MXPA96006226A - Transformation of g - Google Patents

Abstract

The invention relates to a method for the transformation and regeneration of legumes of the genus Cyamopsis, in particular to the transformation of guar mediated by Agrobacterium (Cyamopsis tetragonoloba, by introducing a recombinant DNA sequence, into at least one cell or protoplast). and the generation of genetically modified explants, using at least one root selection or growth medium, comprising at least one compound selected from an auxin inhibitor, for example 2- (p-chlorophenoxy) -2-methylpropionic acid (PCIB) ), a la-lactamase inhibitor, for example sulbactam, and an ethylene inhibitor, for example silver thiosulfate, to obtain a genetically modified plant or part thereof, containing in the genome, at least one recombinant DNA sequence to the genetically modified plants produced by the method, and to the use of substances such as la-lactamase inhibitor, sulbactam, for faci litar the transformation of guar and other plant

Description

TRANSFORMATION OF GUAR FIELD OF THE INVENTION The present invention relates to a method for the transformation, regeneration and selection of legumes of the Cyamopsi s genus, in particular the guar transformation mediated by Agroba cteri um • *. (Cyamopsi s t etragonol oba), to genetically modified plants produced by the method, as well as to the use of substances such as the sulbactam of the β-lactamase inhibitor, to facilitate the transformation of guar and other plants.

BACKGROUND OF THE INVENTION TRANSFORMATION OF VEGETABLES The Faba cea e (Leguminosae) family is the 0 most important family of dicotyledonous plants in the world. Due to its great economic significance, much effort has been invested in improving agronomic properties through genetic engineering. The transformation mediated by Agrobac terium, 5 is a common method used for the transformation of REF: 23657 genes in plants. The plant species that until now have been successfully transformed by Agrobacterium um are exclusively dicotyledonous (as opposed to monocotyledons), but not all dicots are easily transformed. Some plant families, for example Sol anaceae, have been considered particularly appropriate for gene transfer mediated by Agrobacterium, while other families, such as Fabaceae, are notorious for being recalcitrant. The production of transgenic soybean plants. { Gl i cine max) has been tried in a variety of ways. The leaves and protoplasts have been used as sources of explant, but, regeneration has not been obtained within the transformed plants, in this way. The cotyledons of soybean inoculated with Agrobac teri um t umefaci ens resulted in transgenic plants, but only one of the numerous genotypes treated was successfully transformed in this way (Hinchee et al., Bio / Technology 6: 915, 1988). WO 94/02620 describes a method for the production of transgenic soy plants, using hypocotyls or cotyledon nodes and a series of specially designed steps for soybean transformation, including temperatures, pH values and particular concentrations of Agrobac teri. um. The use of cotyledons as explants, however, is not generally applicable to the transformation of legumes, and other explant sources have been used in most cases. For example, for the transformation of peas (Pi sum sa ti vum), the explants from shoot cultures and seedling epicotypes have been used as explants, and the transgenic callus obtained in this way, was 6 months later regenerated to plants ( Puonti-Kaerlas et al., Plant Cell Rep. 8: 321, 1989). For the transformation of white clover (Tri fol i um repens), the tips of the shoots were inoculated with Agrobacteria, and transgenic plants were obtained (Voisey et al., Plant Cell Rep. 13: 309, 1994). Attempts have been made to transform a number of other legumes. For example, the cotyledon nodules of Pha seolus vulgaris and the hypocotyls incubated with Agrobact erium tamefaci ens resulted in transgenic callus but not transgenic plants (Mc-Clean et al., Plant Cell, Tissue &Org. Cult. 24: 131, 1991). In the same way with the genus Vi gna (García et al., Plant Science 48:49, 1986). No transgenic peanut plants (Arachi s Hipogaea) have been reported despite considerable effort. The present inventors have tried to transform the guar using the soybean cotyledon process described by Hinchee et al., But they were not successful. Together with the results reported with other researchers, this shows that the choice of the transformation method for legumes is completely empirical, and that general, scientifically based rules can not be deduced. In this way, the transformation procedure and the source of the explant for the transformation of a legume, has to be developed according to the particular requirements, of the genus, species or even genotype, in question. The numerous attempts reported to obtain transgenic plants of various legumes, clearly show that the transformation of legumes is very difficult, even for a scientist skilled in the art. This is further evidenced by the fact that of the approximately 100 species of legumes of commercial interest, less than 5 species have been transformed. In this way, the successful transformation of a previously untransformed genus or legume species is nothing other than routine.

