Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
  • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
  • C12N15/8205—Agrobacterium mediated transformation

Definitions

• Genetic transformation of higher plants promises to have a major impact on crop improvement, as well as many other areas of biotechnology. Genetic transformation can be used to produce transgenic plants carrying new genetic material stably integrated into the genome and to engineer 'designer' crops with specific traits.
• Various methods of genetic transformation have been developed and applied to a growing number of plant species. However, the ease and success rate of genetic transformation methods varies widely among plant species [Muller, et al. 1987. "High meiotic stability of a foreign gene introduced into tobacco by gr ⁇ b.3ctert-.m-mediated transformation," Mol Gen Genet 207:171-175; Gasser, C.S. and Fraley, R.T. 1989.
• Agrobacterium tumefaciens The most common and widely used method of transformation of dicotyledonous plants utilizes a bacterium, Agrobacterium tumefaciens, to effect gene transfer.
• Agrobacterium tumefaciens is a gram-negative, soil dwelling plant pathogen that infects its plant host and subsequently delivers and integrates part of its genetic material into the plant genome.
• the transferred portion of DNA is termed the T-DNA fragment, and additional genetic material can be added to the T-DNA.
• the additional genetic material will then be integrated into the genome along with the T-DNA. In this way, Agrobacterium can be used to facilitate the transfer of new genes into the plant genome
• stably transformed transgenic plants involves two processes: transformation of plant cells and then regeneration of those transformed cells to whole plants.
• a plant tissue explant is incubated with Agrobacterium carrying a T-DNA containing a selectable marker gene and a 'gene of interest'.
• a proportion of the cells in the explant will become transformed, and whole plants are then regenerated from these cells via somatic embryogenesis or direct organogenesis.
• Transformants are selected by inclusion of the appropriate selective conditions in the regeneration media.
• the choice of tissue explant depends on the plant species. Leaf, cotyledons, hypocotyls, cotyledonary meristems, and embryos are among those that have been used successfully.
• transgenic plant line In order to develop a transgenic plant line expressing a new trait, it is desirable to produce a large number of transgenic plants from which the best expressing line can be selected.
• the requirement for a number of plant lines stems from 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 gene integrated into different sites of the genome. Due to this phenomenon termed 'position effect', the various transgenic lines will vary in the levels of expression of the introduced gene (Ulian, et al. 1994. "Expression 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 for those expressing the introduced gene at a high level.
• Arabidopsis thaliana a model plant used widely for genetic and molecular analyses of plant developmental processes.
• a direct method of transformation has been developed for Arabidopsis thaliana (Bechtold, et al. 1993. "In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants," Comptes Renus de TAcademie des Sciences Serie III Sciences de la vie 316.).
• the plant is (1) grown to maturity, (2) immersed in a suspension of Agrobacterium cells, (3) held under vacuum for a short period of time, and then (4) allowed to set seed. A proportion of the progeny is transformed.
• Leguminous crops such as peas, soybean, bean, alfalfa, peanut, chick pea, pigeon pea and clover have widespread economic importance throughout the world.
• Legumes are an important source of protein as grain and forage legume crops for animals and as grain legumes for humans.
• soybeans Glycine max
• soybean oil is the most widely used edible oil in the world.
• the productivity, and therefore value, of a wide range of leguminous crops could be increased by the introduction of traits such as disease resistance, herbicide resistance, insect resistance, reduced levels of tannins and lignin (forage legumes), and improved protein and lipid quality.
• the soybean cyst nematode causes losses in yield of up to one billion United States dollars per year.
• Medicago truncatula Gaertn. is a diploid, autogamous, annual medic that is grown as a pasture legume in a number of regions throughout the world, including Mediterranean areas, South Africa and Australia (Crawford, et al. 1989.
• Medicago truncatula also is emerging as a model legume for studies of the nitrogen-fixing Rhizobium/legume symbiosis and the arbuscular mycorrhizal symbiosis [Cook, et al. 1995. "Transient induction of a peroxidase gene in Medicago truncatula precedes infection by Rhizobium meliloti," Plant Cell 7:43-55; van Buuren, et al. 1998. "Novel genes induced during an arbuscular mycorrhizal (AM) symbiosis between M. truncatula and G. versiforme," MPMI 12: 171-181]. The attributes that make M.
