Reproduction of the Three-line Genic Male Sterile Line Parent Mian 7MB-1 （Brasscia Napus L.） and Seed Production of F1 Based on Somatic Tissue Culture

[Objective] The aim was to study the reproduction of the three-line genic male sterile （GMS） lineparent Mian7MB-1 （B. NapusL.） and the seed production of F1 through somatic tissue culture. [Methed] Through hybridization, a new breeding material Mian 7MB-1 in three-line genic temporary maintainer line propagated by tissue culture was used to improve the sterile plant rate of rapeseed in dual-purpose recessive GMS line, such as Mian 7AB type, S45AB type, and etc. And then the variety comparative test was performed. [Result] In order to avoid some fertility restoration phenomena occurring during the process of self-reproduction, Mian 7AB was propagated in bulk with somatic tissue culture of temporary maintainer line plant stem. The propagated temporary maintainer line seedlings were applied to the breeding and seed production of net room male sterile line parent, promoting the sterile plant rate of the male sterile line parent to 91.7% -93.5%. The male sterile line parents per hectare were enough for the seed production of hybrid F1 in 7 500 -15 000 hm^2. [ Conclusion ] Compared with the original dual-purpose GMS line, the seed production ultilizing male sterile line with high sterile plant rate greatly reduced the labor, significantly improved the seed yield, ensuring the seed quality and forming a perfect breeding and seed production system.

甘蓝型油菜BnaSLY1基因进化分析及功能研究

赤霉素调控植物表皮细胞增长、茎叶伸长以及株型建成。拟南芥SLY1属于F-box蛋白,它通过靶向泛素化赤霉素信号转导路径的负向调控因子——DELLA蛋白来影响植物生长发育,然而油菜中BnaSLY1的基因功能尚未揭示。本研究对BnaSLY1进行了表达特征和进化树分析,利用CRISPR/Cas9技术创制了BnaSLY1不同拷贝数的突变体,结合RNA-Seq技术对BnaSLY1的生物学功能及其对油菜生长发育的影响进行了研究。结果表明,甘蓝型油菜Westar中有2个SLY1同源拷贝BnaA01.SLY1和BnaA06.SLY1,它们表达模式基本相同,为组成型表达基因,其蛋白定位在细胞核,且在不同的油菜品种及十字花科植物间序列保守。与对照相比,单突bnaa01sly1和bnaa06sly1开花时间推迟,株高显著降低,而双突bnasly1还表现出深绿色光叶表型,叶片厚度增加,开花期比单突进一步推迟,株高也进一步降低。RNA-Seq结果显示,双突与Westar之间的差异表达基因显著富集在生长素信号转导路径以及蜡质合成通路,多个开花时间相关基因表达也发生显著变化。本研究表明, BnaSLY1除影响植物株高和开花时间等生长发育进程,还影响表皮蜡质合成,为探索赤霉素信号转导路径在甘蓝型油菜生长发育中的重要作用奠定了理论基础。

CNGC20 plays dual roles in regulating plant growth and immunity in Brassica napus

Inflorescence architecture is determined by inflorescence length,branch angles and the density of siliques,which affects planting density,lodging resistance and mechanical operation in rapeseed.However,the molecular mechanisms controlling inflorescence architecture are poorly understood,restricting the progress of breeding varieties with ideal plant architecture in oilseed rape.In this study,we have identified and characterized a rapeseed inflorescence development mutant,reduced inflorescence length(ril),which exhibits determinate and shortened inflorescences,reduced plant height,compact branches,and increased silique density.Through BSA-seq and map-based cloning,we find that RIL encodes a cyclic nucleotide-gated channel 20(BnaA01.CNGC20).A substitution of proline at the 304th position to leucine(P304L)was identified in the conserved transmembrane domain of BnaA01.CNGC20.This P304L substitution neither affects the subcellular localization and self-assembly of BnaA01.CNGC20,nor disrupts the interactions with BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1(BAK1),which interacts with CNGC20 and phosphorylates it to regulate Ca^(2+)channel stability.However,the P304L substitution increases channel activity and Ca^(2+)influx,which in turn induces immune responses such as cell death,H2O2 accumulation,upregulation of pathogenesis-related genes,and pattern-triggered immunity.The enhanced immunity improves the resistance to Leptosphaeria biglobosa and Sclerotinia sclerotiorum.Transcriptome analysis further revealed that CNGC20 plays dual roles in regulating plant growth and immunity via the brassinosteroid and auxin signaling pathways.The findings in this study provide deeper insights into the intricate relationship between cytosolic Ca^(2+)level and plant development and immunity,as well as the trade-off between immunity and the performance of yield-related traits in the heterozygous plants(+/ril),which may serve as a guide for balancing yield and disease resistance in oilseed rape breeding.

