Table I.Nutrient partitioning in different culture conditions. 3.Davies,M.E.1972.Polyphenol synthesis in cell suspension cultures of Paul's scarlet rose.Planta 104:50-65. Yield coefficient Control N rich C rich 4.Davies,M.E.1972.Effects of auxin on polyphenol accumulation and the development of phenylalanine ammonia-lyase activity in YXIN 0.671±0.12 0.374±0.06 0.53±0.26 darkgrown suspension cultures of Paul's scarlet rose.Planta 104 YxIC 0.16±0.05 0.06±0.006 0.017±0.003 66-77. YPix 0.067±0.02 0.004±0.001 0.12±0.02 5.Do,C.B.,Cormier,F.1991.Effects of low nitrate and high Individual yield coefficients represent the averages from five sets of data sugar concentrations on anthocyanin content and composition of for control conditions and four sets of N-rich or C-rich data.YxIN g grape (Vitis vinefera L.)cell suspension.Plant Cell Rep.9: dry weight cells/mmol NO3.Yxic=g dry weight cells/mmol sucrose. 500-504. Yp/x mg shikonin derivatives/g dry weight cells. 6.Fujita,Y.,Hara,Y.,Suga,C..Morimoto,T.1981.Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon.II.A new medium for the production of shikonin exclusiveness of growth and shikonin production.Thus, derivatives.Plant Cell Rep.1:61-63. the stimulation of shikonin production by C-rich medium 7.Fujita,Y.1988.Shikonin production by plant (Lithospermum ery- cannot be explained entirely by diversion of precursors throrhizon)cell cultures.pp.225-236 In:Y.P.S.Bajaj (ed.),Biotech- nology in agriculture and forestry,vol.4,Medicinal and aromatic for growth into the shikonin biosynthetic pathways as plants I.Springer-Verlag,Berlin,Heidelberg. suggested by Mizukami et al.3.The lack of repeated stimu- 8.Heide,L.,Tabata,M.1987.Enzyme activities in cell-free extracts lation by C-rich medium indicated a saturation of metabolic of shikonin producing Lithospermum erythrorhizon cell suspension activity.We also observed an 8-11%drop in cell viability cultures.Phytochemistry 26:1645-1650. between days 24 and 36 from the value on day 24 (71% 9.Heide,L,Nishioka,N.,Fukui,H.,Tabata,M.1989.Enzymatic regulation of shikonin biosynthesis in Lithospermum erythrorhizon measured by phenosafranin dye exclusion).It is also pos- cell cultures.Phytochemistry 28:1873-1877. sible that successive additions of C-rich medium resulted 10.Janglova,Z.,Suchy,J.,Vanck,Z.1969.Regulation of biosynthesis in altered cellular physiology.Interim cultivation in N-rich of secondary metabolites.7.Intracellular adenosine-5'-triphosphate medium alleviated this problem. concentration in Streptomyces aureofaciens.Folia Microbiol.13 Although the literature is rich with instances of hor- 208-210. 11.Madry,N.,Sprinkmeyer,R.,Pape,H.1979.Regulation of tylosin monal regulation and regulation by specific precursors of synthesis in Streptomyces:Effects of glucose analogs and inorganic secondary metabolites,the effect of carbon and nitrogen phosphate.Eur.J.Appl.Microbiol.Biotechnol.7:365-370. source is still significant.As the relatively small changes 12.Merillon,J.M.,Rideau,M.,Chenieux,J.C.1984.Influence of sucrose in cell and product yields following switching between on levels of ajmalicine,serpentine and tryptamine in Catharanthus roseus cells in vitro.Planta Med.50:497-501. media demonstrates,it is possible to significantly improve 13.Mizukami,H.,Konoshima,M.,Tabata,M.1977.Effect of nutritional overall shikonin production by cycling the environment of factors on shikonin derivative formation in Lithospermum erythrorhi L.erythrorhizon cell cultures between N-rich and C-rich zon callus cultures.Phytochemistry 16:1183-1186. states.It is expected that by cultivation under semicontin- 14.Sarkissian,G.S.,Fowler,M.W.1986.The metabolism and uous or continuous conditions,better control over product utilization of carbohydrates by suspension cultures of plant cells. and cell yields can be achieved.Such studies are being pp.151-181 In:M.J.Morgan (ed.).Carbohydrate metabolism in cultured cells.Plenum,New York and London. actively considered. 