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)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
• • 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
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• This application relates to a plant transformation method and transgenic plants and plant cells originating therefrom.
• 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 Agrobacterium- ediated 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.
• 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 Natl AcadSci USA 80:4803-4807).
• transformation i Agrobacterium has worked well for a number of model species such as tobacco and petunia, the approach is subject to a number of limitations. Some plant species, including many monocotyledonous plant species, are not readily susceptible to infection by Agrobacterium (Potrykus, I. 1990. "Gene transfer to cereals: an assessment," Bio/Technology 8:535-542). In these cases, alternative approaches have been used, including particle bombardment and direct gene transfer into protoplasts via electroporation, microinjection, or polyethylene glycol mediated uptake (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 (Glycine max) by particle acceleration," Bio/Technology 6:923-926; Bommineni, et al. 1994.
• 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 plants are needed. For example, in order to develop a 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. Integration of the T-DNA fragment into the plant genome is a random event, and, therefore, transgenic plants will vary in the levels of expression of the introduced gene due to position effects
• 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 I'Academie 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 as protein sources for plants and animals, and in association with rhizobia, they are also an essential component of the global nitrogen cycle.
• 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.
• 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. "Breeding annual Medicago species for semiarid conditions in Southern Australia,” Adv Agron 42:399-437). In Australia, the annual medics are 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 cultivar of M. truncatula was sown in 1938, and this species has been favored due to its ability to tolerate both low rainfall and high lime soils
• 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.
• AMD arbuscular mycorrhizal
• M. truncatula a useful model plant for molecular and genetic analyses
• 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 R/ztzobtum-legume symbiosis,"
• Plant Mol Biol Rep 8:40-49 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 1 1 :113-117; Chabaud, et al. 1996. Plant Cell Rep 15:305-310; Trieu and Harrison. 1996.
• Agrobacterium-mediated transformation with regeneration via somatic embryogenesis or direct organogenesis is a viable approach, these methods are very labor intensive, not very efficient, and in some cases, very slow. While these approaches may be suitable for the generation of small numbers of transgenic plants, they cannot be used to generate the large numbers of lines required for many genetic approaches and high through-put systems, such as T-DNA mutagenesis or activation tagging.
• An -4gr ⁇ b ⁇ cterz '* Mm-mediated in planta transformation method has now been found wherein plants are exposed directly to Agrobacterium cells, eliminating the need for preparation of cell or callus cultures, explants or other materials for treatment.
• transgenic progeny can be selected from nontransgenic progeny by use of appropriate selection systems, such as the bar gene and PPT herbicide.
• the direct in planta transformation method of the present invention provides high efficiency, low labor input, and large numbers of transgenic plants representing independent transformation events without problems associated with preparation and transformation of isolated cells or tissues and without difficulties associated with subsequent regeneration of whole plants such as by somatic embryogenesis or direct organogenesis.
• Fig. 1 A-1C are schematic representations of the T-DNAs used in the transformation experiments.
• Fig. 1 A is a schematic representation of the pSKIOO ⁇ construct.
• Fig. IB is a schematic representation of the pSLJ525 construct.
• Fig. 1C is a schematic representation of the ⁇ p TNmgfp-ER-bar construct.
• the present invention is a method for direct plant transformation using plants and Agrobacterium comprising: contacting the aerial portions of at least one plant at the time of flowering 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 plant; and applying a vacuum to the plant or plant portions 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 plant such that the
• Agrobacterium cells transfer the T-DNA to cells of the plant at a second point in time to form a transformed plant, 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 transformation of a plant comprising: vernalizing and germinating initial seed to form a plant, contacting the aerial portions of the plant at the time of flowering 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 plant; and applying a vacuum to the plant or plant portions 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 plant such that the Agrobacterium cells transfer the T-DNA to cells of the plant at a second point in time to form a transformed plant, 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 plants and Agrobacterium comprising: contacting the aerial portions of at least one plant at the time of flowering 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 plant; applying a vacuum to the plant or plant portions 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 plant such that the Agrobacterium cells transfer the T-DNA to cells of the plant at a second point in time to form a transformed plant, wherein the first point in time and the second point in time are either the same or different; allowing the transformed plant to grow to maturity and set seed; germinating the seed to form progeny; and selecting for progeny expressing the transferred gene.
