Generation of new cisgenic apple lines resistant to either apple scab or fire blight

MAIN CONCLUSION: Cisgenic introduction of Rvi15 or FB_MR5 into 'Gala' generated single-copy lines with ≤ 201 bp vector remnants and high disease resistance, supporting cisgenesis as a powerful tool for commercial apple improvement. Cisgenesis can be used to transfer disease resistance genes from wild apple germplasm into established apple cultivars, reducing their susceptibility to diseases. This approach allows combining disease resistance and fruit quality traits without compromising the desirable characteristics of the original cultivar. In our study, we used the binary plasmid vector pMF1 to create three apple scab-resistant cisgenic 'Gala' lines with the apple scab resistance gene Rvi15, and three fire blight-resistant cisgenic 'Gala' lines with the fire blight resistance gene FB_MR5. Quantitative PCR revealed that five of six cisgenic lines had a single integrated T-DNA copy, and targeted locus amplification enabled the identification and characterization of insertion sites in five lines. All characterized lines showed T-DNA trimming at the borders, with a total length of non-endogenous sequences at their respective insertion sites ranging from 135 to 201 base pairs. The presence of such residual vector sequences can influence the regulatory status of cisgenic plants, as some countries may classify these lines as genetically modified plants. All cisgenic lines exhibited increased disease resistance compared to the untransformed control genotype 'Gala'. However, the achieved resistances are based on single genes, and it will be necessary to develop combinations of different resistance genes for more durable resistance. Resistance, durability, and evolving regulatory frameworks are crucial factors that together will determine the success of the cisgenic approach in achieving both high fruit quality and resistance for sustainable apple production.