CA2250966A1 - Monitoring of transgenic plants with in vivo markers - Google Patents
Monitoring of transgenic plants with in vivo markers Download PDFInfo
- Publication number
- CA2250966A1 CA2250966A1 CA 2250966 CA2250966A CA2250966A1 CA 2250966 A1 CA2250966 A1 CA 2250966A1 CA 2250966 CA2250966 CA 2250966 CA 2250966 A CA2250966 A CA 2250966A CA 2250966 A1 CA2250966 A1 CA 2250966A1
- Authority
- CA
- Canada
- Prior art keywords
- plant
- plant cell
- plants
- marker gene
- transgenic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A DNA construct that codes for the expression of a heterologous DNA and an in vivo marker gene in a plant cell is described. Plants containing such DNA constructs and methods of making such plants are also described. Further described are methods of detecting transgene flow from transgenic plants to non-transgenic plants, and monitoring transgenic plants.
Description
CA 022~0966 1998-10-02 W O 97/40178 PCT~US97/06637 MONITORING OF TRANSGENIC PLANTS
WITH IN VIVO MP ~TCT;~
Field of the Invention The present invention relates to methods of monitoring transgenic plants, and more specifically to monitoring the escape of transgenes into non-transgenic plant populations.
Background o~ the Invention The potential release and widespread use of transgenic plants have precipitated both regulatory concern and intensive scientific research. One primary issue is the possible escape of transgenes into the natural environment, along with the ramifications of transgene escape. One such ramification is the possibility of increased invasiveness and competitiveness of transgenic genotypes, as compared with conspecifics and congeners and the surrounding vegetation. For example, there is growing evidence that for crop plants in many areas of the world, there is a rapid transfer of genes to weedy relatives. In North America, such transfers have been observed in cultivated plants that are native, such as cranberry, blackberry and poplar, and in cultivated plants with wild relatives such as rice, sorghum, sunflower, and canola.
Transgene escape in canola (predominately Brassica napus) is especially problematic as herbicide-resistant canola has already been commercialized in Canada and Europe, and canola has complex breeding relationships with multiple weedy relatives.
Because of such observations, a major segment of plant biotechnology risk assessment research has therefore SU~ 111 UTE SHEET (RULE 26) CA 022~0966 1998-10-02 W O 97/40178 PCT~US97/06637 Because of such observations, a major segment of plant biotechnology risk assessment research has therefore focused on the potential escape of transgenes and the fate of plants populations containing those transgenes. Most of this research has focused on the phenomenon of gene flow, as this is the first tier of risk assessment for individual plant species. Unfortunately, most methods of estimating gene flow are labor- and resource-intensive, which limits the scope of research that may be performed. Furthermore, most existing studies typically involve the use of genes that are not neutral (e.g. herbicide-resistant genes) or markers already resident in plants (i.e. phenotypic or molecular markers). These markers have the significant disadvantage of not being universally identical amon~
species. Additionally, once transgenes have escaped from the source transgenic plant to the surrounding ecosystem, there currently is no reliable and effective method to monitor their progression through ecosystems.
It would be highly desirable to develop a practical and cost-efficient system to monitor genetically engineered plants containing potentially ecologically important transgenes. Such a system would be beneficial for use in basic and applied research and for monitoring commercial releases.
S-lmm~ry of the In~rention The approach of the present invention involves a novel technology in which the escape of transgenes and the flow of genes from transgenic plant populations to wild-type plant populations may be monitored. This transgene flow monitoring is accomplished in plant cells by linking one or more ecologically important transgenes with a second transgene encoding a detectable in vivo marker.
In view of the foregoing, a first aspect of the present invention is a DNA construct that codes for the - expression of a heterologous DNA and an in vivo marker gene in a plant cell.
SlJ~S 111 UTE SHEET (RULE 26) CA 022~0966 1998-10-02 WO97/40178 PCTrUS97/06637 A second aspect of the present invention is a plant transformation vector carrying a DNA construct as glven above.
A third aspect of the present invention is a plant cell containing a heterologous DNA construct, which construct codes for and expresses a heterologous DNA and an in vivo marker gene in said plant cell.
A fourth aspect of the present invention is a recombinant plant comprising transformed plant cells as given above.
A fifth aspect of the present invention is a method of making transgenic plant cells that contain heterologous DNA and an in vivo marker. The method comprises providing a plant cell capable of regeneration, then transforming the plant cell with a DNA construct that codes for and expresses a heterologous DNA and an in vivo marker gene in the plant cell.
A sixth aspect of the present invention is a method of monitoring for the escape of a transgene in plants. The method comprises planting transgenic plants in a predetermined area, wherein the transgene is linked with a detectable in vivo marker gene; then permitting the transgenic plants to grow in the predetermined area; and finally detecting the presence or absence of the detectable in vivo marker gene in plants growing outside of the predetermined area.
A seventh aspect of the present invention is a method of detecting transgene flow from transgenic plants to wild-type plants. The method comprises planting transgenic plants together with wild-type plants together in a shared area, wherein transgenes of the transgenic plants are operatively associated with a detectable in vivo marker gene; then allowing the transgenic and non-transgenic plants to mate; and finally detecting the presence or absence of the in vivo marker in the progeny of the transgenic and non-transgenic plants.
SUBSTITUTE SHEET (RULE 26) .
CA 022~0966 1998-10-02 W O 97/40178 PCTnUS97/06637 The foregoing and other objects and aspects of the present invention are explained in detail below, in the drawings herein and the specification set forth below.
Brief Description of the DrawingR
Figure 1 is a schematic representation of a bullseye design for growing a mixture of transgenic and nontransgenic plants.
Figure 2 is a schematic representation of the pGPF/Bt binary expression vector useful for in vivo monitoring of transgenic plants. In this figure, Bt means a synthetic Bacillus thurgiensis gene cryIAc; HPH means a hygromycin phosphotransferase gene; sGFP means a synthetic green fluorescent protein, and 35S indicates a promoter sequence. Restriction sites are indicated as follows: B=
BamHI; C= ClaI; H= HinDIII; K= KpnI; N= NcoI; P= PstI;
S=SalI; and X= XhoI.
Detailed Description of the Invention As summarized above, the present invention provides a method of monitoring the escape of transgenes from genc cally engineered plants to surrounding, non-transgenl plants by utilizing in vivo genetic markers.
The term "plants" as used herein refers to vascular plants, including both monocots and dicots, and both gymnosperms and angiosperms.
The term "operatively associated," as used herein, refers to DNA sequences on a single DNA molecule which are associated so that the function of one is affected by the other. Thus, a transcription initiation region is operatively associated with a structural gene when it is capable of affecting the expression of that structural gene (i.e., the structural gene is under the transcriptional control of the transcription initiation region). The transcription initiation region is said to be ~ "upstream" from the structural gene, which is in turn said Sl. ~;~111 UTE SHEET (RULE 26) CA 022~0966 1998-10-02 W O 97/40178 PCTrUS97/06637 to be "downstream" from the transcription initiation region.