• truncatula a useful model plant for molecular and genetic analyses include its small genome (4.5 times larger than Arabidopsis), rapid life cycle, and relatively small physical size (Barker, et al. 1990. "Medicago truncatula, a model plant for studying the molecular genetics of the Rhizobium-leg e 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.
• An Agrobacterium-mediated transformation method has now been found wherein seedlings, rather than flowering plants or tissue explants, are utilized as the subject biological material for exposure to Agrobacterium cells. Moreover, following maturation of treated plants and seed set, transgenic plants are selected directly from a population of progeny representing various insertional events. This seedling transformation method provides high efficiency, low labor input, and large numbers of transgenic plants without all the problems associated with transformation of flowering plants or tissue explants and regeneration via somatic embryogenesis or direct organogenesis.
• Fig. 1 depicts a map of the T-DNA from the binary vector pBI121-b ⁇ r.
• Fig. 2(a) is a Southern blot of H/ ' «dIII 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
• Samples 3.1-3.5 are from Treatment 3 (40- second vacuum infiltration followed by 20-second hold).
• the pBI121-b ⁇ r plasmid DNA is included to the left of the blot. This part of the blot was excised and exposed for a shorter time than the rest of the blot to prevent overexposure.
• C is DNA from a non-transformed control M. truncatula plant.
• Fig. 2(b) is a Southern blot of Hind III 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 (1.5-minute vacuum infiltration); and Samples 3.1-3.5 are from Treatment 3 (40-second vacuum infiltration followed by 20-second hold). The pBI121-b ⁇ r plasmid DNA is included to the left. This part of the blot was excised and exposed for a shorter time than the rest of the blot to prevent overexposure.
• C is DNA from a non-transformed control M. truncatula plant.
• Fig. 3(a) is a Southern blot of Hindlll digested DNA from transgenic plants
• Fig. 3(b) is a Southern blot of HmdIII digested DNA from transgenic plants
• Sample 1.6-1.20 are from Treatment 1 (1-minute vacuum infiltration); and Sample 2.12 and 2.13 are from Treatment 2 (1.5-minute vacuum infiltration).
• C is DNA from a non-transformed control M. truncatula plant.
• Fig. 4 is an agarose gel showing a portion of the bar gene that has been amplified from DNA from transgenic soybean plants via PCR with bar specific primers. The arrow points to a 423bp amplified fragment. The lane labeled "M" contains 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.
• the present invention is a method for direct plant transformation using seedlings and Agrobacterium comprising: (a) contacting at least one seedling with Agrobacterium cells which harbor a vector that enables the Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment to the seedling and (b) applying a vacuum to the seedling in contact with the Agrobacterium cells at one point in time, the vacuum being of sufficient strength to force the Agrobacterium cells into intimate contact with the seedling such that the Agrobacterium cells transfer the T- DNA to cells of the seedling at a second point in time, wherein the first point in time and the second point in time are either the same or different.
• the vector comprises a selectable marker gene.
• a preferred selectable marker gene is a herbicide resistance gene.
• a preferred herbicide resistance gene is a bar gene.
• the present invention is a method for direct plant transformation using seedlings and Agrobacterium comprising: (a) contacting at least one seedling with a mixture of Agrobacterium cells, the mixture comprising cells from a Agrobacterium strain harboring a vector with a first DNA fragment and cells from the Agrobacterium strain harboring the vector with a second DNA fragment, wherein the vector enables the Agrobacterium cells to transfer the T-DNA to cells of the seedling; and (b) applying a vacuum to the seedling in contact with the mixture of Agrobacterium cells at a first point in time, the vacuum being of sufficient strength to force the Agrobacterium cells into intimate contact with the seedling such that the Agrobacterium cells transfer at least one gene to cells of the seedling at a second point in time, wherein the first point in time and the second point in time are the same or different.
• the vector comprises a selectable marker gene.
• a preferred selectable marker gene is a herbicide resistance gene.
