CA2352488A1

CA2352488A1 - Plant transformation process

Info

Publication number: CA2352488A1
Application number: CA002352488A
Authority: CA
Inventors: Maria J. Harrison; Anthony T. Trieu
Original assignee: Individual
Current assignee: Roberts Samuels Noble Foundation Inc
Priority date: 1998-12-23
Filing date: 1999-12-23
Publication date: 2000-06-29
Also published as: WO2000037663A3; JP2002533090A; AU2594300A; WO2000037663A2; EP1141356A2

Abstract

A method of Agrobacterium-mediated genetic transformation using seedlings has been found which is applicable to dicots and monocots capable of being transformed by Agrobacterium. The transformation method utilizes vacuum-infiltration to introduce the 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

PLANT TRANSFORMATION PROCESS
BACKGROUND
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-mediated transformation," Mol Gen Genet 207:171-175; 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 kanamycin 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 medic (Medicago truncatula Gaertn.) by Agrobacterium tumefaciens and regeneration via somatic embryogenesis of transgenic plants with MtENODl2 nodulin promoter fused to the gus reporter gene," Plant Cell Rep 15:305-310; Kar, et al.
1996."Efficient transgenic plant regeneration through Agrobacterium-mediated transformation of chickpea (Cicer arientinum L.)," Plant Cell Rep 16:32-37;
Kim, J.W.
and Minamikawa,T. 1996. "Transformation and regeneration of French bean plants by the particle bombardment process," Plant Sci 117:131-138; Trieu, A.T. and Harrison, M.J. 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-S 19; Cheng, et al. 1997. "Genetic transformation of wheat mediated by Agrobacterium tumefaciens," Plant Physiol 115:971-980 ; Cheng, et aI. 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-mediated barley transformation," Plant) 11:1369-1376.]
The most common and widely used method of transformation of dicotyledonous plants utilizes a bacterium, Agrobacterium tumefaciens, to effect gene transfer.
s 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, 1 o 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 Acad Sci USA 80:4803-4807).
While transformation with Agrobacterium has worked well for a number of model species such as tobacco and petunia, the approach is subject to a number of 15 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," BiolTechnology 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 2o 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," BiolTechnology 6:923-926;
Bornmineni, et al. 1994. "Expression of GUS in somatic embryo cultures of black spruce after microprojectile bombardment," JExp Bot 45:491-495; Christou, P. 1995.
"Strategies 25 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 Natl Acad Sci USA 85:8502-8505].

