NL8801444A - Genetic transformation of eukaryotic cells - esp. plant cells, by treating dried cells with DNA soln. - Google Patents

Abstract

Genetic transformation of cells of a multicellular eukaryotic organism is effected by introducing foreign DNA (I) into the cells and integrating it into the organism's genome. This is done by treating the cells while in a dry abiotic state with an aq. medium contg. DNA contg. a transposon comprising (I) between transposase recognition sequences. Pref. the cells may be pretreated to increase their permeability, e.g., with acetone, ether, CHCl3, CCl4, nonionic surfactants and/or enzymes such as phospholipases. The DNA may also contain a sequence coding for the relevant transposase between the transposase recognition sequences. It is in linear double-stranded form, opt. flanked by histones or basic proteins such as cytochrome C or E.coli recA protein. The treatment is effected in the presence of a chelating agent and an immobilised protease, eg., papain.

Description

Method for genetically transforming cells of a multicellular eukaryotic organism.

The invention is in the field of DNA recombinant technology and more particularly in the field of the genetic transformation of cells of a multicellular eukaryotic organism, in which foreign DNA is introduced into the cells and integrated into the genome.

Various methods have been described to introduce foreign DNA into cells, such as conjugation, transformation, transfection, etc. Some of these known transformation methods (here and further on the term "transformation" in the broad sense of alteration of the genetic information are used) are based on on natural mechanisms of DNA uptake, such as exist in certain types of bacteria, or on the use of viruses that can introduce RNA or DNA into certain host cells, and on the use of Agrobacterium tumefaciens bacteria to introduce DNA into cells of certain types of plants .

Methods have also been described which allow DNA uptake by the cells to be transformed by treating these cells with, for example, calcium chloride or polyethylene glycol, or by using protoplasts. It has also been proposed to introduce DNA directly into the cells to be transformed by microinjection.

In theory, some of these known methods should also be useful for transforming reproductive cells (cells of the germinative line) or totipotent cells (cells that can differentiate into different types of cells) from multicellular eukaryotic organisms, so that a fully transformed organism can eventually are formed, but in practice each of the known methods appears to have one or more serious drawbacks. For example, microinjection of cells is a time-consuming method, making it impossible to treat many thousands of cells at once. In addition, it requires expensive equipment. Protoplast transformation has the disadvantage of being limited to those host cell types that can be regenerated from protoplasts. Likewise, A. tumefaciens technology has the major drawback of being limited to those species of plants susceptible to infection with Agrobacterium strains, while also the need to eliminate the bacteria after transformation.

For the integration of the foreign DNA into the genome of the host cells, so-called transposons can be used, as described in US patent 4,670,388. A transposon is a DNA fragment terminated with nucleotide sequences recognized by a transposase enzyme and thereby enabling the entire fragment to be integrated somewhere in the genome by an existing integration system comprising the transposase. In said US patent it is explained that for the transformation of cells of multicellular organisms, such as of Drosophila melanogaster (fruit flies), a mixture of a transposon can be used, comprising the foreign DNA to be integrated between sites that can be recognized by a transposase, and an active transposon, ie a DNA fragment comprising the transposase encoding DNA between the transposase recognition sequences. This mixture of transposons is introduced into the cells to be transformed by the microinjection method.

The present invention also uses transposons to realize an integration of foreign DNA into the genome of host cells of multicellular eukaryotic organisms, but avoids the drawbacks of the microinjection method using the understanding that cells from dry tissues of a negative osmotic gradient are able to absorb water and DNA molecules contained therein.

The invention provides a method of genetically transforming cells of a multicellular eukaryotic organism, wherein foreign DNA is introduced into the cells and integrated into the genome, characterized by treating cells in a dry abiotic state, which survive such a dry abiotic state, with a DNA-containing aqueous medium containing a transposon, comprising the foreign DNA to be integrated between recognition sequences of a transposase.

The invention utilizes negative osmotic potentials of dry tissues which, after mechanical and / or chemical and / or enzymatic treatment, can take up nucleic acids along with the water flow due to an osmotic gradient. This is possible due to the permeability of the membranes of dry cells, which can be further enhanced by treatment with chemicals or enzymes that attack the membranes, but without killing the cells.

Preferably, plant seed embryo tissues are used, but in principle the technique can be applied to any type of cell, tissue or organism that can survive a dry abiotic state, such as microorganisms, spores, pollen, eggs, other tissues from seeds and even animal organisms, such as mosquitoes (Tardigrada) and rotifers (Rotifera).

