Establishment And Optimization Of Systems For Protoplasts Isolation Of Three Plants That Used In Subcellular Location

Plant protoplasts are bare cells with their cell walls removed but surrounded by plasma membrane.Plant protoplasts are more fragile compared with the intact plant cells,but they contain the complete genetic material and have totipotent as well as intact cells,they can potentially regenerate to new plants.Protoplasts can efficiently absorb DNA,organelles,viruses and other substances beyond them,and can fusion with other protoplasts that are incompatible with them thus differentiate into new plants.Plant protoplasts are of great significances in theoretical study of biological basis,crop varieties improvement,transient gene expression and subcellular localization of proteins study.Plant cell wall is mainly composed of cellulose,hemicellulose and pectin.Chickpea,soybean and sacsaoul,which are widely researched in our team and with good foreground in research are used as materials to isolate protoplasts.Effects on protoplasts isolation of different enzyme combinations,concentration of Mannitol in enzymatic hydrolysate,pretreatment time,pH of enzymatic hydrolysate and enzymolysis time are studied in order to prepare protoplasts with high yield and vigor of these three plants and provide technical support for further researches in protoplasts culture,fusion,transient expression and subcellular localization.Specific results are:1.A system for rapid isolation of protoplasts from chickpea Kabuli leaves has been established.Chickpea leaves from seedling about 10 to 30 days are selected as materials for protoplasts isolation.The best condition for protoplasts isolation is to plasmolyze the plant materials in CPW-M13 solution for 3 hours and then transfer them into enzymatic hydrolysate enzymolyse for 7 to 8 hours under temperature of 27? and rotate speed of 45 rpm on water bath shaker.The optimum combination of enzyme consists of Onozuka R-10(0.5%),Hemicellulase(0.8%),Macerozyme R-10(0.8%),MES(0.1%)and Mannitol(10%)dissolved in CPW solution with pH 4.8.2.A system for rapid isolation of protoplasts from soybean Xiangdou No.3 leaves has also been established.Materials were cut from Xiangdou No.3 seedlings of about 10 to 30 days with new euphyllas.The euphyllas were plasmolyzed for 2 hours in CPW-M13 solution and then enzymolyzed under temperature of 27? and rotate speed of 45 rpm for 6h.Onozuka R-10(0.5%),Hemicellulase(0.8%),Macerozyme R-10(0.8%)in combination with Pectolyase Y-23(0.4%)dissolving in CPW solution with MES(0.1%)and Mannitol(10%),pH 6.0 was found best for protoplasts isolation of Xiangdou No.3 leaves.3.Another system for protoplasts isolation from sacsaoul assimilating branches has also formed.Assimilating branches of about 2 to 3 cm long were chosen as materials to isolate protoplasts.The optimized condition for sacsaoul protoplasts isolation is enzymolyzing in CPW solution containing Onozuka R-10(1.0%),Macerozyme R-10(0.4%),MES(0.1%)and Mannitol(10%)with pH 5.6 for 10 to 11 hours.And the enzymolysis circumstance is under temperature of 27°C and rotate speed of 45rpm on a water bath shaker.