Evaluation of different strategies to develop cisgenesis in grapevine

Cisgenesis may be conceived as a technological evolution of the traditional genetic engineering to introduce the desired trait in planta with a minimal impact on its genomic asset. This technology is a great opportunity for plant breeding in the near future and its application in the most important crops is becoming a crucial goal for scientific and commercial research. We took up this challenge and tried to set up an effective system in grapevine. One of the requirements for a cisgenic plant is the absence, in the genome, of selectable marker genes. To this aim, we evaluated two strategies both based on site-specific recombination to excise undesired sequences from the plant genome after the selection phase. The first exploits the nptII marker gene to obtain an effective selection of the modified plants and allows to remove it when desired by means of an heat-shock induction. The second avoids the use of antibiotic resistance genes and is based on the use of a ‘reporter gene’ Md_Myb10 which produces a red-violet colour in plant tissue. After a chemical induction with the hormone -estradiol, this gene may be removed along with undesired sequences. Gene transfer experiments on different grapevine cultivars were carried out via Agrobacterium tumefaciens carrying the pB-Npt-Hsp-Flp-Gus or the pMF1 vectors respectively for the first and the second strategy and plants with integrated T-DNA were obtained. The proper conditions for an effective removal of undesired DNA are currently under evaluation.