Preferably, use is made of cells or tissues which form the basis of the development of a complete organism, such as totipotent cells or cells of the germinative line or tissues from which germinative cells can originate, such as apical meristems or flower bud merrants. However, it is also possible to use parts of dry embryos and then regenerate them into complete organisms by in vitro culture.

Preferably, use is made of dry tissues, the cells of which are interconnected by plasmodesmata, which are exposed after removal of the epidermal cells or parts thereof. These plasmodesmata are found in plant cells, which are channels with an opening of 20 to 40 nm, which even virus particles can pass through. The connecting channels between animal cells have a smaller diameter: 2 nm in mammalian cells and 3 nm in insect cells. In principle, these connecting channels in animal cells could only allow naked single-stranded DNA molecules.

Preferably, substances are used which increase the permeability of the cell membranes. Dry cells as found in seeds, for example, will lose intracellular substances upon water absorption (imbibition); ions, amino acids, sugars, organic acids, phenols and even proteins. This indicates leakage of the membranes, which are fragmented by the inflowing water (gradients of about 1000 bar). Studies with dyes (such as Evan's blue), which are not taken up by normal living cells, have shown that these dyes penetrate up to 4 cell layers deep into the tissues of the embryo within a period of two hours.

It is believed that the cells of the outermost layer of the embryo break open by the water flow, but that the deeper cells of the embryo absorb water through the plasmodesmata so that the waterfront gradually enters the embryo. The course of the electrical conductivity of the water in which the seeds or embryos lie indicates that the leakage of intracellular substances continues until the embryo is fully imbibed. Embryos, protected in seeds by the seed coat, absorb water more slowly and are less damaged. Embryos, which are exposed to water vapor instead of water, also absorb water more slowly and show much less loss of intracellular substances. Embryos can absorb water, be dried again and take up water again without losing their germination. However, there is a stage during the water uptake that should not be reached, because then the germination would be lost when drying again.

This stage is different from species to species (McRersie and Tomes (1980) Can J. Bot. 58_: 471-476). The membranes of the embryo cells are still in the laminar phase (Vigil et al. (1982) Plant Physiology, 69 (suppl) 3). Presumably the membranes are only damaged at the level of the piasmodes by the inflowing water. Further damage to the membranes can be achieved by using: 1. grease solvents such as diethyl ether, acetone, chloroform, carbon tetrachloride, etc.

2.non-ionic detergents such as Triton-X100, octylgluco-side, Nonidet, Brij, etc.

3. enzymes such as phospholipases.

The method that can best be used differs from species to species and must be tested beforehand to determine the germination rate after treatment.

Preferably, linear double-stranded DNA molecules are used surrounded by histones or basic proteins such as cytochrome C, or recA protein from E. coli, in the form of 5-10 nm fibers. However, one can also use single stranded DNA surrounded by DNA binding proteins such as the bacteriophage T4 gene 32 protein. Furthermore, it is also possible to use naked double-stranded DNA in the presence of chelates that bind bivalent metal ions and proteases bound to 1 µm oxirane-acrylic spheres to prevent the degradation of the DNA by nucleases.

It is also possible to use double-stranded DNA surrounded by protamine (Korohoda and Strzalka (1979) Z. Pflanzen-physiol 94: 95-99).

Preferably, use is made of a mixture of DNA molecules containing an active transposon and DNA molecules containing the gene to be transferred, located between sequences recognized by the transposase of the active transposon. However, it is also possible to work with a DNA molecule on which these two entities are present together or a DNA molecule in which the gene to be transferred lies within the limits of the active transposon. However, the latter possibility requires consideration of the instability of the inserted gene. If the receptor cells already contain an active transposon, it is sufficient to use a DNA molecule on which the gene to be transferred is located between the sequences recognized by the transposase of the active transposon. Another possible method uses the single stranded DNA, loaded with proteins, which can be isolated from transfer stimulated Agrobacterium strains where the gene to be transferred lies between the border sequences of the T-DNA (Agrobacterium tumefaciens, Agrobacterium rhizoqenes, Agrobacterium rubi) ; see J. Bacteriol. 169 (1987) 5035-5045; PNAS 84 (1987) 9006-9010.

It goes without saying that the transposons resp. the transposon contain one or more markers (selection genes), thereby facilitating the selection of transformed cells or organisms.