The influence of Lactobacillus plantarum fermentation in selenium-enriched Brassica napus L.:changes in the nutritional constituents,bioactivities and bioaccessibility

Selenium(Se)-enriched Brassica napus L.is a valuable organic Se supplement.In this study,the fermentation broth enriched with organic Se(FFS)was prepared using Lactobacillus plantarum to ferment the substrate of Se-enriched Brassica napus L.Significant increases were observed after fermentation in total sugars,reducing sugars,soluble proteins,total phenolic content(TPC),and total flavonoid content(TFC).The organic Se was retained at a concentration of 54.75 mg/g in the freeze-dried sample.Principal component analysis and cluster analysis showed good separation between the FFS and unfermented(FS)groups.Fragrant 2-ethyloxetane had the highest content among all volatiles,while sinapine had the highest content among all phenolic compounds.The fermentation process showed remarkable improvement in the abundance and concentration of volatile compounds and phenolic contents,making FFS exhibit strong antioxidant activity and inhibitory capacity againstα-glucosidase activity.The bioaccessibility of phenolic compounds was significantly greater in FFS compared to FS.ADMET analysis revealed that the majority of phenolic compounds contained in FFS did not exhibit mutagenicity toxicity,hepatotoxicity,skin sensitization,or blood-brain barrier penetration,indicating a favorable level of biosafety.Overall,our study provides a new insight into the further utilization of Se-enriched Brassica napus L.in foods.

Effect of Foliar Fertilizer Spraying at Seedling Stage on the Growth Physiological Characteristics and Yield of Brassica napus L.

In order to select a suitable foliar fertilizer for Brassica napus L.at the seedling stage,using‘Fengyou 958’as the material,different foliar fertilizers including BR,Se,Si,BR+Si,BR+Se,Se+Si and BR+Se+Si were sprayed at the seedling stage to study their effect on the physiological characteristics,growth and yield of Brassica napus L..The results showed that the growth,chlorophyll content,soluble sugar content,soluble protein content,and yield of different treatments at the budding stage improved compared with the control.The effect of the Si+Se treatment was the best,followed by the Se and Si treatments.The chlorophyll content of the flower decreased continuously during the whole flowering period,and the chlorophyll content of the mature silique peel was higher than that of the seed.The content of soluble sugar in flowers was the highest in the early flowering stage,and the content of soluble sugar in leaves at the flowering stage was higher than that at the budding stage.The soluble sugar content in the harvested silique peel decreased gradually with the increase of time,and that in the silique peel and seed was similar at 35 d after pollination.The soluble protein content in the silique peel and seed decreased gradually at the mature stage,and the soluble protein content in the silique peel was higher than that in the seed at the same stage.Si+Se foliar fertilizer spraying at the seedling stage can promote the growth and yield of Brassica napus L.and can be applied in Brassica napus L.production.

