【作者】 刘良宏；

【导师】 石淑稳；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2000， 硕士

【摘要】 菌核病（Sclerotinia sclerotiorum）是我国油菜的主要病害之一，每年使油菜遭受直接经济损失达30亿元以上，且至今尚未找到菌核病的抗源。为寻找抗性材料，本试验以甘蓝型油菜为试材，采用⁶⁰Coγ—射线辐射和草酸离体筛选方法，对抗菌核病材料的筛选技术做了初步的研究，得到以下结论： 1、采用160—320Krad剂量的γ—射线处理油菜种子后，再将种子接种于含有O—6.0mM草酸的B₅培养基上。试验结果表明，辐射和草酸毒素对种子发芽率、下胚轴的生长、幼苗根长及幼苗成苗率等都有着明显的影响： A、对种子发芽率的影响随着B₅培养基所含草酸浓度的增加，发芽率急剧下降，当草酸浓度为3.0mM时，其发芽率降低一半，而当草酸浓度大于5.0mM时，种子几乎不能发芽：种子经过辐射处理后，其发芽率也会降低，而且随着辐射剂量的增大，种子耐酸能力有所下降，这可能是种子辐射损伤后，生长势受到严重抑制的结果。通过相关分析发现，种子发芽率与草酸浓度之间呈直线相关，相关系数高达0.9543。 B、对幼苗下胚轴生长的影响当辐射剂量在160Krad时，在不同浓度草酸培养基上，幼苗的下胚轴平均长度为26.3mm，仅为对照（51.7mm）的50.9％；在草酸浓度低于4.0mM范围内，培养基草酸浓度每增加1.0mM，其下胚轴长度下降约30％，当草酸浓度在3.0mM时，幼苗下胚轴的平均长度仅为对照的13.5％。差异显著性检验表明，不同草酸浓度之间和不同辐射剂量之间，幼苗下胚轴的平均长度均达到了显著水平。 C、辐射和草酸复合处理对幼苗主根生长的影响 随草酸浓度的增加及辐射剂量的提高，幼苗主根长度都有逐渐缩短的趋势，这与对幼苗下胚轴长度的影响是相似的。但差异显著性检验发现，只有不同辐射剂量之间，主根长度的差异才达到了显著水平。 D、对幼苗生长发育的影响 种子经辐射处理后接种于B₅培养基上，7天后，剪掉幼苗的下胚轴，将带有子叶的顶芽转入B₅培养基进行培养。结果表明，辐射剂量为0、80、120Krad的处理成苗率分别为99.2％、91.8％和90.1％，但当辐射剂量达到160Krad时，成苗率仅7.3％。推测高剂量辐射使大部分种子的分生细胞受损死亡，因此，这些种子的萌发仅仅表现为胚根、胚轴的伸长和子叶的伸展；将带有子叶的顶芽剪下转入B₅培养基上进行培养，大多数难以长出真叶，即使有少数能长出一、二片真叶，一段时间以后，生长点的生长也停止，不能形成正常苗：将萌发后的幼苗直接转入B₅培养基，幼芽同样没能形成真叶。 2、一至三片真叶的幼苗对草酸的耐性 用含有不同浓度草酸的B₅培养基处理一至三片真叶期的幼苗96小时，结果发现，一至三叶期的幼苗对草酸的忍耐能力没有多大的差异。当草酸浓度达到3.0mM时，98.4％的幼苗茎基部全部腐烂，幼苗死亡，这同时也说明幼苗不及种子更能忍耐草酸毒素。油菜抗（耐）菌核病材料离体筛选方法的初步研究 3、叶片接种菌丝体进行抗病鉴定将菌丝体接种在用草酸筛选种子所形成的试管苗叶片上，其发病状况是:幼苗虽然都表现为感病，但发病程度存在差异，即叶片病斑的直径与筛选采用的草酸浓度之间呈明显的负相关，相关系数r=0.9554。 4、抗菌核病材料的离体筛选程序可采用两条途径筛选抗性材料，一条途径是:种子辐射处理一种子草酸筛选一幼苗草酸筛选^{幼苗再次草酸筛选}一幼苗离体叶片接种鉴定筛选;另一条途径是将辐射处理的种子培养成苗后，直接从幼苗期开始进行草酸筛选。辐射适宜的剂量为8任一一¹20Krad，适宜种子处理的草酸浓度为5.0爪M，适宜幼苗筛选的草酸浓度为3.OmM。 5、离体筛选得到耐菌核病的植株筛选得到了9株抗（耐）性表现较好的幼苗，其中8株表现较抗（耐），其离体片接种24小时后未见有明显病斑出现（对照苗病斑直径为5.2Inln），接种36小时后的病斑直径仅为对照的45.9%:另一株表现为高抗（耐），接种36小时后也未见明显病斑，接种72小时后的病斑仅6.0^。更多还原

【Abstract】 Sclerotinia sclerotiorum is the main disease on oilseed rape (Brassica ssp), which has caused 30 millions directed damages to the rapeseed production in China every year and the sourse of resistance to Sclerotinia sclerotiorum has not been found so far. In order to acquiring some new disease-restance materials, premiliminary studies on induced mutation with -raysand sreening in vitro were done with Brassica napus .The results were summarised below:1. The dry rapeseeds were treated with y -rays dose ranging from 160 to 320 Krad and then were seeded in B5 medium added oxalic acid(OA) with concentration ranging from 0 to 6.0 mM.The results showed the effects on the germination capacity of seeds, hypocotyl length, root length and the survial rate of the seedlings after the seeds were treated with V -rays and OA. Following were the conclusions.A. The germination percentage of the seeds was rapidly decreased with the increase of the concentration of OA added in medium. When the concentration reached to 3.0 mM, the germination rate reduced by half and the seeds almost cant germinate in 65 medium. The seed germination also deduced after the rapeseeds were irradiated by the Y -rays and the tolerant of seeds to the OA became weak. Correlation analysis revealed that negative relation between the seed germination pecentage and the concentration of OA added in the medium reached significant level(r=0.9543).B. The hypocotyl length were significant shorter than that of controls. When seeds were treated with 160 Krad Y -rays, the average length of hypocotyl was 26.3 mm in OA-added medium,which was only 50.9% compared with that of controls.When the OA’s the concentration varied from 0 to 4.0 mM,the hypocotyl length hardly reduced by 30 pecent while the concentration rised every one mM. Test usfng Duncan’s method indicated that the difference of hypocotyl length between the OA’s congcentration- and between dose of Y -rays were significant.C. The effect on growth stage of main root was similar to that on the hypocotyl length. The primary root’s length of seedling was shorter and shorter gradually with the increase of OA’s concentration and dose of Y -rays.But test using Duncan’s method demostrated that the difference was significant only between the dose of Y -rays.D. A test about culture in vitro with apical buds along with cotyledons cut from the seedlings was done.The results showed that the survial rate of seedling was 99.2%,91.8% and 90.1% respectively when the dry seeds had been exposed with 0,80 and 120 Krad Y -rays.But the survial rate was only 7.3% when the dose of Y -rays was 160 Krad. Result showed that the LD5 o of Huashuang No.3 was between 120 and 160 Krad. Because meristematic cells of the seed had suffered from irradiation injury and died ,the germination of seeds were only the straight growth of corculum and embrynonal and the extension of cotyledon.So there were no euphylis to be grown when the apical buds were translated into medium. Even if there were one or two euphylises to be form, they would not grow any more and die after a period. The same result had been gained when the seedling were derectly translateded into the Bs medium.2. A culture in vitro in OA-added medium seedlings at one-leaf stage,two-leaf stage and three-leaf stage were done. The results showed that the seedlings’ capacity of tolerance to OA were not significant between the seedling at different stage. When the concentration of OA reached to 3.0 mM, the stem of 98.4% of seedlings rot away and the seedlings died after one month. This also demostrated that the seedlings’ capacity of tolerance to OA toxin were inferior to that of seeds.3. Identification for the seedlings’ resistance to Sclerotinia sclerotiorum by detached-leaves inoculated with mycelium were done. The results showed that the seedlings screened with OA-added medium at seed-stage all had suffered the disease, but there were significant difference between the seedlings from different medium and negative relation between the更多还原