Effect of Combined Use of Boron and Manganese Fertilizers on the Nutritional Quality and Physiological Indices of Brassica campestris L.ssp.chinensis L.var.utilis

[Objective] The purpose of this study is to determine the effects of com-bined use of boron and manganese fertilizers on the nutritional quality and physio-logical indices of Brassica campestris. [Method] In the nutrient solutions for growing B. campestris by hydroponics, boric acid and manganese sulfate were added at 0.5, 2.5, and 7.5 mg/L respectively. Another treatment without boron and manganese was prepared as the control. Quality and physiological indices of B. campestris in the 10 treatments were measured. [Result] Boron and manganese shared obvious in-teraction in improving the quality and physiological indices of B. campestris. To cul-tivate B. campestris with high quality and strong resistance, the optimum concentra-tions of boron and manganese in the nutrient solution should be 2.5 mg/L boric acid and 2.5-7.5 mg/L manganese sulfate. [Conclusion] The findings wil provide refer-ence for studying effects of trace elements on nutrient composition of vegetables.

Preliminary Study on Tissue Culture Technique of Brassica campestris L. ssp. chinensis (L.) Makino var. utilis

In order to establish a rapid propagation system for Brassica campestris L. ssp. chinensis var. utilis, the anther and cotyledon-cotyledonary petioles were used as explants to conduct tissue culture research. The results showed that not bloomed flower buds with higher styles than petals were appropriate for anther cul- ture. Moreover, most microspores were at the mid-late uninucleate stage at that time. The pollen germination rates of Brassica campestris L. ssp. chinensis var. u- tills were low and the germination rates in autumn and winter were higher than that in summer. The induction medium of anther callus of Brassica campestris L. ssp. chinensis var. utilis was MS＋1.0 mg/L KT＋1.0 mg/L 2, 4-D＋3% sugar＋6 g/L agar＋ 8% coconut milk and the induction media of adventitious bud were MS＋2.0 mg/L 6-BA＋ 0.5 mg/L NAA＋I.0 g/L activated carbon＋2% sugar＋6 g/L agar or MS＋2.0 mg/L ZT＋ 0.5 mg/L IAA＋0.5 g/L AgNO3＋1.0 g/L activated carbon＋2% sugar＋6 g/L agar. The induction percentage of adventitious bud by anther culture was 36.7%. Browning phenomenon appeared during the culture of adventitious bud and regeneration plant could not be formed. The plant regeneration rate reached 80% when cotyledon- cotyledonary petioles were used as explant.

Effect of Nitrogen and Sulfur Supply on Glucosinolates in Brassica campestris ssp.chinensis

Glucosinolates （GSs） are a group of plant secondary metabolites containing abundant nitrogen （N） and sulfur （S） mainly in Brassica and have the beneficial effects on human health including anti-carcinogenic, cholesterol-reducing and other pharmacological effects. The objective of this study was to investigate the effect of different concentrations of N （5, 10, and 20 mmol L-a, denoted by N5, N10 and N20） and S （0,5, 1, and 2 mmol L^-1, denoted by S0.5, S1 and S2） on the yield and GSs in pakchoi （Brassica campestris L. ssp. chinensis var. communis） in hydroponics. Results showed that N10 and N20 significantly enhanced the yield compared with N5, however, N20 had a negative effect relative to N10. Only with N10 and N20 low S supply （S0.5） reduced the yield. The concentrations of aliphatic GSs, aromatic GS and total GSs were enhanced by N5 and indolyl GSs were enhanced by N20. S2 enhanced the concentration of individual GS and total GSs. The concentrations of indolyl GSs were maximized in N20S2 treatment, whereas the highest concentrations of aliphatic GSs, aromatic GS and total GSs were found in N5S2 treatment. Effects of N and S on aliphatic GSs were higher than on indolyl GSs. The results suggest that the accumulation of aliphatic GSs and aromatic GS could be enhanced by low N and high S and restricted by high N while that of indolyl GSs could be enhanced by high N and high S.

