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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  i n the Department of Food Science  We accept t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September, 1972  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree the  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 reference  and  study.  I f u r t h e r agree 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 copying of t h i s for  s c h o l a r l y purposes may  by h i s r e p r e s e n t a t i v e s . .  be  granted by  thesis for financial  written  permission.  gain  s h a l l not be  PQ-Q-Q^ S  CAJL/VVC5L  The U n i v e r s i t y of B r i t i s h Vancouver 8 , Canada  Columbia  thesis  Department or  I t i s understood t h a t copying or  of t h i s  Department of  the Head of my  that  publication  allowed without  my  ii  ABSTRACT The fleshy hips of the b r i a r rose, Rosa rugosa were cultured on a modified medium developed by Gamborg (1963) to produce callus and suspension cultures. very l i g h t , almost white.  These c e l l s were  Reduced ascorbic acid, determined  by the 2,6-dichlorophenolindophenol t i t r a t i o n method, was found i n the suspension cultures. This finding prompted an investigation into culture techniques f o r obtaining an optimum concentration of ascorbic acid.  During the growth of the c e l l s , the pH rose and ascorbic  acid concentration increased a f t e r the sixth day.  Agecof  culture was also an important factor as., cultures older than three: or four weeks contained v i r t u a l l y no ascorbic acid. Possible precursors of ascorbic a c i d i n plants were added to cultures and t h e i r effect on the ascorbic a c i d l e v e l 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 s l i g h t  increase and a comparatively large increase was obtained with L-galactono-Y-lactone.  Confirmation f o r the l a t t e r was  obtained using the 2,4-dinitrophenylhydrazine method to rule out the p o s s i b i l i t y o r microbial contamination and to be sure  that  another metabolite  reducing  capacity  of  was  the  not  medium d e c r e a s e d  the  level  of  the  acid  of  medium by  from the  cell  acid.  one-half  concentration,  sugar  total  ascorbic  f o r the  increased  system.  Omission of myoinositol the  responsible  yield  vitamin  solution  but  no  Decreasing  caused  a  had  the  effect  sucrose  sharp decrease  indicating that  an  adequate  i s a p r e r e q u i s i t e f o r optimum p r o d u c t i o n  of  content  in  ascorbic  supply of  on  of  ascorbic  acid. Growing resulted  liquid  suspensions  i n increased  ascorbic  transfer;  however, t o t a l  addition,  there  the  no  acid values  with  y i e l d s decreased  evidence  of  each  successive  each time.  chlorophyll production  In in  cultures. This  in  was  cell  under c o n t r o l l e d i l l u m i n a t i o n  the  This  work  t i s s u e culture of  culture for  exploratory  may  ascorbic  be  useful  Rosa rugosa.  coupled  environment of  Rosa r u g o s a t o  which  with the  the  cells  produce a s c o r b i c  make i t s r e c o v e r y  ascorbic  industrially  i s as  complete yet  e f f e c t s of may  acid  acid  Therefore,  f o r e l u c i d a t i n g the  acid production  information  physical  shows t h a t  s t i l l  is  present  tissue pathway unclear.  altering  direct cultures  the of  i n quantities that  attractive.  would  iv  TABLE OF CONTENTS page INTRODUCTION  1  LITERATURE REVIEW  4  MATERIALS AND METHODS1.  Plant o r i g i n , c u l t u r a l conditions and preparat i o n of a single c e l l suspension  14  2.  Measurement of c e l l growth  16  3.  pH measurement  17  4.  Ascorbic a c i d measurement  17  5.  Comparison with l i v i n g tissue  20  6.  Location of ascorbic a c i d  20  7.  Comparison between age and colour of cultures and ascorbic acid content  21  3.  9. 10.  Possible methods of varying ascorbic a c i d content of cultures  21  Confirmation  of precursor studies  23  Determination of t o t a l ascorbic acid, dehydroascorbic a c i d and diketogulonic a c i d  24  RESULTS AND DISCUSSION 1.  Growth of cultures  26  2.  Growth curve  30  3.  Location of ascorbic a c i d  33  V  RESULTS AND DISCUSSION (cont.) 4.  page  Comparison between age and colour of cultures and ascorbic a c i d 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  45  £.  C e l l s grown under controlled illumination  46  9.  E f f e c t of sucrose  4#  SUMMARY  50  LITERATURE CITED  51  vi  LIST OF FIGURES page 1.  Possible pathways of ascorbic acid synthesis i n plants  5  2.  Mechanisms of determining t o t a l and reduced ascorbic acid  9  3.  Composition of PRL-4 medium  15  4.  Typical c a l l u s culture of Rosa rugosa cultured on PRL-4-C-CM medium  27  5.  Typical f l a s k of c e l l s of Rosa rugosa cultured on PRL-4-C-CM medium  23  6.  Microscopic examination of t y p i c a l c e l l populations of Rosa rugosa from the suspension culture flasks  29  Results of growth curve showing pH, fresh weight and ascorbic acid content  31  7. 8. 9.  10.  Results of growth curve showing pH,  dry weight  and ascorbic acid content  32  E f f e c t of the addition of selected precursors on the reducing power calculated as ascorbic acid on rose c e l l s  37  E f f e c t of the addition of selected precursors on the reducing power calculated as ascorbic acid on rose c e l l s  38  11.  Thin layer chromatographs showing the r e s u l t s of  12.  the DNPH method of detecting ascorbic a c i d Paper chromatographs showing the disappearance of L-galactono-J*-lactone over time  41 42  vii  LIST OF TABLES page 1.  Comparison of ascorbic acid values of l i v i n g tissue and plant c e l l s  34  2. 3.  Average ascorbic acid content of f r u i t s Comparison between age and colour of cultures and ascorbic acid content  34  4.  T i t r a t i o n values of precursors and precursors dissolved i n media  35  5.  Comparison of dry weights of dark and l i g h t grown cells .  44  6.  Effect of a l t e r i n g the environment on f i n a l weight of tissue and ascorbic a c i d content (12th day) ...  44  7. 3.  35  Spectrophotometric readings f o r " t o t a l " ascorbic a c i d and dehydroascorbic + diketogulonic acids ...  47  Summary of r e s u l t s obtained from attempts to vary the ascorbic a c i d content of rose tissue culture  47  INTRODUCTION The propagation of whole plants from i s o l a t e d fragments has been carried out for centuries.  The majority of these  techniques were concerned with organ propagation. t i v e l y new  technique f o r the production  c a l l e d plant tissue culture.  A rela-  of c e l l s per se i s  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 s t e r i l e conditions i n a vessel on an a r t i f i c i a l medium.  The r e s u l t i n g c e l l s were  capable of d i v i s i o n with a r e s u l t i n g increase i n the plasma mass (3$).  The growth of the tissue was  cell  thought to  take place as a consequence following wounding of a plant organ.  L i v i n g parenchymatous c e l l s adjacent to the wound  frequently became meristematic forming masses of "undifferentiated" c e l l s (44).  