Particular preference is given to a method according to the invention in which cells of apical meristem tissue of dry plant seed embryos are treated.

In addition, it is further preferred that the dry plant seeds of embryos are pretreated to make the apical meristem tissue more accessible to the DNA-containing aqueous medium.

Some possibilities for such mechanical pre-treatment are: 1. Incision of dry embryos into the meristematic tissues.

2. Soften embryos by incubation in air saturated with water vapor before incision into the meristematic tissues.

3. Drilling a 0.1 mm hole into the meristematic tissues of dry embryos.

4. Freezing and thawing of seeds or embryos before cutting.

5. Grinding of dry seeds into the rudistematic tissues (in monocotyledonous plants, the primary root can be almost completely abraded off because final secondary roots emerge from the cotyledonary node fairly quickly) 6. Crushing of embryos in the dry state and after uptake of DNA induce secondary embryos in vitro.

The method according to the invention therefore preferably consists of three steps, namely a first step in which dry embryos from plant seeds into incised meristem tissue are cut, truncated or otherwise mechanically damaged, a second step in which the permeability of the cells is increased, and finally a third step in which the uptake of DNA takes place.

The invention can be used to introduce herbicide resistance, resistance to pathogens or resistance to environmental factors, or to realize metabolism changes that increase efficiency. The invention makes it possible to improve the nutritional value of certain plant species and to realize other or better production routes for secondary metabolites. The invention can also be used to transfer properties from one species or variety to another species or variety, which gives the breeding of agricultural crops, horticultural crops and ornamental plants a new dimension of variability.

The invention is further elucidated by means of the following examples.

Example 1

Expression of the E-glucichonidase gene of Escherichia coli under control of the 35S promoter of CaMV and the termination signals of the nos gene of Agrobacterium tumefaciens C58 in maize (Zea mays).

Step 1

The transposon Ac7 of maize was isolated by the group of Prof. dr. P. Starlinger, Institut für Genetik, Cologne, Germany; its sequence is fully determined. (Müller Neumann et al. (1984) Mol. Gen. Genet. 198: 19-24; Pohlmann et al. (1984) Cell 3 ^ 7: 635-643; and erratum in Mol. Gen Genet. (1985) 198: 540).

This transposon contains a large ORF α (open reading frame) with four introns presumably encoding the transposase (Kunze et al. (1987) EMBO J 1555-1563) (Fig. 1). The transposon Ac7 was obtained via Prof. dr. Van Montagu, Lab for Genetics, University of Ghent, Belgium, and is located in the plasmid pMHIO (fig.

1). By cutting the plasmid pMHIO with EcoRl and partial digestion with HindII, a linear plasmid was isolated after agarose gel electrophoresis which has lost the 0.9 kb nucleotide sequence from position Ac 1292 to position Ac 2197. The B-Fluoronidase gene (GUS) of E_j_ coli was donated and sequenced by R. Jefferson's group at the Plant Breeding Institute in Cambridge, England. The plasmid pBI 221 containing the GüS gene was obtained from him under the control of the 35S promoter of CaMV (Cauliflower Mosaic Virus) and with the termination signals of the nos gene from pTiC58 of Aqrobacterium tumefaciens C58 (Fig. 2). The complete chimeric GUS gene was excised from the vector with EcoRI and HindIII and donated into the above linear pMHIQ plasmid and transformed into the E. coli strain HB101 (J. Mol. Biol. 41 (1969) 459-472). The thus obtained plasmid pMG15 (fig. 3) was further transformed into the Agrobacterium tumefaciens strain C58 Cl (pGV3850) [deposited on 21-12-1983 with the Deutsche Saromlung von Mikroorganismen under DSM number 2798 and described in EP-A-1167183 in which much of the T DNA has been replaced by the pBR322 sequence (Zambryski et al.

(1983) EMBO J..2: 2143-2150). The plasmid pMG15 has retained a 3234 bp sequence of pBR322 which allows integrative recombination to obtain a cointegrate of pMGl5 in pGV3850. Agrobacteria with this cointegrate are resistant to carbenicillin (pBR322) and spectinomycin (pMHIO) and produce β-Glucuronidase due to the activity of the CaMV 35S promoter in Agrobacterium.