DNA constructs, or "expression cassettes," of the present invention, code for the expression of a heterologous DNA and the in vivo marker gene in plant ~ cells. Such constructs preferably include, 5' to 3' in the direction of transcription, a transcription initiation region, a heterologous DNA operatively associated with the transcription initiation region, and a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal for polyadenylation (e.g., the nos terminator). All of these regions should be capable of operating in the cells to be transformed. The termination region may be derived from the same gene as the transcription initiation region, or may be derived from a different gene. The in vivo marker gene may be positioned upstream or downstream from (i.e., 5' or 3' to) the heterologous DNA and is either provided with its own regulatory regions or is operably associated with the same regulatory regions as provided for the heterologous DNA to be expressed. The in vivo marker gene is located on the same DNA molecule as the heterologous DNA to be expressed.
Suitable in vivo marker genes include those encoding fluorescent proteins, such as green fluorescent protein (GFP), apoaequorin, and analogs and derivatives thereof. Green fluorescent protein is derived from the jellyfish Aequorea victoria and has been expressed in a wide variety of microbial, plant, insect and mammalian cells. A. Crameri et al., Nature Biotech. 14, 315-319 (1996).
The transcription initiation region, which preferably includes the RNA polymerase binding site (promoter), may be native to the host organism to be transformed or may be derived from an alternative source, where the region is functional in the host. Other sources include the Agrobacterium T-DNA genes, such as the transcriptional initiation regions for the biosynthesis of SIJ~S 111 UTE SHEET (RULE 26) CA 022~0966 1998-10-02 W O 97/40178 PCTrUS97/06637 nopaline, octapine, mannopine, or other opine transcriptional initiation regions, transcriptional initiation regions from plants, transcriptional initiation regions from viruses (including host specific viruses), or partially or wholly synthetic transcription initiation regions. Transcriptional initiation and termination regions are well known. See, e. g., dGreve, J. Mol . Appl .
Genet. 1, 499-511 (1983); Salomon et al., EM~O ~. 3, 141-146 (1984); Garfinkel et al., Cell 27, 143-153 (1983); and Barker et al., Plant Mol. Biol. 2, 235-350 (1983).
The transcriptional initiation regions may, in addition to the RNA polymerase binding site, include regions which regulate transcription, where the regulation involves, for example, chemical or physical repression or induction (e.g., regulation based on metabolites or light) or regulation based on cell differentiation (such as associated with leaves, roots, seed, or the like in plants). Thus, the transcriptional initiation region, or the regulatory portion of such region, is obtained from an appropriate gene which is so regulated. ~or example, the 1,5-ribulose biphosphate carboxylase gene is light-induced and may be used for transcriptional initiation. Other genes are known which are induced by stress, temperature, wounding, pathogen effects, etc.
The heterologous DNA may encode a structural gene, an antisense agent, a ribozyme, etc. Structural genes are those portions of genes which comprise a DNA
segment coding for a protein, polypeptide, or portion thereof, possibly including a ribosome binding site and/or a translational start codon, but lacking a transcription initiation region. The term can also refer to introduced copies of a structural gene where that gene is also naturally found within the cell being transformed. The structural gene may encode a protein not normally found in the cell in which the gene is introduced or in combination with the transcription initiation region to which it is operationally associated, in which case it is termed a SU~;~ UTE SHEET (RULE 26) CA 022~0966 l998-l0-02 W O 97/40178 PCT~US97/06637 heterologous structural gene. Genes which may be operationally associated with a transcription inltiation region of the present invention for expression in a plant species may be derived from a chromosomal gene, cDNA, a synthetic gene, or combinations thereof. Any structural gene may be employed. Where plant cells are transformed, the structural gene may encode an enzyme to introduce a desired trait, such as glyphosphate resistance; a protein such as a Bacillus thuringiensis protein (or fragment thereof) to impart insect resistance; or a plant virus protein or fragment thereof to impart virus resistance.
The expression cassette may be provided in a DNA
construct which also has at least one replication system.
For convenience, it is common to have a replication system functional in Escherichia coli, such as ColE1, pSC101, pACYC184, or the like. In this manner, at each stage after each manipulation, the resulting construct may be cloned, sequenced, and the correctness of the manipulation determined. In addition, or in place of the E. coli replication system, a broad host range replication system may be employed, such as the replication systems of the P-1 incompatibility plasmids, e.g., pRK290. In addition to the replication system, there will frequently be at least one marker present, which may be useful in one or more hosts, or different markers for individual hosts. That is, one marker may be employed for selection in a prokaryotic host, while another marker may be employed for selection in a eukaryotic host, particularly a plant host. The markers may be protection against a biocide, such as antibiotics, toxins, heavy metals, or the like; provide complementation, for example by imparting prototrophy to an auxotrophic host; or provide a visible phenotype through the production of a novel compound. Exemplary genes which may be employed include neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (HPT), chloramphenicol acetyltransferase (CAT), nitrilase, and the gentamicin resistance gene. For plant host selection, non-limiting examples of suitable SUBSTITUTE SHEET (RULE 26) CA 022~0966 l998-l0-02 W O 97/40178 PCTnUS97/06637 markers are ~-glucuronidase, providing indigo production, luciferase, providing visible light production, NPTII, providing kanamycin resistance or G418 resistance, HPT, providing hygromycin resistance, and the mutated aroA gene, providing glyphosate resistance.
Vectors that may be used to carry out the present invention include Agrobacterium vectors. Numerous Agrobacterium vectors are known. See, e.g., U.S. Patent No. 4,536,475 to Anderson, U.S. Patent No. 4,693, 977 to Schliperoort et al.; U.S. Patent No. 4,886,937 to Sederoff et al.; T. Hall et al., EPO Application 0122791; R. Fraley et al., Proc Natl. Acad. Sci. USA 84, 4803 (1983); L.
Herrera-Estrella et al., EMBO .J 2, 987 (1983); G. Helmer et al., Bio/Technology 2, 520I (1984); N. Murai et al., Science 222, 476 (1983). In general, such vectors comprise an agrobacteria, typically Agrobacterium tumefaciens, that carries at least one tumor-inducing (or "Ti") plasmids.
When the agrobacteria is Agrobacterium rhizogenes, this plasmid is also known as the root-inducing (or "Ri") plasmid. The Ti (or Ri) plasmid contains DNA referred to as "T-DNA" that is transferred to the cells of a host plant cells when that plant is infected by the agrobacteria. In an Agrobacterium vector, the T-DNA is modified by genetic engineering techniques to contain the "expression cassette", or the gene or genes of interest to be expressed in the transformed plant cells, along with the associated regulatory sequences. The agrobacteria may contain multiple plasmids, as in the case of a "binary" vector system. Such Agrobacterium vectors are useful for introducing foreign genes into a variety of plant species, and are particularly useful for the transformation of dicots.