Guar Guar (Cyamopsi s tetragonol oba) is a legume of significant commercial interest, due to the high content of galactomannan in the seeds. Guar galactomannan is also known as guar gum, and is used as a viscosity enhancer both for food and non-food purposes. The galactomannan is found in the endosperm, which constitutes approximately 35% of the dry weight of the seed, being 80-90% pure galactomannan. Large endosperms are an unusual feature in Fabaceae, where the endosperm fraction of the seeds is predominantly absent or rudimentary; instead food reserves for germination in legumes are more frequently deposited in enlarged cotyledons.

It has not been reported that any of the legume species with endosperms that contain a large amount of galactomannan, have been genetically transformed.

Sulbactam An inherent drawback of the genetic transformation mediated by Agrobacterium um is the fact that bacteria continue to develop after transformation. In order to prevent the overgrowth of plant material, bacteria must be effectively eliminated, usually by the addition of an antibiotic similar to penicillin (ß-lactams) such as carbenicillin, cefotaxime, etc. Penicillin-like substances are chosen because they are, in principle, non-toxic to plant tissues. In practice, however, these compounds frequently exert a considerable toxic effect on the explants. A possible reason for the phytotoxicity, in addition to the possible direct toxic effects, may be that the antibiotics are gradually degraded during the prolonged incubation time in the presence of bacteria and plant tissues. An example of an undesirable degradation product is from the widely used carbenicillin antibiotic, which can be degraded to phenylacetic acid. Phenylacetic acid possesses properties similar to auxin, and consequently gives increased callus growth on the explant, which in turn can deteriorate regeneration. Thus, it may be highly desirable to make it possible to use smaller amounts of antibiotics and / antibiotics that do not have such unwanted side effects. In the course of their work on the transformation of guar, the inventors found that the β-lactamase inhibitor, sulbactam, dramatically reduces the required concentrations of substances similar to penicillin, by improving the transformation efficiency and reducing the costs significantly. This new procedure to control the growth of Agrobacteria is generally applicable to the transformation of plants, because this is related to the bacteria that must always be eliminated during the transformation, and in this way is not limited to any particular plant species. .

BRIEF DESCRIPTION OF THE INVENTION In one aspect, the present invention relates to a genetically modified plant or part thereof, of the genus Cyamopsi s, said plant or part of the plant in its genome comprising at least one recombinant DNA sequence. Yet another aspect of the invention relates to a method for the production of a genetically modified plant or part thereof, of the genus Cyamopsi s, comprising the steps of introducing a recombinant DNA sequence into at least one cell or protoplast and generating genetically modified explants, using at least one shoot growth medium or selection, comprising at least one compound selected from an auxin inhibitor, a β-lactamase inhibitor and an ethylene inhibitor, to obtain a plant or part of the same, genetically modified, which contains in its genome at least one recombinant DNA sequence. A further aspect of the invention relates to a method for the production of a genetically modified plant, in which at least one medium used for the selection or growth of the cells, protoplasts, callus or parts of the plant, comprises at least one substance that inhibits bacterial growth or that increases the effect of a bacterial growth inhibitor, without have no substantial toxic effect for the plant or for the regulation of growth. In a further aspect, the present invention relates to chimeric plants capable of producing transgenic seeds and obtained by grafting a genetically modified shoot grown in vi tro on a non-in vi tro cultivated plant.

DETAILED DESCRIPTION OF THE INVENTION The terms "genetically modified plant" and "transgenic plant" in the context of the present application, refer to the meanings generally encompassed by these terms in the art, for example plants that have been altered in such a way that their genome comprises at least one recombinant DNA sequence. The "recombinant DNA sequence" will typically be one that is capable of being expressed or which affects the expression of the gene in the plant, but may also be, for example, a sequence that can serve as a marker, without necessarily being expressed or affect the expression of the gene. The sequences that are expressed or that affect the expression of the gene will often be genes that are foreign to the plant in question in its native form, but may also be, for example, a slightly altered form of the native gene or for example a promoter sequence. or regulatory that results in the altered expression of the native gene. The method described herein for the production of genetically modified plants is directed to genetic transformation in general, and is not limited to the incorporation of any particular category of DNA sequences. The term "plant parts" generally refers to any part of the plant, for example, tissue or organ, which is not a complete plant, including the undifferentiated callus as well as the differentiated parts of the plant such as shoots, leaves, roots, fruits, seeds, etc. As indicated above, the invention relates in particular to genetically modified plants of the Cyamopsi s genus, and more particularly to plants of the species C. te tragonol oba (guar). Genetically modified Cyamopsi plants can be produced by the method mentioned above, in which the first step is the introduction of a recombinant DNA sequence into at least one cell or protoplast. The introduction of recombinant DNA can be achieved by methods commonly employed for the production of genetically engineered plants, including Agrobacterium um-mediated transfer, for example by means of a Ti-plasmid of A. Tumefa ci ens or a Ri-plasmid of A. rhi zogenes as a vector, as well as by, for example, microinjection, eletroporation or bombardment of particles. A preferred method (described later in the Examples) is gene transfer mediated by Agrobacterium. As explained below, good results have been obtained by the transformation of cotyledons using Agrobac t eri um t umefaci ens, although the cotyledons used were from seeds that had been germinated for a relatively long period of time, such as 11-12 days .