15.Seitz,H.U.,Eilert,U.,De Luca,V.,Kurz,W.G.W.1989.Elicitor We thank Dr.Liu Chang Ryul at the Genetic Research Institute in mediated induction of phenylalanine ammonia lyase and tryptophan Daeduck,Korea for providing the Lithospermum erythrorhizon cell decarboxylase:Accumulation of phenols and indole alkaloids in cell line and the National Science Foundation for its partial support of suspension cultures of Catharanthus roseus.Plant Cell Tissue and this research. Organ Culture 18:71-78. 16.Srinivasan,V.,Ryu,D.D.Y.1992.Enzyme activity and shikonin production in Lithospermum erythrorhizon cell cultures.Biotechnol. References Bioeng.40:69-74. 17.Tabata,M.,Mizukami,H.,Hiraoka,N.,Konoshima,M.1973.Pig- 1.Bradford,M.M.1976.A rapid and sensitive method for the quan- ment formation in callus cultures of L.erythrorhizon.Phytochemistry titation of microgram quantities of protein using the principles of 13:927-932. protein-dye binding.Anal.Biochem.72:248-254. 18.Tabata,M..Fujita,Y.1985.Production of shikonin derivatives 2.Cormier,F.,Crevier,H.A.,Do,C.B.1990.Effects of sucrose by plant cell cultures.pp.207-218 In:M.Zaitlin,P.Day,and concentration on the accumulation of anthocyanins in grape (Vitis A.Hollaender (eds.)Biotechnology in plant science.Academic, vinifera)cell suspension.Can.J.Bot.68:1822-1825. Orlando,FL. SRINIVASAN AND RYU:ALTERNATING CARBON AND NITROGEN FEEDING 799Table I. Nutrient partitioning in different culture conditions. Yield coefficient Control N rich C rich YXIN 0.671 0.12 0.374 f 0.06 0.53 z 0.26 YXlC 0.16 * 0.05 0.06 * 0.006 0.017 * 0.003 YPlX 0.067 2 0.02 0.004 f 0.001 0.12 -c 0.02 Individual yield coefficients represent the averages from five sets of data for control conditions and four sets of N-rich or C-rich data. YXIN = g dry weight cells/mmol NO3-. YX/C = g dry weight cells/mmol sucrose. Yplx = mg shikonin derivatives/g dry weight cells. exclusiveness of growth and shikonin production. Thus, the stimulation of shikonin production by C-rich medium cannot be explained entirely by diversion of precursors for growth into the shikonin biosynthetic pathways as suggested by Mizukami et al.13. The lack of repeated stimu￾lation by C-rich medium indicated a saturation of metabolic activity. We also observed an 8-11% drop in cell viability between days 24 and 36 from the value on day 24 (71% measured by phenosafranin dye exclusion). It is also pos￾sible that successive additions of C-rich medium resulted in altered cellular physiology. Interim cultivation in N-rich medium alleviated this problem. Although the literature is rich with instances of hor￾monal regulation and regulation by specific precursors of secondary metabolites, the effect of carbon and nitrogen source is still significant. As the relatively small changes in cell and product yields following switching between media demonstrates, it is possible to significantly improve overall shikonin production by cycling the environment of L. erythrorhizon cell cultures between N-rich and C-rich states. It is expected that by cultivation under semicontin￾uous or continuous conditions, better control over product and cell yields can be achieved. Such studies are being actively considered. We thank Dr. Liu Chang Ryul at the Genetic Research Institute in Daeduck, Korea for providing the Lithospermum erythrorhizon cell line and the National Science Foundation for its partial support of this research. References 1. 