Vectors which may be used to transform plant tissue with DNA constructs of the present invention also include non-Agrobacterium vectors, particularly ballistic vectors, as well as vectors suitable for DNA-mediated transformation.
SU~ 1 1 1 lJTE SHEET (RULE 26J
CA 022~0966 1998-10-02 W O 97/40178 PCT~US97/06637 Microparticles carrying a DNA construct of the present invention, which microparticles are suitable for the ballistic transformation of a cell, are also useful for transforming cells according to the present invention. The microparticle is propelled into a cell to produce a transformed cell. Where the transformed cell is a plant cell, a plant may be regenerated from the transformed cell according to techniques known in the art. Any suitable ballistic cell transformation methodology and apparatus can be used in practicing the present invention. Exemplary apparatus and procedures are disclosed in Stomp et al., U.S. Patent No. 5,122,466; and Sanford and Wolf, U.S.
Patent No. 4,945,050 (the disclosures of all U.S. Patent references cited herein are incorporated herein by reference in their entirety). When using ballistic transformation procedures, the expression cassette may be incorporated into a plasmid capable of replicating in the cell to be transformed. Examples of microparticles suitable for use in such systems include 1 to 5 ~m gold spheres. The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.
Plant species may be transformed with the DNA
construct of the present invention by the DNA-mediated transformation of plant cell protoplasts and subsequent regeneration of the plant from the transformed protoplasts in accordance with procedures well known in the art.
The various fragments comprising the various constructs, expression cassettes, markers, and the like may be introduced consecutively by restriction enzyme cleavage of an appropriate replication system, and insertion of the particular construct or fragment into the available site.
After ligation and cloning the DNA construct may be isolated for further manipulation. All of these techniques are amply exemplified in the literature and find particular exemplification in Sambrook et al., Molecular Cloning: A
SU~;~ JTE S~IEET (RULE 26) CA 022~0966 1998-10-02 W O 97/40178 PCTrUS97/06637 Laboratory M~n~ , (2d Ed. 1989)(Cold Spring Harbor Laboratory, Cold Sprlng Harbor, NY).
Any plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may 5 be transformed with a vector of the present invention. The term "organogenesis," as used herein, means a process by which shoots and roots are developed sequentially from meristematic centers; the term "embryogenesis," as used herein, means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristems, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
Plants of the present invention may take a variety of forms. The plants may be chimeras of transformed cells and non-transformed cells; the plants may be clonal transformants (e.g., all cells transformed to contain the expression cassette); the plants may comprise grafts of transformed and untransformed tissues (e.g., a transformed root stock grafted to an untransformed scion in citrus species). The transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, first generation (or T1) transformed plants may be selfed to give homozygous second generation (or T2) transformed plants, and the T2 plants further propagated through classical breeding techniques. A dominant selectable marker (such as npt II) can be associated with the expression cassette to assist in breeding.
Plants which may be employed in practicing the present invention include (but are not limited to) tobacco SUBSTITUTE SHEET (RULE 26) .
CA 022~0966 1998-10-02 (Nicotiana tabacum), canola (Brassica spp.), potato (Solanum tuberosum), soybean (glycine max), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum~, sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris~, corn (Zea mays), wheat, oats, rye, barley, rice, vegetables, ornamentals, and conifers. Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuea sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Pisum spp.) and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C.
cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (dianthus caryophyllus), poinsettia (Euphorbia pulcherima), and chrysanthemum. Gymnosperms which may be employed to carrying out the present invention include conifers, including pines such as loblolly pine (Pinus taeda), Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); etc.
The present invention additionally provides a method which consists of linki~g one or more ecologically important transgenes, such as genes conferring herbicidal, disease or insect resistance to a second transgene coding a real time, in vivo marker, such as green fluorescent protein (GFP) gene. This method provides means for estimating gene flow from transgenic pollen-donor SIJ~S 111 UTE SHEET (RULE 26) CA 022~0966 1998-10-02 W O 97/40178 PCTrUS97/06637 populations to non-transgenic pollen-recipient populations (e.g., escape of transgenic genes within a plant population comprising a mixture of transgenic and nontransgenic plants). The fate of the transgenes may be traced by visual observation of transgenic/non-transgenic hybrids under an ultraviolet or blue light. In one embodiment of the invention, a mixture of plants are planted in a bullseye design, as illustrated in Figure 1. The mixture of plants may comprise plants of one species, but preferably comprises plants of multiple species. In the bullseye design, transgenic plants are grown in the center or "bullseye" of the plot. Non-transgenic plants are grown within the concentric rings which surround the center.
After planting, the plants are allowed to mate. After mating, seeds from a number of plants are samples from sampling points in the ring. These seeds are germinated and then examined and scored for the presence or absence of fluorescence using an ultraviolet lignt. The results of this scoring is used to determine crossing frequency and rates. A nested analysis of variance (e.g., ANOVA (SAS
Institute, Cary, NC)) is used to detect significant differences among species, distance, and location within distance.
In a particularly preferred embodiment of the invention, the method of estimating gene flow as described above is used to monitor the transference of genes between selected genetically engineered crop species and weed species that are sexually compatible with the transgenic crop species. A partial listing of crops species and their corresponding weed species is provided below in Table l.
Crop Species Weed Species (sexually compatible with crop species) Canola: Brassica napus and B. campestris B. napus, B. campestris, B. juncea, B.
kaber, B. oleracea, B. adepressa, Sinapsis Other Brassica cole crops such as cabbage alba, 5. arvensis and cauliflower Sunflower: Helianthis annuus H. annuus SUBSTITUTE SHEET (RULE 26) CA 022~0966 1998-10-02 W O 97/40178 PCT~US97/06637 Corn: Zea mays Teosinte: Z. mays, Z. diploperennis, Z.
perennis, Z. Iuxurians Tomato: Lycopersicum esculentium L. esculentium, L. chmielewski, L., pimpinellifolium Potato: Solanum tuberosum S. tuberosum, S. ajanuiri, S. curilohum, S:
~ demissum, S. goniocalys, S. sucrense, S.
vernsi, S. stentomum x sparsipulum Rice: Oryza sativa O. sativa, O. perennis Alfalfa: Medicago sativa M. sativa Soybean: Glycine max ~;. max, G. soja Ryegrass: Lolium perenne L. perenne, Festuca spp.
White clover: Trifolium repens T. repens Carrot: Daucus carota D. carota Radish: Raphinus sativus Raphanus sativus, R. raphanastrum Table 1: Partial list of crop species and sexually compatible weeds.
The examples which follow are set forth to illustrate the present invention, and are not to be 15construed as limiting thereof.
