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A competitive polymerase chain reaction to quantify DNA of Leptosphaeria maculans during blackleg development in oilseed rape.

An assay based on the competitive polymerase chain reaction technique was developed to quantify Leptosphaeria maculans during blackleg disease development in oilseed rape leaves. By means of primers specific to the highly virulent type of L. maculans, a heterologous internal control template was prepared by amplifying and cloning DNA from Leptosphaeria korrae under low-stringency annealing conditions. Coamplification of L. maculans with the internal control DNA provided accurate quantification of 1 to 10(9) copies of target DNA. The assay was applied to a comparative study of L. maculans colonization of resistant and susceptible rape cultivars. The assay revealed that lesion size was associated with the quantity of L. maculans DNA during the first 12 days after inoculation of the susceptible cultivar Westar and the moderately resistant cultivar Legend. In these cultivars, the quantity of DNA per lesion increased during the first 12 days after inoculation and then declined. This decline in detectable fungal DNA coincided with abundant sporulation, rapid necrosis, and the onset of leaf senescence. Trace amounts of L. maculans DNA were detected in the resistant cultivar Glacier, in which lesion size was similar to that in the wounded, uninoculated check. The assay is rapid, accurate, and very sensitive and can be incorporated into conventional disease screening programs.

Ascomycota↗

Complex formation of myrosinase isoenzymes in oilseed rape seeds are dependent on the presence of myrosinase-binding proteins.

The enzyme myrosinase (EC 3.2.3.1) degrades the secondary compounds glucosinolates upon wounding and serves as a defense to generalist pests in Capparales. Certain myrosinases are present in complexes together with other proteins such as myrosinase-binding proteins (MBP) in extracts of oilseed rape (Brassica napus) seeds. Immunhistochemical analysis of wild-type seeds showed that MBPs were present in most cells but not in the myrosin cells, indicating that the complex formation observed in extracts is initiated upon tissue disruption. To study the role of MBP in complex formation and defense, oilseed rape antisense plants lacking the seed MBPs were produced. Western blotting and immunohistochemical staining confirmed depletion of MBP in the transgenic seeds. The exclusive expression of myrosinase in idioblasts (myrosin cells) of the seed was not affected by the down-regulation of MBP. Using size-exclusion chromatography, we have shown that myrosinases with subunit molecular masses of 62 to 70 kD were present as free dimers from the antisense seed extract, whereas in the wild type, they formed complexes. In accordance with this, MBPs are necessary for myrosinase complex formation of the 62- to 70-kD myrosinases. The product formed from sinalbin hydrolysis by myrosinase was the same whether MBP was present or not. The performance of a common beetle generalist (Tenebrio molitor) fed with seeds, herbivory by flea beetles (Phyllotreta undulata) on cotyledons, or growth rate of the Brassica fungal pathogens Alternaria brassicae or Lepthosphaeria maculans in the presence of seed extracts were not affected by the down-regulation of MBP, leaving the physiological function of this protein family open.

Animals↗

Identification of Catabolites of Chlorophyll-Porphyrin in Senescent Rape Cotyledons.

Developing shoots of rape seedlings (Brassica napus L.) were excised and fed with 4-[14C]5-aminolevulinic acid to label the pyrroles in chlorophyll (Chl) synthesized during the final phase of expansion and greening of the cotyledons. About 80% of 14C taken up into the cotyledons was incorporated into Chl. The subsequent incubation of labeled shoots in permanent darkness caused the rapid loss of labeled Chl while increasing proportions of 14C appeared in the fraction of water-soluble compounds. Reversed-phase high performance liquid chromatography resolved three nonfluorescent polar catabolites of Chl-porphyrin that were progressively accumulated as senescence advanced. At intermediate stages of senescence, the cotyledons contained a fluorescent radio-active derivative of Chl that was also detectable, together with traces of other putative fluorescent catabolites, in isolated senescent chloroplasts. The nonfluorescent catabolites, identified by means of radiolabeling, were also found to accumulate in attached cotyledons senescing under photoperiod; under these conditions, one of the compounds, NCC-1, was particularly abundant. The catabolites of rape exhibited the same ultraviolet spectra, characterized by a maximum at 320 nm, as a previously reported secoporphinoid catabolite from barley (B. Krautler, B. Jaun, W. Amrein, K. Bortlik, M. Schellenberg, P. Matile [1992] Plant Physiol Biochem 30: 333-346). Different polarities suggest, however, that the structures may be different. A terminology for Chl catabolites is proposed because present knowledge suggests that a large number of different structures results from species-specific processing of breakdown products and may require a suitable nomenclature.

