MXPA01006531A - Plant transformation process - Google Patents

Abstract

A method of i(Agrobacterium)-mediated genetic transformation using seedlings has been found which is applicable to dicots and monocots capable of being transformed by i(Agrobacterium). The transformation method utilizes vacuum-infiltration to introduce the i(Agrobacterium) T-DNA carrying a gene of interest into the seedlings. Upon maturity, seeds collected from the infiltrated seedlings are germinated, and progeny carrying the transgene are selected. This transformation method produces progeny exhibiting stable inheritance of the transgene without the need for regeneration methods such as somatic embryogenesis or organogenesis.

Description

PROCEDURE FOR TRANSFORMING PLANTS BACKGROUND OF THE INVENTION The genetic transformation of higher plants promises to have a major impact on crop improvements, as well as many other areas of biotechnology. Genetic transformation can be used to produce transgenic plants that carry new genetic material stably integrated into the genome or to manipulate "design" crops with specific characteristics. Several methods of genetic transformation have been developed and applied to a recent number of plant species. However, the relationship of ease and success of genetic transformation methods varies widely among plant species [Muller, et al. 1987. "High meiotic stability of a foreign gene introduced into tobaceo by Agrobacierium-med'iaXed transformation," Mol Gen Gene207: 171-175; Gasser, C.S. and Fraley, R.T. 1989. "Genetically engineering plants for crop improvement," Science 244: 1293-1299; Umbeck, et al. 1989. "Inheritance and expression of genes for kanamyein and chloramphenicol resistance in transgenic cotton plants," Crop Science 29: 196-201; Gordon-Kamm, et al. 1990. "Transformation of maize cells and regeneration of fertile transgenic plants," Plant Cell 2: 603-618; Chabaud, et al. 1996. "Transformation of barrel medie (Medicago truncatula Gaertn.) By Agrobacterium tumefaciens and regeneration via somatic embryogenesis of transgenic plants with MtENOD12 nodulin promoter fused to the gus repórter gene, "Plant Cell f? ep15: 305-310; Kar, et al., 1996." Efficient transgenic plant regeneration through /.grobacíer/um-mediated transformation of chickpea (Cicer arietinum L.), "Plant Cell Rep 16: 32-37; Kim, JW and Minamikawa, T. 1996." Transformation and regeneration fo French bean plants by the particle bombardment process, "Plant Sci 117: 131-138; Trieu, AT and Harrison, MJ 1996. "Rapid transformation of Medicago truncatula: regeneration via shoot organogenesis," Plant Cell Rep 16: 6-11, Bean, et al., 1997. " A simple system for pea transformation, "Plant Cell Rep 16: 513-519; Cheng, et al., 1997." Genetic transformation of wheat mediated by Agrobacterium tumefaciens, "Plant Physiol 115: 971-980; Cheng, et al., 1997. "Expression and inheritance of foreign genes in transgenic peanut plants generated by Agrobacterium-mediated transformation," Plant Cell Rep 16: 541-544, and Tingay, et al., 1997. "Agrobacterium tumefaciens-med ated barley transformation," Plant J 11: 1369-1376.] The most common and widely used method of transformation of dicotyledonous plants uses a bacterium, Agrobacterium tumefaciens, to effect gene transfer Agrobacterium tumefaciens is a gram-negative plant pathogen that lives in the soil and infects the host plants and subsequently administer and integrate part of their genetic material into the plant genome.The transferred portion of DNA is called the T-DNA fragment, and additional genetic material can be added to the T-DNA. The additional genetic material will be integrated into the genome together with the T-DNA. In this way, Agrobacterium can be used to facilitate the transfer of new genes into the plant genome (Fraley, et al., 1983. "Expression of bacterial genes in plant cells," Proc Nati Acad Se 'USA 80: 4803-4807). Although the transformation with Agrobacterium has worked well for a number of model species such as tobacco and petunia, the method is subject to a number of limitations. Some plant species, including many monocotyledonous plant species, are not easily susceptible to infection by Agrobacterium (Potrykus, I. 1990. "Gene transfer to cereals: an assessment," Bio / Technology 8: 535-542). In these cases, alternative methods have been used, including particle bombardment and direct gene transfer within protoplasts via electroporation, microinjection or polyethylene glycol-mediated capture [Klein, et al. 1987. "High velocity microprojectiles for delivering nucleic acids into living cells," Nature 327: 70-73; McCabe, et al. 1988. "Stable transformation of soy bean (Clycine max) by particle acceleration," Bio / Technology 6: 923-926; Bommineni, et al. 1994. "Expression of GUS in somatic embryo cultures of black spruce after microprojectile bombardment," J. Exp Bot 45: 491-495; Christou, P. 1995. "Strategies for variety-independent genetic transformation of important cereals, legumes and woody species utilizing particle bombardment," Eupytica 85: 13-27; Kim and Minamikawa. 1996. Plant Sci 117: 131-138; Klein et al. 1998. "Stable genetic transformation in intact Nicotania cells by the particle bombardment process, "Proc Nati Acad Sci USA 85: 8502-8505." Regardless of the method of administration of the novel genetic material, it is necessary to regenerate complete fertile plants from the transformed cells. Transformed involves two processes: transformation of plant cells and then regeneration of those transformed cells into whole plants.In the majority of cases, a plant tissue expiator is incubated with Agrobacterium carrying a T-DNA containing a selection marker gene and a "gene of interest." A portion of the cells in the explant will be transformed, and most plants are regenerated from these cells via somatic embryogenesis or direct organogenesis.The transformants are selected by inclusion of the conditions of appropriate selection in the regeneration medium.The choice of the tissue explant gone depends on the plant species. Among those that have been successfully used are leaves, cotyledons, hypocotyledons, cotyledonary meristems, and embryos. Because neither transformation and regeneration are 100% effective, the opportunity to obtain a transformed plant depends on these two processes occurring consecutively in the same cell. In many cases, the production of transgenic plants is prevented due to the inability to regenerate plants from those tissues susceptible to transformation. For the species in which somatic embryogenesis It is a viable method of plant regeneration, there are other limitations. Plants regenerated via somatic embryogenesis may show significant somatic variation, altered ploidy, genotypic abnormalities, and poor fertility (Etean, et al., 1997. Plant Cell Rep 16: 513-519). While regeneration via direct organogenesis overcomes some of these problems, not all plants can regenerate in this way. Finally, although the transformation of many plant crops is possible, it is usually achieved in highly regenerable lines or cultivars, and the selected agriculturally important lines are usually not amenable to transformation. Therefore, the introduction of a desired characteristic within the selected lines has been limited to subsequent methods of traditional crossing following the transformation of parent lines. In order to develop a transgenic plant line that expresses a new characteristic, it is desirable to produce a large number of transgenic plants from which the line that best expresses itself can be selected. The requirement for a plant line number lies in the fact that the integration of the T-DNA fragment into the plant genome is a random event, and therefore, each transgenic plant will contain the new integrated gene within different sites of the plant. genome Due to this phenomenon called "position effect", the various transgenic lines will vary in the expression levels of the introduced gene (Ulian, et al, 1994. "Express on and inheritance pattern of two foreign genes in petunia." Theor Appl Genet 88 : 433-440). Therefore, it is desirable to produce a large number of transgenic lines in order to select from those that express the gene introduced at a higher level. The only plant that has successfully been transformed with a high degree of ease and efficiency is Arabidopsis thaliana, a model plant widely used for genetic and molecular analysis of plant development processes. A direct method of transformation has been developed for Arabidopsis thaliana, (Bechtold, et al, 1993. "In plant Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants", Comptes Renus de lAcademie des Sciences Series lll Sciences de la vie 316) . In this transformation process, the plant (1) grows to maturity, (2) it is immersed in a suspension of Agrobacterium cells, (3) it is kept under vacuum for a short period of time, and then (4) It allows you to produce seeds. A proportion of the progeny is transformed. Recent data suggest that the progenitor of the gametophyte, gametophyte, or fertilized embryos are the targets (Bechtold, N. and Pelletier, G. 1998. "In plant transformation of adult Arabidopsis thaliana plants by vacuum infiltration, "Methods Mol Biol 82: 259-266.) Although the Bechtold method has been tried in other species including Brassica napus and / 3efa vulgaris, these attempts have continuously been reported as unsuccessful ( Siemens, J. Scheiler, O. 1996. "Transgenic plants: genetic transformation-recent developments and the state of the art," Plant Tissue Culture and Biotechnology 2: 66-75).

