Research on the embryonic development and propagation culture techniques of Litsea cubeba

Objective As an economically important woody oilseed species in China，Litsea cubeba（Lour.）Pers. faces significant bottlenecks in conventional propagation methods.This study aims to establish an efficient somatic embryogenesis system for L. cubeba to achieve mass propagation，thereby overcoming the limitations of traditional breeding techniques characterized by long cycles and low multiplication rates. Method Somatic embryogenesis stage：The single factor experimental method was used to explore the effect of activated carbon concentration on somatic embryogenesis，and the embryos of L. cubeba from May to August were used as explants，and 7 collection times were set to explore the effects of different collection times on somatic embryogenesis.In this study，the effects of high-concentration sucrose solution immersion，sucrose and acid hydrolyzed casein content on somatic embryogenesis were investigated by three-factor and three-level orthogonal experiments.Besides，the effects of different concentrations of 6-BA，2，4-D and IAA on somatic embryogenesis were investigated by using a three-factor and four-level orthogonal experimental method.Secondary embryo proliferation stage：The effects of different concentrations of 6-BA，NAA，inositol and cyclodextrin on the proliferation of secondary embryos were investigated by a four-factor three-level experimental method.In this way，the most suitable medium and culture conditions for the somatic embryogenesis and secondary embryogenesis of L. cubeba and its secondary embryonic proliferation were determined. Result During somatic embryogenesis（cultured at 24±2 ℃ in darkness for 90 days），embryos developed through direct or indirect pathways.The optimal collection time was 75 days after full bloom，when seeds exhibited appropriate differentiation and stress resistance with high embryogenic potential.Activated charcoal at 150 mg/L showed the best effect，while higher concentrations inhibited embryogenesis.Orthogonal tests with three factors6-BA（0.5，1.0，1.5，and 2.0 mg/L），2，4-D（0.2，0.4，0.6，and 0.8 mg/L），and IAA（0.25，0.5，0.75，and 1.0 mg/L） revealed the primary-secondary order of effects as IAA > 6-BA > 2，4-D.The optimal induction medium was MS + 30 g/L sucrose + 7.0 g/L agar + 700 mg/L casein hydrolysate + 2 mg/L 6-BA + 0.6 mg/L 2，4-D + 0.5 mg/L IAA，achieving a maximum somatic embryogenesis rate of 23.3%.For secondary embryo proliferation（cultured at 24±2 ℃ in darkness for 30 days），6-BA（PPConclusion This study significantly improves the efficiency of somatic embryogenesis and secondary embryo proliferation in L. cubeba，providing a technical foundation for its large-scale propagation.