DEVELOPMENT OF in vitro REGENERATION AND Agrobacterium MEDIATED TRANSFORMATION SYSTEMS FOR Moringa oleifera

Abstract

The present investigations were aimed to develop a high efficiency of in vitro regeneration and genetic transformation systems of Moringa oleifera Lam from nodal segments of young aseptically grown seedlings using Agrobacterium-mediated transformation approach. Frequency of responded explants and number of shoots per explant were recorded during the course of the regeneration experiment. Regeneration capacity of nodal segments was evaluated on Murashige and Skoog (MS) media supplemented with 18 different combinations of plant growth regulators of benzylaminopurine (BAP), Zeatine (Zea) and naphthaleneacetic acid (NAA). Application of 1.0 mg/l BAP individually was found to be superior in terms of highest number of responded explants (95.7%) as well as the highest average (6.6) of axillary shoot developments per explant with direct emerging of adventitious shoots escaping callus formations. Well developed shoots subjected to rooting media supplemented with IAA or IBA or their different combinations. The most successful rooting events (100%) for regenerated shoots were obtained on rooting media containing ½ MS salts and supplemented with 1.0 mg/l IBA along with 0.5 mg/l IAA within three weeks maximum. The plant transformation vector pBIN121 harbors both the uidA (GUS) and NPTII (kanamycin resistant) genes were used to establish the Agrobacterium-mediated transformation experiment. Number of Putative transformed young shoots that developed onto Kanamycin selective regeneration medium were recorded representing 39.6% transformation efficiency. PCR analysis was carried out to verify successful transformation and gene integration for both GUS and NPTll genes in randomly selected young shoots while Histochemical GUS assay confirmed the successful expression of GUS gene in different parts of the putative transgenic plantlets.