MXPA96002691A - Selectable markers of the tfda gene in plantasy use of the mis - Google Patents

Abstract

The present invention relates, in general, to cells of transgenic plants and plants. In particular, the present invention relates to: 1) a method for selecting a transgenic plant cell, which comprises transforming one or more plant cells with a tfdA gene, 2) a plant cell comprising a tfdA gene, where this plant cell is free of other foreign marker genes, and 3) a sweetgum plant cell comprising a tf gene

Description

8ELECTABLE MARKERS OF THE TDAA GENE IN PLANTS AND USE THEMSELVES Reference to Related Requests This is a partial continuation of the Request for Patent of the United States of America with Number of Series 08 / 179,667, filed on January 11, 1994, the contents of which are incorporated in full as a reference to the V. $ - present.

Field of the Invention The present invention relates, in general, to cells of transgenic plants and plants. In particular, the The present invention relates to: 1) a method for selecting a transgenic plant cell comprising transforming one or more plant cells with a tfdA gene; 2) a plant cell comprising a tfdA gene, wherein this plant cell is free of other foreign marker genes; and 3) a liquida bar plant cell comprising a tfdA gene.

BACKGROUND OF THE INVENTION 2,4-Dichlorophenoxyacetic acid (2,4-D) is a herbicide used to control leaf weeds wide. The gene coding for the first one in the degradation path of 2,4-dichlorophenoxyacetic acid (Figure 1) is tfdA. This gene encodes a monooxygenase that catalyzes the conversion of 2,4-dichlorophenoxyacetic acid to 2,4-dichlorophenol (DCP). Transgenic tobacco and cotton plants containing the tfdA gene have been shown to have a higher tolerance to 2,4-dichlorophenoxyacetic acid (Streber et al., Bio / technology 7: 811-816 (1989) and Bayley et al., Theor. Appl. Genet 83: 645-649 (1992) respectively), so far, attempts to use the tfdA gene as a selectable marker to identify transformed plants have failed. Streber et al., Bio / technology 7: 811-816 (1989), theorize that "in a chimeric tissue, the transformed cells can not develop into shoots, because they are overcultivated or otherwise inhibited by the untransformed stem that is develops rapidly in the presence of 2,4-dichlorophenoxyacetic acid. " Summary of the Invention The invention provides a method for selecting a transgenic plant cell. The invention further provides a method for selecting a transgenic plant cell comprising: a) transforming one or more plant cells with a tfdA gene that can be expressed in the plant cell, b) culturing the cell of the transformed plant with an amount of 2,4-dichlorophenoxyacetic acid which inhibits adventitious bud formation or regeneration from untransformed plant cells; and c) selecting a plant cell that exhibits growth. The invention also provides a method for selecting a transgenic plant, which comprises: a) transforming one or more plant cells with a tfdA gene that can be expressed in the plant cell, b) culturing the cell of the transformed plant with an amount of 2,4-dichlorophenoxyacetic acid which inhibits adventitious shoot formation or regeneration from non-transformed plant cells, c) selecting a plant cell that exhibits growth, and d) regenerating the plant cell in a plant. The invention further provides a plant cell comprising a tfdA gene that can be expressed in the plant cell, wherein the plant cell is free of other foreign marker genes; a plant regenerated from the plant cell; progeny or a propagule of the plant; and seed produced by the progeny. The invention further provides a liquidambar plant cell comprising a tfdA gene that can be expressed in the cell of the plant; a plant regenerated from the cell of the liquidambar plant; progeny of the plant; a plant propagule; and seeds produced by the progeny. Herbicide resistance (and more specifically, resistance to 2,4-dichlorophenoxyacetic acid) in Liquidambar would greatly reduce the costs of site preparation., and possibly make the cultivation of Liquidambar (Liguidáírubar styraciflua L.) economically in the plantations. Although transgenic tobacco and cotton plants containing the tfdA gene have been shown to have a higher tolerance to 2,4-dichlorophenoxyacetic acid (Streber et al., Bio / technology 7: 811-816 (1989) and Bayley et al., Theor. Appl. Genet 83: 645-649 (1992), respectively), it was not known if tolerance could be conferred in the liquidambar. The present invention provides cells from transgenic sweetgum plants that contain the tfdA gene. The invention also relates to a method for obtaining, and to a method for handling, a hard planting, which comprises: (a) transforming one or more plant cells with a polynucleotide comprising a gene for resistance to herbicides that is can express in that plant cell; ~ (b) regenerate the plant cell in a plant; (c) growing the regenerated plant with an amount of herbicide that will kill the non-transformed plants; (d) selecting a plant that exhibits growth; (e) propagate the plant to produce many plants; (f) induce the formation of the root in these plants; (g) cultivating the rooted plants to a size of planting material; (h) plants plants of planting material size in a trimmed and defoliated site, and (i) apply herbicide over the entire site to suppress competitive growth until the planting material can develop without competitive growth control. The invention also relates to a method for obtaining, and to a method for handling, a hardwood plantation, which comprises: (a) transforming one or more plant cells with a polynucleotide comprising a gene for herbicide resistance and when minus a second gene encoding a foreign selectable marker that can be expressed in that plant cell; (b) culturing the plant cell with an amount of a chemical that inhibits regeneration of the adventitious bud from cells of untransformed plants with the foreign selectable marker gene; (c) selecting a plant cell that exhibits regeneration of the adventitious bud; (d) regenerating the plant cell in a plant; (e) propagate the plant to produce many plants; (f) induce root formation in these plants; (g) cultivating the rooted plants to a size of planting material; (h) plant plants of planting material size in a trimmed and defoliated site, and (i) apply herbicide over the entire site to suppress competitive growth until the planting material can develop without competitive growth control. The invention also relates to a method for obtaining, and to a method of handling, a hardwood plantation, which comprises: (a) transforming one or more plant cells with a polynucleotide comprising a herbicide resistance gene, and at least one second gene encoding a foreign selectable marker that can be expressed in that plant cell; (b) culturing the plant cell with an amount of a chemical that inhibits the regeneration of the adventitious bud from non-transformed plant cells with the foreign selectable marker gene; (c) selecting a plant cell that exhibits regeneration of the adventitious bud; (d) regenerating the plant cell in a plant; (e) growing the regenerated plant with an amount of herbicide that kills non-transformed plants; (f) select a plant that exhibits growth; (g) regenerating that plant cell in a plant; (h) propagate the plant to produce many plants; (i) induce root formation in these plants; (j) cultivating the rooted plants to a size of planting material; (k) planting plant size plants in a trimmed and defoliated site, and (1) applying the herbicide over the entire site to suppress competitive growth until the planting material can develop without competitive growth control. Preferably, the herbicide resistance gene is the tfdA gene and the herbicide is 2,4-dichlorophenoxyacetic acid, the herbicide resistance gene is the mutant acetohydroxy acid synthase gene of Arabidopis, or the herbicide resistance gene is the 5-enolpyruvylshikimate-3-phosphate synthase gene and the herbicide is glyphosate. The invention also relates to a method for obtaining, and a method for handling, a hardwood plantation, which comprises: planting plants of planting material size in a cut and defoliated site, and applying herbicide over the entire site to suppress competitive growth until the planting material can be developed without competitive growth control, where the plant is a hardwood comprising a gene for herbicide resistance. The invention also relates to a plantation of hardwood trees comprising a gene for resistance to herbicides. Preferably, the herbicide resistance gene is the tfdA gene, the mutant acetohydroxy acid synthase gene of AraJidopis, or the 5-enolpyruvylshikimate-3-phosphate synthase gene. Other objects and advantages of the present invention will become clear from the following description.

Brief Description of the Figures Figure 1. The path of degradation of 2,4-dichlorophenoxyacetic acid. The gene designations are shown in parentheses for each enzyme. Figure 2. Construction of pUCWlOl. Figure 3. Construction of pUC 200. Figure 4. Construction of pBI121. Figure 5. Liquidambar 2040 ELISA selected on kanamycin.

Figure 6. Liquidase 2027 ELISA selected on 2,4-dichlorophenoxyacetic acid or cannabin. Figure 7. Sequence of a tfdA gene of Figure 3 of Streber et al., ". Of Bacteriology 169: 2950-2955 (1987).

Definitions Plant should be understood to refer to a differentiated multicellular organism capable of photosynthesis, including angiosperms (monocotyledons and dicots) and gymnosperms. Plant cell must be understood to refer to the structural and physiological unit of plants. The term "plant cell" refers to any cell that is part of, or derived from, a plant. Some examples of cells encompassed by the present invention include differentiated cells that are part of a living plant; Differentiated cells in culture; undifferentiated cells in culture; undifferentiated tissue cells such as corns or tumors. Progeny of the plant cell should be understood to refer to any cell or tissue derived from plant cells, including calluses; parts of plants such as stems, roots, fruits, leaves, or flowers; plants; plant seeds; pollen; and plant embryos.

The propagules should be understood to refer to any plant material capable of spreading sexually or asexually, or of spreading in vivo or in vitro. These propagules preferably consist of the protoplasts, cells, calluses, tissues, embryos, or seeds of the regenerated plants. Transgenic plant must be understood to refer to a plant that has stably incorporated exogenous DNA into its genetic material. The term also includes exogenous DNA which can be introduced into a cell or protoplast in different forms, including, for example, naked DNA in a circular, linear, or superspiral form, DNA contained in nucleosomes or chromosomes or cores or parts thereof, DNA complexed or associated with other molecules, DNA encased in liposomes, spheroplasts, cells, or protoplasts.

