Development of a new initial breeding material in sunflower (Helianthus annuus L.) using direct organogenesis and somatic embryogenesis

Summary Immature zygotic embryos from three inbred lines of sunflower (Helianthus annuus L.) were used as donor material for the induction of direct organogenesis and somatic embryogenesis. The scope of the spontaneously appearing somaclonal variation has been studied among the regenerants produced according to the method of Freyssinet & Freyssinet (1988), and was compared to the results obtained after gamma ray mutagenesis (7 and 10 Gy). Freshly excised immature zygotic embryos were used for irradiation treatment. Genetic changes occurring spontaneously during the regeneration procedure were observed for date of flowering, vegetation period, plant height, head diameter, 1000 seed weight, kernel oil content, and fatty acid composition. The mutagenic treatment had a marked stimulatory effect on the frequency of the regenerants produced and the degree of the observed changes. The 7 Gy treatment was most efficient for the majority of the characters studied. The type of changes was correlated with the genotype. The degree and type of somaclonal variation, with or without mutagenic treatment, should be sufficient for an application in the production of new breeding material.     

Variation of prezygotic barriers in the interspecific hybridization between oilseed rape and wild radish

Escape of transgenes from genetically modified oilseed rape, Brassica napus, into wild radish, Raphanus raphanistrum, depends on sexual compatibility. The variation in prezygotic barriers of two different cultivars for interspecific hybridization with a population of wild radish was investigated by hand crossing and fluorescence microscopy of pistils. Significant differences were observed between oil seed rape cultivars in their ability to accept wild radish pollen germinating onto their stigma and the rate of fertilization of ovules. Some differences among the pollen donor plants were also detected. These results suggest that the rate of interspecific hybridization in the field would depend upon the oilseed rape cultivar and the genotype composition of the local wild populations. The implication of S-related genes, as revealed through identification by pistil tissue prints of class I and II S-types of SLG (S-Locus Glycoprotein) and SLR1 (S-Locus Related),and immuno-IEF, was not significant. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

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.     

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.     

Variation for agronomic characteristics in Crambe hispanica, a wild relative of Crambe abyssinica

Summary In order to assess the potential of Crambe hispanica, either in breeding programmes of C. abyssinica or as an oilseed crop in itself, 36 accessions of C. hispanica (29 of var. hispanica and 7 of var. glabrata) were grown in a greenhouse and evaluated for morphological characteristics, earliness, plant habit, seed characteristics and fatty acid composition. Four lines of C. abyssinica were included for reference. The 29 accessions of var. hispanica showed significant variation for all observed characteristics. Besides morphological characteristics, large variation was found for earliness, number of primary branches, seed yield, 1000 seed weight and volume, and linoleic, linolenic and erucic acid content. Morphological characteristics, earliness and plant habit did not show any high correlations with seed characteristics or fatty acid composition, except for seed hull (pericarp) mottling, which was related to a high oil and erucic acid content. The seven accessions of var. glabrata showed little variation. The large genetic variation in combination with promising figures for several characteristics, found in C. hispanica, may be useful in breeding programmes of the oilseed crop C. abyssinica, for which the available genetic variation is limited. Prospects of selection for high-erucic acid genotypes are discussed. Compared to C. abyssinica, both botanical varieties of C. hispanica are characterized by a cordate shape of the basal leaves, lack of seed retention and a lower DNA content. Plants of var. glabrata differed from var. hispanica in a sparsely hispid upper leaf surface and round stems and branches covered with a waxy layer. These clear differences and lack of success in intercrossing both varieties of C. hispanica strongly suggest that their taxonomic classification should be reconsidered.     

Degradation of oxalic acid by transgenic oilseed rape plants expressing oxalate oxidase

Summary Oxalic acid is thought to have a primary role in the pathogenicity of several plant pathogens, notably Sclerotinia selerotiorum. A gene coding for the enzyme oxalate oxidase was isolated from barley roots and introduced into oilseed rape as a means of degrading oxalic acid in vivo. This report describes the production of several transgenic plants of oilseed rape and the characterisation of these plants by Southern, Western and enzyme activity assays. Plants were shown to contain an active oxalate oxidase enzyme and were tolerant of exogenously supplied oxalic acid.     

