| Mustard is a characteristic vegetable of Brassica,which is widely planted in southern China.It originated in China and has a long history of cultivation in China.There are many kinds of mustard,including leaf,stem,root and bolting,which can be roughly divided into 16 varieties.Mustard can be eaten in a variety of ways,rich in nutrients,fresh and salted,so it is favored by consumers and vegetable processing enterprises.With the increasing diversification of consumer demand,including nutritional quality of fresh mustard,functional components and flavor quality of processed mustard,mustard breeders are committed to cultivating new mustard varieties with high quality,stress resistance and suitable processing.Cytoplasmic male sterile line(CMS)is an important way to utilize the Heterosis of cruciferous vegetable crops.Presently,the most widely used CMS systems in cruciferous vegetables are pol CMS and ogu CMS.The cytoplasmic male sterility type(hau CMS)of mustard(Brassica juncea L.)was discovered in 1999 by Huazhong Agricultural University as a new type of cytoplasmic male sterility source in Brassica juncea L.with stable sterility and complete abortion.In the process of transferring the male sterile source of oilseed mustard rape to the male sterile line of mustard vegetable through hybridization and backcross breeding,some of the male sterile lines of mustard mutant hau CMS had problems of POD-SETTING deformity and curvature,the seed production of mustard vegetable hybrids was affected,and the application and popularization of hau cytoplasmic male sterile hybrid varieties of mustard were restricted.In this study,the POD-SETTING characteristics of hau cytoplasmic male sterile lines of different mustard varieties were investigated in the field,by counting pod deformities,analyzing the genetic diversity of existing mustard varieties by molecular biomarkers,and screening out the hau cytoplasmic male sterile lines with good podding traits.At the same time,the development of floral organs and Anther Abortion of hau cytoplasmic male sterile line of mustard were observed by cytology,and the formation mechanism of podding abnormality of hau cytoplasmic male sterile line of mustard was preliminarily revealed.Finally,the pod-deformed mustard male sterile lines were improved by cell fusion technology,and the stable and normal pod-bearing mustard male sterile lines were expected to be obtained.The results of this study will provide sterile resources and theoretical basis for the improvement of hau cytoplasmic male sterile line in mustard,and accelerate the popularization and application of hau CMS in cruciferous vegetables.The main results are as follows:1.Morphological investigation on pods of mustard vegetables.The POD-SETTING morphology of 30 cytoplasmic male sterile lines and 29 corresponding maintainer lines of mustard were investigated,the pods of 29 mustard maintainer lines were plump and erect while the pods of 30 cytoplasmic male sterile lines of mustard were different.The pods of some male sterile materials(such as stem mustard,etc.)are as full and erect as those of maintainer lines,while the pods of some male sterile lines(such as mustard tuber,etc.)showed distorted deformity in varying degrees.2.Identification of sterile genes in male sterile lines of mustard vegetables.Twenty-nine male sterile lines of mustard were identified.The results showed that seven of them were ogu male sterile lines of mustard,and the other 23 were hau cytoplasmic male sterile lines of mustard.According to the results of pod morphology investigation,the pod morphology of ogu cytoplasmic male sterile line of mustard was identical,all of them were erect and plump,and there was no abnormality.The podding of hau cytoplasmic male sterile lines in mustard was uneven.Five of them had good podding performance.They were basically upright and plump with very few deformities.They are 17 W 165,17 W 171,17 W 505,17 W 509 and 17 W 69,respectively.The other 18 materials had podding deformity in varying degrees,which needed to be improved.3.Investigation of POD-SETTING characteristics of cytoplasmic male sterile lines in mustard vegetables.The podding characteristics of 30 cytoplasmic male sterile lines of mustard were investigated in the field.From the view of pod deformity rate,11 materials had low deformity rate,about 0-1%,while the deformity rate of mustard ogu CMS materials was 0.The statistical results of the number of seeds per pod are divided into two parts: The number of seeds per pod and the number of grains per pod.From the number of seeds per pod,the average number of seeds per pod was 13.0.The highest number of seeds per pod was 17W153,and the lowest number was 17W159.The average number of grains per pod was 9.9.The highest number of grains per pod was 17W103,and the lowest number was 17W101.Statistical data showed that the podding traits of ogu CMS were excellent,while hau CMS only had 4 excellent podding traits.In terms of the number of seeds,podding deformity would affect seed production.4.Analysis of genetic diversity of mustard vegetable maintainer lines.Ten pairs of primers with clear bands and good repeatability were screened by cluster analysis of 29 mustard maintainer lines using SSR markers.Cluster analysis showed that the genetic similarity coefficient of 29 mustard maintainer lines ranged from 0.6 to 1.0,at the genetic similarity coefficient of 0.74.Twenty nine mustard maintainer lines were divided into 6 groups: The first is material 17W154,the second category is materials 17 W 102 and 17 W 508,the third category is material 17W504,the fourth category is materials 17W158 and 17W182,the fifth category is material 17W176.And the sixth category covers all the remaining materials.5.Cytological observation of cytoplasmic male sterile lines in mustard vegetables.Cytological observation on floral organ development and Anther Abortion of ogu cytoplasmic male sterile line in mustard was carried out.It was found that the floral organs of the ogu cytoplasmic male sterile line of mustard developed normally and the anthers began to abort at the late tetrad stage.Abortion was characterized by early degradation of tapetum,followed by complete degradation of microspore and anther abortion.Cytological observation on floral organ development and Anther Abortion of hau cytoplasmic male sterile line of mustard(normal podding)was carried out.It was found that the pistils were basically normal and the stamens were basically carpitized.A few stamens without complete carpitization still had a pharmacy development in the corner.The abortion of the residual chamber can be observed during the tetrad stage,when the tapetum is abnormally elongated,hypertrophic and late degraded,followed by the degradation of nutrient-deficient microspores and complete abortion of anthers.Cytological observation on floral organ development and Anther Abortion of hau cytoplasmic male sterile line(pod malformation)in mustard was carried out.It was found that the morphological development of both pistils and stamens were abnormal,the pistils were completely carpelled,and the pistils and stamens formed a pistil-stamen chimera.The number of ovules in the pistils was large,and the endocarpized stamens also had ovules.The abortion of stamens begins when the primordia of stamens occur,and the carpel tissue does not produce the primordia of stamens,and continues to grow and develop in the state of a carpel.6.Study on cell fusion technology of hau cytoplasmic male sterile line in mustard vegetables.The improvement of podding characteristics of mustard was preliminarily explored by means of cell engineering.The way of protoplast chemical fusion was attempted,and the methods and conditions suitable for the technology of protoplast chemical fusion of mustard were explored.The results showed that the SCM solution extraction method combined with the sucrose solution layered purification had a good effect.After 14 hours of shading enzymatic hydrolysis with 0.5% cellulase+0.1% pectinase+0.2 mmol/L mannitol+80 mmol/L CaCl rammer,the product was purified.The formula of sucrose purification solution is 0.5M sucrose+1mM MES.PEG chemical fusion method was used for fusion.The formula of the fusion solution is 15% PEG + 60 mM CaCl ramming + 25 mM mannitol + 10% DMSO.The fusion method was that protoplast mixed droplets were cultured on the PEG fusion solution in the dark and then diluted.A large number of protoplast fusion hybrids of mustard can be obtained from the above results. |
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