Overexpression of Ginkbilobin-2 homologous domain gene to enhance the tolerance to Phytophthora cinnamomi in plants of European chestnut

Castanea sativa , a species of high ecological and economic relevance in Europe, faces severe threats from root rot caused by Phytophthora cinnamomi . To explore genetic strategies for enhancing disease tolerance, we investigated the functional role of a chestnut gene homologous to Ginkgo biloba ’s ginkbilobin-2 ( Cast_Gnk2-like ), known for its antifungal properties. Using Agrobacterium tumefaciens -mediated transformation, the Cast_Gnk2-like gene was introduced into somatic embryos from two embryogenic chestnut lines. Transformation efficiency was genotype-dependent, and varied from 14.2% to 2.5%. Twelve independent transgenic lines were confirmed by PCR, and each was estimated to carry a single copy of the transgene. Gene expression analysis revealed significant Cast_Gnk2-like transcript levels in two transgenic lines. Following cold storage and germination treatment, viable transgenic plants were regenerated. Disease tolerance assays demonstrated that Cast_Gnk2-like overexpression significantly reduced root necrosis and symptom severity, indicating enhanced tolerance to P. cinnamomi . These findings highlight the potential of targeted gene overexpression to improve disease resilience in chestnut through genetic engineering.