Gamma ray-induced tissue responses and improved secondary metabolites accumulation in Catharanthus roseus

Abstract

The present study investigated the impact of gamma ray irradiation on callus biomass growth and the yield of vincristine and vinblastine of in vitro grown tissues of Catharanthus roseus. The biochemical alteration underlying the synthesis of secondary metabolites has also been studied and a comparison of yield was prepared. The embryogenic tissues were exposed to 20, 40, 60, 80, and 100 Gy gamma ray doses and the callus biomass fresh weight, the embryogenesis (the embryo numbers, germination, plant regeneration), the alteration of protein, proline, and sugar attributes at different morphogenetic stages were monitored. The callus biomass growth was maximum (1.65 g) in 20 Gy exposed tissues and was less in 100 Gy treatment (0.33 g). The gamma-irradiated embryogenic tissues differentiated into embryos but the embryogenesis % and somatic embryo number per culture reduced with increasing doses. It was least in 80 Gy where very low numbers of embryos were formed (3.45 and 3.30 mean torpedo and cotyledonary embryo numbers per callus mass, respectively) which later germinated into plantlets. Protein, proline, sugar, and different antioxidant enzymes, i.e., superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) activities, were investigated as the tissues were exposed to gamma ray elicitation/signaling, evoking cellular stress. Increased 80 Gy gamma dose inhibited a 42.73% decrease in protein accumulation at initiation stages of embryogenic tissue. Soluble sugar level also declined gradually being least in 80 Gy treated tissues (14.51 mg gm^-1 FW) compared to control (20.2 mg gm^-1 FW). Proline content, however, increased with increasing gamma doses, maximum at 80 Gy (8.28 mg gm^-1 FW). The SOD, APX, and CAT activity increased linearly with enhanced level of gamma doses and maximum, i.e., 3.91 EU min^-1 mg^-1, 1.71 EU min^-1 mg^-1, and 4.89 EU min^-1 mg^-1, protein activity was noted for SOD, APX, and CAT, respectively, at 80 Gy gamma rays treated tissues. The quantification of vinblastine and vincristine in gamma ray elicitated tissues was made by using high-pressure thin layer chromatography (HPTLC). Somatic embryo-regenerated plant's leaves had the maximum yield of vinblastine (15.13 µgm gm^-1 DW) at 40 Gy irradiation dose compared to control (13.30 µgm gm^-1 DW)-the increased yield % is 13.75. The stem is also rich source producing 11.98 µgm gm^-1 DW of vinblastine. Among the various developmental stages of embryos, vinblastine content was highest in germinating stage of embryos (10.14 µgm gm^-1 DW) compared to other three, i.e., initiation, proliferation, and maturation embryo stages. Similarly, highest accumulation of vincristine (6.32 µg gm^-1 DW) was noted at low gamma irradiation dose (20 Gy) in leaf tissues. The present study indicates that the synthesis of vinblastine and vincristine was growth- and development-specific and the lower 20-40 Gy gamma levels were more effective in enriching alkaloids while higher doses declined yield. KEY POINTS: • Vinblastine and vincristine yield was quantified in in vitro grown tissues and leaves of embryo regenerated Catharanthus roseus after gamma ray treatment. • The accumulation of vinblastine and vincristine was maximum in regenerated leaves; low doses were more efficient in improving yield. • Gamma ray irradiation impacted biochemical profiles, caused cellular stress, and perhaps responsible for improved alkaloid yield.

Keywords: Embryogenic tissue; Gamma irradiation; HPTLC; Stress; Vinblastine; Vincristine.