Sand-Wounding of Shoot and Petiole Explants Enhances Transformation Efficiency in Sugar Beet (Beta vulgaris L.) by Agrobacterium-mediated Transformation

Abstract

Genetic transformation of recalcitrant species, like sugar beet, has been always a challenging task. In this study, a selectable marker gene (phosphomannose isomerase) and a reporter gene (synthetic green fluorescent protein) were transferred to sugar beet via Agrobacterium-mediated transformation by using sand-wounded shoot and petiole explants of two different diploid sugar beet genotypes. Considering the overall means of genotypes and explant types, a clear genotypic variation was evident as ELK345 produced a mean of 5.7% transient expression and 1.97% stable transformation efficiency while it was 4.3% and 1.28% for the genotype M1195. When explant types were compared independently from other parameters, it was shown that petiole explants were more productive than shoot explants, with an overall mean of 5.3% versus 4.7% and 1.77% versus 1.48% transient expressions and stable transformation efficiencies, respectively. On the other hand, when a comparison was made without taking into account the genotypes, the effect of wounding treatment on transient expression resulted in about 25% (4.7% vs. 5.9%) and 20% (4.3% vs. 5.1%) increases in petiole and shoot explants, respectively. Such a promoting effect of wounding was even more prominent when the stable transformation efficiencies were compared; petiole explants achieving almost a 60% increase (1.38% vs. 2.16%) while shoot explants doubled (0.98% vs. 1.97%) their efficiencies. The optimized protocol reported here can be employed for further increasing the transformation efficiency in recalcitrant plant species including sugar beet. The advantages of using GFP as a reporter and mannose as a positive selection are also discussed.