【作者】 胡甦；

【导师】 王永清；

【作者基本信息】 四川农业大学 ， 园林植物与观赏园艺， 2010， 硕士

【摘要】 本研究以三角紫叶酢浆草叶色变异株系为材料,研究其组织培养技术体系,并以野生型材料为对照,用RAPD和ISSR标记对其进行DNA检测鉴定。主要研究结果如下：1.形态发生能力：比较继代1次、5次和10次材料,叶片和叶柄的不定芽和愈伤组织的分化率均随继代次数增加而降低,不定根发生率随继代次数增加而升高。连续继代10次以内,选择叶片做外植体(培养物)更有利于保持相对稳定的愈伤组织和不定芽分化能力。2.增殖：TDZ能明显促进变异株系芽苗的增殖,增殖效果随其浓度增加而增加。一定量的TDZ能显著提高变异株系芽苗叶片花色素苷的积累。3.壮苗：适当提高不含生长调节物质的MS培养基中的磷、钾元素含量有利于芽苗的生长和叶片花色素苷的积累。当培养基中KH2PO4的浓度为680 mg/L(即4倍磷钾元素含量)时,试管苗的苗重、苗高和叶片数均达最大值。4.生根培养与鳞茎发生：NAA作用优于IBA,但两者配合使用效果最好。培养基中不加入6-BA有利于增加发根数和鳞茎数。最佳生根和鳞茎诱导配方均为：1/2MS+NAA 0.2 mg/L+IBA0.2 mg/L(蔗糖40g/L,琼脂粉7g/L,pH5.8-6.0)。5.炼苗移栽：变异株系的生根苗和带根鳞茎的移栽成活率都很低,分别为2.56%和27.27%。6.蔗糖浓度对野生型和变异型材料叶片颜色的影响：三角紫叶酢浆草野生型和变异型材料的叶片颜色对蔗糖浓度变化都很敏感。(1)野生型叶绿素总量、叶绿素a含量、叶绿素b含量以及Chla/Chlb的比值均随蔗糖浓度提高而迅速升高；除Chla/Chlb比值有所下降外,上述其他指标数值均在蔗糖浓度高于30g/L后逐渐趋于稳定。变异型材料的叶绿素总量、叶绿素a、叶绿素b含量以及Chla/Chlb比值随蔗糖浓度升高均没有显著的变化。类胡萝卜素含量变化趋势与叶绿素基本一致。不同的是,变异型的类胡萝卜素含量也随蔗糖浓度提高而显著升高,但其增长幅度远低于野生型。相同蔗糖浓度下,变异型的上述所有色素指标的数值都远低于野生型。(2)野生型和变异型材料的花青素含量随蔗糖浓度增加而显著升高。蔗糖浓度为10-30g/L,野生型的增长速率大于变异型,超过30g/L后,野生型增长速率逐渐缓慢,而变异型出现并保持指数型增长趋势。7.采用改良CTAB法提取材料的DNA。优化了三角紫叶酢浆草的RAPD反应体系,首次建立并系统优化了三角紫叶酢浆草ISSR反应体系和扩增参数。优化后的RAPD反应体系为：反应液的总体积25μl, 10×Buffer 2.5μl, Mg2+浓度2.0 mmol/L, Taq酶1U,引物浓度0.3μmol/L, dNTP Mixture 0.25 mmol/L,模板DNA 80-100ng。RAPD-PCR反应程序为：94℃预变性4min；94℃变性45s,37℃退火1min,72℃延伸1.5min,40次循环；72℃延伸5 min；4℃保存。优化后的ISSR反应体系为：25μl反应液体系中含10×Buffer 2.5μl, Mg2+浓度1.25 mmol/L, Taq酶0.9U,引物浓度0.3μmol/L, dNTP Mixture 0.25 mmol/L,模板80ng。ISSR-PCR反应程序为：94℃预变性5min；94℃变性50s,退火时间60s(不同引物的退火温度不同),72℃延伸1.5min,40次循环；72℃延伸7min；4℃保存。8. RAPD和ISSR检测结果表明,在本研究涉及的引物检测范围内,变异株系继代5次与继代10次材料的基因组DNA扩增产物之间没有明显的差异,野生型花纹叶片材料与无花纹叶片材料的扩增结果之间也没有显著的差异。9.从70个RAPD随机引物和100个ISSR随机引物中分别筛选出2个RAPD特异引物(S76,S1t8)和2个ISSR特异引物(UBC818, UBC868),分别扩增出114和96个片段,多态性片段数为18和12,多态性百分率为15.79%和12.50%。其中差异性条带有5种,均能区分野生型和变异株系。变异株系的差异性缺失带S118 700-800测序结果序列包含编码叶绿体光系统Ⅰ第Ⅶ亚基的基因。证明了变异株系叶片颜色的变化的确与总基因组DNA的变化相关,变异株系叶绿素缺失性状可能与上述基因的缺失或者非正常表达有关。更多还原

