Isolation And Fusion Of Mesophyll Protoplasts And Transient Expression Of GFP In Phalaenopsis

Phalaenopsis has high ornamental and economic value and is an important flower cultivated world-wide.Breeders have produced large number of phalaenopsis cultivars by conventional cross breeding,but some traits still need to be improved in efficient ways.Protoplast fusion system can promote the fusion of genetic traits from different species and genus.However,protoplast fusion technique is not yet appicable in phalaenopsis,which hinders the fast creation of new gemplasm.With the release of phalaenopsis genome sequences in 2014,the transgenosis and functional identification of key gene in phalaenopsis become especially important.The transient expression is an important method to study the structure and function of genes,which is based on high quality protoplasts.Therefore,establishment of stable protoplast transient expression system for phalaenopsis will speed up the gene function study.In this study,mesophyll protoplasts were isolated from young leaves of phalaenopsis plantlets with enzymolysis method.And then exogenous genes were transiently expressed in isolated protoplasts through PEG-mediated transformation.Our results will promote the research on genetic engneering in phalaenopsis.The main results are as follows:1.The optimal condition for isolating phalaenopsis protoplasts.Young leaves of phalaenopsis plantlets were applied to protoplast isolation and purification.Through the orthogonal test,we identified the effects of Cellulase R-10,Macerozyme R-10,Mannitol and enzymatic hydrolysis time on the mesophyll protoplasts yield and viability of phalaenopsis.The result showed that the cellulase concentration and the enzymatic hydrolysis time were the key factors influencing the yield of viable protoplasts.The optimal condition for protoplast isolation was 1.0%Cellulase,0.7%Macerozyme,0.4M Mannitol and enzymatic hydrolysis for 6 hours,giving the highest survival rate with an average yield of 5.944x 106g-1FW,and the least cell debris numbers.2.The optimal condition for phalaenopsis protoplast fusion.Two varieties of phalaenopsis 'CQY' and '780' with different color were employed in protoplast fusion test.Effects of different type and concentration of PEG,and different fusion time on the phalaenopsis protoplast fusion were analyzed.Our results indicated that the highest(35.93%)protoplast fusion rate could be obtained after protoplasts treated by 35%PEG6000-high Ca2+-high pH for 20 minutes.3.3.The establishment of gene transient expression system with phalaenopsis mesophyll protoplast.The PEG-mediated method was used to study the transient expression of pUC-GFP and pGreen-GFP in phalaenopsis protoplast.The effects of PEG concentration,incubation time,plasmid concentration and protoplast density on transformation efficiency were evaluated.Our results suggested that the transformation efficiency was raised with the increased of final PEG concentration,plasmid concentration and incubating time.The transformation efficiency could reach up to the maximum when PEG final concentration was 20%,plasmid concentration was 20ug,protoplast density was 2×105 per/ml,incubating time was 30 min.