Detailed Description of the Invention The present invention relates to a method for selecting a transgenic plant cell. In one embodiment, the present invention relates to a method for selecting a transgenic plant cell, which comprises: a) transforming one or more plant cells with a tfdA gene that can be expressed in the plant cell, b) cultivar the plant cell transformed with an amount of 2,4-dichlorophenoxyacetic acid which inhibits the formation of adventitious shoots from non-transformed plant cells, and c) selecting a plant cell that exhibits growth. In another embodiment, the present invention relates to a method for selecting a transgenic plant, which comprises: a) transforming one or more plant cells with a tfdA gene that can be expressed in that plant cell, b) culturing the cell of plant transformed with an amount of 2,4-dichlorophenoxyacetic acid that inhibits the formation of adventitious shoots from non-transformed plant cells, c) selecting a plant cell that exhibits growth, and d) regenerating that plant cell in a plant . All plants that can be transformed are intended to be included within the scope of the invention (preferably, dicotyledonous plants). These plants include, for example, species of the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscya us, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthue, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Sencia, Salpiglossis, Cucumis, Browalia, Glycine, Lolium, Zea, Triticum, Sorghum, Malus, Apium, and Datura. Suitable species of dicotyledonous forest trees (hardwoods) include, for example, species from the genera Acacia, Acer, Actinidia, Albizzia, Alnus, Amelanchier, Atriplex, Betula (Birch), Brachycome, Broussonetia, Camellia, Carya , Castanea (Castaño), Catalpa, Cinchona, Corylus (Hazel) Diospyrus, Eucalyptus, Fagus (Haya), Ficus, Fraxinus (Fresno), Gleditsia, Hamamelia, Hedera, Ilex, Kalmia, Liquidambar (Liquidambar), Liriodendron, Moghania, Morus , Paulownia, Populus, Prunus, Quercus (Oak), Rhododendron, Robinia, Salix, Santalum, Sapium, Simmondeia, Tectona, Tupidanthus, Ul us (Olmo), and Vaccinium. Plant transformation techniques are well known in the art, and include direct transformation (which includes, but is not limited to: microinjection (Crossway, Mol. Gen. Genetics 202: 179-185 (1985)), glycol transformation of polyethylene (Krens et al., Nature 296: 72-74 (1982)), high velocity ballistic penetration (Klein et al., Nature 327: 70-73 (1987)), fusion of protoplasts with other entities, either mini-cells, cells, lysosomes, or other fusible lipid surface bodies (Fraley et al., Proc. Nati, Acad. Sci. USA 79: 1859-1863 (1982)), electroincorporation (Fromm et al., Proc. Nati. Acad. Sci. USA 82: 5824 (1985)), and the techniques set forth in U.S. Patent No. 5,231,019, and transformation mediated by Agrobacterium turne faciens (Hoeke et al., Nature 303: 179 (1983), by Framond et al., Bio / technology 1: 262 (1983), Fraley et al., WO 84/02913, WO84 / 02919, and WO84 / 02920, Zambryski et al., EP 116,718, Jordán et al., Plant Cell Reports 7: 281-284 (1988), Leple et al., Plant Cell Reports 11: 137-141 (1992 ), Stomp et al., Plant Phyeiol. 92: 1226-1232 (1990), and Knauf et al., Plasmid 8: 45-54 (1982)). A preferred method of transformation is the leaf disc transformation technique as described by Horsch et al., Science 227: 1229-1230 (1985). The transformation techniques described above can use a tfdA gene or a fragment thereof that can be expressed in plants. Within the scope of a tfdA gene are included the functional derivatives of tfdA, as well as variants, analogs, species, and allelic and mutational derivatives. A "fragment" of a molecule means any portion of the genetic sequence of amino acids or nucleotides. A "functional derivative" of a sequence is a molecule that possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of the protein or nucleic acid sequence. A "variant" of a nucleic acid means a molecule substantially similar in structure and biological activity to the nucleic acid, or to a fragment thereof. Accordingly, since two molecules have a common activity, and can substitute one another, variants are considered as this term is used herein, even when the nucleotide sequence is not identical. An "analogue" of a protein or genetic sequence means a protein or genetic sequence substantially similar in function to a protein or genetic sequence described herein. An "allele" is an alternative form of a gene that occupies a given place on the chromosome. A "mutation" is any detectable change in the genetic material that can be transmitted to daughter cells, and possibly even to successive generations, giving rise to mutant cells or mutant individuals. If the descendants of a mutant cell give rise only to somatic cells in multicellular organisms, a mutant spot or cell area is present. Mutations in the germline of sexually reproducing organisms can be transmitted by gametes to the next generation, resulting in an individual with the new mutant condition in both somatic and germ cells. A mutation can be any (or a combination of) detectable unnatural change that affects chemical or physical constitution, mutability, replication, phenotypic function, or recombination of one or more deoxyribonucleotides; Nucleotides can be added, deleted, replaced, inverted, or transported to new positions with and without investment. Mutations can occur spontaneously, and can be induced experimentally by the application of mutagens. A mutant variation of a nucleic acid molecule results from a mutation. A mutant polypeptide can result from a mutant nucleic acid molecule. A "species" is a group of natural populations that are really or potentially intertwined. A variation of species within a nucleic acid or protein molecule is a change in the nucleic acid or amino acid sequence that occurs between the species, and can be determined by sequencing the DNA of the molecule in question. The tfdA gene illustrated in Figure 7 (IDENTIFICATION SEQUENCE NUMBER: 1) may be altered by substitutions, additions, or deletions that provide functionally equivalent molecules. Due to the degeneracy of the nucleotide coding sequences, other DNA sequences that encode substantially the same amino acid sequence illustrated in Figure 7 (IDENTIFICATION OF SEQUENCE NUMBER: 2) can be used in the practice of the present invention. These include, but are not limited to, nucleotide sequences comprising all or portions of the tfdA gene illustrated in Figure 7 (IDENTIFICATION OF SEQUENCE NUMBER: 1) that are altered by the substitution of different codons that encode a functionally equivalent amino acid residue. within the sequence, thus producing a silent change. These functional alterations of a given nucleic acid sequence provide an opportunity to promote a secretion and / or processing of hotcroloqar protein. encoded by the foreign nucleic acid sequences fused thereto. All variations of the nucleotide sequence of the tfdA gene and fragments thereof allowed by the genetic code, therefore, are included in the present invention. In addition, the tfdA gene may comprise a nucleotide sequence resulting from the addition, deletion, or substitution of at least one nucleotide for the 51 end and / or the 3 'end of the nucleic acid formula shown in the SEQUENCE IDENTIFICATION NUMBER: 1 or a derivative thereof. In this aspect, any nucleotide or polynucleotide can be used, since its addition, deletion, or substitution does not alter the amino acid sequence of the SEQUENCE IDENTIFICATION NUMBER: 2 which is encoded by the nucleotide sequence. The tfdA gene may have, as necessary, restriction endonuclease recognition sites aggregated at its 5 'end and / or its 3' end. In addition, it is possible to suppress codons or substitute one or more codons for different codons that degenerate the codons to produce a structurally modified polypeptide, but one that has substantially the same utility or activity of the polypeptide produced by the unmodified nucleic acid molecule. As recognized in this field, the two polypeptides are functionally equivalent, as well as the two nucleic acid molecules, which give rise to their production, even though the differences between the nucleic acid molecules are not related to the degeneracy of the genetic code. The tfdA gene is preferably operably linked to a functional promoter region in plants, a transcription initiation site, and a transcription termination sequence. The particular promoter used in the expression cartridge is a non-critical aspect of the invention. Any of a number of promoters that direct transcription in a plant cell is suitable. The promoter can be constitutive or inducible. Some examples of functional promoters in plants include the nopaline synthase promoter and other promoters derived from native Ti plasmids, viral promoters including the 35S and 19S RNA promoters of cauliflower mosaic virus (Odell et al., Nature 313: 810 812 (1985), and numerous plant promoters General methods for selecting cells from transgenic plants containing a selectable marker are well known and are described, for example, by Herrera-Estrella, L., and Simpson, J., "Foreign Gene Expression in Plants," in Plant Molecular Biology, A Practical Approach, ch Sha, ed., IRL Press, Oxford, England (1988), pages 131-160.To use the tfdA gene as a selectable marker, the amount of 2,4-dichlorophenoxyacetic acid that inhibits the adventitious bud formation from cells of non-transformed plants, and that allows the formation of the adventitious bud from cells of trans plants formed, can be determined by: 1) coating untransformed cells on a medium containing different concentrations of 2,4-dichlorophenoxyacetic acid, and 2) determining the lowest concentration of 2,4-dichlorophenoxyacetic acid that will inhibit the formation of the outbreak adventitious by plant cells. This lower concentration can then be used to select the cells of transformed plants. In general, the solubilized 2,4-dichlorophenoxyacetic acid should be present in an amount of about 0.001 to 5 milligrams / liter of culture medium. With respect to the cells of liquidambar plants transformed with the tfdA gene, 2,4-dichlorophenoxyacetic acid should preferably be present in an amount of about 0.01 to 0.5 milligrams / liter of culture medium. A preferred amount of 2,4-dichlorophenoxyacetic acid is from about 0.01 to 0.2 milligrams / liter of culture medium. The amount of 2,4-dichlorophenoxyacetic acid to be used for the selection of transformed shoot cultures is determined by identifying the minimum concentration of 2,4-dichlorophenoxyacetic acid that will inhibit the adventitious bud formation. The expanding leaves of a selected liquidambar clone are surface sterilized and cut into small pieces. The pieces of sheet are then placed on WPM 0.1 milligrams / liter NAA, 2.5 milligrams / liter BA containing 2,4-dichlorophenoxyacetic acid at concentrations '< ._ from 0.0 to 5.0 milligrams / liter. The leaf pieces are incubated until the control pieces (without 2,4-dichlorophenoxyacetic acid) regenerate the shoots. Concentration The ideal 2, 4-dichlorophenoxyacetic acid for the selection of transformants is the lowest concentration of 2,4-dichlorophenoxyacetic acid that will not allow regeneration. In another embodiment, the present invention relates to a plant cell comprising a tfdA gene that can be Expressing in the plant cell, wherein the plant cell is free of other foreign marker genes (preferably, other foreign selectable marker genes); a plant regenerated from the plant cell; the progeny or a propagule of the plant, and the seeds produced by the progeny. Plant regeneration techniques are well known in the field, and include those stipulated in Handbook of Plant Cell Culture, Volumes 1 to 3, Evans et al., Eds., Macmillan Publiehing Co., New York, NY (1983, 1984, 1984, respectively); Predieri and Malavasi, Plant Cell, Tissue, and Organ Culture 17: 133-142 (1989); James, D.J., and collaborators, J. Plant Physiol. 132: 148-154 (1988); Fasolo, F. et al., Plant Cell, Tissue, and Organ Culture 16: 75-87 (1989); Valobra and James, Plant Cell, Tissue, and Organ Cul ture 21: 51-54 (1990); Srivastava, P.S. and collaborators, Plant Science 42: 209-214 (1985); Rowland and Ogden, Hort. Science 27: 1127-1129 (1992); Park and Son, Plant Cell, Tiseue and Organ Cul ture 15: 95-105 (1988); Noh and Minocha, Plant Cell Reports 5: 464-467 (1986); Brand and Lineberger, Plant Science 57: 173-179 (1988); Bozhkov, P.V. and collaborators, Plant Cell Reports 11: 386-389 (1992); Kvaalen and von Arnold, Plant Cell, Tissue, and Organ Cul ture 27: 49-57 (1991); Tremblay and Tremblay, Plant Cell, Tissue, and Organ Culture 27: 95-103 (1991); Gupta and Pullman, Patent of the United States of North America Number 5,036,007; Michler and Bauer, Plant Science 77: 111-118 (1991); etzstein, H.Y. and collaborators, Plant Science 64: 193-201 (1989); McGranahan, G.H. and collaborators, Bio / technology 6: 800-804 (1988); Gingas, V.M. Hort. Science 26: 1217-1218 (1991); Chalupa, V., Plant Cell Reports 9: 398-401 (1990); Gingas and Lineberger, Plant Cell, Tissue, and Organ Cul ture 17: 191-203 (1989); Bureno, M.A. and collaborators, Phys. Plant. 