After the introduction of the desired recombinant DNA into the chosen plant material (e.g., tissue, cells or protoplasts), the genetically modified explants are generated using at least one seedling growth or selection means comprising at least one compound selected from an auxin inhibitor, a β-lactamase inhibitor and an ethylene inhibitor, since it has been found that the presence of one or more of these compounds in the selection medium and / or outbreak growth leads to less callus and an increased frequency of regenerated and transformed shoots. Preferably, the selection medium comprises at least one auxin inhibitor, a β-lactamase inhibitor, and the shoot growth medium comprises at least one β-lactamase inhibitor. More preferably, the selection medium comprises an auxin inhibitor, a β-lactamase inhibitor and an ethylene inhibitor, and the growth medium of shoots comprises an auxin inhibitor and an ethylene inhibitor. The inhibitors (auxin inhibitor, β-lactamase inhibitor, ethylene inhibitor) can function either by eliminating or reducing the amount of the respective compounds (for example by inhibiting the biosynthesis of the compounds or by degradation of the compounds), or by inhibiting the action of the compounds. While not wishing to be committed to any particular theory, it is believed that the effect of the inhibitors is, at least in part, related to the anti-auxin effects, or to the inhibition of the "auxin-like" effects, since that the presence of auxin leads to increased growth of the callus and therefore to a lower frequency of regeneration of the shoot and isolation of the transformed shoots. This is only the case for those compounds that work directly as auxin inhibitors. As for β-lactamase inhibitors, it was previously explained by way of example that an alleged defect of sulbactam is the elimination of the degradation product of phenylacetic acid (from the antibiotic carbenicillin), with phenylacetic acid having undesirable properties similar to auxin which result in increased callus growth. Similarly, the ethylene inhibitor, in addition to its direct effect on ethylene that presumably serves to prevent premature senescence in developing shoots, is also believed to have a beneficial influence due to the fact that ethylene is known to be associated with the answers to the auxin. The use of an auxin inhibitor and the ethylene inhibitor will be advantageous notwithstanding the type of gene transfer employed, for example, with bacterial mediated transfer such as Agrobacterium um transfer, as well as other methods such as microinjection, electrophoresis. and bombardment of particles, while the use of the B-lactamase inhibitor is particularly suitable for procedures employing gene transfer through Agrobacterium um or other strains of bacteria that produce β-lactamase. A preferred auxin inhibitor is 2- (p-chlorophenoxy) -2-methyl-propionic acid (PCIB), which can be used in the selection medium and optionally also in the shoot development medium in a concentration of about 0.01-10 mg / l, • typically about 0.05-5 mg / l, for example about 0.1-2 mg / l. Other auxin inhibitors that can be used are, for example, 2, 3, 5-triiodobenzoic acid (TIBA), N-naphthyphthalamic acid (NPA), morfactins, 2,4,6-trichlorophenoxyacetic acid and 7-chloroindolacetic acid.

