Nucleolar dominance does not occur in root tip cells of allotetraploid Brassica species.

Using in situ hybridization and silver staining methods, the numbers of active and inactive rDNA loci have been established for three allotetraploid species of Brassica (B. napus, B. carinata, and B. juncea) and their diploid ancestors (B. campestris, B. nigra, and B. oleracea). The allotetraploid species have chromosome numbers equal to the sum of the numbers in their diploid relatives, but have fewer rDNA loci. All species investigated have lower numbers of active NORs (AgNORs, nucleolar organizer regions) compared with the numbers of rDNA sites revealed by in situ hybridization. The number of active rDNA loci of the allotetraploid species is equal to the number of AgNORs in their diploid ancestors, indicating the absence of nucleolar dominance in amphidiploid Brassica species, at least in root meristematic cells.     

Physical mapping of rDNA loci in Brassica species.

The number of major rDNA loci (the genes coding for 18S-5.8S-26S rRNA) was investigated in the economically important Brassica species and their wild relatives by in situ hybridization of an rDNA probe to metaphase chromosomes and interphase nuclei. The diploid species B. nigra (B genome) has two major pairs of rDNA loci, B. oleracea (C genome) has two major pairs and one minor pair of loci, while B. campestris (A genome) has five pairs of loci. Among the three tetraploid species arising from these three diploid ancestors, B. carinata (BBCC genomes) has four loci, B. juncea (AABB genomes) has five major pairs and one minor pair of loci, and B. napus (AACC genomes) has six pairs of loci, indicating that the number of loci has been reduced during evolution. The complexity of the known rDNA restriction fragment length polymorphism patterns gave little indication of number of rDNA loci. It is probable that chromosome rearrangements have occurred during evolution of the amphidiploid species. The data will be useful for physical mapping of genes relative to rDNA loci, micro- and macro-evolutionary studies and analysis of aneuploids including addition and substitution lines used in Brassica breeding programs.     

Growth and mineral nutrition of six rapid-cycling Brassica species in response to seawater salinity

The growth of six rapid-cycling lines of Brassica species, B. napus, B. campestris, B. nigra, B. juncea, B. oleracea and B. carinata was inhibited by seawater salinity. Based on the change in dry matter reduction relative to the control at varying concentrations of salts (4, 8 and 12 dS m^-1), the relative salt tolerance of these species was evaluated. B. napus and B. carinata were the most tolerant and most sensitive species, respectively, while the other four species were moderately tolerant. The influence of seawater on the concentrations of 12 elements including macronutrients and micronutrients in the shoots of these Brassica plants was characterized to determine the relationship between nutritional disturbance and relative salt tolerance. It was found that seawater salinity had a significant effect on the concentrations of Ca, Mg, K, Cl, Na and total N in the shoots of these plants but only the change in Ca concentration was significantly related to the relative salt tolerance of these six rapid-cycling Brassica species according to a rank analysis of the data. This finding indicates that Ca may play a regulatory role in the responses of Brassica species to saline conditions.     

Immunopurification and Immunocharacterization of the Glucosinolate Biosynthetic Enzyme Thiohydroximate S-Glucosyltransferase