Legume crops such as chicharros, soybean, bean, alfalfa, peanut, chickpea, geranium and clover have a wide economic importance to the entire width of the world. Legumes are an important source of proteins such as grains and legume crops for animals and as grain legumes for humans, for example, soy (Gi'ycine max) is a main source of protein in food for animals and humans, and soybean oil is the most widely used edible oil in the world. Productivity, and therefore the value of a wide range of legume crops can be increased by introducing characteristics such as disease resistance, herbicide resistance, insect resistance, reduced levels of tannins, and lignin (forage legumes) , and improved proteins and lipid quality, for example, the soy nematode cyst causes loss in the field of more than one trillion dollars in the United States per year. With the recent cloning of the gene for resistance to the nematode cyst of sugar beet and a gene for resistance to potato nematode cyst (Williamson, VM 1999. Curr Opin Plant Biol 2: 327-31), strategies have been exploited for genetic engineering of resistance in plants. Although there have been some attempts to introduce these characteristics into legume crops via genetic manipulation, current transformation methods involve tissue culture that is labor intensive and inefficient. In particular, in legumes with large grains of seed, such as horse mackerel and soybeans, provided much difficulty for the transformation, and tissues susceptible to transformation have proven difficult to regenerate. (Bingham et al, 1975"Breeding alfalfa which regenerates from callus tissue in culture, Crop Science 15: 719-721; Hinchee, et al, 1988." Production of transgenic soybean plants using Agrobacterium-med \ ated transfer DNA, "Bio / Technology 6: 915-922; Schroeder, et al, 1993. "Transformation and regeneration of two cultivars of pea (Pisum sativum L)" Plant Physiol 101: 751-757; Chabaud, et al, 1996. Plant Cell Rep 15: 305-310; Kar, et al, 1996. Plant Cell Rep 16: 32-37, Kim and Mlnamikawa, 1996. Plant Sci 117: 131-138, Trieu and Harrison, 1996. Plant Cell Rep 16: 6-11; , et al, 1997. Plant Cell Rep 16: 513-519; Cheng, et al., 1997. Plant Cell Rep 16: 541-544; and Dillen, et al, 1997. "Exploiting the presence of regeneration capacity in the Phaseolus gene. pool for Agrobacterium-meditate gene transfer to the common bean, Mededelingen-Faculteri-Landbou / vkundige-en-Toegepaste-Biologische-Wetenschappen-Unversiteit-Gent 62: 1397-1402). In an alternative method, it was shown that the cells within the soy meristem can be transformed by particle bombardment. However, this leads to chimeric plants with transformed sectors. Some of these sectors will eventually give rise to seeds, and the seeds will carry the transgene (McCabe, et al, 1988. Bio / Technology 6: 923-926; Chowrira, et al, 1995. "Electroporation-mediated gene transfer intact nodal meristems in plant: generating transgenic plants without in vitro tissue culture ", Molecular Biotechnology 3: 17-23, and Chowrira, et al, 1996." Transgenic grain legumes obtained by in plant electroporation-mediated gene transfer ", Molecular Biotechnology 5: 85-96) .While this procedure has enabled the production of transgenic soy, it is a very intensive work due to the numerous meristems that are needed to bomb for that they had a realistic probability to obtain any type of transgenic seed .. Medicago truncatula Gaertn. (barrel alfalfa) is a diploid alfalfa., autogamética, annual that grows as a legume of pasture in a number of regions throughout the world, including Mediterranean area, South Africa and Australia (Crawford, et al, 1989. "Breeding annual Medicago species for semiarid conditions in Southern Australia", Adv Agron 42: 399-437). In Australia, annual alfalfa is the main legume found on over 50 million hectares of agricultural land, and a variety of species and ecotypes have been developed. The first commercial crop of M. truncatula was shaded in 1938, and this species has been favored due to its ability to tolerate both soils with little rain and high amounts of lime (Crowford, et al, 1989, Adv Agron 42: 399- 437). Medicago truncatula has also emerged as a model legume for studies of the symbiosis of Rhizobium nitrogen fixer / legume and mycorrhizal arbuscular symbiosis [Cook, et al, 1995. "Transient induction of a peroxidase gene in Medicago truncatula precedes infection by Rhizobium melilotí ' Plant Cell 7: 43-55; van Buuren, et al. 1998. "Novel genes induced during an aruscular mycorrhizal (AM) symbiosis between M. truncatula and G. versiforme", MPMI 12: 171-181].

