【作者】 张彦妮；

【导师】 卓丽环；

【作者基本信息】 东北林业大学 ， 园林植物与观赏园艺， 2005， 博士

【摘要】 本研究通过不定芽和愈伤组织两种途径,首次建立了复叶槭的组织培养再生体系,并采用农杆菌介导法和针刺法首次对槭树科植物进行了抗虫基因的遗传转化,得到了经PCR和PCR-Sourthern检测呈阳性的转基因愈伤组织和转基因植株。本论文研究的主要内容和结果如下: 1.复叶槭不定芽途径再生体系的建立 (1) 以茎节、顶芽为外植体,通过比较不同激素种类和浓度对增殖率和分化率的影响,筛选出适合不同外植体的最佳培养基和激素组合,从而建立起复叶槭不定芽再生体系。结果表明,适合茎节和顶芽的增殖培养基均是MS基本培养基附加0.1mg/L 6-BA和0.01mg/L NAA,分化培养基分别是附加0.1 mg/L TDZ和0.01 mg/L 2,4-D的MS培养基和附加0.01 mg/L TDZ的MS培养基,最佳的生根培养基为MS基本培养基附加0.1mg/LNAA,生根率为89%。 (2) 以茎节为外植体,比较了液体培养和固体培养的效果,结果发现,在TDZ同一浓度下,液体培养比固体培养的分化率高,产生不定芽的时间相对较早。 2.复叶槭茎段等诱导的愈伤组织途径再生体系的建立 以茎段、叶片、子叶、叶柄为外植体,通过比较不同激素种类和浓度对不同外植体愈伤组织的诱导和分化率的影响,筛选出适合不同外植体的最佳培养基和激素组合,从而通过愈伤组织再生途径初步建立起复叶槭的组织培养再生体系。结果表明,不同外植体在不同培养基上诱导愈伤组织的颜色和质地略有差异,脱分化和再分化能力不同。茎段的脱分化和再分化能力相对最强,叶柄最差。适合茎段再分化的最佳培养基是MS基本培养基附加0.0005 mg/L TDZ和0.01 mg/L NAA,分化率是22.8%,其他外植体脱分化后没能再分化。 3.茎段等诱导的愈伤组织解剖学研究 通过组织切片技术研究了不同颜色和质地的愈伤组织以及茎节在不同时期诱导的愈伤组织的内部结构,结果表明,不同颜色和质地的愈伤组织的细胞排列方式、细胞大小等不同,浅棕色疏松等浅色系的愈伤组织多为胚性愈伤组织,从而为从颜色和质地上确定复叶槭胚性愈伤组织奠定基础。茎节在不同时期的细胞分裂有一定差异,通过组织学观察到了不定芽的形成多是经过愈伤组织途径。 4.复叶槭遗传转化的初步研究 (1) 筛选了适合复叶槭不同转化体的卡那霉素浓度。复叶槭的叶片、茎段和茎节、愈伤组织分别选用15mg/L、25mg/L、15mg/L作为筛选浓度较为适宜。比较了不同菌液浓度和感染时间对转化率的影响。结果表明,用LB液体培养基和过夜培养的菌液以1∶1稀释后感染10min转化率相对较高。 (2) 通过农杆菌介导法和针刺法,首次将蜘蛛神经毒蛋白杀虫肽基因Toxin和Bt基因导入复叶槭,经分子生物学检测,获得了复叶槭抗虫转基因愈伤组织和转基因植株。更多还原

【Abstract】 In this study, tissue culture technique and regeneration system for Acer negundo L. were first established. Moreover, the insect-resistant genes were first transformed into Acer negundo via Agrobecterium-mediaXed transformation and needle-sticking method transformation in the aceraceous plants. Positive calli and plants were obtained by PCR amplification and PCR-Southern analysis. The main contents and results are listed as follows:1. The establishment of regeneration system by the route of adventitious buds of Acer negundo(1) A regeneration protocol through axillary buds has been developed for Acer negundo. Stalk nodus and tip shoot explants were placed on MS medium supplemented with different hormone or different concentration and then compared their ratio of proliferation and differentiation. The best medium and hormone combinations for tip shoot and stalk nodus were established. The result showed that MS medium containing 0.1 mg/L 6-BA and 0.01 mg/L NAA suited proliferation culture for stalk nodus and tip shoot. The best differentiation medium for them were separately MS solid medium with 0.1 mg/L TDZ and 0.01 mg/L 2,4-D and MS solid medium supplemented with 0.01 mg/L TDZ. The best rooting medium was MS medium with 0.1 mg/L NAA. The rooting ratio was 89%.(2) Nodal segments were separately cultured MS liquid medium and solid medium supplemented with different TDZ concentration. The effect of differentiation in liquid medium was better than in solid medium and adventitious shoot produced earlier on the medium with the same TDZ concentration.2. The establishment of regeneration system by the route of callus from stem et al. of Acer negundoA tissue culture system for Acer negundo by the route of inducing calli were primarily established through screening out the best media and hormone combinations for different explants (stem segment, leaf, cotyledon, petiole). The calli induced from different explants had a little difference in color and texture. Their capability of induced calli and differentiation were different and stem segment was the strongest and petiole was poor. The best medium for differentiation of stem segment was MS basal medium supplemented with 0.0005 mg/L TDZ and 0.01 mg/L NAA. The ratio of differentiation was 22.8 %. The else explants did not regenerate.3. The study of anatomy of calli induced from stem et al.The inside tissue of calli from different color and texture was observed. Moreover, the histological events during adventitious bud development in stalk nodus have been examined. The results showed the arrange mode and size of different origin calli were different slightly. Tint brown or tin calli were embryogenic callis in majority. The research would establish basis for identification embryogenic calli from color and texture. Meristematic activity in thesuperficial layers led to bud primordial formation.4. The primary study on genetic transformation of Acer negundo(1) The concentration of kanamycin was confirmed for screening out transformed explants from different explants. It was fitting that leaves , stem and stalk nodus , calli were selected by 15mg/L、 25mg/L、 15mg/L of kanamycin separately. The effects of different concentration of bacterium solution and infecting times on the transformant ratio were studied. As a result, the bacterium liquid diluted with the same volume LB liquid medium infected explants for 10 minutes would have the higher transformant ratio.(2) The Toxin gene and Bt gene were first transformed into Acer negundo by Agrobacterium-mediated method and needle-sticking method. Transgenic calli and transgenic plants were obtained by PCR and PCR-Southern hybridization analysis.更多还原