Glyphosate-tolerant CP4 and GOX genes as a selectable marker in wheat transformation

Abstract

The lack of alternative selectable markers in crop transformation has been a substantial barrier for commercial application of agricultural biotechnology. We have developed an efficient selection system for wheat transformation using glyphosate-tolerant CP4 and GOX genes as a selectable marker. Immature embryos of the wheat cultivar Bobwhite were bombarded with two separate plasmids harboring the CP4/GOX and GUS genes. After a 1 week delay, the bombarded embryos were transferred to a selection medium containing 2 mM glyphosate. Embryo-derived calli were subcultured onto the same selection medium every 3 weeks consecutively for 9-12 weeks, and were then regenerated and rooted on selection media with lower glyphosate concentrations. Transgenic plants tolerant to glyphosate were recovered. ELISA assay confirmed expression of the CP4 and GOX genes in R0 plants. Southern blot analysis demonstrated that the transgenes were integrated into the wheat genomes and transmitted to the following generation. The use of CP4 and GOX genes as a selectable marker provides an efficient, effective, and alternative transformation selection system for wheat.

Callus induction and plant regeneration by Brazilian new elite wheat genotypes

“…The results presented here demonstrated the different capacity of callus induction and plant regeneration of the evaluated genotypes, which makes it possible to choose the best genotypes to achieve such characteristics, by optimizing the growing process of immature embryo culture and the plant regeneration process. (Murashige e Skoog 1962) e MS modificado -MMS (Zhou et al 1995) Euphytica 108: 161-167.…”

“…Nine Brazilian wheat (Triticum aestivum L.) genotypes were cultivated in vitro to evaluate the embryogenetic capacity. The explants (immature zygotic embryos) were tested in two different culture media, MS (Murashige and Skoog 1962) and modified MS -MMS (Zhou et al 1995) …”

Variation in transformation frequencies among six common wheat cultivars through particle bombardment of scutellar tissues.

“…Scutellar tissues of immature embryos have been recently used as a target to deliver a foreign DNA through particle bombardment (Weeks et al, 1993;Vasil et al, 1993;Nehra et al, 1994;Becker et al, 1994;Zhou et al, 1995;Takumi and Shimada, 1996). In previous wheat transformation studies, seven common wheat cultivars, i.e., Bobwhite, Pavon, RH770019, Fielder, Florida, Chinese Spring, and Akadaruma, have been used to generate transgenic plants.…”

Genetic transformation technology in the Triticeae

“…The 5-enol-pyruvyl shikimate-3-phosphate synthase (EPSPS) encoded by the aroA gene from Agrobacterium strain CP4 confers resistance to the non-selective herbicide Glyphosate. This selectable marker system was implemented for the production of transgenic wheat by Zhou et al (1995) using biolistic gene transfer and by Hu et al (2003) employing Agrobacterium. Another selection system that has been utilized in biolistic transformation of wheat rests on the rice acetolactate synthase (ALS or AHAS) gene (Ogawa et al 2008, Kempe et al 2009).…”