Extraction Of Potato Protoplasts And Optimization Of Transient Transformation System

Potato is an important food crop in the world with comprehensive nutrition,and is also an important food and vegetable crop in China.It has great potential in application and development.Transient transformation combined with reporter genes can be used in subcellular localization,functional studies of transcriptional elements such as promoters,and cloning and screening of genes resistance to plant disease.Protoplasts obtained from leaves are widely available and easy to prepare.PEG mediated transient transformation is one of the transformation methods with high frequency because of its low cost,simple operation and low requirement on instruments and equipment.In this study,the effects of enzymatic hydrolysis times and enzyme concentrations on protoplast extraction,as well as the effects of PEG concentrations and transformation times on the protoplast transformation efficiency were researched,a more efficient protoplast transformation system was expected to obtain,which will lay a good foundation for the subsequent verification of the function of genes or DNA elements.The main results are as follows:Ⅰ.The effects of enzyme concentrations,enzymatic hydrolysis times and mannitol concentrations on activities and yields of potato protoplasts were investigated with potato in vitro plantlets as experimental materials.The results showed that the activities of DM protoplasts increased with the increasing of cellulase concentrations when pectase concentration was 0.1%and enzymolysis time was 4h.When cellulase concentration reached 1.50%,the activity of DM protoplasts was 68%,and the yield was 3.30x10⁶ g^-1FW.After that,the activity of DM protoplasts remained stable.When pectinase concentration was 0.1%and cellulase concentration was 1.5%,the activity of DM protoplasts reached the highest value of 93.00%after 3h treatment,and the yield was3.19x10⁶ g^-1 FW at this time,and then the activity decreased continuously.After that,when the enzymatic hydrolysis time was 3h and the concentration of mannitol was 0.4M,DM extraction effect was the best.When cellulose concentration was set at 1.5%,the highest activity and yield at 3h for Favorita protoplasts were 85%,3.59 x10⁶ g^-1 FW,respectively.When the concentration of mannitol was 0.3M and 0.4M,the extraction yield and activity of Favorita protoplasts were the highest when the enzymatic hydrolysis time was 3h,but there were fewer fragments produced when the enzymatic hydrolysis time was 0.4M,and the yield was 2.61 X10⁶ g^-1 FW and the activity was 84%.Ⅱ.On the basis of the optimal protoplast extraction system,the effects of different vectors,PEG concentrations,transformation times,plasmid concentrations and varieties on the protoplast transformation efficiency were further investigated.The transformation efficiencies of different constructs are different.Under the same conditions,the transformation efficiency of p KGWFS7.1-YFP vector was 11%,while the transformation efficiency of PAN580.1-YFP vector was very low.Even when the amount of plasmid DNA was increased to 30 ng,the transformation efficiency was only 1～2%.When the PEG concentration were 12.5%and 25%,the highest transformation efficiency were 8.35%and18.42%,respectively.When the PEG concentration was 40%,the maximum transformation efficiency was about 1～2%.When the PEG concentration was up to 50%,the protoplasts were basically damaged and the transformation efficiency was not obtained.In addition,it was also found that although the transformation time was different,there was no obvious difference between different transformation times,which may be caused by the small span of time gradient we set.Under the same transformation conditions,the transformation efficiency of different varieties were different:DXR was 4.8%,while Favorita was 7.4%.