Nitrogen Dioxide-Induced Responses in Brassica campestris Seedlings: The Role of Hydrogen Peroxide in the Modulation of Antioxidative Level and Induced Resistance

This article investigates the responses of Brassica campestris seedlings to an acute level of nitrogen dioxide （NO2） exposure in a plant growth chamber, and examines whether pretreating plants with hydrogen peroxide （H2O2） will alleviate NO2-caused injury. Twenty-eight-day-old B. campestris plants sprayed with 10 mmol L^-1 H2O2 aqueous solution （corresponding to approximate 1.0 mg H2O2 per single plant） were exposed to different concentrations of NO2 （0.25, 0.5, 1.0, and 2.0 μL L^-1, respectively） for 24 h under controlled environment. To measure the plant biomass, the plants were fumigated with the same NO2 concentrations as mentioned above for 7 h per day （8.00-15.00） for 7 days. As a control, charcoal filtered air alone was applied. Data were collected on plant biomass, total chlorophyll, photosynthetic rate, stomatal conductance, nitrate and nitrate reductase （NR）, antioxidative enzymes, ascorbate （ASA）, and malondialdehyde （MDA）, immediately after exposure. The results showed that exposure to a moderate dose of NO2 （e.g., 0.25 μL L^-1） had a favorable effect on plants, and the dry weight of the above-ground part increased, whereas the exposure to high NO2 concentrations （e.g., 0.5 μL L^-1 or higher） caused a reduction in the plant biomass and the total chlorophyll, when compared with the control. In addition, at 0.5 μL L^-1 or higher NO2 concentrations, prominent increases in the MDA level and superoxide dismutase （SOD） and NR activities were observed. Exposure to 1 μL L^-1 and higher NO2 resulted in necroses appearing on older leaves, and an increase in catalase （CAT） activity, decrease in ASA content, increased accumulation of NO3^-, and reduction in photosynthesis, when compared with the controls. No changes were detected in stomatal conductance under NO2 fumigation. The pretreatment with 10 mmol L^-1 H2O2 alleviated significantly NO2- caused biomass decrease and photosynthetic inhibition when compared with H2O2-untreated plants. Under NO2 fumigation, further induction in SOD and CAT activities occurred in H2O2 treated plants when compared with H2O2- untreated plants. The effect of NO2 on the ASA and MDA contents was also absent in H2O2-treated plants. However, the H2O2 treatment did not alter the nitrate content and NR activity in plants under NO2 fumigation. The H2O2 treatment caused a lower rate of stomatal conductance. Taken together, these data suggest that fumigation with an acute level of NO2 causes oxidative damage to B. campestris seedlings. The H2O2 pretreatment markedly protects plants against NO2 stress and this may be associated with inducible antioxidative level. NO2 fumigation contributes, at least in part, to the enhanced levels of nitrate in B. campestris leaves.

菜心BclUBE2基因的克隆与表达分析

采用同源克隆法从菜心中获得cDNA全长459 bp的E2基因,命名为BclUBE2。该基因编码153个氨基酸,分子量为17.24ku,理论等电点为5.37,为亲水性非分泌蛋白。结构分析显示,该蛋白含有一个泛素结合酶E2活性位点、一个WD重复序列和一个高度保守的半胱氨酸。进化分析发现,菜心BclUBE2蛋白与芸薹属芜菁和油菜的亲缘关系较近。qRT-PCR分析表明,菜心BclUBE2基因在根、茎、叶、叶柄中均有表达,且表达丰度和变化趋势不同。在低温胁迫下,根中的表达量呈现先降低后升高的变化趋势,且在处理1 h时表达量最低;在茎、叶、叶柄中均呈现先升高后降低的表达趋势,茎和叶柄处理6 h时的表达量最高,叶片处理1 h时的表达量最高。说明菜心BclUBE2基因在响应低温胁迫中发挥作用。

菜心cDNA-AFLP分析体系的优化与建立

以菜心的顶端分生组织为试材,对影响菜心cDNA-AFLP体系的关键因子进行了探讨,以期建立适于菜心转录组差异表达的cDNA-AFLP分析体系。结果表明:Trizol法适用于菜心总RNA提取;SMART双链cDNA合成法效率较高,其中第二链cDNA合成的最佳循环数为24个;Taq I/Ase I分步各酶切3 h可使双链cDNA酶切完全;预扩增体系以模板稀释20倍,27个循环扩增效果较好,退火温度对扩增结果影响不大,但Mg2+对PCR扩增影响较大,经比较加入2.5μL Mg2+(25 mmoL/L)的预扩及选扩电泳的效果均最佳;预扩产物稀释20倍用于选择性扩增,其PAGE电泳可以获得丰富且重复性好的带型。cDNA-AFLP分析体系的优化与建立为菜心抽薹开花分子机理进一步研究奠定了基础。

