Immunocytochemical localization of myrosinase in Brassica napus L.

The cytological and intracellular localization of myrosinase (EC 3.2.3.1) has been studied by immunochemical techniques using paraffin-embedded sections of radicles and cotyledons from seeds of Brassica napus L. cv. Niklas. For immunolabelling, sections were sequentially incubated with a monoclonal anti-myrosinase antibody and with peroxidase-and fluorescein-isothiocyanate-conjugated secondary antibodies. Enzyme and fluorescence label was present in typical myrosin cells both in radicles and in cotyledons. With higher magnification, fluorescence label revealed that the intracellular localization of myrosinase was associated with the tonoplast-like membrane surrounding the myrosin grains in the myrosin cells. The results also indicate that a large proportion of the positive myrosin cells are located in the second-outermost cell layer of the peripheral cortex region of the radicles.Abbreviations FITC fluorescein isothiocyanate - PBS phosphate-buffered saline - PBS-T PBS with 0.5% (v/v) Tween-20 (polyoxyethylene sorbitane monolaurate) This work was supported by The Norwegian Research Council for Science and the Humanities. We wish to thank Professor Med. O.A. Haugen, Department of Pathology, University of Trondheim, Norway, for the skilful assistance provided regarding fixation and sectioning.     

Melliferous potential of Brassica napus L. subsp. napus (Cruciferae)

Morphophysiological characteristics of oilseed rape flowers, such as features of the nectaries, nectar production, and observations on honey bee visits and honey and seed yield were studied with the aim to evaluate the melliferous potential of this crop as well as its attractiveness to pollinators. Calculation of the theoretical maximal honey yield revealed that the actual amount of extracted honey was much lower than the potential yield, indicating that this bee pasture is underutilized. We found that honey bee pollination increased oilseed rape yield, i.e., seed production, by 12 % compared with the treatment in which pollinators were excluded.     

甘蓝型油菜花粉离体培养

通过对甘蓝型油菜花粉发育阶段和活力的检测确定花粉发育的时期,分离出单核晚期花粉进行离体培养.结果表明,(1)筛选出适合油菜小孢子花粉离体培养的液体培养基为T_1+怀特维生素(White's vitamins)+2%椰子汁+0.5 mol/L麦芽糖,在此培养基上花粉的成熟率可达25.1%,萌发率达6.3%.(2)筛选出适合成熟花粉离体萌发液体培养基为0.6 mol/L麦芽糖+1.6 mmol/L硼酸+2.9 mmol/L硝酸钙+29.6 μmol/L VB_1,在此培养基上,自然成熟花粉的萌发率可达75.2%.将离体培养成熟的花粉培养在萌发培养基,萌发的花粉占成熟花粉的66.3%.     

Analysis of RFLP mapping inaccuracy in Brassica napus L.

 We identified sources of mapping inaccuracy during the construction of RFLP linkage maps from one F₂ population and two F₁ microspore-derived populations from the same cross of oilseed Brassica napus. The genetic maps were compared using a total of 145 RFLP marker loci including 82 loci common to all three populations. In the process, we identified a series of mapping events that could lead to ambigous conclusions. Superimposed restriction fragments could be mistaken as a single dominant restriction fragment in a F₂ population and, when analyzed as such, would yield inaccurate linkage information. Residual heterozygosity in parental lines resulted in complicated allelic assignment and yielded subsequent difficulties in linkage determination. Loose and spurious linkages occurred during mapping and were identified by comparing maps derived from different populations. LOD scores and χ² test of independence were compared for their capacity to detect loose linkages or generate spurious ones. Extreme segregation distortions towards the same parental allele also contributed to an additional source of spurious linkage. Small but significant segregation distortions resulted in reduced estimates of the recombination fraction. The use of the same ‘probe× enzyme’ combinations in doubled haploid populations allowed the identification of the correct allele assignment as well as loose and spurious linkages. A translocation between two homoeologous linkage groups was observed. The consequences of such a chromosomal event as a source of error in mapping applications are discussed. Received: 7 September 1996/Accepted: 25 October 1996     

Regulation of Cadmium-Induced Proteomic and Metabolic Changes by 5-Aminolevulinic Acid in Leaves of Brassica napus L.