Regardless of the method of delivery of the new genetic material, it is necessary to regenerate whole fertile plants from the transformed cells. The production of stably transformed transgenic plants involves two processes: transformation of plant cells and then regeneration of those transformed cells to whole plants. In most cases, 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.
Because neither the transformation nor the regeneration are 100% effective, the chance of obtaining a transformed plant depends on these two processes occurring t5 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 species in which somatic embryogenesis is a viable method of regenerating plants, there are other limitations. Plants regenerated via somatic embryogenesis may show significant somatic variation, altered ploidy, phenotypic 2o abnormalities and poor fertility (Bean, et al. 1997. Plant Cell Rep 16:513-519). While regeneration via direct organogenesis overcomes some of these problems, not all plants can be regenerated in this way. Finally, although transformation of many crop plants is possible, it is usually achieved in highly regenerable lines or cultivars, and the elite agriculturally important lines are not usually amenable to transformation.
Therefore, 25 introduction of a desired trait into the elite lines has been limited to subsequent traditional breeding methods following transformation of parental lines.
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 3o 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 WO 00/37663 PCT/US99l30972 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.
The only plant which has been successfully transformed with a high degree of ease and efficiency is Arabidopsis thaliana, a model plant used widely for genetic and molecular analyses of plant developmental processes. A direct method of 1o transformation has been developed forArabidopsis thaliana (Bechtold, et al.
1993. "In plants Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants," Comptes Renus de 1'Academie des Sciences Serie 111 Sciences de la vie 316.). In this transformation process, the plant is (1) grown to maturity, (2) immersed in a suspension ofAgrobacterium cells, (3) held under vacuum for a short period of 15 time, and then (4) allowed to set seed. A proportion of the progeny is transformed.
Recent data suggest that the gametophyte progenitor, gametophyte, or fertilized embryos are the targets (Bechtold, N. and Pelietier, G. 1998. "In plants Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration,"
Methods Mol Biol 82:259-266). Although Bechtold's method has been tried in other 2o species including Brassica napus and Beta vulgaris, these attempts reportedly have been 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).
Leguminous crops such as peas, soybean, bean, alfalfa, peanut, chick pea, 25 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. For example, soybeans (Glycine max) are a major source of protein in animal and human food, and soybean oil is the most widely used edible oil in the world. The productivity, and therefore value, of a wide range of 30 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. For example, the soybean cyst nematode causes losses in yield of up to one billion United States dollars per year.
With the recent cloning of a beet cyst nematode resistance gene and a potato cyst nematode resistance gene (Williamson, V.M. 1999. Curr Opin Plant Biol 2:327-31), strategies are now being explored for genetically engineering resistance in plants.
While there have been some attempts to introduce these traits into leguminous crops via genetic engineering, the current transformation methods involving tissue culture are exceedingly labor intensive and inefficient. In particular, the large seed grain legumes, such as pea, bean, and soybean have proved very difficult to transform, to 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-mediated DNA transfer," BiolTechnology 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 Minamikawa.
1996.
Plant Sci 117:131-138; Trieu and Harrison. 1996. Plant Cell Rep 16:6-11; Bean, 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 2o gene pool for Agrobacterium-meditated gene transfer to the common bean.
Mededelingen-Faculteit-Landbouwkundige-en-Toegepaste-Biologische-Wetenschappen-Universiteit-Gent 62:1397-1402].
In an alternative approach, it was shown that cells within a soybean 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 seed, and the seed will carry the transgene (McCabe, et al. 1988. BiolTechnology 6:923-926;
Chowrira, et al. 1995. "Electroporation-mediated gene transfer into intact nodal meristems in planta: generating transgenic plants without in vitro tissue culture,"
Molecular Biotechnology 3:17-23; and Chowrira, et al. 1996. "Transgenic grain legumes obtained by in planta electroporation-mediated gene transfer,"
Molecular Biotechnology 5:85-96). While this procedure has enabled the production of transgenic soybean, it is very labor intensive because numerous meristems need to be bombarded to have a realistic chance of obtaining any transgenic seeds.
Medicago truncatula Gaertn. (barrel medic) 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 1 o species has been favored due to its ability to tolerate both low rainfall and high lime soils (Crawford, et al. 1989. Adv Agron 42:399-437). Medicago truncatula also is emerging as a model legume for studies of the nitrogen-fixing Rhizobium/Iegume 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 2o al. 1990. "Medicago truncatula, a model plant for studying the molecular genetics of the Rhizobium-legume 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:I 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 isolated through cell culture selection," Mol Plant-Microbe Interact 10:307-315).
3o Although 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 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 t o 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.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 depicts a map of the T-DNA from the binary vector pBI121-bar.
Fig. 2(a) is a Southern blot of HindIII digested DNA from transgenic plants (progeny of infiltrated plants) hybridized with a bar probe. Samples 1.3-1.8 are from 2o Treatment 1 (I-minute vacuum infiltration); Samples 2.2-2.7 are from Treatment 2 (1.15-minute vacuum infiltration); and Samples 3.1-3.5 are from Treatment 3 (40-second vacuum infiltration followed by 20-second hold). The pBI121-bar 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 infltration); Samples 2.2-2.7 are from Treatment (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-bar 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 HindIII digested DNA from transgenic plants (progeny of infiltrated plants) hybridized with a npt II probe. Samples 1.6-1.20 are from Treatment l (1-minute vacuum infiltration) and Samples 2.12 and 2.13 are from Treatment 2 (l.l S-minute vacuum infiltration). C is DNA from a non-transformed control M. truncatula plant.
Fig. 3(b) is a Southern blot of HindIII digested DNA from iransgenic plants (progeny of infiltrated plants) hybridized with a bar probe. 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.
t5 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 by marker is indicated. Samples 6-27 are soybean transformants that survived the herbicide treatment. Transformants 6, 13, 14, 1 S, and 16 show an amplified fragment of the correct size.
SUMMARY OF THE INVENTION
In one aspect, 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-3o 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. In a preferred method, 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.
In another aspect, 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;
1 o 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 15 different. In a preferred method, 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.
In another aspect, the present invention is a method for direct plant transformation using seedlings and Agrobacterium comprising: (a) contacting at least 2o 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 ofAgrobacterium cells at a first point in time, the vacuum being of sufficient strength to force the Agrobacterium cells into intimate contact with 25 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) 3o 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. In a preferred method, the selectable marker gene is a herbicide resistance gene. A preferred herbicide resistance gene is a bar gene.
In yet another aspect, the present invention is a plant transformed according to the above-described methods of seedling transformation.
In yet another aspect, the present invention is a seed from a plant transformed according to the above-described methods of seedling transformation.
In yet another aspect, the present invention is a progeny plant from a seed obtained from a plant transformed according to the above-described methods of seedling transformation.
DETAILED DESCRIPTION
