Genetic control of seed weight in flax (Linum usitatissimum) and possible implications

Summary Mean seed weight data were obtained from the F₁ and F₂ of a six-by-six diallel cross with flax (Linum usita-tissimum L.). Pronounced reciprocal differences appeared in the F₁, but had largely disappeared by the F₂. The genetic control of mean seed weight was examined using two types of analysis of variance. The models underlying both analyses were fitted to the data by matrix methods supplying weighted least-squares estimates of the parameters in the models. Weights, the use of which dealt with the problem of variation in the reliability of means, were the reciprocals of the variances of individual cell (cross/self) means in the diallel data table. Elimination of redundant parameters supplied the minimum adequate models for each analysis type. Dominance was apparently masked by the large transient maternal effects in the F₁, but surfaced in the F₂, where dominance was towards larger mean seed weight. This may be coupled with findings elsewhere of possible advantages for larger seed weights to speculate on a role in preserving infrequent hybrid progeny among inbreeding (flax) species. Maternal effects producing larger seed size, plus dominance with the same result might be valuable, in conjunction with growth and competitive advantages conferred by larger seed, in preventing early elimination of rare hybrids.     

A possible role of prolyl oligopeptidase during Linum usitatissimum (flax) seed development

Involvement of prolyl oligopeptidases (POPs) in the control of several mammalian peptide hormone signalling pathways has been studied extensively in recent years. POPs are ubiquitous enzymes, but little attention has been paid to understanding their function in plants. Using a cDNA-AFLP approach, two flax (Linum usitatissimum) POP ESTs were identified as being specifically expressed in the early stages of flax seed development. This specific expression was confirmed using real time RT-PCR and in situ hybridisation approaches. Seed expression of Arabidopsis POP genes was measured and showed no specificity. Comparison between results obtained with flax and Arabidopsis is discussed in order to address a hypothetic function for POPs during seed formation. These results provide the first insights into POP gene expression and hypothetical function in plants.     

Characterization of transgenic sulfonylurea-resistant flax (Linum usitatissimum)

Summary Fourteen transgenic flax (Linum usitatissimum) lines, carrying a mutant Arabidopsis acetolactate synthase (ALS) gene selected for resistance to chlorsulfuron, were characterized for resistance to two sulfonylurea herbicides. Progeny of 10 of the 14 lines segregated in a ratio of 3 resistant to 1 susceptible, indicating a single insertion. Progeny of 1 line segregated in a 151 ratio, indicating two insertions of the ALS gene at independent loci. Progeny from 3 lines did not segregate in a Mendelian fashion and were likely the products of chimeric shoots. Resistance to chlorsulfuron was stably inherited in all lines. At the enzyme level, the transgenic lines were 2.5 to more than 60 times more resistant to chlorsulfuron than the parental lines. The transgenic lines were 25–260 times more resistant to chlorsulfuron than the parental lines in root growth experiments and demonstrated resistance when grown in soil treated with 20 g ha^-1 chlorsulfuron. The lines demonstrated less resistance to metsulfuron methyl; in root growth experiments, the transgenic lines were only 1.6–4.8 times more resistant to metsulfuron methyl than the parental lines. Resistance was demonstrated in the field at half (2.25 g ha^-1) and full (4.5 g ha^-1) rates of metsulfuron methyl.     

SSR and morphological trait based population structure analysis of 130 diverse flax (Linum usitatissimum L.) accessions

A total of 130 flax accessions of diverse morphotypes and worldwide origin were assessed for genetic diversity and population structure using 11 morphological traits and microsatellite markers (15 gSSRs and 7 EST–SSRs). Analysis performed after classifying these accessions on the basis of plant height, branching pattern, seed size, Indian/foreign origin into six categories called sub-populations viz. fibre type exotic, fibre type indigenous, intermediate type exotic, intermediate type indigenous, linseed type exotic and linseed type indigenous. The study assessed different diversity indices, AMOVA, population structure and included a principal coordinate analysis based on different marker systems. The highest diversity was exhibited by gSSR markers (SI = 0.46; He = 0.31; P = 85.11). AMOVA based on all markers explained significant difference among fibre type, intermediate type and linseed type populations of flax. In terms of variation explained by different markers, EST-SSR markers (12%) better differentiated flax populations compared to morphological (9%) and gSSR (6%) markers at P = 0.01. The maximum Nei's unbiased genetic distance (D = 0.11) was observed between fibre type and linseed type exotic sub-populations based on EST-SSR markers. The combined structure analysis by using all markers grouped Indian fibre type accessions (63.4%) in a separate cluster along with the Indian intermediate type (48.7%), whereas Indian accessions (82.16%) of linseed type constituted an independent cluster. These findings were supported by the results of the principal coordinate analysis. Morphological markers employed in the study found complementary with microsatellite based markers in deciphering genetic diversity and population structure of the flax germplasm.     

