Efficient soybean regeneration and Agrobacterium‐mediated transformation using a whole cotyledonary node as an explant

Abstract An optimized regeneration and Agrobacterium ‐mediated transformation protocol based on whole cotyledonary node explants was developed in soybean ( Glycine max ) cultivar Zhong Huang 13. Adding 6‐benzylaminopurine (BAP) in a germinating medium could significantly increase regeneration efficiency; the optimal BAP concentration for shoot formation was 0.5 mg/L. The concentrations of plant growth regulators in a shoot induction medium were optimized by the orthogonal test [L 9 (3 3 )]. The best combination for shoot regeneration was a medium of Murashige & Skoog salts with B5 vitamins (MSB) supplemented with 3.5 mg/L BAP, 0.2 mg/L indole‐3‐butyric acid (IBA), and 0.2 mg/L kinetin (KT). Under this favorable condition, one node could regenerate 28–30 shoots. Soybean whole cotyledonary nodes were transformed by inoculation with A. tumefaciens strain EHA105 harboring a vector pBI121 containing a β‐glucuronidase gene ( gus ). GUS assay, polymerase chain reaction, and Southern blot analysis indicated that the gus gene was transformed into soybean plants with 23.1% transformation efficiency. Transgenic plants could be obtained within 5–6 weeks, which was about 4 weeks less than that of a traditional single cotyledonary node method.