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USRE41943E1 - Glyphosate-resistant plants - Google Patents

Glyphosate-resistant plants Download PDF

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USRE41943E1
USRE41943E1 US12/633,520 US63352009A USRE41943E US RE41943 E1 USRE41943 E1 US RE41943E1 US 63352009 A US63352009 A US 63352009A US RE41943 E USRE41943 E US RE41943E
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glyphosate
epsps
seq
sequence
dna fragment
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Bruce Held
Herbert Wilson
Philip E. Dykema
Carol J. Lewnau
Janelle C. Eby
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Mertec LLC
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Mertec LLC
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    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C12N15/8275—Glyphosate

Definitions

  • This invention relates to glyphosate-resistant transgenic plants and methods of making the same.
  • Glyphosate is a widely used component in herbicides. Glyphosate inhibits 5-enolpyruvyl-3-phosphoshikimic acid synthase (EPSP synthase, or EPSPS), which is involved in the synthesis of aromatic amino acids in plant cells. Inhibition of EPSPS effectively disrupts protein synthesis and thereby kills the affected plant cells. Because glyphosate is non-selective, it kills both weeds and crop plants. Accordingly, there is a need to produce transgenic crop plants that are resistant to glyphosate.
  • EEPSP synthase 5-enolpyruvyl-3-phosphoshikimic acid synthase
  • mutant EPSP synthases that are glyphosate-resistant. These mutant EPSP synthases can be transformed into plants and confer glyphosate-resistance upon the transformed plants. Examples of mutant EPSP synthases and glyphosate-resistant transgenic plants are illustrated in U.S. Pat. Nos. 6,040,497 and 5,554,798, 5,310,667 and WO 00/66748.
  • chimeric expression vectors include regulatory sequences, such as enhancers or promoters, that are heterologous to the EPSPS genes.
  • regulatory sequences such as enhancers or promoters, that are heterologous to the EPSPS genes.
  • WO 00/66748 fuses enhancers from CaMV 35S, FMV 35S, rice actin 1, rice GOS2, maize polyubiquitin, or barley plastocyanin genes to a glyphosate-resistant EPSPS coding sequence in order to enhance the expression of the glyphosate-resistant EPSPS in transformed plant cells.
  • the expression cassette of the EPSP synthase gene consists of a native EPSPS 5′ regulator sequence, a coding sequence (with or without introns) encoding a glyphosate-resistant EPSPS which includes a native transit peptide, and a native EPSPS 3′ regulatory sequence (such as an EPSPS transcriptional terminator).
  • Suitable sources of EPSP synthase genes include dicotyledonous plants, such as Arabidopsis thaliana, and mono-cotyledonous plants, such as Zea mays, Arabidopsis thaliana has two EPSP synthase genes (epm1 and epm2).
  • the present invention includes use of one or both of mutated epm1 and epm2 to confer resistance to glyphosate.
  • Mutated EPSP synthase genes from Zea mays or other plants can also be used for transforming plant cells to make glyphosate-resistant plants.
  • a DNA fragment which comprises an EPSPS 5′ regulatory sequence and a glyphosate-resistant EPSPS coding sequence is introduced into regenerable plant cells.
  • the DNA fragment does not contain a non-EPSPS enhancer.
  • Cells are selected for stable transformation. The selected cells are then used to regenerate glyphosate-resistant, transgenic plants.
  • the DNA fragment used for transformation comprises a modified plant genomic sequence.
  • the unmodified plant genomic sequence comprises at least part of an EPSPS gene, and includes an EPSPS 5′ regulatory sequence and a glyphosate-sensitive EPSPS coding sequence (including a chloroplast transit peptide coding sequence).
  • the glyphosate-sensitive EPSPS coding sequence is modified to make the encoded EPSPS glyphosate-resistant.
  • the DNA fragment comprising the modified plant genomic sequence is stably transformed into plant cells, from which glyphosate-resistant plants are regenerated.
  • the DNA fragment used for transformation comprises SEQ ID NO: 2.
  • the DNA fragment used for transformation comprises SEQ ID NO: 4.
  • the DNA fragment comprises SEQ ID NO: 6.
  • any two sequences selected from SEQ ID. NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6 are used to transform plant cells.
  • the transgenic plant comprises transformed SEQ ID. NO: 2 and SEQ ID NO: 4.
  • a DNA fragment is introduced into regenerable, glyphosate-sensitive recipient plant cells.
  • the DNA fragment comprises an EPSPS 5′ regulatory sequence, and a coding sequence encoding a glyphosate-resistant EPSPS.
  • the EPSPS 5′ regulatory sequence is operably linked to the EPSPS coding sequence.
  • the glyphosate-resistant EPSPS includes a chloroplast transit peptide.
  • the DNA fragment does not contain a non-EPSPS enhancer.
  • the recipient plant cells are selected for glyphosate-resistance and stable transformation. The cells thus selected can be used to regenerate glyphosate-resistant plants.
  • a “DNA fragment” may be either linear or circular.
  • the DNA fragment used for transformation is a linear DNA fragment.
  • a “coding sequence” encoding an EPSPS refers to a nucleic acid sequence transcription and translation of which produce a functional EPSPS. The boundaries of the coding sequence are generally determined by a translation start codon at its 5′ end and a translation stop co don at its 3, end.
  • a coding sequence of EPSPS may be a cDNA, or a plant genomic sequence which consists of all of the exons and introns of an EPSPS gene.
  • An EPSPS gene refers to the plant genomic sequence which includes the EPSPS 5′ regulatory sequence, the EPSPS coding sequence (including the sequence encoding the chloroplast transit peptide), and the EPSPS 3′ regulatory sequence (such as an EPSPS transcriptional terminator).
  • a “plant genomic sequence” refers to a nucleotide sequence found in the genome of the plant.
  • a chloroplast transit peptide functions post-translationally to direct a polypeptide to chloroplast.
  • Either endogenous or heterologous chloroplast peptides can be used in the present invention.
  • heterologous means derived from a different source
  • endogenous means derived from the same source.
  • the endogenous transit peptide encoded by a native EPSPS gene is used.
  • an EPSPS 5′ regulatory sequence refers to a nucleotide sequence located upstream (5′) to the start codon of the EPSPS coding sequence in an EPSPS gene in a plant or plant cell which has not been subject to genetic engineering.
  • the 5′ regulatory sequence generally includes an EPSPS promoter which directs the transcription of the EPSPS gene.
  • the EPSPS 5′ regulatory sequence comprises one or more EPSPS enhancers operably linked to the promoter.
  • the 5′ regulatory sequence comprises at least 200 bp.
  • the 5′ regulatory sequence comprises at least 400, 600, 800, 1000, 1,200 or 1,800 bp.
  • An EPSPS 3′ regulatory sequence refers to a nucleotide sequence located downstream (3′) to the stop codon of the EPSPS coding sequence in an EPSPS gene in a plant or plant cell which has not been subject to genetic engineering.