85: 30-34 (1992); and Roberts, D.R. and collaborators, Can. J. Bot. 68: 1086-1090 (1990). The herbicide-resistant plant makes it possible for the farmer to plant a herbicide-tolerant crop, and then treat the field with herbs without adversely affecting the crop. In addition, the herbicide tolerant plant makes it possible for the farmer to grow crops in fields that have been treated with herbicides. These herbicide treated fields will contain a certain amount of the herbicide in the soil, and therefore, a "herbicide forcer" is seen (U.S. Patent No. 4,795,374). Other foreign marker genes (ie, exogenously introduced genes) typically used include selectable markers such as a neo gene (Potrykus et al., Mol.Gen. Gen. 199: 183-188 (1985)), which encodes resistance to kanamycin; a bar gene that codes for bialaphos resistance; a mutant EPSP synthase gene (Hinchee et al., Bio / technology 6: 915-922 (1988)), which codes for glyphosate resistance; a nitrilase gene that confers resistance to bromoxynil (Stalker et al., J. Biol. Chem. 263: 6310-6314 (1988)); a mutant acetolactate synthase (ALS) gene, which confers resistance to imidazolinone or sulphonic urea (European patent application number 154,204); a DHFR gene resistant to methotrexate (Thillet et al., J. Biol. Chem. 263: 12500-12508), and classifiable markers that include β-glucuronidase (GUS) or an R-locus gene, alone or in combination with a gene C-locus (Ludwig et al., Proc. Nati, Acad. Sci. USA 86: 7092 (1989), PazAres et al., EMBO J. 6: 3553 (1987)). Plants containing the tfdA gene and no other foreign marker gene are convenient in that it may be impossible to remove the foreign marker gene, once inserted into the plant, without also removing the tfdA gene. The absence of the foreign marker gene is desired to minimize the number of foreign genes expressed. A plasmid containing only tfdA between the boundaries of the Ti plasmid can be constructed in several ways. One method is to first partially digest the plasmid pUCW200 with EcoRI, such that only the EcoRI site is cut on the right side of the NOS terminator (Figure 4). The ends are then made blunt by the use of standard molecular methods (Maniatis, T. et al., Molecular Cloning: A. Laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)). This linear plasmid is digested with the restriction endonuclease HindIII. The fragment containing tfdA under the control of the CaMV 35S promoter is purified by resolving it and cutting it from a low melting temperature agarose gel. This HindlII-EcoRI fragment is ligated into the binary vector pBIN19 (Bevan, M., Nucleic Acids Research 12: 8711-8721 (1984)), which has been first digested with the restriction endonuclease SstII and the ends become blunt, and then digested with HindIII. The resulting plasmid is then transformed into E. coli S17-1 with selection for kanamycin resistance conferred by the kanamycin resistance gene located on the plasmid pBIN19, but outside the left and right boundaries of the Ti plasmid. Then this plasmid is mobilized towards Agrojacterium tumefaciens LBA 4404, and is used to transform the target plant tissue. In another embodiment, the present invention relates to a liquidgum plant cell comprising a tfdA gene that can be expressed in the plant cell; a plant regenerated from the liquidambar plant cell; progeny of the plant; a plant propagule; and seeds produced by the progeny. Historically, the establishment of hardwood plantations over cut forest land, old agricultural fields, and lowland rivers has only been successful when very intensive site preparation has been employed prior to planting (Hunt, Hardwood Short Course, North Carolina State University (1973), pages 63-71). An example of the required site preparation methods is described in "Hardwood Plantation Management" (Malac, BF and Heeren, RD, Southern Journal of Applied Forestry 3: 3-6 (1979).) Typically site preparation requires a regiment of clipping, raking, and formation of discs or beds The prepared planting site must be clean and level, in such a way that a targeted application of the herbicide is possible, for competitive control.The targeted application of the herbicide is necessary to avoid annihilation of hardwood plants Not only is this intense preparation of the site costly, but it also results in site degradation due to soil compaction by heavy machinery and superior soil loss due to raking. Site degradation results in lower yields. Site preparation requirements will be greatly reduced if wood is planted s that are genetically designed to be resistant to herbicides. The herbicide can be applied by aerial spraying. Therefore, the site would not have to be level. This would eliminate the need for raking, and the formation of disks. Also, the amount of mowing and cultivation required for competitive control would be reduced. The lower site preparation would have the additional benefit of less soil compaction and less top soil loss, and consequently, less site degradation and higher yields will result. The planting of hardwoods resistant to herbicides would also have several advantages over the planting of conventional seedlings or rooted cuts. These advantages include manual planting, and an opportunity to increase the number of stems per acre. Manual planting would be possible because, with the application of aerial herbicide, no straight grooves are needed for the tractors. If restrictions on the separation required to operate the tractors between the rows are no longer necessary, more stems per acre could be planted, which would result in higher yields. The tolerance to the herbicide of 2,4-dichlorophenoxyacetic acid can be conferred to the liquidambar by the introduction of a tfdA gene that can be expressed on liquidambar, in the genome of the liquidambar. The tfdA gene can be introduced into the sweetgum by the transformation techniques illustrated above, or more preferably as stipulated in Chen, Z. and Stomp, A. (1991) "Transformation of Liguidamber styraciflua L. (Sweetgu) using AgroJbasterium Tumefaciens" , In: Proceedings 21st. Southern Forest Treß Improve ent Conference. June 17 to 20, 1991, Knoxville, TN. A tfdA gene is preferably contained on a plasmid wherein the tfdA gene is operably linked to a functional promoter region in plants, a transcription initiation site, and a transcription termination sequence (examples of which are given above). In a preferred embodiment, the tfdA gene binds to a foreign marker gene (described above). Accordingly, the invention also relates to a method for managing a hardwood plantation, which comprises: (a) transforming one or more plant cells with a polynucleotide comprising a herbicide resistance gene that can be expressed therein plant cell; (b) regenerating the plant cell in a plant; (c) growing the regenerated plant with an amount of herbicide that will kill the non-transformed plants; (d) selecting a plant that exhibits growth; (e) propagate this plant to produce many plants; (f) induce root formation in these plants; (g) cultivating the rooted plants to a size of planting material; (h) plants plants of planting material size in a trimmed and defoliated site, and (i) apply herbicide over the entire site to suppress competitive growth until the planting material can develop without competitive growth control. The invention also relates to a method for managing a hardwood plantation, which comprises: (a) transforming one or more plant cells with a polynucleotide comprising a herbicide resistance gene, and at least a second gene encoding a strange selectable marker that can be expressed in that plant cell; (b) culturing the plant cell with an amount of a chemical that inhibits regeneration of the adventitious bud from cells of untransformed plants with the foreign selectable marker gene; (c) selecting a plant cell that exhibits regeneration of the adventitious bud; (d) regenerating the plant cell in a plant; (e) propagate the plant to produce many plants; (f) induce root formation in these plants; (g) cultivating the rooted plants to a size of planting material; (h) plant plants of planting material size in a trimmed and defoliated site, and (i) apply herbicide over the entire site to suppress competitive growth until the planting material can develop without competitive growth control. The invention also relates to a method for managing a hardwood plantation, which comprises: (a) transforming one or more plant cells with a polynucleotide comprising a herbicide resistance gene, and at least a second gene encoding a strange selectable marker that can be expressed in that plant cell; (b) culturing that plant cell with an amount of a chemical that inhibits regeneration of the adventitious bud from cells of non-transformed plants with the foreign selectable marker gene; (c) selecting a plant cell that exhibits regeneration of the adventitious bud; (d) regenerating the plant cell in a plant; (e) growing the regenerated plant with an amount of herbicide that will kill the non-transformed plants; (f) select a plant that exhibits growth; (g) regenerating the plant cell in a plant; (h) propagate the plant to produce many plants; (i) induce root formation in these plants; (j) cultivating the rooted plants to a size of planting material; (k) planting plant size plants in a trimmed and defoliated site, and (1) applying the herbicide over the entire site to suppress competitive growth until the planting material can develop without competitive growth control. The invention also relates to a method for managing a hardwood plantation, which comprises: planting plants of plantation material size in a trimmed and defoliated site, and applying herbicide over the entire site to suppress competitive growth until the Planting material can be developed without a competitive growth control, where this plant is a hardwood that comprises a gene for resistance to herbicides. The invention also relates to a plantation of hardwood trees comprising a gene resistant to herbicides. Preferably, hardwood trees are liquidambar. In general, hardwoods resistant to herbicides can be constructed by transforming genes to encode proteins that detoxify the herbicide, such as the tfdA gene, or which encode proteins that are not sensitive to the action of the herbicide, such as the synthase gene. mutant acetohydroxy acid of AraJidopis (European Patent Application Number 91119254.0, by American Cyanamid Company). Another example of a gene that can confer resistance to herbicides is the 5-enolpyruvylshikimate-3-phosphate synthase gene (U.S. Patent Number 4,940,835, Monsanto Company), which is tolerant to the glyphosate herbicide. The selection for the transformation of these genes into the target plant can be made by selection by the presence of a selectable marker gene, such as the kanamycin resistance gene, which binds to the herbicide resistance gene. This is done by incubating the target tissue on a medium containing kanamycin levels that inhibit the regeneration of transformed cells. Alternatively, selection can be made by selecting for regeneration in the presence of the herbicide at concentrations that inhibit the regeneration of untransformed cells, or allowing regeneration to take place in the absence of selection, and then classifying the plants regenerated by the presence of the herbicide resistance gene by cultivating putative transformants on media containing herbicide levels that inhibit the growth of non-transformed plants. The preferred method of selection for the transformation of the herbicide resistance genes, other than the tfdA gene, is to first select for regeneration in the presence of kanamycin, and then to classify the regenerated plants by incubation on a medium containing herbicide levels that are toxic to non-transformed plants. The plantations are a group of a large number of trees in cultivation. Preferably, nutrients are added, such as triple superphosphate or diammonium phosphate, to the site soil. (See, Davey, CB, Hardwood Short Course, North Carolina State University (1973), pages 72-75), and "Hardwood Plantation Management" (Malac, BF and Heeren, RD, Southern Journal of Applied Forestry 3: 3-6 (1979)). In addition, the site can be raked before planting the planting material. * - The invention also relates to a method for producing or managing a hardwood plantation, which comprises planting plants of planting material size in a trimmed and defoliated site, and apply herbicide over the entire site to suppress competitive growth until the planting material can be grown without competitive growth control, where the plant is a hardwood plant comprising a tfdA gene . From preference, the plant is liquidambar.