The A. tumefaciens strain C58 Cl (pGV3850) itself does not produce β-Glucuronidase. The tests for 6-glucuronidase were performed according to the procedure of the BactidentR test, which is marketed by Merck, Darmstadt.

To test the activity of the Ac transposon, the pMHIO plasmid was transferred to A. tumefaciens C58 Cl (pGV3850) and strains with the pGV3850ï: pMH10 coincinate isolated as colonies resistant to carbenicillin and spectinomycin, The activity of the Ac transposon was tested in Nicotiana tabacum W38 after transformation of leaf discs with the tumefaciens strains C58 Cl (pGV3850: pMG15) and (pGV3850 :: pMH10), screening for nopaline on leaf tissue of the regenerated shoots (NB nopaline spreads in the symplast via the plasmo-desmata) and Southern hydridization of DNA from different transformed plants with a GÜS probe (the GUS insert of pBI221) and an Ac probe (internal HindIII fragment of Ac in pMHIO).

Step 2

Corn seeds were removed from the seed coat above the embryo with a scalpel, after which these seeds were incubated for 4 hours at 30 ° C in a closed container with 90% relative humidity. The embryos were then incised with a fine scalpel along their longitudinal axis, through coleoptile and first leaves into the aplcal meristem, and dried again in an air stream at 25 ° C.

The seeds were placed in acetone for 2 to 3 minutes and dried again. Using a transferpettor (Brandt) equipped with a glass capillary pipette, the tip of which was narrowed by heat stretching (Capillary puller, Narashige Scientific Instrument Lab., Japan), 50 to 100 nl DNA solution was cut. This DNA solution consisted of SalI linearized pMHIO and pMG15 plasmids in 1 mM Na2EDTA, 10 mM ΚΗΡΟ4, pH 6.8, to which insoluble papain bound to oxirane-acrylic spheres of 1 µm, approximately 10 units per ml, was added. The seeds were germinated at 25 ° C and 1000 lux on sand, saturated with Murashige and Skoog medium, to compensate for the loss of ions by draining and EDTA treatment. The combination of EDTA and papain prevented rapid degradation of the naked linear double-stranded DNA. Alternatively, instead of papain, cytochrome C (1 mg / ml) was used to protect the DNA from degradation. The E. coli recA protein (100 µg / ml) could also be used.

Step 3 T ° generation 1 week after treatment, half of the shoot was removed, cut into small pieces and in a solution of X-glucuronide (5-bromo-4-chloro-3-indolyl-B D glucuronide, 40 micrograms / ml) incubated in ethylene glycol monoethyl ether in the dark at 30 ° C overnight. Shoots that convert the X-glucuronide into the blue dye 5-bromo-4-chloro-3-indole were considered positive and further acclimatized in Jiffy pots in the greenhouse and transferred into the greenhouse or field as soon as possible depending on the season.

Step 4 T ° generation

Before flowering, the corncobs were covered with plastic bags and the panicles with paper bags. When the pollen was ripe, the flasks were normally pollinated with the pollen leaving the flask covered with the paper bag containing the pollen. The ripe flasks were harvested and dried in a warm air stream.

After this, the seeds were removed from the flasks and stored at 4 ° C.

Step 5 fluorescent generation

Two weeks after germination, pieces of leaf were cut from the harvested seed and extracted with sand in a porcelain mortar with pestle in extraction buffer: 50 mM NaH2PO4 pH 7.10 mM Na2EDTA, 0.1% Triton X100, 10 mM mercaptoethanol, 1 mM PMSF. 50 microliters of extract was added to 50 microliters of extraction buffer with 10 micromolar 4-methylumbelliferyl-B-D glucuronide and incubated in microtiter plates for 2 hours at 37 ° C. The positive extracts gave intense fluorescence under UV illumination (365 nm). By adding Na2CO3 solution to final 0.1 M, fluorescence was further increased by a factor of 7 (Jefferson et al. (1987) EMBO J 6: 3901-3907).

Step 6 Tl and T2 generation

Positive plants were further grown and the seed harvested as in step 4. Plants, homozygous for GUS, did not break down GUS seeds. The heterozygous plants gave both GUS + and GUS seeds.

The tests were performed as described in step 5. Homozygous plants were further grown and the seed harvested as in step 3.