Such a p r o l i f e r a t i n g tissue  was  called a callus. Culture of c e l l s i s a combination of an a r t and a science.  After reaching a c r i t i c a l s i z e , the c a l l u s may  transferred to a l i q u i d medium where i t w i l l grow as a suspension culture.  Growth i n agitated l i q u i d media i s  generally faster than that of s t a t i c c a l l u s cultures,  be  2 presumably because the nutrients are more e f f e c t i v e l y absorbed and toxic exudates are dispersed by virtue of the greater contact area and development of convection (45).  currents  To obtain a c t i v e l y growing suspension cultures,  constant a g i t a t i o n either by shaking culture f l a s k s , forced aeration or magnetic s t i r r i n g i s practised (24). Production  of c e l l s can be stepped up to an i n d u s t r i a l  scale as are microbial fermentations with r e l a t i v e ease. One major difference i s that maximum plant c e l l y i e l d requires a longer growth period, one to ten weeks, as compared to bacteria which generally a t t a i n maximum y i e l d under i d e a l conditions within one to two days.  The p a r t i c u l a r p o s s i b i l i t y  of large scale plant c e l l production has caused speculation as to the p o s s i b i l i t y of using plant c e l l s as an a l t e r n a t i v e i n helping to a l l e v i a t e the world protein shortage.  The  economic f e a s i b i l i t y of such an operation would be more a t t r a c t i v e i f other metabolites  could also be recovered.  For example, there has been i n t e r e s t i n using plant c e l l s f o r secondary product biosynthesis, biogenesis or biotransformation.  S p e c i f i c examples s t i l l at the research  stage are the production of a n t i b i o t i c s i n lettuce and cauliflower tissues (6) and the biotransformation  of pro-  gesterone and pregnenolone by plant suspension cultures (11). To t h i s point, however, the p r a c t i c a l use of tissue cultures i n industry i s highly speculative (23).  3 P o t e n t i a l l y , the techniques of plant tissue culture are well suited for studying environmental and n u t r i t i v e control and chemical pathways involved i n the synthesis of (45).  p a r t i c u l a r metabolites  I t i s a well established f a c t ,  however, that the metabolic pathways present i n the parent plant are not necessarily operative i n the tissue culture of that plant (46, Therefore,  50).  i n selecting a p a r t i c u l a r metabolite  for  study, i t would seem reasonable to choose one that i s present i n appreciable quantity i n the parent to increase the probab i l i t y of detection i n culture. Rose hips have long been known as an excellent source of ascorbic a c i d .  In t h i s investigation these organs have  been cultured quite s a t i s f a c t o r i l y to form both calluses and suspension cultures.  A very l i g h t coloured, almost white,  c e l l suspension (which rapidly darkens as the c e l l s  age)  results. The present study was  undertaken to explore the possible  metabolic pathway f o r ascorbic acid i n rose tissue culture and to investigate methods that might increase i t s production.  4  LITERATURE REVIEW Vitamin C has the d i s t i n c t i o n of being the f i r s t n u t r i t i o n a l adjunct whose deficiency i n the human diet was recognized as a cause of disease.  Probably as early as 1700,  i t was observed that a lack of fresh f r u i t s and vegetables resulted i n scurvy and that t h i s disease could be prevented and  cured by the proper d i e t .  Gradual recognition  of the  "antiscorbutic p r i n c i p l e " followed. In 1928, Szent-Gyorgyi (47) i s o l a t e d from cabbage and l a t e r from paprika a substance which he c a l l e d hexuronic acid.  I t was l a t e r shown to be i d e n t i c a l to the antiscorbutic  factor.  The proof that ascorbic a c i d was indeed the a n t i -  scorbutic factor was provided by Reichstein,  Griissner and  Oppenauer (7) who synthesized L-ascorbic acid and showed that t h e i r product was p h y s i o l o g i c a l l y a c t i v e . 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 a c i d i s an e s s e n t i a l factor i n the normal growth, development and n u t r i t i o n of the human (49).  Owing  to i t s vitamin a c t i v i t y and since i t i s r e a d i l y oxidized and reduced, i t i s thought to play a r o l e i n a large number of metabolic processes.  I t i s a prerequisite f o r the formation  5  0-C— I I H-C-OH H-C-OH C-H 0 0 I 0 I Cr HO-C-H — C H i HO-C-?H -7 180° I I H-CH-C-OH H-C-OH 0 I 0 I I H-C HO-C-H H-C ! I -C=0 HO' H CH 0H D-glucurono-ylactone D-glucose 2  H  /OH C-  H-C-OH  H-C-OH  I  I  HO-C-H  HO-C-H —>  '  N  HO-C-H I H-C-OH I CH OH 2  D-galactose  Figure 1.  I  HO-C-H  1  H-C  COOR  COOR  I  0  G* o  180°  I HO-C-H  I  HO-C-H  HO 0  I  H-C— I HO-C-H  I  CH OH 2  L-gulonoy-lactone  o=c  HO-C-H — Cy HO H  D-galacturonic acid methyl ester  Hc  .0-0  I H-C— HO-C-H  1  I  CH 0H 2  L-ascbrbic acid  0-C-  I  I  C-H HO-C-H I I o H-C-OH H-C-OH I — \ I H-C-OH ' H-C —  I  :n  o=c  0=C  I  HO-C-H CH 0H 2  L-galactonoy-lactone  HO-C  II  0  HO-C H-C-  I  HO-C-H  I  CH 0H 2  L-ascorbic acid  Possible pathways of ascorbic acid synthesis i n plants. From Loewus, F.A. Tracer studies on ascorbic acid formation i n plants. Phytochem. 2, 109-123, 1963.  6  of collagen, i n t e r c e l l u l a r cement, dentine,  cartilage, callus,  osteoid tissue of bones, blood vessel walls and tissue ( 9 ) .  It i s also indispensable  connective  i n the healing of  wounds and the uniting of fractures. Ascorbic a c i d must be supplied to the body as man l o s t the a b i l i t y to synthesize  i t himself.  has  I t i s produced  b i o l o g i c a l l y to a greater or l e s s e r degree i n a l l plants. Ascorbic a c i d may i s ingested.  then be passed to man  when plant material  The greatest concentrations  of t h i s compound  are found i n areas d i r e c t l y concerned with plant  cell  growth ( 2 9 ) . The complete biosynthetic pathway of ascorbic a c i d i n plants i s s t i l l not clear ( 3 0 ) . i t can be represented  From the evidence to date,  by one or both of the following  sequences ( 3 4 , 2 9 , 26) as also i l l u s t r a t e d i n F i g . 1. (1) D-glucose-> D-glucurono-y-lactone —^ L-gulono-y-lactone —^ L-ascorbic a c i d (2) D - g a l a c t o s e D - g a l a c t u r o n i c a c i d methyl e s t e r — } L-galactono-y-lactone r-^L-ascorbic a c i d In general, the evidence f o r the formation from hexose sugars i s suggestive  of ascorbic a c i d  rather than conclusive.  Sugars are interconvertible and once incorporated into the metabolic cycle, give r i s e to a variety of compounds, among which substances acting as precursors might be found  (31).  The ascorbic acid of plants i s formed continually i n the green organs of the plant.,. A number of workers have  7 established a close r e l a t i o n s h i p between photosynthesis ascorbic acid formation  (8).  and  Numerous attempts have been  made to i d e n t i f y chloroplasts as the s i t e and chlorophyll as the necessary agent f o r synthesis of the Asselbergs  vitamin.  (2), f o r example, found a c o r r e l a t i o n between  the ascorbic acid content of apple leaves and t h e i r surface area.  