The use of the two T-DNA binary system to derive marker-free transgenic soybeans

Abstract

A binary vector, pPTN133, was assembled that harbored two separate T-DNAs. T-DNA one contained a bar cassette, while T-DNA two carried a GUS cassette. The plasmid was mobilized into the Agrobacterium tumefaciens strain EHA101. Mature soybean cotyledonary node explants were inoculated and regenerated on medium amended with glufosinate. Transgenic soybeans were grown to maturity in the greenhouse. Fifteen primary transformants (T 0 ) representing 10 independent events were characterized. Seven of the 10 independent T 0 events co-expressed GUS. Progeny analysis was conducted by sowing the T 1 seeds and monitoring the expression of the GUS gene after 21 d. Individual T 1 plants were subsequently scored for herbicide tolerance by leaf painting a unifoliate leaf with a 100 mg l 21 solution of glufosinate and scoring the leaf 5 d post application. Herbicide-sensitive and GUS-positive individuals were observed in four of the 10 independent events. Southern blot analysis confirmed the absence of the bar gene in the GUS positive/herbicide-sensitive individuals. These results demonstrate that simultaneous integration of two T-DNAs followed by their independent segregation in progeny is a viable means to obtain soybeans that lack a selectable marker.

Evaluation of the Escherichia coli threonine deaminase gene as a selectable marker for plant transformation

“…However, to detect minor fluctuations of other amino acid levels, which may be manifested as a result of the expression of the E. coli TD, may require large sample sizes, due to high inherent variation. Therefore, if the E. coli TD gene can be optimized for use in a selection scheme, given the potential for negative developmental abnormalities being generated ( Figure 2) and/or alteration of intracellular amino acid profile as a consequence of constitutive perturbation of the aspartate family biosynthetic pathway, additional measures, such as recombination approaches (Dale and Ow 1991;Zuo et al 2001) or the simultaneous delivery of two T-DNA elements (Komari et al 1996;Xing et al 2000), would undoubtedly need to be taken for the subsequent removal of the TD transgene from the genome.…”

Sorghum Transformation: Overview and Utility

“…If integrated at unlinked positions, the T-DNAs, will segregate in the progeny. This strategy has been successfully used to derived maker-free transgenic plants in a number of systems (Daley et al 1998;Jacob and Veluthambi 2002;Komari et al 1996;Sato et al 2004b;Xing et al 2000), including sorghum (Zhao et al 2003). The integration of unlinked T-DNA alleles in sorghum is exemplified in Figure 2.…”

Soybean: Plant Manipulation to Agrobacterium Mediated Transformation

“…Tight selection procedure (selection of explants on selective agent before infection) increases transformation efficiency and occurrence of less escape (Chen, 2004). Xing and his colleagues produced marker free plants by introducing two T-DNA binary systems (Xing et al, 2000). Integration of two T-DNA followed by their independent segregation in progeny is a viable mean to produce maker free soybean transgenic plants.…”