Confirmation of QTL controlling seed yield in spring canola (Brassica napus L.) hybrids

Allelic effects observed in QTL discovery experiments must be confirmed to be useful in subsequent breeding efforts. Two QTL affecting seed yield of spring hybrid canola (Brassica napus L.) were previously identified in two populations of inbred backcross lines (IBLs) containing germplasm introgressed from a winter cultivar. The effects of favorable alleles at these QTL were retested by crossing two selected IBLs (M5 and M31) to three spring canola lines having different genetic backgrounds. Doubled haploid (DH) lines derived from each F₁ were genotyped with RFLP markers flanking the QTL and grouped into the four possible QTL genotypes. For the first field experiment, DH lines derived by crossing the M5 line to one spring line were crossed to two female testers and evaluated as individual testcross progenies in one environment. QTL genotypes had large variances and were not significantly different. A second field experiment was conducted using the DH lines from the first experiment and two other sets of DH lines derived from the M31 line crossed to two different spring canola lines. Individual lines within each QTL genotype of each set were bulked and crossed to the same testers used in Experiment 1. Bulked hybrid seeds of each QTL genotype were planted in a split-split plot randomized block design and 12 replicates. QTL genotypes had smaller variances in this experiment, and the effects of one QTL were confirmed in some genetic backgrounds. These results suggest that bulking of QTL genotypes and use of an appropriate experimental design with many replicates are needed to detect small differences between QTL genotypes.     

Optimization of Brassica napus (canola) explant regeneration for genetic transformation

Brassica napus (canola) is the second largest oilseed crop in the world. It is among the first crops to be genetically transformed, and genetically modified cultivars are in commercial production at very significant levels. Despite the early lead with respect to transgenesis, there remain cultivars that are recalcitrant to transformation. To address this, we have conducted an elaborate investigation of the conditions for regenerating shoots from hypocotyl explants from four genetic lines: Invigor 5020, Westar and Topas as well as a microspore culture derived line of Topas (Line 4079). We analyzed the effect of hormonal combinations in regeneration medium, donor plant age and explant type on the regeneration capacity of these plants. The analysis showed that hypocotyls of eight-day-old seedlings grown on media supplemented with 1mg/L dinitrophenylhydrazine (2,4-D) produced the most shoots. Globular somatic embryos emerged following two weeks of 2,4-D treatment. When transferred to the medium containing 5mg/L benzyladenine (BA), approximately 82% of embryos produced shoots within six weeks. Invigor plants were shown to regenerate more efficiently than Topas; the number of plantlets regenerated from Invigor was approximately 40-50% more as compared to Topas or Line 4079. When hypocotyl explants were co-cultivated with the Agrobacterium strain GV3101 harboring a binary vector carrying a firefly luciferase reporter gene (LUC), significant numbers of plantlets were LUC-positive in a luciferase assay. Frequency of such plants were: Invigor 5020 (54.2 ± 2.5%), Westar (53.7 ± 5.3), Topas (16.0 ± 0.24) and Line 4079 (13.4 ± 4).     

Root pruning and transplanting increase zinc requirements of canola (Brassica napus)

