Agrobacterium-mediated transformation of sunflower (Helianthus annuus L.) in vitro and in planta using Lba4404 strain harboring binary vector pBi2E with dsRNA-suppressor of proline dehydrogenase gene

Abstract

To estimate the efficiency of proline dehydrogenase gene suppression aimed at increasing sunflower (Helianthus annuus L.) tolerance level to water deficiency and salinity, we employed the LBA4404 strain harboring pBi2E with double-stranded RNA-suppressor, produced based on the ProDH1 gene of Arabidopsis. Techniques for Agrobacterium-mediated transformation in vitro and in planta during fertilization of sunflower have been proposed. The genotype-dependent integration of T-DNA in the sunflower genome was demonstrated. PCR-analysis showed that ProDH1 is present in the genomes of inbred lines transformed in planta, as well as in T1- and T2-generations. Significantly, increased levels of accumulation of free L-proline during early stages of in vitro cultivation under normal conditions were demonstrated in transgenic regenerants. The application of lethal doses of stressors (0.4 M mannitol and 2.0% sea salts) caused increase of L-proline level in transgenic regenerants and its decline during the recovery period. This data indicates the effectiveness of suppression of the sunflower ProDH1 gene for increased osmotolerance.