ES2292265B1 - IMPROVEMENTS INTRODUCED IN MAIN PATENT N. 9902552, ETIOPE MOSTAZA SEED OIL WITH HIGH CONTENT IN OLEIC ACID. - Google Patents

Abstract

Improvements introduced in main patent No. 9902552, Ethiopian mustard seed oil with high oleic acid content. The present invention relates to an Ethiopian mustard seed oil (scientific name Brassica carinata A.Braun) lacking erucic acid (less than 0.5% by weight with respect to the total fatty acid content of the oil) and with an oleic acid content between 80% and 90% by weight of the total fatty acids. This type of oil is not produced in nature by Ethiopian mustard plants and has been obtained through a biotechnological procedure. The Ethiopian mustard oil, object of the present invention, is always produced independently of the growing conditions, and the lack of erucic acid and the high oleic acid content of this oil constitute heritable characteristics.

Description

Improvements introduced in main patent no. P 9902552: "Ethiopian mustard seed oil with high oleic acid content ".

Technical sector

The present invention is part of the sector of agriculture, because it is a new type of oil produced by genetically improved plant seeds. This New type of oil has a wide range of applications, both in the food sector, where it provides clear nutritional benefits and technological, as in the industrial sector, mainly as biofuel and biolubricant.

State of the art

Ethiopian mustard is an indigenous vegetable species from Ethiopia, where it is grown on a small scale as a vegetable and as an oil plant, because its seeds contain high oil content. This oil has a low oleic acid content, between 8% and 13%, and a high percentage of erucic acid, between 38% and 45% (A. Getinet "Review on breeding of Ethiopian mustard, Brassica carinata A . Braun ", Proceedings 7th International Rapeseed Conference, p. 593-597, 1987). At the end of the 50s the possible existence of harmful effects arising from the use of oils rich in erucic acid in human nutrition was raised, an approach that was accentuated following a series of studies carried out during the 70s, and which showed that C22: 1 fatty acids were cardiotoxic in animals, and therefore potentially toxic to man. For this reason, oils with a high content of erucic acid should not be used for human consumption. (FAO / WHO, "Fats and oils in human nutrition", FAO Study: Food and Nutrition, No. 3, FAO, Rome,
1977).

Work done in different parts of the world has demonstrated the advantages of Ethiopian mustard over other species such as rapeseed ( Brassica napus ) and Indian mustard ( Brassica juncea ), when grown in semi-arid regions, due to its greater resistance to drought (E. Fereres, JM Fernández Martínez, I. Mínguez and J. Domínguez: Productivity of Brassica juncea and Brassica carinata in relation to rapeseed, B. napus . I. Agronomic Studies. In: Proc. 6th Int. Rapeseed Conf., Paris , France 1983, page 293-298). Another advantage of Ethiopian mustard over other oilseed Brassicas is its greater resistance or tolerance to pests and diseases (IJ Anand, JP Sing and RS Malik: B. carinata a potential oilseed crop for rainfed agriculture. Eucarpia Cruciferae Newsletter 10: 76 -78. 1985; RK Gugel, G. Seguin-Swartz and A. Petrie: Pathogenicity of three isolates of Leptosphaeria maculans on Brassica species and other crucifers. Can. J. Plant Pathol. 12: 75-82. 1990).

Two types of Ethiopian mustard seeds that produce oil with low erucic acid content, potentially usable in human food, have been developed to date. The first type was obtained through a program of crossings between different Ethiopian mustard plants with intermediate levels of this fatty acid (LC Alonso et al. "The outset of a new oilseed crop: Brassica carinata with low erucic acid content", Proceedings 8th International Rapeseed Conference, p. 170-176, 1991). The second type was obtained by interspecific crossings between Ethiopian mustard plants high in erucic acid and Indian mustard plants ( Brassica juncea [L.] Czern.) Lacking erucic acid (A. Getinet et al. "Development of zero erucic Ethiopian acid mustard through interspecific cross with zero erucic acid Oriental mustard ", Can. J. Plant Sci. 74: 793-795, 1994). The fatty acid composition of both types of seeds, as well as the normal type, is presented in Table 1. The oleic acid content of both types of Ethiopian mustard seeds lacking erucic acid was lower than
40%