• the present invention is a method for direct transformation of a plant comprising: vernalizing and germinating initial seed to form a plant; contacting the aerial portions of the plant at the time of flowering 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 plant; applying a vacuum to the plant or plant portions 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 plant such that the Agrobacterium cells transfer the T-DNA to cells of the plant at a second point in time to form a transformed plant, wherein the first point in time and the second point in time are either the same or different; allowing the transformed plant to grow to maturity and set seed; germinating the seed to form progeny; and selecting for progeny expressing the transferred gene.
• the vector comprises a selectable marker gene.
• a preferred selectable marker gene is a herbicide resistance gene.
• a preferred herbicide resistance gene
• the present invention is a method for direct plant transformation using plants at the time of flowering and Agrobacterium comprising: contacting the aerial portions of at least one plant 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 T-DNA to cells of the plant; applying a vacuum to the plant portions 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 plant such that the Agrobacterium cells transfer T-DNA to cells of the plant at a second point in time to form a transformed plant, 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
• the present invention is a method for direct transformation of a plant at the time of flowering comprising: vernalizing and germinating initial seed to form the plant; contacting the aerial portions of the plant 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 T-DNA to cells of the plant; applying a vacuum to the plant portions 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 plant such that the Agrobacterium cells transfer T-DNA to cells of the plant at a second point in time to form a transformed plant, 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 gene.
• the present invention is a method for direct plant transformation using plants at the time of flowering and Agrobacterium comprising: contacting the aerial portions of at least one plant 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 T-DNA to cells of the plant; applying a vacuum to the plant portions 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 plant such that the Agrobacterium cells transfer T-DNA to cells of the plant at a second point in time to form a transformed plant, wherein the first point in time and the second point in time are the same
• the present invention is a method for direct transformation of a plant at the time of flowering comprising: vernalizing and germinating initial seed to form the plant; contacting the aerial portions of the plant with a mixture of
• 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 plants at the time of flowering and Agrobacterium comprising: contacting aerial portions of at least one such plant at the time of flowering 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 plant; applying a vacuum to the plant portions 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 plant such that the Agrobacterium cells transfer the T-DNA to cells of the plant at a second point in time to form a transformed plant, wherein the first point in time and the second point in time are the same or different; allowing the transformed plant to grow to maturity and set seed; germinating the seed to form progeny; exposing the progeny to an agent enabling detection of selectable marker gene expression; and selecting for progeny expressing the selectable marker gene and at least one gene, wherein
• the present invention is a method for direct transformation of a plant at the time of flowering comprising: vernalizing and germinating initial seed to form the plant; contacting aerial portions of the plant at the time of flowering 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 plant; applying a vacuum to the plant portions 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 plant such that the Agrobacterium cells transfer the T-DNA to cells of the plant at a second point in time to form a transformed plant, wherein the first point in time and the second point in time are the same or different; allowing the transformed plant to grow to maturity and set seed; germinating the seed to form progeny; exposing the progeny to an agent enabling detection of selectable marker gene expression; and 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 plant transformation.
• the present invention is a seed from a plant transformed according to the above-described methods of plant transformation.
• the present invention is a progeny plant from a seed obtained from a plant transformed according to the above-described methods of plant transformation.
• a plant transformation process has now been found which utilizes vacuum infiltration of plants grown from vernalized seed to flowering to introduce Agrobacterium T-DNA carrying a selectable marker gene and the gene(s) of interest into the plants.
• a plant at flowering as used herein is defined as a plant from about the beginning of first flower bud formation to about the time of last flower set.