RFLP and RAPD mapping in flax (Linum usitatissimum)

A map of flax (Linum usitatissimum) using restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNAs (RAPDs), and comprising 15 linkage groups containing 94 markers, has been developed covering about 1000 cM. The mapping populations were the F₂ populations from two crosses between diverse cultivars. From one cross, CI1303 and Stormont Cirrus, 20 RFLP and 520 RAPD markers were analyzed. Thirteen RFLP and 80 RAPD markers were on the 15 linkage groups, in addition to one sequence-tagged site (STS). Seven polymorphic RAPD markers were found to have unusual segregation patterns. RAPDs were expressed as dominant markers, but for these markers a prevalence of the progeny lacked a band rather than the expected one-fourth ratio. However, these exceptions may be related to the instability of the genome of Stormont Cirrus in which stable and heritable genomic changes can be induced by environmental factors. The current map could be used for the identification of markers linked to loci controlling the ability to generate heritable changes in response to environmental growth conditions, and to develop anchor loci with STSs for a more general application. Received: 20 March 1999 / Accepted: 16 December 1999     

Metabolism of polyunsaturated fatty acids and their toxicity to the microflora of the rumen

Ruminal microorganisms hydrogenate polyunsaturated fatty acids (PUFA) present in forages and thereby restrict the availability of health-promoting PUFA in meat and milk. The aim of this study was to investigate PUFA metabolism and the influence of PUFA on members of the ruminal microflora. Eleven of 26 predominant species of ruminal bacteria metabolised linoleic acid (LA; cis-9,cis-12–18:2) substantially. The most common product was vaccenic acid (trans-11–18:1), produced by species related to Butyrivibrio fibrisolvens. α-Linolenic acid (LNA; cis-9,cis-12,cis-15–18:3) was metabolised mostly by the same species. The fish oil fatty acids, eicosapentaenoic acid (EPA; 20:5(n − 3)) and docosahexaenoic acid (DHA; 22:6(n − 3)) were not metabolised. Cellulolytic bacteria did not grow in the presence of any PUFA at 50 μg ml⁻¹, nor did some butyrate-producing bacteria, including the stearate producer Clostridium proteoclasticum, Butyrivibrio hungatei and Eubacterium ruminantium. Toxicity to growth was ranked EPA > DHA > LNA > LA. Cell integrity, as measured using propidium iodide, was damaged by LA in all 26 bacteria, but to different extents. Correlations between its effects on growth and apparent effects on cell integrity in different bacteria were low. Combined effects of LA and sodium lactate in E. ruminantium and C. proteoclasticum indicated that LA toxicity is linked to metabolism in butyrate-producing bacteria. PUFA also inhibited the growth of the cellulolytic ruminal fungi, with Neocallimastix frontalis producing small amounts of cis-9,trans-11–18:2 (CLA) from LA. Thus, while dietary PUFA might be useful in suppressing the numbers of biohydrogenating ruminal bacteria, particularly C. proteoclasticum, care should be taken to avoid unwanted effects in suppressing cellulolysis.     

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.     

Correlations between gametophytic (pollen) and sporophytic (seed) generations for polyunsaturated fatty acids in oilseed rape Brassica napus L.

Summary Lipids were extracted from the diploid seed and haploid pollen of Brassica napus L. Two fractions of pollen lipids, namely the diploid-specified pollen-coat and the haploid-specified internal cytoplasmic lipids were obtained. Significant correlations exist between pollen and seed generations for linoleic (182) and linolenic (183) acids. In pollen internal storage lipids, the level of 183 is positively correlated and the level of 182 is negatively correlated with the level of 183 in seed lipids. Evidence is presented that strongly supports the hypothesis that lipid biosynthesis occurs within the pollen and that synthesis is specified by haploid genes. These data support the concept of pollen selection, so that selecting among living pollen grains for superior individuals has potential as a new plant breeding tool for improving seed oil quality.     

