Genetic transformation of clone rootstock of stone fruits 146-2 using the green fluorescent protein reporter gene

Abstract

For the form of dwarf winter-hardy clonal rootstocks of stone crops 146-2 (Prunus pumila L.xP.tomentosa Thunb.), system of regeneration and genetic transformation using green fluorescent protein (GFP) has been developed. For eff ective regeneration of accessory shoots, no pre-treatment with 6-benzylamine-purine (BA) and auxin was required. Stimulation of the regeneration of shoots from leaf explants required 2-3 weeks of a dark period. Th e best percentage of regeneration (greater than 75 %) was observed with a combination of 3 mg/L BA and 0.75 mg/L IBA. The achieved regeneration effi ciency made it possible to develop a protocol for genetic transformation, mediated by Agrobacterium, for rootstock 146-2. Whole leaves from in vitro-cultured shoots were used as explants for transformation by the A. tumefaciens strain CBE21, with the binary vector pBINmGFP5ER containing the nptII encoding neomycin phosphotransferase II as a plant-selectable marker under the control of the NOS promoter (nopalin synthase) and the reporter gfp gene encoding a green fluorescent protein under the control of the cauliflower mosaic virus (CaMV) promoter 35S. Th e integration of nptII and gfp into transgenes was confirmed by PCR. Expression of the green fluorescent protein was observed using fluorescence microscopy. The efficiency of transformation based on PCR analysis of independent lines resistant to kanamycin was 0.41-0.83 %. All transgenic lines showed resistance to kanamycin at a concentration of 40 mg/L. They were rooted and acclimatized to greenhouse conditions. Th e developed protocols will be used to produce Plum pox virus (PPV) resistant plants.