• the transformation methods described herein can be applied to flowering portions 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 a 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 above-ground portions of plants are immersed in the Agrobacterium cell suspension and subjected to vacuum infiltration whereby the Agrobacterium cells are introduced into the plants, resulting in infiltrated plants that subsequently produce transformed seed from which a transformed plant is obtained.
• Agrobacterium strains useful in the transformation of a plant 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 plants 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, pBINmgfp-ER Haseloff et al., 1997.
• Ignite® (AgroEvo, Frankfurt, Germany). This selectable marker enables easy selection of transformed progeny 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 are vernalized to prepare the resulting plants 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.
• Vernalization is an essential step in the transformation process of the present invention. It is expected that any vernalization method can be used.
• the preferred vernalization method is to place the rinsed seeds onto sterile filter paper moistened with sterile distilled water and to incubate the seeds at 4°C for 14 days in the dark, periodically adding water to the filter paper to keep it damp.
• the germinating seeds are planted in a medium capable of supporting growth of plants to flowering.
• a preferred method is to plant seeds in 11 cm diameter pots containing Metro Mix 250 (W.R. Grace Co.,
• the optimum age of plants for vacuum infiltration may vary for different species. In general, plants with some flowers and some unopened flower buds are sufficiently mature. However, since plants develop at different rates, vacuum infiltration can be optimized for a specific plant by screening plants at various stages of flowering 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 4 weeks after the vernalized, germinating seeds of M. truncatula are planted in growth medium, plants have begun to flower and 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 ASE1 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 ASE1.
• 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 250 rpm overnight. It is essential to use fresh Agrobacterium to achieve transformation.
• 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.
• sufficient volume of Agrobacterium suspension in the vacuum infiltration medium is added to a container sufficiently large to accommodate immersion of the above ground portions of plants in the Agrobacterium suspension.
• 250 ml of suspension is added to an open container having the approximate dimensions of 15 cm x 10 cm, such as the cover from a blue tip pipette box.
• the container is placed in a vacuum chamber, and a pot containing a plant of interest is inverted and supported over the container in such a way that the aerial portions of the plant are submerged in the Agrobacterium suspension.
• 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 plants. Approximately 28 rnmHg is sufficient for transforming M.
• the time and manner in which the vacuum is applied to the plants depends upon the plant and has to be determined empirically. For M. truncatula, vacuum is held for about 3 minutes and then released quickly. During the vacuum process, the Agrobacterium suspension bubbles profusely.
• the pots containing the infiltrated plants are removed from the vacuum chamber and placed on their sides in a growth chamber in such a manner as to avoid contamination of the soil mix with Agrobacterium suspension.
• the plants are then incubated at about 18°C and 95% humidity with long days of about 16 hours of light.
• Light quality and intensity does not appear to be critical as either cool white lights (Sylvania F48T12, 115W) or Super Spectra lights (Sylvania) can be used. Plants are held in the growth chamber for about a week and will appear sickly during this incubation period. It is important to apply water, if necessary, only to the soil mix.
• Water dripping from the aerial plant portions may transfer Agrobacterium to the soil mix, thereby greatly increasing plant mortality.
• the vacuum infiltration process is then repeated on the same plants according to the procedures given above. Again, the plants are held in the growth chamber for a week, with care taken during watering to avoid transferring Agrobacterium to the soil mix and roots. After the second infiltration, the plants appear unhealthy with many dead leaves, and some plants may die.
• Treated plants are then grown under optimum growing conditions and allowed to set seed. For M. truncatula, this requires about 6 to 8 weeks. During this growth period, additional flowers may form and set seed. Regardless, all seeds from treated plants are collected. Progeny plants are grown from this seed, and many of these progeny are transformed plants.
• 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 progeny 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.
• M. truncatula plants were transformed to incorporate the bar gene into the plant's genome using the transformation process of the present invention.