The attributes that make M. truncatula a model plant useful for molecular and genetic analysis include its small genome (4.5 times larger than Arabidopsis), the rapid life cycle and its relatively small physical size (Barker, et al, 1990. "Medicago truncatula, a model plant for studying the molecular genetics of Rhizobium egume symbiosis ", Plant Mol Biol Rep 8: 40-49). In addition, it can be transformed via Agrobacterium and regenerated via somatic embryogenesis or alternatively by direct organogenesis (Thomas, et al, 1992. "Genetic transformation of Medicago truncatula using Agrobacterium with genetically modified Ri and disarmed Ti plasmids," Plant Cell Rep 11: 1 13-117, Chabaud, et al, 1996. Plant Cell Rep 15: 305-310, Trieu and Harrison, 1996. Plant Cell Rep 16: 6-11, Hoffmann, et al, 1997. "A new Medicago truncatula line with superior in vitro regeneration, transformation, and symbiotic properties solated through cell culture selection, "Mol Plant-Microbe interact 10: 307-315). Although the transformation mediated by Agrobacterium with regeneration via somatic embryogenesis or direct organogenesis is a viable method, these methods are very intense tasks that are not very efficient, and in some cases, very slow. Although these methods may be suitable for the generation of a small number of transgenic plants, they can not be used to generate the large numbers of lines required for many genetic methods and high performance systems, such as T-DNA mutagenesis. or activation by marking. A method of transformation mediated by Agrobacterium has now been found where the shoots, instead of flowering plants or tissue explants, are used as the biological material submitted for exposure to Agrobacterium cells. In addition, following the maturation of the treated plants and the seed set, the transgenic plants are directly selected from a progeny population that represents several insertion events. This method of transformation of shoots provides high efficiency, low input labor and a large number of t ansgenic plants without all the problems associated with transformation of flowering plants or tissue explants and regeneration via somatic embryogenesis or direct organogenesis.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a map of the T-DNA from the binary vector pBI21-bar. Figure 2 (a) is a Southern blot of DNA digested with H / ndIII from transgenic plants (progeny of infiltrated plants) hybridized with a bar probe. Samples 1.3-1.8 are from treatment 1 (1-minute vacuum infiltration); samples 2.2-2.7 are from treatment 2 (1.15-vacuum infiltration mirutos) and samples 3.1-3.5 are from treatment 3 (40-seconds vacuum infiltration followed by 20-seconds maintenance). The plasmid DNA pB1121-bar is included to the left of the blot. It's part of the blot was cut and exposed for a while longer short that. the rest of the blots to prevent overexposure. C is the DNA from a non-transformed plant M. truncatula control. Figure 2 (b) is a Southern blot of Hind digested DNA from transgenic plants (progeny of infiltrated plants) hybridized with a bar probe. Samples 1.3-1.8 are from treatment 1 (1-minute vacuum infiltration); samples 2.2-2.7 are from treatment 2 (1.15-minutes of vacuum infiltration) and samples 3.1-3.5 are from treatment 3 (40-seconds vacuum infiltration followed by 20-seconds maintenance). The pBI121-bar plasmid DNA is included on the left. This part of the blot was cut and exposed for a shorter time than the rest of the blots to prevent overexposure. C is DNA from a non-transformed M. trunc & control plant. Figure 3 (a) is a Southern blot of DNA digested with Hind \\\ from transgenic plants (progeny of infiltrated plants) hybridized with an npt II probe. Samples 1.6-1.20 are from treatment 1 (1-minute vacuum infiltration) and samples 2.12 and 2.13 are from treatment 2 (1.15-minutes vacuum infiltration). C is DNA from an untransformed M. truncatula control plant. Figure 3 (b) is a Southern blot of Hind digested DNA from transgenic plants (progeny of infiltrated plants) hybridized with a bar probe. Samples 1.6-1.20 are from treatment 1 (1-minute vacuum infiltration) and samples 2.12 and 2.13 are from treatment 2 (1.15- minutes of vacuum infiltration). C is DNA from an untransformed M. truncatula control plant. Figure 4 is an agarose gel showing a portion of the bar gene that has been amplified from the DNA of transgenic soybean plants via PCR with specific primers for bar. The arrow points to an amplified fragment of 423 bp. The line marked "N" contains the molecular weight markers. The 500 bp marker is indicated. Samples 6-27 are soybean transformants that survived the herbicide treatment. Transformants 6, 13, 14, 15 and 16 show an amplified fragment of the correct size.

BRIEF DESCRIPTION OF THE INVENTION In one aspect, the present invention is a method for directing plant transformation using shoots and Agrobacterium comprising: (a) contacting at least one shoot with Agrobacterium cells that have a vector that enables Agrobacterium cells to transfer DNA -T that contains at least one gene or fragments of the gene to the shoots and (b) apply a vacuum to the shoots in contact with the Agrobacterium cells at a point of time, the vacuum being strong enough to force the cells of Agrobacterium towards intimate contact with the shoots in such a way that the Agrobacterium cells transfer the T-DNA to the cells of the shoots and at a second point, in time, where the first point in the time and the second point in time are either the same or different. In a preferred method, the vector comprises a selection marker gene. A preferred selection marker gene is a gene for herbicide resistance. A preferred herbicide resistance gene is a bar gene. In another aspect, the present invention is a method for directing plant transformation using shoots and Agrobacterium comprising: (a) contacting at least one shoot with a mixture of Agrobacierium cells, the mixture comprising cells from a strain of Agrobacterium containing a vector with an initial DNA fragment and cells from the Agrobacterium strain that contains the vector with a second DNA fragment, where the vector enables the Agrobacterium cells to transfer the T-DNA to the cells of the outbreak and (b) applying a vacuum to the shoots in contact with the mixture of Agrobacterium cells at a first point in time, the vacuum being strong enough to force the Agrobacterium cells into intimate contact with the shoot in such a way that Agrobacterium cells transfer at least one gene to the bud cells at a second point in time, where the first point in time and the second point in time are the same or different. In a preferred method, the vector comprises a selection marker gene. A preferred selection marker gene is a gene for herbicide resistance. A preferred herbicide resistance gene is a bar gene. In another aspect, the present invention is a method for directing plant transformation using shoots and Agrobacterium comprising: (a) contacting at least one outbreak with Agrobacterium cells that have a vector that enables Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragments to the outbreaks and (b) apply a vacuum to the outbreaks in contact with the Agrobacterium cells at a point of time, the vacuum being strong enough to force the Agrobacterium cells into intimate contact with the shoots in such a way that the Agrobacterium cells transfer the T-DNA to the bud cells and at a second point, in time, where the first point in time and the second point in time are either the same or different; (c) allow the transformed shoots to grow to maturity and produce seeds; (c) germinate the seeds to form progeny; (e) exposing the progeny to an agent that enables the detection of the expression of a selection marker gene; and (f) selecting for the progeny expressing the selection marker gene and at least one gene, wherein the expression of the selection marker gene and at least one gene indicates the gene transfer. In a preferred method, the selection marker gene is a herbicide resistance gene. A preferred herbicide resistance gene is the bar gene. In still another aspect, the present invention is a plant transformed in accordance with the above-described methods of shoot transformation. In still another aspect, the present invention is a seed from a transformed plant in accordance with the methods described above for shoot transformation.

In still another aspect, the present invention is a plant progeny from a seed obtained from a transformed plant in accordance with the methods described above for transformation of shoots.

DETAILED DESCRIPTION OF THE INVENTION A plant transformation process that has now been found uses spike vacuum infiltration to introduce Agrobacterium T-DNA carrying a selection marker gene and the gene (s) of interest within the outbreaks. An outbreak as used herein is defined as a plant from about the beginning of seed germination to around the time it develops true leaves. The transformation methods described here can be applied to shoots of any plant, including dicotyledons and monocots that have been successfully transformed by Agrobacterium-mediated gene transference. In particular, legume plants are transformed at high efficiency ratios. The transformation method described herein is generally achieved by growing the Agrobacterium strain carrying a gene (s) of interest under selection conditions in liquid culture until it reaches its exponential growth phase. The Agrobacterium cells are then concentrated by centrifugation and resuspended in a vacuum infiltration medium. The shoots are immersed in the cell suspension of Agrobacterium and are subjected to vacuum infiltration where the Agrobacterium cells are introduced into the shoots, resulting in infiltrated plants that subsequently produce transformed seeds from which a transformed plant is obtained. The transformation of shoots is achieved through gene transfer mediated by Agrobacterium. The Agrobacterium strains useful in the transformation of the outbreak include any aggressive strain which, in contact with a transformable plant cell, is capable of transferring T-DNA into the cell for integration into the plant genome. In the transformation method described herein, the Agrobacterium strain can carry a plasmid with multiple genes of interest. Alternatively, the transformation is carried out using a mixture of Agrobacterium cells in which the vector carries different DNA fragments, for example, fragments selected from a specific DNA library. To achieve the optimum transformation elation in a given plant, the strain of Agrobacterium is selected that provides the largest number of transformed shoots. For legume plants, the strains of Agrobacterium tumefaciens EHA105, ASE1, and Gv3101 are preferably used. The gene (s) of interest can be transformed into Agrobacterium by any means known in the art. For example, a DNA fragment modified to contain the gene (s) of interest can be inserted into the T-DNA of a Ti plasmid of Agrobacterium which also contains genes required to generate the transformed state.

