Selection For A New Chemical Hybridization Agent And Study On Mechnism Of Male Sterility Induced By The Chemical Hybrid Agent In Rapeseed (Brassica Napus L.)

Rapeseed (Brassica napus L.) is one of the most important oil crops in China as well inworld. There existed significant heterosis in rapeseed. Chemical hybridization agent (CHA) isbecoming one of the most effective ways to utilize the heterosis. Three new sulphonylureaherbicides, monosulfuron-ester sodium, monosulfuron-ester and monosulfuron, designed byNankai University (Tianjin, China), were use as candidate chemicals, to evaluate their effectsof inducing male sterility in rapeseed. Based on these results, the cytological and proteomicchanges were studied in male sterile rapeseed plants induced by monosulfuron-ester sodium(the most effective chemical hybridization agent) treatment with suitable concentration. Theaims of the present study were to select for a new efficient chemical hybridization agent andexplore the mechanism of monosulfuron-ester sodium induced male sterility in rapeseed. Themajor results are as follows:1、The male sterility-inducing ability of three new chemicals in rapeseedThe data of two years experiment showed that spraying10mL of0.05-0.10μg/mlmonosulfuron-ester sodium (MES) or monosulfuron-ester (ME) solution into per rapeseedplants with buds1-2mm in size, can result in completely male sterility. Considering theeffects of MES-or ME-treatment on agronomic traits, such as plant height, seed number forper plant and yield for per plant, the effect of MES is more desirable for being used as a CHAthan ME. But monosulfuron solution has low ability of inducing male sterility in rapeseed-and greated side effect on agronomic traits.Rapeseed plants of forty-nine different genotypes were treated with0.10μg/mL of MES(10ml for per rapeseed plant) when they were with buds of1-2mm in size, to investigate theinteraction of the selected CHA-MES with rapeseed genotypes. The result indicated that allgenotypes can be induced male sterility with exception of one genotype,‘220’. Among the48genotypes of male sterility induced by MES-treatment18genotypes had the rate ofmale-sterile more90%, and7genotypes had the time of sterile lasted for whole time of theiranthesis. Among four agronomic traits tested, only plant height and seed number per siliqueof the treated plants were decreased significantly, but the seed yield per plant were notsignificantly affect. 2、Cytological changes of anthers from male sterile rapeseed plants induced byMES-treatmentCytological investigation revealed that0.10μg/ml of MES treatment resulted in complexchange in cytological structure of rapeseed anther of different development stage, variousdefects occur in different developmential stages. At pollen mother stage, abnormal tapetumand microspore were observed. At the tetrad stage, tapeta was defected or abnormallyexpended and microspore collapsed. At the middle-and vacuolated-microspore stages,defective tapetum and some deformed microspores were also detected. Finally, at mature-stage, all microspores were unviable.3、The proteomic changes in male sterile plant induced by MES treatmentThe2-DE system for anther proteome of oilrapeseed was optimized. The total proteins ofanthers from rapeseed could be efficiently separated by using17cm linear IPG strips with pHranging from4to7,800μg of protein sample and colloidal coomassie brilliant blue (CCB) asdyeing methods, and the system was also suitable for separating the proteins of leaves andbuds from rapeseed.The changes in proteomes of leaves, little buds, anthers from middle buds and anthersfrom large buds of MES-treat plant and Mock-treated (control) plants were investigated bythe2-DE techniques developed above. The result showed that9proteins were differentiallyexpressed in leaves,8in little buds,24in anthers from middle buds and100in anthers fromlarge buds. By using MALDI-TOF MS method,9proteins in leaves,8in little buds,24inanthers from middle buds and90in anthers from large buds were successful identified. Basedon metabolic and functional features, all identified proteins were classified into severalfunctional categories, including cell rescue, defense and virulence (13.8%), proteinsynthesis/assembly/degradation (12.5%), cytoskeleton dynamics (6.9%), carbohydratemetabolism (10.0%), energy (7.5%), cellular transport (9.4%), signal transduction (4.4%),plant development/differentiation (4.4%), wall remodelling/metabolism (3.9%), DNAprocessing (3.8%) as well as amino acid (5.0%), lipid (3.1%), nucleotide (5.0%) andsecondary metabolism (5.0%). Analysis of the differetially expressed proteins revealed thatsix proteins were up-regulated and three proteins down-regulated in leaves of the MES-treatedplants, the up-regulated proteins mainly related to stress responses and plant repair, while thedown-regulated proteins involved in energy metabolism, and the growth and biochemicalmetabolism in leaves of MES-treated plants were not significantly affected. Eight proteinswere differentially expressed in abundance between the small buds of MES-treated plants andcontrol plants, including three up-regulated and five down-regulated proteins. Some proteinsrelated to plant development/differentiation and cytoskeleton dynamics were down-regulated, resulting in defective tapetal cells and abnormal pollen mother cells occasionally observed inanthers during the PMC stage. In anthers of middle buds from MES-treated plants, fourproteins were up-regulated and twenty proteins were down-regulated, the up-regulatedproteins being related to stress responses and the down-regulated proteins mainly functionedin plant development/differentiation, cytoskeleton dynamics, cell wall remodelling, cellulartransport and metabolism of carbohydrate, lipid as well as energy. Many proteins related togene expression regulation, intracellular trafficking and cell wall remodelling/metabolismwere down-regulated, leading to deformed microspores and defective tapetum often detectedin anthers from tetrad stage to the vacuolated-microspore stage. In anthers of large buds fromMES-treated plants, twenty-three proteins were up-regulated and seventy-seven proteins weredown-regulated, the up-regulated proteins being involved in the degradation of lipid,carbohydrate and DNA, and the down-regulated proteins related to cell rescue, defense andvirulence, wall remodelling/metabolism, cellular transport, cytoskeleton dynamics, andbiosynthesis of lipid, carbohydrates, protein and DNA. Some proteins involved in thedegradation of lipid, carbohydrate and DNA were up-regulated, whereas numerous proteinsrelated to the synthesis of these macromolecules were down-regulated, resulting pollendevelopment metabolic disorders and abnormal unviable mature pollen formed. Based onthese cytological and proteomic analysis results, a simple model of physiological andbiochemical metabolism change network was proposed to preliminary explain themechanisms of MES-induced male sterility of rapeseed.Overall, this research first reported that,0.05-0.10μg/mL of monosulfuron-ester sodiumand monosulfuron-ester could be as chemical hybridization agent to induce male sterile ofrapeseed. This study also revealed the cytological characteristics of anthers abortion inducedby MES. The changes in proteomes of leaves and different male gametophyte development(little buds, anthers from middle buds and anthers from large buds) of the control andMES-induced male sterility plant were analyzed by2-DE. On the basis of this study, a simplephysiological and biochemical metabolic network and the mechanisms of MES-induced malesterility of rapeseed were first proposed. These research results provided theory basis for thedevelopment and application of MES in rapeseed, and provided important information forreveal the molecular mechanism of the MES induced male sterility of rapeseed.