Improved in planta genetic transformation efficiency in bitter gourd (Momordica charantia L.)

Abstract

Production of transformed bitter gourd plants through in vitro regeneration is a laborious practice, which may also result in somaclonal variations. Thereby, in the current study, we have established a less tedious in planta protocol for more efficient genetic transformation of bitter gourd var. NBGH-470 (Apurva) using the seed as a target material. Bitter gourd seeds were transformed by Agrobacterium tumefaciens strain LBA4404 bearing the pCAMBIA1301 binary vector, and the transformed plants were selected against hygromycin B. The putatively transformed bitter gourd (To) plants were examined by GUS assay. Two parameters, including vacuum infiltration and sonication improving the in planta transformation, have been investigated in the present work. Amongst several time durations examined, the highest transformation rate (37%) was achieved upon subjecting the pre-cultured bitter gourd seeds to sonication for 15 min, followed by vacuum infiltration for 6 min in LBA4404 culture suspension. The transformed bitter gourd (T1) plants were selected against hygromycin B, and the transgene integration was evaluated by polymerase chain reaction (PCR), Southern hybridization, and reverse transcriptase PCR (RT-PCR). The developed protocol in the current study is suitable to genetically engineer the bitter gourd plants with disease and pest-resistant traits.