 seconds. A rapid t i t r a t i o n and short-time end point were desirable because of the possible interfering action of other constituents of the solution. In general, such interfering materials reacted more slowly with the dye than did ascorbic acid; therefore, their effect was kept to a minimum by rapid t i t r a t i o n , ( i i i ) Calculation Ascorbic acid was calculated according to the following formula: V, * T x l^C" _ mg. ascorbic acid per 100 gm. *™ sample 20 V = ml. dye used for t i t r a t i o n of aliquot of diluted sample T = ascorbic acid equivalent of dye solution expressed as mg. per ml. of dye W = gm. of sample in aliquot titrated This was calculated using both the fresh weight and the calculated dry weights from the freeze dried samples. Comparison with l i v i n g tissue Approximately 2 gm. fresh weight samples of actively growing tissue of the briar rose were examined for their ascorbic acid content. Rose hips, stems and leaves were used. These values were compared with the maximum values obtained in tissue culture and with common values of some fr u i t s . As chlorophyll production and ascorbic acid values seem to be related, comparisons between light grown cultures and the li v i n g tissue were also carried out. Location of ascorbic acid To confirm that ascorbic acid was located only intra-cellular l y , determinations were run on the cell-free f i l t r a t e . Equal volumes of the media in which the cells had been suspended and 3% HPO3 were mixed and the resulting solution was titrated. 21 7. Comparison between age and colour of cultures and ascorbic acid content  Flasks of ce l l s , one, two, three and five weeks old, were compared visually for colour. Then ascorbic acid determinations were made on the contents of each of the flasks. 8. Possible methods of varying ascorbic acid content of cultures  A. Addition of possible precursors Several possible precursors as chosen from the hypothesized metabolic pathways were added at various concentrations by dissolving the test metabolite in 25 ml. of the medium in which the cells had been growing for 12 days. This time interval was chosen as by this time there was a good c e l l population and the cells f i l t e r e d rapidly, a necessity for this determination. Then the f i l t e r e d cells were re-suspended in this medium and returned to their original environment. At specific time intervals (0, 2, 4, 6 and 24 hr.) small portions of the flask contents ( 4 - 5 gm.) were taken and their ascorbic acid content determined. 22 PRECURSORS ADDED QUANTITY 1 gm. .5 gm. .25 gm. D-glucurono-v-lactone X X L-gulono-K-lactone X X L-galactono-tf-lactone X X X D-glucose X X D-galactose X X D-I-)-levulose X X There has been some controversy about the -possible role of myoinositol in ascorbic acid pro-duction (25, 26). As this compound i s present in appreciable.quantity in the vitamin solution of the PRL-4-C-CM medium, i t was l e f t out to see what effect i t might have on ascorbic acid level. The c e l l populations were transferred twice and once again determinations were performed on Day 12. B. Light Rose suspension cultures contained in six 250 ml. erlenmeyer flasks were grown under identical condi-tions as previously described except that three of the cultures were grown in the light. The light was pro-vided by 3 high intensity fluorescent lamps that produced a total illumination output of 9#40 lumens (at 40% rated l i f e ) . The lamps were suspended 52 cm. above the erlenmeyer flasks. After 12 days of incu-bation, ascorbic acid determinations were made. 23 G. Influence of level of sucrose The level of sucrose in the medium was varied (10 gm. - 20 gm./l.) and, after the cells had grown 12 days in light, visual appraisal of chlorophyll level (42) and determination of ascorbic acid were carried out. 9. Confirmation of precursor studies With the addition of precursors which increased the reducing power of the system, i t seemed prudent to verify that ascorbic acid was indeed the substance causing this increase. To rule out the possibility of microbial contamination, a solution of 0 . 