Sonication and vacuum infiltration assisted genetic transformation in bhendi [Abelmoschus esculentus (L.) Moench] using cry2Aa gene for pest resistance

Abstract

Somaclonal variation and recalcitrance nature were the major bottlenecks of agrobacterium mediated genetic transformation in Bhendi (Abelmoschus esculentus (L.). Therefore, Agrobacterium mediated in planta transformation protocols was standardized using sprouted seed explants of bhendi cv. MDU-1, an important vegetable. agrobacterium tumifaciens-mediated transformation was performed using EHA105 strain harboring the binary vector pBinAR carrying cry2Aa gene under the control of CaMV35s promoter and npt II gene as a selectable marker. The transformation event consisted of sonicating the explants for 3 minutes, vacuum infiltration (750 mm of Hg) for 2 minutes in Agrobacterium (pBinAR-cry2Aa) and co-cultivation for 3 days in MS medium with acetosyringone (100µM) showed transformation efficiency of 12.5%. The presence and integration of npt II and cry2Aa transgenes into the bhendi genome was confirmed by polymerase chain reaction (PCR). Work is in progress to grow the T0 seeds for molecular characterization of the inserted transgene using southern and western blot methods.