Mapping loci controlling the concentrations of erucic and linolenic acids in seed oil of Brassica napus L.

The quality of plant oil is determined by its component fatty acids. Relatively high levels of linolenic acid reduce the oxidative stability of the oil, and high levels of erucic acid in the diet have been associated with health problems. Thus, oilseed Brassica napus cultivars with low linolenic and low erucic acid contents are highly desirable for edible oil production. In order to identify genes controlling the levels of erucic and linolenic acids, we analyzed the oil composition of 99 F₁-derived doubled haploid lines from a cross between cv Major (high levels of erucic and linolenic acids) and cv Stellar (low levels of both fatty acids). A molecular marker linkage map of 199 loci for this population was used to identify quantitative trait loci (QTL) controlling oil composition. We identified two regions that accounted for nearly all of the phenotypic variation in erucic acid concentration and one region that accounted for 47% of the variation in linolenic acid concentration. The QTL associated with linolenic acid concentration mapped near a RFLP locus detected by a cDNA clone encoding an omega-3 desaturase, suggesting that the low linolenic acid content of Stellar may be due to a mutation in this gene.     

Mapping the genome of rapeseed (Brassica napus L.). II. Localization of genes controlling erucic acid synthesis and seed oil content

A F₁ microspore-derived DH population, previously used for the development of a rapeseed RFLP map, was analysed for the distribution of erucic acid and seed oil content. A clear three-class segregation for erucic acid content could be observed and the two erucic acid genes of rapeseed were mapped to two different linkage groups on the RFLP map. Although the parents of the segregating DH population showed no significant difference in seed oil content, in the DH population a transgressive segregation in oil content was observed. The segregation closely followed a normal distribution, characteristic of a quantitative trait. Using the program MAPMAKER/QTL, three QTLs for seed oil content could be mapped on three different linkage groups. The additive effects of these QTLs explain about 51% of the phenotypic variation observed for this trait in the DH population. Two of the QTLs for oil content showed a close association in location to the two erucic acid genes, indicating a direct effect of the erucic acid genes on oil content.     

Synthesis of high erucic acid rapeseed (Brassica napus L.) somatic hybrids with improved agronomic characters

Novel Brassica napus somatic hybrids have been created through protoplast fusion of B. oleracea var. botrytis and B. rapa var. oleifera genotypes selected for high erucic acid (22:1) content in the seed oil. Fifty amphidiploids (aacc) and one putative hexaploid (aacccc) hybrid were recovered in one fusion experiment. Conversely, only one amphidiploid and numerous regenerates with higher DNA contents were produced in a similar fusion using a different B. rapa partner. Hybridity was confirmed by morphology, isozyme expression, flow cytometry, and DNA hybridization. Analysis of organellar DNA revealed a distinct bias toward the inheritance of chloroplasts from the B. rapa (aa) genome. All amphidiploids set self-pollinated seed. A erucic acid content as high as 57.4% was found in the seed oil of one regenerated plant. Fatty acid composition was stable in the R₁ generation and was coupled with increased female fertility. Other novel agronomic characters in the hybrids recovered include large seed size, lodging resistance, and non-shattering seed pods.     

Inheritance and variation of erucic acid content in a transgenic rapeseed (Brassica napus L.) doubled haploid population