Development of a KASP Marker on Chromosome A05 for Seed Oil Content of Brassica napus

In order to identify the molecular markers that can be widely used in the breeding of Brassica napus L.varieties with high seed oil content under different genetic backgrounds,we developed a Kompetitive Allele Specific PCR(KASP)marker for seed oil content on the basis of the results from available studies.The verification in the F_(2) population showed that the marker was closely linked to the quantitative trait locus(QTL)for oil content on chromosome A05.The findings helped to breed the‘Fengyou’varieties with high seed oil content in the middle reaches of the Yangtze River.

甘蓝型油菜(Brassica napus L.)千粒重性状遗传体系分析

通过遗传差异较大的 2个甘蓝型油菜 (BrassicanapusL )纯系亲本组合 (HSTC14×宁油 7号 )衍生后代的世代家系群体分析 ,应用主基因 +多基因家系世代联合分离分析方法研究油菜千粒重的遗传体系。结果表明 ,甘蓝型油菜HSTC14×宁油 7号组合千粒重遗传体系系由一对主基因 +多基因构成 ,主基因中只有加性效应 (d =0 10 6 2 ) ,不存在显性效应 ;多基因存在加性 ([d]=0 2 2 2 8)和显性效应 ,但显性效应为负值 ([h]=- 0 2 80 0 )。表明该组合千粒重主要由主基因加性效应和多基因效应决定 ,但无杂种优势。F2∶3家系群体主基因遗传力为 83 39% ,多基因遗传力为 11 71%。HSTC14×宁油 7号组合千粒重的改良可能受主基因无显性效应 ,多基因显性效应为负值的制约。

锰胁迫对甘蓝型油菜Brassica napus L.种子活力和幼苗抗氧化系统的影响

用不同质量分数的锰溶液(0.2%,0.4%,0.8%和1.2%Mn2+)处理甘蓝型胜利油菜Brassica napuscv.Shengli,研究锰对其种子活力、幼苗细胞膜透性、脂质过氧化及抗氧化剂的影响.结果表明:锰胁迫降低了种子活力、幼苗抗坏血酸(Vc)质量分数、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性,破坏了细胞膜的完整性,并使丙二醛(MDA)质量分数、脯氨酸(Pro)质量分数和过氧化物酶(POD)活性增加.

Correlation Analysis of SPAD Value with Chlorophyll Content and Economic Yield Traits of Brassica napus L.

[Objective] The aim was to compare differences of SPAD value, chloro- phyll content, agronomic characters, economic characters and yield traits to analyze correlation of SPAD value with other indices and establish regression functions. [Method] Based on 34 Brassica napus L. varieties, SPAD value, chlorophyll content, agronomic characters, economic characters and yield traits were measured and re- gression functions were established according to correlations. [Result] SPAD value, chlorophyll content, agronomic and economic characters and yield traits all achieved significant level in differences among 34 varieties. Specifically, SPAD value was of extremely significant correlation with chlorophyll a and b, total chlorophyll and carotenoid, and the correlation from high to low was chl-b〉chl-z〉chl-a〉chl-x. SPAD value was of significantly positive correlation with total pod number per plant, plant height, seed number per pod, yield per plant and harvest yield, and of insignificant correlation with branch point height, effective branch number, pod density of main stem, and pod length. [Conclusion] It is simple and rapid to predict chlorophyll con- tent, economic characters and yields of Brassica napus L. with SPAD value and re- gression functions.

激素对甘蓝型油菜(Brassica napus L)矮化突变体幼苗生长及内源GA_3含量的影响

以甘蓝型油菜矮化突变体"DDF-1"为材料,研究了赤霉素(GA3)、生长素(IAA)和油菜素内酯(BR)3种激素对突变体幼苗的影响,并用石蜡切片观察其细胞形态,用酶联免疫吸附技术(ELISA)检测下胚轴内源激素GA3含量.结果表明,外加一定浓度的GA3(7 mg/L)对矮化突变体"DDF-1"的下胚轴伸长作用显著,但不能使之恢复到野生型的长度;萌发早期,BR有明显的促进伸长作用,萌发后期效果不明显;突变体对IAA敏感性较弱.三种激素交互作用对矮秆下胚轴伸长的影响也不显著.施加外源GA3的矮化突变体"DDF-1"的细胞也有明显的伸长.矮化突变体"DDF-1"的内源激素GA3含量高于野生型高秆,但低于施加外源GA3的矮化突变体.矮化突变体"DDF-1"为赤霉素GA3的非应答型矮化突变体.

