Refined plant transformation

BACKGROUND OF THE INVENTION

0001The ability to transform plants by integrating and expressing desirable polynucleotides in plant cells makes it possible to efficiently introduce agronomic and quality traits into a variety of plant species. Transgenic plants that are produced by current transformation methods, however, require extensive tissue culture manipulations, which are time consuming and species specific. Furthermore, such methods do not only integrate the desirable polynucleotide(s) into a plant's genome, but also additional and superfluous nucleic acids. When making a genetically engineered food, the superfluous nucleic acids may be undesirable because they are from non-food sources, such as viruses and bacteria and are, therefore, undesirable.

0002Existing plant transformation methods rely on the use of <i>Agrobacterium</i> for DNA transfer. These methods typically comprise (1) preparing tissue explants, (2) infecting explants with at least one disarmed <i>Agrobacterium</i> strain, (3) culturing and selecting the transformed plant cells on tissue culture media, and (4) inducing proliferation and subsequent regeneration to generate whole plants. Examples of these methods are described in <patcit id="pcit0001" dnum="US5591616A"><text>U.S. Patent Nos. 5,591,616</text></patcit>, <patcit id="pcit0002" dnum="US6051757A"><text>6,051,757</text></patcit>, <patcit id="pcit0003" dnum="US5164310A"><text>5,164,310</text></patcit>, and <patcit id="pcit0004" dnum="US5693512A"><text>5,693,512</text></patcit>, and <patcit id="pcit0005" dnum="EP0672752A1"><text>EP 0 672 752 A1</text></patcit>. However, explant preparation is a laborious process that requires extensive resources, especially for many monocotyledonous plant species including maize, wheat, barley, and oats.

0003Furthermore, the subsequent process of proliferation and regeneration is also very laborious, taking at least 12 months to develop a primary transformed plant. Since different plants require different concentrations of salts, minerals, and hormones, including auxins and cytokinins, for proliferation and regeneration, the applicability of typical transformation methods is limited to one species or only a few cultivars of one species.

0004Even by optimizing cultivar-specific transformation methods, successful transformation has been accomplished for only a very few cultivars of important crop species, such as for the maize inbred lines H99, Oh43, and B73, the spring wheat variety Bobwhite, and the cotton cultivar Coker 312. The introduction of foreign DNA into elite germplasm often requires the transformation of inferior cultivars followed by conventional multi-year breeding programs to introgress the DNA into the desired material.

0005Tissue culture manipulations can be avoided by either vacuum infiltrating plants with an <i>Agrobacterium</i> suspension or emerging such plants in suspensions that also contain approximately 0.05% Silwet L-77 (<nplcit id="ncit0001" npl-type="s"><text>Bechtold et al., Acad Sci Paris Life Sci 316: 1194-1199, 1993</text></nplcit>; <nplcit id="ncit0002" npl-type="s"><text>Clough &amp; Bent, Plant J 16: 735-743, 1998</text></nplcit>). However, this method is only applicable to the model plant systems <i>Arabidopsis thaliana, Arabidopsis lasiocarpa,</i> and <i>Raphanus sativus.</i> Transgenic plants can also be obtained for a fourth plant species, <i>Medicago trunculata,</i> by vacuum infiltrating seedling with <i>Agrobacterium</i> suspensions.

0006Such <i>in planta</i> transformation systems are of limited utility, however, and not applicable to commercially relevant crop plants. Efforts to broaden such applicability to encompass a larger variety of crops have failed because of the inaccessibility of those crops to <i>Agrobacterium-</i>mediated transformation, and/or the resultant, detrimental physiological responses, such as flower abscission and <i>Agrobacterium-induced</i> necrosis.

0007Alternative transformation systems include direct DNA delivery systems like particle bombardment (<patcit id="pcit0006" dnum="US4945050A"><text>U.S. Patent No. 4,945,050</text></patcit>), polyethylene glycol treatment (<patcit id="pcit0007" dnum="US6143949A"><text>U.S. Patent No. 6,143,949</text></patcit>), microinjection (<patcit id="pcit0008" dnum="US4743548A"><text>U.S. Patent No. 4,743,548</text></patcit>), whiskers (<patcit id="pcit0009" dnum="US5302523A"><text>U.S. Patent No. 5,302,523</text></patcit>), and electroporation (<patcit id="pcit0010" dnum="US5284253A"><text>U.S. Patent No. 5,284,253</text></patcit>). Whereas DNA transfer mediated by <i>Agrobacterium</i> is often limited to one to three copies of foreign DNA, direct DNA delivery systems usually result in the transfer of many more copies, which may integrate randomly throughout the plant genome. The unnecessary abundance of insertions is undesirable and may negatively affect the plant genome's integrity.

0008Sonication was shown to greatly enhance the efficiency of both <i>Agrobacterium-mediated</i> transformation and direct DNA delivery (<patcit id="pcit0011" dnum="US5693512A"><text>U.S. Patent 5,693,512</text></patcit>). The ultrasound vibrations are believed to disrupt cell walls and thereby facilitate foreign DNA transfer. Sonication reduces the viability of tissue explants, and any increase in transformation frequency may be compromised by an increase in non-viable or dying plants.

0009These, as well as more conventional transformation methods, introduce a variety of viral and bacterial genetic elements into plant cells. At least four different genetic elements, derived from bacteria, are typically used to transform plants (<nplcit id="ncit0003" npl-type="s"><text>During, Transgenic Research 3: 138-40, 1994</text></nplcit>). Such elements include regulatory sequences such as promoters and terminators to promote appropriate transgene expression in plants. An example of a frequently used foreign promoter is the 35S "super" promoter of Cauliflower Mosaic Virus (CaMV), which is able to not only induce high levels of expression of the transgenes but also enhance the expression of native genes in its vicinity (<nplcit id="ncit0004" npl-type="s"><text>Weigel et al., Plant Physiol., 122: 1003-13, 2000</text></nplcit>).

0010Other strong viral promoters include those from rice tungro bacilliform virus, maize streak virus, cassava vein virus, mirabilis virus, peanut chlorotic streak caulimovirus, figwort mosaic virus and chlorella virus. Other frequently used promoters are derived from bacterial species and include the promoters of the nopaline synthase and octopine synthase gene. Only a few strong and constitutive promoters are derived from food sources. Examples of such promoters are the promoters of the maize Ubiquitin-1 gene (<patcit id="pcit0012" dnum="US6054574A"><text>U.S. Patent 6,054,574</text></patcit>; and <patcit id="pcit0013" dnum="WO0194394A"><text>WO 01/94394</text></patcit>), the sugarcane Ubiquitin-4 gene (<patcit id="pcit0014" dnum="US0046415A" dnum-type="L"><text>U.S. Patent application 02/0046415</text></patcit>), and the potato Ubiquitin-7 gene (<patcit id="pcit0015" dnum="US6448391B1"><text>Garbarino et al., U.S. Patent 6,448,391 B1, 2002</text></patcit>). The applicability of most other plant promoters is limited because of low activity, tissue specificity, and/or poor developmental regulation. Typical terminators are those associated with the nopaline synthase and octopine synthase genes from <i>Agrobacterium.</i>

0011Also required for transformation is the <i>Agrobacterium-derived</i> transfer DNA, <i>i.e.,</i> the T-DNA, which transfers desired polynucleotide(s) from <i>Agrobacterium</i> into plant cell genomes. Thus, transgenic plants of the conventional art contain much superfluous foreign DNA. Furthermore, the infidelity of DNA transfer can result in co-integration of bacterial plasmid sequences that are adjacent to the T-DNA. In fact, about 75% of transformation events in plants such as tomato, tobacco, and potato may contain such superfluous plasmid backbone DNA (<nplcit id="ncit0005" npl-type="s"><text>Kononov et al., Plant J. 11: 945-57, 1997</text></nplcit>). The presence of backbone sequences is undesirable because they contain bacterial origins of replication and/or encode for antibiotic resistance genes.

0012Thus, there is a need for accelerated and species-independent methods for transferring and expressing desired polynucleotides into plant cells and genomes. There is also a need to limit the co-transfer of superfluous, undesirable DNA, if the target plant is a food crop. Such methods are provided herein. To optimize DNA transfer from <i>Agrobacterium</i> to individual plant cell nuclei, plant tissues such as seedlings are agitated in an <i>Agrobacterium</i> suspension. To optimize the subsequent integration of the transferred DNAs into the genome of plant cell nuclei, the plant tissues are exposed to chemicals that induce double strand breaks. Vectors are used that are designed to limit the transfer of undesirable DNA.

SUMMARY OF THE INVENTION

0013According to the present disclosure, a method ("method 1 ") for producing a transgenic plant is provided. The method comprises (a) agitating a solution comprising a germinating plant seedling, or explant thereof, and at least one <i>Agrobacterium</i> strain that harbors a plasmid vector carrying a desired polynucleotide; (b) cultivating the seedling to produce a plant; and (c) screening the plant to determine if the desired polynucleotide is integrated into the genome of at least one cell of the plant, wherein the plant is stably transformed, and wherein the step of agitating the solution does not comprise sonication.

0014<nplcit id="ncit0006" npl-type="b"><text>Rohini and Sankara Rao (Annals of Botany 86; 2000; 1043-1049</text></nplcit>) describe a practical approach for realizing transgenic plants of Safflower (<i>Carthamus tinctorius L.</i>). According to said report, a gene transfer system was developed that ensured recovery of whole plant transformants. Embryo axes of germinating seeds with one the cotyledons removed were pricked with a sterile needle and infected by gentle agitation for 10 minutes.

0015In <patcit id="pcit0016" dnum="WO9856932A"><text>WO 98/56932</text></patcit> a process for producing genetically modified plant material is disclosed. Here, DNA sequences (plant material) are subjected to an <i>Agrobacterium</i>-mediated transfer, which transfer is carried out in an aerated liquid culture medium in which the plant material is cultivated. The culture medium submerging said plant material is generally agitated or otherwise moved.

0016The authors of <patcit id="pcit0017" dnum="WO9849332A"><text>WO 98/49332</text></patcit> disclose an <i>Agrobacterium</i>-mediated transformation of sorghum. This process includes infection, co-culture, optional resting, selection, and regeneration. In the infection step the cells to be transformed are contacted, exposed to a suspension of Agrobacterium.

0017Another plant transformation method is disclosed in <patcit id="pcit0018" dnum="US6162965A"><text>US-6,162,965</text></patcit>. Accordingly, an improved process of Agrobacterium-transformation of plants, particularly of Gramineae is provided, which process uses inhibition of <i>Agrobacterium</i>-induced necrosis.

0018According to the present invention, a method for producing a transgenic plant is provided, comprising (a) vortexing a solution comprising a germinating plant seedling and at least one Agrobacterium strain that harbors a vector carrying a desired polynucleotide; (b) transferring said Agrobacterium-transformed seedling to soil, and exposing said transformed seedling to conditions that promote growth; and (c) screening said plants to determine if said desired polynucleotide is integrated into the genome of at least one cell of said plant, wherein a plant comprising said desired polynucleotide in said genome is a transgenic plant.

0019In preferred embodiments of the present method said germinating plant seedling are from a monocotyledenous plant or from a dicotyledenous plant. In other preferred embodiments the expression of said desired polynucleotide confers a trait to plants that comprise said desired polynucleotide in their genome, wherein said trait is selected from the group consisting of increased drought tolerance, enhanced cold and frost tolerance, improved vigor, enhanced color, enhanced health and nutritional characteristics, improved storage, enhanced yield, enhanced salt tolerance, enhanced heavy metal tolerance, increased disease tolerance, increased insect tolerance, increased water-stress tolerance, enhanced sweetness, improved vigor, improved taste, improved texture, decreased phosphate content, increased germination, increased micronutrient uptake, improved starch composition, improved flower longevity, and production of novel proteins or peptides.

0020In yet another preferred embodiment said desired polynucleotide is selected from the group consisting of a gene or part thereof, the 5'- untranslated region of said gene, the 3'-untranslated region of said gene, the leader sequence associated with said gene, or the trailer sequence associated with said gene, or combinations of such sequences, wherein the orientation of any of said sequences is either in the sense or antisense orientation.

0021In another preferred embodiment the step of screening comprises detecting the presence of said desired polynucleotide in cells of said transgenic plant.

0022According to another preferred embodiment the method further comprises producing progeny from said transgenic plant and detecting the presence of said desired polynucleotide in cells of said progeny.

0023In another preferred embodiment said solution is vortexed from 60 seconds to several hours; or for 5 minutes to 30 minutes.

0024In another preferred embodiment said seedling is exposed to chemical prior to, during, and/or after infection with a chemical that induces double stranded DNA breaks in the genome of a cell of said plant tissue or double-stranded breaks are induced in a genome in a cell of said plant seedling by exposing said plant seedling to ionizing radiation or heavy ions prior to, during, and/or after infection.

0025In further embodiments of the present invention said method further comprises, when said germinating plant seedling are from a monocotyledenous plant, said monocotyledenous plant is selected from the group consisting of turfgrass, wheat, maize, oat, barley, wherein preferably said turfgrass is selected from the group consisting of Agrostis spp., Poa pratensis, Lolium spp., Kentucky Bluegrass And Perennial Ryegrass Mix; Festuca arundinacea, Festuca rubra commutata, Cynodon dactylon, Pennisetum clandestinum, Stenotaphrum secundatum,Zoysia japonica, and Dichondra micrantha; or when said germinating plant seedling are from a dicotyledenous plant, said dicotyledenous plant is selected from the group consisting of cotton, tobacco, Arabidopsis, tomato, lettuce, pepper; or when said desired polynucleotide is selected from the group consisting of a gene or part thereof, the 5'- untranslated region of said gene, the 3'-untranslated region of said gene, the leader sequence associated with said gene, or the trailer sequence associated with said gene, or combinations of such sequences, wherein the orientation of any of said sequences is either in the sense or antisense orientation.

0026According to another preferred embodiment said desired polynucleotide is operably linked to a promoter and a terminator.

0027According to yet another preferred embodiment, when said seedling is exposed to chemical prior to, during, and/or after infection with a chemical that induces double stranded DNA breaks in the genome of a cell of said plant tissue, said chemical is selected from the group consisting of methyl methane sulfonate, HO-endonuclease, bleomycin, neocarzinostatin, camptothecan, and cisplatin.

