The incidence of viruses in wild Brassica nigra in Dorset (UK)

Using enzyme‐linked immunosorbent assays, the frequency of occurrence of six viruses was determined in Brassica nigra collected from five coastal sites in Dorset, spanning approximately 24 km. During 1998–2000, the viruses detected were: Turnip mosaic virus (genus Potyvirus) (TuMV), Turnip yellow mosaic virus (genus Tymovirus) (TYMV), Turnip crinkle virus (genus Carmovirus) (TCV), Turnip rosette virus (genus Sobemovirus) (TRoV), Beet western yellows virus (genus Polerovirus) (BWYV) and Cauliflower mosaic virus (genus Caulimovirus) (CaMV). Multiple infections were detected in some individuals (48/447). TuMV was detected infrequently over the three‐year period (5/597). A representative isolate of each virus was tested for its effects on glasshouse‐grown individuals from different half‐sib families of B. nigra from four of the sites. Whether inoculated manually or via aphids (Myzus persicae), TuMV caused a rapid (within 10 days) lethal systemic necrosis in the B. nigra seedlings except when they were near flowering at the time of inoculation. Each of the other viruses invaded systemically but were not lethal. Indeed, BWYV systemically invaded 13/19 glasshouse‐grown B. nigra seedlings but did not produce any visible symptoms. Otherwise, the isolates tested differed in their pathogenicity and in the symptoms they produced in infected B. nigra. With TYMV or TCV viral antigen concentration was closely linked to pathogenicity; for TRoV or CaMV, there was little or no difference in virus concentration between plants with and without symptoms. Substantial and reproducible differences were observed in sensitivity/susceptibility among B. nigra genotypes from different sites in Dorset challenged with the same virus isolate.     

Agrobacterium tumefaciens-mediated transformation of greenhouse-grown Brassica rapa ssp. oleifera

Greenhouse-grown plants of turnip rape Brassica rapa ssp. oleifera (syn. B. campestris) cv. Valtti and Sisu were transformed by Agrobacterium tumefaciens infection. Of the three A. tumefaciens strains tested (C58C1, EHA105 and LBA4404), LBA4404 gave the best results. Segments excised from one to two upper internodes of an inflorescence-carrying stem served as explants for the Agrobacterium infection. Cultivation of the explants horizontally during the first 3 days of co-cultivation with A. tumefaciens following immediate selection of transformed tissue of the stem segments placed vertically basal side down were critical. Use of silver nitrate (5–10 mg/l) in the culture medium and Micropore (3 M) paper tape for sealing plates was also beneficial. Transgenic shoots were recovered using either hygromycin or kanamycin (20–25 mg/l) selection. Hygromycin was preferable, as the proportion of `escapes' was 90% under kanamycin and 10% under hygromycin selection. Regeneration was achieved by culturing the explants for 3–6 days on 0.5 mg/l of 2,4-di-chlorophenoxyacetic acid and 1–2 weeks on 2–3 mg/l of 6-benzyl aminopurine with/without 0.05 mg/l α-naphthaleneacetic acid. Recovered shoots were then cultured on hormone-free MS medium. This culture program gave 60–80% shoot regeneration. Regenerants were tested by histological β-glucuronidase staining and Southern blotting. The recovery rate of transgenic shoots was 4–9% of the number of explants used in the experiments. Received: 28 November 1997 / Revision received: 25 March 1998 / Accepted: 22 November 1998     

The incidence and distribution of viruses infecting lettuce, cultivated Brassica and associated natural vegetation in Spain

A virus survey was conducted during the spring and autumn of 2001 and 2002 to determine the presence, prevalence and distribution in Spain of the viruses that are most commonly found infecting lettuce and Brassica worldwide. Crop plants showing virus symptoms from the principal lettuce and Brassica-growing regions of Spain, and some samples of the annual and perennial flora nearby, were tested by enzyme-linked immunosorbent assays using specific commercial antibodies against the following viruses: Alfalfa mosaic virus (AMV), Broad bean wilt virus 1 (BBWV-1), Beet western yellows virus (BWYV), Cauliflower mosaic virus (CaMV), Cucumber mosaic virus (CMV), Lettuce mosaic virus (LMV), Pea seed-borne mosaic virus (PSbMV), Turnip mosaic virus (TuMV) and Tomato spotted wilt virus (TSWV). Samples were also tested with a Potyvirus genus antibody. Virus incidence was much lower in spring than in autumn, especially in 2001. In spring 2002, CMV and LMV were the most prevalent viruses in lettuce, while CaMV was the most important virus present in Brassica crops grown in Navarra, followed by CMV and BWYV. In the autumn, the spectrum of viruses was different; potyviruses were widespread in lettuce grown in Madrid, but TSWV and BWYV were predominant in the Murcia region. The prevalent Potyvirus detected in lettuce fields was LMV, but none of the samples collected were positive for PSbMV or TuMV. In Brassica crops, TSWV was the most abundant in autumn-sown crops, especially in the Navarra region. All of the viruses present in lettuce and Brassica were also frequently detected in their associated natural vegetation at the same time, suggesting that they probably play an important role as virus reservoirs. Sonchus spp. were particularly common and were frequently infected with CMV, LMV and BWYV. Another common species, Chenopodium album, was often infected with TSWV and BWYV. Multiple infections were common, especially in non-crop plants, and the most common combination was BWYV and TSWV. The role of weeds in the epidemiology of viruses that infect lettuce and Brassica crops in Spain is discussed.     