EX~PLE 1 Transgenic tobacco was produced that fluoresces green under an ultraviolet light. The plasmid mGFP4 (a 20gift from Jim Haseloff, England) which contains a mutagenized version of the GFP gene from the jellyfish Aequoria victoria under the control of the CaMV35S promoter and a kanamycin selectable marker, was engineered into tobacco (Nicotiana tabacum cv. Xanthi). The method is an 25efficient Agrobacterium tumefaciens-mediated transformation of leaf discs, followed by kanamycin selection and induced in vitro organogenesis (Schardl et al., Gene 61, 1-11 1987). The Agrobacterium strain GV3850 was used for all transformations.
30Of 25 transgenic lines, 2 were recovered in which entire plants fluoresced under an ultraviolet light.
- These two plants were randomly placed among a group of other transgenic tobacco plants of approximately the same SUBSTITUTE SHEET (RULE 26) CA 022~0966 1998-10-02 W O97/40178 PCTrUS97/06637 size , in a darkened room. At separate times, three individuals using a hand-held W light (365 nm, W P model 100 AP, Upland, California) were able to correctly distinguish the green fluorescing transgenic plants from the red-fluorescing non transgenic plants.
EX~MPLE 2 In this experiment, transgenic plants are produced in which the GFP marker is linked to an additional trait, in this case, insect resistance. An improved GFP
gene, sGFP (gift of Jen Sheen, Harvard University) is linked to a synthetic Bacillus thuringiensis transgene (Bt cryIAc), which construction is illustrated in Figure 2. Bt cryIAc codes for an insecticidal endotoxin protein which kills lepidopteran insects, such as corn earworm (Helicoverpa zea). Tobacco is transformed using the method described in Example 1, and canola using the procedure described in A. Mehra-Palta et al., Rapeseed in a Changing World: GCIRC Congress 1108-1115 (Saskatoon, Canada 1991).
Transgenic lines are selected that both fluoresce and express high levels of Bt cryIAc.
It is shown that the two genes are linked and functionally operative together. Twenty transgenic plants containing both genes (confirmed by using conventional molecular methods, e.g. Stewart et al. 1996) together with twenty non-transgenic plants of each crop are planted in a field. One half of the plants (both transgenic and non-transgenic) are exposed to insects, in which 50 eggs of Helicoverpa zea are placed on each plant. The remaining plants are not exposed to insects. The eggs are monitored for hatching. After one week, insect numbers are surveyed and defoliation of plants are estimated by visual ex~min~tion. Concurrently, all plants, both exposed and non-exposed, are observed at night with hand-held W
lights. Plants are scored as GFP positive or negative, - which scores are compared with defoliation measurements.
There is a significant positive correlation between the two SUBSTITUTE SHEET (RULE 26) W O 97/40178 PCT~US97106637 variables, indicating both linkage and functionality of both genes in the field.
The foregoing examples are illustrative of the present invention, and are not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.
SUBSTITUTE SHEET (RULE 26)
WITH IN VIVO MP ~TCT;~
Field of the Invention The present invention relates to methods of monitoring transgenic plants, and more specifically to monitoring the escape of transgenes into non-transgenic plant populations.
Background o~ the Invention The potential release and widespread use of transgenic plants have precipitated both regulatory concern and intensive scientific research. One primary issue is the possible escape of transgenes into the natural environment, along with the ramifications of transgene escape. One such ramification is the possibility of increased invasiveness and competitiveness of transgenic genotypes, as compared with conspecifics and congeners and the surrounding vegetation. For example, there is growing evidence that for crop plants in many areas of the world, there is a rapid transfer of genes to weedy relatives. In North America, such transfers have been observed in cultivated plants that are native, such as cranberry, blackberry and poplar, and in cultivated plants with wild relatives such as rice, sorghum, sunflower, and canola.
Transgene escape in canola (predominately Brassica napus) is especially problematic as herbicide-resistant canola has already been commercialized in Canada and Europe, and canola has complex breeding relationships with multiple weedy relatives.
Because of such observations, a major segment of plant biotechnology risk assessment research has therefore SU~ 111 UTE SHEET (RULE 26) CA 022~0966 1998-10-02 W O 97/40178 PCT~US97/06637 Because of such observations, a major segment of plant biotechnology risk assessment research has therefore focused on the potential escape of transgenes and the fate of plants populations containing those transgenes. Most of this research has focused on the phenomenon of gene flow, as this is the first tier of risk assessment for individual plant species. Unfortunately, most methods of estimating gene flow are labor- and resource-intensive, which limits the scope of research that may be performed. Furthermore, most existing studies typically involve the use of genes that are not neutral (e.g. herbicide-resistant genes) or markers already resident in plants (i.e. phenotypic or molecular markers). These markers have the significant disadvantage of not being universally identical amon~
species. Additionally, once transgenes have escaped from the source transgenic plant to the surrounding ecosystem, there currently is no reliable and effective method to monitor their progression through ecosystems.
It would be highly desirable to develop a practical and cost-efficient system to monitor genetically engineered plants containing potentially ecologically important transgenes. Such a system would be beneficial for use in basic and applied research and for monitoring commercial releases.
S-lmm~ry of the In~rention The approach of the present invention involves a novel technology in which the escape of transgenes and the flow of genes from transgenic plant populations to wild-type plant populations may be monitored. This transgene flow monitoring is accomplished in plant cells by linking one or more ecologically important transgenes with a second transgene encoding a detectable in vivo marker.
In view of the foregoing, a first aspect of the present invention is a DNA construct that codes for the - expression of a heterologous DNA and an in vivo marker gene in a plant cell.
SlJ~S 111 UTE SHEET (RULE 26) CA 022~0966 1998-10-02 WO97/40178 PCTrUS97/06637 A second aspect of the present invention is a plant transformation vector carrying a DNA construct as glven above.
A third aspect of the present invention is a plant cell containing a heterologous DNA construct, which construct codes for and expresses a heterologous DNA and an in vivo marker gene in said plant cell.
A fourth aspect of the present invention is a recombinant plant comprising transformed plant cells as given above.
A fifth aspect of the present invention is a method of making transgenic plant cells that contain heterologous DNA and an in vivo marker. The method comprises providing a plant cell capable of regeneration, then transforming the plant cell with a DNA construct that codes for and expresses a heterologous DNA and an in vivo marker gene in the plant cell.
A sixth aspect of the present invention is a method of monitoring for the escape of a transgene in plants. The method comprises planting transgenic plants in a predetermined area, wherein the transgene is linked with a detectable in vivo marker gene; then permitting the transgenic plants to grow in the predetermined area; and finally detecting the presence or absence of the detectable in vivo marker gene in plants growing outside of the predetermined area.
A seventh aspect of the present invention is a method of detecting transgene flow from transgenic plants to wild-type plants. The method comprises planting transgenic plants together with wild-type plants together in a shared area, wherein transgenes of the transgenic plants are operatively associated with a detectable in vivo marker gene; then allowing the transgenic and non-transgenic plants to mate; and finally detecting the presence or absence of the in vivo marker in the progeny of the transgenic and non-transgenic plants.
SUBSTITUTE SHEET (RULE 26) .