Previous field and glasshouse studies suggested that oilseed rape (Brassica napus L.) was especially sensitive to zinc (Zn) deficiency in the recovery period following transplanting. However, it is not clear whether transplanting, per se, or root damage during transplanting was primarily responsible. Three glasshouse experiments were carried out to test the hypothesis that transplanting increases external Zn requirement of canola cv. Hyola 42 during its post-transplanting recovery. Canola was either directly sown into Zn-treated soils or transplanted at four-leaf stage, and grown until harvest at 7- and 10-leaf stages. In a second experiment with chelate-buffered solution culture, direct-sown and transplanted plants were treated with three concentrations of Zn. In the third experiment, plants were given three levels of Zn supply, and either direct-sown into soils or transplanted at four-leaf stage with pruned (50% of roots removed) or unpruned root systems. Transplanted plants required higher soil Zn supply for maximum root length and root dry weight than direct-sown plants. By contrast, shoots required similarly low external Zn for maximum dry weight in both direct-sown and transplanted plants in soil. Direct-sown plants were more efficient in utilizing soil supplied Zn than transplanted plants particularly compared to those transplanted with a pruned root system, and achieved maximum growth at 100 μg Zn kg^?1 soil compared to 500 μg Zn kg^?1 required by transplanted plants. Since the higher external Zn requirement for the growth of transplanted plants was also obtained in well-stirred solution culture, it was concluded that it was related to the time required for transplanted plants to recover from root injury and re-establish a favourable shoot: root ratio rather than to rhizosphere modification processes. Both transplanting, per se, and root damage during transplanting appeared to contribute to higher external Zn requirements for canola growth compared to direct-sown plants.     

Study on salt tolerance with YHem1 transgenic canola (Brassica napus)

5‐Aminolevulinic acid (5‐ALA) has been suggested for improving plant salt tolerance via exogenous application. In this study, we used a transgenic canola (Brassica napus), which contained a constituted gene YHem1 and biosynthesized more 5‐ALA, to study salt stress responses. In a long‐term pot experiment, the transgenic plants produced higher yield under 200 mmol L^?1 NaCl treatment than the wild type (WT). In a short‐term experiment, the YHem1 transformation accelerated endogenous 5‐ALA metabolism, leading to more chlorophyll accumulation, higher diurnal photosynthetic rates and upregulated expression of the gene encoding Rubisco small subunit. Furthermore, the activities of antioxidant enzymes, including superoxide dismutase, guaiacol peroxidase, catalase and ascorbate peroxidase, were significantly higher in the transgenic plants than the WT, while the levels of O₂·^? and malondialdehyde were lower than the latter. Additionally, the Na⁺ content was higher in the transgenic leaves than that in the WT under salinity, but K⁺ and Cl^? were significantly lower. The levels of N, P, Cu, and S in the transgenic plants were also significantly lower than those in the WT, but the Fe content was significantly improved. As the leaf Fe content was decreased by salinity, it was suggested that the stronger salt tolerance of the transgenic plants was related to the higher Fe acquisition. Lastly, YHem1 transformation improved the leaf proline content, but salinity decreased rather than increased it. The content of free amino acids and soluble sugars was similarly decreased as salinity increased, but it was higher in the transgenic plants than that in the WT.     

Chlorophyllase and peroxidase activity during degreening of maturing canola (Brassica napus) and mustard (Brassica juncea) seed

Chlorophyllase and peroxidase activities were measured in relation to seed maturation and degreening in canola ( Brassica napus cvs Westar and Alto) and mustard ( Brassica juncea cvs Cutlass and Lethbridge 22A). Samples of seed collected at the same moisture content were pooled, then divided and used for each assay. During maturation the green pigment (chlorophyll and related pigments) content of canola seed decreased linearly and was lower than that measured in mustard at all moisture contents studied, except for the highest and lowest moisture contents. Chlorophyllides and pheophorbides were detected in canola and were essentially absent in mustard. This difference in accumulation of dephytylated pigments infers differences in the pigment degradation pathways in Brassica species. Interspecific differences in the enzymology of degreening were found. Green pigment degradation was associated with increased chlorophyllase activity and low peroxidase activity in canola and low Chlorophyllase and high perosidase activity in mustard. The possible role of ethylene in seed degreening is discussed.     

Protection of canola (Brassica napus) against fungal pathogens by strains of biocontrol rhizobacteria

Strains of Pseudomonas fluorescens and Bacillus amyloliquefaciens, isolated from soybean rhizosphere, inhibited mycelial growth of Botrytis cinerea and Sclerotinia sclerotiorum, in vitro. Leaves from Brassica napus seedlings, pre-inoculated with either of these bacteria, exhibited systemic protection against fungal pathogens.     

