Efficient genetic transformation of L. by microprojectile bombardment.

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 0.6 µm 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 () and neomycin phosphotransferase II gene ( 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 L. were determined as follows: 650 psi helium pressure and 6 cm 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 (50 mg l kanamycin) round. Finally, the transformants produced were subjected to histochemical assay, and integration of the transgenes ( and 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.