Organelle Simple Sequence Repeat Markers Help to Distinguish Carpelloid Stamen and Normal Cytoplasmic Male Sterile Sources in Broccoli

We previously discovered carpelloid stamens when breeding cytoplasmic male sterile lines in broccoli (Brassica oleracea var. italica). In this study, hybrids and multiple backcrosses were produced from different cytoplasmic male sterile carpelloid stamen sources and maintainer lines. Carpelloid stamens caused dysplasia of the flower structure and led to hooked or coiled siliques with poor seed setting, which were inherited in a maternal fashion. Using four distinct carpelloid stamens and twelve distinct normal stamens from cytoplasmic male sterile sources and one maintainer, we used 21 mitochondrial simple sequence repeat (mtSSR) primers and 32 chloroplast SSR primers to identify a mitochondrial marker, mtSSR2, that can differentiate between the cytoplasm of carpelloid and normal stamens. Thereafter, mtSSR2 was used to identify another 34 broccoli accessions, with an accuracy rate of 100%. Analysis of the polymorphic sequences revealed that the mtSSR2 open reading frame of carpelloid stamen sterile sources had a deletion of 51 bases (encoding 18 amino acids) compared with normal stamen materials. The open reading frame is located in the coding region of orf125 and orf108 of the mitochondrial genomes in Brassica crops and had the highest similarity with Raphanus sativus and Brassica carinata. The current study has not only identified a useful molecular marker to detect the cytoplasm of carpelloid stamens during broccoli breeding, but it also provides evidence that the mitochondrial genome is maternally inherited and provides a basis for studying the effect of the cytoplasm on flower organ development in plants.     

Overexpression of an Oil Radish Superoxide Dismutase Gene in Broccoli Confers Resistance to Downy Mildew

Superoxide dismutases (SOD) play important roles in plant disease resistance. In this study, a manganese superoxide dismutase gene, designated RsrSOD, was isolated from oil radish (Raphanus sativus var. raphanistroides). RsrSOD was 696?bp in length predicted to encode 231 amino acids. The deduced amino acid sequence contained four putative Mn-binding sites. Under the control of the CaMV35S promoter, RsrSOD was introduced into broccoli (Brassica oleracea var. italica) via Agrobacterium tumefaciens-mediated transformation. Six transgenic lines were obtained out of 35 independent shoots. Both gene expression and enzyme activity of SOD increased significantly in transgenic lines when challenged with Hyaloperonospora parasitica. Three lines, L19, L23, and L25, exhibited the highest resistance against downy mildew with disease symptoms restricted completely. These highly resistant lines would serve as good broccoli breeding materials.     

Changes in Sulforaphane and Selenocysteine Methyltransferase Transcript Levels in Broccoli Treated with Sodium Selenite

Broccoli (Brassica oleracea L. var. italica planck) has been highly valued because of its nutrient content, which has been attributed to both sulforaphane, a sulfur-containing compound, and selenium enrichment ability. The sulforaphane synthesis may be affected by Se/S metabolism because sulfate and selenate share the same initial pathway for uptake in plants. Selenocysteine methyltransferase (SMT) plays a critical role in the Se/S metabolism system. In the present work, we analyzed the SMT expression level and sulforaphane content in transgenic broccoli under the conditions of overexpression and RNAi knockdown of SMT under normal and selenium-stressed conditions. The relative expression value of SMT in the overexpression line is 13.93 % higher than that in the untransformed control. Interestingly, the sulforaphane content of the overexpression line was 14.09 % lower after selenium treatment, while that of the empty vector (pCAMBIA1301) transformed and untransformed plants was 59.04 and 66.56 % lower than that of the non-selenium-treated, respectively. In the SMT-RNAi line, which has a relative expression value for SMT 15.60 % lower than that of the untransformed, we cannot detect sulforaphane after selenium treatment. These results showed that SMT plays a key role in sulforaphane synthesis in a selenium-rich environment. Specifically, overexpression of SMT decreases the negative effect of selenium on sulforaphane synthesis, while knockdown of SMT by RNAi enhances the negative effect.     