0028According to the present invention a method for producing a transgenic plant is provided, comprising (a) vortexing a solution comprising a germinating plant seedling and at least one Agrobacterium strain that harbors a vector carrying a desired polynucleotide; (b) (i) producing callus from said transformed seedling; (ii) inducing shoot and root formation from said callus to produce a plantlet; (c) growing said plantlets into plants; and (d) screening said plants to determine if said desired polynucleotide is incorporated into the genome of at least one cell of said plant, wherein a plant comprising said desired polynucleotide in said genome is a transgenic plant.

0029In preferred embodiments of the present invention said method further comprises that said germinating plant seedling are from a monocotyledenous plant; or that said germinating plant seedling are from a dicotyledenous plant; or that expression of said desired polynucleotide confers a trait to plants that comprise said desired polynucleotide in their genome, wherein said trait is selected from the group consisting of increased drought tolerance, enhanced cold and frost tolerance, improved vigor, enhanced color, enhanced health and nutritional characteristics, improved storage, enhanced yield, enhanced salt tolerance, enhanced heavy metal tolerance, increased disease tolerance, increased insect tolerance, increased water-stress tolerance, enhanced sweetness, improved vigor, improved taste, improved texture, decreased phosphate content, increased germination, increased micronutrient uptake, improved starch composition, improved flower longevity, and production of novel proteins or peptides.

0030According to another preferred embodiment said method further comprises that said desired polynucleotide is selected from the group consisting of a gene or part thereof, the 5'-untranslated region of said gene, the 3'-untranslated region of said gene, the leader sequence associated with said gene, or the trailer sequence associated with said gene; or that said desired polynucleotide is operably linked to a promoter and a terminator; or that said vector comprises (a) a T-DNA or a P-DNA that comprises (i) said desired polynucleotide, and (ii) a selectable marker gene operably linked to a terminator that is not naturally expressed in plants ; and (b) a backbone integration marker gene, wherein said desired polynucleotide and said selectable marker gene are positioned between said border sequences of said T-DNA or between said border-like sequences of said P-DNA, and wherein said backbone integration marker gene is not positioned within said T-DNA or within said P-DNA.

0031According to yet another preferred embodiment the step of screening comprises detecting the presence of said desired polynucleotide in cells of said transgenic plant; or the method further comprises producing progeny from said transgenic plant and detecting the presence of said desired polynucleotide in cells of said progeny.

0032According to yet another preferred embodiment said method comprises vortexing said from 60 seconds to several hours; or for 5 minutes to 30 minutes.

0033According to yet another embodiment said seedling is exposed to chemical prior to, during, and/or after infection with a chemical that induces double stranded DNA breaks in the genome of a cell of said plant tissue; or double-stranded breaks are induced in a genome in a cell of said plant seedling by exposing said plant seedling to ionizing radiation or heavy ions prior to, during, and/or after infection.

0034In preferred embodiments of the present invention said method further comprises, when said germinating plant seedling are from a monocotyledenous plant, said monocotyledenous plant is selected from the group consisting of turfgrass, wheat, maize, oat, barley, wherein preferably said turfgrass is selected from the group consisting of Agrostisspp., Poa pratensis, Lolium spp., Festuca arundinacea, Festuca rubra commutata, Cynodon dactylon, Pennisetum clandestinum, Stenotaphrum secundatum, Zoysia japonica, and Dichondra micrantha; or when said germinating plant seedling are from a dicotyledenous plant, said dicotyledenous plant is selected from the group consisting of cotton, tobacco, Arabidopsis, tomato, lettuce, pepper; or when said desired polynucleotide is operably linked to a promoter and a terminator, the sequences of said promoter and said terminator naturally occur in the genome of plants.

BRIEF DESCRIPTION OF THE DRAWINGS

0035<dl id="dl0001" compact="compact"><dt>Figure 1:</dt><dd>Schematic flowchart of the inventive methods and compositions.</dd><dt>Figure 2:</dt><dd>Alignment of the <i>CAH</i> gene from <i>Myrothecium verrucaria</i> with a new cyanamide tolerance gene isolated from <i>Aspergillus</i> (CAH-H1) and a non-functional yeast <i>CAH</i> homolog (CAH-H2)</dd><dt>Figure 3:</dt><dd>Alignment between a new ubiquitin-like promoter (UbiN) and the corresponding partial sequence of the sugarcane Ubiquitin-4 promoter</dd></dl>

DETAILED DESCRIPTION

0036The present disclosure provides methods for producing transgenic plants and transformation vectors.

0037The present disclosure can be applied to many species of plants, including those that are difficult to transform by applying conventional transformation methods. The present disclosure provides methods for integrating a desired polynucleotide into a plant genome to alter the expression of a plant trait, or to produce a product, such as a pharmaceutically relevant or important protein, and methods for readily selecting and screening for cells and plants that comprise the desired polynucleotide in their genome.

0038In particular, the transformation methods include transforming germinating seedling with a vector comprising a desired polynucleotide, and then either (1) planting the seedling directly into soil; (2) transferring the seedling to culture media, without inducing a callus phase, and then planting the seedling directly into soil; or (3) transferring the seedling to culture media, inducing a callus phase, and shoot and root formation, and then planting the seedling directly into soil.

0039<figref idref="f0001">Figure 1</figref> illustrates such methods. Plant tissues (<figref idref="f0001">Figure 1</figref>, box "(a)") may be transformed by vortexing (<figref idref="f0001">Figure 1</figref>, box "(c)"), and then planted directly into soil (<figref idref="f0001">Figure 1</figref>, box "(e) ") and then grown into the desired transgenic plant (<figref idref="f0001">Figure 1</figref>, box "(h)").

0040Alternatively, after vortexing (<figref idref="f0001">Figure 1</figref>, box "(c)"), the transformed plant tissues may be nurtured on tissue culture medium (<figref idref="f0001">Figure 1</figref>, box "(d)"), planted directly into soil (<figref idref="f0001">Figure 1</figref>, box "(e)"), and then grown into the desired transgenic plant (<figref idref="f0001">Figure 1</figref>, box "(h)").

0041Finally, after vortexing, and nurturing on tissue culture medium, the plant tissue can be induced to undergo callus formation (<figref idref="f0001">Figure 1</figref>, box "(g)"), and shoot and root growth, prior to being grown into the desired transgenic plant.

0042The <i>Agrobacterium</i> vector that can be used in any one of such methods is illustrated in <figref idref="f0001">Figure 1</figref>, box "("f)". The vector may, or may not, include a selectable/screenable marker for identifying transformed, transgenic plants, parts thereof, or transformed cells. For instance, when a plant tissue, such as a seedling, is transformed according to the present methods and planted directly into soil without the induction of a callus phase, the vector does not need to contain a selectable marker gene. Alternatively, after culturing the transformed seedling, or other plant tissue, on callus-inducing tissue culture media, one may select for successful transformants by including in the tissue media, a substance(s) to which only plant cells that contain a selectable marker gene are resistant to, or can tolerate.

0043The present invention uses terms and phrases that are well known to those practicing the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, microbial culture, cell culture, tissue culture, transformation, transfection, transduction, analytical chemistry, organic synthetic chemistry, chemical syntheses, chemical analysis, and pharmaceutical formulation and delivery. Generally, enzymatic reactions and purification and/or isolation steps are performed according to the manufacturers' specifications. The techniques and procedures are generally performed according to conventional methodology (<nplcit id="ncit0007" npl-type="b"><text>Sambrook &amp; Russel, MOLECULAR CLONING: A LABORATORY MANUAL, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001</text></nplcit>).

0044<i><b>Agitation:</b></i> "agitiation" means to cause movement with violence or sudden force. With respect to the present invention, "agitation" refers to a violent and sudden physical vibration of a solution. "Agitation," as used herein, does not encompass the disruption of a solution by treatment with high-frequency sound waves, such as those produced by sonication.

0045<i><b>Agrobacterium:</b></i> as is well known in the field, <i>Agrobacteria</i> that are used for transforming plant cells, are disarmed and virulent derivatives of, usually, <i>Agrobacterium tumefaciens</i> or <i>Agrobacterium rhizogenes</i> that contain a vector. The vector typically contains a desired polynucleotide that is located between the borders of a T-DNA or, according to the present invention, between the border-like sequences of a "plant-DNA" ("P-DNA"), see definition below, which border (like) sequences are capable of transferring the desired polynucleotide into a plant genome.

0046<b><i>Border and Border-like sequences:</i></b> "border sequences" are specific <i>Agrobacterium-derived</i> sequences. Typically, a left border sequence and a right border sequence flank a T-DNA and function as recognition sites for <i>virD2</i>-catalyzed nicking reactions. The sequences of the left and right border sequences may or may not be identical. Their sequences may or may not be inverted repeats of one another. Such activity releases nucleic acid that is positioned between such borders. See Table 1 below for examples of border sequences. The released nucleic acid, complexed with virD2 and virE2, is targeted to plant cell nuclei where the nucleic acid is often integrated into the genome of the plant cell. Usually, two border sequences, a left-border and a right-border, are used to integrate a nucleotide sequence that is located between them into another nucleotide sequence. It is also possible to use only one border, or more than two borders, to accomplish integration of a desired nucleic acid in such fashion.

0047A "border-like" sequence is isolated from a plant, and functions like the border sequence of an <i>Agrobacterium-derived</i> T-DNA. That is, a border-like sequence promotes and facilitates the transfer of a polynucleotide to which it is linked from Agrobacterium to plant cell nuclei, and the subsequent stable integration of this polynucleotide into the plant genome. A plant-DNA, <i>i.e</i>., P-DNA, is delineated by border-like sequences.

0048A border-like sequence of a P-DNA is between 5-100 bp in length, 10-80 bp in length, 15-75 bp in length, 15-60 bp in length, 15-50 bp in length, 15-40 bp in length, 15-30 bp in length, 16-30 bp in length, 20-30 bp in length, 21-30 bp in length, 22-30 bp in length, 23-30 bp in length, 24-30 bp in length, 25-30 bp in length, or 26-30 bp in length.

0049The border-like sequences can be isolated from any plant. See SEQ ID NO.: 3 for a DNA fragment isolated from potato that contains, at either end, a border-like sequence. Thus, P-DNA border-like sequences of use for the present invention are isolated from a plant. A P-DNA border-like sequence is not identical in nucleotide sequence to any known <i>Agrobacterium</i>-derived T-DNA border sequence. Thus, a P-DNA border-like sequence may possess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides that are different from a T-DNA border sequence from an <i>Agrobacterium</i> species, such as <i>Agrobacterium tumefaciens</i> or <i>Agrobacterium rhizogenes.</i> That is, a P-DNA border, or a border-like sequence has at least 95%, at least 90%, at least 80%, at least 75%, at least 70%, at least 60% or at least 50% sequence identity with a T-DNA border sequence from an <i>Agrobacterium</i> species, such as <i>Agrobacterium tumefaciens</i> or <i>Agrobacterium rhizogenes,</i> but not 100% sequence identity. As used herein, the descriptive terms "P-DNA border" and "P-DNA border-like" are exchangeable.

0050A native P-DNA border sequence is greater than or equal to 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51 % or 50% similar in nucleotide sequence to a <i>Agrobacterium</i> a T-DNA border sequence. A border-like sequence can, therefore, be isolated from a plant genome and be modified or mutated to change the efficiency by which they are capable of integrating a nucleotide sequence into another nucleotide sequence. Other polynucleotide sequences may be added to or incorporated within a border-like sequence. Thus, a P-DNA left border or a P-DNA right border may be modified so as to possess 5'- and 3'-multiple cloning sites, or additional restriction sites. A P-DNA border sequence may be modified to increase the likelihood that backbone DNA from the accompanying vector is not integrated into the plant genome.

0051Table 1 below depicts the sequences of known T-DNA border sequences and sequences identified herein as border-like sequences. By aligning sequences with known T-DNA border sequences, new "border-like" sequences were identified that existed in plant genomes. The "potato" border-like sequences of Table 1 were isolated herein, using degenerate primers in polymerase chain reactions on potato genomic template DNA. The use of such potato P-DNA border-like elements for transferring a desired polynucleotide into the genome of a plant cell is also encompassed. <tables id="tabl0001" num="0001"><table frame="all"><title><b>Table 1. "Border" and "Border-Like" sequences</b></title><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="84mm" /><colspec colnum="2" colname="col2" colwidth="47mm" /><thead><row><entry namest="col1" nameend="col2" align="left" valign="top"><b><i>Agrobacterium</i> T-DNA borders</b></entry></row></thead><tbody><row><entry>TGACAGGATATATTGGCGGGTAAAC (SEQ ID NO.12)</entry><entry>Agro. nopaline strains (RB)</entry></row><row><entry>TGGCAGGATATATTGTGGTGTAAAC (SEQ ID N0.13)</entry><entry>Agro. nopaline strains (LB)</entry></row><row><entry>TGGCAGGATATATACCGTTGTAATT (SEQ ID NO.14)</entry><entry>Agro. octopine strains (RB)</entry></row><row><entry>CGGCAGGATATATTCAATTGTAATT (SEQ ID NO.15)</entry><entry>Agro. octopine strains (LB)</entry></row><row><entry>TGGTAGGATATATACCGTTGTAATT (SEQ ID NO.16)</entry><entry>LB mutant</entry></row><row><entry>TGGCAGGATATATGGTACTGTAATT (SEQ ID NO.17)</entry><entry>LB mutant</entry></row><row><entry>YGRYAGGATATATWSNVBKGTAAWY (SEQ ID NO.18)</entry><entry>Border motif</entry></row><row><entry namest="col1" nameend="col2" align="left"><b>Border-like sequences</b></entry></row><row><entry>TGACAGGATATATGGTAATGTAAAC (SEQ ID NO.19)</entry><entry>potato (border-like sequence)*</entry></row><row><entry>TGGCAGGATATATACCGATGTAAAC (SEQ ID NO.20)</entry><entry>potato (border-like sequence)*</entry></row></tbody></tgroup><tgroup cols="2" rowsep="0"><colspec colnum="1" colname="col1" colwidth="84mm" /><colspec colnum="2" colname="col2" colwidth="47mm" /><tbody><row><entry namest="col1" nameend="col2" align="justify">Y=C or T; R=A or G; K= G or T; M=A or C; W = A or T; S = CorG;V= A,C, or G; B= C, G, or T. *potato border-like sequences were obtained and isolated according to the presentty-described methods.</entry></row></tbody></tgroup></table></tables>

0052<b><i>Callus formation</i>:</b> typically, young roots, stems, buds, and germinating seedlings are a few of the sources of plant tissue that can be used to induce callus formation. Callus formation is controlled by growth regulating substances present in tissue culture medium, such as auxins and cytokinins. The specific substances, and concentrations of those substances, that induce callus formation varies between plant species. Occassionally, different sources of explants require different culturing conditions, even if obtained from the same plant or species. Accordingly, a cocktail of various growth substances can be added to tissue culture medium in order to induce callus formation from a variety of plant species that are incubated on such media. Other factors, such as the amount of light, temperature, and humidity, for instance, are important in establishing a callus. Once established, callus cultures can be used to obtain protoplasts, or study somatic embryogenesis, organogenesis, and secondary metabolite production.

