Influence of in vitro culture age on morphology, antioxidant activities, reserpine production, and genetic fidelity in Indian snakeroot (Rauvolfia serpentina (L.) Benth. ex Kurz.)

Abstract

The present study was carried out on Rauvolfia serpentina (L.) Benth. ex Kurz., for the first time, to assess the impact of in vitro culture age on morphology, antioxidant activities, and reserpine production. Fresh in vitro multiple shoot culture was established in Murashige and Skoog semisolid medium fortified with 1 mg/l N6-benzyladenine, while another pre-established in vitro multiple shoot culture was maintained in similar medium for two years. Comparisons were made among two-year-old in vitro culture, six-month-old in vitro culture and six-month-old ex vitro plantlet, wherein six-month-old ex vitro plantlet recorded significantly longer shoots than in vitro cultures. The internode lengths of six-month-old ex vitro plantlet, six-month-old in vitro and two-year-old in vitro cultures were significantly different from each other. The leaf area of six-month-old ex vitro plantlet was highest, followed by that of the six-month-old in vitro and two-year-old in vitro cultures, respectively. Reduction of superoxide dismutase activity was recorded in six-month-old in vitro culture than six-month-old ex vitro plantlet, but was found to be comparable to two-year-old in vitro culture. Marginal increase in peroxidase activity was detected in two-year-old in vitro culture when compared to six-month-old in vitro culture. High-performance thin-layer chromatography analysis detected least reserpine accumulation from six-month-old ex vitro plantlet, whereas, a surge in reserpine was recorded from both six-month-old in vitro and two-year-old in vitro cultures. Eventually, inter simple sequence repeats and start codon targeted polymorphism primers revealed a 6.79% of genetic variation in two-year-old in vitro culture from six-month-old ex vitro plantlet and six-month-old in vitro cultures. The study revealed that for morphological traits and antioxidant activities, a small-scale effect was imposed by tissue culture condition for at least up to two years. However, for enhanced (two-fold) accumulation of reserpine, long-term in vitro culture would be of commercial importance but in the cost of certain extent of genetic variation.