Sur, Roy and Guha (7) investigated synthesis of the  vitamin by germinating seeds of Phaseolus radiatus found that i n sunlight about 60%  and  more ascorbic a c i d was  produced than by growth i n the dark.  Giroud, Ratsimamanga  and Leblond (13), by extraction methods, found a d i r e c t r e l a t i o n s h i p between the concentration of ascorbic a c i d and the presence of chlorophyll. Plant c e l l s should be a good means to study t h i s phenomenon as they are generally cultured i n darkness so that l i t t l e pigmentation i s observed.  Therefore, a comparison  between l i g h t and dark grown cultures should provide some information in. this-regard..  Certain problems may a r i s e ,  however, i n that when exposed to l i g h t , many cultures become only pale green or may  have no pigmentation at a l l  (45).  Ai supposed p a r a l l e l i s m between growth rate and ascorbic a c i d content of higher plants has l e d to the suggestion  that  i t might function as a growth f a c t o r or even as an i n d i s pensable phytohormone, e s p e c i a l l y 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 i n the mind of many b i o l o g i s t s although the action of some vitamins has l i t t l e to do with c e l l enlargement or c e l l d i v i s i o n (39).  Apart from the i n d i r e c t evidence  afforded by high concentrations of ascorbic acid i n rapidly growing tissues and organs of embryos, young plants, animals and man u t i l i z e more ascorbic a c i d than old animals.  There  i s l i t t l e evidence either i n vivo or i n culture f o r a direct e f f e c t of ascorbic acid on any b i o l o g i c a l phenomena one associates with the complex process of growth.  Also, evidence  i n favour of a relationship between ascorbic a c i d and growth must be weighed against the fact that some non-growing tissues i n plants, such as c i t r u s f r u i t s and rose hips, have a r e l a t i v e l y high ascorbic a c i d content (39). Actual quantitative determination of ascorbic acid i n plant extracts i s a problem i n that absolute certainty of r e s u l t s 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 e n t i t i e s that possess Vitamin C a c t i v i t y but exclude materials devoid of Vitamin C a c t i v i t y . The s i t u a t i o n regarding chemical analyses remains dynamic. The complex b i o l o g i c a l relationship between the compounds possessing Vitamin C a c t i v i t y , as well as the chemical  9  Dye  (pink)  L-ascorbic acid  , Dye (colourless)  (A) Reduced ascorbic acid by  0-G  o-=c-  I  HO-C  -2H  i)!i  o f2H  HO-C J  H-C HO-C-H  I  CH OH 2  L-ascorbic acid  I  o=c  0  0=C I H-C — 1  HO-C-H I  CH20H Dehydroascorbic acid  H20  Dehydroascorbic acid  2,6-dichlorophenolindophenol  O-C-OH I 0=C I Q-C  O-G-OH I .DNPH RHNN-C > I ' RHNN^C I I H-C-OH H-G-OH I I HO-C-H HO-C-H I i CH OH GH OH 2  Diketo-Lgulonic acid  2  bis-2,4-dinitrophenylnydrazone  (B) Total ascorbic acid by 2,4-dinitrophenylhydrazine  Figure 2 .  Mechanisms of determining t o t a l and reduced ascorbic a c i d .  10 s i m i l a r i t y of these compounds to others which are i n a c t i v e , has made the existence of a single, simple and s p e c i f i c method as yet impossible. Chemical analyses can be divided into two groups: 1) determination of reduced form; 2) determination of " t o t a l " 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 a n i l i n e derivatives to form coloured hydrazides, while the l a t t e r i s based on oxidation to diketogulonic acid and subsequent formation of a highly coloured hydrazone (3).  The mechanisms are shown  i n F i g . 2. A l l of these methods suffer from lack of s p e c i f i c i t y to one extent or another.  For t h i s work, the oxidation-reduction  method using 2,6-dichlorophenolindophenol as indicator was chosen as oxidation methods measure substances other than reduced ascorbic acid, i n p a r t i c u l a r dehydroascorbic acid  (DHA)  and diketogulonic a c i d ( F i g . 2). The r e l a t i v e l y l a b i l e nature of DHA (35)  suggests that once ascorbic acid i n a food has  been oxidized to t h i s compound, the value of the product as a source of Vitamin C has been impaired (3).  Also DHA i s  usually found i n plant tissues In low concentrations and i s always associated with ascorbic acid i n much higher concentrations (23). Studies indicate that the indophenol,method i s r e l i a b l e f o r the estimation of ascorbic acid i n most plant tissues (41)*  11 The v i s u a l t i t r a t i o n method i s based upon the reduction of the dye by an a c i d solution of ascorbic acid.  In the  absence of i n t e r f e r i n g 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 d i r e c t l y proportional to the ascorbic a c i d content.  A serious error might be expected i n  the analysis of preparations  that contain iron or copper as  these two metals are e f f i c i e n t catalysts i n hastening oxidation of the ascorbic a c i d (14).  the  The most e f f e c t i v e  a c i d i n preventing t h i s l a t t e r oxidation i s metaphosphoric (3).  It has been found that the s t a b i l i t y of ascorbic a c i d  solutions i s not merely a function of pH but depends also on the nature of the a c i d (37).  In addition to acting to  prevent ascorbic a c i d oxidation, metaphosphoric a c i d i s also a protein precipitant and thereby aids i n 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-  a c e t i c a c i d (EDTA) added to the metaphosphoric acid-plant extract solution had further ascorbic a c i d s t a b i l i z i n g ability  (10).  T i t r a t i o n s must be carried out as rapidly as possible to minimize interference from other reducing substances such as cysteine, glutathione, sodium s u l f i d e and reductones i f they are  present. To obtain information about t o t a l ascorbic acid as a  12 comparison, another approach i s required.  Methods of reducing  DHA to ascorbic a c i d so that i t may be determined by t i t 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 i r r e v e r s i b l y destroyed by H2S treatment.  King (21) also found that many  aldehydes, ketones and quinones give r i s e to i n t e r f e r i n g 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 i n part due to reduction of n i t r a t e to n i t r i t e .  On a c i d i f i c a t i o n ,  the nitrous acid formed r a p i d l y oxidizes the ascorbic a c i d . For these reasons, to estimate " t o t a l " ascorbic a c i d , the 2,4-dinitrophenylhydrazine  (DNPH) was employed (3).  The  t o t a l ascorbic a c i d 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 p r e f e r e n t i a l l y protect the reduced ascorbic acid present. Currently, ascorbic, a c i d 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 c e l l s o f f e r p o t e n t i a l i n t h i s  respect. Methods of increasing the quantity of ascorbic a c i d formed i n rose c e l l s were investigated.  Many of the pre-  cursors found e f f e c t i v e f o r 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 c e l l s under controlled illumination and by decreasing the myoinositol and sucrose concentrations i n 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 o r i g i n , c u l t u r a l conditions and preparation of a single c e l l suspension A.  