| 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. |
PDF Full Text Request