Comparing 17-β-estradiol supply strategies for applying the XVE-<i>Cre/loxP</i> system in grape gene transfer (<i>Vitis vinifera</i> L.)

Abstract

Assays for enhancing the performance of 17-β-estradiol induction in the XVE-Cre/loxP system were performed on two transgenic 'Brachetto' plants obtained with the pX6-pKcpGVA construct, which is derived from the chemical-inducible pX6 vector carrying the neomycin phosphotransferase (nptII) gene and the XVE-Cre/loxP sequence. The 17-β-estradiol supply is expected to induce Cre recombinase expression resulting in nptII gene removal. We compared different hormone supply strategies during shoot organogenesis from meristematic proliferative tissue (MPT) or from the cut surface between leaf and petiole (SOLP) or during micropropagation from bud (MB). The effectiveness of the estradiol induction was evaluated on different tissues of the regenerated plantlets by means of nptII copy number quantification with Real time PCR. Results showed that the Cre/loxP inducible system functions effectively – however with different efficiencies- in both root and leaf tissues, and that micropropagation from buds combined with constant wetting with 17-β-estradiol is the most efficient and reproducible strategy for effective in vivo hormone induction.