Origin of the plant The plant used throughout the studies was a b r i a r rose, Rosa rugosa (36).  Samples were obtained  from a plant a c t i v e l y 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 i s l i s t e d i n F i g . 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 coconuts purchased at a l o c a l market.  The milk was  drained, f i l t e r e d through Whatman No. 1 paper and stored frozen i n p l a s t i c b o t t l e s . The complete media was s t e r i l i z e d at 15 p s i f o r 20 minutes before use. Difco agar was added.  For s o l i d media, 1$ Bacto-  15  Ingredient  mg./l. 90 30 300 200 250 1000 150 .75 28 (5 ml.) 1.0 ml. 10.0 ml. 20 gm. 2.0 mg. 2.0 mg. 6.2  NaH2P0/..H20 Na2HPOi. KC1 {NH ) S04 MgS0WH20 KNO3 CaCl2'2H20 KI ^ Iron* Micronutrients** Vitamins*** Sucrose N-Z Amine Type A 2,4-D F i n a l pH 4  2  Fe EDTA stock solution Dissolved i n 100 ml.: FeS04.7H20 Na2EDTA Keep frozen jkjfc  ..  278 mg. 372 mg.  .......  Stock s o l u t i o n : Dissolved i n 100 ml. H2O: 1 gm. MnSOA^O, 300 mg. H0BO0, 300 mg. ZnSO. .7H 0, 25 mg. Na MoO. .2H 0, 25 mg. GuS0 , 25 mg. CaCl »oH 0 * 2  4  2  2  2  2  Stock s o l u t i o n : Dissolved i n 100 ml. H2O: 10 mg. n i c o t i n i c 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 i n suspension cultures of wheat and barley. Can. J . Biochem. 46, 417, 1968.  16 C.  Callus formation Hips of Rosa rugosa were s t e r i l i z e d i n 5$ sodium hypochlorite f o r 20 minutes and quickly rinsed i n d i s t i l l e d water.  They were then dissected with a  s t e r i l e s c a l p e l i n a s t e r i l e p e t r i e dish. Small portions of the inner contents were transferred to the surface of 10 ml. of PRL-4-C-CM agar medium 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 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 i n 100 ml. of PRL-4-C-CM l i q u i d medium i n a 250 ml. erlenmeyer f l a s k .  The culture  was incubated on a rotary shaker (Gyrotary  shaker)  at a speed of 160 revolutions per minute i n a 1" c i r c u l a r o r b i t at 26°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 a new medium at regular time i n t e r v a l s i n order to maintain culture vigour.  2.  Measurement of c e l l growth The growth of the c e l l s was measured by following c e l l dry weight every other day f o r two weeks.  The c e l l s  17 from duplicate cultures were c o l l e c t e d by f i l t r a t i o n through Mira-cloth and a weighed portion was constant weight i n a V i r t i s freeze dryer. c e l l s were used i n ascorbic a c i d  dried to  The remaining  determination.  pH measurement The f i l t r a t e from the c e l l s used f o r dry weight determination  was  used f o r t h i s 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 t i t r a t i o n method (3). A*  Standardization of the dye (daily) A 5-ml aliquot of the standard ascorbic a c i d solution (containing 1 mg. with 5 ml. 3$ HPO3.  I t was  ascorbic acid) was d i l u t e d t i t r a t e d with the  dye  solution to a pink colour which persisted f o r 15 seconds.  Since t h i s volume of dye represented  of ascorbic acid, the ascorbic acid equivalent 1 ml. of the dye solution was  1  mg.  (T) of  equal to 1 divided by  the volume in, 1 ml. of the dye solution used i n t h i s titration.  18 B.  Procedure (  i ) Extraction A sample of c e l l s l e f t a f t e r  filtering  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 c e l l s were  quickly placed i n a j a r and 4 0 ml. of 6% HPO-jEDTA was added.  The j a r was attached to an  Osterizer and blended at 1 0 , 4 0 0 rpm. f o r one minute to mascerate the c e l l s .  This s l u r r y  was then transferred to a 1 0 0 ml. volumetric f l a s k and d i l u t e d 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 filtrate.  A phase contrast microscope was used  to check 1 0 0 $ e f f i c i e n c y 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 erlenmeyer f l a s k ( 5 0 ml.).  The solution was t i t r a t e d  immediately with the standardized solution of 2,6-dichlorophenolindophenol to a f a i n t pink end point which persisted f o r 15 seconds.  The  colour of the dye dissolved i n the d i l u t e sodium bicarbonate solution was blue, but i n  19 an a c i d medium such as the s t a b i l i z i n g solution used i n t h i s determination, pink colour.  the dye assumed a  Therefore, the colour change i n  t h i s method, at the end point, was less to pink.  from colour-  In the t i t r a t i o n s of the  extracts with the dye, i t was  desirable to add  the dye quite r a p i d l y to a point where the r e s u l t i n g pink colour did not immediately disappear. was  As rapidly as possible, the  dye  added dropwise with constant mixing of the  solution u n t i l the f a i n t pink colour of the solution r e s u l t i n g from the unoxidized persisted f o r 15 seconds. and short-time  dye  A rapid t i t r a t i o n  end point were desirable because  of the possible i n t e r f e r i n g action of other constituents of the s o l u t i o n .  In general, such  i n t e r f e r i n g materials reacted more slowly with the dye than did ascorbic acid; therefore, t h e i r e f f e c t was  kept to a minimum by r a p i d  titration, ( i i i ) Calculation Ascorbic a c i d was  calculated according to  the following formula: V, * *™  T  x l^C" _ g . ascorbic a c i d per 100 sample m  gm.  20 V = ml. dye used for t i t r a t i o n of aliquot of d i l u t e d sample T = ascorbic a c i d equivalent of dye solution expressed as mg. per ml. of dye W  =  gm. of sample i n aliquot t i t r a t e d  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 a c t i v e l y growing tissue of the b r i a r rose were examined f o r t h e i r ascorbic acid content. used.  Rose hips, stems and leaves were  These values were compared with the maximum values  obtained i n tissue culture and with common values o f some fruits.  As chlorophyll production and ascorbic  acid  values seem to be related, comparisons between l i g h t grown cultures and the l i v i n g tissue were also carried out.  Location  of ascorbic  acid  To confirm that ascorbic a c i d was located only i n t r a c e l l u l a r l y , determinations were run on the c e l l - f r e e filtrate.  Equal volumes of the media i n which the c e l l s  had been suspended and 3% HPO3 were mixed and the r e s u l t i n g solution was t i t r a t e d .  21  7.  Comparison between age and colour of cultures and ascorbic acid content Flasks of c e l l s , one, two,  three and f i v e weeks  old, were compared v i s u a l l y for colour. acid determinations  Then ascorbic  were made on the contents of each  of the f l a s k s .  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 i n 25 ml. of the medium i n which the c e l l s had been growing f o r 12 days. i n t e r v a l was  This time  chosen as by t h i s time there was  good c e l l population and the c e l l s f i l t e r e d a necessity f o r t h i s determination.  