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 genes) 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 divots and monocots which can be successfully transformed by Agrobacterium-mediated gene transfer. In particular, leguminous plants are transformed at high rates of efficiency.
2o The transformation method described herein is generally accomplished by growing the Agrobacterium strain carrying a genes) 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.
The transformation of seedlings is accomplished through Agrobacterium-mediated gene transfer. Agrobacterium strains useful in the transformation of a _ WO 00/37663 PCT/US99/30972 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. In the transformation method described herein, the Agrobacterium strain can carry one plasmid with multiple genes) of interest. Alternatively, 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 Agrohacterium strain which provides the greatest number of transformed seedlings is selected. For leguminous plants, Agrobacterium tumefaciens EHA105, ASE1, and Gv3101 strains are preferably utilized. The genes) of interest can be transformed into the Agrobacterium by any means known in the art. For example, a DNA fragment modified to contain the genes) of interest can be inserted into the T-DNA of an Agrobacterium Ti plasmid which also contains genes required to generate the transformed state.
Other modifications of the Agrobacterium plasmid T-DNA can be made to 15 assist in the transformation process. For example, to distinguish seedlings which are successfully transformed, 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 2o transformed from non-transformed progeny. Exemplary selectable markers include a neomycin transferase gene or phosphinothricin acetyl transferase (bar) gene.
For example, a preferable selection marker is the bar gene encoding phosphinothricin acetyl transferase which confers resistance to phosphinothricin-based herbicides.
Preferably, the selection marker gene and genes) of interest are incorporated into any 25 vector suitable for use with transforming Agrobacterium strains. For example, 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 HindIII site of the T-DNA.
The bar gene encodes phosphinothricin acetyl transferase which confers resistance to 3o 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.
In the seedling transformation process, 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. Preferably, the seeds are sterilized in a solution of 30% sodium hypochlorite and 0.1 % Tween 20 for approximately 5 minutes 1 o and then thoroughly rinsed to remove the sterilizing solution. Preferably, 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 15 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.
Preferably, a treatment in concentrated sulfuric acid for approximately 10 minutes followed by thorough rinsing to remove the acid is utilized. Preferably, sterile double distilled or deionized water, or water with reduced oxidizable carbon following reverse 20 osmosis, ion exchange and/or activated charcoal treatment is used to rinse the seeds.
After pretreatment, the seeds are placed on a medium capable of supporting germination and subsequent growth of the seedlings. For example, the seeds can be placed on the surface of sterile filter paper or paper towels. Preferably, the seeds are spread on the surface of firm, sterile water agar in petri plates. The seeds are then 25 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 3o plants. In general, seedlings in which the radical has emerged and grown to at least about 1 cm are sufficiently mature. However, since plants develop at different rates, 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.
A few days prior to vacuum infiltration, the transforming Agrobacterium is subcultured on a general plated growth medium preferably containing appropriate antibiotics to distinguish transformed Agrobacterium cells. For example, to Agrobacterium tumefaciens EHA105 and Gv1301 carrying the bar gene are preferably cultured on YEP medium as defined in Example 1 containing rifampicin (20mg/I) and kanamycin (SO mg/I). The Agrobacterium cultures are grown at about 28°C
for about 2-3 days.
One day prior to the vacuum infiltration, a liquid Agrobacterium culture is 15 prepared by aseptically transfernng 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 forAgrobacterium EHA105 and Gv1301. The liquid cultures are grown under conditions which provide the Agrobacterium to reach exponential growth.
2o Preferably, 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.
To provide optimal conditions for transformation, the vacuum infiltration is preferably performed using the transforming Agrobacterium liquid culture in 25 exponential growth phase (OD6~ = 1.6). 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 15m1 liquid culture, pelleted by centrifugation, and then resuspended in 30m1 vacuum infiltration medium). Any plant growth medium capable of supporting the infiltration process and the Agrobacterium 3o within the plant while being compatible with plant growth can be used as the vacuum infiltration medium. More preferably, the vacuum infiltration medium comprises acetosyringone which induces the vir genes of the Agrobacterium. For leguminous plants, the vacuum infiltration medium defined in Examples 1 and 2 is preferably utilized.
To perform the vacuum infiltration, 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 to container to wet and partially cover the seedlings. For a standard petri plate, approximately 1 Oml 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.
15 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 2o 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.
Following vacuum infiltration, the Agrobacterium suspension is decanted, and the seedlings are blotted on sterile filter paper or blotting paper. The seedlings can then 25 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. Preferably, the plants are kept at a humidity, temperature, duration of photo period, and spectrum of light which favor plant growth. To increase the viability of the transformed plants and to improve the transformation efficiency, the seedlings are 30 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 - WO 00/37b63 PCT/US99/30972 can be used. For leguminous plants, 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.
Several methods known in the art can be used to distinguish the progeny exhibiting stable inheritance of the transgene. For transgenic plants wherein the genes) of interest results in a visible phenotypic change, the selection can be based upon visual examination of the progeny. For plant transformations involving 1 o Agrobacterium carrying plasmids containing a selectable marker gene, the appropriate selectable agent can 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 in the genome of the transformed plants.
The seedling transformation processes of the present invention are further 1 s illustrated in detail in the examples provided below. While these examples describe the invention, it is understood that modifications to the methods to optimize transformation of a specific plant are well within the skill of one in the art, and such modifications are considered within the scope of the invention.
Exarnnle 1: Transformation of M. truncatula by Vacuum-infiltration of Seedlings 2o 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 Prior to transformation, a modified version of the binary vector, pBI121 vector 25 (Clontech, Palo Alto, CA) was made by inserting a copy of a phosphinothricin acetyl transferase (bar) gene, under the control of a 35S promoter and octopine synthase 3' sequences, into the HindIII site of the T-DNA to create a plasmid called pBI121-bar (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.
30 "New A,grobacterium 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-bar 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.
Day 1:
The M. truncatula seed was sterilized and germinated as follows. The seeds were soaked in conc. HzS04 for approximately 10 min. The acid was removed, and the seeds were rinsed extensively in sterile cold double distilled water. This treatment was 1 o 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. 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 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 fox 15 days. This vernalization step was used to promote early flowering of M. truncatula.
About Day 12:
2o Agrobacterium tumefaciens EHA105 carrying pBI121-bar was subcultured for isolation onto a fresh agar plate containing YEP medium [1 liter: l Og Bacto-peptone (Difco, Detroit, MI); lOg yeast extract; Sg NaCI; 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.
About Day 14:
One loop, or approximately 3 large colonies of the Agrobacterium subculture was inoculated into about 15 ml TY liquid medium [1 liter: Sg tryptone, 3g yeast extract, 0.88g CaC12~2Hz0 at pH=7] containing rifampicin (20 mg/1) and kanamycin (50 mg/1) and incubated on a 28°C shaker at 250 rpm overnight.

