WO1998038294A1 - Growth-enhanced transgenic plants - Google Patents

Abstract

The present invention relates, in general, to transgenic plant cells and plants, and further relates to, for example, (1) a method of growing a growth-enhanced transgenic perennial plant, comprising cultivating a transgenic plant precursor, wherein cells in the transgenic plant precursor express a 2,4-dichlorophenoxyacetic acid monooxygenase gene, particularly the tfdA gene, at a growth-enhancing level; (2) a method of growing a yield-enhanced plantation, comprising cultivating a plot of growth-enhanced transgenic perennial plants; (3) a growth-enhanced transgenic plant comprising a transgenic plant wherein cells in the plant express a 2,4-dichlorophenoxyacetic acid monooxygenase gene, particularly the tfdA gene, at a growth-enhancing level; and (4) a yield-enhanced plantation, comprising a plot of growth-enhanced transgenic perennial plants.

Description

GROWTH-ENHANCED TRANSGENIC PLANTS

The present invention relates, in general, to transgenic plants and plant precursors, and further relates to, for example, (1 ) a method of growing a growth-enhanced transgenic perennial plant, comprising cultivating a transgenic plant precursor, wherein cells in the transgenic plant precursor express a 2,4-dichlorophenoxyacetic acid monooxygenase gene at a growth- enhancing level; (2) a method of growing a yield-enhanced plantation, comprising cultivating a plot of growth-enhanced transgenic perennial plants; (3) a growth-enhanced transgenic perennial plant or plant precursor comprising a transgenic plant or plant precursor wherein cells in the plant or plant precursor express a 2,4-dichlorophenoxyacetic acid monooxygenase gene at a growth-enhancing level; and (4) a yield-enhanced plantation, comprising a plot of growth-enhanced transgenic perennial plants.

2,4-dichlorophenoxyacetic acid (2,4-D) is a herbicide used to control broadleaf weeds. It can be degraded according to the pathway shown in Figure 1. The first step of the degradation pathway is catalyzed by a monooxygenase which catalyzes the conversion of 2,4-D to 2,4-dichlorophenol (DCP). The tfdA gene, isolated from the soil bacterium Alcaligenes eutrophus, encodes a 2,4-dichlorophenoxyacetic acid monooxygenase which catalyzes the conversion of 2,4-D to DCP. Transgenic tobacco and cotton plants transformed with the tfdA gene have been shown to have increased tolerance to 2,4-D (Streber et al., Bio/technology 7: 811-816, 1989 and Bayley et al., Theor. Appl. Genet. 83: 645-649, 1992, respectively). In U.S. Application Serial No. 08/358,117, it was taught how the tfdA gene can be used directly as a selectable marker to identify transgenic plants.

l - The present invention relates to the cultivation of growth-enhanced plants and yield-enhanced plantations, particularly perennial plants. It has been unexpectedly discovered that plants transformed with a 2,4-dichlorophenoxyacetic acid monooxygenase such as the tfdA gene that encodes enhanced growth as compared to plants not transformed with the gene.

Summary of the Invention

The invention provides a method of obtaining a growth-enhanced transgenic perennial plant, comprising cultivating a transgenic plant precursor, wherein cells in the transgenic plant precursor express a 2,4-dichlorophenoxyacetic acid monooxygenase gene at a growth-enhancing level. The methods of the invention further include a method for obtaining a yield-enhanced plantation, comprising cultivating a plot of growth-enhanced transgenic perennial plants, wherein cells in the transgenic plants express a 2,4-dichlorophenoxyacetic acid monooxygenase gene at a growth-enhancing effective level. In preferred embodiments, the 2,4-dichlorophenoxyacetic acid monooxygenase gene is the tfdA gene.

In another preferred embodiment of the invention, the growth enhancement is measurable after more than one growing season.

In yet other preferred embodiments, the plant precursor or plant is a woody perennial plant or plant precursor. Preferably, the plant or precursor is of the genera Acacia, Acer (Maple), Actinidia, Albizzia, Alnus, Amelanchier, Atriplex, Betula (Birch), Brachycome, Broussonetia, Camellia, Camptobera, Carya, Castanea (Chestnut), Catalpa, Cinchona, Citrus, Coffea, Corylus (Hazelnut), Diospyrus, Eucalyptus, Fagus (Beech), Ficus, Fraxinus (Ash), Gingko, Gleditsia, Gmelina, Hamamelia, Hedera, Ilex, Juglans, Kalmia,

Lexceana, Liquidambar (Sweetgum), Liriodendron, Malus, Moghania, Morus, Olea, Paulownia, Populus, Prunus, Quercus (Oak), Rosa, Rhododendron, Robinia, Salix, Santalum, Sapium, Simmondsia, Sycamore, Tectona, Theobrama, Tupidanthus, Ulmus (Elm), Vaccinium, or Vitus, or of the order Coniferales. More preferably, the precursor or plant is sweetgum. The invention also includes a growth-enhanced transgenic perennial plant or plant precursor comprising a transgenic plant or plant precursor, wherein cells in the plant or plant precursor express a 2,4-dichlorophenoxyacetic acid monooxygenase gene at a growth-enhancing effective level. In preferred embodiments, the 2,4-dichlorophenoxyacetic acid monooxygenase gene is the tfdA gene.

In another preferred embodiment, the plant or plant precursor is a woody perennial. Preferably the woody perennial is of the genera Acacia, Acer (Maple), Actinidia, Albizzia, Alnus, Amelanchier, Atriplex, Betula (Birch), Brachycome, Broussonetia, Camellia, Camptobera, Carya, Castanea

(Chestnut), Catalpa, Cinchona, Citrus, Coffea, Corylus (Hazelnut), Diospyrus, Eucalyptus, Fagus (Beech), Ficus, Fraxinus (Ash), Gingko, Gleditsia, Gmelina, Hamamelia, Hedera, Ilex, Juglans, Kalmia, Lexceana, Liquidambar (Sweetgum), Liriodendron, Malus, Moghania, Morus, Olea, Paulownia, Populus, Prunus, Quercus (Oak), Rosa, Rhododendron, Robinia, Salix, Santalum, Sapium, Simmondsia, Sycamore, Tectona, Theobrama, Tupidanthus, Ulmus (Elm), Vaccinium, or Vitus, or of the order Coniferales. More preferably, the woody perennial is sweetgum.

The invention also includes a yield-enhanced plantation, comprising a plot of growth-enhanced transgenic perennial plants, wherein cells in the transgenic plants express a 2,4-dichlorophenoxyacetic acid monooxygenase gene at a growth-enhancing effective level.

In a preferred embodiment, the 2,4-dichlorophenoxyacetic acid monooxygenase gene is the tfdA gene. In another preferred embodiment, the plant or plant precursor is a woody perennial. Preferably the woody perennial is of the genera Acacia, Acer (Maple), Actinidia, Albizzia, Alnus, Amelanchier, Atriplex, Betula (Birch), Brachycome, Broussonetia, Camellia, Camptobera, Carya, Castanea (Chestnut), Catalpa, Cinchona, Citrus, Coffea, Corylus (Hazelnut), Diospyrus, Eucalyptus, Fagus (Beech), Ficus, Fraxinus (Ash), Gingko, Gleditsia, Gmelina, Hamamelia, Hedera, Ilex, Juglans, Kalmia, Lexceana, Liquidambar (Sweetgum), Liriodendron, Malus, Moghania, Morus, Olea, Paulownia, Populus, Prunus, Quercus (Oak), Rosa, Rhododendron, Robinia, Salix, Santalum, Sapium, Simmondsia, Sycamore, Tectona, Theobrama, Tupidanthus, Ulmus (Elm), Vaccinium, or Vitus, or of the order Coniferales. More preferably, the woody perennial is sweetgum.