a rapidly,  Then the  f i l t e r e d c e l l s were re-suspended i n t h i s medium and returned to t h e i r o r i g i n a l environment. time intervals  At s p e c i f i c  (0, 2, 4, 6 and 24 hr.) small  portions of the f l a s k contents  (4-5  gm.)  were  taken and t h e i r ascorbic acid content determined.  22  PRECURSORS ADDED  QUANTITY 1 gm.  D-glucurono- -lactone L-gulono-K-lactone L-galactono-tf-lactone D-glucose D-galactose D-I-)-levulose  .5 gm.  .25 gm.  X X X X X X  v  X  There has been some controversy  X X X X X X  about the  -  possible r o l e of myoinositol i n ascorbic acid production (25, 26). As t h i s compound i s present i n appreciable.quantity  i n 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 l e v e l .  The  c e l l populations were transferred twice and once again determinations were performed on Day 12. B.  Light Rose suspension cultures contained  i n s i x 250 ml.  erlenmeyer f l a s k s were grown under i d e n t i c a l conditions as previously described except that three of the cultures were grown i n the l i g h t .  The l i g h t was pro-  vided by 3 high i n t e n s i t y fluorescent lamps that produced a t o t a l illumination output of 9#40 lumens (at 40% rated l i f e ) .  The lamps were suspended 52 cm.  above the erlenmeyer f l a s k s .  After 12 days of incu-  bation, ascorbic acid determinations were made.  23 G.  Influence of l e v e l of sucrose The l e v e l of sucrose i n the medium was varied (10 gm. - 20 gm./l.) and, a f t e r the c e l l s had grown 12 days i n l i g h t , v i s u a l appraisal of chlorophyll l e v e l (42) and determination of ascorbic a c i d 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 v e r i f y that ascorbic acid was indeed the substance causing t h i s increase.  To rule out the p o s s i b i l i t y of microbial  contamination, a solution of 0 . 5 gm. L-galactono-tflactone dissolved i n 2 ml. s t e r i l e d i s t i l l e d water was f i l t e r s t e r i l i z e d , added to a culture (approximately 30 gm. c e l l wet weight), suspended i n approximately 25 ml. media and incubated under standard conditions f o r 24 hours. At the end of t h i s time, ascorbic acid was  determined  by the 2,4?-dinitrophenylhydrazine (DNPH) method ( 3 ) , using t h i n layer chromatography.  Analyses were performed  on standard ascorbic acid and L-galactono-/-lactone solutions, a control f l a s k of c e l l s , and the f l a s k to which the possible precursor L-galactono-y-lactone had been added.  This method was s l i g h t l y modified with the  substitution of the solvent system chloroform-ethyl acetate (50-50) (5) f o r separation on the chromatographic plate. Paper chromatography was used to detect the disappearance 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 s p e c i f i c time i n t e r v a l s (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 t h i s time the sugars were detected using the standard ammoniacal s i l v e r n i t r a t e spray (43).  10.  Determination of t o t a l ascorbic acid, dehydroascorbie acid and diketogulonic acid The method used f o r 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 s l i g h t l y modified i n that samples were blended with 40 ml. 10$ HPO3 and then d i l u t e d to 100 ml. with 5% HPO3. exactly.  Thereafter the procedure was followed  For determining DHA. and diketogulonic acid,  samples were ground with 10 ml. 5$ HPO3 and 1.0 gm.  25 stannous chloride and d i l u t e d to 200 ml. From t h i s 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  determined as follows. was  a c i d was  A 20 gm. fresh weight sample  incubated with 0.25 gm. ascorbic acid f o r 24 hours  and a 4 gm. sample of t h i s was used to assess t o t a l ascorbic  acid, DHA and diketogulonic a c i d .  The r e s u l t s  were compared with a 4 gm. control sample. In the event that too high a concentration of ascorbic acid was used i n 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 t o t a l ascorbic acid, DHA and diketogulonic acid.  These r e s u l t s 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 c a l l u s cultures from the b r i a r rose Rosa rugosa were yellow or white i n colour and very compact. shows a t y p i c a l example.  Fig. 4  To maintain callus vigour, the  calluses were transferred to fresh media about once a month s t a r t i n g i n August, 1970. The suspension cultures were also l i g h t i n colour and developed quite heavy growth as can be seen i n Figure §.  Transferring of the suspension culture c e l l s  to new media was much more frequent than that required for callus tissue.  I t was done at l e a s t every two weeks.  The cultures f o r experimental work were started i n l i 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 July Sept. Oct. Oct. Nov. Nov. Dec. Dec.  £, 1971 30 10 6 l£ 2 17 4 17  Jan. Jan. Feb. Feb. March March March April  6, 1972 25 4 20 3 14 24 3  A - clump of c e l l s (lOx)  B - chain of c e l l s (lOx) Figure 6.  Microscopic examination of t y p i c a l c e l l populations of Rosa rugosa from the suspension culture flasks  30 Microscopic examination of the suspended c e l l s revealed some differences within the culture.  The vast  majority of the c e l l s showed a tendency to clump together as shown i n Figure 6; however, there was a small proportion that formed chains (Fig. 6).  In the microscopic  f i e l d s examined, free l i v i n g single c e l l s were not observed.  2.  Growth curve From F i g . 7 and 8, i t can be seen that the c e l l s increase i n population u n t i l Day 16.  After Day 6, growth  picks up considerably with a concomitant r i s e i n pH.  The  hydrogen ion concentration seems to be quite c r i t i c a l f o r rapid growth of the plant c e l l s . u n t i l Day 16.  Generally, the pH r i s e s  Mapson (31) found that an a l k a l i n e s h i f t  i n pH increases the e f f i c i e n c y of the conversion of hexose sugars to ascorbic a c i d .  Results from t h i s growth  curve also tend to confirm the l a t t e r observation as shown by the comparatively  large increase i n ascorbic  a c i d from Day.6 to Day 8.  Thereafter, the ascorbic a c i d  value increases slowly to Day 14 and by Day 16 s t a r t s to drop o f f s l i g h t l y .  This s i t u a t i o n may  be somewhat  analogous to whole plants where i t has been found that ascorbic acid concentration continues to increase r i g h t up to anthesis and then drops o f f .  31  Time pB o  (days)  ascorbic acid •  weight  A  Values are the average o f two d e t e r m i n a t i o n s F i g u r e 7. R e s u l t s o f growth curve showing pH, Afresh weight and a s c o r b i c a c i d content  32  Time  (days)  pH o a s c o r b i c a c i d E l 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 Figure  8,  R e s u l t s o f g r o w t h c u r v e s h o w i n g pH, ascorbic acid content  dry weight  and  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 t h i s time and the c e l l s were e a s i l y and r a p i d l y filtered.  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 i n the determination  of ascorbic a c i d .  There was also a good measurable quantity of ascorbic acid present t h i s time.  i n the cultures to serve as a control at Unless otherwise stated, future experiments  were performed i n duplicate.  3.  