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TABLE 1 Fatty acids in mustard oil Ethiopian

one

At present, vegetable oils are sought that have a high content of oleic acid, both for use in human food and for industrial use. In human nutrition, an oil high in oleic acid has several advantages. First, this type of oil is very stable against oxidation. During the same, substances that confer unpleasant taste and smell are produced, which considerably reduces the half-life of the oil in the market. Also, some of the substances produced as a result of the oxidation of the oil are related to the formation of peroxides, which cause the formation of free radicals that in turn have been linked to carcinogenic processes (Vles and Gottembos "Nutritional characteristics and food uses of vegetable oils "Oil Crops of the World, McGraw-Hill, USA, page 63-86, 1989). Second, clinical studies have shown that oils rich in oleic acid reduce levels of total cholesterol and low-density lipoprotein (LDL) in blood (Mattson and Grundy "Comparison of effects of dietary saturated, monounsaturated and polyunsaturated fatty acids on plasma lipids and lipoproteins in man "J. Lipid Res. 26: 194-202, 1985). Third, oil rich in oleic acid is highly thermostable, so its use in fried foods is advisable (MC Dobarganes et al. "Thermal stability and frying performance of genetically modified sunflower seed ( Helianthus annuus L.) oils" J. Agr. Food. Chem. 41: 678-681, 1993). In the industrial field, oils with high oleic acid content are highly demanded, especially by the lubricating oil industry, precisely because of their high thermostability (S. Harold et al. "Varieties of rapeseed oil and derived products for use in fuels and lubricants "Proceedings 9th International Rapeseed Conference, p. 1341-1344, 1995).

Rape varieties ( Brassica napus L.) (US-5955623; US-5625130; US-5840946; US-5638637) and napina ( Brassica rapa L. = Brassica campestris L.) (US-5638637; US-5840946 have been developed ) with a modified composition of fatty acids, containing at least 80% by weight of oleic acid and less than 0.1% by weight of erucic acid, always referring to the total content of fatty acids. The invention applied to plant material developed in US-5625130 has been directed to obtaining plants of Brassica napus (genome AACC 2n = 38), Brassica campestris (genome AA 2n = 20) or Brassica juncea (genome AABB, 2n = 38) , the procedure for obtaining Brassica carinata plants (BBCC genome, 2n = 34) whose seeds produce an oil with the characteristics of low erucic acid and high oleic acid content shown by the plants object of The present invention. The genomic differences between Brassica carinata and the rest of the oilseed Brassicas prevents genetic transfer between them, and therefore the Brassica carinata plants object of the present invention can be grown in the vicinity of other species of oilseed Brassicas without danger of contamination and decrease in high oleic content or modification of the pattern of the remaining fatty acids in the seed.

Explanation of the invention.

One of the objects of the invention is a vegetable oil obtained from Ethiopian mustard seeds (scientific name Brassica carinata A. Braun) modified biotechnologically. This oil lacks erucic acid (less than 0.5% by weight), which allows it to be used in human food. Likewise, this vegetable oil has a high oleic acid content, between 80% and 90% by weight of the total fatty acids. The remaining fatty acids present in the Ethiopian mustard oil object of the present invention are comprised between 2% and 8% by weight of palmitic acid; between 0.5% and 3% by weight of stearic acid; between 2% and 6% by weight of linoleic acid; between 2% and 10% by weight of linolenic acid; and less than 4% by weight of eicosenoic acid. The content of other minor fatty acids (myristic, palmitoleic, behenic, nervous) is always less than 2% by weight. The ratio of oleic acid: linoleic acid is always greater than 15, with oleic acid contents exceeding 85% by weight being customary. The content of erucic acid may be less than 0.1% and even less than 0.05% by weight.

