WO2018104596A1

WO2018104596A1 - High omega -3 content flax plants

Info

Publication number: WO2018104596A1
Application number: PCT/FR2017/000238
Authority: WO
Inventors: Vivien SARAZIN; Véronique TRAMCOURT; Xavier GUILLOT; Patrice LABOULET
Original assignee: Etablissements Andre Laboulet
Priority date: 2016-12-09
Filing date: 2017-12-06
Publication date: 2018-06-14
Also published as: FR3059870A1; WO2018104596A4

Abstract

The invention relates to a method for increasing the content of C18:3 fatty acid or alpha-linolenic acid in flaxseed, and to the resulting plants, which method involves increasing the expression of the Δ15 desaturase responsible for the conversion of C18:2 into C18:3 in the seed within the plant.

Description

LINEN PLANTS WITH HIGH O EGA-3 CONTENT

The present invention relates to a process for increasing the C18: 3 fatty acid content, or alpha-linolenic acid, of a flaxseed, as well as the resulting plants, via increased expression. of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed within the plant.

Flax cultivation is one of the oldest utilitarian cultures, the first traces of its use dating back to 8000 BC. JC in Turkey. Its name Linum usitatissimum, translated from the Latin "lin most useful", refers to its multiple outlets. Flax is currently used in the textile (fiber), food (seed and oil) and chemical (oil) industries. The peculiarities of flaxseed are its oil content (35 to 45%) and the fatty acid profile of its predominantly alpha-linolenic acid oil (C18: 3, 48 to 65%). The triple unsaturation of this fatty acid gives the oil a high reactivity with respect to oxygen and therefore a high degree of siccativity. This ability of the oil to polymerize and dry rapidly when deposited in a thin layer is of interest for oleochemical applications. However, in the context of food use, this chemical reactivity induces rapid organoleptic degradation of the oil. Flaxseeds are used for food and feed (respectively 5% and 18% in the European Union). But in these areas, France has a different profile from its European counterparts. Indeed the recent evolutions related to the demands of the breeders, the organizations of health and especially of the consumers, led to a drastic change of its use. In France, for example, 75 to 95% of flaxseeds produced and used in France are used for animal feed. This trend is therefore due to a growing demand for foods rich in Omega-3 (eggs, meat, milk). At this level, the Bleu-Blanc-Cœur sector has set itself the task of promoting and controlling the quality of food products from the sectors (especially linseed) to ensure consumers a high quality in the animal, environmental and of human health. For Omega-3 in flax, ie alpha-linolenic acid (ALA), in this Bleu-Blanc-Cœur food chain, the accepted low limit is 48% of C18: 3 in the diet. oil from the harvested lot. In below this low limit the reference price is subject to a discount of 30 euros / tonne. On the other hand, from a C18: 3 content of 56%, a premium of 3 euros / ton is applied relative to the reference price. From 60% it is 15 euros / ton in addition to the reference price. This premium can reach 30 euros / tonne compared to the reference price for a content higher than 62% in C18: 3. It is easy to understand the advantage of having high flax C18: 3 producer. In 2013, France produced more than 16,000 tonnes of linseed on 8,510 hectares, which was only enough to cover half of its needs.

Oilseed flaxseed production is a niche market but tends to grow strongly in the coming years. Currently contracts between producers, collectors and industrialists have set a minimum price for the farmer of 350 € / t (Omega-3 <56%). The price of linseed varies often, but it can be noted that it is currently quite high (525 € / t at the beginning of February 2015).

Linseed oil, because of its drying properties, is mainly used in oleochemistry. It is used in varnishes, paints, inks and as a coating agent. In France, flaxseed oil was banned in the human diet from 1906 to 2008 because of its difficult conservation. Since then, the Ministerial Decree of 12/07/2010 issued following the favorable opinions issued by the French Agency for Sanitary Security - Order of 12 July 2010 amending the Decree of 4 December 2008 fixing the conditions of use of oil flax for food use in: JORF No. 0166 of 21 July 2010, Text No. 37, pp. 13441 of the Foods (ref. Nos. 2008-SA-0392 and 2010-SA-0109) - authorized its consumption as virgin oil. However, to be marketed, the virgin linseed oil must be packaged in opaque containers of low capacity (250mL), and have undergone inerting under nitrogen. The order also states that the shelf life of the oil must be less than three months after opening. As a result, in France the annual production of linseed oil remains low.

Oleaginous linseed is reinforced in the context of ongoing social developments, due to its agronomic and environmental advantages, as well as the high quality of its oil and fiber. It is a "green" crop that can be introduced in winter or spring version in a wide range of rotations. Flax could reclaim a sole of 20 to 30 000 hectares in France with the development of the valorization of whole seeds in France. animal nutrition, based on the emphasis on respect for the environment and the nutritional balance of the consumer. Other demanding quality markets based on the direct use of flax products for human consumption (following the order of 12 July 2010) could also contribute to a renewed vitality for this sector.

In order to improve ALA levels in flaxseed, breeders carry out a classic genealogical selection based on the cross-breeding of lineages with higher alpha-linolenic acid content. Selection is then made within the offspring of these crosses taking into account multiple criteria such as lodging resistance, yield, oil content, in addition to the ALA content.

It is considered by the entire industry that there is a natural metabolic barrier to achieving levels greater than 66.7% ALA in oilseed flaxseed. The inventors of the present invention have overexpressed the Δ15 desaturase enzyme responsible for the conversion of C18: 2 to C18: 3 in a seed within a flax plant, which leads to a considerable increase in the content of C18: 3 fatty acid in the seed.

The overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, the last enzyme in the C18: 3 biosynthetic pathway, results in increasing the levels of this fatty acid produced by the plant. .

A significant increase in the amount of C18: 3 in the seeds of flax plants is observed in plants overexpressing the Δ15 desaturase responsible for the conversion of C 8: 2 to C18: 3 in the seed. The seeds of plants overexpressing the Al5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed contain more C18: 3 than those of plants not overexpressing this enzyme.

Under unfavorable environmental conditions, the plants according to the invention, overexpressing the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, produce more C18: 3 than plants that do not overexpress Δ15 desaturase, the amount of C18: 3 in the seed is much lower.

The overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed in plants according to the invention makes it possible to dispense with particular culture conditions and specifically directed towards the overproduction of fatty acids by the flaxseed, in particular C18: 3 alpha-linolenic acid.

The present invention thus relates to:

A method for increasing the amount of alpha-linolenic acid (C18: 3) in a plant of Linum usitatissimum, said method comprising overexpressing the Δ 15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of said plant.

The method according to the invention is characterized in that it comprises a modification of the Δ15 desaturase gene.

The method according to the invention is also characterized in that the modification of the Δ15 desaturase gene comprises a modification at the level of the promoter region of said gene.

It is an object of the invention to provide a method according to the invention, characterized in that the modification at the promoter region of said gene is a mutation.

In the method according to the invention, the mutation is characterized in that the mutation is a deletion.

The method according to the invention is also characterized in that the gene comprises SEQ ID No. 1.

The method according to the invention is also characterized in that the deletion is directed to a sequence between positions 10 and 20 of SEQ ID No. 2.

The method according to the invention is also characterized in that the deletion concerns a sequence between the positions 12 and 17 of SEQ ID No. 2. The nucleotides of positions 2 and 17 are included in the deletion.

The method according to the invention is also characterized in that the deletion comprises the sequence AAAGGG li is thus illustrated, on the sequence of SEQ ID No. 2 corresponding to the standard plant, the underlined part and in bold corresponding to the portion of deleted sequence in the plant VL8.29.3 (or HT3) in which the complete sequence corresponds in SEQ ID Ν.

The method according to the invention is also characterized in that it comprises the transformation of a plant cell of Linum usitatissimum with at least one nucleic acid which encodes the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed and is under the control of a promoter ensuring the overexpression of the ΔΙ 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of said plant and the crop of the plant at maturity.

The method according to the invention is also characterized in that the at least one nucleic acid comprises a nucleic acid according to SEQ ID No. 1.

The method according to the invention is also characterized in that the overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of said plant is carried out by introgression of a genetic element coding for overexpression of Δ15 desaturase.

