LU86372A1 - PROCESS FOR TREATING PLANT MATERIAL IN ORDER TO OBTAIN EXPRESSION OF AT LEAST ONE GENE, AND PLANT MATERIAL IN WHICH THIS GENE EXPRESSES - Google Patents

Description

* * - 2 - The invention relates to a process for treating plant material in order to obtain the expression of at least one gene in plant cells, according to which the plant material is first sterilized and then put to be incubated in a DNA solution having at least one foreign gene to be expressed and / or the cDNA of this gene.

It is already known that the introduction into plants of foreign genes can be carried out by direct absorption of DNA by plant cells.

In particular, the transformation of plant protoplasts has already been carried out by simple incubation with a plasmid having the foreign gene.

Thus by working on isolated protoplasts, it has already been possible to transform monocotyls such as the cereal TRITICIUM MONOCOCCUM (Mol.Gen.Genet.199, p. 178 by LÖRZ H., BAKER B. and SCHELL J. - 1985) or the grass LOLIUM MULTIFLORUM (Mol.Gen.Genet 199, p. 183 by POTRYKUS I., SAUL MW, PETRUSKA J., PASZKOWSKI J. and SHILLITO D.-1985). In both cases, as is often the case with transformed protoplasts, it has not yet been possible to regenerate plants.

It seems easier to be able to process whole plant material such as the seed or the excised embryo and research has been done in this direction.

It was already known that, although the seed is a complex system by, in particular, the presence of cell walls and the possible exudation, in certain species, of nucleases in the incubation medium, exogenous DNA could penetrate into the seed cells and in the embryo (J. Mol. Biol. 43, 243 - LEDOUX L. and HUART L.? 1969) and that only a small percentage of DNA was degraded when taken if the seed variety was well chosen (Europ.

J. Biochem. 23., 96 LEDOUX L. et al; 1971).

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Thiamine mutants of ARABIDOPSIS THALïANA have already been corrected by treatment with adequate bacterial DNA (Nature 249, 17 LEDOUX L. et al. 1974 and Canad. J. Genet.Cyt.2_l, 515; LEDOUX L. et al; 1979).

It had been deduced therefrom that the bacterial DNA incubated with seeds could be, after taking, expressed by the cells of the plant.

A method of treating seeds in a plasmid DNA solution has been described in Arch. Int.

Physiol. Biochem. 90, B 44 (LEDOUX L. et al -1982). This article describes the direct transfer and expression of plasmid DNA derived from the plasmid pBR 325 carrying the ampicillin resistance gene coding for β-lactamase.

The methods for treating the plant material as described in the publications mentioned above, however, only allow those skilled in the art to obtain good results in rare cases. In particular, the absorption of DNA by monocots is in most cases difficult to achieve.

The object of the invention is to provide a simple process which is generally applicable and which therefore makes it possible to obtain the expression of a gene by a large number of varieties of plants.

For this purpose, at least one dry seed from an upper plant, at least one dry spore from a lower plant or at least one dry excised plant embryo whose moisture content is less than 10% by weight, is incubated. until the embryo has tripled in size, in a DNA solution comprising the complete foreign gene and / or the cDNA of that gene.

It has been found that by applying this process DNA is absorbed by both monocotylates and dicots.

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Preferably, at least one seed, spore or embryo is incubated with a moisture content of less than 5% by weight.

Although under the aforementioned conditions the nuclease activity is insignificant, it may be useful to select from a given plant the varieties with the lowest nuclease content.

The sterilization required before incubation may result in the moisture content of the plant material becoming too high. In this case, or in the case where the plant material to be treated already has too high a moisture content by its nature, the plant material is dried before it is incubated in the DNA solution.

In a particular embodiment of the invention, peelable seeds are incubated and peeled before incubation in the DNA solution.

In a remarkable embodiment of the invention, a DNA solution comprising at least one foreign eukaryotic gene and / or the cDNA of this gene is used as DNA solution.

This eukaryotic gene is for example the gene coding for human growth hormone (hGH).