- WO 00/376b3 PCT/US99/30972 About Day 15:
The Agrobacterium liquid culture was grown until an exponential phase (ODboo 1.6) was reached. The Agrobacterium cells were pelleted by centrifugation and resuspended in 30 ml of vacuum-infiltration medium (VIM) [1 liter: l Oml PDM
salt solution at 100X concentration (400m1 at 100X: 100g KN03; 12g NHQHZP04; 16g MgS04~7H20; 0.4g MnS04~H20; 0.2g H3B03; 0.008g CuS04~5H20; 0.04g KI; 0.004g CoC12~6Hz0; 0.04g ZnS04~7H20, and 0.004g Na2Mo04~2H20; filter sterilized and stored at room temperature); l Oml PDM iron and vitamins (1 liter at 100X:
O.Sg nicotinic acid; O.OSg pyridoxine~HCl; 0.5g thiamine~HCl; 100g myo-inositol;
1.5g 1o FeS04~7H20; and 2g Na2EDTA; filter sterilized; stored for immediate use at 4°C; long term storage at -20°C); 0.2g CaC12~H20; 1.5m1 of IOmM benzylaminopurine (BAP; 0.0565g in 0.15m12N NaOH and 24.85m1 double distilled H20 stored at 4°C);
0.05m1 of IOmM alpha-naphthaleneacetic acid (NAA; 0.0465g in 3m195% ethanol and 22m170% ethanol stored at 4°C); lOg sucrose; and O.ImI acetosyringone (AS; 1M in DMSO stored at -20°C ), wherein PDM salts, PDM iron and vitamins, CaCl2~Hz0 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, BAP, NAA, and AS are added).
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.
2o Approximately 1 Oml 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 l, a vacuum was drawn to 28mmHg for approximately 1 min, released rapidly, redrawn to 28mmHg for approximately 1 min, and finally released rapidly. In 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. In Treatment 3, a vacuum was drawn to 28mmHg for approximately 40 seconds, held for 20 seconds, and finally released rapidly. For all treatments, the 3o seedlings were then blotted on sterile filter paper or blotting paper and spread onto petri plates containing co-cultivation medium(CM) [1 liter: l Oml PDM salt solution at 10X

