Preliminary Study On Efficient Regeneration System Of Tissue Culture And Expression Analysis Of WRKY Gene In Eggplant

Solanum melona L.,belonging to Solanaceae,is an important vegetable crop.Eggplant has weak resistance to diseases and insect pests,especially to Verticillium wilt.Using transgenic technology to breed new varieties is one of the most effective solutions.At present,transgenic technology is rarely used in eggplant research.The most difficult problem is that there is no complete and efficient genetic regeneration system.At the same time,WRKY gene is a kind of plant specific transcription factor,which plays an important role in disease response.Therefore,it is very important to find an efficient eggplant regeneration system and carry out the research work on WRKY gene related to Verticillium wilt resistance in eggplant.In this study,‘Yunqie 6’was used as material,different antibiotic treatments were used to promote seed germination.And hypocotyls,cotyledons and stem segments with buds were used as explants to study effect of various hormone treatments on regeneration in different stages of genetic transformation,and to establish a suitable genetic regeneration system for eggplant.20 wild eggplant resources were used as materials,the relative expression of WRKY1,WRKY22 and WRKY33 genes was detected by real-time fluorescence quantitative PCR.The correlation between WRKY genes expression and disease index of tested materials was analyzed to explore the relationship between WRKY gene expression and Verticillium wilt resistance of eggplant.The main results are as follows:1.Soaking seeds with gibberellin under warm water treatment is helpful to improve the germination ability of’Yunsha No.6’seed,the seed germination rate is73.33%.Adding 750 μl/L streptomycin sulfate and 1000 μl/L cephalosporin to MS medium had better germination effect,and the germination rate was 66.67%.In addition,soaking seeds with gibberellin and disinfecting with 10% NaClO for 2⁴ min in MS medium were beneficial to the germination of eggplant seeds.MS + TDZ 3 mg/L +NAA 0.1 mg/L was the best medium for the callus induction of ‘Yunqie 6’,and the induction rate was 85.92%.The best medium for inducing bud differentiation was MS +IAA 0.1 mg/L + ZT 4.0 mg/L + 6-BA 2.0 mg/L,and the rate of budding was 86.11%.Adding 0.04 g/L activated carbon to MS medium was beneficial to rooting of ‘Yunqie 6’,and activated carbon promoted the rooting of eggplant,resulting in many strong roots.2.WRKY gene expression analysis showed that WRKY1 and WRKY22 both contained one WRKY conservative domain,while WRKY33 contained two WRKY conservative domains,one PPR₂conservative domain and one P-loop NTPase conservative domain.A pair of qRT-PCR primers for WRKY1,WRKY22 and WRKY33 were determined according to the results of dissolution curve.Three WRKY genes were detected in all the tested materials.The relative expression quantity of WRKY1,WRKY22 and WRKY33 genes in 20 materials was different.In all the wild eggplants,the highest expression quantity of WRKY1 gene was in 245-4-2,the highest expression quantity of WRKY22 gene was in 246-3-1,and the highest expression quantity of WRKY33 gene was in 245-1-3.There was a significant negative correlation between the relative expression of WRKY22 gene and the disease index of wild eggplants,and the correlation coefficient was-0.445.However,there was no significant correlation between the relative expression of WRKY1 and WRKY33 genes and the disease index of wild eggplants,and the correlation coefficients were-0.110 and 0.237,respectively.