It is evident from previous reports that 5-aminolevulinic acid (ALA), like other known plant growth regulators, is effective in countering the injurious effects of heavy metal-stress in oilseed rape (Brassica napus L.). The present study was carried out to explore the capability of ALA to improve cadmium (Cd²⁺) tolerance in B. napus through physiological, molecular, and proteomic analytical approaches. Results showed that application of ALA helped the plants to adjust Cd²⁺-induced metabolic and photosynthetic fluorescence changes in the leaves of B. napus under Cd²⁺ stress. The data revealed that ALA treatment enhanced the gene expressions of antioxidant enzyme activities substantially and could increase the expression to a certain degree under Cd²⁺ stress conditions. In the present study, 34 protein spots were identified that differentially regulated due to Cd²⁺ and/or ALA treatments. Among them, 18 proteins were significantly regulated by ALA, including the proteins associated with stress related, carbohydrate metabolism, catalysis, dehydration of damaged protein, CO2 assimilation/photosynthesis and protein synthesis/regulation. From these 18 ALA-regulated proteins, 12 proteins were significantly down-regulated and 6 proteins were up-regulated. Interestingly, it was observed that ALA-induced the up-regulation of dihydrolipoyl dehydrogenase, light harvesting complex photo-system II subunit 6 and 30S ribosomal proteins in the presence of Cd²⁺ stress. In addition, it was also observed that ALA-induced the down-regulation in thioredoxin-like protein, 2, 3-bisphosphoglycerate, proteasome and thiamine thiazole synthase proteins under Cd²⁺ stress. Taken together, the present study sheds light on molecular mechanisms involved in ALA-induced Cd²⁺ tolerance in B. napus leaves and suggests a more active involvement of ALA in plant physiological processes than previously proposed.     

Evidence for spontaneous diploid androgenesis in Brassica napus L.

Summary Seeds of androgenetic origin were obtained among the F₁ progenies of two crosses between resynthesized and cultivated forms of Brassica napus. The high-erucic, white-flowered, resynthesized line No7076 acted as the female, and the zero-erucic, yellow-flowered, cultivars Topas and Puma, as males. No androgenetic seeds were obtained in the reciprocal crosses. Resynthesized rape could thus be of potential use for the production of androgenetic plants. Of special interest is the high frequency (21%) of spontaneous androgenesis observed in one of the two crosses. One plant, determined from erucic acid content and flower colour analysis as androgenetic, had a diploid chromosome number. Further knowledge about the genetic control of spontaneous androgenesis in the present material and the origin of the cytoplasm in androgenetic plants are required in order to exploit this phenomenon in practical plant breeding.     

甘蓝型油菜正季与反季材料小孢子的培养

本文以甘蓝型油菜Westar的F1代为供试材料,通过对8个正季和同样的8个反季材料进行小孢子培养对比实验。结果表明,相同材料(基因型)正季能获得胚状体的反季一样能获得胚状体,但是相同材料的出胚数反季要比正季少50%,并且出胚时间要晚5～8d。正季与反季材料的成苗百分率相同,平均达到93%,加倍率基本一样,达到80%。由此可见,用反季节材料培养小孢子同样能获得成功,对特殊材料可以利用此法进一步加速育种进程。     

Studies of the Biochemical Background to Differences in Glucosinolate Content in Brassica napus L. III. Further Studies to Localize Metabolic Blocks

To localize the metabolic block(s) in the biosynthesis of glucosinolates in the Brassica napus L. cv. Bronowski, 5-methylthiopentanal oxime-1-¹⁴C was synthesized and fed to this cultivar and cv. Regina II, which has average levels of glucosinolates. The aldoxime was as efficient as a precursor of 3-butenylglucosinolate in both cultivars, but less efficient as a precursor of 2-hydroxy-3-butenylglucosinolate in Bronowski. These results suggest that there is a block in the biosynthesis of 3-butenylglucosinolate in Bronowski that is situated before the synthesis of the intermediate 5-methylthiopentanal oxime, as well as a block in the hydroxylation step. The silique walls of Bronowski contained increased amounts of total sulphur and inorganic sulphate.     

Intergeneric Somatic Hybridization Between Brassica napus L. And Sinapis alba L.