The invention also relates to a method in which the introgression of a genetic element coding for overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of said plant is obtained by protoplast fusion.

The invention also relates to a method in which the introgression of a genetic element coding for overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of said plant is obtained by embryo rescue. .

Another subject of the invention relates to a method for increasing the amount of alpha-linolenic acid (C18: 3) in a plant of Linum usitatissimum, comprising the detection of the presence of a genetic element, in particular a sequence of nucleic acid, linked to the overexpression of the ΔΙ 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of said plant in a donor plant and the transfer of the genetic element, in particular a nucleic acid sequence, linked to the overexpression of ΔΙ 5 desaturase thus detected, from the donor plant to a recipient plant.

In a particularly preferred embodiment, the genetic element, in particular the nucleic acid sequence, linked to the overexpression of Δ15 desaturase is identical or complementary to the sequence present in the sample of Linum usitatissimum VL8.29.3 deposited under the NCIMB Number 42681 on October 24, 2016.

The method according to the invention is characterized in that the detection is carried out using at least one molecular marker.

The method according to the invention is characterized in that the at least one molecular marker comprises a mutation at the promoter region of the Δ15 desaturase gene responsible for the conversion of C18: 2 to C18: 3 in the seed of said plant. .

The method according to the invention is characterized in that the mutation is a deletion.

The method according to the invention is characterized in that the molecular marker comprises the sequence SEQ ID No. 1. Such a sequence can be found in the Linum usitatissimum sample VL8.29.3 filed as NCIMB No. 42681 on October 24, 2016.

The invention is further directed to a method for increasing the amount of alpha-linolenic acid (C18: 3) in a plant of Linum usitatissimum, comprising providing a plant population and selecting individuals in the population that exhibit expression. of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the highest possible seed.

In one embodiment of the method according to the invention, the plant population is a population of mutant plants.

The method according to the invention is characterized in that said method comprises the selection, within a plant population, of at least one plant exhibiting an overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed compared to the expression of Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of parent plants.

In a particular embodiment, the method according to the invention is characterized in that the introgression comprises:

a) The provision of a plant with a given level of expression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, b) The supply of a plant with an increased level of expression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed relative to the plant provided in a),

(c) The crossing of the plant provided in (a) with the plant provided in (b); (d) The generation of offspring from crossing (c),

e) Selection within the offspring of at least one plant with a level of expression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed higher than that of the plant provided in b) .

In a particular embodiment, the method as described above comprising an additional step of crossing the plant selected in e) with the plant provided in b) followed by an additional step of selection within the offspring obtained from minus one plant with an expression level of Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed higher than that of the plant selected in e).

In a particular embodiment, the plants a and b) are Linum usitatissimum flax plants.

A plant with an increased level of expression of Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed relative to the plant provided in a), can be the plant Linum usitatissimum VL8.29.3 deposited under the NCIMB Number 42681 on October 24, 2016.

The invention also relates to a plant selection method of Linum usitatissimum, characterized in that it comprises the search for an allele of the Δ 15 desaturase gene responsible for the conversion of C18: 2 to C18: 3 in the seed having a mutation resulting in an increase in the amount of alpha-linolenic acid (C18: 3) in a plant of Linum usitatissimum. The invention also relates to a plant that can be obtained by a method according to one of the preceding embodiments.

The invention also relates to the use of a plant obtainable by a method according to one of the preceding embodiments or a part of such a plant, for the preparation of a composition intended for the food or feed.

Finally, the invention also relates to a plant of Linum usitatissimum, characterized in that the content, in% by weight, of alpha-linolenic acid (C18: 3) of the seed oil of the plant is greater than 66.7%, particularly greater than 67%, more particularly greater than 67.5%, more particularly greater than 68%, more particularly still greater than 69%, more particularly greater than 70%, more particularly still greater than 70.5%, more particularly still greater than 71%, more particularly greater than 72%, more particularly still greater than 72.5% and more particularly still greater than 73%. ^"The content, in% by weight alpha-linolenic acid (C18: 3) of the seed oil of the plant is less than 76%, particularly less than 75%, more particularly less than 74%.

A plant according to the invention is characterized in that the germination rate is greater than 90%.

A plant according to the invention is characterized in that the yield is between 85 and 110% of the average value of control reference varieties following evaluation under similar conditions in France.

In a particular case, the invention is characterized in that the yield is between 85 and 10%, between 90 and 110%, between 95 and 110%, and even between 100 and 110% of the value of at least one variety. reference reference selected from the group consisting of FESTIVAL, CTPS France 2010 registration, and OMEGALIN, CTPS France 2012 registration and evaluated in France under similar conditions. In particular, the reference value can be obtained by averaging the yield of the two control varieties in questions evaluated in France under similar conditions. The assessment conditions correspond to standard flax growing conditions in France implemented in the context of the evaluation of flax varieties according to the VATE criteria required for inclusion in the national catalog, for example by the CTPS / GEVES or Inovia lands in the network Behavior.

A plant according to the invention is characterized in that it comprises a modification of the Δ15 desaturase gene responsible for the conversion of C18: 2 to C18: 3 in the seed.

A plant according to the invention is characterized in that the modification of the Δ 15 desaturase gene comprises a modification at the level of the promoter region of said gene.

A plant according to the invention is characterized in that the modification at the level of the promoter region of said gene is a mutation.

A plant according to the invention is characterized in that the mutation is a deletion.

A plant according to the invention is characterized in that the gene comprises SEQ ID No. 1.

A plant according to the invention is characterized in that the deletion is directed to a sequence between positions 10 and 20 of SEQ ID No. 1.

A plant according to the invention is characterized in that the deletion is directed to a sequence between the positions 12 and 17 of SEQ ID No. 1.

A plant according to the invention is characterized in that the deletion comprises the sequence AAAGGG.

Advantageously, a plant according to the invention is a progeny of the plant Linum usitatissimum VL8.29.3 deposited under the NCIMB Number 42681 on October 24, 2016. This plant contains all the genotypic elements causative of the phenotype of the invention necessary for the transfer to any another flax plant devoid of these elements to ensure conversion to a flax plant with a high C18: 3 fatty acid content. In the context of the present invention, the expression "yield between 85 and 110% of the value of reference reference varieties following evaluation under similar conditions in France" is understood as follows.

The yield can be between 85 and 110%, between 86 and 110%, between 87 and 110%, between 88 and 110%, between 89 and 110%, between 90 and 110%, between 91 and 110%, 92 and 110% between 93 and 110%, between 95 and 110%, between 96 and 110%, between 100 and 110%, in particular between 101 and 110% of the value of reference reference varieties following evaluation under similar conditions in France . The value of reference varieties can be understood as the average value of one or more control varieties following evaluation under similar conditions in France.

The plant according to the invention is cultivated under the given environmental and agroclimatic conditions corresponding to the standard flax crop conditions, in France, implemented in the context of the evaluation of flax varieties according to the VATE criteria necessary for the registration in the national catalog for example by the CTPS / GEVES.

The growing conditions can also correspond to those of the official network "flax behavior" of Terres Inovia which are quite close to those of the CTPS / GEVES.

In all cases the announced yield can be obtained, however the Terres Inovia protocol gives better results for both the plants according to the invention than for the reference plants and this, taking into account the ancillary treatments and supplements implemented.

It is understood, whatever the crop conditions used, that it is the same for the plants according to the invention and for the plants acting as control varieties which are all grown in the same area and in the same areas. same conditions in order to compare their performance.

The tests are conducted according to standard farming practices under given environmental and agroclimatic conditions. The tests are usually conducted in 3 to 6 repetitions. These tests make it possible to describe the cultural value of a plant in the main environmental and agroclimatic contexts encountered in France as well as the use value of the harvested products.

These tests make it possible to evaluate the yield as well as a certain number of characters, and to provide samples for the appreciation of the technological value. In the context of the present invention, the characteristic serving as a reference for comparison is the yield as measured according to the protocol indicated above (CTPS / GEVES).

The yield obtained is compared with that obtained under the same conditions with one or more control reference varieties of the market before or at the time of filing the present application. In the case of using more than one reference variety, the comparison yield is the average yield of said reference varieties.

