Importance of sepals in fruit and seed development in linseed (Linum usitatissimum L.)

Summary The green sepals in linseed (Linum usitatissimum L.) persist till almost the time of fruit ripening. Since the plant retains very few leaves and the fruits are better exposed to receive sunlight, the green sepals may be important in fruit and seed development. Influence of the removal of 0, 1, 3 and 5 sepals on the day of anthesis, 7, 15 and 25 days after anthesis on fruit and seed development was examined. Excision of all the fine sepals on the day of anthesis stopped fruit and seed development in the varieties M 10 and NP (RR) 45 but in Mukta the reduction was about 55%. Removal of three sepals also decreased fruit growth in Mukta and NP (RR) 45. The effect of removal of sepals was more pronounced on the number of seeds per fruit than on seed weight. Sepals fixed ¹⁴CO₂ and translocated ¹⁴C to seeds. However, the ¹⁴CO₂ fixation was maximum at early stages of fruit development, whereas a higher percentage of the fixed ¹⁴C was translocated at later stages of growth. It is suggested that the sepal size in linseed could be a useful selection criterion.     

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.     

Inheritance of resistance to Oidium liniSkoric in linseed (Linum usitatissimum L.)

Summary Resistance to powdery mildew (Oidium lini) in each of the four linseed cultivars, viz., LC 216, LC 255, LC 256 and LC 269 was found to be conditioned by one dominant gene. Crosses between the resistant parents indicated that the same gene was present in all the cultivars and is designated as Ol.     

Genetic analysis of the response of linseed (Linum usitatissimum L.) somatic tissue to in vitro cultivation

The response of linseed somatic tissue to in vitro cultivation can be improved if the gene action involved is known. Nine homozygous lines, seven F₁ and fifty F₂ plants were cultivated in MS basal medium with 0.1 mg/l BAP. Callogenesis and regeneration analyses were done using a random model in order to separate environmental and genotypic effects at 10, 20 and 30 days after culture initiation. Genetic variability for callogenesis and regeneration was observed, that could be used in a breeding program. The appropriate period for the evaluation of callogenesis was at 20 days after culture initiation. For regeneration, this moment would depend on the selection criterion adopted. To select genotypes on the basis of earliness in shooting at 10 days would be preferable, while for ability of producing shoots, 30days would be more suitable. For both characters the analyses of genetic action indicated that, non-additive effects were involved in the culture response, however the expression of callogenesis and regeneration ability had different genetic basis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparison of pedigree selection and single seed descent for oil yield in linseed (Linum usitatissimum L.)

Summary Four populations of linseed derived from five parents were advanced from the F₂ to the F₆ generation by SSD and to the F₅ by pedigree selection. These populations were used to compare the efficiency of the two methods in order to provide superior genotypes with respect to grain yield and oil content, i.e. oil yield. The results showed minimum differences between pedigree and SSD lines for grain yield, where in only one cross the SSD lines were significantly superior to the pedigree lines. Since pedigree selection was carried out for both, seed yield and oil content, a positive response to selection was expected. However, early selection for yield, a character with low heritability, was not successful. On the contrary, significant differences for oil content were detected between the two groups of lines in three of the four crosses studied. In these cases the pedigree lines were superior to the SSD lines. These results demonstrate that strict selection in early generations for oil content, a character with comparatively high heritability, is feasible and successful in linseed. However, selection for seed yield should be postponed to later inbred generations. Consequently, in breeding for maximum oil yield of linseed a two-step selection procedure is recommended.Abbreviations PS pedigree selection - SSD single seed descent - TGW thousand grain weight     

Parametric and nonparametric measures of phenotypic stability in linseed (Linum usitatissimum L.)

A combined analysis with three parametricand two nonparametric measures to assess G × E interactions and stability analyses toidentify stable genotypes of linseed across18 environments in Ethiopia wereundertaken. The combined analysis ofvariance for environments (E), genotypes(G) and G × E interaction was highlysignificant (p<0.01), suggestingdifferential responses of the genotypes andthe need for stability analysis. Theparametric stability measures ofcoefficient of variability and thestability variance showed that R12-N10D wasthe most stable genotype, whereascultivars' superiority measure indicatedChilalo to be the most stable cultivar.Like most of the parametric methods, thenon-parametric measures revealed thatR12-N10D had the smallest changes in ranksand thus was the most stable genotype incontrast to R12-D24C, which was unstableand the lowest yielder. A comparison of thefive stability measures showed that thecoefficient of variability, stabilityvariance and variance of ranks were similarin assessing the relative stability of thegenotypes, whereas cultivars' superioritymeasure deviated from the others. Thestability variance and variance of rankswere significantly rank correlated, andwere the best in determining thecomparative stability of linseed genotypes.The coefficient of variability was alsorelatively better than the cultivar'ssuperiority measure. Further studies ofrepeatability tests are, however, needed todetermine the best methods. The stabilitystatistics generally identified R12-N10D,followed by Chilalo, as the most stablevarieties, whereas R12-D24C and R11-M20Gwere the least stable varieties.     

