Efficient genetic transformation of Momordica charantia L. by microprojectile bombardment.

Abstract

Here, we report the optimized conditions for biolistic particle delivery-mediated genetic transformation of bitter melon using petiole segments. In this study, DNA-coated gold particles of 0.6µm were used for optimizing the parameters of transformation and eventually regeneration of bitter melon putative transgenics. Initially, biolistic parameters namely helium pressure and macrocarrier to target tissue distance, were optimized using binary vector pBI121 carrying both β-glucuronidase gene (GUS) and neomycin phosphotransferase II gene (npt II) as a reporter and as a selectable marker gene, respectively. The effect of optimized physical parameters on the frequency of transient (79.2±1.52%) and stable (41.9%) expressions has been investigated. The optimized biolistic parameters for petiole segments of Momordica charantia L. were determined as follows: 650 psi helium pressure and 6cm target distance. Using the optimized parameters, transformation of bitter melon was carried out for generation of putative transformants from bombarded tissues on SRM-K medium, with a mean number of 50.3 explants surviving at the end of the final selection (50mgl-1 kanamycin) round. Finally, the transformants produced were subjected to GUS histochemical assay, and integration of the transgenes (GUS and npt II) into the nuclear genome was confirmed by PCR analysis. DNA blot analysis confirmed the transgene integration in the transformed plantlet genomes. The present study may be used for developing transplastomic technology in this valuable medicinal plant for enhanced metabolic engineering pathways and production of biopharmaceuticals.