Journal Article↗

Modeling nitrogen uptake in oilseed rape cv Capitol during a growth cycle using influx kinetics of root nitrate transport systems and field experimental data.

The use of kinetic equations of NO3- transport systems in oilseed rape (Brassica napus), determined by 15NO3- labeling under controlled conditions, combined with experimental field data from the INRA-Châlons rape database were used to model NO3- uptake during the plant growth cycle. The quantitative effects of different factors such as day/night cycle, ontogenetic stages, root temperature, photosynthetically active radiation, and soil nitrate availability on different components of the constitutive high-affinity transport systems, constitutive low-affinity transport systems, inducible low-affinity transport systems, and inducible high-affinity transport systems of nitrate were then determined to improve the model's predictions. Simulated uptake correlated well with measured values of nitrogen (N) uptake under field conditions for all N fertilization rates tested. Model outputs showed that the high-affinity transport system accounted for about 89% of total NO3- uptake (18% and 71% for constitutive high-affinity transport systems and inducible high-affinity transport systems, respectively) when no fertilizer was applied. The low-affinity transport system accounted for a minor proportion of total N uptake, and its activity was restricted to the early phase of the growth cycle. However, N fertilization in spring increased the duration of its contribution to total N uptake. Overall, data show that this mechanistic and environmentally regulated approach is a powerful means to simulate total N uptake in the field with the advantage of taking both physiologically regulated processes at the overall plant level and specific nitrate transport system characteristics into account.

Biological Transport, Active↗

Lipid storage metabolism is limited by the prevailing low oxygen concentrations within developing seeds of oilseed rape.

The aim of this study was to investigate whether endogenous restrictions in oxygen supply are limiting for storage metabolism in developing oilseed rape (Brassica napus) seeds. Siliques were studied 30 d after flowering, when rapid lipid accumulation is occurring in the seeds. (a). By using microsensors, oxygen concentrations were measured within seeds and in the silique space between seeds. At ambient external oxygen (21% [v/v]) in the light, oxygen fell to 17% (v/v) between and 0.8% (v/v) within seeds. A step-wise reduction of the external oxygen concentration led within 2 h to a further decrease of internal oxygen concentrations, and a step-wise increase of the external oxygen concentration up to 60% (v/v) resulted in an increase in internal oxygen that rose to 30% (v/v) between and 8% (v/v) within seeds. (b). The increase in oxygen levels in the seeds was accompanied by a progressive increase in the levels of ATP, UTP, and the ATP to ADP and UTP to UDP ratios over the entire range from 0% to 60% (v/v) external oxygen. (c). To investigate metabolic fluxes in planta, 14C-sucrose was injected into seeds, which remained otherwise intact within their siliques. The increase in oxygen in the seeds was accompanied by a progressive increase in the rate of lipid (including triacylglycerol), protein and cell wall synthesis, and an increase in glycolytic flux over a range from sub- to superambient oxygen concentrations. In contrast to lipid synthesis, starch synthesis was not significantly increased at superambient oxygen levels. The levels of fermentation products such as lactate and glycerol-3P increased only at very low (0%-4% [v/v]) external oxygen concentrations. (d). When 14C-acetate or 14C-acetyl-coenzyme A (CoA) was injected into seeds, label incorporation into triacylglycerol progressively increased over the whole range of external oxygen concentrations from 0% to 60% (v/v). (e). Stimulation of lipid synthesis was accompanied by an increase in sugar levels and a decrease in the levels of hexose-phosphates and acetyl-CoA, indicating sucrose unloading and the use of acetyl-CoA as possible regulatory sites. (f). Increased lipid synthesis was also accompanied by an increase in the maximal activities of invertase and diacylglycerol acyltransferase. (g). The developmental shift from starch to lipid storage between 15 and 45 d after flowering was accompanied by an increase in the seed energy state. (h). The results show that at ambient oxygen levels, the oxygen supply is strongly limiting for energy metabolism and biosynthetic fluxes in growing rape seeds, affecting lipid synthesis more strongly than starch synthesis. The underlying mechanisms and implications for strategies to increase yield and storage product composition in oilseed crops are discussed.

Acetates↗

Glucosinolate Biosynthesis (Further Characterization of the Aldoxime-Forming Microsomal Monooxygenases in Oilseed Rape Leaves).

