CORRELATION BETWEEN BREAKDOWN PRODUCTS OF GLUCOSINOLATES AND THE AMOUNT OF RAPESEED MEAL INTRODUCED IN THE DIETS OF 80 LAMBS AND 32 BULLS. EVOLUTION OF 5-VINYL-1,3-OXAZOLIDINE-2-THIONE (5-VOT) AND THIOCYANATE IONS IN BIOLOGICAL FLUIDS AND ORGANS
Cold acclimation results in increased sucrose content which acts with other adaptations to confer freezing tolerance to plants. Inorganic pyrophosphate is a by-product of two reactions, pyrophosphate:fructose-6-phosphate-1-transferase and UDP-glucose pyrophosphorylase, leading to sucrose synthesis. Removal of pyrophosphate by overexpression of an inorganic pyrophosphatase (Ppase) was attempted to elevate sucrose levels of canola seedlings in response to low temperatures. A vector containing chimeric fusions of a cold inducible promoter (Bn115) with an E. coli ppa gene was constructed and introduced into the cytosol of B. napus L. cv. Westar by Agrobacterium-mediated transformation. After cold treatment for 7 days, ppase activity of the transformants was increased on average by 64 fold, whereas that of wild type remained unchanged. Cold acclimated transgenic seedlings also had higher levels of sucrose in cotyledons than did the wild type. There is a relatively strong correlation between ppase activity and sucrose content among the transformants (r=0.62). Freezing tolerance, determined as electrolyte leakage (%), was increased in the selected transformants after cold acclimation.
Transgenic (genetically modified) canola types being developed in Australia includes both those with modified crop production traits (herbicide tolerance, insect resistance) and those with modified product quality traits (e.g. high oleic acid). There are currently no commercial transgenic canola crops grown in Australia. The Genetic Manipulation Advisory Committee (GMAC) oversees gene technology research in Australia. GMAC has set out guidelines and advice on how genetically modified organisms can be handled. GMAC is to be replaced by an Office of Gene Technology Regulation (OGTR) which is expected to have new statutory powers to give approval for commercial release. An Institutional Biosafety Committee (IBC) must be set up for each transgenic type. The IBC ensures that GMAC guidelines and advice are followed for that particular type. The 1998 requirements for field trials in Australia were that the trial must be 400 metres from any other Brassica crop, the trial area including the buffer and a 50 metre area surrounding the trial must be kept free of Brassica and related weeds, a 15 metre canola buffer is to be planted around the trial as a pollen trap, and canola cannot be sown on the trial area for three seasons after the trial. With Australia’s production approaching 2 million hectares, it is becoming very difficult to meet GMAC requirements for isolation and rotation. There is a huge potential for transgenic canola in Australia and in the long term, general release will make the testing and selection of new transgenic cultivars much easier.
The objective of this research was to determine whether chlorophyll in green canola seed could be decreased under normal seed maturation conditions and following frost, through the use of antisense RNA, without affecting the quality and quantity of storage protein and oil. We intended to limit the level of chlorophyll through the production of the antisense form of Type I CAB of photosystem II in transgenic Brassica napus, cv Westar seeds. We used the seed specific promoter for napin, which directs a high level of gene transcription.
The results from the T5 generation indicate that the amount of chlorophyll a and b in the seed was significantly decreased under control conditions. The reduction was correlated with suppression of both CAB transcripts and protein. Following exposure to frost, the seed often undergoes renewed pigment synthesis. This increase was again suppressed in the transgenics. The combination of decreased accumulation under control conditions and decreased synthesis following frost led to low levels of pigment in the seed shortly after the frost. More importantly, mature seed from the transgenics contained little or no pigment, whereas the wild type controls were green.
Freshly isolated microspores from B. juncea (Indian Mustard) were cultured in NLN medium at a density of 3.5x104/ml in dark at 30oC formed embryos in 10-15 days time. Addition of activated charcoal resulted in 0.1% of the cultured microspores forming embryos. Higher or lower culture density of the microspores adversely affected development of embryos. Strong genotypic effect was also observed in microspore embryogenesis. Microspore embryos were allowed to grow and regenerate on B5 medium supplemented with 0.01 mg/l GA3. Chromosome doubling was carried out with Colchicine and ca. 60% of the plants transferred to green house were fertile and set selfed seeds. Microspore embryogenesis was also possible in B. rapa using almost similar methodology with certain modifications. The microspores cultured at a density of 2.0x104/ml in dark at 25oC formed embryos in 10-15 days time. The microspore embryogenesis response was comparable to B. juncea and genotypic differences were evident.
