Effects And Mechanism Of Male Sterility Induced By Tribenuron-Methyl In Rapeseed (Brassica Napus L.)

Many researches and practice showed that rape has a significant heterosis. At present, the main approaches of heterosis utilization in rapeseed consist of cytoplasmic male sterility, nuclear male sterility, self incompatibility and utilization of chemical hybridizing agents. Among them,there are many advantages of chemical hybridizing agents in producing hybrids, such as more flexible in parental choice, extrication from the tedious process of the parents supporting and maintaining the fertility, and without adverse cytoplasmic effects. So the approach of using chemical hybridizing agents to produce hybrids will be an important way in rapeseed heterosis utilization. In this study, the effects of five chemicals including Tribenuron-methyl,Aijiang,Salicylic,Maiji and Shengbiding, on male sterility-inducing ability of rape (Brassica napus L.) was investigated by spraying these chemicals onto leaves of rapes in two growth seasons. An effective chemical hybridizing agent——tribenuron-methyl was selected. Further, cytological observation of the stanmen of male sterillty induced by the efficient chemical hybridizing agent tribenuron-methyl was conducted. The changes of acetolactate synthase (ALS) activity were measured in different tissues and different varieties. Structure of acetolactate synthase ALS1, ALS2 and ALS3 gene was analyzed by PCR amplication and DNA sequencing. These results will provide theortical basis and techniques for utilization of chemical hybridizing agents in rapeseed hybrid breeding. The main results obtained were obtained as follows:1. Tribenuron-methyl was screened out with the best effect of inducing male sterility in rapeseed. 0.075-0.1μg/mL tribenuron-methyl was sprayed onto leaves of rape twice , the first time was in bolting period with 1-2 mm length bud , and 15-20mL solution per plant, and the second time was after 10 days the first spraying, and 8-10mL solution per plant being sprayed onto each plant. By this was, full sterile plants rate reached 94-100%, and the sterile period lasted for 21-25 days. This method could be applied in making hybrid in the field. With regards to the treatment of smearing chemical on the main stem, the optimal concentration of tribenuron-methyl for inducing male sterility was 0.3-0.5μg/mL,the rate of full sterile plants reached 97-100%,and sterile period lasted for 14-20 days. As for the smearing onto the primary branches, the optimal concentration of tribenuron-methyl was 0.3-0.6μg/mL. The later two methods can be used in making crosses instead of hand emasculation in rapeseed breeding.2. Microscopic and ultrastructural observations revealed that there were distinct cytologic features between male sterility induced by tribenuron-methyl and genetic male sterility. Male sterility induced by tribenuron-methyl may occur during various development periods of the anther, and had complex phenomenon. Various organizations of anther were affected by tribenuron-methyl treatment. The abnormal tapetum is likely to lead to anther abortion.3. ALS enzyme activity of rape decreased after treated with tribenuron-methyl, reaching the lowest at ninth day after treated by tribenuron-methyl. There were some differences in ALS activity inhibited by tribenuron-methyl among different tissues of the plant. The inhibited order was: buds> upper leaves> middle leaves and lower leaves. There were different effects of tribenuron-methyl on fifty rapeseed cultivars. ALS enzyme activity was inhibited in both sensitive cultivars and non-sensitive cultivars after treated by tribenuron-methyl, however, ALS enzyme activity of sensitive cultivars were inhibited greater. There was no nucleoside sequence difference in the coding region of ALS1 and ALS3 between 82089 (not sensitive cultivar) and zhongshuang NO.9 (sensitive cultivar). But there exsit some differences in ALS2 gene between 82089 and zhongshuang NO.9. ALS2 gene-specific primers could amplify a desired product in 82089, but not in zhongshuang NO. 9. Further, ALS2 gene-specific primers were used to perform PCR amplification in 50 varieties, the results showed that some varieties had PCR product, but the others not. However, this polymorphic amplication results have no relationship with the male sterility-inducing ability by tribenuron-methyl. .