Studies On Anther Development And Transcriptional Regulation Of Cytoplasmic Male Sterile In Alfalfa (Medicago Sativa L.)

As one of the most important forage legume in the world,alfalfa(Medicago sativa L.)has high productivity,rich nutrition,strong adaptability,high tolerance to harvest as well as good palatability,and also plays a decisive role in such ecological managements as soil improvement,water and fertilizer conservation.Male sterility can be found in higher plants and the commercialization of the hybrids with male sterility makes it available to utilization of heterosis.Therefore,the discovery of male sterility of alfalfa casts great significance on the utilization of heterosis of forage.This study selected ourprevious screened alfalfa cytoplasmic male sterile mutant CMS1 as research materials,sampling observation comparative analysis of fertility control and CMS1 for two consecutive years(WT)and plant anther pollen number,pollen vitality,through the method of paraffin section of different stages of sporophyte and gametophyte cytological observation to observe the difference,the anther and pollen in different developmental stages of the structure by scanning electron microscopy,and use high-throughput sequencing analysis of mutant and fertile differences on transcriptome of flower plant at different developmental stages,so as to further determine the molecular mechanism of cytoplasmic male sterile line CMS1 of alfalfa pollen abortion.The main results are as follows:1)The pollen amount of CMS1 was measured through blood-cell counter in the year of 2015 and 2016.The results showed that the pollen amount of both mutants were significantly lower than the wild-type counterpart WT with the pollen amount of WT?CMS1 being 2838 and 304 respectively.The pollen amount of WT is 9 times of that of other two mutants.The method of KI-I2 was applied to test the pollen vitality.It can be found through observation that a large number of pollen grains were ellipsoid or deformed and turned into light brown or yellow after being stained by KI-I2.The statistical results showed that the pollen viability of CMS1 was 10.1%,which was much lower than 92.4% of WT.It was concluded that CMS1 pollen belonged to complete sterility.2)Cytological observation was carried out on tapetum and microspores at different developmental phases of male ferity mutants CMS1 and the strain WT.The results indicated that the anthers of CMS1 in early period are not in four edges during the microspore cell development stage.In the tetrad period,the microspores were not arranged in the form of tetrahedrons,and there was no callose wall around the microspores of the mutants.At the free microspore stage,there was no significant difference between CMS1 and WT in the development of microspore and tapetum.At the stage of vacuolization and binucleation stages,the tapetum of both CMS1 degraded at a slower pace during which the degree of vacuolization of the microspore cells reached the maximum,suggesting that the PCD of tapetum has the feature of time and its delay severely affected the fertility of microspores.At the stage of maturation,the anthers of CMS1 were in a state of shrinkage,and the parenchyma cells and vascular bundles of the septum were abnormal.The abnormality of anther development in CMS1 has occurred in the early stage,and the delayed degradation of tapetum has hindered the formation of pollen wall,leading to pollen abortion.3)Observation on anthers of CMS1 in different developmental stages had been conducted through scanning electron microscope,it came to conclusion that at the tetrad stage the anthers of CMS1 was flattened and the surface of the connective was folded with the anther deformed as a whole.At the free microspore stage,the anthers were further shrunk and the sporangiate was withered.At the same time,the surface of the anthers was enlarged to be scaly and that of the connective produced a reticular structure.All these suggested that nutrients and waster of anther wall and anther septum were missing,thus affecting the development of microspores.At the stage of maturation,the whole anthers of CMS1 was withered and the degree of anther surface wrinkle reached maximum.At the same time,the anther wall did not crack and the development of the septum became abnormal compared with that of WT.It can be predicted that the outer structure of anther wall and abnormal development of the connective can lead to pollen abortion.Observation on the pollen of CMS1 in different developmental stages through scanning electron microscope suggested that at the tetrad stage the pollen both CMS1 was deformed and there was no difference in strips on the surface of pollen compared with WT.Therefore,it was predicted that the wavy stripes may be the outer wall structure of the pollen.From the free microspore stage to the maturation stage,the pollen grains of WT increased and gradually became full.Such compounds as carbohydrate,lipide and protein assembled on the surface of pollen outer wall with the help of ubisch body,making the surface smoother.On the contrary,there was no such features as mentioned in the pollen of CMS1,which illustrated that PCD failed to occur in the tapetum of CMS1 on time.Therefore,ubisch body cannot assemble on the surface of pollen so there was no way to produce skin layer on the pollen wall,resulting in pollen abortion and male sterile phenotype.4)CMS1 and WT were spliced out of 170968 unigenes,72.19% of which can be annotated in 7 database;along with the development of the archesporial cell to uninucleate stage,the expression of the two genes increased in uninucleate stage reached the maximum value,the difference of 306 genes,which further the uninucleate stage anther and pollen cell activity is the most vigorous development,the degradation process of the tapetum and pollen are closely relat.In conclusion,alfalfa mutant strains CMS1 showed male sterility in the early developmental stage of microspore.The main reason behind the pollen abortion is the missing of surface structure of pollen outer wall caused by PCD delay of anther tapetum.The development of anther and phlegm also played a key role in pollen development and anther cracking.This research could provide a theoretical basis for the study of molecular biology of siRNA in male sterility of alfalfa.What's more,it will offer convenience for the utilization of alfalfa heterosis.