Suspension Culture and Somatic Embryogenesis of Korean Pine

Abstract

Korean pine is an important afforestation tree species in Northeast China, which has a high ecological and economic value. Although regeneration of somatic embryogenesis using immature zygotic embryos of Korean pine as explants has been successful, it cannot be applied to automation and large-scale production. Therefore, we urgently need a method that can increase the output of somatic embryos (SEs) to meet the needs of large-scale production. We used Korean pine 1-1 and 1-100 cell lines as research materials to evaluate the effects of inoculum-density, culture time, orbiting speed, vessel volume, plant growth regulator (PGR) concentration, and carbon source on the proliferation of embryogenic tissue (ET). The somatic embryogenesis ability of ET cultured in different liquid suspension media was also evaluated. We found that during liquid suspension culture of Korean pine ET, the sedimented cell volume (SCV), fresh weight (FW) and dry weight (DW) were affected by inoculumdensity, culture time, orbiting speed, 2,4-D concentration, 6-BA concentration and carbon source type. Fourty mg ⋅ mL −1 ET were transferred to a 200 mL Erlenmeyer flask containing 20 mL liquid medium, and cultured at 100 rpm/min for 14 days to obtain the maximum proliferation. In addition, we also found that SCV, FW and DW were higher when PGRs were reduced in the liquid suspension medium. The substitution of maltose for sucrose resulted in slow growth of cultures and limited SE yield (13 SEs g −1 FW). Although culture proliferation was high at 50 rpm, SE yield was inhibited by 48% compared with 100 rpm (50 rpm = 33 SEs g −1 FW; 100 rpm/min = 70 SEs g −1 FW). Cultivation in low-concentration PGR(1.15 μM ⋅ L −1 2,4-D, 0.25 μM ⋅ L −1 6-BA) and sucrose liquid medium at 100 rpm/min (80 SEs g −1 FW) could not only promote culture proliferation but also increase SE yield. The determination of the suspension culture scheme of Korean pine ET provides a reference for further expansion to bioreactor culture in the future and lays a foundation for the automation and scale of somatic embryogenesis of Korean pine.

A method for permanent genetic transformation using embryonic callus of Pinus koraiensis

“…The approach embryo maturation was adapted from Peng 35 with some changes. Three grams of various transgenic Pinus koraiensis embryonic callus samples were placed into 20 mL of liquid medium (DCR+30 g•L -1 sucrose) devoid of any hormones and incubated for seven days.…”

Long-Term Successional Subculture Dynamics and Their Effects on the Proliferation Efficiency, Embryogenic Potential, and Genetic Stability of Embryogenic Tissues in Larix principis-rupprechtii Mayr

“…Factors impacting diverse stages of somatic embryogenesis (embryogenic tissue induction, proliferation culture, pre-treatment, maturation treatment, etc.) and techniques to enhance culture efficiency across various Pine species have been explored [10,[12][13][14][15][16][17][18][19][20]. Furthermore, with the advancement of bioinformatics, researchers are increasingly focusing on the role of genes involved in the various stages of somatic embryogenesis [8,[21][22][23].…”