Further objects and advantages of the present invention will be clear from the description that follows.

Brief Description of the Drawings

FIGURE 1. A 2,4-D degradative pathway. Gene designations are shown in parentheses for each enzyme.

FIGURE 2. Construction of pUCW101.

FIGURE 3. Construction of pUCW200. FIGURE 4. ELISA of sweetgum 2027 selected on 2,4-D or kanamycin.

FIGURE 5. Bar graph of the average heights of five categories of sweetgum clones (ten trees per category).

FIGURE 6. Bar graph of the average diameters at breast heights of five categories of sweetgum clones (ten trees per category).

Definitions

An analog of a protein or genetic sequence should be understood as referring to a protein or genetic sequence substantially similar in function to a protein or genetic sequence described herein. Exogenous DNA should be understood as referring to DNA introduced into a cell or organism, or into an ancestor of such a cell or organism, by a recombinant technology. As will be understood by those of ordinary skill, an exogenous DNA remains in an ancestor if the original introduction to the parental cell or organism created a heritable form of the DNA within the cell or organism.

A fragment of a molecule should be understood as referring to any portion of the amino acid or nucleotide genetic sequence.

A functional derivative should be understood as referring to a molecule that possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of a protein or nucleic acid sequence.

Growing season should be understood to mean the period of a year commencing with bud break of a plant and continuing until dormancy of the plant, during which period the bulk of a plant's growing occurs. Generally, there is one growing season per calendar year.

A mutation should be understood as referring to any detectable change in the genetic material which can be transmitted at least one generation, that is, to daughter cells 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 area of cells arises. Mutations in the germ line of sexually reproducing organisms which are transmitted by the gametes to the next generation can result in an individual with the new mutant condition in both its somatic and germ cells. A mutation can be any (or a combination of) detectable, unnatural change affecting the chemical or physical constitution, mutability, replication, phenotypic function, or recombination of one or more deoxyribonucleotides; nucleotides may be added, deleted, substituted for, inverted, or transposed to new positions with and without inversion. Mutations can occur spontaneously or can be induced experimentally by application of mutagens. A mutant variation of a nucleic acid molecule results from a mutation. A mutant polypeptide may result from a mutant nucleic acid molecule.

Plant should be understood as referring to a multicellular differentiated organism capable of photosynthesis including angiosperms (monocots and dicots) and gymnosperms. Plant cell should be understood as referring to the structural and physiological unit of plants. The term "plant cell" refers to any cell which is either part of or derived from a plant.

Plant cell progeny should be understood as referring to any cell or tissue derived from plant cells including callus; plant parts such as stems, roots, fruits, leaves or flowers; plants; plant seed; pollen; and plant embryos. Plant precursor should be understood as referring to any biological matter from which a plant can be generated or grown, including, but not limited to, one or more plant cells, plant cell progeny, propagules, seeds, seedlings, cuttings, plantlets and plants. Plantation should be understood as referring to a group of plants under cultivation.

Propagules should be understood as referring to any plant material capable of being sexually or asexually propagated, or being propagated in vivo or in vitro. Such propagules preferably consist of the protoplasts, cells, calli, tissues, embryos or seeds of the regenerated plants.

To transform or the transformation of a plant or plant precursor should be understood to mean the process of introducing exogenous DNA into a plant or plant precursor to produce a transgenic plant.

Transgenic plant should be understood as referring to a plant having stably incorporated exogenous deoxyribonucleic acid ("DNA") in its genetic material. The term also includes plants where the exogenous DNA which was introduced into a cell or protoplast, or an ancestor of such a cell or protoplast, in various forms, including, for example, naked DNA in circular, linear or supercoiled form, DNA contained in nucleosomes or chromosomes or nuclei or parts thereof, DNA complexed or associated with other molecules, DNA enclosed in liposomes, spheroplasts, cells or protoplasts. A variant of a nucleic acid should be understood as referring to a molecule substantially similar in structure and biological activity to the nucleic acid, or to a fragment thereof. Thus, provided that two molecules possess a common activity and may substitute for each other, they are considered variants as that term is used herein even if the nucleotide sequence is not identical.

A woody perennial should be understood to mean a perennial which is a tree, bush or liana.

Detailed Description of the Invention

The present invention relates, among other things, to a method of growing a growth-enhanced transgenic perennial plant, comprising cultivating a transgenic plant precursor, wherein cells in the precursor express a 2,4- dichlorophenoxyacetic acid monooxygenase at a growth-enhancing effective level.

A "growth-enhanced" plant should be understood to mean a plant which exhibits a greater increase in volume, for example, as measured by height and diameter of trunk or stem, over a growing season than a non- growth-enhanced plant. An "effective level" of expression of the 2,4- dichlorophenoxyacetic acid monooxygenase gene in cells of a transgenic plant precursor should be understood to mean a level of expression sufficient to cause the plant to be a growth-enhanced plant. Expression by "cells" in the plant precursor should be understood to mean expression in a sufficient number of cells to cause the plant to be a growth-enhanced plant.

In a preferred embodiment, the growth enhancement of the transgenic plant is measurable after more than one growing season.

Preferably, the transgenic plant precursor is a woody perennial. More preferably, the woody perennial is of the genera Acacia, Acer (Maple), Actinidia, Albizzia, AInus, Amelanchier, Atriplex, Betula (Birch), Brachycome, Broussonetia, Camellia, Camptobera, Carya, Castanea (Chestnut), Catalpa, Cinchona, Citrus, Coffea, Corylus (Hazelnut), Diospyrus, Eucalyptus, Fagus (Beech), Ficus, Fraxinus (Ash), Gingko, Gleditsia, Gmelina, Hamamelia, Hedera, Ilex, Juglans, Kalmia, Lexceana, Liquidambar (Sweetgum), Liriodendron, Malus, Moghania, Morus, Olea, Paulownia, Populus, Prunus, Quercus (Oak), Rosa, Rhododendron, Robinia, Salix, Santalum, Sapium, Simmondsia, Sycamore, Tectona, Theobrama, Tupidanthus, Ulmus (Elm), Vaccinium, or Vitus, or of the order Coniferales (collectively referred to here as "Preferred Plants"). Even more preferably, the woody perennial is sweetgum. In other preferred embodiments, the 2,4-dichlorophenoxyacetic acid monooxygenase gene is the tfdA gene.

The invention further provides a method of growing a yield-enhanced plantation, comprising cultivating a plot of growth-enhanced transgenic perennial plants, wherein cells in the transgenic plants express a 2,4- dichlorophenoxyacetic acid monooxygenase gene at a growth-enhancing effective level.

A "yield-enhanced" plantation should be understood to mean a plantation which exhibits more product per acre, for example, more wood, than a plantation comprising similar age and number of non-growth-enhanced plants. Preferably, the plants are woody perennials, more preferably, Preferred Plants, and even more preferably, sweetgum. It should be understood that a plantation can comprise any combination of perennials.