Screening of Drought-tolerant Brassica napus L.Varieties and Analysis on Their Physiologic and Biochemical Variations under Drought Stress

[Objective] The aim of this study was to screen drought-tolerant Brassica napus L. germplasm resources by analyzing their physiological and biochemical changes under drought stress. [Method] Forty varieties of B. napus varieties were cultured under PEG-6000 osmotic stress and extreme drought stress in pots, re- spectively. Then, the contents of chlorophyll, carotenoid, proline, malondialdehyde （MDA）, soluble sugar, soluble protein, and the activities of superoxide dismutase （SOD）, catalase（CAT）, peroxidase（POD） were measured under drought stress. [Result] Sever- al drought-tolerant varieties of B. napus were screened out： YAU200908, Xiangyou No.15, YAU200903, YAU200907, YAU200906 and YAU200904. Physiological and biochemical analysis showed that, the contents of chlorophyll and carotenoid de- creased with drought stress increasing; the contents of proline, soluble sugar, solu- ble protein, MDA and the activities of SOD,CAT, POD raised with drought stress in- creasing. [Conclusion] In the rapeseed varieties with stronger drought tolerance, the decrease in carotenoid content and the increase in proline content, soluble sugar content, MDA content, SOD activity, CAT activity were more obvious, so all these physiological and biochemical indices can be used to evaluate the drought tolerance of rapeseed.

Effects of ABA on Photosynthetic Characteristics of Pods and Yield of Brassica napus

[Objective] The aim was to evaluate the effects of different concentrations of ABA on photosynthetic characteristics of pods and yield of Brassica napus and analyze the relationships between ABA concentration and yield components, to provide theoretical basis for cultivation of high-yield rapeseed. [Method] At the end of flowering stage, the rape plants were separately sprayed with 1, 2, 100 and 200μmol/L of ABA; photosynthetic parameters, water use efficiency （WUE）, yield and yield components of the rape plants were investigated 0, 5, 10 and 15 d later, respectively. [Result] ABA treatment with different concentrations significantly changed pod photosynthetic parameters and yield components of rapeseed. Low concentra- tions of ABA could improve pod＇s net photosynthetic rate and yield components while high concentrations had the contrary effect. [Conclusion] Whether ABA func- tioned in inhibiting or promoting role might depend on the interaction between endogenous hormone levels and exogenous ABA concentration. Improvement of WUE would be the main reason for yield increasing.

Cloning of Brassica napus EIN3 Gene and Its Expression Induced by Sclerotinia sclerotiorum

[Objective] The study was to investigate roles of Brassica napus EINB in （ BnEIN3 ） resistance to Sclerotinia sclerotiorum. [ Methods] Genomic PCR and RT-PCR were carded out to isolate genomic DNA and cDNA sequences of BnEIN3 from oilseed rape, based on the highly conserved region of EIN3 gene from Arabidopsis thaliana and the homologous sequences of oilseed rape ESTs. Expression levels of BnEIN3 were detected in three varieties of oilseed rape inoculated with S. sclerotiorum by real-time quantitative PCR.[Results] A 1 947 bp DNA fragment was obtained from oilseed rape. The fragment shared 82% identity to A. thaliana EIIV3, encoded 614 amino acids containing an EIN3 domain, and was named as BnEIN3. Real-time PCR results showed that expression patterns of BnEIN3 were drastically different in different varieties. In highly resistant oilseed rape variety D083, BnEIN3 expression level was significantly increased 72 h after S. sclerotiorum inoculation whereas in middle resistant and susceptible varieties Zhongshuang 9 and 84039, BnEIN3 expression was suppressed. [ Conclusion ] BnEIIV3 may play an important role in oilseed rape resistance to S. sclerotiorum.

