Establishment&Application Of Citrus Protoplast Transient Transformation System

As the number one fruit in the world,citrus has some special biological characteristics,resulting in slowing down the conventional breeding.The sweet orange genome database provides superior platform for breeding via genetic engineering,however,the system of stable genetic transformation and gene function identification are still immature.The transient expression can achieve the goal that the genes express in host cells at a short time,as provide a convenient method for gene-protein study.In this research,citrus mesophyll protoplasts were used as the main host cell,gene gfp was used as the report gene for testing,and the transformation was conducted with PEG-Ca.Based on the optimized protoplast isolating technology,the study set a series of variables:types of PEG,concentrations of PEG,concentrations of mannitol in PEG-Ca solution,heating treatment time during transformation,plasmid amount and co-culture time etc.,aiming at improving the citrus protoplast transient transformation efficiency.The results are as follows:1.Establishment of the citrus protoplast transient transformation system.In this research,HB pomelo leaves protoplasts were isolated by enzyme-mediated vacuum infiltration,which shortened the action time apparently,protected the protoplasts from bio-enzyme and improved the protoplasts uniformity.Moreover,peel and juice sac protoplasts were isolated for the first time.PEG-Ca transformation optimized conditions were as follows:PEG type was PEG4000,and its concentration was 40%,mannitol concentration 0.3M in the PEG-Ca solution,heating temperature was 47? for 7min,transformed plasmid amount was 15 ?g in 3×105 protoplasts,and protoplasts co-culture time was 36h after transformation.Finally,the efficiency of citrus mesophyll protoplast transient transformation reached to 52.1%.2.Application of the citrus protoplast transient transformation system.At present,the mentioned technology above has been applied in sub-cellular localization experiment for several proteins expressed by citrus genes.To detail,organelles include nucleus,cytoplasm,chloroplast,mitochondria etc.,especially to deserve to be mentioned,this paper can realize co-localization in nucleus level.