  • the 3, regulatory sequence generally includes a transcription terminator which controls the termination of the transcription of the EPSPS gene.
  • “Operably linked” refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a 5′ regulatory sequence is operably linked to a coding sequence if the 5′ regulatory sequence functions to initiate transcription of the coding sequence.
  • the DNA fragment used for transformation does not include a non-EPSPS enhancer.
  • a “non-EPSPS enhancer” refers to an enhancer which is not used by an EPSPS gene in a plant or plant cell which has not been subject to genetic engineering.
  • Non-EPSPS enhancers include, but are not limited to, enhancers that are associated with CaMV 35S, FMV 35S, rice actin 1, rice GOS2, maize polyubiquitin, or barley plastocyanin genes.
  • a “glyphosate-resistant” cell or plant refers to a cell or plant that can survive or continue to grow in the presence of certain concentrations of glyphosate that typically kill or inhibit the growth of other cells or plants. Growth includes, for instance, photosynthesis, increased rooting, increased height, increased mass, or development of new leaves.
  • a glyphosate-resistant cell can grow and divide on a culture medium containing 50 mg/l or more glyphosate.
  • a glyphosate-resistant cell can grow and divide on a culture medium containing 100 mg/l or more glyphosate, such as 200 mg/l, 300 mg/l or 400 mg/l glyphosate.
  • a glyphosate-resistant cell can grow and divide on a culture medium containing 500 mg/l or more glyphosate, such as 600 mg/l.
  • glyphosate includes any herbicidally effective form of N-phosphonomethylglycine (including any salt thereof) and other forms which result in the production of the glyphosate anion in plants.
  • Regenerable glyphosate-resistant plant cells may be used to regenerate glyphosate-resistant plants.
  • the glyphosate-resistant plant thus regenerated can survive or continue to grow after being sprayed with glyphosate at a rate of 25 g/ha (grams per hectare) or more.
  • the glyphosate-resistant plant thus regenerated can survive or continue to grow after being sprayed with glyphosate at a rate of 50 g/ha or more, such as 100 g/ha, 200 g/ha, 400 g/ha, or 800 g/ha.
  • the glyphosate-resistant plant thus regenerated can survive or continue to grow after being sprayed with glyphosate at a rate of 1000 g/ha or more, such as 2000 g/ha and 3000 g/ha.
  • the spray may preferably be carried out at or after the growth stage of v2, such as v3, v4, v5 or later stages.
  • the regenerated glyphosate-resistant plant can tolerate the spray of glyphosate at between 0.1 M and 0.4 M.
  • a “glyphosate-resistant” EPSPS refers to an EPSPS the expression of which in a plant cell confers glyphosate resistance upon the plant cell.
  • An EPSPS is “glyphosate-sensitive” if it does not confer glyphosate-resistance when being expressed in plant cells.
  • EPSPS mutations have been known to be glyphosate-resistant and capable of conferring glyphosate resistance upon transformed plants.
  • EPSPS of Zea mays (GenBank Accession No. X63374) can be mutated at amino acid residues 102 (substitution of He for Thr) and 106 (substitution of Ser for Pro).
  • EPSPS encoded by epm1 gene of Arabidopsis thaliana can be mutated at amino acid residues 179 (substitution of He for Thr) and 183 (substitution of Ser for Pro).
  • EPSPS encoded by epm2 gene of Arabidopsis thaliana can be mutated at amino acid residues 177 (substitution of He for Thr) and 182 (substitution of Ser for Pro). These mutated EPSPSs are glyphosate-resistant and capable of conferring glyphosate resistance upon transformed plants.
  • Other mutated or modified EPSPSs such as those described in U.S. Pat. Nos. 5,310,667, 5,866,775, 6,225,114, and 6,248,876, or natural EPSPS variants showing glyphosate-resistance, can be used in the present invention.
  • bacteria-derived, glyphosate-resistant EPSPSs, after fusion with a chloroplast transit peptide can also be used.
  • the DNA fragment comprising the EPSPS 5′ regulatory sequence and the glyphosate-resistant EPSPS coding sequence can be stably transformed into a regenerable plant cell.
  • stable transformation refers to integration of the DNA fragment into the genome of the transformed plant cell.
  • the EPSPS 5′ regulatory sequence in the DNA fragment used for transformation comprises an EPSPS enhancer and an EPSPS promoter.
  • the DNA fragment used for transformation further comprises an EPSPS 3′ regulatory sequence, such as an EPSPS transcriptional terminator, which is operably linked to the coding sequence encoding the glyphosate-resistant EPSPS.
  • the DNA fragment used for transformation comprises a modified plant genomic sequence that encodes a glyphosate-resistant EPSPS. Without modification, the plant genomic sequence encodes a glyphosate-sensitive EPSPS. Modifications that are capable of converting a glyphosate-sensitive EPSPS to a glyphosate-resistant EPSPS are known in the art.
  • the DNA fragment used for transformation is modified from a plant genomic sequence.
  • the plant genomic sequence comprises an EPSPS regulatory sequence, a coding sequence encoding a glyphosate-sensitive EPSPS which includes a chloroplast transit peptide, and preferably an EPSPS 3, regulatory sequence, such as an EPSPS transcriptional terminator.
  • the genomic sequence may be obtained by fragmenting the genome of a plant of interest, or isolated from bacterial artificial chromosome clones. Other methods for obtaining genomic sequences can also be used, such as PCR or DNA synthesis.
  • the EPSPS-coding sequence in this plant genomic sequence is then subject to nucleotide modification(s) to render the encoded EPSPS glyphosate resistant. Suitable modifications for this purpose, such as nucleotide substitutions, are well known in the art.
  • the DNA fragment comprising the genomic sequence thus modified can be stably transformed into glyphosate-sensitive recipient plant cells. These transformed plant cells are selected for glyphosate resistance and then used to regenerate glyphosate-resistant plants.
  • the recipient plant cells are regenerable. They can be derived from immature embryos or meristematic tissues which contain cells that have not yet terminally differentiated. Juvenile leaf basal regions, immature tassels and gametic cells can be used to provide regenerable recipient cells for Zea mays.
  • the preferred source of recipient cells for soybean includes the immature cotyledon.
  • two or more DNA fragments can be stably transformed into a recipient plant cell.
  • Each of these DNA fragments includes an EPSPS 5′ regulatory sequence, a coding sequence encoding a glyphosate-resistant EPSPS which contains a chloroplast transit peptide, and preferably an EPSPS 3′ regulatory sequence (such as an EPSPS transcriptional terminator).
