【作者】 凤婷；

【导师】 赵密珍；

【作者基本信息】 安徽大学 ， 细胞生物学， 2013， 硕士

【摘要】 本实验以江苏省农科院园艺研究所草莓资源圃保存的‘红颊’草莓品种的组培苗为外植体进行实验。‘红颊’是一种优质大果型草莓品种,其颜色鲜艳,着色一致,外形美观,香味口感极佳,但是炭疽病发病率高已经成为其产量增长的重要影响因素之一。本实验目的是通过EMS诱变筛选出定向突变的抗炭疽病新植株。首先探讨激素,放置方式,暗培养时间,温度,不同部位等条件对其再生的影响,筛选出最佳组合以优化其再生体系,运用最小显著极差法处理实验数据,探讨不同植物生长调节剂组合(IBA, NAA, IAA)、暗培养时间(5,10,15,20d)、放置方式(正面朝上,反面朝上)、不同取材部位(叶柄,叶片)对其再生的影响。结果表明：MS+TDZ(2.0mg·L-1)+NAA(0.1mg·L-1)为最适宜的不定芽分化培养基,不定芽分化率最高可达到100.0%,一周(7天)是最佳的暗培养时间,叶片正面朝上放置较好。叶柄再生能力比叶片强,两者都是基部比顶部分化率高,诱导能力从强到弱可依次排列为叶柄基部>叶片基部>叶片顶部>叶柄顶部。最佳生根培养基为1/2MS+IBA (0.1mg-L-1),20天后100%的不定芽可长出3-4cm长的根,移栽成活率达到98.06%。由此建立起‘红颊’草莓品种的高效再生体系。然后配置浓度分别为0.05%,0.10%,0.2%,0.4%,0.8%的5个梯度EMS诱变液,设空白对照。将诱变材料放入不同浓度诱变液1h探讨不同浓度对其影响。同时将诱变材料放入0.1%的诱变液处理时间分别为0.5,1.0,1.5,2.0h,以探讨不同诱变时间对其影响。还将诱变材料放入0.1%的诱变液处理1.0h,以探讨处理后其内部不同活性指标的变化。根据PPO、SOD等活性物质的变化,推测EMS较低浓度,较短时间处理下,细胞能自身修复,一旦超过临界点,则伤害无法得到修复。0.8%浓度的EMS诱变液配以长时间处理,短时间内实验材料褐化死亡,而在0.2%浓度的EMS诱变液配以短时间处理,处理后的材料无褐化死亡现象或者褐化率过低。得出诱变组合为浓度为0.2%,处理时间为80min；浓度为0.4%,处理时间为60min；浓度为0.4%,处理时间为60min这三种组合均可。将诱变得到的再生芽转接到Ms培养基培养中培养,待其长大后移入生根培养基。当根长到3-4cm长时移到穴盘内进行培养,待其长成后用用浓度为每毫升7×106个孢子的炭疽菌孢子悬浮液喷洒植株表面,初步筛选出表型为抗炭疽病的草莓植株5株。这些抗性植株的遗传基因需进一步进行分子学验证。更多还原

【Abstract】 Strawberry cultivar’ Benihoppe’ is a high-quality type of strawberry cultivar with beautiful appearance and excellent fragrance taste. But anthracnose become one of the important factors affecting its production growth. The purpose of this study is to obtain new plants which resistant to anthracnose through the EMS mutagenesis screening.The plantlets kept in vitro were used as test materials to study the influences of various combinations of plant growth regulators, culture durations under dark, positioning explant styles, and samplings on strawberry regeneration with the experimental data being analyzed by the shortest significant range method. The results showed that the optimal medium for adventitious bud regeneration was MS+TDZ (2.0mg·L-1)+NAA (0.1mg·L-1) with the highest differentiation rate of100.0%, for which7days of dark time and face up positioning were necessary. It was noticed that the adventitious bud regeneration capacity from petiole was better than that from leaf blade, and both of them could have better regeneration capacity at their bases. The best rooting medium was1/2MS+IBA (0.1mg·L-1), on which100%of the adventitious buds could grow3-4cm roots after20days of culture. After transplanting,98.06%of the rooted shoots could survived well in greenhouse.The leaves used as mutagenic materials were treated with different concentrations of EMS solutions for different time to test the physiologically active substance and the degree of injury. The results indicated that the damage of the mutagenic materials was growing with concentration and time of EMS solutions increasing.0.8%concentration of EMS is too high. Most of all material was browning and died in short time under the0.8%concentration, on other side, browning of the materials was not heavy under0.2%of EMS.0.4%concentration of EMS for60min was the best combination of mutation.According to the above results,many leaves used as mutagenic materials were treated with0.4%concentrations of EMS solutions for60min,then transferred the leaves into induction medium (MS+TDZ (2.0mg·L-1)+NAA (0.1mg·L-1)). to get regeneration bud. Take regeneration bud to the rooting medium. When the plantlets’ root reach3-4cm length, transplant them in plug. Spraying plant with the7X106/ml concentrations of the anthrax spore suspension when the plants grew up, five plant showed relatively resistant to anthracnose. These resistant plants should be done further molecular genetic identification.更多还原