关键词: 落叶松, 长白落叶松, 杂种落叶松, 胚性愈伤组织, 体细胞胚发生, 悬浮培养

Abstract: [Objective] Using embryogenic lines of larch, a suspension culture system suitable for embryogenic callus development was established and optimized to explore the main factors affecting the tissue proliferation. On the basis of these, somatic embryo maturation induction of suspension tissue was carried out. It aims to set up a basis for rapid proliferation of embryogenic callus and large-scale propagation of somatic embryos.[Method] The subculture of embryogenic calli of Larix olgensis, L. gmelinii ×kaempferi and L. kaempferi ×olgensis was carried out, and the fresh proliferated tissues were selected for the suspension culture. In order to optimize the appropriate culture conditions, the orthogonal array of L₉(3⁴) was designed and verified by a test, in which the proliferation amount and the proliferation rate of the embryogenic callus were taken as the response values. The embryogenic tissue obtained from suspension proliferation was used for somatic embryogenesis, and the number of somatic embryos was counted.[Result] In the process of larch suspension culture, the influence of initial inoculation quantity, shaking intensity and suspension culture time on the proliferation of the embryogenic callus was very significant. The proliferation amount and the proliferation rate of the embryogenic suspension tissue were decreased with the increase of inoculation amount, and a tendency of first increase followed by a decrease with increase of shaking intensity was exhibited; with the increase of incubation time, the amount and rate of proliferation were both increased. The 4 g·L^-1embryogenic callus was cultured in the BM medium containing 2,4-D 0.15 mg·L^-1, 6-BA 0.05 mg·L^-1 and KT 0.05 mg·L^-1 (SCM)under the condition of light avoidance and the 120 r·min^-1 shaking intensity. The three lines of Larix embryogenic callus can proliferate rapidly and steadily. The proliferation rates of the three lines after 15 days suspension culture were 2 569.42%, 4 189.96% and 3 001.67% respectively, showing a significant interspecific differences. The mature cultivation mode has significant effects on the amount of somatic embryos in the suspension culture(P=0.000). The embryogenic callus obtained from the suspension culture was inoculated to the solid proliferation medium(PCM) containing agar 6 g·L^-1 for 15 days, then transferred to 1/4 BM medium with inositol 10 g·L^-1(TCM) for 14 days, and then transferred to the somatic embryo maturation medium containing ABA 20 mg·L^-1, AgNO₃ 5 mg·L^-1, and PEG₄₀₀₀ 80 g·L^-1 for 8 weeks. The results showed that the amount of somatic embryogenesis increased significantly(P=0.000). The number of somatic embryogenesis of the three Larix embryogenic lines OO-A1,GK-F1 and KO-H was (101.69±11.19), (93.09±9.34) and (5.78±1.47) embryo·g^-1respectively.[Conclusion] Suspension culture can obtain a large scale of Larix embryogenic calli with uniform dispersion and high quality in the short term, without affecting the somatic embryogenesis and maturation. After 15 days of dark culture, in the BM liquid medium(SCM) the fresh weight of the embryogenic tissue of Larix can be increased by 26.99-42.90 times when the inoculation amount of callus was 4 g·L^-1 and the shaking intensity was 120 r·min^-1. Before the somatic maturation induction, the embryogenic suspension callus was first cultured in the solid proliferation medium(PCM) for 15 days and then transferred into the transitional medium(TCM) for 14 days, after which the amount of the somatic embryogenesis could be significantly increased.

Key words: Larix, Larix olgensis, hybrid larch, embryogenic calli, somatic embryogenesis, suspension culture