A preferred β-lactamase inhibitor is sulbactam (available from Pfizer under the trade name Betamaze). The sulbactam can be used in the selection medium or in the shoot development medium, at a concentration of about 10-1000 mg / l, typically about 20-500 mg / l, for example about 50-200 mg / l. l. A preferred ethylene inhibitor is silver thiosulfate, which is typically used in the selection medium or in the shoot growth medium, at a concentration of about 50 μm, typically about 1-10 μm, for example from 0.5-5 μm. Other ethylene inhibitors that can be used are, for example, aminoethoxyvinylglycine (AVG), cobalt and norborneol. In addition to the compounds described above, certain other compounds having a beneficial effect have also been used, when used in the selection and / or growth or shoot medium. For example, it has been found that an improved frequency of transformation is obtained when a nickel salt is added to the selection medium. Thus, the selection medium preferably contains a nickel salt, for example, NiCl; 6H0, for example at a concentration of approximately 0.1-10 mg / L, for example 0.5-5 mg / L. It has also been found that the benzyladenine purine (BAP) leads to an improved result. The selection medium thus contains, preferably BAP, for example in a concentration of approximately 0.1-10 mg / l, such as 1-5 mg / l. BAP may also be present in the growth medium or shoots at similar concentrations. The presence of kanamycin, for example in the form of kanamycin sulfate, at a concentration of about 50-300 mg / l, typically about 100-200 mg / l, for example about 130-160 mg / l, in the means of selection, has also been shown to have a beneficial effect when the inserted DNA sequence includes a gene for kanamycin resistance. It has also been found that good results are obtained when the explants, typically explants from which the shoots have been harvested, are transferred to a second selection medium with a lower concentration of kanamycin than that of the first selection means, preferably not greater than 125 mg / l, typically 20-100 mg / l, for example 30-70 mg / l. Similarly, other amino-glycoside antibiotics such as hygromycin, neomycin, streptomycin and gentamicin may be employed in the selection medium, together with an inserted DNA sequence comprising the gene relevant for antibiotic resistance. Other examples of selection, for example herbicides or positive selection agents such as mannose or xylose, can also be used. After the selection and harvest of '* • regenerated shoots, the presence of outbreaks H) genetically transformed can be determined by various methods. One of these (in addition to the use of an antibiotic together with an antibiotic resistance gene as described above) is by means of the reporter gene of β-glucuronidase, the use del. which is described below, as well as in WO 93/05163. The resultant transgenic Cyamopsi outbreaks can then be regenerated in whole plants by known methods, for example already either by direct root formation on the shoots or by grafting the transgenic shoots on the established rooted plants. The last method, the grafting of the transgenic shoots on the stems of the established plants (which by themselves can be transgenic or not) has been found to be appropriate, resulting in chimeric plants capable of producing transgenic seeds. It has also been found that particularly good results are obtained when outbreaks transgenics are grafted onto the seedlings, for example seedlings 7-28 days old, typically 12-21 days old. As mentioned above, another aspect • r * of the present invention relates to a method for the production of a genetically modified plant in which, at least one medium used for the selection or growth of the cells, protoplasts, callus or parts of the plant comprises at least one substance that inhibits bacterial growth or that increases the effect of a bacterial growth inhibitor, without having any substantial toxic effect for the plant or for the regulation of the growth of the plant, for example, an inhibitor of the β-lactamase. This aspect is related to the fact of that the advantageous effect of the use of, for example, a β-lactamase inhibitor such as sulbactam, is not limited to the transformation and selection of guar plants, but rather is generally applicable to mediated gene transfer by Agrobacteri um (or in the presence of other strains of ß-lactamase-producing bacteria), in any plant in order to eliminate unwanted bacterial growth subsequent to transformation. A significant practical and economic benefit of this procedure is that the amount of antibiotics similar to penicillin, used in the means of selection and development of shoots, can be greatly reduced, for example to a level of approximately 10% of that which is necessary in the absence of the ß-lactamase inhibitor. When the β-lactamase inhibitor is sulbactam, it is used in the amounts given above. The invention is further illustrated by the following non-limiting examples.

EXAMPLES General procedure for the transformation of guar Seedlings Guar seeds were sterilized in a sodium chlorite solution containing 2.5% free chlorine, pH 7.0, and two drops of Tween 80 per 100 ml of solution. The seeds (approximately 10 g per 100 ml) were stirred for 25 min., Washed 5 times with sterile water and dried on filter paper overnight. The seeds were then sown on germination medium and placed in the dark at 25 ° C for 4 days. Subsequently, germination was continued at 12h / 12h day / night for 7 days. The rich germination medium used, resulted in high quality guar seedlings.

Germination medium: 4.43 g / 1 MSMO (Sigma M6899) 20 g / 1 sucrose 8.0 g / 1 agar pH 5.8 (adjusted with KOH) Suspension of Agrobac t eri um t umefaci ens The suspension of Agrobact erium was prepared as an overnight culture (incubation for 17-18 h) in L-B medium. Acetosyringone was not added to the bacteria culture.