0053The skilled artisan is well aware of various protocols, media, and conditions that can be modified to induce callus formation from a particular explant. The FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS' Agricultural Services Bulletin No. 108, entitled, "PLANT TISSUE CULTURE: AN ALTERNATIVE FOR PRODUCTION OF USEFUL METABOLITE" by Masanaru Misawa of Bio International Inc., Toronto, Canada (http://www.fao.org/docrep/t0831e/t0831e00.htm#con) lists such conditions in Chapter 4. There, one learns that the successful production of callus depends upon plant species and their qualities. Dicotyledons, for example, are quite amenable to callus formation, compared to monocotyledons. Suitable tissue culture media for inducing callus formation from an explant may include inorganic salts, carbon sources, vitamins, phytohormones, and organic supplements. See for additional information: Plant Cell Tissue and Organ Culture, Fundamental Methods, Gamborg and Phillips, eds, 1995 (Springer Verlag, New York)

0054<i><b>Desired Polynucleotide:</b></i> a desired polynucleotide is a genetic element, such as a promoter, enhancer, or terminator, or gene or polynucleotide that is to be transcribed and/or translated in a transformed cell that comprises the desired polynucleotide in its genome. If the desired polynucleotide comprises a sequence encoding a protein product, the coding region may be operably linked to regulatory elements, such as to a promoter and a terminator, that bring about expression of an associated messenger RNA transcript and/or a protein product encoded by the desired polynucleotide. Thus, a "desired polynucleotide" may comprise a gene that is operably linked in the 5'- to 3'- orientation, a promoter, a gene that encodes a protein, and a terminator. Alternatively, the desired polynucleotide may comprise a gene or fragment thereof, in an "antisense" orientation, the transcription of which produces nucleic acids that may form secondary structures that affect expression of an endogenous gene in the plant cell. A desired polynucleotide may also yield a double-stranded RNA product upon transcription that initiates RNA interference of a gene to which the desired polynucleotide is associated. A desired polynucleotide may be positioned within a T-DNA or P-DNA, such that the left and right T-DNA border sequences, or the left and right border-like sequences of the P-DNA, flank or are on either side of the desired polynucleotide. The disclosure envisions the stable integration of one or more desired polynucleotides into the genome of at least one plant cell. A desired polynucleotide may be mutated or a variant of its wild-type sequence. It is understood that all or part of the desired polynucleotide can be integrated into the genome of a plant. It also is understood that the term "desired polynucleotide" encompasses one or more of such polynucleotides. Thus, a P-DNA or T-DNA of the present invention may comprise one, two, three, four, five, six, seven, eight, nine, ten, or more desired polynucleotides.

0055According to the disclosure, a desired polynucleotide also may be used to alter a trait (see definition below) associated with a plant. In a situation where the plant is a food crop for consumption, it is preferable that the plant is not transformed so as to integrate undesirable DNA into its genome. A desired polynucleotide also may be used for pharmaceutical purposes, to express in plants a product of pharmaceutical relevance or importance. In that situation, any foreign, native, or undesirable nucleic acids may be used to express the desired polynucleotide. Examples of pharmaceutically relevant desired polynucleotides include those that encode peptides, nutraceuticals, vaccines, growth factors, and enzymes.

0056<b><i>Dicotyledonous plant (dicot)</i>:</b> a flowering plant whose embryos have two seed halves or cotyledons. Examples of dicots include but are not limited to, cotton, tobacco, <i>Arabidopsis,</i> tomato, potato sugar beet, broccoli, cassava, sweet potato, pepper, poinsettia, bean, alfalfa, soybean, carrot, strawberry, lettuce, oak, maple, walnut, rose, mint, squash, daisy, geranium, avocado, and cactus.

0057<b><i>Food source</i>:</b> the present invention contemplates to improve food crops by introducing DNA that is mainly or exclusively derived from human food sources into the genomes of these crops and plants. Examples of edible food sources preferably includes baker's yeast and plants that produce edible fruits, vegetables, and grains. Preferably, DNA is not obtained from animals, bacteria, viruses, and fungi. Accordingly, genetic elements such as promoters, terminators, genes, and selectable markers, introduced into a plant genome, may be preferably derived from, or isolated from, plants that produce edible foods or organisms, such as yeast.

0058<b><i>Foreign</i>:</b> "foreign," with respect to a nucleic acid, means that that nucleic acid is derived from non-plant organisms, or derived from a plant that is not the same species as the plant to be transformed or is not derived from a plant that is not interfertile with the plant to be transformed, does not belong to the species of the target plant. According to the disclosure, foreign DNA or RNA represents nucleic acids that are naturally occurring in the genetic makeup of fungi, bacteria, viruses, mammals, fish or birds, but are not naturally occurring in the plant that is to be transformed. Thus, a foreign nucleic acid is one that encodes, for instance, a polypeptide that is not naturally produced by the transformed plant. A foreign nucleic acid does not have to encode a protein product. According to the present disclosure, a most desired transgenic plant is one that contains minimal, if any, foreign nucleic acids integrated into its genome. The present disclosure also encompasses transgenic plants that do contain non-plant species nucleic acids in their genomes.

0059<i><b>Gene:</b></i> A gene is a segment of a DNA molecule that contains all the information required for synthesis of a product, polypeptide chain or RNA molecule, that includes both coding and non-coding sequences.

0060<i><b>Genetic element:</b></i> a "genetic element" is any discreet nucleotide sequence such as, but not limited to, a promoter, gene, terminator, intron, enhancer, spacer, 5'-untranslated region, 3'-untranslated region, or recombinase recognition site.

0061<i><b>Genetic modification:</b></i> stable introduction of DNA into the genome of certain organisms by applying methods in molecular and cell biology.

0062<i><b>Introduction:</b></i> as used herein, refers to the insertion of a nucleic acid sequence into a cell, by methods including infection, transfection, transformation or transduction.

0063<i><b>Monocotyledonous plant (monocot):</b></i> a flowering plant whose embryos have one cotyledon or seed leaf. Examples of monocots include, but are not limited to turfgrass, maize, rice, oat, wheat, barley, sorghum, orchid, iris, lily, onion, and palm. Examples of turfgrass include, but are not limited to <i>Agrostis spp.</i> (bentgrass species including colonial bentgrass and creeping bentgrasses), <i>Poa pratensis</i> (kentucky bluegrass), <i>Lolium spp.</i> (ryegrass species including annual ryegrass and perennial ryegrass), <i>Festuca arundinacea</i> (tall fescue) <i>Festuca rubra commutata</i> (fine fescue), <i>Cynodon dactylon</i> (common bermudagrass varieties including Tifgreen, Tifway II, and Santa Ana, as well as hybrids thereof); <i>Pennisetum clandestinum</i> (kikuyugrass), <i>Stenotaphrum secundatum</i> (st. augustinegrass), <i>Zoysia japonica</i> (zoysiagrass), and <i>Dichondra micrantha.</i>

0064<i><b>Native:</b></i> a "native" genetic element refers to a nucleic acid that naturally exists in, originates from, or belongs to the genome of a plant that is to be transformed. Thus, any nucleic acid, gene, polynucleotide, DNA, RNA, mRNA, or cDNA molecule that is isolated either from the genome of a plant or plant species that is to be transformed, or is isolated from a plant or species that is sexually compatible, or interfertile with the plant species that is to be transformed, is "native" to, <i>i.e</i>., indigenous to, the plant species. In other words, a native genetic element represents all genetic material that is accessible to plant breeders for the improvement of plants through classical plant breeding. For instance, native DNA incorporated into cultivated potato (<i>Solanum tuberosum</i>) can be derived from any genotype of S. <i>tuberosum</i> or any genotype of a wild potato species that is sexually compatible with <i>S. tuberosum</i> (<i>e.g., S. demissum</i>). Any variants of a native nucleic acid also are considered "native" in accordance with the present invention. In this respect, a "native" nucleic acid may also be isolated from a plant or sexually compatible species thereof and modified or mutated so that the resultant variant is greater than or equal to 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61 %, or 60% similar in nucleotide sequence to the unmodified, native nucleic acid isolated from a plant. A native nucleic acid variant may also be less than about 60%, less than about 55%, or less than about 50% similar in nucleotide sequence.

0065A "native" nucleic acid isolated from a plant may also encode a variant of the naturally occurring protein product transcribed and translated from that nucleic acid. Thus, a native nucleic acid may encode a protein that is greater than or equal to 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, or 60% similar in amino acid sequence to the unmodified, native protein expressed in the plant from which the nucleic acid was isolated.

0066<i><b>Naturally occurring nucleic acid:</b></i> this phrase means that the nucleic acid is found within the genome of a selected plant species and may be a DNA molecule or an RNA molecule. The sequence of a restriction site that is normally present in the genome of a plant species can be engineered into an exogenous DNA molecule, such as a vector or oligonucleotide, even though that restriction site was not physically isolated from that genome. Thus, the disclosure permits the synthetic creation of a nucleotide sequence, such as a restriction enzyme recognition sequence, so long as that sequence is naturally occurring in the genome of the selected plant species or in a plant that is sexually compatible with the selected plant species that is to be transformed.

0067<b><i>Operably linked:</i></b> combining two or more molecules in such a fashion that in combination they function properly in a plant cell. For instance, a promoter is operably linked to a structural gene when the promoter controls transcription of the structural gene.

0068<i><b>P-DNA:</b></i> P-DNA ("plant-DNA") is isolated from a plant genome and comprises at each end, or at only one end, a T-DNA border-like sequence. Thus, a P-DNA may comprise a left border-like sequence and a right border-like sequence. The border-like sequences preferably share at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90% or at least 95%, but less than 100% sequence identity, with a T-DNA border sequence from an <i>Agrobacterium</i> species, such as <i>Agrobacterium tumefaciens</i> or <i>Agrobacterium rhizogenes.</i> Thus, P-DNAs can be used instead of T-DNAs to transfer a desired polynucleotide from <i>Agrobacterium</i> to a plant chromosome. The desired polynucleotide may or may not be native to the plant species to be transformed. That is, a P-DNA may be used to transfer foreign, as well as native, nucleic acids into a plant cell. Accordingly, the vectors can be used to transfer a desired polynucleotide (see definition above for "desired polynucleotide") into a plant genome. It is understood that all or part of the P-DNA containing the desired polynucleotide can be integrated into a plant genome by <i>Agrobacterium-mediated</i> transformation.

0069A P-DNA may be modified to facilitate cloning and should preferably not naturally encode proteins or parts of proteins. The P-DNA can be modified to reduce the frequency of vector backbone integration into a transformed plant genome.

0070A P-DNA is characterized in that it contains, at each end, at least one border sequence, referred to herein as a P-DNA "border-like" sequence, because its sequence is similar to, but not identical with, conventional T-DNA border sequences. See the definition of a "border sequence" and "border-like" above.

0071A desired polynucleotide and selectable marker may be positioned between the left border-like sequence and the right border-like sequence of a P-DNA. The desired polynucteotide and a selectable marker may comprise a gene operably linked to a variety of different nucleic acids, such as to promoter and terminator regulatory elements that facilitate their expression, <i>i.e</i>., transcription and/or translation of the DNA sequence encoded by the desired polynucleotide or selectable marker.

0072Thus, the P-DNA may be used to transfer foreign DNA into plant genomes, as well as polynucleotides that are endogenous to plants. Accordingly, the "desired polynucleotide" that is transferred to a plant genome can be foreign, or native, or from a food-source, and may represent a gene that is useful for producing a pharmaceutical product, such as a hormone or enzyme. The desired polynucleotide contained within the P-DNA also may be used to alter a trait associated with the transformed plant.

0073<i>Plant tissue:</i> a "plant" is any of various photosynthetic, eukaryotic, multicellular organisms of the kingdom <i>Plantae</i> characteristically producing embryos, containing chloroplasts, and having cellulose cell walls. A part of a plant, <i>i.e</i>., a "plant tissue" may be treated according to the methods of the present invention to produce a transgenic plant. Preferably, the plant tissue that is transformed using an <i>Agrobacterium-derived</i> vector is a germinating seedling. The methods described herein, however, are not limited to the transformation of only germinating seedling. Other suitable plant tissues can be transformed according to the present invention and include, but are not limited to, pollen, leaves, stems, calli, stolons, microtubers, and shoots. Thus, the present disclosure envisions the transformation of angiosperm and gymnosperm plants such as turfgrass, wheat, maize, rice, barley, oat, sugar beet, potato, tomato, tobacco, alfalfa, lettuce, carrot, strawberry, cassava, sweet potato, geranium, soybean, oak, eucalyptus, walnut, and palm. According to the present invention "plant tissue" also encompasses plant cells. Plant cells include suspension cultures, callus, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, seeds and microspores. Plant tissues may be at various stages of maturity and may be grown in liquid or solid culture, or in soil or suitable media in pots, greenhouses or fields. A plant tissue also refers to any clone of such a plant, seed, progeny, propagule whether generated sexually or asexually, and descendents of any of these, such as cuttings or seed. Of particular interest are Kentucky bluegrass, creeping bentgrass, maize, and wheat, and dicots such as cotton, tomato, lettuce, <i>Arabidopsis,</i> tobacco, and geranium.

0074<i><b>Plant transformation and cell culture:</b></i> broadly refers to the process by which plant cells are genetically modified and transferred to an appropriate plant culture medium for maintenance, further growth, and/or further development. Such methods are well known to the skilled artisan.

0075<b><i>Progeny</i>:</b> a "progeny", such as the progeny of a transgenic plant, is one that is born of, begotten by, or derived from a plant or the transgenic plant. Thus, a "progeny" plant, <i>i.e.,</i> an "F1" generation plant is an offspring or a descendant of the transgenic plant produced by the inventive methods. A progeny of a transgenic plant may contain in at least one, some, or all of its cell genomes, the desired polynucleotide that was integrated into a cell of the parent transgenic plant by the methods described herein. Thus, the desired polynucleotide is "transmitted" or "inherited" by the progeny plant. The desired polynucleotide that is so inherited in the progeny plant may reside within a P-DNA or T-DNA construct, which also is inherited by the progeny plant from its parent. The term "progeny" as used herein, also may be considered to be the offspring or descendants of a group of plants.