This Ethiopian mustard seed oil can Be used in human and animal feed. Can also be Used as biolubricant and biofuel.

Likewise, another object of the present invention is the seeds, genetically modified plants that give rise to the oil free of erucic acid and with a high content of oleic acid, mentioned in the previous paragraphs, and the method of obtaining said plants. Said process for obtaining Ethiopian mustard plants ( Brassica carinata A. Braun), comprises the following stages:

to)

Treatment of normal seeds of Ethiopian mustard, previously embedded in water for 16 hours, with a solution of the methyl sulphonate mutagenic agent of ethyl (EMS), prepared at a concentration between 0.1% and 2% (vol / vol), for a period of time included between 0.5 and 6 hours.

b)

Cultivation of treated seeds according to section a), selection of those that show an increase in the oleic acid content of the oil compared to normal plant seeds, maintaining an acid content similar to those.

C)

Crossing of Ethiopian mustard plants ( Brassica carinata A. Braun) with normal erucic acid content of the oil with plants of a rapeseed line ( B. napus L.) with less than 2% by weight of erucic acid in the oil of its seeds , followed by backcrossing, self-fertilization, and selection of Ethiopian mustard plants with less than 2% by weight of erucic acid in the oil of their seeds.

d)

Crossing of Ethiopian mustard plants ( Brassica carinata A. Braun) with normal erucic acid content of the oil with plants of an Indian mustard line ( B. juncea [L.] Czern.) With less than 2% by weight of erucic acid in the oil of its seeds, followed by backcrossing, self-fertilization, and selection of Ethiopian mustard plants with less than 2% by weight of erucic acid in the oil of its seeds.

and)

Crossing mustard plants Ethiopian obtained according to section c) with Ethiopian mustard plants obtained according to section d), followed by self-fertilization and selection of Ethiopian mustard plants from the aforementioned crossing with less than 2% by weight of erucic acid in the oil of its seeds.

F)

Genetic recombination between plants Ethiopian mustard obtained in the chemical mutagenesis program [stages a) and b)] and Ethiopian mustard plants obtained in the interspecific crossings program [stages c), d) and e)], followed by self-fertilization and seed selection with low Erucic acid content and high acid content oleic

g)

Sowing the selected seeds in stage f), followed by self-fertilization and selection to obtain a stable line of Ethiopian mustard with low acid content Erucic and high oleic acid content.

h)

Genetic recombination between plants Ethiopian mustard obtained in stages a) to g) and mustard plants Ethiopian characterized by standard content in oleic acid and low linolenic acid content, followed by self-fertilization and selection of F 2 seeds that have a higher acid content oleic and lower linoleic acid and linolenic acid content than those from plants developed in stages a) to g).

Detailed description of the invention

To obtain the modified plants genetically whose seeds produce the oil object of the present invention a long process of improvement has been carried out genetics consisting of (1) chemical mutagenesis program, (2) interspecific crossings program, (3) program of genetic recombination between products obtained in the program of chemical mutagenesis and products obtained in the program of interspecific crossings, and (4) crossings program intraspecific aimed at further improving the developed material.

The chemical mutagenesis program consisted of the treatment of normal seeds with a chemical with mutagenic properties, that is, capable of inducing mutations in the Plant DNA For these mutations to be useful, they must be inheritable, so the mutagenic treatment was followed by multi-year process consisting of identification and fixation of those inheritable mutations that were transmitted from generation to generation As a result of this program, they obtained Ethiopian mustard plants whose seeds had a increase in oleic acid content compared to seeds of normal plants, although they maintained a content in erucic acid similar to normal plant seeds. The program of mutagenesis was carried out for five generations, after which demonstrated the genetic stability of the high character high eric bottom oleic, independent of the conditions of plant cultivation

