An Efficient Agrobacterium-Mediated Transformation Protocol for Black Pepper (Piper nigrum L.) UsingEmbryogenic Mass as Explant

Abstract

A protocol was developed for an efficient Agrobactertium mediated transformation of black pepper plants through somatic embryogenesis. Embryogenic mass derived from primary somatic embryos that were obtained from micropylar region of mature germinating seeds of black pepper was found to be the ideal target tissue for transformation. Genetic fidelity test of embryogenic mass derived plantlets by RAPD using 23 random primers revealed no genetic variation among the progenies and the parent plant. Among the antibiotics used for selection of transformants, cefotaxime at100 µg ml-1 was found to be optimum to control Agrobacterium besides its ability to promote somatic embryo proliferation. In case of kanamycin, a step wise increase in concentration from 25 to 50 and then to100 µg ml-1 were found to be optimum. Embryogenic mass co cultivated with Agrobacterium carrying the β-glucuronidase (GUS) reporter gene were cultured on plant growth regulator free Schenk and Hildebrandt (SH) medium and transformants were selected in selection medium containing cefotaxime and step wise increase in kanamycin concentration. The transient GUS gene expression was determined histochemically. Transformants that survived in the selection medium were hardened in the green house. An average of nine hardened putative plantlets was obtained per gram of embryogenic mass. Presence of transgene in these plantlets was assayed by PCR, dot blot and Southern blot hybridization. Results presented demonstrated for the first time an efficient transformation and regeneration of black pepper without the use of growth regulators. This simple efficient procedure would allow transformation of black pepper with genes of desirable characters.