Establishment Of Tissue Culture System Of Embryoids And Calli From Hybrid Progenies Of High Potassium Flue Cured Tobacco Lines

Anther tissue culture can quickly shorten the breeding time and improve the breeding efficiency,while leaf tissue culture can be used for propagation.In this study,anthers and leaves of F1 generation hybrid of HKDN high potassium line were used as explants,the tissue culture system of embryoids and calli was established by two ways: Anthers directly induce embryoids,anthers and leaves first induce callus and then differentiate into buds.In order to lay the technical foundation for genetic transformation and screening resistant mutants at the cellular level.The results are as follows:1.Induction of anther embryoids(1)Basic media: The best basic medium for anther culture was H medium,and the highest embryoid induction rate was 19.2%,growth well.(2)Genotypes:The embryoid induction rate of F1 generation of different materials was different,especially H11(♀HKDN-2×♂Coker371)had the highest induction rate and the best growth.(3)Casein hydrolysate: With the increase of casein hydrolysate concentration,the induction rate of embryoids decreased,which indicated that casein hydrolysate was not conducive to the increase of induction rate;However,the addition of 0.1g/L casein hydrolysate in subculture could promote the growth of embryoids.(4)Inoculation density: When 20 and 25 anthers per dish were inoculated,the induction rate was better,and the embryoid growth was better.In order to save the cost of the experiment,25 anthers per dish was the best inoculation density.(5)Drying treatment:The longer the drying time,the lower the anther induction rate of each material,so the material anther is not suitable for drying treatment.(6)Multi factor interaction: Under the interaction of hormone,genotype and dark culture days,the best combination of embryoid induction was H11 + 0.2mg/l2,4-D + dark culture for 7d,the induction rate was 70%,and the growth was also the best.2.Callus induction and differentiation of anther and leaf(1)Callus induction: 0.4 mg / L 2,4-D + 0.3 mg / L 6-BA + dark culture for 9 days was the best culture of callus induced by anther;When callus was induced by leaves,the best medium for callus induction was MS medium,that is,without any hormone,the callus induction rate was high and the structure was loose.(2)Callus differentiation:The best medium for callus differentiation was MS + 0.2mg/L NAA + 0.2mg/L 6-BA,the germination rate reached 65%,and the young buds grew well and the color was green.(3)Bud rooting:The best medium for rooting was MS + 0.1mg/LIAA,and the rooting rate was 100%,and the average root length was 6.8cm.In conclusion,the best medium for anther induction of high potassium flue-cured tobacco F1 was H + 0.2mg/L2,4-D,the optimal culture conditions were dark culture for 7days,inoculation density of 25 seeds / dish,and 0.1g/L casein hydrolysate could be added in the subsequent culture to promote the rapid propagation of embryoids;The optimal medium for callus induction from anthers and leaves was H + 0.4mg/l 2,4-D + 0.3mg/l 6-BA and MS + without hormone,respectively;The best medium for callus differentiation was MS +0.2mg/l NAA + 0.2mg/l 6-BA;The optimum medium for bud rooting was MS + 0.1mg/l IAA...