Influence of genotype, growth regulators, sucrose level and preconditioning of donor plants on flax (Linum usitatissimum L.) anther culture

The effect of genotype, growth regulators and preconditioning of donor plants on callus induction in anther culture of flax was investigated. Anthers were cultured on modified MS medium supplemented with five different combinations of plant growth regulators. The results suggested that specific combinations of growth regulators must be designed for each genotype. Major differences between the present results and previous reports are discussed. The influence of sucrose concentration was also investigated. For flax cultivar, 'Mikael', callus induction was higher in medium supplemented with 1 mg l(-1) BAP and 2 mg l(-1) 2,4D containing 6% sucrose, while this combination of growth regulators significantly increased callogenesis in cultivars 'Lirina', 'Barbara' and 'Szaphir' when supplemented with 9% or 12% sucrose. The preconditioning of donor plants influenced callogenesis in subsequently isolated anthers. Anthers from donor plants grown at a lower temperature (18/14 degrees C) significantly increased callus induction over those from plants grown at a higher temperature (22/18 degrees C), although each genotype still required optimization of growth regulator combinations in the induction medium. Only 'Mikael' regenerated shoots when the callus was from induction medium supplemented with 2 mg I(-1) BAP and 1 mg l(-1) NAA.     

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     

Effect of culture medium composition on callus formation and regeneration in oil flax anther culture

Regeneration ability in callus cultures from anthers of two hybrid genotypes of oil flax was studied on N6 and LMA-1 nutrient media at various concentrations of cytokinine 6-benzylamynopurine (BAP). It was shown that callus grew and developed better at BAP concentrations of 2 mg/l, comparing with 4 and 6 mg/l. Shoot and root regeneration was observed in F1 genotype 6-8-gnezdny x M22 only and did not depend on BAP concentration in the medium and on the medium composition itself. Transfer onto fresh medium often stimulated dedifferentiation of the regenerated structures.     

Effect of genotype and medium on wheat (Triticum aestivum L.) anther culture

Four winter wheat (Triticum aestivum L.) and two spring wheat cultivars were evaluated in anther culture on three to four different media for their ability to initiate callus and green plants. Five media were used in the experiment: stored-potato medium with Ficoll 400, fresh-potato medium with Ficoll 400, fresh-potato medium with agar, fresh-potato liquid medium without agar or Ficoll 400, and a one tep 85D12-3 medium. Greatly different frequencies of calli and/or green plants were obtained from different cultivars and media. The callus initiation frequency varied from 2.7% for Arapahoe to 52% for Pavon, both on the stored potato medium with Ficoll 400. The frequency of green plant regeneration ranged from 0% for Arapahoe and Siouxland on the stored-potato medium with Ficoll 400 and 0% for Redland and Arapahoe in the fresh-potato medium with Ficoll 400 to 12% for Chris in the 85D12-3 medium (one-step procedure). Chris and Centurk 78, previously reported as having high levels of response, had significantly higher (P < 0.05) frequencies of green plant regeneration on the 851312-3 medium than the other cultivars. An unexpected observation is that wet MSC^– medium enhanced callus regeneration more than a drier MSC^– medium.     

亚麻原生质体培养及植株再生

Shoot protoplasts of four fiber flax (Linum usitatissimum) varieties (7309, 948, Belinka and Viking) were isolated and cultured. The optimal condition for higher protoplast yield 1.8 x 10(6)/gFW and activity 85.5% (c.v. 948) were from 10 day old seedings. Culture in V-KM Agroase-island medium led to first divisions after 3 days (c. v. 948), and after twenty days with an efficiency of 36% of divided cells and 5.2% in plating efficiency. Plant regeneration was obtained in 7309 and Belinka on agar media B5-2, MS3 containing 0.6 mg/L 6-BA and 0.1 mg/L NAA. Roots and leaves regeneration were observed in Viking and 948 respectively.     

