A high-efficiency Agrobacterium tumefaciens mediated transformation system using cotyledonary node as explants in soybean (Glycine max L.)

A high-efficiency Agrobacterium tumefaciens mediated transformation system was established through testing and optimising a range of variables using soybean cotyledonary as explants and glufosinate as selective agent. Seven cultivars (Heihe 19, Heihe 25, Heinong 37, Ha 03-3, YC-1, YC-2, and Zhonghuang 39) were used to screen the optimal cultivars. As a result, cultivars YC-1 and YC-2 were screened, which had the highest transient GUS expression percentage and shoot induction frequency. The cell density of Agrobacterium was found to be related with the transient GUS expression percentage; its optimal cell density was OD 0.8–1.0. It was beneficial to A. tumefaciens infection at 23 °C and dim light co-cultivation condition. Interestingly, the infection time showed no relation to the GUS transient percentage. Three types of antioxidant solutions were tested on their effects of alleviating cotyledonary explant browning and necrosis. The results showed that addition of 20 μM lipoic acid on solid CCM had the lowest cotyledonary explant necrosis and a high transient GUS expression rate. Following the optimized A. tumefaciens mediated transformation system, the bar gene was successfully transferred into soybean with the transformation efficiency reaching 14.71 %, more than 50 times increase from the un-optimized procedure. Southern and T 1 progeny analysis showed the exogenous gene had been integrated into the soybean genome and could be inherited next progeny