0076<i><b>Seed:</b></i> a "seed" may be regarded as a ripened plant ovule containing an embryo, and a propagative part of a plant, as a tuber or spore. Seed may be incubated prior to <i>Agrobacterium-mediated</i> transformation, in the dark, for instance, to facilitate germination. Seed also may be sterilized prior to incubation, such as by brief treatment with bleach. The resultant seedling can then be exposed to a desired strain of <i>Agrobacterium.</i>

0077<b><i>Seedling</i>:</b> a young plant that is grown from a seed. Certain parts of a seedling, such as part or all of the scutellum may be removed prior to exposing the seedling to a solution comprising an <i>Agrobacterium</i> strain.

0078<b><i>Selectable</i>/</b><i><b>screenable marker:</b></i> a gene that, if expressed in plants or plant, tissues, makes it possible to distinguish them from other plants or plant tissues that do not express that gene. Screening procedures may require assays for expression of proteins encoded by the screenable marker gene. Examples of such markers include the beta glucuronidase (<i>GUS</i>) gene and the luciferase (LUX) gene. The instant invention demonstrates that cyanamide tolerance genes such as <i>CAH</i> can also be used as a marker. Thus, a gene encoding resistance to a fertilizer, antibiotic, herbicide or toxic compound can be used to identify transformation events. Examples of selectable markers include the cyanamide hydratase gene (<i>CAH</i>) streptomycin phosphotransferase (SPT) gene encoding streptomycin resistance, the neomycin phosphotransferase (<i>NPTII</i>) gene encoding kanamycin and geneticin resistance, the hygromycin phosphotransferase (HPT or <i>APHIV</i>) gene encoding resistance to hygromycin, acetolactate synthase (<i>als</i>) genes encoding resistance to sulfonylurea-type herbicides, genes (<i>BAR</i> and/or <i>PAT</i>) coding for resistance to herbicides which act to inhibit the action of glutamine synthase such as phosphinothricin (Liberty or Basta), or other similar genes known in the art.

0079<b><i>Sequence identity</i>:</b> as used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified region. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, <i>e.g.,</i> according to the algorithm of <nplcit id="ncit0008" npl-type="s"><text>Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988</text></nplcit>) <i>e.g.,</i> as implemented in the program PC/GENE (Intelligenetics, Mountain View, California, USA). As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

0080Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of <nplcit id="ncit0009" npl-type="s"><text>Smith and Waterman, Adv. Appl. Math. 2: 482 (1981</text></nplcit>); by the homology alignment algorithm of <nplcit id="ncit0010" npl-type="s"><text>Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970</text></nplcit>); by the search for similarity method of <nplcit id="ncit0011" npl-type="s"><text>Pearson and Lipman, Proc. Natl. Acad. Sci. 85: 2444 (1988</text></nplcit>); by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, California; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin, USA; the CLUSTAL program is well described by <nplcit id="ncit0012" npl-type="s"><text>Higgins and Sharp, Gene 73: 237-244 (1988</text></nplcit>); <nplcit id="ncit0013" npl-type="s"><text>Higgins and Sharp, CABIOS 5: 151-153 (1989</text></nplcit>); <nplcit id="ncit0014" npl-type="s"><text>Corpet, et al., Nuclei Acids Research 16: 10881-90 (1988</text></nplcit>); <nplcit id="ncit0015" npl-type="s"><text>Huang, et al., Computer Applications in the Biosciences 8: 155-65 (1992</text></nplcit>), and <nplcit id="ncit0016" npl-type="s"><text>Pearson, et al., Methods in Molecular Biology 24: 307-331 (1994</text></nplcit>).

0081The BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, <nplcit id="ncit0017" npl-type="b"><text>Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995</text></nplcit>); <nplcit id="ncit0018" npl-type="s"><text>Altschul et al., J. Mol. Biol., 215:403-410 (1990</text></nplcit>); and, <nplcit id="ncit0019" npl-type="s"><text>Altschul et al., Nuclei Acids Res. 25:3389-3402 (1997</text></nplcit>).

0082Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M = 5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (<i>see</i><nplcit id="ncit0020" npl-type="s"><text>Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915</text></nplcit>).

0083In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (<i>see, e.g.,</i><nplcit id="ncit0021" npl-type="s"><text>Karlin &amp; Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5877 (1993</text></nplcit>)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.

0084BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (<nplcit id="ncit0022" npl-type="s"><text>Wooten and Federhen, Comput. Chem., 17:149-163 (1993</text></nplcit>)) and XNU (<nplcit id="ncit0023" npl-type="s"><text>Claverie and States, Comput. Chem., 17:191-201 (1993</text></nplcit>)) low-complexity filters can be employed alone or in combination.

0085Multiple alignment of the sequences can be performed using the CLUSTAL method of alignment (<nplcit id="ncit0024" npl-type="s"><text>Higgins and Sharp (1989) CABIOS. 5:151-153</text></nplcit>) with the default parameters (GAP PENALTY =10, GAP LENGTH PENALTY = 10). Default parameters for pairwise alignments using the CLUSTAL method are KTUPLE 1, GAP PENALTY = 3, WINDOW = 5 and DIAGONALS SAVED = 5.

0086<i><b>Trait</b>:</i> a "trait" is a distinguishing feature or characteristic of a plant, which may be altered according to the present invention by integrating one or more "desired polynucleotides" and/or screenable/selectable markers into the genome of at least one plant cell of a transformed plant. The "desired polynucleotide(s)" and/or markers may confer a change in the trait of a tranformed plant, by modifying any one of a number of genetic, molecular, biochemical, physiological, morphological, or agronomic characteristics or properties of the transformed plant cell or plant as a whole. Thus, expression of one or more, stably integrated desired polynucleotide(s) in a plant genome, may alter a trait that is selected from the group consisting of, but not limited to, increased drought tolerance, enhanced cold and frost tolerance, improved vigor, enhanced color, enhanced health and nutritional characteristics, improved storage, enhanced yield, enhanced salt tolerance, enhanced heavy metal tolerance, increased disease tolerance, increased insect tolerance, increased water-stress tolerance, enhanced sweetness, improved vigor, improved taste, improved texture, decreased phosphate content, increased germination, increased micronutrient uptake, improved starch composition, and improved flower longevity.

0087<b><i>Transcription and translation terminators:</i></b> The expression vectors typically have a transcriptional termination region at the opposite end from the transcription initiation regulatory region. The transcriptional termination region may be selected, for stability of the mRNA to enhance expression and/or for the addition of polyadenylation tails added to the gene transcription product.

0088<b><i>Transfer DNA (T-DNA):</i></b> an <i>Agrobacterium</i> T-DNA is a genetic element that is well-known as an element capable of integrating a nucleotide sequence contained within its borders into another nucleotide. In this respect, a T-DNA is flanked, typically, by two "border" sequences. A desired polynucleotide and a selectable marker may be positioned between the left border-like sequence and the right border-like sequence of a T-DNA. The desired polynucleotide and selectable marker contained within the T-DNA may be operably linked to a variety of different, plant-specific <b>(<i>i.e</i>.,</b> native), or foreign nucleic acids, like promoter and terminator regulatory elements that facilitate its expression, <i>i.e</i>., transcription and/or translation of the DNA sequence encoded by the desired polynucleotide or selectable marker.

0089<b><i>Transformation of plant cells:</i></b> A process by which a nucleic acid is stably inserted into the genome of a plant cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of nucleic acid sequences into a prokaryotic or eukaryotic host cell, including <i>Agrobacterium-mediated</i> transformation protocols, viral infection, whiskers, electroporation, microinjection, polyethylene glycol-treatment, heat shock, lipofection and particle bombardment.

0090<i><b>Transgenic plant:</b></i> a transgenic plant is one that comprises at least one cell genome in which an exogenous nucleic acid has been stably integrated. According to the present disclosure, a transgenic plant is a plant that comprises only one genetically modified cell and cell genome, or is a plant that comprises some genetically modified cells, or is a plant in which all of the cells are genetically modified. A transgenic plant of the present invention may be one that comprises expression of the desired polynucleotide, <i>i.e</i>., the exogenous nucleic acid, in only certain parts of the plant. Thus, a transgenic plant may contain only genetically modified cells in certain parts of its structure.

0091<b><i>Undesirable DNA:</i></b> any DNA that is not derived from a common food source and is not essential for expression of a beneficial trait in a transgenic plant, when making a genetically engineered food crop. Under these circumstances, undesirable DNA is DNA from viruses, bacteria, fungi, animals, and non-edible plants.

0092<i><b>Vortexing, turbo-vortexing:</b></i> either term refers to the abrupt agitation of plant tissues, such as germinating seedling, using a standard vortex or other device. According to the present invention, plant tissues may be vortexed from 60 seconds to several hours. Preferably, the plant tissue is vortexed for about 5 to about 30 minutes. It is well within the purview of the skilled artisan to determine a suitable length of time to vortex plant tissues from various monocotyledon and dicotyledon plant species.

0093<b><i>Variant:</i></b> a "variant," as used herein, is understood to mean a nucleotide or amino acid sequence that deviates from the standard, or given, nucleotide or amino acid sequence of a particular gene or protein. The terms, "isoform," "isotype," and "analog" also refer to "variant" forms of a nucleotide or an amino acid sequence. An amino acid sequence that is altered by the addition, removal or substitution of one or more amino acids, or a change in nucleotide sequence, may be considered a "variant" sequence. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, <i>e.g.,</i> replacement of leucine with isoleucine. A variant may have "nonconservative" changes, <i>e.g.</i>, replacement of a glycine with a tryptophan. Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted may be found using computer programs well known in the art such as Vector NTI Suite (InforMax, MD) software. "Variant" may also refer to a "shuffled gene" such as those described in Maxygen-assigned patents. For instance, a variant may include variants of sequences and desired polynucleotides that are modified according to the methods and rationale disclosed in <patcit id="pcit0019" dnum="US6132970A"><text>U.S. 6,132,970</text></patcit>,

0094It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a gene" is a reference to one or more genes and includes equivalents thereof known to those skilled in the art and so forth. Indeed, one skilled in the art can use the methods described herein to express any native gene (known presently or subsequently) in plant host systems.

0095A surprising discovery of the present invention is that a germinating seedling that is agitated in a solution containing <i>Agrobacterium</i> cells harboring a vector that contains a desired polynucleotide can be planted into soil according to the methods described herein, and grown into a plant that contains cells that are stably transformed with the desired polynucleotide. Accordingly, the first, most basic method of the present invention entails vortexing germinating seedling with an <i>Agrobacterium</i> strain containing an appropriate vector, and then simply planting the vortexed seedling in soil, under conditions that promote growth.

0096The efficiency of stable transformation can be further enhanced by inducing double strand breaks in the chromosomes of germinating seedling before, during, and/or after infection. Such double strand breaks can be generated by, for instance, subjecting seedlings to low doses of chemicals such as methyl methane sulfonate (MMS), HO-endonuclease, bleomycin, neocarzinostatin, camptothecan, and cisplatin, or by using ionizing radiation or heavy ions. Similar effects may also be accomplished by temporarily blocking the cell's own double strand gap repair mechanism. Mutations that may inadvertently arise from these treatments can be easily removed by back-crossing transgenic plants with untransformed plants.

0097Accordingly, the methodology may entail vortexing a plant tissue with an <i>Agrobacterium</i> vector to optimize transfer of the vector and desired polynucleotide(s) into plant cells, and also the induction of double stranded breaks in plant chromosomes to increase the frequency of stably transforming, <i>i.e</i>., integrating, the plant genome with the desired polynucleotide(s).

0098The transgenic plant is crossed or self-fertilized to transmit the desired gene or nucleotide sequence to progeny plants. Seedlings of this next generation of transgenic plants can be screened for the presence of a desired polynucleotide using standard techniques such as PCR, enzyme or phenotypic assays, ELISA, or Western blot analysis. Alternatively, if the transformation vector comprises a selectable/screenable marker(s), the plant progeny may be selected for resistance or tolerance to a particular substance, as is described in detail below.

0099The second method entails transferring the <i>Agrobacterium-transformed</i> seedling to soil only after the seedling has been nurtured on minimal tissue culture medium (<i>e.g</i>. <nplcit id="ncit0025" npl-type="s"><text>MS - Murashige &amp; Skoog, Physiol. Plant, 15: 473-479, 1962</text></nplcit>), without the induction of a callus. The "pre-planting" nurturing step helps to boost the strength, nutrients, and resources available to the seedling prior to planting directly in soil.

0100The third inventive method encompasses inducing the transformed seedling to undergo a callus phase, stimulating the growth of shoots and roots, and then planting directly in soil. To perform the latter, a novel <i>Agrobacterium</i> transformation vector is provided, that may, or may not, be used in conjunction with the novel vortex method for transforming seedlings.

0101The novel transformation vector comprises an alternative to the Agrobacterium-derived T-DNA element , which is characterized by a "left border" at its 5'-end, and a "right border" at its 3'-end. According to the invention, the alternative transfer DNA may be isolated from an edible plant in order to minimize the quantity of undesirable nucleic acids introduced into the target plant genome. Such a plant transfer DNA (P-DNA) also is delineated by left and right border-like sequences that support the transfer of one polynucleotide into another. For the purposes of the present invention, either T-DNA or P-DNA constructs can be used to transfer a desired polynucleotide into a plant cell. The skilled artisan would understand that, in some instances, it is desirable to reduce the amount and number of undesirable genetic elements that are introduced into a plant genome via <i>Agrobacterium-</i>mediated transformation. Accordingly, the skilled artisan could use the P-DNA of the present invention in such instances, because the P-DNA, and its border-like sequences, is isolated from a plant genome.

0102According to the present disclosure, a desired polynucleotide is positioned within such a P-DNA or T-DNA and is operably linked to a promoter and a terminator, that can express it. In order to further minimize the quantity of foreign nucleic acid introduced into a plant genome after successful transformation, the promoter and terminator linked to the desired polynucleotide may be promoters and terminators that naturally occur in a plant genome.

0103If required, a selectable marker that confers a detectable trait to plant cells containing it, can be positioned within the T-DNA/P-DNA of the inventive vector. Such a selectable marker may encode proteins that confer tolerance to herbicides such as glyphosate-N-acetyltransferase <i>(GAT)</i> or 5-enolpyruvylshikimate-3-phosphate synthase <i>(EPSPS).</i> A preferred selectable marker gene confers antibiotic resistance to transgenic plants, such as the neomycin phosphotransferase gene. Another preferred selectable marker gene provides cyanamide tolerance. One example of a cyanamide tolerance gene is the <i>Myrothecium verrucaria</i> cyanamide hydratase (<i>CAH</i>) gene. The instant invention demonstrates that distant homologs of the <i>CAH</i> gene, derived from soil fungi such as <i>Aspergillus, Cladosporium,</i> and <i>Penicillium</i> (but not from the yeast species <i>Saccharomyces cereviseae</i>) also function as cyanamide tolerance genes.