The objective of this research is to reveal the possibility of using microspore culture technology in the breeding of rapeseed hybrids. Doubled-haploid lines through microspore culture and chromosome doubling were produced from 4 lines of maintainers and restorers respectively of pol-CMS in B. napus L.. It was indicated that the restorer or maintainer genes of pol-CMS were not genetically related to the embryogenesis ability of microspore culture. Several improved restorer lines were obtained through the testing of doubled-haploid plants from the microspore culture of F2 offspring between the crossing of different restorers. The breeding of rapeseed hybrids could become more efficient with the application of microspore culture technology in the selection of restorers.
Two types of mutagenes were used to generate genetic variation in microspore culture of oilseed rape: ultraviolet (UV) radiation and chemical mutagen N-methyl-N-nitroso urea (MNU). Embryos derived from mutated microspores were placed in liquid medium B5 with addition of the selective agent phosphinothricin (PPT), an active ingredient of herbicide Liberty (AgrEvo). Within a few days only surviving embryos were transferred to agar plates for further development. The next resistance test was conducted on M1 progeny of DH mutant lines.
Genetically modified (GM) oilseed rape have been tested in UK registration trials since 1995. Herbicide tolerance is the major trait but modified fatty acid profiles are also under test.
GM testing and release is subject to regulation by the European Union (EU) directives and the UK authorities and is the subject of much public scrutiny. GM material must undergo the same testing requirements as conventional varieties but pollen barriers, isolation from field crops, the exclusion of produce from the food chain and post trial monitoring are some of the additional conditions and costs of the testing process. Consequently, GM trials are grown in isolation which also helps reduce the risk of vandalism to conventional trials.
Concerns about the release of GM material into the environment and the food chain have delayed introduction on the farm. At the time of writing there has been no EU clearance for commercial planting of any GM material. The debate on GM release in Europe continues to be a very live issue.
The Agrobacterium rhizogenes transformation system was evaluated as an alternative to the commonly applied Agrobacterium tumefaciens transformation. Initiation and establishment of hairy root cultures and regeneration of 5 spring and 2 winter rapeseed cultivars were attempted with three A. rhizogenes agropine strains and one mannopine strain. After inoculation of aseptic stem segments the ability of hairy root formation was scored for a period of six weeks.
No hairy root clones were obtained with the mannopine strain 8196, whereas the three agropine strains 15834, A4RS and LBA 9402 were successful and differed significantly in their transformation ability. Mean transformation frequency ranged from 22% (for 15834) up to 79% (for A4RS) over 7 rapeseed genotypes. No significant difference for the transformation frequency was found between the rapeseed genotypes.
A4RS and LBA 9402 transformed root segments of two spring- and two winter-rapeseed cultivars were used as explants in shoot regeneration experiments. The shoot regeneration rate ranged from 2.4 up to 43.3%. 30 different transformed plants showed in the greenhouse to varying degrees typical characters of hairy root derived plants. Some transformed genotypes showed significant differences in plant height, flowering time, chlorophyll-content of the leaves and fertility, whereas others were not different from the controls.
The cotransforamtion rate of two spring rapeseed cultivars with LBA 9402 TL-DNA and the 35S GUS gene showed an average of more than 40%.
Following oilseed rape (Brassica napus L.) cultivation increased nitrate levels in the soil have been frequently observed during late autumn and winter. This may contribute to leaching of nitrate to the groundwater. Breeding for improved nitrogen efficiency in rapeseed is therefore of considerable ecological but also economical interest. There are several molecular strategies that can be followed to elucidate the role of specific candidate genes in the expression of the complex trait 'N efficiency'. In the present investigation an asparagine synthetase gene from E. coli (AsnA, E. C. 18.104.22.168) was expressed under control of the CaMV35S-promoter in the spring rapeseed cv. Drakkar to study possible effects on the N-metabolism. Transgenic plants showed an up to 12-fold higher asparagine synthetase activity. However, the amino acid content in leaf samples of plants grown in hydroponic culture was not different from the control cv. Drakkar. In a greenhouse experiment, the plants were grown under high and low N fertilizer supply. At harvest the seed yield, the total dry matter as well as the N content in the straw and seeds were determined. The two transgenic clones did not show improved characters compared to the control plants, indicating that the expression of the AsnA gene has no greater effect on the N metabolism in rapeseed.
