ES2245216A1 - Obtaining plants comprises genetic transformation of cellular embryo using microbial vectors, taking cork oak immature embryos and placing in an aseptic culture and transferring the matured embryos to a chamber with illumination - Google Patents

Abstract

Obtaining plants comprising genetic transformation of cellular embryo using microbial vectors, taking cork oak immature embryos and placing in an aseptic culture with macronutrients mineral salts and micronutrient solution, conducting an infection treatment with a bacterial solution, leaving for 2 to 4 days, adding antibiotic bacteria, washing with distilled water and transferring the matured embryos to a chamber with illumination for stimulating the germination.Method for obtaining plants through the genetic modification of cork oak embryos. The method consists of the genetic modification of embryo cells using microbial vectors, in a way that once the desired gene is incorporated in the plant cells, plants can then be regenerated from these cells. For this, immature cork oak embryos are taken and conditioned in aseptic cultivation medium, with macronutrient mineral salts and micronutrient solutions, subsequently they undergo an infection treatment with a bacterial solution and then pass to the cultivation medium for 2 to 4 days, after which the bacteria are eliminated using antibiotic and washing with distilled water, for the final passing of the embryos to the proliferation medium, from where the new embryos introduced, once they have reached maturity and broken their dormancy, are transferred to a lighted chamber in order to stimulate germination.

Description

Method for obtaining plants by genetic transformation of cork oak embryos.

Object of the invention

The present invention is based on an application from modern plant biotechnology to the genetic improvement of cork oak, aimed at obtaining plants through genetic transformation of embryos.

The object of the invention is the production of genetically transformed plants through the introduction of genes in plant cells, using vectors microbial, so that once the desired gene is incorporated into the plant cells, plants are regenerated from of said cells.

Background of the invention

The cork oak ( Quercus suber L. ) is a plant species with great interest for the production of cork, raw material used in different industrial sectors, with very diverse applications. But such production is currently threatened by the appearance of different types of insects such as lepidoptera and beetles, which are dedicated to excavating galleries in the trunks of the cork oaks, causing the consequent stroption in the cork, so that it is useless for its subsequent application. In this way, obtaining cork is decreasing, as cork oaks are increasingly affected by the aforementioned insects, so new cork oak plants are needed, which are also not affected by the different kinds of harmful insects.

In this type of plant species, the sexual reproduction, or propagation by seeds, is a slow method when it comes to obtaining new crosses and varieties, more so if what required is to make crossings with subsequent selection of individuals On the other hand, the fruiting of the cork oak is vecera, with a recurrence of two or three years, which makes it difficult to classical genetic improvement, already slow, since they need several years for plants to reach sexual maturity.

Therefore the cork oak is a plant species in which the reproductive cycles are very long and make it unfeasible in the practice of obtaining crossings for the incorporation of Desirable genetic characters in select individuals.

Therefore, an alternative has been sought in the modern plant biotechnology, as never before had obtained genetically transformed cork oak material by biotechnological methods

Description of the invention

The method for obtaining plants by genetic transformation of cork oak embryos than the invention proposes to solve the problem fully satisfactorily previously exposed. It is a new method that allows Obtain genetic recombination immediately, without need to resort to sexual reproduction.

The method object of the invention consists in obtaining genetic transformation of embryonic cells using as a transformation vector an Agrobacterium sp . Plasmid, a technology that is applicable to the genetic improvement of the cork oak, so that once the desired gene is incorporated to plant cells, plants are regenerated from said cells.

To carry out the development of the method proposed here, immature embryos are taken from cork oak and are initially grown in aseptic conditions. He culture medium will be constituted from a series of nutrients and cofactors that allow the development of embryos of cork oak.

On the one hand the medium has macronutrient mineral salts, such as SO 4 (NH 4) 2, NO 3 K or NO 3 Na, Cl 2 Ca \ cdot
2H 2 O, SO 4 Mg • 7 O 2 O, KCl and PO 4 H 2 Na • O 2 or PO 4 HNa 2.

As solution of micronutrients, the solution of Murashige and Skoog (1962) is used, to which Chelated iron in the form of Fe-EDTA is also added, adding SO 4 Fe c Fe 7 H 2 O and sodium ethylenediamine tetraacetate Na_ { 2}
EDTA

The cofactors used are the following: ascorbic acid, nicotinic acid, calcium pantothenate, pyridoxine, thiamine and glutamine.

In addition to all this, embryos require a carbon source, which in an embodiment of the invention may be sucrose, and the culture medium should be keep between pH values between 5.5 and 5.7.

