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林业科学 ›› 2018, Vol. 54 ›› Issue (7): 146-154.doi: 10.11707/j.1001-7488.20180716

• 研究简报 • 上一篇    下一篇

落叶松胚性愈伤组织悬浮培养体系的优化

宋跃, 李淑娟, 张含国, 白晓明, 毕显禹, 董实伟, 董昊   

  1. 林木遗传育种国家重点实验室(东北林业大学) 哈尔滨 150040
  • 收稿日期:2017-10-19 修回日期:2018-02-07 出版日期:2018-07-25 发布日期:2018-08-11
  • 基金资助:
    国家"863"计划项目(2013AA102704-04-03);黑龙江省科学基金项目(C2017008)。

Establishment and Optimization of Embryogenic Callus Suspension Culture System of Larix

Song Yue, Li Shujuan, Zhang Hanguo, Bai Xiaoming, Bi Xianyu, Dong Shiwei, Dong Hao   

  1. State Key Laboratory of Tree Genetics and Breeding(Northeast Forestry University) Harbin 150040
  • Received:2017-10-19 Revised:2018-02-07 Online:2018-07-25 Published:2018-08-11

摘要: [目的]以落叶松胚性系为研究对象,建立和优化适合落叶松胚性愈伤组织增殖的悬浮培养体系,在此基础上对悬浮组织进行体细胞胚的成熟诱导。旨在为实现落叶松胚性愈伤组织的快速增殖及体细胞胚的规模化繁育奠定基础。[方法]对已诱导获得的长白落叶松、兴安×日本杂种落叶松及日本×长白杂种落叶松胚性愈伤组织进行继代培养,取新鲜增殖的组织进行悬浮培养。采用L9(34)正交试验设计,以胚性愈伤组织的增殖量及增殖率为响应值对悬浮培养条件进行筛选和优化,并对选出的最适培养条件进行验证。以悬浮增殖的胚性组织进行体细胞胚成熟培养,统计体胚发生量。[结果]在落叶松胚性愈伤组织的悬浮培养过程中,接种量、震荡强度及培养时间对胚性组织增殖具有显著的影响。在一定范围内,落叶松胚性组织的增殖量及增殖率随初始接种量的增加而降低,随震荡强度的升高呈先增加后下降,而随培养周期的延长而增加。以4 g·L-1接种于含2,4-D 0.15 mg·L-1、6-BA 0.05 mg·L-1及KT 0.05 mg·L-1的BM培养基(SCM),在120 r·min-1避光条件下悬浮培养,3个落叶松胚性系的胚性组织均能快速、稳定地增殖,培养15天的增殖率分别为2 569.42%、4 189.96%及3 001.67%,表现出较明显的种间差异。成熟培养方式对落叶松胚性悬浮组织的体胚发生量影响极显著(P=0.000)。悬浮培养获得的胚性组织接种到含琼脂6.0 g·L-1的固体增殖培养基(PCM)上继代15天后,转接到添加肌醇10 g·L-1且无生长调节剂的1/4BM过渡培养基(TCM)上培养14天,再转入含有ABA 20 mg·L-1、AgNO3 5 mg·L-1、PEG4000 80 g·L-1的体胚成熟培养基上培养8周,体胚发生量显著提高(P=0.000)。在此条件下,3个落叶松胚性系OO-A1、GK-F1及KO-H的体胚发生量分别为(101.69±11.19)、(93.09±9.34)及(5.78±1.47)个·g-1。[结论]悬浮培养能在短期内获得大量分散均匀、质量较高的落叶松胚性愈伤组织,且不会影响体细胞胚的发生和成熟。在BM液体培养基(SCM)中,接种量为4 g·L-1、震荡强度为120 r·min-1、暗培养15天,落叶松胚性组织的鲜质量可增加26.99~42.90倍。悬浮培养获得的胚性组织经固体增殖培养基(PCM)继代15天及过渡培养基(TCM)预培养14天后,再进行体胚成熟诱导可明显提高其体胚发生量。

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

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