Flax Transformation Research Articles

An Apical Meristem-Targeted in planta Transformation Method for the Development of Transgenics in Flax (Linum usitatissimum): Optimization and Validation.

Efficient regeneration of explants devoid of intrinsic somaclonal variations is a cardinal step in plant tissue culture, thus, a vital component of transgenic technology. However, recalcitrance of economically important crops to tissue culture-based organogenesis ensues a setback in the use of transgenesis in the genetic engineering of crop plants. The present study developed an optimized, genotype-independent, nonconventional tissue culture-independent in planta strategy for the genetic transformation of flax/linseed. This apical meristem-targeted in planta transformation protocol will accelerate value addition in the dual purpose industrially important but recalcitrant fiber crop flax/linseed. The study delineated optimization of Agrobacterium tumefaciens-mediated transformation and stable T-DNA (pCambia2301:GUS:nptII) integration in flax. It established successful use of a stringent soilrite-based screening in the presence of 30 mg/L kanamycin for the identification of putative transformants. The amenability, authenticity, and reproducibility of soilrite-based kanamycin screening were further verified at the molecular level by GUS histochemical analysis of T0 seedlings, GUS and nptII gene-specific PCR, genomic Southern hybridization for stable integration of T-DNA, and expression analysis of transgenes by sqRT-PCR. This method resulted in a screening efficiency of 6.05% in the presence of kanamycin, indicating amenability of in planta flax transformation. The strategy can be a promising tool for the successful development of transgenics in flax.

Establishment of the 'imbibed seed piercing' method for Agrobacterium-mediated transformation of jute and flax bast fibre crops via phloem-specific expression of the β-glucuronidase Gene

Jute (Corchorus capsularis) and flax (Linum usitatissimum) have been established as important sources of bast fibres (phloem fibres) worldwide. These long and lustrous natural fibres are used for various commercial and industrial purposes and hold immense future prospects. To meet the worldwide demand for bast fibres, improvement in the production and quality of jute and flax is essential. Molecular breeding is an expeditious approach for varietal improvement and requires an efficient gene transformation system. The available methods for jute and flax transformation are highly specific to species and variety, requiring a long time and rigorous preparation of media, explants, and screening procedures. In this study, we used a fast and stable method for Agrobacterium tumefaciens-mediated transformation of jute (variety JRC321) and flax (variety FT-897), known as the ‘imbibed seed piercing’ method (ISPM), where seeds were used as explants. A phloem-specific Arabidopsis sucrose-proton H+ symporter 2 (AtSUC2) promoter-driven gus gene (β-glucuronidase) was used for the assessment of genetic transformation of jute and flax plants. Transformed seedlings were selected following three consecutive selection pressure tests conducted at two-week intervals using kanamycin-supplemented culture media. Transgene integration was verified using Southern blotting and PCR-based molecular characterization of jute and flax plants. The gus gene expression levels in the transgenic jute and flax tissue samples were analysed by semi-quantitative and quantitative RT-PCR. The GUS protein expression in transgenic plant tissues was confirmed by the development of blue colour in the histochemical GUS assay. Blue colouration was observed only in phloem cells of the tested plant tissues. Transgenic progeny plants of subsequent generations followed the 3:1 transgene segregation pattern and exhibited phloem-specific GUS expression. This fast, easy, and stable ISPM could also be applied to other fibre producing industrial crops and could form a part of a modern CRISPR/Cas9-based genome editing tool by efficiently delivering CRISPR vectors in jute and flax plants.

Physicochemical and Mechanical Performances of Technical Flax Fibers and Biobased Composite Material: Effects of Flax Transformation Process

Physicochemical and Mechanical Performances of Technical Flax Fibers and Biobased Composite Material: Effects of Flax Transformation Process

The use of phosphomannose isomerase selection system for Agrobacterium-mediated transformation of tobacco and flax aimed for phytoremediation

ABSTRACTA plant selection system based on the phosphomannose isomerase gene (pmi) as a selectable marker is often used to avoid selection using antibiotic resistance. Nevertheless, pmi gene is endogenous in several plant species and therefore difficult to use in such cases. Here we evaluated and compared Agrobacterium-mediated transformation of Linum usitatissimum breeding line AGT-952 (without endogenous pmi gene) and Nicotiana tabacum var. WSC-38 (with endogenous pmi gene). Transformation was evaluated for vectors bearing transgenes that have the potential to be involved in improved phytoremediation of contaminated environment. Tobacco regenerants selection resulted in 6.8% transformation efficiency when using a medium supplemented with 30 g/L mannose with stepwise decrease of the sucrose concentration. Similar transformation efficiency (5.3%) was achieved in transformation of flax. Relatively low selection efficiency was achieved (12.5% and 34.8%, respectively). The final detection of efficient pmi selection was conducted using PCR and the non-endogenous genes; pmi transgene for flax and todC2 transgene for tobacco plants.