The initial steps in glucosinolate biosynthesis are thought to proceed from amino acids, via N-hydroxy amino acids, to aldoximes. We showed previously that microsomes from green leaves of oilseed rape (Brassica napus cv Bienvenu) contain two distinct monooxygenases that catalyze the conversion of homophenylalanine and dihomomethionine to their respective aldoximes. Further characterization of these enzymes has now demonstrated that the latter enzyme catalyzes the NADPH-dependent oxidative decarboxylation of two higher homologs of methionine, in addition to dihomomethionine. No activity was found for either enzyme with L-methionine, DL-homomethionine, L-phenylalanine, L-tyrosine, or L-tryptophan. Both of these rape monooxygenase activities are dependent on O2, not requiring any other O2 species or radical. The presence of an unoxidized sulfur atom and its relative position in the side chain of the aliphatic substrates are important for binding to the active site of the methionine-homolog enzyme. Neither enzyme has any characteristics of a cytochrome P450-type enzyme, and antiserum raised against cytochrome P450 reductase did not significantly inhibit monooxygenase activity.

Journal Article↗

Regulation of apoplastic NH4+ concentration in leaves of oilseed rape

Regulation of apoplastic NH4+ concentration in leaves of oilseed rape (Brassica napus L.) was studied using a vacuum-infiltration technique that allowed controlled manipulations of the apoplastic solution. In leaves infiltrated with NH4+-free solution, the apoplastic NH4+ concentration returned in less than 1.5 min to the preinfiltration level of 0.8 mM. Infiltrated 15NH4+ was rapidly diluted by 14NH4+/14NH3 effluxed from the cell. The exchange rate of 15N/14N over the apoplast due to combined 14N efflux from the symplast and 15N influx from the apoplastic solution was 29.4 &mgr;mol g-1 fresh weight h-1 between 0 and 5 min after infiltration. The net uptake of NH4+ into the leaf cells increased linearly with apoplastic NH4+ concentrations between 2 and 10 mM and could be partially inhibited by the channel inhibitors La3+ and tetraethylammonium and by Na+ and K+. When apoplastic pH increased from 5.0 to 8.0, the steady-state apoplastic NH4+ concentration decreased from 1.0 to 0.3 mM. Increasing temperature increased the rate of NH4+ net uptake and reduced the apoplastic steady-state NH4+ concentration. We conclude that the apoplastic solution in leaves of oilseed rape constitutes a highly dynamic NH4+ pool.

Journal Article↗

The Biosynthesis of Some Isothiocyanates and Oxazolidinethiones in Rape (Brassica campestris L.).

The incorporation of the radioactivity from acetate-1-(14)C, acetate-2-(14)C, dl-methionine-1-(14)C, dl-methionine-2-(14)C, dl-methionine-3,4-(14)C, dl-homomethionine-2-(14)C, dl-allyl-glycine-2-(14)C, and dl-2-amino-5-hydroxyvalerate-2-(14)C into the aglycones of progoitrin, gluconapin, and glucobrassicanapin of maturing rape plants (Brassica campestris L.) was investigated. Radioactivity from dl-methionine-2-(14)C, dl-methionine-3,4-(14)C, dl-homomethionine-2-(14)C, and acetate-2-(14)C were incorporated into the 3 major thioglucosides. The other organic compounds were poorly incorporated except for dl-allylglycine-2-(14)C into glucobrassicanapin. The results obtained suggest that the rape plant can synthesize amino acids by the condensation of acetate (as acetyl CoA) to alpha-keto acids to yield a homologue of the original amino acid. These newly formed amino acids are then employed to synthesize the 3 major thioglucosides.

Journal Article↗

The role of phosphorus in aluminium-induced citrate and malate exudation from rape (Brassica napus).