Current transformation methods result in the gene of interest being introduced into selected genotypes amenable to transformation. The introduced gene is then transferred to the breeding line of choice by conventional cross-pollination. In this paper we describe the first part of a study designed to elucidate the genetic variation for a number of factors that affect transformation in Brassica oleracea.
As part of a programme to develop more repeatable and reliable Agrobacterium tumefaciens mediated transformation, more than 100 genotypes of six subspecies of B.oleracea were screened for adventitious shoot formation. We describe variation within and between subspecies of B.oleracea for adventitious shoot regeneration from hypocotyl segments and cotyledonary petioles of cabbage, cauliflower, broccoli, brussels sprouts, kale and kohl rabi. The frequency of shoot regeneration varied significantly between genotypes within subspecies from both explant types. Frequencies of shoot formation from cotyledon and hypocotyl explants from the same genotype were compared and the speed of shoot regeneration recorded. The benefits of using a standardized protocol to screen breeding lines for use in transformation programmes are discussed
Studies were developed in order to attempt extraction of B. rapa (AA, 2n=20) and B. oleracea (CC, 2n=18) diploid genomes from the allotetraploid B. napus (AACC, 2n=38).
Observations performed on pollen mother cells (PMCs) at the anaphase I stage of meiosis of haploid oilseed rape (AC, 2n=19) often show unbalanced separations with 9 chromosomes to one side and 10 to the other. As these chromosome numbers correspond to the haploid forms of B. oleracea and B. rapa respectively, microspore cultures were carried out from haploid oilseed rape plants. Five different varieties at an haploid level were used . They showed : (1) PMCs at the metaphase I stage of meiosis either with a high level of chromosome pairing (5 to 9 bivalents) or a low level of chromosome pairing (1 to 6 bivalents) according to the original oilseed rape mother genotype, (2) few pollen grains stained by aceto-carmin Belling solution (range : 0 to 24%), (3) mainly uninucleate embryo sacs (57%).
1 to 11% of the microspores observed from haploid plants were identical in size and shape to those of dipoid genotypes. 72% of the microspore derived embryos had a chromosome number ranging from 18 to 25. Isozyme analysis were carried out to determine if deletions occurred.
In order to precise the genomic structure of female and male gametes produced from haploid plants, selfing and crosses either by a diploid or an haploid plant were performed.
The efficiency of this method to extract aneuploid oilseed rape lines is discussed.
Among the most frequent weeds of oilseed rape (Brassica napus, AACC, 2n=38) analyzed under French conditions, we identified wild radish (Raphanus raphanistrum, RrRr, 2n=18) as the weed presenting the highest rate of interspecific hybrid production and a high frequency of chromosome pairing in ACRr (2n=28) F1 hybrids.
In order to compare our first results obtained on the production of F1 interspecific hybrids under optimal conditions (e.g. using male sterile oilseed Basta® resistant as female and wild radish as pollinator) to the one obtained under normal agronomic conditions, we grew a 1 ha field of a Basta® resistant variety, ‘Synergy’. Wild radish plants were transplanted either as clusters or as isolated plants in the middle, the border or the margin of the field. The frequency of F1 interspecific hybrid production ranged from 10-7 to 3.10-5 from the seedlings obtained from wild radish seeds and from 2 10-5 to 5 10-4 for the smallest seeds harvested on oilseed rape, with 95% confidence limit.
From the F1 interspecific hybrid produced under agronomic conditions, we observed that the levels of fertility were determined by the genomic structure of the plants and by the cytoplasm. In the following generations, « BC1 » to « BC4 », obtained under optimal conditions from ACRr hybrids, the limiting factors for transgene establishment in natural wild radish populations are the original cytoplasm of the hybrids and the possibilities of recombination between the genomes. The impacts of the different factors will be discussed.