Mutants of Arabidopsis with alterations in seed lipid fatty acid composition

Summary A diverse collection of mutants of Arabidopsis with altered seed lipid compositions was isolated by determining the fatty acid composition of samples of seed from 3,000 mutagenized lines. A series of mutations was identified that caused deficiencies in the elongation of 181 to 201, desaturation of 181 to 182, and desaturation of 182 to 183. In each of these cases the wild type exhibited incomplete dominance over the mutant allele. These results, along with results from earlier studies, point to a major influence of gene dosage in determining the fatty acid composition of seed lipids. A mutation was also isolated that resulted in increased accumulation of 183. On the basis of the effects on fatty acid composition, the nature of the biochemical lesion in three of the mutants could be tentatively attributed to deficiencies in activities of specific enzymes. The other mutant classes had relatively less pronounced changes in fatty acid composition. These mutants may represent alterations in genes that regulate lipid metabolism or seed development. The availability of the mutants should provide new opportunities to investigate the mechanisms that control seed lipid fatty acid composition.Abbreviations FAMES (fatty acid methyl esters) - PC (phosphatidylcholine) - 181 (oleic acid) - 182 (linoleic acid) - 183 (linolenic acid) - 201 (eicosenoic acid) Supported in part by grants from the USDA (No. 89-37262-4388), USDA/NSF/DOE Plant Science Center Program, the U.S. Department of Energy (No. AC02-76ER01338), Karlshamns Research Foundation, and the WSU Research and Arts Committee     

Novel seed lipid phenotypes in combinations of mutants altered in fatty acid biosynthesis inArabidopsis

Summary The seed fatty acid (FA) composition of various single mutant combinations ofArabidopsis thaliana that affect FA biosynthesis has been examined. Double mutant combinations offae, a mutation affecting CIS elongation, and a series of four other FA biosynthetic mutants were synthesized. The four other single mutants were:fad2 andfad3, which are deficient in 181 and 182 desaturation, respectively;fab1, which is elevated in 160 and decreased in 181; andfab2, which is elevated in 180 and decreased in 181. The superimposition of two blocks in the FA biosynthetic pathway leads to dramatic changes in the FA content of the double mutants. The tenArabidopsis stocks analyzed to date (wild-type, five single mutants, and four double mutants) make seed oils with a wide range of FA compositions, and illustrate the diversity of oils it is possible to obtain from a single plant species.     

Variation within flax (Linum usitatissimum) and barley (Hordeum vulgare) in response to allelopathic chemicals

Summary A possible method of manipulating allelopathy would be to develop crop varieties showing an increased tolerance to allelopathic chemicals. We therefore examined four flax (Linum usitatissimum) varieties and two wild Linum species in the presence of p-coumaric acid and four barley (Hordeum vulgare) varieties in the presence of p-coumaric acid, scopoletin and wild oat (Avena fatua) extract. Analysis of variance indicates significant interaction between variety and treatment for shoot and root growth for seedling flax, shoot growth for older flax, and root growth for seedling barley. These differences in tolerance between varieties could be exploited to develop-varieties with greater tolerances to the allelochemicals produced by weeds or in crop residues and therefore potentially more tolerant of the presence of weeds.     

Enzymatic hydrolysis of soybean oil using lipase from different sources to yield concentrated of polyunsaturated fatty acids

The ability of three commercially available lipases to mediate the hydrolysis of the soybean oil to yield concentrated of essential fatty acids was evaluated. The tested lipases were from microbial (Candida rugosa and Thermomyces lanuginosa) and animal cells (Porcine pancreatic lipase). In terms of free fatty acids, microbial lipases were more effective to promote the enzymatic hydrolysis of the soybean oil (over 70%) than the porcine pancreatic lipase (24%). In spite of this, porcine pancreatic lipase (PPL) showed the most satisfactory specificity towards both essential fatty acids and was, therefore, chosen to carry out additional studies. An experimental design was performed taking into consideration the enzyme and NaCl amounts as independent variables. The main effects were fitted by multiple regression analysis to a linear model and maximum fatty acids concentration could be obtained using 3.0 wt% of lipase and 0.08 wt% of NaCl. The mathematical model representing the hydrolysis degree was found to describe adequately the experimental results. Under these conditions, concentrations of 29.5 g/L and 4.6 g/L for linoleic and linolenic acids, respectively, were obtained.     

The biosynthesis of cyanogenic glucosides in Linum usitatissimum (linen flax) in Vitro

Microsomal preparations from dark-grown Linum usitatissimum (linen flax) seedlings synthesize acetone cyanohydrin, the precursor of the cyanogenic glucoside linamarin, from valine in the presence of NADPH. N-Hydroxyvaline and isobutyraldoxime, which are predicted intermediates in the pathway, are also converted into products. These microsomal preparations also convert isoleucine into 2-butanone cyanohydrin the precursor of lotaustralin. The biosynthetic activity is located exclusively in the developing cotyledons.     