Preparing homogeneous UDP-glucose:thiohydroximate S-glucosyltransferase (S-GT), the penultimate biosynthetic enzyme of glucosinolates, by standard chromatographic methods has yielded too little protein for adequate purity evaluation, identity verification, and structural analysis. The low yields were apparently due to low abundance in source tissues, aggravated by enzyme instability. Here we describe an immunological method for purification of workable quantities from florets of Brassica oleracea ssp. botrytis (cauliflower). Florets that had undergone browning due to exposure to sunlight contained higher S-GT activities than are normally found in Brassica tissues. S-GT was adsorbed from crude tissue extracts onto an agarose-monoclonal antibody complex. Elution from the complex required harsh alkaline conditions (pH 11.5), giving extremely variable activity recoveries (maximum 20%). The eluate contained two proteins that could be separated readily by preparative polyacrylamide gel electrophoresis or anion-exchange chromatography. The overall S-GT protein recovery was estimated at less than 200 [mu]g/kg of cauliflower tissue. Molecular weight determinations with homogeneous cauliflower S-GT gave relative molecular weight (Mr) values of 55,500 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 57,600 by gel chromatography; isoenzymes with isoelectric point values of 4.80 and 4.95 were identified. A polyclonal antibody raised against denatured enzyme showed broad cross-reactivity in immunoblots with S-GT from a number of Brassica species and other crucifers. The monoclonal antibody that was used in the immunopurification was much more specific; it exclusively precipitated S-GT isoenzymes that had their genomic origin in the primary diploids B. oleracea and Brassica campestris. Thus, all of the S-GT was precipitated from the amphidiploid Brassica napus, which is a hybrid of B. orleracea and B. campestris. About half of the S-GT was precipitated from the amphidiploids Brassica carinata and Brassica juncea, which have B. oleracea and B. campestris as one of their parents, respectively. It was shown that the S-GT isoenzymes of B. juncea with Mr 55,500 and about 57,000 originate from the parents B. campestris and B. nigra, respectively.     

Identification of individual chromosomes and parental genomes in Brassica juncea using GISH and FISH

The three diploid (B. nigra, B. oleracea, B. campestris) and three allotetraploid (B. carinata, B. juncea, B. napus) species of Brassica, known as the "U-triangle" are one of the best model systems for the study of polyploidy. Numerous molecular investigations have provided a wealth of new insights into the polyploid origin and changes during the evolution of Brassica, but there are still many controversial aspects of their relationship and evolution. Interpretation of genome changes during evolution requires individual chromosome identification within the genome and clear distinction of genomes within the allotetraploid. The aim of this study was to identify individual chromosomes of B. juncea (genome AABB; 2n = 4x = 36) and to determine their genomic origin. Fluorescence in situ hybridization with 5S and 45S rDNA probes enabled discrimination of a substantial number of chromosomes, providing chromosomal landmarks for 20 out of 36 chromosomes of B. juncea. Additionally, along with double target genomic in situ hybridization, it allowed assignment of all chromosomes to either the A or B genomes.     

Chromosomal localization and characterization of rDNA loci in the Brassica A and C genomes.

Using fluorescence in situ hybridization, we located ribosomal DNA loci on prometaphase chromosomes of the diploid species Brassica rapa and Brassica oleracea and their amphidiploid Brassica napus. Based on comparisons of chromosome morphology and hybridization patterns, we characterized the individual B. napus rDNA loci according to their presumed origins in the Brassica A and C genomes. As reported in other studies, the sum of rDNA loci observed on B. rapa (AA genome) and B. oleracea (CC genome) chromosomes was one greater than the total number of loci seen in their amphidiploid B. napus (AACC). Evidence is presented that this reduction in B. napus rDNA locus number results from the loss of the smallest A genome rDNA site in the amphidiploid.     

Karyotyping of Brassica amphidiploids using 5S and 25S rDNA as chromosome markers

Species of Brassica have small, morphologically similar chromosomes, which makes karyotyping difficult using conventional cytogenetic methods. Molecular cytogenetic methods, like fluorescence in situ hybridisation (FISH) have the potential to improve karyotyping through the use of chromosome- or genome-specific markers. Simultaneous application of more than one DNA probe can greatly enrich the results obtained compared with separate single target FISH experiments. This paper demonstrates the use of multicolour fluorescence in situ hybridisation with 5S and 25S rDNA for karyotyping three amphidiploid species: B. napus, B. juncea and B. carinata. Using this method, it was possible to identify eight out of nineteen pairs of chromosomes in B. napus, ten out of eighteen pairs in B. juncea and six out of sixteen pairs in B. carinata. Additionally, rDNA sites allow the determination of the genomic origin of all marked chromosomes in B. napus and B. juncea.     