In preferred embodiments, the 2,4-dichlorophenoxyacetic acid monooxygenase gene is the tfdA gene. The invention also provides a growth-enhanced transgenic perennial plant or plant precursor, wherein cells of the plant or precursor express a growth-enhancing effective level of a 2,4-dichlorophenoxyacetic acid monooxygenase gene. Preferably, the plants are woody perennials, more preferably Preferred Plants, and even more preferably, sweetgum. In preferred embodiments, the 2,4-dichlorophenoxyacetic acid monooxygenase gene is the tfdA gene. The invention also provides a yield-enhanced plantation, comprising a plot of growth-enhanced transgenic perennial plants, wherein cells of the plants express a 2,4-dichlorophenoxyacetic acid monooxygenase gene at a growth-enhancing effective level. Preferably, the plants are woody perennials, more preferably, the plants are Preferred Plants, and even more preferably, the plants are sweetgum.

In preferred embodiments, the 2,4-dichlorophenoxyacetic acid monooxygenase gene is the tfdA gene.

For all aspects of the invention, the transgenic plants and plant precursors can be cultivated in a greenhouse, or in the field, meaning outside of any protective enclosure such as a greenhouse. In a preferred embodiment, as further described in Example 6, after transformation the plants are grown in a greenhouse until they reach planting size, and are then planted in the field. The invention relates to the unexpected discovery that plants transformed with an expressible 2,4-dichlorophenoxyacetic acid monooxygenase gene grow faster than corresponding untransformed plants. The transgenic plants which are the subject of the invention are created by genetic engineering methods of transformation appropriate for plants, which generally means that at least a parent of the plants was generated from cells treated by a method intended to insert a 2,4-dichlorophenoxyacetic acid monooxygenase gene into the cells, as discussed further below. Generally, the potentially transformed plant will be monitored or selected for expressing the 2,4-dichlorophenoxyacetic acid monooxygenase gene. This can, for example, be done by: (a) direct selection on 2,4-dichlorophenoxyacetic acid, as described below; (b) monitoring for the presence of 2,4-dichlorophenoxyacetic acid monooxygenase activity extracted from plant tissue, for instance using the HPLC method of detecting 2,4-dichlorophenol (DCP) set forth below; (c) monitoring for the presence of the monooxygenase in plant extract, for instance using an immuno-detection assay; (d) monitoring for the presence of a marker gene that was concurrently introduced into the plant with the 2,4-dichlorophenoxyacetic acid monooxygenase gene; or (e) selecting for transformants using a selectable marker gene that was concurrently introduced into the plant with the 2,4-dichlorophenoxyacetic acid monooxygenase gene. The gene will generally be present in cells found throughout the plant, though the level of expression in different tissue types will generally vary depending on, for example, the differences in utilization in the various tissues of the promoter driving the expression of the 2,4- dichlorophenoxyacetic acid monooxygenase gene.

All plants which can be transformed are intended to be included within the scope of the invention. Such plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia,

Pelargonium, Panicum, Pennisetum, Ranunculus, Sencia, Salpiglossis, Cucumis, Browalia, Glycine, Lolium, Tea, Triticum, Sorghum, Malus, Apium, Datura, and the like.

Suitable woody perennials include, for example, the Preferred Plants, and the like.

Plant transformation techniques are well known in the art and include but are not limited to, microinjection (Crossway, Mol. Gen. Genetics 202: 179-185, 1985), polyethylene glycol transformation (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 minicells, cells, lysosomes, or other fusible lipid-surfaced bodies (Fraley et al., Proc. Natl. Acad. Sci. USA 79: 1859-1863, 1982), electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82: 5824, 1985) and techniques set forth in U.S. Patent No. 5,231 ,019)) and Agrobacterium tumefaciens mediated transformation (Hoekema et al., Nature 303: 179, 1983, de Framond et al., Bio/Technology 1 : 262, 1983, Fraley et al. WO84/02913, WO84/02919 and WO84/02920, Zambryski et al. EP 116,718, Jordan et al., Plant Cell Reports 7: 281-284, 1988, Leple et al., Plant Cell Reports 11 : 137-141 , 1992, Stomp et al., Plant Physiol. 92: 1226-1232, 1990, and Knauf et al., Plasmid 8: 45-54, 1982). One preferred method of transformation is the leaf disc transformation technique as described by Horsch et ai, Science 227: 1229-1230, 1985. The above-described transformation techniques can utilize a 2,4- dichlorophenoxyacetic acid monooxygenase gene, such as the tfdA gene or fragment thereof. Included within the scope of the tfdA gene is the tfdA gene of nucleotide sequence set forth in Streber et al., J. of Bacteriology 169:2950- 2955, 1987, and also in GenBank Accession No.: M16730, functional derivatives of a tfdA gene, as well as variant, analog and mutational derivatives.

The tfdA gene that is depicted in Streber and GenBank Accession No.: M16730, can be altered by substitutions, additions or deletions that provide for functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence, such as a tfdA gene product depicted in GenBank Accession No.: M 16730, may 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 depicted in Streber and GenBank Accession No.: M16730, which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.

Such functional alterations of a given nucleic acid sequence afford an opportunity to promote secretion and/or processing of heterologous proteins encoded by foreign nucleic acid sequences fused thereto. All variations of the nucleotide sequence of the relevant gene and fragments thereof permitted by the genetic code are, therefore, included in this invention. In addition, the dichlorophenoxyacetic acid monooxygenase gene comprises a nucleotide sequence which results from the addition, deletion or substitution of at least one nucleotide to the 5'-end and/or the 3'-end of the nucleic acid formula shown in Streber and GenBank Accession No.: M16730 or a derivative thereof. Any nucleotide or polynucleotide may be used in this regard, provided that its addition, deletion or substitution does not destroy the enzymic activity of the encoded gene. In a more preferred embodiment, the change does not alter the amino acid sequence of Streber and GenBank Accession No.: M16730 which is encoded by the nucleotide sequence. The tfdA gene may, as necessary, have restriction endonuclease recognition sites added to its 5'-end and/or 3'-end.

Further, it is possible to delete codons or to substitute one or more codons by codons other than degenerate codons to produce a structurally modified polypeptide, but one which has substantially the same utility or activity of the polypeptide produced by the unmodified nucleic acid molecule. As recognized in the art, the two polypeptides are functionally equivalent, as are the two nucleic acid molecules which give rise to their production, even though the differences between the nucleic acid molecules are not related to degeneracy of the genetic code.

The term "2,4-dichlorophenoxyacetic acid monooxygenase gene" includes any nucleotide sequence encoding for a polypeptide which functions as a 2,4-dichlogophenoxyacetic acid monooxygenase. The 2,4- dichlorophenoxyacetic acid monooxygenase gene used for transformation is preferably operably linked to a promoter region functional in plants, a transcription initiation site, and a transcription termination sequence. The particular promoter used in the expression cassette is a noncritical aspect of the invention. Any of a number of promoters which direct transcription in a plant cell is suitable. The promoter can be either constitutive or inducible. Some examples of promoters functional in plants include the nopaline synthase promoter derived from the Agrobacterium Ti plasmid (available in vectors pB1101 , pBI101.2 and pBI101.3 from Clontech, Palo Alto, CA) 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, with the 35S promoter available in vector pBI1221 from Clontech), the p1'2' mas bidirectional promoter (Velten et al., EMBO J. 3: 2723-2730,1984, where one orientation is referred to as the "mas V promoter" and the reverse orientation as the "mas 2' promoter"), the light- inducible promoter of gene ST-LS1 (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84: 7943-7947, 1987), and numerous plant promoters. A promoter that is well suited for a particular use can be determined by standard subcloning methods and the plant transformation and propagation methods taught herein. Efficiency for a particular purpose will often vary on the strength of the promoter and the tissue-by-tissue variations in promoter utilization. Plants transformed using various promoters can be compared for growth enhancement.

