Suberoylanilide hydroxamic acid induced microspore embryogenesis and promoted plantlet regeneration in ornamental kale (Brassica oleracea var. acephala)

Protoplasma - Isolated Microspore Culture (IMC) is an efficient method to obtain the homozygous strain; however, it is difficult to apply in ornamental kale due to its low rate of microspore...     

l-Ascorbic acid sodium salt promotes microspore embryogenesis and chromosome doubling by colchicine in ornamental kale (Brassica oleracea var. acephala)

Isolated microspore culture (IMC) represents a potential alternative technique in the plant breeding process, as it allows the effective production of doubled haploid (DH) homozygous lines. However, the implementation of this technique is limited by a low rate of embryogenesis, high level of embryo death, and low frequency of chromosome doubling. Thus, we investigated the effects of using different concentrations of L-ascorbic acid sodium salt (VcNa), which has never been applied for kale, to enhance the embryogenesis and regeneration by IMC. Specifically, 1 to 5 μM VcNa was added to the NLN-13 medium of four kale genotypes, while control was grown on VcNa-free medium. Overall, 1–4 μM VcNa at pH 5.84 increased embryogenesis, with 4 μM VcNa being the optimum concentration (12.92-fold increase). The proportion of embryo deaths declined when using appropriate VcNa concentrations. To increase the frequency of chromosome doubling, an artificial chromosome doubling protocol was developed for kale microspore-derived haploids. This protocol involved dipping roots of haploid plantlets in colchicine solution and adding colchicine treatment to solid Murashige and Skoog (MS) medium. Optimum chromosome doubling of haploids was achieved by dipping their roots in 750 mg/L colchicine solution for 4–6 h and 1000 mg/L colchicine solution for 2 h (doubling for nearly 50% of haploids). In conclusion, this study delineated an effective tissue culture process in promoting chromosomal ploidy of microspore-derived regenerated plants, allowing more microspores to be maintained that have excellent ornamental characteristics through crossbreeding.

     

Selection for high and low thiocyanate ion content in kale (Brassica oleracea var. acephala)

In 1978 genetical variation for thiocyanate ion (SCN-) was found in a kale population being selected for improved digestible organic-matter yield. Four generations of half-sib family selection for high and low SCN^- content in young leaves resulted in subpopulations with almost a twofold difference in their means, no overlap in their distributions, no observable reduction in their genetical variation, and only small differences in other traits. The total observed divergence in SCN^-content was 82% of that expected for a random mating population in linkage equilibrium in the absence of genotype x environment interactions. Average heritability on a single plot basis was low, namely 30%, but was increased to 46% with two replicates. This maximised the predicted response to selection for a fixed number of families selected and a fixed total number of plots.
Bulks of the high and low subpopulations were assessed in a trial in 1988. Averaged over September and December harvests of young and mature leaves, SCN- contents were 104.5 and 58.5 mg/100 g DM, respectively. Of eight controls, cv. Merlin had the lowest content, 63.5, and cv. Proteor had the highest content, 89.6. Cultivar x harvest date, cultivar x leaf age and cultivar x harvest date X leaf age interactions were removed by a logarithmic transformation of the data. Stem contents were very low and variable with an overall mean of 10.9 in September and > 5 in December, and no significant differences between cultivars or populations.
The subpopulations will be valuable for research into possible effects of SCN content on the disease and pest resistance of kale and on the performance of animals fed kale.     

羽衣甘蓝种子主要品质性状的表现及其相关性

随机挑选148份羽衣甘蓝种质资源和高世代材料,分析了成熟种子的含油量、蛋白质、硫苷和7种主要脂肪酸成分的表现特征及其相关性。结果表明:羽衣甘蓝成熟种子平均含油量为29.48%,平均蛋白质含量为45.13%,含油量和蛋白质总量为74.61%。硫苷含量的变幅最大,变异系数为31.72%。7种主要脂肪酸成分中,油酸和芥酸的含量较高,其次为亚油酸,棕榈酸和硬脂酸的含量较低。除硫苷含量和硬脂酸含量外,其余9个性状的表现均呈单峰正态分布。相关性分析表明,大多数性状间都具有显著或极显著的相关性,这与对甘蓝型、白菜型和芥菜型3种类型油菜的研究结果相一致。在羽衣甘蓝中存在一些优异的种质资源,通过筛选可以在油菜优质育种中加以利用。     

