Efficient Agrobacterium-Mediated Transformation and Analysis of Transgenic Plants in Hybrid Black Poplar (Populus × euromericana Dode Guinier)

Abstract

Black poplar ( Populus× euramericana Dode Guinier) is an industrially important tree with broad applications in wood and paper, biofuel and cellulose-based industries as well as plant breeding programs and soil phytoremediation approaches. Here, we have focused on development of direct shoot regeneration and Agrobacterium -mediated transformation protocols using the in vitro internodal stem tissue from hybrid black poplar. To obtain efficient plant regeneration, the internodal stem explant was cultured on SIM and RIM medium containing different concentrations of BAP × IBA and IBA × NAA, respectively. The crucial factors involved in genetic transformation have been evaluated to achieve Agrobacterium- mediated transformation protocol. We achieved fast and highly potential shoot regeneration from the explants cultured on SIM containing BAP 0.5mg/L and IBA 0.05mg/L with 28.57 shoots per explant. The normal roots developed from the plantlets cultured on RIM containing IBA 0.1mg/L and NAA 0.05mg/L and 100% of the regenerated plants were hardened and transferred to the greenhouse condition. Our results indicated that 0.5 µM Basta® could provide a stringent selection for the inhibition of non-transformed cells. We also obtained the highest transformation efficiency of 93.33% through preculturing the explants for 6 days and dipping into IM medium containing A. tumefaciens strain LBA4404 (OD 600 = 0.6) and 100 µM AS for 10 min. The Southern blotting analysis, RT-PCR and GUS histochemical analysis were confirmed the stable single or two-copies gus transgenesis in the genomic DNA and its expression in the selected T0 generation plants. The findings indicate that these protocols could be used for genetic engineering approaches in hybrid black poplar.