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林业科学 ›› 2017, Vol. 53 ›› Issue (12): 41-49.doi: 10.11707/j.1001-7488.20171205

• 论文与研究报告 • 上一篇    下一篇

抗松材线虫病赤松体细胞胚的发育和成熟萌发

许建秀, 吴小芹, 叶建仁, 朱丽华, 吴静   

  1. 南京林业大学林学院 南方现代林业协同创新中心 南京 210037
  • 收稿日期:2016-10-23 修回日期:2017-09-16 出版日期:2017-12-25 发布日期:2018-01-13
  • 基金资助:
    江苏省科技支撑计划项目(BE2014405);国家林业公益性行业科研专项(201204501);江苏高校优势学科建设工程项目(PAPD)。

Development, Maturation and Germination of Somatic Embryo of Nematode-Resistant Pinus densiflora

Xu Jianxiu, Wu Xiaoqin, Ye Jianren, Zhu Lihua, Wu Jing   

  1. Co-Innovation Center for Sustainable Forestry in Southern China College of Forestry, Nanjing Forestry University Nanjing 210037
  • Received:2016-10-23 Revised:2017-09-16 Online:2017-12-25 Published:2018-01-13

摘要: [目的]研究脱落酸、PEG、肌醇、碳源、凝固剂及培养方式等对抗松材线虫病赤松体细胞胚发育及成熟萌发的影响。[方法]通过对抗松材线虫病赤松种质资源库的调查与分析,选用诱导和增殖状态良好的抗松材线虫病赤松无性系22#-1和13#-1为供试材料,通过不同培养条件的对比试验,筛选出最优的抗病赤松体细胞胚发育及成熟萌发条件。采用SPSS17.0等软件对体细胞胚进行统计分析。[结果]添加15 mg·L-1 ABA和140 g·L-1 PEG8000的LP培养基上,正常体细胞胚数达到171个·g-1,高于其他处理;在渗透剂方面,肌醇的浓度为8.0 g·L-1时,抗病赤松的正常体细胞胚数最多,达到179个·g-1,胚性愈伤组织直接放置于添加了肌醇的成熟培养基中,没有在中间过渡培养基中培养,因此可以缩短2~3周培养时间,在8周前后就形成了成熟的子叶胚;在碳源方面,添加60 g·L-1麦芽糖的LP培养基上,正常的体细胞胚数达到235个·g-1,高于其他处理;在凝固剂方面,植物凝胶浓度为3.0 g·L-1时,培养基凝固,软硬适中,产生的子叶胚生长良好,而培养基中添加了聚乙二醇(PEG),凝固剂琼脂从6.0 g·L-1添加至12.0 g·L-1时,培养基均不能凝固,愈伤组织无法生长,也无子叶胚形成;在培养方式方面,采用液-固增殖-固成熟方式培养时,胚性愈伤组织经11~12周发育为成熟体细胞胚,其中22#-1的正常体细胞胚数最多达到239个·g-1,高于其他处理;在体细胞胚萌发培养基上产生的体细胞胚的萌发率和植株转化率分别为67.2%和46.5%,再生植株移栽3个月后成活率为32.7%。[结论]将抗松材线虫病赤松胚性愈伤组织置于15.0 mg·L-1ABA、140 g·L-1 PEG8000、8.0 g·L-1肌醇、60 g·L-1麦芽糖、3.0 g·L-1植物凝胶的LP培养基上,以液-固增殖-固成熟培养方式培养,成功获得了正常的体细胞胚和再生植株,同时可缩短2~3周培养时间,加快抗松材线虫病赤松体细胞胚胎发生的进程。研究结果可为抗病赤松的大规模繁殖和工厂化生产提供技术支持。

关键词: 赤松, 松材线虫, 胚性愈伤组织, 体细胞胚, 成熟, 萌发

Abstract: [Objective] Exogenous hormones, penetrant, sugar, coagulant and culture model that have impacts on somatic embryogenesis of nematode-resistant Pinus densiflora were studied.[Method] On the basis of investigation and analysis against nematode-resistant P. densiflora germplasm resources database, two clones 22#-1 and 13#-1 of nematode-resistant P. densiflora that the induction and proliferation of somatic embryos at a good state were tested. Somatic embryogenesis was determined by comparative tests of different culture methods and different culture media. The optimum medium and method for somatic embryogenesis and germination and plantlet regeneration were screened. SPSS17.0 and other software were used to analyze the somatic embryos.[Result] The number of normal somatic embryos reached 171 embryo·g-1, higher than other treatments on the LP medium added with 15 mg·L-1ABA+140 g·L-1 PEG8000. Regarding the penetrant, the number of normal somatic embryos of nematode-resistant P. densiflora was up to 179 embryo·g-1, higher than other treatments on 8 g·L-1 inositol concentration. Embryogenic callus was directly placed on the maturation medium adding inositol. Embryogenic callus was not cultured on the middle medium. Therefore, the culture time could be shortened by 2-3 weeks. Mature cotyledon embryos were formed around 8 weeks. Regarding the carbon source, cultured on LP medium with 60 g·L-1 maltose, the number of normal somatic embryos reached 235 embryo·g-1, higher than other treatments; For the coagulant, when plant gel concentration was 3 g·L-1, solidification of medium displayed a moderate hardness and cotyledon embryo grew well. Supplemented with polyethylene glycol (PEG) and adding coagulant agar concentration from 6 g·L-1 to 12 g·L-1, the medium was not solidified. Callus was unable to grow and no cotyledon embryos were formed. Regarding the culture model, after 11-12 weeks the embryogenic callus developed into mature somatic embryos on liquid-solid proliferation-solid maturation. The number of normal somatic embryos of 22#-1 was up to 239 embryo·g-1, higher than other treatments. The somatic embryo germination rate was 67.2% and plantlet conversion rate from the germinated somatic embryos was 46.5%. Up to 32.7% of the transplanted plantlets were successfully survived three months in soil.[Conclusion] Embryogenic callus of nematode-resistant P. densiflora was on the LP medium with 15.0 mg·L-1 ABA +140 g·L-1 PEG8000+ 8 g·L-1 inositol+60 g·L-1 maltose+3 g·L-1 plant gel. The culture method was liquid-solid proliferation-solid maturation culture. Normal mature somatic embryos were successfully obtained and regenerated plantlets were transplanted to survive. At the same time, the culture duration could be shortened by 2-3 weeks, and the process of somatic embryogenesis of nematode-resistant P. densiflora was speeded up. Regenerated plants survived through transplanting. This study provides a feasible technique for large-scale propagation and mass production of nematode-resistant P. densiflora.

Key words: Pinus densiflora, Bursaphelenchus xylophilus, embryogenic callus, somatic embryogenesis, maturation, germination

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