观赏羽衣甘蓝SSR标记分型与亲缘关系研究

观赏羽衣甘蓝凭借优良的观赏特性和抗逆性已经成为重要的冷季观赏植物。国内观赏羽衣甘蓝育种起步较晚,并且缺乏对种质资源遗传背景的系统研究。本研究应用SSR标记对不同类型的观赏羽衣甘蓝材料进行标记分型和亲缘关系分析。从99对均匀分布于甘蓝基因组的SSR引物中筛选出46对多态性好的引物,对27份不同类型的观赏羽衣甘蓝材料进行标记分型,共扩增出210个多态性位点,平均PIC值为0.58。进一步利用标记分型结果进行STRUCTURE群体结构、UPGMA聚类和聚类热图分析,结果显示3种分析结果基本一致,可以将27份材料分为圆叶、羽叶和皱叶3种类型,其中圆叶和羽叶类型的亲缘关系更近,与皱叶类型的亲缘关系较远;STRUCTURE分析还可以将双亲为不同类型的杂交种材料进行区分;聚类热图分析可以将标记分型结果形象的展示出来。本研究为进一步建立观赏羽衣甘蓝分子指纹图谱,明确种质资源的遗传背景,建立观赏羽衣甘蓝分子标记辅助选择育种体系,培育具有自主知识产权的新品种奠定基础。     

Bottom-up and top-down herbivore regulation mediated by glucosinolates in Brassica oleracea var. acephala

Quantitative differences in plant defence metabolites, such as glucosinolates, may directly affect herbivore preference and performance, and indirectly affect natural enemy pressure. By assessing insect abundance and leaf damage rate, we studied the responses of insect herbivores to six genotypes of Brassica oleracea var. acephala, selected from the same cultivar for having high or low foliar content of sinigrin, glucoiberin and glucobrassicin. We also investigated whether the natural parasitism rate was affected by glucosinolates. Finally, we assessed the relative importance of plant chemistry (bottom-up control) and natural enemy performance (top-down control) in shaping insect abundance, the ratio of generalist/specialist herbivores and levels of leaf damage. We found that high sinigrin content decreased the abundance of the generalist Mamestra brassicae (Lepidoptera, Noctuidae) and the specialist Plutella xylostella (Lepidoptera, Yponomeutidae), but increased the load of the specialist Eurydema ornatum (Hemiptera, Pentatomidae). Plants with high sinigrin content suffered less leaf injury. The specialist Brevicoryne brassicae (Hemiptera, Aphididae) increased in plants with low glucobrassicin content, whereas the specialists Pieris rapae (Lepidoptera, Pieridae), Aleyrodes brassicae (Hemiptera, Aleyrodidae) and Phyllotreta cruciferae (Coleoptera, Chrysomelidae) were not affected by the plant genotype. Parasitism rates of M. brassicae larvae and E. ornatum eggs were affected by plant genotype. The ratio of generalist/specialist herbivores was positively correlated with parasitism rate. Although both top-down and bottom-up forces were seen to be contributing, the key factor in shaping both herbivore performance and parasitism rate was the glucosinolate concentration, which highlights the impact of bottom-up forces on the trophic cascades in crop habitats.     

Pigment variation and transcriptional response of the pigment synthesis pathway in the S2309 triple-color ornamental kale (Brassica oleracea L. var. acephala) line

Ornamental kale is popular because of its colorful leaves and few studies have investigated the mechanism of color changes. In this study, an ornamental kale line (S2309) with three leaf colors was developed. Analysis of the anthocyanin, chlorophyll, and carotenoid contents and RNA-seq were performed on the three leaf color types. There was less chlorophyll in the white leaves and purple leaves than in the green leaves, and the anthocyanin content was greatest in the purple leaves. All the downregulated DEGs related to chlorophyll metabolism were detected only in the S2309_G vs. S2309_W comparison, which indicated that the decrease in chlorophyll content was caused mainly by the inhibition of chlorophyll biosynthesis during the leaf color change from green to white. Moreover, the expression of 19 DEGs involved in the anthocyanin biosynthesis pathway was upregulated. These results provide new insight into the mechanisms underlying the three-color formation.     

