Hybridization of radish (Raphanus sativus L.) and oilseed rape (Brassica napus L.) through a flower-culture method

Summary Hybridization between radish and oilseed rape has been cumbersome, requiring elaborate embryo rescue techniques. With a modified flower culture method, we have achieved successful hybridization between radish and (transgenic) oilseed rape without the laborious and technically demandingin vitro ovule or embryo rescue techniques.The hybrid nature of the intergeneric hybrids was demonstrated using morphological traits, and DNA analyses. The described method will facilitate the generation ofRaphanobrassica hybrids useful for biosafety studies of the potential for transgenes to spread in weedyCruciferae as well as for breeding programs aimed at introducing useful radish genes, e.g. nematode resistance genes, into oilseed rape.     

A comparison of traditional and haploid-derived breeding populations of oilseed rape (Brassica napus L.) for fatty acid composition of the seed oil

Summary Microspore embryogenesis technology allows plant breeders to efficiently generate homozygous micros-pore-derived breeding populations of oilseed rape (Brassica napus L.) without traditional generations of inbreeding. This study was conducted to compare the frequency distribution of microspore-derived population and single seed descent populations with respect to fatty acids of seed oil. Both microspore-derived populations and single seed descent populations were produced from each of three crosses made between selected parents containing contrasting amount of erucic, oleic, linoleic and linolenic acids. The fatty acid content of F₃ plants derived lines (F₅ seed) developed by single seed descent was compared to that of microspore-derived populations. The means, ranges and distribution pattern of seed fatty acid contents were similar in both populations for each fatty acid studied, although a few heterozygous lines were observed in the single seed descent populations. The results indicated that microspore-derived population form random, homozygous F₁ plant derived gametic arrays for all fatty acids evaluated. Selection for altered fatty acid composition in microspore-derived and single seed descent homozygous populations should be equally efficient, in the absence of linkage of traits investigated.     

Effect of donor plant age and inflorescence age on microspore culture of Brassica napus L.

Summary Effect of age of donor plants and age of inflorescence on embryogenesis in microspore culture of B. napus was examined. Microspores isolated from buds of older plants had a higher embryo yield than those of younger ones. The effect of the age of inflorescence showed a different pattern. In older plants, a higher embryogenesis response was observed in microspores isolated from buds of new inflorescences, while in young plants, microspores isolated from buds of old inflorescences showed high embryo yield. These different responses were considered to be attributable to a difference in the developmental stage of pollen at the time of microspore isolation. Our results indicated that microspores collected from older inflorescences and older plants have sufficient embryogenic potential when the optimum developmental stage of pollen was used. Frequency of embryo to plant conversion was influenced by the size of embryos subcultured, but not by donor plant age or the age of the inflorescence.     

Heterosis and inbreeding depression in forage rape (Brassica napus L.)

Summary Heterosis and inbreeding depression for total fresh and dry weights were studied over two seasons in 15 crosses of six cultivars of forage rape (Brassica napus L.). Some of the hybrids exhibited considerable heterosis over both mid-parent and better parent; followed by depression in the F₂. The cross Windal × Canard had 62% and 25% greater dry matter yield over the best cultivar used in this experiment in 1978 and 1979, respectively. Possibilities of the utilization of this hybrid superiority are discussed.     

Relationships between seed yield and growth characters,yield components,seed quality of summer-type oilseed rape (Brassica napus L.)

Summary Five genotypes of rapeseed were observed in the field on a single plant and plot basis to evaluate growth characteristics, yield, yield components and seed quality. Observations were made in two years at two locations on three seeding date and three seeding rate treatments. Correlation of seed yield with growth characters demonstrated no consistent trend, indicating that an early maturity and high seed yield should be possible. Harvest index was strongly correlated with seed yield. Percent seed oil and protein were not directly related to seed yield.     

Self-incompatibility as a pollination control mechanism for spring oilseed rape,Brassica napus L.