• a preferred herbicide resistance gene is a bar
• the present invention is a method for direct plant transformation using seedlings and Agrobacterium comprising: (a) contacting at least one seedling with Agrobacterium cells which harbor a vector that enables the
• Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment and a selectable marker gene to the seedling ; (b) applying a vacuum to the seedling in contact with the mixture of Agrobacterium cells at a first point in time, the vacuum being of sufficient strength to force the Agrobacterium cells into intimate contact with the seedling such that the Agrobacterium cells transfer the T-DNA to cells of the seedling at a second point in time, wherein the first point in time and the second point in time are the same or different; (c) allowing the transformed seedling to grow to maturity and set seed; (d) germinating the seed to form progeny; (e) exposing the progeny to an agent enabling detection of selectable marker gene expression; and (f) selecting for progeny expressing the selectable marker gene and at least one gene, wherein expression of the selectable marker gene and at least one gene indicates gene transfer.
• the selectable marker gene is a herbicide resistance gene.
• a preferred herbicide resistance gene is a bar gene.
• the present invention is a plant transformed according to the above-described methods of seedling transformation.
• the present invention is a seed from a plant transformed according to the above-described methods of seedling transformation.
• the present invention is a progeny plant from a seed obtained from a plant transformed according to the above-described methods of seedling transformation.
• a plant transformation process has now been found which utilizes vacuum infiltration of seedlings to introduce Agrobacterium T-DNA carrying a selectable marker gene and the gene(s) of interest into the seedlings.
• a seedling as used herein is defined as a plant from about the beginning of seed germination to about the time true leaves develop.
• the transformation methods described herein can be applied to the seedlings of any plant, including dicots and monocots which can be successfully transformed by Agrobacterium-mediated gene transfer. In particular, leguminous plants are transformed at high rates of efficiency.
• the transformation method described herein is generally accomplished by growing the Agrobacterium strain carrying a gene(s) of interest under selective conditions in liquid culture until it reaches exponential growth phase.
• the Agrobacterium cells are then pelleted by centrifugation and resuspended in a vacuum infiltration medium.
• the seedlings are immersed in the Agrobacterium cell suspension and subjected to vacuum infiltration whereby the Agrobacterium cells are then introduced into the seedlings, resulting in infiltrated plants that subsequently produce transformed seed from which a transformed plant is obtained.
• Agrobacterium- mediated gene transfer The transformation of seedlings is accomplished through Agrobacterium- mediated gene transfer.
• Agrobacterium strains useful in the transformation of a seedling include any aggressive strain which, upon contact with a transformable plant cell, is capable of transferring T-DNA into the cell for integration into the plant's genome.
• the Agrobacterium strain can carry one plasmid with multiple gene(s) of interest.
• transformation is performed using a mixture of Agrobacterium cells in which the vector carries different fragments of DNA, e.g., selected fragments from a specific DNA library. To achieve the optimum transformation rate in a given plant, the Agrobacterium strain which provides the greatest number of transformed seedlings is selected.
• Agrobacterium tumefaciens EHA105, ASE1, and Gv3101 strains are preferably utilized.
• the gene(s) of interest can be transformed into the Agrobacterium by any means known in the art.
• a DNA fragment modified to contain the gene(s) of interest can be inserted into the T-DNA of an Agrobacterium Ti plasmid which also contains genes required to generate the transformed state.
• Agrobacterium plasmid T-DNA can be made to assist in the transformation process.
• a selectable marker gene can be incorporated into the T-DNA of Agrobacterium plasmid.
• Selectable marker genes useful in the transformation methods described herein include any selectable marker gene which can be incorporated into the Agrobacterium T-DNA and upon expression, can distinguish transformed from non-transformed progeny.
• Exemplary selectable markers include a neomycin transferase gene or phosphinothricin acetyl transferase (bar) gene.
• a preferable selection marker is the bar gene encoding phosphinothricin acetyl transferase which confers resistance to phosphinothricin-based herbicides.
• the selection marker gene and gene(s) of interest are incorporated into any vector suitable for use with transforming Agrobacterium strains.
• the binary vector, pBI121 vector (Clontech, Palo Alto, CA) can be modified wherein a copy of a phosphinothricin acetyl transferase (bar) gene is inserted, under the control of a 35S promoter and octopine synthase 3' sequences, into the H dIII site of the T-DNA.
• the bar gene encodes phosphinothricin acetyl transferase which confers resistance to phosphinothricin-based herbicides, such as Ignite® (AgroEvo, Frankfurt, Germany).
