Application Of Introducing Exogenous DNA Into Wheat After Self-pollination For Wheat Yellow Rust Disease Resistance

To solving the problems of lacking the resources of resistant gene and not enough ideal resistance materials in wheat, the resistance genes of yellow rust (Puccinia striiformis) from the relative of wheat were introduced into wheat. By taking oat (Avena saliva L.) and common wheat (Ningchun No.4) as donor and recipient, total DNA prepared from oat was introduced into Ningchun No.4 through pollen tubing. After 3-6 hours self-pollination, the yellow rust resistant gene from oat was inoculated into common wheat, therefore creating the new variant strains for common wheat breeding.1. Four different plant DNA extracting methods were compared using common wheat, violet wheat and corn. The results showed that all 4 methods were comparable to the quality standard of DNA with infused gene; the density of DNA also fulfilled the molecular breeding requirements of no less than 200 ug/ml. The amount of extracted DNA was different with different plants by using different extraction methods. The variance analysis indicates that the mutual effects between plant species and extraction methods were highly significant.2. The resistance of donor, recipient and all progenies to yellow rust were identified by inoculating mixed raced with different virulence at adult stage during 1996 -1999. The results showed that the donor was immune, whereas the recipient was seriously susceptible. Six of the progenies were highly resistant to five physiological races of yellow rust. The result also indicated that the new genes resistant to yellow rust may be carried on oat DNA, which have been integrated into the genome of wheat and expressed steadily.3. After introducing oat total DNA into Ningchun No.4, the progenies have many variations such as morphological character, chemophysiology, quality trait etc. Comparing to the recipient, the height of most progenies was higher; The leaf area of plant increased 5-8cm~2; The blade color of some variant strains were green-blue; The blades were thick and hard; The spike length, spikelet number, grain number of main spike and grain weight of main spike were generally increased; 1000-grain weight increased up to 3-4g; The shape of seeds changed from egg circular to oval and their furrow became deeper; The seeds color of some progenies turned into white; The variance analysis indicated that all the agronomic traits of the variant strains except biological yield had changed.4. The isoenzyme patterns between donor, recipient and their progenies showed obviously difference. The strains having variance in morphological characters all have difference in isoenzyme patterns. There are special bands of donor in the enzyme patterns of progenies of both esterase isoenzyme and isoenzyme peroxidase. Some new hybrid enzyme bands, as well as the tendency towards donor or recipient or complete complementary enzyme bands appeared in the isoenzyme peroxidase and the activities of enzyme also took place great change.5. While introducing oat total DNA into Ningchun No.4, the violet wheat total DNA was also introduced into Ningchun No.4, one variant plant with purple of grain and stem appeared in D2 generation. The height of the plant was far higher than Ningchun No.4's, but a little lower than violet wheat's. The color of grain was pale purple. Second year, the seeds of variant plant were planted one row and harvested with single-plant. Both variant traits can be inheritance, which showed that the DNA of violet wheat had been introduced into wheat, and the technology of exogenous DNA introduction was indeed carried out.6 The Random Amplified Polymorphism DNA (RAPD) analysis showed that three of 64 tested random primers (S1402, S1466, S1468) amplified the polymorphic DNA in variant strains (99-05, 99-07, 99-08), which the same as oat. The Simple Sequence Repeat (SSR) analysis indicated that the random primer xgwml40 of wheat amplified the polymorphic DNA in donor oat, receptor Ningchun No.4, variant strains (99-02, 99-03, 99-07) and their disease resistant and susceptible generations with Ningchun No.4, which showed they were the transformed progenies of oat total DNA into the genome of Ningchun No.4.