A simple and efficient genetic transformation system for soybean ( Glycine max (L.) Merrill) targeting apical meristem of modified half-seed explant

The present study is an attempt to establish a fast, highly reproducible transformation with a simplified regeneration system in soybean targeting the apical meristem. The modified half-seed explants from soybean cultivar (cv.) JS335 were subjected to different time intervals of sonication (0, 1, 10, 20, and 30 min) and vacuum infiltration (0, 1, 10, 20, and 30 min) in the presence of Agrobacterium tumefaciens strain EHA105 harbouring pCAMBIA1301. The explants were then co-cultivated and subjected to a modified plant regeneration process that involves only two steps (1) primary shoot regeneration, and (2) in vitro rooting of primary shoot. The rooted plantlets were hardened and maintained in the greenhouse until maturity. Sonication treatment of 10 min, followed by plant regeneration using a modified method, recorded the highest transformation efficiency of 26.3% compared to other time duration tested. Furthermore, 10 min of vacuum infiltration alone resulted in even higher transformation efficiency after regeneration, reaching 28.0%. Interestingly, coupling sonication and vacuum infiltration for 10 min respectively produced the highest transformation efficiency after regeneration of 38.0%. The putative transformants showed gus expression in mature leaves, trifoliate leaves, flowers, and pods. The presence of hpt II was also confirmed in putative transformants, with an amplicon size of 500 bp. Quantitative real-time PCR confirmed the existence of hpt II as one to two copies in the soybean genome of T0 plants. Furthermore, the segregation pattern was observed in the T1 generation soybean plants which were confirmed using PCR for hpt II . The optimized protocol when tested with other Indian soybean cultivars showed an enhanced transformation efficiency ranging from 19.3% (cv. MAUS47) to 36.5% (cv. CO1). This optimized protocol could provide a reliable platform to overcome the challenges that are associated with the genetic engineering of soybean.