Step 7 T2 generation

Total DNA was prepared from leaves of the homozygous plants according to the method of Lemmers et al. (1980) J. Mol. Biol. 144: 353-376. Aliquots of 10 micrograms of DNA were digested with EcoRl and HindlII

separately. These restriction enzymes each cut one of the ends of the chimeric GUS gene, so that in the collection of generated fragments, some fragments showed the GUS gene at one of their ends. Southern hybridization with the EcoRlHindlII fragment from pBI221 containing the chimeric GUS gene yielded two classes of band patterns: class A, in which the GUS gene was found in a limited number of bands, and class B, in which the GUS gene with many bands hybridized.

Class A plants are considered to be homozygous GUS + plants that have lost the active transposon or in which the transposon has become inactive (Chomet et al. (1987) EMBO J. 6 .: 295-302).

Class B plants are considered to be homozygous GUS + plants that still contain an active transposon or have contained it for at least one generation.

Example 2

Expression of the chloramphenicol acetyltransferase gene from the pBR325 plasmid under control of the nos promoter and termination signals of Agrobacterium tumefaclens C58 in Kool (Brassica oleracea).

Step 1

As in Example 1, use was made of the transposon Ac 7 of maize active in Brassicceae (Van Sluys et al. (1987) EMBO J._6: 3881-3889).

The chloramphenicol acetyltransferase gene (CAT) with the control elements of the nos gene of A. tumefaciens C58 was donated by De Block et al. (1984) EMBO J.3: 1681-1689. From pNCAT4 (Fig. 4) the 5600 bp Stul-Sall fragment was isolated. The plasmid pMH10 was digested partially with Bell and then partially with BamHI, after which an 8.1 kb linear plasmid, which lost the sequence between Ac 1531 and Ac 4498, was isolated by agarose gel electrophoresis. After ligation, a pMHIO Ds plasmid was obtained that still has the ends of Ac, but lost most of the ORF α (Fig. 5). This plasmid was transformed into the 13. coli strain HB101. After partial cutting with PvuII and then Xhol, the 7.8 kb linear plasmid pMHIO Ds PX was isolated by agarose gel electrophoresis and ligated with the 5.6 kb Stul-Sall fragment of pNCAT4 containing the chimeric CAT gene and a procaryotic gene. The 13.3 kb plasmid pMC34 thus obtained was transformed to HB101 (Fig. 6). Transformants were resistant to spectinomycin and kanamycin.

The activity of the chimeric CAT gene and of the transposon was tested on Nicotiana tabacum W38. For this, the plasmid pMHIO was transferred to A. tumefaciens C58 Cl (pGV3850) and colonies containing the pGV3850: βMHIO co-integrated were isolated on carbenicillin, spectinomycin plates. The plasmid pMC34 was also similarly transferred and the colonies with the cointegrate pGV3850 :: pMC34 were isolated on kanamycin, carbenicillin plates.

Leaf disks from tobacco plants were transformed with both A. tumefaciens strains. After induction of callus on callus-inducing medium, the young calli were tested on the same medium with 75 mg / l chloramphenicol.

The resistant calli were transferred to shoot-inducing medium and after 1 month transferred to root-inducing medium, all as described by De Block et al. (1984) EMBO J. 3, 1681-1689. Young plants were acclimatized in the greenhouse and further grown in pots with topsoil.

Total DNA was prepared from young leaves according to the method of Lemmers et al. (1980) J. Mol. Biol. 144: 353-376.

Southern hybridization of BqlII and SalI digests this DNA with the probes CAT (Sau3A fragment of pBR325) and Ac (internal Hindii fragment of Ac from pMHIO) found three classes of plants: those containing the CAT gene, but no Ac ( integration edited by A. tumefaciens), those containing CAT and Ac on a limited number of bands, and those containing CAT and Ac on a large number of bands. This proved that the transposon was active in both cis and trans.

Step 2

Seeds of Brassica oleracea (rapid cycling populations, Crucifer Genetics Cooperative, University of Wisconsin, obtained from Prof. PH Williams) were stripped of their seed coat at the apical meristem. After absorption of water vapor for 2 hours, the apical meristem was cut and air-dried. After treatment with acetone for 2 to 3 minutes and drying again, about 50 nl of DNA solution was placed in the cut.

This DNA solution contained Sall linearized plasmids pMHIO and pMC34 preincubated for 10 minutes at 20 ° C in a buffer (pH 6.8) containing 10 mM ΚΗΡΟ4, 1 mM Na2EDTA and 1 mg / ml cytochrome C. The seeds were germinated in petri dishes on dry sand in an atmosphere of 90% relative humidity. This prevented further damage to the embryo and the meristematic cells.