Once the crop contains all nutrients and is in the right conditions for good development of cork oak embryos, a sterilization of the same, operation performed in an autoclave, at a pressure between 0.5 and 1 atmospheres, and at a temperature that is between 115 and 120ºC, during a 20 minute time period.

Subsequently the crops thus prepared, are kept in a chamber under ambient temperature conditions which will be between 18 and 25 ° C.

The embryos are then subjected to an infection treatment consisting of immersing them for 25 to 35 minutes in a suspension of Agrobacterium tumefaciens LBA4404 / p35S GUS INT / pCAMBIA 1301. Then they are dried with filter paper and passed to culture medium during a period of time between two and four days. Bacteria are subsequently removed by adding antibiotic, for example cefotaxime, followed by several washes in sterile distilled water, and the embryos are passed to culture medium with glutamine, cefotaxime and hygromycin.

Once this treatment is finished, approximately one month later, the embryos are passed in the middle of proliferation.

The induced embryos, once reached the maturity, undergo a new treatment, in this case of rupture of the lethargy they have undergone in the previous stage, for this, This new process consists of storing them at a low temperature, between 2 and 5ºC, for a period of time between two and ten weeks

The next stage of the proposed method consists of in moving the embryos to a chamber where the temperature at about 25ºC and with enough lighting to be able to Stimulate germination. During this stage the development of Stem of cork oak plants is improved by adding 6-benzyladenine (BA), in a proportion comprised between 0.4 and 0.5 µM, to the germination medium.

Finally the plants thus germinated are already ready to be transplanted to containers intended for the cultivation of plants, its initial acclimatization in greenhouses being convenient before being transplanted into its final place of growth and Obtaining the cork.

This method offers important advantages since besides allowing genetic recombination to be obtained Immediately, the requirements to carry it out are minimal in as for the starting material, the cultivation space and the potential cost reduction. On the other hand, one of the main problems that are solved is obtaining plants of material selected and adapted to ecological conditions of the environment where the life of the cork oak is going to develop.

Example of embodiment of the invention

According to a preferred embodiment of the invention become immature embryos of said plant and it grown in aseptic conditions, in a medium with the following Macronutrient mineral salts formula:

SO4 (NH4) 2 0.13 to 0.20 g / l NO_ {3} K or NO_ {Na} 0.75 to 1.00 g / l Cl_ {Ca} \ Cdot2H_ {2} O 0.12 to 0.15 g / l SO 4 Mg • 7H 2 O 0.25 to 0.31 g / l KCl 0.30 to 0.95 g / l PO_H2 {Na} \ H2 O 0.09 a 0.15 g / l PO 4 HNa 2 (optional) 0.03 g / l

As a micronutrient solution, that of Murashige and Skoog (1962), which is formed by the following compounds:

BO_ {3} H_ {3} 6.20 mg / l SO_ {4} Mn \ cdotH_ {O} 6.90 mg / l SO_ {4} Zn \ cdot2H2 {O} 10.59 mg / l IK 0.83 mg / l MoO_ {Na} {2} \ cdot2H2 {O} 0.25 mg / l SO_ {4} Cu \ cdot5H_ {O} O 0.025 mg / l Cl_2 Co \ 6d2H2 O 0.025 mg / l

In addition, chelated iron is added in the form of Fe-EDTA, adding 27.8 mg / l of SO 4 Fe • 7H 2 O and 37.3 mg / l Na 2 EDTA (sodium ethylenediamine tetracetate).

On the other hand in the treatment a series of cofactors listed below:

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Acid ascorbic 10 µM Acid nicotinic 10 µM Pantothenate calcic 5 µM Pyridoxine 5 \ muM Thiamine 3 \ muM Glutamine 3 mM

As a carbon source sucrose is used in 80 to 90 mM ratio, keeping the pH of the culture medium in a value of 5.6. Sterilization is performed in autoclave between 0.5 and 1 atmospheres and at a temperature between 115 and 120ºC for 20 minutes

After this stage of preparation and establishment, crops are kept in a low chamber ambient temperature conditions of 18 to 25 ° C.

The embryos are then immersed for 30 minutes in a suspension of Agrobacterium tumefaciens LBA
4404 / p35S GUS INT / pCAMBIA 1301. They are then dried on filter paper and passed to culture medium for two days. Bacteria are removed by adding 628 µM cefotaxime, followed by three washes in sterile distilled water, and the embryos are passed to culture medium with 3 mM glutamine, 628 µM cefotaxime and 94 µM hygromycin. A month later, embryos are passed through proliferation. The new induced embryos, once they reach maturity, undergo a lethargy breakdown treatment, consisting of storing them at a low temperature (2 to 5ºC) for a period of two to ten weeks. Subsequently they are transferred to a chamber at 25 ° C and with lighting, to stimulate germination. Stem development is improved by adding 6-benzylamine (BA) from 0.4 to 0.5 µM to the germination medium.

