Establishment of regeneration and genetic transformation protocols for Castanea seguinii Dode

Somatic embryogenesis serves as a prevalent method for germplasm enhancement, yet it encounters challenges such as the low germination rate in the somatic embryogenesis of Castanea mollissima Blume. Seeking a solution, the potential of C. seguinii Dode, a closely related species known for its short juvenile period, was explored. A comparative analysis demonstrated that the propagation coefficient of adventitious shoots in C. seguinii was twice that of C. mollissima . Recognizing the promise of C. seguinii in expediting the chestnut research and breeding program, a protocol for somatic embryo regeneration and genetic transformation of C. seguinii was established by using immature embryos as explants. Our optimized protocol demonstrated a callus induction rate of 31.6%, and the germination rate of the cotyledon embryo was 12.5%, with the entire process completed in 17 weeks (from callus induction to somatic embryo germination). Notably, the transient transformation rate using Agrobacterium tumefaciens reached 19.9%, the stable transformation efficiency could reach 13.04% within 12 weeks timeframe (from the initial infection of the callus, through the acquisition of transgenic callus, to the final generation of transgenic seedlings). Further, the roots of these transgenic plants were able to establish symbiotic relationship with Scleroderma citrinum as wild type. This study contributes an alternative experimental platform supporting molecular design breeding and fundamental research of ectomycorrhizal symbiosis within woody plants, especially in the field of Castanea plants.