Genotypic and exogenous factors affecting shoot regeneration from anther callus of linseed (Linum usitatissimum L.)

Summary The objective of this study was to investigate factors affecting the regeneration capacity of linseed anther culture. Four different environmental conditions in a phytotron were tested with regard to their effects on anther donor plants of cv. Hella. Anther response and shoot regeneration from anther callus was maximal when donor plants were grown in a 16 hrs-day at 14°C day/8°C night temperature. Anthers of four linseed genotypes were cultured on different media. Maximum shoot regeneration was achieved when the induced calli were transferred onto a modified N6 medium containing zeatin (1 mg l^-1). Most of the calli regenerated shoots in the second subculture on regeneration media. Shoots were rooted on modified B5 or MS media containing NAA (0.1 mg l^-1). Cytological examinations of incubated anthers and root tips of regenerated plants indicated that the anther calli were derived from microspores.Abbreviations B5 Gamborg's (1975) medium - BAP 6-benzylaminopurine - 2,4D dichlorophenoxyacetic acid - N6 Chu's (1978) medium - NAA -naphthaleneacetic acid - MS Murashige & Skoog's (1962) medium - ZEA zeatin     

Breeding for Modified Fatty Acid Composition by Induced Mutations in Linseed (Linum usitatissimum L.)

Seeds of linseed (Linum usitatissimum L.) cultivars ‘Raulinus’ and ‘Bionda’ were treated mutagenically with ethyl methanesulfonate (EMS) in order to broaden the variation of fatty acid composition in the seed oil and to select mutants with reduced linolenic acid (C18: 3) content. The M₂ generation was evaluated for fatty acid composition of the oil. Genetic variation for unsaturated fatty acids was demonstrated to be extended in the M₂ generation as compared to the controls. Finally in the M₅ generation of cv. ‘Raulinus’, lines with reduced C18:3 (< 40% vs. 55%) and correspondingly increased linoleic or oleic acid contents, respectively, could be selected. These mutants proved to be genetically stable as demonstrated by parent-offspring (M₄/M₅) regression.     

Comparison of Mahalanobis's method and metroglyph technique in the study of genetic divergence in Linum usitatissimum L. germ plasm collection

Summary A group of fifty-seven varieties of Linum usitatissimum L. collected from different geographics sources was studied to understand the nature of genetic divergence using Mahalanobis's statistical distance (D²). Varieties having genetic similarity were grouped into fifteen clusters. Most of the clusters at individual level included varieties of different geographical regions. However, Argentine and Indian types formed separate and distinct constellations. The Indian group was situated at a maximum distance from Argentine and US groups, and was nearest to the Australian group. For improvement of Indian linseed, hybridization of indigenous types with Argentine and US types might result in superior recombinants.84.64% of the total divergence was attributable to plant height and seed size, which are based characteristics used in classifying the species into oil and flax types.Since it is difficult to compute D² values among large numbers of germplasm collections, another relatively simple and easy method, the metroglyph technique, was tried upon the same set of fifty-seven varieties. Comparisons between the results of the two analyses revealed the striking similarity as regards to group arrangement. It has been suggested that the metroglyph technique would be suitable for preliminary grouping prior to undertaking D² analysis.     

In vitro breeding of high-linolenic, doubled-haploid lines of linseed (Linum usitatissimum L.) via androgenesis

In the course of a breeding programme for high-linolenic-acid linseed (oilflax, Linum usitatissimum L.) doubled-haploid lines (DH-lines) of three F₁ hybrids were generated using an anther-culture technique. A total of 82 DH-lines were generated and multiplied in 1993, of which 39 were suitable for testing in a repeated field trial in 1994. Yield, fat content and fatty-acid composition were determined. The yield of the DH-lines was not significantly different from that of respective mid-parent values. Fatty-acid composition and fat content showed remarkable variation, and DH-lines exceeded cross parents in some cases for both fat content and linolenic-acid proportion. Superior DH-lines surpassed mid-parent linolenic-acid-content values by 4.5% and fat content by 1.9%. The results indicate that anther culture is a useful tool in linseed breeding.     