The interspecific cross-breeding program consisted of the transfer of genes responsible for the absence of erucic acid from rapeseed public material (scientific name Brassica napus L.) and Indian mustard (scientific name Brassica juncea [L.] Czern.) Free of Erucic acid (less than 2% by weight of erucic acid) to a selected line of Ethiopian mustard. The interspecific crossings program was carried out through three subprograms: (a) interspecific cross between rape material free of erucic acid and a selected Ethiopian mustard line, followed by a backcross generation towards the Ethiopian mustard parental and three additional generations of self-fertilization; (b) interspecific cross between Indian mustard material free of erucic acid and a selected Ethiopian mustard line, followed by a generation of backcrossing towards the Ethiopian mustard parental and three additional generations of self-fertilization; (c) genetic recombination between Ethiopian mustard plants from interspecific crossings with rape material free of erucic acid and Ethiopian mustard plants from interspecific crossings with Indian mustard material free of erucic acid. Said genetic recombination was carried out for six generations, after which the stability of the erectic zero character was demonstrated, independent of the growing conditions of the plants.

The genetic recombination program between products obtained in the chemical mutagenesis program and products obtained in the interspecific crossings program it took place over four generations, after which demonstrated the stability of the erect and high zero characters oleic, independent of the growing conditions of the plants.

Ultimately, a program was carried out of intraspecific crossings aimed at achieving an increase additional oleic acid content of the seeds by introduction of genes that cause a partial blockage of the desaturation of linoleic acid to linolenic acid. When meeting previously partially blocked the desaturation of oleic acid to linoleic acid, the introduction of the new genes was translated in a reduction of linoleic acid and acid contents linolenic acid and an increase in oleic acid content in the seed oil This additional increase in content in oleic acid is stably expressed regardless of plant growing conditions.

Embodiment of the invention

First

Chemical Mutagenesis Program

Ethiopian mustard seeds of the line C-101, with 8.5% oleic acid, were submerged in water at room temperature for 16 hours to promote softening of the outer shell and, therefore, favor the penetration of the mutagenic agent. After this time, it submerged for a period of 2 hours at room temperature and with continuous stirring at 75 r.p.m. in an agent solution mutagenic ethyl methyl sulphonate (EMS), prepared at a concentration of 1% (vol / vol) in 0.1 M phosphate buffer at pH 7. After the mutagenic treatment the seeds were washed during 10 hours in running water, dried, and seeded in the field. The corresponding plants were harvested individually, and the seeds of each of the harvested plants were sown in countryside. The corresponding plants were harvested individually and its seeds were analyzed to know their acid composition fatty From these analyzes, a plant with a abnormally high content of oleic acid (19.3%). In successive generations was made selection to increase and stabilize their oleic acid content. Finally, a line was obtained in the fifth generation with maximum and minimum values of 27.9% and 17.0%, respectively, of oleic acid. The line was called N2-3591. Its average fatty acid composition, compared to the line from which it was obtained, C-101, is shown in Table 2.

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TABLE 2 Fatty Acids in%

2

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Second

Interspecific Crossings Program

Ethiopian mustard plants (scientific name Brassica carinata A. Braun) have a genome in the nucleus of their cells that is of BBCC configuration. Indian mustard plants (scientific name Brassica juncea [L.] Czern) have a genome in the nucleus of their cells that is of AABB configuration. Rapeseed plants (scientific name Brassica napus L.) have a genome in the nucleus of their cells that is of AACC configuration. In order to obtain BBCC genome plants with the oil of their seeds lacking erucic acid, a program of interspecific crossings was carried out between a public material plant of erectic rapeseed, with less than 2% by weight of erucic acid ( B. napus L cv. "Duplo"), a public material plant of zero erutic Indian mustard, with less than 2% by weight of erucic acid ( B. juncea cv. "Zem-1"), and an Ethiopian mustard plant with levels normal erucic acid, between 39% and 49% by weight erucic acid ( B. carinata cv. "C-101"). This program was carried out through three subprograms:

2.1. Subprogram of crossings between C-101 and Duplo

An Ethiopian mustard plant C-101 crossed with a Duplo rapeseed plant. The seeds obtained from this cross were sown in pots and several of the resulting plants were crossed again with plants of C-101. The resulting seed plants of the crossing they self-fertilized for three generations, after which ABCC genomic configuration plants were obtained, with the Energetic zero genes from rapeseed incorporated.