General methods for selecting transformed plant cells containing a selectable marker are well known and taught, for example, by Herrera-Estrella, L. and Simpson, J., "Foreign Gene Expression in Plants," in Plant Molecular Biology, A Practical Approach, C.H. Shaw, ed., IRL Press, Oxford, England, 1988, pp. 131-160. For example, for use of a 2,4-D gene as a selectable marker, the amount of 2,4-D which inhibits adventitious shoot formation from non-transformed plant cells and allows adventitious shoot formation from transformed plant cells can be determined by 1 ) plating non- -transformed cells on media containing various concentrations of 2,4-D and 2) by determining the lowest concentration of 2,4-D which will inhibit adventitious shoot formation by the plant cells. This lowest concentration can then be used to select transformed plant cells. In general, solubilized 2,4-D should be present in an amount ranging from about 0.001 to about 5 mg/L culture medium. With regard, for example, to the sweetgum plant cells transformed with a tfdA gene, 2,4-D should preferably be present in an amount ranging from about 0.01 to about 0.5 mg/L culture medium. A preferred amount of 2,4-D is about 0.01 to about 0.2 mg/L culture medium. The determination of the lowest amount of 2,4-D which will inhibit adventitious shoot formation is taught in U.S. Application Serial No. 08/358,117.

Other foreign marker genes (i.e., exogenously introduced genes) typically used in selection of transformed cells include selectable markers such as a neo gene (Potrykus et al., Mol. Gen. Genet 199: 183-188, 1985), which codes for kanamycin resistance; a bar gene which codes for bialaphos resistance; a mutant EPSP synthase gene (Hinchee et al., Bio/technology 6: 915-922, 1988), which encodes glyphosate resistance; a nitrilase gene which confers resistance to bromoxynil (Stalker et al., J. Biol. Chem. 263: 6310-6314, 1988); a mutant acetolactate synthase gene (ALS) which confers imidazolinone or sulphonylurea resistance (EP application number 154,204); a methotrexate resistant DHFR gene (Thillet et al., J. Biol. Chem. 263: 12500-12508) and screenable markers which include β-glucuronidase (GUS) or an R-locus gene, alone or in combination with a C-locus gene (Ludwig et al., Proc. Natl. Acad. Sci. USA 86: 7092, 1989; Paz-Ares et al., EMBO J. 6: 3553, 1987).

A 2,4-dichlorophenoxyacetic acid monooxygenase gene may be introduced into sweetgum by the transformation techniques outlined above or more preferably as set forth in Chen, Z. and Stomp, A., 1991 "Transformation of Liquidamber styraciflua L. (Sweetgum) using Agrobacterium Tumefaciens, " In: Proceedings 21^st Southern Forest Tree Improvement Conference. June 17-20, 1991 Knoxville, TN. The 2,4-dichlorophenoxyacetic acid monooxygenase gene is preferably contained on a plasmid wherein the gene is operably linked to a promoter region functional in plants, a transcription initiation site, and a transcription termination sequence (examples of which are provided above). In one preferred embodiment, the gene is linked to a foreign marker gene (described above). After selection, the transformed plant cells can be regenerated into transgenic plants. Plant regeneration techniques are well known in the art and include those set forth in the Handbook of Plant Cell Culture, Volumes 1-3, Evans et ai, eds., Macmillan Publishing Co., New York, NY, 1983, 1984, 1984, respectively; Predieri and Malavasi, Plant Cell, Tissue, and Organ Culture 17: 133-142, 1989; James, D.J., et al., J. Plant Physiol. 132: 148-154, 1988; Fasolo, F., et ai, Plant Cell, Tissue, and Organ Culture 16: 75-87, 1989; Valobra and James, Plant Cell, Tissue, and Organ Culture 21 : 51-54, 1990; Srivastava, P.S., et ai., Plant Science 42: 209-214, 1985; Rowland and Ogden, Hort. Science 27: 1127-1129, 1992; Park and Son, Plant Cell, Tissue, and Organ Culture 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., et ai., Plant Cell Reports 11 : 386-389, 1992; Kvaalen and von Arnold, Plant Cell, Tissue, and Organ Culture 27: 49-57, 1991 ; Tremblay and Tremblay, Plant Cell, Tissue, and Organ Culture 27: 95-103, 1991 ; Gupta and Pullman, U.S. Patent No. 5,036,007; Michler and Bauer, Plant Science 77: 111-118, 1991 ; Wetzstein, H.Y., et al., Plant Science 64: 193-201 , 1989; McGranahan, G.H., et ai., 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 Culture 17: 191-203, 1989; Bureno, M.A., et ai., Phys. Plant. 85: 30-34, 1992; and Roberts, D.R., et ai., Can. J. Bot. 68:1086-1090, 1990.

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

Examples The following protocols and experimental details are referenced in the examples that follow.

a. Strain Sources and Growth Conditions.

The bacterial strains and plasmids used herein are listed in Table 1 , and media formulas are shown in Table 2 and 3. Pseudomonas aeruginosa PAOIc containing plasmid pRO101 or plasmid pR01727 were grown on TNA plates containing 50 μg/ml tetracycline (TC⁵⁰) at 37°C. P. aeruginosa PAOIc (pUCW101 ; Figure 2) was grown on TNA containing 500 μg/ml carbeniciliin (Cb⁵⁰⁰) at 37°C. P. putida PPO300 (pUCW200; Figure 3), Agrobacte um tumefaciens LBA4404 (pUCW200; Figure 3), Escherichia co// HB101 (pBI121 , Figure 4), and E. coli S17-1 (pUCW200; Figure 3) were grown on TNA containing 50 μg/ml kanamycin (Km50). The P. putida and A. tumefaciens strains were grown at 30°C, and the E. coli strains were grown at 37°C.

Growth of P. aeruginosa PAOIc (pUCW101 ), P. putida PP0300 (pUCW200), and A. tumefaciens LBA4404 (pUCW200) for analysis of 2,4-D conversion to 2,4-dichlorophenol was done by inoculating 50 ml of

Burk's/CAA media containing 1 mM 2,4-D with a loop of culture from an overnight TNA plate containing the appropriate antibiotic. These liquid cultures were shaken at 30°C for 4 hours, and then filter sterilized. The sterile filtrate was analyzed by HPLC as described below.

a: Abbreviations: Sm: streptomycin. Cb: carbenicillin.

Tc: tetracycline. Km: kanamycin. Gus: β-glucuronidase.

b: Holloway et al., Microbiol. Rev. 43: 73-102, 1979. c: ATCC 17514, American Type Culture Collection, Rockville, MD. d: Simon, 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 Tryptone 5.0 g/L

Yeast [Extract 2.5 g/L

NaCI 8.5 g/L

Glucose 1.0 g/L

Agar 20.0 g/L

Autoclave and temper to 50°C. Add antibiotic if required and pour plates

LB Luria Broth Base 15.5 g/L

Agar 20.0 g/L

Autoclave and temper to 50°C. Add antibiotic if required and pour plates.

