In Vitro Leaf-Based Method for Agrobacterium-Mediated Genetic Transformation of Sugar Beet

Sugar beet, one of the most important natural sources of sugars in the world, is well known as a recalcitrant crop for genetic transformation. In the present study, several key components of Agrobacterium-mediated transformation of sugar beet have been studied. The correct choice of explant and plant regeneration potential of domestic breeding lines was evaluated; however, most attention was paid to the search for the most efficient selectable marker gene and selection agents. To produce transgenic plants, we applied a method based on the agrobacterial inoculation of wounded morphogenic structures previously initiated on in vitro cultivated leaves. Four selective marker genes conferring antibiotic or herbicide resistance were evaluated. In the case of selection using kanamycin or G418 (nptII gene controlled by the nos promoter), no transgenic plants were obtained, while the addition of the aminoglycoside antibiotic hygromycin (hpt gene, driven by the nos promoter) to the medium ensured the successful production of transgenic plants from three breeding lines with a frequency ranging from 1.5 to 5.1%. The selection of transgenic tissues using herbicides such as phosphinothricin and glyphosate after transformation with the bar and cp4-epsps genes (both controlled by the CaMV 35S promoter) also ensured the obtaining of transgenic plants, but the transformation efficiency was significantly low, reaching only 1.0 and 0.4%, respectively. Primary transgenic sugar beet plants grown in the greenhouse demonstrated enhanced resistance to herbicides in dosages commonly used in the field. In addition, after self-pollination of the primary T0 transgenic lines, homozygous T2 offspring were successfully selected, which demonstrated stable resistance to glyphosate due to the constitutive expression of the introduced cp4-epsps gene.