| Objective:Our reasearch is basis on the experiment in mature embryos in vitro propagation ofAndrographispaniculat(Burm.f)Nees,by using colchicine-induced A paniculata variousexperimental materials,and establishing programs to identify polyploid,screening the best polyploid induction programs.And then,we through real-time fluorescence quantitative PCR method to study two different cell ploidy level of A.andrographolide ApCPS,which controlling gene expression relative to explore changes in ploidy level of synthesis of andrographolide,for the purpose of selecting fine germplasm research provide experimental basis.Method:1.Establishment of rapid propagation system in vitro embryos mature of A paniculata.Using plant tissue culture techniques in vitro to A.paniculata mature embryos as experimental materials,establishing A.paniculata mature embryos in vitro rapid propagation system,these provide useful materials for the later experiments induced polyploidy.2.The method of inducing polyploidy.Colchicine is a chemical inducer selected on the basis of A.paniculata mature embryos in vitro propagation system established on seed Andrographis,after seed germination process Andrographitis just germinated mature embryos,colchicime added buds induction other four buds inducing medium induced manner.After inducing material selection study,the concentration of inducing agent,induction of treatment on select time of the three factors on the survival rate of the experimental material,the induction rate,and the experimental material growth conditions were recorded.3.The establishment of polyploidy level identification program.With the blank treatment of experimental materials as the control group,we selecte the presence of the presence of variation in leaf morphological character of the experimental material as a preliminary screening induced changes in ploidy level,combined stoma characteristics observed,the size of the main observation stoma,stoma density.With existence stoma characteristics seeglings,chromosome numbers were observed,and eventually flow cytometry ploidy were used for the detection polidy level4.Preliminary comparative study of the different treatment groups experimental material morpholbgy.After induction of colchicine after A.paniculata mature embryos,mature embryos after germination,the germination of mature embryos just completed research through experimental material change growth patterns induced mainly observed experimental material growth apical meristem changes,changes in hypocotyl changes in blade thickness,stoma characteristics,changes in the stem cross-section characteristics were record.5.The relative expression of two ploidy level of A.paniculata ApCPS gene.Taking tetraploid seelings with diploid seedlings as control group,taking leaves from different growth stages of experimental materials to ApCPS for the purpose of gene,and UBS as an internal primers to detect the relative expression of the target gene.Results:1.A.paniculata vitro propagation system for in the results.With the seeds treat with 200 mg/L of GA3 soaking for 48 h,the sterilization system is 75%EtOH(45 s)+HgCl2(8 min).1/2 MS is the based medium,with 0.1 mg/L 6-BA + 0.1 mg/LNAA is the best buds induced conditions.1/2 MS medium + 0.1 mg/L NAA is rooting medium,and sand:vermiculite stone = 1:1 is the best hardening matrix.2.The results of the establishment of polyploidy level identification program.In this study,the establishment of plant ploidy level identification scheme,combined with the leaf morphological character of stoma characteristics and initial screening,which will eventually flow cytometry to determine ploidy level.Since diploid A paniculata(2n = 2X=50)chromosome number 50,which corresponds to the number of homologous tetraploid chromosome(2n = 4X = 100)is 100,the number of chromosomes is large,which is not conducive to count under the microscope,and the number of root tip chromosome counting procedure is more complicated,it is not the choice of this method for identification of plant ploidy level3.Polyploid induction results.The optimal induction program of A.paniculata is:with 200 mg/Lof GA3 soaking its mature embryos for 24 h after inoculation culture 11 d-14 d in 1/2 MS medium,then selecting the germinated with one pair of cotyledons from mature embryos at 0.05%,0.075%colchicine solution soak for 24 h,and then inoculation in new 1/2 MS medium and cultured,and ultimately through flow cytometry by polidy level test.we get four Andrographis autotetraploid A.paniculata.In addition,(1)Colchicine induced A paniculata seeds did not get tetraploid materials,0.1%colchicine treatment 72 h,0.2%of colchicine 48h.72 h,0.4%of colchicine processing 48 h,morphological variation exists blade was chimera,and 0.4%of colchicine-induced 72 h to obtain cells containing octoploid levei guard cell,inducing the processing time of less than 24 h treatment groups not the existence of morphological differences no seedlings.(2)in order to directly induce GA3 colchicine-treated seed,further characterized by leaf morphology characteristic variation no seedlings,no homozygous tetraploid no seedings,colchicine at 0.4%48 h the process to obtain two sub octoploid s guard cell stoma cells chimera(3)In order to induce multiple shoots as material experiments,as the toxic effects of colchicine-induced buds material.resulting in even lower concentrations under colchicine induction treatment,the explants were all dead,so the this method is not conducive to the continuation of the experiment,and thus abandoned as the buds induced experimental material4.Effect of colchicine-induced material growth.(1)Morphology.Variation exists between no colchicine treaded and colchicine-treated materials,the thickness of the blade,dark in color,while the blade folds obvious were recorded.Variation of stoma characteristics exist,its porosity characteristics,including the size of the stomata,stoma density,the number contained in the chloroplasts,there were significant differences(p<0.01).(2)Toxicity performance.High concentrations of the toxic effects of colchicine to induce seed material mainly after seed hypocotyl,can cause radicle browning or necrosis leading to death of seeds;high concentrations of colchicine on the germination just mature embryo toxicity mainly in the growth cone toxic effects,cause growth cones swelling and even death.In addition,the role of colchicine on the morphology of the stem,the stem can cause irregular shape.5.The result oftwo ploidy level seedlings witn its ApCPS relative gene expression analysis.Take the growth duration of 56 d and 84 d tetraploid,diploid as material,the 56 d growth cycle in two materials,with 56 d in the growth cycle of two materials,the material is higher than the expression of diploid tetraploid material,but in the growth cycle of two polidy level of the material 84 d,the tetraploid material is higher than the diploid material(p<0.05).Conclusions:1.The main result of t this experiment.In this studies,we choice the colchicine as polyploidy inducing agent,test a variety of materials induction treatment,select the most suitable material Finally,four sterile autotetraploid A.paniculata seedlings were obtained.At the same time,the systerm of establishing ploidy level identification,which can quickly and accurately identify the presence of induced mutation of the experimental material from a large number of experimental materials.After identificating the different ploidy level seedlings,there are some differences in plant morphology characteristics such as leaf color,leaf thickness,stem diameter,pore characteristics and so on.In addition,our study analysis ApCPS gene expression in different growth periods and different polidy level seedlings of A paniculata,which is for the poupose of indicating different metabolic with in A paniculata as mentioned.2.The significance of this study.Up on this study,mature embryos in vitro propagation is on the establishment of colchicine treatment methods and identification of programs,and the use of molecular biology of the gene within the real-time PCR analysis control andrographolides compound relative expression.In this study,in order to change A.paniculata germplasm and biological diversity,as for severe degradation cleistogamous caused,and the low extinction may provide feasible solutions,we try to obtain high yield and high levels of expression of the active ingredient varieties.It provides an experimental basis for the research on molecular level of for selecting excellent breeding lines in A.paniculata. |
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