Yield is defined as seed yield (in quintals per hectare) for a seed moisture content of about 6 to 9% by weight. This yield is thus compared, under the standard culture conditions described above, with control varieties such as FESTIVAL, CTPS France 2010 registration, and OMEGALIN, CTPS France 2012 registration for spring linens.

In a particular embodiment, a method according to the invention is a method for increasing the amount of C18: 3 fatty acid in the seed of a flax plant, which method comprises the transformation of a plant cell with at least one acid a nucleic acid that codes for the ΔΙ5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, thereby generating a plant that overexpresses the Δ15 desaturase responsible for the conversion of C 8: 2 to C18: 3 in the seed, and the crop of the plant at maturity.

The present invention thus relates to a method for increasing the amount of C18: 3 fatty acid in the seed of a flax plant, said method comprising overexpressing the ΔΙ5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed which results in an increase in C18: 3 amounts. In another embodiment, the method according to the present invention aims to increase the amount of C18: 3 fatty acid in the seed of a flax plant, which method comprises the transformation of a plant cell with at least one acid nucleic acid which codes for the Δ Ι 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, thus generating a plant which overexpresses the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, and growing the plant at maturity.

In a particular embodiment, the nucleic acid comprises the sequence SEQ ID No. 1.

In such a method according to the present invention the at least one nucleic acid which codes for the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed is under the control of a promoter ensuring the overexpression of Δ15 desaturase. responsible for the conversion of C18: 2 to C18: 3 in the seed. In one embodiment, the promoter comprises a mutation.

In a particular embodiment, the mutation comprises a deletion.

In a particular embodiment, the nucleic acid comprises SEQ ID NO: 1.

In another preferred embodiment of the present invention the at least one nucleic acid comprises a nucleic acid selected from the group consisting of SEQ ID No. 1.

In one embodiment of the present invention, overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of a flax plant is achieved by introgression of a genetic element coding for overexpression. Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of said flax plant.

The plants according to the invention thus have an increase in the amounts of alpha-linolenic fatty acid C18: 3.

It is also an object of the present invention to provide a method in which the introgression of a genetic element coding for overexpression of the Δ15 desaturase responsible for the conversion of C18.2 to C18: 3 in the seed of the flax plant is obtained by protoplast fusion.

It is also an object of the present invention to provide a method in which the introgression of a genetic element coding for overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of the Flax plant is obtained by embryo rescue.

The genetic element corresponds to a nucleotide sequence corresponding to that present in the plant Linum usitatissimum VL8.29.3 filed under the number NCIMB 42681 on October 24, 2016. The present invention also relates to a method for the production of plant having an amount of acid C18: 3 in the seed of an increased flax plant, method comprising detecting the presence of a genetic element, in particular a nucleic acid sequence, related to the overexpression of the Δ15 desaturase responsible for the conversion of the C18: 2 in C18: 3 in the seed in a donor flax plant and the transfer of the genetic element, in particular a nucleic acid sequence, related to the overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 at C18: 3 in the seed thus detected, from the donor flax plant to a recipient flax plant.

The overexpression of the ΔΙ 5 desaturase responsible for the conversion of C18: 2 to C8: 3 in the seed of the flax plant results in an increase in the amount of alpha-linolenic acid in the seed of the flax plant .

In a particular embodiment of a method according to the invention, the detection is carried out using at least one molecular marker. In an alternative embodiment of a method according to the invention, the detection is carried out by measuring the enzymatic activity of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed in the plant of donor flax.

The present invention also relates to a method for increasing the amount of C18: 3 fatty acid in the seed of a flax plant comprising providing a flax plant population and the selection of individuals from the population that exhibit Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the highest possible seed.

In a particular embodiment of the invention, the plant population is a population of mutant plants.

In particular, the population of mutant plants is obtained by TILLING.

In particular, the mutant plant population is obtained by CRISPR / Cas9.

It is also an object of the present invention to provide a method which comprises selecting, within a population of flax plants, at least one plant exhibiting overexpression of the ΔΙ 5 desaturase responsible for the conversion of C18: 2 in C18: 3 in the seed relative to the expression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of the parent flax plants.

In a particular embodiment of the present invention, the introgression comprises: a) providing a flax plant having a given level of expression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in seed, b) The provision of a flax plant with an increased level of expression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed relative to the plant provided in a), c ) The crossing of the plant provided in a) with the plant provided in b), d) The generation of an offspring resulting from crossing c), e) The selection within the offspring of at least one plant having a level expression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, higher than that of the plant provided in b). In a particular embodiment, the method described above comprises an additional step of crossing the plant selected in e) with the plant provided in b) followed by an additional step of selection within the offspring obtained from at least one plant having an expression level of Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed higher than that of the plant selected in e).

It is also an object of the present invention to provide a flax plant selection method, characterized in that it comprises the search for an allele of the Δ15 desaturase enzyme gene responsible for the conversion of C18: 2 in C18: 3 in the seed having a mutation resulting in an increase in the amount of alpha-linolenic acid in the seed of the flax plant.

In one embodiment, the mutation corresponds to a nucleotide sequence similar to that present in the plant Linum usitatissimum VL8.29.3 filed as NCIMB No. 42681 on October 24, 2016.

The present invention finally relates to a flax plant that can be obtained by a method according to one of the embodiments described here.

Finally, the present invention also relates to the use of a flax plant as obtained by a method according to one of the embodiments described herein, or a derivative of such a plant, for the preparation of a composition intended for human consumption, animal feed or the preparation of biofuels.

The invention also relates to a plant obtained or obtainable from a donor plant comprising a genetic element controlling the conversion of C18: 2 to C18: 3 in the seed, in particular from the plant Linum usitatissimum VL8 .29.3 filed under No. NCIMB No. 42681 on October 24, 2016, by introgressing said genetic element into a recipient plant.

An object of the present invention is therefore the use of at least one nucleic acid which encodes the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, and the Δ15 desaturase encoded protein responsible for the conversion of C18: 2 to C18: 3 in the seed, to obtain a flax plant with at least one increase in the amount of alpha-linolenic acid in the seed of the flax plant via overexpression of the ΔΙ5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed.

The invention provides a method for obtaining an increase in the amount of alpha-linolenic acid in the seed of a flax plant, including the step of overexpressing the ΔΤ5 desaturase responsible for C18: 2 conversion. in C18: 3 in the seed in cells of said plant.

The overexpression of the ΔΙ 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed in the cells of the plant can be obtained by various means available to those skilled in the art, whether by transgenesis, by transformation, by introgression, by selection, by marker-assisted selection, by random or directed mutagenesis followed or not by selection, for example.

A particular object of the invention is a process for producing a flax plant, or part of a flax plant, having an increase in the amount of alpha-linolenic acid in the seed of the plant of lin, which method comprises transforming a plant cell with at least one nucleic acid which encodes the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, and generating from such a cell, a plant that overexpresses the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed.

The method according to the invention for improving the amount of alpha-linolenic acid in the seed of the flax plant comprises transforming a flax plant cell with at least one nucleic acid which encodes the Δ15 desaturase responsible for the conversion. C18: 2 C18: 3 in the seed, and the generation from such a cell, of a flax plant which overexpresses the ΔΙ 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed .

In a particular embodiment, the at least one nucleic acid which codes for the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of the flax plant is under the control of a promoter providing overexpression of the Δ15 desaturase responsible for the conversion of C18.2 to C18: 3 in the seed. Another object of the invention is a process for producing a flax plant cell, a flax plant or a part of a flax plant, having an increased amount of alpha-linolenic acid in the seed of the flax plant, which method comprises the step of transforming a flax plant cell with at least one nucleic acid that encodes the Δ1-desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, and the additional step of generating of a plant therefrom that overexpresses the Δ Ι 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed. This strategy of overexpression of a genetic sequence represents a new genetic improvement that allows plants to exceed their maximum production potential of C18: 3.

In a particular embodiment of a method according to the invention, the nucleic acid which codes for the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed is under the control of a promoter providing the overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of the plant.