Other modifications of the Agrobacterium T-DNA plasmid can be born to assist in the transformation process. For example, to distinguish shoots that have been successfully transformed, a selection marker gene can be incorporated into the Agrobacxerium T-DNA plasmid. Selection marker genes useful in the transformation methods described herein include any selectable marker gene that can be incorporated into the T-DNA of Agrobacterium and that its subsequent expression can distinguish transformed from non-transformed progeny. Exemplary selection markers include a neomycin transfer gene or a phosphinotricinyl transferase (ba) gene. For example, a preferable selection marker is the bar gene encoding phosphinothricin acetyl transferase that confers resistance to phosphinothricin-based herbicides. Preferably, the selection marker gene and the gene (s) of interest are incorporated into any suitable vector for use with strains of. Agrobacterium transformation. For example, the binary vector, vector pB1121 (Clontech, Palo Alto, CA) can be modified where a copy of the phosphinotricin-acetyl transferase (bar) gene is inserted, under the control of the 35S promoter and the 3 'sequences of octopinsynthase, within the site Hind \\\ of T-DNA. The bar gene encodes phosphinotricin acetyl transferase which confers resistance to phosphinothricin-based herbicides, such as Ignite® (AgroEvc, Frankfurt, Germany). This selection marker enables the easy selection of transformed plants: after spraying the plants with a herbicide containing phosphinothricin (PPT), only the plants Transforms containing the bar gene survive the exposure of the herbicide. In the process of transformation of shoots, the initial seeds can be pretreated to optimize their germination and to prepare the resulting shoots for the transformation. The sterilization of the surface of the seeds is preferred to remove any microorganism that interferes or that can infect the seed in germination. Any means of sterilization which does not deleteriously affect the seeds can be used. Exemplary methods include the use of 20-30% sodium hypochlorite or aqueous 70% ethanol. Preferably, the seeds are sterilized in a solution of 30% sodium hypochlorite and 0.1% Tween 20 for approximately 5 minutes and then extensively rinsed to remove the sterilization solution. Preferably, doubly sterile or deionized water is used, or water with reduced oxidizable carbon following reverse osmosis, ion exchange and / or treatment with activated carbon to rinse the seeds. Some seeds, for example M. truncatula, experience a prolonged latency period that results in delayed germination. These seeds can be treated by a scarification process capable of breaking dormancy. For example, it can be used by breaking or scraping the seed coat, soaking the seed to soften the seed coat, or a controlled acid treatment. Preferably a treatment with concentrated sulfuric acid is used for approximately 10 minutes followed by extensive rinsing to remove the acid. Preferably, double water is used sterilized distilled or deionized, or oxidized reduced carbon water followed by reverse osmosis, ion exchange and / or treatment with activated carbon to rinse the seeds. After pretreatment, the seeds are placed on a medium capable of maintaining the germination and the subsequent growth of the shoots. For example, seeds can be placed on the surface of sterile filter paper or paper towels. Preferably, the seeds are spread on the agar surface in sterile, firm water, in petri dishes. The seeds are then placed under environmental conditions capable of inducing germination and maintaining the development of the seeds. Vemalization may be preferred for certain plants such as M. truncatulal to promote early flowering. Incubation of the resulting shoots is continued until the shoots reach an appropriate stage of development for vacuum infiltration. The optimum age of the shoots for vacuum infiltration varies for different plants. In general, the shoots in which the roots have emerged and grow to at least about 1 cm are sufficiently mature. However, since the plants develop at different speeds, vacuum infiltration can be optimized for specific plants by selecting the shoots at various stages of development using the methods here and determining the state at which the transformation efficiency is maximal. The incubation time and temperature can also be adjusted to provide optimal conditions for a specific variety. For example, approximately 15 days after the seeds have been placed on medium germination, the shoots of M. truncatula and soy are enough? ripe for vacuum infiltration. A few days before the vacuum infiltration, the transformed Agrobacterium is subcultured on a medium of general growth in a dish preferably containing appropriate antibiotics to distinguish transformed Agrobacierium cells. For example, Agrobacterium tumefaciens EHA105 and Gv1301 carrying the bar gene are preferably cultured on YEP medium as defined in example 1 containing rifampicin (20 mg / l) and canamine (50 mg / l). Agrobacterium cultures are grown at around 28 ° C for about 2-3 days. One day before vacuum infiltration, an Agrobacterium culture is prepared by aseptically transferring an appropriate inoculum into general growth medium suitable for Agrobacterium growth. TY liquid medium and YEP liquid medium containing appropriate antibiotics to select transformed Agrobacterium are preferred for Agrobacterium EHA105 and Gv1301. Liquid cultures are grown under conditions that provide Agrobacterium to reach exponential growth. Preferably, the liquid culture is incubated at about 28 ° C in a shaking incubator at about 250 rpm overnight. It is essential to use fresh Agrobacterium to achieve the transformation. To provide optimal conditions for transformation, vacuum infiltration is preferably carried out using the liquid culture of Agrobacierium transformed into exponential growth phase (OD6oo = 1 -6). The Agrobacterium cells in the liquid culture are concentrated by centrifugation and resuspended in two volumes of a vacuum infiltration medium (for example, the Agrobacterium cells grown in 15 ml of liquid culture are concentrated by centrifugation, and then resuspended in 30 ml of vacuum infiltration medium). Any medium of plant growth capable of maintaining the infiltration process and the Agrobacterium inside the plant while being compatible with the growth of the plant can be used as a vacuum infiltration medium. More preferably, the vacuum infiltration medium comprises ketosyringone which induces the Agrobacierium genes. For legume plants, the vacuum infiltration medium defined in Examples 1 and 2 is preferably used. To carry out the vacuum infiltration, the shoots are removed from the germination / incubation medium and placed in any clean container capable of maintaining several shoots as well as a volume of vacuum infiltration medium that partially covers the shoots. Petri dishes are useful for this purpose, using about 3-40 shoots per box. The Agrobacterium suspension in the vacuum infiltration medium is added to the container to moisten and partially cover the shoots. For a standard petri dish, approximately 10 ml of the suspension is sufficient. The petri dish containing the suspension shoots of Agrobacterium is placed in a vacuum chamber. The preferred amount of vacuum to be used in the transformation process is the minimum amount necessary to force Agrobacterium towards the apoplastic spaces of the shoots. Approximately the Agrobacterium was sufficient for the transformation of M. truncatula the time and manner in which the vacuum is applied to the shoots depends on the plant and has to be determined empirically. The vacuum can be applied and then released. Alternatively, the vacuum can be applied, released, reapplied and then released again. The duration of the vacuum may vary from about 0.1 to about 5 minutes more preferably from 0.5 to about 2 minutes, and more preferably from about 1 minute. For M. truncatula the plants are kept under vacuum for 0.5 minutes and for 2 minutes to give rise to transgenic plants. But the plants are kept under vacuum for 1 minute for maximum transformation efficiency. Following the vacuum infiltration, the Agrobacterium suspension is decanted, and the shoots are placed on sterile filter paper or blot paper. The shoots can then be planted within a mixture of complete earth that will allow the complete growth and development of the plant and the production of seed. The shoots are then allowed to mature and seeds are established. Preferably, the plants are kept in humidity, temperature, photoperiod duration, and light spectrum that favor the growth of the plant. To increase the viability of the transformed plants and to improve the transformation efficiency, the shoots are optionally incubated on a co-cultivation medium for 2-3 days before being planted in a complete soil mixture. Any one can be used middle of. cocultivation that maintains the growth of the outbreaks. For legume plants, the co-cultivation medium given in Examples 1 and 2 is preferred. The plants are then allowed to develop to maturity and produce seeds. A portion of the seeds will carry the transgene in their genomes. The seeds are germinated and the resulting progeny exhibiting stable heritability of the transgene is selected. Various methods known in the art can be used to distinguish progeny exhibiting stable heritability of the transgene. For transgenic plants where the gene (s) of interest results in a visible phenotypic change, selection may be based on visual examination of the progeny. For plant transformations involving plasmids carrying Agrobacterium containing a selection marker gene, the appropriate selection agent may be applied to the plants to select the transformants. Optionally, Southern blot analysis or PCR analysis can be used to verify the presence of the transferred gene within the genome of the transformed plants. The shoot transformation processes of the present invention are further illustrated in detail in the examples provided below. While these examples describe the invention, it is understood that modifications to methods for optimizing transformation to a specific plant are also within the skill of one skilled in the art, and such modifications are considered to be within the scope of the invention.