Burk's Salts*

Stock Solutions: a. MgS0₄-7H₂0 39.90 g/L b. FeSo₄-7H₂0 0.01 g/L c. NaMo0₄-2H₂0 0.05 g/L d. (NH₄)₂S0₄ 100.00 g/L e. 1 M Potassium Phosphate buffer, pH 7.1

Autoclave stock solution and store at room temperature.

Burk's/CAA

To 1 L of sterile distilled water containing 0.3% casamino acids add: 5 ml of stock solutions a, b, and c; 10 ml of stock solutions d and e.

Burk's/succinate plates

To 1 L of distilled water containing 0.2% succinate and 2% noble agar, which has been autoclaved and tempered to 50°C add:

5 ml of stock solutions a, b, and c;

10 ml of stock solutions d and e. Add appropriate antibiotic if desired and pour plates.

'Page et ai., J. Bacteriol. 125: 1080-1087, 1975 TABLE 3

WPM 0.1 mg/L NAA, per liter

2.5 mg/L BA^a

100 ml

WPM-macro 10 ml

WPM-macro 10 ml

WPM-Ca 10 ml

Inositol (10 mg/ml) 10 ml

Chelated Iron⁰ 1 ml

WPM Vitamin 20 ml

Sucrose 2.5 ml

NAA (0.1 mg/ml)^c

BA (0.1 mg/ml)^d

Bring volume to one liter with distilled H₂0, pH to 5.8, add 7 g agar, and autoclave.

WPM-macro g/L WPM-micro g/L

NH₄N0₃ 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 WPM-Vitamin g100 ml

Ca(N0₃)₂-4H₂0 5.56 Thiamine HCI 0.1

CaCI₂-H₂0 0.96 Nicotinic acid 0.05

Pyridoxine HCI 0.05

Glycine 0.2

a. Lloyd et al., Comb. Proc. Inter. Plant. Prop. Soc. 30: 421-427,

1980. b. Chelated Iron = Na₂ EDTA 3.73 g/L; FeS0₄ - 7H₂0 2.73 g/L. c. NAA = Napthaleneacetic acid. d. BA = Benzylamino purine. b. Molecular Biology Methods.

Plasmids were isolated by harvesting the bacterial growth of

10 TNA plates containing the appropriate antibiotic by suspending the growth from each plate in 5 ml of TE buffer (50 mM Tris-HC1 , 20 mM EDTA, pH 8.0), pooling the solutions in a 250 ml centrifuge bottle and pelleting the cells by centrifugation at 10,000 x g for five minutes. The pellet was resuspended in 20 ml of lysis buffer (50 mM Tris-HC1 , pH 5 8.0, 20 mM EDTA, 50 mM glucose, 2 mg/ml lysozyme), and incubated at room temperature for five minutes. Freshly prepared (40 ml) alkaline-SDS solution (0.2M NaOH, 1 % SDS) was added. The cells were lysed by gentle inversion and incubated in an ice water bath for 10 minutes. Potassium acetate (30 ml of a 5 M solution) was added,

10 the solution was mixed by gentle 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 to a clean centrifuge bottle and the DNA was precipitated by the addition of two volumes of 95% ethanol and incubation in an ice water bath for 1 hour.

15 The precipitate was collected by centrifugation at 10,000 x g for 30 minutes at 4°C. The resulting pellet was resuspended in 10 ml of ice cold TE buffer by slowly passing the mixture through a pipet. After the pellet was resuspended, 5 ml of 7.5 M ammonium acetate was mixed in by gentle inversion. This solution was incubated in an ice water bath

20 for 20 minutes and then centrifuged for 10 minutes at 10,000 x g and 4°C. The supernatant was decanted to a 50 ml centrifuge tube and 0.313 volumes of 42% polyethylene glycol (MW 6000-8000) was mixed in by gentle inversion. The DNA was allowed to precipitate from 4 hours to overnight at 4°C. The DNA was collected by centrifugation at

25 10,000 x g for 10 minutes at 4°C. The pellet was resuspended in 8 ml of ice cold TE buffer and then added to 8 g of cesium chloride. After the cesium chloride was in solution, 0.6 ml of a 10 mg/ml solution (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,

30 using a Ti50 rotor. The plasmid band from this cesium chloride-ethidium bromide gradient was drawn off using a pasteur pipet. The ethidium bromide was removed by several extractions with water saturated n-butanol and then dialyzed for 24 hours, with two buffer changes, in a TE buffer solution. The purified DNA was stored at -20°C.

Routine analysis of strains for the desired plasmid was done by mini-prep. A loop of culture taken from a TNA antibiotic plate was suspended in 100 μl of lysis buffer by vortexing. After 5 minutes of incubation at room temperature, 200 μl of alkaline-SDS solution was mixed in by gentle inversion and the lysed cells were incubated in an ice water bath for 10 minutes. Potassium acetate (150 μl of a 5M solution) was mixed in by gentle inversion and incubation in the ice water bath was continued for 5 minutes. The lysate was cleared by microfugation at 4°C for 5 minutes, and the supernatant was decanted to a fresh tube. The DNA was precipitated by adding 1 ml of 95% ethanol and incubating the mixture at -70°C for 30 minutes, followed by microfugation for 30 minutes. The pellet was resuspended in 100 μl of ice cold, sterile distilled water, and 50 μl of 7.5 M ammonium acetate was mixed in by gentle inversion of the tube. This mixture was incubated in an ice water bath for 10 minutes, microfuged for 2 minutes, and the supernatant was decanted to a fresh tube. The DNA was precipitated by addition of 300 μl of 95% ethanol, incubation at -70°C for 30 minutes, and microfugation for 30 minutes. The DNA pellet was dried by vacuum desiccation for 10 minutes, and resuspended in 40 μl of TE buffer. 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 1-2 μl 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 7.5 M ammonium acetate, and two volumes of 95% ethanol. The DNA in this solution was precipitated by incubation at -70°C for 30 minutes and then centrifugation for 30 minutes at 4°C in the microfuge. The pellet was resuspended in 100 μl of ice cold sterile distilled water by vortexing 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 desiccation for 10 minutes, resuspended in 16 μl of ice cold sterile distilled water and 4 μl of SX Gibco-BRL ligase buffer was added. Gibco-BRL T4 ligase was added to 1 Wiess unit. The ligation mixture was incubated at room temperature for 2 hours, and stopped by addition of 30 μl of ice cold, sterile distilled water.

Analysis of plasmids and DNA fragments was done by agarose gel electrophoresis. The gel is made by adding agarose to a final concentration of 0.7% in TAE buffer (40 mM Tris-acetate, 0.1 mM EDTA). A 15 cm² gel had a total volume of 100 ml and a mini-gel had a total volume of 25 ml. The agarose buffer solution was melted in the microwave, tempered to 50°C and then poured into the gel mold and allowed to solidify for 20 minutes. The cast gel was then placed in the gel box, and submerged in TAE buffer. The DNA was loaded into the wells, and electrophoresed for 2.5 hours at 100 volts when 15 cm² gels were run, and 45 minutes at 130 volts when mini-gels were run. The DNA was visualized by staining the gel in 300 ml of water containing 40 μl of 10 mg/ml ethidium bromide solution for 20 minutes, and then exposing the gel to UV light at 305 nm. The gel was photographed using a Fisher Brand photodocumentation system and Polaroid 660 film.