0104Calcium cyanamide is an environment-friendly nitrogen fertilizer. Because nitrogen is released only gradually, it poses less risk of nitrate pollution to groundwater than do the popular urea-based or ammonium-nitrate-based fertilizers. Furthermore, it provides beneficial additional effects because both the lime and cyanamide breakdown products such as dicyandiamide limit growth of undesirable fungi and parasites including <i>Sclerotinia, Pythium, Erysiphe</i> and nematodes, whereas it stimulates growth of the beneficial fungi <i>Aspergillus</i> and <i>Penicillium.</i>

0105One reason calcium cyanamide is not widely used in agriculture is that it can only be applied pre-emergence. However, tolerance to cyanamide makes it possible now to apply cyanamide during and after emergence. By using cyanamide-tolerant transgenic plants, calcium cyanamide can be applied both as a pre- and post-emergence fertilizer to increase yield and quality of crops and other agronomically important plants.

0106A novel combination of cyanamide fertilizer and cyanamide-tolerant plants to reduce the prevalence of soil-borne fungi, nematodes and insects, thereby increasing crop yield and quality. Enhanced disease and pest control can be obtained by not only applying before emergence but also during the growth phase of the plant.

0107The post-emergence application of calcium cyanamide is also predicted to limit the growth of undesirable plants, such as weeds, that are not naturally cyanamide tolerant. Such an application would limit the growth of multiple weeds including annual bluegrass, goosegrass, crowfootgrass, dollarweed, purple nutsedge, torpedograss, kyllinga, and alligatorweed on lawns planted with cyanamide-tolerant turf grass.

0108The present invention eliminates the need for explant starting material, such as immature plant embryos. Thus the methodology is species-independent, cost-effective, and less labor intensive, than conventional species-dependent methods that require selection, proliferation, and regeneration of individually transformed somatic cells.

<i>Seedling characteristics</i>

0109The methodology utilizes a seedling that has only just begun to germinate and which is characterized, in a monocotyledonous or dicotyledonous plant, by a just-emerging coleoptile or cotyledon at the surface of the seed coat.

0110There may be an optimal stage of cotyledon emergence, <i>i.e</i>., germination, in seeds that provides a high frequency of transformation. For tobacco seeds, for instance, a high level of transformation frequency via agitation is observed when the cotyledon is one-half to three-quarters emerged from the seed coat. The time it takes to establish the optimal cotyledon emergence stage will vary depending on the specific dicotyledon species and the environmental conditions during germination, such as light, moisture, temperature, and the emergence medium (soil, artificial medium, sand, etc.).

0111One skilled in the art would know how to systematically define these environmental parameters for each dicotyledon seed species in order to determine the optimal cotyledon emergence stage. In this fashion, one may optimize when to agitate a germinating seed so as to obtain a high frequency of transformation. One may quantify the level of transformation by monitoring transient GUS expression assays or by stable transformation. For monocotyledon plants, such as turf and wheat, one would develop a timing of transformation based upon optimal coleoptile emergence instead of cotyledon emergence.

0112A seedling that is at such an early-stage of germination will possess cells that are rapidly proliferating as the seed develops. Furthermore, certain cells of the coleoptile may be progenitors of germ line cells, which means that transforming these cells in particular will increase the likelihood of obtaining an inheritable, but artificial or modified, trait. Accordingly, the present disclosure makes use of this naturally-occurring state of cell multiplication and development by exposing these seedlings to an <i>Agrobacterium</i> vector that contains a gene or nucleotide sequence that the skilled artisan wishes to integrate into cells of the germinating seedling.

<i>Agitation</i>

0113In particular, a seedling that is characterized by a just-emerging coleoptile or cotyledon may be agitated in a solution that contains an <i>Agrobacterium</i> strain. For instance, such a seed may be placed into a tube or some other vessel that contains an <i>Agrobacterium</i> solution, which is then vortexed in a standard bench-top vortex for a short period of time. A tube containing a seedling in solution may be turbo-vortexed. Alternatively, the seedling may be submerged into a solution that is mixed for some period of time with a magnetic stir-bar using a standard bench-top mixing device.

<i>Vortexing</i>

0114The vortexing step described above may be enhanced by adding a small amount of sand to the <i>Agrobacterium</i>-containing solution. In experiments with tobacco and geranium, for example, the inclusion of a small amount of sand in the transfection solution during vortexing greatly increased the frequency of transformation. Other materials in place of sand that act in an abrasive fashion may be added to the <i>Agrobacterium-</i>containing transfection solution, such as, but not limited to, small glass beads, silicon, plastic grains, or stone. Turbo-vortexing also may be employed to facilitate transformation.

0115Depending on the size of the germinating seedling and the intensity of the agitation, different seedlings from different plant species, may be vortexed for different periods of time, such as anywhere from a few seconds, or 1-15 minutes, 5-10 minutes, 1-5 minutes, 15-20 minutes, an hour, or several hours. Small germinating seedlings from plants such as tobacco, turfgrass and <i>Arabidopsis,</i> for instance, may require less agitation than larger germinating seedlings such as wheat, maize and cotton.

<i>Removing plants that comprise vector backbone sequences</i>

0116It is possible that DNA from the vector portion flanking the P-DNA or T-DNA of a transformation construct is incorporated into the host plant genome while agitating a germinating seed in the <i>Agrobacterium-containing</i> transformation solution. Thus, it is necessary to distinguish plants that contain only the desired polynucleotide insert integrated into their genome and from plants that also contain regions of the plasmid vector (<i>i.e.,</i> "backbone DNA") after transformation. Backbone DNA is the part of an <i>Agrobacterium</i> binary vector that excludes the T-DNA/P-DNA.

0117In order to facilitate identification of plants that contain backbone DNA, a "backbone integration marker," which alters some morphological feature of the plant, is placed upstream and/or downstream of the T-DNA/P-DNA. Thus, it is possible a backbone integration marker gene that changes the shape of the transformed plant's leaves, roots, stem, height or some other morphological feature, that is not attributable to an effect of the desired polynucleotide, can be used to identify plants that contain vector backbone sequences. The color, texture or other traits of a plant may be similarly altered. "Morphological" refers to the form and structure of an organism without particular consideration of function, or which relates directly to the form and structure of an organism or its parts.

0118Thus, a transformed plant that has a morphologically altered feature as compared to a non-transformed or wild-type plant of that plant species, is indicative of a plant that contains backbone vector DNA in its genome.

0119Accordingly, an <i>Agrobacterium</i> vector may also carry an operable cytokinin gene upstream and/or downstream of the insertion DNA that will alter some morphological feature of the plant if it is integrated into the plant genome. Thus, it is straightforward to distinguish between desired and undesired transformation events. Transformed plants that exhibit such an altered morphological feature can be removed from the pool of desired plants, because they must contain undesirable, <i>i.e</i>., backbone, DNA sequences integrated into the genome. In this way, plant genomes that contain integrated and undesirable vector sequences, as well as an integrated desired polynucleotide, can be identified by detecting the expression of the cytokinin gene. Thus, transgenic plants produced by the method of the present invention that display a cytokinin-overproducing phenotype can be discarded, while those that are indistinguishable from untransformed plants can be maintained for further analysis. A preferred cytokinin gene is the <i>Agrobacterium</i> isopentenyl phosphotransferase (IPT) gene. Another cytokinin gene is, for instance, the <i>Agrobacterium</i> transzeatine synthase (<i>TZS</i>) gene. The present invention is not limited to the use of only a cytokinin gene. Any gene that alters a morphological feature of a plant can be used similarly.

0120Another strategy for identifying plants stably transformed with only desired DNA is to PCR amplify genomic DNA prepared from the plant using combination of primer pairs designed to the desired and to backbone vector DNA sequences. Genomes from plants that produce PCR products using primers designed to the backbone vector sequences are from plants that contain integrated backbone DNA.

0121Thus, by either using the expression of a gene to change a morphological feature of a plant, or by screening for stably integrated foreign DNA in a transformed plant, plants stably transformed with only desired DNA sequences can be identified and selected.

0122Similarly, while the stable integration of marker genes into the genomes of plant cells facilitates the identification of transformation events, such modifications of plant genomes are undesirable because marker genes usually represent foreign DNA that can be harmful to the plant, and to elements in the surrounding environment. Use of a marker gene can be avoided through modification of conventional <i>Agrobacterium-</i>based methods.

0123It is known that plant cells exposed during agitation to two different <i>Agrobacterium</i> strains, can receive T-DNAs from both strains. One of the <i>Agrobacterium</i> strains used for plant infection may contain a mutant <i>virD2</i> gene. This mutant <i>Agrobacterium</i> strain is capable of transferring T-DNAs to plant nuclei but most of these T-DNAs will fail to integrate into the plant genome (<nplcit id="ncit0026" npl-type="s"><text>Shurvinton et al., Proc. Natl. Acad. Sci. U.S.A., 89: 11837-11841, 1992</text></nplcit>; <nplcit id="ncit0027" npl-type="s"><text>Mysore et al., Mol. Plant Microbe Interact., 11 : 668-683, 1998</text></nplcit>). The mutant <i>Agrobacterium</i> strain can further contain a marker gene such as the neomycin phosphotransferase (<i>NPTII</i>) gene, operably linked to a promoter and followed by a termination signal, between T-DNA borders. Infection of explants with this mutant strain will result in temporary marker gene expression in some plant cells. Only plant cells that transiently express the marker gene are able to survive media that contain a selection agent such as kanamycin.

0124The virulent <i>Agrobacterium</i> strain that contains a wild-type <i>virD2</i> gene carries the recombinant DNA molecule of interest but lacks a marker gene. Upon co-infection, some plant cells will contain both a non-integrating T-DNA with the marker gene and an integrating carrier DNA with the sequences of interest. In fact, 65% of tobacco cells containing at least one T-DNA derived from one of the strains have been shown to also contain at least one T-DNA from the other strain (<nplcit id="ncit0028" npl-type="s"><text>De Neve et al., Plant J., 11:15-29, 1997</text></nplcit>; <nplcit id="ncit0029" npl-type="s"><text>De Buck et al., Mol. Plant Microbe Interact., 11: 449-57, 1998</text></nplcit>).

0125After about 5 to 10 days, the infected seedlings or explants are transferred to media lacking the selection agent to support further growth of events that had survived the temporary selection period. A significant percentage of these events contain the T-DNA carrying a recombinant DNA molecule of interest and lack the T-DNA with a selectable marker gene for transformation.

0126<i>Agrobacterium</i> strains that contain a functional <i>virD2</i> gene instead of mutant <i>virD2</i> for transient marker gene expression may also be used for selection of plant transformants. However, the frequency of obtaining genetically modified plants lacking a marker gene is generally low compared to use of the mutant <i>virD2</i> gene.

0127Cells that transiently express a marker gene can be discriminated from cells that don't express such a gene using a variety of selection systems. However, not all these selection systems are equally suitable. In potato and tobacco, the most preferred selection agents are kanamycin (about 100 mg/L) and paramomycin (about 25-50 mg/L) because they arrest untransformed cells within 5 to 10 days. Other selection agents include hygromycin, glyphosate, glufosinate and cyanamide. The marker genes corresponding to these various agents encode neomycin phosphotransferase (<i>NPTII</i>) for kanamycin or paramomycin resistance, hygromycin phosphotransferase (<i>HPTII</i>) for resistance to hygromycin, 5-enolpyruvul-3-phosphoshikimic acid synthase (<i>EPSPS</i>) for glyphosate resistance, phosphinothricin acetyltransferase (<i>PAT</i>) for glufosinate resistance, and cyanamide hydratase (<i>CAH</i>) for cyanamide resistance.

0128An alternative way to develop transgenic plants lacking a selectable marker gene is based on excision of the marker gene cassette after plant transformation. Such excision can be accomplished by, <i>e.g.,</i> placing a constitutively expressed marker gene together with an inducible <i>Cre</i> gene between two lox sites. Induction of the <i>Cre</i> gene would then in certain cases result in excision of all sequences between the lox sites. One example of an inducible promoter is the sunflower Ha hsp17.7 G4 promoter (<nplcit id="ncit0030" npl-type="s"><text>Coca et al., Plant Mol. Biol., 31: 863-76, 1996</text></nplcit>). By subjecting regenerating plantlets to a mild heat shock, induction of the heat shock promoter will lead to <i>Cre</i> gene expression and subsequent ejection of the region between the lox sites in some of the transformants.

0129The integration, for example, of any desired polynucleotide into a cell of a plant using the inventive methods is contemplated. Particularly preferred desired polynucleotides that can be integrated into a plant genome and expressed according to the methodologies described herein, include, but are not limited to, (i) the synthetic peptide gene D4E1 (<patcit id="pcit0020" dnum="US6084156A"><text>U.S. Patent 6,084,156</text></patcit>; <patcit id="pcit0021" dnum="US6018102A"><text>U.S. Patent 6,018,102</text></patcit>) to confer bacterial resistance to transgenic plants such as geranium; (ii) the HOS1 gene homologs to enhance cold, freezing and salt tolerance in transgenic plants through gene silencing (<nplcit id="ncit0031" npl-type="s"><text>Lee et al., Gene and Develop., 15: 912-924, 2001</text></nplcit>); (iii) the <i>Vitreoscilla</i> hemoglobin gene (<patcit id="pcit0022" dnum="US5959187A"><text>U.S. Patent 5,959,187</text></patcit>) to develop greener and insect tolerant turfgrass that displays increased seed germination and enhanced vigor; and (iv) genes involved in the lignin biosynthetic pathway.

0130Other plant traits whose expression can be modified, introduced, reduced, or increased by integrating a foreign or native desired polynucleotide or variant thereof into a plant genome by the methodology, include traits selected from the group consisting of, but not limited to, increased drought tolerance, enhanced cold and frost tolerance, improved vigor, enhanced color, enhanced health and nutritional characteristics, improved storage, enhanced yield, enhanced salt tolerance, enhanced heavy metal tolerance, increased disease tolerance, increased insect tolerance, increased water-stress tolerance, enhanced sweetness, improved vigor, improved taste, improved texture, decreased phosphate content, increased germination, increased micronutrient uptake, improved starch composition, and improved flower longevity.