油菜(B.napus L.)硼素营养与结实性的研究

硼营养不足,油菜植株各器官含硼量均下降,下降幅度因器官而异,而以花器官下降幅度最大。缺硼时,花器发育异常,雄蕊在解剖生理上的异常现象尤为突出,严重时花粉败育,花蕾脱落,甚至出现颗粒无收的现象。花粉败育表现为花粉囊空瘪,核质解体,无孢原组织分化,绒毡层发育异常,小孢子原生质团溢出药壁外等现象。硼营养不足降低了花药中可溶性糖、淀粉、游离脯氨酸和可溶性蛋白质含量。同时花粉活力和呼吸强度也降低。研究还表明油菜品种对硼营养反应存在基因型差异。宁油8号比宁油7号对缺硼反应更为敏感。缺硼条件下,改善硼营养后,油菜各器官含硼量提高最多的是花器,并使有效分枝数、有效角果数和每角平均实粒数增加,从而提高油菜籽粒产量。

Optimization of Culture Techniques for DH Line in Brassica napus L.

[Objective] The aim of this study is to reveal the disinfectants and disinfection methods,medium components,and embryoid culture method on dissociative microspore culture.[Method] B5 as basic medium appended with different concentrations of sucrose,agar and different hormone combinations was used to optimize the culture technique for DH line in Brassica napus L.[Result] Both the 15 min disinfection of NaClO containing 5% Cl-and 10 min disinfection of 0.1% HgCl2 performed well in disinfection and subsequent embryo production;in the extraction process of dissociative microspores,B5 medium containing 2% sucrose could achieve a good embryo production effect;under dark condition microspores were firstly incubated at 32 ℃ 5-7 d,then at 25 ℃ 12-15 d,and finally transferred to 25 ℃ oscillator(60-65 r/min)for 3-7d,when the embryoid would become full ripeness;1/2MS medium appended with 1.2% agar,0.02% NAA,2.0 mg/L 6-BA,3.4 mg/L AgNO3 and 2% sucrose was helpful for embryoid differentiation and plantlet generation,presenting low degree of browning and slight vitrification.[Conclusion] The results may facilitate DH Line in rape production in large scale and high efficient transformation system.

抗倒伏甘蓝型油菜(Brassica napus L.)根和茎解剖学结构分析

对甘蓝型油菜(Brassica napus L.)6个抗倒伏材料和5个易倒伏材料的苗期和苔期的根部和茎部采用石蜡切片的方法切出6μm厚的薄片,用番红-固绿双重染色,借助Olympus显微镜观察不同材料茎的表皮、皮层、厚角组织、维管束和髄的结构,以及根的木质部、导管等,并对茎和根的结构特点进行了比较。结果表明:①在苗期的茎中,与易倒伏材料相比,抗倒伏材料有较多的厚角组织;抗倒伏材料维管束的面积占茎基部横切面面积之比比易倒伏材料平均高13.1个百分点;抗倒伏材料和易倒伏材料间维管束的个数差异不大,但抗倒伏材料的维管束排列相对比较紧密。②在苔期的茎中,抗倒伏材料的厚角组织和厚壁组织比易倒伏材料发达;虽然两者间维管束的个数和面积无明显差异,但抗倒伏材料维管束排列紧密。③在苗期的根部,抗倒伏材料和易倒伏材料木质部的面积占根部横切面面积之比差异不显著;但抗倒伏材料导管数目比易倒伏材料平均多74.7个,易倒伏材料中较大导管的数目多于抗倒伏材料。④在苔期的根部,抗倒伏材料木质部占根横切面面积的之比比易倒伏材料高8.33个百分点,抗倒伏材料单位面积导管的数目多于易倒伏材料。因此,较多的厚角组织和厚壁组织、较多的导管数和排列紧密的维管束是甘蓝型油菜抗倒伏材料苗期和苔期重要的解剖学特点。