Summary Self-incompatibility was shown to be an effective method of pollination control in spring rapeseed (B. napus L. ssp. oleifera (Metzg.)) by comparing the yield of a Westar-Topas syn-1 produced by crossing two SI lines with the yield of the corresponding syn-1 produced by hand pollination. Although the trial showed high-parent heterosis in the syn-1s, there was insufficient replication to determine the level of heterosis.Abbreviations SI self-incompatible - SC self-compatible     

Direct plant development from microspore-derived embryos of winter oilseed rape Brassica napus L. ssp. oleifera (DC.) Metzger

The influence of temperature/photoperiod treatment and gibberellic acid concentration (0, 0.1 or 1.0 mg/l) on direct conversion of microspore-derived embryos (MDEs) to plantlets of winter oilseed rape was investigated. Physiologically mature, 21-day-old MDEs were transferred to a solid B5 medium supplemented with gibberellic acid, and cultured at 24 ^°C, 4 ^°C or 1 ^°C for 14 days, and then at 24 ^°C for the next 21 days. Low temperature was linked with short photoperiod (8 h light/16 h dark), and high temperature was linked with long photoperiod (16 h light/8 h dark). The highest embryo conversion rate was at 1 ^°C with over 70%, compared to<20% at 4 ^°C. Two-way analysis of variance confirmed the significance of the effect of temperature/photoperiod treatment. By contrast, gibberellic acid concentration had no significant effect on stimulation of shoot development from apical meristems of MDEs. Roots developed from apical root meristems of MDEs very easily. The best obtained conversion rate of MDEs induced with cold treatment at1 ^°C for 14 days was 86.5%. Observations on morphological development of MDEs showed clear differences in reaction at various temperature/photoperiod treatments. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro selection for oleic and linoleic acid content in segregating populations of microspore derived embryos of Brassica napus

Microspore derived embryos (MDEs) in Brassica napuscontain large amounts of storage lipids which show a genotype specific fatty acid composition (FAC). One cotyledon of regenerating emblyos can be dissected at an early stage during the in vitro culture and used for fatty acid analysis. Thus, in breeding programmes to modify oil quality, only MDEs having the desired FAC need to be regenerated to plantlets and transferred to the greenhouse. In the present study the applicability of this method for the selection of a high oleic acid content and a low linoleic acid content in the seed oil has been tested by crossing a Brassica napus mutant line having a high oleic acid (C18:1) content in the seed oil (75%) with a wild type doubled haploid line with 62% C18:1 in the seed oil. Microspore culture was applied to the F1 plants. In total 59 MDEs were obtained, from which 31 were cultured with and 28 without 15μM abscisic acid for 3 weeksin vitro. One cotyledon was dissected under aspetic conditions and used for fatty acid analysis. The remaining part of the embryos were further regenerated to plantlets and transferred to the greenhouse to obtain seeds after self pollination. Seeds harvested from the doubled haploid lines in the greenhouse were used for fatty acid analysis and also for growing in the field. The abscisic acid treatment of the MDEs generally improved the correlations for linoleic and oleic acid between the MDEs and the seeds harvested in the greenhouse and the field. The correlations ranged from 0.68** to 0.81**.This indicates that selection for high oleic acid can be started already during an early stage of the in vitro culture. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic analyses of agronomic and seed quality traits of doubled haploid population in Brassica napus through microspore culture

