Junzan Huang

The walnut cultivar ‘Yunxin No. 14’ is an early fruiting, high-yielding, and widely adaptable fruit tree with compact growth and superior nuts. Establishing a successful tissue culture system for this cultivar is crucial for its rapid clonal propagation and as a foundation for future genetic transformation. Using young fruits as explants, 3% NaClO sterilization for 20 min effectively controlled contamination and browning. Somatic embryos induced from zygotic embryos cultured on DKW medium with 30 g·L⁻¹ sucrose showed high proliferation and minimal browning. After a 4-day dehydration treatment using saturated NH₄NO₃, mature somatic embryos germinated rapidly on differentiation medium (DKW containing 1 mg·L⁻¹ 6-BA and 0.1 mg·L⁻¹ IBA), reaching 90.0% germination. Optimal shoot multiplication was achieved on DKW medium supplemented with 2 mg·L⁻¹ 6-BA and 0.3 mg·L⁻¹ IBA, yielding a proliferation rate of 91.1% and a proliferation index of 3.1. For rooting, shoots (~3 cm) treated with Clonex^® rooting gel were transferred to a low-cost, sugar-free vermiculite medium with gaseous CO₂ as the sole carbon source. Root initiation occurred within two weeks at a rate of 54.2%, significantly shortening the rooting phase. Rooted plantlets were acclimatized in a peat:perlite:vermiculite (2:2:1, v/v/v) mixture under high humidity for two weeks before outdoor transfer, achieving an 88.6% survival rate. This study provides a reliable protocol for the micropropagation of ‘Yunxin No. 14’ and a valuable reference for other difficult-to-root woody species.