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Biotechnol. Prog. 1991, 7, 288-290
Growth and Anthocyanin Accumulation in Carrot Cell Suspension Cultures Growing on Fructose, Glucose, or Their Mixtures S. K. Zwayyed,+G. C. Frazier,t and D. K. Dougall'J Departments of Chemical Engineering and Botany, University of Tennessee, Knoxville, Tennessee 37996-1100
Carrot suspension cultures have been grown on glucose, fructose, and mixtures of these two sugars and the effects of these sugars on growth and anthocyanin accumulation measured. Several parameters of growth decrease with increasing glucose in the medium. The anthocyanin accumulated remains relatively low and constant until the glucose exceeds 50 % of the sugar, when the anthocyanin accumulation increases with increased glucose. T h e data suggest that glucose and fructose are metabolized differently in these carrot cultures despite the interconversion of their 6-phosphates in glycolysis. Ways in which the metabolism of these sugars might differ are discussed, as is the possibility of increasing the accumulation of anthocyanin by alterations in the way these sugars are made available to the cultures.
Introduction We have shown that our carrot cell culture hydrolyzes sucrose in two days or less and then uses the fructose in preference to the glucose. Further, with sucrose as the carbon source the culture grew faster and accumulated more biomass but less anthocyanin than when glucose was the carbon source (Dougalland Frazier, 1989). These data suggest that fructose released from the sucrose led to the differences between cultures grown on glucose and on sucrose. To verify this suggestion and to investigate the possibility that the volumetric productivity of the cultures could be increased by alteration in the carbohydrate supplied to the cultures, we have examined the growth and anthocyanin accumulation of the carrot cell suspension cultures with glucose alone, fructose alone, mixtures of glucose and fructose, and sucrose alone as the carbon source.
Materials and Methods The wild carrot (Daucus carota) clone used in these experiments and in previous experiments (Dougall and Frazier, 1989)was WC63-1-9-1-13-1. The detailed growth experiments with glucose, with fructose, with mixtures of glucose and fructose, and with sucrose as carbon sources at 15 pL-' were performed as described (Dougall and Frazier, 1989) except that (a) media were filter-sterilized by using 0.22-pm filter capsules (Gelman Sciences) and (b) evaporation losses were corrected every day by weighing all the flasks and returning each to its original weight with sterile distilled water. The five treatments were examined in three separate experiments, two treatments in each of two experiments and the remaining one in a separate experiment. The data for each growth experiment were plotted semilogarithmically, and the period of exponential growth was identified by inspection. The line of best fit to In (dryweight) versus days for that period was calculated by the method of least squares. The slope of the line of best fit is the growth rate in days-'. The anthocyanin content of the cultures in each experiment is given as the mean and standard deviation of all values obtained after the growth of a culture was no longer exponential.
* Author to whom correspondence should be addressed. Department of Chemical Engineering. Department of Botany.
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All the treatments were compared directly in one experiment in which dry weight and anthocyanin were measured at day 11, i.e., when the culture had reached stationary phase and anthocyanin was maximal. In this experiment each treatment was replicated five times and each of these separate flasks was sampled in duplicate for measurement of dry weight and anthocyanin. Data for this experiment are given as the mean and standard deviation of the 10 samples for each treatment.
Results The growth rates, the anthocyanin contents, and other data obtained with the carrot clone used here when it was grown on different ratios of glucose and fructose are given in Table I. These data show that the growth rate is highest with fructose alone as the carbon source and decreases to a constant value (ca. 80% of the maximum) when glucose is 50% or more of the carbohydrate supplied. In addition, days to maximum biomass increased as the percentage of glucose in the medium increased to 50 ?6 and then remained constant at higher proportions of glucose. The maximum biomass achieved decreased with increased glucose in the medium, and it appears to be minimum when glucose is 75% of the supplied carbohydrate. The anthocyanin content of the cultures in these experiments remained constant during the lag in the increase in biomass and for 1-2 days beyond. Anthocyanin then increased exponentially while the biomass was increasing exponentially. When the increase in biomass ceased to be exponential, anthocyanin content ceased to increase. These features can be seen in the data given by Dougall and Frazier (1989) and in part in Dougall et al. (1983). Because anthocyanin accumulation ceases when the biomass ceases to increase exponentially, the maximum anthocyanin content of the tissue in these experiments is expressed as the mean and standard deviation of anthocyanin of all the samples taken after the exponential increase in biomass. The standard deviations shown in Table I are 20-25% of the means. The source of this variability is not known. The level of anthocyanin accumulated in the tissue was constant when glucose was 50% or less of the carbohydrate in the medium; it approximately doubled from this value when the carbohydrate supplied was 75% glucose and trebled when the carbohydrate was 100% glucose.
