A strategy for establishing an efficient somatic embryo regeneration system in Castanea mollissima Blume

Chinese chestnut (Castanea mollissima Blume), an economic forest species, has important economic value and ecological function. The innovation, improvement, reproduction and utilization of clonal germplasm are the critical foundation for industrial development of Chinese chestnut. Somatic embryogenesis (SE) is an important method to obtain clonal plants for large-scale production. To establish a stable and efficient somatic embryo regeneration system, we introduced the liquid suspension culture, notably increasing the induction rate of embryogenic callus (EC) from 45.73% to 84.23%, increasing the proliferation coefficient of EC from 3.2 to 15.3, and shortening the developmental process of somatic embryos by three weeks. Synchronization rates of somatic embryos at four developmental stages were significantly enhanced by screening via steel sieve after liquid suspension culture. After the selection of mature cotyledonary embryos, the germination rate was increased from 2.2% to 13.3%. We also identified high-embryogenic-competence lines among tested embryogenic cell lines. Furthermore, GRF7 and GIF1 were dramatically highly expressed in the high-embryogenic-competence lines at EC stage, offering effective marker genes to early identify high-embryogenic-potential lines. Based on liquid suspension culture, uniformity screening and early detection of high-embryogenic-potential lines by marker genes, this efficient somatic embryo regeneration system will make it available for large-scale and synchronic reproduction of clonal germplasm and rapid genetic transformation in Chinese chestnut. Key message Based on liquid suspension culture, uniformity screening, and early identification of high-embryogenic-potential lines by GRF7 and GIF1, a modified somatic embryo regeneration system was established in Chinese chestnut.