Constrution Of Tobacco DH Population And Directional Selection Hybrid Progeny By Black Shank Toxin-Stress

Tobacco is an important economic crop all over the world,which plays a critical role in the sustained and stable development of national revenue and economy.At present,tobacco production is facing many deficiencies including serious pests and diseases and low resistance of main varieties.As an important genetic analysis group,the double haploid?DH?population has been widely used in the fields for QTL analysis and genetic map construction of crop important traits;Black shank disease is a fungal disease that easily occurs in tobacco production and has serious harm.Numerous studies have shown that cultivation of disease-resistant varieties is the most economical and effective measure in the process of controlling crop diseases.In this study,the induction medium was designed and optimized for studying the conditions and factors affecting the direct androgenesis development of tobacco anthers,the doubling effect of the medium with colchicine on haploid tobacco chromosomes and the construction of tobacco DH populations.On the other hand,to study feasibility about directed selection technology of toxin stress in tobacco black shank resistance breeding,the culture of tobacco black shank strain,toxin extraction,targeted disease-resistant genotype selection of F₂ generation under toxin stress and disease-resistant identification were carried out.The following main research results were obtained:1. The optimal medium for direct androgenesis development of tobacco anthers that does not depend on the donor genotype is:Nitsch H+15%coconut milk+0.1 mg/L IAA+0.1 mg/L 6-BA+30.0 g/L sucrose+6.5 g/L agar?p H 5.6?.The induction rate of F₁ pollen embryo was separately 23.28%,26.64%and 28.07%in the three hybrid combinations?TMK-12×Jiyan 5,K326×Shuangkang 70 and NC71×Jingyehuang?,which were 15.47%,19.27%,and 13.96%higher than the control,respectively.2. Colchicine has a toxic effect on tobacco haploid seedlings.With the increases of colchicine treatment concentration and the extension of treatment time,the survival rate of tobacco seedlings for the three hybrid combinations showed a downward trend.High concentration of colchicine is seriously toxic to haploid seedlings and the chromosome doubling effect is poor.Treatment with 0.02%colchicine for 4 days has the best chromosome doubling effect on tobacco haploid,reaching 50.60%.3. After induction of direct androgenesis development of tobacco anthers,haploid chromosome doubling,ploidy identification and secondary doubling,two tobacco DH populations containing 92 and 53 DH strains were successfully established,respectively.4. The pathogenic toxins of tobacco black shank inhibit the growth of tobacco seed radicles,which is related to the resistance of tobacco varieties to black shank disease.The toxins have poor ability to inhibit resistant varieties.The radicle growth of various resistant tobacco varieties is significantly different after their seeds being stressed by 50%pathogenic toxin for 15 days,suggesting that this treatment method can be used as an important indicator that effectively distinguishs the tobacco disease-resistant genotype.5. There were separately 41.75%,35.13%and 24.50%seedling radicles of F₂generation seeds growing normally for the three tobacco hybrid combinations?NC55-1×K326,K326×Yunyan 99,NC71×Yunyan 99?under the 50%pathogenic toxin stress.Further,the resistance characteristics to black shank disease of above normal seedlings at the seedling stage were identified in the greenhouse.Compared with the control,the incidence of black shank disease was reduced by 15.32%,22.22%,and 19.29%,the disease index were decreased by 12.59%,15.43%,and 14.18%in the three hybrid combinations,respectively.