Medium LB: 10 g / 1 tryptone Bacto 10 g / 1 NaCl 5.0 g / 1 yeast extract pH 7.4 (adjusted with NaOH) Transformation and co-culture The bacterial suspension was in most cases diluted to an OD of 0.1 (660 nm) with LB medium, but good results have also been obtained at an OD of approximately 1. The cotyledons with approximately 2 irtm of hypocotyl were removed from the 12-day-old plants. The cotyledons were then separated, using forceps to create wound surfaces and placed in the Agrobac terium suspension for 30 min. The preferred method for co-culture was the so-called sandwich method, where the explants were placed on the filter paper, which in turn was placed on co-culture medium. The filter paper soaked in a co-culture liquid medium was also placed on top of the explants, in order to prevent the explants from drying out.

Co-culture medium: 0.43 g / 1 of MS basal salt mixture (Sigma M5524) g / 1 sucrose 100 mg / l myo-inositol 0.1 mg / l thiamine, hydrochloride 0.5 mg / l pyridoxine, hydrochloride 0.5 mg / l nicotinic acid 1.0 μm silver thiosulfate 8.0 g / 1 agar pH 5.1 The co-culture proceeded for 3 days at 25 ° C and with a regimen of 12 h / 12 h day / night. After co-culture of the explants, they were washed with MS 1/10 medium, to which 100 mg / l of carbenicillin, 100 mg / l of cefotaxime and 1000 mg / l of lysozyme had been added, 2-3 times per 45 min., While stirring at 100 rpm.

Selection The explants were transferred to the selection medium and incubated as described above (25 ° C, 12 h of day / 12 h of night).

Selection medium: 3.2 g / 1 Gamborg B5 (Sigma G5893) 20 g / 1 sucrose 1.0 mg / l benzylaminopurine 0.05 mg / l gibberellic acid (GA3) 1.0 μM silver thiosulfate 1.0 mg / l NiCl2, « 6H20 0.5 mg / l of 2- (p-chlorophenoxy) -2-methylpropionic acid (PCIB) 50 mg / l of cefotaxime 50 mg / l of carbenicillin 100 mg / l of sulbactam (Betamaze) 145 mg / l kanamycin sulfate pH 5.7 Harvest of the transgenic shoots First harvest: After 4 weeks, shoots larger than 3 mm were harvested and transferred to the shoot medium. After 10-14 days the shoots were tested for the activity of the reporter gene of β-glucuronidase (GUS), see below. Positive outbreaks to the GUS were transferred to the fresh outbreak medium, while negative outbreaks to the GUS were discarded.

Bud medium: 3.2 g / 1 Gamborg B5 (Sigma G5893) 20 g / 1 sucrose 0.1 mg / l benzylaminopurine 1.0 μM silver thiosulfate 0.1 mg / l gibberellic acid (GA3) 100 mg / l cefotaxime 100 mg / l of sulbactam (Betamaze) 8.0 g / 1 of agar pH 5.7 Second harvest: After the first harvest, the explants (from which the shoots had been harvested) were transferred to a second selection medium with a lower concentration of kanamycin. (50 mg / l). After another 4 weeks the shoots were harvested and tested for the GUS. The positive shoots were transferred to fresh medium, while the negative shoots were discarded.

Analysis of transgenic shoots The tips of young leaves were excised and transferred to a multiple well plate containing 200 μl of X-gluc solution. After incubation for 16 h at 35 ° C, the tips of the leaves were destained with 96% ethanol, and the degree of staining with blue was determined under a microscope.

Solution X-gluc (50 ml): 0.2 M Na2HP04 15.5 ml 0.2 M NaH, P04 9.5 ml H20 19.5 ml 0.1 M K3 (Fe (CN) 6) 0.25 ml 0.1 M K4 (Fe (CN) # 3H20 0.25 ml 0.1 M Na - EDTA 5.0 ml X-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronate cyclohexyl ammonium) 50 mg Graft Root formation of transgenic shoots was achieved by grafting. Sprouts with a healthy green appearance and with a length of 0.5-1.0 cm were selected for grafting on guar plants over 1.5-2 months of age developed at a rate of 32 ° C / 25 ° C 14 h / 10 h day / night. Before grafting all the leaves except the one on the highest part, two leaves were removed, and the transgenic shoots were grafted onto the almost vertical cuts in the stem in the nodules. The grafted plants were transferred to a humidity chamber for 5-6 days.

Transgenic plants • "'The grafted plants were subsequently transferred to a development chamber, for further growth of the transgenic shoots. The conditions of development were as described above. After approximately 2 months, mature pods 15 were harvested with numerous transgenic seeds.

Example 1 Transformation of different varieties of guar 0 This example shows the transformation of several varieties of guar, using the method described above.