Salinity Tolerance in Brassica Oilseeds

Brassica oilseed species now hold the third position among oilseed crops and are an important source of vegetable oil. The most common Brassica oil-seed crops grown for commercial purposes are rape seeds, (Brassica campestris L. and B. napus L.) and mustards (B. juncea (L.) Czern. & Coss. and B. carinata A.Br.). The other Brassica species such as B. nigra (L.) Koch and B. tournefortii Gouan are grown on a very small scale. Brassica napus, B. juncea, and B. carinata are amphidiploids, whereas B. campestris and B. nigra are diploid. Most of the Brassica species have been categorized as moderately salt tolerant, with the amphidiploid species being the relatively salt tolerant in comparison with the diploid species. Due to the higher salt tolerance of the amphidiploids, it has been suggested that their salt tolerance has been acquired from the A (B. campestris) and C (B. oleracea L.) genomes. However, significant inter- and intraspecific variation for salt tolerance exists within brassicas, which can be exploited through selection and breeding for enhancing salt tolerance of the crops. There are contrasting reports regarding the response of these species to salinity at different plant developmental stages, but in most of them it is evident that they maintain their degree of salt tolerance consistently throughout the plant ontogeny. The pattern of uptake and accumulation of toxic ions (Na⁺ and Cl^?), in tissues of plants subjected to saline conditions appears to be mostly due to mechanism of partial ion exclusion (exclusion of Na⁺ and/or Cl^?) in most of the species, although ion inclusion in some cases at intraspecific levels has also been observed. Maintenance of high tissue K⁺/Na⁺ and Ca^{2 +}/Na⁺ ratios has been suggested as an important selection criterion for salt-tolerance in brassicas. Osmotic adjustment has also been reported in Brassica plants subjected to saline conditions, but particularly to a large extent in salt-tolerant species or cultivars. The roles of important organic osmotica such as total soluble sugars, free amino acids, and free proline, which are central to osmotic adjustment, have been discussed. In canola, B. napus, no positive relationship has been observed between salt tolerance and erucic acid content of seed oil in different cultivars. Furthermore, glucosinolate content of the seed meal in canola generally increases with an increase in salt level of the growth medium. This review highlights the responses of potential Brassica crops to soil salinity from the whole plant to the molecular level. It also describes the efforts made during the past millennium in uncovering the mechanism(s) of salinity tolerance of these crops both at the whole plant and cellular levels. The important selection criteria, which are used by researchers to enhance the degree of salinity tolerance in brassicas, are summarized. In addition, the vital role of genetic engineering and molecular biology approaches to the improvement of salt tolerance in brassicas is emphasized.     

Phytoextraction of zinc, copper, nickel and lead from a contaminated soil by different species of Brassica

In a pot culture experiment, five different species of Brassica (Brassica juncea, Brassica campestris, Brassica carinata, Brassica napus, and Brassica nigra) were grown for screening possible accumulators of heavy metals, viz. Zn, Cu, Ni, and Pb. The plants were grown to maturity in a soil irrigated with sewage effluents for more than two decades in West Delhi, India. The soil analysis showed enhanced accumulation of Zn, Cu, Ni, and Pb in this sewage-irrigated soil. Among all species, B. carinata showed the highest concentration (mg kg(-1)) as well as uptake (microg pot(-1)) of Ni and Pb at maturity. Although B. campestris showed a higher concentration of Zn in its shoots (stem plus leaf), B. carinata extracted the largest amount of this metal due to greater biomass production. However, B. juncea phytoextracted the largest amount of Cu from the soil. In general, the highest concentration and uptake of metal was observed in shoots compared to roots or seeds of the different species. Among the Brassica spp., B. carinata cv. DLSC1 emerged as the most promising, showing greater uptake of Zn, Ni, and Pb, while B. juncea cv. Pusa Bold showed the highest uptake of Cu. The B. napus also showed promise, as it ranked second with respect to total uptake of Pb, Zn, and Ni, and third for Cu. Total uptake of metals by Brassica spp. correlated negatively with available as well as the total soil metal concentrations. Among the root parameters, root length emerged as the powerful parameter to dictate the uptake of metals by Brassica spp. Probably for the first time, B. carinata was reported as a promising phytoextractor for Zn, Ni, and Pb, which performed better than B. juncea.     