Electrically induced protoplast fusion was used to produce somatic hybrids between Brassica napus L. and Sinapis alba L. Seven hybrids were obtained and verified by the simple sequence repeat and cleaved amplified polymorphic sequence analysis of the genefael, indicating that the characteristic bands from S. alba were present in the hybrids. The hybridity was also confirmed by chromosome number counting because the hybrids possessed 62 chromosomes, corresponding to the sum of fusion-parent chromosomes. Chromosome pairing at meiosis was predominantly normal, which led to high pollen fertility,ranging from 66% to 77%. All hybrids were grown to full maturity and could be fertilized and set seed after self-pollination or back-crosses with B. napus. The morphology of the hybrids resembled characteristics from both parental species. An analysis of the fatty acid composition in the seeds of F1 plants was conducted and the seeds were found to contain different amounts of erucic acid, ranging from 11.0% to 52.1%.     

Synthesis and phosphorylation of pollen proteins during the pollen-stigma interaction in self-compatible Brassica napus L. and self-incompatible Brassica oleracea L.

A technique is described which permits the in vivo study of protein synthesis and phosphorylation in the pollen of Brassica spp. during the early stages of the pollen-stigma interaction. In Brassica napus and B. oleracea, compatible pollination is followed by a dramatic activation of protein synthesis in the pollen involving the synthesis of approximately 40 proteins. After incompatible pollinations in B. oleracea, virtually no newly synthesised polypeptides were detected in the pollen except for a small group of high molecular weight proteins which were not normally synthesised during compatible pollinations. Both compatible and incompatible pollinations were followed by the appearance of newly phosphorylated proteins in the pollen; these fell into four distinct groups. In B. oleracea, the number of phosphorylated proteins and the degree of phosphorylation of individual proteins within the four groups differed between compatible and incompatible pollinations. One group of phosphorylated proteins appeared to correspond with the small group of high molecular weight polypeptides which were synthesised in pollen after incompatible pollinations. These findings are discussed in the perspective of cell signalling during the pollen-stigma interaction in Brassica and also in terms of their possible implication in sporophytic self-incompatibility.     

甘蓝型油菜高效离体再生体系的建立

以甘蓝型油菜(Brassica napusL.)HC8为材料,从无菌苗苗龄、预培养基激素浓度、预培养天数、6-BA及NAA的浓度等方面对影响油菜组织培养的因素进行了分析研究,建立了甘蓝型油菜品系HC8的离体再生技术体系。结果表明,该油菜组织培养的最佳苗龄为5 d;最佳预培养时间为5 d,最佳2,4-D浓度为1.0 mg/L;子叶最佳诱导培养基为MS+2.0 mg/L 6-BA+0.05 mg/LNAA+3.5 mg/L AgNO3或MS+3.0 mg/L 6-BA+0.1 mg/L NAA+3.5 mg/L AgNO3,该条件下子叶愈伤组织诱导率最高可达100%,再生频率及分化频率分别可达88.0%和108.33%;下胚轴最佳诱导培养基为MS+4.0 mg/L 6-BA+0.05 mg/L NAA+3.5 mg/L AgNO3,子叶愈伤组织诱导率最高可达95.24%,再生频率及分化频率分别可达81.82%和104.55%;最佳生根培养基为MS+0.5 mg/L NAA,生根率最高为90.0%。     

Transfer and segregation of triazine tolerant chloroplasts in Brassica napus L.

Summary Hypocotyl protoplasts of 45 different genotypes of German winter oilseed rape Brassica napus L. (double zero quality: high in yield, seeds low in erucic acid and glucosinolate content) were regenerated to plants. Triazine/triazinone (tri)-tolerant chloroplasts of the Canadian spring oilseed rape variety OAC Triton were introduced into some winter oilseed rapes by means of protoplast fusion. X-ray irradiation was used to limit the transfer of nuclear DNA of Triton protoplasts and to promote the selective transfer of tri-tolerant chloroplasts. Regenerated cybrid plants survived a treatment rate of 1000 g/ha metribuzin. The presence and segregation of the tri-tolerant chloroplasts in winter oilseed rape plants, regenerated from fusion products and their progeny, was investigated by restriction fragment length polymorphism (RFLP). Our results indicate that chloroplast segregation was not completed in plants regnerated from fusion products derived from X-irradiated OAC Triton mesophyll protoplasts and German winter oilseed rape hypocotyl protoplasts. In regenerants and their progeny both chloroplast types can still be present. Chloroplasts derived from wintertype protoplasts can outcompete tritolerant chloroplasts during plant development. In some instances, even progeny plants not kept under selective conditions (metribuzin) lost tri-tolerant chloroplasts. A homogenous population of tri-tolerant chloroplasts was necessary to obtain stable tri-tolerant winter oilseed rape plants.     