• This selectable marker enables easy selection of transformed plants: upon spraying the plants with phospinothricin (PPT) containing herbicides, only transformed plants containing the bar gene survive exposure to the herbicide.
• PPT phospinothricin
• the starter seeds can be pretreated to optimize their germination and to prepare the resulting seedlings for transformation.
• Surface-sterilization of the seeds is preferred to remove any interfering microorganisms which might infect the germinating seed.
• Any sterilization means which does not deleteriously affect the seeds can be used. Exemplary methods include use of aqueous 20-30% sodium hypochlorite or 70% ethanol.
• the seeds are sterilized in a solution of 30% sodium hypochlorite and 0.1% Tween 20 for approximately 5 minutes and then thoroughly rinsed to remove the sterilizing solution.
• sterile double distilled or deionized water, or water with reduced oxidizable carbon following reverse osmosis, ion exchange and/or activated charcoal treatment is used to rinse the seeds.
• Some seeds for example M. truncatula, experience a prolonged dormancy period resulting in delayed germination. These seeds can be treated by a scarification process capable of breaking the dormancy. For example, cracking or scratching the seed coat, soaking the seed to soften the seed coat, or a controlled acid treatment can be utilized.
• a treatment in concentrated sulfuric acid for approximately 10 minutes followed by thorough rinsing to remove the acid is utilized.
• sterile double distilled or deionized water, or water with reduced oxidizable carbon following reverse osmosis, ion exchange and/or activated charcoal treatment is used to rinse the seeds.
• the seeds are placed on a medium capable of supporting germination and subsequent growth of the seedlings.
• the seeds can be placed on the surface of sterile filter paper or paper towels.
• the seeds are spread on the surface of firm, sterile water agar in petri plates.
• the seeds are then placed under environmental conditions capable of inducing germination and supporting development of seedlings. Vernalization may be preferred for certain plants such as M. truncatula to promote early flowering. Incubation of the resulting seedlings is continued until the seedlings reach an appropriate stage of development for vacuum infiltration. The optimum age of seedlings for vacuum infiltration varies for different plants.
• vacuum infiltration can be optimized for a specific plant by screening seedlings at various stages of development using the methods disclosed herein and determining the stage at which the transformation efficiency is maximized.
• the time and temperature of incubation can also be adjusted to provide optimum conditions for a specific variety. For example, approximately 15 days after the seeds are placed on germination medium,
• M. truncatula and soybean seedlings are sufficiently matured for vacuum infiltration.
• the transforming Agrobacterium is subcultured on a general plated growth medium preferably containing appropriate antibiotics to distinguish transformed Agrobacterium cells.
• Agrobacterium tumefaciens EHA105 and Gvl301 carrying the bar gene are preferably cultured on YEP medium as defined in Example 1 containing rifampicin (20mg/l) and kanamycin (50 mg/1).
• the Agrobacterium cultures are grown at about 28°C for about 2-3 days.
• a liquid Agrobacterium culture is prepared by aseptically transferring an appropriate inoculum into a general growth medium suitable for growing Agrobacterium.
• TY liquid medium and YEP liquid medium containing appropriate antibiotics to select for the transformed Agrobacterium are preferred for Agrobacterium EHA105 and Gvl301.
• the liquid cultures are grown under conditions which provide the Agrobacterium to reach exponential growth.
• the liquid culture is incubated at about 28°C in a shaker incubator at about
• the Agrobacterium cells in the liquid culture are pelleted by centrifugation and resuspended in two volumes of a vacuum infiltration medium (e.g., Agrobacterium cells grown in 15ml liquid culture, pelleted by centrifugation, and then resuspended in 30ml vacuum infiltration medium).
• a vacuum infiltration medium e.g., Agrobacterium cells grown in 15ml liquid culture, pelleted by centrifugation, and then resuspended in 30ml vacuum infiltration medium.
• Any plant growth medium capable of supporting the infiltration process and the Agrobacterium within the plant while being compatible with plant growth can be used as the vacuum infiltration medium.
• the vacuum infiltration medium comprises acetosyringone which induces the vir genes of the Agrobacterium.
• the vacuum infiltration medium defined in Examples 1 and 2 is preferably utilized.
