Production and in vitro assessment of transgenic plums for resistance to Plum pox virus: a feasible, environmental risk-free, cost-effective approach

During the production and assessment of transgenic plants resistant to quarantine viruses, the need to contain genetically modified plants (GMPs) and pathogens severely limits working options. Moreover, in the case of fruit trees, acclimatisation and viral inoculation are very time-consuming, thus a quick and safe method to assess the resistance to quarantine viruses, such as Plum pox virus (PPV), is desirable. This article focuses on the production of transgenic plums together with a contained and rapid evaluation in vitro for PPV resistance. The plum Stanley was transformed by Rhizobium radiobacter (syn. Agrobacterium tumefaciens) using a PPV-M derived hairpin construct (h-UTR/P1) that had previously been shown to confer high and broad-spectrum PPV resistance in model plant. Two transgenic clones, St24 and St28, were obtained. To assess their ability to resist PPV infection, micropropagated shoots of the transgenic clones were micrografted in vitro onto PPV-D infected GF305 rootstock. Following successful grafting, the transgenic scions were analysed by immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) for PPV detection. A total of 97% (47/48) of St24 and 73% (17/23) of St28 tested plants were resistant to the heterologous strain of PPV. In line with an RNA silencing mediated resistance mechanism, the St24 clone was shown to accumulate higher concentration of PPV UTR/P1-specific small interfering RNAs (siRNAs) than St28 one. The results are of practical interest not only developing plum clones that are highly resistant to PPV, but also for setting up quick and contained inoculation test procedure.