Identification of molecular markers associated with oleic and linolenic acid in spring oilseed rape (Brassica napus)

The quality of the oil derived from oilseed rape is determined by its fatty acid composition. Breeding oilseed rape for enhanced oil quality includes the development of cultivars with high oleic and low linolenic acid. Random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) techniques were investigated for the development of molecular markers for genes controlling oleic and/or linolenic acid. Markers that were identified were converted to sequence characterized amplified region (SCAR) markers for use in breeding. Molecular markers associated with these two fatty acids were identified in a doubled haploid population derived from a cross between the oilseed rape lines TO99‐5318‐20, very high oleic (>79%) and very low linolenic acid (<2%) × DH12075, high oleic (68%) and higher linolenic acid (>7%). Eight RAPD markers were associated with oleic and linolenic acid contents. The RAPD marker UBC 2₈₃₀ accounted for 43% and 13% of the genetic variation for oleic and linolenic acid levels, respectively. The RAPD marker UBC 153₅₅₀ accounted for 19% of the genetic variation for linolenic acid. The UBC 2₈₃₀ fragment was converted to a SCAR marker. The markers identified in this study should be useful tools for the early generation selection of high oleic and low linolenic acid genotypes in oilseed rape breeding programmes.     

Doubled haploid lines of Brassica carinata with modified erucic acid content through mutagenesis by EMS treatment of isolated microspores

Brassica carinata is a potential oilseed crop for the Mediterranean area. Chemical mutagenesis has been applied to microspores of B. carinata with the purpose of identifying lines with altered erucic acid content. From a population of nearly 400 doubled haploid plants recovered, nine lines have been identified that exhibit promising useful changes in erucic acid concentration in the seed oil. Three lines showed erucic acid contents below 25%, with a minimum of 17.1%, and in six lines the level of this fatty acid was greater than 52%. Changes in other fatty acids are also described and discussed.     

Leaf glucosinolate profiles and their relationship to pest and disease resistance in oilseed rape

Summary Glucosinolates are sulphur-containing glycosides which occur within vegetative and reproductive tissue of oilseed rape. Following tissue damage, glucosinolates undergo hydrolysis catalysed by the enzyme myrosinase to produce a complex array of products which include volatile isothiocyanates and several compounds with goitrogenic activity. Many of these products have been implicated in the interaction betweenBrassica and their pests and pathogens and some may have a role in defence mechanisms. Low glucosinolate (00) oilseed rape cultivars have been shown to possess similar concentrations of leaf glucosinolates as high glucosinolate (0) oilseed rape cultivars. Likewise, despite considerable speculation to the contrary, 00 cultivars have been shown not to be more susceptible to pests and pathogens than 0 cultivars. The potential to enhance pest and disease resistance of oilseed rape by manipulating the leaf glucosinolate profile without reducing seed quality is discussed.     

The application of chemical mutagenesis and biotechnology to the modification of linseed (Linum usitatissimum L.)

Summary In the early 1980s the phenomenon of somaclonal variation induced by cell culture was exploited to produce genetic variation in linseed. The linseed variety Andro, derived from the widely grown Canadian variety McGregor, was selected in saline culture and was released for production in Canada. Andro possesses traits very different from its parent, such as increased seedling vigour and tolerance to heat stress. Additional stable somaclonal variation in characters such as yield, days to maturity, seed weight and oil content were subsequently induced in McGregor. However, despite extensive screening of the somaclonal variants, no significant variation in the fatty acid profile was found.Chemical mutagenesis using ethyl methanesulphonate was, however, succesful in modifying the fatty acid profile of McGregor. Initial screening of M₂ seed by the thiobarbituric acid colourimetric procedure was followed by gas chromatography to select half-seeds with atypical fatty acid profiles. Two independent, partially dominant genes were identified that were responsible for reducing the linolenic acid (18 : 3) from 50% to 2% while increasing linoleic acid (18 : 2) to 70%. A single, partially dominant gene, inherited independently of the linolenic acid genes, increased palmitic acid (16 : 0) from 7% to 30% and palmitoleic acid (16 : 1) from trace amounts to 4%. Agrobacterium-mediated transformation of linseed has also been successful. Herbicide tolerance genes for glyphosate, sulfonylurea and phosphinothricin have been incorporated into Canadian varieties. Commercially useful levels of tolerance to sulfonylurea herbicides have been achieved with no adverse agronomic affect. It is expected that a transgenic variety containing this resistance will be registered for commercial production in Canada in 1994.Standard breeding techniques, the application of antisense technology and the overexpression of fatty acid synthesis genes are being used to further modify the fatty acid profile of linseed, as well as for the transfer of abiotic stress-related genes identified in bromegrass.     