• the seedlings are removed from the germination/incubation medium and placed in any clean container capable of holding several seedlings as well as a volume of vacuum infiltration medium to partially cover the seedlings.
• Petri plates are useful for this purpose, using about 30-40 seedlings per plate.
• the Agrobacterium suspension in the vacuum infiltration medium is added to the container to wet and partially cover the seedlings. For a standard petri plate, approximately 10ml of the suspension is sufficient.
• the petri plate containing the seedlings in Agrobacterium suspension is placed in a vacuum chamber.
• the preferred amount of vacuum to be used in the transformation process is the minimal amount necessary to force the Agrobacterium into the apoplastic spaces of the seedlings. Approximately 28mmHg was sufficient for transforming M. truncatula and soybean.
• the time and manner in which the vacuum is applied to the seedlings depends upon the plant and has to be determined empirically.
• the vacuum can be applied then released. Alternately, the vacuum can be applied, released, reapplied, and then released again.
• the duration of vacuum can vary from about 0.1 to about 5 min, more preferably from about 0.5 to about 2 min, and most preferably for about 1 min. For M. truncatula, plants held under vacuum for 0.5 min and for 2 min gave rise to transgenic plants, but plants held under vacuum for 1 min gave the maximum transformation efficiency.
• the Agrobacterium suspension is decanted, and the seedlings are blotted on sterile filter paper or blotting paper.
• the seedlings can then be planted into a complete soil mix that will allow full growth and development of the plant and the production of seed.
• the seedlings are then permitted to mature and set seed.
• the plants are kept at a humidity, temperature, duration of photo period, and spectrum of light which favor plant growth.
• the seedlings are optionally incubated on a co-cultivation medium for 2-3 days prior to planting in a complete soil mix. Any co-cultivation medium which supports growth of the seedlings can be used.
• the co-cultivation medium given in Examples 1 and 2 is preferred.
• the plants are then permitted to develop to maturity and set seed. A portion of the seeds will carry the transgene in their genomes. The seeds are germinated, and the resulting progeny which exhibit stable inheritance of the transgene are selected.
• transgenic plants wherein the gene(s) of interest results in a visible phenotypic change, the selection can be based upon visual examination of the progeny.
• the appropriate selectable agent can be applied to the plants to select the transformants.
• Southern blot analysis or PCR analysis can be used to verify the presence of the transferred gene in the genome of the transformed plants.
• Example 1 Transformation of M. truncatula by Vacuum-infiltration of Seedlings M. truncatula seedlings were transformed to incorporate the bar gene and the nptll gene into the plant's genome using the transformation process of the present invention. Preliminary
• pBI121 vector Prior to transformation, a modified version of the binary vector, pBI121 vector (Clontech, Palo Alto, C A) was made by inserting a copy of a phosphinothricin acetyl transferase (bar) gene, under the control of a 35 S promoter and octopine synthase 3' sequences, into the Hindlll site of the T-DNA to create a plasmid called pBI121-b ⁇ r
• FIG. 1 The construct was confirmed by restriction analysis and PCR analysis, and then transformed into an Agrobacterium tumefaciens strain EHA105 (Hood, et al. 1993. "New Agrobacterium helper plasmids for gene transfer to plants," Trans Res 2:208- 218). Additional constructs in Agrobacterium were also obtained as given in Table III. While the following procedure is presented for the pBI121-b ⁇ r in the EHA105 Agrobacterium strain, the same procedure was followed for the other constructs with the exception that the growth medium was supplemented with specific antibiotics necessary for maintaining the plasmid.
• the M. truncatula seed was sterilized and germinated as follows. The seeds were soaked in cone. H 2 SO 4 for approximately 10 min. The acid was removed, and the seeds were rinsed extensively in sterile cold double distilled water. This treatment was used to break dormancy in M. truncatula.
• the seeds were then surface-sterilized by soaking the seeds in a sterilizing solution such as 30% Clorox / 0.1% Tween 20 solution for approximately 5 min with gentle agitation.
• a sterilizing solution such as 30% Clorox / 0.1% Tween 20 solution for approximately 5 min with gentle agitation.
• the seeds were rinsed extensively with sterile cold double distilled water.
• the seeds were then spread on a firm water-agar (for example, 0.8%) (Sigma
• the Agrobacterium liquid culture was grown until an exponential phase (OD 6 oo 1.6) was reached.