0 1991 American Chemical Society and American Instltute of Chemlcal Engineers
Biotechd. Rog., 1991, Vol. 7, No. 3
Table I. Growth Rate, Maximum Biomass Achieved, Days to Maximum Biomass, and Anthocyanin Accumulated by Wild Carrot Subclone WC63-1-9-1-13-1Grown on Media with Different Ratios of Fructose and Glucose. 76 glucose 0 25 50 75 100 experiment A B C B A growth rata, days-' 0.46 0.41 0.38 0.36 0.38 nb 5 6 7 6 5 maximum biomass, g-L-1 5.5 5.2 4.8 4.3 4.8 9 12 13 13 13 days to max biomass anthocyanin, pmol (L of culture)-' 7c 19b 36a meanC 1Oc llc 2 4 7 2 2 SD
289 I
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90
80
Relative to glucose, fructose as a sole carbon source for the carrot cell cultures used here gives the higher growth rate and consequently the least time to maximum biomass, the higher biomass accumulation, and the lower anthocyanin accumulation. These two hexoses have a number of different effects on the culture. Glucose and fructose have different effects on other cultures also. With an embryogenic wild carrot suspension culture, Verma and Dougall(1977) found that after 13 days of growth in the presence of 2,4-dichlorophenoxyaceticacid (2,4-D), three times as much biomass accumulated with fructose as the carbon source as with glucose or sucrose. In the absence of 2,4-D, growth on fructose was 50% greater than on glucose but half that on sucrose. Growth for a further 1or 2 weeks removed these differences. In media without 2,4-D, the number of embryos present at each time was
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Total hexoses supplied at 15 gL-l. Number of data points at approximately 24-hour intervals used to calculate the growth rate. In all cases the correlationcoefficient was greater than 0.998. Means followed by the same letter are not significantly different (5%)by Duncan's multiple range test.
Discussion
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The conclusions on the effects of glucose/ fructose mixtures on the growth rate, biomass accumulation, and anthocyanin accumulation by the tissue are derived from experiments conducted at different times. They were verified by an experiment in which all the treatments were carried out a t the same time to rule out differences in inoculum, etc., as an explanation of the observed effects. The data from this experiment are shown in Figure 1. In addition to the mixtures of glucose and fructose, sucrose was included in this experiment to determine whether it would give the same results as the equimolar mixture of glucose and fructose. The results show that these two treatments were not distinguishable. The dry weight accumulated by the cultures declined significantly and progressively as the proportion of fructose in the carbohydrate supplied to the cultures decreased. This is consistent with the decreases in maximum biomass accumulation with decreasing fructose in the supplied carbohydrate shown in Table I. As the fructose in the supplied carbohydrate decreased to 5096, the anthocyanin in the cultures was not significantly changed. Further decrease in the fructose led to significant increases in the accumulated anthocyanin. The response of anthocyanin to changes in the proportion of fructose in the supplied carbohydrate shown in Figure 1is parallel to that seen in Table I but is 4-5 times greater. The reason for this quantitative difference may lie in the observation that different cultures of clone WC63-1-9-1-13-1 maintained separately for a year accumulate different amounts of anthocyanin (Ilan and Dougall, unpublished results). The experiments shown in Table I were all performed with tissue from one culture species, while that in Figure 1was performed with another culture series.
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Glucose as Percent of Total Initial Sugars Figure 1. Effect of sugar composition on growth of and anthocyanin accumulation in wild carrot cell cultures at day 11, with constant total initial sugars of 15 g/L. Dry weight ( 0 )and anthocyanin( 0 ) ;dry weight (A) andanthocyanin(A) withsucrose only. Points are the means of five replicate cultures. Bars represent f l standard deviation. Points with the same letter on each curve are not different at the 5% level of significance by Duncan'smultiplerange test. For sucrosethe standarddeviation of the dry weight measurements is f0.6 g/L, and for anthocyanin, *4 pmol/L of culture.