  • EPSPS 5′ regulatory sequence a coding sequence encoding a glyphosate-resistant EPSPS which contains a chloroplast transit peptide
  • EPSPS 3′ regulatory sequence such as an EPSPS transcriptional terminator
  • Transformation of plant cells can be carried out using various methods. These methods include, but are not limited to, Agrobacterium tumefaciens mediated DNA transfer, PEG or liposome mediated DNA transfer, electroporation, micro-injection, microprojectile or particle bombardment, receptor-mediated DNA transfer, and viral or other vector mediated DNA transfer. Preferably, transformation is carried out using aerosol beam injection as described in U.S. patent application Ser. No. 09/450,226, which is incorporated herein by reference.
  • stably transformed plant cells can be performed using methods as appreciated by one of ordinary skill in the art.
  • the transformed cells can be grown and selected on media containing glyphosate.
  • the introduced DNA fragment is stably transformed and integrated into a chromosome of the transformed plant cell.
  • assays can be used to confirm stable transformation. Suitable assays include molecular biological assays, such as Southern and Northern Blotting and PCR, or biochemical assays, such as ELISA and Western Blot.
  • plant part assays such as leaf and root assays, or analysis of the phenotype of the whole regenerated plant, can be used to confirm stable transformation.
  • Plants can be regenerated from the selected, stably transformed cells. Progeny can be recovered from the regenerated plants and tested for glyphosate resistance. Seeds or other parts of the regenerated transgenic plants can also be obtained. In one embodiment, glyphosate-resistant plants are made by crossing.
  • the glyphosate-resistant EPSPS coding sequence can be derived from either monocotyledonous or dicotyledonous plants.
  • the representative monocotyledonous and dicotyledonous plants used in the present invention include, but are not limited to, Oryza sativa, Zea mays, Hordeum vulgare, Triticum aestivum, Avena sativa, turf grasses including species of the genera Poa, Festuca, Lolium, Zoysia, and Cynodon among others, Glycine max, Gossypium hirsutii, Lycopersicum esculentum, Solanum tuberosum, Phaseolus species, Beta vulgaris, and Brassica species.
  • F27K7 and F4L23 Two bacterial artificial chromosome (BAC) clones, F27K7 and F4L23, were obtained from the Arabidopsis Biological Resource Center, DNA Stock Center, at the Ohio State University.
  • F27K7 and F4L23 contain the EPSPS genes found on chromosome 1 and 2 of Arabidopsis thaliana, respectively.
  • the F27K7 clone was digested using Sac II and Bam HI restriction enzymes to produce a 4.7 kb fragment, the sequence of which is shown as SEQ ID NO: 1.
  • the 4.7 kb fragment comprises the complete EPSPS gene (epm1) found on chromosome which includes an EPSPS 5′ regulatory sequence (the sequence before nucleotide residue 1290), an EPSPS coding sequence (from nucleotide residue 1290 to nucleotide residue 3729), and an EPSPS 3, regulatory sequence (the sequence after nucleotide residue 3729).
  • the EPSPS coding sequence also encodes a chloroplast transit peptide (from nucleotide residue 1290 to nucleotide residue 1612). The sequence encoding this chloroplast transit peptide can be predicted using the computer program PSORT maintained on the public accessible GenomeNet at Kyoto University, Japan.
  • the 4.7 kb fragment was cloned into a pbluescript II vector (Stratagene), and two nucleotide substitutions were introduced into the EPSPS coding sequence using QuikChange® Site-Directed Mutagenesis Kit (Stratagene) according to the instructions of the manufacturer.
  • the two nucleotide substitutions are a cytosine to thymine substitution at nucleotide 2007 and a cytosine to thymine substitution at nucleotide 2018.
  • the mutated sequence is shown as SEQ ID NO: 2.
  • the mutated sequence encodes a glyphosate-resistant EPSPS which has, as compared to the EPSPS encoded by SEQ ID NO: 1, a Thr to Ile mutation at amino acid 179 and a Pro to Ser mutation at amino acid 183.
  • the amino acid sequence of the glyphosate-resistant EPSPS is shown as SEQ ID NO: 7.
  • the pbluescript II vector containing SEQ ID NO: 2 is referred to as epm1 vector.
  • the BAC F4L23 clone was digested using Eco RI restriction enzyme to produce a 5.2 kb fragment, the sequence of which is shown as SEQ ID NO: 3.
  • the 5.2 kb fragment comprises the complete EPSPS gene (epm2) from chromosome 2, which includes an EPSPS 5 ′regulatory sequence (the sequence before nucleotide 1515), and EPSPS coding sequence (from nucleotide 1515 to nucleotide 3872), and an EPSPS 3′ regulatory sequence (the sequence after nucleotide 3872).
  • the EPSPS coding sequence also encodes a chloroplast transit peptide (from nucleotide 1515 to nucleotide 1665). The sequence encoding this chloroplast transit peptide can be predicted using the computer program PSORT maintained on the public accessible GenomeNet at Kyoto University, Japan.
  • SEQ ID NO: 4 shows the mutated sequence which has two nucleotide substitutions in the EPSPS coding sequence as compared to SEQ ID NO: 3.
  • the two substitutions are a cytosine to thymine substitution at nucleotide 2134 and a cytosine to thymine substitution at nucleotide 2145.
  • the mutated sequence encodes a glyphosate-resistant EPSPS which has, as compared to the EPSPS encoded by SEQ ID NO: 3, a Thr to Ile mutation at amino acid 178 and a Pro to Ser mutation at amino acid 182.
  • the amino acid sequence of the (putative) glyphosate-resistant EPSPS is shown as SEQ ID: 8.
  • the pbluescript II vector containing SEQ ID NO: 4 is referred to as epm2 vector.
  • the Bam HI/Sac II fragment (SEQ ID NO: 2) of epm1 vector and the Eco RI fragment (SEQ ID NO: 4) of epm2 vector were used to transform soybean embryogenic callus using an aerosol beam injector as described in U.S. patent application Ser. No. 09/450,226, which is incorporated herein by reference. These fragments comprised mutant epm1 and mutant epm2 which encode (putative) glyphosate-resistant EPSPSs. These fragments were used either alone or, preferably, together.
  • the transformed tissue was is selected for glyphosate resistance using the method described below.
  • Table 1 shows the composition of B1-30 3Co5My0.01PA medium.
  • the tissue was is then transferred to the same medium but now containing 300 mg/l glyphosate. After a number of passages (up to 5 passages, each passage may last for about a month) on this latter medium, resistant clonal material may be identified. After an optional further few passages on B1-30 3Co5My0.01PA medium but containing 500 mg/l glyphosate, the growing tissue was is transferred to a regeneration media as described in U.S. patent Ser. No. 09/450,226. Regenerated plants were are transferred to pots in a greenhouse. These plants and their progenies were are sprayed with glyphosate at commercial rates, and complete resistance to glyphosate was is expected to be observed. Progenies segregated are expected to segregate 3:1 for glyphosate resistance as would be expected for Mendelian inheritance of a transgene.
