Enhancement of Opium Alkaloids Production in Callus Culture of Papaver Rhoeas Linn

Indian Journal of Biotechnology Vol 2, April 2003, pp 271-272

Enhancement of Opium Alkaloids Production and P. rhoeas (Khanna & Sharma, 1977). Effect of in Callus Culture of Papaver rhoeas Linn. ascorbic acid, tyrosine and auxins on the production of alkaloids is also studied in callus culture of P. Renu Sarin* somniferum (Khanna et al, 1978, Sarin et al, 1983). Tetraploid, tracheid containing callus of P. Department of Botany, University of Rajasthan, Jaipur 302 004, India somniferum reportedly produced codeine, morphine and thebaine (Erdelsky, 1978). The high yielding Received 29 May 2002; accepted 12 August 2002 tissues having large cells containing amorphous Callus culture of Papaver rhoeas Linn. established on alkaloid contents were observed in P. somniferum and revised tobacco medium showed presence of three opium P. rhoeas (Sarin & Khanna, 1989). Effect of tyrosine, alkaloids namely morphine, thebaine and narcotine. The a known precursor of opium alkaloids on the colour of the tissue varied from light grey, dark grey and production of high yielding cell lines of P. finally black depending on the age of the tissue. The high yielding cell lines of dark grey color were isolated and somniferum was also studied (Sarin, 1996). In the maintained as suspension culture on revised Murashige and present investigation various concentrations of Skoog's (1962) medium. These high yielding tissues were fed tyrosine were fed to the high yielding cell lines of P. with different concentrations (12.5, 25, SOand 100 mg/100 011) rhoeas for the enhancement of opium alkaloids for of tyrosine, a known precursor of opium alkaloids in order to commercial exploitation. further increase the alkaloid content of the tissue. The dark grey tissues grown on RT liquid medium supplemented with The callus tissue of P. rhoeas was established from 12. 5 mg/100 011 tyrosine yielded maximum percentage of seeds on revised Murashige and Skoog's tobacco alkaloids and therefore this concentration of tyrosine is medium (Khanna & Staba, 1968) supplemented with considered as the most suitable condition for the enhancement of alkaloids in Papaver rhoeas tissue culture. 1 ppm of 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 1% agar (RT). The tissue was a mixture of light Keywords: Papaver rhoeas, alkaloids, tyrosine, high-yielding grey, dark grey and finally black colour depending cell lines upon the age of the tissue. The tissue was maintained on RT medium as static culture for seven years by Papaver rhoeas Linn. (Raktapost; Lalposta) is a frequent subculturings after every 4-6 weeks and common garden plant, cultivated in gardens subsequently clumps of different colours were throughout the plains of northern India. The latex of separated, multiplied and maintained on RT liquid capsules is narcotic and slightly sedative as it contains medium for six months by frequent subculture after some opium alkaloids. Opium, the inspissated milky every 14 days. The tissues of different colours grown latex from immature capsules of Papaver somniferum, on RT liquid medium for 6 months were harvested contains major opium alkaloids such as morphine, separately after 14 days, dried and extracted for major codeine, thebaine, narceine, narcotine and papaverine. opium alkaoids (Longman & De Bussy, 1972). Among these alkaloids morphine is purely narcotic Three such replicates of these samples were and is normally reserved for severe pain when other examined and mean values taken. Detection and analgesics have failed to give relief. Thebaine has determination of three opium alkaloids, morphine, almost the same analgesic effect as morphine but it is thebaine and narcotine was done in all the samples by the most poisonous of the opium alkaloids. It is co-chromatography, mp and superimposable IR scarcely used as such in therapy but is used in the (Perkin-Elmer 337) spectra of the isolated compounds form of its derivatives. Narcotine is widely used in the with their respective authentic samples by the preparation of cough linctus (Jain & Dahiya, 1974). methods described earlier (Khanna & Khanna, 1976; Production of opium alkaloids has been reported Khanna & Sharma, 1977). Total alkaloid content of from callus tissue cultures of Papaver somniferum each sample of the tissue was calculated on dry Linn. (Khanna & Khanna, 1976, Staba et al, 1982) weight basis. The high yielding cell lines of dark grey colour *Tel: 0141-2701875 were separately transferred to RT liquid medium E-mail: [email protected] supplemented with different concentrations (12.5, 25, 272 INDIAN 1 BIOTECHNOL, APRIL 2003