The DNA can contain the foreign gene as such (optionally with introns and promoter) for example a synthetic or engineered gene or the cDNA as such of this gene.

It can also be an expression vector, for example a plasmid, a cosmid or a phage comprising the foreign gene and / or the cDNA of this gene.

The invention also relates to the plant material obtained by the above process and in which the gene is expressed.

- 5 - * "The invention thus relates, inter alia, to a seed, a spore or an excised plant embryo in which or which expresses a foreign gene, this seed or spore or this embryo having absorbed DNA comprising the foreign gene to be expressed or the cDNA of this gene according to the above method.

In particular, the plant material has absorbed DNA comprising the complete gene encoding human growth hormone or the cDNA of this gene.

The invention also relates to plants, calluses or cells obtained from a seed, a spore or an embryo treated by the process according to the above invention in which the foreign gene is thus expressed as the descendants including hybrids of this plant.

Other particularities and advantages of the invention will emerge from the description of a process for treating plant material in order to obtain the expression of at least one gene, and of the plant material in which this gene is expressed, according to the invention; this description is given below only by way of nonlimiting example and with reference to the accompanying drawings.

FIG. 1 represents the structure of an expression vector which can be used in the method according to the invention.

FIG. 2 represents the structure of another expression vector which can be used in the method according to the invention.

According to the invention, the dry sterile plant material is germinated in a DNA solution comprising the foreign gene which it is desired to have expressed by plant cells.

The aforementioned plant material consists of a seed, a spore or an excised embryo.

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The seed or the embryo can be as well of a monocotylée, for example rice or timothy, as of a dicotylée like Arabidopsis Thaliana.

We start by sterilizing the plant material by an adequate chemical means, with or without an adjuvant such as a surfactant.

Then we put the germinating plant material in the DNA solution.

Incubation should be done at least until the visible start of the seedling or more particularly at least until the embryo has tripled in size.

This size tripling occurs in most cases after the first 72 hours and in some cases already after the first 48 hours of seedling development from the germ.

It has been found that at this stage of germination, a stage which is very specific for metabolism, nucleases do not form and the cells accept, inter alia, DNA with the foreign gene, provided however that these cells are sufficiently dry. When the cells have hydrated, they close for foreign DNA.

The maximum admissible water content varies with the variety of plant but is in any case less than 10% by weight and preferably less than 5% by weight.

If necessary, we start by drying the plant material, either in the open air, or by chemical drying or even by freeze-drying.

Such drying may be necessary when the plant material contains too much moisture of its nature, but especially if the plant material has absorbed moisture during sterilization by the wet method.

If the plant material is a seed, it is peeled by removing glumes and lemons, before incubation each time it is possible. This promotes the absorption of DNA.

Such a coat is for example indicated for most cereals such as barley. To promote this coat, the seed is first wetted for 30 minutes in water. This normally has the consequence that it is necessary to dry or re-dry the seed before incubating it in the DNA solution.

The aqueous DNA solution can be a saline solution or a buffered solution.

DNA can be in linear form as well as in circular or superhelical form. It contains one or more complete foreign genes (possibly with promoter and introns) and / or the cDNA of this gene.

This gene can also be a gene manipulated to promote its expression, for example according to a known technique by which it is placed downstream of particular promoters or UAS (Upstream Activating Sequences) sequences and upstream of polyadenylation or termination sequences.

The DNA can be the foreign gene, manipulated or not, as such or the cDNA as such of this gene.

The DNA can also consist of an expression vector comprising this gene and / or the cDNA of this gene.

The foreign gene or the cDNA of this gene can thus be inserted into at least one plasmid, in particular a plasmid or starting vector of the pBR series (pBR 322,325, 328 etc.), at least one cosmid, at least one phage or one combination of these vectors.

A DNA solution with a concentration of at least 1 pg / ml is used. The minimum concentration depends on the plant material, but in most cases a concentration close to 400 to 500 jug / ml is preferred.

