| Transgenic technology has the potential to revolutionize agricultural production.Agrobacterium-mediated genetic transformation is the most widely used method for soybean genetic transformation.However,compared with other cultivars,the low conversion efficiency has been a bottleneck in the study of soybean gene function,which restricts the commercial development of transgenic soybean.Moreover,the genetic transformation efficiency of soybean has a strong genotype dependence,which limits the application of genetic transformation technology in commercial soybean varieties with good traits.In this study,Agrobacterium-mediated soybean cotyledons genetic transformation and soybean protoplast transformation were carried out to study the differences and regulatory mechanism of exogenous genes expression between soybean cultivars,and the effects on resistance shoots regeneration efficiency.The main results were:（1）Soybean varieties with high or low genetic transformation efficiency were screened.17 soybean cultivars were transformed using Agrobacterium-mediated soybean cotyledon genetic transformation system,and a total of 168 transgenic soybean plants were obtained.According to the genetic transformation efficiency of different soybean cultivars,Dongnong50（DN50）,Williams82（Wm82）,Bert（Br）and Shennong9（SN9）were classified as HIGH-efficiency transformation soybean cultivars（transformation efficiency is higher than 5.5%）,while Kottman（Kt）,General（Gr）,Zhonghuang35（ZH35）and Liaodou16（LD16）were classified as LOW-efficiency transformation soybean cultivars（transformation efficiency is lower than 5.5%）.In the Agrobacterium-mediated genetic transformation of soybean cotyledons,when the medium contained screening agent(5mg·L-1 PPT),the efficiency of resistance shoots regeneration in HIGH-efficiency soybean cultivars was more than 75%,while that in LOW-efficiency soybean cultivars was less than 20%.There was no significant difference in shoot elongation efficiency（about 80%）and root regeneration efficiency（about85%）between soybean cultivars.（2）The soybean protoplasm isolation,purification,transformation and expression systems were established and optimized.Soybean leaves were cultured in dark environment at 28℃until the true leaves were fully expanded for 4 days;CPW solution containing 0.4m mannitol was used to pretreat the tissue material for 30min;Enzymatic solution containing 1.6%cellulase,0.8%isolase and 1.5%pectinase was used to oscillate for 10 hours at 130r/min×28℃under dark conditions;Soybean protoplast cells were purified by 300-mesh sieve and 100 g centrifugation,and the yield of the obtained soybean protoplast cells was about 1.12×107·g·FW-1,and the activity of isolated soybean protoplast cells was more than 92%.The plant expression vectors pEarleyGate 104 and pER8-GFP were transformed into soybean protoplast cells by 20%PEG4000-mediated method and EYFP/GFP was used as fluorescence reporter protein.The results showed that the transformation efficiency（proportion of fluorescent cells）of different soybean cultivars was above 75%on the second day after transformation（2DAT）.（3）There has a significantly difference on the expression of exogenous genes between high/low genetic transformation efficiency soybean culitivar protoplas.The transformation efficiency of exogenous genes in soybean protoplasm cells was increased first and then decreased with the extension of culture time,the maximum value was observed on 2DAT.There was no significant difference in the transformation efficiency of exogenous genes between soybean cultivar protoplast.However,on 3DAT,exogenous gene transformation efficiency and gene expression level in LOW-efficiency soybean protoplast were significantly lower than those in HIGH-efficiency soybean protoplast.（4）The endogenous protease hydrolysis pathway is not the main reason for the decrease of exogenous gene expression in soybean protoplasts.Compared with the control,protease inhibitors MG132（20μM）,AEBSF（50μM）and MG115（200μM）treatment had no significant effect on the transformation efficiency and gene expression level of exogenous genes at 3DAT in soybean cultivar protoplasts.（5）Methylation inhibitor 5-Azac significantly affected the expression level of exogenous genes.Compared to 2DAT,the expression level of methylase encoding gene MET1 in LOW-efficiency soybean protoplasts were significantly increased on 3DAT,and significantly higher than that of HIGH-efficiency soybean protoplast.Furthermore,compared to 2DAT,methylation levels of EYFP promoter and coding regions in LOW-efficiency soybean protoplasts were increased on 3DAT,and significantly higher than that in HIGH-efficiency soybean protoplast.10μM 5-Azac treatment significantly reduced the methylation levels of EYFP promoter and coding regions in LOW-efficiency soybean protoplasts on 3DAT,and increased the expression levels of exogenous genes.（6）Methylation inhibitors 5-Azac significantly increased resistant shoots induction efficiency in soybean cultivars with low genetic conversion efficiency.In the Agrobacterium-mediated soybean genetic transformation system,10μM 5-Azac was added into shoot regeneration medium after 1 week of resistance shoot regeneration.5-Azac treatment significantly improve the efficiency of resistant shoot regeneration in most soybean cultivars（19/22）,especially for LOW-efficiency soybean cultivars.Taken together,high methylation levels of EYFP promoter and coding regions in LOW-efficiency soybean protoplasts inhabited the expression of exogenous genes,which resulting the low efficiency of resistant shoot regeneration.Methylation inhibitor 5-Azac treatment can effectively reduce the methylation levels of EYFP promoter and coding regions,and increased the efficiency of resistant shoot regeneration.Since shoot regeneration is the key for high efficiency of Agrobacterium-mediated soybean transformation,these results provide new venue for improving the transformation efficiency of LOW soybean cultivar in Agrobacterium-mediated genetic transformation systems. |
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