• Agrobacterium tumefaciens strain ⁇ A105 Hood, et al. 1993. "New Agrobacterium helper plasmids for gene transfer to plants/' Trans Res 2:208-218). Additional constructs in Agrobacterium strains were also obtained as given in Table I.
• Medicago truncatula Gaertn 'Jemalong' (line A17) was used for all of the experiments.
• the M. truncatula seed was sterilized and germinated as follows. The seeds were soaked in cone. H 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 vernalized on a firm water-agar (for example, 0.8%) (Sigma Chemical Co., St. Louis, MO) in petri plates.
• the water-agar petri plates containing the seeds were wrapped with aluminum foil and kept at 4°C for about 15 days.
• Metro-Mix 250 or 350 (W.R. Grace Co., Cambridge, MA) with 9 plants per pot.
• the light level was 150 ⁇ mol/m 2 /s with 18 hours light/25°C and 6 hours dark/22°C.
• the plants were fertilized with MIRACLE-GRO® (Stern's Nurseries, Inc.) when necessary.
• Agrobacterium tumefaciens carrying the appropriate construct was subcultured for isolation onto a fresh agar plate containing YEP medium [1 liter: lOg Bacto-peptone (Difco, Detroit, MI); lOg yeast extract; 5g NaCl; and 15g Bacto-agar (Difco, Detroit, MI) at pH-6.8 without adjusting] containing rifampicin (20 mg/1) and kanamycin (50 mg/1). and the subculture was incubated at approximately 28°C for about 2-3days.
• 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 flower infiltration medium.
• Flower infiltration media is 0.5X MS salts [IX MS salt per liter: 1.65g NH 4 NO 3 ; 0.33g CaCl 2 ; 0.18g MgSO 4 ; 1.9g KNO 3 ; 0J7g KH 2 PO 4 ; 6.2mg H 3 BO 3 ; 0.025mg CoCl 2 • 6H 2 O; 0.025mg CuSO 4 ⁇ 5H 2 O; 37.3mg Na-EDTA; 16.9mg MnSO 4 • H 2 O; 0.25mg Na 2 MoO 4 ; 0.83mg KI; 27.8mg FeSO 4 • 7H 2 O; and 8.6mg ZnSO 4 ⁇ 7H 2 O], IX Gamborg's vitamins [per liter: l
• Vernalized seedlings were grown until the plants had small flower buds and a few opened flowers. This occurs approximately 4 weeks after planting. The plants were watered heavily on the day before infiltration took place. To infiltrate the plants, the pots were inverted and the above ground portion of the plant submerged in a container filled with a suspension of Agrobacterium in flower infiltration media. Usually the soil is held in the pot by the roots; however; if the soil appeared loose the pot was packed with cotton wool to ensure that the soil did not fall out. The pot and tray were placed in a vacuum chamber and a vacuum drawn to 25 mmHg and held for 3 minutes. The vacuum was released very rapidly and the procedure repeated once. Following two exposures to vacuum some of the plant leaves became dark and water soaked. The pots were removed from the Agrobacterium and placed on their sides in a tray to prevent Agrobacterium from entering the soil. The tray and pots were transferred to a growth chamber set at
• the infiltrated plants were permitted to mature and seed.
• the seeds were then collected and germinated under conditions optimal for germination, i.e., a short cold treatment (4°C) for three-four days on a damp filter paper, left at room temperature for 1 - 2 days, and then planted in soil (Metro-Mix 350).
• a short cold treatment (4°C) for three-four days on a damp filter paper, left at room temperature for 1 - 2 days, and then planted in soil (Metro-Mix 350).
• the progeny seedlings had grown to the first trifoliate stage (approximately 15 days old), they were sprayed with Ignite (AgrEvo, Wilmington, DE) diluted to contain 80 mg/L phosphinothricin (PPT) (-1/7000 dilution of 600mg/ml solution stored at -20°C).
• PPT phosphinothricin
• a Selectable marker gene b ⁇ r gene. b
• the majority of the transformants have been analyzed by Southern blot analysis and, in some instances, progeny analysis.