2.2. Subprogram of crossings between C-101 and Zem-1

An Ethiopian mustard plant C-101 crossed with an Indian mustard plant Zem-1 The seeds obtained from this cross they were planted in pots and several of the resulting plants were They crossed again with C-101 plants. The plants of the seeds resulting from the crossing self-fertilized for three generations, after which plants were obtained from BBAC genomic configuration, with genes for erect zero from Indian mustard incorporated.

2.3. Subprogram of crossings between plants ABCC and BBAC genomic configuration

ABCC and BBAC genomic configuration plants are They crossed each other. The seeds from the crosses are they sowed. The plants they gave rise to were self-fertilized and They were harvested individually. Its seeds were analyzed for Know the fatty acid composition of your oil. From a total of 507 plants analyzed, 45 of them presented a low content in erucic acid (less than 2% by weight of this fatty acid). The Seeds of five of these plants were sown, after which He made a selection process for three generations. The best of these five lines was called 25X-1. its fatty acid composition, compared to their parents C-101, Duplo, and Zem-1 is shown in Table 3

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TABLE 3 Fatty Acids in%

3

Third

Crossing program between N2-3591 and 25X-1

Plants of the high oleic mutant in the high erect background, N2-3591, were crossed with plants of the 25X-1 erect zero line. The seeds obtained from the cross were sown in pots and the corresponding plants were self-fertilized and harvested individually. The seeds from these plants were analyzed for the fatty acid composition of their oil. From a total of 300 seeds analyzed, five were identified that combined the erectic zero character with the high oleic character. These seeds were sown and self-fertilized, proceeding with a selection for two generations for high oleic acid content and low erucic acid content. After this selection, the IAS-3 line was obtained, which contains less than 2% by weight of erucic acid and between 70% and 80% by weight of oleic acid. The fatty acid composition of IAS-3, compared with the lines from which it was obtained and with other lines of erutic zero Ethiopian mustard developed to date, is shown in the
Table 4

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TABLE 4 Fatty Acids in%

4

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Fourth

Crossing program between IAS-3 and IAS-21

Plants of the IAS-3 line (patent application number 9902552) were crossed with plants of the IAS-21 line (patent application number 9902553), characterized by a standard content of oleic acid and a low content of linolenic acid in The oil of its seeds. The seeds obtained from the cross were sown in pots and the corresponding plants were self-fertilized and harvested individually. The seeds from these plants were analyzed for the fatty acid composition of their oil. From a total of 1250 seeds analyzed, six seeds were identified that presented an additional increase in oleic acid content along with a reduction in linoleic acid and linolenic acid content. These recombinant seeds were sown, a selection process being carried out for three generations to ensure the fixation of the new fatty acid profile. This profile was demonstrated completely fixed and stable from the second generation, expressing itself independently of the growing conditions of the plants. The line thus obtained, called IAS-245, contains less than 2% by weight of erucic acid and between 80% and 90% by weight of oleic acid. The average fatty acid composition of IAS-245, compared to the lines from which it was obtained, is shown in the
Table 5.

A seed deposit of the IAS-245 line in the NCIMB (National Collections of Industrial, Food and Marine Bacteria), with NCIMB number 41157 and deposit date February 13, 2003.

