Use of the phosphomannose isomerase (PMI) gene for agrobacteriummediated transformation of <i>Prunus domestica</i> L. leaf explants without the use of selective antibiotic resistance genes

Abstract

We developed an efficient system for agro-bacterial transformation of plum (Prunus domestica L.) leaf explants using the PMI/mannose and GFP selection system. Th e variety `Startovaya` was transformed using Agrobacterium tumefaciens strain CBE21 carrying the vector pNOV35SGFP. Leaf explants were placed onto a nutrient medium containing various concentrations and combinations of mannose and sucrose to develop an efficient selection system. Nine independent transgenic lines of plum plants were obtained on a regeneration medium containing 20 g/L sucrose and 15 g/L mannose. The highest transformation frequency (1.40 %) was produced using a delayed selection strategy. Starting from the 1st days after transformation and ending by regeneration of shoots from the transgenic callus, selection of transgenic cells was monitored by GFP fluorescence that allowed avoid ing formation of escapes. Integration of the manA and gfp transgenes was confi rmed by PCR and Southern blotting. On the whole, no direct correlation between the fluorescence level and the copy numbers of the transgenes was found in the present study, though the most intensive fluorescence was observed in line #9 with a single-copy insert. The difference of GFP expression level may have been caused by the integration site or by other factors such as DNA methylation and varying copy number. The described transformation protocol using a positive PMI/mannose system is an alternative selection system for production of transgenic plum plants without genes of antibiotic and herbicide resistance, and the use of leaf explants enables retention of variety traits of plum plants.