Another subject of the invention is a plant cell, a plant or part of a plant, which is transgenic for at least one nucleic acid which encodes the Δ Ι 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of the plant and that it overexpresses.

A nucleic acid encoding the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed can be any nucleic acid encoding the functional enzyme such that when introduced into a host cell and under the To control a suitable promoter, the amount of Δ 15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed and / or its enzymatic activity within the cell is increased.

By way of example, a nucleic acid coding for the Δ 15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed may be the nucleic acid according to the sequence SEQ ID No. 1. This sequence SEQ ID No. 1 corresponds to a nucleic acid encoding the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed itself and comprises a mutated promoter portion.

Such a sequence can be found in the plant Linum usitatissimum VL8.29.3 filed as NCIMB No. 42681 on October 24, 2016.

More particularly, in the context of the present invention, the nucleic acid encoding the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed: i) has a sequence having at least 55% homology with the SEQ ID No. 1 or its complementary sequence, or ii) has a sequence that hybridizes to SEQ ID No. 1 or its complementary sequence, under stringent conditions, and which encodes the Δ15 desaturase responsible for the conversion of the C18: 2 in C18: 3 in the seed.

In a particular embodiment, the nucleic acid encoding the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed has a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 97%, at least 98% or at least 99% homology with SEQ ID ^No. 1.

The nucleic acid encoding the ΔΙ5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed to be transferred in order to overexpress the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed can also be a nucleic acid homologous to the host plant chosen from the nucleic acids of plants, microorganisms or algae.

A suitable nucleic acid can be found in the plant Linum usitatissimum VL8.29.3 filed as NCIMB No. 42681 on October 24, 2016. The term "homologue" or "homology" refers to any nucleic acid, or protein, having one or more sequence modifications with respect to all or part of the sequence of SEQ ID No. 1 while retaining most or all the totality of Δ15 desaturase activity responsible for the conversion of C18: 2 to C18: 3 in the seed.

In one embodiment of the present invention, overexpression of the Δ15 desaturase responsible for the conversion of C18.2 to C18: 3 in the seed in plants can be achieved using the sequence SEQ ID No. 1.

In a particular embodiment of the present invention, the nucleic acid encoding the ΔΙ 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed comprises a nucleic acid selected from the group consisting of SEQ ID NO: 1 .

In a particular embodiment, the nucleic acid encoding the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed has a sequence having at least 60%, at least 65%, at least 70%, at least 75%), at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96% , at least 97%, at least 98%, at least 99% homology with the sequence of a nucleic acid selected from the group consisting of SEQ ID No. 1. The present invention further provides an expression cassette comprising a plant-expressible promoter operably linked to a coding region containing at least one nucleic acid encoding a Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed. wherein said promoter is not a ΔΙ 5 desaturase promoter responsible for the conversion of C18: 2 to C18: 3 in the seed. Said promoter may be a 35S promoter, a ubiquitin promoter or an actin promoter, for example.

The term "transgenic" means that the plant or plant cell comprises in its genome at least one nucleic acid encoding the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed which is foreign to this plant or plant cell, or which comprises in its genome at least one endogenous coding sequence of the Δ15 desaturase responsible for the conversion of the C18.2 to C18.3 in the seed operably linked to at least one regulatory region, for example, a promoter, which is not present in the endogenous gene of this plant or plant cell. In general, the foreign nucleic acid is stably integrated into the genome such that the polynucleotide is passed on to successive generations. The term transgenic also includes the case in which the plant cell or the plant comprises in its genome two or more endogenous or exogenous nucleic acids of the species of the cell or plant encoding the ΔΙ 5 desaturase responsible for the conversion. C18: 2 C18: 3 in the seed; thus allowing overexpression of L-asparate oxidase. The term "overexpression" is intended here to mean both an increase in the amount of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed relative to the amount expressed in a control plant, and a ectopic expression of this enzyme, in a tissue or compartment and / or at a stage of development where it is not normally expressed. It encompasses the situation in which the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed is endogenous or heterologous, ie when it is an organism, such as a plant, different from the host cell, or wherein at least one transcriptional regulatory region of the ΔΙ 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed is not present in the endogenous gene. The amount and / or activity of the Δ15 desaturase protein responsible for the conversion of C18: 2 to C18: 3 in the seed expressed in a plant cell can be determined by any known assay technique available to humans of career. More particularly, the Δ15 desaturase activity responsible for the conversion of C18: 2 to C18: 3 in the seed can be evaluated by gas chromatographic (GC) analysis by analyzing the% C 8: 2 and C 18: 3 in the oil.

The term "overexpression" also means that the enzymatic activity of L-aspartate oxidase produced within the host cell after introduction of the sequence encoding the ΔΙ 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, with an equal amount of enzyme produced, is greater than the enzymatic activity of the ΔΙ 5 desaturase responsible for the conversion of C18: 2 to C18: 3 of the host cell before the introduction of said sequence. Such overactivity specific may be due to a difference in primary, secondary or tertiary structure of the protein due to a difference in the encoding nucleic sequence.

Overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed results in an increase in the amount of alpha-linolenic acid in the seed of the flax plant.

By the term "control plant" in the context of the invention is meant a flax plant which has the same genetic background as a flax plant according to the present invention in which the control plant does not have the nucleic acid or genetic element allowing overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed according to the present invention; overexpression due to an increase in the amount of alpha-linolenic acid in the seed of the flax plant.

A control plant is cultivated for the same length of time and under the same conditions as a plant according to the present invention. The expression "variety", "cultivar" or "plant variety" is here understood according to the definition of UPOV. Thus, a control plant may be a variety, a pure line or a hybrid, provided that it has the same genetic background as the plant according to the present invention with the exception of the nucleic acid, or the genetic element, allowing the improvement of the amount of alpha-linolenic acid in the seed of the flax plant according to the present invention and related to the overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed.

In the methods or plants of the invention, the nucleic acid or genetic element encoding the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed can be heterologous with respect to the plant in which it is introduced or may belong to the same species, insofar as it can be expressed in plants in quantities greater than the quantity conventionally produced by an unprocessed plant or a plant which did not contain the sequence or the element introduced or introgressed. Thus, any nucleotide sequence encoding the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed can be used, for example a wild-type sequence for the Δ15 desaturase gene responsible for the conversion of C18: 2 to C18: 3 in the seed, a mutated sequence at the coding portion of the enzyme resulting in a higher specific activity or a mutated sequence at the level of the promoter region resulting in a higher production of the protein and thus an overexpression of the enzyme, or even a combination of both cases, and therefore a better conversion of C18: 2 to C18: 3 in the seed.

Advantageously, a sequence coding for the ΔΙ 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, used to transform the cells or plants according to the invention comprises a nucleic acid of SEQ ID No. 1 .

One embodiment of the invention comprises overexpressing the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed in a flax plant, overexpressing the ΔΙ 5 desaturase responsible for C18: 2 conversion. at C18: 3 in the seed encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 1, in flax plant cells.

Those skilled in the art know how to identify nucleic acid sequences encoding the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed within different species, by comparison of SEQ ID NO: 1 with the sequences of other species, with a computer program such as BLAST (www.ncbi.nlm.nih.gov) and fast DB with the default settings. Those skilled in the art may also use one of the nucleic acid sequences selected from the group consisting of SEQ ID No. 1 to perform sequence comparisons to identify and find sequences encoding the Δ! Desaturase responsible for the conversion of C18: 2 to C18: 3 in the appropriate seed. These algorithms are described in "Current methods of sequencing and synthesis methods and applications," pages 127-149, 1988, in Alabama R. Liss, Inc.

The homologous sequences are preferably defined as follows: i) DNA sequences which show a similarity or identity of at least 55%, preferably at least 70%, preferably at least 80%, still more preferably at least 90%. %, more preferably and still at least 95% with the sequence SEQ ID No. 1; ii) sequences which hybridize with the sequence of SEQ ID NO: 2, with its complementary sequence, under stringency hybridization conditions, for example of low stringency, or

iii) sequences encoding an Δ15 desaturase enzyme responsible for the conversion of C18: 2 to C18: 3 in the seed comprising the amino acid sequence of SEQ ID NO: 1, or a homologous amino acid sequence, by for example, any amino acid sequence with an Δ15 desaturase enzymatic activity responsible for the conversion of C18: 2 to C18: 3 in the seed and having at least 60%, preferably at least 70%, preferably at least 80%, still more preferably at least 90%, more preferably at least 95% sequence identity with the sequence of SEQ ID NO: 1.