CA 022~0966 1998-10-02 W O 97/40178 PCTnUS97/06637 The foregoing and other objects and aspects of the present invention are explained in detail below, in the drawings herein and the specification set forth below.
Brief Description of the DrawingR
Figure 1 is a schematic representation of a bullseye design for growing a mixture of transgenic and nontransgenic plants.
Figure 2 is a schematic representation of the pGPF/Bt binary expression vector useful for in vivo monitoring of transgenic plants. In this figure, Bt means a synthetic Bacillus thurgiensis gene cryIAc; HPH means a hygromycin phosphotransferase gene; sGFP means a synthetic green fluorescent protein, and 35S indicates a promoter sequence. Restriction sites are indicated as follows: B=
BamHI; C= ClaI; H= HinDIII; K= KpnI; N= NcoI; P= PstI;
S=SalI; and X= XhoI.
Detailed Description of the Invention As summarized above, the present invention provides a method of monitoring the escape of transgenes from genc cally engineered plants to surrounding, non-transgenl plants by utilizing in vivo genetic markers.
The term "plants" as used herein refers to vascular plants, including both monocots and dicots, and both gymnosperms and angiosperms.
The term "operatively associated," as used herein, refers to DNA sequences on a single DNA molecule which are associated so that the function of one is affected by the other. Thus, a transcription initiation region is operatively associated with a structural gene when it is capable of affecting the expression of that structural gene (i.e., the structural gene is under the transcriptional control of the transcription initiation region). The transcription initiation region is said to be ~ "upstream" from the structural gene, which is in turn said Sl. ~;~111 UTE SHEET (RULE 26) CA 022~0966 1998-10-02 W O 97/40178 PCTrUS97/06637 to be "downstream" from the transcription initiation region.
DNA constructs, or "expression cassettes," of the present invention, code for the expression of a heterologous DNA and the in vivo marker gene in plant ~ cells. Such constructs preferably include, 5' to 3' in the direction of transcription, a transcription initiation region, a heterologous DNA operatively associated with the transcription initiation region, and a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal for polyadenylation (e.g., the nos terminator). All of these regions should be capable of operating in the cells to be transformed. The termination region may be derived from the same gene as the transcription initiation region, or may be derived from a different gene. The in vivo marker gene may be positioned upstream or downstream from (i.e., 5' or 3' to) the heterologous DNA and is either provided with its own regulatory regions or is operably associated with the same regulatory regions as provided for the heterologous DNA to be expressed. The in vivo marker gene is located on the same DNA molecule as the heterologous DNA to be expressed.
Suitable in vivo marker genes include those encoding fluorescent proteins, such as green fluorescent protein (GFP), apoaequorin, and analogs and derivatives thereof. Green fluorescent protein is derived from the jellyfish Aequorea victoria and has been expressed in a wide variety of microbial, plant, insect and mammalian cells. A. Crameri et al., Nature Biotech. 14, 315-319 (1996).
The transcription initiation region, which preferably includes the RNA polymerase binding site (promoter), may be native to the host organism to be transformed or may be derived from an alternative source, where the region is functional in the host. Other sources include the Agrobacterium T-DNA genes, such as the transcriptional initiation regions for the biosynthesis of SIJ~S 111 UTE SHEET (RULE 26) CA 022~0966 1998-10-02 W O 97/40178 PCTrUS97/06637 nopaline, octapine, mannopine, or other opine transcriptional initiation regions, transcriptional initiation regions from plants, transcriptional initiation regions from viruses (including host specific viruses), or partially or wholly synthetic transcription initiation regions. Transcriptional initiation and termination regions are well known. See, e. g., dGreve, J. Mol . Appl .
Genet. 1, 499-511 (1983); Salomon et al., EM~O ~. 3, 141-146 (1984); Garfinkel et al., Cell 27, 143-153 (1983); and Barker et al., Plant Mol. Biol. 2, 235-350 (1983).
The transcriptional initiation regions may, in addition to the RNA polymerase binding site, include regions which regulate transcription, where the regulation involves, for example, chemical or physical repression or induction (e.g., regulation based on metabolites or light) or regulation based on cell differentiation (such as associated with leaves, roots, seed, or the like in plants). Thus, the transcriptional initiation region, or the regulatory portion of such region, is obtained from an appropriate gene which is so regulated. ~or example, the 1,5-ribulose biphosphate carboxylase gene is light-induced and may be used for transcriptional initiation. Other genes are known which are induced by stress, temperature, wounding, pathogen effects, etc.
The heterologous DNA may encode a structural gene, an antisense agent, a ribozyme, etc. Structural genes are those portions of genes which comprise a DNA
segment coding for a protein, polypeptide, or portion thereof, possibly including a ribosome binding site and/or a translational start codon, but lacking a transcription initiation region. The term can also refer to introduced copies of a structural gene where that gene is also naturally found within the cell being transformed. The structural gene may encode a protein not normally found in the cell in which the gene is introduced or in combination with the transcription initiation region to which it is operationally associated, in which case it is termed a SU~;~ UTE SHEET (RULE 26) CA 022~0966 l998-l0-02 W O 97/40178 PCT~US97/06637 heterologous structural gene. Genes which may be operationally associated with a transcription inltiation region of the present invention for expression in a plant species may be derived from a chromosomal gene, cDNA, a synthetic gene, or combinations thereof. Any structural gene may be employed. Where plant cells are transformed, the structural gene may encode an enzyme to introduce a desired trait, such as glyphosphate resistance; a protein such as a Bacillus thuringiensis protein (or fragment thereof) to impart insect resistance; or a plant virus protein or fragment thereof to impart virus resistance.
The expression cassette may be provided in a DNA
construct which also has at least one replication system.
For convenience, it is common to have a replication system functional in Escherichia coli, such as ColE1, pSC101, pACYC184, or the like. In this manner, at each stage after each manipulation, the resulting construct may be cloned, sequenced, and the correctness of the manipulation determined. In addition, or in place of the E. coli replication system, a broad host range replication system may be employed, such as the replication systems of the P-1 incompatibility plasmids, e.g., pRK290. In addition to the replication system, there will frequently be at least one marker present, which may be useful in one or more hosts, or different markers for individual hosts. That is, one marker may be employed for selection in a prokaryotic host, while another marker may be employed for selection in a eukaryotic host, particularly a plant host. The markers may be protection against a biocide, such as antibiotics, toxins, heavy metals, or the like; provide complementation, for example by imparting prototrophy to an auxotrophic host; or provide a visible phenotype through the production of a novel compound. Exemplary genes which may be employed include neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (HPT), chloramphenicol acetyltransferase (CAT), nitrilase, and the gentamicin resistance gene. For plant host selection, non-limiting examples of suitable SUBSTITUTE SHEET (RULE 26) CA 022~0966 l998-l0-02 W O 97/40178 PCTnUS97/06637 markers are ~-glucuronidase, providing indigo production, luciferase, providing visible light production, NPTII, providing kanamycin resistance or G418 resistance, HPT, providing hygromycin resistance, and the mutated aroA gene, providing glyphosate resistance.