Location of ascorbic acid Determination of ascorbic a c i d i n the  filtrate  revealed that very l i t t l e free excreted ascorbic a c i d was  present since only 0.03  required (Table 4).  ml. of indicator dye  was  T i t r a t i o n of f r e s h l y prepared  unincubated media showed the same r e s u l t , which would indicate that there were no i n t e r f e r i n g reducing compounds.  Therefore, ascorbic acid seems to be located  intracellularly.  4.  Comparison between age and colour of cultures and ascorbic acid content Examination of the r e s u l t s i n Table 3 quickly shows that the colour of the culture i s not as good an i n d i c a t i o n of the presence of ascorbic a c i d as i s the age of the  34 Table 1.  Comparison of ascorbic a c i d values of l i v i n g tissue and plant c e l l s  Tissue  Average ascorbic a c i d values Fresh weight Dry weight (mg./lOO,,gm) (mg./lOO gm)  Rose hips  240  130G  Rose stem and leaves  290  1300  Rose plant c e l l s grown i n dark  13  320  Rose plant c e l l s grown i n l i g h t : 1st transfer 2nd transfer 3rd transfer  17 26 35  Table 2. Fruit Apples  Average ascorbic acid, content, of f r u i t s Ascorbic a c i d (mg./lOO gm. fresh weight) 2-10  Grapefruit  40  Lemon  50  Orange  50  Strawberry  60  Rose hips  up to 1% of fresh weight  From: Mapson, L.W. Vitamins i n f r u i t i n The Biochemistry of F r u i t s and Their Products, V o l . 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 1  week  2  Ascorbic acid value fresh weight (mg./KM)., gin,)  Visual appraisal of colour  8  almost white  weeks  13  almost white  3h weeks  1  beige coloured  5  weeks  1  chocolate brown  12  weeks  0  beige to dark brown  0  very bright yellow  13 weeks Table 4 .  T i t r a t i o n values of precursors and precursors dissolved i n media  Solution  Volume of indicator required i n 3fo HPO3 . 2 5 gm. media + 25 ml. HPO3 1 mg./lO ml. i n 25 ml. at beginning .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.  af-lactone  . 0 3 ml.  .1 ml.  .1  ml.  L-gulonoi'-lactone  . 0 3 ml.  .1 ml.  .1  ml.  L-galactonoV- lactone  .03 ml.  .1 ml.  .1  ml.  D-glucurono-  L-ascorbic acid D-(-)-levulose  1  7.4  ml.  . 0 3 ml.  1900 ml. (extrapolated) .1 ml.  .35 ml. .1  ml.  36 culture.  I t also i l l u s t r a t e s the existence of b i o l o g i c a l  v a r i a t i o n i n cultures, p a r t i c u l a r l y comparing the two cultures that were both approximately  three months o l d .  Generally, ascorbic acid i s found i n larger quantities i n very l i g h t coloured cultures.  5.  Comparison with other l i v i n g tissue As can be seen i n Table 1, the amount of ascorbic a c i d found i n dark grown c e l l s i s considerably l e s s than that of fresh tissue (13 mg.  compared to 240 mg.).  Expo-  sure of the c e l l s 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 cont r i b u t e to the reducing power of the cultures, standard solutions of the precursors were prepared and t i t r a t e d . As shown i n Table 4, the dye was oxidized with the addition of 0.03  ml. so that t h i s p o s s i b i l i t y was  pre-  cluded as a possible source of interference. T i t r a t i o n of medium containing the same weights of precursors used experimentally showed that there was interaction between the precursor and the medium that  no  to  37'  80; -P  fctO , u  10  to  40  •H CD  O CO 50 h£)<H  a  Ho  •H  <DOiD AO  « 9 i ° -O-Cr  t i o~i_i-tJ—  -a  Time (hr.) D-glucose  •5 .25  -a  Terror gm. gm. 7  d o  -o  Time (hr.) D-galactose  T  so 70-  -P  0  CD  •H £  > Xi  t6H  o a)  CU  CD  •H  •  U  oa  50  5<3 id-  30;  H  ao P  lolv-O  trrr  Time (hr.) D-glucurono-Y-lactone Figure 9.  /  Time (hr.) D-(-)levulose  XT  E f f e c t of the addition of selected precursors on the reducing power calculated as ascorbic a c i d on rose cells. 5  70 So  ;80  70 •H  to  Q> 2=  O  03  P-i  hO<H •H O 0)  o IV  Time ( h r . ) L-gulono-#-lactone  t—i_  4¥  1.0 .5 ^ .25  gm. gm. gm.  XV-  Time ( h r . ) • L-galactono-y-lactone A • O  Time (hr.) • • L-ascorbic acid igure  10.. E f f e c t o f the a d d i t i o n o f s e l e c t e d p r e c u r s o r s on .th» r e d u c i n g power c a l c u l a t e d as a s c o r b i c a c i d on r o s e cells.  39 4)..  might influence the volume of dye required (Table  From t h i s 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 i n t r a c e l l u l a r l o c a t i o n of ascorbic acid. Fig.  9 indicates that there i s no appreciable change  in the reducing power of rose tissue c e l l s with the addition of D-glucose, D-(-)-levulose, D-galactose and D-glucurono-V-lactone at the 1% and 2% l e v e l s . explanations  are possible.  Two  The f i r s t i s that the possible  precursor remained i n the medium, or, that i t did enter the c e l l but perhaps was  diverted to another pathway as  could c e r t a i n l y occur with the addition of D-glucose. Also the ascorbic acid could be transient and  converted  to DHA- or diketogulonic a c i d before detection was  possible.  Further c l a r i f i c a t i o n about t h i s point might provide information about the biosynthetic pathway of ascorbic acid i n t i s s u e culture.  After 24 hours, there was  a  s l i g h t increase i n reducing power with L-gulono-^-lactone; but the most spectacular increases occur with t i o n of L-galactono-fc'-lactone  administra-  to the cultures ( F i g . 10).  In another paper an account i s given of the enzymic conversion of L-galactono-V-lactone to L-ascorbic a c i d by extracts of plant tissues (34).  Jackson et a l . (19) also  demonstrated t h i s increase by addition of t h i s compound to s l i c e s of rose hip t i s s u e .  40 Figures from the l i t e r a t u r e show that, using excised plant sections, conversion of L-galactono-Y-lactone to ascorbic a c i d i s 80-90% e f f i c i e n t (27).  However, the  comparatively small increases shown here and lack of i n h i b i t i o n even when one gram of precursor i s added does not at f i r s t sight support t h i s observation. That the L-galactono-^-lactone  did disappear from  the medium i s shown from the r e s u l t s of the paper chromatograph (Fig. 12).  After 24 hours, the spot f o r t h i s  compound i s barely v i s i b l e .  I t i s also of interest that  the spot corresponding to D-(-)-levulose has completely.  disappeared  However, as can be seen from F i g . 9, the  addition of D-(-)-levulose to the culture does not bring about any change i n the t i t r a t i o n value.  Therefore, i t  would seem that D-(-)-levulose i s not responsible f o r the increase. To confirm that the increase i n ascorbic a c i d values was not the r e s u l t of microbial contamination  or the  presence of some other metabolite i n the preparations, the alternate method f o r determining ascorbic acid with DNPH was  carried out.  The aseptic techniques  described  i n the methods were followed and the determination  was  made a f t e r 24 hours. Fig. 11 i l l u s t r a t e s the r e s u l t s obtained from the chromatographic plate.  Comparing the sample and the  Control  Band f o r ascorbic acic  With L-galactonoY-lactone added  Band f o r ascorbic acic  Figure 11.  —  Thin layer chromatographs showing the r e s u l t s of the DNPH method of detecting ascorbic a c i d using the solvent system chloroform-ethyl acetate ( $ 0 - 5 0 )  42  ft  B  Figure 1 2 .  