Agrobacterium-mediated genetic transformation and stable transgene expression in Muscat Ottonel grape (Vitis...

Grapevine (Vitis vinifera L.) is an economically significant fruit crop, but its genetic improvement through conventional breeding is limited due to high heterozygosity and long generation periods. Genetic engineering offers a promising alternative for enhancing grapevine traits without altering key varietal characteristics. This study reports the successful Agrobacterium tumefaciens-mediated transformation of Muscat Ottonel grape plants, focusing on an efficient protocol using leaf explants and optimized culture conditions. Transgenic Muscat Ottonel grape plants were generated through an optimized protocol involving pre-cultured leaf explants on regeneration medium with 100 µM acetosyringone, followed by co-cultivation with A. tumefaciens strain LBA4404 containing the pCAMBIA2301 vector with gus and nptII genes. The transformed explants were selected on media with 30 mg/L kanamycin and 200 mg/L cefotaxime. Shoot organogenesis was enhanced by 4 g/L phytoagar and 1.5 g/L gelrite, resulting in a transformation efficiency of 66.66%. Histochemical GUS assays, PCR, Southern blot, and Northern blot analyses confirmed stable gene integration and expression in four out of six transgenic lines. Notably, transgenic plants displayed stable gus expression without phenotypic deviations. This study establishes a reliable protocol for the Agrobacterium-mediated transformation of Muscat Ottonel grape, demonstrating high efficiency and stable gene expression. The absence of a hypersensitive response in Muscat Ottonel during transformation contributed to the efficiency. This protocol is valuable for future grapevine genetic studies and breeding programs to introduce beneficial traits, like disease resistance, into elite cultivars.