Distribution of glucosinolates and sulphur-rich cells in roots of field-grown canola (Brassica napus)

To investigate the role played by the distribution pattern of glucosinolates (GSLs) in root systems in the release of biocides to the rhizosphere, GSLs have been localized, for the first time, to specific regions and cells in field-grown roots. GSL concentrations in separated tissues of canola (Brassica napus) were determined by chemical analysis, and cell-specific concentrations by extrapolation from sulphur concentrations obtained by quantitative cryo-analytical scanning electron microscopy (SEM). In roots with secondary growth, GSL concentrations in the outer secondary tissues were up to 5x those of the inner core. The highest GSL concentrations (from sulphur measurements) were in two cell layers just under the outermost periderm layer, with up to 100x published concentrations for whole roots. Primary tissues had negligible GSL. Release and renewal of the peripheral GSLs is probably a normal developmental process as secondary thickening continues and surface cells senesce, accounting for published observations that intact roots release GSLs and their biocide hydrolosates to the rhizosphere. Absence of myrosin idioblasts close to the root surface suggests that GSLs released developmentally are hydrolysed by myrosinase in the rhizosphere, ensuring a continuous localized source of biotoxic hydrolysates which can deter soil-borne pests, and influence microbial populations associated with long-lived components of the root system.     

Stable expression of Phytase (phyA) in canola (Brassica napus) seeds: towards a commercial product

TheAspergillus niger gene encoding phytase(phyA) was expressed in canola (Brassicanapus). Phytase expression is controlled by the seed-specificcruciferin (CruA) promoter. Secretion of the enzyme was aimed for byincorporating the cruciferin signal peptide in the expression construct.Transgenic canola lines were generated by Agrobacteriummediated transformation using nptII as the selectable marker. Ninety-fiveindependent transgenic events were generated. Phytase expression in the T1seedsranged from 0 to 600 U/g seed. Single-copy lines were selected(based on segregation for kanamycin resistance, phytase expression and Southernanalyses) from originally multi-copy transgenic lines. Phytase was expressed inthese sub-lines up to 103 U/g. Expression levels were monitoredthrough an additional 3–4 generations (in the greenhouse and in thefield)and the accumulation of phytase appeared to be fairly stable. In the expressionrange studied, phytase expression was gene-dosage dependent.     

Peroxidase-mediated chlorophyll bleaching in degreening canola (Brassica napus) seeds and its inhibition by sublethal freezing

Peroxidase activity in isolated thylakoids from degreening canola ( Brassica napus cv. Westar) seeds was demonstrated. The enzyme catalyzes the degradation of thylakoid-bound pigments in the presence of H₂O₂ and 2,4-dichlorophenol. Peroxidase activity is related to degreening, with periods of rapid degreening associated with high enzyme activity. Both de novo synthesis and substrate availability appear to control enzyme activity. Peroxidase is initially inhibited and then stimulated by sublethal freezing. Therefore, inhibition of peroxidase activity following sublethal freezing may be responsible, in part, for a failure of the seed to degreen.     

Seasonal changes in the rhizosphere microbial communities associated with field-grown genetically modified canola (Brassica napus)

The introduction of transgenic plants into agricultural ecosystems has raised the question of the ecological impact of these plants on nontarget organisms, such as soil bacteria. Although differences in both the genetic structure and the metabolic function of the microbial communities associated with some transgenic plant lines have been established, it remains to be seen whether these differences have an ecological impact on the soil microbial communities. We conducted a 2-year, multiple-site field study in which rhizosphere samples associated with a transgenic canola variety and a conventional canola variety were sampled at six times throughout the growing season. The objectives of this study were to identify differences between the rhizosphere microbial community associated with the transgenic plants and the rhizosphere microbial community associated with the conventional canola plants and to determine whether the differences were permanent or depended on the presence of the plant. Community-level physiological profiles, fatty acid methyl ester profiles, and terminal amplified ribosomal DNA restriction analysis profiles of rhizosphere microbial communities were compared to the profiles of the microbial community associated with an unplanted, fallow field plot. Principal-component analysis showed that there was variation in the microbial community associated with both canola variety and growth season. Importantly, while differences between the microbial communities associated with the transgenic plant variety were observed at several times throughout the growing season, all analyses indicated that when the microbial communities were assessed after winter, there were no differences between microbial communities from field plots that contained harvested transgenic canola plants and microbial communities from field plots that did not contain plants during the field season. Hence, the changes in the microbial community structure associated with genetically modified plants were temporary and did not persist into the next field season.     