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In order to allow, after the application of the process, a selection of the plant material transformed by the process, it is possible to incubate the plant material to be treated in a DNA solution not comprising only these genes coding for a desired protein and / or cDNA of this gene but also a gene coding for resistance to a toxic substance for example to an antibiotic, to heavy metals, to organochlorines or to herbicides.

The selection of the transformed plant material can advantageously be carried out on the progeny of the plants obtained from the treated plant material.

The foreign gene in question can be either eukaryotic, prokaryotic or viral.

A eukaryotic gene of interest to be expressed is the gene for human growth hormone (hGH).

Although when DNA processing takes place at the very start of germination, the cells form few nucleases, it has been found that for a given plant some varieties of this plant produce fewer nucleases than others. It is therefore useful to select, above all by biochemical determination, the varieties of a plant that are the poorest in nucleases.

After the incubation of the plant material in the DNA solution, the treated material is placed in an appropriate culture medium allowing the development of plants or calluses formed from the treated plant material or parts, for example cells isolated from this material.

The plants or calluses are grown until the seeds or spores are harvested, which in turn is grown. The descendants of the plants are then treated to extract by known methods the protein expressed by the foreign gene absorbed by the plant material originally treated.

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The examples given below illustrate the invention in more detail.

Example 1 1. Preparation of seeds:

Shake barley seeds (HORDEUM VULGARE

L. var. Trumph) for 15 min in a solution of 10 / il

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of polyoxyethylene sorbitan monolaureate (Tween 20) for 20 ml of water.

The seeds are then carefully peeled.

They are sterilized for 60 min with sodium hypochlorite (13% active chlorine) added with 10 μl of Tween 20 per 20 ml.

After washing with sterile water to a pH of 6, the seeds are dried in a sterile medium at room temperature until the moisture content is less than 5% by weight.

When the seeds are fresh and the embryo appears to be very hydrated, the seeds can be dried overnight on drierite or they can be lyophilized overnight before sterilizing them.

2. Preparation of the DNA solution.

A bacterial culture is prepared from the E.Coli K 12-HB 101 strain containing the plasmid or expression vector c hGH 800 / pBR 322 (hereinafter called pc hGH 800) in a complete Luria medium (medium L ), to which was added, after adjusting the pH to 7.5 with 1 N NaOH and sterilization, 50 mg / 1 of ampicillin filtered through millipore.

The preparation of the above plasmid is described in detail in Science 205, 602-607 (1979) by J.A. MARTIAL, R.A. HALLEWELL, J.D. BAXTER and H.M. GOODMAN.

This expression vector was prepared by inserting the 800 bp DNA fragment (base pairs) comprising the 800 bp of cDNA of the hGH gene, i.e. the gene * 4 -lode human growth hormone, at the Hind III site of pBR 322 inserted into the strain E. Coli CM 240. This DNA fragment was placed in the same reading direction as the gene coding for the resistance to ampicillin. The structure of the pc hGH 800 expression plasmid vector is represented in FIG. 1 in which £

Pl is the main promoter of the bla (Ap) gene in pBR 322, P3 is the natural promoter of this gene (Pl and P2 are part of the "promoter" area of the Te gene), and ori is the origin of replication.

The culture is prepared in the following manner.

A preculture is first prepared with 40 ml of medium L and 50 mg / 1 of Ap. The mixture is allowed to stir at 37 ° C overnight. The next day, 1 liter of this same medium is seeded with the strain. The bacteria are allowed to multiply until AggQ reaches 0.4 to 0.6. Chloramphenicol is then added at a rate of 250 mg / l.

This antibiotic has the effect of blocking protein synthesis and preventing the replication of chromosomal DNA. Plasmid DNA, on the other hand, continues to replicate. The plasmids are amplified for 12 to 16 hours.

Next, cell lysis of the bacteria is carried out by the large-scale alkaline lysis method described by MANIATIS et al., In "Molecular Closing" Cold-Spring Harbor Laboratory (1982).

The plasmid DNA is then isolated and purified in a CsCl gradient in the presence of ethidium bromide.