Heat-stress effects on reproduction and seed set in Linum usitatissimum L. (flax)

Heat stress can detrimentally affect the reproductive capacity of many plants. The effect of a 7 or 14 d heat stress on flowering, seed set, pollen viability and germinability of flax (Linum usistatissimum L.) was assessed under growth chamber conditions. An incremental (2 °C/h), cyclical (daytime high 40 °C and night‐time low 18 °C) heat stress was applied 12 d after the initiation of flowering. Although flower formation in flax was not affected by heat stress, boll formation and seed set were reduced with onset of the heat stress. On removal of heat stress the stressed plants showed a compensatory response, flowering and producing bolls at a greater rate than the control plants. Heat stress significantly prolonged flowering by 17 d. Boll weight and seed weight were reduced with heat stress and the number of malformed, sterile seed increased three‐fold after 14 d of heat stress. Pollen viability and appearance were negatively affected after 6 and 10 d of heat stress, respectively. Pollen germinability decreased by the sixth day of heat stress, with no pollen germinating by the tenth day. Effects of heat stress on pollen viability and germinability alone, which did not occur until after the sixth day of the stress, could not account for the decreased boll formation due to heat stress in flax. These observations suggest that a combined effect of heat stress on both pollen and ovules contributes to decreased boll formation and seed set in flax.     

Isolation and characterisation of RNA from flax (Linum usitatissimum L.)

A method for isolation of flax RNA is described; properties of the isolated RNA are given. RNA degradation was held to a minimum through a high pH (9.5) extraction buffer, diethylpyrocarbonate (4%) as a nuclease inhibitor, a high concentration (1.5%) of sodium dodecylsulphate, 2 mm Mg²⁺, and separation of the RNA from contaminating materials on Sephadex G-50.     

Development and analysis of EST-SSRs for flax (Linum usitatissimum L.)

A set of 146,611 expressed sequence tags (ESTs) were generated from 10 flax cDNA libraries. After assembly, a total of 11,166 contigs and 11,896 singletons were mined for the presence of putative simple sequence repeats (SSRs) and yielded 806 (3.5%) non-redundant sequences which contained 851 putative SSRs. This is equivalent to one EST-SSR per 16.5 kb of sequence. Trinucleotide motifs were the most abundant (76.9%), followed by dinucleotides (13.9%). Tetra-, penta- and hexanucleotide motifs represented <10% of the SSRs identified. A total of 83 SSR motifs were identified. Motif (TTC/GAA)n was the most abundant (10.2%) followed by (CTT/AAG)n (8.7%), (TCT/AGA)n (8.6%), (CT/AG)n (6.7%) and (TC/GA)n (5.3%). A total of 662 primer pairs were designed, of which 610 primer pairs yielded amplicons in a set of 23 flax accessions. Polymorphism between the accessions was found for 248 primer pairs which detected a total of 275 EST-SSR loci. Two to seven alleles were detected per marker. The polymorphism information content value for these markers ranged from 0.08 to 0.82 and averaged 0.35. The 635 alleles detected by the 275 polymorphic EST-SSRs were used to study the genetic relationship of 23 flax accessions. Four major clusters and two singletons were observed. Sub-clusters within the main clusters correlated with the pedigree relationships amongst accessions. The EST-SSRs developed herein represent the first large-scale development of SSR markers in flax. They have potential to be used for the development of genetic and physical maps, quantitative trait loci mapping, genetic diversity studies, association mapping and fingerprinting cultivars for example. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Integrated consensus genetic and physical maps of flax (Linum usitatissimum L.)

Three linkage maps of flax (Linum usitatissimum L.) were constructed from populations CDC Bethune/Macbeth, E1747/Viking and SP2047/UGG5-5 containing between 385 and 469 mapped markers each. The first consensus map of flax was constructed incorporating 770 markers based on 371 shared markers including 114 that were shared by all three populations and 257 shared between any two populations. The 15 linkage group map corresponds to the haploid number of chromosomes of this species. The marker order of the consensus map was largely collinear in all three individual maps but a few local inversions and marker rearrangements spanning short intervals were observed. Segregation distortion was present in all linkage groups which contained 1–52 markers displaying non-Mendelian segregation. The total length of the consensus genetic map is 1,551?cM with a mean marker density of 2.0?cM. A total of 670 markers were anchored to 204 of the 416 fingerprinted contigs of the physical map corresponding to ~274?Mb or 74?% of the estimated flax genome size of 370?Mb. This high resolution consensus map will be a resource for comparative genomics, genome organization, evolution studies and anchoring of the whole genome shotgun sequence.     