羽衣甘蓝柱头酵母双杂交cDNA文库的构建与分析

目的:构建应用于酵母双杂交系统的羽衣甘蓝柱头cDNA文库。方法:以羽衣甘蓝S13-bS13-b自交不亲和系为材料,提取柱头的总RNA,用亲和层析法分离纯化mRNA,利用CytoTrapXR建库试剂盒构建羽衣甘蓝柱头cDNA文库。结果:羽衣甘蓝柱头cDNA原始文库的库容量为2.5×105;扩增后文库的库容量约为4×108,重组率为96%,插入片段大小为0.4～3kb,平均长度在0.8kb左右。结论:构建了应用于酵母双杂交系统的羽衣甘蓝自交不亲和系柱头的cDNA文库,为探讨芸苔属植物自交不亲和的分子机理奠定了基础。     

羽衣甘蓝SEPALLATA-like基因的系统发育与表达分析

E类MADS-box基因SEPALLATA (SEP)-like在被子植物生殖生长特别是花器官发育方面具有重要作用。为分析羽衣甘蓝E功能MADS-box基因SEP-like基因的序列特征及其在花发育过程中的时空表达模式,以羽衣甘蓝品系‘14 line’为试材,利用cDNA末端快速扩增(Rapid amplification of cDNA ends,RACE)技术克隆了SEP直系同源基因BroaSEP1/2/3 (GenBank登录号:KC967957、KC967958、KC967960)。序列和系统进化树分析表明,这3个基因分别与野甘蓝(Brassica oleracea var. oleracea)、芜菁Brassica rapa、萝卜Raphanus sativus、甘蓝型油菜Brassica napus的SEP1、SEP2、SEP3基因具有很高的同源性。推导的氨基酸序列显示,这些基因编码的蛋白质都包含高度保守的MADS结构域、I结构域和K结构域,每一基因都有其亚家族特异的C-末端功能域SEPⅠ和SEPⅡ基序。BroaSEP1、BroaSEP2、BroaSEP3基因的开放阅读框长...     

NIRS定量分析羽衣甘蓝油含量、蛋白质含量数学模型的建立

应用近红外反射光谱技术(NIRS)采集807份羽衣甘蓝种子光谱数据,然后挑选250份材料,用经典化学方法测试种子油含量和饼粕蛋白质含量.通过偏最小二乘法(PLS)、改良最小二乘法(MPLS)和不同光谱散射及数学处理技术来建立NIRS定量分析数学模型.种子油含量和饼粕蛋白质含量模型的交叉检验相关系数(1-VR)分别为0 9180、0 9062,交叉检验标准差(SECV)分别为1 0606、0 8720,校正标准差(SEC)分别为0 9267、0 8119.两种模型的外部检验相关系数(RSQ)分别为0 949、0 915,相对误差分别小于3 60%、2 70%,预测标准差(SEP)分别为0 881、0 779,检验偏差(BIAS)均较小分别为-0 054、-0 062.研究表明:这两个数学模型的分析结果具有较高的精确度,在品质育种中具有较好的应用前景.     

