B i o t e c h n o l o g y Letters Vol 13 No 2 R e c e i v e d as r e v i s e d 24th J a n u a r y
125-130
(1991)
D E V E L O P M E N T OF M E D I A FOR G R O W T H OF D I O S C O R E A D E L T O I D E A CELLS AND IN VITRO DIOSGENIN PRODUCTION: INFLUENCE OF MEDIA CONSTITUENTS AND NUTRIENT STRESS
Autotrophic
G.A. RAVISHANKAR Cell Culture Discipline, Central Food Technological Institute ( C S I R ) , M y s o r e - 570 0 1 3 , I n d i a
Research
S. G R E W A L
Plant Tissue
Culture Lab, Regional Research Jammu-Tawi 180 001, India
Laboratory
(CSIR),
SUMMARY
Callus culture of Dioscorea deltoidea produced diosgenin and sterols during stationary phase. Ammonium nitrate (420 mg Nitrogen/l) as sole nitrogen source supported better growth than a combination of ammonium nitrate and potassium nitrate (totally equivalent to 840 mg Nitrogen/l). The production of diosgenin increased under low phosphate concentration (100 mg/l) whereas high phosphate concentration (240 mg/!) promoted growth. Micronutrients, when used at 189 strength, enhanced growth and diosgenin production. Depletion of nitrogen increaseci the diosgenin synthesis by a factor of 2. Adoption of a two staBe culture method enhanced the diosgenin production in cultured cells by eight-fold. INTRODUCTION
Production of pharmaceutically important compounds using plant ceils cultured in vitro has been a subject of intense study (Misawa, 1985). Diosgenin, a steroicl, is commercially obtained from Dioscorea tubers, and is an important steroidal base for the manufacture of pharmaceutically useful compounds. The development of a two stage culture system, with suitably desiqned media for growth and metabolite prociuction by cultured plant ceils, has led to improved growth rate and production (Sasse et al., 1982). In this communication, we report the development of media for growth of D. deltoidea cells and tissues, and for in vitro diosgenin production, with a view to enhance yield potentials. MATERIALS AND METHODS
Cultural conditions: T h e callus of D. deltoidea was initiated from hypocotyls of 10 day ohi seedlings on 40 ml MS medium (Murashige and Skoog , 1962) supplemented with i mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 3% sucrose. Callus was maintained on the same medium b~ regular subcultures at 45 day intervals. Cultures were incubated at 25 + 2 C in continuous light (3000 Lux)~ Changes in the constituents of the experimental media were effected as described in the "Results" section. Initial inoculum was 500 + 20 mg fresh tissue, and cultures were incubated under the above conditions. Growth: Growth of ceils and of callus dry-weight. Dry weight was determined to constant weight.
tissues was measured in terms of fresh and after drying, in a hot-air oven at 60~
Diosgenin quantitation: Extraction of diosgenin and sterols from dry cells was carried out by the method of Heble and Staba (1980). The sterol and diosgenin was quantitated by spectrophotometric method followed by preparative TLC (Sanchez et al., 1972). Nutrient Stress:
For studies
* To whom correspondence
involving
should
nutrient
be addressed
125
stress,
200 + 30 mg of callus was
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Table
I. Effect of nitrate/phosphate stress on the growth of D. deltoidea r and diosgenin production.
Growth (30th day) Treatment
I. DD-Control 2. DD-NS 3. DD-PS
Fresh Weight (mg/20 ml)
Dry Weight (mg/20 ml)
3840 + 150 3630 ~ 155 3610 + 115
234 + 22 220 ~ 16 225 + 17
Diosgenin % on dry weight basis (30th day)
0.42 + 0.03 0.81 ~ 0.04 0.48 + 0.03
Inoculum size 200 + 12 mg fresh tissue. In all the above treatments, callus was grown for 24 days on standard MS medium, before transfer to control or nutrient stress media. The growth values on 24th day was fresh weight 2265 + 130 mg/20 ml medium, or equivalent dry weight was 210 +--20 mg/20 ml medium; and the diosgenin content was 0.29 ~ 0.02% on dry weight basis. The data in this table were recorded 6 days after transfer, i.e., on 30th day of culture. - DD-control: Standard medium; DD-NS: Nitrate Stress Medium; DD-PS: Phosphate Stress Medium. q
Table 2.
