Effects Of Proline Metabolic Pathway On Pollen Development Of Cytoplasmic Male Sterile Lines In Wucai

Brassica campestris L.ssp.chinesis(L.)Makino var.rosularis Tsen et Lee is a cruciferous,biennial herb.Because of its good taste and rich nutritional value,wucai is very popular in autumn and winter.However,its long conventional breeding cycle,and some varieties are prone to problems such as weak resistance and degeneracy,the breeding of wucai has entered a bottleneck.The hybrid varieties of cytoplasmic male sterile lines not only increase yield and enhance variety resistance,but also reduce timeandcost of the production.Nevertheless,how to rapidly select sterile lines remains to be further studied.To further understand the sterility mechanism of cytoplasmic male sterility,the cytoplasmic male sterile lines of wucai,4-1A and 4-2A and their maintainer lines 4-1B and4-2B with Ogu CMS sterility were selected as experimental materials,compared the morphological and pollen development cytological differences of the CMS lines and their maintainer lines,observed the effects of exogenous spraying of proline on the pollen viability of the CMS lines,and measured the free amino acids and proline contents with the different tissues during the growth and development of the plants,the enzyme activities of the rate-limiting enzyme in the proline metabolic pathway and relative gene expression levels.The main results are as follows:1.There was no significant difference in plant height,root length and flowering time between the male sterile lines and the maintainer lines of wucai.However,the buds of the sterile lines had stunted growth,abnormal floral organs,and the stamens shriveled and no significant pollen.The pistilswere short and thick and the petals were small.2.Observated cytological of anthers development between the sterile lines and its maintainer lines,it showed that the abortive stage of the sterile lines occurred in the early tetrad stage(stage II).The main manifestation was that the tapetum cells disintegrated prematurely.Abnormal meiosis did not form a tetrad,and the microspore develops abnormally due to lack of nutrients,resulting in the failure to form mature pollen grains,thereby pollen abortion,and no obvious pollen production.3.The contents of the free amino acids and proline in the leaves of the sterile lines were significantly higher than their maintainer lines.During the anther development,started from the stage II(stage II),the free amino acids and proline contents in the sterile lines were significantly lower than that of its maintainer,and presumably owing to the route of transport of proline from the vegetative organs to the reproductive organs was hindered,affecting the normal development of flower buds.4.In order to further verify the speculation,spraying exogenous proline on the leaves and inflorescences of the sterile lines and their maintainers,and observed changes of pollen viability.The results showed that direct spraying of proline on the sterile inflorescence,could restore some the pollen viability.However,spray on leaves of the sterile lines,there was no effect on fertility,verifying the possibility of speculation.5.Measured the activities of rate-limiting enzymes in the proline metabolism pathway in different tissues of the sterile lines and their maintainer lines,there was no significant difference in ProDH activity of the proline degradation pathway,the activities of P5 CS and OAT of the proline synthesis pathway of sterile lines buds were significantly lower than their maintainer lines.And the low relative gene expression levels of P5 CS and OAT in the sterile lines,indicating that the ornithine pathway is also involved in the development of pollen except that the glutamate pathway.