5 gm. L-galactono-tf-lactone dissolved in 2 ml. sterile d i s t i l l e d water was f i l t e r s t e r ilized, added to a culture (approximately 30 gm. c e l l wet weight), suspended in approximately 25 ml. media and incubated under standard conditions for 24 hours. At the end of this time, ascorbic acid was determined by the 2,4?-dinitrophenylhydrazine (DNPH) method ( 3 ) , using thin layer chromatography. Analyses were performed on standard ascorbic acid and L-galactono-/-lactone solutions, a control flask of cells, and the flask to which the possible precursor L-galactono-y-lactone had been added. This method was slightly modified with the substitution of the solvent system chloroform-ethyl acetate (50-50) (5) for separation on the chromatographic plate. Paper chromatography was used to detect the disap-pearance of L-galactono-},-lactone from the culture medium. The standards D-glucose, D-(-)-levulose and L-galactono-ft'-lactone were compared with the compounds obtained from the media at specific time intervals (0, 2, 4, 6 and 24 hours). Separation of the sugars was carried out on Whatman No. 1 paper using a solvent system of ethyl acetate, pyridine and water (10-4-3) (15, 16). The papers were allowed to develop 20 hours and at the end of this time the sugars were detected using the standard ammoniacal silver nitrate spray (43). 10. Determination of total ascorbic acid, dehydroascorbie acid and diketogulonic acid  The method used for the determination of ascorbic, dehydroascorbic and diketogulonic acids was.a modification of the DNPH technique (3). The determination of "t o t a l " ascorbic acid was slightly modified in that samples were blended with 40 ml. 10$ HPO3 and then diluted to 100 ml. with 5% HPO3. Thereafter the procedure was followed exactly. For determining DHA. and diketogulonic acid, samples were ground with 10 ml. 5$ HPO3 and 1.0 gm. 25 stannous chloride and diluted to 200 ml. From this point, the procedure was followed as outlined. The entire determination was performed on two separate occasions using a 4 gm. and a 7 gm. sample respectively. The fate of added ascorbic acid was determined as follows. A 20 gm. fresh weight sample was incubated with 0.25 gm. ascorbic acid for 24 hours and a 4 gm. sample of this was used to assess total ascorbic acid, DHA and diketogulonic acid. The results were compared with a 4 gm. control sample. In the event that too high a concentration of ascorbic acid was used in the f i r s t instance, another 20 gm. sample of cells, was incubated with 0.1 gm. ascorbic acid for 24 hours and.a 7 gm. sample was taken to determine total ascorbic acid, DHA and diketogulonic acid. These results were also compared to a 7 gm. control sample. A l l spectrophotometric readings were made on a Bausch and Lomb Spectronic 20. 26 RESULTS AND DISCUSSION 1. Growth of cultures The callus cultures from the briar rose Rosa rugosa were yellow or white in colour and very compact. Fig. 4 shows a typical example. To maintain callus vigour, the calluses were transferred to fresh media about once a month starting in August, 1970. The suspension cultures were also light in colour and developed quite heavy growth as can be seen in Figure §. Transferring of the suspension culture cells to new media was much more frequent than that required for callus tissue. It was done at least every two weeks. The cultures for experimental work were started in li q u i d medium on June £, 1971* Once established, 10 ml. aliquots of the c e l l culture were transferred regularly to new medium,according tp the following schedule. TRANSFER DATES June £, 1971 Jan. 6, 1972 July 30 Jan. 25 Sept. 10 Feb. 4 Oct. 6 Feb. 20 Oct. l£ March 3 Nov. 2 March 14 Nov. 17 March 24 Dec. 4 Apr i l 3 Dec. 17 A - clump of cells (lOx) B - chain of cells (lOx) Figure 6. Microscopic examination of typical c e l l populations of Rosa rugosa from the suspension culture flasks 30 Microscopic examination of the suspended cells revealed some differences within the culture. The vast majority of the cells showed a tendency to clump together as shown i n Figure 6; however, there was a small pro-portion that formed chains (Fig. 6). In the microscopic fields examined, free l i v i n g single cells were not observed. 