The efficiency of short-distance isolation was assessed specifically regarding honey bee movements because this insect is the predominant pollen vector in our region (Brittany, France). The trial consisted of two identical strips (6m x 30m) of a winter oilseed rape line. The design was a V truncated in such a way that the zone, without any vegetation, between the two strips varied from 3 m to 12 m width. A hive was placed at 8 m from the top of the V. Three equal sectors (60 m²) were identified in each strip. Honey bees were marked with different colours according to each sector of one strip, named the « Donor » strip. Almost all honey bees were marked until their number was nearly balanced between the three sectors. Then, marked and unmarked honeybees were counted on the « Donor » and « Recipient » strips. Results showed that the foraging area in a given strip is about 120 m² and that a mean proportion of 2.3% of marked bees/total number of honeybees was recovered in the « Recipient » strip. It can be concluded that a barren zone from 3m to 12 m width is not sufficient to completely discourage honey bees to cross between two identical resources but that it can significantly reduce cross pollination.
Hormone and enzyme activities during pod development and senescence were investigated in order to help identify biochemical and developmental processes for genetic manipulation of for the production of shatter resistant plants. Cell separation in the dehiscence zone (DZ) was delayed and more force was required to open fully mature pods produced on plants treated with the auxin mimic 2-methy-4-chlorophenoxyacetic acid (4-CPA). When indole-3-acetic acid (IAA) levels were low , small amounts of ethylene produced by the pod wall of parthenocarpic pods were sufficient to trigger the processes leading to cell separation in the DZ. Correlation was also obtained between the activity of an endo-acting isoform of polygalacturonase (PG35-8) and the breakdown of the middle lamella of DZ cells. The processes identified as targets for genetic manipulation for the production of shatter resistant plants were (i) suppression of activity of cell wall degrading enzymes in the DZ and, (ii) sustained activity of IAA.
Genetic engineering of crop plants relies on the development of efficient methods for the regeneration of viable shoots from cultured tissues. The objective of the present study was to develop a protocol for efficient shoot and plant regeneration from seedling explants of commercial oil seed (Brassica napus L.) cultivars and compare the regeneration capacity of the most commonly used explants: cotyledon, hypocotyl and root. Using the protocol developed in this study, we were able to regenerate shoots from hypocotyl, cotyledon and root explants of all the 7 commercial cultivars of B. napus tested. No difference in regeneration potential was found between two cotyledons of a seed and regeneration capacity of the three hypocotyl sections. The cultivars showed a varied response to shoot regeneration. The in vitro regenerated shoots were successfully rooted and acclimatised to glasshouse conditions. Our study shows that seedling explants of commercial cultivars of Australian canola are amenable to multiple shoot formation with high regeneration frequencies, and could be used for genetic transformation experiments.
Protoplasts from different genotypes were isolated from leaves of in vitro cultured plants. For somatic hybridization, protoplasts were mixed 1:1, resuspended in a fusion solution and fused by use of the cell fusion system KRÜSS CFA 500. After fusion, protoplasts were cultured in liquid medium for microcallus regeneration. In a second step regenerated microcalli were transferred to different solid media for shoot regeneration. At this point we could establish an electrical fusion method which resulted in plant regeneration from the combination Brassica napus + Brassica juncea.
Determining fatty acid composition of oil in oleaginous seeds previously requires extraction in mild conditions to avoid breakdown or polymerization. A rapid and gentle method of extraction is compared to three other standardized methods involving hot solvent or extractor apparatus and commonly used for oil content determination or characterization. Several types of oleaginous seeds are studied: sunflower seeds with low or high content of oleic acid, rapeseed seeds with low or high content of erucic acid, mustard seeds with high content of erucic acid, soya bean seeds, camelina sativa (gold of pleasure) seeds with high content of erucic acid, linseed seeds with high content of linolenic acid. Results of the comparison between the four extraction methods show that fatty acid composition can be determined with a simple cold extraction of the oil according to the reproducibility values for the GC analysis given by ISO 5508. Nevertheless, little differences are observed for linoleic and erucic acids.
The rapeseed Brassica napus (cv. Brutor) contains several distinct serine proteinase inhibitors. One of them, the protein RTI, Rapeseed Trypsin Inhibitor, shows similar characteristics to those of a homologous protein isolated from seeds of another Cruciferae: the white mustard (Sinapis alba). A protein highly similar to RTI is expressed in the late stages of seed maturation, some of other inhibitors could be constitutive proteins.
