Transformation of <i>Brassica napus</i> and <i>Brassica oleracea</i> Using <i>Agrobacterium tumefaciens</i> and the Expression of the <i>bar</i> and <i>neo</i> Genes in the Transgenic Plants

TLDR

The study established a genotype‑independent transformation method for Brassica napus and Brassica oleracea using neo or bar selectable markers. Hypocotyl explants were infected with Agrobacterium carrying chimeric neo or bar genes, with AgNO₃ required for shoot regeneration, and vitrification was avoided by reducing medium water potential, vessel humidity, and cytokinin concentration. Rooted transformed shoots were obtained with 30 % efficiency in 9–12 weeks; Southern blotting and progeny analysis revealed 1–3 gene copies per transformant, variable expression levels, and up to 25 % of transformants lacked detectable enzyme activity despite being transformed.

Abstract

An efficient and largely genotype-independent transformation method for Brassica napus and Brassica oleracea was established based on neo or bar as selectable marker genes. Hypocotyl explants of Brassica napus and Brassica oleracea cultivars were infected with Agrobacterium strains containing chimeric neo and bar genes. The use of AgNO(3) was a prerequisite for efficient shoot regeneration under selective conditions. Vitrification was avoided by decreasing the water potential of the medium, by decreasing the relative humidity in the tissue culture vessel, and by lowering the cytokinin concentration. In this way, rooted transformed shoots were obtained with a 30% efficiency in 9 to 12 weeks. Southern blottings and genetic analysis of S1-progeny showed that the transformants contained on average between one and three copies of the chimeric genes. A wide range of expression levels of the chimeric genes was observed among independent transformants. Up to 25% of the transformants showed no detectable phosphinotricin acetyltransferase or neomycin phosphotransferase II enzyme activities although Southern blottings demonstrated that these plants were indeed transformed.