Hodified media for growth and production of diosgenin in cultured cells of D deltoidea
Constituents
Ammonium nitrate Potassium nitrate KH~PO 4
CuSO, NaMo~ 4 CoCI 2 Vitamins Iron (Chelated) Sucrose 2,4-D
Growth medium mg/l 420 N Nil 240 9.3 0.41 0.05 0.50 0.05 Full Strength 189 strength 30,000 1.0
Other constituents as in MS medium
127
Production medium mg/l Nil Nil I00 9.3 0.41 0. {)5 0.50 0.05 189 strength Full strength 30,000 1.0
Other constituents as in MS medium
grown on a filter paper (Whatman No.l) platform placed in I00 ml Erlenmeyer flasks containing 20 ml MS liquid medium with 1.0 mg/l 2,4-D and 3% sucrose. The medium was decanted out after 24 days of growth of callus, and replaced by 20 ml of stress medium i.e. MS medium lacking nitrate or phosphate. Controls received standard MS salts with 1.0 mg/l 2,4-D and 3% sucrose. The growth and diosgenin content were recorded (Table i) from five replicate cultures on 3Oth day. Two stage culture of cells: In the first stage of cultures, Dioscorea cells of 500 mg (+ 30mg) fresh weight were inoculated into a modified liquid growth medium (Table 2~. They were incubated in continuous light, for 25 days, on a rotary shaker running at i00 RPM. Later, the cells were filtered in a sterilized setup, using Whatman No.l filter paper, in the second stage, they were transferred to the production medium (Table 2). The growth and diosgenin content were recorded (Table 3). RESULTS Growth of callus and steroid production: Dioscorea callus entered a stationary phase after 35 days and accelarated synthesis of diosgenin or sterols followed this phase. On the 9Oth day the maximal production of diosgenin was 0.52%, or 2622 ~g/40 ml medium. Total sterols constituted 0.62% of dry weight of callus and a sterol production of 3250 ~g/40 ml medium was recorded on 90th day (Fig. IA and IB). Influence of nitrogen sources: The callus tissues were fed with ammonium nitrate or potassium nitrate (Fig. Z) as nitrogen sources, at three concentrations, viz:420, 84{) and 1260 mg/l nitKogen (N) equivalents. Standard ~IS medium contains ammonium nitrate and potassium nitrate, totally equivalent to 840 mg/l of N. Controls received standard MS salts. Ammonium nitrate or potassium nitrate, at 420 or 840 mg/l N concentration, enhanced the growth as compared to the control, but diosgenin yield was reduced on percentage or absolute basis. The combination of ammonium nitrate and potassium nitrate (MS medium) was found to be superior in promoting diosgenin production in a single stage culture system. Ammonium nitrate, at ~20 mg/l nitrogen concentration, favoured growth, and this concentration was later adopted for the growth medium, in the two-stage culture system. Influence of phosphate: The standard MS medium contains 170 mg/l KH~PO I . In this set of experiments, levels of KI[zPO4 used were: 100, 170 and 240~mg/l (Fi~. 3A). Growth at 240 mg/l KH?PO 4 was superior to control, whereas, at the lower level (100 mg/l), diosgenin prSduction was increased by a factor of 1.5. This indicated that the growth medium should contain a hiBh level of KHZ?Od, a low level of KH2PO 4 is preferable for diosgenin production. Influence of minor elements, vitamins and iron: Standard MS medium (control) contains 6.2 mg/l H3BO3, 0.83 mg/L KI, 0.025 mg/l CuSO 4 SHOO, 0.25 mg/l of NaMoO 4 2H~O and 0.025 mg/l CoCI~ 6H.O. When various levels of Ehe minor elements were administered (Figs. 3A &~3B)f it became clear that higher levels of H3BO3, CuSO 4 and NaMoO. favoured higher diosgenin production, as compared to the control The optimal levels of minor elements for growth were HqBO 3 9.3 mg/l, Ki 0.~I mg/l, CuSO, 0.05 mg/l, NaMoO. 0.50 mg/l and CoCI 2 0.05 mgTl. These level were also foun~ suitable for d~osgenin production. Elevation of vitamin or iron concentration to 1 8 9 strength of MS medium marginally enhanced diosgenin content and growth, respectively. Whereas their reduction to 89 strength redhced growth and diosgenin content (Fig.4). Effect of nitrate/phosphate stress on g r o w t h and d i o s g e n i n production in two stage culture: Growth of cells on standard medium (control), under the experimental conditions showed a nearly ten fold increase [n 24 days (Table I).
128
Table 3.