To each ml of the solution, 1.0 g of cesium chloride is added. 4.5 ml of ethidum bromide at 10 mg / ml are added. The final density of the solution is adjusted with the refractometer: the refractive index must be equal to 1.386. Aggregates are formed which are removed 1 ‘+ - 11 - by centrifugation.

A first centrifugation is carried out for 48 hours at 36,000 rpm at 25 ° C (MARTIN CHRIST Omega II, rotor 65 Ti).

Plasmid DNA is isolated using a syringe and a second ultracentrifugation is performed at 45,000 rpm for 48 hours. The plasmid DNA band is isolated. 2 volumes of water-saturated isobutanol are added thereto and the mixture is stirred several times to remove the ethidum bromide soluble in isobutanol. After centrifugation the isobutanol is removed. This operation is repeated 4 times. The elimination of CsCl is carried out by dialysis for several hours against a solution of 0.01 M Na Cl renewed 2 to 3 times.

The DNA is precipitated by adding 1/10 volume of 5M NaCl and two volumes of ethanol. The mixture is left at -20 ° C. for 1 h and centrifuged for 10 minutes at 10,000 rpm (Sorvall RC-5.4 ° C). The alcohol is eliminated and the pellet is dissolved in a volume of sterile 0.01 M Nacl so as to obtain a final DNA concentration close to 400 μg / ml. The plasmid c hGH 800 is kept in the presence of chloroform at 4 ° C.

3. Treatment of the seeds in the DNA solution.

The seeds are placed individually in the cells of a porcelain plate in 150 μl of plasmid solution.

The absorbent papers on which the porcelain plate rests are moistened to avoid evaporation of the solutions. We then seal the petri dish with insulation paper.

The Petri dish is wrapped in aluminum foil to avoid too rapid germination. The Petri dish is kept at 20 ° C for 3 days. After * 4.

- 12 - »two days, a sterility check is carried out by placing a platinum loop between the seeds and placing part of the seeds in the medium of Luria containing per liter: - 10 gr. of bactotryptone - 5 gr. of bacto-yeast extract “5 gr. of Nacl - 1 gr. glucose

The solidification of the medium was carried out by the addition of 15 g / l of agar.

A Petri dish containing a rich agar medium is then streaked and placed at 27 ° C. The conditions allow the development of most molds and bacteria that can colonize the seeds. We keep the box under observation for several days.

4. Cultivation of plants.

When the incubation is complete, the seeds are taken up and placed in a tube containing 6 ml of agar culture medium.

This culture medium is the "Brown" medium (described by. Miksche J.P. and Brown J.A.M. in Am.J. Bot 52, 533 (1965)) at pH 5.8 allowing the development of plants from the seeds.

The medium is solidified with 7.8 g / l of ager (oxoid) which is dissolved by heating for 3 minutes at 110 ° C. The medium is distributed in tubes at the rate of 6 ml in each tube and sterilized (15 minutes at 110 ° C).

The culture conditions are controlled at three levels, that is to say at: - the temperature (22 ° C) - the light intensity (10,000 lux and 16 hours per day) - the humidity (65% ) - · Λ - 13 - * 5. Extraction of growth hormone.

The tissues of the plants are placed in a "micro-Potter" tube where they are ground in the presence of 200 μl of 0.1 M ice-cold phosphate buffer pH 6.8, Triton x-100 0.25%. The tube in which the homogenization takes place is immersed in another tube containing ice. The grinding is done using a homogenizer (TRI-R STIR-R, S63C at 1200 rpm). 50 passages are made and then centrifuged for 2 minutes at 9000 g (BECKMAN Microfuge B), which makes it possible to remove part of the cellular debris. The supernatant is collected and placed in an eppendorf tube placed in ice. The pellet is taken up in 200 μl of phosphate-triton buffer and resubmitted at 50 passages. This operation is repeated until the base is white.

The supernatant is collected.

Human growth hormone is precipitated between 25% and 60% of ammonium sulfate saturation.

The precipitate is dissolved in a phosphate buffer and the hormone is isolated by chromatography on SEPHADEX G 75.