similar on both sugars. With another carrot cell suspension culture, Nagarajan et al. (1989) found that fructose gave slightly higher biomass accumulation and much lower anthocyanin accumulation than did glucose. With Anchusa officinalis suspension cultures, De-Eknamkul and Ellis (1985)found that fructose gave higher biomass and higher rosmarinic acid accumulation than did glucose. With Dioscorea deltoidea suspension cultures, Tal et al. (1982) found that fructose gave slightly higher biomass but identical amounts of diosgenin when compared to glucose. Kat0 et al. (1981) showed with tobacco cell suspension cultures that the growth rate was lower when fructose was the carbon source than when glucose was the carbon source. The fresh weight of loblolly pine callus cultures grown on fructose was approximately 10% higher than that of similar cultures grown on glucose (Vuke and Mott, 1987). Fructose gave twice as many alfalfa somatic embryos as did glucose (Strickland et al., 1987). It is to be noted here that sucrose, which on hydrolysis gives an equal mixture of glucose and fructose, gave the same results as an equal mixture of glucose and fructose. This result cannot be generalized to other plant tissue cultures. The papers cited above in the comparison of glucose and fructose effects in different cell cultures also include sucrose as a carbon source and show that a wide range of responses to each of these three sugars is found in plant tissue cultures. Improvement or optimization of the production of a secondary product in plant tissue cultures by altering the proportion of two sugars in the medium has been achieved in only one case that we could find. Nagarajan et al. (1989) found that cell yield and anthocyanin content of their carrot cell culture was highest on galactose when sugars were tested individually as carbon sources. They showed that when the total sugar was kept constant and part of the galactose was substituted with glucose or sucrose, increases in anthocyanin productivity were achieved and
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the cost of the medium decreased. They did not examine the kinetics of biomass or anthocyanin accumulation in their cultures. Here we were not able to improve the growth or anthocyanin accumulation by using mixtures of glucose and fructose. While growth was best on fructose and anthocyanin accumulation was best on glucose, on an equimolar mixture of glucoseand fructose the culture here displayed the worst features of its performance on each sugar (Table I and Figure 1). The exponential increase in anthocyanin seen in these experiments, together with the cessation of anthocyanin accumulation when growth ceases, suggests that anthocyanin accumulation is growth associated. This association makes it unlikely that major improvement in yield of anthocyanin can be achieved by a two-stage culture protocol in which cells would be grown first on fructose to give greatest biomass in the shortest time followed by growth on glucose to give greatest accumulation of anthocyanin in the biomass. Some improvement in yield may occur in the two-stage protocol as a result of the decreased time to maximum biomass and increased biomass found in growth on fructose, but this possibility has not been tested. The reason for the difference in growth rate and in biomass accumulated on fructose versus glucose and for the decrease in growth rate with increasing proportions of glucose in the sugar provided is not known, but several explanations appear plausible. One is that, relative to fructose, glucose provides only a limiting level of a compound required for growth and that, relative to glucose, fructose provides only a limiting level of a compound required for anthocyanin synthesis. Another explanation is the result of the observed accumulation of polysaccharides in the medium by the cells. This diverts some sugar and some metabolic energy into an activity that does not contribute to the growth of the cells. Perhaps more extracellular polysaccharide synthesis and less growth occurs with glucose as the carbon source than with fructose. The increase in anthocyanin with increased glucose in the
medium suggests further that glucose gives a higher level of an intermediate that is required for anthocyanin synthesis than does fructose. Both of these explanations suggest that the metabolism of glucose is a t least quantitatively different from the metabolism of fructose.
Literature Cited De-Eknamkul,W.; Ellis,B. E. Effectsof macronutrients on growth and rosmarinic acid formation in cell suspension cultures of Anchusa officinalis. Plant Cell Rep. 1985, 4, 46-49.
Dougall, D. K.; Frazier, G. C. Nutrient utilization during biomass and anthocyanin accumulation in Suspensioncultures of wild carrot cells. Plant Cell, Tissue Organ Cult. 1989, 18, 95-104.
Dougall, D. K.; LaBrake, S.; Whitten, G. H. Growth and anthocyanin accumulation rates of carrot suspension cultures grown with excessnutrients after semicontinuousculture with different limiting nutrients at several dilution rates, pHs, and temperatures. Biotechnol. Bioeng. 1983, 25, 581-594. Kato, A,;Tsuji,K. Growth-substrate relationship of tobaccocells in suspension culture. J. Ferment. Technol. 1981,59,33-36. Nagarajan, R. P.; Keshavarz, E.; Gerson, D. F. Optimization of anthocyanin yield in a mutated carrot cellline (Daucuscar0 t o ) and its implications in large scale production. J. Ferment. Bioeng. 1989,68, 102-106.
Strickland, S. G.; Nichol, J. W.; McCall, C. M.; Stuart, D. A. Effect of carbohydrate source on alfalfa somatic embryogenesis. Plant Sci. 1987, 48,113-121. Tal, B.; Gressel,J.;Goldberg,I. The effect of medium constituents on growth and diosgenin production by Dioscorea deltoidea cells grown in batch cultures. Planta Med. 1982,44,111-115. Verma, D. C.; Dougall, D. K. Influence of carbohydrates on quantitative aspects of growth and embryo formation in wild carrot suspension cultures. Plant Physiol. 1977, 59, 81-85. Vuke, T. M.; Mott, R. L. Growth of loblolly pine callus on a varietyof carbohydratesources. Plant Cell Rep. 1987,6,153156.
Accepted April 9, 1991. Registry No. Glucose, 50-99-7; fructose, 57-48-7.
