Study On Screening Cabbage Double Haploid Strains Resistant To Black Rot Mutant In Vitro Culture

Cabbage black rot had become one of the diseases that affected the cabbage planting. Itwas the main part of the cabbage disease-resistant breeding that speeding up the screening ofcabbage disease-resistant material and promoting the main part of the new varieties breeding.We got cabbage black rot bacterial strain and extracted crude toxin after separation andpurification on the experiment. In order to obtain back rot disease-resistant mutants, we tookadvantage of crude toxin two-step screening methods in vitro to screen DH cabbage leafcallus which was DH33-24、DH25-2and DHX-1three materials. After disease-resistantidentification of mutant strains in the seeding stage, we chose the excellent resistance andgood agronomic traits mutant strains as material. The main results were as follows:1. The result showed that the medium was pH7.0, NaCl5g which the most suitable forbacterial growth and the optimum temperature was28℃by observing the growth of thebacterial colonies, after changing the NaCl content in culture medium, the regulation ofdifferent pH value and set different bacterial culture temperature.2. Collecting cabbage leaves infected black rot in the field, then separated and purifiedthe black rot germs. Observing pathogen morphology through plate culture, opticalmicroscope observation gram stain and scanning electron microscope, and it conformed to thepathogen features. In order to make Koch’s postulation verification, we used spray method inseedling period, and then it proved that we got purification and cabbage black rot bacteria.3. After bacteria liquid culturing, extracted crude toxin from cabbage black rot.According to cabbage seeds germination test, the crude toxin had certain stability to hightemperature and pressure. Compared to high temperature and pressure sterilization, the filtersterilization made more inhibition to cabbage seeds germination which implied the hightemperature and pressure had damaged the crude toxin activity.4. In order to confirm mutant screening concentration, we used different concentration ofcabbage black rot pathogens crude toxin to screen DH33-24, DH25-2and DHX-1plant stemleaf in vitro. The optimal screening disease-resistant plant crude toxin concentration ofDH33-24, DH25-2and DHX-1was40%,30%and20%respectively, by observing thegrowth of cabbage callus. 5. Using two step selection methods which adding crude toxin to medium in callusformation and adventitious bud rooting period to screen disease-resistant mutation. DH33-24DH strain blade callus induction rate was9.17%, adventitious bud induction rate was221%and mutant regenerated plants were18; DH25-2DH strain blade callus induction rate was17.88%, adventitious bud induction rate was186%and mutant regenerated plants were19;DHX-1DH strain blade callus induction rate was14.77%, adventitious bud induction ratewas156%and mutant regenerated plants were13.6. We made DH mutant regeneration plant which from DH33-24, DH25-2and DHX–1three DH strain leaf in vitro culture disease resistance identify in seedling stage. The numberof disease-resistant mutant regeneration plants from DH33-24, DH25-2and DHX-1in vitroculture is15,11and9strains respectively. Combining with agronomic traits identification,good comprehensive characters regeneration plant number was4,3, and3strainsrespectively.