EXAMPLES

Example 1

Development of a species-independent method to obtain transgenic plants without the need for plant cell proliferation and regeneration

0131Binary vectors that were created to develop a species-independent transformation method carry an intron-containing beta glucuronidase (<i>GUS</i>) gene (Genbank accession number AF354045) operably linked to a promoter and terminator. The MMV24P promoter of mirabilis mosaic virus (<patcit id="pcit0023" dnum="US6420547B"><text>Maiti et al., US Patent 6420547, 2002</text></patcit>), and the promoter of the sugarcane ubiquitin-4 gene (<patcit id="pcit0024" dnum="US20020046415A1"><text>Albert and Wei, US Patent 20020046415A1, 2002</text></patcit>) were used to transform dicotyledonous and monocotyledonous plants, respectively. The binary vectors were introduced into <i>Agrobacterium</i> by incubating competent LBA4404 cells (50 µL) with 1 µg of vector DNA for 5 minutes at 37°C, freezing for about 15 seconds in liquid nitrogen (about -196°C), and incubating again at 37°C for 5 minutes. After adding 1 mL of liquid broth (LB), the treated cells were grown for 3 hours at 28°C and plated on LB/agar containing streptomycin (100 mg/L) and kanamycin (100 mg/L). The vector DNAs were then isolated from overnight cultures of individual LBA4404 colonies and examined by restriction analysis to confirm their integrity.

0132The resulting <i>Agrobacterium</i> strains were used to successfully transform eight different plant systems.

1. <i><u>Arabidopsis thaliana</u></i>

0133First, seed of the <i>Arabidopsis thaliana</i> ecotype Columbia was sterilized by turbo-vortexing with 20% bleach. The sterile seed was then incubated for 2 days at room temperature in the dark to allow germination. The germinating seedlings were then emerged into an <i>Agrobacterium</i> suspension, which was obtained by resuspending precipitated cells of an overnight-grown culture in MS medium to obtain an optical density of 0.6 - 0.75. The mixture was turbo-vortexed using a high-performance microcentrifuge tube attachment for the Vortex-Genie 2 Mixer (Part # SI-0563) manufactured by Scientific Industries, Inc., Airport Orville Drive, Bohemia, NY 11716 at a speed setting of "4" for 5 to 30 minutes. The treated seedlings were transferred to either soil or MS medium not containing any hormones, and incubated at 25°C. After 3 weeks, plants were sampled to assay for GUS expression (<nplcit id="ncit0032" npl-type="s"><text>Jefferson et al., EMBO J 6: 3901-3907, 1987</text></nplcit>). Approximately 13% of tested plants (168 of 1274) displayed a blue color in significant portions of both petioles and leaves (Table 2). GUS assays on control plants that had been infected without vortexing were negative. A total of 10 randomly chosen GUS-positive plants were grown for 4 more weeks at 25°C to allow seed set. The resulting seed was sterilized and germinated on MS medium, and progenies were then GUS assayed to determine the frequency of transgene transmission to the next generation. These analyses demonstrated that up to 78% of progeny plants represented stably transformed lines (Table 3).

2. <u><i>Nicotiana tabacum</i> (tobacco)</u>

0134[0129] Second, seed of the <i>Nicotiana tabacum</i> (tobacco) variety SR-1 was sterilized by turbo-vortexing with 20% bleach. The sterile seed was then incubated for 5 days at room temperature in the dark to allow germination. Seedlings were turbo-vortexed with <i>Agrobacterium</i> as described above. After 2 days of co-cultivation, the treated seedlings were transferred to either soil or MS medium not containing any hormones, and incubated for about 3 weeks at 25°C. Treated seedlings were then assayed for GUS expression. As shown in Table 4, a 5-minute vortex-period resulted in a frequency of GUS-expressing seedlings of approximately 7% (44 of 628 seedlings); a 30-minute vortex-period resulted in a slightly lower efficiency (Table 4). Four randomly chosen GUS-positive seedlings were grown for 12 more weeks at 25°C to allow seed set. The resulting seed was sterilized and germinated on MS medium, and progenies were then GUS assayed to determine the frequency of transgene transmission to the next generation. To confirm the presence of the GUS gene, DNA was extracted from T1 seedlings and used to perform a PCR analysis. These phenotypic and molecular analyses demonstrated that 21 % of the progeny plants represented stably transformed lines (Table 5).

3. <u><i>Gossypium hirsutum</i> (cotton)</u>

0135[0130] Third, seed of the <i>Gossypium hirsutum</i> (cotton) variety Coker-312 was sterilized by turbo-vortexing with 20% bleach. After removal of seed coat and cotyledons, the sterile seed was incubated for 2 days at room temperature in the dark to allow germination. Seedlings were then <i>Agro</i>-infected in a similar way as described above, except that turbo-mixing was carried out for 15 minutes. The treated seedlings were transferred to MS medium not containing any hormones, and incubated at 25°C. After 3 weeks, samples of individual seedlings were assayed for <i>GUS</i> expression. A very high percentage of these leaves (50%) developed an intense blue color in stems, petioles and leaves, indicating that a high proportion of cells stably expressed the <i>GUS</i> gene. These seedlings are allowed to grow into mature plants and set seed. The frequency of transformation events that is transmitted to the next generation can be determined by screening progeny plants for GUS expression. Approximately 5-75% of progeny plants is predicted to represent stably transformed lines.

4. <u><i>Lactuca sativa</i> (lettuce)</u>

0136[0131] Fourth, seed of the <i>Lactuca sativa</i> (lettuce) variety "Royal Oak Leaf" was sterilized, germinated for 3 days, and turbo-vortexed with <i>Agrobacterium</i> as described above. After 2 days of co-cultivation, the treated seedlings were transferred to MS medium not containing any hormones, and incubated for about 3 weeks at 25°C. Treated seedlings were then assayed for GUS expression. Seventy percent of lettuce seedlings displayed GUS activity, demonstrating that the marker-free transformation method is particularly effective in this crop system. About 5-75% of progeny plants are expected to contain a transmitted transgene.

5. <u><i>Lycopersicon esculentum</i> (tomato)</u>

0137Fifth, seed of the <i>Lycopersicon esculentum</i> (tomato) variety variety "Juliet hybrid" was sterilized, germinated for 4 days, and turbo-vortexed with <i>Agrobacterium</i> as described above. After 2 days of co-cultivation, the treated seedlings were transferred to MS medium not containing any hormones, and incubated for about 3 weeks at 25°C. Treated seedlings were then assayed for GUS expression. Ninety percent of tomato seedlings displayed GUS activity, demonstrating that the marker-free transformation method is particularly effective in this crop system. About 5-75% of progeny plants are expected to contain a transmitted transgene.

6. <u><i>Agrostis palustris</i> (creeping bentgrass)</u>

0138Sixth, seed of the <i>Agrostis palustris</i> (creeping bentgrass) variety L-93 was sterilized by turbo-vortexing with 20% bleach. The sterile seed was incubated at room temperature in the dark to allow germination. After 1 week, the germinating seedlings were turbo-vortexed with <i>Agrobacterium</i> for approximately 30 minutes. The infected seedlings were transferred to either soil or MS medium not containing any hormones, and incubated at 25°C. At several time points, the seedlings were assayed for GUS expression. Three days post-infection, all seedlings displayed a uniformly blue color in all tissues, indicating that the <i>GUS</i> gene was transferred effectively to the nuclei of a large proportion of plant cells. Even after 3 weeks, a high frequency of seedlings (22 of 106) still displayed a blue color in all tissues, indicating that most or all the cells of these seedlings contained the <i>GUS</i> gene stably integrated in their genomes. The frequency of seedlings that developed at least some blue color at the latter time point was 35% (37 of 106). This experiment was repeated several times with similar results. Seedlings that tested positive for uniform GUS expression were grown for an additional three weeks and subsequently transferred to a vernalization chamber set at 2°C. After a 2-month incubation period, the plants can be transferred to another growth chamber, and grown for 2 months at 25°C with a 16-hour photoperiod to allow flowering and seed set. The harvested progeny seed can be planted in soil, and 2-week old plants can be PCR analyzed for the presence of the <i>GUS</i> gene. Approximately 5-75% of progeny plants derived from GUS-positive TO plants is predicted to contain the transmitted <i>GUS</i> gene.

7.<u><i>Triticum aestivum</i> (wheat)</u>

0139[0138] Seventh, seed of the <i>Triticum aestivum</i> (wheat) variety "Bobwhite" was sterilized by vortexing with 20% bleach. The sterile seed was incubated for 2 days at room temperature in the dark to allow germination. After removal of the scutellum, seedlings were turbo-vortexed with an <i>Agrobacterium</i> strain carrying the GUS vector. Surprisingly, these treated seedlings only comprising coleoptile and coleorhiza developed vigorously on MS medium not containing any hormones, and could be transferred to soil within three weeks. Almost all seedlings displayed a blue color after three days, indicating transient <i>GUS</i> gene expression. Approximately 4.5% of leaves still displayed large blue sectors on leaves and petioles, even after 3 weeks, indicating that many cells of these leaves contained the <i>GUS</i> gene stably integrated into their genomes. This experiment was repeated with similar results. GUS-positive seedlings were allowed to grow into mature plants and flower. DNA extracted from these flowers confirmed the presence of the <i>GUS</i> gene in at least some of the flower cells. Approximately 5-75% of progeny plants derived from GUS-positive flowers is predicted to contain the transmitted <i>GUS</i> gene.

8.<u><i>Zea mays</i> (maize)</u>

0140[0139] Eighth, seed of the recalcitrant <i>Zea mays</i> (maize) variety "Bonus" was sterilized by vortexing with 20% bleach. The sterile seed was incubated for 2 days at room temperature in the dark to allow germination. After removal of the scutellum, seedlings were infected with an <i>Agrobacterium</i> strain carrying pSIM115 or similar vectors. The treated seedlings were transferred to MS medium not containing any hormones, and incubated at 25°C. Of all seedlings transiently expressing the <i>GUS</i> gene three days after infection, about 5.5% still displayed an intense blue color 3 weeks later. Thus, a relatively high proportion of transferred DNAs succeeded in stably integrating into the plant genome. GUS-positive seedlings were transferred to the greenhouse, and are allowed to grow into flowering plants. PCR analysis is expected to confirm the presence of the <i>GUS</i> gene in about 5% of the flowers. Approximately 5-75% of progenies derived from these flowers are predicted to represent transgenic events.

0141[0141] The above experiments demonstrate that vortex-mediated seedling transformation is an effective and generally-applicable method to generate transgenic monocotyledonous and dicotyledonous plants. Transgenic plants developed through this species-independent method do not contain undesirable marker genes.

Example 2

(Not part of the invention)

Optimized integration of transferred DNAs

0142Example 1 demonstrates that the transfer of DNA from <i>Agrobacterium</i> to individual plant cell nuclei can be optimized for many different plant species by agitating seedlings in <i>Agrobacterium</i> suspensions. This example also shows that not all the transferred DNAs subsequently integrate into the plant cell genome. To optimize the second phase of the transformation process, 100 maize seedlings were infected as described in Example 1, and placed on media that contain low levels (50 parts per million) of methyl methane sulfonate (MMS), from 1 day prior to infection until 1 day after infecton. An additional 100 seedlings were placed on control media that lack MMS. Approximately 2 weeks after infection, seedlings were assayed for stable GUS expression. Interestingly, 25% of MMS-treated seedlings contained multiple blue sectors on all assayed tissues whereas only 2.5% of control seedlings contained an occasional blue spot. Thus, the frequency of stable transformation can be increased at least 12.5-fold by using agents that trigger double strand breaks.

Example 3 (Not part of the invention)

Fertilizer tolerance genes as screenable and selectable markers

0143As alternative to the transformation method described in Example 1, which eliminates the need for an undesirable marker gene, a transformation method that relies on the use of a marker gene was developed.

0144The first step in developing this method was to identify a gene that not only makes it possible to select or screen for transformed plant cells but one which also confers a new and beneficial trait to resulting transgenic plants. One example of such a gene provides herbicide tolerance. A more preferred example confers tolerance to cyanamide fertilizers. To identify sources of cyanamide tolerance, a selection of soil fungi were plated on potato dextrose agar (PDA) media containing 35 mg/L cyanamide. Fungi that grew vigorously on these media include <i>Aspergillus</i> sp., <i>Penicillium</i> sp., and <i>Cladosporium</i> sp.

0145A putative fungal cyanamide tolerance gene was amplified from <i>Aspergillus</i> DNA with HotMaster Taq DNA Polymerase (Eppendorf). The primer pair used in these reactions was 5'-TCTAGATGTCACAGTACGGATTTGTAAG -3', and 5'-GGTCACCTCACTGCCCATCAGGGTGCCGGCTTC -3'. The amplified fragments were both inserted into the yeast expression vector pNMT1-TOPO (Invitrogen) and the bacterial vector pGEM-T (Invitrogen). Sequence analysis of the new cyanamide tolerance gene inserted into pGEM-T (designated <i>CAH-H1;</i> see SEQ ID No.: 1) revealed less than 50% homology with both the previously identified <i>Myrothecium verrucaria</i> cyanamide hydratase (<i>CAH</i>) gene (<nplcit id="ncit0033" npl-type="s"><text>Maier-Greiner et al., Angew Chem Int Ed Engl, 30: 1314-1315, 1991</text></nplcit>), and a <i>CAH</i> homolog of the highly cyanamide-sensitive species <i>Saccharromyces cereviseae</i> (<figref idref="f0002">Figure 2</figref>). The PNMT1-TOPO vector carrying <i>CAH-H1</i> was introduced into <i>Saccharomyces pombe</i> by using the S.c. EasyComp Transformation Kit (Invitrogen). Functional activity of the homolog was demonstrated by growing transformed cells on Edinburgh minimal medium (Invitrogen) containing 100 mg/L ampcilin and 50 mg/L cyanamide at 30°C. After 4 days, numerous colonies were observed on plates containing <i>S</i>. <i>pombe</i> cells transformed with pNMT1:<i>CAH-H1</i>, whereas no colonies were observed on pNMT1 control plates. The new cyanamide tolerance gene can be used as selectable marker gene for plant transformation by inserting it between a functional promoter and terminator, and introducing the resulting expression cassette into plant cells.

