A multi-stage protocol for indirect somatic embryogenesis and plantlet regeneration from leaf explants of Quercus chungii

Somatic embryogenesis (SE) is pivotal for mass clonal propagation in forestry, yet oak species remains recalcitrant. This study established an indirect SE protocol for Quercus chungii plantlet regeneration using leaf explants from two-year-old seedlings. Callus was induced on MS medium with 2,4-D (1.0 mg·L−1) and 6-BA (1.0 mg·L−1) (6–8 weeks) with or without AC (3.0 g·L−1), followed by PGR-free 4-week interval, and embryogenic stimulation via NAA (1.0 mg·L−1) and 6-BA (1.0 mg·L−1) for 4–5 weeks, another PGR-free 3–4 weeks intervals and then reduced NAA (0.25 mg·L−1) and 6-BA (0.25 mg·L−1) for sustaining callus proliferation, then triggered the proembryogenic masses developmental process by the removal of NAA but keeping 6-BA (0.5 mg·L−1) in darkness for 4–5 weeks, leading to early embryo formation. Embryos matured on 0.1 mg·L−1 ABA-supplemented ½ MS medium under a 16/8 h photoperiod. A percentage of 51.1 % callus induction rate and 77 % normal embryos maturation were achieved, yielding viable plantlet with 5.67 % somatic embryos conversion. The conceptual framework for developing SE protocol in Q.chungii and the obtained multi-stage protocol enhance SE efficiency in Q.chungii, aiding mass clonal propagation potential for the development of clonal forestry and the conservation and utilization of Q.chungii and related oaks.