2. Growth curve From Fig. 7 and 8, i t can be seen that the cells increase in population until Day 16. After Day 6, growth picks up considerably with a concomitant rise in pH. The hydrogen ion concentration seems to be quite c r i t i c a l for rapid growth of the plant c e l l s . Generally, the pH rises u n t i l Day 16. Mapson (31) found that an alkaline shift in pH increases the efficiency of the conversion of hexose sugars to ascorbic acid. Results from this growth curve also tend to confirm the latter observation as shown by the comparatively large increase in ascorbic acid from Day.6 to Day 8. Thereafter, the ascorbic acid value increases slowly to Day 14 and by Day 16 starts to drop off slightly. This situation may be somewhat analogous to whole plants where i t has been found that ascorbic acid concentration continues to increase right up to anthesis and then drops off. 31 Time (days) pB o ascorbic a c i d • weight A Values are the average of two determinations Figure 7. Results of growth curve showing pH, Afresh weight and ascorbic a c i d content 32 Time (days) pH o a s c o r b i c a c i d El w e i g h t L\ V a l u e s a r e t h e a v e r a g e o f two d e t e r m i n a t i o n s F i g u r e 8, R e s u l t s o f growth c u r v e showing pH, d r y w e i g h t and a s c o r b i c a c i d c o n t e n t 33 Day 12 was chosen as the day to conduct a l l sub-sequent experiments as there was a good c e l l population at this time and the cells were easily and rapidly f i l t e r e d . F i l t r a t i o n was an important consideration as speed i s c r i t i c a l in the determination of ascorbic acid. There was also a good measurable quantity of ascorbic acid present in the cultures to serve as a control at this time. Unless otherwise stated, future experiments were performed in duplicate. 3. Location of ascorbic acid Determination of ascorbic acid in the f i l t r a t e revealed that very l i t t l e free excreted ascorbic acid was present since only 0.03 ml. of indicator dye was required (Table 4). Titration of freshly prepared unincubated media showed the same result, which would indicate that there were no interfering reducing com-pounds. Therefore, ascorbic acid seems to be located intracellularly. 4. Comparison between age and colour of cultures and ascorbic acid content  Examination of the results in Table 3 quickly shows that the colour of the culture i s not as good an indication of the presence of ascorbic acid as i s the age of the 34 Table 1. Comparison of ascorbic acid values of l i v i n g tissue and plant cells Tissue Average ascorbic acid values Fresh weight Dry weight (mg./lOO,,gm) (mg./lOO gm) Rose hips 240 130G Rose stem and leaves 290 1300 Rose plant cells grown in dark 13 320 Rose plant cells grown in li g h t : 1st transfer 2nd transfer 3rd transfer 17 26 35 Table 2. Average ascorbic acid, content, of fruits Fruit Ascorbic acid (mg./lOO gm. fresh weight) Apples 2-10 Grapefruit 40 Lemon 50 Orange 50 Strawberry 60 Rose hips up to 1% of fresh weight From: Mapson, L.W. Vitamins in f r u i t in The Bio-chemistry of Fruits and Their Products, Vol. 1, ed. Hulme, A.C, Academic Press,, London and New York,369-3^4; 1970. 35 Table 3« Comparison between age and colour of cultures and ascorbic acid content Age of culture Ascorbic acid value fresh weight (mg./KM)., gin,) Visual appraisal of colour 1 week 8 almost white 2 weeks 13 almost white 3h weeks 1 beige coloured 5 weeks 1 chocolate brown 12 weeks 0 beige to dark brown 13 weeks 0 very bright yellow Table 4 . Titration values of precursors and precursors dissolved in media Volume of indicator required Solution in 3fo HPO3 1 mg./lO ml. . 2 5 gm. in 25 ml. at beginning media + 25 ml. HPO3 .after 24 hours 3% HPO3 . 0 3 ml. media . 0 3 ml. D-glucose . 0 3 ml. .1 ml. .1 ml. D-galactose . 0 3 ml. .1 ml. .1 ml. D-glucurono-af-lactone . 0 3 ml.1 .1 ml. .1 ml. L-gulono-i'-lactone . 0 3 ml. .1 ml. .1 ml. L-galactono-V- lactone .03 ml. .1 ml. .1 ml. L-ascorbic acid 7 . 4 ml. 1900 ml. (extrapolated) .35 ml. D-(-)-levulose . 0 3 ml. .1 ml. .1 ml. 36 culture. It also illustrates the existence of biological variation in cultures, particularly comparing the two cultures that were both approximately three months old. Generally, ascorbic acid i s found in larger quantities in very light coloured cultures. 