In the oil-rape (Brassica napus var. oleifera) seed, three classes of protein proteinase inhibitors have been identified. RTI-I and RTI-II belong to the Soybean family, RTI-III being the prototype of a new family of serine proteinase inhibitors from Cruciferae. The three-dimensional model of the 5-oxoPro1-Gly62-RTI-III isoinhibitor, built mainly on the basis of the similarity of the connectivity of disulphide bridges to those of snake venom toxins (e.g. erabutoxin b), consists of three-finger shaped loops extending from the cross-over region, and including the two short two-stranded anti parallel β-sheets. The Cys5-Cys27 and Cys18-Cys31 disulphides link finger I to II, the Cys42-Cys52 disulphide connects finger II to III, and the Cys54-Cys57 disulphide links the C-terminus to finger III. In such a three-dimensional organisation, the Cys18(P4)-Cys27(P6’) reactive site loop of the 5-oxoPro1-Gly62-RTI-III isoinhibitor is strongly connected to the protein core by the Cys5-Cys27 and Cys18-Cys31disulphide bridges. Moreover, Pro20(P2) and Pro24(P3’) residues may play a central role in the achievement of the proper conformation for the Arg21(P1)-Ile22(P1’) scissile peptide bond.
The rapid Canadian and Australian adoption of herbicide tolerant Brassica napus, resulting from both, mutation and gene insertion, indicates a wide acceptance of this technology by producers and consumers in North and Cental America as well as Asia. Concern remains, however, as to the potential impact this new technology may have for evolutionary change in agriculture and natural habitats. Studies have shown that B. napus and B. rapa plants exhibiting herbicide tolerance are no more invasive of cultivated or natural habitats than herbicide susceptible plants of the same species, unless the relevant herbicide is applied to remove competing vegetation. The possible escape of herbicide tolerant genes from B. napus and B. rapa into related weedy species such as Sinapis arvensis (wild mustard), Raphanus raphanistrum (wild radish), Hirschfeldia incana (Hoary mustard), B. nigra (black mustard) and Erucastrum gallicum (dog mustard) is still under investigation. Data to date indicate that natural barriers to introgression into the genomes of S. arvensis, R. raphanistrum and H. incana are strong. Further studies with E. gallicum are underway. Pollen outflow from fields of B. napus and B. rapa by wind and insects can be substantial and extend over long distances. Such pollen movement is an important factor in assessing the risks of both inter and intraspecific crossing. Outcrossing among fields of the same species can endanger the purity of seed stocks, raise concerns of organic growers and result in gene stacking among volunteer canola plants. The Canadian experience as to the extent of natural outcrossing among different sized pollen donor and recipient populations and the effectiveness of distance isolation will be examined. A case of gene stacking in volunteer B. napus will be reviewed and the need for more intensive agronomic management and grower education outlined.
Agrobacterium rhizogenes-mediated transformation was used to produce transgenic swede (Brassica napus var. rapifera). Hypocotyl explants of six cultivars or breeding lines were co-cultivated with strain A4T containing the binary vector pMOA4 (Barrell and Conner 1995). The T-DNA of pMOA4 contains one gene in the T-DNA region for transfer into plant cells: NOS-BAR-pAg7, a chimeric gene conferring resistance to phosphinothricin, the active ingredient in the herbicide Basta (Buster). This binary vector is a minimal T-DNA vector and contains the minimum features required for efficient plant transformation. There is no extraneous DNA in the T-DNA region, therefore the bar gene is tightly flanked by the T-DNA left and right borders.
The co-cultivation protocol was based on Henzi G. et al. (1999) and included use of a B. campestris feeder layer. Hairy root cultures were selected by their characteristic phenotype of rapidly branching plagiotropic growth (Christey 1997). High rates of transformation were obtained with all six lines producing numerous hairy root cultures. PCR analysis for the rolC gene confirmed the transgenic nature of 21 independent hairy root cultures representing five cultivars, but of these three did not contain the bar gene. Regeneration of transgenic shoots from hairy root cultures has proven difficult with shoots only obtained from six independent transgenic lines from three cultivars. Shoot regeneration was achieved by transfer of roots to the thidiazuron (TDZ)-containing media of Christey et al. (1997) followed by transfer to similar media usually containing 0.1mg/l TDZ and 10mg/l BA. PCR analysis for the rolC and bar genes has confirmed the presence of both genes in the four shoot cultures tested.
Agrobacterium rhizogenes, Brassica napus var. rapifera, forage brassicas.