The ubiquitin-encoding multigene family of flax, Linum usitatissimum.

Ubiquitin (Ubq), a 76-amino acid (aa) protein, is found in all eukaryotic organisms and is one of the most conserved proteins so far studied. It is implicated in many cellular processes. The Ubq-encoding genes (ubq) are generally present as a multigene family. In flax, we have estimated that this multigene family contains at the most ten members. The initial flax ubq sequences were isolated from a flax genomic library in lambda EMBL4 using a heterologous Arabidopsis thaliana ubq probe. An 916-bp fragment from one of the phage clones was subcloned and sequenced. The aa sequence derived from the nucleotide sequence of this fragment is identical to that of other plant Ubqs. This fragment was then used to isolate additional flax ubq clones. In all, eleven phage lambda clones, which represent six members of the gene family, were restriction-mapped and characterized. These six members are represented as three monomers, three poly-Ubqs, one hexamer and two tetramers. They can be present at either a single locus (two of the monomers and one of the poly-Ubqs) or at two loci (the remaining three genes). The other four members of the family are yet to be cloned and characterized.     

Peroxidase Activity in Linum usitatissimum L.

Peroxidase activity was measured at three stages, from seedlingto maturity, in stem tissue of two genotypes of Linum usitatissimum,and their reciprocal F₁ hybrids. The plants were grown throughoutin growth chambers, allowing close control over the environmentalconditions. There were large and consistent differences betweenthe activities of the parental genotypes, and between dialysedand undialysed extracts. Activities in both parents and theF₁ were expressed on a logarithmic scale. The activity of theF₁ fell, with one exception, between the activities of the twoparents. The relationship of F₁ activity to the mean of theparental activities fluctuated with the stage of growth.     

Effect of medium osmotic potential on callus induction and shoot regeneration in flax anther culture

Development of an efficient and cost-effective doubled haploid production system in flax (Linum usitatissimum L.) is the prerequisite for the application of doubled haploid technology in a practical breeding program. Pre-culture of anthers on a medium containing 15% sucrose for 2–7 days before transfer to the same medium containing 6% sucrose for a total of 28 days culture period significantly increased shoot regeneration for all four genotypes evaluated. Moreover, pre-culture of anthers on medium containing 15% sucrose for 2–7 days was sufficient to dramatically reduce the frequency of shoot regeneration from somatic tissues and thereby to increase the frequency of microspore-derived plants in flax anther culture. Furthermore, replacing 15% sucrose with 6% sucrose and 9% polyethylene glycol (PEG), or 3% sucrose and 12% PEG, in pre-culture medium did not significantly affect callus induction and shoot regeneration. The results indicate that sucrose may act as carbon/energy source as well as an osmotic regulator in flax anther culture. Sucrose as an osmotic regulator may be replaced by a non-metabolizable osmoticum: PEG. The implication of this study in flax anther culture and breeding is discussed.     

The influence of genotype and medium on rye (Secale cereale L.) anther culture

Anthers of three rye inbred lines - L9, L318, Dw28, one synthetic hybrid - F₁(5) and one variety, Dakowskie Zote (DZ), were cultured on two media based on N6, and two based on P2 components. The induction rate significantly depended on genotype - the best results were obtained for line L318 and the lowest percentage of responding anthers was noticed in line L9. There was no universal medium for all tested genotypes. The highest induction rate (IR) for lines L318, L9 and hybrid F₁(5) was obtained on medium CI (11.94, 0.71 and 1.75 respectively). For DZ, the P2I medium was better than the others while for Dw28 CIP turned to be as suitable as CI. A highly significant interaction between genotype and medium was proved. Single anthers of DZ, L318 and L9 produced embryos on CI, CIP and P2I media. They did not develop into plants but after transferring them to CS1.7 medium, a secondary, embryogenic callus was obtained. Such a reaction has not been described in rye until now. In spite of a relatively high IR, plant regeneration was rather poor. An elaboration of this step in haploid production is needed. Most of the analyzed calluses and microspore derived regenerants proved to be haploids, according to flow cytometry analysis. Plants treated with colchicine were doubled haploids.     