Selection for reduced linolenic acid content in Ethiopian mustard (Brassica carinata Braun)

Reducing linolenic acid content is one of the most important objectives for the development of Ethiopian mustard lines with high oil quality. This work was aimed at searching for variability of the fatty acid composition of oil within a germplasm collection of Ethiopian mustard. A total of 217 lines were analysed by gas-liquid chromatography (GLC) in 1991, and one was selected as having reduced values of both linolenic acid content (10.2% versus 14.0% of total fatty acids as the collection average) and linoleic acid desaturation ratio (LDR, 0.34 versus 0.45). After 3 years of pedigree selection for low linolenic acid content, this line showed, in 1995, average values of this fatty acid of 5.4% and 2.4% in two different environments, compared with 11.6% and 8.3%, respectively, in the control. The values of the LDR were 0.18 and 0.09, respectively, compared with 0.36 and 0.27 in the control line.     

Seed colour, seed weight and seed oil content in Linum usitatissimum accessions held by Plant Gene Resources of Canada

The purpose of this study was to investigate variation of and relationships among seed colour, seed weight and seed oil content in cultivated flax (Linum usitatissimum L. ssp. usitatissimum). Seed from 2934 flax genebank accessions recently grown at Saskatoon, SK, Canada, originating from 72 countries was used to describe the variation of the seed characters. The dominant seed colour of the accessions was medium brown (2730 accessions, 93.0%), followed by yellow (126 accessions, 4.3%). Based on single observations for all accessions, the overall mean and standard deviation was 5.95 ± 1.22 mg/seed for seed weight and 38.3 ± 1.74% for oil concentration. Within three infraspecific groups of flax, seed weight, oil concentration and oil amount per seed increased in the following order: fibre flax (convar. elongatum), intermediate flax (convar. usitatissimum), large‐seeded flax (convar. mediterraneum). The world collection exceeded the range of variation of seed weight and oil concentration found in 52 North American cultivars. There was a weak, positive association of higher oil concentration with higher seed weight (r² = 0.32; P < 0.001). Yellow‐seeded flax had a higher seed weight (6.31 vs. 5.92 mg/seed) and oil concentration (39.4% vs. 38.3%) than brown‐seeded flax. There was a tendency for yellow seed colour to be associated with higher oil concentration in all seed weight classes. The results suggested that indirect selection for increased seed oil concentration in flax is possible by selection for higher seed weight and yellow seed colour.     

Isolation of induced mutants in Ethiopian mustard (Brassica carinata Braun) with low levels of erucic acid

Ethiopian mustard (Brassica carinata Braun) is a potential oil crop for the rain-fed Mediterranean area. However, its usage is limited by the high erucic and high glucosinolate content of the oil and meal, respectively. In the course of a mutagenesis programme, an agronomically good line of Ethiopian mustard was treated with EMS in order to widen the natural variability of nutritional traits in this species. As a result of this programme several low erucic mutants were isolated; two of these mutants showed erucic acid values in the M4 generation in the range 5–10% of total fatty acids. Near-infrared reflectance spectroscopy (N1RS) was successfully applied as a rapid screening method for erucic acid in this breeding programme.     

Embryonic heterosis in the linolenic acid content of Matthiola incana seed oil

Summary The fatty acid composition of seed-oil of breeding lines and F₁ hybrids of Matthiola incana was analyzed, using direct esterification and gas chromatography. The breeding lines tested differed significantly with respect to the levels of palmitic, oleic, linoleic and linolenic acids. Embryonic-stage heterosis in linolenic acid concentration was demonstrated by F₁ hybrid seeds, derived from mating horticulturally different lines of M. incana. Linolenic acid content was negatively correlated with both oleic acid content (r=–0.85) and linoleic acid content (r=–0.66). None of the breeding lines or the F₁ hybrids significantly passed the limit of 67% linolenic acid. Possible genetic and biochemical explanations for the above phenotypic data are discussed.     

Allelic variation in linolenic acid content of high erucic acid Ethiopian mustard and incorporation of the low linolenic acid trait into zero erucic acid germplasm

Zero erucic acid germplasm of Ethiopian mustard is characterized by high levels of linolenic acid (about 21%). Two genetic sources of low linolenic acid (N2‐4961 and HF‐186, about 5%), have been developed in a high erucic acid background. The objectives of this research were to study the genetic relationship between the two low linolenic acid lines and to transfer the trait to zero erucic acid germplasm. F1 seed generations from crosses between both lines had higher average linolenic acid concentration than both parents. F2 seeds segregated for linolenic acid content following a continuous variation from 1.8 to 7.4%, exceeding the limits of the parental distribution ranges. Transgressive recombinants with very low linolenic acid concentration (0.7‐2.7%) were confirmed in the F3 seed generation. The results suggested that N2‐4961 and HF‐186 possess alleles for low linolenic acid at different loci. Transgressive low linolenic acid F3 plants were crossed with plants of a zero erucic acid line and a selection for zero erucic, low linolenic acid was conducted. As a result, a zero erucic acid F3:4 line containing 1.5 ± 0.7% linolenic acid was developed.     

