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本论文首次报道了马尾松(Pinus massoniana Lamb.)幼胚体细胞胚胎发生的完整发育过程,并对影响马尾松胚性愈伤组织诱导的因素如球果采种期、球果冷藏处理时间、外植体处理方式等进行了探讨,统计胚性愈伤组织诱导率,进行增殖评价,探讨ABA浓度梯度对马尾松体细胞胚分化成熟的影响,试验数据用SPSS16统计分析软件进行方差分析、差异显著性检验。结果表明:1)2008~2009连续2年内15个采种期得到的幼胚,胚性愈伤组织诱导和增殖有显著性差异,最适宜的马尾松球果采种期是6月下旬至7月下旬,诱导率在9.66%~22.59%之间;2)球果冷藏处理时间,对胚性愈伤组织诱导有显著性差异,其中4℃冷藏球果15d有利于幼胚胚性愈伤组织诱导;3)雌配子体包含幼胚的接种处理方式是可取的;4)胚性愈伤组织经稳定增殖培养后,转入分化成熟培养基,得到体细胞胚状体“爆发式”分化成熟,数量多,质量好。适宜体胚成熟转化的培养基为:成熟LP培养基添加ABA5.0mg·L-1+60.0g·L-1蔗糖,并附加L-谷氨酰胺和水解酪蛋白;5)成熟体细胞胚在无激素萌发型LP培养基上正常萌发,并转化为结构完整的小植株。本研究首次建立了马尾松幼胚体细胞胚胎发生技术平台,为马尾松遗传改良种质创新、缩短育种周期奠定了研究基础。
This paper reports for the first time the complete development of somatic embryogenesis in Pinus massoniana Lamb. The effects of different factors on embryogenic callus induction such as seed collection, conch cooling, Implanted callus induction rate and proliferation evaluation, to explore the effect of ABA concentration gradient on the differentiation and maturation of P. massoniana embryos. The experimental data were analyzed by SPSS16 statistical analysis software, the difference was significant Sexual test. The results showed that: 1) There were significant differences in the induction and proliferation of immature embryos and embryogenic callus obtained from 15 seeding times in 2008 ~ 2009 for two consecutive years. The most appropriate seed-setting period for cones was from late June to late July , And the induction rate was between 9.66% and 22.59%. 2) The storage time of cones was significantly different from that of embryogenic callus induction. Cold storage of cones at 4 ℃ for 15 days was beneficial to embryogenic callus induction; 3) It is advisable to inoculate the female gametophyte which contains the immature embryos. 4) After the embryogenic callus has been stably proliferated and cultured, it is transferred to the differentiated mature medium to obtain the differentiation and maturation of the somatic embryoid body “explosive” A large number, good quality. The medium suitable for maturation of somatic embryos is as follows: mature LP medium is added with ABA 5.0 mg · L -1 + 60.0 g · L -1 sucrose, and L-glutamine and hydrolyzed casein are added; 5) mature somatic embryos Hormone-free germ-type LP medium normal germination, and transformed into a structurally intact plantlets. In this study, we established the technology platform of somatic embryogenesis for P. massoniana for the first time, which lays the foundation for genetic improvement of germplasm and shortening the breeding cycle of P. massoniana.