Transgenic flax/linseed (Linum usitatissimum L.) - expectations and reality

This review summarizes the history, important milestones, current status and perspectives of biotech flax/linseed (Linum usitatissimum L.), supplemented with some of our original research, breeding and data on environmentalsafety. We show how recent biotechnology methods and genetic engineering contributed to the flax/linseed breeding in order to speed up the breeding process (doubled haploids technology; in vitro selection with the use of pathogenic toxins or heavy metals; genetic transformation) and for the creation of new flax/linseed cultivars. The focus is laid on genetic engineering which represents an excellent technology to enrich the flax/linseed genepool with genes of interest, which are not naturally present in the flax/linseed genome. Different methods of flax transformation are mentioned, as well as various genes of interest that have been used for flax transformation to date aimed at improving transgenic flax properties, affecting both qualitative and quantitative traits. The fatty acid content and composition, the lignan (especially secoisolariciresinol diglucoside - SDG) content, flax fibre quality, tolerance to herbicides and resistance to diseases belong, among others, to flax traits that have already been modified by genetic engineering. Selection genes, reporter genes and also promoters that have been used for the vector construction are also summarized. This paper describes different fields of utilization of genetically modified (GM) flax with different improved properties. The history of the only so far officially registered transgenic linseed cultivar Triffid is described in detail. Finally, potential risks and benefits of flax modification are evaluated and also the prime expectations of GM flax and real current state of this technology compared. Unfortunately, the products created by this technology are under strict (albeit not scientifically-based) legislative/political control in the European Union (EU), which prevents the access of products, created by breeders using this top technology, to the EU market.

Floral-dip transformation of flax (Linum usitatissimum) to generate transgenic progenies with a high transformation rate.

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax (<em>Linum usitatissimum</em>) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation. In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Optimization of flax shive-cementitious composites: Impact of different aggregate treatments using linseed oil

This study investigates the effect of flax shive treatments on physico-chemical characteristics of shive and physico-mechanical properties of flax shive cementitious composites. In the context of sustainable development and biorefinery, the main objectives of this work are to optimize a lightweight lignocellulosic concrete and to upgrade a product and a by-product of flax transformation, respectively linseed oil and flax shives. To improve the compatibility between cementitious matrix and vegetable aggregates, flax shives have been treated with raw and emulsified linseed oil. A more marked decrease in water absorption capacity and hydro soluble molecule release has been obtained for shives coated with raw linseed oil. Compared to standard shive composite, whatever the linseed oil treatment, the setting times have been reduced, the mechanical strengths improved and the thermal conductivity slightly increased. In the case of dimensional variations, the coating with raw linseed oil has led to a slight better improvement compared to emulsion coating. The flax shive-cementitious composites obtained exhibit the properties of insulating bearing lightweight aggregate concretes.

ER disruption and GFP degradation during non-regenerable transformation of flax with Agrobacterium tumefaciens

Flax is considered as plant species susceptible to Agrobacterium-mediated genetic transformation. In this study, stability of flax transformation by Agrobacterium rhizogenes versus Agrobacterium tumefaciens was tested by using combined selection for antibiotic resistance and visual selection of green fluorescent protein (GFP)-fusion reporter targeted to the endoplasmic reticulum (ER). Transformation with A. rhizogenes was stable for over 2 years, whereas transformation by A. tumefaciens resulted in non-regenerable stable transformation which was restricted solely to transgenic callus and lasted only 6-8 weeks. However, shoots regenerated from this callus appeared to be non-transgenic. Importantly, callus and root cells stably transformed with A. rhizogenes showed typical regular organization and dynamics of ER as visualized by GFP-ER marker. On the other hand, callus cells transformed with A. tumefaciens showed disintegrated ER structure and impaired dynamics which was accompanied with developmental degradation of GFP. Consequently, shoots which regenerated from such callus were all non-transgenic. Possible reasons for this non-regenerable flax transformation by A. tumefaciens are discussed.

Agrobacterium-mediated transformation of flax with a mutant tubulin gene responsible for resistance to dinitroaniline herbicides

Agrobacterium tumefaciens was used to transform fiber flax with the pBITUBA8 plasmid carrying the mutant alpha-tubulin gene imparting resistance to dinitroaniline herbicides and the nptII selective marker gene imparting resistance to canamycin. The transformants were selected in parallel on media containing canamycin and trifluralin (a dinitroaniline herbicide). The transgenic nature of the resultant regenerants resistant to dinitroaniline herbicides was confirmed by means of Southern blotting and polymerase chain reaction (PCR) analysis using specific probes for the ntpll gene and the gene of alpha-tubulin.