EXAMPLE 1 Transformation of M. truncatula by infiltration to shoot vacuum.

The M. truncatula shoots were transformed to incorporate the bar gene and the nptll gene into the plant genome using the transformation process of the present invention.

Preliminary Before transformation, a modified version of the binary vector, vector pBI121 (Clontech, Palo Alto, CA) was made by inserting a copy of a phosphinotricin-acetyl transferase (bar) gene, under the control of a 35S promoter and a sequence 3 'octopinsynthase within the Hind site of the T-DNA to create a plasmid called pBI121-¿> ar (figure 1). The construction was confirmed by restriction analysis and PCR analysis, and then transformed into a strain of Agrobacterium tumefaciens EHA105 (Hood, 1993: "New Agrobacterium helper plasmids for gene transfer to plants," Trans Res 2: 208 -218). Additional constructions in Agrobacterium were also obtained as given in Table 3. While the following procedure was presented for pBII21-bar in the Agrobacterium strain EHA105, the same procedure was followed for the other constructions with the exception that the Growth medium was supplemented with specific antibiotics needed to maintain the plasmid.

Day 1: The M. truncatula seed was sterilized and germinated as follows. The seeds were soaked in concentrated H2SO4 for approximately 10 minutes. The acid was removed, and the seeds were rinsed extensively in sterile double distilled cold water. This treatment was used to break the latency of M. truncatula. The seeds were then sterilized on the surface by soaking the seeds in a sterile solution such as Clorox / 30% Tween 20 0.1% solution for approximately 5 minutes with gentle agitation. The seeds were rinsed extensively with cold sterile double distilled water. The seeds were then spread on an agar in firm water (eg, 0.8%) (Sigma C hemical Co. St. Louis, MO) in a petri dish. The petri dishes with agar in water containing the seeds were covered with aluminum foil and kept at 4 ° C for 15 days. This step of vernalization was used to promote the early flowering of M. truncatula.

Around day 12: Agrobacterium tumefaciens EHA 105 carrying pBII121-bar was subcultured for isolation on a fresh agar plate containing YEP medium [1 liter: 10 g Bacto-peptone (Difco, Detroit, Ml); 10 g of yeast extract; 5 g NaCl and 15 g Bacto-agar (Difco, Detroit, Ml) pH = 6.8 unadjusted] containing rifampicin (20 mg / l) and kanamycin (50 mg / l), and the subculture was incubated at approximately 28 ° C for about 2-3 days.

Around day 14: One loop, or about 3 large colonies of the Agrobacterium subculture were inoculated into about 15 ml of liquid medium TY [1 liter: 5 g tryptone, 3 g yeast extract, 0.88 g CaCl2-2H2O at pH = 7] which contains rifampicin (20 mg / l) and kanamycin (50 mg / l) and incubated on a shaker at 28 ° C at 250 rpm throughout the night.

Around day-15: The liquid culture of Agrobacterium was grown until it reached an exponential phase (OD6oo 1 -6). The Agrobacterium cells were concentrated by centrifugation and resuspended in 30 ml of vacuum infiltration medium (VIM) [1 liter: 10 ml PDM saline at 100 X concentration (400 ml at 100X: 100 g KNO3; 12 g NH4H2PO4, 16 g MgSO4-7H2O, 0.4 g MnSO4 20, 0.2 g H3BO3, 0.008 g CuSO4-5H20, 0.04 g Kl, 0.004 g CoCl2-6H2O, 0.04 g ZnSO4-7H2O, and 0.004 g Na2MoO4-2H20, filtered, sterilized and stored at room temperature); 10 ml of iron PDM and vitamins (1 liter at 100X: 0.5 g nicotinic acid, 0.05 g pyridoxine-HCl, 0.5 g thiamine HCl, 100 g, myo-inositol, 1.5 g FeSO4-7H2O, and 2g Na2EDTA, filtered sterilized, stored for immediate use at 4 ° C, and for long-term storage at -20 ° C); 0.2 g CaCl2? 20; 1.5 ml of 10 mM benzylaminopurine (BAP, 0.0565 g in 0.15 ml 2N NaOH and 24.85 ml double distilled H2O stored at 4 ° C); 0.05 ml of 10 mM alpha-naphthaleneacetic acid (NA, 0.0465 g in 3 ml of 95% ethanol and 22 ml of 70% ethanol stored at 4 ° C); 10 g sucrose; Y 0. 1 ml of; acetosyringone (AS; 1 M in DMSO stored at -20 ° C), where the PDM salts, the PDM iron and the vitamins, CaCl? 2O and the sucrose are combined, the pH was adjusted to 5.8 with KOH and put into autoclave in liquid cycle for 20 minutes, and when the medium is cooled to 50 ° C, BAP, NAA and AS are added.] The shoots are removed from the agar plates in water and placed in a clean petri dish standard to approximately 30-40 shoots of M. truncatula per petri dish. Approximately 10 ml of the Agrobacterium suspension was added in the vacuum infiltration medium to the petri dish at a volume sufficient to moisten and partially cover the shoots. The petri dishes containing the shoots moistened with Agrobacterium suspension were placed in a vacuum chamber. Three methods of vacuum infiltration were tested. In treatment 1, a vacuum of 28 mmHg was applied for approximately one minute, rapidly released, reapplied at 28 mmHg for approximately 1 minute and finally released quickly. In treatment 2, vacuum was applied at 28 mmHg for approximately 1.5 minutes, released quickly, reapplied at 28mmHg for approximately 1.5 minutes, and finally released quickly. In Treatment 3, vacuum was applied at 28 mmHg for approximately 40 seconds, maintained for 20 seconds, and finally released quickly. For all treatments, the shoots were then dried on sterile filter paper or blot paper and spread on petri dishes containing co-culture medium (CM) [1 liter: 10 ml PDM saline at 10X concentration, 10 ml of iron PDM and vitamin 0.2 g CaCl2-H2O; 10 g sucrose 7.5 g agar-agar (Sigma Chemical Co., St. Louis, MO); and 0.1 ml AS, where the PDM salts, the PDM iron and vitamins, CaCl2? 2O, agar-agar and sucrose are combined, the pH adjusted to 5.8 with KOH and autoclaved in liquid cycle for 20 minutes, and when the medium is cooled to 50 ° C, AS is added]. The shoots were incubated in a growth chamber under the conditions given in Table 1 for approximately 2-3 days.

