Isolation And Flow Purification Of Endosperm Protoplast From Developing Seed Of Maize

Endosperm is the crucial factor for the formation of maize yield. In the growth of maizeendosperm, there is a special cell division, which is called endoreduplication, is the criticalpower of seed growth and economic yield formation of maize. And we used Flow Cytometerto conduct the separation and purification of endosperm cell protoplast and the extraction oftranscriptome. On the basis of predecessors’ research, this study optimized a set of effectivecorn grain endosperm protoplast isolation method by adjusting enzyme type as well as theconcentration, and by using plasma membrane stabilizer and osmotic pressure stabilizers, andpurifies protoplast by flow separation technology. The main results were as follows:1. Establishing the maize endosperm protoplasts system, and achieving effective dynamicprotoplasts.（1） Taking corn inbred Zheng58as the material, this experiment stripped8-10days afterpollination fresh grain ear using eight different concentrations of three enzymes which iscellulase, hemicellulase andmacerozyme, which aimed to enzymatic endosperm cell walls toobtain maize endosperm protoplasts. The results showed that enzyme solution, whichcombined1%cellulase,0.5%macerozyme, with0.5%hemicellulase, was to obtain thelargest number of protoplasts, and the greatest impact for segregation enzymes on protoplastyield.（2） In the process of protoplast separation, it was important to balance the osmoticpressure due to losing the protection of cell wall. Taking osmotic regulator as an sucrose, thisresearch studied the yield of protoplasts and vitality on the condition of four concentrationswhen treating the endosperm tissue overnight enzymatic hydrolysis by the optimal enzymetreatment combination. The results showed that when hydrolyzate sucrose concentrationbetween0.7-0.8mol.L^-1, the yield and concentration of protoplast reached maximum（3） The plasma membrane stabilizer can protect the integrity of protoplast membrane orpromote the regeneration of the cell walls when culturing the protoplast. Taking CaCl₂·2H₂Oand MES as membrane stabilizer, this research studied the yield of protoplasts and enzymaticactivity on four different concentration ratio. The results showed that, with the increase ofconcentration of the plasma membrane stabilizer, protoplast yield and viability weresignificantly improved. Whether higher concentrations of plasma membrane stabilizers could further improve the yield and viability of protoplasts or not remained to be furtherexperimental study.（4） Hydrolysis time are also very important for the protoplast on yield and viability.Accordance with the best combination of enzyme treatment, the optimum osmotic pressure,and the optimum concentration of the plasma membrane stabilizer, this research reconfiguredhydrolyzate, and counted the total number of living protoplasts protoplasts number bysampling interval of2hours until14h.The results show that for maize endosperm materials,enzymatic hydrolysis time of4h is the best time.2. Purification of ProtoplastsRough protoplasts after hydrolysis contain a variety of particles and cell debris, and theyneed purification. The results show that conventional purification methods can obtain pureprotoplasts, but the number of protoplasts loss a lot, which effected subsequent experiments.Separation technology using flow cytometry can achieve a large number of viable protoplasts ina short time to achieve effective separation. GM technology has important practical significance on achieving the target traitimprovement, shortening the breeding cycle and improving the breeding efficiency.Usinggenetically modified （GM） technology has produced a large number of excellent new varietiesof crops, which has brought huge benefits to agricultural production. While we obtain hugeeconomic benefits, people pay more and more attention on the safety of GM foods.International puts forward strict rules on genetically modified （GM） detection. There has strictrules on the planting and sale of the genetically modified （GM） crops in china, any varietiesof maize with insect resistance and herbicide resistance components can’t through the reviewand approval. So during the process of breeding, we need detect the genetically modifiedingredients of germplasm, to determine the fate of these materials, to prevent unnecessarypollutionIn this study, according to SN/T1196-2003（GM maize qualitative PCR detection method） testing standards, the insect and herbicide composition detection of12numbered as44-2,44-3,44-4,57-1,57-2,146-2,146-3,146-4,146-5,166-1,166-3,166-4inbred lines,and the PCR product was sequenced verification, the main results are as follows:1.146-2,146-4,146-5,166-1,166-3,166-4sample for herbicide resistance.2.44-44-44-2,3,4,1,57-2,57-146-3sample for insect resistance and herbicideresistance.3. Based on the second step of PCR screening test standard on maize transgenic qualitativecomponent, it may lead to false negatives phenomenon. Directly judged by the third step ofidentification testing would avoid false negative phenomenon. This proved the steps oftransgenic ingredients Qualitative PCR detection method can be optimized.