Triacylglycerol Bioassembly in Microspore-Derived Embryos of Brassica napus L. cv Reston

Erucic acid (22:1) was chosen as a marker to study triacylglycerol (TAG) biosynthesis in a Brassica napus L. cv Reston microspore-derived (MD) embryo culture system. TAGs accumulating during embryo development exhibited changes in acyl composition similar to those observed in developing zygotic embryos of the same cv, particularly with respect to erucic and eicosenoic acids. However, MD embryos showed a much higher rate of incorporation of ¹⁴C-erucoyl moieties into TAGs in vitro than zygotic embryos. Homogenates of early-late cotyledonary stage MD embryos (14-29 days in culture) were assessed for the ability to incorporate 22:1 and 18:1 (oleoyl) moieties into glycerolipids. In the presence of [1-¹⁴C]22:1-coenzyme A (CoA) and various acyl acceptors, including glycerol-3-phosphate (G-3-P), radiolabeled erucoyl moieties were rapidly incorporated into the TAG fraction, but virtually excluded from other Kennedy Pathway intermediates as well as complex polar lipids. This pattern of erucoyl incorporation was unchanged during time course experiments or upon incubation of homogenates with chemicals known to inhibit Kennedy Pathway enzymes. In marked contrast, parallel experiments conducted using [1-¹⁴C]18:1-CoA and G-3-P indicated that ¹⁴C oleoyl moieties were incorporated into lyso-phosphatidic acids, phosphatidic acids, diacylglycerols, and TAGs of the Kennedy Pathway, as well as other complex polar lipids, such as phosphatidylcholines and phosphatidylethanolamines. When supplied with l-[2-³H(N)]G-3-P and [1-¹⁴C]22:1-CoA, the radiolabeled TAG pool contained both isotopes, indicating G-3-P to be a true acceptor of erucoyl moieties. Radio-high-performance liquid chromatography, argentation thin-layer chromatography/gas chromatography-mass spectrometry, and stereospecific analyses of radiolabeled TAGs indicated that 22:1 was selectively incorporated into the sn-3 position by a highly active diacylglycerol acyltransferase (DGAT; EC 2.3.1.20), while oleoyl moieties were inserted into the sn-1 and sn-2 positions. In the presence of sn-1,2-dierucin and [1-¹⁴C]22:1-CoA, homogenates and microsomal preparations were able to produce radiolabeled trierucin, a TAG not found endogenously in this species. A 105,000g pellet fraction contained 22:1-CoA:DGAT exhibiting the highest specific activity. The rate of 22:1-CoA:DGAT activity in vitro could more than account for the maximal rate of TAG biosynthesis observed in vivo during embryo development. In double label experiments, G-3-P was shown to stimulate the conversion of [³H]phosphatidylcholines to [³H]diacylglycerols, which subsequently acted as acceptors for ¹⁴C erucoyl moieties. In vitro, 22:1 moieties did not enter the sn-1 position of TAGs by a postsynthetic modification or transacylation of preformed TAGs.     

Non-zygotic embryos of Brassica napus L. contain embryo-specific storage proteins

The storage-protein content of non-zygotic and zygotic embryos of B. napus was compared, using antibodies to guantitate 12S storage protein in extracts by rocket immunoelectrophoresis. Non-zygotic embryos were induced from microspores in anther culture and on the hypocotyls of zygotic embryos in culture. All embryo-like structures were found to contain 12S storage protein, whereas preculture anthers, anthers from which embryos had been removed, and regenerated shoots did not have detectable 12S storage protein. In zygotic embryos, 12S storage protein was first detected at the cotyledon stage, but microsporic embryos contained storage protein at the globular and heart stages. Storage protein levels in microsporic and hypocotyl embryos were low relative to those in zygotic embryos. The largest microsporic embryo had a storage protein concentration of 13 g mg^-1 fresh weight, almost 10 times lower than a mature zygotic embryo. Thus, although storage proteins are present in both zygotic and non-zygotic embryos, the timing and extent of accumulation differ.     