Effects of turnip mosaic virus and turnip yellow mosaic virus on the survival, growth and reproduction of wild cabbage (Brassica oleracea)

Wild plants of Brassica oleracea (wild cabbage) are commonly infected with turnip mosaic poty virus (TuMV), turnip yellow mosaic tymovirus (TYMV) and several other viruses. A field experiment in which plants were inoculated either with TuMV or TYMV showed that virus infection significantly reduced survival, growth and reproduction. Relative to water inoculated-controls, plants infected with TYMV had greater mortality, were shorter, had a smaller leaf area and number, showed a greater amount of damage from herbivory and chlorosis, were less likely to flower and produced fewer pods and lower total seed output. Plants infected with TuMV did not appear to be adversely affected at first; however, mortality after 18 months was higher than control plants. Although TuMV infection had no effect on the number of plants flowering, the infected plants did produce fewer pods and a lower total seed output. We conclude that both viruses can significantly affect vegetative and reproductive performance of wild cabbage and hence that introgression of virus resistance (particularly when conferred by a major gene or a transgene) from a crop might increase plant fitness in natural populations of this species. Ecological risk assessments of virus resistance transgenes must do more than survey adult plants in natural populations for the presence of the target virus. Failure to detect the virus could be due to high mortality on infection with the virus.     

The prevalence and spatial distribution of viruses in natural populations of Brassica oleracea

We report a survey of four viruses (beet western yellows luteovirus (BWYV), cauliflower mosaic caulimovirus (CaMV), turnip mosaic potyvirus (TuMV), turnip yellow mosaic tymovirus (TYMV)) in five natural populations of Brassica oleracea in Dorset (UK). All four viruses were common; 43% of plants were infected with BWYV, 60% with CaMV, 43% with TuMV and 18% with TYMV. For each virus there were significant differences in the proportion of infected plants among populations, which were not completely explained by differences in the age of plants. Multiple virus infections were prevalent, with 54% of plants having two or more virus types. There were statistically significant associations between pairs of viruses. The CaMV was positively associated with the other three viruses, and BWYV was also positively associated with TuMV. There was no detectable association between BWYV and TYMV, whereas TuMV and TYMV were negatively associated. We suggest these associations result from BWYV, CaMV and TuMV having aphid vectors in common, as aphids are attracted to plants that already have a virus infection. Infected plants were distributed randomly or were very weakly aggregated within populations. The implications of widespread multiple virus infections in natural plant populations are discussed with respect to the release of transgenic plants expressing virus-derived genes.     

Identification and preliminary analysis of a new PCP promoter from Brassica rapa ssp. chinensis

The promoter of Brassica campestris Male Fertile 5 (BcMF5), a pollen coat protein member, class A (PCP-A) gene family, was isolated from Brassica rapa L. ssp. chinensis Makino (Chinese cabbage-pak-choi) by Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR). Sequence analysis suggested that the 605-bp promoter of BcMF5 appears to be a pollen promoter. In an attempt to confirm the promoter activity of BcMF5 promoter, −609 to +3 bp and −377 to +3 bp fragments of the upstream sequence of BcMF5 were inserted at the site upstream of the coding region of the uidA gene in the sense orientation to construct two deletion expression vectors. Transient expression analysis in onion epidermal cells by particle bombardment showed that both −609 to +3 bp and −377 to +3 bp fragments of BcMF5 promoter were capable of driving β-glucuronidase gene expression. Furthermore, by Agrobacterium-mediated genetic transformation method, Arabidopsis transgenic Kan^R plants were obtained. GUS assay analysis revealed that the promoter of BcMF5 induced gene expression at the early stage of anther development and drove high levels of GUS expression in anther walls, upper regions of petals, pollen, and pollen tubes in the middle and late stage of anther development, but did not drive any expression in sepals and pistils.     

结球白菜下胚轴原生质体培养及其体细胞胚植株再生

结球白菜（Brassica campestris ssp．pekinensis）的原生质体培养由于基因型依赖性强,细胞易褐化,愈伤组织的芽诱导率低等而难于再生植株。本实验以结球白菜的下胚轴原生质体为试材,研究了影响其细胞分裂及愈伤组织形成的因素,探索了经过体细胞胚发生途径获得再生植株的技术。结果表明,试材的基因型及培养基组成影响细胞分裂及褐化;KM8P是结球白菜原生质体培养更适宜的培养基,能显著减轻细胞的褐化;液体培养基中一定浓度的活性炭能在一定程度上减轻细胞褐化进程,并有利于星状细胞团的形成;基因型Asko中,愈伤组织形成体细胞胚的结构,其发生的频率约为5％,该类体细胞胚能全部顺利地发育成完整植株。本技术具有再生植株形成容易、频率较高且通过体细胞胚发生途径等优点。     