1. 2. 3. 4. 5.  D-glucose i n water L-galactono-V-lactone i n water pure media media and L-galactono-y-lactone (0 hours)  1. 2. 3. 4. 5.  D-glucose i n water D-(-)-levulose i n water L-galactono-|j -lactone i n water media + L-galactono-^-lactone (4 hours) " tt tt it (6 hours)  1. 2. 3. 4.  D-glucose i n water D-(-)-levulose i n water L-galactono-y-lactone i n water media +• L-galactono-i -lactone ( 2 4 hours)  n  it  tt  tt  it  (2  M  )  /  /  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 c l e a r l y there i s an increase i n t o t a l ascorbic a c i d . 1 cm.  The width of the band of the sample was  compared to h cm. f o r the control.  In addition,  the i n t e n s i t y of the coloured derivative was much greater f o r the sample.  The calculated Rf value f o r t h i s  p a r t i c u l a r solvent system was 0.26 ture value of 0.25  (5).  compared to the l i t e r a -  To determine i f any of the other  coloured bands on the plate were perhaps derivatives of L-galactono-V-lactone, a standard of t h i s compound was also run.  Under the conditions of t h i s experiment, no  coloured derivative was  formed.  Addition of pure ascorbic a c i d to a culture showed some i n t e r e s t i n g r e s u l t s . the addition of 0.25  gm.  As can be seen i n Table 4>  to 25 ml. of media, the t i t r a t i o n  value of the medium dropped from a calculated 1900 to about 0.35  with  ml. a f t e r 24 hours.  ml.  The c e l l s at t h i s  time were a very white colour as they were with addition of L-galactono-V-lactone,  but the increase i n ascorbic  acid value of the c e l l s c e r t a i n l y did not p a r a l l e l the disappearance of ascorbic acid from the medium (Fig. 10). Since the t i t r a t i o n method measures only reduced ascorbic acid, the p o s s i b i l i t y was raised that there may  be a marked increase i n the amount of ascorbic  acid present as the oxidized forms DHA acid.  and  diketogulonic  44 Table 5.  Comparison of dry weights of dark and l i g h t grown c e l l s  Treatment  Fresh weight (gm.)  Light Light  30.9 29.4  Dark Dark  27.8 42.5  Table 6.  Dry weight (gm.) .85 .8 .8 1.0  E f f e c t of a l t e r i n g the environment on f i n a l weight of tissue and ascorbic acid content (12th day) Ascorbic acid values fresh weight mg./lOO gm.  Tissue from f l a s k fresh weight gm.  9-13  30-40  ii 9  13^1 16.7  10 12  10.5 10.7  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  Treatment  Control - Myoinositol 1st transfer 2nd transfer  +Light 1st transfer  45 Table 7 shows the spectrophotometric determination  r e s u l t s of the  of " t o t a l " ascorbic acid and dehydro-  ascorbic and diketogulonic acids.  In the case of normal  c e l l s the majority i s present as the reduced form (the difference between t o t a l ascorbic a c i d and that contributed by DHA and diketogulonic a c i d ) .  However, with  the addition of large amounts of ascorbic a c i d , DHA and diketogulonic acid r i s e 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 t h i s conversion could r e s u l t i n greater accumulation of ascorbic acid, thereby increasing yield.  7.  Omission of myoinositol from the medium Omission of t h i s compound from the vitamin solution had a profound effect on the growth of the cultures.  On  three separate occasions growth was much l e s s dense than the control c e l l s . also.  There was much more clumping of c e l l s  By the second transfer, the c e l l s had become  s l i g h t l y darker i n colour than the controls. From Table 6, i t can be seen that ascorbic a c i d 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 i n c e l l y i e l d .  I t seems  46 then that myoinositol has l i t t l e influence on ascorbic a c i d production as also noted by Loewus (26) who ruled out t h i s compound as an important precursor of ascorbic acid.  8.  C e l l s grown under controlled i l l u m i n a t i o n Growing rose plant c e l l s under constant i l l u m i n a t i o n brought about physical changes i n the c e l l s themselves. After the f i r s t 12 days, the c e l l s had a rather bleached appearance compared to the controls.  However., as  suggested by Street (45), there was no green, pigmentation whatsoever.  The c e l l s had a tendency to clump together  much more than those c e l l s grown i n the dark. By the second and t h i r d transfers the,light grown f l a s k s showed small bright yellow b a l l s or calluses which were quite separate and d i s t i n c t compared to the more uniformly suspended dark grown controls.  After f i l t e r i n g ,  the l i g h t grown c e l l s seemed to adsorb more l i q u i d ; however, t h i s was an o p t i c a l 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 i n Table 5. In the case of l i g h t grown f l a s k s , with each successive transfer, the t o t a l f l a s k fresh weight decreased as shown i n Table 6.  A possible solution to. t h i s problem  might be more frequent transferring as c e l l processes  47 Table 7.  Spectrophotometric readings f o r " t o t a l " ascorbic acid and dehydroascorbic + diketogulonic acids  Sample  Absorbance "Total" DHA + ascorbic a c i d diketogulonic a c i d  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 Table 8.  1.4  Summary of r e s u l t s obtained from attempts to vary the ascorbic a c i d content of rose tissue culture Total Treatment Ascorbic Acid Value cell 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  :  slight  increase  tt  •+ L-galactono-V- lactone  increase  - Myoinositol  remains the same  dec.  t Light - 1st transfer 2nd transfer 3rd transfer  s l i g h t increase increase increase  same dec. dec.  - g Sucrose  decrease  same  * D-(-)-levulose  remains the same  same  tt  4* probably proceed at a much greater rate i n l i g h t than i n darkness. quickly.  Hence nutrients would be depleted much more Also development of the optimum medium f o r  l i g h t grown cultures would help here. In contrast, with each successive transfer of c e l l s grown under l i g h t , the ascorbic acid concentration increased.  Various workers such as Mapson (32) and King  (22) have also found that ascorbic a c i d synthesis depends on photosynthetic  activity.  Kefford and Chandler (20)  have noted that, i n the northern hemisphere, the highest ascorbic acid values were found on the southern exposure of the tree where the f r u i t s received the most sunlight. It can also be seen that the presence of chlorophyll i s not an absolutely necessary factor f o r increasing ascorbic a c i d synthesis, as i t s concentration  did r i s e i n  tissue culture with no v i s i b l e pigmentation whatever. It may well be, however, that, i f pigmentation were to occur, the ascorbic a c i d content might increase much more dramatically.  9.  E f f e c t of sucrose Table 6 shows that a decrease i n the amount of sucrose d i d not appreciably a l t e r the c e l l  population  compared to l i g h t grown second transfer c e l l s which were started at the same time.  Further transfers would  49 probably a f f e c t t h i s . c e l l s was  The physical appearance of the  similar to the l i g h t grown c e l l s .  