Erucic acid (22:1) is a valuable renewable resource for the oleochemical industry. Currently available high erucic acid rapeseed cultivars contain only about 50% erucic acid in the seed oil. A substantial increase of the erucic acid content of the rapeseed oil could increase market prospects. The transgenic line TNKAT, over expressing the rapeseed fatty acid elongase gene (fae1) and expressing the Ld-LPAAT gene from Limnanthes douglasii was crossed with the line 6575-1 HELP (high erucic and low polyunsaturated fatty acid). A from the F₁ plants produced population of 90 doubled haploid (DH) lines was tested in a greenhouse with three replicates. Parental lines TNKAT and 6575-1 HELP contained 46 and 50% erucic acid in the seed oil, respectively. In the DH population the erucic acid content ranged between 35 and 59%. The Ld-LPAAT + Bn-fae1.1 transgene showed a 1:1 segregation. The transgenic DH lines contained up to 8% trierucolyglycerol, but surprisingly had a by 2.3% lower erucic acid content compared to the non-transgenic segregants. Results indicated that the ectopically expressed fae1.1 gene may not be functional. The DH population also showed a large quantitative variation for PUFA content ranging from 6 to 28% (TNKAT: 21%, 6575-1 HELP: 8%). Regression analysis showed that in the DH population a 10% reduction in PUFA content led to a 4.2% increase in erucic acid content. Development of locus specific PCR primers for the two resident erucic acid genes fae1.1 (A-genome) and fae1.2 genes (C-genome) of rapeseed allowed sequencing of the respective alleles from TNKAT and 6575-1 HELP. Single nucleotide polymorphisms were only found for the fae1.1 gene. Use of allele specific fae1.1 PCR primers, however, did not reveal a significant effect of the fae1.1 allele from either parent on erucic acid content. The high erucic acid low polyunsaturated fatty acid DH lines and the fae1 locus specific primers developed in the present study should be useful in future studies aimed at increasing erucic acid content in rapeseed.     

A comparative assessment of purification techniques for mesophyll protoplasts of Brassica napus L.

The effects of three different general purification protocols have been assessed quantitatively using mesophyll protoplasts of Brassica napus. Within the initial sample two distinct sub-populations were determined. The methods used influenced the ratio of the vacuolated to chloroplastic type protoplast sub-populations. Overall recovery rates of the initial sample varied according to the method used from 38% to 27%, but the relative recovery of the sub-populations varied considerably with a purified ratio of between 1.0:0.78 to 1.0:7.0. Size distribution profiles of the initial and purified populations are also presented.     

Genetic control of linolenic acid concentration in seed oil of rapeseed (Brassica napus L.)

Summary Results from a diallel mating of two rapeseed lines with distinctly different linolenic acid concentration show that this trait is mainly under control of nuclear genes of the embryo. However, significant differences in reciprocal F₁, BC₁ and BC₂ indicate maternal control, which is realized by interaction between maternal genotype and nuclear genes of the embryo. Additionally, temperature exerts considerable influence on the degree of maternal control. Since no reciprocal differences are detectable in F₂, cytoplasmic factors seem not to be involved in the inheritance of linolenic acid concentration. Hypotheses on the physiological nature of maternal control of this trait are discussed.     

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

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

Gene transferability from transgenic Brassica napus L. to various subspecies and varieties of Brassica rapa

Gene transferability from transgenic rapeseed to various subspecies and varieties of Brassica rapa was assessed in this study. Artificial crossability was studied in 118 cultivars of 7 B. rapa subspecies and varieties with the transgenic rapeseed GT73 (Brassica napus) as the pollen donor. On average 5.7 seeds were obtained per pollination, with a range from 0.05 to 19.4. The heading type of B. rapa L. showed significantly higher crossability than non-heading types of B. rapa. The spontaneous outcrossing rate between B. rapa (female) and the transgenic rapeseed Ms8 × Rf3 (B. napus) (male) ranged from 0.039 to 0.406%, with an average of 0.19%. The fertilization process and the development of the hybrid seeds as shown by fluorescent staining techniques indicated that the number of adhered pollens on the stigma was reduced by 80%, the number of pollen tubes in the style was reduced by 2/3 and the fertilization time was delayed by over 20 h when pollinated with the transgenic rapeseed Ms8 × Rf3 in comparison with the bud self-pollination of B. rapa as control. About 10–70% of the interspecific hybrid embryos were aborted in the course of development. Some seeds looked cracked in mature pods, which showed germination abilities lower than 10%. The spontaneous outcrossing rates were much lower than the artificial crossability, and their survival fitness of the interspecific hybrid was very low, indicating that it should be possible to keep the adventitious presence of the off-plants under the allowed threshold, if proper measures are taken.     