Vectors that may be used to carry out the present invention include Agrobacterium vectors. Numerous Agrobacterium vectors are known. See, e.g., U.S. Patent No. 4,536,475 to Anderson, U.S. Patent No. 4,693, 977 to Schliperoort et al.; U.S. Patent No. 4,886,937 to Sederoff et al.; T. Hall et al., EPO Application 0122791; R. Fraley et al., Proc Natl. Acad. Sci. USA 84, 4803 (1983); L.
Herrera-Estrella et al., EMBO .J 2, 987 (1983); G. Helmer et al., Bio/Technology 2, 520I (1984); N. Murai et al., Science 222, 476 (1983). In general, such vectors comprise an agrobacteria, typically Agrobacterium tumefaciens, that carries at least one tumor-inducing (or "Ti") plasmids.
When the agrobacteria is Agrobacterium rhizogenes, this plasmid is also known as the root-inducing (or "Ri") plasmid. The Ti (or Ri) plasmid contains DNA referred to as "T-DNA" that is transferred to the cells of a host plant cells when that plant is infected by the agrobacteria. In an Agrobacterium vector, the T-DNA is modified by genetic engineering techniques to contain the "expression cassette", or the gene or genes of interest to be expressed in the transformed plant cells, along with the associated regulatory sequences. The agrobacteria may contain multiple plasmids, as in the case of a "binary" vector system. Such Agrobacterium vectors are useful for introducing foreign genes into a variety of plant species, and are particularly useful for the transformation of dicots.
Vectors which may be used to transform plant tissue with DNA constructs of the present invention also include non-Agrobacterium vectors, particularly ballistic vectors, as well as vectors suitable for DNA-mediated transformation.
SU~ 1 1 1 lJTE SHEET (RULE 26J
CA 022~0966 1998-10-02 W O 97/40178 PCT~US97/06637 Microparticles carrying a DNA construct of the present invention, which microparticles are suitable for the ballistic transformation of a cell, are also useful for transforming cells according to the present invention. The microparticle is propelled into a cell to produce a transformed cell. Where the transformed cell is a plant cell, a plant may be regenerated from the transformed cell according to techniques known in the art. Any suitable ballistic cell transformation methodology and apparatus can be used in practicing the present invention. Exemplary apparatus and procedures are disclosed in Stomp et al., U.S. Patent No. 5,122,466; and Sanford and Wolf, U.S.
Patent No. 4,945,050 (the disclosures of all U.S. Patent references cited herein are incorporated herein by reference in their entirety). When using ballistic transformation procedures, the expression cassette may be incorporated into a plasmid capable of replicating in the cell to be transformed. Examples of microparticles suitable for use in such systems include 1 to 5 ~m gold spheres. The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.
Plant species may be transformed with the DNA
construct of the present invention by the DNA-mediated transformation of plant cell protoplasts and subsequent regeneration of the plant from the transformed protoplasts in accordance with procedures well known in the art.
The various fragments comprising the various constructs, expression cassettes, markers, and the like may be introduced consecutively by restriction enzyme cleavage of an appropriate replication system, and insertion of the particular construct or fragment into the available site.
After ligation and cloning the DNA construct may be isolated for further manipulation. All of these techniques are amply exemplified in the literature and find particular exemplification in Sambrook et al., Molecular Cloning: A
SU~;~ JTE S~IEET (RULE 26) CA 022~0966 1998-10-02 W O 97/40178 PCTrUS97/06637 Laboratory M~n~ , (2d Ed. 1989)(Cold Spring Harbor Laboratory, Cold Sprlng Harbor, NY).
Any plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may 5 be transformed with a vector of the present invention. The term "organogenesis," as used herein, means a process by which shoots and roots are developed sequentially from meristematic centers; the term "embryogenesis," as used herein, means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristems, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
Plants of the present invention may take a variety of forms. The plants may be chimeras of transformed cells and non-transformed cells; the plants may be clonal transformants (e.g., all cells transformed to contain the expression cassette); the plants may comprise grafts of transformed and untransformed tissues (e.g., a transformed root stock grafted to an untransformed scion in citrus species). The transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, first generation (or T1) transformed plants may be selfed to give homozygous second generation (or T2) transformed plants, and the T2 plants further propagated through classical breeding techniques. A dominant selectable marker (such as npt II) can be associated with the expression cassette to assist in breeding.
Plants which may be employed in practicing the present invention include (but are not limited to) tobacco SUBSTITUTE SHEET (RULE 26) .
CA 022~0966 1998-10-02 (Nicotiana tabacum), canola (Brassica spp.), potato (Solanum tuberosum), soybean (glycine max), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum~, sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris~, corn (Zea mays), wheat, oats, rye, barley, rice, vegetables, ornamentals, and conifers. Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuea sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Pisum spp.) and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C.
cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (dianthus caryophyllus), poinsettia (Euphorbia pulcherima), and chrysanthemum. Gymnosperms which may be employed to carrying out the present invention include conifers, including pines such as loblolly pine (Pinus taeda), Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); etc.
The present invention additionally provides a method which consists of linki~g one or more ecologically important transgenes, such as genes conferring herbicidal, disease or insect resistance to a second transgene coding a real time, in vivo marker, such as green fluorescent protein (GFP) gene. This method provides means for estimating gene flow from transgenic pollen-donor SIJ~S 111 UTE SHEET (RULE 26) CA 022~0966 1998-10-02 W O 97/40178 PCTrUS97/06637 populations to non-transgenic pollen-recipient populations (e.g., escape of transgenic genes within a plant population comprising a mixture of transgenic and nontransgenic plants). The fate of the transgenes may be traced by visual observation of transgenic/non-transgenic hybrids under an ultraviolet or blue light. In one embodiment of the invention, a mixture of plants are planted in a bullseye design, as illustrated in Figure 1. The mixture of plants may comprise plants of one species, but preferably comprises plants of multiple species. In the bullseye design, transgenic plants are grown in the center or "bullseye" of the plot. Non-transgenic plants are grown within the concentric rings which surround the center.
After planting, the plants are allowed to mate. After mating, seeds from a number of plants are samples from sampling points in the ring. These seeds are germinated and then examined and scored for the presence or absence of fluorescence using an ultraviolet lignt. The results of this scoring is used to determine crossing frequency and rates. A nested analysis of variance (e.g., ANOVA (SAS
Institute, Cary, NC)) is used to detect significant differences among species, distance, and location within distance.
In a particularly preferred embodiment of the invention, the method of estimating gene flow as described above is used to monitor the transference of genes between selected genetically engineered crop species and weed species that are sexually compatible with the transgenic crop species. A partial listing of crops species and their corresponding weed species is provided below in Table l.