The isolated hormone is then purified according to known techniques.

Example 2.

Example 1 is repeated, but instead of placing the seeds treated with DNA in a Brown culture medium, they are placed in Petri dishes containing a B5 medium which induces the formation of calluses from seeds. This medium is described by GAMBORG B.L., MILLER R and 0JINA K. in Exper. Cell.Res. 50, 151 - 1968). We put about 20 seeds per box.

The growth hormone is extracted in the manner described in Example 1 from calluses, from plants obtained from these calluses or from the descendants of plants obtained from these calluses.

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Examples 3 and 4

Examples 1 and 2 are repeated, but instead of the expression vector pc hGH 800, the expression vector "hGH gene subcloned in pBR 322" is used (hereinafter called pg hGH N), the preparation of which is described in Nucleic acids Res. 9 (1981) 3719 - 3730 by M. DE ΝΟΤΟ, D.D. MOORE and H. GOODMAN.

This pg hGH N vector is due to the insertion of the hGH gene at the ECO RI site of pBR 322.

The length of the inserted fragment which contains the gene is 2,600 bp.

The gene contains 5 exons and its natural promoter. It is inserted in the opposite direction to that of cDNÂ in the vector pc hGH 800 of Examples 1 and 2.

The structure of the vector pg hGH N is represented in FIG. 2 in which 1,2,3,4 and 5 represent exons and a, b, c and d represent introns.

The host of this plasmid is also the E. Coli K 12-HB 101 strain.

Examples 5 to 8

The envelope of beet seeds (BETA VULGARIS L. var. Mavigo) is broken and the seeds are removed.

The seeds are treated for 2 min with 2% ^ £ 0 ^

After five washes with sterile water, the seeds are treated for 30 minutes with sodium hypochlorite (13% active chlorine). The sterilized seeds are then washed with sterile water to a pH of 6. They are then dried in a sterile atmosphere at room temperature until the moisture content of the seeds is less than 5% by weight.

A DNA solution is then prepared, the seeds are treated with this solution and the growth hormone is extracted as described in Examples 1 to 4.

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Examples 8 to 12

Examples 1 to 4 are repeated, but replacing the barley seeds with green pepper seeds (CAPTICUM ANNUUM L., var. Fasciculatum) which are treated in the following manner before being incubated in the manner described in the examples 1 to 4 in the DNA solution.

The seeds are placed for 40 minutes in sodium hypochlorite (13% active chlorine).

The seeds are washed with sterile water to a pH of 6.

If the moisture content is less than 5% by weight further drying is not necessary.

Examples 12 to 16

Examples 1 to 4 are repeated, but replacing the barley seeds with soybeans (GLYCINE MAX., L. var. Japan black).

Before incubating them in the DNA solution as described in examples 1 to 4, they are put for a maximum of 9 minutes in 2% and subjected to ten rapid washes with sterile water.

The humidity of the seeds to be incubated is less than 5% by weight.

Examples 16 to 20.

Examples 1 to 4 are repeated, but replacing the barley seeds with seeds of ARABIDOPSIS TBALIANA. (mutant V 131 st. pyr.gl.) which is treated as follows.

The seeds are placed in porcelain cups and covered with a 5% calcium hypochlorite solution previously filtered through millipore (0.45 μm). After 10 minutes, remove the calcium hypochlorite solution and rinse the seeds 4 to 5 times with sterile water. The sterilized seeds are then transferred, under sterile conditions, to the wells of a porcelain plate placed in a sterile appropriately sized glass petri dish.

The moisture content of the seeds is less than 5% by weight.

The treated seeds are incubated, in batches of several, as described in Examples 1 to 4, in the DNA solution but by adding thiamine to the "Brown" culture medium to which agar has already been added. until you get a final concentration. -5 middle of 5.10 M.

Examples 20 to 24

Examples 1 to 4 are repeated, but replacing the barley seeds with rice seeds (ORYZA SATIVA, var. Japonica, cv Honnen Wase) and treating them as follows before incubation in the DNA solution .