A simple method to estimate the percentage of hybridity in canola (Brassica napus) F1 hybrids

We have developed an efficient PCR-based system that uses RAPD markers for the certification of F₁ hybrids of canola. These markers were selected by screening five parental lines used in three crosses X, Y and Z with 131, 131 and 322 primers respectively. Stable DNA fragments that were homozygous and specific to the male inbreds were used to certify F₁ hybrid populations. The hybrid production system was based on self-incompatibility (SI) alleles that prevent self-pollination of the female parent. The efficiency of two S-alleles was compared under both field and greenhouse conditions. The percentage of hybridity was estimated in different F₁ populations. We found a significant difference between the two alleles for their efficiency in controlling selfing; both alleles were stable under greenhouse conditions, one allele appeared less reliable under field conditions.     

The role of chlorophyllase in degreening canola (Brassica napus) seeds and its activation by sublethal freezing

Chlorophyllase mediates dephytylation of chlorophylls and pheophytins during seed degreening in canola ( Brassica napus cv. Westar). Degreening can be correlated with chlorophyllase activity in vitro, but it is difficult to demonstrate in vivo activity because of low levels of the dephytylated breakdown products during rapid degreening. If, however, degreening is inhibited by sublethal freezing, chlorophyllide and pheophorbide accumulation can be related to the action of chlorophyllase. Changes in the rate of in vitro dephytylation during degreening and the dramatic increase following freezing may indicate enzyme activation and de novo enzyme synthesis. Evidence from Western blots is presented in support of de novo synthesis. It is concluded that failure of the seed to degreen following sublethal freezing does not result from a reduction in chlorophyllase activity.     

Plasmodiophora brassicae: a review of an emerging pathogen of the Canadian canola (Brassica napus) crop

Plasmodiophora brassicae causes clubroot disease in cruciferous plants, and is an emerging threat to Canadian canola (Brassica napus) production. This review focuses on recent studies into the pathogenic diversity of P. brassicae populations, mechanisms of pathogenesis and resistance, and the development of diagnostic tests for pathogen detection and quantification. TAXONOMY: Plasmodiophora brassicae is a soil-borne, obligate parasite within the class Phytomyxea (plasmodiophorids) of the protist supergroup Rhizaria. DISEASE SYMPTOMS: Clubroot development is characterized by the formation of club-shaped galls on the roots of affected plants. Above-ground symptoms include wilting, stunting, yellowing and premature senescence. DISEASE CYCLE: Plasmodiophora brassicae first infects the root hairs, producing motile zoospores that invade the cortical tissue. Secondary plasmodia form within the root cortex and, by triggering the expression of genes involved in the production of auxins, cytokinins and other plant growth regulators, divert a substantial proportion of plant resources into hypertrophic growth of the root tissues, resulting in the formation of galls. The secondary plasmodia are cleaved into millions of resting spores and the root galls quickly disintegrate, releasing long-lived resting spores into the soil. A serine protease, PRO1, has been shown to trigger resting spore germination. PHYSIOLOGICAL SPECIALIZATION: Physiological specialization occurs in populations of P. brassicae, and various host differential sets, consisting of different collections of Brassica genotypes, are used to distinguish among pathotypes of the parasite. DETECTION AND QUANTIFICATION: As P. brassicae cannot be cultured, bioassays with bait plants were traditionally used to detect the pathogen in the soil. More recent innovations for the detection and quantification of P. brassicae include the use of antibodies, quantitative polymerase chain reaction (qPCR) and qPCR in conjunction with signature fatty acid analysis, all of which are more sensitive than bioassays. RESISTANCE IN CANOLA: Clubroot-resistant canola hybrids, recently introduced into the Canadian market, represent an important new tool for clubroot management in this crop. Genetic resistance must be carefully managed, however, as it has been quickly overcome in other regions. At least three resistance genes and one or two quantitative trait loci are involved in conferring resistance to P. brassicae. Root hair infection still occurs in resistant cultivars, but secondary plasmodia often remain immature and unable to produce resting spores. Fewer cell wall breakages occur in resistant hosts, and spread of the plasmodium through cortical tissue is restricted. More information on the genetics of clubroot resistance in canola is needed to ensure more effective resistance stewardship. USEFUL WEBSITES: http://www.canolacouncil.org/clubroot/resources.aspx, http://tu-dresden.de/die_tu_dresden/fakultaeten/fakultaet_mathematik_und_naturwissenschaften/fachrichtung_biologie/botanik/pflanzenphysiologie/clubroot, http://www.ohio.edu/people/braselto/plasmos/     