concentration; l Oml PDM iron and vitamins; 0.2g CaC12~H20; lOg sucrose; 7.Sg agar-agar (Sigma Chemical Co., St. Louis, MO); and 0.1 ml AS, wherein PDM salts, PDM
iron and vitamins, CaC12~H20, 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 I for approximately 2-3 days.
Table I: Growth Chamber Conditions Temp. Humidity photo-period Light ( C) ( /o) SET 20 90 16 hrs Top light only (Day & (8am-l2midnight) (1/2 fluorescent, no Night) incandescent) 4 light bulbs Sylvania 115W, F48T1 About Day 17:
1o The seedlings were washed twice with HZO 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 that are suitable for optimal plant growth, i.e., at 22-25°C with eighteen hour days.
About Day 40 The plants began to flower at approximately 24 days after planting in soil.
The resulting seeds were collected and germinated under conditions optimal for 2o 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. When the seedlings had a few leaves (approximately 15 days old), they were sprayed with 80 mg/L PPT ( dilution of 600mg/ml solution stored at -20°C), and the results are presented below.
1s In this study, 120 M. truncatula seedlings were vacuum infiltrated with Agrobacterium tumefaciens strain EHA105 carrying the pBI121-bar plasmid as described above. Three different treatments were used, and forty seedlings were infiltrated per treatment. The treatments varied only in the length of time of the vacuum infiltration treatment. The infiltrated plants were permitted to mature and set seed. The progeny seedlings were sprayed with Ignite (PPT) and resistant seedlings were further analyzed for the presence of the bar, nptll and B-Glucuronidase (GUS) genes. The results are shown in Table II . Fig. 2A, 2B, 3A, and 3B present data obtained in Experiments T-84-1, T84-2, and T84-3 of Table II. These results show the to efficiency of transformation ranging from 2.9% to 27.6% for the various transformation experiments.
Table II: Transformants Resulting from Transformation of M. truncatula via Infiltration of Seedlings with Agrobacterium Approx. No.
of Construct No. of No. No. of Seedlings% % indepen-and of Experi-Agrobac- Plants Seed Seedlingsresistanttransfor-dent trans-ment' teriumb InfiltratedCollectedGerminatedto PPT mation formants (*) T84-1 pB1121-bar /EHA 105 40 1326 329 22 6.7 78 T84-2 pBI121-bar /EHA 105 40 1263 302 16 5.3 ND' T84-3 pBI121-bar /EHA 105 40 1214 173 S 2.9 ND