NaCl胁迫对不同生育期红菜薹的影响

研究了NaCl胁迫对不同生育时期红菜薹[Brassica campestris L.ssp.chinensis(L.)Makino var.utilis Tsen et Lee]的影响。结果表明,在种子萌发期,300 mmol/L的NaCl处理显著降低了红菜薹种子的发芽率、发芽势和发芽指数;在幼苗期,不同浓度的NaCl处理对红菜薹的地上部鲜重、根鲜重、根干重、丙二醛含量、根系活力均无显著影响,在300 mmol/L的NaCl胁迫下红菜薹的地上部干重反而显著增加;在成株期,不同浓度的NaCl处理对红菜薹的叶片数、主薹高度、主薹茎粗、侧薹数、单株产量、丙二醛含量、根系活力均无显著影响。

Study on Genetic Relationship of Purple Tsai-tai Germplasms with SSR Markers

[Objective] This study aimed to explore the genetic relationship of purple tsai-tai germplasms using SSR molecular markers. [Method] SSR analysis of 45 purple tsai-tai samples was conducted with 65 pairs of primers selected from cabbage primers, and the cluster analysis was carried out. [Result] A total of 23 pairs of SSR primers were screened; cluster analysis showed that the genetic relationship of purple tsai-tai germplasms had relatively significantly locality, and the 45 purple tsaitai samples can be divided into three groups of Sichuan, Hunan and Hubei; furthermore, the similarity coefficients of 45 purple tsai-tai samples were all greater than 0.5 （ranging from 0.547 0 to 0.910 7）, indicating that the closer the genetic relationship among purple tsai-tai samples is, the narrower the genetic basis will be. [Conclusion] This study provides a theoretical and technical basis for the identification, protection and breeding of the resources of local varieties of seaweed sprouts. This study provided theoretical and technical basis for the identification, protection, breeding and utilization of local purple tsai-tai resource.

Factors Affecting Transformation Efficiency by Micro-Injecting Agrobacterium into Flower Bud of Chinese Cabbage

Three main parameters were selected to study their importance in transformation by budmicroinjection in non-head Chinese cabbage [Brassica campestris ssp. chinensis (L.) Makinovar. communis Tsen et Lee]. The results showed that the developmental stage of floral bud, theconcentrations of sucrose and surfactant Silwet L-77 were critical for the successfultransformation by this new method. The suitable bud size is 2-3 mm in length, the favorableconcentration of sucrose and surfactant Silwet L-77 are 8 and 0.02% respectively. When thesucrose concentration was greater than 10% or that of Silwet L-77 was above 0.1%, the treatedbuds became yellow and finally blighted. 4/6 T1 seedlings resistant to kanamycin were positiveby PCR analysis, and T2 progeny of all these positive T1 plants have one or more hybridizingbands by Southern blot. Under 5% sucrose, 0.02% Silwet L-77 and grade 2 bud (2-3 mm in itslength) parameters, the most favorable transformation efficiency is about 0.56%, and meanefficiency reaches 0.16% in all experiments indicating that bud microinjection is potentialtransformation way in non-head Chinese cabbage.

Differential expression of salt tolerance related genes in Brassica campestris L. ssp. chinensis (L.) Makino var. communis Tsen et Lee

We examined salt tolerance responsive genes in Pak-choi under salt stress and analyze their potential function. The LRNA differential display was used to screen the transcript derived fragments （TDFs） related to salinity tolerance in tolerant and Loderately tolerant Pak-choi germplasm. Seventy-eight primer combinations generated 101 differential eDNA fragments, which ere divided into 10 expression types. Seven cDNA sequences （GenBank accession Nos. DQ006915-DQ006921） obtained and ,~quenced were highly homologous to some known expression genes or the genes related to the signaling pathways in plants under ifferent abiotic stress.