5. Comparison with other l i v i n g tissue As can be seen in Table 1, the amount of ascorbic acid found in dark grown cells i s considerably less than that of fresh tissue (13 mg. compared to 240 mg.). Expo-sure of the cells to constant illumination does increase the ascorbic acid considerably over time, almost to the point where i t compares favourably with f r u i t s reputed to be good sources of ascorbic acid (Table 2). 6. Addition of precursors to cultures To insure that the precursors themselves did not con-tribute to the reducing power of the cultures, standard solutions of the precursors were prepared and titrated. As shown in Table 4, the dye was oxidized with the addition of 0.03 ml. so that this possibility was pre-cluded as a possible source of interference. Titration of medium containing the same weights of precursors used experimentally showed that there was no interaction between the precursor and the medium that to 80; -P fctO , u •H CD O CO to h£)<H a •H <DOiD AO « 9 i ° -O-Cr -a Time (hr.) D-glucose 10 40 50 Ho 37' t i o~i_i-tJ— Terror -a -o • 5 gm. .25 gm. d o Time (hr.) D-galactose so -P •H 0 £ CD > Xi o a) CU CD U •H • o a H t6H 5<3 i d -P lolv-O trrr Time (hr.) D-glucurono-Y-lactone 7T 70-50 30; ao / Time (hr.) D-(-)levulose XT Figure 9. Effect of the addition of selected precursors on the reducing power calculated as ascorbic acid on rose c e l l s . 5 Q> 2= O P-i 70 ;80 •H 03 hO<H •H O 0) o IV So 70 to Time (hr.) L-gulono-#-lactone 4 ¥ t— i _ 1.0 gm. .5 ^  gm. .25 gm. Time (hr.) • L-galactono-y-lactone A • O XV-igure 10.. Time (hr.) • • L-ascorbic acid E f f e c t of the addition of selected precursors on .th» reducing power calculated as ascorbic a c i d on rose c e l l s . 39 might influence the volume of dye required (Table 4).. From this same table, i t can be seen that t i t r a t i o n of the medium at the end of the incubation period showed no difference, further confirming the intracellular location of ascorbic acid. Fig. 9 indicates that there i s no appreciable change in the reducing power of rose tissue cells with the addition of D-glucose, D-(-)-levulose, D-galactose and D-glucurono-V-lactone at the 1% and 2% levels. Two explanations are possible. The f i r s t i s that the possible precursor remained in the medium, or, that i t did enter the c e l l but perhaps was diverted to another pathway as could certainly occur with the addition of D-glucose. Also the ascorbic acid could be transient and converted to DHA- or diketogulonic acid before detection was possible. Further c l a r i f i c a t i o n about this point might provide information about the biosynthetic pathway of ascorbic acid in tissue culture. After 24 hours, there was a slight increase in reducing power with L-gulono-^-lactone; but the most spectacular increases occur with administra-tion of L-galactono-fc'-lactone to the cultures (Fig. 10). In another paper an account i s given of the enzymic con-version of L-galactono-V-lactone to L-ascorbic acid by extracts of plant tissues (34). Jackson et a l . (19) also demonstrated this increase by addition of this compound to slices of rose hip tissue. 40 Figures from the literature show that, using excised plant sections, conversion of L-galactono-Y-lactone to ascorbic acid is 80-90% efficient (27). However, the comparatively small increases shown here and lack of inhibition even when one gram of precursor i s added does not at f i r s t sight support this observation. That the L-galactono-^-lactone did disappear from the medium i s shown from the results of the paper chro-matograph (Fig. 12). After 24 hours, the spot for this compound is barely visible. It i s also of interest that the spot corresponding to D-(-)-levulose has disappeared completely. However, as can be seen from Fig. 9, the addition of D-(-)-levulose to the culture does not bring about any change in the t i t r a t i o n value. Therefore, i t would seem that D-(-)-levulose i s not responsible for the increase. To confirm that the increase in ascorbic acid values was not the result of microbial contamination or the presence of some other metabolite in the preparations, the alternate method for determining ascorbic acid with DNPH was carried out. The aseptic techniques described in the methods were followed and the determination was made after 24 hours. Fig. 11 illustrates the results obtained from the chromatographic plate. Comparing the sample and the Control Band for ascorbic acic With L-galactono-Y-lactone added Band for ascorbic acic — Figure 11. Thin layer chromatographs showing the results of the DNPH method of detecting ascorbic acid using the solvent system chloroform-ethyl acetate ( $ 0 - 5 0 ) 42 ft B 1. 