T87-1 pGA482-bar /EHA 105 40 ND 217 10 4.6 86 T87-2 pGA482-bar /EHA105 40 ND 502 89 17.7 78 T87-3 pKYLX7IGus _ /EHA105 40 ND ND ** ND ND

T87-4 pBINmgfp-ER-Gar l EHA 40 ND 382 13 3.4 8 T8?-7 pBINmgfp-ER-bar and pKYLX7IGus40 ND 210 58 27.6 ND

/ EHA I
OS -T88 pSKI015/

Gv3101 70 ND 565 40 7.1 ND

a The following treatment methods were used: T84-l, T87-I, T87-2, T87-3, T87-4, T87-7, and T88, infiltration for I min (2X); T84-2, infiltration forl.5 min (2X);
T84-3, infiltration for 40 sec and hold 20 sec.
~ The following 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.
1: 285-297) or pSKI006 (http://www.salk.edu /LABS/pbio-w/). A. tumefaciens strain EHA105, carrying pBI121-bar or PKYLX7-Gus (Franklin et al. 1993 "Genetic 1 o transformation of green bean callus via Agrobacterium mediated DNA
transfer," Plant Cell Rep 12:74-79), or pBINmgfp-ER-bar, or pGA482-bar. A. tumefaciens strain Gv3 I O1 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 HindIIIlHpaI
fragment containing the 35S-bar-OCS 3'sequences cassette was excised from pSLJ525 and inserted between the HindIII and HpaI sites of pGA482 to create pGA482-bar.
The same HindIIIlHpaI fragment was used to produce pBI121-bar and pBINmgfp-ER-bar.
For each of these vectors, the HpaI site was converted to a HindIII site by the addition of an NpaI-HindIII linker and then the HindIII fragment was then inserted into the 2o HindIII site of pBI121 or pBINmgfp-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. USA 94: 2122-21271997) to create pBI121-bar and pBINmgfp-ER-bar respectively.
ND=not determined.
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 HindIII. The digested DNA was separated by electrophoresis 3o and blotted to nylon membranes. The membranes were probed with an internal DNA
fragment (450bp) of the bar gene labeled with 32P-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-bar 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. As expected 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 32P-dATP. The nptll gene is o carried on the pBI121-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 bar-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. Again, the non-transformed control plant does not contain DNA capable of hybridizing to this probe (Fig. 3A and 3B). The pBI121-bar 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 2o 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.
Seed from the transgenic plants were collected and germinated, and the resulting seedlings were sprayed with PPT herbicide. The progeny of the transgenic plants were also highly resistant to PPT, indicating that the transgenes are stable and 3o inherited by the following generation. As shown in Table III, 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 T1 generation.
Table III: Segregation Analysis (Phosphinothricin Resistance) of Progeny from a Selection of Transformants Prepared by Infiltration of Seedlings Number of plants Number of plantsChi-square Progeny resistant to sensitive to test from phosphinothricinphosphinothricinagainst (R) (S) transformants ratios (p)a 3R:1 1 SR:1 638:1 S S S

T84-1.14 83 0 -T84-1.19 44 20 ** _ _ T84-1.20 38 14 ** _ _ T84-2.6 44 21 * * _ _ a * * = p value of >_ 0.05; * = p value of z 0.01; - = p value of 5 0.01.
Example 2: Transformation of Soybean by Vacuum-infiltration of Seedling-s Soybean seedlings were transformed to incorporate the bar gene into the plant's genome using the transformation process of the present invention.
to Day 1:
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.
s The seeds were then spread 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 18-20°C for 15 days.
About Day 12:
Agrobacterium tumefaciens Gv3101 which carries the SKI015 vector with a 2o copy of the bar gene was subcultured for isolation onto a fresh agar plate containing YEP medium [1 liter: I Og Bacto-peptone (Difco); 1 Og yeast extract; Sg NaCI;
and 1 Sg Bacto-agar (Difco) at pH=6.8 without adjusting] containing rifampicin (10 mg/1), kanamycin (SO mg/1), carbenicillin (SO mg/1), and gentamicin (20 mg/1), and the subculture was incubated at approximately 28°C for about 2-3days.
About Day 14:
One loop, or approximately 3 large colonies of the Agrobacterium subculture was inoculated into about 15 ml TY liquid medium [1 liter: Sg tryptone, 3g yeast extract, 0.888 CaC12~2Hz0 at pH=7] containing rifampicin {10 mg/1), kanamycin (50 mg/1), carbenicillin (50 mg/1), and gentamicin (20 mg/1) and incubated on a 28°C
shaker at 250 rpm overnight.
About Day 15:
1 o The Agrobacterium liquid culture was grown until an exponential phase (OD6oo 1.6) was reached. The Agrobacterium cells were pelleted by centrifugation and resuspended in 30 ml of vacuum-infiltration medium (VIM) [1 liter: l Oml PDM
salt solution at lOX concentration; l Oml PDM iron and vitamins; 0.28 CaC12~H20;
1.$ml of IOmM benzylaminopurine (BAP; 0.05658 in O.15m12N NaOH and 24.85m1 double 15 distilled H20 stored at 4°C); O.OSmI of l OmM alpha-naphthaleneacetic acid (NAA;
0.04658 in 3ml 95% ethanol and 22m170% ethanol stored at 4°C); l Og sucrose; and O.lml acetosyringone (AS; 1M in DMSO stored at -20°C ), wherein PDM
salts, PDM
iion and vitamins, CaC12~H20 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, 20 BAP, NAA, and AS are added].
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 20m1 of the Agrobacterium suspension in the vacuum infiltration medium was added to the petri plate, a volume sufficient to wet and partially cover the 25 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 30 liter: lOml PDM salt solution at 10X concentration; lOml PDM iron and vitamins; 0.28 CaC12~H20; l Og sucrose; 7.Sg agar-agar (Sigma Chemical Co., St. Louis, MO);
and O.lml AS, wherein PDM salts, PDM iron and vitamins, CaCI2~H20, agar-agar, and sucrose are combined, the pH adjusted to 5.8 with KOH and autoclaved on liquid cycle for 20 rnin, 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.
Table IV: Growth Chamber Conditions Temp. Humidity photo-period Light ( C) ( /o) SET 20 90 16 hrs Top light only (Day & (8am-l2midnight) (1/2 fluorescent, no Night) incandescent) 4 light bulbs Sylvania 115W, F48T1 1o About Day 17:
The seedlings were washed twice with HZO 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 Da~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.
2o When the seedlings had one leaf, they were sprayed with 100 ml/1 PPT ( dilution of 600mg/ml solution in 0.1 % Tween 20 stored at -20°C), and the results are presented below.
In this study, 30 soybean seedlings were vacuum infiltrated with Agrobacterium tumefaciens strain Gv3101 carrying the SKI015-bar plasmid as described above.
Fourteen of the thirty infiltrated soybean seedlings survived, were transplanted into WO 00/37663 PC'T/US99/30972 pots, and allowed to mature and set seed. Approximately 700 progeny seeds were collected, germinated and grown in vermiculite. After the seedlings developed at least one true leaf, they were sprayed with PPT herbicide. PCR analysis was performed to confirm that the plants which survived the PPT herbicide carried the bar gene in their genomes. DNA was extracted from each surviving plant and used as a template in a PCR reaction with bar specific primers. A fragment of the expected size was amplified from the 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%.
1o In summary, the seedling transformation process described herein is more efficient and less labor intensive than previously reported methods. In addition, somatic alterations are avoided, and direct introduction of genetic material into elite lines is made possible. Large numbers of transgenic plants representing diverse integration events can be generated very rapidly and efficiently, and the transgenes are t 5 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 subsequent regeneration of Agrobacterium transformed cells is problematic.