The present invention is described in greater detail in the following non-limiting examples.

Examples ~ The following protocols and experimental details are referenced in the following examples. to. Sources of Strain and Conditions of Cultivation. The bacterial strains and plasmids used herein are listed in Table 1, and the media formulas are shown in Tables 2 and 3. Peeudomonae aeruginoea PAOlc containing plasmid pROlOl or plasmid pR01727 is grown on TNA plates containing 50 micrograms / milliliter of tetracycline (TC50) at 37 ° C. P. aeruginosa PAOlc (pUCWlOl, Figure 2) was cultured on TNA containing 500 micrograms / milliliter of carbenicillin (Cb500) at 37 ° C. P. putida PPO300 (pUCW200; Figure 3), Agro £ > acteriu / n tumefaciens LBA4404 (pUCW200; Figure 3), Escherichia coli HB101 (pBI121, Figure 4), and E. coli S17-1 (pUC 200; Figure 3), on TNA containing 50 micrograms / milliliter of kanamycin (Km5). Strains of P. putida and A. tumefaciens were grown at 30 ° C, and strains of E. coli were grown at 37 ° C. The culture of P. aeruginosa PAOlc (pUCWlOl), P. putida PPO300 (pUCW200), and A. tumefaciens LBA4404 (pUCW200), for the analysis of the conversion of 2,4-dichlorophenoxyacetic acid to 2,4-dichlorophenol, was done by inoculating 50 milliliters of Burk / CAA medium containing 1 mM of 2,4-dichlorophenoxyacetic acid with a culture cycle from a TNA plate overnight containing the appropriate antibiotic. These liquid cultures were shaken at 30 ° C for 4 hours, and then sterilized with filter. The sterile filtrate was analyzed by HPLC as described below. a: Abbreviations: Sm: streptomycin. Cb: carbenicillin. Tc: tetracycline. Km: kanamycin. Gus: ß-glucuronidase. b: Holloway et al., Microbiol. Rev. 43: 73-102 (1979). c: ATCC 17514, American Type Culture Collection, Rockville, MD. d: Simón, R. et al., Bio / technology 1: 784-791 (1983). e: Clontech Laboratories, Inc., Palo Alto, CA. f: Cuskey et al., J. Bacteriol. 169: 2398-2404 (1987). g: Harker et al., J. Bacteriol. 171: 314-320 (1989).

TABLE 2 TNA Triptona 5.0 grams / liter Yeast Extract 2.5 grams / liter NaCl 8.5 grams / liter Glucose 1.0 grams / liter Agar 20.0 grams / liter Autoclave and tempered at 50 ° C. Add antibiotic if required and pour dishes.

LB Luria Broth Base 15.5 grams / liter Agar 20.0 grams / liter Autoclave. and tempered at 50 'C. Add antibiotic if required and pour dishes.

Burk Salts Material Solutions: a. gS? 4-7H20 39.9 grams / liter b. FeS? 4 ~ 7H2? 0.01 grams / liter c. NaMo04-2H20 0.05 grams / liter d. (NH4) 2S0 100.00 grams / liter e. Potassium phosphate 1 M regulator, pH 7.1 Autoclave the material solution and store at room temperature.

Solution of Burk / CAÁ To 1 liter of sterile distilled water containing 0.3 percent casamino acid, add: 5 milliliters of material solutions a, b, and c: 10 milliliters of material solutions d and e.

Burk dishes / succinate To 1 liter of distilled water containing 0.2 percent succinate and 2 percent noble agar, which has been autoclaved and tempered at 50 ° C, add: 5 milliliters of material solutions a, b, and e, 10 milliliters of material solutions d and e. Add the appropriate antibiotic if desired, and pour dishes.

Page and collaborators, J. Bacteriol. 125: 1080-1087 (1975) TABLE 3 WPM 0.1 mg / 1 NAA, per liter 2.5 mg / 1 BAa 100 ml WPM-macro 10 ml WPM-icro 10 ml WPM-Cs 10 ml Inositol (10 mg / ml) 10 ml Iron Queladob 1 ml Vitamin WPM 20 ml Sucrose 2.5 ml NAA (0.1 mg / ml) c BA (0.1 mg / ml) d Bring the volume up to 1 liter with distilled H2O, pH up to 5.8, add 7 grams of agar, and autoclave.

WPM-macro g / i WPM-micro g / i NH4NO3 4.0 H3BO3 0.67 K2S04 9.9 ZnS04-7H20 0.86 KH2P04 1.7 MnS04-H20 1.69 MgS04-7H20 3.7 Na2Mo04-2H20 0.025 CuS04-5H20 0.025 WPM-Ca g / 100 ml Vitamin WPM g / 100 ml Ca (N03) 2-4H20 5.56 Thiamine HCl 0.1 CaCl2-H20 0.96 Nicotinic Acid 0.05 Pyridoxine HCl 0.05 Glycine 0.2 to. Lloyd et al., Comb. Proc. inter. Plant. Prop. Soc. 30: 421-427 (1980). b. Chelated Iron = Na2 EDTA 3.73 grams / liter; FeS04 - 7H20 2.73 grams / liter. c. NAA = Naphthaleneacetic acid. d. BA = Benzylaminopurine. b. Molecular Biology Methods The plasmids were isolated by harvesting the 10-well TNA bacterial culture containing the appropriate antibiotic, by suspending the culture of each dish in 5 milliliters of TE buffer (50 mM Tris-HCl, 20 mM EDTA, pH 8.0), group the solutions in a 250 milliliter centrifuge bottle, and granulate the cells by centrifugation at 10,000 xg for 5 minutes. The pellet was resuspended in 20 milliliters of lysis buffer (50 mM Tris-HCl, pH 8.0, 20 mM EDTA, 50 mM glucose 2 mg / ml lysozyme), and incubated at room temperature for 5 minutes. The freshly prepared SDS-alkaline solution (40 milliliters) (0.2M NaOH, 1% SDS) was added. The cells were lysed by gentle inversion and incubated in a bath of ice water for 10 minutes. Potassium acetate (30 milliliters of a 5M solution) was added, the solution was mixed by light inversion and incubated in an ice water bath for 10 minutes. This solution was centrifuged for 10 minutes at 10,000 x g, 4 ° C. The supernatant was decanted into a clean centrifuge bottle, and the DNA was precipitated by the addition of two volumes of 95 percent ethanol and incubation in an ice water bath for 1 hour. The precipitate is. collected by centrifugation at 10,000 x g for 30 minutes at 4 ° C. The resulting granule was resuspended in 10 milliliters of ice cold TE regulator by slowly passing the mixture through a pipette. After the granule was resuspended, 5 milliliters of 7.5 M ammonium acetate were mixed by light inversion. This solution was incubated in a bath of ice water for 20 minutes, and then centrifuged for 10 minutes at 10,000 x g and at 4 ° C. The supernatant was decanted into a 50 milliliter centrifuge tube, and 0.313 volumes of 42 percent polyethylene glycol (molecular weight 6000-8000) were mixed by light inversion. The DNA was allowed to precipitate from 4 hours until overnight at 4 ° C. The DNA was collected by centrifugation at 10000 x g for 10 minutes at 4 ° C.

