High transformation frequencies obtained from a commercial wheat (Triticum aestivum L. cv. 'Combi') by microbombardment of immature embryos followed by GFP screening combined with PPT selection

Improved gene transfer techniques are necessary to obtain adequate numbers of stable transgenic wheat plants needed for practical purposes. Considering that wheat transformation is genotype-dependent, we used cv. Combi in all experiments, which had been selected from agronomically important German spring wheat cultivars because of its high transformation ability. In most wheat gene transfer attempts, immature embryos or embryogenic scutellar calli were microbombarded. We compared both methods under optimised conditions, using bar, uidA, and gfp as markers in co-transformation attempts. Integration of the genes mentioned above was proven by Southern blotting, expression levels were measured by assays on phosphinothricin acetyltransferase and beta-glucuronidase activities, and by monitoring for green fluorescent protein in most developmental stages. Following bombardment of scutellar calli, an average transformation frequency of 0.13% was attained. Using immature embryos, mean transformation frequency (1.06%) was 8-fold higher. In addition, embryo techniques were over 2 weeks faster than scutellar callus procedures. Introducing gfp as a vital marker led to an improvement of embryo-based techniques. In a first screening, transient gfp-expressing embryos were transferred to phosphinothricin containing callus medium. Only gfp-expressing calli which developed on it were cultured further on phosphinothricin containing regeneration medium. Shoots obtained from gfp-expressing calli were rooted on phosphinothricin-free medium, and cultured ex vitro. Average transformation frequency (4.93%) was 38-fold higher than with scutellar callus techniques. Differences between the transformation strategies used were of high statistical significance. Combining green fluorescent protein screening with phosphinothricin selection in embryo-based techniques offers a promising system to obtain high wheat transformation frequencies.