STANDARDIZATION OF AN EFFICIENT AND REPRODUCIBLE EMBRYOGENIC CELL SUSPENSION CULTURE PROTOCOL FOR PRODUCTION OF SECONDARY METABOLITES IN PLUMBAGO ZEYLANICA LINNS.L. Patidar, M.K. Tripathi, G. Tiwari , R.P. Patel3 and Ashok Ahuja
In this study, a simplified procedure for establishment of embryogenic cell suspension culture from the friable embryogenic callus of Plumbago zeylanica L. was accomplished by transferring 6-8 weeks-old embryogenic calli achieved from nodal segment and leaf disc explants in liquid media. The cultures obtained were swamped with clumps of proliferating embryos of different developmental stages with modest nonembryogenic tissues. The number and size of somatic embryos/cell clumps was recorded to calculate growth rate of embryogenic tissues under different conditions. Initiation and proliferation of embryogenic cell suspension culture was manipulated by supplementation of diverse exogenous plant growth regulators to culture medium at variable concentration. For the establishment of cell suspension cultures, MS medium fortified with 2.0-3.0 mgL-1 2,4-D with 0.5 mgL-1 BAP was found to be the most effective. For subsequent subculturing, the media containing with 2.0-mgL-1 2,4-D or the reduced level of 2,4-D (1.0 mgL-1) in combination with 0.5 mgL-1 BAP supported somatic embryogenesis at a faster rate. Frequent and efficient plantlet regeneration occurred on MS medium amended with 0.5 mgL-1, each of BAP, TDZ and NAA. Full strength MS medium added with either of IBA or NAA at the concentration of 0.1 mg.L-1 was found to be optimum for exhibiting higher in vitro rooting response i.e. root proliferation, number of root(s) and root of higher length. Regenerated plantlets showed normal development and established successfully in the field after hardening with normal phenotypic appearance.
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