Summary The results showed that the F1 genotype from the cross (Brassica napus cv. Zheshuang 758 × cv. Z-4115) had good response to embryogenesis, and their embryo yield and rate of plant regeneration reached 69.8 embryo/bud and 46.9%, respectively. Characters from the doubled haploid (DH) populations in B. napus were analyzed and it was showed that the means of each agronomic trait were between their parents, but they were nearer to the paternal in 6 agronomic traits (plant height, branch position, number of pods in the main raceme, length of pod, number of pods/plant and number of seeds/pod). The number of genes controlling each agronomic trait was analyzed based on the DH populations. The results showed that the number of genes controlling number of pods in the main raceme was the highest (15.6), and the least number of genes was involved for stem width (only 7.9). According to estimated coefficients of skewness and kurtosis of the traits tested, gene interaction was found to be absent for stem width, plant height, length of main raceme, number of primary and secondary branches, pod density in the main raceme and seed weight/plant. Complementary interaction was also observed in five agronomic traits (number of pods in the main raceme, length of pod, number of pods/plant, number of seeds/pod and 1000-seed weight). A significantly positive correlation was observed between seed yield/plant and four agronomic traits (length of main raceme, length of pod, number of pods/plant and 1000-seed weight). The experiment also showed that the erucic acid, glucosinolate, oil and protein contents of DH populations were 34.23%, 87.09 μmol/g, 44.09% and 42.67%, respectively. The numbers of genes controlling each quality trait were 7.8, 9.7, 9.4 and 8.7, respectively. Partial correlations between the seed quality traits and the agronomic characters of DH populations were analyzed. In this experiment, the partial correlations among seed quality traits were also analyzed and it was found that the oil content had a negative correlation with the other three seed quality traits.     

Rapid and efficient screening of phosphinothricin tolerant oilseed rape (Brassica napus) with a novel germination test

A novel screening test is described for the discrimination of transgenic phosphinothricin tolerant oilseed rape from non transgenic rape seedlings. The method is based on the germination of rape seeds on filter paper soaked with a 0.005% phosphinothricin solution. Under these conditions inhibition of seedling development by the herbicide can be observed after 10 days. The germination test gains an advantage over the routinely used herbicide spraying, because it is rapid, needs little space and allows efficient screening of huge numbers of seeds. The assay has been successfully applied to the screening of different transgenic and non transgenic rapeseed varieties/lines and has been compared to other methods such asBasta® spray test, drop test, ELISA-technique and PCR-amplification of the pat gene. This test allows on one hand large screening programmes to monitor the foreign gene in the environment and on the other quality control of seedlots before market introduction of herbicide tolerant oilseed rape.     

Selection for fast germination in rapeseed (Brassica napus L. and B. Campestris L.)

Summary Eleven populations of Brassica napus L. and twelve populations of B. campestris L. were subjected to three cycles of selection for fast germination at 2°C and at 25°C. The seeds from the selected populations, and unselected control populations grown in the same environment as the selected populations, were examined for germination behaviour at 2°C and 25°C, and for growth behaviour at 10°C. The populations in both species responded differently in terms of germination behaviour to selection for fast germination. In most of the populations that did respond positively to selection, selection practised at 2°C was superior to selection at 25°C in improving percent germination at 2°C, and was as good as the selection at 25°C in improving germination rate at the higher temperature. Selection for fast germination had no effect on growth characteristics of B. napus and B. campestris populations grown at 10°C. Thus, selection for fast germination at one low temperature may lead to improvement in germination characteristics over a range of temperatures, but will not necessarily lead to improved growth performance of the selected populations.     

Superior performance of two homozygous diploid lines from naturally occurring polyhaploids in oilseed rape (Brassica napus)

Summary Homozygous diploids in oilseed rape were produced by pipetting drops of 0.1 per cent colchicine into the leaf axils of rooted cuttings from naturally occurring polyhaploids after removing the waxy covering with a scalpel. Trial results from spring and winter rape showed that occasional homozygous diploid lines yielded consistently more oil per ha than the parental varieties. Prospects for breeding oilseed rape as a self-fertilised crop are discussed.     

The transfer of characters from Brassica campestris L. to Brassica napus L.: Production of clubroot-resistant oil-seed rape (B. napus ssp oleifera)

Summary Four oil-seed rape lines were crossed with a clubroot resistant Brassica campestris line from the European Clubroot Differential sct. The allotriploid hybrids were backcrossed to the rape lines to introgress clubroot resistance into oil-seed rape. Using a combination of screening for disease resistance and chromosome number, a high proportion of 38-chromosome, clubroot resistant selections were obtained.     