Crop Species Weed Species (sexually compatible with crop species) Canola: Brassica napus and B. campestris B. napus, B. campestris, B. juncea, B.
kaber, B. oleracea, B. adepressa, Sinapsis Other Brassica cole crops such as cabbage alba, 5. arvensis and cauliflower Sunflower: Helianthis annuus H. annuus SUBSTITUTE SHEET (RULE 26) CA 022~0966 1998-10-02 W O 97/40178 PCT~US97/06637 Corn: Zea mays Teosinte: Z. mays, Z. diploperennis, Z.
perennis, Z. Iuxurians Tomato: Lycopersicum esculentium L. esculentium, L. chmielewski, L., pimpinellifolium Potato: Solanum tuberosum S. tuberosum, S. ajanuiri, S. curilohum, S:
~ demissum, S. goniocalys, S. sucrense, S.
vernsi, S. stentomum x sparsipulum Rice: Oryza sativa O. sativa, O. perennis Alfalfa: Medicago sativa M. sativa Soybean: Glycine max ~;. max, G. soja Ryegrass: Lolium perenne L. perenne, Festuca spp.
White clover: Trifolium repens T. repens Carrot: Daucus carota D. carota Radish: Raphinus sativus Raphanus sativus, R. raphanastrum Table 1: Partial list of crop species and sexually compatible weeds.
The examples which follow are set forth to illustrate the present invention, and are not to be 15construed as limiting thereof.
EX~PLE 1 Transgenic tobacco was produced that fluoresces green under an ultraviolet light. The plasmid mGFP4 (a 20gift from Jim Haseloff, England) which contains a mutagenized version of the GFP gene from the jellyfish Aequoria victoria under the control of the CaMV35S promoter and a kanamycin selectable marker, was engineered into tobacco (Nicotiana tabacum cv. Xanthi). The method is an 25efficient Agrobacterium tumefaciens-mediated transformation of leaf discs, followed by kanamycin selection and induced in vitro organogenesis (Schardl et al., Gene 61, 1-11 1987). The Agrobacterium strain GV3850 was used for all transformations.
30Of 25 transgenic lines, 2 were recovered in which entire plants fluoresced under an ultraviolet light.
- These two plants were randomly placed among a group of other transgenic tobacco plants of approximately the same SUBSTITUTE SHEET (RULE 26) CA 022~0966 1998-10-02 W O97/40178 PCTrUS97/06637 size , in a darkened room. At separate times, three individuals using a hand-held W light (365 nm, W P model 100 AP, Upland, California) were able to correctly distinguish the green fluorescing transgenic plants from the red-fluorescing non transgenic plants.
EX~MPLE 2 In this experiment, transgenic plants are produced in which the GFP marker is linked to an additional trait, in this case, insect resistance. An improved GFP
gene, sGFP (gift of Jen Sheen, Harvard University) is linked to a synthetic Bacillus thuringiensis transgene (Bt cryIAc), which construction is illustrated in Figure 2. Bt cryIAc codes for an insecticidal endotoxin protein which kills lepidopteran insects, such as corn earworm (Helicoverpa zea). Tobacco is transformed using the method described in Example 1, and canola using the procedure described in A. Mehra-Palta et al., Rapeseed in a Changing World: GCIRC Congress 1108-1115 (Saskatoon, Canada 1991).
Transgenic lines are selected that both fluoresce and express high levels of Bt cryIAc.
It is shown that the two genes are linked and functionally operative together. Twenty transgenic plants containing both genes (confirmed by using conventional molecular methods, e.g. Stewart et al. 1996) together with twenty non-transgenic plants of each crop are planted in a field. One half of the plants (both transgenic and non-transgenic) are exposed to insects, in which 50 eggs of Helicoverpa zea are placed on each plant. The remaining plants are not exposed to insects. The eggs are monitored for hatching. After one week, insect numbers are surveyed and defoliation of plants are estimated by visual ex~min~tion. Concurrently, all plants, both exposed and non-exposed, are observed at night with hand-held W
lights. Plants are scored as GFP positive or negative, - which scores are compared with defoliation measurements.
There is a significant positive correlation between the two SUBSTITUTE SHEET (RULE 26) W O 97/40178 PCT~US97106637 variables, indicating both linkage and functionality of both genes in the field.
The foregoing examples are illustrative of the present invention, and are not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.
SUBSTITUTE SHEET (RULE 26)
Claims (35)
1. A DNA construct that codes for the expression of a heterologous DNA and an in vivo marker gene in a plant cell.
2. A DNA construct according to claim 1, wherein said heterologous DNA encodes a Bacillus thuringiensis insect resistance protein.
3. A DNA construct according to claim 1, wherein said in vivo marker gene encodes a fluorescent protein.
4. A plant transformation vector carrying a DNA
construct that codes for the expression of a heterologous DNA and an in vivo marker gene in a plant cell.
construct that codes for the expression of a heterologous DNA and an in vivo marker gene in a plant cell.
5. A plant transformation vector according to claim 4, wherein said vector is an Agrobacterium vector.
6. A plant transformation vector according to claim 4, wherein said vector is a microparticle.
7. A plant cell containing a heterologous DNA
construct, which construct that codes for and expresses a heterologous DNA and an in vivo marker gene in said plant cell.
construct, which construct that codes for and expresses a heterologous DNA and an in vivo marker gene in said plant cell.
8. A plant cell according to claim 7, which plant cell is a dicotyledonous plant cell.
9. A plant cell according to claim 7, which plant cell is a monocotyledonous plant cell.
10. A recombinant plant comprising transformed plant cells, said transformed plant cells containing a heterologous DNA construct, which construct that codes for and expresses a heterologous DNA and an in vivo marker gene in said plant cell.
11. A recombinant plant according to claim 10, which plant is a monocot.
12. A recombinant plant according to claim 10, which plant is a dicot.
13. A recombinant plant according to claim 9, which plant is a dicot selected from the group consisting of tobacco, potato, soybean, peanuts, cotton, and vegetable crops.
14. A recombinant plant according to claim 10, which plant is canola.
15. A method of making transgenic plant cells, said method comprising:
providing a plant cell capable of regeneration;
transforming said plant cell with a DNA
construct that codes for and expresses a heterologous DNA
and an in vivo marker gene in said plant cell.
providing a plant cell capable of regeneration;
transforming said plant cell with a DNA
construct that codes for and expresses a heterologous DNA
and an in vivo marker gene in said plant cell.
16. A method according to claim 15, wherein said transforming step is carried out by infecting said plant cell with an Agrobacterium vector that transfers said DNA construct into said plant cell.
17. A method according to claim 15, wherein said transforming step is carried out by bombarding said plant cell with microparticles carrying said expression cassette.
18. A method according to claim 15, wherein said plant cell resides in a plant tissue capable of regeneration.
19. A method according to claim 15, further comprising the step of regenerating shoots from said transformed plant cells.