Exudation of organic anions is believed to be a common tolerance mechanism for both aluminium toxicity and phosphorus deficiency. Nevertheless, which of these stresses that actually elicit the exudation of organic anions from rape (Brassica napus L) remains unknown, and the combined effects of Al toxicity and P deficiency on rape have not been reported before. Therefore, in the current study, Brassica napus var. Natane nourin plants grown with or without 0.25 mM P were exposed to 0 or 50 micro M AlCl(3) and several parameters related to the exudation of organic anions from the roots were investigated. Eight days of P deficiency resulted in a significant growth reduction, but P deficiency alone did not induce exudation of organic anions. In contrast, Al strongly induced organic acid exudation, while simultaneously inhibiting root growth. Increased in-vitro activity of citrate synthase (CS, EC 4.1.3.7), malate dehydrogenase (MDH, EC 1.1.1.37) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), together with reduced root respiration, indicated that the Al-induced accumulation and subsequent exudation of citrate and malate were associated with both increased biosynthesis and reduced metabolism of citric and malic acid. Phosphorus-sufficient plants showed more pronounced aluminium-induced accumulation and exudation of organic anions than P-deficient plants. A divided root chamber experiment showed the necessity of direct contact between Al and roots to elicit exudation of organic anions. Prolonged exposure (10 days) to Al resulted in a decrease in the net exudation of citrate and malate, and the rate of decrease was much more rapid in P-deficient plants than in P-sufficient plants. It is concluded that P nutrition affects the level of Al-induced synthesis and exudation of organic anions. However, the mechanism needs further investigation.

Journal Article↗

Impact of oilseed rape expressing the insecticidal serine protease inhibitor, mustard trypsin inhibitor-2 on the beneficial predator Pterostichus madidus.

Abstract Insect-resistant transgenic plants have been suggested to have deleterious effects on beneficial predators feeding on crop pests, through transmission of the transgene product by the pest to the predator. To test this hypothesis, effects of oilseed rape expressing the serine protease inhibitor, mustard trypsin inhibitor -2 (MTI-2), on the predatory ground beetle Pterostichus madidus were investigated, using diamondback moth, Plutella xylostella as the intermediary pest species. As expected, oilseed rape expressing MTI-2 had a deleterious effect on the development and survival of the pest. However, incomplete pest mortality resulted in survivors being available to predators at the next trophic level, and inhibition studies confirmed the presence of biologically active transgene product in pest larvae. Characterization of proteolytic digestive enzymes of P. madidus demonstrated that adults utilize serine proteases with trypsin-like and chymotrypsin-like specificities; the former activity was completely inhibited by MTI-2 in vitro. When P. madidus consumed prey reared on MTI-2 expressing plants over the reproductive period in their life cycle, no significant effects upon survival were observed as a result of exposure to the inhibitor. However, there was a short-term significant inhibition of weight gain in female beetles fed unlimited prey containing MTI-2, with a concomitant reduction of prey consumption. Biochemical analyses showed that the inhibitory effects of MTI-2 delivered via prey on gut proteolysis in the carabid decreased with time of exposure, possibly resulting from up-regulation of inhibitor-insensitive proteases. Of ecological significance, consumption of MTI-2 dosed prey had no detrimental effects on reproductive fitness of adult P. madidus.

Animals↗

Local and systemic changes in arginine decarboxylase activity, putrescine levels and putrescine catabolism in wounded oilseed rape.

* Here we report the effect of mechanical wounding on putrescine biosynthesis and catabolism in oilseed rape (Brassica napus ssp. oleifera). * The lamina of first leaves was wounded by crushing with forceps, and first and second leaves were harvested at various intervals over a 24 h period. Levels of free polyamines were measured and activities of enzymes of polyamine biosynthesis and catabolism were assayed in the harvested tissue. * Mechanical wounding of the first leaves led to significant, but transient, increases in arginine decarboxylase (ADC) activity and levels of free putrescine in the wounded first leaf and in unwounded second leaves. The increased putrescine appeared to be the result of a combination of increased ADC activity, coupled with reduced putrescine catabolism, as activity of the oxidative enzyme diamine oxidase was significantly reduced following wounding, both locally and systemically. * The role of the increased free putrescine in the wound response of oilseed rape is not known, although the possibility that it is used to form putrescine conjugates is worthy of further investigation.

Amine Oxidase (Copper-Containing)↗

Homosexual rape and sexual selection in acanthocephalan worms.

Acanthocephalan males have cement glands associated with the vas deferens, secretions from which seal the female vagina with a cap after copulation and so prevent subsequent insemination. Homosexual rape results in the male victim having the genital region sealed off with cement and effectively removed from the reproductive population. Sperm and cement are transferred to females during copulation, but apparently only cement is transferred to males during homosexual rape. Acanthocephalans conform to a parental investment model, and we interpret the evolution of the cement gland and sexual behavior as the result of sexual selection.

Acanthocephala↗

Mapping the mosaic of ancestral genotypes in a cultivar of oilseed rape (Brassica napus) selected via pedigree breeding.