The Effect of A Genome Substitution on the Resistance of Brassica napus to Infection by Leptosphaeria maculans

Six accessions belonging to four subspecies of Brassica rapa, including three accessions of B. rapa subsp. sylvestris, were crossed with B. oleracea subsp. alboglabra in order to develop a series of synthetic B. napus lines with a common C genome but contrasting A genomes. Different A genomes had significant effects on the efficiency of B. napus resynthesis and the sexual compatibility of the synthetic lines with oilseed rape cultivars. The synthetic lines were used to investigate the effect of A genome substitution on the resistance of B. napus to infection by Leptosphaeria maculans, and to explore the potential for the use of wild forms of B. rapa in oilseed rape breeding programmes. Synthetic lines derived from two wild accessions of B. rapa, and their F₁ hybrids with oilseed rape cultivars, expressed high levels of resistance to L. maculans in glasshouse experiments. One of these lines also expressed high levels of resistance in field experiments in England and Australia when exposed to a genetically diverse pathogen population. All other synthetic lines and cultivars were highly susceptible in both glasshouse and field experiments. F₁ hybrids between oilseed rape cultivars and synthetic lines derived from B. rapa subsp. chinensis were significantly more susceptible than either parent.     

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.     

Combination of resistance to Verticillium longisporum from zero erucic acid Brassica oleracea and oilseed Brassica rapa genotypes in resynthesized rapeseed (Brassica napus) lines

Resynthesized (RS) forms of rapeseed (Brassica napus L.; genome AACC, 2n = 38) generated from interspecific hybridization between suitable genotypes of its diploid progenitors Brassica rapa L. (syn. campestris; genome AA, 2n = 20) and Brassica oleracea L. (CC, 2n = 18) represent a potentially useful resource to introduce resistance against the fungal pathogen Verticillium longisporum into the gene pool of oilseed rape. Numerous cabbage (B. oleracea) accessions are known with resistance to V. longisporum; however, B. oleracea generally has high levels of erucic acid and glucosinolates in the seed, which reduces the suitability of resulting RS rapeseed lines for oilseed rape breeding. In this study resistance against V. longisporum was identified in the cabbage accession Kashirka 202 (B. oleracea convar. capitata), a zero erucic acid mutant, and RS rapeseed lines were generated by crossing the resistant genotype with two spring turnip rape accessions (B. rapa ssp. olerifera) with zero erucic acid. One of the resulting zero erucic acid RS rapeseed lines was found to have a high level of resistance to V. longisporum compared with both parental accessions and with B. napus controls. A number of other zero erucic acid RS lines showed resistance levels comparable to the parental accessions. In the most resistant RS lines the resistance and zero erucic acid traits were combined with variable seed glucosinolate contents. Erucic acid‐free RS rapeseed with moderate seed glucosinolate content represents an ideal basic material for introgression of quantitative V. longisporum resistance derived from B. oleracea and B. rapa into elite oilseed rape breeding lines.     

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.     

Effect of hardening on frost tolerance and fatty acid composition of leaves and stems of a set of faba bean (<Emphasis Type="Italic">Vicia faba</Emphasis> L.) genotypes

