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 crops146-2 (Prunus pumilaL.xP.tomentosaThunb.), 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 byAgrobacterium, for rootstock 146-2. Whole leaves from in vitro-cultured shoots were used as explants for transformation by theA. tumefaciensstrain CBE21, with the binary vector pBINmGFP5ER containing thenptIIencoding neomycin phosphotransferase II as a plant-selectable marker under the control of the NOS promoter (nopalin synthase) and the reportergfpgene encoding a green fluorescent protein under the control of the cauliflower mosaic virus (CaMV) promoter 35S. Th e integration ofnptIIandgfpinto 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 producePlum pox virus(PPV) resistant plants.