与大白菜霜霉病抗性主效QTL连锁的分子标记开发

霜霉病是危害大白菜的三大病害之一,该病的发生会严重影响大白菜的产量及品质,因而研究与霜霉病抗性QTL紧密连锁的分子标记对大白菜抗病新品种培育具有重要意义。该研究在前期工作的基础上,选用高感霜霉病株系91-112、高抗霜霉病株系T12-19以及由二者为双亲构建的DH群体为实验材料,针对大白菜霜霉病抗性主效QTL——BrDW所在的标记区间,利用已有的大白菜基因组信息发展与抗性QTL紧密连锁的分子标记,通过Blast和IMap分析,将与BrDW连锁的RAPD标记K14-1030定位于大白菜KBrB058M10上(位于Contig214上),根据KBrB058M10附近的BAC及BAC-end序列设计引物,结合限制性内切酶酶切及HRM分析方法,筛选得到5个与BrDW连锁的分子标记,包括1个Indel标记Brb062-Indel230,3个CAPS标记Brb094-DraⅠ787、Brb094-AatⅡ666和Brb043-BglⅡ715,1个SNP标记Brh019-SNP137;同时,通过筛选与目标区域具有同源性的Unigene序列得到了1个与BrDW紧密连锁的SSR标记bru1209。标记Brb062-Indel230、Brb094-DraⅠ787、Brb094-AatⅡ666、Brb043-BglⅡ715、Brh019-SNP137和bru1209与RAPD标记K14-1030之间的遗传距离分别为4.3 cM、1.7 cM、5.9 cM、5.9 cM、4.6 cM和0.8 cM,在对DH群体中的抗性株系选择上准确率分别为69.7%、70.9%、72.4%、72.4%、58.3%和74.2%,可应用于分子标记辅助选择,为霜霉病抗性分子育种奠定了良好基础。     

Control of Lepidopteran insect pests in transgenic Chinese cabbage (Brassica rapa ssp. pekinensis) transformed with a synthetic Bacillus thuringiensis cry1C gene

 A synthetic Bacillus thuringiensis cry1C gene was transferred to three Korean cultivars of Chinese cabbage via Agrobacterium tumefaciens-mediated transformation of hypocotyl explants. Hygromycin resistance served as an efficient selective marker. The transformation efficiency ranged from 5% to 9%. Transformation was confirmed by Southern blot analysis, PCR, Northern analysis, and progeny tests. Many transgenic plants of the closed-head types (lines Olympic and Samjin) flowered in vitro. Over 50 hygromycin-resistant plants were successfully transferred to soil. The transgenic plants and their progeny were resistant to diamondback moths (DBM, Plutella xylostella), the major insect pest of crucifers world-wide, as well as to cabbage loopers (Trichoplusia ni) and imported cabbage worms (Pieris rapae). Both susceptible Geneva DBM and a DBM population resistant to Cry1A protein were controlled by the Cry1C-transgenic plants. The efficient and reproducible transformation system described may be useful for the transfer of other agriculturally important genes into Chinese cabbage. Received: 12 June 2000 / Revision received: 21 August 2000 / Accepted: 22 August 2000     

红菜薹雄性不育系花药败育的细胞形态学观察

采用石蜡切片技术,在光学显微镜下系统研究了红菜薹(Brassica campestris L.ssp．chinensis L.var．utilis TsenetLee．)波里马胞质雄性不育系(Polima CMS)、红菜薹萝卜胞质雄性不育系(Ogura CMS)及相应保持系花药发育过程的细胞形态学特征。观察结果表明：红菜薹Polima CMS花药发育受阻于孢原细胞阶段,不形成花粉,属无花粉型,此不育系花药不形成绒毡层和中层;而红菜薹Ogura CMS花药败育发生于小孢子母细胞期或四分体时期,表现为绒毡层细胞异常,挤压四分体,导致四分体和绒毡层同时解体而败育。     

诱导二倍体和同源四倍体白菜细胞质雄性不育系不定芽培养基的筛选

以二倍体和四倍体白菜细胞质雄性不育系的子叶为外植体,采用正交设计研究TDZ(0.1、0.5、1.0 mg·L-1)、NAA(0.1、0.5、0.8 mg·L-1)和AgNO3(0、5、10mg·L-1)三因素三水平诱导不定芽的结果表明:二倍体的不定芽再生优化培养基为MS 1.0 mg·L-1TDZ 0.8mg·L-1NAA 5 mg·L-1AgNO3(其再生率为80.7%),三因素影响的顺序为:NAA＞AgNO3＞TDZ;四倍体的不定芽再生优化培养基为MS 0.5 mg·L-1 TDZ 0.5 mg·L-1 NAA 10 mg·L-1 AgNO3(再生率为83.3%),三因素影响的顺序为:AgNO3＞NAA＞TDZ.后者所需TDZ/IAA的比值低于前者.