Recent oilseed rape breeding has produced low glucosinolate cultivars that yield proteinaceous meal suitable for animal feed. The low glucosinolate character was introduced into modern cultivars from Brassica napus 'Bronowski', a cultivar that is agronomically inferior in most other respects. Residual segments of 'Bronowski' genotype in modern cultivars probably cause reduced yield, poorer winter hardiness, and lower oil content. The quantity and distribution of the 'Bronowski' genotype in the modern oilseed rape cultivar Brassica napus 'Tapidor' was investigated using a segregating population derived from a cross between 'Tapidor' and its high glucosinolate progenitor. This population was analyzed with 65 informative Brassica RFLP probes and a genetic linkage map, based on the segregation at 77 polymorphic loci, was constructed. The mapping identified 15 residual segments of donor genotype in 'Tapidor', which together occupy approximately 29% of the B. napus genome. Mapping the loci that control variation for the accumulation of total seed glucosinolates in the segregating population has identified three loci that together explain >90% of the variation for this character. All of these loci are in donor segments of the 'Tapidor' genome. This result shows the extent to which conventional breeding programmes have difficulty in eliminating residual segments of donor genotype from elite material.

Agriculture↗

Response of spring rape (Brassica napus var. oleifera L.) to inoculation with plant growth promoting rhizobacteria containing 1-aminocyclopropane-1-carboxylate deaminase depends on nutrient status of the plant.

Responses of rape (Brassica napus var. oleifera L.) to inoculation with plant growth promoting rhizobacteria, Pseudomonas putida Am2, Pseudomonas putida Bm3, Alcaligenes xylosoxidans Cm4, and Pseudomonas sp. Dp2, containing 1-aminocyclopropane-l-carboxylate (ACC) deaminase were studied using growth pouch and soil cultures. In growth pouch culture, the bacteria significantly increased root elongation of phosphorus-sufficient seedlings, whereas root elongation of phosphorus-deficient seedlings was not affected or was even inhibited by the bacteria. Bacterial stimulation of root elongation of phosphorus-sufficient seedlings was eliminated in the presence of a high ammonia concentration (1 mM) in the nutrient solution. Bacterial effects on root elongation of potassium-deficient and potassium-sufficient seedlings were similar. The bacteria also decreased inorganic phosphate content in shoots of potassium- and phosphorus-sufficient seedlings, reduced ethylene production by phosphorus-sufficient seedlings, and inhibited development of root hairs. The effects of treatment with Ag+, a chemical inhibitor of plant ethylene production, on root elongation, ethylene evolution, and root hair formation were similar to bacterial treatments. The number of bacteria on the roots of phosphorus-deficient seedlings was not limited by phosphorus deficiency. In pot experiments with soil culture, inoculation of seeds with bacteria and treatment with aminoethoxyvinylglycine, an inhibitor of ethylene biosynthesis in plants, increased root and (or) shoot biomass of rape plants. Stimulation of plant growth caused by the bacteria was often associated with a decrease in the content of nutrients, such as P, K, S, Mo, and Ba, in shoots, depending on the strain used. The results obtained show that the growth-promoting effects of ACC-utilizing rhizobacteria depend significantly on the nutrient status of the plant.

Alcaligenes↗

Identification of allergens in oilseed rape (Brassica napus) pollen.

BACKGROUND: Pollen from oilseed rape (OSR), Brassica napus, an increasingly cultivated oilplant from the Brassicaceae, has been recognized as a potential cause of allergic sensitization. Allergens have been hardly investigated. METHODS: We characterized IgE binding proteins in OSR pollen by immunoblot, immunoblot inhibition and specific monoclonal antibodies using sera from 89 patients sensitized to OSR. RESULTS: Two low-molecular-weight allergens of 6/8 kD and 14 kD as well as a high molecular-weight cluster (27-69 kD) comprising six cross-reactive peptides could be identified. The three allergens were recognized by 50, 34 and 80% of patients, respectively. Immunoblot IgE binding to OSR could be totally inhibited by rye pollen and moderately by birch pollen (6/8 and 14 kD) while mugwort had little effect. An anti-profilin-specific monoclonal antibody bound specifically to a 14-kD protein in OSR. Binding to the 6/8-kD rape allergen could be effectively inhibited by rAln g 2, a calcium-binding protein from alder. Periodate treatment led to a significant reduction in IgE binding to the 27 to 69-kD OSR allergens indicating that carbohydrate determinants are involved in IgE binding. OSR proteins were capable to quench IgE binding to timothy grass pollen proteins of >/=60 kD suggesting that grass pollen group 4 allergens cross-react with the 27 to 69-kD cluster in OSR. CONCLUSIONS: The data demonstrate that OSR pollen is allergenic and indicate that the identified allergens represent cross-reacting homologues of well-known pollen allergens, i.e. calcium-binding proteins, profilins, and high-molecular-weight glycoproteins. Via cross-reactivity, exposure to OSR pollen may be a prolonging and aggravating factor in underlying birch and grass pollen allergy.