The granule was resuspended in 8 milliliters of ice-cold TE buffer, and then added to 8 grams of cesium chloride. After the cesium chloride was in solution, 0.6 milliliters of a solution of 10 milligrams / milliliter (in distilled water) of ethidium bromide was added. The solution was centrifuged in an ultracentrifuge at 40,000 rpm for 42 hours at 20 ° C, using a Ti50 rotor. The plasmid band from this gradient of cesium chloride-ethidium bromide was plotted using a pasteur pipette. Ethidium bromide was removed by several extractions with normal butanol saturated with water, and then dialysed for 24 hours, with two changes of regulator, in a TE buffer. The purified DNA was stored at -20 ° C. Routine analysis of the strains for the desired plasmid was done by inipreparation. A cycle of the culture taken from the TNA antibiotic dish was suspended in 100 microliters of lysis buffer by swirling. After 5 minutes of incubation at room temperature, 200 microliters of alkaline-SDS solution was mixed, by light inversion, and the Used cells were incubated in an ice water bath for 10 minutes. Potassium acetate (150 microliters of a 5 M solution) was mixed by gentle inversion, and incubation in the ice water bath was continued for 5 minutes. The lysate was clarified by microfugation at 4 ° C for 5 minutes, and the supernatant was decanted into a fresh tube. The DNA was precipitated by the addition of 1 milliliter of 95 percent ethanol, and incubating the mixture at -70 ° C for 30 minutes, followed by microfugation for 30 minutes. The granule was resuspended in 100 milliliters of ice sterile distilled water, and 50 microliters of 7.5M ammonium acetate was mixed by light inversion of the tube. This mixture was incubated in a bath of ice water for 10 minutes, microfuged for 10 minutes, and the supernatant was decanted into a fresh tube. The DNA was precipitated by the addition of 300 microliters of 95 percent ethanol, incubation at -70 ° C for 30 minutes, and microfugation for 30 minutes. The DNA granule was dried by vacuum drying for 10 minutes, and resuspended in 40 microliters of TE buffer. The analysis was done by agarose gel electrophoresis as described below. Restriction endonuclease digestion was done by incubating the purified plasmid DNA in the appropriate Boehringer Mannheim buffer with 1 to 2 microliters of the required Boehringer Mannheim restriction endonuclease at 37 ° C for 1 hour. The reaction was inactivated by incubation at 70 ° C for 10 minutes, followed by incubation in an ice water bath for 10 minutes. DNA ligation was performed by mixing the two restriction endonuclease-digested DNA fragments to be ligated, adding 1/10 volume of 7.5 M ammonium acetate, and two volumes of 95 percent ethanol. The DNA of this solution was precipitated by incubation at -70 ° C for 30 minutes, and then centrifugation for 30 minutes at -4 ° C in the icrofugo. The granule was resuspended in 100 microliters of ice-cold sterile distilled water by swirling for 15 seconds. The resuspended DNA was reprecipitated by the ammonium acetate-ethanol method described above. After the second precipitation, the DNA pellet was dried by vacuum drying for 10 minutes, resuspended in 16 microliters of ice cold sterile distilled water, and 4 microliters of 5X Gibco-BRL ligase regulator was added. Gibco-BRL ligase T4 was added to a Wiess unit. The ligation mixture was incubated at room temperature for 2 hours, and stopped by the addition of 30 microliters of frozen sterile distilled water. The analysis of plasmids and DNA fragments was made by agarose gel electrophoresis. The gel is made by adding agarose to a final concentration of 0.7 percent in TAE regulator (40 mM Tris-acetate, 0.1 mM EDTA). A gel of 15 cm2 had a total volume of 100 milliliters, and a mini-gel had a total volume of 25 milliliters. The agarose buffer solution was melted in a microwave, warmed to 50 ° C, and then poured into the gel mold and allowed to solidify for 20 minutes. The empty goal was then placed in the gel box, and immersed in the TAE regulator. The DNA was loaded into the wells, and electrophoresed for 2.5 hours at 100 volts when the 15 cm2 gels were tested, and 45 minutes at 130 volts when the mini-gels were tested. The DNA was visualized staining the gel in 30 milliliters of water containing 40 microliters of ethidium bromide solution at 10 milligrams / milliliter for 20 minutes, and then exposing the gel to ultraviolet light at 305 nanometers. The gel was photographed using a "Fisher Brand photodocumentation system and a Polaroid 660 film. The low melting temperature agarose gels were tested as described above, except that the amount of the low melting point agarose was 1 per cent. cent, or the gels were tested at 4 ° C. The DNA was visualized by staining with ethidium bromide, and the desired fragment was cut out of the gel. The cut fragment was eluted from the gel matrix by the addition of 100 microliters of TE buffer and incubation at 70 ° C for 10 minutes. An equal volume of phenol saturated with TE was added, and mixed by light inversion. The phases were separated by microfuge from the sample for 3 minutes at 4 ° C. The upper (aqueous) layer was collected, and the phenol layer was extracted twice more with an equal volume of TE buffer. The aqueous phases were pooled and extracted once with a 1: 1 mixture of phenol: chloroform, and once with chloroform. The DNA was precipitated by the addition of 1/10 volume of 7.5 M ammonium acetate, two volumes of 95 percent ethanol, incubation at -70 ° C for 30 minutes, and microfugation for 30 minutes. The granule was dried under vacuum for 10 minutes, and then resuspended in 100 microliters of TE buffer. c. Methods of Transformation and Conjugation The transformation of P. aeruginosa PAOlc was as described by Mercer et al (Mercer, A.A. and Loutit, J.S., J. Bacteriol.140: 37-42 (1979)). The e . coli S17-1 was transformed as described by Maniatis et al. (Maniatis, T. et al., Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. The transfer of plasmid pUCW200 from E. coli S17-1 to P. putative PP0300, or P. tumefaciens LB? 4404 by conjugation, was made by culturing the strains at 30 ° C in LB medium, or LB containing 50 micrograms / milliliter of kanamycin (E. coli S17). -1), and then mixing equal volumes of each culture, filtering the mixture through a sterile 0.22 micron filter, and placing the filter on a plate of LB. The plates were incubated overnight at 30 ° C, followed by filter washing with 5 milliliters of sterile distilled water, and coating dilutions of the cell suspension on Burk's salts containing 0.2 percent succinate and 50 micrograms / milliliter of kanamycin.These dishes were incubated at 30 ° C for 48 hours, and the transconjugados purified by re-scratching on identical media. d. HPLC analysis by 2,4-dichlorofenoxyacetic acid and 2,4-dichloro phenol. The HPLC analysis was done using a Supelco C8 column, a mobile phase of 70:30 methanol: water at 1 milliliter / minute, and detection at 280 nanometers of a 20 microliter injection. The peaks were identified by comparing the retention times with those of known standards.

Example 1. Construction of Plasmid pUCWlOl. To construct the pUCWlOl (Figure 2), plasmid pROlOl, which encodes all enzymes for the degradation of 2,4-dichlorophenoxyacetic acid in chloro aleylacetic acid, was digested with the restriction endonucleases Ba / nHI and HindIII. The DNA fragment containing the tfdA gene was isolated from a low melting temperature agarose gel, and ligated into the plasmid of vector pR01727 that had been digested with the same restriction endonucleases. The ligated DNA was transformed into P. aeruginosa PAOlc, and the transformants containing the desired insert were selected by coating for the culture on TNA Cb 00, followed by replica coating to Tc50 DNA and Cb500 TNA. Strains with the correct phenotype of sensitivity at Tc (due to inactivation by insertion) and resistance to Cb, they were further characterized by isolation of the plasmid DNA and digestion with Ba / nHI and HindIII. The digested plasmid DNA was analyzed by agarose gel electrophoresis to confirm that the desired fragment had been cloned. This plasmid was designated pUCWlOl, Figure 2. The expression of tfdA on plasmid pUCWlOl in P. aeruginosa PAOlc was confirmed by the growth of this 2,4-dichlorophenoxyacetic acid strain and the detection of 2,4-dichlorophenol by HPLC.

Example 2. Construction of Plasmid pUCW200. The binary vector pBI121 of A. tumefaciens (Clontech Laboratories, Inc., Palo Alto, CA., shown in Figure 4) was used to transfer and express "stably tfdA in liquidambar." PBI121 contains the transcription initiation sequences and translation of the 35S cauliflower mosaic virus promoter, and the transcription and polyadenylation termination sites, and the translation stop codons from the nopaline sintar.n gene (NOS) .PBI121 also contains the limits of Left and right T-DNA (LB and RB) which are the sequences used for the transformation of a plant cell (see Zambryski et al., Cell 56: 193-201 (1989) and Zambryski et al., Annu, Rev. Plant Physiol . & Plant Mol. Biol. 43: 465-490 (1992)) .The DNA between these limits will be inserted and then replicated with the plant chromosomal DNA.The subcloning of tfdA into the pBI121 plasmid creating the plasmid pUCW200 will be performed as diagram in Figure 3. Plasmid pUCWlOl was digested with the restriction endonucleases Xbal and Sacl. The DNA fragment containing the tfdA gene was isolated from a low melting temperature agarose gel, and ligated into plasmid pBI121, which had been cut with the same enzymes. This mixture was transformed into E. coli S17-1, and the transformants were selected by growth on LB Km at 37 ° C. The transformants were collected, cultured overnight on an identical medium, and analyzed by inserts by minipreparation analysis. A strain that appeared to contain the appropriate insert was further characterized by purification of the plasmid DNA as described above, and shaping the insert by digestion with XbaJ and Sacl. This plasmid was designated pUCW200, Figure 3.

