Hypocotyl-based Agrobacterium-mediated transformation of soybean (Glycine max) and application for RNA interference

Abstract

An efficient system of gene transformation is necessary for soybean [Glycine max (L.) Merrill] functional genomics and gene modification by using RNA interference (RNAi) technology. To establish such system, we improved the conditions of tissue culture and transformation for increasing the frequency of adventitious shoots and decreasing the browning and necrosis of hypocotyls. Adding N(6)-benzylaminopurine (BAP) and silver nitrate in culture medium enhanced the shoot formation on hypocotyls. BAP increased the frequency of the hypocotyls containing adventitious shoots, while silver nitrate increased the number of shoots on the hypocotyls. As a result, the number of adventitious shoots on hypocotyls cultured in medium containing both BAP and silver nitrate was 5-fold higher than the controls. Adding antioxidants in co-cultivation medium resulted in a significant decrease in occurrence of browning and necrosis of hypocotyls and increase in levels of beta-Glucuronidase (GUS) gene expression. Histochemical assays showed that the apical meristem of hypocotyls was the "target tissue" for Agrobacterium tumefaciens transformation of soybean. Gene silencing of functional gene by using RNAi technology was carried out under above conditions. A silencing construct containing an inverted-repeat fragment of the GmFAD2 gene was introduced into soybean by using the A. tumefaciens-mediated transformation. Several lines with high oleic acid were obtained, in which mean oleic acid content ranged from 71.5 to 81.9%. Our study demonstrates that this transgenic approach could be efficiently used to improve soybean quality and productivity through functional genomics.

Silver Nitrate and Putrescine Enhance in vitro Shoot Organogenesis in Polygonum tinctorium

“…Shoot organogenesis of Gloxinia Sinningia speciosa Baill. has been enhanced by ethylene inhibitors 22 , and other studies confirmed that they improve shoot regeneration and transformation efficiency [23][24][25] . In this study, we developed an efficient method for the regeneration of P. tinctorium plants from internode explants by using different concentrations of an ethylene inhibitor and polyamine.…”

Soybean: Plant Manipulation to Agrobacterium Mediated Transformation

“…Embryonic tips were also found sensitive against kanamycin treatment at level higher then 10 mg/l. Addition of antioxidant in co-cultivation medium result in significant decrease in browning and necrosis of hypocotyls and increased GUS expression (Wang & Xu, 2008). Embryogenic tips showed better response for hypervirulent strain KYRT1 than EHA105 and LBA4404 when infected for 20 hours (Dang & Wei, 2007).…”

Application of somatic embryos to rapid and reliable analysis of soybean seed components by RNA interference-mediated gene silencing

“…However, conventional breeding is dependent on genetic resources, takes many years, and may be hindered by linkage drag in some instances (Boerma and Walker 2005). In contrast, RNAi-mediated gene silencing has been applied for modification of soybean seed components including enzymes and other proteins that are encoded by multiple gene copies (Chen et al 2006;Flores et al 2008;Nunes et al 2006;Schmidt and Herman 2008;Shi et al 2007;Wang and Xu 2008). RNAi, together with overexpression of exogenous or endogenous genes, is thus a powerful strategy for soybean improvement.…”