Differential sensitivity of canola (Brassica napus) seedlings to ultraviolet-B radiation,water stress and abscisic acid

Responses of canola (Brassica napus L.) seedlings to three ultraviolet (UV)-B levels [0 (zero), 5 (ambient) and 10 (enhanced) kJ m^?2 d^?1], two watering regimes (well-watered and water-stressed), and two abscisic acid (ABA) levels (with and without application) were investigated. Overall, enhanced UVB and water stress negatively affected plant growth and physiology, but ABA had very little effect. Enhanced UVB decreased stem height, leaf area, plant dry matter, water use efficiency and wax content, but increased concentrations of chlorophyll a, carotenoids and flavonoids, and ethylene evolution. Water stress reduced stem height and diameter, leaf area, plant dry matter, leaf weight ratio and shoot:root weight ratio under zero and ambient UVB. Water stress also reduced chlorophyll a and carotenoids in plants exposed to enhanced UVB. ABA with watering regime had significant interactive effects only on leaf dry matter and wax content. We found that enhanced UVB and water stress adversely affected B. napus seedlings. Interaction between these two factors affected plant performance. In this interaction, ABA had little significant role. Also, optimum vegetative growth and biomass were achieved under ambient UVB.     

Genetic and physical mapping of flowering time loci in canola (Brassica napus L.)

We identified quantitative trait loci (QTL) underlying variation for flowering time in a doubled haploid (DH) population of vernalisation—responsive canola (Brassica napus L.) cultivars Skipton and Ag-Spectrum and aligned them with physical map positions of predicted flowering genes from the Brassica rapa genome. Significant genetic variation in flowering time and response to vernalisation were observed among the DH lines from Skipton/Ag-Spectrum. A molecular linkage map was generated comprising 674 simple sequence repeat, sequence-related amplified polymorphism, sequence characterised amplified region, Diversity Array Technology, and candidate gene based markers loci. QTL analysis indicated that flowering time is a complex trait and is controlled by at least 20 loci, localised on ten different chromosomes. These loci each accounted for between 2.4 and 28.6 % of the total genotypic variation for first flowering and response to vernalisation. However, identification of consistent QTL was found to be dependant upon growing environments. We compared the locations of QTL with the physical positions of predicted flowering time genes located on the sequenced genome of B. rapa. Some QTL associated with flowering time on A02, A03, A07, and C06 may represent homologues of known flowering time genes in Arabidopsis; VERNALISATION INSENSITIVE 3, APETALA1, CAULIFLOWER, FLOWERING LOCUS C, FLOWERING LOCUS T, CURLY LEAF, SHORT VEGETATIVE PHASE, GA3 OXIDASE, and LEAFY. Identification of the chromosomal location and effect of the genes influencing flowering time may hasten the development of canola varieties having an optimal time for flowering in target environments such as for low rainfall areas, via marker-assisted selection.     

