High efficient protocol for Callus induction and Regeneration of a medicinal plant Orthosiphon stamineus

Abstract

Callus induction and indirect organogenesis protocol were developed using different explants of Orthosiphon stamineus such as leaf, petiole, and internode on MS medium supplemented with different plant growth regulators. For callus induction, MS medium with 2, 4-D, NAA and IBA alone or in combinations with BAP were used. High-efficiency callus induction was obtained (100%) in petiole compared with other explants at the concentrations of 5mg/l 2, 4-D hormone, and 99% callus induction was obtained in leaf and internode at the concentration of 4.0 mg/l 2,4-D hormone. Similarly, 100% callus induction was obtained at the concentrations of 4.0 mg/l NAA with 0.5 mg/l BAP combinations in petiole and internode whereas 99% of callus induction was obtained from the same concentration. The induced callus of leaf, petiole, and internode was transferred to shooting medium fortified with plant growth regulators at 5.0 mg/l BAP and 0.5 mg/l NAA. Among these, internode has shown maximum percentage of the shoot (60%) regeneration than petiole and leaf callus. Shoot regenerated plantlets were then transferred to ¼MS medium fortified with 1.0 mg/l NAA hormone results in high efficient rooting (20) in vitro plantlets without inducing basal callus. Further, root-induced healthy plantlets were subjected to hardening in the greenhouse and established in natural environmental condition with a survival rate of 95% without any morphological changes of true type plants. Hence the developed protocol was optimized to yield a high frequency of callus induction and regeneration in different explants of O.stamineus with different concentrations of plant growth regulator. Keywords: