【作者】 张小红；

【导师】 陈耀锋；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2006， 博士

【摘要】 利用体细胞无性系变异及赤霉菌毒素离体筛选技术,创造新抗源,培育抗赤霉病新品种,已成为小麦赤霉病抗性改良的重要途径。本研究以普通小麦品种为试材,通过幼胚培养毒素筛选及抗毒素细胞变异系再生植株的抗性筛选,获得了两个综合农艺性状与其各自供体亲本基本一致,但抗性与其供体亲本相比显著提高的抗赤霉突变体,并对获得的两个抗赤霉突变体的分子差异、生理生化特性及细胞组织学特性进行了分析。获得以下结果:1、以普通小麦幼胚为外植体对离体培养毒素筛选体系的研究表明,胚发育时间对离体培养有较大影响,花后10d～13d的幼胚愈伤组织和胚性愈伤组织的诱导率较高;蔗糖在20～50 g/L浓度范围内对胚性愈伤组织的诱导效果较好;6.0g/L的禾谷镰刀菌粗毒素是小麦幼胚离体筛选的一个敏感浓度,对愈伤组织诱导有明显抑制,对胚性愈伤组织的形成和苗分化产生强烈抑制,筛选中粗毒素浓度不宜高于这一浓度或连续使用,否则难以获得再生绿苗。2、对筛选获得的抗毒素细胞系再生植株不同世代的抗赤性表现及分离特点的研究表明,R0代再生植株抗赤性存在差异,经毒素筛选的后代抗病植株比例与未经毒素筛选的相比明显提高;抗病性主要在R1代分离,并趋向于感病方的分离;抗病性在R2代仍存在部分分离,在R3代稳定。对再生植株不同世代的农艺性状变异特点研究表明,变异可发生在株高、穗长、小穗数、穗型、株型、熟性和小穗密度等多个方面,不同基因型发生表型变异的频率不同;再生植株农艺性状变异的分离主要发生在R1代,部分性状在R2代继续分离,在R3代稳定。总体变异趋于矮杆、早熟和穗密度增加。通过田间抗性筛选,本实验最终在西农1376和花育888的抗毒素细胞变异系再生植株中分别获得一个抗赤霉病性明显改良的变异系,其抗赤霉病性与高抗扩展的苏麦3号和西农85相比呈中等抗性,但与各自感病亲本相比抗性显著提高。农艺性状鉴定结果显示,两个抗赤霉变异系与各自供体亲本基本相似。3、抗赤霉变异系RAPD检测结果显示,抗赤霉变异系与它们供体亲本相比在多个位点都发生了变异,变异率为2.10%～2.86%,从分子水平上证明了变异的真实性。抗赤霉变异系R西农1376-2和亲本西农1376间检测出的多态片段S3471220和S3751360,抗赤霉变异系R花育888和亲本花育888间检测到的多态片段S149620都可在抗扩展品种苏麦3号上找到,说明离体条件下发生变异的抗性位点具有特异性,初步判断这三个多态片段S3471220、S3751360和S149620与小麦赤霉病抗性有关。4、以典型抗扩展品种和突变体供体亲本为抗、感对照,对抗赤霉突变体的健康穗及接种穗的生理生化特性研究结果显示,在健康穗中,抗性突变体的PAL活性、总酚、类黄酮及MDA含量及变化趋势与其感病亲本相一致,但在病原菌侵染后,这些生理生化物质的含量与变化程度与其亲本有了明显的差异,其变化范围介于典型高抗品种和感病亲本之间,变化趋势与亲本或抗性品种一致。说明离体筛选的抗赤霉突变体的生理生化抗性机制与一般抗性品种类似。生理生化特性研究还表明,不同基因型健康穗或接种穗PAL活性、总酚及类黄酮含量存在显著差异,与材料的抗赤性无一定顺序关系。材料抗赤性强弱与接种穗与健康穗之间的PAL活性及总酚、类黄酮含量的增加幅度呈负相关。健康穗MDA含量在基因型间无差异,但在接种穗中,随处理时间延长在感病品种中MDA含量持续增加,在突变体与抗扩展品种中前期增加,第7d后下降,第11d后接种穗MDA含量高低与材料抗赤性强弱呈负相关。这些特性在典型抗、感品种间表现明显。5、对抗赤霉突变体的健康穗及接种穗的穗轴细胞组织学研究结果显示,抗性突变体与其感病亲本的花期健康穗轴在皮层厚壁组织、绿色组织及维管组织结构上基本一致,不同发育时间的变化趋势也一致;接种处理后,抗性突变体与其感病亲本的穗轴组织在各方面的变化趋势相一致,变化量也无明显差异。主要差异在于病菌侵染后抗性突变体穗轴组织中的菌丝量较少,受侵染程度较轻。据此可以认为,对供试的抗性突变体而言,其抗赤霉病性的提高与其穗轴组织结构无关。实验结果还证明,高抗品种和感病品种的穗轴组织结构确实存在一定差异,在健康穗中,主要表现在高抗品种的皮层厚壁细胞壁和皮层厚壁组织较厚,皮层厚壁细胞层数较多,维管束数目较多,穗轴表皮下绿色组织面积较小,抗、感品种间的差异达显著水平,随发育时间延长,高抗品种的皮层厚壁细胞壁厚度、厚壁组织厚度和厚壁细胞层数增加的幅度较大;在病穗中,主要表现在高抗品种的皮层厚壁组织细胞层数和厚壁细胞壁厚度增加的幅度较大。6、利用农艺性状和分子标记相结合的方法,对来自国内外的30多份小麦抗赤霉种质资源及本实验筛选的两个抗赤霉突变体的遗传多样性进行了分析,结果表明,供试材料总体表现植株较高、穗较长,小穗数少,穗密度较稀,穗粒数较多等相似的形态特征,各农艺性状的遗传变异程度由高到低依次为株高〉穗长〉小穗数〉穗粒数〉反应指数〉有效分蘖,变异系数在8.69%～28.75%之间,多样性指数在1.74～1.95之间,说明农艺性状的遗传多样性较丰富;RAPD分析结果表明,16种引物共扩增出176条带,多态性位点比例为84.09%,平均遗传相似系数0.7069,遗传距离0.3524,基于材料间的遗传距离,供试材料可聚为五类,地理来源相近的基本上聚为一类,亲缘关系较近的也被聚在一起,说明RAPD能充分反应材料间的遗传差异,能较好将供试材料进行分类,同时反映出材料间遗传关系与小麦生态环境和地理来源高度相关。更多还原

【Abstract】 Using somatic clone variation and toxin-selecting method to produce new resistant strains and to cultivate new varieties has become the key method to improve Scab-resistance in wheat. The study, applying Fusarium Graminearum crude toxin to select embryogenic calli and to regenerate plants, was obtained two wheat mutants with Scab-resistance improved significantly and similar agronomical characters to their donors, and analyzed difference of the mutants at the molecular, physiological and biochemical, histological and cellular levels. The results showed as follows:1. The study of immature embryo culture and toxin-selecting system showed that inductivity of embryogenic calli was higher during 10-13 days after blooming date for young embryo culture; that suitable sugar concentration was 20- 50g/L. When the selective dose of Fusarium Graminearum crude toxin was beyond 6.0g/L, formation of embryogenic calli and differentiation of plantlets were inhibited intensively, so the toxin-selecting dose should not exceed this sensitive value or apply the dose successively, otherwise, the plantlets of regenerated could not obtained. The author suggested that a preferable dose for toxin selection should not affect growth of callus, at the same time, controlling embryonic callus formation and seedling differentiation effectively, and to a certain degree, without losing ability of differentiation.2. Scab-resistance and characteristics segregation of four generations of regeneration plants obtained from toxin-selecting somatic clones showed that Scab-resistance among regeneration plants of intra-generation of R0 was different, the percentage of anti-disease progenies selected by toxin was higher than that of unselected ones; that resistance segregation mainly appeared in generation R1 and inclined to the susceptible direction, continuing in some progenies in generation R2 and ending in generation R3.Agronomical characters variance among different generations of regenerated plants indicated that variance could take place on plant height, head length, the number of spikelet, spike shape, plant form, maturity and spike density, moreover, different genotypes had different frequency of phenotypes; that segregation of agronomical characters mainly appeared in generation R1, continuing in some progenies in R2 generation and ending in R3 generation, and the general trend of agronomical characters was dwarf, early-ripening and spike-densed.Through field resistance evaluation and selection of regeneration plants obtained from toxin-selecting somatic clones, a scab-resistant mutant was obtained from variety Xi’nong 1376 and Huayu 888, respectively. Compared with high-resistant checks, Sumai No.3 and Xi’nong 85, the mutants had middle-resistance to scab, which were improved more significantly than their susceptible donors. Agronomical characters of two mutants were similar to their donor parents, respectively.3. RAPD test of scab-resistant mutants showed that, the mutants were different to their donors in many loci with the variant rate from 2.10% to 2.86%, which verified the existence of mutation at the molecular level. Polymorphic fragment S3471220 and S3751380 detected from the mutant RXi’nong1376-2 and its donor Xi’nong1376, as well as polymorphic fragment S149620 detected from the mutant RHuayu888 and its donor Huayu888, was all detected in the high-resistant variety Sumai No.3, which suggested that resistant mutation loci under the condition of isolated culture had specificity and three polymorphic fragments may relate to scab- resistance.4. The dynamic changes of physiological and biochemical indexes of healthy spikes and inoculation ones of the resistant mutants indicated that PAL activity and content and changing tendency of total phenolate and flavoniods and MDA in the healthy spikes of the mutants were similar to that of their susceptible donors, whereas, for the inoculated spikes, content and changing range of these indexes existed significant difference between two kinds of strains, changing from high-resistant variety (Sumai3 ) to their susceptible donors. The result implied that resistance mechanism of the mutants and the normal resistant variety was alike at physiological and biochemical level.The result also showed that, compared with high-resistant variety PAL activity and content of total phenolate and flavoniods in the inoculated spikes of the mutants and their susceptible donors were increased significantly, which were also different within the healthy or inoculated spikes of different genotypes, yet, there was no relationship between scab-resistance and absolute value of PAL activity or content of total phenolate and flavoniods at a certain stage. But changing range of PAL activity or content of total phenolate and flavoniods between healthy spikes and infected spikes were related negatively to scab-resistance. Content of MDA in healthy spikes showed no difference among genotypes in inoculated spikes, however, in the spikes of susceptible variety, which increased continuously, but in the spikes of the mutants and high-resistant variety, which increased during the early period and decreased at the 7th-day, after 11 days later, content of MDA were negatively related to scab-resistance. This tendency was noticeable between the typical resistant and susceptible varieties.5. Histological and cellular research on rachis of healthy and inoculated spikes of the mutants at the different stages showed that, structure and developed tendency of cortical sclerenchyma tissue and green tissue and fibrovascular tissue in the rachis were alike between the mutants and their donors at anthesis stage, which also showed no apparent difference in the inoculated spikes’rachis between the mutants and their donors, major difference was that the mutants had less hyphae number than their donors. So it was considered that the increase of resistant ability in the mutants had no relationship to their rachis structure.This experiment also confirmed that there was different between rachis structure of high-resistant variety and that of susceptible variety. Healthy spikes of high-resistant varieties had thicker cell wall and tissue of cortical sclerenchyma, more number of cortical sclerenchyma layers, more number of fibrovascular bundles, smaller area of green subcutaneous tissue in rachis, and theses differences, except number of fibrovascular bundles, enlarged over time. Considering inoculated spikes, cell wall and tissue layers of cortical sclerenchyma displayed different, and to how much extent the relationship between this difference and anti-extension ability of variety had not determined.6. On the analysis of agronomical characters and molecular marking technology, more than 30 alien anti-Gibberella wheat materials and two mutants coming from this experiment were evaluated their hereditary multiplicity. The result indicated that agronomical characters, which were relatively rich in hereditary multiplicity with diversified index ranging between 1.74 and 1.95, the order of hereditary variance listing as plant height>spike length>number of spikelet>number of spike grains>reactive index>number of effective tillers, coefficient of variation changing from 8.69% to 28.75%, had not showed significant relationship to disease resistance based on correlation analysis; RAPD analysis indicated that 16 primers produced 176 amplification bands, percentage of polymorphism loci was 84.09%, mean hereditary resemblance coefficient 0.7069, hereditary distance 0.3524 and on which the materials was categorized 5 groups, the result of RAPD analysis revealed hereditary differences effectively because the materials coming from the same geographical areas and related varieties were grouped together, which also concluded that hereditary relationship among the materials were high related to wheat growth ecotope and original geographical area.更多还原