There was,  however, quite a difference i n the  ascorbic acid values, 7 mg. and 27 mg.  and  5 mg.  compared to 26  mg.  for illuminated f l a s k s determined at the same  time and 9 mg.  f o r the dark control.  found that ascorbic acid synthesis  Mapson (31)  also  depends on an adequate  supply of hexose.sugars and that conditions which promote the synthesis of sucrose appear to favour the of ascorbic a c i d .  synthesis  Chinoy, Patel and Suthar (8) found  that the biosynthesis siderably by sucrose.  of ascorbic a c i d i s enhanced con-  50  SUMMARY Table 8 gives a summary of the effectiveness of the various methods of a l t e r i n g the ascorbic a c i d content of the tissue culture of Rosa rugosa. administered,  Of the test compounds  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 y i e l d but had p r a c t i c a l l y no effect on the ascorbic a c i d value. Addition of l i g h t to the cultures had a pronounced effect on the ascorbic a c i d value.  I t increased with each  successive transfer while the t o t a l c e l l y i e l d f e l l appreciably with continued propagation.  More frequent t r a n s f e r r i n g may  help overcome t h i s problem. Decreasing  the amount of sucrose i n 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 f o r ascorbic acid  production.  51  LITERATURE CITED 1.  Aberg, B. Vitamins as growth factors i n higher plants. i n Encyclopedia of Plant Physiology, Vol. 14, ed. Ruhland, W., Springer, B e r l i n , 418, 1961.  2.  Asselbergs, E.A.M. Studies on the formation of ascorbic a c i d i n detached apple leaves. Plant Physiol. 32, 326329, 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 a c i d and dehydroascorbie a c i d i n turbid and coloured solutions i n the presence of other reducing substances. J . B i o l . Chem. 126, 771-784, 1938.  5.  B o l l i g e r , H.R. Vitamins, i n 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 i n v i t r o and t h e i r production of antimicrobial metabolites. Can. J . M i c r o b i o l . 11,  7.  785-739, 1965.  Ghayen, J . Ascorbic a c i d and i t s i n t r a c e l l u l a r l o c a t i o n , with s p e c i a l reference to plants. Int. Rev. Cytol. 2, 77-131, 1953.  8.  Chinoy, J . J . , Shah Hemlata T. Patel, C K . and. Suthar, H.K. Role of auxin and g i b b e r e l l i n i n the synthesis of ascorbic acid and growth of tissue explants. Biologica Plantarum 9, 182-194, 1967.  9.  Doby, G. Plant Biochemistry. London and Budapest. 1965.  Interscience Publishers,  10.  Freebairn, H.T. Determination and s t a b i l i z a t i o n of reduced ascorbic a c i d i n extracts from plant material. Anal. Chem. 31, 1850-1851, 1959.  11.  Furuya, T., Hirotani, M., and Kawaguchi, K. Biotransformation 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 i n suspension cultures of wheat and barley. Can. J . Biochem. 4 6 , 417-421, 1968.  13.  Giroud, A., Ratsimamanga, A.R. and Leblond, C-P. Relationship between ascorbic acid and chlorophyll. B u l l . Soc. Chim. b i 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. I s o l a t i o n of a b a c t e r i a l lipopolysaccharide from Xanthomonas campestris containing 3-acetamido-3,6-dideoxy-D-galactose and D-rhamnose. J . B i o l . Chem. 241, 1424-1428, 1966.  16.  Hough, L. and Jones, J.K.N. Chromatography on paper. i n Methods i n 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 i n the estimation of dehydroascorbic a c i d . Biochem. J . 64, 203-208, 1956.  18.  I s l e r , 0. Developments i n 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 a c i d by plants. Nature 191, 282-283. 1961.  20.  Kefford, J.F. ahd Chandler, B.V.... The Chemical Constituents of Citrus F r u i t s . 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, a c i d . 'Physiol. Rev. 16, 238-262,,1936.  23.  K l e i n , R.M. Plant tissue cultures, a possible source of plant constituents. Econ. Bot. 14, 286-289, I960.  24.  Lamport, D.T.A. C e l l suspension cultures of higher plants: i s o l a t i o n and growth energetics. Expt. C e l l Res. 1964, 195-206, 1964.  53  25.  Loewus, F. I n o s i t o l metabolism and c e l l wall formation i n plants. Fed. Amer. Soc. Exp. Bio., Federation Proceedings 24, "1^5-3527 1 9 o T .  26.  Loewus, F.A. Tracer studies on ascorbic a c i d formation i n plants. Phytochem. 2, 109-128, 1963.  27.  Loewus, F. and Baig, M.M. Biosynthesis and degradation of i s o t o p i c a l l y l a b e l l e d ascorbic acid, i n Methods i n 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-Lascorbic acid i n plant tissues by the Roe and Kuether procedure. Biochem. J . 80, 459-461, 1961.  29.  Mapson, L.W. Function of ascorbic acid i n plants, i n Vitamins and Hormones. Advances i n Research and Applications, V o l . 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 i n plants. Part 1. Function. Ann. Rev. Plant Physiol. 9, 119-150, 1958.  31.  Mapson, L.W. The biosynthesis of ascorbic acid, i n Vitamins and Hormones. Advances i n Research and Applications, V o l . 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 i n f r u i t , i n The Biochemistry of F r u i t s and Their Products, V o l . 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 f o r 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 a c i d : the conversion of Lgalactono-V-lactone into L-ascorbic a c i d by plant mitochondria. 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 i n 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 a c i d 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 i n d u s t r i a l p o t e n t i a l of plant c e l l culture, i n Progress i n I n d u s t r i a l Microbiology, V o l . 9, ed. Hockenhull, D.J.D., Gordon and Breach, London, 14-39, 1971.  39.  R i n a l d i n i , L.M. The e f f e c t of Vitamin C on c e l l s and tissues i n culture, i n 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 f o r the determination of L-ascorbic a c i d . Ann. NjY. Acad. S c i . 92, 277-283, 1961.  41.  Roe, J.H. and Oesterling, M.J. The determination of dehydroascorbie acid and ascorbic acid i n plant tissues by the 2,4-dinitrophenylhydrazine method. J . B i o l . Chem. 152,  511-517, 1944.  42.  Sestak, Z., Catsky, J . and J a r v i s , P.G. Plant Photosynthetic 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. i n Plant Physiology, V o l . VB, ed. Steward, F.C., Academic Press, New York, 113-181, 1969.  45.  Street, H.E. The n u t r i t i o n and metabolism of plant tissue and organ cultures, i n C e l l s and Tissues i n Culture: Methods, Biology and Physiology, V o l . 3, ed. Willmer, E.N., Academic Press, London and New York, 602, 1966.  46.  Street, H.E., Henshaw, G.G. and B u i a t t i , M.C. The culture of i s o l a t e d 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, C P . and Krishman, P.S. Loss of ascorbic a c i d during estimation by the Roe-Kuether method. J . Food S c i . 26,  11-14,  1961.  49.  Vitamin C Merck Service B u l l e t i n , Merck and Co. N.J., 1956.  50.  Weinstein, L.H., N i e k e l l , L.G., Laurencot, J r . , H.J. and Tulecke, W. Biochemical and p h y s i o l o g i c a l 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.  Inc.,  

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