Sensitivity of different flax (Linum usitatissimum L.) genotypes to salinity determined by GE biplot

Plants respond differently to salt stress depending on their genetic structure and the severity of the stress. Salinity reduces seed germination, delays plant emergence, and inhibits seedling growth. The selection of the tolerant genotypes, however, plays a vital role in increasing agricultural output since various genotypes greatly vary for their tolerance to salinity. Therefore, this study determined the impact of five different NaCl levels (i.e., 0, 50, 100, 150 and 200 mM) on seed germination and growth attributes of 10 flax (Linum usitatissimum L.) genotypes. The germination and growth characteristics of the genotypes under study were examined using the biplot approach at varied salt levels. The results indicated that individual and interactive effects of genotypes and salinity levels significantly (p ≤ 0.01 or p ≤ 0.05) affected several seed germination traits. The relations of genotype × germination traits indicated that ‘G4′ and ‘G6′ were the most stable genotypes with the highest performance regarding seed germination characteristics. The genotype ‘G2′ was associated with shoot length, while ‘G7′ was linked with salinity tolerance index. The biplot divided the germination characteristics into five different groups according to sector analysis. Most of the germination parameters had higher values under 100 mM, while some of the parameters had better values under 0, 50 and 200 mM NaCl levels. The tested genotypes varied for their seed germination and growth response depending on the NaCl levels. The genotypes ‘G4′, ‘G5′ and ‘G6′ proved more tolerant to high NaCl levels. Therefore, these genotypes can be used to improve flax productivity under saline soils.     

Inheritance of low linolenic acid content of the seed oil of a mutant in Glycine max

Summary Linolenic acid content of the oil from F₁, F₂, and F₃ seeds was compared with the parental values from a cross between a soybean cultivar with high (7.0%) and a mutant line with low (3.4%) linolenate (183). Linolenic acid content of F₁ seeds was intermediate to that of selfed seeds from the two parents and values from reciprocal crosses were essentially the same. This demonstrated that in this cross, linolenic acid content of the oil was controlled by the embryo rather than by the maternal parent. The distribution of linolenic acid in F₂ seeds from F₁ plants was trimodal and extended across the range of parental values. High and low linolenate F₂ plants bred true for 183 content and the F₃ distribution of seeds from F₂ plants with intermediate levels of 183 was similar to the F₂ distribution. The data were consistent with a model for two alleles with additive effects at a single locus controlling percent linolenic acid in these progenies. The simply-inherited alleles for low linolenate could be readily transferred to agronomically superior soybean cultivars, which would improve the fatty acid composition of the oil.Cooperative Investigations of the Northern Regional Research Center, ARS, USDA, Peoria, Illinois and the Purdue Univ. Agric. Exp. Stn. Journal Paper No. 10,020 of the Purdue Univ. Agric. Exp. Stn.     

Histological study of embryo-like structures initiated from hypocotyl segments of flax (Linum usitatissimum L.)

Cultivation of flax hypocotyl segments on MS medium supplemented with auxin (2,4-d, NAA) and combination of auxin (NAA) and cytokinin (BAP, zeatin) resulted in production of callus on the cut ends of segments and prolonged cultivation in globular structures resembling early stages of somatic embryos. Embryo-like structures protruded on the surface directly from the subepidermal layers of hypocotyl segments. Despite these globular structures closely resembling somatic embryos, histological observations did not reveal their embryogenic character–organogenesis was the predominant developmental morphogenic pathway. Based on our experiments, as well as on critical revision of existing reports on flax somatic embryogenesis, we conclude, that there has not yet been convincing histological proof of somatic embyogenesis from flax hypocotyl segments.     

Identification of microspore-derived plants in anther culture of flax (Linum usitatissimum L.) using molecular markers

The microspore origin of anther-culture-derived plants of flax was determined using inter-simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) markers. Polymorphic fragments between the two parents of the F₁ donor plants were identified and their segregation patterns in anther-culture-derived plants were used to elucidate the origin of those plants and to determine the degree of independence of plants regenerated from the same callus. Using one ISSR primer (UBC 889) and two RAPD primers (UBC 556 and 561), 12 out of 16 plants were unequivocally identified as being derived from microspores. Plants derived from the same callus had identical PCR patterns at five polymorphic loci and thus were likely derived from the same microspore. Therefore, it is proposed that the number of calli forming shoots be used to describe the anther culture efficiency in flax. Received: 3 February 1998 / Revision received: 8 June 1998 / Accepted: 8 July 1998