2 . 3 . 4 . 5 . 1 . 2 . 3 . 4 . 5 . 1 . 2 . 3 . 4 . D-glucose in water L-galactono-V-lactone in water pure media media and L-galactono-y-lactone (0 hours) n it tt tt it (2 M ) D-glucose in water D-(-)-levulose in water L-galactono-|j/-lactone in water media + L-galactono-^-lactone (4 hours) " tt tt it (6 hours) D-glucose in water D-(-)-levulose in water L-galactono-y-lactone in water media +• L-galactono-i/-lactone (24 hours) Figure 1 2 . Paper chromatographs showing the disappearance of L-galactono-V-lactone over time using the solvent system ethyl acetate, pyridine and water ( 1 0 - 4 - 3 ) 43 control, quite clearly there i s an increase in total ascorbic acid. The width of the band of the sample was 1 cm. compared to h cm. for the control. In addition, the intensity of the coloured derivative was much greater for the sample. The calculated Rf value for this particular solvent system was 0.26 compared to the l i t e r a -ture value of 0.25 (5). To determine i f any of the other coloured bands on the plate were perhaps derivatives of L-galactono-V-lactone, a standard of this compound was also run. Under the conditions of this experiment, no coloured derivative was formed. Addition of pure ascorbic acid to a culture showed some interesting results. As can be seen in Table 4> with the addition of 0.25 gm. to 25 ml. of media, the titration value of the medium dropped from a calculated 1900 ml. to about 0.35 ml. after 24 hours. The cells at this time were a very white colour as they were with addition of L-galactono-V-lactone, but the increase in ascorbic acid value of the cells certainly did not parallel the disappearance of ascorbic acid from the medium (Fig. 10). Since the tit r a t i o n method measures only reduced ascorbic acid, the possibility was raised that there may be a marked increase in the amount of ascorbic acid present as the oxidized forms DHA and diketogulonic acid. 44 Table 5. Comparison of dry weights of dark and light grown cells Treatment Fresh weight (gm.) Dry weight (gm.) Light Light 30.9 29.4 .85 .8 Dark Dark 27.8 42.5 .8 1.0 Table 6. Effect of altering the environment on f i n a l weight of tissue and ascorbic acid content (12th day) Treatment Ascorbic acid values fresh weight mg./lOO gm. Tissue from flask fresh weight gm. Control 9-13 30-40 - Myoinositol 1st transfer i i 9 13^1 16.7 2nd transfer 10 12 10.5 10.7 +Light 1st transfer 16 18 39.2 40.4 2nd transfer 26 27 25.0 25.4 3rd transfer 35 41 15.4 17.6 - i sucrose 7 23.0 - £ sucrose 5 19.0 45 Table 7 shows the spectrophotometric results of the determination of "total " ascorbic acid and dehydro-ascorbic and diketogulonic acids. In the case of normal cells the majority i s present as the reduced form (the difference between total ascorbic acid and that con-tributed by DHA and diketogulonic acid). However, with the addition of large amounts of ascorbic acid, DHA and diketogulonic acid rise dramatically. This explains why the t i t r a t i o n value does not increase as greatly as might be expected. Addition of some blocking agent into the system to prevent this conversion could result in greater accumulation of ascorbic acid, thereby increasing yield. 