This contains the tfdA gene in the binary vector of Agrobacterium pBI121. The expression of tfdA on the plasmid pUCW200 was tested by first transferring the plasmid from E. coli S17-1 to P. putida PPO300 by conjugation, and then cultivating P. putida (pUCW200) in the presence of 2,4-dichlorophenoxyacetic acid and detecting 2,4-dichlorophenol t by HPLC. Plasmid pUCW200 was mobilized from S17-1 to A. tumefaciene LBA4404 as described above.

The presence of plasmid pUCW200 in A. tumefaciene LBA4404 was confirmed by minipreparation analysis. The expression of tfdA in this strain was confirmed by cultivating A. tumefaciens LBA4404 (pUCW200) in the presence of 2,4-dichlorophenoxyacetic acid, and demonstrating the accumulation of 2,4-dichlorophenol in the medium by HPLC.

Example 3. Toxicity of 2,4-dichloro-enoxyacetic Acid for Sweetgum The effect of 2,4-dichlorophenoxyacetic acid on the regeneration of the adventitious bud from pieces of liquidambar leaves was examined. The results of the 2,4-dichlorophenoxyacetic acid toxicity test are shown in Table 4. Two concentrations of 2,4-dichlorophenoxyacetic acid were tested with two liquidambar clones. The highest concentration (1.0 milligram / liter) resulted in leaf pieces that formed callus tissue, while the lowest concentration (0.1 milligram / liter) resulted in the formation of callus and root on the leaf pieces. No shoots were observed on the leaf pieces at any concentration. Therefore, adventitious bud formation can be used in the presence of 0.1 milligrams / liter of 2,4-dichlorophenoxyacetic acid as an indicator of resistance to 2,4-dichlorophenoxyacetic acid.

TABLE 4 TOXICITY OF 2, 4-DICHLOROPHENOXYACEAL ACID FOR LIQUIDAMBAR Clone of (2, 4-D) # roots / piece Liquidgum mg / L of leaf Callo 2040 1.0 0.00 yes 2040 0.1 6.53 yes 2071 1.0 0.00 yes 2071 0.1 3.94 yes Example 4. Transformation of Liquidámbar with pBI121. The transformation mediated by Agrobacterium tumefaciens was used to transform liquidambar. Agrobacterium tumefaciens LB4404 has the ability to transfer the vector plasmid pI3I121 (Figure 4) into a plant cell. Once inside the plant cell, the DNA between the right (RB) and left (LB) limits is integrated (randomly) into the plant chromosome. Then it replicates as if it were a part of the plant's genome, and therefore, when this cell divides and differentiates into an outbreak, all cells in this adventitious bud contain the transformed gene in the original objective cell. The liquidambar transformation method used is described below: 1. The expanding leaves of a known liquid gum clone were surface sterilized by first rinsing them with soapy water and then stirring them in a 10 percent bleaching solution (in sterile water) for 10 minutes. minutes, followed by three rinses (for 2 minutes each) with sterile distilled water. 2. Each leaf was aseptically cut into small pieces (5 to 10 millimeters). Some of the pieces were placed over WPM 0.1 / 2.5 (Table 2). These pieces acted as the regeneration control. 3. Agrobacterium tumefaciens LB4404, which contained plasmid pBI121, was grown overnight at 30 ° C in 50 milliliters of LB containing 50 milligrams / liter of kanamycin. The next morning, this culture was inoculated in 500 milliliters of the identical medium, and the strain was cultured for 4 hours at 30 ° C. The cells were harvested by centrifugation (5 minutes at 10,000 x g), washed once with LB, and finally resuspended in 100 milliliters of fresh LB. 4. Leaf pieces were cocultivated with Agrobacterium for 30 minutes at room temperature. Then they were stained dry on a sterile Whatman No. 3 filter paper, and placed over WPM 0.1 / 2.5. The dishes were sealed with parafilm and incubated in the culture chamber. 5. After 3 days, the leaf pieces were transferred to WPM 0.1 / 2.5 containing 500 milligrams / liter of carbenicillin (Cb). Carbenicillin, an antibiotic, was used to kill residual Agrobacterium. 6. After two weeks, the pieces of leaf that were not of control were transferred to a selective medium. In this case, WPM 0.1 / 2.5 Cb500, kanamycin 75 milligrams / liter (Km 5). E1 plasmid pBI121 contains the resistance gene to kanamycin, NPT-II (Figure 4). Liquidambar is sensitive to kanamycin at 75 milligrams / liter, and does not regenerate in its presence. Accordingly, adventitious buds formed in the presence of kanamycin may contain the resistance gene. 7. The shoots, regenerated under selective pressure, were cut from the leaf piece and transferred to WPM 0.01 / 2.0 Cb500, Km75. Putatively transformed liquidambar buds were obtained at 24 hours, which grew on WPM 0.01 milligrams / liter NAA, 2.0 milligrams / liter BA, Cb500, Km75, in an experiment following the transformation protocol described above.

Example 5. Transformation of Sweetgum with pUCV / 200. A liquidambar transformation experiment was carried out with Agrobacterium tumefaciens LBA4404, which contained the plasmid pUCW200, as described above, except that the selection was altered (in step 6). Half of sheet pieces were placed on WPM 0.1 / 2.5 Cb500csn 0.1 milligrams / liter of 2,4-dichlorophenoxyacetic acid, and the other half was placed on the normal WPM 0.1 / 2.5 Cb500, Km75. The results of this selection are shown in Table 5.

TABLE 5 EFFECT OF THE SELECTION OF 2, 4-DICHLOROPHENOXY ACID ON THE FREQUENCY OF TRANSFORMATION OF LIQUIDAMBER Average Clone of # shoots / piece Ratio of sheet selection (buds / piece of leaf) 2027 (control) Cb, control 73/18 4.06 2027 (pUCW200) Cb, 2, 4-D 38/72 0.53 2027 (pUCW200) Cb, Km 3/85 0.04 2040 (control) Cb, control 85/30 2.83 2040 (pUCW200) Cb, 2, 4-D 77/66 1.17 2040 (PUCW200) Cb, Km 16/49 0.33 Abbreviations: Cb = carbenicillin. Km = kanamycin.

Example 6: ELISA Analysis of Transformed Liquidambar Clones An analytical method used to confirm the transfer of selected genes in liquidambar clones is an enzyme linked immunosorbent assay (ELISA) for the detection of the NPTII protein encoded by the resistance gene. kanamycin on plasmid PBI121. (ELISA Kit NPTT II, Prime Report 3 (2): 3 (1991)). The tissue of the plant (100 to 800 milligrams of fresh weight) was placed in 3 milliliters of extraction buffer (0.25 M Tris-HCl, pH 7.8, phenylmethylsulfonyl fluoride 0.1 mM), and homogenized using a Tekmar Tissuizer, model TR-10 (equipped with a microprobe), during the two impulses of 30 minutes each. An additional 2 milliliters of extraction buffer is added, and the cellular waste is removed by ultracentrifugation at 50,000 RPM for 20 minutes at 4 ° C. The supernatant is collected, and 4 volumes of ice-cold acetone are added, followed by incubation at -20 ° C for 4 hours, or overnight. Precipitated proteins are harvested by centrifugation at 4 ° C, 10,000 RPM for 20 minutes. The granule is resuspended in 1 milliliter of extraction regulator. This sample is used in the ELISA NPT-II kit purchased at 5 Prime-3 Prime, Inc., Boulder, CO. The ELISA method involves the use of an antibody specific for the kanamycin resistance protein (neomycin phosphotransferase, NPT-II) to detect this protein in the cytoplasmic fraction of the putative liquidambar transformants. The presence of this protein is an indication of the transformation, because the gene encoding it is located on the vector plasmid pBI121 of Agrobacterium tumefaciens LBA4404. This plasmid is transferred to the objective plant cell by means of A. tumefaciens LBA4404, where it is integrated into the genome of the plant and expresses its genes. Since the genes on this plasmid are physically linked, the presence of one of the gene products is evidence of the presence of the other genes located on the plasmid. For example, the presence of the NPT-II protein in plant extracts transformed with the pUCW200 plasmid, the 2,4-dichlorophenoxyacetic acid resistance plasmid, which also contains the kanamycin resistance gene, is positive evidence of the presence of the 2,4-dichlorophenoxyacetic acid resistance gene.

Example 7. ELISA results from the Sweetámbar Constructions / pBI 121. Of the 24 previously described outbreaks obtained from the transformation experiments of pBI121, only 15 survived another selection on kanamycin. These 15 were tested for the presence of NPT-II by ELISA. Only one clone, 2040.2hr (pAG121), gave a positive result in this trial (Figure 5). The ELISA results of Figure 5 are shown as A4o5n? Ff the reciprocal of the antigen dilution. The antigen in this case is the extract of plant cells, which is serially diluted down the microtitre plate. A positive reaction is one in which the sample exhibits a decrease in absorbance in correlation with the dilution of the antigen. The minimum absorbance value considered as positive, after correction by the bottom, is an O.D.4o53e 0.1. The frequency of transformation in these experiments was 6.7 percent.

