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 inoculum-density, 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.
Highlights
Korean pine (Pinus koraiensis Sieb.et Zucc.) is the dominant species of broad-leaved Korean pine forests and it has an extremely important ecological value
We evaluated the proliferation rate of embryogenic tissue (ET) under different inoculum-density, orbiting speed, plant growth regulator (PGR) concentration, and carbon source, and came up with a culture plan suitable for effective proliferation of Korean pine ET
After 14 d of culture, the ET was transferred to a maturation medium to obtain the highest somatic embryos (SEs) yield (80 SEs ⋅ g−1 fresh weight (FW))
Summary
Korean pine (Pinus koraiensis Sieb.et Zucc.) is the dominant species of broad-leaved Korean pine forests and it has an extremely important ecological value. It is the main afforestation tree species in Northeast China, with excellent wood and seeds, and extremely high economic value [1]. The development of breeding technology for large-scale clone production is imminent to restore and expand the resources of P. koraiensis, and give full play to the ecological function and economic value of this species. The embryogenic tissue obtained from the immature zygotic embryos of Korean pine has the ability of somatic embryogenesis [8]. We urgently need a method that can increase the yield of SEs to meet large-scale production
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