Somatic embryogenesis of masson pine (Pinus massoniana): initiation, maturation and genetic stability analysis at SSR loci

Pinus massoniana planted widely in southern China in recent decades. The pinewood nematode (PWN) causes pine wilt disease (PWD), which threatens pine forests, including masson pine. Resistance breeding to pinewood nematodiasis has become an important way of preventing damage caused by nematodes. In masson pine, somatic embryogenesis (SE) was established, but still restricted by many factors. In this study, whole megagametophyte were used for embryogenic callus induction. The effects of explant maturity and family on the initiation of embryogenic callus, as well as the effects of maltose and long-term subculture on the somatic embryo maturation, were investigated. The maturity of zygote embryos gradually increased as the seed collection date increased, and the initiation rate of embryogenic suspensor masses (ESM) decreased. Four types of extruded ESM were observed, and the extrusion type was related to explant maturity. The megagametophytes had the highest initiation rates when the immature zygotes were mainly concentrated in stage 3. Seed family GX4 was the female parent with highest ESM initiation rate. Long-term subculture significantly affected the somatic embryo yield. Addition of 40.0 g/L maltose to maturation medium yielded 12.7 ± 0.9 somatic embryos per milliliter. We additionally choose 16 regenerated plants and analyzed the genetic stability using SSR molecular marker. The average variation rate of per locus was 2.78%. This report presents a successful protocol for establishing SE of masson pine and provides meaningful suggestions for optimizing the large-scale propagation of masson pine. Key message We provided meaningful suggestions for optimizing the mass propagation of masson pine and we accelerates the progress of breeding for PWN resistance.