Claims

WE CLAIM:

1. A method for direct plant transformation using leguminous seedlings and Agrobacterium comprising:
(a) contacting at least one leguminous seedling with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling said Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment to said seedling;
(b) applying a vacuum to said seedling 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 seedling such that said Agrobacterium cells transfer said T-DNA to cells of said seedling at a second time, wherein said fast and second time are the same or different.

2. The method of Claim 1, further comprising:
(c) allowing said seedling to grow to maturity and set seed;
(d) germinating said seed to form progeny; and {e) selecting for progeny expressing said transferred gene.

3. The method of Claim 1, wherein said vector comprises a selectable marker gene.

4. The method of Claim 2, wherein said vector comprises a selectable marker gene.

5. The method of Claim 3, wherein said selectable marker gene comprises a herbicide resistance gene.

6. The method of Claim 4, wherein said selectable marker gene comprises a herbicide resistance gene.

7. The method of Claim 5, wherein said herbicide resistance gene comprises a bar gene.

8. The method of Claim 6, wherein said herbicide resistance gene comprises a bar gene.

9. A method for direct plant transformation using leguminous seedlings and Agrobacterium comprising:
(a) contacting at least one leguminous seedling with a mixture of Agrobacterium cells, said mixture comprising cells from a Agrobacterium strain harboring a first vector wherein said first vector enabling said cells to transfer T-DNA containing a first DNA
fragment to said seedling and cells from the same Agrobacterium strain harboring a second vector wherein said second vector enabling said cells to transfer T-DNA
containing a second DNA fragment to said seedling; and (b) applying a vacuum to said seedling 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 seedling such that said Agrobacterium cells transfer T-DNA to cells of said seedling at a second time, wherein said first and second time are the same or different.

10. The method of Claim 9, further comprising:
(c) allowing said seedling to grow to maturity and set seed;
(d) germinating said seed to form progeny; and (e) selecting for progeny expressing said transferred gene.

11. The method of Claim 9, wherein said vector comprises a selectable marker gene.

12. The method of Claim 10, wherein said vector comprises a selectable marker gene.

13. The method of Claim 11, wherein said selectable marker gene comprises a herbicide resistance gene.

14. The method of Claim 12, wherein said selectable marker gene comprises a herbicide resistance gene.

15. The method of Claim 13, wherein said herbicide resistance gene comprises a bar gene.

16. The method of Claim 14, wherein said herbicide resistance gene comprises a bar gene.

17. A method for direct plant transformation using leguminous seedlings and Agrobacterium comprising:
(a) contacting at least one leguminous seedling with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling said Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment and a selectable marker gene to said seedling;
(b) applying a vacuum to said seedling 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 seedling such that said Agrobacterium cells transfer said T-DNA to cells of said seedling at a second time, wherein said first and second time are the same or different;
(c) allowing said transformed seedling to grow to maturity and set seed;
(d) germinating said seed to form progeny;
(e) exposing said progeny to an agent enabling detection of selectable marker gene expression;
(f) selecting for progeny expressing said selectable marker gene and at least one gene, said expression of said selectable marker gene and at least one gene indicating gene transfer.

