Recombinase Based Stacking of Multiple Disease Resistance Genes in Granola Potato

Abstract

Phytophthora infestans, Potato Virus Y (PVY), and Potato Leafroll Virus (PLRV) are major diseases that severely affect potato production and quality worldwide, including Indonesia. These pathogens pose significant threats to crop security, and effective, durable control methods are still limited. This study aimed to establish an efficient transformation system using Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY (GAANTRY)-based gene stacking technology to generate multi-pathogen-resistant potato lines, which may reduce reliance on chemical control and support environmentally sustainable cultivation. Two Granola potato clones, Granola Indonesia (Gra I) and Granola MSU (Gra M), were evaluated for their in vitro growth and regeneration performance. Gra M exhibited vigorous vegetative growth, while Gra I showed the highest regeneration efficiency using mid-stem node explants cultured on GNZ medium. Transformation using Agrobacterium rhizogenes strain ArPORT1 harboring the multigene vector pMSU2DR-02 resulted in 13% transformation efficiency and 100% regeneration with a 10-minute infection period. PCR confirmed stable integration of five resistance genes (RpiAmr1, RpiAmr3, RpiVnt1, RySto, and Rladg) without vector backbone fragments. These results demonstrate that GAANTRY-based gene stacking is an effective approach for producing potato cultivars with single T-DNA insertions carrying multiple resistance genes, providing broad-spectrum and durable resistance as a sustainable alternative to chemical control for long-term crop protection. The resulting transgenic potato lines have the potential to be utilized to improve disease resistance and productivity among smallholder farmers in Indonesia.