Agrobacterium rhizogenes-mediated transformation was used to produce transgenic forage kale (Brassica oleracea var. acephala) and forage rape (B. napus var. biennis) with resistance to the herbicide Basta (Buster). Hairy root cultures were selected by their characteristic phenotype of rapidly branching plagiotropic growth (Christey 1997). Root tips were transferred to the thidiazuron-containing media of Christey et al. (1997) for shoot regeneration. Due to problems with rooting in vitro hairy root derived forage kale, use of increased levels of Phytagel (Sigma) were required. An increase in Phytagel from the usual 0.3% to 0.45% or 0.6%, gave a dramatic increase in the number of in vitro cuttings initiating roots. Prior to transfer to the greenhouse, putative transgenic shoots were checked for Basta resistance by either conducting a leaf-piece assay or by growing apical cuttings on medium containing 10mg/l Basta.
Selfed seed collected from the original transgenic forage kale and forage rape plants was planted in a contained greenhouse. As seedlings were segregating, Basta was applied with a paint brush to one leaf per plant to select transgenic plants for field testing. As a control, non-transgenic plants were also planted. A total of 329 eight-week-old seedlings, including controls, were transplanted to the field. Six transgenic lines were represented including three independent transgenic lines of Conds forage kale, and one line each of Striker, H103d and 95A2/5 forage rape. Throughout the trial observations and measurements were conducted on several aspects of plant development including plant phenotype, survival and days to flowering. At the completion of the trial, biomass was determined.
Microdissection and microcloning of satellite from Brassica campestris were studied. Ten satellites were isolated by using glass needles and their DNA was amplified by linker-adaptor-PCR method. Chromosomal fluorescence in site hybridization (FISH) confirmed that the PCR products originated from the satellite. PCR products were cloned into T-vector to construct satellite DNA library. Dot hybridization using the plasmids from 41 clones showed that among clones 77.5% carried high copy and 22.5% low or single copy. The average insert-DNA sizes in 36 clones analyzed by PCR were 660 bp and Southern blotting showed that the clones with high repetitive and those with low or single copy were 69% and 31%, respectively.
microdissection, microcloning, FISH, dot hybridization, southern blotting
Agrobacterium tumefaciens mediated transformation of four Australian commercial Brassica napus cultivars is reported. Explants from 4 day old seedlings were inoculated and cocultivated with Agrobacterium tumefaciens strain, LBA4404 harbouring a binary vector containing the neomycin phosphotransferase-II gene, permitting transformed shoots to be selected on kanamycin containing medium. After 3-4 months rooted transformed plantlets were successfully transferred and grown under glasshouse conditions. Transgenic plants were regenerated from all types of explants tested but the number of plants regenerated per explant type varied between the cultivars. Higher numbers of transformed plants were obtained from cotyledon and hypocotyl explants than root explants, presumably indicating these seedling explants of canola are more amenable to genetic transformation. Cotyledons from young seedlings were the best target for transformation of RK7 and westar cultivars, while RI25 hypocotyl explants produced maximum number of shoots. Of the five cultivars tested, RK7 gave best response while RI25 produced least number of shoots. Integration and expression of the introduced transgene was analysed by DNA gel blot analysis, GUS staining and NPT-II expression assay.
Allergy to Brassica pollen has been reported in some countries. We have cloned a cDNA encoding IgE-binding proteins from expression libraries of anthers of Brassica rapa L. and B. napus L. using serum IgE from a patient who was allergic to Brassica pollen. One of the Brassica pollen allergen Bra r 1 belongs to a new family of Ca2+-binding proteins, characterized by the presence of two potential EF-hand calcium-binding domains. Disruption of these EF-hand motifs by amino acid substitutions demonstrated that both domains of Bra r 1 constitute functional Ca2+-binding sites. Calcium-binding deficient mutants displayed significantly reduced IgE binding activity. Injection of these mutated Bra r 1 variants into a murine model system showed that mouse IgG raised against the mutants recognized native Bra r 1 in Brassica pollen extracts suggesting the potential use of the engineered allergens for effective immunotherapy.