TABLE 1 Conditions of the growth chamber Around day 17: The shoots were washed twice with H2O and then planted in pots in Metromix 200 soil mixture. To allow the plants to slowly adjust to the humidity of the environment the following procedure was followed: the pots that contained the buds were initially covered with a plastic cover; After a week, the cover was opened and after a couple of days, the cover was completely removed. The plants were allowed to mature under conditions which are suitable for optimum plant growth, ie at 22-25 ° with days of eighteen hours.

Around day 40: The plants started to flower approximately 24 days after being sown on land. The resulting seeds were collected and germinated under optimal conditions for germination, that is, a short cold treatment for 4 days on a soaked filter paper, left at room temperature for 1-2 days, and then planted on land. When the shoots had a few leaves (approximately 15 days old), they were sprayed with 80 mg / L of PPT (~ 1/7000 dilution of 600 mg / ml solution stored at -20 ° C), and the results are presented then. In the study, 120 shoots of M. truncatula were infiltrated under vacuum with Agrobacterium tumefaciens strain EHA105 carrying the plasmid pB1121-bar as described above. Three different treatments were used, and forty outbreaks per treatment were infiltrated. The treatment varied only in the length of the vacuum treatment time. The infiltrated plants were allowed to mature and produce seeds. The shoot progeny was sprinkled with Ignite (PPT) and the resistant shoots were further analyzed for the presence of the bar, nptll and B-glucuronidase (GUS) genes. The results are shown in table II. Figures 2A, 2B, 3A, and 3B present the data obtained, in the experiments T-84-1, T84-2, and T84-3 of Table II. These Results show the transformation efficiency in a range of 2.9% to 27.6% for the various transformation experiments.

TABLE II: Transformants resulting from the transformation of M. truncatula via shoot infiltration with Agrobacterium a The following treatment methods were used: T84-1, T87-1, T87-2, T87-3, T87-4, T87-7, and T88, infiltration for one minute (2X); T84-2, infiltration for 1.5 minutes (2X); T84-3, infiltration for 40 seconds and maintenance for 20 seconds. b The following strains of Agrobacterium tumefaciens and binary vectors were used in these experiments: A. tumefaciens strain ASE1 carrying the binary rector PSLJ525 (Jones et al., 1992. "Effective vectors for transformation, expression of heterologous genes, and assaying transposon excision in transgenic plants, "Trans. Res. 1: 285-297) or pSKI006 (http: // w \? w.salk.edu/LABS/pbio-w/). A. tumefaciens strain EHA105, which carried pBI121-bar or PKYLX7-Gus (Franklin et al., 1993"Genetic tranformation of green bean callus via Agrobacterium mediated DNA transfer," Plant Cell Rep 12: 74-79), or pBINmgfp-ER- bar, or pGA482-¿> ar. A. tumefaciens strain Gv3101 carrying pSKI015 (Kardailsky et al., 1999. "A pair of related genes with antagonistic roles in floral induction," Science 286, 1962-1965). The addition of the bar gene to a number of vectors was achieved as follows. A HindWMHpal fragment containing the 35S-bar-OCS 3 'sequence cassette was excised from pSLJ525 and inserted between the HindlW and Hpal sites of pGA482 to generate pGA482-j ar. The same H / 'nc.lll / Hpal fragment was used to produce pB1121-bar and pBINmg / p- £ f? -bar. For each of these vectors, the Hpal site was converted to a Hind site by the addition of a Hpal-Hind adapter and then the fragment was inserted into the site of pBI121 or Pbinmgfp-ER (Haseloff et al. 1997"Removal of a cryptic intron and subcellulose localization of green fluorescent protein are required to mark transgenic Arabidopsis plants. Brightly," Proc. Nati. Acad. Sci. USA 94: 2122-21271997) to create pBI121-¿> ary pBI f-imgfp-er-bar respectively. c ND = not determined.

Plants transformed from treatments T84-1, T84-2, and T84-3 were also analyzed by Southern blot analysis to demonstrate the presence of the transgene within the plant. The DNA was isolated from 22 transgenic plants (12 plants from the T84-1 treatment, 6 plants from the T84-2 treatment, and 4 plants from the T84-3 treatment) and digested with the restriction enzyme. Hind \\. The digested DNA was separated by electrophoresis and adhered to nylon membranes. The membranes were assayed with an internal DNA fragment (450 bp) of the bar gene labeled with 32 P-dATP. All the transformed plants containing the DNA fragments hybridized to the bar probe indicating that this gene is integrated into the genome (Fig. 2A and 2B). The plasmid pBI121-j ar DNA was included in the biol: as a positive control, and a DNA sample was included from non-transformed M. truncatula plants as a negative control. As expected, the hybrid bar probe with the plasmid DNA but not the DNA from the untransformed M. truncatula plant (Fig. 2A and 2B). The expected size of the hybridization fragment from the plasmid is 1.6 kb; however, all the fragments were larger, probably due to a rearrangement towards the left end. The blots were cut into strips and retested with an internal fragment of the npill gene (766 bp) labeled with 32P-dATP. The npfll is carried in plasmid pB1121-bar between the right border of the T-DNA and the bar gene (Fig. 1). All transformed plants containing the DNA fragment that hybridize with the rjpfll probe indicate that this gene is also integrated into the genome. This combination of digestion and probe provides a right edge analysis and demonstrates the presence of independent transformants. For example, the unique bar hybrid fragments shown with transformants 1.6, 1.10, 1.11, 1.14, 1.13, and 2.12 provide evidence that these are independent transformants. Again, the untransformed control plants do not contain DNA capable of hybridizing to this probe (Fig. 3A and 3B). The T-DNA pB1121 -bar also contains a copy of the GUS gene between the bar gene and the left border (Fig. 1); however, this gene could not be detected in the transformed plants. The loss of genes located between the selection marker and the left border has been previously reported; thus, the loss of the GUS gene in the transformed plants confirms these findings. These results were consistent with the Southern bar analysis and offered an explanation of the more than expected bar hybridization fragment. Thus, it was shown that the GUS gene or any gene of interest must be inserted into the plasmid between the bar gene and the right border (at the location of the npfll gene) to ensure integration. Seeds from transgenic plants were collected and germinated, and the resulting shoots were sprayed with PPT herbicide. The progeny of the transgenic plants were also highly resistant to PPT, indicating that the transgenes are stable and inheritable by the next generation. As shown in Table III, we obtained data showing that transgenes were heritable in a Mendelian manner stable. The results show that the lines can propagate past generation T1.

TABLE III: Analysis of segregation (resistance to phosphinotricin) of the progeny from a selection of transformants prepared by infiltration of shoots. a "_ = p value of> 0.05; * = value of> 0.01; - = p value of <value of 0.01.

