A rapid and efficient protocol for in vitro propagation of Hybanthus enneaspermus (L.) F. Muell. (Violaceae) has been developed from the shoot tip and nodal explants. The explants were cultured on MS basal medium supplemented with different concentrations of cytokinins, viz., BAP and Kin, ranging from 5 µM to 25 µM, either individually or in combinations of both these cytokinins for shoot induction. Shoot buds of both the explants proliferated on MS medium supplemented with both cytokinins. The best response was observed on MS medium containing 15 µM BAP. Subsequently the optimum concentration of BAP (15 µM) was combined with different concentrations of Kin ranging from 2 µM to 10 µM. Maximum number of 28.6 ± 0.90 and 36.8 ± 1.54 shoots were produced on MS medium containing 15 µM BAP + 6 µM Kin from the shoot tip and nodal explants respectively. The regenerated shoots were transferred to rooting medium containing auxins at different concentrations ranging from 2 µM to 10 µM of IAA, IBA or NAA. The highest number of roots were observed on half strength MS medium fortified with 4 µM IBA. The plantlets were then hardened and acclimatized in soil. About 80% of plantlets were survived in the field condition. A completely randomized design was used in all experiments and analysis of variance and mean separations were carried out using Duncan’s Multiple Range Test. Each treatment factor consists of 10 replicates repeated for 5 times. This protocol would help ex situ conservation of this medicinal plant.
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