Inter-plot competition in yield trials of swedes (Brassica napus ssp. rapifera L.)

Summary Two experiments were done to evaluate the use of single-row plots for assessing dry-matter yield of swedes. In the first experiment the correlation between the dry-matter yields of 20 cultivars in unguarded single-row plots and those in guarded double-row plots was poor (r=0.39). It was improved (r=0.58) by adjusting the single-row plot fresh-weight yields for inter-plot competition, through an analysis of covariance in which the covariate was the mean of the adjacent plot yields, and then estimating the pure stand dry-matter yields. However, the correlation was still lower than that between unguarded double-row plots and the guarded double-row plots (r=0.73), and this latter correlation was also improved (r=0.82) by adjusting the fresh-weight yields of the unguarded plots for inter-plot competition.The second experiment provided an explanation for the partial success of the covariance adjustment. The individual neighbour effects of six cultivars were determined. Doon Major and Ruta Øtofte decreased the yields of adjacent cultivars whereas Dryden and Melfort increased them. Angela and Magres had little effect. The correlation between the dry-matter yield neighbour effects of cultivars and their fresh-weight yields was high (r=–0.81), but not complete. So although competitive ability was associated with root fresh-weight yield, other factors must also be involved.It was concluded that a two-replicate trial with unguarded double-row plots is preferable to a four-replicate trial with unguarded single-row plots for dry-matter yield assessment of early generation families in a pedigree swede breeding programme.     

RAPD markers linked to microspore embryogenic ability in Brassica crops

In order to identify the markers linked to microspore embryogenic ability in Brassica crops, RAPD segregation analyses were performed in a microspore-derived (MD) population and a F₂ population derived from F₁between ‘Ho Mei’ (high responsive parent in microspore embryogenesis) and ‘269’ (low responsive parent) in Chinese cabbage, and between ‘Lisandra’ (high responsive parent) and ‘Kamikita’ (low responsive parent) in oil seed rape. After 230 and 143 primers were screened, a total of 148 and 52markers were detected to be polymorphic between the parents in Chinese cabbage and oilseed rape, respectively. Twenty-seven percent of the markers in the MD population showed a significant segregation distortion in both crops. Of the markers showing segregation distortion in the MD population, 71–75% of the markers followed the expected Mendelian segregation ratio in the F₂ population. When the relationships between such distorted markers and microspore embryogenesis of the F₂ population were examined, 7 and 3 markers were identified to be associated with embryogenic ability in Chinese cabbage and oilseed rape, respectively. These markers showed additive effects on embryo yields, and the plants having more alleles of the high responsive parent produced higher embryo yields. These markers maybe useful in marker-assisted selection for improving microspore responsiveness straits in Brassica crops. This revised version was published online in July 2006 with corrections to the Cover Date.     

The introgression of S alleles into forage rape,Brassica napus L. from turnip. Brassica campestris L. ssp. Rapifera

Summary Forage rape. B. napus, is self-compatible, the work described illustrates the introgression of functional incompatibility alleles into B. napus from turnips, B. campestris, in two generations. By grading seed, produced by backcrossing turnip/rape hybrids to rape, the frequency of 2n=38 semi articial B. napus recovered, amply justifies the cytological screening involved in such breeding programmes.     

Self-incompatibility interactions in Brassica napus

Summary The interactions of nine S-gene lines of Brassica napus were examined. Seven different S-genes were involved in the crosses, with three lines of different origin having the same S-gene. In general, the reactions on selfing the heterozygotes were as expected from the results of test crosses with the homozygous parent lines. All types of dominance relationships were found, including reversal of dominance in pollen and pistil. Weakening of activity of one or both genes was found in several cases. Background genotype was found have to some effect and reciprocal differences in reactions were also observed.     

Abundance, polymorphism and genetic mapping of microsatellites in oilseed rape (Brassica napus L.)