EXAMPLE 2: Transformation of soybean by infiltration to the vacuum of shoots The bean sprouts were transformed by incorporating the bar gene into the plant genome using the transformation process of the present invention.

Day 1: The seeds were sterilized on the surface by immersing the seeds in 20% sodium hypochlorite for approximately 5 minutes with gentle agitation. The seeds were rinsed eight times in sterile double distilled water. The seeds were placed in a large volume of water and allowed to soak at room temperature for 3-12 hours. The seeds were then spread on an agar in solid water (eg, 0.8%) (Sigma Chemical Co., St. Louis, MO) in a petri dish. The petri dishes with agar in water containing the seeds were covered with aluminum foil and kept at 18 to 20 ° C for 15 days.

Around day 12: Agrobacterium tumefaciens Gv3101 carrying the vector SK1015 with a copy of the bar gene was subcultured for isolation on a fresh agar plate containing YEP medium [1 liter: 10 g Bacto-peptone (Difco, Detroit, Ml); 10 g of yeast extract; 5 g NaCl and 15 g Bacto-agar (Difco, Detroit, Ml) pH = 6.8 unadjusted] containing rifampin (10 mg / l) and kanamycin (50 mg / l), carbenicillin (50 mg / ml) and subculture it was incubated at approximately 28 ° C for about 2-3 days.

Around day 14: One loop, or about 3 large colonies of the Agrobacterium subculture were inoculated into about 15 ml of liquid medium TY [1 liter: 5 g tryptone, 3 g yeast extract, 0.88 g CaCl2-2H2O at pH = 7] which contains rifampicin (20 mg / l), kanamycin (50 mg / l), carbenicillin (50 mg / ml) and gentamicin (20 mg / ml) and were incubated on a shaker at 28 ° C at 250 rpm overnight.

Around day 15: The liquid culture of Agrobacterium was grown until it reached an exponential phase (OD600 1.6). The Agrobacterium cells were concentrated by centrifugation and resuspended in 30 ml of vacuum infiltration medium (VIM) [1 liter: 10 ml PDM saline at a concentration 10 X; 10 ml of iron PDM and vitamins; 0.2 g CaCI H2O; 1.5 ml of 10 mM benzylaminopurine (BAP, 0.0565 g in 0.15 ml 2N NaOH and 24.85 ml of double distilled H2O stored at 4 ° C); 0.05 ml of 10 mM alpha-naphthaleneacetic acid (NA, 0.0465 g in 3 ml of 95% ethanol and 22 ml of 70% ethanol stored at 4 ° C); 10 g sucrose; and 0.1 ml acetosyringone (AS; 1 M in DMSO stored at -20 ° C), where the PDM salts, the PDM iron and the vitamins, CaCl H2O and the sucrose are combined, the pH was adjusted to 5.8 with KOH and it was autoclaved in liquid cycle for 20 minutes, and when the medium is cooled to 50 ° C, BAP, NAA and AS are added.] The shoots were removed from the agar plates in water and placed in a box of standard clean petri to approximately 10-20 shoots of M. truncatula per petri dish. Approximately 20 ml of the Agrobacterium suspension was added in the vacuum infiltration medium to the Petri dish at a sufficient volume to moisten and partially cover the shoots. The petri dishes containing the shoots moistened with Agrobacterium suspension were placed in a vacuum chamber. Vacuum was applied at 28 mmHg for approximately 2 minutes, released quickly, reapplied at 28 mmHg for approximately 2 minutes and finally released quickly. The shoots were then dried on sterile filter paper or blot paper and spread on petri dishes containing coculture medium (CM) [1 liter: 10 ml PDM saline solution at 10X concentration, 10 ml iron PDM and vitamins 0.2 g CaCl2 H2?; 10 g sucrose 7.5 g agar-agar (Sigma Chemical Co., St. Louis, MO); and 0.1 ml AS, where the PDM salts, the PDM iron and vitamins, CaCl2? 2O, agar-agar and sucrose are combined, the pH adjusted to 5.8 with KOH and autoclaved in liquid cycle for 20 minutes, and when the medium is cooled to 50 ° C, AS is added]. The shoots were incubated in a growth chamber under the conditions given in Table IV for approximately 6-7 days until Agrobacterium can be seen growing around the shoots on the medium.

TABLE IV Conditions of the growth chamber Around day 17: The shoots were washed twice with H2O and then planted in pots in Metromix 200 soil mixture. To allow the plants to adjust slowly to the humidity of the environment the following procedure was followed: the pots containing the buds were initially covered with a plastic cover; After a week, the cover was opened and after a couple of days, the cover was completely removed. The plants were allowed to mature under conditions that are suitable for plant growth in a greenhouse.

Around day 40: The plants began to bloom, and the resulting seeds were collected and germinated by soaking them in water for three hours followed by immediate planting on land. When the shoots had a leaf, they were sprayed with 100 ml / L of PPT (~ 1/6000 dilution of 600 mg / ml solution in 0.1% of Tween 20 stored at -20 ° C), and the results are presented below . In this study, 30 soybean shoots were infiltrated by vacuum with Agrobacterium tumefaciens strain Gv3101 carrying the plasmid SK1015- £ > ar as described above. Fourteen of the thirty infiltrated soybeans survived, were transplanted into pots, and allowed to mature and produce seeds. Approximately 700 seeds of the progeny were collected, germinated and grown in vermiculite. After the buds developed at least one true leaf, these were sprayed with PPT herbicide. The PCR analysis was carried out to confirm that the plants that survived the PPT herbicide carried the bar gene in their genome. The DNA was extracted from each surviving plant and used as a template in a PCR reaction with specific primers for bar. A fragment of the expected size was amplified from DNA samples from at least eleven of the transformed plants, indicating that these plants had been transformed. The frequency of transformation was around 1.57%. In summary, the outbreak transformation process described here is more efficient and less laborious than the methods reported previously. In addition, somatic alterations are avoided, and the direct introduction of genetic material within the selected lines is possible. Large quantities of transgenic plants representing diverse integration events can be generated very rapidly and efficiently, and the transgenes are stable and inheritable by subsequent generation. The greatest difficulty with the regeneration of cells transformed by Agrobacterium through tissue culture is avoided in the transformation process of the present invention, making it useful for legumes such as soybean, bean and peas for which the subsequent generation of transformed cells with Agrobacterium is problematic.

Claims (41)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for direct plant transformation using legume shoots and Agrobacterium comprising: (a) contacting at least one legume shoot with Agrobacterium cells, said Agrobacterium cells contain a vector, said vector enables said d3 Agrobacterium cells to transferring T-DNA containing at least one gene or fragment of the gene to said outbreak; (b) applying a vacuum to said shoot in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said shoot in such a manner that said Agrobacterium cells transfer said DNA -T to the cells of said outbreak in a second time, wherein said first and second time are the same or different.

2. The method according to claim 1, character 2.ado also because it comprises: (c) allowing said bud to grow to maturity and produce seeds; (d) germinating said seed to form progeny; and (e) selecting for the progeny expressing said transferred gene.

3. - The method according to claim 3, further characterized in that said vector comprises a selection marker gene.

4. The method according to claim 2, further characterized in that said vector comprises a selection marker gene.

5. The method according to claim 3, further characterized in that said selection marker gene comprises a gene for resistance to herbicide.

6. The method according to claim 4, further characterized in that said selection marker gene comprises a herbicidal resistance gene.