Preferably, such a homologous nucleotide sequence hybridizes specifically to sequences complementary to the sequence SEQ ID No. 1 under rigorous conditions. The parameters that define the stringency conditions depend on the temperature (Tm) at which 50% of the paired strands separate. Low stringency hybridization conditions are those in which hybridization is observed using a hybridization temperature of 5-10 ° C below Tm, and the hybridization buffers are solutions of high ionic strength, by example a 6 x SSC solution.

The terms "sequence similarity" or "sequence identity" or "sequence homology" are used interchangeably herein and mean in the context of two or more nucleic acid or protein sequences that are the same or have a percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum match, as measured using one of the following sequence comparison algorithms or by visual inspection, if two sequences to be compared between they have different lengths, a sequence identity preferably relates to the percentage of the nucleotide residues of the shortest sequence, which are identical to the nucleotide residues of the longer sequence. Sequence identity can be determined conventionally with the use of computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison 575 Science Drive, WI 53711). Bestfit uses Smith and Waterman's (1981) local homology algorithm to find the segment with the highest sequence identity between the two sequences. When Bestfit or any other sequence alignment program is used to determine whether a particular sequence has, for example, 95% identity with a reference sequence of the present invention, the parameters are preferably adapted in such a way that the percent identity is calculated over the entire length of the reference sequence and homology discrepancies up to 5% of the total number of nucleotides in the reference sequence are allowed.

A nucleic acid sequence is homologous to a sequence, such as the sequence (coding sequence, CDS) shown in SEQ ID NO: 1 for example, as used herein, when it comprises a nucleotide sequence which differs from that sequence. by mutation, insertion, deletion or substitution of one or more bases, or by degeneracy of the genetic code, provided that it encodes a polypeptide having the activity of the Δ15 desaturase enzyme responsible for the conversion of C18 : 2 in C18: 3 in the seed. A protein is homologous to the Δ15 desaturase responsible for the conversion of C18: 2 to C8: 3 in the seed represented in SEQ ID NO: 1, when it comprises an amino acid sequence which differs from the sequence SEQ ID N 1, by mutation, insertion, deletion or substitution of one or more of the amino acids, as long as it is a polypeptide having the activity of the Δ15 desaturase enzyme responsible for the conversion of C18: 2 to C18: 3 in the seed.

The Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed is an enzyme that catalyzes the chemical conversion reaction of linoleic acid (C18: 2 co6) to linolenic acid (C18: 3 ω3).

The expression "the activity of the enzyme ΔΙ 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed" or "the Δ15 desaturase enzymatic activity responsible for the conversion of C18: 2 to C18: 3 in the seed, as used herein, refers in particular to its carbon dehydrogenation activity located in delta-15 positioin (ie the 15th carbon closest to the hydroxyl group) of linoleic acid which can be determined by GC analysis of the fatty acid profile transesterified oil extracted from flax seeds. A detailed measurement protocol is described in the experimental section later.

The nucleic acid which codes for the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed is generally inserted, in single or multiple copies, into a nucleotide construct, called an expression cassette, in which it is functionally linked to elements allowing its expression, more particularly its overexpression and possibly its regulation.

These elements include transcription promoters, activators and / or terminators. In a particular embodiment, the plant cell is transformed with an expression cassette comprising at least one nucleotide sequence coding for the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed and a promoter specific for a tissue. Expression in lignin-containing or inflorescent tissues may be of particular interest, as well as selective or preferential expression in flowers or seeds. A specific promoter of the root may also be useful. Expression in root tissues can be accomplished using the acid chitinase gene (Samac et al., 1990), or the specific deep subdomains of the Ca V35S promoter that have been identified (Benfield et al., 1989). . Among the transcription promoters that may be employed are: a 35S promoter, or the Caesar mosaic virus (CaMV) double constitutive 35S promoter (pd35S), as described in Kay et al., 1987; a radiot cruciferin PCRU promoter which directs the expression of the associated sequences only in the seeds of the transgenic plant; promoter PGEA1 and PGEA6 which correspond to the 5 'non-coding region of the Arabidopsis thaliana seeds storage protein genes (GEA1 and GEA6, respectively) which direct specific expression in the seeds; the chimeric PSP promoter (Ni et al., 1995) which is a fusion of a triple repetition of a transcriptional activator element of the octopine synthase gene promoter in Agrobacterium tumefaciens; a rice actin promoter, optionally followed by the rice actin intron (PAR-IAR), for example; the promoter contained in the plasmid pAct1-F4 (Me Elroy et al, 1991) the HMGW wheat promoter (high molecular weight glutenin); the promoter of the maize zein gene (β-zein) contained in the plasmid p63, which directs the expression in the albumen seeds.

Other suitable plant-expressible promoters in accordance with the present invention include, but are not limited to: promoters from the ubiquitin family (e.g., the maize ubiquitin promoter of EP 0 342 926), a rice actin promoter such as the promoter described by Me Elroy et al., (Already mentioned above) or the promoter described in US 5,641,876; any of the promoters of the cassava mosaic virus vein (WO 97/48819), any of the pPLEX series of underground clover Stunt virus (WO 96/06932), or an alcohol dehydrogenase promoter for example, pADH 1 S (GenBank accession numbers X04049, X00581).

Among the terminators that can be used in the constructs of the invention, mention may be made especially of the 3 'end of the Agrobacterium tumefaciens nopaline synthase gene. The expression cassette may be inserted into a nucleotide vector, such as a plasmid, which may further comprise a marker gene, for example a gene for selecting a transformed plant from a plant that does not contain foreign DNA. transfected. As a marker gene, this may in particular consist of a gene which confers antibiotic resistance or herbicide resistance, or resistance to an amino acid, for example.

The vector thus constructed can be used to transform host cells, according to techniques known to those skilled in the art.

These include methods for direct gene transfer into plant cells, such as transformation with Agrobacterium tumefaciens, direct microinjection into plant embryoids (Neuhaus et al., 1987), vacuum infiltration (Bechtold et al. et al., 1993) or electroporation (Chupeau et al., 1989), or alternatively, direct precipitation with PEG (Schocher et al., 1986) or bombardment of DNA-coated particles plasmid of interest, using a gun (Fromm M. et al., 1990), for example. According to another embodiment of the method of the invention, the plant cells are transformed with a vector as defined above, transferred into a host cell capable of infecting said plant cells by allowing the integration, in the genome of the latter, nucleotide sequences of interest initially contained in the genome of the aforementioned vector. Advantageously, the cellular host used is a bacterial strain, such as Agrobacterium tumefaciens, or Agrobacterium rhizogenes, for example.

The flax plants thus obtained overexpress the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed.

Such flax plants or parts of a plant are advantageously obtained by the method described above, in which a plant cell is transformed with at least one nucleic acid which encodes the Δ15 desaturase enzyme responsible for the conversion of C18: 2 in C18: 3 in the seed, and cultured, whereby one obtains a plant that overexpresses the ΔΙ 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed.

The transgenic plants according to the invention encompass both the plants of the first generation as well as their descendants containing the expression cassette allowing the overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed according to the invention. the invention (lineage varieties or hybrid varieties, in particular).

Parts of the plant include any tissue or organ, such as roots, flowers, stems, leaves, fruits, reserve organs or seeds.

The present invention particularly comprises seeds which have an increased expression of Δ, Ι 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, obtained by specific overexpression of an Δ15 desaturase coding sequence responsible for the conversion of C18: 2 to C18: 3 in the seed.

