Genetic transformation and regeneration of transgenic plants from protocorm-like bodies of vanilla (Vanilla planifolia Andrews) using Agrobacterium tumefaciens

Abstract

An efficient transformation protocol was developed for vanilla (Vanilla planifolia) using protocorm-like bodies (PLBs) derived from shoot tips as explants. Of the ten media tested, Murashige and Skoog (MS) medium containing 0.45 µM thidiazuron (TDZ) produced maximum PLBs per shoot tip. Genetic fidelity of PLB-derived plantlets was confirmed by random amplified polymorphic DNA (RAPD) using 23 random primers. PLB’s were co-cultured with Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pBI121 containing the beta-glucuronidase (gusA) and neomycin phosphotransferase II (npt II) genes for three days in MS medium supplemented with acetosyringone and transferred to selective regeneration medium containing 4.43 µM benzyladenine (BA), 2.68 µM Naphthalene acetic acid (NAA) supplemented with 50 mgl-1kanamycin and 250 mgl-1 cefotaxime. After 15 days of culture, the surviving explants were transferred to the same regeneration medium but with a higher concentration of kanamycin (75 mgl-1). Finally, explants surviving after 30 days were subjected to more stringent selection in the regeneration medium supplemented with 100 mgl-1 of kanamycin. A strong β glucuronidase activity was detected in the transformed plantlets by histochemical assay. Integration of T-DNA into nuclear genome of transgenic plant was confirmed by polymerase chain reaction and Southern hybridization while expression of transgene was confirmed by northern hybridization. This protocol allows effective and high frequency transformation of vanilla