正交设计优化菜薹ISSR反应体系研究

菜薹原产中国,是华南地区重要的特产蔬菜之一。目前,分子标记技术已广泛应用于多种作物研究,但在菜薹上的应用研究刚刚起步。ISSR主要是由单一引物且以重复序列为主要引物序列的PCR标记,其反应条件受模板DNA、Taq DNA聚合酶、Mg2+、dNTP和引物等因素的影响。正交试验设计能明确各因素间的互作效应,建立最优化的反应体系。本研究利用正交试验设计,从Taq DNA聚合酶、dNTP、引物、Mg2+4种因素3个水平对菜薹ISSR反应体系进行了优化,确立了适合菜薹的ISSR反应体系并在7个菜薹品种中进行了验证。在20μl反应体系中,含Taq DNA聚合酶1U、dNTP250μmol/L、引物0.25μmol/L、1×PCRbuffer、Mg2+2.5mmol/L、模板DNA30ng。通过梯度PCR测验,确定了适宜的退火温度。这一体系的建立为今后利用ISSR技术进行菜薹种质资源分类、遗传图谱构建和基因定位奠定了技术基础。

乌塌菜ISSR-PCR反应体系的建立及优化

为获得乌塌菜ISSR-PCR的最佳反应体系,采用单因素浓度梯度试验和正交优化设计相结合的方法,研究了引物浓度、dNTP浓度、Mg2+浓度、Taq DNA聚合酶用量对PCR反应的影响。并在此基础上对退火温度和循环数进一步优化,最终确立了适合乌塌菜ISSR-PCR反应的最佳体系和程序。即20μL PCR反应体系含:30 ng模板DNA,0.50μmol.L-1引物,0.25 mmol.L-1 dNTP,1 mmol.L-1 Mg2+,1.0 U Taq DNA聚合酶。PCR扩增程序为:94℃预变性3 min;94℃变性30 s,50℃退火1 min,72℃延伸90 s,35个循环;72℃延伸7 min,4℃保存。这一优化的ISSR-PCR反应体系的建立为今后利用ISSR技术对乌塌菜进行种质资源的分类鉴定奠定了基础。

我国乌塌菜研究进展

在植物学分类上,乌塌菜〔Brassica campestris L.ssp.chinensis(L.)Makino var.rosularis Tsenet Lee〕属于十字花科芸薹属白菜亚种的一个变种,别名塌菜、塌棵菜、塌地菘、太古菜、黑菜等,其营养价值较高,含有丰富的矿物质和维生素。本文综述了我国乌塌菜种质资源收集鉴定及主要种类、新品种选育、育种技术、栽培技术、生理相关研究以及分子技术等方面取得的进展和研究成果,分析了目前我国乌塌菜研究中存在的主要问题,同时对未来工作进行了展望。

不同青菜品种吸锌能力差异及与根系分泌物的关系

青菜 [Brassicacampestrisvar chinensis(L )Makino]不同品种对土壤中锌的累积吸收有明显差异[1] ,我们推断根系分泌物在青菜吸收锌的过程中可能起着重要作用。因此在本项研究中 ,对 2个不同锌利用效率的青菜品种的根系分泌物组成、数量及其与锌吸收的关系进行了探讨。结果表明 ,2种锌水平下 ,五月慢根系分泌物中低分子量有机酸及氨基酸的总量均明显高于黑油白菜 ,缺锌时品种间差异达 0 0 5显著水平 ;缺锌条件下 ,五月慢根系分泌物中草酸、丙氨酸的含量同样显著高于黑油白菜。五月慢在缺锌时有较高的根系分泌物总量及其草酸、丙氨酸含量都显著增加 ,这可能是五月慢具有较高累积吸锌量及锌利用效率的重要原因之一。

安徽乌塌菜种质资源形态特征多样性分析

对安徽省乌塌菜48份材料17个形态性状进行统计分析。结果表明,安徽乌塌菜具有丰富的形态多样性,变异系数8.67%～55.33%。通过主成分分析,将17个性状综合为6个主成分,第一主成分的方差贡献率为30.998 2%,第二主成分的方差贡献率为29.208 2%。叶柄长、展幅、株型、叶片长、外叶色、叶面皱缩度、心叶色、叶片数等8个形态性状累积贡献率达到71.821 5%,可反映出乌塌菜形态性状的基本信息。基于形态性状的聚类分析把48份材料聚为3大类,第1类14份材料,第2类25份材料,第3类仅9份材料,同一类群包括不同地方的品种。本研究为发挥安徽乌塌菜种质资源优势,进行种质资源创新及杂种优势利用提供依据。

