Dual-cytokinin synergistic strategy for the micropropagation of wild and rare Piper species (Piper sarmentosum, Piper retrofractum and Piper macropiper)
摘要
Several wild relatives of genus Piper are rich in bioactive compounds, having high medicinal importance. Piper sarmentosum Roxb., Piper retrofractum Vahl., and Piper macropiper Pennant are three such wild species, rare in distribution and not widely cultivated. Efficient and reproducible micropropagation protocols have been developed in these three species using nodal segments as explants. Three different cytokinins, namely 6-benzylaminopurine (BAP), Kinetin (Kn), and meta-Topolin (mT), were used alone or in combination with each other for in vitro shoot multiplication. Synergistic treatment of BAP along with 4.65 µM Kn in Murashige and Skoog (MS) medium was found to elicit the best multiplication response in all three species. Maximum number of shoots per nodal explant was observed as 7.50 ± 0.06 in P. sarmentosum on MS medium supplemented with 11.11 µM BAP and 4.65 µM Kn, 7.08 ± 0.07 in P. retrofractum on 2.22 µM BAP and 4.65 µM Kn supplemented medium, and 6.91 ± 0.52 in P. macropiper on 6.66 µM BAP and 4.65 µM Kn containing medium. The combination of BAP and Kn was found to induce longer shoots as well. In vitro shoots rooted well on MS medium devoid of any phytohormones. All rooted plantlets were effectively hardened and acclimatized with a survival rate of 80%. Clonal fidelity of the micropropagated plants was confirmed using ISSR markers. The developed micropropagation protocol can be used for mass propagation of true-to-type plants facilitating their cultivation for conservation as well as commercial purposes. Further, this study provided a foundation for future studies on micropropagation of rare and endemic Piper species.