The method according to claim 5, further characterized in that said herbicidal resistance gene comprises a bar gene.

8. The method according to claim 6, further characterized in that said herbicidal resistance gene comprises a bar gene.

9. A method for direct plant transformation using legume shoots and Agrobacterium comprising: (a) contacting at least one legume shoot with a mixture of Agrobacterium cells, said mixture comprising cells of an Agrobacterium strain containing a first vector wherein said first vector enables said cells to transfer T-DNA containing a first DNA fragment to said outbreak and cells from the same Agrobacterium strain containing a second vector wherein said second vector enables said cells to transfer into T-DNA containing a second DNA fragment to said outbreak; and (b) applying a vacuum to said shoot in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells in intimate contact with said shoot so that said Agrobacterium cells transfer the DNA- T cells of said outbreak in a second time, wherein said first and second time are the same or different.

10. The method according to claim 9, further characterized in that: (c) allowing said bud to grow to maturity and produce seeds; (d) germinating said seed to form progeny; and (e) selecting for the progeny expressing said transferred gene.

11. The method according to claim 9, further characterized in that said vector comprises a selection marker gene.

12. The method according to claim 10, further characterized in that said vector comprises a selection marker gene.

13.- The method of. according to claim 11, further characterized in that said selection marker gene comprises a herbicidal resistance gene.

14. - The method according to claim 12, further characterized in that said selection marker gene comprises a herbicidal resistance gene.

15. The method according to claim 13, further characterized in that said herbicide resistance gene comprises a bar gene.

16. The method according to claim 14, further characterized in that said herbicide resistance gene comprises a bar gene.

17. A method for direct plant transformation using legume shoots and Agrobacterium comprising: (a) contacting at least one legume shoot with Agrobacterium cells, said Agrobacterium cells contain a vector, said vector enables said cells to Agrobacterium to transfer T-DNA containing at least one gene or fragment of the gene to said outbreak; (b) applying a vacuum to said shoot in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said shoot in such a manner that said Agrobacterium cells transfer said DNA -T to the cells of said outbreak in a second time, wherein said first and second time are the same or different; (e) exposing said progeny to an agent that enables the detection of the expression of the selection marker gene; (f) selecting for the progeny expressing said selection marker gene and at least one gene, said expression of said selection marker gene and at least one gene indicates the transfer of the gene.

18. The method according to claim 17, further characterized in that said selection marker gene comprises a gene for herbicide resistance.

19. The method according to claim 18, further characterized in that said herbicide resistance gene comprises a bar gene.

20. A plant transformed according to the method of claim 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19.

21. The seed from a transformed plant according to the method of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18 or 19.

22. A plant progeny from a seed obtained from a transformed plant according to the method of claim 1, 2,3,4,5,6,7,8,9 , 10, 11,12, 13,14,15,16,17, 18 or 19.

23.- A method for direct plant transformation using Medicago truncatula sprouts comprising: (a) contacting at least one outbreak of Medicago truncated with Agrobacterium cells, said Agrobacterium cells contain a vector, said vector enables said Agrobacterium cells to transfer T-DNA containing at least one gene or fragment of the gene to said bud of Medicago truncatula; (b) apply a vacuum to said bud of Medicago truncatula in contact with said cells of Agrobacterium at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said bud of Medicago truncatula in such a manner that said Agrobacterium cells transfer said T-DNA to the cells of said Medicago truncatula bud in a second time, wherein said first and second time are the same or different.

24. The method according to claim 23, further comprising: (c) allowing said bud of Medicago truncatula to grow to maturity and produce seeds; (d) germinate said seed so that it forms progeny; and (e) selecting for the progeny expressing said transferred gene.

25. The method according to claim 23, characterized in that said vector comprises a selection marker gene.

26. The method according to claim 24, characterized in that said vector comprises a selection marker gene.

27. The method according to claim 25, further characterized in that said selection marker gene comprises a gene for resistance to herbicide.

28. The method according to claim 26, further characterized in that said selection marker gene comprises a gene for resistance to herbicide.

29. - The method according to claim 27, further characterized in that said herbicide resistance gene comprises a bar gene.

30. The method according to claim 28, further characterized in that said herbicide resistance gene comprises a bar gene.

31.- A method for direct plant transformation using buds of Medicago truncatula and Agrobacterium comprising: (a) contacting at least one outbreak of Medicago truncatula with a mixture of Agrobacterium cells, said mixture comprising Agrobacterium cells containing a first vector, said vector enables said cells to transfer T-DNA containing at least a first DNA fragment to said bud of Medicago truncatula and cells from the same strain of Agrobactarium containing a second vector wherein said second vector enables said cells for transferring T-DNA to the cells of said Medicago truncatula outbreak at a second time, wherein said first and second time are the same or different; and (b) applying a vacuum to said bud of Medicago truncatula in contact with said Agrobacterium cells at a first time, said vacuum of sufficient force to force said Agrobacterium cells into intimate contact with said bud of Medicago truncatula in such a way that said Agrobacterium cells transfer said T-DNA to the cells of said outbreak of Medicago truncatula in a second time, where said first and second time are the same or different.

32. The method according to claim 31, further comprising: (c) allowing said bud of Medicago truncatula to grow to maturity and produce seeds; (d) germinate said seed so that it forms progeny; and (e) selecting for the progeny expressing said transferred gene.

33. The method according to claim 31, further characterized in that said vector comprises a selection marker gene.

34. The method according to claim 32, further characterized in that said vector comprises a selection marker gene.

35. The method according to claim 33, further characterized in that said selection marker gene comprises a gene for resistance to herbicide.

36. The method according to claim 34, further characterized in that said selection marker gene comprises a gene for resistance to herbicide.

37. The method according to claim 35, further characterized in that said herbicide resistance gene comprises a bar gene.

38. - The method according to claim 36, further characterized in that said herbicide resistance gene comprises a bar gene.

39.- A method for direct plant transformation using Medicago truncatula and Agrobacterium sprouts comprising: (a) contacting at least one outbreak of Medicago truncatula with Agrobacterium cells, said Agrobacterium cells containing a vector, said vector enables said Agrobacterium cells to transfer T-DNA containing at least one gene or a fragment of the gene and a selection marker gene to said outbreak of Medicago truncatula; (b) applying a vacuum to said bud of Medicago truncatula in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said bud of Medicago truncatula in such a way that said Agrobacterium cells transfer said T-DNA to the cells of said outbreak of Medicago truncatula in a second time, wherein said first and second time are the same or different; (c) allow the said bud of Medicago truncatula to grow to maturity and produce seeds; (d) germinating said seed to form progeny; (e) exposing said progeny to an agent that enables detection of the expression of a selection marker gene; (f) selecting for the progeny expressing said selection marker gene and at least one gene, said expression of said selection marker gene and at least one gene indicating gene transfer.

40. - The method according to claim 39, further characterized in that said selection marker gene comprises a gene for resistance to herbicide.

41. The method according to claim 40, further characterized in that said herbicide resistance gene comprises a bar gene. 42.- A Medicago truncatula plant transformed according to the method of claim 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 , 40 or 41. 43.- A seed of Medicago truncatula transformed according to the method of claim 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 41. 44.- A plant progeny from a seed obtained from a Medicago truncatula plant transformed according to the method (ie claim 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 41.