Comparison of yield of cell biomass and diosgenin production in single stage and two stage cultures a
Nature of culture
Age
25th day
Growth cells
of
Fresh Wt 3200 + 150mg
Diosgenin content
% 0.390 + 0.02 900 + 10--~g/40 ml medium
Dry wt. Control (single stage,
230 + 5 mg 35th day
MS Medium)
Fresh wt. 4100 + 100mg
=' 0.420 + 0.02 1640 + 3~ ~ g / 4 0
ml medium
Dry wt. 390 + 15 mg
First stage
25th day on modified ~rowth medium
Fresh wt. 7230 + 48 m E
~'
0.410 + C).04
3000 + 145 ~ g / 4 0
ml medium
Dry wt. 730 + 8 mg
Two
stage Seconci stage
Subsequently for i0 days on production medium
Fresh Wt. 7700 + 40 mg
% 1.825 + 0.07 15510 + 1-30 ~ g / 4 0
mi medium
Dry wt. 850 + 15 mg
a
Inoculum
: 500 + 25 mg fresh weight of cells; Voiume of culture medium an average of i0 replicates with standarcl errors.
40 ml; Data represents
s
129
was
Under stress conditions, the growth of callus on the experimental medium (DD-NS; DD-PS) was lower than in the control medium (DD-Control). Diosgenin yield was almost doubled, in the nitrate-stressed medium, whereas, only a marginal increase in diosgenin synthesis was obtained in the phosphate-stressed medium (Table i). Modified media for growth and diosgenin production were formulated (Table 2), based on the data obtained in single stage culture (Figures 2 to ~) and stress inductive medium (Table I), the callus grown on modifiedrmedium showed nearly double the growth (Table 3), while diosgenin production was increased more than four-fold on percentage basis, and about nine-fold on absolute basis. DISCUSSION
A combination of different nitrates and reduction of phosphate level increased diosgenin production in the single stage system. We have reported that an altered ratio of carbon-nitrogen-phosphorus also influenced growth and diosgenin synthesis (Ravishankar and Grewal,1988). An earlier report with Acer pseudoplatanus Cells (Simpkin et al., 1970) showed good growth on a nitrate salt as sole nitrogen source. Similarly, improved growth of tobacco ~ells (Ikeda et al., 1977) and ubiquinone formation were obtained with a nitrate salt, as the sole nitrogen source. Our results indicate that nitrate or ammonia nitrogen, when used alone, is less effective for diosgenin production in a single-stage culture, A higher KH PO (240 mg/l) level was found suitable for growth, whereas, a lower level2(l~0 mg/l) was more suitable for diosgenin production. Nitrogen was required for optimal formation of caffeoyl putrescine (Knobloch and Berlin, 1981) and alkaloids in Paganum. On' the other hand it repressed the induction of alkaloid biosynthesis in Catharanthus (Knobloch and Berlin, 1980). In this study, the profound influence of nitrogen stress on diosgenin production has been revealed. Phosphate has been shown to be taken up by Nicotiana and Catharanthus in the first 18 11 of the growth cycle. The accumulated Pi is then distributed among the dividing cells (Sasse et al., 1982). Knobloch et ai., (1981) opine that, only when intracellular phosphate is sufficiently depleted, do the cells become capable of secondary metabolite formation in an induction medium. Talet al., (1983) have demonstrated repressive effect of phosphate added during the post-exponential phase. Our study has demonstrated that low phosphate favours diosgenin production in a sinBle-stage system. On the other hand, phosphate stress did not improve the tissue's ability to synthesise diosgenin. The growth and production media reported here will be very useful in scaling up this system for diosgenin production. REFERENCES
Heble, M.R., and Staba, E.J. (1980). Planta Medica Suppl. 124-128. Ikeda l.,Matsumoto,T.,and Noguchi,M. (1977). Agric Biol Chem. 41, 1197-1202. Knobloch, K.H., and Berlin, J. (1980). Z. Naturforsch. 35C, 551-556. ~nobloch, K.H., and Berlin, J. (1981). Planta Mediea 42, 167-172. Knoblor K.H., Beutflagel, G., and Berlin, J. (1981). Planta 153, 582-585. Misawa, M. (1985) In: Advances in Biochemical Engineering/Biotechnology Vol.31, A. Fiechter, ed. pp. 59-88. Berlin: Springer-Verlag. Murashige, T., and Skoog, F. (1962). Physiol. Plant. 15, 473-497. Ravishankar, G.A., and Grewal, S. (1988). Curr Sci. 57, 679-681. Sanchez L.G., Acevedo J.C.M., and Soto, R.R. (1972). Analyst 97, 973-976. Sasse, F., Knobloch, K.H., and Berlin, J. (1982). In: Proc. 5th Intl. Cong. Plant Tissue & Cell culture, A. Fujiwara, ed. pp 343-344. Tokyo: Abe Photoprinting Co. Ltd. Simpkin,J.,Collin, H.A.,and Street H.E. (1970). Physiol. Plant. 23, 385-396. Tal, B.,Rokem, J.S., and Goldberg, I. (1983). Plant Cell Reports 2, 219-222.
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