0146To develop transformation methods that include a screening step for cyanamide tolerance, vectors were created that contain the <i>CAH</i> gene (<patcit id="pcit0025" dnum="US6268547B"><text>U.S. Patent No. 6,268,547</text></patcit>). <i>Agrobacterium</i> strains carrying such a fertilizer tolerance gene driven by the sugarcane ubiquitin-4 promoter were used to infect germinating bentgrass seedlings as described above. The infected seedlings were then planted in soil and allowed to grow for six weeks in a growth chamber (25°C with a 16-hour photoperiod). The resulting plants were spray-treated with a 2% Dormex solution (Siemer and Associates Inc, Fresno, CA), which contains 1% hydrogen cyanamide.

0147About a third of the plants (84 of 250) displayed a high level of tolerance, whereas the remainder of the plants developed severe leaf necrosis. The cyanamide-tolerant plants were grown to maturity, and DNA was then extracted from flowers of these plants for PCR analysis. Using the CAH-specific primer pair 5'- CCA ACG GAT GGA CTG CCG TTC CAG TC -3', and 5'- CAT GGA GTG ATT GTA GGT TTC GGG AC - 3', a 1 80-bp DNA fragment was amplified successfully from DNA of all of cyanamide-tolerant plants, indicating that the analyzed flowers contained the <i>CAH</i> gene stably integrated into the genomes of at least some of their cells. Thus, the data demonstrate that the <i>CAH</i> gene is an effective new screenable marker gene.

0148The eighty-four cyanamide-tolerant flowering plants were allowed to further mature and set seed. Progeny seedlings of some of these lines were planted in soil and analyzed for the presence of the <i>CAH</i> gene by performing PCR reactions on DNA isolated from these seedlings. This experiment demonstrated that an average of 20% of progeny plants contained the <i>CAH</i> gene stably integrated into their genomes (Table 6). Interestingly, this frequency is similar to those found for tobacco and <i>Arabidopsis</i> frequencies (21 % and 53%, respectively), and implies the general applicability of vortex-mediated transformation methods that do not require a selection-step..

0149Seed of the more recalcitrant plant species <i>Poa pratensis</i> (Kentucky bluegrass) was also successfully transformed with the <i>CAH</i>-vector. Seed of the bluegrass variety Liberator was sterilized by turbo-vortexing with 20% bleach. The sterile seed was incubated for 6 days at room temperature in the dark to allow germination. Seedlings were infected with an <i>Agrobacterium</i> strain carrying the <i>CAH</i> gene as described in Example 2. The treated seedlings were transferred to soil and grown for 3 weeks at 25°C with a 16-hour photoperiod. To screen for plants that contain the <i>CAH</i> gene in a significant portion of plant cells, plants were then sprayed with 2% Dormex. Approximately 10% (6 of 70) of plants displayed full tolerance to this spray-treatment. These plants are being vernalized and will be permitted to flower and set seed. Progenies will be tested phenotypically and molecularly to determine the frequency of plants that contain the <i>CAH</i> gene stably integrated into their genomes. This frequency is expected to be about 5-75%.

0150[0142] The method described above was slightly modified to include a selection step for cyanamide tolerance. Seed of the creeping bentgrass variety L-93 was sterilized, germinated, and infected with an <i>Agrobacterium</i> strain carrying a <i>Cah</i>-vector as described in Example 1. Instead of planting the treated seedlings into soil, they were transferred to tissue culture media containing auxin 2,4-D (2 mg/L) and cyanamide (37.5 mg/L), to induce callus formation, and to select for transformation events, respectively. Surprisingly, a large percentage of seedlings (20%) developed rapidly proliferating cyanamide-tolerant callus tissue on their shoot apices, mostly around the crown region, within about 4 weeks. These calli were transferred to new MS media with a lower concentration of 2,4-D (0.01 mg/L) to induce shoot formation. Emerging shoots that arose from calli within about two weeks were transferred to MS medium lacking 2,4-D to induce root formation. After two more weeks, sufficient root mass was established, and plantlets were transferred to soil. The resulting regenerated plants displayed high levels of tolerance to spray-treatment with Dormex, and were shown by PCR to contain the <i>CAH</i> gene stably integrated into their genomes. This is the first time that whole seedlings have been used effectively as 'explant' material for the efficient transformation and subsequent proliferation and regeneration of individual plant cells. Thirty-six cyanamide tolerant plants were vernalized and allowed to set seed. Progenies derived from 2 plants were assayed by PCR to confirm the transmission of the <i>CAH</i> gene to the next generation. As shown in Table 6, the majority of tested T1 plants (5 of 6) showed positive for the transgene, implying the efficacy of this transformation method (standard 3:1 segregation ratios predict a maximum of 75% transgene-transmission to selfed progenies).

0151The very high transformation efficiencies that can be obtained by using whole seedlings as explant material for vortex-mediated transformation make this a preferred method for applications that require high-throughput transformation procedures such as functional genomics. This method is also desirable for, for example, "proof-of-concept" experiments, and for projects related to the overexpression of pharmaceutical and nutraceutical proteins and peptides in plants.

Example 3

(Not part of the invention)

New binary vectors for transformation of plants

0152Current methods to express a foreign gene in crop plants result in the introduction of various nucleic acids that are derived from non-food sources. The introduction of such DNA in the food supply is undesirable and should be limited or avoided. The current invention provides tools and methods to (1) replace the <i>Agrobacterium</i>-derived T-DNA with a DNA fragment derived from a food source, (2) prevent transformation events that contain bacterial vector backbone sequences from developing into whole plants, (3) replace the frequently used nopaline synthase (nos) terminator derived from <i>Agrobacterium</i> with a terminator derived from a food source, and (4) replace frequently used virus promoters with promoters derived from food sources.

<u>1. New transfer DNA</u>

0153The <i>Agrobacterium</i>-derived T-DNA is delineated by a 25-bp left-border (LB) and right-border (RB) repeat, which function as specific recognition sites for virD2-catalyzed nicking reaction (<patcit id="pcit0026" dnum="US4940838A"><text>Schilperoort et al., U.S. Patent 4940838, 1990</text></patcit>). The single stranded DNA released by these nicking reactions is transferred to plant cell nuclei where it often successfully integrates into the plant genome. Advanced BLAST searches of public databases including those maintained by The National Center For Biotechnology Information and SANGER failed to identify any border sequences in plants. It was therefore necessary to consider plant DNA sequences that are similar but not identical to T-DNA borders, designated here as "border-like". The challenge in trying to replace T-DNA borders with border-like sequences is that border sequences are highly conserved (see Table 1). A large part of these sequences is also highly conserved in the nick regions of other bacterial DNA transfer systems such as that of IncP, PC194, and fX174, indicating that these sequences are essential for conjugative-like DNA transfer (<nplcit id="ncit0034" npl-type="s"><text>Waters et al., Proc Natl Acad Sci 88: 1456-60, 1991</text></nplcit>). Because there are no reliable data on border sequence requirements, the entire border seems therefore important in the nicking process. A single study that attempted to address this issue by testing the efficacy of border mutants in supporting DNA transfer is unreliable because negative controls did not appear to function appropriately (<nplcit id="ncit0035" npl-type="s"><text>van Haaren et al., Plant Mol Biol 13: 523-531, 1989</text></nplcit>). Furthermore, none of the results of this study were confirmed molecularly. Despite these concerns, two possibly effective border mutants are shown in Table 1 as well.

0154Based on the homology among border sequences, a T-DNA border motif was identified (Table 1). Although this motif comprises 13,824 variants, many of which may not function -or may be inadequate-in transferring DNA, it represents the broadest possible definition of what a T-DNA border sequence is or may be. This border motif was then used to search publicly available DNA databases for homologs using the "Motif Alignment and Search Tool" (<nplcit id="ncit0036" npl-type="s"><text>Bailey and Gribskov, Bioinformatics 14: 48-54, 1998</text></nplcit>) and "advanced BLASTN" ("penalty for nucleotide mismatch" = -1; "expect" = 105; <nplcit id="ncit0037" npl-type="s"><text>Altschul et al., Nuclei Acids Res 25: 3389-3402, 1997</text></nplcit>). Again, these searches did not identify any identical matches in organisms other than <i>Agrobacterium.</i>

0155To try and increase the chance of isolating a plant DNA fragment containing border-like sequences that correspond to the border motif, DNA was isolated from 100 genetically diverse potato accessions (the so-called "core collection," provided by the US Potato Genebank, WI). This DNA was pooled and used as template for polymerase chain reactions using a variety of oligonucleotides designed to anneal to borders or border-like sequences. Amplified fragments were sequence analyzed, and the sequence was then confirmed using inverse PCR with nested primers. One of the potato DNA fragments that was of particular interest contains a novel sequence without any major open reading frames that is delineated by border-like sequences (Table 1). One of the border-like sequences of this fragment contains 5 mismatches with the closest T-DNA border homolog; the other border-like sequence contains 3 mismatches with the closest homolog. Although both sequences contain one mismatch with the border motif, they were tested for their ability to support DNA transfer. For that purpose, the fragment was first reduced in size to 0.4-kilo basepairs by carrying out an internal deletion (SEQ ID NO.: 2). The resulting fragment was designated "P-DNA" (plant DNA) to distinguish it from the <i>Agrobacterium</i>-derived T-DNA.

0156To test the efficacy of P-DNA transfer from Agrobacterium to plant cells, an expression cassette for the neomycin phosphotransferase (NPTII) gene was inserted within the P-DNA sequence, located on a T-DNA-free plasmid that can be maintained in both <i>E. coli</i> and <i>A. tumefaciens.</i> An <i>Agrobacterium</i> strain carrying the resulting vector was used to infect stem explants of 4-week-old in vitro grown plantlets of the potato variety Russet Ranger. The infected stems were incubated for 2 days on co-culture medium (1/10 MS salts, 3% sucrose, pH 5.7) containing 6 g/L agar at 22°C in a Percival growth chamber (16 hrs light) and subsequently transferred to callus induction medium (CIM, MS medium supplemented with 3% sucrose 3, 2.5 mg/L of zeatin riboside, 0.1 mg/L of naphthalene acetic acid, and 6g/L of agar) containing timentine (150 mg/L) and kanamycin (100 mg/L). After 1 month of culture on CIM, explants were transferred to shoot induction medium (SIM, MS medium supplemented with 3% sucrose, 2.5 mg/L of zeatin riboside,0.3 mg/L of giberelic acid GA3, and 6g/L of agar) containing timentine and kanamycin (150 and 100 mg/L respectively). After 3-4 weeks, the number of explants developing transgenic calli and/or shoots was counted. More calli were observed on potato stem explants infected with an <i>Agrobacterium</i> strain containing the P-DNA vector (0.59 calli/explant) than on explants infected with the control T-DNA vector pBI121 (Genbank accession number AF85783) (0.31 calli/explant).

0157Turf seedlings were also infected with a modified P-DNA vector comprising a ubiquitin-4 promoter driving GUS expression. GUS assays on the transformed plants showed that transformation efficiency were similar to those with control T-DNA vectors.

<u>2. <i>Cytokinin genes as backbone-integration markers</i></u>

0158To make it possible to select against the frequent occurrence of backbone integration events, an expression cassette comprising the <i>Agrobacterium</i> isopentenyl transferase (<i>IPT</i>) gene driven by the Ubi3 promoter and followed by the Ubi3 terminator (SEQ ID NO.: 3) was inserted as 2.6 kbp SacII fragment into the backbone of the P-DNA vector described above.

0159Transformed shoots, generated by infecting potato leaf explants as described above, could be grouped into two different classes. The first class of shoots (55 of 193) was phenotypically indistinguishable from control shoots transformed with LBA::pBI121. The second class of shoots (138 of 193) displayed an IPT phenotype. Shoots of the latter class were stunted in growth, contained only very small leaves, displayed a light-green to yellow color, and were unable to root upon transfer to hormone-free media. To confirm that shoots with an IPT phenotype contained the IPT gene stably integrated in their genomes, all shoots were transferred to Magenta boxes containing MS medium supplemented with 3% sucrose and timentine 1 50 mg/L, allowed to grow for 3 to 4 additional weeks, and used to isolate DNA. This plant DNA served as template in PCR reactions with an oligonucleotide pair designed to anneal to the IPT gene: 5'- GTC CAA CTT GCA CAG GAA AGA C-3', and 5'-CAT GGA TGA AAT ACT CCT GAG C-3'. This PCR experiment confirmed a strict correlation between IPT phenotype and presence of the <i>IPT</i> gene. A second PCR experiment was carried out to test whether IPT-free plants did not contain any other backbone sequences. Because the IPT expression cassette is positioned close to the left border-like sequences, the oligonucleotide pair for this experiment was designed to anneal to backbone sequences close to the right border-like sequence: 5'-CAC GCT AAG TGC CGG CCG TCC GAG-3', and 5'-TCC TAA TCG ACG GCG CAC CGG CTG-3'. Data from this experiment confirm that plants that are positive for the <i>IPT</i> gene are also positive for this other part of the backbone.

<i><u>3. New terminators</u></i>

0160Instead of the frequently used bacterial terminator of the nopaline synthase gene, a new sequence derived from a food source was used to terminate transcription of a selectable marker gene. This terminator is the yeast alcohol dehydrogenase-1 (ADH1) terminator (Genbank accession number V01292, SEQ ID NO. 4). Surprisingly, this specific yeast terminator was shown to function effectively in plant cells by Agro-infecting potato stem explants with different binary vectors that carry an intron-containing <i>GUS</i> gene operably linked to the Ubi7 promoter and followed by either that terminator or the yeast CYC1 terminator. Five days after infection, high levels of transient GUS expression were monitored with the ADH1 terminator, whereas almost no GUS expression was detected with the CYC1 terminator. To terminate transcription of a desired polynucleotide in dicotyledonous plant species, the potato Ubiquitin-3 terminator was used (SEQ ID NO.:5). For transcriptional termination in monocotyledonous plant species, a new terminator was amplified from DNA of the rice variety "Lemont", where it is associated with the actin-1 gene, with the primer set: 5'-GGATCCTCGTCATTTACTTTTATCTTAATGAGC -3' and 5'-GAATTCACATTATAAGCTTTATATTACCAAGG -3' (SEQ ID NO.:6). Functional activity of this rice terminator was demonstrated by operably linking it to a promoter-GUS fusion. Five days after infecting bentgrass seedlings with an <i>Agrobacterium</i> strain containing the resulting expression cassette between borders of a binary vector, transient GUS expression levels were equally high as with a control experiment based on a similar vector carrying the frequently used terminator of the bacterial nopaline synthase gene.