Low melting temperature agarose gels were run as described above, except the amount of low melting agarose was 1 % and the gels were run at 4°C. The DNA was visualized by ethidium bromide staining, and the desired fragment was cut out of the gel. The excised fragment was eluted from the gel matrix by adding 100 μl of TE buffer and incubating at 70°C for 10 minutes. An equal volume of TE saturated phenol was added and mixed in by gentle inversion. The phases were separated by microfuging the sample for 3 minutes at 4°C. The top (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 adding 1/10 volume of 7.5 M ammonium acetate, 2 volumes of 95% ethanol, incubation at -70°C for 30 minutes, and microfugation for 30 minutes. The pellet was vacuum dried for 10 minutes and then resuspended in 100 μl of TE buffer.

c. Transformation and Conjugation Methods.

Transformation of P. aeruginosa PAOIc was as described by Mercer et al. (Mercer, A.A. and Loutit, J.S., J. Bacteriol. 140: 37-42, 1979). E. coli S17-1 was transformed as described by Maniatis eif al. (Maniatis, T. et al., Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982.

Transfer of plasmid pUCW200 from E. coli S17-1 to P. putida PP0300 or A. tumefaciens LBA4404 by conjugation was done by growing the strains at 30°C in LB media or LB containing 50 μg/ml kanamycin (E. coli S17-1 ) and then mixing equal volumes of each culture, filtering the mixture through a sterile 0.22 μm filter and placing the filter on an LB plate. The plates were incubated overnight at 30°C, followed by washing the filter with 5 ml of sterile distilled water and plating dilutions of the cell suspension on Burk's salts containing 0.2% succinate and 50 μg/ml kanamycin. These plates were incubated at 30°C for 48 hours and the transconjugates were purified by restreaking to identical media. d. HPLC Analysis for 2,4-D and

2,4-Dichlorophenol.

HPLC analysis was done using a Supelco C8 column, a mobile phase of 70:30 methanol.water at 1 ml/min., and detection at 280 nm of a 20 μl injection. Peaks were identified by comparison of retention times to those of known standards.

Example 1. Construction of Plasmid pUCW101.

To construct pUCW101 (Figure 2), plasmid pR0101 , which encodes all the enzymes for the degradation of 2,4-D to chloromaleylacetic acid, was digested with restriction endonucleases SamHI and Hind\\\. The DNA fragment containing the tfdA gene was isolated from a low melting temperature agarose gel and ligated into vector plasmid pR01727 which had been digested with the same restriction endonucleases. The ligated DNA was transformed into P. aeruginosa PAOIc and transformants which contained the desired insert were selected by plating for growth on TNA Cb⁵⁰⁰, followed by replica plating to DNA Tc⁵⁰ and TNA Cb⁵⁰⁰. Strains with the correct phenotype of Tc sensitivity (due to insertional inactivation) and Cb resistance were characterized further by isolation of the plasmid DNA and digestion with SamHI and Hind\\\. The digested plasmid DNA was analyzed by agarose gel electrophoresis to confirm that the desired fragment had been cloned. This plasmid was designated pUCW101 , Figure 2.

Expression o fdA on plasmid pUCW101 in P. aeruginosa PA01 c was confirmed by growth of this strain in the presence of 2,4-D and detection of 2,4-dichlorophenol by HPLC. Example 2. Construction of Plasmid pUCW200.

The A. tumefaciens binary vector pBI121 (Clontech Laboratories, Inc., Palo Alto, CA.; shown in Figure 3) was used to transfer and stably express tfdA in sweetgum. pBI121 contains the transcriptional and translational start sequences of the cauliflower mosaic virus 35S promotor, and the transcriptional termination and polyadenylation sites and the translational stop codons from the nopaline synthase (NOS) gene. pBI121 also contains left and right T-DNA borders (LB and RB) which are sequences used for 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). DNA between these boundaries will be inserted and then replicated with the plant's chromosomal DNA. Subcloning tfdA into plasmid pBI121 creating plasmid pUCW200 was accomplished as diagramed in Figure 3. Plasmid pUCW101 was digested with restriction endonucleases Xba\ and Sad. 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 transformants were selected for growth on LB Km⁵⁰ at 37°C. Transformants were picked, grown overnight on identical media and analyzed for inserts by mini-prep analysis. One strain which seemed to contain the proper insert was further characterized by purifying the plasmid DNA as described above and confirming the insert by digestion with Xbal and Sac\. This plasmid was designated pUCW200, Figure 3. It contains a tfdA gene in the Agrobacterium binary vector pBI121.

Expression of tfdA on plasmid pUCW200 was tested by first transferring the plasmid from E. co// S17-1 into P. putida PPO300 by conjugation, and then growing P. putida (pUCW200) in the presence of 2,4-D and detecting 2,4-dichlorophenol by HPLC. Plasmid pUCW200 was mobilized from E. coli S17-1 to A. tumefaciens LBA4404 as described above. The presence of plasmid pUCW200 in A. tumefaciens LBA4404 was confirmed by mini-prep analysis. Expression of tfdA in this strain was confirmed by growing A. tumefaciens LBA4404 (pUCW200) in the presence of 2,4-D and demonstrating the accumulation of 2,4-dichlorophenol in the media by HPLC.

Example 3. Transformation of Sweetgum with pUCW200.

Agrobacterium tumefaciens-med ated transformation was used to transform sweetgum. Agrobacterium tumefaciens LB4404 has the ability to transfer plasmid pUCW200 into a plant cell. Once inside the plant cell, DNA between the right (RB) and left (LB) borders integrates (randomly) into the plant's chromosome. It is then replicated as if it were a part of the genome of the plant, and thus, when this cell divides and differentiates into a shoot, all the cells of this adventitious shoot contain the gene transformed into the original target cell.

The sweetgum transformation method used is outlined below: 1. The expanding leaves from a known sweetgum clone were surface sterilized by first rinsing them with soapy water and then stirring them in 10% bleach solution (in sterile water) for 10 minutes, followed by three rinses (for 2 minutes each) with sterile distilled water.

2. Each leaf was aseptically cut into small (5 to 10 mm) pieces. Some of the pieces were placed on WPM 0.1/2.5 (Table 2).

These pieces acted as the regeneration control.

3. Agrobacterium tumefaciens LB4404, containing plasmid pUCW200, was grown overnight at 30°C in 50 ml of LB containing 50 mg/L kanamycin. The next morning this culture was inoculated into 500 ml of the identical media, and the strain was grown for 4 hours at 30°C. The cells were harvested by centrifugation (5 min at 10,000 x g), washed once with LB, and finally, resuspended in 100 ml of fresh LB. 4 The leaf pieces were co-cultivated with Agrobacterium for 30 minutes at room temperature They were then blotted dry on sterile Whatman No. 3 filter paper and placed on WPM 0.1/2.5 The plates were sealed with parafilm and incubated in the growth chamber 5 After three days, the leaf pieces were transferred to

WPM 0.1/2 5 which contained 500 mg/L carbenicillin (Cb⁵⁰⁰) Carbenicillin, an antibiotic, was used to kill the residual Agrobacterium.

