【作者】 贾彩凤；

【导师】 李悦；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2005， 硕士

【摘要】 华山松(Pinus armandii Franch.)是重要的乡土速生针叶树种,有较高的综合利用价值,探索该树种的离体繁殖技术,建立快速繁殖体系,对该树种的遗传改良研究和开展生物工程工作奠定良好的理论和技术基础。本文创新点在于以成熟胚为外植体,成功建立了华山松的直接器官发生体系及其植株再生体系;并分别以幼胚和成熟胚为外植体,对华山松的体细胞胚胎发生途径进行了探索,国内外尚未见到相关报道。主要研究结果有: 1 直接器官发生的研究 采用70%酒精0.5min和0.1%升汞10min对华山松种子灭菌处理后,剥取种胚垂直接种于培养基表面。华山松的不定芽诱导培养基为:DCR+BA 3.0～5.0mg/L+蔗糖30g/L,诱导率最高达到67.57%;分化培养基为:DCR+0.1mg/L IBA+2g/L AC;生根培养基为:1/2DCR+4.0～5.0mg/L IBA,从诱导的愈伤组织内可以分化出较粗短的不定根,但这种生根苗移栽时较难成活。 2 成熟胚的体细胞胚胎发生研究 华山松成熟胚诱导愈伤组织的培养基成分为:DCR+2,4-D10mg/L+BA 4mg/L+CH,肌醇各1000mg/L+Gln500mg/L,其中类似胚性愈伤组织诱导率达到21.58%。培养2w后将愈伤转至与诱导培养基组分相似,但植物生长调节剂降低10倍的固体或液体培养基上进行继代培养,愈伤组织最终褐化死亡。 3 未成熟胚的体细胞胚胎发生研究 华山松未成熟胚诱导愈伤组织的培养基成分为: DCR+2,4-D10mg/L+BA,KT各4mg/L+CH,肌醇各1000mg/L+Gln500mg/L; 1/2LM+2,4-D2mg/L+BA lmg/L+CH,肌醇各1000mg/L+Gln500mg/L; 将幼胚接种在DCR培养基上只能诱导出愈伤组织,其中类似胚性愈伤组织率最高达到47.62%。以1/2LM为基本培养基时,子叶胚前期的幼胚诱导愈伤组织的胚性可高达52.71%。但幼胚诱导的愈伤组织经固体继代培养和液体悬浮培养后,都没有胚状体的分化。华山松的子叶期幼胚在1/2LM培养基上发生早熟萌发现象,诱导出具有根和茎的完整植株,且诱导率达92%以上。 总之,华山松的组培苗生根不易,应加强组培苗瓶内瓶外的生根诱导处理;体细胞胚胎发生困难,应对如何有效的保持和增殖胚性愈伤组织,诱导胚状体的分化及体细胞胚的萌发等进行深入研究。更多还原

【Abstract】 Pinus armandii Franch. is one of the most important fast-growing conifers in China with highly value of general utilization . There are great theoretical and practical importances on developing the genetic improvement and biology engineering to explore the generation technology in vitro and establish the rapid propagation system of Pinus armandii Franch. The innovations of this paper lied in that we have established the direct organogensis and plantlet regeneration system through mature embryos , and explored the influencing elements of somatic embryogenesis of Pinus armandii Franch., while no relevant reports about that in domestic and overseas studies. The main results are as following:1 Studies of direct organogensisSeeds of Pinus armandii Franch. were soaked in 70% alcohol for 30s, then being sterilized into 0.1% HgCl₂ for 10 min. The mature embryos were inoculated vertically in the DCR medium for induction. The contents of medium inducing the adventitious buds are : DCR+BA3.0⁵.0mg/L+sucrose30g/L,with the highest frequency of 67.57%; while the differentiation medium were: DCR+IBA 0.1mg/L+AC2 g/L;and the rooting medium were :1/2DCR+IBA 4.0⁵.0 mg/L,on which the stubbed adventitious roots can be induced from the calli, but the seedlings are difficult to survive after transplanted .2 Somatic embryogenesis by mature embryosThe contents of medium inducing the calli from mature embryos of Pinus armandii Franch.were: DCR+2,4-D10mg/L+BA4mg/ L +CH,Myoinositol 1000mg/L individually +Gln 500mg/L,with the highest frequency of similar embryonic calli of 21.58%. After 2 weeks calli were transferred to the medium whose contents were similar as induction medium but hormones reduced to 10 times, but the subculture substances were browned and dead.3 Somatic embryogenesis by immature embryosThe contents of medium were as following: DCR+2,4-D 10mg/L +BA,KT 4mg/L individually+CH,Myoinositol 1000mg/L individually+Gln500mg/L;1/2LM+2,4-D 2mg/L+BA 1mg/L+CH,Myoinositol 1000mg/Lindividually+Gln500mg/L. The immature embryos of Pinus armandii Franch.inoculated in the DCR medium can induce calli only, with the frequency of similar embryonic calli of 47.62%. While the highest frequency of similar embryonic calli can reach 52.71% when the precotyledonary embryos were inoculated in 1/2LM medium. But all the calli induced by immature embryos did not differentiate the embryoids through solid and liquid subculture. The whole plantlets with root and stem can be formed through precocious germination when the cotyledonary embryos of Pinus armandii Franch. were inoculated in 1/2LM medium with the induction frequency of 92%.In a word, the rooting induction from cultured seedlings of Pinus armandii Franch. is not easy, so we should strengthen the rooting studies from inside and outside of the culture containers. The somatic embryogenesis system establishment through immature embryos and mature embryos is very difficult, which needs further research about how to keep and propagate the embryonic calli efficiently, and induce the embryoids differentiation and somatic embryos germination.更多还原