The number of GUS + shoots (GUS-positive, for example transformed) is calculated per 1000 transformed explants, using the C58 strain of Agrobac terium nopalina. The American variety Lewis gave the highest number of transgenic shoots harboring the GUS gene as a marker for successful transformation, and this variety was the only one used in the subsequent examples.

Example 2 Agrobacteria A number of different strains of Agroba cteri um t umefaci ens were tested for the transformation of guar and all were found to be appropriate. For example, 500 explants of guar per bacterial strain were treated with each of the four different strains (strain LBA 4404 of octopine, Ditta et al, Proc. Na t.Acad.Sci. 11: 7347, 1980 and three strains derived from C58). For each strain, 11-26 regenerated shoots were produced, of which 1-3 were positive for GUS. Similarly, another strain of Agrobacterium um (EHA 101 strain of L, L-succinamopin), which was used to treat 2500 explants, resulted in 25 regenerated shoots, of which 8 were GUS positive. LBA 4404 was also used to treat 2000 explants, resulting in 67 regenerated shoots, of which 17 were positive to GUS. The strains of A. t umefa ci ens employed, contained in the genes of the T-DNA region coding for the β-glucuronidase (for the GUS assay) and the neomycin-phosphotransferase (for selection on the media containing kanamycin).

Example 3 Selection of kanamycin In this example, the optimum concentration of kanamycin sulfate in the selection medium was 145 mg / l, but efficient transformation was also obtained using other concentrations in the range of 125-145 mg / l. The use of kanamycin at 100 mg / l or less, gave regeneration frequencies close to 100%, although only very few transgenic shoots were obtained.

For each treatment, a total of 1600 explants were transformed with strain EHA 101 of Agrobacterium. Approximately 50% of the transgenic shoots were found in the second harvest, where the concentration of kanamycin was reduced. When the concentration of kanamycin was reduced to 125-145 mg / l, only a few transgenic shoots were found in the second harvest.

Example 4 BAP and NiCl2 This example shows the beneficial effects of the addition of purine benzyl-adenine (BAP) and NiCl2 to the selection medium.

For each treatment, a total of 1200 explants were transformed using strain EHA 101 of Agrobacterium. The addition of 5 mg / l of BAP increased the number of transformants, but also the total number of regenerated shoots, which were approximately twice as high at 5 mg / l as at 1 mg / l. The addition of 1 mg / l of NiCL2 »6H20 resulted in a transformation frequency of 2-4 times higher, probably due to the absence of nickel in the employed MS and Gamborg B5 media.

Example 5 Silver thiosulfate This example shows that silver thiosulfate (STS) significantly improves the frequency of transformation.

The number of GUS + shoots is calculated per 1000 explants transformed using EHA 101. A concentration of 2.5 μM silver thiosulfate resulted in significantly more transformants than silver thiosulfate 0, 5.0 or 10.0 μM. The increased transformation frequency caused by STS was due to the significantly improved quality of the shoots. In the absence of STS, the transgenic shoots were atrophied and yellowish, whereas the presence of STS supported the growth. Since it is known that plant ions inhibit the action of ethylene, the beneficial effects of STS could be due to a reduced effect of ethylene in the containers, preventing premature senescence.

Example 6 PCIB PCIB (2- (p-chlorophenoxy) -2-methylpropionic acid) has an anti-auxin effect and can inhibit callus formation. In the absence of PCIB, callus formation was extensive during the selection procedure, deteriorating regeneration. The addition of 0.1-2 mg / l of PCIB reduced the amount of callus significantly and improved the regeneration and the number of transgenic shoots.

Example 7 Sulbactam This example shows that the β-lactamase inhibitor, sulbactam significantly reduces the amounts of carbenicillin and cefotaxime, required for selection, and increases the frequency of transformation.

For each treatment, a total of 800 explants were transformed using EHA 101. All 3 treatments resulted in the elimination of Agrobacteria. The transgenic shoots on carbenicillin at 800 mg / l, developed poorly and had a yellowish appearance. These outbreaks did not recover after the transfer to the outbreak medium, and many transgenic outbreaks sooner or later died. Transgenic shoots selected on carbenicillin at 50 or 100 mg / l in the presence of 100 mg / l of sulbactam, developed well, had a normal green appearance and could be kept intact for long periods.

Example 8 Tidiazuron The addition of the cytokinin thidiazuron (TDZ) significantly improved the transformation frequencies. As shown in the table below, the optimal concentrations of TDZ in the selection media were in the range of 0.3-3.0 mg / l, which increased the transformation frequencies by 1.5-1.8 times.

In addition to increasing the frequency of transformation, thidiazuron was also beneficial for the subsequent cloning of transgenic shoots.