6. After two weeks, the non-control leaf pieces were transferred to selective media. Half of the leaf pieces were placed on WPM 0.1/2.5 Cb⁵⁰⁰ with 0.1 mg/L 2 4-D and the other half were placed on the normal WPM 0.1/2.5 Cb⁵⁰⁰, Kanamycin 75 mg/L (Km⁷⁵). The results of this selection are shown in Table 5. Plasmid pUCW200 contains the kanamycin resistance gene, NPT-II (Figure 3). Sweetgum is sensitive to kanamycin at 75 mg/L, and will not regenerate in its presence. Therefore, adventitious shoots formed in the presence of kanamycin may contain the resistance gene.

7 Shoots, regenerated under selective pressure, were excised from the leaf piece and transferred to WPM 0.01/2.0 Cb⁵⁰⁰ Km⁷⁵.

TABLE 5 EFFECT OF 2,4-D SELECTION ON SWEETGUM TRANSFORMATION FREQUENCY

Selection # shoots/#leaf Ratio (shoots/leaf

Clone Media pieces piece)

2027 (control) Cb, control 73/18 4 06

2040 (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 4. ELISA Analysis of Transformed Sweetgum Clones.

An analytical method used to confirm the transfer of selected genes into sweetgum clones is an enzyme linked immunosorbant assay (ELISA) for the detection of the NPTII protein encoded by the kanamycin resistance gene on plasmid pUCW200. (NPTT II ELISA Kit, Prime Report 3(2): 3, 1991. Plant tissue (from 100 to 800 mg fresh weight) is placed in 3 ml of extraction buffer (0.25 M Tris-HCI, pH 7.8, 0.1 mM phenylmethylsulfonyl fluoride) and homogenized using a Tekmar Tissuizer, model TR-10 (equipped with a microprobe), for the two pulses of 30 seconds each. An additional 2 ml of extraction buffer is added and the cellular debris 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 four hours, or up to overnight. The precipitated proteins are collected by centrifugation at 4°C, 10,000 RPM for 20 minutes. The pellet is resuspended in 1 ml of extract buffer. This sample is used in the NPT-II ELISA kit purchased from 5 Prime - 3 Prime, Inc., Boulder, CO. The ELISA method involves using an antibody specific for the kanamycin resistance protein (neomycin phosphotransferase, NPT-II) to detect this protein in the cytoplasmic fraction of putative sweetgum transformants. The presence of this protein is an indication of transformation because the gene encoding it is located on plasmid pUCW200. This plasmid is transferred into the target plant cell by A. tumefaciens LBA4404 where it integrates into the plant's genome 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. The presence of the NPT-II protein in plant extracts transformed with plasmid pUCW200, the 2,4-D resistance plasmid, which also contains the kanamycin resistance gene, is positive evidence for the presence of the 2,4-D resistance gene. Example 5. ELISA Results of Sweetgum/pUCW200 Transformation.

The transformation frequencies of pUCW200 isolates selected on 2,4-D were compared to those selected on kanamycin. ELISA for NPT-II was used as a measure of transformation frequency. Four isolates selected on 2,4-D and four isolates selected on kanamycin were assayed. The results are shown in Figure 4. All four of the 2,4-D selected clones (designated 2027 (pUCW200)-TA, -TC, -TD and -TE) were positive for NPT-II, indicating that they are transformed. However, none of the kanamycin selected clones (designated 2027 (pUCW200)-KA, -KB, -KC, and -KD) were positive. The fact that 2,4-D selection did not yield any false positive isolates demonstrates that it is an outstanding selectable marker. Also, experiments have shown that sweetgum leaf pieces placed on 2,4-D containing media do not regenerate adventitious shoots. Since co-cultivated leaf pieces did regenerate adventitious shoots in the presence of 2,4-D and 100% of those shoots were transformed, these shoots must be using the 2,4-D resistance gene product to convert the 2,4-D in the media to 2,4-dichlorophenol. This indicates that these isolates are expressing the resistance gene.

Example 6. Description of Propagation Method.

Sweetgum were propagated by growing the adventitious shoots on WPM containing 0.01 mg/L napthaleneacetic acid and 2.0 mg/L benzylamino purine. The original shoot formed new shoots on this media. The new shoots were aseptically excised from the original shoot and grown independently. This propagation was repeated until the required number of shoots were generated. These shoots were then elongated by incubating them on WPM containing 0.5 mg/L benzylamino purine. Elongated shoots of greater than 1 cm in length were then incubated on root induction media consisting of 1/3 strength WPM containing 0.1 mg/L IBA (indole-3-butyric acid). When roots began to form, the plantlets were transferred to plug trays containing a soilless mix consisting of equal parts peat, perlite, and vermiculite and incubated in 100% relative humidity until new growth appeared. The plantlets were then transplanted to 10 cubic inch leach tubes and grown in a greenhouse until they reach planting size. The plants are then hardened off to outdoor conditions until dormant, removed from tubes, and planted at the prepared site.

Example 7. A 5 X 10 Plantation of Sweetgum Trees

A sweetgum plantation was established at the Union Camp Nursery, Belleville, Georgia with ten trees each of four clonal propagations (as described in Example 6) of different tfdA transformants of the same parent tree line, with the different transformants labelled "TA", "TC", "TD" and "TE", as described in Example 5. A further ten trees were propagated from an adventitious shoot derived from cells of the parent tree line. The trees of each category were grown in a single row. As described in Example 6, for their first growth season, the shoots were initially grown in the greenhouse and transferred to outdoor conditions. In this experiment, the ground was not treated with 2,4-dichlorophenoxyacetic acid. As a consequence of the tissue culture propagation method, as would be known to one skilled in the art, the trees initially varied in size. After the second growth season, the trees of each category showed the average heights illustrated in Figure 5. The p-values for the average height increases shown by the TA and TD clones compared to the controls were in excess of 0.9. The p-value for the average height increase shown by the TE clones compared to the controls was in excess of 0.95. The TC clones showed no average height increase and were believed to have stopped expressing or to poorly express the tfdA gene. Figure 6 shows the average diameters at breast height ("DBH") for the five clones. The p-values for the average HBD increases shown by the TA and TD clones compared to the controls were in excess of 0.9. The p-value for the average HBD increase shown by the TE clones compared to the controls was in excess of 0.95. The TC clones showed no average HBD increase.

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims.

Claims

What Is Claimed Is:

1. A method for obtaining a growth-enhanced transgenic perennial plant, the method comprising cultivating a transgenic plant precursor, wherein cells in the precursor or in the plant express a 2,4- dichlorophenoxyacetic acid monooxygenase gene at a growth- enhancing effective level.

2. The method of claim 1 , wherein the growth enhancement of the transgenic plant is measurable after more than one growing season.

3. The method of claim 1 , wherein the transgenic plant precursor is a precursor of a woody perennial.

4. The method of claim 3, wherein the woody perennial is of the genera Acacia, Acer (Maple), Actinidia, Albizzia, AInus, Amelanchier, Atriplex, Betula (Birch), Brachycome, Broussonetia, Camellia, Camptobera, Carya, Castanea (Chestnut), Catalpa, Cinchona, Citrus, Coffea, Corylus (Hazelnut), Diospyrus, Eucalyptus, Fagus (Beech), Ficus, Fraxinus (Ash), Gingko, Gleditsia, Gmelina, Hamamelia, Hedera, Ilex, Juglans, Kalmia, Lexceana, Liquidambar (Sweetgum), Liriodendron, Malus, Moghania, Morus, Olea, Paulownia, Populus, Prunus, Quercus (Oak), Rosa, Rhododendron, Robinia, Salix, Santalum, Sapium, Simmondsia, Sycamore, Tectona, Theobrama, Tupidanthus, Ulmus (Elm), Vaccinium, or Vitus, or of the order Coniferales.

