Open Access

ARTICLE

Suspension Culture and Somatic Embryogenesis of Korean Pine

by Chunxue Peng^1,2, Fang Gao^1,2, Hao Wang^1,2, Iraida Nikolaevna Tretyakova³, Alexander Mikhaylovich Nosov^4,5, Hailong Shen^1,2,*, Ling Yang^1,2,*

1 State Key Laboratory of Tree Genetics and Breeding, School of Forestry, Northeast Forestry University, Harbin, 150040, China
2 State Forestry and Grassland Administration Engineering Technology Research Center of Korean Pine, Harbin, 150040, China
3 Laboratory of Forest Genetics and Breeding, Institution of the Russian Academy of Sciences, V. N. Sukachev Institute of Forest Siberian Branch of RAS, Krasnoyarsk, 660036, Russia
4 Department of Cell Biology and Institute of Plant Physiology, K. A. Timiryazev Russian Academy of Sciences, Moscow, 127276, Russia
5 Department of Plant Physiology, Biological Faculty, Lomonosov Moscow State University, Moscow, 119991, Russia

* Corresponding Authors: Hailong Shen. Email: ; Ling Yang. Email:

(This article belongs to the Special Issue: Tree Somatic Embryogenesis and Application)

Phyton-International Journal of Experimental Botany 2022, 91(1), 223-238. https://doi.org/10.32604/phyton.2022.015523

Received 25 December 2020; Accepted 09 April 2021; Issue published 16 August 2021

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.

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