Reproducible and expedient rice regeneration system using in vitro grown plants

Abstract

Inevitable prerequisite for expedient regeneration in rice is the selection of totipotent explant and developing an apposite combination of growth hormones. Here, we reported a reproducible regeneration protocol in which basal segments of the stem of the in vitro grown rice plants were used as ex-plant. Using the protocol, callus was developed from the exposed cells of root segments and cortical tissues of basal part of the stem. Various levels of 2,4-dichlorophenoxyacetic acid (2,4-D) were used, where 1 mg/L was found as best level for callus induction. Further, 25 combinations of kinetin and naphthalene acetic acid (NAA) were developed to investigate the regeneration response of the calli. The root-derived calli did not respond to any combination at all, whereas the nodular calli derived from the stem segments responded variably to kinetin and NAA combinations in the Murashige and Skoog (MS) medium from base to top. Higher kinetin to NAA ratios promoted embryogenesis, whereas lower ratios exhibited rhizogenesis. A combination of 3 mg/L kinetin and 1 mg/L NAA was established to be the best combination for plant regeneration through embryogenesis in rice from in vitro grown plants. Plants regenerated in vitro were successfully acclimatized in the pots, where they exhibited phenotypically indistinguishable normal growth when compared with plants developed from seed, hence the developed regeneration system for rice in these studies may be treated as one of the best strategies to in vitro clonal propagation and purification, ahead of seasonal growth of plants in the field or green house.

Key words: Rice, in vitro regeneration, explants, naphthalene acetic acid (NAA), kinetin, rhizogenesis.