Example 8. Results of Liquidámbar Transformation ELISA / pUCW200 The transformation frequencies of selected pUCW200 isolates on 2,4-dichlorophenoxyacetic acid were compared with those selected on kanamycin. ELISA was used for NPT-II as a measure of the frequency of transformation. Four selected isolates were tested on 2,4-dichlorophenoxyacetic acid and four isolates selected on kanamycin. The results are shown in Figure 6. The four clones selected with 2,4-dichlorophenoxyacetic acid (designated as 2027 (PUCW200) -TA, -TC, -TD, and -TE) were positive for NPT-II, indicating that transform. However, none of the clones selected with kanamycin (designated as 2027 (pUCW200) -KA, -KB, -KC, and -KD) were positive. Plasmid pUCW200 differs from plasmid pBI121 in that pUCW200 has the resistance gene to 2,4-dichlorophenoxyacetic acid substituting the GUS gene of pBI121. Since transformation experiments using pBI121 with selection on kanamycin gave a transformation frequency of 6.5 percent, it is not surprising that none of the four kanamycin-selected isolates tested in this experiment was positive. However, these results establish the frequency of transformation of pUCW200 in liquidambar, with a selection of 2,4-dichlorophenoxyacetic acid, at 100 percent. It is clear that this method of selection is much better than the selection with standard kanamycin. The fact that selection with 2,4-dichlorophenoxyacetic acid did not produce false positive isolates demonstrates that it is an outstanding selectable marker. Also, experiments have shown that pieces of liquidambar leaf placed on medium containing 2,4-dichlorophenoxyacetic acid do not regenerate adventitious shoots. Since the cocultivated leaf pieces regenerated adventitious buds in the presence of 2,4-dichlorophenoxyacetic acid, and 100 percent of those shoots were transformed, these shoots must be using the product of the 2,4-dichlorophenoxyacetic acid resistance gene product 2, 4-dichlorophenoxyacetic acid in the medium in 2,4-dichlorophenol. This indicates that these isolates are expressing the resistance gene.

Example 9. Description of the Propagation Method Liquidambar was propagated by growing adventitious shoots on WPM containing 0.01 milligrams / liter of NAA and 2.0 milligrams / liter of BA. The original bud formed new shoots on this medium. The new shoots were aseptically cut from the original shoot and grown independently. This propagation was repeated until the required number of outbreaks was generated. These shoots were then lengthened by incubation on WPM containing 0.5 milligrams / liter of BA. Elongated shoots greater than 1 centimeter in length were then incubated on a root induction medium consisting of WPM at a concentration of 1/3 containing 0.1 milligrams / liter of IBA (indol-3-butyric acid). When the roots began to form, the seedlings were transferred to covered trays containing a soilless mixture consisting of equal parts of peat, perlite, and vermiculite, and incubated at a relative humidity of 100 percent until the new growth appeared. . Then the seedlings were transplanted into 163.87 cm leaching tubes, and were grown in a greenhouse until they reached plantation size. Then the plants harden for outdoor conditions until they are dormant, they are removed from the tubes, and planted in the prepared site. At a specified time before and / or after planting, the site is sprayed with 2,4-dichlorophenoxyacetic acid. The time to spray the site and the concentration of 2,4-dichlorophenoxyacetic acid used is readily determined by one skilled in the art. More specifically, these factors are determined by the growth of different plants from the plants transformed at the site. The amount of 2,4-dichlorophenoxyacetic acid sprayed on the site is an amount sufficient to: 1) inhibit the growth of untransformed plants at the site, and 2) allow the transformed plants to grow.

Example 10. Transformation and Propagation of Potatoes Potatoes (preferably Russet Burbank potatoes) transformed with the tfdA gene are produced essentially as described by De Block, Theor. Appl. Genet 76: 767-774 (1988) except that: 1) the regeneration medium includes 0.1 milligrams / liter of 2,4-dichlorophenoxyacetic acid, and 2) no kanamycin was used in the experiments with the tfdA gene. Briefly, the procedure described by De Block, supra, is as follows: Plant Materials Sterile plants of cvs 'Bintje1,' Desiree1, 'Berolina', or 'Russet Burbank' are propagated in vitro, by transferring the upper buds or pieces of 1 cm long stem explants, together with an e to help the medium YES. The shoots are grown at 23 ° C with 1 day of 16 hours under light intensity of 3000 lux (a mixture of "lumilux white" and "natura" of Osra, FRG).

Medium SI. Medium B5 (Gamborg et al., Exp. Cell. Res. 50: 151-158 (1968)) with 20 grams / liter of sucrose and supplemented with 150 milligrams / liter of CaCl2 »2H2 ?, 0.4 percent agarose, pH 5.8. S2: MS medium (Murashige and Skoog, Physiol. Plant 15: 473-479 (1962)) with 30 grams / liter of sucrose, and supplemented with 0.5 grams / liter of MES, pH of 5.5, 20 grams / liter of mannitol . S3: MS medium without sucrose, and supplemented with 200 milligrams / liter of glutamine, 0.5 grams / liter of MES, pH of 5.7, 0.5 grams / liter of PVP, 20 grams / liter of mannitol, 20 grams / liter of glucose, 40 milligrams / liter of adenine-S04, 0.5 percent agarose, 1 milligram / liter of trans-zeatin, 0.1 milligrams / liter of NAA, 1 gram / liter of carbenicillin, or 0.5 grams / liter of cefotaxime. S4: S3 supplemented with 10 milligrams / liter of AgN03. S5: Medium S3 without NAA and half the concentration of antibiotics. S6: S5 supplemented with 10 milligrams / liter of AgN? 3. S7: S5 supplemented with 0.01 milligrams / liter of GA3; 250 milligrams / liter of carbenicillin, or 150 milligrams / liter of cefotaxime. S8: S5 supplemented with 0.1 milligrams / liter of GA3 and 10 milligrams / liter of Ag? 3, - 250 milligrams / liter of carbenicillin or 150 milligrams / liter of cefotnime.

Antibiotics, hormones, and AgN? 3 were added after autoclaving. Ag2S2? 3 was added as 10 milligrams / liter of AgN0 + 117 milligrams / liter of Na2S2? 3 «5K > 2 Minimum medium A is as described (Miller, J.H., Experiments in molecular genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1972)).

Transformation, Selection, and Regeneration Leaves (3 to 10 millimeters) are cut from buds of 3 to 4 weeks of age in the base. The leaves are not hurt additionally. About 10 wounded leaves are floated face down on 10 milliliters of S2 infection medium contained in a Pet'ri dish of 9 centimeters, and 30 microliters of Agrobacterium tumefaciens LBA4404 containing the plasmid pUCW200, which had been grown in medium, are added. Luria wines for later registration. Then the dishes are incubated at a low light intensity (500 lux). After 2 days, the leaves are washed with S2 medium containing 1 gram / liter of carbenicillin or 0.5 grams / liter of cefotaxime, dried on a filter paper, and placed face up on the S3 medium containing 0.1 milligrams / liter of 2,4-dichlorophenoxyacetic acid. The Petri dishes are sealed with tape that allows the diffusion of gas (ribbon "urgo pore", Urgo, Chenove, France) and are incubated at a high intensity of light (3000 lux, a mixture of "white lumilux" and "natura"). "from Osram, FRG). After 1 week, the leaves are transferred to a fresh medium. After 2 more weeks, many small stems are formed on the damaged edges of the leaves, and the leaves are transferred to a selective medium S5 for 'Berolina', 'Bintje', and 'Desiree', and S6 for 'Russet Burbank' . After 2 to 3 more weeks, the leaves with callus are transferred to an S7 medium for 'Berolina', Bintje ', and' Desiree ', and S8 for' Russet Burbank '. From this point forward, glass jars of 250 milliliters are used. When cefotaxime is used in the medium, it is important to transfer the leaves to the fresh medium every 10 days. After 2 weeks, the first shoots are isolated (0.5 centimeters high) and transferred to a medium for root formation SI that contains 100 milligrams / liter of carbenicillin or cefotaxime. Normally, buds take root in about 1 week. When they do not root after 2 weeks, a thin slice of the base of the stem is cut: most of these clipped buds take root after 1 week. All shoots are harvested within a period of 3 weeks. In order to avoid isolating identical buds, never take two buds of the same callus or closely linked calluses.

When the roots begin to form, the seedlings are transferred to covered trays containing a soilless mixture consisting of equal parts of peat, perlite, and vermiculite, and incubated at a relative humidity of 100 percent until the new growth appears. . The seedlings are then transplanted into 163.87 cm3 leach tubes, and grown in a greenhouse until they reach planting size. Then the plants harden for outdoor conditions, until they are dormant, they are removed from the tubes, and planted in the prepared site. At a specified time before and / or after planting. The site is sprayed with 2,4-dichlorophenoxyacetic acid. The time to spray the site and the concentration of 2,4-dichlorophenoxyacetic acid used are easily determined by one skilled in the art. More specifically, these factors are determined by the growth of other plants different from the plants transformed at the site. The amount of 2,4-dichlorophenoxyacetic acid sprayed on the site is an amount sufficient to: 1) inhibit the growth of untransformed plants at the site, and 2) allow the transformed plants to grow.

Example 11. Transformation and Propagation of Soybeans Soy beans (preferably Winchester soybeans) processed with the tfdA gene are produced essentially as described by Hinchee et al., Bio / Technology 6: 917-921 (1988) except that: 1) the regeneration medium includes 0.1 milligrams / liter of 2,4-dichlorophenoxyacetic acid, and 2) kanamycin was not used in the experiments with the tfdA gene. Briefly, the procedure described by Hinchee et al., Supra, is as follows: Regeneration Yeast bean seedlings (Glycine max (L.) Winchester) are germinated aseptically from 4 to 10 days on Difco purified agar at 0.8 percent at 25 ° C under a photoperiod of 16: 8 (cold white fluorescent light at 40 μEn / s). After washing in soapy water, rinsing in distilled water, and placing in 70 percent ethanol for 2 minutes, the seeds are transferred to 50 percent Clorox for 10 to 13 minutes, followed by rinsing five times with sterile distilled water. The seeds are soaked in an aqueous Captan solution for 1 hour before transferring to the germination medium. After germination, the cotyledons are removed and placed with the adaxial side down on a B5BA medium. The B5BA medium is composed of B5 salts (GaBorg et al., Exp. Cell, Ree. 50: 152-158 (1968)), 20 milligrams / liter of sucrose, 1.15 milligrams / liter of benzylic adenine (BA), 0.1 milligrams / liter of 2,4-dichlorophenoxyacetic acid, and 8 grams / liter of Difco purified agar, at a pH of 5.8, before autoclaving. The cotyledon explants are transferred to B5O medium (identical to B5BA but without BA) after 3 to 4 weeks. These and all the cultivated explants are kept under the same environmental conditions as the germinating seedlings. The cotyledon explants that produce shoots are subcultured every 4 weeks on fresh B5O medium. The elongating shoots are removed and placed on medium I / 2B5O (half of the major and minor salts of the B5O medium) in corked glass jars or sterile disposable 50 milliliter disposable plastic centrifuge tubes. The seedlings (rooted shoots) are moved to vermiculite in 5 cm pots after several new leaves are produced. These seedlings are then placed in a plastic container, whose lid is gradually opened to harden them before being grown in the greenhouse. The seedlings that produce new leaves after hardening are transplanted to the soil and grown in the greenhouse for the establishment of flowering and seeds.