Interspecific hybrids between a transgenic rapeseed (Brassica napus) and related species: cytogenetical characterization and detection of the transgene.

In interspecific hybrids produced between a transgenic rapeseed, an allotetraploid species, resistant to herbicide, phosphinotricin, and five diploid related species, the risk for gene introgression in weed genomes was explored through cytogenetic and bar gene characterizations. Among the 75 hybrids studied, most had the expected triploid structure, with the exception of B. napus - B. oleracea amphidiploid plants and one B. napus - S. arvensis amphidiploid plant. In triploid hybrid plants, the reciprocal hybrids did not exhibit any difference in their meiotic behavior. The comparison of the percentage of chromosome pairing in the hybrids with that of haploid rapeseed permit to conclude that allosyndesis between AC genomes and related species genomes took place. This possibility of recombination was confirmed by the presence of multivalent associations in all the interspecific hybrids. Nevertheless, in B. napus - B. adpressa hybrids a control of chromosome pairing seemed to exist. The possibility of amphidiploid plant production directly obtained in the F1 generation increased the risk of gene dispersal. The B. napus - B. oleracea amphidiploid plant presented a meiotic behavior more regular than that of the B. napus - S. arvensis amphidiploid plant. Concerning the herbicide bar gene characterization, the presence of the gene detected by DNA amplification was correlated with herbicide resistance, except for two plants. Different hypotheses were proposed to explain these results. A classification of the diploid species was established regarding their gene dispersal risk based on the rate of allosyndesis between chromosomes of AC genomes of rapeseed and the genomes of the related species.     

传粉导致的转基因白菜与其近缘种属材料间的基因流动

从形态、染色体及分子水平上证实,转基因不结球白菜(Brassica campestris ssp．chinensis var．utilis Tsen et Lee)中编码除草剂Basta抗性的bar基因能在田间条件下,经自然传粉,以较高频率侵入芜菁(B．campestris ssp．rapifera)、结球白菜(B．campestris／sssp．pek／nens／s)和不结球白菜(B．campestrs ssp．ch／nens／s)的基因组中,也能少量侵入同属异种的甘蓝型油菜(B.napus)基因组中;在温室人工辅助授粉条件下,除在上述种中的基因漂移率提高外,bar基因尚能以一定频率侵入同属的黑芥(B．nigra)、埃塞俄比亚芥(B．car／nata)、芥菜(B．juncea)基因组中,但始终未能得到转基因白菜与结球甘蓝(B．oleracea)、萝卜(尺．sativus)的杂种。转基因白菜与十字花科的7种常见杂草经温室人工辅助授粉,也均未得到抗性杂种。     

Towards identifying Brassica proteins involved in mediating resistance to Leptosphaeria maculans: a proteomics-based approach

To better understand the pathogen-stress response of Brassica species against the ubiquitous hemi-biotroph fungus Leptosphaeria maculans, we conducted a comparative proteomic analysis between blackleg-susceptible Brassica napus and blackleg-resistant Brassica carinata following pathogen inoculation. We examined temporal changes (6, 12, 24, 48 and 72 h) in protein profiles of both species subjected to pathogen-challenge using two-dimensional gel electrophoresis. A total of 64 proteins were found to be significantly affected by the pathogen in the two species, out of which 51 protein spots were identified using tandem mass spectrometry. The proteins identified included antioxidant enzymes, photosynthetic and metabolic enzymes, and those involved in protein processing and signaling. Specifically, we observed that in the tolerant B. carinata, enzymes involved in the detoxification of free radicals increased in response to the pathogen whereas no such increase was observed in the susceptible B. napus. The expression of genes encoding four selected proteins was validated using quantitative real-time PCR and an additional one by Western blotting. Our findings are discussed with respect to tolerance or susceptibility of these species to the pathogen.     