A Seed Treatment for Eliminating Non-hybrid Plants when using Atrazine Resistance as a Genetic Marker for Hybrid Seed Production

Brassica plants, with a combination of chloroplasts encodingtriazine resistance and mitochondria encoding cytoplasmic malesterility, offer new opportunities for hybrid seed production.Such plants can be grown as a random mixture with a male parent,thereby allowing more effective pollen transfer for greaterefficiency of hybrid seed production. Harvested seed consistsof hybrid seed from the ‘female’ parent, which istriazine-resistant, and non-hybrid, triazine-sensitive seedresulting from self pollination of the ‘male’ parent. This study demonstrates that triazine-sensitive broccoli (BrassicaoleraceaL. var.italica) progeny can easily be eliminated byallowing the seed to imbibe a solution of 10 g l^-1atrazine overnightthen drying-back the seed for subsequent germination. Such atreatment prevents triazine-sensitive broccoli from developingbeyond the cotyledon stage following germination, and remainseffective for at least 3.5 years after seed treatment. The growthof triazine-resistant broccoli plants is unaffected by thisseed treatment. Atrazine; Brassica oleracea; broccoli; hybrid seed; triazine resistance; seed treatment     

Agrobacterium tumefaciens-mediated transformation to alter ethylene and cytokinin biosynthesis in broccoli

Broccoli (Brassica oleracea var. italica) deteriorates rapidly following harvest. Postharvest treatment of broccoli with 6-benzylaminopurine delays senescence, whilst exogenous ethylene has been shown to accelerate this process following harvest. To alter ethylene biosynthesis, broccoli was transformed, using Agrobacterium tumefaciens-mediated transformation, with an antisense ACC oxidase gene from broccoli driven by the asparagine synthetase promoter from asparagus. In addition, broccoli was transformed with the chimeric gene construct SAG12-IPT to alter cytokinin biosynthesis during harvest-induced senescence. Transformation was achieved using both hypocotyl and cotyledonary petiole explants. The presence of an antisense ACC oxidase gene enhanced transformation efficiency, but Ag⁺ incorporated into the medium did not. The transgenic nature of these plants was confirmed by PCR and Southern analyses.     

Broccoli growth parameters and level of head infestations in simple and mixed plantings: Impact of increased flora diversification

The impacts of interplanting broccoli, Brassica oleracea var. italica L., with chili pepper, Capsicum annuum L., or yellow sweetclover (YSC), Melilotus officinalis L., on broccoli growth parameters and levels of lepidopteran head infestations were studied during two field seasons (winter & summer). During both seasons, several broccoli growth characteristics (e.g. plant vertical area, whole plant biomass, etc.) were significantly reduced in broccoli‐YSC habitats. However, broccoli heads in broccoli‐YSC habitats did not differ significantly in size from other treatment habitats. Additionally, these heads contained the lowest mean lepidopteran contaminants per broccoli crown during the winter season. During this period, the average number of lepidopteran contaminants per broccoli head was more than twice that in monoculture and pepper intercropped broccoli than in broccoli‐YSC habitats. During the summer experiment, the average number of lepidopteran contaminants per broccoli head was greater in pepper intercropped broccoli than in both monoculture and YSC interplanted broccoli. Potential causes of broccoli growth differences and their relationship to lepidopteran ovipositional behavior are discussed.     

Isolation and characterization of the 54-kDa and 22-kDa chitinase genes of Serratia marcescens KCTC2172

A DNA fragment (pCHI5422) containing two genes encoding a 54-kDa and a 22-kDa chitinase was isolated from a cosmid DNA library of Serratia marcescens KCTC2172. The complete nucleotide sequence of pCHI5422 consisting of 4581 bp was determined. The nucleotide sequence of the 22-kDa chitinase consists of 681 bp of open reading frame encoding 227 amino acids and is located 1422 bp downstream of the translation termination codon of the 54-kDa chitinase sequence. The 54-kDa chitinase gene consisted of 1497 bp in a single open reading frame encoding 499 amino acids. The genes encoding the 54-kDa and 22-kDa chitinase were separately subcloned in Escherichia coli and the individual chitinases were expressed and purified from the culture broth using chitin affinity chromatography. When chitohexaose was used as substrate, the major product of the enzymatic reaction of both the 54-kDa and 22-kDa chitinases was a (GlcNAc)₂ dimer with a minor amount of monomer. The specific activity of the 54-kDa and 22-kDa chitinases were 300 μM (min)⁻¹ mg⁻¹ and 17 μM (min)⁻¹ mg⁻¹ on the natural swollen chitin, respectively.     

