Two critical factors are required for efficient transformation of multiple soybean cultivars: Agrobacterium strain and orientation of immature cotyledonary explant

Abstract

An efficient transformation system was developed for multiple soybean [Glycine max (L.) Merrill.] cultivars using Agrobacterium-mediated gene transfer. A significantly high number of hygromycin-resistant somatic embryos (SEs) was obtained when immature zygotic cotyledons were inoculated with Agrobacterium tumefaciens strain KYRT1 and when the abaxial side of explants was oriented upwards (i.e., the adaxial side of explants was in contact with the medium). Most hygromycinresistant SEs on selective medium were induced along the periphery of the abaxial side of cotyledonary explants. Extended periods of selection (up to 10 weeks postcocultivation) increased the frequency of somatic embryogenesis, and more than 50% of selected SEs tested positive for b-glucuronidase (GUS). Following maturation and regeneration of selected SEs, ten independent transgenic soybean plants of cv Jack were obtained, and the overall transformation frequency ranged from 1.1 to 1.7%. Six and two transgenic plantlets were obtained from cvs Dwight and Williams, respectively. In addition, transgenic suspension lines were established from cvs Jack, Williams, Dwight, Rend and Ina. Molecular analysis of embryogenic lines and/or transgenic plants, established from different cultivars, confirmed stable integration, expression, and/or inheritance of transgenes in both T 0 and T 1 plants.

Strategies for Improvement of Soybean Regeneration via Somatic Embryogenesis and Genetic Transformation

“…Even after more than two decades, the stable transformation of soybean cannot yet be considered as routine because it depends on the ability to bring together efficient transformation and regeneration techniques. Two methods have been successfuly used: particle bombardment Finer & McMullen, 1991;Christou & McCabe, 1992;Finer et al, 1992;Stewart et al, 1996;Aragão et al, 2000;Droste et al, 2002;Homrich et al, 2008;Wu et al, 2008;Li et al, 2009;Hernandez-Garcia et al, 2009;Xing et al, 2010;Viana et al, 2011) and Agrobacterium tumefaciens system Trick & Finer, 1998;Aragão et al, 2000;Yan et al, 2000;Ko et al, 2003Ko et al, , 2004Paz et al, 2006;Hong et al, 2007;Miklos et al, 2007;Liu et al, 2008;Wang & Xu, 2008;Wiebke-Strohm et al, 2011).…”

“…Studies with tumorigenic Agrobacterium strains (Pedersen et al, 1983;Droste et al, 1994;Mauro et al, 1995) and transient assays (Meurer et al, 1998;Droste et al, 2000) showed that soybean can be readily transformed by this bacterium. Currently, it is known that addition of acetosyringone during bacterial infection, combination of appropriate Agrobacterium strain and soybean cultivar, as well as development of super virulent bacterium strain and suitable plasmids increase efficiency of soybean transformation (Somers et al, 2003;Ko et al, 2003Ko et al, , 2004Wiebke-Strohm et al, 2011).…”

“…Still using zygotic cotyledons as target, a new approach was developed in which formation of secondary somatic embryos was allowed under continued selection system. The unicellular origin of secondary somatic embryos permitted recovery of complete, stable and fertile transgenic plants, whose progeny also displayed these characteristics (Yan et al, 2000;Ko et al, 2003).…”

Transformation of HBsAg (hepatitis B surface antigen) gene into tomato mediated by Agrobacterium tumefaciens

“…High-frequency transformation relies on the highly efficient T-DNA delivery from Agrobacterium into plant cells (Ko et al 2003).…”

Soybean: Plant Manipulation to Agrobacterium Mediated Transformation

“…However, longer sonication time may damage plant tissue (Santarem et al 1998). Way of placement of explant (adaxial side incontact with medium) on medium (Ko et al, 2003); exposure of soybean explants to AgNO 3 throughout shoot induction and shoot elongation (Olhoft et al, 2004); explant preparation in the presence of Agrobacterium culture and varying level of kanamycin during selection and regeneration (Zia et al, 2010b) are important for better recovery of transformants. Instead of kanamycin resistant plant, glufosinate resistant (bar gene) plants were produced by Zhang et al (1999) and Clemente et al (2000) using cotyledonary node explants of 5 days old seedlings.…”