Inheritance of reduced linolenic acid content in the Ethiopian mustard mutant N2-4961

The zero erucic acid Ethiopian mustard lines developed so far are characterized by an exceptionally high linolenic acid content in the seed oil. The mutant line N2‐4961, expressing low linolenic acid content in a high erucic acid background, was developed through chemical mutagenesis. The objective of this research was to study the inheritance of low linolenic acid content in this mutant. Line N2‐4961 was reciprocally crossed with its parent line C‐101 and the linolenic acid content of the reciprocal F₁, F₂ and BC₁ generations was studied. No maternal, cytoplasmic or dominance effects were detected in the analysis of F₁ seeds and F₁ plants from reciprocal crosses. Linolenic acid content segregated in 1: 2: 1 ratios in all the F₂ populations studied, suggesting monogenic inheritance. This was confirmed with the analysis of the reciprocal backcross generation. The simple inheritance of low linolenic acid content in N2‐4961 will facilitate the transference of this trait to zero erucic acid lines of Ethiopian mustard.     

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.     

The effect of T-DNA on the agronomic performance of transgenic flax plants

Summary Registration and commercial release of crop cultivars improved using recombinant DNA technologies will require extensive field testing of the transgenic lines. In addition to testing the efficacy of the transferred gene(s), regulatory bodies and farmers need data from large scale agronomic studies to provide assurance that the foreign DNA does not adversely affect normal agronomic productivity or quality of the crop. Here, five transgenic sulfonylurea resistant flax (Linum usitatissimum) lines were included in a multiple location cultivar registration trial, evaluated and compared with four standard commercial cultivars and 24 other (conventionally produced) breeding lines. The transgenic lines did not appear to be affected by T-DNA for any quality or agronomic parameter tested, including yield.     

Impact of low linolenic acid content on seed yield of winter oilseed rape (Brassica napus L.)

An essential quality improvement of rapeseed oil can be obtained by reduction of its linolenic acid (C18:3) content from about 10% to less than 3% of the total fatty acids. Genotypes low in C18:3 have been developed by mutagenesis. The initial summer rapeseed mutant had been low yielding and highly susceptible to various diseases. It has been debated whether the low C18:3 character can be successfully combined with high seed yield for physiological reasons. Therefore, the low linolenic character of mutant M48 was transferred into high-yielding genotypes by repeated backcrossing to well-adapted low erucic acid, low glucosinolate (00-) winter rapeseed cultivars. Lines with low C18:3 content were selected from BC3 and BC4 generations and examined in 1990–95. Positive selection response for seed yield was shown to continue over the years. Presently, the best lines are yielding as well as the control cultivars being equivalent also in oil and glucosinolate contents. In order to test the effect of a low C18:3 content on seed yield, plants with low and with high C18:3 content, respectively, were selected from 16 segregating BC5-F₂ populations and bulked to form 32 F₃ populations. These ‘isogenic’ bulk populations were tested for field performance at four locations in 1995. The results show that C18:3 content of the seed oil is not associated with seed yield, oil content, beginning of flowering, plant height and disease resistance. Means of relative seed yield for the high and the low linolenic F₃ bulk populations were not significantly different with 88.0% and 86.9% of the control cultivars, respectively. There was a significant interaction between genotypes with high or low C18:3 content and location. This shows that under specific environmental conditions a low C18:3 content may be either favourable or unfavourable. The results indicate that the low C18:3 character of the original mutants per se does not cause a decrease in seed yield, oil content or general field performance.     

Identification of RAPD markers linked to linolenic acid genes in rapessed

Summary The inheritance of the low linolenic acid content (derivated from mutant lines) in rapeseed was investigated. Molecular techniques of gene mapping through RAPD markers were applied on a microspore-derived progeny from a high × low linolenic acid F₁ hybrid. Bulked segregant analysis made it possible to test rapidly number of RAPD primers. Two linkage groups of 6 markers (72.7 cM and 75.6 cM) were determined. Each corresponded to a major QTL which explained 24% and 30.7% of the total phenotypic variation of the linolenic acid content. It was confirmed that two independant mutations were implied in the low linonenic acid content.