Winter Brassicas must be exposed to extended periods of low temperature to flower and set seed. This low temperature exposure also results in the acquisition of freezing tolerance. To investigate the relationship between freezing tolerance and vernalization, homozygous microspore-derived lines were developed from reciprocal crosses between two winter cultivars of Brassica napus, Cascade and Rebel. One line, Vern-, which has very high freezing tolerance was identified. Further physiological and morphological characterization of this line, including determination of photoperiod responses, field studies and SEM of reproductive development, verified that Vern- is a true spring type, having completely lost the vernalization requirement. In order to determine the genetic basis of the phenotype, molecular analysis of the parents and Vern- was undertaken. The results show that Rebel, although traditionally classified as a winter cultivar, carries spring alleles at the major and some of the minor loci that control vernalization requirement and flowering time. Vern- appears to have inherited these alleles from Rebel, while inheriting freezing tolerance alleles from Cascade. Thus, Vern- has lost the vernalization requirement, despite the very high degree of both inherent and acclimation-specific freezing tolerance. The results indicate that the linkage between freezing tolerance and vernalization can be broken in winter B. napus.
Production of rapeseed for canola oil has increased over the past few decades. Areas where rapeseed had not been grown previously are now being considered for production. Some potential production areas have soils with low pH and toxic levels of aluminum (Al). Cultivars of several crops have been developed that are more tolerant to Al and are capable of higher yields with reduced levels of lime. A screening procedure was based on procedures developed for other crops. Six days after seeds were imbibed, seedlings were suspended in a liquid medium containing high levels of Al. On day 7, roots of the seedlings were placed in a hematoxylin solution to identify damage from Al. Root tips were scored from 0 (no staining, no damage) to 4 (dark staining, severe damage). Available released cultivars and the Plant Introduction collection (total of 546 lines) were screened for reaction to Al. Most of the recently developed cultivars were rated susceptible to highly susceptible for Al damage, but several PI lines with tolerance to Al were identified.
The capacity for vernalization varies among genotypes of spring rape, Brassica napus var. annua, and affects their suitability for cultivation in the mediterranean environment of Western Australia. The inheritance of the vernalization response was investigated in crosses among five fully inbred genotypes of spring rape derived from a cross between two cultivars differing in their vernalization response. Generation mean analysis of time to flowerig and other related characters indicated that additive, dominance and non-allelic interaction effects all play an important role in the vernalization responses of these characters. Estimates of broad- and narrow-sense heritability indicated that the vernalization responses can be fairly readily eliminated by selection. The potential of a combined molecular and cytogenic approach to the mapping of loci that confer a vernalization response was also explored in backcrosses of B. napus (genomic constitution AACC) to B. rapa (AA). The RFLP probe/enzyme combination wg7b3/HindIII co-segregated with lateness at a locus on the C genome. Screening of a larger number of backcross progeny would permit the identification of the C-genome chromosome conferring the vernalization response, which would facilitate marker assisted selection to eliminate the vernalization requirement for early flowering.
Breeding for improved N-efficiency in rapeseed is gaining increasing interest for ecological and economical reasons. 18 different German winter rapeseed cultivars and breeding lines were tested for two years in multi-locational field trials for their yield under three different N-fertilizer regimes, namely nil, medium and high. Significant genotypic differences were found for grain, oil and protein yield as well as for total N-uptake. Genotypes giving a superior yield under limiting N-supply were identified. The tested hybrid cultivars gave superior yields not only at the high but also at the medium and low N-fertilizer levels. Genotype x N-fertilizer interactions were significant for grain yield (P<0.05) and for total N-uptake (P<0.01). A close correlation between total N-uptake and grain yield was found at low and high N-fertilizer supply, indicating the importance of N-uptake efficiency.
The objective of this study is to investigate the genetic variation in N efficiency, its two components (uptake efficiency and utilization efficiency) and its inheritance in a genetically broad material of winter oilseed rape. Ninety genotypes (1998) from different groups (lines, hybrids, old land varieties, resyntheses and crosses between Falcon and resyntheses) are investigated under field conditions at various locations in Northern Germany. The experimental design includes two rates of nitrogen application (0 or 240 kg N/ha) and two replications.
Several characters were evaluated: chlorophyll content, plant height, fresh weight, dry weight, seed yield, N amount in the seeds, straw yield and N amount in the straw, N content in dropped leaves, oil and glucosinolat content in the seed. The harvest indices and the N harvest indices were calculated.
The resyntheses were the group with lowest grain yield and lowest N harvest index. N content of dropped leaves showed significant reaction on nitrogen supply and a large genetic variation at both N levels. No general difference can be observed among genotype groups for this trait.