结球甘蓝和青花菜小孢子胚植株再生

结球甘蓝（Brassica oleracea var.capitata）和青花菜（Brassica oleracea var.italica）小孢子胚再生植株频率低是目前影响游离小孢子培养技术有效应用的关键问题之一,研究其小孢子胚植株再生频率的影响因素,提高胚再生植株频率,对促进游离小孢子培养技术在甘蓝类蔬菜育种中更好地应用具有重要意义。该文以结球甘蓝中甘11和青花菜TI-111等基因型为试材,对影响游离小孢子胚再生成植株的固体培养基类型、琼脂浓度、胚的类型及胚在液体培养基中的滞留时间等因素进行了研究。结果表明：游离小孢子培养25天的子叶胚在琼脂浓度为1%–1.25%的B5培养基上植株再生频率最高。进一步通过8个不同基因型对上述实验结果进行了验证,结果显示,游离小孢子培养25天的子叶胚在1%琼脂浓度的B5培养基上植株再生频率达77.8%–97.2%。     

羽衣甘蓝类蛋白质二硫键异构酶PDIL1-2的基因克隆及蛋白表达分析

以羽衣甘蓝（Brassica oleracea var.acephala）自交不亲和系（S13-bS13-b）为试材,利用RT-PCR和RACE的方法从柱头中分离类蛋白质二硫键异构酶BoPDIL1-2基因。将BoPDIL1-2编码区的序列构建到原核表达载体pET-14b上,并转化到大肠杆菌中进行原核表达与纯化;用纯化后的蛋白免疫小鼠,制备BoPDIL1-2多克隆抗体;通过免疫印迹法检测BoPDIL1-2在不同组织及不同发育阶段柱头的表达情况。氨基酸序列比对分析结果显示,羽衣甘蓝BoPDIL1-2与油菜BnPDIL1-2、拟南芥AtPDIL1-2的一致性分别是97.3%和85.5%。SDS-PAGE结果显示,在分子量58 kD处有BoPDIL1-2蛋白特异性地诱导表达。免疫印迹结果显示BoPDIL1-2在羽衣甘蓝柱头中特异表达,而且在柱头发育早期表达量较低,在开花期柱头表达量较高。     

结球甘蓝和青花菜小孢子胚植株再生

结球甘蓝(Brassica oleracea var. capitata)和青花菜(Brassica oleracea var. italica)小孢子胚再生植株频率低是目前影响游离小孢子培养技术有效应用的关键问题之一, 研究其小孢子胚植株再生频率的影响因素, 提高胚再生植株频率, 对促进游离小孢子培养技术在甘蓝类蔬菜育种中更好地应用具有重要意义。该文以结球甘蓝中甘11和青花菜TI-111等基因型为试材, 对影响游离小孢子胚再生成植株的固体培养基类型、琼脂浓度、胚的类型及胚在液体培养基中的滞留时间等因素进行了研究。结果表明: 游离小孢子培养25天的子叶胚在琼脂浓度为1%–1.25%的B5培养基上植株再生频率最高。进一步通过8个不同基因型对上述实验结果进行了验证, 结果显示, 游离小孢子培养25天的子叶胚在1%琼脂浓度的B5培养基上植株再生频率达77.8%–97.2%。     

Selection for resistance to clubroot (Plasmodiophora brassicae) in marrowstem kale (Brassica oleracea var. acephala L.)

Ninety-six cultivars of Brassica oleracea were screened for clubroot resistance in a seedling test using two populations of Plasmodiophora brassicae. The most resistant cultivars were kales. Sixteen resistant marrowstem kale cultivars of diverse geographical origin were used to start a selection programme for clubroot resistance. Four generations of selection, involving single plants, half-sib and full-sib families, reduced a disease index averaged over six clubroot populations from 41.2 to 12.5. This was lower than the most resistant cultivar in the original population, cv. Mixti 28.8, and as good as a German landrace of cabbage noted for its resistance, Bohmerwaldkohl 10.5. In comparison, the mean of five kale controls, cvs Bittern, Canson, Condor, Kestrel and Merlin, was 61.1 and the value for the most susceptible control, cabbage cv. Septa, was 89.3. In the final assessment, there were no clubroot population x B. oleracea genotype interactions and in the initial assessment of cultivars there were only small interactions which could be removed by an angular transformation of the data. It was concluded that a high level of non-differential resistance had been achieved and that it may prove durable. It was also concluded from a small field trial that this level of resistance would prevent serious yield losses in practice.     

cDNA cloning and characterization of a new stress-responsive gene BoRS1 from Brassica oleracea var. acephala