Chelate-assisted phytoextraction using canola (Brassica napus L.) in outdoors pot and lysimeter experiments

Phytoextraction is an emerging technology for non-destructive remediation of heavy metal-polluted soils. This study was conducted to test chelate-assisted phytoextraction of Cu, Pb and Zn using EDTA and canola (Brassica napus L. cv. Petranova) on a moderately polluted industrial soil (loamy sand) in the sub-continental climate of Eastern Austria. The effects of the rate (up to 2.1 g kg^–1 soil) and mode (single versus split) of EDTA application on the biomass, water contents and metal concentrations in shoots and roots were investigated along with changes of metal lability in soil and leaching from the root zone in parallel outdoors pot and lysimeter experiments. Labile (1 M NH₄NO₃-extractable) metal concentrations in soil increased considerably upon application of EDTA, indicating enhanced phytoavailability. However, this was also associated with enormously increased metal concentrations in the leachates collected below the root zone. Enhanced metal labilities and leachate concentrations persisted for more than 1 year after harvest. Metal lability was more enhanced by EDTA in rhizosphere relative to bulk soil, indicating interactions of EDTA with root activities. Shoot biomass and water contents of canola were virtually unaffected by EDTA, revealing that canola can tolerate excessive metal concentrations in soil pore water. Metal concentrations in shoots were increased considerably, but were insufficient to obtain reasonable extraction rates. Split applications were generally more effective than the same amounts of EDTA added at once. Metal concentrations in roots decreased after each application of EDTA, possibly indicating metal removal from roots by free protonated EDTA, but increased again within several days. As the application of chelate-assisted phytoextraction is limited by the risk of groundwater pollution, further work should focus on natural, continuous phytoextraction technologies.     

Dynamics of infection by Leptosphaeria maculans on canola (Brassica napus) as influenced by crop rotation and tillage

Abstract

This study, intending to understand the effects of crop rotation and tillage on blackleg disease, was conducted in a field at Carman, Manitoba, Canada, from 1999 – 2002. Canola, wheat and flax were among the rotated crops. Rotations were performed under conventional or zero-till conditions. The number of infected plants, infected leaves per plant, lesions per plant, and percentage of leaf coverage with lesions decreased when canola was rotated with wheat and flax under zero till. The number of lesions per plant and percentage of leaf coverage with lesions were strongly correlated with stem disease severity, and the number of infected plants with stem disease incidence. Ascospores and pycnidiospores of Leptosphaeria maculans were reduced by crop rotation and tillage. This study suggests that the appropriate combination of rotation and tillage may lower airborne inoculum and reduce infection of canola plants by L. maculans.     

Slow vacuolar channels in vacuoles from winter and spring varieties of rape (Brassica napus)

Currents passing through slowly activating vacuolar channels (SV) in isolated vacuoles from winter (Górczański) and spring (M?ochowski) varieties of rape (Brassica napus) were examined using the patch-clamp technique. Eight-week-long vernalization at 5/2 degrees C (day/night) was applied to obtain the generative stage of winter rape. SV channels of vacuoles isolated from vegetative (rosette) and generative leaves of both varieties were examined in order to investigate a possible role of these ion channels in rape flowering. Single SV channel conductance measured in a vacuole-attached configuration (natural cell sap) ranged from 60 to 83 pS. Lower values were observed in the generative leaves of both varieties. Unitary conductance measured in excised cytoplasm-out membrane patches did not differ significantly among the experimental variants, with the exception of spring generative vacuoles, where it was significantly lower. There was also no difference in SV current densities measured in the whole-vacuole configuration. Gibberellic acid (GA(3)) (2mg/l) caused lowering of macroscopic SV currents by 20%, and had no significant effect on the single channel conductance. We conclude that SV channels play a role in rape vernalization and flowering owing to their multifactor regulation abilities rather than structural changes.