【作者】 李茜；

【导师】 张存旭；

【作者基本信息】 西北农林科技大学 ， 林木遗传育种， 2006， 硕士

【摘要】 白皮松（Pinus bungeana Zucc.）为我国特有的乡土树种,也是我国北方园林绿化与生态工程造林的优良树种。本研究以白皮松未成熟合子胚以及成熟合子胚为外植体,诱导体细胞胚胎发生。系统研究了白皮松体胚发生所涉及的各种影响因素,以及体胚发生过程中的生理生化特性。研究结果将为白皮松优良无性系繁殖、基因保存、遗传改良以及人工种子生产等方面奠定坚实的理论基础。具体结果如下:1. BM1、DCR1和改良MS三种培养基相比,接种于DCR1的未成熟合子胚生长迅速,愈伤组织较大,分化能力较强。DCR1培养基的平均胚性愈伤组织诱导率达到56.59%,说明DCR1培养基最适合进行白皮松胚性愈伤组织的诱导。2.白皮松胚性愈伤组织在含有较高浓度生长素2,4-D和较高浓度的细胞分裂素6-BA的培养基上诱导率较高。不同浓度的2,4-D和6-BA对白皮松胚性愈伤组织的诱导率均存在极显著差异,2,4-D和6-BA的组合存在交互作用并对其胚性愈伤组织诱导有显著影响。多重比较可知6 mg·L^-12,4-D+2 mg·L^-16-BA为诱导胚性愈伤组织的最佳激素浓度组合。3.不同发育时期的未成熟合子胚诱导率差异很大。6月30日采摘合子胚的胚性愈伤组织诱导率明显高于其他四个时期,最高达到97.22%,平均诱导率为76.23%。4. 7月30日和8月10日采集的白皮松成熟合子胚很难诱导出胚性愈伤组织。个别外植体产生的愈伤组织经继代培养后生长势也较弱,不能进一步分化成胚性细胞。5. 0.3 mg·L^-12,4-D+0.2 mg·L^-1NAA为胚性愈伤组织保持和增殖的最佳激素组合浓度。2,4-D与NAA的交互作用对胚性愈伤组织增殖存在显著影响。6.继代方式对胚性愈伤组织增殖率有显著影响。以固体/液体交替培养的方式进行增殖培养,增殖率可达88.89%。7.培养在附加9000 mg·L^-1肌醇和0.1 mg·L^-1IBA的DCR1增殖培养基上的胚性愈伤组织将形成后期原胚。加入低浓度的IBA,能使更多的胚性细胞团转化成具胚柄胚性细胞团。8.蔗糖浓度的变化对白皮松体细胞胚的成熟发育有着极显著影响。含有50 g·L^-1蔗糖和0.1 mg·L^-1IBA的DCR1培养基最适合白皮松体细胞胚的成熟诱导,该组合明显提高了白皮松体细胞胚的成熟频率。9.在10 mg·L^-1ABA+75 g·L^-1PEG的DCR1培养基上,体细胞成熟率最高,达到更多还原

【Abstract】 Pinus bungeana is a native tree species in China. It is widely used garden green and ecological afforestation in north China. In this thesis, immature zygotic embryos were used as a source of initial explants. Factors affecting somatic embryogenesis as well as physiological and biochemical characteristics difference between embryogenic callus (EC) and non–embryogenic callus (N–EC) were investigated. The results would lay a good foundation for propagation excellent clones, gene preservation, genetic improvement and large-scale artificial seed production for Pinus bungeana.The results were as follows:1. Embryogenic callus can be induced on DCR1, BM1 and MSG medium with immature zygotic. Compareing with BM1 and MSG medium, the callus produced on DCR1 growed fast, and had a strong differentiation ability. The induction rates reached 56.59%.2. Higher inducing rates of EC were obtained on DCR1 medium with high 2,4-D and 6-BA concentration combination. The different concentration combination of 2,4-D and 6-BA , as well as their interaction had a significant effect on induction rates of EC. The result of Duncan’s multiple range test showed that 6 mg·L-12,4-D + 2 mg·L-16-BA was more favorable to induce EC.3. The different development stage of immature zygotic embryos have significant effect on. induction rates. More efficient induction of embryogenic cultures was achieved from the immature zygotic embryos sampled on June 30, Inducing rates reached 97.22%. The average induction rates was 76.23%.4. The mature zygotic embryos sampled on July 30 and August 10 were difficult to induce EC. EC produced from one or two explants growth potential became weak after subculture and could not further differentiated embrionic cell.5. 0.3 mg·L-1 2,4-D + 0.2 mg·L-1 NAA was more favorable hormone combination for EC maintain and proliferation.6. The subculture manners had a remarkable influence on EC multiplication rate. 88.89% multiplication rate achieved with solid/liquid alternate manner.7. The late stage proembryos were induced in EC of multiplication medium with 9,000 mg·L-1myo–inositol and 0.1 mg·L-1 IBA. More EC group could change into embryogenic suspensored cell mass with low concentration IBA.更多还原