青花菜Ogu不育胞质分子鉴定和序列分析

雄性不育是农作物利用杂种优势、进行轮回选择和群体改良的重要手段,在农作物生产中具有巨大的利用价值。该研究为了鉴定青花菜细胞质雄性不育材料的不育胞质类型,以期今后为青花菜种质资源的收集、利用及分子标记辅助育种提供新的不育标记。根据Gen Bank中orf138基因保守序列设计特异引物,对20个青花菜种质资源基因组DNA进行PCR扩增。结果表明:特异引物P1/P2在12个青花菜雄性不育基因型中均扩增出392 bp的片段,在8个可育基因型中未扩增出条带,与田间育性鉴定结果相符。获得青花菜Ogu胞质雄性不育的特异基因orf138序列,Gen Bank中的登录号为HQ149728;用Blastn在Gen Bank中进行同源性比对分析,发现12个不育材料的特异片段与已报道的萝卜Ogu CMS所具有的Ogu orf138基因(Genbank登录号:Z18896.1)同源度高达100%。序列同源比对发现orf138基因存在变异位点。研究结果可为青花菜雄性不育细胞质的分子鉴定、进一步阐明胞质雄性不育败育机理,以及指导青花菜新型不育系的创建和杂种优势高效利用提供理论依据。     

绵羊CAST基因2型和4型转录本的克隆及特性分析

钙蛋白酶抑制蛋白(Calpastatin, CAST)是一种内源性的需要Ca²⁺激活的钙蛋白酶抑制剂, 在肌肉组织的蛋白质降解过程中起重要的调节作用。文章利用牛^CAST基因的mRNA序列, 通过逆转录RT-PCR首次克隆获得绵羊CAST基因2型转录本和4型转录本的部分cDNA序列, 并对序列进行生物信息学分析。CAST基因2型转录本的扩增片段为4 385 bp, 完整的开放阅读框为2 361 bp, 编码786个氨基酸; CAST基因4型转录本的扩增片段为1 467 bp, 完整的开放阅读框为1 317 bp, 编码438个氨基酸。CASTⅡ型蛋白序列存在4个保守结构域, CASTⅣ型蛋白序列存在3个保守结构域; 两者的二级结构均以螺旋为主, 富含疏水区域, 其氨基酸序列存在多个磷酸化位点以及蛋白激酶C(Protein kinase C, PKC)的磷酸化位点。通过RT-PCR分析CAST基因2型转录本和4型转录本的组织表达谱, 结果表明CAST基因2型转录本在所检测的10个组织中均表达, CAST基因4型转录本仅在睾丸组织中表达。     

Explaining curd and spear geometry in broccoli, cauliflower and `romanesco': quantitative variation in activity of primary meristems

Brassica oleracea L. is highly polymorphic and includes varieties which exhibit a headed phenotype (a large preinflorescence): the curd of cauliflower and `romanesco' (var. botrytis), and the spear of broccoli (var. italica). This headed phenotype results from highly iterative patterns of activity at the primary meristems. Differences in the morphology of curds and spears are accounted for by three quantitative variables: the rate of production of branch primordia on the flanks of the apical meristems (RPP); the number of branch primordia produced before the first formed begin producing their own branch primordia (the iteration interval, ITI); and the duration of the preinflorescence stage (before production of flower primordia). Relatively stable iteration parameters (RPP and ITI) during curd development lead to the production of semi-spherical curds with a smooth surface in cauliflower and broccoli, whereas in `romanesco' RPP and ITI increase throughout curd development, inducing a pyramidal curd with an angular surface. A relatively long preinflorescence stage in cauliflower and `romanesco' results in the curd surface being composed largely of branch primordia, whereas in broccoli this stage is short and the spear surface is made up of flower buds. Simplified growth models for these three headed types are presented. The implications for the genetic control of the B. oleracea L. headed phenotype and the relationships between shoot apical meristem size, phyllotaxis and curd/spear morphology are discussed. Received: 11 September 1997 / Accepted: 12 November 1997     

Biotechnological applications in in vitro plant regeneration studies of broccoli (<Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">italica</Emphasis>), an important vegetable crop

Biotechnology holds promise for genetic improvement of important vegetable crops. Broccoli (Brassica oleracea L. var. italica) is an important vegetable crop of the family Brassicaceae. However, various biotic and abiotic stresses cause enormous crop yield losses during commercial cultivation of broccoli. Establishment of a reliable, reproducible and efficient in vitro plant regeneration system with cell and tissue culture is a vital prerequisite for biotechnological application of crop improvement programme. An in vitro plant regeneration technique refers to culturing, cell division, cell multiplication, de-differentiation and differentiation of cells, protoplasts, tissues and organs on defined liquid/solid medium under aseptic and controlled environment. Recent progress in the field of plant tissue culture has made this area one of the most dynamic and promising in experimental biology. There are many published reports on in vitro plant regeneration studies in broccoli including direct organogenesis, indirect organogenesis and somatic embryogenesis. This review summarizes those plant regeneration studies in broccoli that could be helpful in drawing the attention of the researchers and scientists to work on it to produce healthy, biotic and abiotic stress resistant plant material and to carry out genetic transformation studies for the production of transgenic plants.     