The objective of the present study was to estimate the abundance and degree of polymorphism of simple sequence repeat (SSR) markers in rapeseed. By screening about 45000 clones of a small inserts library of rapeseed total DNA the abundances of GA/TC and CA/TG simple sequence repeats in the rapeseed genome were estimated to be approximately one repeat every 100 kb and 400 kb, respectively. After sequencing 13 positive clones, primer pairs could be designed for 11 microsatellite loci. Seven of these primer pairs produced reproducible amplification products in a set of 31 rapeseed genotypes, with one pair amplifying two independent products, giving a total of eight amplified loci. The different microsatellite loci displayed between one and three visible alleles. At four loci, additional null alleles were observed. With up to four alleles, polymorphic microsatellite markers show significantly higher allele numbers in rapeseed than restriction fragment length polymorphism (RFLP) markers. Four of the eight microsatellite markers could be mapped on four different linkage groups of an RFLP map of the rapeseed genome.     

Evaluation of cytoplasmic effects on agronomic and seed quality traits in two doubled haploid populations of Brassica napus L.

Cytoplasmic effects have been occasionally implicated in the inheritance of several traits in oilseed rape (Brassica napus L.), including linolenic acid concentration (18:3) in the oil. It is important that these be considered when choosing the direction of cross for producing new breeding populations. To study this phenomenon, a reciprocal cross was made between two genotypes of oilseed rape, Reston and LL09, which differed for their erucic and linolenic acid concentrations in the seed oil. Two DH populations, which were produced by microspore culture from reciprocal F₁ plants, were evaluated in the growth room for one generation and in the field at two locations in Southern Ontario in 1993and 1994. Field notes were taken on days to flower, days to maturity,plant lodging, plant height and, seed quality traits. In the growth room study, the phenotypic distribution of 18:3 differed significantly between the two reciprocal DH populations. In the field, significant reciprocal differences between the population means were detected for 18:3,flowering date and protein content in both years and for days to maturity and oil content in 1993 only. To further study the parental lines,chloroplast (cp) and mitochondrial (mt) DNA from parental lines were isolated and subjected to RFLP and RAPD analysis. Several random primers revealed reproducible DNA polymorphism (RAPD) between the parental mt DNA. It is concluded that the direction of cross should be taken into consideration by oilseed rape breeders relying solely on doubled haploids for developing genotypes with modified seed quality traits in Brassica napus L. This revised version was published online in July 2006 with corrections to the Cover Date.     

AFLP and SCAR markers linked to the suppressor gene (Rf) of a dominant genetic male sterility in rapeseed (Brassica napus L.)

Rs1046AB is a line which is true breeding for a dominant genetic male sterility gene (Ms) but which is a mixture of male fertile and sterile individuals (a two-type line) because it is segregating for a dominant suppressor gene (Rf). This system provides a promising alternative to the CMS system for hybrid breeding in Brassica napus. In order to identify molecular markers linked to the rf gene, a near-isogenic line (NIL) population from the cross between a sterile individual (MsMsrfrf) and a fertile individual (MsMsRfrf) in Rs1046AB was subjected to amplified fragment length polymorphism (AFLP) analysis, with a combination of comparing near isogenic lines (NILs) and bulked segregant analysis (BSA). From 2,816 pairs of AFLP primers, six fragments showing polymorphism between the fertile and sterile bulks as well as the individuals of the bulks were identified. Linkage analysis indicated that the six AFLP markers are tightly linked to the Rf gene and all are distributed on the same side. The minimum genetic distance between the Rf gene and a marker was 0.7 cM. Since the AFLP markers are not suitable for large-scale application in MAS (marker-assisted selection), our objective was to develop a fast, cheap and reliable PCR-based assay. Consequently, three of the four closest AFLP markers were converted directly to sequence characterized amplified region (SCAR) markers. For the other marker a corresponding SCAR marker was successfully obtained after isolating the adjacent sequences by PCR Walking. The available SCAR markers of the Rf gene will greatly facilitate future breeding programs using dominant GMS to produce hybrid varieties.