TABLE 5 Fatty Acids in%

5

Claims (10)

1. Ethiopian mustard seed oil with a high oleic acid content, which includes an erucic acid content of less than 0.5% by weight with respect to the total fatty acids of the oil, a linoleic acid content between 2% and 6 % by weight, a linolenic acid content between 2% and 10% by weight, a palmitic acid content between 2% and 8% by weight, a stearic acid content between 0.5% and 3% by weight, and an eicosenoic acid content of less than 4%, characterized in that the oleic acid content of the oil is between 80% and 90% by weight, also referring to the total fatty acids of the oil. 2. Ethiopian mustard seed oil with high oleic acid content according to claim 1, characterized in that the ratio of oleic acid to linoleic acid is always higher than 15. 3. Ethiopian mustard seed oil with high oleic acid content according to claims 1 and 2, characterized in that the oleic acid content is greater than 85%. 4. Ethiopian mustard seed oil with a high oleic acid content according to claims 1 to 3, characterized in that the erucic acid content of the oil is less than 0.1% by weight with respect to the total fatty acids of the oil. 5. Ethiopian mustard seed oil with a high oleic acid content according to claim 4, characterized in that the erucic acid content of the oil is less than 0.05% by weight with respect to the total fatty acids of the oil. 6. Ethiopian mustard seeds that have a oil with high oleic acid content according to claims 1-5. 7. Ethiopian mustard plants ( Brassica carinata A. Braun) which, when self-fertilized, produce seeds that have an oil with a high oleic acid content according to claims 1-5. 8. Method for obtaining Ethiopian mustard plants ( Brassica carinata A. Braun), according to claim 7, characterized in that it comprises the following steps:

to)

Treatment of normal seeds of Ethiopian mustard, previously embedded in water for 16 hours, with a solution of the methyl sulphonate mutagenic agent of ethyl (EMS), prepared at a concentration between 0.1% and 2% (vol / vol), for a period of time included between 0.5 and 6 hours.

b)

Cultivation of treated seeds according to section a), selection of those that show an increase in the oleic acid content of the oil compared to normal plant seeds, maintaining an acid content similar to those.

C)

Crossing of Ethiopian mustard plants ( Brassica carinata A. Braun) with normal erucic acid content of the oil with plants of a rapeseed line ( B. napus L.) with less than 2% by weight of erucic acid in the oil of its seeds , followed by backcrossing, self-fertilization, and selection of Ethiopian mustard plants with less than 2% by weight of erucic acid in the oil of their seeds.

d)

Crossing of Ethiopian mustard plants ( Brassica carinata A. Braun) with normal erucic acid content of the oil with plants of an Indian mustard line ( B. juncea [L.] Czern.) With less than 2% by weight of erucic acid in the oil of its seeds, followed by backcrossing, self-fertilization, and selection of Ethiopian mustard plants with less than 2% by weight of erucic acid in the oil of its seeds.

and)

Crossing mustard plants Ethiopian obtained according to section c) with Ethiopian mustard plants obtained according to section d), followed by self-fertilization and selection of Ethiopian mustard plants from the aforementioned crossing with less than 2% by weight of erucic acid in the oil of its seeds.

F)

Genetic recombination between plants Ethiopian mustard obtained in the chemical mutagenesis program [stages a) and b)] and Ethiopian mustard plants obtained in the interspecific crossings program [stages c), d) and e)], followed by self-fertilization and seed selection with low Erucic acid content and high acid content oleic

g)

Sowing the selected seeds in stage f), followed by self-fertilization and selection to obtain a stable line of Ethiopian mustard with low acid content Erucic and high oleic acid content.

h)

Genetic recombination between plants Ethiopian mustard obtained in stages a) to g) and mustard plants Ethiopian characterized by standard content in oleic acid and low linolenic acid content, followed by self-fertilization and selection of F 2 seeds that have a higher acid content oleic and lower linoleic acid and linolenic acid content than those from plants developed in stages a) to g).

9. Use of a seed oil of Ethiopian mustard according to claims 1 to 5, in compositions for human and animal food. 10. Use of a seed oil of Ethiopian mustard according to claims 1 to 5, in compositions Biolubricants and biofuels.