<i><u>4. New promoters</u></i>

0161Instead of viral promoters such as the 35S promoter of cauliflower mosaic virus, new plant promoters were developed and used to express genes in transgenic plants. For some important dicotyledonous plants including potato and cotton, a new promoter was isolated from the potato genome. This new promoter represents a small part (492-bp) of the previously described 1 220-bp and 1788-bp promoters of the potato Ubiquitin-7 gene (<patcit id="pcit0027" dnum="US6448391B1"><text>Garbarino et al., U.S. Patent 6,448,391 B1, 2002</text></patcit>).

0162This conveniently-sized fragment (SEQ ID NO.: 7) was tested for its efficacy to promote high-level expression of transgenes by Agro-infecting tobacco explants with a binary vector carrying the fragment operably linked to the <i>NPTII</i> gene, and placing the infected explants on MS media containing 100 mg/L kanamycin. Within two weeks, a large number of calli developed on these explants, whereas explants infected with a control strain did not contain any calli. Apart from tobacco, the small new promoter was also shown to be active in potato and cotton. An alternative promoter that can be used to drive high-level expression represents 1,026-bp of the Ubi7 promoter (SEQ ID NO.: 8).

0163For monocotyledonous plants, a promoter was developed that resembles the sugarcane ubiquitin-4 promoter. The sequence of this small promoter, designated UbiN, is shown in SEQ ID NO.:9; its homology with the corresponding part of the original Ubiquitin-4 promoter is shown in <figref idref="f0003">Figure 3</figref>. The functional activity of UbiN was assessed by first inserting it between a small Hindlll - Sall 0.2-kbp DNA fragment (SEQ ID NO.: 10) isolated from a modified maize matrix attachment region using the primer set: 5'- AAG CTT AAT AGC TTC ACC TAT ATA ATA -3', and 5'- GTC GAC GGC GTT TAA CAG GCT -3', and a modified EcoRI - BamHl 1.4-kbp fragment containing an intron associated with a sugarcane ubiquitin gene, using the primer set 5'- GAA TTC CCT TCG TCG GAG AAA TTC ATC GAA G -3', and 5'- GGA TCC CTG CAA GCA TTG AGG ACC AG -3' (SEQ ID NO.: 11). The fused DNA fragments were then operably linked to the <i>CAH</i> gene followed by a terminator, and a binary vector containing this expression cassette was used to Agro-infect bentgrass seedlings as described in Example 1. Vigorously growing calli demonstrated that the sugarcane-derived promoter is effective in promoting transgene expression.

<u>5. New vectors</u>

0164As shown in <figref idref="f0001">Figure 1</figref>, a vector may comprise, in 5'- to 3'- orientation, (i) a cytokinin gene (the backbone integration marker) operably linked to elements that can express it, (ii) a first border(-like) P-DNA sequence, (iii) a desired polynucleotide that is operably linked to a promoter and terminator, (iv) an optional selectable marker that is operably linked to a promoter and a terminator, which is associated with a gene that is not naturally expressed in plants, and (v) a second border(-like) P-DNA sequence. A vector also may comprise another desired polynucleotide operably linked to a promoter and terminator, preferably derived from food sources, and inserted within the T-DNA or P-DNA sequence.

Tables

0165<tables id="tabl0002" num="0002"><table frame="all"><title>Table 2. <i>Arabidopsis</i> transformation in TO</title><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="22mm" /><colspec colnum="2" colname="col2" colwidth="21mm" /><colspec colnum="3" colname="col3" colwidth="25mm" /><colspec colnum="4" colname="col4" colwidth="38mm" /><thead><row><entry valign="top"><i>Experiment</i></entry><entry valign="top"><i>Transgene</i></entry><entry valign="top"><i>Treatment</i></entry><entry valign="top"><i>GUS-positive seedlings</i></entry></row></thead><tbody><row><entry>85-6</entry><entry><i>GUS</i></entry><entry>5-min. vortex</entry><entry>15% (11 of 74)</entry></row><row><entry>90-1</entry><entry><i>GUS</i></entry><entry>5-min. vortex</entry><entry>21% (16 of 75)</entry></row><row><entry>95-1</entry><entry><i>GUS</i></entry><entry>5-min. vortex</entry><entry>17% (30 of 181)</entry></row><row><entry>91-1</entry><entry><i>GUS</i></entry><entry>5-min. vortex</entry><entry>5% (3 of 62)</entry></row><row><entry>92-1</entry><entry><i>GUS</i></entry><entry>5-min. vortex</entry><entry>18% (15 of 183)</entry></row><row><entry>90-3</entry><entry><i>GUS</i></entry><entry>5-min. vortex</entry><entry>16% (14 of 87)</entry></row><row><entry rowsep="0"><i>AVERAGE</i></entry><entry rowsep="0"><i>GUS</i></entry><entry rowsep="0">5-min. vortex</entry><entry rowsep="0"><i>13% (89 of 662)</i></entry></row><row><entry /><entry /><entry /><entry /></row><row><entry>85-5</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>15% (11 of 74)</entry></row><row><entry>90-2</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>7% (5 of 69)</entry></row><row><entry>91-2</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>9% (4 of 47)</entry></row><row><entry>91-4</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>3% (2 of 80)</entry></row><row><entry>90-4</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>1% (1 of 72)</entry></row><row><entry>92-4</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>11% (14/123)</entry></row><row><entry>63-2</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>32% (27/84)</entry></row><row><entry>63-3</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>24% (15/63)</entry></row><row><entry rowsep="0"><i>AVERAGE</i></entry><entry rowsep="0"><i>GUS</i></entry><entry rowsep="0">30-min. vortex</entry><entry rowsep="0"><i>13%</i> (<i>79 of 612)</i></entry></row><row><entry /><entry /><entry /><entry /></row></tbody></tgroup></table></tables><tables id="tabl0003" num="0003"><table frame="all"><title>Table 3. Transgenic <i>Arabidopsis</i> plants in selfed progeny</title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="29mm" /><colspec colnum="2" colname="col2" colwidth="28mm" /><colspec colnum="3" colname="col3" colwidth="39mm" /><thead><row><entry valign="top"><i>Experiment</i></entry><entry valign="top"><i>Transgene</i></entry><entry valign="top"><i>GUS-positive seedlings</i></entry></row></thead><tbody><row><entry>63-2-67</entry><entry><i>GUS</i></entry><entry>37% (43 of 117)</entry></row><row><entry>63-6-16</entry><entry><i>GUS</i></entry><entry>51% (55 of 108)</entry></row><row><entry>63-3-57</entry><entry><i>GUS</i></entry><entry>71% (36 of 51)</entry></row><row><entry>63-3-60</entry><entry><i>GUS</i></entry><entry>64% (54 of 85)</entry></row><row><entry>78-8-34</entry><entry><i>GUS</i></entry><entry>56% (53 of 94)</entry></row><row><entry>63-2-22</entry><entry><i>GUS</i></entry><entry>48% (73 of 153)</entry></row><row><entry>63-3-12</entry><entry><i>GUS</i></entry><entry>48% (70 of 147)</entry></row><row><entry>69-2-60</entry><entry><i>GUS</i></entry><entry>78% (53 of 68)</entry></row><row><entry><i>AVERAGE</i></entry><entry><i>GUS</i></entry><entry><i>53% (437 of 823)</i></entry></row></tbody></tgroup></table></tables><tables id="tabl0004" num="0004"><table frame="all"><title>Table 4. Tobacco transformation in TO</title><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="22mm" /><colspec colnum="2" colname="col2" colwidth="21mm" /><colspec colnum="3" colname="col3" colwidth="25mm" /><colspec colnum="4" colname="col4" colwidth="38mm" /><thead><row><entry valign="top"><i>Experiment</i></entry><entry valign="top"><i>Transgene</i></entry><entry valign="top"><i>Treatment</i></entry><entry valign="top"><i>GUS-positive seedlings</i></entry></row></thead><tbody><row><entry>94-1</entry><entry><i>GUS</i></entry><entry>5-min. vortex</entry><entry>4% (4 of 94)</entry></row><row><entry>91-5</entry><entry><i>GUS</i></entry><entry>5-min. vortex</entry><entry>0% (0 of 74)</entry></row><row><entry>94-2</entry><entry><i>GUS</i></entry><entry>5-min. vortex</entry><entry>7% (7 of 100)</entry></row><row><entry>91-6</entry><entry><i>GUS</i></entry><entry>5-min. vortex</entry><entry>1% (1 of 75)</entry></row><row><entry>75-1</entry><entry><i>GUS</i></entry><entry>5-min. vortex</entry><entry>8% (15 of 194)</entry></row><row><entry>78-5</entry><entry><i>GUS</i></entry><entry>5-min. vortex</entry><entry>19% (17 of 91)</entry></row><row><entry><i>AVERAGE</i></entry><entry><i>GUS</i></entry><entry>5-min. vortex</entry><entry><i>7%(44 of 628)</i></entry></row><row><entry>85-2</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>0% (0 of 23)</entry></row><row><entry>92-6</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>0% (0 of 127)</entry></row><row><entry>73-2</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>10% (16 of 155)</entry></row><row><entry>73-1</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>5% (7 of 135)</entry></row><row><entry>70-3</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>8% (4 of 51)</entry></row><row><entry>68-3</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>0% (0 of 49)</entry></row><row><entry>60-1</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>2% (2 of 83)</entry></row><row><entry>68-1</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>8% (5 of 61)</entry></row><row><entry>80-1</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>2% (2 of 97)</entry></row><row><entry>80-3</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>7% (4 of 54)</entry></row><row><entry>85-1</entry><entry><i>GUS</i></entry><entry>30-min. vortex</entry><entry>4% (1 of 27)</entry></row><row><entry rowsep="0"><i>AVERAGE</i></entry><entry rowsep="0"><i>GUS</i></entry><entry rowsep="0">30-min. vortex</entry><entry rowsep="0"><i>5% (41 of 862)</i></entry></row><row><entry /><entry /><entry /><entry /></row></tbody></tgroup></table></tables><tables id="tabl0005" num="0005"><table frame="all"><title>Table 5. Transgenic tobacco plants in selfed progeny</title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="27mm" /><colspec colnum="2" colname="col2" colwidth="26mm" /><colspec colnum="3" colname="col3" colwidth="38mm" /><thead><row><entry valign="top"><i>Experiment</i></entry><entry valign="top"><i>Transgene</i></entry><entry valign="top"><i>GUS-positive seedlings</i></entry></row></thead><tbody><row><entry>62-3-11</entry><entry><i>GUS</i></entry><entry>12% (10 of 85)</entry></row><row><entry>70-3-18</entry><entry><i>GUS</i></entry><entry>15% (16 of 110)</entry></row><row><entry>70-3-23</entry><entry><i>GUS</i></entry><entry>27% (74 of 275)</entry></row><row><entry>70-4-49</entry><entry><i>GUS</i></entry><entry>22% (20 of 91)</entry></row><row><entry><i>AVERAGE</i></entry><entry><i>GUS</i></entry><entry><i>21% (120 of 561)</i></entry></row></tbody></tgroup></table></tables><tables id="tabl0006" num="0006"><table frame="all"><title><b>Table 6. Transgenic creeping bentgrass in selfed progeny</b></title><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="24mm" /><colspec colnum="2" colname="col2" colwidth="23mm" /><colspec colnum="3" colname="col3" colwidth="34mm" /><colspec colnum="4" colname="col4" colwidth="39mm" /><thead><row><entry valign="top"><i>Experiment</i></entry><entry valign="top"><i>Transgene</i></entry><entry valign="top"><i>Treatment in TO</i></entry><entry valign="top"><i>CAH-positive seedlings</i></entry></row></thead><tbody><row><entry>5G-23</entry><entry><i>CAH</i></entry><entry><i>Dormex screen</i></entry><entry>4 of 21</entry></row><row><entry>3B-7</entry><entry><i>CAH</i></entry><entry><i>Dormex screen</i></entry><entry>1 of 3</entry></row><row><entry>3B-14</entry><entry><i>CAH</i></entry><entry><i>Dormex screen</i></entry><entry>0 of 1</entry></row><row><entry rowsep="0"><i>AVERAGE</i></entry><entry rowsep="0"><i>CAH</i></entry><entry rowsep="0"><i>Dormex screen</i></entry><entry rowsep="0"><i>20% (5 of 25)</i></entry></row><row><entry /><entry /><entry /><entry /></row><row><entry>5J-18</entry><entry><i>CAH</i></entry><entry><i>Cyanamide selection</i></entry><entry>1 of 1</entry></row><row><entry>5J-23</entry><entry><i>CAH</i></entry><entry><i>Cyanamide selection</i></entry><entry>4 of 5</entry></row><row><entry><i>AVERAGE</i></entry><entry><i>CAH</i></entry><entry><i>Cyanamide selection</i></entry><entry>~<i>83% (5 of 6)</i></entry></row></tbody></tgroup></table></tables>

<u>SEQ ID NOs.</u>

0166<tables id="tabl0007" num="0007"><table frame="none"><tgroup cols="2" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="26mm" /><colspec colnum="2" colname="col2" colwidth="140mm" /><tbody><row><entry>SEQ ID NO.: 1</entry><entry>Cyanamide tolerance gene from <i>Aspergillus</i> sp.</entry></row><row><entry>SEQ ID NO.:2</entry><entry>Potato P-DNA. The bold underlined portions represent the left (5'-) and right (3'-) border-like sequences of the P-DNA respectively.</entry></row><row><entry>SEQ ID NO.:3</entry><entry>Expression cassette for the cytokinin <i>IPT</i> gene</entry></row><row><entry>SEQ ID NO.:4</entry><entry>Terminator associated with the yeast <i>ADH1</i> gene</entry></row><row><entry>SEQ ID NO.:5</entry><entry>Terminator associated with the potato Ubiquitin-3 gene</entry></row><row><entry>SEQ ID NO.:6</entry><entry>Terminator associated with the rice actin-1 gene</entry></row><row><entry>SEQ ID NO.:7</entry><entry>Short 0.5-kbp promoter associated with the potato Ubiquitin-7 gene</entry></row><row><entry>SEQ ID NO.:8</entry><entry>Short 1.0-kbp promoter associated with the potato Ubiquitin-7 gene</entry></row><row><entry>SEQ ID NO.:9</entry><entry>Plant-like promoter</entry></row><row><entry>SEQ ID NO.:10</entry><entry>Part of a maize matrix-associated region</entry></row><row><entry>SEQ ID NO.:11</entry><entry>Intron associated with the sugarcane Ubiquitin-4 gene</entry></row></tbody></tgroup></table></tables><img file="EP1560484B1_D0001.tif" /><img file="EP1560484B1_D0002.tif" /><img file="EP1560484B1_D0003.tif" />