Transformation of Soybean (Glycine max) by Infecting Germinating Seeds with Agrobacterium tumefaciens

Abstract

The transfer of genetic material into soybean tissue was accomplished by using an avirulent strain of Agrobacterlum tumefaciens which contained the binary vector pGA482. The method used for transformation requires no tissue culture steps as it involves the inoculation of the plumule, cotyledonary node, and adjacent cotyledon tissues of germinating seeds. The identification of neomycin phosphotransferase (NPT) II enzyme activity in the tissues of 16 (Ro) soybean plants indicated that the plant expressible Nos-NPT II gene, contained within the T-DNA region from pGA482, had been transferred at least into somatic tissues.Putative transformed Ro soybean plants were advanced to produce R1 plants which were also assayed for the presence of the transferred Nos-NPT II gene. The combined results of these assays indicated that about 0.7% of the surviving inoculated seeds yielded transformed tissues in the Ro plant, and that about 1/10 of these plants yielded transformed R1 plants. The presence of the Nos-NPT II gene in DNAs isolated from both Ro and R1 plant was demonstrated by using genomic blot hybridization and polymerase chain reaction methods. Integration of this gene into the soybean genome was demonstrated for three R1 soybean plants.

Tissue culture-based Agrobacterium-mediated and in planta transformation methods

“…The advantage of wounding, compared to acetosyringone, is that besides increasing the accessibility to target tissue for Agrobacterium, it can also enhance other inducers of T-DNA transfer. Different types of wounding include simple wounds made during the normal course of explant prepara-doi: 10.17221/177/2016-CJGPB tion (Horsch et al 1985), particle gun-mediated micro-wounding (Bidney et al 1992), using syringes filled with Agrobacterium (Chee et al 1989), and the use of sonication (Trick & Finer 1997). Sonication can overcome barriers of the host specificity and the inability of Agrobacterium to reach the proper cells in the target tissue and thus enhances DNA transfer (Trick & Finer 1997).…”

Advances in Agrobacterium-mediated plant transformation with enphasys on soybean

“…Chee et al (1989) used a needle to inoculate the plumule, cotyledonary node, and adjacent cotyledonary tissue with Agrobacterium. Only 0.7% of the inoculated seeds yielded transformed tissues.…”

“…Limited success has been reported with Agrobacterium-mediated transformation of seeds, somatic embryos, colyledonary nodes, and immature zygotic cotyledons (Hinchee et al, 1988;Chee et al, 1989;Parrott, et al 1989;Townsend & Thomas, 1993;Meurer, 1998;Yan et al, 2000;Gelvin, 2003).…”

Transformation of Soybean Embryogenic Cultures by Microprojectile Bombardment.

“…Soybean transformation has been difficult, but several methods have been recently developed using Agrobacterium [1][2][3], microprojectile bombardment [4][5][6][7], or electroporation.…”