Notes on Thysanoptera found on flax (Linum usitatissimum L.) in the British Isles

The following eighteen species of Thysanoptera Terebrantia have been found on flax in the British Isles: Melanthrips fuscus (Sulzer), Aeolothrips fasciatus (L.), Anaphothrips obscurus (Müler), Aptinothrips rufus (Gmelin), Chirothrips manicatus Hal., Limothrips cerealium Hal., L. denticornis Hal., Stenothrips graminum Uzel, Taeniothrips atratus (Hal.), T. vulgatissimus (Hal.), Thrips angusticeps Uzel, T. discolor Hal., T. flavus Schrank, T. fuscipennis Hal., T. major Uzel, T. minutissimus L., T. physapus L., T. tabaci Lindeman. Each species is described briefly with notes on habits of adults and larvae, place of pupation, number of generations in the year, hibernation, time of occurrence on plants, plants and objects on which found, host plants of larvae and adults, importance to flax, record of locality and collector on flax, distribution, including altitudes, in the British Isles. More species occur in the south than in the north of Great Britain, and species common to both regions usually occur in greater numbers in the south. The insects breed on certain species of crop plants, weeds or trees of arable land. No damage of economic importance to flax by Thysanoptera has been proven in the British Isles, and the flax thrips, Thrips lini Ladureau, has not been found. Taeniothrips vulgatissimus (Hal.) may breed on flax and its adults, and those of T. atratus (Hal.) may cause superficial damage to petals of flowers. Thrips angusticeps Uzel and T. tabaci Lindeman will probably breed on flax.     

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.     

A lipoxygenase-divinyl ether synthase pathway in flax (Linum usitatissimum L.) leaves

Incubation of linoleic acid with an enzyme preparation from leaves of flax (Linum usitatissimum L.) led to the formation of a divinyl ether fatty acid, i.e. (9Z,11E,1'Z)-12-(1'-hexenyloxy)-9,11-dodecadienoic [(omega5Z)-etheroleic] acid, as well as smaller amounts of 13-hydroxy-9(Z),11(E)-octadecadienoic acid. The 13-hydroperoxide of linoleic acid afforded the same set of products, whereas incubations of alpha-linolenic acid and its 13-hydroperoxide afforded the divinyl ether (9Z,11E,1'Z,3'Z)-12-(1',3'-hexadienyloxy)-9,11-dodecadienoic [(omega5Z)-etherolenic] as the main product. Identification of both divinyl ethers was substantiated by their UV, mass-, (1)H NMR and COSY spectral data. In addition to the 13-lipoxygenase and divinyl ether synthase activities demonstrated by these results, flax leaves also contained allene oxide synthase activity as judged by the presence of endogenously formed (15Z)-cis-12-oxo-10,15-phytodienoic acid in all incubations.     

Peroxidase activity in Linum usitatissimum L.

Summary Crosses were made, in all combinations, between 2 parental genotypes of Linum and their reciprocal F ₁ hybrids. The parents and progeny obtained were grown in controlled environmental conditions and sampled at 35 and 70 days after germination to determine, on an individual plant basis, total plant fresh weight and peroxidase activity of main stem tissue. Peroxidase activity required transformation to a log₁₀ scale, whereas the original linear scale was satisfactory for plant weight. There was no correlation between plant weight and corresponding peroxidase activity. Pronounced heterosis appeared in the F ₁'s for both characters at sample 1, but this heterosis had declined at sample 2 and in the F ₂'s. Heterosis operated in a positive direction for plant weight and in a negative direction for peroxidase activity. No consistent differences were found amongst the variances of segregating or non-segregating generations for either character.