【Abstract】 An in vitro culture protocol was developed for the leaf color mutational strain of Oxalis triangularis. RAPD and ISSR markers were used for its identification with the wild type as check. The results were as follows.1. Morphogenetic ability:by comparing tissues subcultured for 1,5 and 10 times, the occurrence rates of adventitious buds and calluses coming from leaf blades and leaf stalks reduced, while the differentiation rates of adventitious roots rose, with the increase in subculture times. It was beneficial to remain relatively stable differentiation rates of adventitious buds and calluses to choose leaf blades as explants within 10 times subculture.2. Proliferation:a low concentration of TDZ,0.02 to 0.40 mg/L, remarkably promoted proliferation of the mutational strain, and the multiplication rate went up with TDZ concentration increase. A certain amount of TDZ could significantly enhance the anthocyanin accumulation.3. Plantlet strengthening:it was helpful for strengthening plantlet growth and enhancing the anthocyanin accumulation to appropriately increase the amount of phosphorus and potassium in MS without any plant growth regulator. When the KH2PO4 concentration was 680 mg/L, i.e.,4 times of original amount of phosphorus and potassium, the increment of plantlet weight, height and leaf number reached the maximum values.4. Rooting and bulb formation:NAA was better than IBA in the effect on rooting and bulb formation. Nevertheless, the effect was the best by combining use of NAA and IBA. Root number and bulb number increased when 6-BA was not added in the medium. The best medium for rooting and bulb formation was 1/2MS+ NAA 0.2 mg/L+IBA 0.2 mg/L (sucrose 40g/L, agar 7g/L, pH5.8-6.0).5. Seedling hardening and transplanting:the survival rate of the both rooted seedlings and bulbs with roots were very low, and were, respectively,2.56% and 27.27%.6. Effects of the sucrose concentration on leaf color of the wild and mutant strain:the wild type and the mutant type of Oxalis triangularis were both sensitive to the change of sucrose concentration. (1) The total chlorophyll content, Chl.a content, Chl.b content and the Chl.a/Chl.b ratio of the wild type all increased rapidly when the sucrose concentration gradually rose. All the indcies above, except the Chl.a/Chl.b ratio, tended to remain stable after sucrose concentration reaching 30 g/L. But there were no significant changes of the total chlorophyll content, Chl.a content, Chl.b content and the Chl.a/Chl.b ratio of the mutant type when the sucrose concentration gradually rose. The carotenoid content had a similar trend to the chlorophyll content, except that the carotenoid content of the mutant type increased significantly when the sucrose concentration rose, but by a much narrower range than the wild type. Growing on media with the same sucrose concentration, the mutant type had much lower values of all the leaf pigment indcies above than the wild type. (2) Anthocyanidin contents of both the wild type and mutation type increased with the increase in sucrose concentration. The rate of rise of the wild type was faster than the mutant type when the sucrose was within the concentration of 10-30 g/L. However, the rate of the wild type turned slow after the sucrose concentration exceeded 30 g/L, while the rate of the mutant type started and maintained an exponential increasing trend.7. Improved CTAB method was suitable for DNA extraction. RAPD-PCR reaction system for Oxalis triangularis was optimized. A suitable ISSR-PCR reaction system for Oxalis triangularis was established and optimized systematically for the first time. The optimal RAPD-PCR reaction system was as follows. The total 25μl reaction system contained 2.5μl 10×buffer,2.0 mmol/L Mg2+,1U Taq DNA polymerase,0.3μmol/L primer, 0.25mmol/L dNTP mixture, and 80-100ng template DNA. The suitable reaction program was:94℃for 4min, followed by 40 cycles of 94℃for 45s, annealing at 37℃for lmin, 72℃for 1.5min,72℃for 5min. The ISSR-PCR reactions were performed in a 25μl volume containing 2.5μl 10×buffer,1.25mmol/L Mg2+,0.9U Taq DNA polymerase, 0.3μmol/L primer,0.25mmol/L dNTP mixture, and 80ng template DNA. The optimized reaction program was:94℃for 5min, followed by 40 cycles of 94℃for 50s, annealing at suitable temperature for different primers for 60s,72℃for 1.5min, and at last extension at 72℃for 7min. All amplification products were stored at 4℃.8. The result of RAPD and ISSR marker identification showed that within the test range of primers used in this study there were no significant differences between genome DNA of the mutant type materials subcultured for 5 times and 10 times, and neither were the genome DNA of the wild type with stripes on leaf blades and the wild type without stripes.9. Two specific RAPD primers, i.e., S76 and S118, and two specific ISSR primers i.e.,UBC818 and UBC868, were selected from 70 RAPD primers and 100 ISSR primers, respectively, the amplification products of which contained 114 and 96 clear bands, respectively, including 18 and 12 polymorphic fragments, and the percentage of polymorphism were 15.79% and 12.50%, respectively. There were 5 types of specific bands, which could identify the wild type and the mutational strain of Oxalis triangularis. The sequencing result of the absent specific band, S118 700-800, in the mutant type contained a gene that coded for subunit VII, i.e., PsaC protein, in photosystem I. The evidences above proved that the leaf color change of the mutational strain of Oxalis triangularis was closely related to the change of the total genome DNA, and the chlorophyll absence trait of the mutational strain might be associated with the absence or abnormal expression of the gene mentioned above.更多还原