20. A method according to claim 16, further comprising the step of regenerating roots from said transformed plant cells.
21. A method according to claim 16, further comprising the step of regenerating a plant from said transformed plant cells.
22. A method according to claim 16, wherein said plant cell is a monocot cell.
23. A method according to claim 16, wherein said plant cell is a dicot cell.
24. A method of monitoring for the escape of a transgene in plants, comprising:
(a) planting transgenic plants in a predetermined area, wherein said transgene is linked with a detectable in vivo marker gene;
(b) permitting said transgenic plants to grow in said predetermined area; and (c) detecting the presence or absence of said detectable in vivo marker gene in plants growing outside of said predetermined area, or inside said predetermined area at different times.
(a) planting transgenic plants in a predetermined area, wherein said transgene is linked with a detectable in vivo marker gene;
(b) permitting said transgenic plants to grow in said predetermined area; and (c) detecting the presence or absence of said detectable in vivo marker gene in plants growing outside of said predetermined area, or inside said predetermined area at different times.
25. A method according to claim 25, wherein said predetermined area is an agricultural field.
26. A method according to claim 25, wherein said in vivo marker gene encodes a fluorescent protein.
27. A method according to claim 26 wherein said in vivo marker gene encodes green fluorescent protein.
28. A method according to claim 25 wherein said transgene encodes insect resistance.
29. A method of detecting transgene flow from transgenic plants to wild-type plants, comprising:
(a) planting transgenic plants together with wild-type plants together in a shared area, wherein transgenes of the transgenic plants are operatively associated with a detectable in vivo marker gene;
(b) allowing said transgenic and non-transgenic plants to mate; and (c) detecting the presence or absence of the in vivo marker in the progeny of said transgenic and non-transgenic plants.
(a) planting transgenic plants together with wild-type plants together in a shared area, wherein transgenes of the transgenic plants are operatively associated with a detectable in vivo marker gene;
(b) allowing said transgenic and non-transgenic plants to mate; and (c) detecting the presence or absence of the in vivo marker in the progeny of said transgenic and non-transgenic plants.
30. A method according to claim 30, wherein said in vivo marker gene encodes a fluorescent protein.
31. A method according to claim 30 wherein said in vivo marker gene encodes green fluorescent protein.
32. A method according to claim 30 wherein said transgene encodes insect resistance.
33. A method according to claim 30 wherein the transgenic and wild-type plants comprises multiple species.
34. A method according to claim 30 wherein said transgenic plant is tobacco or canola.
35. A method according to claim 30, wherein said planting step includes the step of planting said wild-type plants together with said transgenic plants in said shared area.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US1623796P | 1996-04-19 | 1996-04-19 | |
| US60/016,237 | 1996-04-19 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2250966A1 true CA2250966A1 (en) | 1997-10-30 |
Family
ID=21776087
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2250966 Abandoned CA2250966A1 (en) | 1996-04-19 | 1997-04-18 | Monitoring of transgenic plants with in vivo markers |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0904388A1 (en) |
| JP (1) | JP2000509274A (en) |
| AU (1) | AU2679097A (en) |
| CA (1) | CA2250966A1 (en) |
| WO (1) | WO1997040178A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6426185B1 (en) | 1998-01-16 | 2002-07-30 | Large Scale Biology Corporation | Method of compiling a functional gene profile in a plant by transfecting a nucleic acid sequence of a donor plant into a different host plant in an anti-sense orientation |
| US6303848B1 (en) | 1998-01-16 | 2001-10-16 | Large Scale Biology Corporation | Method for conferring herbicide, pest, or disease resistance in plant hosts |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987007299A1 (en) * | 1986-05-29 | 1987-12-03 | Calgene, Inc. | Transformation and foreign gene expression in brassica species |
| WO1995007463A1 (en) * | 1993-09-10 | 1995-03-16 | The Trustees Of Columbia University In The City Of New York | Uses of green fluorescent protein |
| US5474929A (en) * | 1993-10-28 | 1995-12-12 | National Research Council Of Canada | Selectable/reporter gene for use during genetic engineering of plants and plant cells |
| GB9504446D0 (en) * | 1995-03-06 | 1995-04-26 | Medical Res Council | Improvements in or relating to gene expression |
-
1997
- 1997-04-18 EP EP97918766A patent/EP0904388A1/en not_active Withdrawn
- 1997-04-18 CA CA 2250966 patent/CA2250966A1/en not_active Abandoned
- 1997-04-18 AU AU26790/97A patent/AU2679097A/en not_active Abandoned
- 1997-04-18 JP JP9538242A patent/JP2000509274A/en active Pending
- 1997-04-18 WO PCT/US1997/006637 patent/WO1997040178A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| AU2679097A (en) | 1997-11-12 |
| EP0904388A1 (en) | 1999-03-31 |
| JP2000509274A (en) | 2000-07-25 |
| WO1997040178A1 (en) | 1997-10-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU719306B2 (en) | Method for reducing expression variability of transgenes in plant cells | |
| EP0663921B1 (en) | Method for increasing expression and reducing expression variability of foreign genes in plant cells | |
| US6544733B1 (en) | Method of identifying non-host plant disease resistance genes | |
| US20140366219A1 (en) | Increasing Soybean Defense Against Pests | |
| CA3128376A1 (en) | Plant explant transformation | |
| Pipatpanukul et al. | Transformation of indica rice (Oryza sativa L.) cv. RD6 mediated by Agrobacterium tumefaciens | |
| US8962916B2 (en) | Viral promoter, truncations thereof, and methods of use | |
| US9157087B2 (en) | Inducible plant promoters and the use thereof | |
| US8895716B2 (en) | Viral promoter, truncations thereof, and methods of use | |
| US7276596B2 (en) | Promoter from maize invertase inhibitor gene | |
| CA2250966A1 (en) | Monitoring of transgenic plants with in vivo markers | |
| Kudo et al. | TRANSFORMATION OF CHRYSANTHEMUM (DENDRANTHEMA GRANDI-FLORUM (RAMAT.) KITAMURA) VIA AGROBACTERIUM TUMEFACIENS | |
| US6433248B1 (en) | Trans-activation of transcription from viral RNA | |
| US8344206B2 (en) | Viral promoter, truncations thereof, and methods of use | |
| KR100877889B1 (en) | Process for producing herbicide resistant transgenic sweetpotato plants by Agrobacterium-mediated transformation | |
| EP0945508A1 (en) | The insect-resistant use of sweet potato sporamin gene and method for controlling pests using the gene | |
| US20110055977A1 (en) | Enhancement of Reproductive Heat Tolerance in Plants | |
| EP1003849A1 (en) | Method for reducing expression variability of transgenes in plants cells | |
| Burke et al. | Selection procedure for identifying transgenic cells, embryos, and plants without the use of antibiotics | |
| Ko et al. | Production of Transgenic Petunia hybrida cv. Rosanpion Using Agrobacterium-mediated Transformation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Dead |