Interspecific somatic hybridization between Brassica napus and Brassica hirta (Sinapis alba L.)

Summary Somatic hybridization between Brassica napus and B. hirta (or Sinapis alba) is described. No cybrid plant with B. napus nucleus exhibiting cytoplasmic male sterility was recovered. Somatic hybrids were identified morphologically and, for some of them, by cytological observations. They were also characterised by Southern hybridization of nuclear rDNA. Chloroplast and mitochondrial DNA restriction analysis showed that 2 plants out of 14 have B. hirta ctDNA, one the B. napus mtDNA and the other a hybrid. Nine possess B. napus ctDNA with a hybrid mtDNA. For six of them, mtDNA patterns present novel bands, suggesting intergenomic recombination during fusion. These hybrids will be included in the breeding program.     

Agrobacterium-mediated transformation of thin cell layer explants from Brassica napus L.

Summary Agrobacterium-mediated transformation of thin cell layer explants (Klimaszewska and Keller 1985) yielded large numbers of transgenic plants of a major Canadian rapeseed cultivar Brassica napus ssp. oleifera cv Westar. The morphology and fertility of these plants were indistinguishable from controls. The Ti plasmid vector, pGV3850 (Zambryski et al. 1983) was used as a cis vector and as a helper plasmid for the binary vector pBin19 (Bevan 1984). Selectable marker genes that conferred resistance to high levels of kanamycin (Km) on Nicotiana tabacum were less efficient in the selection of transgenic B. napus. At low levels of Km (15 g/ml) large numbers of transgenic plants (50%) were identified among the regenerants by nopaline synthase activity and several of these were confirmed by Southern blot analyses. Only a small number were resistant to higher levels of Km (80 g/ml). Preliminary analyses indicated that resistance to Km was transmitted to the selfed progeny. Chimeric chloramphenicol acetyl transferase genes were ineffective biochemical markers in transgenic B. napus.Contribution No. 1092 Plant Research Centre, Ontario, Canada     

An isozyme marker for resistance to race 3 of Fusarium oxysporum f. sp. lycopersici in tomato

Summary The levels of erucic acid and other fatty acids in seeds of microspore-derived spontaneous diploid plants from crosses between low and high erucic acid parents were examined. The analysis confirmed that erucic acid is simply inherited and is determined by two genes that act in an additive manner. The effects of the genes for erucic acid on the levels of the other fatty acids was also determined and many significant correlations were found. In particular, erucic acid levels were negatively correlated with oleic acid and linoleic acid levels. The study also illustrates several advantages of using haploidy to analyze the inheritance of agronomically important traits. In particular, the number of phenotypic classes is smaller in androgenic populations and differences between classes are greater than in an F₂ population.     

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

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

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

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

Physiological and yield effects of uniconazole on winter rape (Brassica napus L.)

Uniconazole at various concentrations on rape, at the three-leaf stage, was examined for physiologic and yield effects. Foliar sprays of 10, 25, and 50 mg/liter significantly reduced seedling height, and increased shoot width (stem width before elongation), number of green leaves, and total dry weight at transplanting. Chlorophyll content, superoxide dismutase and catalase activities, root oxidizability (capacity for root oxidation), and ethylene production were also increased. Additionally, the number of branches and pods/plant were increased; and a 7.4, 8.5, and 4.3% increase of seed yield over the controls was observed with treatments at 10, 25, and 50 mg/liter uniconazole, respectively. No significant effects were observed on plant maturity, the seed oil content, or the erucic acid and glucosinolate content. Total oil production significantly increased with 10, 25, and 50 mg/liter by 9.9, 10.6, and 6.8%, respectively, over the controls. These results suggested that uniconazole-induced high productivity was accompanied by increased levels of activities of various antioxidants, including superoxide dismutase and catalase, and by the improvement of root oxidizability and plant vigor.Abbreviations SOD superoxide dismutase - CAT catalase - NBT nitro blue tetrazolium - TTC red tetrazolium - IAA indoleacetic acid     