  • mutant epm1 such as SEQ ID NO: 2
  • mutant epm2 such as SEQ ID NO: 4
  • a corn (B 73) BAC library was screened with a probe containing a sequence of a corn EPSPS gene published in Genbank accession number X63374 by Incyte Genomics Inc. Four BAC clones were identified. Southern blot analysis indicated that all four clones contained the same EPSPS gene.
  • One BAC clone was further characterized by nucleotide sequencing which resulted in identification of a 6.0 kb genomic fragment flanked by unique Cla I and Eco RV sites. The sequence of the 6.0 kb fragment was shown as SEQ ID NO: 5.
  • the 6.0 kb fragment includes an EPSPS 5′ regulatory sequence (the sequence before nucleotide 1868), an EPSPS coding sequence (from nucleotide 1868 to nucleotide 5146), and an EPSPS 3, regulatory sequence (the sequence after nucleotide 5146).
  • the EPSPS coding sequence also encodes a chloroplast transit peptide (from nucleotide 1868 to nucleotide 2041). The sequence encoding this chloroplast transit peptide can be predicted using the computer program PS ORT maintained on the public accessible GenomeNet at Kyoto University, Japan.
  • the 6.0 kb fragment was cloned into the Cla I and Eco RV sites of a pbluescript vector, and then subject to site directed mutagenesis using QuikChange Site-Directed Mutagenesis Kit (Stratagene).
  • Two mutations were introduced into the EPSPS coding sequence: the first mutation being a cytosine to thymine substitution at nucleotide 2886 and the second mutation being a cytosine to thymine substitution at nucleotide 2897.
  • the mutated sequence is shown as SEQ ID NO: 6.
  • the mutations changed the encoded amino acid residue Thr to Ile at position 164 and Pro to Ser at position 168.
  • This mutated EPSPS amino acid sequence is shown as SEQ ID NO: 9.
  • the mutated EPSPS is glyphosate-resistant.
  • the pBluescript vector comprising SEQ ID NO: 6 is referred to as HCEM.
  • the Cla I and Eco RV fragment (SEQ ID NO: 6) of HCEM was introduced into cultured immature corn embryos using an aerosol beam injector according to U.S. patent application Ser. No. 09/450,226.
  • the Cla I-Eco RV fragment comprised the glyphosate-resistant EPSPS coding sequence.
  • DN62A100RR a medium containing 100 mg/l glyphosate.
  • Table 3 lists the composition of DN62A100RR and other media. After two 14-day passages on DN62A100RR, actively growing tissue was is transferred to DN62A300RR medium which contains 300 mg/l (Table 3). After two 14-day passages on this medium, tissue was finally transferred to DN62540RR medium which contains 540 mg/l glyphosate (Table 3). Stable transformation allowed continued growth on 540 mg/l glyphosate. Regeneration was is carried out as described in U.S. patent application Ser. No. 09/450,226.
  • Transformation can also be accomplished using Agrobacterium-mediated DNA delivery.
  • the transformation and regeneration were are performed according to the methods as described in U.S. patent application Ser. No. 09/203,679, which is herein incorporated by reference. Briefly, after culturing on DN62A0S20GLC (Table 2) for five days, co-cultivated embryos were are transferred to DN62ALC180RR medium which contains 180 mg/l glyphosate (Table 3). After two 14-day passages on this medium, actively growing tissue was is transferred to DN62540RR medium containing 540 mg/l glyphosate (Table 3). Stable transformation allowed will allow continued growth on 540 mg/l glyphosate. Regeneration was is carried out as described in U.S. Ser. No. 09/203,679.

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Abstract

This invention relates to glyphosate-resistant transgenic plants and methods of making the same. In a preferred embodiment, a DNA fragment which comprises an EPSPS 5′ regulatory sequence and a glyphosate-resistant EPSPS coding sequence is introduced into regenerable plant cells. The encoded EPSPS has a chloroplast transit peptide. The DNA fragment does not contain a non-EPSPS enhancer. Cells are selected for stable transformation, and the selected cells can be used to regenerate glyphosate-resistant transgenic plants. The DNA fragment used for transformation preferably comprises a modified plant genomic sequence, such as SEQ ID NO: 2, SEQ ID NO:4 or SEQ ID NO: 6. In one embodiment, two DNA fragments of this invention are stably transformed into a plant to confer glyphosate-resistance.

Description

TECHNICAL FIELD
This invention relates to glyphosate-resistant transgenic plants and methods of making the same.
BACKGROUND
Glyphosate is a widely used component in herbicides. Glyphosate inhibits 5-enolpyruvyl-3-phosphoshikimic acid synthase (EPSP synthase, or EPSPS), which is involved in the synthesis of aromatic amino acids in plant cells. Inhibition of EPSPS effectively disrupts protein synthesis and thereby kills the affected plant cells. Because glyphosate is non-selective, it kills both weeds and crop plants. Accordingly, there is a need to produce transgenic crop plants that are resistant to glyphosate.
Recombinant DNA technology has been used to create mutant EPSP synthases that are glyphosate-resistant. These mutant EPSP synthases can be transformed into plants and confer glyphosate-resistance upon the transformed plants. Examples of mutant EPSP synthases and glyphosate-resistant transgenic plants are illustrated in U.S. Pat. Nos. 6,040,497 and 5,554,798, 5,310,667 and WO 00/66748.
Current plant transformation technology employs chimeric expression vectors. These vectors include regulatory sequences, such as enhancers or promoters, that are heterologous to the EPSPS genes. For instance, WO 00/66748 fuses enhancers from CaMV 35S, FMV 35S, rice actin 1, rice GOS2, maize polyubiquitin, or barley plastocyanin genes to a glyphosate-resistant EPSPS coding sequence in order to enhance the expression of the glyphosate-resistant EPSPS in transformed plant cells.
No one has used a complete expression cassette of the EPSP synthase gene isolated from the genome of a donor plant and mutated to give glyphosate resistance. In one embodiment of the present invention, the expression cassette of the EPSP synthase gene consists of a native EPSPS 5′ regulator sequence, a coding sequence (with or without introns) encoding a glyphosate-resistant EPSPS which includes a native transit peptide, and a native EPSPS 3′ regulatory sequence (such as an EPSPS transcriptional terminator). The fact that such an expression cassette is sufficient to provide glyphosate resistance is surprising. Moreover, the use of the native EPSPS 5′ and/or 3′ regulatory sequences simplifies the process of constructing expression vectors suitable for plant transformation.
Suitable sources of EPSP synthase genes include dicotyledonous plants, such as Arabidopsis thaliana, and mono-cotyledonous plants, such as Zea mays, Arabidopsis thaliana has two EPSP synthase genes (epm1 and epm2). The present invention includes use of one or both of mutated epm1 and epm2 to confer resistance to glyphosate. Mutated EPSP synthase genes from Zea mays or other plants can also be used for transforming plant cells to make glyphosate-resistant plants.