Enrichment of carotenoids in flaxseed (Linum usitatissimum) by metabolic engineering with introduction of bacterial phytoene synthase gene crtB

Linseed flax (Linum usitatissimum L.) is an industrially important oil crop, which includes large amounts of alpha-linolenic acid (18:3) and lignan in its seed oil. We report here the metabolic engineering of flax plants to increase carotenoid amount in seeds. Agrobacterium-mediated transformation of flax was performed to express the phytoene synthase gene (crtB) derived from the soil bacterium Pantoea ananatis (formerly called Erwinia uredovora 20D3) under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter or the Arabidopsis thaliana fatty acid elongase 1 gene (FAE1) seed-specific promoter. As a result, eight transgenic flax plants were generated. They formed orange seeds (embryos), in which phytoene, alpha-carotene, and beta-carotene were newly accumulated in addition to increased amounts of lutein, while untransformed flax plants formed light-yellow seeds, in which only lutein was detected. Interestingly, despite the control of the CaMV 35S promoter, the expression of crtB was not observed in the leaves but in the seeds in the transgenic flax plants. Total carotenoid amounts in these seeds were 65.4-156.3 microg/g fresh weight, which corresponded to 7.8- to 18.6-fold increase, compared with those of untransformed controls. These results suggest that the flux of phytoene synthesis from geranylgeranyl diphosphate was first promoted by the expressed crtB gene product (CrtB), and then phytoene was consecutively decomposed to the downstream metabolites alpha-carotene, beta-carotene, and lutein, as catalyzed by endogenous carotenoid biosynthetic enzymes in seeds. The transgenic flaxseeds enriched with the carotenoids could be valuable as nutritional sources for human health.

Development of transformation vectors based upon a modified plant α‐tubulin gene as the selectable marker

A plant transformation and selection system has been developed utilizing a modified tubulin gene as a selectable marker. The vector constructs carrying a mutant alpha-tubulin gene from goosegrass conferring resistance to dinitroaniline herbicides were created for transformation of monocotyledonous and dicotyledonous plants. These constructs contained beta- and/or mutant alpha-tubulin genes driven either by ubiquitin or CaMV 35S promoter. The constructs were used for biolistic transformation of finger millet and soybean or for Agrobacterium-mediated transformation of flax and tobacco. Trifluralin, the main representative of dinitroaniline herbicides, was used as a selective agent in experiments to select transgenic cells, tissues and plantlets. Selective concentrations of trifluralin estimated for each species were as follows: 10 microM for Eleusine coracana, Glycine max, Nicotiana plumbaginifolia and Nicotiana sylvestris; 3 microM for Linum usitatissimum. PCR and Southern blotting analyses of transformed lines with a specific probe to nptII, alpha-tubulin or beta-tubulin genes were performed to confirm the transgenic nature of regenerated plants. Band specific for the mutant alpha-tubulin gene was identified in transformed plant lines. Results confirmed the stable integration of the mutant tubulin gene into the plant genomes. The present study clearly demonstrates the use of a plant mutant tubulin as a selective gene for plant transformation.

The use of the phosphomannose isomerase gene as alternative selectable marker for Agrobacterium-mediated transformation of flax (Linum usitatissimum)

In order to meet the future requirement of using non-antibiotic resistance genes for the production of transgenic plants, we have adapted the selectable marker system PMI/mannose to be used in Agrobacterium-mediated transformation of flax (Linum usitatissimum L.) cv. Barbara. The Escherichia coli pmi gene encodes a phosphomannose isomerase (E.C. 5.1.3.8) that converts mannose-6-phosphate, an inhibitor of glycolysis, into fructose-6-phosphate (glycolysis intermediate). Its expression in transformed cells allows them to grow on mannose-selective medium. The Agrobacterium tumefaciens strain GV3101 (pGV2260) harbouring the binary vector pNOV2819 that carries the pmi gene under the control of the Cestrum yellow leaf curling virus constitutive promoter was used for transformation experiments. Transgenic flax plants able to root on mannose-containing medium were obtained from hypocotyl-derived calli that had been selected on a combination of 20 g L(-1) sucrose and 10 g L(-1) mannose. Their transgenic state was confirmed by PCR and Southern blotting. Transgene expression was detected by RT-PCR in leaves, stems and roots of in vitro grown primary transformants. The mean transformation efficiency of 3.6%, that reached 6.4% in one experiment was comparable to that obtained when using the nptII selectable marker on the same cultivar. The ability of T1 seeds to germinate on mannose-containing medium confirmed the Mendelian inheritance of the pmi gene in the progeny of primary transformants. These results indicate that the PMI/mannose selection system can be successfully used for the recovery of flax transgenic plants under safe conditions for human health and the environment.

Genetic transformation of flax (Linum usitatissimum) by Agrobacterium tumefaciens: regeneration of transformed shoots via a callus phase

Genetic transformation of flax (Linum usitatissimum) has been achieved using an A. tumefaciens strain carrying a non-oncogenic Ti plasmid-derived vector containing a chimaeric npt-II gene and a wild type nopaline synthase gene. Fertile, transformed shoots were most easily obtained from Km(r) callus developing on hypocotyl sections. The totipotency of the Km(r) callus was dependent upon its origin. T-DNA was visualised by Southern blotting in all Km(r) tissues. Efficient expression of nopaline synthase and the chimaeric npt-II gene was found in transformed Km(r) callus and regenerated shoots.