The dry seeds are first carefully peeled using the forceps. After removing the pericarp, the seeds are treated with sodium hypochlorite (13% active chlorine) added with Tween 20 (10 / μl / 20 ml) for 40 minutes after which the seeds are washed with sterile water to a pH of 6.

The moisture content of the washed seeds is less than 5% by weight. It has been found that a concentration of DNA in the solution of 200 μg / rol is sufficient.

Examples 24 to 28

Timothy seeds (PHLEUM PRATENSE var. Errecta, cv RVP) are shaken for 3 hours in water, then peeled under a binocular magnifier. The seeds are then sterilized with sodium hypochlorite (13% active chlorine). The seeds are washed and aseptically dried at room temperature for two hours until the moisture content of the seeds is less than 5% by weight.

The seeds are then treated in the manner described in - Examples 1 to 4, apart from the treatment of the seeds before incubation.

Claims (17)

1. A method of treating plant material in order to obtain the expression of at least one gene in plant cells, according to which the plant material is first sterilized and then it is incubated in a DNA solution having at least one foreign gene to be expressed and / or the cDNA of this gene / characterized in that at least one dry seed of an upper plant, at least one dry spore of lower plant or at least one embryo of excised plant is incubated dry, with a moisture content of less than 10% by weight, at least until the embryo has tripled in size, in an aqueous solution of DNA comprising the complete foreign gene and / or the cDNA of this gene . 2. Treatment method according to claim 1, characterized in that at least one seed, at least one spore or at least one embryo is incubated, the moisture content of which is less than 5% by weight. 3. Method according to either of claims 1 and 2, characterized in that the plant material is dried before incubating it in the DNA solution until the moisture content of the plant material is lower than the maximum content mentioned above. 4. Method according to either of claims 1 to 3, characterized in that peelable seeds are incubated and peeled before being incubated in the DNA solution. 5. Method according to either of claims 1 to 4, characterized in that incubates in the DNA solution during the first 72 hours of development of the seedling from the germ. = "· 10 - 19 - r 6. Method according to either of claims 1 to 5 / characterized in that it is incubated in a saline DNA solution. 7. Method according to either of claims 1 to 6, characterized in that a DNA solution comprising at least one foreign eukaryotic gene and / or the cDNA of this gene is used as the DNA solution. 8. Method according to claim 7, characterized in that a DNA solution is used comprising the gene coding for human growth hormone (hGH) and / or cDNA this gene. 9. Method according to either of claims 1 to 8, characterized in that a DNA solution comprising the foreign gene and / or the cDNA of this gene inserted in a vector is used. 10. Method according to either of claims 1 to 9 / characterized in that a DNA solution is used comprising not only the foreign gene and / or the cDNA of this gene but also a selection gene and / or the cDNA of such a gene. 11. Method according to claims 8 to 10, characterized in that a solution comprising the plasmid pc hGH 800 comprising the gene for 1'hGH at the HIND III site of pBR 322 is used as DNA solution. 12. Method according to claims 8 to 10, characterized in that a solution comprising the plasmid pg hGH N comprising the hGH gene inserted at the Eco RI site of pBR 322 is used as the DNA solution. 13. Method according to either of claims 1 to 12, characterized in that the seed or the spore is put in a DNA solution comprising at least 1 μg of DNA per ml of solution. 14. Method according to claim 13, characterized in that the plant material to be incubated in * - 20 - * · 4 »V a DNA solution comprising at least 400 DNA per ml. 15. Seed, spore or embryo of excised plant in which or which expresses a foreign gene, this seed or spore or this embryo having absorbed according to the method according to either of claims 1 to 14 of DNA comprising the foreign gene to be expressed or the cDNA of this gene. 16. Seed, spore or plant embryo according to claim 15, characterized in that he or she has absorbed DNA comprising the complete gene coding for human growth hormone or the cDNA of this gene. 17. Plants, calluses or cells obtained from a seed, a spore or an excised plant embryo, treated according to the method according to either of claims 1 to 14, and derivatives or descendants of these plants.