SUMMARY OF THE INVENTION
In accordance with one aspect of the present invention, a DNA fragment which comprises an EPSPS 5′ regulatory sequence and a glyphosate-resistant EPSPS coding sequence (including a chloroplast transit peptide coding sequence) is introduced into regenerable plant cells. The DNA fragment does not contain a non-EPSPS enhancer. Cells are selected for stable transformation. The selected cells are then used to regenerate glyphosate-resistant, transgenic plants.
In one embodiment, the DNA fragment used for transformation comprises a modified plant genomic sequence. The unmodified plant genomic sequence comprises at least part of an EPSPS gene, and includes an EPSPS 5′ regulatory sequence and a glyphosate-sensitive EPSPS coding sequence (including a chloroplast transit peptide coding sequence). The glyphosate-sensitive EPSPS coding sequence is modified to make the encoded EPSPS glyphosate-resistant. The DNA fragment comprising the modified plant genomic sequence is stably transformed into plant cells, from which glyphosate-resistant plants are regenerated.
In a preferred embodiment, the DNA fragment used for transformation comprises SEQ ID NO: 2. In another preferred embodiment, the DNA fragment used for transformation comprises SEQ ID NO: 4. In yet another preferred embodiment, the DNA fragment comprises SEQ ID NO: 6. In a further preferred embodiment, any two sequences selected from SEQ ID. NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6 are used to transform plant cells. In one embodiment, the transgenic plant comprises transformed SEQ ID. NO: 2 and SEQ ID NO: 4.
Other features, objects, and advantages of the present invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating preferred embodiments of the invention, are given by way of illustration only, not limitation. Various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description.
DETAILED DESCRIPTION
This invention relates to methods of making glyphosate-resistant plants. In accordance with one aspect of the invention, a DNA fragment is introduced into regenerable, glyphosate-sensitive recipient plant cells. The DNA fragment comprises an EPSPS 5′ regulatory sequence, and a coding sequence encoding a glyphosate-resistant EPSPS. The EPSPS 5′ regulatory sequence is operably linked to the EPSPS coding sequence. The glyphosate-resistant EPSPS includes a chloroplast transit peptide. The DNA fragment does not contain a non-EPSPS enhancer. The recipient plant cells are selected for glyphosate-resistance and stable transformation. The cells thus selected can be used to regenerate glyphosate-resistant plants. As used herein, a “DNA fragment” may be either linear or circular. Preferably, the DNA fragment used for transformation is a linear DNA fragment. A “coding sequence” encoding an EPSPS refers to a nucleic acid sequence transcription and translation of which produce a functional EPSPS. The boundaries of the coding sequence are generally determined by a translation start codon at its 5′ end and a translation stop co don at its 3, end. A coding sequence of EPSPS may be a cDNA, or a plant genomic sequence which consists of all of the exons and introns of an EPSPS gene. An EPSPS gene refers to the plant genomic sequence which includes the EPSPS 5′ regulatory sequence, the EPSPS coding sequence (including the sequence encoding the chloroplast transit peptide), and the EPSPS 3′ regulatory sequence (such as an EPSPS transcriptional terminator). A “plant genomic sequence” refers to a nucleotide sequence found in the genome of the plant.
A chloroplast transit peptide functions post-translationally to direct a polypeptide to chloroplast. Either endogenous or heterologous chloroplast peptides can be used in the present invention. As used herein, “heterologous” means derived from a different source, and “endogenous” means derived from the same source. In a preferred embodiment, the endogenous transit peptide encoded by a native EPSPS gene is used.
As used herein, an EPSPS 5′ regulatory sequence refers to a nucleotide sequence located upstream (5′) to the start codon of the EPSPS coding sequence in an EPSPS gene in a plant or plant cell which has not been subject to genetic engineering. The 5′ regulatory sequence generally includes an EPSPS promoter which directs the transcription of the EPSPS gene. Preferably, the EPSPS 5′ regulatory sequence comprises one or more EPSPS enhancers operably linked to the promoter. In one embodiment, the 5′ regulatory sequence comprises at least 200 bp. Preferably, the 5′ regulatory sequence comprises at least 400, 600, 800, 1000, 1,200 or 1,800 bp.
An EPSPS 3′ regulatory sequence refers to a nucleotide sequence located downstream (3′) to the stop codon of the EPSPS coding sequence in an EPSPS gene in a plant or plant cell which has not been subject to genetic engineering. The 3, regulatory sequence generally includes a transcription terminator which controls the termination of the transcription of the EPSPS gene.
“Operably linked” refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a 5′ regulatory sequence is operably linked to a coding sequence if the 5′ regulatory sequence functions to initiate transcription of the coding sequence.
Preferably, the DNA fragment used for transformation does not include a non-EPSPS enhancer. As used in the present invention, a “non-EPSPS enhancer” refers to an enhancer which is not used by an EPSPS gene in a plant or plant cell which has not been subject to genetic engineering. Non-EPSPS enhancers include, but are not limited to, enhancers that are associated with CaMV 35S, FMV 35S, rice actin 1, rice GOS2, maize polyubiquitin, or barley plastocyanin genes.
As used herein, a “glyphosate-resistant” cell or plant refers to a cell or plant that can survive or continue to grow in the presence of certain concentrations of glyphosate that typically kill or inhibit the growth of other cells or plants. Growth includes, for instance, photosynthesis, increased rooting, increased height, increased mass, or development of new leaves. In one embodiment, a glyphosate-resistant cell can grow and divide on a culture medium containing 50 mg/l or more glyphosate. Preferably, a glyphosate-resistant cell can grow and divide on a culture medium containing 100 mg/l or more glyphosate, such as 200 mg/l, 300 mg/l or 400 mg/l glyphosate. More preferably, a glyphosate-resistant cell can grow and divide on a culture medium containing 500 mg/l or more glyphosate, such as 600 mg/l. For purposes of the present invention, the term “glyphosate” includes any herbicidally effective form of N-phosphonomethylglycine (including any salt thereof) and other forms which result in the production of the glyphosate anion in plants.
Regenerable glyphosate-resistant plant cells may be used to regenerate glyphosate-resistant plants. In one embodiment, the glyphosate-resistant plant thus regenerated can survive or continue to grow after being sprayed with glyphosate at a rate of 25 g/ha (grams per hectare) or more. Preferably, the glyphosate-resistant plant thus regenerated can survive or continue to grow after being sprayed with glyphosate at a rate of 50 g/ha or more, such as 100 g/ha, 200 g/ha, 400 g/ha, or 800 g/ha. More preferably, the glyphosate-resistant plant thus regenerated can survive or continue to grow after being sprayed with glyphosate at a rate of 1000 g/ha or more, such as 2000 g/ha and 3000 g/ha. The spray may preferably be carried out at or after the growth stage of v2, such as v3, v4, v5 or later stages. In another embodiment, the regenerated glyphosate-resistant plant can tolerate the spray of glyphosate at between 0.1 M and 0.4 M.