• the Agrobacterium cells were pelleted by centrifugation and resuspended in 30 ml of vacuum-infiltration medium (VIM) [1 liter: 10ml PDM salt solution at 100X concentration (400ml at 100X: lOOg KNO 3 ; 12g NH 4 H 2 PO 4 ; 16g
• VIP vacuum-infiltration medium
• the seedlings were removed from the water agar plates and placed in a clean standard petri dish at approximately at 30-40 M. truncatula seedling per petri plate. Approximately 10ml of the Agrobacterium suspension in the vacuum infiltration medium was added to the petri plate, a volume sufficient to wet and partially cover the seedlings. The petri plates containing the seedlings wetted with Agrobacterium suspension were placed in a vacuum chamber. Three methods of vacuum infiltration were tested. In Treatment 1, a vacuum was drawn to 28mmHg for approximately 1 min, released rapidly, redrawn to 28mmHg for approximately 1 min, and finally released rapidly.
• Treatment 2 a vacuum was drawn to 28mmHg for approximately 1.5 min, released rapidly, redrawn to 28mmHg for approximately 1.5 min, and finally released rapidly.
• Treatment 3 a vacuum was drawn to 28mmHg for approximately 40 seconds, held for 20 seconds, and finally released rapidly.
• the seedlings were then blotted on sterile filter paper or blotting paper and spread onto petri plates containing co-cultivation medium(CM) [1 liter: 10ml PDM salt solution at 10X concentration; 10ml PDM iron and vitamins; 0.2g CaCl 2 ⁇ 2 O; lOg sucrose; 7.5g agar- agar (Sigma Chemical Co., St.
• CM co-cultivation medium
• the plants began to flower at approximately 24 days after planting in soil.
• the resulting seeds were collected and germinated under conditions optimal for germination, i.e., a short cold treatment for four days on a damp filter paper, left at room temperature for 1-2 days, and then planted in soil.
• the seedlings had a few leaves (approximately 15 days old), they were sprayed with 80 mg/L PPT ( -1/7000 dilution of 600mg/ml solution stored at -20°C), and the results are presented below.
• 120 M. truncatula seedlings were vacuum infiltrated with Agrobacterium tumefaciens strain EHA105 carrying the pBI121-b ⁇ r plasmid as described above.
• Agrobacterium tumefaciens strains and binary vectors were used in these experiments: A. tumefaciens strain ASE1 carrying the binary vectors pSLJ525 (Jones et al. 1992. "Effective vectors for transformation, expression of heterologous genes, and assaying transposon excision in transgenic plants," Trans. Res.
• HindllllHpal fragment was used to produce pBI121-b ⁇ r and pB Nmgfp-ER-bar.
• the Hpal site was converted to a HmdIII site by the addition of an Hpal-Hindl ⁇ l linker and then the Hindlll fragment was then inserted into the HmdIII site of pBI 121 or pB ⁇ Nmgfp-ER (Haseloff et al. 1997 "Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly," Proc. Natl. Acad. Sci.
• Transformed plants 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.
• DNA was isolated from 22 transgenic plants (12 plants from Treatment T84- 1, 6 plants from Treatment T84-2, and 4 plants from Treatment T84-3) and digested with restriction enzyme HmdIII. The digested DNA was separated by electrophoresis and blotted to nylon membranes. The membranes were probed with an internal DNA fragment (450bp) of the bar gene labeled with P-dATP.
• All of the transformed plants contained DNA fragments that hybridized to the bar probe indicating that this gene is integrated into the genome (Fig. 2A and 2B).
• the plasmid pBI121-b ⁇ r DNA was included on the blot as a positive control, and a sample of DNA from non-transformed M. truncatula plant was included as a negative control.
• the bar probe hybridized to the plasmid DNA but not to the DNA from the non-transformed M. truncatula plant (Fig. 2A and 2B).
• the expected size of the hybridizing fragment from the plasmid is 1.6kb; however, all of the fragments were larger, probably due to a rearrangement towards the left border.
• the blots were stripped and reprobed with an internal fragment of the nptll gene (766bp) labeled with 32 P-dATP.