Mixoploidy in wild and cultivated Cruciferae species able to hybridize with Brassica napus

Chromosome numbers in root meristem cells of the seedlings of wild and cultivated Cruciferae species able to hybridize with Brassica napus have been studied. The seedlings of Brassica junceae, Diplotaxis tenuifolia and Raphanum raphanistrum showed exclusively diploid metaphases. Up to 5% of B. napus and B. cretica seedlings were mixoploids. Diploid cells prevailed among the mixoploids, but hypo- and hyperdiploid ones were observed as well. Nearly 20 % of B. campestris and R. sativum seedlings were mixoploid, and di-tetraploid chimeras constituted considerable proportion of them. Less than a half of B. nigra seedlings were diploid, while the rest of plants were mixoploid. Among them the seedlings bearing preferentially tetra- and triploid cells dominated. Biological implications and presumable reasons underlying the discovered mixoploidy are discussed.     

Seed coat microsculpturing changes during seed development in diploid and amphidiploid Brassica species

BACKGROUND AND AIMS: Seed coat morphology is known to be an excellent character for taxonomic and evolutionary studies, thus understanding its structure and development has been an important goal for biologists. This research aimed to identify the developmental differences of seed coats between amphidiploids and their putative parents in Brassica. METHODS: Scanning electron microscope (SEM) studies were carried out on six species (12 accessions), three amphidiploids and their three diploid parents. KEY RESULTS: Twelve types of basic ornamentation patterns were recognized during the whole developmental process of the seed coat. Six types of seed coat patterns appeared in three accessions of Brassica rapa, five types in B. oleracea, B. nigra and B. carinata, seven types in B. napus, and eight types in B. juncea. There was less difference among seed coat patterns of the three accessions of B. rapa. The reticulate and blister types were two of the most common patterns during the development of seeds in the six species, the blister-pimple and the pimple-foveate patterns were characteristic of B. rapa, and the ruminate of B. oleracea and B. nigra. The development of seed coat pattern in amphidiploids varied complicatedly. Some accessions showed intermediate patterns between the two putative parents, while others resembled only one of the two parents. CONCLUSIONS: The variation in the patterns of seed coat development could be used to provide a new and more effective way to analyse the close relationship among amphidiploids and their ancestral parents.     

Plant regeneration from peduncle segments of oil seed Brassica species: Influence of silver nitrate and silver thiosulfate

Cultured peduncle segments of B. juncea, B. campestris, B. napus, B. nigra and B. carinata produced shoot buds on Murashige and Skoog medium supplemented with benzyladenine and 1-naphthalene acetic acid. Supplementation of the media with 30 μm silver nitrate or silver thiosulfate enhanced the frequency of shoot regeneration. The regenerated shoots could be rooted at a frequency of 95% and transferred to soil where 75% survived and set seed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Water relations and leaf gas exchange properties in some elite canola (Brassica napus) lines under salt stress

In order to examine whether growth of eight genetically diverse canola (Brassica napus) lines under salt stress is positively associated with their rate of photosynthesis and other gas exchange related attributes, 20‐day old plants of all eight lines were subjected to salinised soil containing 2.4 dS m^?1 NaCl (control), 4 dS m^?1 NaCl, 8 dS m^?1 NaCl or 12 dS m‐¹ NaCl. The lines DGL (non canola) and Dunkeld were found to be salt tolerant and Rainbow and Cyclon salt sensitive with regard to shoot dry matter production and seed yield under saline conditions. In most of the lines there was a negative relationship between growth and net CO₂ assimilation rate. For example, the salt sensitive line Cyclon was the lowest and Con‐III the highest, and the salt tolerant line Dunkeld intermediate in net CO₂ assimilation rate under salt stress. Stomatal conductance was found to be lower in the salt sensitive line Cyclon, followed by the salt tolerant line Dunkeld along with Oscar. Water use efficiency estimated as Pn/E was moderate in the salt sensitive line Cyclon and the salt tolerant line Dunkeld. In conclusion, high salt tolerance of Dunkeld and DGL (non‐canola) was not positively associated with net CO₂ assimilation rate or Pn/E.     