Example 9 Graft Root formation of transgenic shoots was achieved by grafting. Also the grafting on developed plants (of 1.5-2 months of age) is possible, as explained above, although better success rates were obtained by grafting on seedlings that are 12-21 days old. Seedlings are produced by placing sterilized guar seeds on germination media (see above), and subsequently growing them for 12-21 days at 25 ° C at a rate of 13h / llH day / night. The cotyledons were excised and the hypocotyl was cut vertically 0.5-1 cm down. A transgenic shoot was placed in the incision and fastened with a short piece of sterile string. After 5-10 days the rope was removed, and the grafted seedlings were transferred to the soil. Of the 49 transgenic shoots grafted - "* on such seedlings, 42 survived (89%) and resulted in 10 fertile plants with a normal phenotype.

Example 10 Analysis of Southern spotting In order to confirm the presence of the transgenes and the number of gene copies in the transgenic guar lines, the genomic DNA was extracted from leaf samples, digested with HindIII and subjected to electrophoresis on a 0.8% agarose gel. The Southern blotches were made with Hybond N + (Amershan), and the prehybridization and hybridization was at 68 ° C in the recommended buffer by the manufacturer. The DNA probes were either the PMI gene or the GUS gene marked by random priming with the "Ready to go" kit (Pharmacia). 1 x 10 ° CPM / ml of labeled probe was added to the hybridization buffer. Sourthern spotting analysis using the PMI gene as a probe is shown in Figure 1. As can be seen, each band, except band 1, shows an intense band at 2.1 kb, which is the expected fragment size of DNA obtained by digestion by means of HindIII. Band 1 shows a thin band of a similar size. Band 8 is a non-transgenic guar line. Southern blot analysis using the GUS gene as a probe is shown in Figure 2. As can be seen, most bands show an intense band, indicating that these lines contain a copy of the GUS gene. The digestion of the DNA in band 7 has resulted in 4 bands, suggesting that this band contains 4 copies of the GUS gel. Band 8 is a non-transgenic guar line.

Figure 1 shows the genomic Southern analysis of the transgenic guar lines. 10 μg of Genomic DNA from different transgenic guar lines digested with HindIII, subjected to electrophoresis on a 0.8% agarose gel, and DNA stained on Hybond N + and hybridized to a probe consisting of the PMI gene labeled with [32P] dCTP . Markers of [J5P] DNA (Amersham) were used as a molecular weight marker (MW).

Figure 2 shows the Southern genomic analysis of the transgenic guar lines. 10 μg of genomic DNA from different transgenic guar lines digested with HindIII, subjected to electrophoresis on a 0.8% agarose gel and DNA stained on Hybond N + and hybridized to a probe consisting of the GUS gene labeled with [j2P ] dCTP. The markers [- '"" SjADN (Amersham) were used as a molecular weight (MW) marker.

Example 11 Analysis of transgenic progeny The inheritance and segregation of transgenes was studied in some of the independent guar transformants. The primary transformants were self-fertilized and a number of seeds were planted (10-20). The presence of the GUS gene in the second generation plants was demonstrated by the GUS assay (see above).

This table shows that the GUS gene is inherited in a stable manner, and that it is segregated approximately as would be expected for a simple dominant gene. In addition, the GUS gene has retained its activity. , J0.

It is noted that in relation to this date, the best method known by. The applicant for carrying out said invention is the one that is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (33)

1. A plant or part thereof, genetically modified of the genus Cyamopsi s, characterized the plant or part of the plant because it comprises in its genome at least one recombinant DNA sequence.

2. A plant or part of the genetically modified plant, according to claim 1, characterized in that it is of the species C. tetragonol oba.

3 . The seeds, seedlings or parts of the plants, characterized in that they are obtained from a plant in accordance with claim 1 or 2.

4. A method to produce a genetically modified plant or part of the same genus Cyamopsi s, characterized the method because it comprises the steps of introducing a recombinant DNA sequence into at least one cell or protoplast, and the generation of genetically modified explants, using at least one selection or shoot growth means comprising at least one compound selected from an auxin inhibitor, a β-lactamase inhibitor and an ethylene inhibitor, to obtain a genetically modified plant or part thereof, which contains in its genome at least one recombinant DNA sequence.

5. A method according to claim 4, characterized in that the genetically modified shoots are generated using a selection means comprising at least one auxin inhibitor and a β-lactamase inhibitor, and a shoot growth medium comprising at least one a β-lactamase inhibitor.

