Evaluation of MAT-vector system in white poplar (Populus alba L.) and production of ipt marker-free transgenic plants by ‘single-step transformation’

Genetic transformation of an elite white poplar genotype (Populus alba L., cv. ‘Villafranca’) was performed with MAT vectors carrying the ipt and rol genes from Agrobacterium spp. as morphological markers. The effects associated with the use of different gene promoters and distinct in vitro regeneration protocols were evaluated. Poplar plantlets showing abnormal ipt and rol phenotypes were produced only in the presence of exogenous growth regulators. The occurrence of abnormal ipt and rol phenotypes allowed the visual selection of transformants. The ipt-type MAT vector pEXM2 was used to monitor the activity of the yeast site-specific recombination R/RS system in the transformed white poplar cells. Results from these experiments demonstrated that recombinase-mediated excision events occurred during the early stages of in vitro culture, thus causing the direct production of ipt marker-free transgenic plants with normal phenotype at an estimated frequency of 36.4%. Beside this unexpected finding, transgenic ipt-shooty plants were obtained at a frequency of 63.6% and normal shoots were subsequently recovered after a prolonged period of in vitro culture. Although the transformation efficiency observed in this study, using both ipt and nptII genes as selection markers, was similar to that previously reported with standard vectors carrying only the nptII gene, the easy identification of ipt transformants, the early recombinase-mediated excision events and finally the relatively short time period required to produce ipt marker-free transgenic plants support for the choice of MAT vectors as a reliable strategy for the future production of marker-free GM poplars.