Studies On Protoplast Isolation And Purification Of Blueberry

The factors that affect protoplast isolation and purification,including Enzyme,isolation time,pH value of enzyme liquid, osmotic stabilities,physiological state,pre-treatments,genotypes, speed and time,days and generations of cubculture callus induced from leaves,were studied with three blueberry cultivars 'Sunrise', 'Northland' and 'Fundy' in vitro.The main results were as following:1 The fundamental conditions of protoplast isolation with leaves in vitro were same as that of callus induced from leaves. The basic conditions were 1.5%Cellulase R-10+1.5%Macerozyme R-10+0.7mol/L mannitol+5mmol/LMES, isolating time 15hr and pH6.5 of enzyme.2 The result of protoplast isolation was different because of different materials. Leaf in vitro was better than callus. Yield of vigorous protoplast of leaves was 2.07 × 10~5个/g FW ,wheras callus was 1.57×10~5个/g FW. Yield and vigor of protoplast of callus were 1.57×10~5个/g FW and 79.64% after subcultured 14d , which were better than that of 7d,21d,28d. The third generations of subcultured callus could produce more vigorous protoplast than that of the first,second or the forth generations.3 Yield of vigorous protoplast from 1-3th leaves was higher than that from 4-5th leaves, separately 2.07×10~5个/g FW and 0.77×10~5个/g FW .4 4℃ cold treatment or 3hr dark treatment could improve yield and vigor of protoplast in some extent, but diversity is not remarkable.5 The results of protoplast isolation between genotypes were different. Yield of vigorous protoplast from 'Northland' was far higher than 'Sunrise' and 'Fundy'.6 The conditions of protoplast purification were studied in this paper with 'Fundy' leaves in vitro. A plenty of vigorous protoplast could be obtained when centrifugate 7min with 800r/min .7 pH value of enzyme before the protoplast isolated and after isolated were tested. The pH value declined after isolated, but the range was different.8 The variety and concentration of osmotic stability also effected the results of protoplast isolation. Mannitol was more fit for protoplast isolation of blueberry than sucrose, and the optimum concentration was 0.7mol/L.