• the nptll gene is carried on the pBI 121 -bar plasmid, between the right border of the T-DNA and the bar gene (Fig. 1). All of the transformed plants contained DNA fragments that hybridized to the nptll probe indicating that this gene is also integrated into the genome.
• This combination of digest and probe provided a right border analysis and demonstrated the presence of independent transformants. For example, the unique b ⁇ r-hybridizing fragments shown with Transformants 1.6, 1.10, 1.11, 1.14, 1.13, and 2.12 provided evidence that these are independent transformants.
• the non-transformed control plant does not contain DNA capable of hybridizing to this probe (Fig. 3 A and 3B).
• the pBI121-b ⁇ r T-DNA 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. Loss of genes located between the selectable marker and the left border have been previously reported; thus, the lack of the GUS gene in the transformed plants confirmed these findings. These results were consistent with the bar Southern analysis and offered an explanation of the larger than expected bar hybridizing fragment. Thus, it was demonstrated that the GUS gene or any gene of interest must be inserted in the plasmid between the bar gene and the right border (at the location of the nptll gene) to ensure integration.
• Table III Segregation Analysis (Phosphinothricin Resistance) of Progeny from a Selection of Transformants Prepared by Infiltration of Seedlings
• the seeds were then surface-sterilized by soaking the seeds in 20% sodium hypochlorite for approximately 5 min with gentle agitation. The seeds were rinsed for eight times in sterile double distilled water. The seeds were then placed in a large volume of water and allowed to imbibe at room temperature for 3-12 hours. The seeds were then spread on a firm water-agar (for example, 0.8%) (Sigma
• Agrobacterium tumefaciens Gv3101 which carries the SKI015 vector with a copy of the bar gene was subcultured for isolation onto a fresh agar plate containing
• VAM vacuum-infiltration medium
• BAP 0.0565g in 0.15ml 2N NaOH and 24.85ml double distilled H 2 O stored at 4°C
• NAA 1 OmM alpha-naphthaleneacetic acid
• the seedlings were removed from the water agar plates and placed in a clean standard petri dish at approximately at 10-20 soybean seedlings per petri plate. Approximately 20ml of the Agrobacterium suspension in the vacuum infiltration medium was added to the petri plate, a volume sufficient to wet and partially cover the seedlings. The petri plates containing the seedlings wetted with Agrobacterium suspension were placed in a vacuum chamber. A vacuum was drawn to 28mmHg for approximately 2 min, released rapidly, redrawn to 28mmHg for approximately 2 min, and finally released rapidly.
• the seedlings were then blotted on sterile filter paper or blotting paper and spread onto petri plates containing co-cultivation medium(CM) [1 liter: 10ml PDM salt solution at 10X concentration; 10ml PDM iron and vitamins; 0.2g CaCl 2 ⁇ 2 O; lOg sucrose; 7.5g agar-agar (Sigma Chemical Co., St. Louis, MO); and 0.1ml AS, wherein PDM salts, PDM iron and vitamins, CaCl ⁇ 2 O, agar-agar, and sucrose are combined, the pH adjusted to 5.8 with KOH and autoclaved on liquid cycle for 20 min, and when the medium cools to 50°C, AS is added].
• the seedlings were incubated in a growth chamber under the conditions given in Table IV for approximately 6-7 days until the Agrobacterium could be seen growing around the seedlings on the media.
• the seedlings were washed twice with H O and then planted in pots in Metro- mix 200 soil mixture. To allow the plants to adjust slowly to ambient humidity the following procedure was followed: the pots containing the seedlings were initially covered with a plastic cover; after one week, the cover was propped open, and after a couple of days, the cover was removed completely. The plants were allowed to mature under conditions for optimal growth in a greenhouse. About Day 40
• the plants began to flower, and the resulting seeds were collected and germinated by soaking in water for three hours followed by immediate planting in soil. When the seedlings had one leaf, they were sprayed with 100 ml/1 PPT ( -1/6000 dilution of 600mg/ml solution in 0.1% Tween 20 stored at -20°C), and the results are presented below.
• soybean seedlings were vacuum infiltrated with Agrobacterium tumefaciens strain Gv3101 carrying the SKI015-b ⁇ r plasmid as described above.
• Agrobacterium tumefaciens strain Gv3101 carrying the SKI015-b ⁇ r plasmid as described above.

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