Allergens↗

Identification of a Ca2+ binding protein as a new Bermuda grass pollen allergen Cyn d 7: IgE cross-reactivity with oilseed rape pollen allergen Bra r 1.

cDNA clones encoding two isoforms of an allergen from pollen of Bermuda grass (Cynodon dactylon) have been isolated using IgE from allergic patients. Homologous transcripts are present in pollen of 15 other grasses tested. This allergen, tentatively designated as Cyn d 7, contains two calcium binding domains and shows significant sequence similarity with other Ca2+ binding pollen allergens, namely Bet v 4 from birch and Bra r 1 from oilseed rape. Approximately 10% of allergic sera tested showed IgE reactivity to this allergen. IgE cross-reactivity was observed between this allergen and Bra r 1 of oilseed rape. IgE reactivity of this allergen requires protein-bound Ca2+. Using IgE affinity-purified from the recombinant allergen to probe Western blots of pollen extracts Cyn d 7 has been identified as a 12 kDA protein.

Allergens↗

The effect of electric field on callus induction with rape hypocotyls.

The influence of electric field treatment on dedifferentiation and calli formation on rape hypocotyls was investigated. Segments, 10 mm long, of the upper part of rape (Brassica napus L., cv. Góczański) hypocotyls were stimulated by different combinations of voltage/time (1.5 V/120 h, 3 V/3 h, 10 V/15 min and 30 V/30 s) under in vitro conditions. With all electric field treatments, segments oriented with their apical part towards the cathode produced more calli as compared to control (non-treated with electric field). Under opposite orientation slight inhibition of callus growth was observed. As the strongest effect on callus growth was observed after treatment with 30 V/30 s, this electric field treatment was selected for following analyses: the incorporation of [14C]-2,4-D (2,4-dichlorophenoxyacetic acid) and [14C]-BAP (benzylaminopurine) from the culture medium, changes in ACC (1-aminocyclopropane-1-carboxylic acid) level and the redox activity in apical and bottom parts of hypocotyls during 18 d of culture. In contrast to changes in fresh weight, electric field treatment (30 V/30 s) stimulated a higher accumulation of 2,4-D and BAP in basal parts of hypocotyls than in apical ones. Moreover, orienting the apical part towards the cathode resulted in lower uptake of hormones as compared with the opposite orientation. The ACC concentration increased, especially in the basal parts of hypocotyls, independently on electric field application. However, the highest level was observed after electric field treatment with orientation of the apical part towards the anode. The distribution of oxidative substances (measured as the amount of ferric ions) between the apical and bottom part of hypocotyls was not changed when the apical parts were oriented towards the cathode. Under these conditions a decrease in apical and an increase in basal parts was observed during culture. Opposite orientation influenced the redistribution of oxidative substances from the first day of electric field treatment. Based on these results we suggest that electric field action can be connected with its influence on specific concentration of oxidative substances and hormone distribution in cells.

Brassica napus↗

[Studies on transgenic oilseed rape (Brassica napus) plants transformed with beta-1,3-glucanase and chitinase genes and its resistance to Sclerotinia sclerotiorium].

By Agrobacterium-mediated method, the cotyledonary petiole of good quality rape variety H165 was transformed with plant expression vector pBLGC which constitutively express beta-1,3-glucanase and chitinase genes. We obtained some Kanamycin(Kan)-resistant regenerational green shoots, with these shoots, PCR identification was conducted. Results showed that 30% green shoots which grew in medium of Kan 15 mg/L and 53% green shoots in Kan 25 mg/L had positive reaction. We also made dot blot analysis with those green shoots, some of them gave positive signal, indicating that the foreign genes had integrated into rape genome. Fungal challenge of these transgenic plants showed that some plants were much more resistant to Sclerotinia sclerotiorium than non-transgenic control plants.

Ascomycota↗