A new broccoli × broccoli immortal mapping population and framework genetic map: tools for breeders and complex trait analysis

A unique broccoli × broccoli doubled haploid (DH) population has been created from the F₁ of a cross between two DH broccoli lines derived from cultivars Green Duke and Marathon. We genotyped 154 individuals from this population with simple sequence repeat and amplified fragment length polymorphism markers to create a B. oleracea L. var. italica ‘intra-crop’ specific framework linkage map. The map is composed of nine linkage groups with a total length of 946.7 cM. Previous published B. oleracea maps have been constructed using diverse crosses between morphotypes of B. oleracea; this map therefore represents a useful breeding resource for the dissection of broccoli specific traits. Phenotype data have been collected from the population over five growing seasons; the framework linkage map has been used to locate quantitative trait loci for agronomically important broccoli traits including head weight (saleable yield), head diameter, stalk diameter, weight loss and relative weight loss during storage, as well as traits for broccoli leaf architecture. This population and associated linkage map will aid breeders to directly map agronomically important traits for the improvement of elite broccoli cultivars.     

Mediation of host selection and oviposition behavior in the diamondback moth Plutella xylostella and its predator Chrysoperla carnea by chemical cues from cole crops

Host plant-mediated orientation and oviposition by diamondback moth (DBM) Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) and its predator Chrysoperla carnea Stephens (Neuroptera: Chrysopidae) were studied in response to four different brassica host plants: cabbage, (Brassica oleracea L. subsp. capitata), cauliflower (B. oleracea L. subsp. botrytis), kohlrabi (B. oleracea L. subsp. gongylodes), and broccoli (B. oleracea L. subsp. italica). Results from laboratory wind tunnel studies indicated that orientation of female DBM and C. carnea females towards cabbage and cauliflower was significantly greater than towards either broccoli or kohlrabi plants. However, DBM and C. carnea males did not orient towards any of the host plants. In no-choice tests, oviposition by DBM did not differ significantly among the test plants, while C. carnea layed significantly more eggs on cabbage, cauliflower, and broccoli than on kohlrabi. However, in free-choice tests, oviposition by DBM was significantly greater on cabbage, followed by cauliflower, broccoli, and kohlrabi, while C. carnea preferred to oviposit on cabbage and cauliflower, followed by broccoli and kohlrabi. The predation rates of DBM by C. carnea on kohlrabi and broccoli were not significantly different from one another, but were significantly higher than that on cabbage and cauliflower. When two types of plant, intact and injured, were available to adult DBM, female oviposition was significantly greater on injured plant leaves than on intact plants leaves. Similarly, C. carnea oviposition was significantly greater on injured plant leaves than on intact leaves. Implications regarding the possible role of green leaf volatiles in host selection/preference, as well as in tritropic interactions, are discussed.     

Proteomic analysis of stress-related proteins in transgenic broccoli harboring a gene for cytokinin production during postharvest senescence

Our previous study revealed a cytokinin-related retardation of post-harvest floret yellowing in transgenic broccoli (Brassica oleracea var. italica) that harbored the bacterial isopentenyltransferase (ipt) gene. We aimed to investigate the underlining mechanism of this delayed post-harvest senescence. We used 2D electrophoresis and liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry for a proteomics analysis of heads of ipt-transgenic and non-transgenic inbred lines of broccoli at harvest and after four days post-harvest storage. At harvest, we found an accumulation of stress-responsive proteins involved in maintenance of protein folding (putative protein disulfide isomerase, peptidyl-prolyl cis-trans isomerase and chaperonins), scavenging of reactive oxygen species (Mn superoxide dismutase), and stress protection [myrosinase-binding protein, jasmonate inducible protein, dynamin-like protein, NADH dehydrogenase (ubiquinone) Fe-S protein 1 and stress-inducible tetratricopeptide repeat-containing protein]. After four days’ post-harvest storage of non-transgenic broccoli florets, the levels of proteins involved in protein folding and carbon fixation were decreased, which indicates cellular degradation and a change in metabolism toward senescence. In addition, staining for antioxidant enzyme activity of non-transgenic plants after post-harvest storage revealed a marked decrease in activity of Fe-superoxide dismutase and ascorbate peroxidase. Thus, the accumulation of stress-responsive proteins and antioxidant enzyme activity in ipt-transgenic broccoli are most likely associated with retardation of post-harvest senescence.