传粉导致的转基因白菜与其近缘种属材料间的基因流动

从形态、染色体及分子水平上证实,转基因不结球白菜(Brassica campestris ssp. chinensis var. utilis Tsen et Lee)中编码除草剂Basta抗性的bar基因能在田间条件下,经自然传粉,以较高频率侵入芜菁(B. campestris ssp. rapifera)、结球白菜(B. campestris ssp. pekinensis)和不结球白菜(B. campestris ssp. chinensis)的基因组中,也能少量侵入同属异种的甘蓝型油菜(B. napus)基因组中; 在温室人工辅助授粉条件下,除在上述种中的基因漂移率提高外,bar基因尚能以一定频率侵入同属的黑芥(B. nigra)、埃塞俄比亚芥(B. carinata)、芥菜(B. juncea)基因组中,但始终未能得到转基因白菜与结球甘蓝(B. oleracea)、萝卜(R. sativus)的杂种.转基因白菜与十字花科的7种常见杂草经温室人工辅助授粉,也均未得到抗性杂种.     

Complexity and expression of the glutamine synthetase multigene family in the amphidiploid crop Brassica napus

In the amphidiploid genome of oilseed rape (Brassica napus) the diploid ancestral genomes of B. campestris and B. oleracea have been merged. As a result of this crossing event, all gene loci, gene families, or multigene families of the A and C genome types encoding a certain protein are now combined in one plant genome.In the case of the multigene family for glutamine synthetase, the key enzyme of nitrogen assimilation, six different cDNA sequences were isolated from leaf and root specific libraries. One sequence pair (BnGSL1/BnGSL2) was characterized by the presence of amino- terminal transit peptides, a typical feature of all nuclear encoded chloroplast proteins. Two other cDNA pairs (BnGSR1-1/BnGSR1-2 and BnGSR2-1/BnGSR2-2) with very high homology between each other were found in a root specific cDNA library and represent protein subunits for cytosolic glutamine synthetase isoforms.Comparative PCR amplifications of genomic DNA isolated from B. napus, B. campestris and B. oleracea followed by sequence–specific restriction analyses of the PCR products permitted the assignment of the cDNA sequences to either the A genome type (BnGSL1/BnGSR1- 1/BnGSR2-1) or the C genome type (BnGSL2/BnGSR1-2/BnGSR2-2). Consequently, the ancestral GS genes of B. campestris and B. oleracea are expressed simultaneously in oilseed rape. This result was also confirmed by RFLP (restriction fragment length polymorphism) analysis of RT-PCR products.In addition, the different GS genes showed tissue specific expression patterns which are correlated with the state of development of the plant material. Especially for the GS genes encoding the cytosolic GS isoform BnGSR2, a marked increase of expression could be observed after the onset of leaf senescence.     

芸薹属多倍体植物基因组进化的RAPD分析

多倍化是促进高等植物发生进化的重要力量。为了更清楚地了解多倍体在形成之后其基因组是如何进化的,利用38个随机引物对芸薹属Brassica L.禹氏三角（U’Triangle）中的多倍体物种及其祖先二倍体物种进行了研究。根据扩增出的273条带计算了遗传距离,并用UPGMA法进行了聚类分析。结果发现,二倍体物种B.campestris（AA）与B.oleracea（CC）的亲缘关系比与B.nigra（BB）的要近;异源多倍体B.napus（AACC）比起其二倍体祖先之一B.campestris（AA）与另一个