7. Omission of myoinositol from the medium Omission of this compound from the vitamin solution had a profound effect on the growth of the cultures. On three separate occasions growth was much less dense than the control c e l l s . There was much more clumping of cells also. By the second transfer, the cells had become slightly darker in colour than the controls. From Table 6, i t can be seen that ascorbic acid values obtained on the 12th day of incubation show very l i t t l e change as compared to the controls even though there was a dramatic decrease in c e l l yield. It seems 46 then that myoinositol has l i t t l e influence on ascorbic acid production as also noted by Loewus (26) who ruled out this compound as an important precursor of ascorbic acid. 8. Cells grown under controlled illumination Growing rose plant cells under constant illumination brought about physical changes in the cells themselves. After the f i r s t 12 days, the cells had a rather bleached appearance compared to the controls. However., as suggested by Street (45), there was no green, pigmentation whatsoever. The cells had a tendency to clump together much more than those cells grown in the dark. By the second and third transfers the,light grown flasks showed small bright yellow balls or calluses which were quite separate and distinct compared to the more uniformly suspended dark grown controls. After f i l t e r i n g , the light grown cells seemed to adsorb more liquid; however, this was an optical i l l u s i o n as can be seen by the comparisons of dry and fresh weights of samples of both conditions of growth as shown in Table 5. In the case of light grown flasks, with each suc-cessive transfer, the total flask fresh weight decreased as shown in Table 6. A possible solution to. this problem might be more frequent transferring as c e l l processes 47 Table 7. Spectrophotometric readings for "total " ascorbic acid and dehydroascorbic + diketogulonic acids Sample Absorbance "Total" DHA + ascorbic acid diketogulonic acid 4 gm. control 0.06 0.03 4 gm. control + .25 gm. ascorbic acid 2.0 2.0 7 gm. control 0.16 0.02 7 gm. control •+.1 gm. ascorbic acid 1.4 Table 8. Summary of results obtained from attempts to vary the ascorbic acid content of rose tissue culture Treatment Ascorbic Acid Value Total c e l l pop. Control remains the same same D-glucose it it tt tt •+ D-galaetose tt tt tt tt 4. D-glucurono-tf-lactone tt tt tt it + L-gulono-V-lactone : s l i g h t increase tt •+ L-galactono-V- lactone increase tt - Myoinositol remains the same dec. t Light - 1st transfer 2nd transfer 3rd transfer slight increase increase increase same dec. dec. - g Sucrose decrease same * D-(-)-levulose remains the same same 4* probably proceed at a much greater rate in light than in darkness. Hence nutrients would be depleted much more quickly. Also development of the optimum medium for light grown cultures would help here. In contrast, with each successive transfer of cells grown under light, the ascorbic acid concentration increased. Various workers such as Mapson (32) and King (22) have also found that ascorbic acid synthesis depends on photosynthetic activity. Kefford and Chandler (20) have noted that, in the northern hemisphere, the highest ascorbic acid values were found on the southern exposure of the tree where the frui t s received the most sunlight. It can also be seen that the presence of chlorophyll is not an absolutely necessary factor for increasing ascorbic acid synthesis, as i t s concentration did rise in tissue culture with no visible pigmentation whatever. It may well be, however, that, i f pigmentation were to occur, the ascorbic acid content might increase much more dramatically. 9 . Effect of sucrose Table 6 shows that a decrease in the amount of sucrose did not appreciably alter the c e l l population compared to light grown second transfer cells which were started at the same time. Further transfers would 49 probably affect this. The physical appearance of the cells was similar to the light grown c e l l s . There was, however, quite a difference in the ascorbic acid values, 7 mg. and 5 mg. compared to 26 mg. and 27 mg. for illuminated flasks determined at the same time and 9 mg. for the dark control. Mapson (31) also found that ascorbic acid synthesis depends on an adequate supply of hexose.sugars and that conditions which promote the synthesis of sucrose appear to favour the synthesis of ascorbic acid. Chinoy, Patel and Suthar (8) found that the biosynthesis of ascorbic acid i s enhanced con-siderably by sucrose. 