As used herein, a “glyphosate-resistant” EPSPS refers to an EPSPS the expression of which in a plant cell confers glyphosate resistance upon the plant cell. An EPSPS is “glyphosate-sensitive” if it does not confer glyphosate-resistance when being expressed in plant cells.
A variety of EPSPS mutations have been known to be glyphosate-resistant and capable of conferring glyphosate resistance upon transformed plants. For instance, EPSPS of Zea mays (GenBank Accession No. X63374) can be mutated at amino acid residues 102 (substitution of He for Thr) and 106 (substitution of Ser for Pro). EPSPS encoded by epm1 gene of Arabidopsis thaliana can be mutated at amino acid residues 179 (substitution of He for Thr) and 183 (substitution of Ser for Pro). EPSPS encoded by epm2 gene of Arabidopsis thaliana can be mutated at amino acid residues 177 (substitution of He for Thr) and 182 (substitution of Ser for Pro). These mutated EPSPSs are glyphosate-resistant and capable of conferring glyphosate resistance upon transformed plants. Other mutated or modified EPSPSs, such as those described in U.S. Pat. Nos. 5,310,667, 5,866,775, 6,225,114, and 6,248,876, or natural EPSPS variants showing glyphosate-resistance, can be used in the present invention. In addition, bacteria-derived, glyphosate-resistant EPSPSs, after fusion with a chloroplast transit peptide, can also be used.
The DNA fragment comprising the EPSPS 5′ regulatory sequence and the glyphosate-resistant EPSPS coding sequence can be stably transformed into a regenerable plant cell. As used herein, stable transformation refers to integration of the DNA fragment into the genome of the transformed plant cell.
In one embodiment, the EPSPS 5′ regulatory sequence in the DNA fragment used for transformation comprises an EPSPS enhancer and an EPSPS promoter. In another embodiment, the DNA fragment used for transformation further comprises an EPSPS 3′ regulatory sequence, such as an EPSPS transcriptional terminator, which is operably linked to the coding sequence encoding the glyphosate-resistant EPSPS.
In yet another embodiment, the DNA fragment used for transformation comprises a modified plant genomic sequence that encodes a glyphosate-resistant EPSPS. Without modification, the plant genomic sequence encodes a glyphosate-sensitive EPSPS. Modifications that are capable of converting a glyphosate-sensitive EPSPS to a glyphosate-resistant EPSPS are known in the art.
In a preferred embodiment, the DNA fragment used for transformation is modified from a plant genomic sequence. Before modification, the plant genomic sequence comprises an EPSPS regulatory sequence, a coding sequence encoding a glyphosate-sensitive EPSPS which includes a chloroplast transit peptide, and preferably an EPSPS 3, regulatory sequence, such as an EPSPS transcriptional terminator. The genomic sequence may be obtained by fragmenting the genome of a plant of interest, or isolated from bacterial artificial chromosome clones. Other methods for obtaining genomic sequences can also be used, such as PCR or DNA synthesis.
The EPSPS-coding sequence in this plant genomic sequence is then subject to nucleotide modification(s) to render the encoded EPSPS glyphosate resistant. Suitable modifications for this purpose, such as nucleotide substitutions, are well known in the art. The DNA fragment comprising the genomic sequence thus modified can be stably transformed into glyphosate-sensitive recipient plant cells. These transformed plant cells are selected for glyphosate resistance and then used to regenerate glyphosate-resistant plants.
The recipient plant cells are regenerable. They can be derived from immature embryos or meristematic tissues which contain cells that have not yet terminally differentiated. Juvenile leaf basal regions, immature tassels and gametic cells can be used to provide regenerable recipient cells for Zea mays. The preferred source of recipient cells for soybean includes the immature cotyledon.
In another preferred embodiment, two or more DNA fragments can be stably transformed into a recipient plant cell. Each of these DNA fragments includes an EPSPS 5′ regulatory sequence, a coding sequence encoding a glyphosate-resistant EPSPS which contains a chloroplast transit peptide, and preferably an EPSPS 3′ regulatory sequence (such as an EPSPS transcriptional terminator). These DNA fragments can be modified plant genomic sequences. They can be derived from the same or different plant species. They can be derived from the same EPSPS gene, or from different EPSPS genes of the same plant species, such as emp1 and emp2 of Arabidopsis thaliana.
Transformation of plant cells can be carried out using various methods. These methods include, but are not limited to, Agrobacterium tumefaciens mediated DNA transfer, PEG or liposome mediated DNA transfer, electroporation, micro-injection, microprojectile or particle bombardment, receptor-mediated DNA transfer, and viral or other vector mediated DNA transfer. Preferably, transformation is carried out using aerosol beam injection as described in U.S. patent application Ser. No. 09/450,226, which is incorporated herein by reference.
Selection for stably transformed plant cells can be performed using methods as appreciated by one of ordinary skill in the art. For instance, the transformed cells can be grown and selected on media containing glyphosate. Preferably, the introduced DNA fragment is stably transformed and integrated into a chromosome of the transformed plant cell. A variety of assays can be used to confirm stable transformation. Suitable assays include molecular biological assays, such as Southern and Northern Blotting and PCR, or biochemical assays, such as ELISA and Western Blot. In addition, plant part assays, such as leaf and root assays, or analysis of the phenotype of the whole regenerated plant, can be used to confirm stable transformation.
Plants can be regenerated from the selected, stably transformed cells. Progeny can be recovered from the regenerated plants and tested for glyphosate resistance. Seeds or other parts of the regenerated transgenic plants can also be obtained. In one embodiment, glyphosate-resistant plants are made by crossing.
Both monocotyledonous and dicotyledonous plants can be transformed using the methods of the present invention. The glyphosate-resistant EPSPS coding sequence can be derived from either monocotyledonous or dicotyledonous plants. The representative monocotyledonous and dicotyledonous plants used in the present invention include, but are not limited to, Oryza sativa, Zea mays, Hordeum vulgare, Triticum aestivum, Avena sativa, turf grasses including species of the genera Poa, Festuca, Lolium, Zoysia, and Cynodon among others, Glycine max, Gossypium hirsutii, Lycopersicum esculentum, Solanum tuberosum, Phaseolus species, Beta vulgaris, and Brassica species.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture and molecular genetics described herein are those well known and commonly employed in the art. Standard techniques can be used for recombinant nucleic acid methods, polynucleotide synthesis, plant cell culture, cell culture, tissue culture, and plant transformation and regeneration. Generally, enzymatic reactions and purification and/or isolation steps are performed according to the manufacturers' specifications. The techniques and procedures are generally performed according to conventional methodology disclosed, for example, in Molecular Cloning A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), and Current Protocols in Molecular Biology (John Wiley & Sons, Baltimore, Md., 1989).