Finally the germinated plants are transplanted to containers for growing plants and acclimatize in greenhouse.

The described technique is applied industrially by organizing a laboratory where the operations described above. This requires cameras from laminar flow, autoclaves, sterilization stoves, and the handling instruments and culture containers, etc. Saying laboratory should have a greenhouse with capacity sufficient for acclimatization and conditioning of plants produced.

Claims (10)

1. Method for obtaining plants through genetic transformation of cork oak embryos, characterized in that it consists in the genetic transformation of embryonic cells using microbial vectors, so that once the desired gene is incorporated into plant cells, regeneration is carried out of plants from said cell; for this, immature embryos are taken that are conditioned in aseptic culture medium, with macronutrient mineral salts, micronutrient solution, cofactors and a carbon source, subsequently immersed in a bacterial solution for a period of time between 25 and 35 minutes and they then pass to the culture medium for 2 to 4 days, after which the bacteria are removed by adding antibiotic and washing with distilled water, to finally pass the embryos to proliferation medium, from where the new induced embryos, once maturity is reached and broken their lethargy, storing them at a temperature of 2 to 5ºC, they are transferred to a chamber that is maintained at a temperature between 20 and 30ºC and with lighting to stimulate germination. 2. Method for obtaining plants by genetic transformation of cork oak embryos, according to claim one, characterized in that said macronutrient mineral salts are constituted by the following compounds in the corresponding amounts: SO4 (NH4) 2 0.13 to 0.20 g / l NO_ {3} K or NO_ {Na} 0.75 to 1.00 g / l Cl_ {Ca} \ Cdot2H_ {2} O 0.12 to 0.15 g / l SO 4 Mg • 7H 2 O 0.25 to 0.31 g / l KCl 0.30 to 0.95 g / l PO_H2 {Na} \ H2 O 0.09 a 0.15 g / l PO 4 HNa 2 (optional) 0.03 g / l 3. Method for obtaining plants by genetic transformation of cork oak embryos, according to claim 1, characterized in that Murashige and Skoog is used as a micronutrient solution in the following quantities: BO_ {3} H_ {3} 6.20 mg / l SO_ {4} Mn \ cdotH_ {O} 6.90 mg / l SO_ {4} Zn \ cdot2H2 {O} 10.59 mg / l IK 0.83 mg / l MoO_ {Na} {2} \ cdot2H2 {O} 0.25 mg / l SO_ {4} Cu \ cdot5H_ {O} O 0.025 mg / l Cl_2 Co \ 6d2H2 O 0.025 mg / l to which 27.8 mg / l of SO_4 Fe \ cdot7H2O and 37.3 mg / l of Na 2 EDTA. 4. Method for obtaining plants by genetic transformation of cork oak embryos, according to claim 1, characterized in that the cofactors used are the following: Acid ascorbic 10 µM Acid nicotinic 10 µM Pantothenate calcic 5 µM Pyridoxine 5 \ muM Thiamine 3 \ muM Glutamine 3 mM 5. Method for obtaining plants by genetic transformation of cork oak embryos, according to claim 1, characterized in that sucrose is used as a carbon source, in an amount of 80 to 90 mM. 6. Method for obtaining plants by genetic transformation of cork oak embryos, according to claim 1, characterized in that the pH of the culture medium is adjusted to values between 5.5 and 5.7. 7. Method for obtaining plants by genetic transformation of cork oak embryos, according to claim 1, characterized in that the sterilization is carried out in an autoclave, at a pressure between 0.5 and 1 atmospheres and at a temperature of 115 to 120 ° C for 20 minutes 8. Method for obtaining plants by genetic transformation of cork oak embryos, according to claim 1, characterized in that the bacterial solution used in the treatment of embryo infection is a suspension of Agrobacterium tumefaciens LBA4404 / p35S GUS INT / pCAMBIA 1301. 9. Method for obtaining plants by genetic transformation of cork oak embryos, according to claim 1, characterized in that the antibiotics used in the culture medium to eliminate bacteria are caffeotaxime and hygromycin. 10. Method for obtaining plants by genetic transformation of cork oak embryos, according to claim 1, characterized in that the treatment is optionally complemented by improving the development of the stem by adding 0.4 to 0.5 µM 6-benzyladenine to the germination medium.