2. Bradford, M. M. 1976. A rapid and sensitive method for the quan￾titation of microgram quantities of protein using the principles of protein-dye binding. Anal. Biochem. 72: 248-254. Cormier, F., Crevier, H.A., Do, C.B. 1990. Effects of sucrose concentration on the accumulation of anthocyanins in grape (Vitis vinifera) cell suspension. Can. J. Bot. 68: 1822-1825. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. Davies, M. E. 1972. Polyphenol synthesis in cell suspension cultures of Paul’s scarlet rose. Planta 104: 50-65. Davies, M.E. 1972. Effects of auxin on polyphenol accumulation and the development of phenylalanine ammonia-lyase activity in darkgrown suspension cultures of Paul’s scarlet rose. Planta 104: 66-77. Do, C.B., Cormier, F. 1991. Effects of low nitrate and high sugar concentrations on anthocyanin content and composition of grape (Vitis vinefera L.) cell suspension. Plant Cell Rep. 9: 500-504. Fujita, Y., Hara, Y., Suga, C., Morimoto, T. 1981. Production of shikonin derivatives by cell suspension cultures of Lithospermurn erythrorhizon. 11. A new medium for the production of shikonin derivatives. Plant Cell Rep. 1: 61-63. Fujita, Y. 1988. Shikonin production by plant (Lithospermum ery￾throrhizon) cell cultures. pp. 225-236 In: Y. P. S. Bajaj (ed.), Biotech￾nology in agriculture and forestry, vol. 4, Medicinal and aromatic plants 1. Springer-Verlag, Berlin, Heidelberg. Heide, L., Tabata, M. 1987. Enzyme activities in cell-free extracts of shikonin producing Lithospermum erythrorhizon cell suspension cultures. Phytochemistry 26: 1645-1650. Heide, L., Nishioka, N., Fukui, H., Tabata, M. 1989. Enzymatic regulation of shikonin biosynthesis in Lithospermum erythrorhizon cell cultures. Phytochemistry 28: 1873- 1877. Janglova, Z., Suchy, J., Vanck, Z. 1969. Regulation of biosynthesis of secondary metabolites. 7. Intracellular adenosine-5’-triphosphate concentration in Streptomyces aureofaciens. Folia Microbiol. 13: 208 - 21 0. Madry, N., Sprinkmeyer, R., Pape, H. 1979. Regulation of tylosin synthesis in Streptomyces: Effects of glucose analogs and inorganic phosphate. Eur. J. Appl. Microbiol. Biotechnol. 7: 365-370. Merillon, J. M., Rideau, M., Chenieux, J. C. 1984. Influence of sucrose on levels of ajmalicine, serpentine and tryptamine in Catharanthus roseus cells in vitro. Planta Med. 50: 497-501. Mizukami, H., Konoshima, M., Tabata, M. 1977. Effect of nutritional factors on shikonin derivative formation in Lithospermum erythrorhi￾zon callus cultures. Phytochemistry 16: 1183- 1186. Sarkissian, G. S., Fowler, M. W. 1986. The metabolism and utilization of carbohydrates by suspension cultures of plant cells. pp. 151-181 In: M.J. Morgan (ed.), Carbohydrate metabolism in cultured cells. Plenum, New York and London. Seitz, H. U., Eilert, U., De Luca, V., Kurz, W.G. W. 1989. Elicitor mediated induction of phenylalanine ammonia lyase and tryptophan decarboxylase: Accumulation of phenols and indole alkaloids in cell suspension cultures of Catharanthus roseus. Plant Cell Tissue and Organ Culture 18: 71-78. Srinivasan, V., Ryu, D. D. Y. 1992. Enzyme activity and shikonin production in Lithospermum erythrorhizon cell cultures. Biotechnol. Bioeng. 40: 69-74. Tabata, M., Mizukami, H., Hiraoka, N., Konoshima, M. 1973. Pig￾ment formation in callus cultures of L. erythrorhizon. Phytochemistry 13: 927-932. Tabata, M., Fujita, Y. 1985. Production of shikonin derivatives by plant cell cultures. pp. 207-218 In: M. Zaitlin, P. Day, and A. Hollaender (eds.), Biotechnology in plant science. Academic, Orlando, FL. SRlNlVASAN AND RYU: ALTERNATING CARBON AND NITROGEN FEEDING 799