6. A method according to claim 5, characterized in that the selection means comprises an auxin inhibitor, a ß-lactamase inhibitor and an ethylene inhibitor, and the shoot growth medium comprises an auxin inhibitor and an ethylene inhibitor.

7. A method according to any of claims 4 to 6, characterized in that the auxin inhibitor is 2- (p-chlorophenoxy) -2-methylpropionic acid (PCIB) and the β-lactamase inhibitor is sulbactam.

8. A method according to any of claims 4 to 7, characterized in that the means of selection comprises PCIB as the inhibitor of auxin at a concentration of 0.01-10 mg / l and the sulbactam as the inhibitor of the β-lactamase in a concentration of 10-1000 mg / l, and the shoot growth medium comprises sulbactam as the β-lactamase inhibitor, in a concentration of 10-1000 mg / l.

9. A method according to claim 8, characterized in that the selection means comprises PCIB in a concentration of 0.05-5 mg / l, for example 0.1-2 mg / l.

10. A method according to claim 8 or 9, characterized in that the selection medium and / or the shoot growth medium comprises sulbactam in a concentration of 20-500 mg / l, for example 50-200 mg / l.

11. A method according to any of claims 4 to 10, characterized in that the selection means further comprises kanamycin.

12. A method according to claim 11, characterized in that the selection medium comprises kanamycin sulfate in a concentration of 50-300 mg / l, typically 100-200 mg / l, for example 130-160 mg / l.

13. A method according to claim 12, characterized in that the explants are transferred from the selection means to a second selection means comprising kanamycin sulfate, at a concentration lower than that of the first selection means, preferably not greater than 125 mg / l, typically 20-100 mg / l, for example 30-70 mg / l.

14. A method according to any of claims 4 to 13, characterized in that the shoot selection and / or growth medium comprises silver thiosulfate as the ethylene inhibitor at a concentration of up to 50 μm.

15. A method according to claim 14, characterized in that the shoot selection and / or growth medium comprises silver thiosulfate. in a concentration of 0.1-10 μm, for example 0.5-5 μm.

16. A method according to any of claims 4 to 15, characterized in that the shoot selection and / or growth medium further comprises a nickel salt.

17. A method according to any of claims 4 to 16, characterized in that the means for selecting and / or growing shoots also comprises a gibberellin.

18. A method according to any of claims 4 to 17, characterized in that the shoot selection and / or growth medium further comprises an antibiotic similar to penicillin, for example carbenicillin or cefotaxime.

19. A method according to any of claims 4 to 18, characterized in that the transformation is carried out on the cotyledons.

20. A method according to claim 19, characterized in that the cotyledons are from seeds that have been germinated for at least 4 days, typically at least 7 days, for example at least 10 days.

21. A method according to any of claims 4 to 20, characterized in that it is for the production of a genetically modified plant of the species C. tetragonoloja.

22. A method according to any of claims 4 to 21, characterized in that the genetically modified shoots are grafted onto established seedlings or rooted plants.

23. A method for the production of a genetically modified plant, characterized in that at least one means is used for the selection or growth of the cells, protoplasts, calluses or parts of the plant, comprising at least one substance that inhibits bacterial growth or that increases the effect of a bacterial inhibitor, without having any substantial effect on the regulation of plant growth or toxic to the plant.

24. A method according to claim 23, characterized in that the substance is a β-lactamase inhibitor.

25. A method according to claim 24, characterized in that the β-lactamase inhibitor is sulbactam.

26. A method according to claim 25, characterized in that the sulbactam is present in at least one selection or growth medium in a concentration of 10-1000 mg / l, typically 20-500 mg / l, for example 50-200 mg / l.

27. A method according to any of claims 24 to 26, characterized in that the β-lactamase inhibitor is used together with reduced amounts of antibiotics and in the presence of bacterial strains that produce β-lactamase.

28. A method according to any of claims 23 to 27, characterized in that the substance is used in connection with the gene transfer mediated by Agrobacterium.

29. A method according to any of claims 23 to 28, characterized in that the bacterial growth inhibitor is used for the production of a genetically modified plant of the Cyamopsi s species.

30. Chimeric plants capable of producing transgenic seeds, and characterized because they are obtained by grafting a genetically modified shoot, grown in vi tro on a non-in vi tro cultivated plant.

31. The chimeric transgenic plants, according to claim 30, characterized in that they belong to the Faba ceae family.

32. The chimeric transgenic plants, according to claim 31, characterized in that they belong to the Cyamopsi s family.

33. The chimeric transgenic plants, according to claim 32, characterized in that they belong to the species C. te tragonol oba.