A Three-Tier In Vitro Strategy for Accelerated Pine Breeding and Resistance Research Against Pine Wilt Disease

Pine wilt disease (PWD), caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, is a globally destructive threat to coniferous forests, causing severe ecological and economic losses. Conventional resistance breeding is critically hampered by long life cycles of trees and field evaluation challenges. To address these limitations, we developed a three-tier biotechnology pipeline with a dual-output goal (generating both resistant germplasm and mechanistic insights) designed to bridge the in vitro-field gap. This strategy is founded upon the resolution of a longstanding pathogenesis debate, which established aseptic PWNs as a standardized research tool. The pipeline integrates high-throughput in vitro cellular screening (Tier 1), whole-plant validation via organogenesis (Tier 2), and scaled production coupled with mechanistic investigation through somatic embryogenesis (Tier 3). Tier 1 enables rapid phenotypic screening, Tier 2 validates resistance in whole plants, and Tier 3 facilitates mass production and in-depth study. It operates as a closed-loop, knowledge-driven system, simultaneously accelerating PWN-resistant germplasm development and empowering molecular mechanism discovery. Validated across Pinus massoniana and P. densiflora, this work provides a concrete, community-usable model system that directly addresses a core methodological bottleneck in forest pathology. This strategy effectively bridges the in vitro-field gap, offering a replicable model for perennial crop breeding and contributing to resilient forest management.