An improved Agrobacterium mediated transformation and regeneration protocol for successful genetic engineering and genome editing in eggplant

Successful genetic manipulation largely depends on effective transformation and regeneration methods. The Agrobacterium mediated transformation has always been the method of choice for crop improvement through genetic engineering and the recently developed CRISPR/Cas9 based genome editing approaches. In this study, we have developed an improved regeneration and Agrobacterium mediated transformation protocols for three high yielding eggplant varieties BARI begun 2, 4 and 6. Several key parameters were intensively studied which include culture media composition, concentration of growth regulators, compatible antibiotic selection, hyperhydricity and rooting process. For the three different explants used in the study, it was found that MS+2.5 mg/l BAP alone is optimal as a hormone supplement for maximum shoot regeneration. Significant reduction (16.67 +/- 0.11%) in hyperhydricity was observed when casein hydrolysate along with sorbitol was used at a concentration of 0.2 mg/l and 0.1 mg/l respectively in MS+BAP 2.5 mg/l supplemented medium. Transformation efficiency was found to be highest with an Agrobacterium concentration of 0.6 at OD 600 nm, 10 min of infection and two days of co-cultivation. We found 100 mg/l concentration of kanamycin to be suitable as a selection pressure for screening eggplant transformation events. Rooting media composition standardized in this protocol provides a higher rate of root formation (85%) in both in vitro and ex vitro conditions. Using this protocol, existing problems in eggplant genetic engineering can easily be overcome with higher transformation efficiency.