Optimization of somatic embryogenesis system through suspension culture pathway of embryogenic callus in Pinus koraiensis

To establish an optimized suspension culture system for embryogenic callus of Korean pine (Pinus koraiensis) to enhance the proliferation efficiency and somatic embryogenesis (SE) of its embryogenic callus.

Taking the embryonic callus of Korean pine as the material, the optimal conditions for suspension culture were determined by optimizing the initial inoculation amount, the type and concentration of plant growth regulators, the type and addition amount of carbon source, and the culture period; at the same time, the overall embryo maturation mode and ABA treatment concentration were adjusted to optimize the culture method, thereby establishing an optimized scheme for Korean pine SE based on the suspension culture pathway.

1) Based on the test results of the three clonal lines, the initial inoculation amount of embryonic callus was all optimal at 5-10 g·L^-1; 2) The treatment of 2,4-D with 0.5 mg/L had the best effect, the sediment cell volume (SCV), fresh weight (FW) and dry weight (DW) of the embryonic callus reached the highest levels, and the growth rates were 106.89%, 531.98% and 526.34% respectively; 3) The treatment of 6-BA at 0.1 mg/L was the best, its SCV, FW and DW were significantly higher than those of other treatments, and the growth rates were 106.45%, 562.16% and 567.92%, respectively; 4) When the sucrose concentration was 40 g/L, the SCW was small but the cell weight was large; 5) When cultured for 20 days, the maximum callus proliferation amount could be obtained, and its SCV, FW and DW increased by 958.67%, 605.67% and 575.45% respectively; 6) The application of ABA during suspension culture led to a significant reduction in somatic embryo yield as the ABA concentration increased. The embryonic calli that had been suspension-cultured for 20 days was then transferred to mature medium for 14 days of transition culture for somatic embryo induction, which could significantly increase the somatic embryo yield, with an average of 274 embryos/g.

Optimization procedure: mLV liquid medium supplemented with 0.5 mg/L of 2,4-D, 0.1 mg/L of 6-BA and 40 g/L sucrose; 5-10 g·L^-1 callus was initially added and dark cultured for 20 days, then, the suspension-cultured calli were transitional cultured for 14 days and then transferred to mature medium for somatic embryo induction.