Virulence of Agrobacterium tumefaciens strains with Brassica napus and Brassica juncea

Summary Brassica napus and Brassica juncea were infected with a number of Agrobacterium tumefaciens strains. Tumourigenesis was very rapid and extremely efficient on B. juncea with all but one of the strains. Tumourigenesis on B. napus varied widely. It was very efficient with the nopaline strains, was reduced with the succinamopine strain A281 and was very weak with the octopine strains. The latter observation was confirmed with six different B. napus rapeseed cultivars. The selectivity was due to differences in the virulence of Ti plasmids with B. napus, rather than the tumourigenicity of the T-DNA or virulence of the chromosomal genes associated with the strains. An exception was strain LBA4404. The virulence of the octopine strains was increased by coinfection with more virulent disarmed strains and by induction with acetosyringone.     

Uptake and distribution of sinigrin in microspore derived embryos of Brassica napus L

In Brassica napus, glucosinolates are transported from all parts of the plant into the embryo during seed development. In this study we describe the uptake of the alkenyl glucosinolate sinigrin by microspore derived embryos from high and low glucosinolate genotypes. Microspore derived embryos develop completely isolated from maternal tissues unlike zygotic embryos, which contains glucosinolates transported into the embryo synthesised in the vegetative tissues. The sinigrin in the culture medium was almost completely absorbed by the embryos after three days of culture. The embryos of high and low glucosinolate genotypes were equally capable of absorbing sinigrin from the medium. A significant increase in different alkenyl glucosinolates following feeding of sinigrin suggests induction of biosynthetic enzymes in the embryos. Following excess feeding of sinigrin, we found a strong uptake against a concentration gradient and stable accumulation by the embryos. The glucosinolate was detected in single dissected cotyledons by a photometric test and by HPLC. This test could potentially be useful for screening mutants defective in glucosinolate uptake into the embryo.     

Cytoplasmic male sterility in rapeseed (Brassica napus L.)

Summary Restriction patterns of chloroplast (cp) and mitochondrial (mt) DNA in Brassica napus rapeseed reveal the alloplasmic nature of cytoplasmic male sterility in this crop. Both the Shiga and Bronowski systems probably exploit cytoplasmic diversity in B. napus cultivars arising from introgression of cytoplasm from the other rapeseed species, B. campestris. Nuclear genes specific to these systems do not cause sterility in maintainers (Bronowski and Isuzu-natane) because they have a campestris cytoplasm, but give rise to sterility in napus cytoplasms. In the course of hybridization to napus cultivars a line with the triazine resistant cytoplasm (a campestris cytoplasm) has undergone an alteration in the mt genome rendering its restriction pattern more similar than previously to that of napus. The alteration may be an inversion between 7.2 and 3.4 kb in length.     

Artificial Brassica napus flowering in Bangladesh

Summary Natural rapeseed (Brassica napus L.; AACC 2n=38), originated in the temperate climate of the Southwest European Mediterranean region, fails to complete its generative phase in the subtropics and is thus not cultivated in countries like Bangladesh. Adapted agroecotypes are available from the diploid representatives of its genome A (B. campestris/pekinensis, 2n=20) and C (B. oleracea/alboglabra, 2n=18). An artificial resynthesis based on carefully selected progenitor lines was expected to give a photoperiodically better adapted rapeseed. B. pekinensis x B. oleracea/alboglabra gave 2 hybrids and 87 matromorphous plants from 1,448 crossed flowers and the reciprocal combination gave no hybrid but 11 matromorphous plants from 2,228 pollinated flowers. The two true hybrids were vegetatively propagated and chromosome doubled. Part of the F₂ was grown in Sweden (all plants flowered and the most early ones were selected), part in Bangladesh (13 out of 706 plants flowered). The selected F₃ material flowered in Bangladesh and transgressions in earliness could be recorded, some lines were of definite agronomic potential. A correlation in earliness between reaction in Sweden (long day) and Bangladesh (short day) was observed.