50 SUMMARY Table 8 gives a summary of the effectiveness of the various methods of altering the ascorbic acid content of the tissue culture of Rosa rugosa. Of the test compounds administered, only L-galactono-ct'-lac tone appreciably increased the ascorbic acid value, confirmed by alternate methods of analysis. Omitting myoinositol from the vitamin preparation decreased c e l l yield but had practically no effect on the ascorbic acid value. Addition of light to the cultures had a pronounced effect on the ascorbic acid value. It increased with each successive transfer while the total c e l l yield f e l l appreciably with continued propagation. More frequent transferring may help overcome this problem. Decreasing the amount of sucrose in the medium also affected the ascorbic acid value as i t f e l l dramatically. A good supply of sugar seems a necessary condition for ascorbic acid production. 51 LITERATURE CITED 1. Aberg, B. Vitamins as growth factors in higher plants. in Encyclopedia of Plant Physiology, Vol. 14, ed. Ruhland, W., Springer, Berlin, 418, 1961. 2. Asselbergs, E.A.M. Studies on the formation of ascorbic acid i n detached apple leaves. Plant Physiol. 32, 326-329, 1957. 3. Association of Vitamin Chemists. Methods of Vitamin Assay. Interscience Publishers, New York. 1966. 4. Bessey, O.A. A method for the determination of small quantities of ascorbic acid and dehydroascorbie acid in turbid and coloured solutions in the presence of other reducing substances. J. Biol. Chem. 126, 771-784, 1938. 5. Bolliger, H.R. Vitamins, in Thin Layer Chromatography, ed. Stahl, E., Academic Press Inc., New York and London, 246, 1965. 6. Campbell, G., Chan, E.C.S. and Barker, W.G. Growth of lettuce and cauliflower tissue in vitro and their pro-duction of antimicrobial metabolites. Can. J. Microbiol. 11, 785-739, 1965. 7. Ghayen, J . Ascorbic acid and i t s intracellular location, with special reference to plants. Int. Rev. Cytol. 2, 77-131, 1953. 8. Chinoy, J.J., Shah Hemlata T. Patel, CK. and. Suthar, H.K. Role of auxin and gibberellin in the synthesis of ascorbic acid and growth of tissue explants. Biologica Plantarum 9, 182-194, 1967. 9. Doby, G. Plant Biochemistry. Interscience Publishers, London and Budapest. 1965. 10. Freebairn, H.T. Determination and stabilization of reduced ascorbic acid in extracts from plant material. Anal. Chem. 31, 1850-1851, 1959. 11. Furuya, T., Hirotani, M., and Kawaguchi, K. Biotrans-formation of progesterone and pregnenolone by plant suspension cultures. Phytochem. 10(5) 1013-1017, 1971. 52 12. Gamborg, O.L., and Eveleigh, D.E. Culture methods and detection of glucanases in suspension cultures of wheat and barley. Can. J. Biochem. 46, 417-421, 1968. 13. Giroud, A., Ratsimamanga, A.R. and Leblond, C-P. Rela-tionship between ascorbic acid and chlorophyll. Bull. Soc. Chim. bi o l . Paris, 17. 232-251, 1935. 14. Glick, D. Methods of Biochemical Analysis, Vol. 1. Interscience Publishers Inc., New York, 1954. 15. Hickman, J. and Ashwell, G. Isolation of a bacterial lipopolysaccharide from Xanthomonas campestris contain-ing 3-acetamido-3,6-dideoxy-D-galactose and D-rhamnose. J. Biol. Chem. 241, 1424-1428, 1966. 16. Hough, L. and Jones, J.K.N. Chromatography on paper. in Methods in Carbohydrate Chemistry, Vol. 1, ed. Whistler, R.L. and Wolfrom, M.L., Academic Press, New York, 21-31, 1962. 17. Hughes, R.E, The use of homocysteine in the estimation of dehydroascorbic acid. Biochem. J. 64, 203-208, 1956. 18. Isler, 0. Developments in the f i e l d of vitamins. Experientia 26, 225-240, 1970. :,„; 19. Jackson, G.A.D., Wood, R.B. and Prosser, M.V. Conversion of L-galactono-j-lactone into L-ascorbic acid by plants. Nature 191, 282-283. 1961. 20. Kefford, J.F. ahd Chandler, B.V.... The Chemical Constitu-ents of Citrus Fruits. Academic Press, New York and London, 1970. 21. King, C.G.. Chemical methods for, the determination of vitamin C. Ind. Eng. Chem. Anal. Ed. 13, 225-227, 1941. 22. King, C.G. .Vitamin C, ascorbic, acid. 'Physiol. Rev. 16, 238-262,,1936. 23. Klein, R.M. Plant tissue cultures, a possible source of plant constituents. Econ. Bot. 14, 286-289, I960. 24. Lamport, D.T.A. Cell suspension cultures of higher plants: isolation and growth energetics. Expt. Cell  Res. 1964, 195-206, 1964. 53 25. Loewus, F. Inositol metabolism and c e l l wall formation in plants. Fed. Amer. Soc. Exp. Bio., Federation Pro- ceedings 24, "1^5-3527 1 9 o T . 