菜心基因组DNA提取及RAPD反应体系的优化

用改良的CTAB法提取菜心基因组DNA,并利用紫外分光光度法测定其纯度和含量,用琼脂糖凝胶电泳法检测DNA的降解状况和纯度．结果表明:此法提取的DNA具有典型的天然DNA分子的标准紫外吸收光谱特点, 且适于进行RAPD分析．RAPD分析的优化反应体系为:25μL的反应液中含有0．8 U的Taq酶,0．28 μmol／L primer,30 ng的DNA,2．0 mmol／L Mg2+,0．20 mmol／L dNTPs．PCR扩增循环为94℃、5 min;94℃、1 min; 36℃、1 min;72℃、2 min,37个循环;72℃延伸10 min．

27个菜心品种遗传多样性的ISSR分析

利用ISSR技术对27个菜心品种的遗传多样性进行了分析.结果表明,12条引物共检测到103个可重复位点,其中多态位点58个,多态位点百分率(P)为56.31%.平均多样性指数(I)为0.229,遗传相似系数(H)为0.144,观察等位基因数(Na)和有效等位基因数(Ne)分别为1.563和1.225,遗传距离(GD)的范围在0.029～0.344之间,表明菜心遗传多样性较低.经UPGMA聚类分析,将27个菜心品种分为六大类群.

TA29-barnase基因转化菜心

利用根癌农杆菌导入法,以菜心带柄子叶为外植体,对TA29-barnase基因转化菜心进行研究。获得转化植株,进行PCR、Southern blotting杂交和半定量RT-PCR检测,表明目的基因已经整合到转化植株中,并且目的基因在转基因植株花蕾中得到表达,但是表达水平在不同转基因植株间存在差别;转基因植株开花后,均表现雄性不育,不能产生花粉或产生没有活力的少量花粉,自交不能结实;用未转化植株正常花粉对雄性不育植株进行授粉,能够正常结实;保持系(未转化植株)与不育株杂交后代中雄性不育株与可育株的比例为1:1,在杂交后代植株子叶期,喷洒10mg/L的PPT可以完全杀死可育株;利用其他菜心品种为父本与不育株进行杂交,获得的F1植株在生长势和产量方面表现优势,表明开展菜心优势育种具有一定的潜力。

菜心ISSR-PCR反应体系的优化

以菜心（Brassica campestris L．ssp．Chinensis Var．utilis Tssen．et Lee．）为材料，对影响ISSR-PCR扩增结果的因素如Mg^2＋、Taq DNA聚合酶、dNTPs、Primer、模版DNA的浓度及引物退火温度、延伸时间和循环次数进行了探讨，确立了适合菜心ISSR-PCR分析的最佳反应体系和PCR扩增参数：在25pL反应体系中含10×buffer 2．5μL，2．0mmol／LMg^2＋，0．5U Taq DNA聚合酶，0．2mmol／LdNTPs，0．5μmol／L引物，30ng模板DNA．PCR扩增程序：94℃预变性3min；94℃变性1min，49．7～56℃退火（退火温度随引物不同而定）1min，72℃延伸45s，40个循环；72℃延伸5min．

菜薹细胞质不育系小孢子发生的细胞形态学研究

菜薹雄性不育系小孢子发生的细胞形态学观察表明，孢原细胞分化期之前败育的花占５９．６％，不形成花粉囊；有花粉囊发育的花占４０．４％，其中绝大多数花中只有一个雄蕊能形成一个体积很小的花粉囊，雄性不育系小孢子发育在小孢子母细胞期、四分体及单核小孢子期均有败育，败育方式有绒毡层细胞异常肥大，挤压小孢子母细胞；减数分裂异常，无胞质分裂而形成巨细胞；单核小孢子粘连成花粉块，异常膨大等。还有雄蕊雌蕊化等花器异常现象。