Transcriptional Regulation of 10 Mitochondrial Genes in Different Tissues of NCa CMS System in Brassica napus L. and Their Relationship with Sterility

Northern blot analysis was conducted with mitochondrial RNA from seedling leaves, floral buds, and developing seeds of NCa CMS, maintainer line and fertile F1 using ten mitochondrial genes as probes. The results revealed that 9 out of the 10 mitochondrial genes, except for atp6, showed no difference in different tissues of the corresponding materials of NCα CMS system and that they might be constitutively expressed genes. Eight genes, such as orf139, orf222, atpl, cox1, cox2, cob, rm5S, and rm26S, showed no difference among the three tissues of all the materials detected. So the expression of these eight genes was not regulated by nuclear genes and was not tissue-specific. The transcripts of atp9 were identical among different tissues, but diverse among different materials, indicating that transcription of atp9 was neither controlled by nuclear gene nor tissue-specific. Gene atp6 displayed similar transcripts with the same size among different tissues of all the materials but differed in abundance among tissues of corresponding materials and its expression might be tissue-specific under regulation of nuclear gene. Moreover, three transcripts of orf222 were detected in the floral buds of NCa cms and fertile F1, but no transcript was detected in floral buds of the maintainer line.The transcription of orf139 was similar to that of orf222 but only two transcripts of 0.8 kb and 0.6 kb were produced. The atp9 probe detected a single transcript of 0.6 kb in NCa cms and in maintainer line and an additional transcript of 1.2 kb in fertile F1. The relationship of expression of orf222, orf139, and atp9 with NCa sterility was discussed.

甘蓝型油菜（Brassica napus L．）的春化作用及其应用

研究了甘蓝型春性、半冬性和冬性油菜（ＢｒａｓｓｉｃａｎａｐｕｓＬ．）春化时间，不同设备条件和不同生长阶段的春化效果，以及人工春化技术的利用，结果表明春性油菜的开花株率不受春化处理时间的影响，半冬性和冬性油菜的开花株率与春化处理时间有关．半冬性和冬性油菜的萌芽种子在春化室内通过春化阶段，分别需时２０ｄ和４５ｄ；冬性油菜在冰箱冷藏室通过春化阶段需时５０ｄ；三叶期冬油菜通过春化阶段需时３５ｄ．冬油菜在春化室的春化效果比冰箱冷藏室的效果好．适当延长春化时间能提高春化效果。延长每天光照时间能加快油菜的生育进程．利用冰箱冷藏室和生长室可以在上海当地完成半冬性和冬性油菜的春化和夏繁工作。

甘蓝型油菜(B.napus L.)细胞质雄性不育系MICMS的选育

本研究选用“陆奥”油菜为母本,“五十铃”油菜为父本进行品种间杂交,以父本连续回交,育成了新的质核互作结构的甘蓝型油菜雄性不育系 MICMS。该不育系花器官形态和花药解剖结构的变异现象与已知的油菜细胞质雄性不育系相似。通过不同品种测交,已鉴定出对 MICMS 具有较好恢、保能力的品种。

甘蓝型油菜(Brassica napus L.)无花瓣性状的遗传及其育种潜势

将正常品种与无花瓣亲本杂交,正反交F_1代中有瓣对无瓣为完全显性。在F_2及B_1中,有瓣:无瓣分别为255:1与15:1,表明无花瓣性状的遗传受着核中四对隐性基因p_1p_1p_2p_2p_3p_3p_4p_4的制约。无花瓣基因的育种价值主要体现在减耗节能,包括个体节能(减少建成花瓣的损耗)和群体节能(降低黄色冠层对自然光的吸收与反射损失),以及对菌核病的形态避病能力(田间发病率仅为1.75％)。