Direct Plant Regeneration via Multiple Shoot Induction in Stylosanthes seabrana

Abstract

We report an efficient protocol for in vitro direct plant regeneration via multiple shoot induction in Stylosanthes seabrana. Stylosanthes is an important range legume for humid to semi-arid tropics. It is very good fodder for animals, containing about 15% crude protein with 61% in vitro dry matter digestibility. S. seabrana, in particular, is rich in lysine and sulphur-containing amino acids, which are limiting amino acids in ruminants' feed. Limited genetic variability in the germplasm and susceptibility to anthracnose disease are major challenges in Stylosanthes improvement. S. seabrana, being a self-pollinated and diploid species, has been identified as a candidate species for improvement through genetic transformation. In vitro plant regeneration is one of the pre-requisites for development of transgenic Stylosanthes with desirable traits. In the present study, we used apical meristematic axis from in vitro grown seedlings as explant for multiple shoot induction. MS medium supplemented with BAP, Kn and TDZ, in different concentrations and combinations, were used for apical meristem culture. Best response, in terms of the number of shoots (4) per apical meristem, was observed on MS medium supplemented with 3.0 mg/l BAP. Shoot elongation was achieved on MS medium containing 2.0 mg/l GA and best rooting was induced on MS medium supplemented with 2.0 mg/l IAA. Tissue cultured plants showed normal growth, flowering and seed setting. To the best of our knowledge, this is the first report on plant regeneration via multiple shoot induction in S. seabrana. A standardized protocol would be very useful for Agrobacterium-mediated genetic transformation of Stylosanthes with the gene(s) of interest.