It should be understood that the above-described embodiments and the following examples are given by way of illustration, not limitation. Various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from the present description.
EXAMPLE 1 Preparation and Mutation of Arabidopsis Genomic Fragments Containing EPSPS Genes
Two bacterial artificial chromosome (BAC) clones, F27K7 and F4L23, were obtained from the Arabidopsis Biological Resource Center, DNA Stock Center, at the Ohio State University. F27K7 and F4L23 contain the EPSPS genes found on chromosome 1 and 2 of Arabidopsis thaliana, respectively. The F27K7 clone was digested using Sac II and Bam HI restriction enzymes to produce a 4.7 kb fragment, the sequence of which is shown as SEQ ID NO: 1. The 4.7 kb fragment comprises the complete EPSPS gene (epm1) found on chromosome which includes an EPSPS 5′ regulatory sequence (the sequence before nucleotide residue 1290), an EPSPS coding sequence (from nucleotide residue 1290 to nucleotide residue 3729), and an EPSPS 3, regulatory sequence (the sequence after nucleotide residue 3729). The EPSPS coding sequence also encodes a chloroplast transit peptide (from nucleotide residue 1290 to nucleotide residue 1612). The sequence encoding this chloroplast transit peptide can be predicted using the computer program PSORT maintained on the public accessible GenomeNet at Kyoto University, Japan.
The 4.7 kb fragment was cloned into a pbluescript II vector (Stratagene), and two nucleotide substitutions were introduced into the EPSPS coding sequence using QuikChange® Site-Directed Mutagenesis Kit (Stratagene) according to the instructions of the manufacturer. The two nucleotide substitutions are a cytosine to thymine substitution at nucleotide 2007 and a cytosine to thymine substitution at nucleotide 2018. The mutated sequence is shown as SEQ ID NO: 2. The mutated sequence encodes a glyphosate-resistant EPSPS which has, as compared to the EPSPS encoded by SEQ ID NO: 1, a Thr to Ile mutation at amino acid 179 and a Pro to Ser mutation at amino acid 183. The amino acid sequence of the glyphosate-resistant EPSPS is shown as SEQ ID NO: 7. The pbluescript II vector containing SEQ ID NO: 2 is referred to as epm1 vector.
The BAC F4L23 clone was digested using Eco RI restriction enzyme to produce a 5.2 kb fragment, the sequence of which is shown as SEQ ID NO: 3. The 5.2 kb fragment comprises the complete EPSPS gene (epm2) from chromosome 2, which includes an EPSPS 5 ′regulatory sequence (the sequence before nucleotide 1515), and EPSPS coding sequence (from nucleotide 1515 to nucleotide 3872), and an EPSPS 3′ regulatory sequence (the sequence after nucleotide 3872). The EPSPS coding sequence also encodes a chloroplast transit peptide (from nucleotide 1515 to nucleotide 1665). The sequence encoding this chloroplast transit peptide can be predicted using the computer program PSORT maintained on the public accessible GenomeNet at Kyoto University, Japan.
The 5.2 kb fragment was cloned into a pbluescript II vector, and then subject to site-directed mutagenesis using QuikChange® Site-Directed Mutagenesis Kit (Stratagene). SEQ ID NO: 4 shows the mutated sequence which has two nucleotide substitutions in the EPSPS coding sequence as compared to SEQ ID NO: 3. The two substitutions are a cytosine to thymine substitution at nucleotide 2134 and a cytosine to thymine substitution at nucleotide 2145. The mutated sequence encodes a glyphosate-resistant EPSPS which has, as compared to the EPSPS encoded by SEQ ID NO: 3, a Thr to Ile mutation at amino acid 178 and a Pro to Ser mutation at amino acid 182. The amino acid sequence of the (putative) glyphosate-resistant EPSPS is shown as SEQ ID: 8. The pbluescript II vector containing SEQ ID NO: 4 is referred to as epm2 vector.
EXAMPLE 2 Transformation of Soybean
The Bam HI/Sac II fragment (SEQ ID NO: 2) of epm1 vector and the Eco RI fragment (SEQ ID NO: 4) of epm2 vector were used to transform soybean embryogenic callus using an aerosol beam injector as described in U.S. patent application Ser. No. 09/450,226, which is incorporated herein by reference. These fragments comprised mutant epm1 and mutant epm2 which encode (putative) glyphosate-resistant EPSPSs. These fragments were used either alone or, preferably, together.
The transformed tissue was is selected for glyphosate resistance using the method described below. First, the beamed embryogenic callus was is maintained for one month on B1-30 3Co5My0.01PA medium. Table 1 shows the composition of B1-30 3Co5My0.01PA medium.
TABLE 1
Ingredients in 1 liter B1-30 3Co5My0.10PA Medium (pH 5.8)
MS Salts* 4.43 g
NaEDTA 37.3 mg
2,4 dichlorophenoxyacetic acid 30 mg
Phytagar 8 g
Coconut water 30 ml
Myo-inositol 5 g
Phytic acid 10 mg
*Sigma Plant Culture catalogue, reference M5519
The tissue was is then transferred to the same medium but now containing 300 mg/l glyphosate. After a number of passages (up to 5 passages, each passage may last for about a month) on this latter medium, resistant clonal material may be identified. After an optional further few passages on B1-30 3Co5My0.01PA medium but containing 500 mg/l glyphosate, the growing tissue was is transferred to a regeneration media as described in U.S. patent Ser. No. 09/450,226. Regenerated plants were are transferred to pots in a greenhouse. These plants and their progenies were are sprayed with glyphosate at commercial rates, and complete resistance to glyphosate was is expected to be observed. Progenies segregated are expected to segregate 3:1 for glyphosate resistance as would be expected for Mendelian inheritance of a transgene.
Preferably, both mutant epm1 (such as SEQ ID NO: 2) and mutant epm2 (such as SEQ ID NO: 4) are stably transformed into a plant cell, from which glyphosate-resistant plants can be regenerated.
EXAMPLE 3 Preparation and Mutation of Corn Genomic Fragments Containing EPSPS Gene
A corn (B 73) BAC library was screened with a probe containing a sequence of a corn EPSPS gene published in Genbank accession number X63374 by Incyte Genomics Inc. Four BAC clones were identified. Southern blot analysis indicated that all four clones contained the same EPSPS gene. One BAC clone was further characterized by nucleotide sequencing which resulted in identification of a 6.0 kb genomic fragment flanked by unique Cla I and Eco RV sites. The sequence of the 6.0 kb fragment was shown as SEQ ID NO: 5. The 6.0 kb fragment includes an EPSPS 5′ regulatory sequence (the sequence before nucleotide 1868), an EPSPS coding sequence (from nucleotide 1868 to nucleotide 5146), and an EPSPS 3, regulatory sequence (the sequence after nucleotide 5146). The EPSPS coding sequence also encodes a chloroplast transit peptide (from nucleotide 1868 to nucleotide 2041). The sequence encoding this chloroplast transit peptide can be predicted using the computer program PS ORT maintained on the public accessible GenomeNet at Kyoto University, Japan.
