The procedure providing enhanced Agrobacterium-mediated transformation of wheat

Abstract

The examinations of conditions for establishing a variety independent Agrobacterium-mediated transformation procedure for wheat are preferable since many of cultivars and breeding lines remain recalcitrant for biotechnological manipulation, mainly due to low efficiency of plant regeneration in vitro, which is highly genotype specific. This paper describes and discusses an improved protocol for enhanced and low-genotype dependent Agrobacterium-mediated transformation using a super-binary vector LBA4404/pTOK233 carrying reporter gus-intron gene and hygromycin (hpt) and kanamicyn (nptII) selectable marker genes. The protocol was optimized on highly responsive common wheat cv. Vesna. Transient expression monitored by the gus-intron on explants after 3, 6 and 25 days of co-cultivation, followed by GUS expression and hygromycin resistance in whole plants indicated the protocol including a co-cultivation of freshly isolated immature embryos in the presence of ascorbic acid, and acetosyringone added only in the bacteria-containing infection medium combined with a delayed and stepwise increasing hygromycin B selection procedure significantly enhanced the transformation efficiency in cv. Vesna that exceed 7\% of treated explants from previously 0.41\%. Explant pre-cultivation did not additionally improve transformation efficiency. The optimized protocol was successful in evoking satisfactory transformation efficiencies from 3.6\% to 10.8\% in 5 less-responsive wheat genotypes. All 57 T0 hygromycin-resistant and GUS-positive lines were phenotypically normal and fertile. Therefore, the conditions employed in this study may serve as a base to facilitate the transformation in other, particularly recalcitrant wheat cultivars.