Step 3 T ° generation

The young plants were acclimatized in Jiffy pots in the conservatory and further grown in pots with topsoil. After about one month, the plants of the same experiment were fertilized by means of a cotton swab by applying the pollen to the pistils. Seed could be harvested after about 2 months.

Step 4 Tl generation

Dry seeds were sterile extracted from the chickens and germinated on Murashige and Skoog medium with 0.8% agar and 50 mg / l chloramphenicol in petri plates.

About 3% of the seeds developed into green plants, the others had white to pale yellow germ leaves, but plants with white and green spotted germ leaves also occurred. The green plants were further cultivated in the greenhouse. Young leaves were harvested and approximately 100 mg of leaf tissue was ground in 1.5 ml Eppendorf tubes at 4 ° C with 50 microliters of extraction buffer: 0.25M Tris HCl pH 7.8, 10 mM Na2EDTA, 1 mM leupeptin, 10 mM Na- ascorbate, 1 mM cysteine.

After centrifugation in an Eppendorf centrifuge at 13,000 rpm for 10 minutes at 4 ° C, 50 microliters of supernatant was collected. To this was added 5 microliters of chloramphenicol (0.1 microcurie, NEN radioactive Chemicals) and 10 microliters of acetylcoenzyme A (8 mg / ml). After incubation for 1 hour at 37 ° C, the reaction mixture was extracted twice with 0.2 ml of ethyl acetate. The organic phase was dried in a vacuum centrifuge and taken up again in 10 microliters of ethyl acetate. The samples were applied to a thin layer (cellulose) chromatography plate and passed in a closed container with a mixture of methanol / chloroform (5/95 V / V) for 1 hour at room temperature.

After drying, the chromatography plate was stored in a cassette along with an X-ray film for about 14 days. Plants expressing the CAT gene convert chloramphenicol to 3-acetylchloramphenicol which was spontaneously converted to 1-acetylchloramphenicol. Brassicaceae exhibit a low level of chloramphenicol acetyl transferase activity. Transformed plants, however, showed clear activity and were resistant to the antibiotic during germination.

Step 5 T2 generation

The CAT + plants were grown in the greenhouse and crossed with each other. Seed could already be harvested after 2 months and the seed of those plants which yielded only chloramphenicol resistant seeds was resumed. DNA was isolated from leaves according to the method of Lemmers et al. (1980) J. Mol. Biol. 144: 353-376. As described in the tobacco experiments, BglII and Sall digests were examined by Southern hybridization versus the CAT probe and the Ac probe.

Claims (10)

A method of genetically transforming cells of a multicellular eukaryotic organism, in which foreign DNA is introduced into the cells and integrated into the genome, characterized by treating cells in a dry abiotic state, which can have such a dry abiotic state survive, with a DNA-containing aqueous medium containing a transposes, comprising the foreign DNA to be integrated between recognition sequences of a transposase. The method of claim 1, wherein a DNA-containing aqueous medium is used which also contains a transposon comprising transposase encoding DNA between recognition sequences of the transposase. The method of claim 1 or 2, wherein the DNA is used in the form of linear double-stranded DNA molecules surrounded by histones or basic proteins such as cytochrome C or recA protein from E. coli, in the form of fibers, or in the form of naked double-stranded DNA molecules in the presence of a chelating agent and a plastic carrier-bound protease, such as papain. The method according to any one of claims 1 to 3, wherein dry cells are used which have undergone a pretreatment to increase their permeability. The method of claim 4, wherein the permeability of the cells is increased by pretreatment with a fat solvent such as acetone, diethyl ether, chloroform, carbon tetrachloride, etc., a non-ionic detergent such as Triton X100, octyl glucoside, Nonidet, Brij, etc., and / or an enzyme such as a phospholipase. The method according to any one of claims 1 to 5, wherein cells from plant seed dry embryos are treated. The method of claim 6, wherein cells of apical meristem tissue from dry plant seed embryos are treated. The method of claim 7, wherein the dry plant seeds and embryos are pretreated to make the apical meristem tissue more accessible to the DNA-containing aqueous medium. The method of claim 8, wherein the dry plant seeds and embryos are incised or truncated into meristem tissue. Multicellular eukaryotic organism, descended from cells genetically transformed by the method according to any one of the preceding claims.