Heavy metal tolerant transgenic Brassica napus L. and Nicotiana tabacum L. plants

Summary A chimeric gene containing a cloned human metallothionein-II (MT-II) processed gene was introduced into Brassica napus and Nicotiana tabacum cells on a disarmed Ti-plasmid of Agrobacterium tumefaciens. Transformants expressed MT protein as a Mendelian trait and in a constitutive manner. Seeds from self-fertilized transgenic plants were germinated on media containing toxic levels of cadmium and scored for tolerance/ susceptibility to this heavy metal. The growth of root and shoot of transformed seedlings was unaffected by up to 100 M CdCl₂, whereas control seedlings showed severe inhibition of root and shoot growth and chlorosis of leaves. The results of these experiments indicate that agriculturally important plants such as B. napus can be genetically engineered for heavy metal tolerance/sequestration and eventually for partitioning of heavy metals in non-consumed plant tissues.     

Selection of Brassica napus L. embryogenic microspores by flow sorting

Flow cytometry can be used to select and sort microspore subpopulations of Brassica napus cv. Topas. Data obtained from embryogenic microspore populations were used to identify potentially embryogenic microspores from developmentally heterogeneous microspore populations based on differences in forward light scatter and green autofluorescence. Culture enrichment for embryogenic microspores is possible. Frequencies of 8 and 14% microspore embryogenesis were obtained when selected 16 h and 72 h after culture initiation. This represents 5- and 13-fold increase in microspore embryogenesis compared to non-sorted controls.     

Metabolic engineering of isoflavone genistein in Brassica napus with soybean isoflavone synthase

Genistein, 4′,5,7-trihydroxyisoflavone, is an isoflavonoid compound predominantly restricted to legumes and known to possess phyto-oestrogenic and antioxidative activities. The key enzyme that redirects phenylpropanoid pathway intermediates from flavonoids to isoflavonoids is the isoflavone synthase (IFS). Brassica napus is a non-legume oilseed crop with vegetative tissues producing phenylpropanoids and flavonoids, but does not naturally accumulate isoflavones due to the absence of IFS. To demonstrate whether exogenous IFS is able to use endogenous substrate to produce isoflavone genistein in oilseed crop, the soybean IFS gene (GmIFS2) was incorporated into B. napus plants. The presence of GmIFS2 in B. napus was shown to direct the synthesis and accumulation of genistein derivatives in leaves up to 0.72 mg g⁻¹ DW. In addition, expression levels for most B. napus genes in the phenylpropanoid pathway were altered. These results suggest that the heterologous GmIFS2 enzyme is functionally active at using the B. napus naringenin as a substrate to produce genistein in oilseed rape.     

Age-related Changes in the Ultrastructure and Membrane Properties of Brassica napus L. Seeds

The ultrastructure was studied of imbibed non-aged winter rape(Brassica napus L.) seeds in comparison with that of artificiallyaged seeds in which viability was partially or completely impaired.In parallel, measurements were made of lipid-phosphorus content,the leakage of phosphate from the seeds and their vigour andgerminability. Decreases in lipid-phosphorus which accompaniedthe loss of viability corresponded to an increase in phosphateleakage, suggesting damage to cellular membranes. Three ultrastructuralsymptoms possibly related to age-induced membrane deteriorationwere observed: (i) the lowering of electron contrast in allcellular membranes excluding plasmalemma; (ii) coalescence ofsmall storage lipid bodies to larger units presumably as a resultof the degradation of enclosing half-unit membranes; and (iii)the appearance of protoplasmic inclusions inside the storageprotein bodies, possibly resulting from rupture of the enclosingunit membranes. It is suggested that the presence of enlarged fibrillar centresin nucleoli of low viability seeds observed here for the firsttime in aged seed material may be the morphological manifestationof age-induced damage to nucleic acids. Brassica napus L, seeds, accelerated ageing, ultrastructure, leakage