Applications Of Flow Cytometry In Plant Research

Flow cytometry,which formed by liquid flow system,optical system,electronic control system and computer system,is a new type of analytical and sorting technology developed in the 1960 s and 1970 s,Cells and other biological particles were rapid and accurate analyzed and sorted by flow cytometry.In the 1980 s,this technique began to be widely used in plant DNA content,cell cycle,transgene and secondary metabolites accumulation detection.Based on the differences of gene expression of different cells,this paper established the technology system of the maize root separation of protoplast,flow analysis,sorting and nucleic acid extraction by adjusting the digestive enzyme components,digestion time,selection of stationary liquid,slurry preparation,selection of dyes and so on.In the mean time,using five kinds of different types of corn starch as materials,the analysis and sorting of starch from different sources were successfully carried out.The flow system of starch was established for the first time.The main research results are:1.Separation of protoplast and flow analysis of maize root tissueFlow cytometry was used to analyze and sort the protoplasts of corn root tip.The following results were concluded:(1)The maize root tissue was digested for 6 hours in enzyme solution which was CPW solution containing 1.00% cellulose,0.30% macerozyme,13% mannitol,and 5.8 pH.(2)The complete protoplast can be efficiently enriched and purified using 25% sucrose floatation method.(3)The maize root tissue protoplasts need Hoechst 33342 to achieve the best dyeing effect at a temperature of 38 ° C and a pH of 11.59.The maize root tissue protoplasts can be divided into two distinct subpopulations.2.Maize tip fixation and its nucleic acid extractionThe extraction of protoplast nucleic acid from the root tip tissue of immobilized maize was carried out.Three conclusions were found:(1)A large number of protoplasts were obtained by enzymatic hydrolysis of the fixed corn root tip tissue for 4-5 hour at 35℃.The enzyme solution was CPW solution contained 2.8% cellulose,0.60% macerozyme.(2)The protoplasts were purified by 80% high concentration of sucrose.(3)The nucleic acid extraction of maize root tip protoplasts with Carnoy 2 and Methacarn solution fixed is a suitable method for nucleic acid extraction because this method has little effect on the concentration and quality of extracted nucleic acid.3.Flow Analysis and Sorting of Different Types of Corn StarchThrough the flow analysis of different types of corn starch,the following conclusions were found:(1)Different types of maize can be stained with fluorescent dye APTS.0.25% gelzn was the best suspension for starch suspension preparation.(2)Starch from different origins can be divided into different subpopulations according to the size.Common corn was divided into two subgroups.Waxy maize was divided into three subgroups.Sweet corn was divided into three subgroups.High amylsoe corn was divided into one subgroup.And burst corn was divided into two subgroups.(3)Subgroups of starch were clustered successfully comfirmed by scanning electron microscopy.Flow cytometry analysis of starch can be used as a new type of analysis,which aimed to provide more starch characterization information.