Based on the study of both manuscript and archival sources there is at first the introduction and growing of the oleic form of B. campestris L. with the following view to the hypothesis about origin of the oleic form of B. napus and history of its expanding reviewed. Cultivation of B. napus is supported by the evidence since 1578 in western Europe and since 1587 in the region of the Czech Republic.
Evolution and spreading of this group of oilseeds is observed from the old times till nowadays regarding to the beginning of breeding.
Milestones of the development of the modern breeding and technical progress in B. napus are chronologically and briefly mentioned in the period from the 1st International Symposium for the Chemistry and Technology of Rapeseed Oil and Other Cruciferae 1967 in Gdañsk (Poland) till the 10th International Rapeseed Congress in Canberra.
The nectar collecting posture of honey bees on the flowers and their flights between mutant and non mutant plants were compared. Observations were done on small areas in three separated experiments. Three mutants were under study : dwarf, apetalous, and cleistogamous oilseed rape lines. On dwarf plants, because the flower morphology is identical to wild types , honey bees behaved in the same manner, i.e. they crawled over the flower and had contacts with the stamens. Their flights between both types were partially reduced only when the difference in height was higher than 80cm and when they differed in their male-fertility (male-fertile versus male-sterile lines). On apetalous flowers, honey bees exhibited a side working posture which reduced their contacts with stamens. Moreover, they were individually constant to one flower type and this behaviour induced a nearly absolute lack of flights between the apetalous and the petalous types. On cleistogamous flowers, about 43% of the nectar foragers were side-working. The others were able to collect nectar by the small hole at the top of the flower. In some cases pollen gatherers opened the flower during their visits. Flights between cleistogamous and conventional flowers were by two times less frequent than within a same type. Thus, incidences of these mutations on the honey bee mediated pollen transfer are different. Dwarf and tall plants could be intercrossed. On the opposite, intercrossing between apetalous and petalous flowers could not be ensured by honey bees only. Cleistogamy could lead to an increased autogamy but a pollen dispersal by visiting honey bees could not be excluded.
Genetic variation in pod shattering resistance which exceeded that found in a selection of modern cultivars has been identified amongst synthetic lines of B. napus produced at the John Innes Centre. Procedures have been developed to measure the resistance to shattering at the Silsoe Research Institute. These simulate response to impact in the crop canopy and measure the mechanical properties of the dehiscence zone (DZ). Susceptibility to shatter was not necessarily associated with plant morphology or raceme characters and can therefore be selected for independently. Anatomical studies at IACR – Long Ashton have identified two structural differences in lines that appear to be related to increased resistance to opening: (i) increased thickening and prominent vascular tissue on the inner edge of the DZ, and (ii) reduced cell separation in the unthickened cells of the DZ. Lines with the most resistance contained both modifications.
Changes in the structure of the DZ, which are associate with susceptibility to shatter, have been investigated in an irradiation – induced mutant and a population of its parent cultivar, Jet Neuf. Much lignification of groups of cells was found throughout the DZ of the most resistant Jet Neuf plants and in the mutant. In addition, vascular traces are situated close to the inner edge of the DZ in the mutant and help to secure the valves, making it more difficult to open the pods. The changes in pod structure in the mutant appear to be associated with differences in flower structure and raceme architecture. These are likely to be pleiotropic effects caused during initial irradiation treatment.
The development of yellow-seeded forms of Brassica napus has been a major focus of breeding research at AAFC Saskatoon for the last 20 years. The original yellow-seeded germplasm was low yielding, highly susceptible to blackleg disease, and the seed had a low oil content. In 1991, a cross was made between the yellow-seeded line YN90-1016 and the black-seeded, blackleg resistant Australian variety Shiralee, followed by a cross to the high oil content breeding lines N89-17 and N89-53. The two crosses were pedigree selected and five near isogenic yellow/black seeded pairs, consisting of 20 F5 derived sublines for each pair, were field tested from 1996 to 1998. Genetically stable, true breeding yellow-seeded lines were identified that had high oil content were resistant to blackleg and had high yield. The results of the field tests indicated that the yellow-seeded line YN90-1016 can successfully be used a source of the yellow seed trait in crosses with black-seeded germplasm to improve the agronomic performance and seed quality of yellow-seeded B. napus. Selected F7 lines of the yellow-seeded F4 line YN94-663, derived from the above cross, are presently used as parents in crosses with various black-seeded B. napus germplasm to produce commercially competitive, high yielding, high oil content, canola quality B. napus varieties for production in western Canada.