5. The method of claim 4, wherein the woody perennial is sweetgum.

6. The method of claim 1 , wherein the gene is the tfdA gene.

7. The method of claim 6, wherein the growth enhancement of the transgenic plant is measurable after more than one growing season.

8. The method of claim 6, wherein the transgenic plant precursor is a precursor of a woody perennial.

9. The method of claim 8, wherein the woody perennial is of the genera Acacia, Acer (Maple), Actinidia, Albizzia, AInus, Amelanchier, Atriplex, Betula (Birch), Brachycome, Broussonetia, Camellia, Camptobera, Carya, Castanea (Chestnut), Catalpa, Cinchona, Citrus, Coffea, Corylus (Hazelnut), Diospyrus, Eucalyptus, Fagus (Beech), Ficus, Fraxinus (Ash), Gingko, Gleditsia, Gmelina, Hamamelia, Hedera, Ilex, Juglans, Kalmia, Lexceana, Liquidambar (Sweetgum), Liriodendron, Malus, Moghania, Morus, Olea, Paulownia, Populus, Prunus, Quercus (Oak), Rosa, Rhododendron, Robinia, Salix, Santalum, Sapium, Simmondsia, Sycamore, Tectona, Theobrama, Tupidanthus, Ulmus (Elm), Vaccinium, or Vitus, or of the order Coniferales.

10. The method of claim 9, wherein the woody perennial is sweetgum.

11. A method for obtaining a yield-enhanced plantation, the method comprising cultivating a plot of growth-enhanced transgenic perennial plants, wherein cells in the transgenic plants express a 2,4-dichlorophenoxyacetic acid monooxygenase gene at a growth-enhancing effective level.

12. The method of claim 11 , wherein the transgenic perennial plants are woody perennials.

13. The method of claim 12, wherein the woody perennials are of the genera Acacia, Acer (Maple), Actinidia, Albizzia, AInus, Amelanchier, Atriplex, Betula (Birch), Brachycome, Broussonetia, Camellia, Camptobera, Carya, Castanea (Chestnut), Catalpa, Cinchona, Citrus, Coffea, Corylus (Hazelnut), Diospyrus, Eucalyptus, Fagus (Beech), Ficus, Fraxinus (Ash), Gingko, Gleditsia, Gmelina, Hamamelia, Hedera, Ilex, Juglans, Kalmia, Lexceana, Liquidambar (Sweetgum), Liriodendron, Malus, Moghania, Morus, Olea, Paulownia, Populus, Prunus, Quercus (Oak), Rosa, Rhododendron, Robinia, Salix, Santalum, Sapium, Simmondsia, Sycamore, Tectona, Theobrama, Tupidanthus, Ulmus (Elm), Vaccinium, or Vitus, or of the order Coniferales.

14. The method of claim 13, wherein the woody perennials are sweetgum.

15. The method of claim 11 , wherein the 2,4- dichlorophenoxyacetic acid monooxygenase gene is the tfdA gene.

16. The method of claim 15, wherein the transgenic perennial plants are woody perennials.

17. The method of claim 16, wherein the woody perennials are of the genera Acacia, Acer (Maple), Actinidia, Albizzia, AInus, Amelanchier, Atriplex, Betula (Birch), Brachycome, Broussonetia, Camellia, Camptobera, Carya, Castanea (Chestnut), Catalpa, Cinchona, Citrus, Coffea, Corylus (Hazelnut), Diospyrus, Eucalyptus, Fagus (Beech), Ficus, Fraxinus (Ash), Gingko, Gleditsia, Gmelina, Hamamelia, Hedera, Ilex, Juglans, Kalmia, Lexceana, Liquidambar (Sweetgum), Liriodendron, Malus, Moghania, Morus, Olea, Paulownia, Populus, Prunus, Quercus (Oak), Rosa, Rhododendron, Robinia, Salix, Santalum, Sapium, Simmondsia, Sycamore, Tectona, Theobrama, Tupidanthus, Ulmus (Elm), Vaccinium, or Vitus, or of the order Coniferales.

18. The method of claim 17, wherein the transgenic perennial plants are sweetgum.

19. A growth-enhanced transgenic perennial plant or a growth-enhanced transgenic perennial plant precursor comprising a transgenic plant or plant precursor, wherein cells in the plant or precursor express a 2,4-dichlorophenoxyacetic acid monooxygenase gene at a growth-enhancing effective level.

20. The transgenic plant or precursor of claim 19, wherein the 2,4-dichlorophenoxyacetic acid monooxygenase gene is the tfdA gene.

21. The transgenic plant of claim 19, wherein the plant or precursor is a woody perennial.

22. The growth-enhanced transgenic plant or precursor of claim 21 , wherein the woody perennial is of the genera Acacia, Acer (Maple), Actinidia, Albizzia, AInus, Amelanchier, Atriplex, Betula (Birch), Brachycome, Broussonetia, Camellia, Camptobera, Carya, Castanea (Chestnut), Catalpa, Cinchona, Citrus, Coffea, Corylus (Hazelnut), Diospyrus, Eucalyptus, Fagus (Beech), Ficus, Fraxinus (Ash), Gingko, Gleditsia, Gmelina, Hamamelia, Hedera, Ilex, Juglans, Kalmia, Lexceana, Liquidambar (Sweetgum), Liriodendron, Malus, Moghania, Morus, Olea, Paulownia, Populus, Prunus, Quercus (Oak), Rosa, Rhododendron, Robinia, Salix, Santalum, Sapium, Simmondsia, Sycamore, Tectona, Theobrama, Tupidanthus, Ulmus (Elm), Vaccinium, or Vitus, or of the order Coniferales.

23. The plant or precursor of claim 22, wherein the plant or precursor is sweetgum.

24. A yield-enhanced plantation, comprising a plot of growth-enhanced transgenic perennial plants, wherein cells in the transgenic plants express a 2,4-dichlorophenoxyacetic acid monooxygenase gene at a growth-enhancing effective level.

25. The yield-enhanced plantation of claim 24, wherein the 2,4-dichlorophenoxyacetic acid monooxygenase gene is the tfdA gene.

26. The yield-enhanced plantation of claim 24, wherein the plants are woody perennials.

27. The yield-enhanced plantation of claim 26, wherein the woody perennials are of the genera Acacia, Acer (Maple), Actinidia, Albizzia, AInus, Amelanchier, Atriplex, Betula (Birch), Brachycome, Broussonetia, Camellia, Camptobera, Carya, Castanea (Chestnut), Catalpa, Cinchona, Citrus, Coffea, Corylus (Hazelnut), Diospyrus, Eucalyptus, Fagus (Beech), Ficus, Fraxinus (Ash), Gingko,

Gleditsia, Gmelina, Hamamelia, Hedera, Ilex, Juglans, Kalmia, Lexceana, Liquidambar (Sweetgum), Liriodendron, Malus, Moghania, Morus, Olea, Paulownia, Populus, Prunus, Quercus (Oak), Rosa, Rhododendron, Robinia, Salix, Santalum, Sapium, Simmondsia, Sycamore, Tectona, Theobrama, Tupidanthus, Ulmus (Elm), Vaccinium, or Vitus, or of the order Coniferales.

28. The plantation of claim 27, wherein the plants are sweetgum.