A new stress-responsive gene BoRS1 (GenBank accession No. AY373021 ) was isolated from Brassica oleracea var. acephala by rapid amplification of cDNA ends (RACE). The full-length cDNA of BoRS1 was 2076 bp and contained a 1851 bp open reading frame (ORF) encoding 617 amino acids. Sequence analysis indicated that the deduced BoRS1 shared some identities with LTI65 , RD29A, RD29B and COR78 from Arabidopsis thaliana . Southern blot analysis of genomic DNA indicated that other related genes existed and there were two copies of BoRS1 in the genome of B. oleracea . Northern blot analysis revealed that BoRS1 was up-regulated by cold, mannitol, NaCl and abscisic acid (ABA). Expressional fluctuation of time course with ABA implied a two-step induction process. Tissue-specific expression analysis indicated that BoRS1 was expressed in all the tested plant tissues including leaves, stems and roots. Our studies imply that BoRS1 is a new gene that is responsive to environmental stresses such as low temperature, salinity and osmotic stress.     

Comparative analysis of glucosinolate production in hairy roots of green and red kale (Brassica oleracea var. acephala)

Abstract

Glucosinolates (GSLs) are sulfur- and nitrogen-containing secondary metabolites that function in plant defense and provide benefits to human health. In this study, using Agrobacterium rhizogenes R1000, green and red kale hairy roots were established. The expression levels of GSLs biosynthesis genes and their accumulation in both kale hairy roots were analyzed by quantitative real-time PCR and HPLC. The results showed that the expression of most indolic GSLs biosynthesis genes was higher in the hairy roots of green kale than in that of red kale. In contrast, the expression of BoCYP83A1 and BoSUR1 encoding key enzymes aromatic GSL biosynthesis was significantly higher in red kale hairy root. The HPLC analysis identified six GSLs. The levels of 4-methoxyglucobrassicin, glucobrassicin, and 4-hydroxyglucobrassicin were 6.21, 5.98, and 2 times higher, respectively, in green kale than in red kale, whereas the levels of neoglucobrassicin and gluconasturtiin were 16.2 and 3.48 times higher, respectively, in red kale than in green kale. Our study provides insights into the underlying mechanisms of GSLs biosynthesis in kale hairy roots and can be potentially used as “biological factories” for producing bioactive substances such as GSLs.     

羽衣甘蓝BoMAPK4原核表达、抗体制备及蛋白表达分析

以羽衣甘蓝（Brassica oleracea var. acephala）自交不亲和系（S_13-bS_13-b ）为材料,提取柱头RNA,利用RT-PCR分离羽衣甘蓝柱头丝裂原活化蛋白激酶4（MAPK4）基因。结果表明：获得的BoMAPK4 cDNA含有一个 1 122 bp开放阅读框,编码373个氨基酸,具有丝氨酸/苏氨酸结构域,无信号肽和跨膜结构。羽衣甘蓝BoMAPK4与油菜（B. napus）BnMAPK4、芜菁（B. rapa）BrMAPK4、拟南芥（Arabidopsis thaliana）AtMAPK4的氨基酸序列一致性分别为99.7%、99.5%、95.4%。将BoMAPK4编码区的序列构建到原核表达载体pET-14b上,并转化到BL21（DE3）大肠杆菌（Escherichia coli）中进行原核表达。SDS-PAGE结果显示,在分子量大小约45 kDa处有BoMAPK4重组蛋白特异性表达。利用亲和层析的方法获得高纯度的重组BoMAPK4蛋白。利用获得的重组BoMAPK4蛋白免疫小鼠,制备BoMAPK4的多克隆抗体。提取羽衣甘蓝萼片、花瓣、花药、柱头、花柱和子房的总蛋白,利用免疫印迹的方法进行蛋白表达检测,结果发现BoMAPK4在花瓣、花药和子房中表达的较少,在萼片和花柱中表达的较多,在柱头中的表达量最高。这些研究为探讨BoMAPK4的生物学功能奠定了基础。