Classification of Culivar. The soybeans seeds are aseptically germinated for 5 days on Difco purified agar at 0.8 percent. The hypocotyledons are cut into 5-millimeter segments, and inoculated (Horsch et al., Science 227: 1229-1231 (1985)) with A. tumefaciens LBA4404 containing the plasmid pUCW200. The hypocotyledon segments are cocultivated with Agrobacterium for 2 days on 1/10 SH medium (1/10 of the major and minor salts of SH (Schenk and Hildebrandt, Can J, Bot.50: 199-204 (1972))) , before placing on the MS NAA / K medium containing 500 milligrams / liter of carbenicillin with or without 100 milligrams / liter of kanamycin. The MS NAA / K medium is composed of MS and organic salts (Murashige and Skoog, Phyeiol, Plant 15: 473-497 (1962)) with 2.15 milligrams / liter of kinetin and 4.68 milligrams / liter of naphthaleneacetic acid (NAA). Each cultivar sample is represented by 20 to 40 segments. The hypocotyledon segments remain on MS NAA / K for 4 weeks before being labeled. The number of hypocotyledons that produce callus is counted, as well as the number of independent calluses per explant.

Transformation of Cotyledon explants. Cultivated Winchester cotyledon explants are prepared as well as for regeneration. The transformation with A. tumefaciene LBA 4404 containing the plasmid pUCW200 is carried out as described for the hypocotyledons in the cultivar classification. The inoculated cotyledons are cultured as described for cotyledon regeneration, except that the B5BA medium contains 500 milligrams / liter of carbenicillin and 100 milligrams / liter of cefotaxime. Elongated shoots are grown in B5O medium. When the roots begin to form, the seedlings are transferred to covered trays containing a soilless mixture consisting of equal parts of peat, perlite, and vermiculite, and incubated at a relative humidity of 100 percent - until the new increase. The plants are then transplanted into 163.87 cm leach tubes, and grown in a greenhouse until they reach planting size. Then the plants harden for outdoor conditions until they are dormant, they are removed from the tubes, and planted in the prepared site. At a specified time before and / or after planting, the site is sprayed with 2,4-dichlorophenoxyacetic acid. The time to spray the site and the concentration of 2,4-dichlorophenoxyacetic acid used are easily determined by one skilled in the art. More specifically, these factors are determined by the growth of other plants different from the plants transformed at the site. The amount of 2,4-dichlorophenoxyacetic acid sprayed on the site is an amount sufficient to: 1) inhibit the growth of untransformed plants at the site, and 2) allow the transformed plants to grow.

* * * * * All publications mentioned hereinabove are incorporated herein by reference in their entirety. Although the above invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by an expert in the field, from a reading of this description, that different changes in form and detail may be made without departing of the true scope of the invention and the appended claims.

Claims (38)

1. A method for selecting a transgenic plant cell, which comprises: a) transforming one or more plant cells with a polynucleotide comprising a tfdA gene that can be expressed in that plant cell, b) growing the transformed plant cell with an amount of 2,4-dichlorophenoxyacetic acid that inhibits regeneration of the adventitious shoot from untransformed plant cells; and c) selecting a plant cell that exhibits regeneration of the adventitious bud.

2. The method according to claim 1, wherein the plant cell is a dicotyledonous plant cell.

3. The method according to claim 2, wherein the dicotyledonous plant cell is a hardwood plant cell.

4. The method according to claim 3, wherein the hardwood plant cell is a liquidambar plant cell.

5. The method according to claim 1, wherein the polynucleotide further comprises at least one second gene encoding a protein.

6. A method for selecting a transgenic plant, which comprises: a) transforming one or more plant cells with a polynucleotide comprising a tfdA gene that can be expressed in that plant cell, b) culturing the transformed plant cell with an amount of 2,4-dichlorophenoxyacetic acid which inhibits the regeneration of the adventitious shoot from the non-transformed plant cells, c) selecting a plant cell that exhibits the regeneration of the adventitious shoot, and d) regenerating the plant cell in a plant. The method according to claim 6, wherein the plant is a dicotyledonous plant. The method according to claim 7, wherein the dicot plant is a hardwood. 9. The method according to claim 8, wherein the hardwood is a liquidambar. The method according to claim 6, wherein the polynucleotide further comprises at least one second gene encoding a protein. 11. A plant cell comprising a tfdA gene that can be expressed in that cell, wherein the plant cell is free of other foreign selectable marker genes. The plant cell according to claim 11, wherein the plant cell is a dicotyledonous plant cell. 13. The plant cell according to claim 12, wherein the dicotyledonous plant cell is a hardwood plant cell. The plant cell according to claim 13, wherein the hardwood plant cell is a liquidambar plant cell. 15. A plant regenerated from the plant cell according to claim 11. 16. The progeny of the plant according to claim 15, wherein said progeny comprises the tfdA gene. 1

7. A plant propagule according to claim 15, wherein said propagule comprises the tfdA gene. 1

8. A seed produced by the progeny according to claim 16, wherein said seed comprises the tfdA gene. 1

9. A liquidgum plant cell comprising a tfdA gene that can be expressed in that plant cell. 20. A plant regenerated from the liquidambar plant cell according to claim 19. 21. The progeny of the plant according to claim 20, wherein said progeny comprises the tfdA gene. 22. A plant propagule according to claim 20, wherein said propagule comprises the tfdA gene. 23. A seed produced by the progeny according to claim 21, wherein said seed comprises the tfdA gene. 24. A method for producing a hardwood plant, which comprises: a) transforming one or more plant cells with a polynucleotide comprising a tfdA gene that can be expressed in that plant cell; b) regenerating the plant cell in a plant; c) cultivate the regenerated plant with an amount of 2,4-dichlorophenoxyacetic acid that will kill the non-transformed plants; d) select a plant that exhibits growth; e) propagate the plant to produce many plants; f) induce root formation in these plants; g) cultivate the rooted plants to a size of plant material; h) plant the size plants of plantation material in a cut and defoliated site, and i) apply 2,4-dichlorophenoxyacetic acid over the entire site to suppress competitive growth until the planting material can develop without competitive growth control . 25. A method for producing a hardwood plantation, which comprises: a) transforming one or more plant cells with a polynucleotide comprising a tfdA gene and at least a second gene encoding a foreign selectable marker that can be expressed in that plant cell; b) culturing the plant cell with an amount of a chemical that inhibits regeneration of the adventitious bud from the non-transformed plant cells with the foreign selectable marker gene; c) selecting a plant cell that exhibits regeneration of the adventitious shoot; d) regenerating the plant cell in a plant; e) propagate the plant to produce many plants; f) induce root formation in these plants; g) cultivate the rooted plants to a size of planting material; h) plant the size plants of plantation material in a cut and defoliated site, and i) apply 2,4-dichlorophenoxyacetic acid over the entire site to suppress competitive growth, until the planting material can develop without growth control competitive. 26. A method for producing a hardwood plantation, which comprises: a) transforming one or more plant cells with a polynucleotide comprising a tfdA gene and at least a second gene encoding a foreign selectable marker that can be expressed in that plant cell; b) cultivating the plant cell with an amount of a chemical that inhibits the regeneration of the adventitious bud from non-transformed plant cells with the foreign selectable marker gene; c) selecting a plant cell that exhibits regeneration of the adventitious shoot; d) regenerating the plant cell in a plant; e) cultivating the regenerated plant with an amount of 2,4-dichlorophenoxyacetic acid that kills non-transformed plants; f) select a plant that exhibits growth; g) propagate the plant to produce many plants; h) induce root formation in these plants; i) cultivate the rooted plants to a size of planting material; j) planting plant size plants in a trimmed and defoliated site, and k) applying 2,4-dichlorophenoxyacetic acid over the entire site to suppress competitive growth until the planting material can develop without competitive growth control . 27. The method of claim 26, wherein the hardwood is liquidambar. 28. The method of any of claims 24 to 26, which further comprises the step of adding nutrients to the site. 29. The method of any of claims 24 to 26, which further comprises the step of raking the site before planting the planting material. 30. A method to produce a hardwood plantation, which includes: planting plants of plantation material size in a cut and defoliated site, and applying 2,4-dichlorophenoxyacetic acid over the entire site to suppress competitive growth until the planting material can be grown without a competitive growth control, where the plant is a hardwood comprising the tfdA gene. 31. The method of claim 30, wherein the hardwood is liquidambar. 32. A plantation of hardwood trees comprising a tfdA gene. 33. The plantation of claim 32, wherein the hardwood trees are liquidambar. 34. The plant cell of claim 19, which is free of foreign marker genes other than tfdA. 35. The plant of claim 20, which is free of foreign marker genes other than tfdA. 36. The progeny of claim 21, which is free of foreign marker genes other than tfdA. 37. The propagule of claim 22, which is free of foreign marker genes other than tfdA. 38. The seed of claim 23, which is free of foreign marker genes other than tfdA.