Establishment of Physalis grisea genetic transformation system and overexpression of AtPAP1 enhances drought stress

Physalis grisea (P. grisea), commonly referred to as ground lantern, possesses significant medicinal and ornamental value. It is worth noting that orphan crops within the genus Physalis occupy a critical evolutionary position within the Solanaceae family. However, an established transformation system for this species has yet to be developed. This study optimized the regeneration system for P. grisea and established a stable genetic transformation system, thereby facilitating the exploration of critical genes' potential functions in anthocyanin synthesis under abiotic stress using the developed transgenic P. grisea. The results indicated that hypocotyls were effective as explants, yielding induction rates of regenerated buds exceeding 44.15 %. In contrast, when cotyledons and true leaves were employed as explants, the induction rates of regenerated buds were 18.74 % and 3.52 %, respectively. Phenotypic observation and molecular identification revealed that with an optimal optical density (OD₆₀₀) of 0.6, an infection duration of 5 min, and a co-culture time of 2 days, the transgenic positive rate for P. grisea hypocotyls reached its peak at approximately 42.17 %. By establishing a transgenic system of AtPAP1 in the hypocotyls of P. grisea, morphological observations indicated that the heterologous overexpression of the AtPAP1 gene significantly enhanced anthocyanin accumulation in transgenic P. grisea plants. The AtPAP1 overexpressing P. grisea plants exhibited reduced plant height, shortened internodes, and a substantial increase in stem diameter, along with varying degrees of purple pigmentation. Under drought conditions, the accumulation of anthocyanins in AtPAP1 transgenic lines positively contributed to drought resistance. Physiological assays demonstrated that the levels of POD (peroxidase) and RWC (relative water content) were elevated compared to those in WT plants, while the concentrations of MDA (malondialdehyde), O₂^- (superoxide anion free radical), and H₂O₂ (hydrogen peroxide) were reduced. Additionally, RT-qPCR analyses indicated that anthocyanin-related genes, including CHI and CHS, were up-regulated, thereby enhancing the drought tolerance of P. grisea. In summary, the stable Agrobacterium-mediated transformation system represents a valuable tool for genetic engineering and the creation of drought-resistant germplasm in P. grisea. KEY MESSAGE: This study contributes to the establishment of a robust regeneration and genetic transformation system for P. grisea and demonstrates that AtPAP1 can significantly enhance the drought resistance of transgenic P. grisea plants.