26. Loewus, F.A. Tracer studies on ascorbic acid formation in plants. Phytochem. 2, 109-128, 1963. 27. Loewus, F. and Baig, M.M. Biosynthesis and degradation of isotopically labelled ascorbic acid, in Methods in Enzymology XVIII Part A. ed. Colowick, S.P. and Kaplan, N.O. Academic Press, London and New York, 22-28, 1970. 28. Mapson, L.W. A note on the estimation of dehydro-L-ascorbic acid in plant tissues by the Roe and Kuether procedure. Biochem. J . 80, 459-461, 1961. 29. Mapson, L.W. Function of ascorbic acid in plants, in Vitamins and Hormones. Advances in Research and Appli-cations, Vol. XI, ed. Harris, R.S., Marrian, G.F. and Thimann, K.V., Academic Press, London and New York, 1-28, 1953. 30. Mapson, L.W. Metabolism of ascorbic acid in plants. Part 1. Function. Ann. Rev. Plant Physiol. 9, 119-150, 1958. 31. Mapson, L.W. The biosynthesis of ascorbic acid, in Vitamins and Hormones. Advances in Research and Appli-cations, Vol. XIII, ed. Harris, R.S., Marrian, G.F. and Thimann, K.V., Academic Press, London and New York, 71-97, 1955. 32. Mapson, L.W. Vitamins in f r u i t , in The Biochemistry of Fruits and Their Products, Vol. 1, ed. Hulme, A.C., Academic Press, London and New York, 369-384, 1970. 33. Mapson, L.W. and Ingram, M. Observations on the use of Escherichia c o l i for the reduction and estimation of dehydroascorbic acid. Biochem. J . 48, 551-559, 1951. 34. Mapson, L.W., Isherwood, F.A. and Chen, Y.T. Biological synthesis of L-ascorbic acid: the conversion of L-galactono-V-lactone into L-ascorbic acid by plant mito-chondria. Biochem. J. 56, 21-28, 1954. 35. M i l l s , M.B., Damron, C.M. and Roe, J.H. Ascorbic acid, dehydroascorbic acid and diketogulonic acid in fresh and processed foods. Anal. Chem. 21, 707-709, 1949. 54 36. N e i l l , J. Personal communication. 1972. 37. Ponting, J.D. Extraction of ascorbic acid from plant materials. Relative s u i t a b i l i t y of various acids. Ind. Eng. Chem. Anal. Ed. 15, 339, 1943. 38. Puhan, Z. and Martin, S.N. The industrial potential of plant c e l l culture, in Progress in Industrial Micro-biology, Vol. 9, ed. Hockenhull, D.J.D., Gordon and Breach, London, 14-39, 1971. 39. Rinaldini, L.M. The effect of Vitamin C on cells and tissues in culture, in Methods, Biology and Physiology, Vol. 1, ed. Willmer, E.N., Academic Press, London and New York, 680-699, 1965. 40. Roe, J.H. Appraisal of methods for the determination of L-ascorbic acid. Ann. NjY. Acad. Sci. 92, 277-283, 1961. 41. Roe, J.H. and Oesterling, M.J. The determination of dehydroascorbie acid and ascorbic acid in plant tissues by the 2,4-dinitrophenylhydrazine method. J. Biol. Chem. 152, 511-517, 1944. 42. Sestak, Z., Catsky, J. and Jarvis, P.G. Plant Photo-synthetic production. Manual of Methods. Dr. W. Junk N.V. Publishers, The Hague, 1971. 43« Sherma, J. and Zweig, G. Paper Chromatography and Electrophoresis, Vol. 2. Paper Chromatography. Academic Press, New York and London, 1971. 44. Street, H.E. Knowledge gained by culture of organs and tissue explants. in Plant Physiology, Vol. VB, ed. Steward, F.C., Academic Press, New York, 113-181, 1969. 45. Street, H.E. The nutrition and metabolism of plant tissue and organ cultures, in Cells and Tissues in Culture: Methods, Biology and Physiology, Vol. 3, ed. Willmer, E.N., Academic Press, London and New York, 602, 1966. 46. Street, H.E., Henshaw, G.G. and Buiatti, M.C. The culture of isolated plant c e l l s . Chem. and Ind. 1965, 27-33, 1965. 47. Szent-Gydrgi, A. CLXXIII. Observations on the function of peroxidase systems and the chemistry of the adrenal cortex. Description of a new carbohydrate derivative. Biochem. J. 22, 1337-1409, 1928. 55 48. Tewari, CP. and Krishman, P.S. Loss of ascorbic acid during estimation by the Roe-Kuether method. J. Food  Sci. 26, 11-14, 1961. 49. Vitamin C Merck Service Bulletin, Merck and Co. Inc., N.J., 1956. 50. Weinstein, L.H., Niekell, L.G., Laurencot, Jr., H.J. and Tulecke, W. Biochemical and physiological studies of tissue cultures and the plant parts from which they are derived. I. Agava toumeyana- T-r-el. • Contrib. Boyce  Thompson Inst. 20, 239-250, 1959. 

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