• c ND Not determined.
• TS1-TS3 in Table 1 in which three sets of transgenic plants were obtained, three additional experiments were undertaken (TS86-1, TS86-2 and TS86-4). Thirty-six plants were infiltrated in each experiment, and the seed from the 36 plants was collected as a single pool. Overall, the number of seeds collected was low, as was the viability; however, in these experiments
• T86-1, T86-2 and T86-4 between 13 and 76% of the Tl seedlings were resistant to the selective agent, phosphinothricin (PPT) (Table 1), indicating successful transformation. Seedlings arising from a control experiment in which the plants were infiltrated with the infiltration media lacking Agrobacterium did not give rise to any PPT-resistant seedlings (data not shown). Transformants were not recovered from experiments in which the binary vector contained the nptll gene as a selectable marker (data not shown). Although kanamycin has been used successfully as a selective agent in M. truncatula tissue culture transformation procedures (Chabaud, et al. 1996. Plant Cell Rep 15:305-310), the seedlings displayed a high level of resistance to kanamycin, and transformants could not be distinguished easily.
• PPT phosphinothricin
• DNA was extracted from the majority of the transgenic plants from experiments T86-1 , T86-2 and T86-4 and analyzed on Southern blots. DNA was isolated from the transformants and digested with appropriate enzymes as indicated in Fig. lA-l C. For example, DNA from transformants from the T86-2 experiment was digested with 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 32 P-dATP. The corresponding plasmid 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.
• Hwdlll digested DNA from transgenic plants from Experiment T86-2 was also blotted and hybridized with an nptll probe (766bp) labeled with 32 P-dATP to obtain a left border analysis.
• the probe hybridized to DNA from the transformants and not to wildtype M. truncatula (All) DNA.
• the majority of the transformants appeared to have identical hybridization patterns, indicating that there are a large number of sibling transformants.
• Additional left border analyses of a selection of these transformants confirmed these results, and only 3 of the 23 (13%) transformants were determined to be independent.
• the progeny (T2) from four Tl transformants from experiments T86-1 and -2 were grown and analyzed for resistance to phosphinothricin. As shown in Table II, data was obtained which showed that the transgenes were inherited in a stable Mendelian fashion. The results show that the lines can be propagated past the Tl generation. In three cases, the progeny segregated for resistance to phosphinothricin, indicating that the original transformants were hemizygous. However, in one case, all of the progeny were resistant, suggesting that the original transformant was homozygous.
• T86-2J 1 are siblings.
• Table II Segregation Analysis (Phosphinothricin Resistance) of Progeny from a Selection of Transformants Prepared by Infiltration of Plants at Flowering
• the transformation process described herein is more efficient and less labor intensive than previously reported methods.
• somatic alterations are avoided, and direct introduction of genetic material into elite lines is made possible.
• Large numbers of transgenic plants can be generated very rapidly and efficiently, and the transgenes are stable and inherited by the subsequent generation.
• the major difficulty with regeneration of Agrobacterium transformed cells through tissue culture is avoided in the transformation procedures of the present invention, making it useful for legumes such as, soybean, bean and peas for which transformation or subsequent regeneration of Agrobacterium transformed cells is problematic.
• Agrobacterium tumefaciens ASE1 and Agrobacterium tumefaciens EHA105 were utilized in the transformations.
• the following Agrobacterium tumefaciens strains and binary vectors were used in the experiments outlined in Table I: (1)
• the addition of the bar gene to pB ⁇ Nmgfp-ER was achieved as follows.
• a HindllllHpal fragment was excised from pSLJ525.
• the Hp ⁇ l site was converted to a H dIII site by the addition of an Hp ⁇ l- H/ «dIII linker, and then the HmdIII fragment was then inserted into the HmdIII site of pBINmgtj -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 AcadSci USA 94: 2122-21271997) to create pBINmgrp-ER- bar.

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