In one embodiment of the invention, the expression cassette of the invention is used to overexpress the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of a flax plant. This use leads to an increase in the amount of alpha-linolenic acid in the seed of the flax plant. As mentioned above, the nucleic acid coding for overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed within the plants according to the invention may belong to the same species and this in the to the extent that it can be expressed in plants in amounts greater than the amount conventionally produced by a plant which did not contain the nucleotide sequence or introduced genetic element. Thus, any nucleotide sequence encoding the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed can be used, for example, a wild-type sequence for the Δ15 desaturase gene responsible for C18 conversion. : 2 in C18: 3 in the seed, a mutated sequence of a wild-type sequence which codes for the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, or a mutated sequence of the promoter of the D gene; a Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the wild or mutated seed that induces an increase in the amount of Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, or the stability of the messenger RNA of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, or an expression of a ΔΙ 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed with superior enzymatic activity, this p relative to the amount and / or activity of Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed that were produced or expressed within the host plant prior to receiving the introduced sequence.

As indicated above, the overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed can be obtained by various means available to those skilled in the art, whether by transgenesis or by transformation. by introgression, by selection, by marker-assisted selection, by random or directed mutagenesis followed or not by selection, for example.

In a particular embodiment of the method according to the present invention, the overexpression of the ΔΙ5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of the flax plant is carried out by introgression of a coding genetic element. for overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed. The term "genetic element" or "genetic material" as used herein refers to any gene, gene group, QTL, locus, allele, chromosomal fragment, nucleotide sequence, nucleic acid sequence that is capable of contributing to the increase in quantity. of alpha-linolenic acid in the seed of the flax plant, influencing the expression of the Δ15 desaturase responsible for the conversion of C18.2 to C18: 3 in the seed at the level of the DNA itself, at the level of translation, transcription and / or activation of a final polypeptide product, that is, to regulate the metabolism of the plant leading to the phenotypic expression of the genotype. In the context of the present invention, the terms "introgression", "introgressed" and "introgresser" refer to the process by which one or more genetic elements such as a gene or genes, one or more QTLs, one or alleles, one or more chromosomal fragments, or one or more nucleic sequences present in the genome of a species, variety or cultivar are relocated and stably transferred into the genome of another species, variety or cultivar, by sexual crossing. The transfer can be natural or artificial. The process may possibly be completed by backcrossing with a recurrent parent, in which case introgression refers to the introduction of one or more genetic elements, such as one or more genes, one or more alleles, one or more QTL, one or more chromosomal fragments or one or more nucleic sequences of one species in the genetic pool of the other by repeated backcrossing of an interspecific hybrid with one of its parents. Introgression can also be described as the stable integration of heterologous genetic material into the genome of a recipient plant by sex crossing between plants of the same or near, ie, sexually compatible, species. The concept of sexually compatible means that the fertilization of a flower of one plant by the pollen of another plant gives rise to the fertilization of the egg and the production of a fruit containing one or more seeds able to sprout and give a new plant. The concept of sexually compatible also includes cases where the viability of the embryo formed is ensured by one or more embryo rescue techniques. The skilled person has various embryo rescue methods that he can use depending on the species involved. It is thus proposed according to a preferred embodiment of the present invention, a method for increasing the amount of alpha-linolenic acid in the seed in a flax plant comprising providing a population of flax plants, including the providing a population of flax plants derived from crosses, and selecting individuals from the population who exhibit expression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the highest possible seed.

The flax plant population may be a population of mutant plants generated by chemical mutagenesis or by any other means capable of inducing one or more mutations within the genome of the treated plants such as TILLING or CRISPR / Cas9. The mutagenesis treatment results in the voluntary introduction of mutations by the action of chemical or physical mutagenic agents in a DNA sequence, which agents may be a chemical treatment such as, for example, an ethyl methanesulphonate (EMS) treatment. Advantageously, a TILLING population may be used for the selection of individuals that exhibit expression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the highest possible seed.

Typically TILLING technology relies on seed mutagenesis followed by phenotyping and genotyping to identify mutations and allele (s) associated with a given phenotype, preferably an advantageous phenotype. The TILLING population generation can be performed as follows. MO seeds are mutagenized by ethylmethanosulphonate (EMS) treatment. The M1 plants derived from the MO seeds are self-fertilized, the DNA is extracted from the M2 families for high-throughput mutation screening and the M3 seeds are harvested and conserved. The DNA of the M2 families is pooled 8 times and amplified for a target gene. The amplification products are incubated with an endonuclease that preferentially cleaves mismatches in heteroduplexes between wild and mutant. The digestion products are subjected to sequence gel electrophoresis. The LI-COR technology allows double-strand fluorescent labeling (IRDye 700 and 800) which allows rapid visual confirmation because the mutations are detected on the two complementary strands and thus easily distinguished from the artifacts. After detecting a mutation in a pool, the DNA of the individual families is quickly screened by deconvolution of the pool to identify the family carrying the mutation.

In a particular embodiment of the present invention, the introgression of a genetic element coding for overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed is obtained by protoplast fusion.

Thus in such an embodiment of the invention, to increase the amount of alpha-linolenic acid in the seed of the flax plant, the protoplast fusion can be used for the transfer of at least one genetic element to from a donor plant to a recipient plant. Protoplast fusion is an induced or spontaneous union, such as somatic hybridization, between two or more of the protoplasts (the cell walls are removed by enzymatic treatment) to produce a single bi- or multi-nucleated cell. The fused cell, which can also be obtained with plant species that can not be sexually crossed in nature, is grown in a hybrid plant with the combination of desirable characteristics. More specifically, a first protoplast can be obtained from a plant according to the invention and which has an increase in the amount of alpha-linolenic acid in the seed of the flax plant and overexpression of the Δ1 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed. A second protoplast can be obtained from a plant that includes characteristics of commercial value. The protoplasts are then fused using traditional protoplast fusion procedures, which are known in the art.

Alternatively, the embryo rescue can be used in the transfer of a genetic element, particularly a nucleic acid, allowing overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of a donor plant according to the invention to a recipient plant according to the invention. Embryo rescue can be used as a procedure to isolate embryos from crosses in which the plants fail to produce viable seeds. In this process, the fertilized or immature egg of a plant is a tissue of culture to create new plants. It is thus an object of the present invention to provide a method in which the introgression of a genetic element coding for overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed is obtained. by embryo rescue. The present invention also relates to a method for the production of flax plants having an increased amount of alpha-linolenic acid in the seed of the flax plant, comprising detecting the presence of a genetic element, in particular a sequence of nucleic acid, related to the overexpression of the ΔΙ5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed in a donor flax plant and the transfer of the genetic element, in particular a nucleic acid sequence , related to the overexpression of Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed thus detected, from the donor plant to a recipient flax plant. The transfer of the nucleic acid sequence can be accomplished by any of the methods previously described herein.

The transfer can be carried out by a technique chosen from transgenesis, introgression, protoplast fusion, embryo restoration.

An exemplary embodiment of such a method comprises the introgression transfer of the genetic element, in particular the nucleic acid sequence, linked to the overexpression of the ΔΙ 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed from a donor plant to a recipient plant by sexual crossing of the plants. This transfer can therefore advantageously be achieved using traditional crossing and selection techniques.

In a particular embodiment of the method according to the invention, the detection of the presence of a genetic element linked to the overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed is carried out at using at least one molecular marker.

In another embodiment of the method according to the invention, the detection of the presence of the genetic element linked to the overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed is carried out by the measurement of the enzymatic activity of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed in the donor plant.

According to some embodiments, the genetic element responsible for overexpressing the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed can be introgressed into commercial varieties of plants of agronomic interest using of marker-assisted selection (SAM) which involves the use of one or more molecular markers for the identification and selection of offspring plants that contain the genetic element, gene or plurality of genes, sequences of nucleic acid encoding the desired overexpression characteristic of the ΔΙ 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed.