18. The method of Claim 17, wherein said selectable marker gene comprises a herbicide resistance gene.

19. The method of Claim 18, wherein 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. Seed from 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.

22. A progeny plant from a seed obtained from 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.

23. A method for direct plant transformation using Medicago truncatula seedlings and Agrobacterium comprising:
(a) contacting at least one Medicago truncatula seedling with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling said Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment to said Medicago truncatula seedling;
(b) applying a vacuum to said Medicago truncatula seedling 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 Medicago truncatula seedling such that said Agrobacterium cells transfer said T-DNA to cells of said Medicago truncatula seedling at a second time, wherein said first and second time are the same or different.

24. The method of Claim 23, further comprising:
(c) allowing said Medicago truncatula seedling to grow to maturity and set seed;
{d) germinating said seed to forth progeny; and (e) selecting for progeny expressing said transferred gene.

25. The method of Claim 23, wherein said vector comprises a selectable marker gene.

26. The method of Claim 24, wherein said vector comprises a selectable marker gene.

27. The method of Claim 25, wherein said selectable marker gene comprises a herbicide resistance gene.

28. The method of Claim 26, wherein said selectable marker gene comprises a herbicide resistance gene.

29. The method of Claim 27, wherein said herbicide resistance gene comprises a bar gene.

30. The method of Claim 28, wherein said herbicide resistance gene comprises a bar gene.

31. A method for direct plant transformation using Medicago truncatula seedlings and Agrobacterium comprising:
(a) contacting at least one Medicago truncatula seedling with a mixture of Agrobacterium cells, said mixture comprising cells from Agrobacterium strain harboring a first vector wherein said first vector enabling said cells to transfer T-DNA
containing a first DNA fragment to said Medicago truncatula seedling and cells from the same Agrobacterium strain harboring a second vector wherein said second vector enabling said cells to transfer T-DNA containing a second DNA fragment to said Medicago truncatula seedling; and (b) applying a vacuum to said Medicago truncatula seedling 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 Medicago truncatula seedling such that said Agrobacterium cells transfer T-DNA to cells of said Medicago truncatula seedling at a second time, wherein said first and second time are the same or different.

32. The method of Claim 31, further comprising:
(c) allowing said Medicago truncatula seedling to grow to maturity and set seed;
(d) germinating said seed to form progeny; and (e) selecting for progeny expressing said transferred gene.

33. The method of Claim 31, wherein said vector comprises a selectable marker gene.

34. The method of Claim 32, wherein said vector comprises a selectable marker gene.

35. The method of Claim 33, wherein said selectable marker gene comprises a herbicide resistance gene.

36. The method of Claim 34, wherein said selectable marker gene comprises a herbicide resistance gene.

37. The method of Claim 35, wherein said herbicide resistance gene comprises a bar gene.

38. The method of Claim 36, wherein said herbicide resistance gene comprises a bar gene.

39. A method for direct plant transformation using Medicago truncatula seedlings and Agrobacterium comprising:
(a) contacting at least one Medicago truncatula seedling with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling said Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment and a selectable marker gene to said Medicago truncatula seedling;
(b) applying a vacuum to said Medicago truncatula seedling 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 Medicago truncatula seedling such that said Agrobacterium cells transfer said T-DNA to cells of said Medicago truncatula seedling at a second time, wherein said first and second time are the same or different;
(c) allowing said transformed Medicago truncatula seedling to grow to maturity and set seed;
(d) germinating said seed to form progeny;
(e) exposing said progeny to an agent enabling detection of selectable marker gene expression;
(f) selecting for progeny expressing said selectable marker gene and at least one gene, said expression of said selectable marker gene and at least one gene indicating gene transfer.

40. The method of Claim 39, wherein said selectable marker gene comprises a herbicide resistance gene.

41. The method of Claim 44, wherein 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. Seed from 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.

44. A progeny plant from a seed obtained from 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.