Table I-Papaver rhoeas Linn. tissue growth and alkaloid tyrosine (12.5 mgllOO ml) in the medium further content (on dry wt basis) in static cultures, suspension cultures, increased the percentage of total alkaloid content in cell-lines of different colours and high yielding cell lines supplied this cell line (3.6% crude, Table 1). with tyrosine. From the results obtained it is concluded that Type of culture Growth Alkaloid content incorporation of tyrosine, a known precursor of these index (%) alkaloids at a concentration of 12.5 mg/lOO ml in the (i) Static culture- high yielding cell lines of Papaver rhoeas is the most (Age in Weeks) favourable condition for the enhancement of the 2 2.6 1.0 opium alkaloids in vitro. This can be utilized 4 5.6 1.3 6 3.8 1.8 commercially for the production of three opium 8 2.9 0.7 alkaloids viz. morphine, thebaine and narcotine.

(ii) Suspension culture-(14 Days old cell lines of different References colorus) Erdelsky K, 1978. in 4th Int Congr Plant tissue and cell culture, edited by T Thorpe. University of Calgary, Canada. Light Grey 5.5 1.7 Dark Grey 3.5 2.4 Jain G C & Dahiya M S, 1974. Separation and identification of Black 2.2 2.3 opium alkaloids by thin layer chromatography. Curr Sci, 43, 444. (iii) High yielding cell lines incorporated with tyrosine Khanna P & Khanna R, 1976. Production of major alkaloids from (mg/100 ml) in vitro tissue culture of Papaver somniferum Linn. Indian J Control 3.5 2.4 Exp Bioi, 14,628-629. 12.5 3.8 3.6 Khanna P & Sharma G L, 1977. Production of opium alkaloids 25 3.4 3.2 from in vitro tissue culture of Papaver rhoeas Linn. Indian J 50 2.8 2.3 Exp Bioi, 15, 951-952. 100 2.5 1.6 Khanna P & Staba J, 1968. Antimicrobials from plant tissue culture. Lloydia. 31. 180. Final dry wt of the tissue-Initial dry wt of the tissue Khanna et al, 1978. Production of free ascorbic acid and effect of Growth Index = ------exogenous ascorbic acid and tyrosine on production of major Initial dry wt of the tissue opium alkaloids from in vitro tissue cultures of Papaver somniferum Linn. Indian J Exp Bioi. 16. 110-112. Longman & De Bussy J H. 1972. Natural Organic Materials and 50 and 100 mg/lOO ml of tyrosine. The tissues were Related Synthetic Product. Elsevier Publishing Co., London. harvested after 14 days and analyzed quantitatively Pp 730. for their alkaloid contents. Murashige T & Skoog F, 1962. A revised medium for rapid Three-opium alkaloids viz. morphine, thebaine and growth and bioassays with tobacco tissue cultures. Physiol Plant, 16,473-497. narcotine were confirmed in all the samples of tissue. Sarin R, 1996. Effect of tyrosine on the production of alkaloids in The total alkaloid content in static cultures was the high yielding cell lines of Papaver somniferum tissue maximum (1.8 crude, Table 1) in 6 weeks old tissue. culture. J Plant Biochem Biotechnol, 5, 61-62. In the suspension culture of the callus of three colours Sarin R. et al, 1983. Effect of some growth regulators on (light grey, dark grey and black) the amount of production of opium alkaloids in vitro tissue culture of alkaloids was maximum in the dark grey tissue (2.4% Papaver somniferum Linn. J Indian Bot Soc, 62, 73-76. crude). So it is concluded that the dark grey tissue is Sarin R & Khanna P, 1989. High yielding tissues of two Papaver species grown in vitro. J Indian Bot Soc, 68, 95-96. the high yielding cell line and therefore it is selected Staba E J et al, 1982. Alkaloid production from Papaver tissue for the enhancement of alkaloids. Incorporation of cultures. Lloydia, 45. 256.