Frost tolerance is a main component of winter-hardiness and improving it would promote faba bean (Vicia faba L.) cropping in cool-temperate regions. In many species, leaf fatty acid composition was found to be related to frost tolerance. The objective of this study was to determine, in a representative sample of genotypes, the effect of hardening on leaf and stem (1) frost tolerance and (2) fatty acid composition, and to seek correlations between them. First leaf, second leaf and stem of 31 faba bean genotypes were analyzed after hardening and without hardening. High frost tolerance of known winter genotypes and several experimental lines was shown. Hardening had a significant, positive effect on frost tolerance of all three organs. Stems were on average more frost tolerant than leaves. Hardening induced significant changes in the fatty acid composition: oleic acid decreased significantly in leaves by 3.24% and in stems by 1.77%, whereas linolenic acid increased in leaves by 6.28% and in stems by 9.06%. In stems, correlations between frost tolerance and fatty acid composition were not significant. Correlation coefficients strongly indicated that non-hardened oleic acid content, changes in oleic acid and in linoleic plus linolenic acid content in leaves partly explained their frost tolerance; 0.347 (P < 0.1) < |r| < 0.543 (P < 0.01). The results corroborate the importance of using genetic differences in the fatty acid metabolism in breeding grain legumes for frost tolerance.     

Interspecific transfer of Brassica juncea-type high blackleg resistance to Brassica napus

Summary Complete resistance to Leptosphaeria maculans, the cause of blackleg of oilseed rape (Brassica napus), was transferred from B. juncea to B. napus through an interspecific cross. B. juncea-type complete resistance (JR) was recognized first in one F₃ progeny (Onap^JR) by the absence of leaf-lesions on seedlings and canker-free adult plants. The commercially important characters of B. napus were retained in advanced lines of Onap^JR, which combined JR with low erucic acid levels (<0.5%), high seed yield and variable maturity dates.JR appeared to be inherited as a major gene or genes. Segregation for resistance and susceptibility contintied to occur during later generations of selection of Onap^JR. JR was readily transferred from Onap^JR to other suitable B. napus cultivars or lines with partial resistance to blackleg and resulted in highly vigorous carly generation selections adapted to cold, wet situations along with complete resistance to blackleg.     

Phenotypic variation for saturated fatty acid content in soybean

Summary Concern over high saturates in human diets has prompted the development of soybean [Glycine max (L.) Merr.] lines producing oil with reduced saturated fatty acid concentration. To better understand those factors that influence phenotypic expression for palmitic and stearic acid content in soybean, thirty soybean lines random for saturated fatty acid content were grown in eight field environments contrasting for mean temperature during seed-filling. Palmitic and stearic acid content varied significantly (P<0.01) both among genotypes and across environments, while genotype x environment interactions were reflected in changes in line variance and ranking for both traits. Therefore selection of a superior genotype for saturated fatty acid composition may not correlate well from one environment to another. In general, early-maturing lines were less sensitive than later-maturing lines in their response to changes in mean daily temperature for palmitic concentration. However, factors in addition to temperature appeared to influence genotype response for stearic acid content. It appears that genetic systems conditioning palmitic and stearic acids are independent, and that separate breeding strategies need be adopted to make simultaneous improvement for these two oil traits. In summary, development of soybean lines with low or high saturated fatty acid content may be accomplished through evaluation and selection in a few environments contrasting for temperature.Cooperative investigations of the USDA-ARS, and North Carolina Agric. Res. Serv., Raleigh, NC, U.S.A.     

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.     

New mutants of winter rapeseed (Brassica napus L.) with changed fatty acid composition

Seeds of the winter oilseed rape (Brassica napus L.) line PN 3756/93 were treated with ethyl methanesulphonate to induce mutations in the fatty acid biosynthetic pathway. The seed mutagenic treatment was repeated in the M₂ generation. After treatments, individual seed and plant selections were made for changes in fatty acid composition during several generations of inbreeding. Self‐pollinated plants with changed fatty acid compositions were inbred to obtain genetically homozygous and stable mutant lines. Two mutants, M‐10453 and M‐10464, with increased levels of oleic acid (approximately 76%) and reduced linoleic and linolenic acid contents (8.5% and 7.5%, respectively) were selected. Gene or genes controlling desaturation of oleic acid were probably mutated in these plants. The third mutant, M‐681 had a very low linolenic acid content (approximately 2.6%) and increased linoleic acid content (approximately 26%). This would suggest the occurrence of mutations in genes controlling linoleic acid desaturation. The results of selection work during several generations showed that the environment had substantial influence on the composition of seed oil. This made the search for mutants with modify fatty acid compositions difficult. The induced mutants are not directly usable as new varieties, but can be used as parents in crosses for the development of high quality rapeseed varieties.