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  • 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

Definitions

• the present invention relates to methods of monitoring transgenic plants, and more specifically to monitoring the escape of transgenes into non-transgenic plant populations.
• 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.
• 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 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.
• 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 b linking one or more ecologically important transgenes wit a second transgene encoding a detectable in vivo marker.
• 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.
• a second aspect of the present invention is a plant transformation vector carrying a DNA construct as given 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.
• Figure 1 is a schematic representation of a bullseye design for growing a mixture of transgenic and nontransgenic plants.
• FIG. 2 is a schematic representation of the pGPF/Bt binary expression vector useful for in vivo monitoring of transgenic plants.
• Bt means a synthetic Bacillus thurgiensis gene cryJAc
• HPH means a hygromycin phosphotransferase gene
• sGFP means a synthetic green fluorescent protein
• the present invention provides a method of monitoring the escape of transgenes from genetically engineered plants to surrounding, non ⁇ transgenic plants by utilizing in vivo genetic markers.
• plants refers to vascular plants, including both monocots and dicots, and both gymnosperms and angiosperms.
• 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 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)
• Other sources include the Agrobacterium T-DNA genes, such as the transcriptional initiation regions for the biosynthesis of 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 .
• 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) .
• the transcriptional initiation region, or the regulatory portion of such region is obtained from an appropriate gene which is so regulated. For 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 heterologous structural gene.
• Genes which may be operationally associated with a transcription initiation 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.
• 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.
• a replication system functional in Escherichia coli , such as ColEl, pSClOl, pACYC184, or the like.
• the resulting construct may be cloned, sequenced, and the correctness of the manipulation determined.
• a broad host range replication system may be employed, such as the replication systems of the P-l incompatibility plasmids, e.g., pRK290.
• 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 gentamicin resistance gene
• suitable 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 AgroJbacterium vectors. Numerous AgroJbacterium 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.
• such vectors comprise an agrobacteria, typically AgrroJbacterium tume.facie.ns, that carries at least one tumor-inducing (or "Ti") plasmids.
• agrobacteria typically AgroJbacterium 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.
• 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.
• 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.
• the expression cassette may be incorporated into a plasmid capable of replicating in the cell to be transformed.
• 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 Laboratory Manual, (2d Ed. 1989) (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) .
• organogenesis means a process by which shoots and roots are developed sequentially from meristematic centers
• embryogenesis 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.
• 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 Tl) 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 (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 ( Ci trus spp.), cocoa ( Theobroma cacao) , tea (Camellia sinensis) , banana (Afusa spp.), avocado (Persea amer ic ana) , fig (Ficus casica) , guava (Psidium guajava) , mango (Mangifera in
• 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 (HiJbiscus 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.
• 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 linking 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 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.
• 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.
• 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 light. 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.
• 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 1.
• Table 1 Partial list of crop species and sexually compatible weeds.
• Transgenic tobacco was produced that fluoresces green under an ultraviolet light.
• the plasmid mGFP4 (a gift 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 taJbacum cv. Xanthi) .
• the method is an efficient AgroJbacterium tumefaciens-mediated transformation of leaf discs, followed by kanamycin selection and induced in vi tro organogenesis (Schardl et al . , Gene 61, 1-11 1987) .
• the AgroJbacterium strain GV3850 was used for all transformations. Of 25 transgenic lines, 2 were recovered m 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 size , in a darkened room. At separate times, three individuals using a hand-held UV light (365 nm, UVP model 100 AP, Upland, California) were able to correctly distinguish the green fluorescing transgenic plants from the red-fluorescing non transgenic plants.
• 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
• Bt crylAc Bacillus thuringiensis transgene
• Bt crylAc 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 crylAc.

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