In the context of the present invention, such identification and selection is based on the selection of genes, genetic elements or nucleic acid sequences or markers associated therewith. The plants obtained according to these embodiments may advantageously draw the majority of their traits from the recipient plant, and draw at least one phenotypic character of increasing the amount of alpha-linolenic acid in the seed of the donor flax plant, thanks to the overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed. As discussed above, conventional crossover techniques can be used for introgression of nucleic acid sequence responsible for overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, related to an increase in the amount of alpha-linolenic acid in the seed of the flax plant in a recipient plant. In some embodiments, a donor flax plant exhibiting an increase in the amount of alpha-linolenic acid in the seed and comprising a nucleic acid sequence responsible for overexpression of the Δ15 desaturase responsible for C18 conversion. : 2 in C18: 3 in the seed, is crossed with a recipient plant which in some embodiments may have commercially desirable characteristics. The resultant plant population (representing F1 hybrids) is then self-fertilized producing F2 seeds. The F2 plants derived from F2 seeds are then screened for plants with overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, associated with an increase in the amount of alpha acid. -linolenic in the seed of the flax plant; this by methods known to those skilled in the art.

Plant lines exhibiting overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, associated with an increase in the amount of alpha-linolenic acid in the seed of the flax plant can be developed using recurrent and backcrossing, selfing, and / or dihaploid selection techniques, or any other technique used to make parental lines. In a recurrent and backcrossing selection process, the increase in expression of the ΔΙ 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, associated with an increase in the amount of alpha-linolenic acid in the seed of the flax plant can be introgressed into a target recipient plant (the recurrent parent) by crossing the recurrent parent with a first donor plant, which differs from the recurrent parent and is referred to herein as the "non-recurrent parent". The recurrent parent is a plant that does not overexpress the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, associated with an increase in the amount of alpha-linolenic acid in the seed of the plant. flax plant but may have desirable commercial characteristics.

The non-recurrent parent may be any plant variety or pure lineage that is sexually compatible with the recurrent parent. Plants of the offspring of a cross between the recurrent parent and the non-recurrent parent are backcrossed to the recurrent parent. The resulting plant population is then screened for overexpression of the Δ Ι 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, associated with an increase in the amount of alpha-linolenic acid in the seed of the flax plant.

Marker Assisted Selection (SAM) can be performed using hybridization probes or polynucleotides to identify which plants include a nucleic acid sequence or any genetic element causing overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, associated with an increase in the amount of alpha-linolenic acid in the seed of the flax plant. Following screening, hybrid F1 plants that overexpress the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed, associated with an increase in the amount of alpha-linolenic acid in the seed of the flax plant, are then selected and backcrossed with the recurrent parent for a number of generations to allow the plant to become more inbred. This process can be done for two, three, four, five, six, seven, eight, or more generations.

In general, the present invention relates to a process for producing a flax plant having an increase in the amount of alpha-linolenic acid in the seed of the flax plant which may comprise: (a) providing a flax plant exhibiting a given level of expression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed;

(b) providing a second flax plant having a level of expression of Δ Ι 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed higher than that of the plant provided in (a); (c) crossing the plant provided in (a) with the plant provided in (b) to produce plants of progeny F1;

(d) selecting F1 offspring plants which overexpress the Δ 15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed relative to the plant provided in (a); (e) crossing the plants selected in (d) with the plant provided in (a) to produce backcross progeny plants;

(f) selecting plants of the backcross progeny that overexpress the Δΐ 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed relative to the plants selected in (d); (g) repeating steps (e) and (f) two or more times in succession;

(h) possibly self-pollinate backcrossed plants to identify homozygous plants and

(i) cross at least one plant of the backcross progeny or self-pollinated plants with another plant provided in (a) to produce a plant having an increase in the amount of alpha-linolenic acid in the seed of the flax plant when grown under the same environmental conditions.

As indicated, the last generation of backcrossing can be self-pollinated to provide homozygous individuals overexpressing the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed and increasing the amount of acid. alpha-linolenic in the seed of the flax plant.

In accordance with a preferred embodiment of the present invention, the screening step comprises selecting individuals which each contain an allele of the gene which encodes for overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed.

The present invention also relates to a plant selection method, characterized in that it comprises the search for an allele of the Δ15 desaturase enzyme gene responsible for the conversion of C18: 2 to C18: 3 in the seed having a mutation resulting in an increase in the amount of alpha-linolenic acid in the seed of the flax plant.

In addition, according to a preferred embodiment of the present invention, the selection step comprises selecting with a molecular marker for the Δ15 desaturase gene allele responsible for C18: 2 conversion to C18: 3 in the seed.

In addition, according to a preferred embodiment of the present invention, the screening step comprises selecting by measuring the activity of the enzyme ΔΙ 5 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed in young plants and selecting those with high activity of the enzyme. In a particular embodiment, the invention relates to a flax plant selection method having overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed comprising the following steps: (a) providing a population of flax plants;

(b) measuring the activity of the Δ1 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of each individual of the plant population provided;

(c) selecting the plant (s) having the Δ15 desaturase activity responsible for the conversion of C18: 2 to C18: 3 in the highest seed.

In a particular embodiment, the plant population is a population of TILLING or resulting from modification by CRISPR / Cas9,

In another particular embodiment, the plant population is a population derived from intraspecific or interspecific crosses. In another particular embodiment, the plant population is a population of commercial varieties.

In the context of the present invention, the expression "overexpression" with reference to the Δ Ι 5 desaturase responsible for the conversion of C18.2 to C18: 3 in the seed refers to the fact that the nucleic acid sequence encoding the protein is transcribed into RNA in increased amount and / or that the RNAs of the protein are translated into protein in increased amount and / or that the amount or specific activity of the translated protein is increased. In the end, the notion of expression or overexpression refers to the enzymatic activity of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed within the plant and to the fact that it is due to a greater synthesis of the protein and / or more important specific activity.

In the context of the present invention, the terms "sexual crossing" and "sexual reproduction" refer to the fusion of gametes to produce offspring (for example by fertilization, so as to produce seeds by pollination in plants). In some embodiments, a "sexual crossing" or "cross fertilization" is the fertilization of one individual by another (for example, for example, cross pollination of plants). The term "self-fertilization" refers to certain embodiments of seed production by self-pollination or self-pollination, ie pollen and eggs are from the same plant. By the term "character" is meant in the context of the present a characteristic or a phenotype, for example yield, biomass or germination rate. A trait may be inherited dominantly or recessively, or may be monogenic or polygenic.

By the term "donor plant", in the context of the invention is meant a plant which provides at least one genetic element linked to an overexpression of the Δ 15 desaturase responsible for the conversion of C18: 2 to C18: 3 in seed.

By the term "recipient plant", in the context of the present invention is meant a plant which receives at least one genetic element linked to an overexpression of the Δ 15 desaturase responsible for the conversion of C18: 2 to C18: 3 in seed.

In the context of the present invention, the terms "genetic marker", "DNA marker" or "molecular marker" are interchangeable and refer to a characteristic of the genome of an individual (for example a nucleotide or a sequence of d nucleic acid that is present in the genome of an individual), which is linked to one or more loci of interest. Genetic markers include, for example, single nucleotide polymorphisms (SNPs), indels (ie insertions / deletions), simple sequence repeats (SSRs), fragment length restriction polymorphisms (RFLPs), or lengths of amplified fragments (AFLP) for example. Genetic markers can, for example, be used to locate genetic loci containing alleles that contribute to the variability of phenotypic characteristics.

The term "genetic marker" may also refer to a sequence of polynucleotides complementary to a genomic sequence, such as a sequence of a nucleic acid used as a probe. A genetic or molecular marker can be physically located in a position on a chromosome that is distal or proximal to one or more genetic loci with which it is bound (i.e., is intragenic or extragenic, respectively). In some embodiments of the present invention, the one or more genetic markers comprise between one and ten markers, and in some embodiments, the one or more genetic markers comprise more than a dozen genetic markers. In the context of the present invention, the term "genotype" refers to the genetic constitution of a cell or organism. As is known in the art, a genotype can refer to a single locus or multiple loci. In some embodiments, the genotype of an individual relates to one or more genes that are linked by the fact that one or more of the genes are involved in the expression of a phenotype of interest (eg character as defined here). Thus, in some embodiments, a genotype has one or more alleles present in an individual at one or more loci for a trait.

In the context of the present invention, the term "gene" refers to an inherited unit comprising a DNA sequence that occupies a specific location on a chromosome and that contains the genetic instruction for a particular trait or trait in an organism. .