The 6.0 kb fragment was cloned into the Cla I and Eco RV sites of a pbluescript vector, and then subject to site directed mutagenesis using QuikChange Site-Directed Mutagenesis Kit (Stratagene). Two mutations were introduced into the EPSPS coding sequence: the first mutation being a cytosine to thymine substitution at nucleotide 2886 and the second mutation being a cytosine to thymine substitution at nucleotide 2897. The mutated sequence is shown as SEQ ID NO: 6. The mutations changed the encoded amino acid residue Thr to Ile at position 164 and Pro to Ser at position 168. This mutated EPSPS amino acid sequence is shown as SEQ ID NO: 9. The mutated EPSPS is glyphosate-resistant. The pBluescript vector comprising SEQ ID NO: 6 is referred to as HCEM.
EXAMPLE 4 Transformation of Corn
The Cla I and Eco RV fragment (SEQ ID NO: 6) of HCEM was introduced into cultured immature corn embryos using an aerosol beam injector according to U.S. patent application Ser. No. 09/450,226. The Cla I-Eco RV fragment comprised the glyphosate-resistant EPSPS coding sequence.
Selection was carried out as follows: the beamed embryos were allowed to remain on DN62A0S20G medium for 5 days. Table 2 shows the composition of DN62A0S20G medium.
TABLE 2
Ingredients in 1 liter Culture Medium (pH 5.8)
DN62A0S20G DN62A0S20GLC
N6 Salts* 3.98 g 3.98 g
N6 Vitamins 1 ml 1 ml
Asparagine 800 mg 800 mg
Myoinositol 100 g 100 g
Proline 1400 mg 1400 mg
Casamino acids 100 mg 100 mg
2,4 dichlorophenoxyacetic acid 1 mg 1 mg
Glucose 20 g 20 g
Silver nitrate 10 mg 10 mg
Cefotaxime 0 mg 50 mg
*Sigma Plant Culture catalogue, reference C1416
The beamed embryos were then transferred to DN62A100RR a medium containing 100 mg/l glyphosate. Table 3 lists the composition of DN62A100RR and other media. After two 14-day passages on DN62A100RR, actively growing tissue was is transferred to DN62A300RR medium which contains 300 mg/l (Table 3). After two 14-day passages on this medium, tissue was finally transferred to DN62540RR medium which contains 540 mg/l glyphosate (Table 3). Stable transformation allowed continued growth on 540 mg/l glyphosate. Regeneration was is carried out as described in U.S. patent application Ser. No. 09/450,226.
TABLE 3
Ingredients in 1 liter Culture Medium (pH 5.8)
DN62A100R R DN62ALC180R R DN62A300R R DN62540RR
N6 Salts* 3.98 g 3.98 g 3.98 g 3.98 g
N6 Vitamins 1 ml 1 ml 1 ml 1 ml
Asparagine 800 mg 800 mg 800 mg 800 mg
Myoinositol 100 mg 100 mg 100 mg 100 mg
Proline 1400 mg 1400 mg 1400 mg 1400 mg
Casamino acids 100 mg 100 mg 100 mg 100 mg
2,4 1 mg 1 mg 1 mg 1 mg
dichlorophenoxyacetic
acid
Sucrose 20 g 20 g 20 g 20 g
Silver nitrate 10 mg 10 mg 10 mg
Glyphosate 100 mg 180 mg 300 mg 540 mg
Cefotaxime 0 mg 50 mg 0 mg 0 mg
*Sigma Plant Culture catalogue, reference C1416
Transformation can also be accomplished using Agrobacterium-mediated DNA delivery. In this case, the transformation and regeneration were are performed according to the methods as described in U.S. patent application Ser. No. 09/203,679, which is herein incorporated by reference. Briefly, after culturing on DN62A0S20GLC (Table 2) for five days, co-cultivated embryos were are transferred to DN62ALC180RR medium which contains 180 mg/l glyphosate (Table 3). After two 14-day passages on this medium, actively growing tissue was is transferred to DN62540RR medium containing 540 mg/l glyphosate (Table 3). Stable transformation allowed will allow continued growth on 540 mg/l glyphosate. Regeneration was is carried out as described in U.S. Ser. No. 09/203,679.
Resistance to glyphosate in regenerants was is confirmed by spraying them with glyphosate at commercial rates. Seed from the regenerants segregated is expected to segregate 3:1 for resistance as would be expected with Mendelian inheritance of a transgene. Seeds from backcrossed individuals segregated are expected to segregate 1:1. Corn transformation may also be accomplished by other means including, for example, particle bombardment or electroporation of competent cells.

Claims (13)

1. A method of making a glyphosate-resistant plant cell, comprising:
(a) introducing a first DNA fragment into a plurality of regenerable plant cells, the first DNA fragment comprising a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6; and
(b) selecting from said regenerable plant cells a glyphosate-resistant plant cell which is stably transformed with the first DNA fragment.
2. The method according to claim 1, further comprising introducing a second DNA fragment with the first DNA fragment into said regenerable plant cells, wherein the second DNA fragment comprises a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 6, and the sequence of the first DNA fragment is different from the sequence of the second DNA fragment.
3. The method according to claim 2, wherein the first DNA fragment comprises SEQ ID NO: 2, and the second DNA fragment comprises SEQ ID NO: 4.
4. A glyphosate-resistant plant cell made according to the method of claim 3, which is stably transformed with said first DNA fragment and said second DNA fragment.
5. A plant regenerated from the glyphosate-resistant plant cell of claim 4.
6. A regenerable, glyphosate-resistant plant cell comprising an introduced, chromosomally integrated DNA sequence which comprises a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.
7. The glyphosate-resistant plant cell according to claim 6, comprising another introduced, chromosomally integrated DNA sequence, wherein said another introduced, integrated DNA sequence comprises a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.
8. The glyphosate-resistant plant cell according to claim 6, wherein the introduced, chromosomally integrated DNA sequence comprises SEQ ID NO: 2, and wherein the plant cell comprises another introduced, chromosomally integrated DNA sequence which comprises SEQ ID NO: 4.
9. A plant regenerated from the glyphosate-resistant plant cell of claim 6.
10. A plant regenerated from the glyphosate-resistant plant cell of claim 8.
11. An isolated polynucleotide comprising the sequence depicted in SEQ ID NO: 2.
12. An isolated polynucleotide comprising the sequence depicted in SEQ ID NO: 4.
13. An isolated polynucleotide comprising the sequence depicted in SEQ ID NO: 6.
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