Effects of Phytohormones on Growth Phase Study of In Vitro Established Callus of Podophyllum Hexandrum Royle

Kuntal Das, Raman Dang, Rizwan Ahmad and P.E.Rajasekharan

Abstract: The callus culture from shoots (from aseptically germinated seeds) and roots (from cultivated plant) of Podophyllum hexandrum were initiated and maintained on Murashiage and Skoog (MS) basal medium with various concentrations of Phytohormones. The study showed that combined use of Naphthalene Acetic acid (NAA) and 6-Benzyl amino purine (BAP) exhibits better results (NAA at 0.5M to 1.5M and BAP at 1.5 M to 2.5 M ) for shoot callus initiation and maintenance over that of root callus. Growth phase study was measured with fresh weight method for shoot callus and revealed its growth rate correlation with sigmoid curve. Further, TLC chromatogram was developed to identify the presence of podophyllotoxin (active constituent) in shoot callus.
Podophyllum hexandrum Royle (Family: Berberidaceae), is a perennial herb. It is a source of highly valued podophyllotoxin has been subjected to heavy collection from the wild and cultivated land. It grows on the lower slopes of the Himalayas in scrub and forest from Afghanistan eastwards to central China. The ever increasing demand of Podophyllum is mainly due to two semi-synthetic derivatives obtained from rhizomes of podophyllotoxin (lignan derivative) viz. etoposide and teniposide which are used in the treatment of various types of cancer. The anticancer lignan derivative podophyllotoxin in Podophyllum hexandrum is biosynthesized at very low quantities in intact plant, so the biotechnological production of podophyllotoxin has been considered essential.
Literature survey revealed somatic embryogenesis in callus derived from zygotic embryos of P. hexandrum on MS medium supplemented with Phytohormones (Arumugam et al., 1990 and Ewald et al., 1995). Callus formation from root segments, on White medium and B5 medium with phytohormone combination were also reported (Heyenga et al., 1990 and Fujii, 1991). Production of increased levels of podophyllotoxin from cell and suspension cultures has already been initiated from root cultures (Woerdenbag et al., 1990 and Chattopadhyay et al., 2001). In relation to growth phase study, it is required to observe the effects of Phytohormones on callus growth rates in the aseptic medium. Generally the studies are used for quantitative purpose using several methods viz. fresh weight method, cell count method, dry weight method, density cell count, total cell count, mitotic index etc. Few of these methods were used for growth rate study of other species of Podophyllum and Trigonella (Kadkade, 1982 and Hardman & Stevans, 1978).
The present investigation was carried out to compare, establish and standardize condition for in vitro culture and growth rate study of initiated callus of Podophyllum hexandrum and to identify the presence of podophyllotoxin by the method.


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