In the context of the present invention, the terms "nucleic acid" or "oligonucleotide" or "polynucleotide" or "nucleic acid sequence" or grammatical equivalents thereof, mean at least two nucleotides covalently linked together. Oligonucleotides are typically about 7, 8, 9, 10, 12, 15, 18, 20, 30, 40, 50 or up to about 100 nucleotides in length. Nucleic acids, nucleic sequences and polynucleotides are polymers of any length, including the longer lengths, for example, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10000, etc. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some instances nucleic acid analogs are included which may have alternative backbones including, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages. (see Eckstein, 1991), and peptide backbones and nucleic acid links. Mixtures of natural nucleic acids and analogs can be used. In the context of the present invention, the expression "phenotype" or "phenotypic character" refers to the appearance or any other detectable characteristic of an individual, resulting from the interaction of its genome, proteome and / or metabolome with the environment. In the context of the present invention, a "plant" is a plant at any stage of development, particularly a seed plant.

In the context of the present invention, a "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in the form of an isolated single cell or a cultured cell, or as an upper organized unit portion such as, for example, plant tissue, plant organ or whole plant. A plant cell may be able to regenerate a plant or not be able to regenerate a plant.

In the context of the present invention, the term "plant material" refers to leaves, stems, roots, flowers or parts of flowers, fruits, pollen, ova, zygotes, seeds, cuttings, cells or cultures. tissues, or any other part or products of a plant, more particularly seeds.

As used herein, the term "plant part" refers to a part of a plant, comprising single cells and cell tissues such as plant cells that are intact in plants, clumps of cells, and Tissue cultures from which plants can be regenerated or not. Examples of plant parts include, but are not limited to, individual cells and pollen tissues, eggs, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems , shoots and seeds, as well as grafts, rootstocks, protoplasts, cali, and the like, more particularly seeds.

As used herein, the term "population" refers to a genetically heterogeneous set of plants sharing a common genetic derivation.

Figure 1 represents the crossover and selection scheme from lines L1, L2 and L3 crossed with mutant 48. EXAMPLE

The Applicant implemented a program in 2001 to exceed the limit of 66.7% ALA in oils extracted from flaxseed by means of unsupervised mutagenesis. For this the technique of bombardment with gamma rays (400Gr) was chosen. Several lines belonging to the Applicant have been subjected to this treatment, resulting in the production of several transformed lines with ALA contents close to 70%. However, the yields of these lines were too low to hope for commercial exploitation. These lines were used as broodstock for crosses with other lines with superior agronomic qualities.

Following genealogical selection, new varieties combining high ALA content (greater than 66.7%) with strong agronomic qualities were established. The yields of these varieties are comparable to those of the controls used for listings. The same is true for the germination of seeds from these varieties with high ALA content. These last two elements are important because they are the main defects of Lutéa (yellow seeded flax), one of the varieties listed in the French catalog with the highest ALA content (63.5% on average).

In varieties with high levels of ALA, a genetic marker was found upstream of a gene encoding a desaturase responsible for the conversion of C18: 2 to C18: 3 in flaxseed.

Irradiation of flax seeds and crossing pattern Three batches of 1000 seeds from a standard M1 line were placed in an irradiation chamber (Cobalt-60) and received a gamma ray dose of 400 Gy (10 Gy / h).

After 5 days of incubation at 25 ° C. in a petri dish, the seeds were sown in pots placed in a greenhouse. The survival rate was 15%. The seeds of each plant of this descending population M2, M3, M4 ... were harvested individually, then analyzed by GPC in order to obtain their C18: 3 profiles.

The M48 plant was chosen for its very high C18: 3 content (> 70%). After fixation by successive self-pollinations, the M48 line was crossed with three other lines: L1, L2 and L3 (see Figure 1).

L1, L2, L3: Linseed lines linseed standard selection Laboulet Seeds.

M48: high-grade mutated line on M1 genetic background.

In the offspring of these crosses, fixation and selection were carried out over the next 10 generations to create high C18: 3 varieties with agronomic performance.

HT1, HT2, HT3: fixed lines from crosses with the high-grade line.

The plant Linum usitatissimum VL8.29.3 filed under NCIMB No. 42681 on October 24, 2016 corresponds to HT3.

Determination of fatty acids in CPG

The content of the various fatty acids is important for the food markets of the oils. As a result, the C18: 3 content of flaxseed is essential for their commercial value.

A bulk from the same plant is milled, then the oil is extracted from the ground material with isooctane and transesterified with a methanolic solution of hydroxide of potassium. The methyl esters of fatty acids are then analyzed by gas chromatography (GC).

The linolenic acid content is determined by gas chromatography of the fatty acid methyl esters according to the standard of International Organization for Standardization, Animal and vegetable fats and oils 1505508: 1990E.

Sequencing

The extractions of genomic DNA were carried out according to the protocol of the kit DNeasy Plant Mini Kit (Qiagen).

The complete genome of the HT1, HT2, VL8.29.3 and L1 lines were sequenced with a very high speed Hiseq2500 sequencer (Illumina).

Positioning of the deletion · Complete genomes of HT1, HT2, HT3 (VL8.29.3) and L1 were sequenced and aligned.

^• HT1, HT2, HT3 (VL8.29.3) are the only ones to have a deletion (in comparison to L1 and flax accessible databases) upstream of a delta-15 desaturase, explaining the overexpression of this one. · A publication on flax mutants possessing very low C18: 3 contents shows that deletions in the same zone led to a gene silencing of Lus10038321. g (Vrinten et al., 2005).

^• 5 'promoter zone of the Lus10038321 gene .g -> omega-3 fatty acid desaturase; delta-15 desaturase (Linum usitatissimum clone LuBAC395P20, complete sequence).

Results of yield and seed content in C18: 3

The Omegalin and Festival varieties are the two control varieties and the "100%" of reference yield comparing the yield of the other plants tested is the average of these two (ie [02.7 + 97.3] / 2).

It is found that the plant HT1 high content of C18: 3 allows. to maintain a grain yield (in% compared to the average of the yields of the witnesses) in a range quite compatible with agricultural use and in particular in comparison with the varieties Eurodor, Omegalin, Festival and Lutéa grown in France in the same agronomic and environmental conditions, namely according to VATE GE VES as explained in the description .

Deposit of Biological Material: Linum usitatissimum VL8.29.3 was filed as NCIMB Number 42681 on October 24, 2016.

Claims

A method for increasing the amount of alpha-linolenic acid (18: 3) characterized in that it comprises transforming a plant cell of Linum usitatissimum with at least one nucleic acid which encodes the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed and is under the control of a promoter ensuring the overexpression of the Δ15 desaturase responsible for the conversion of C18: 2 to C18: 3 in the seed of said plant.

2. Plant selection method of Linum usitatissimum, characterized in that it comprises the search for an allele of the Δ15 desaturase gene responsible for the conversion of C18: 2 to C18: 3 in the seed having a deletion according to SEQ ID NO: ^s 1 (by comparison to SEQ NO: 2) in the promoter sequence, resulting in an increase in the amount of alpha-linolenic acid (C18: 3) in a plant Linum usitatissimum.

3. Plant of Linum usitatissimum, characterized in that the content, in% by weight, of alpha-linolenic acid (C18: 3) of the oil of the seeds of the plant is greater than 66.7%, that the germination rate is greater than 90% and that the yield is between 85 and 110% of the value of reference reference varieties following evaluation under similar conditions in France.

4. Plant according to claim 3, characterized in that it comprises a modification of the Δ15 desaturase gene responsible for the conversion of C18: 2 to C18: 3 in the seed.

5. Plant according to claim 3, characterized in that the modification of the Δ15 desaturase gene comprises a modification at the promoter region of said gene.

6. Plant according to claim 5, characterized in that the gene comprises SEQ ID No. 1.

Plant obtainable by a method according to one of claims 1 to 2.

8. Use of a plant according to one of claims 3 to 7, or a part of such a plant, for the preparation of a composition intended for human or animal consumption.

9. Plant obtainable by a method according to one of claims 1 to 4.

10. Use of a plant according to one of claims 5 to 9, or a portion of such a plant, for the preparation of a composition for human or animal food.