Monsanto Company

Monsanto Technology LLC

Other

Massachusetts Institute of Technology

Washington University

Robert T Fraley

Stephen G Rogers

Robert B Horsch

Dilip Maganlal Shah

Roger N Beachy

Lee Gehrke

James Scott Elmer

Leslie Brand

David Bisaro

Stephen T Rogers

This invention relates to several plasmids which are useful for genetically transforming plant cells. A first plasmid, such as pMON120, contains a T-DNA border, one or more marker genes, a unique cleavage site, and a region of Ti plasmid homology. A gene which is expressed in plant cells may be inserted into this plasmid to obtain a derivative plasmid, such as pMON128 which expresses neomycin phosphotransferase in plant cells. The derivative plasmid is inserted into a suitable microorganism, such aswhich contains a Ti plasmid. The inserted plasmids recombine with Ti plasmids to form co-integrate plasmids. Only a single crossover event is required to create the desired co-integrate plasmid.cells with co-integrate plasmids are selected and co-cultured with plant cells. The co-integrate Ti plasmids enter the plant cells and insert a segment of T-DNA which does not contain tumorigenic genes into the plant genome. The transformed plant cell(s) may be regenerated into a morphologically normal plant which will pass the inserted gene(s) to its descendants.

Plasmids for transforming plant cells

This invention relates to genetically transformed, non-tumorous plant cells. A modified Ti plasmid is created which contains a left T-DNA border, one or more desired genes, and a right T-DNA border. This region does not contain tumorigenic or phytohormone-altering genes. The Ti plasmid is inserted into plant cells, where the T-DNA region is transferred into the plant genome. The transformed plant cells may be regenerated into morphologically normal plants which will pass the desired gene(s) to their descendants.

Genetically transformed plants

The invention involves recombinant, double-stranded DNA that contains a promoter which functions in plant cells to cause the production of RNA sequences of a plant virus, a DNA sequence that causes the production of an RNA sequence encoding the coat protein of said plant virus, and a 3&prime; non-translated region which functions in plant cells to cause the addition of polyadenylated nucleotides to the 3&prime; end of said RNA sequence; which double-stranded DNA can be used in a method for genetically transforming plants to produce genetically transformed plant cells and plants that are resistant to virus infection.

Protection of plants against viral infection

In one aspect the present invention relates to the use of viral promoters in the expression of chimeric genes in plant cells. In another aspect this invention relates to chimeric genes which are capable of being expressed in plant cells, which utilize promoter regions derived from viruses which are capable of infecting plant cells. One such virus comprises the cauliflower mosaic virux (CaMV). Two different promoter regions have been derived from the CaMV genome and ligated to heterologous coding sequences to form chimeric genes. These chimeric genes have been shown to be expressed in plant cells. This invention also relates to plant cells, plant tissue, and differentiated plants which contain and express the chimeric genes of this invention.

Chimeric genes for transforming plant cells using viral promoters

This invention relates to chimeric genes which are capable of being expressed in plant cells. Such genes contain (a) a promoter region derived a gene which is expressed in plant cells, such as the nopaline synthase gene; (b) a coding or structural sequence which is heterologous with respect to the promoter region; and (c) an appropriate 3&prime; non-translated region. Such genes have been used to create antibiotic-resistant plant cells; they are also useful for creating herbicide-resistant plants, and plants which contain mammalian polypeptides.

Chimeric genes suitable for expression in plant cells

The invention relates to novel plant plasmid vectors comprising geminivirus DNA or a portion thereof having inserted therein a heterologous DNA sequence or gene, to processes and DNA intermediates useful in producing said vectors and to methods utilizing such vectors to replicate and express heterologous DNA sequences or genes in plants. In some embodiments, methods and compositions are provided for Ti plasmid delivery of these novel vectors into plants. In other embodiments, methods and compositions are provided which allow for the generation of geminivirus DNA containing plant plasmids in stably transformed plants. In still other embodiments, methods and compositions are provided for replicating and expressing heterologous DNA sequences or genes in plants employing the geminivirus DNA containing vectors of the present invention without causing disease symptoms.

Plant vectors

In one aspect the present invention relates to the use of viral promoters in the expression of chimeric genes in plant cells. In another aspect this invention relates to chimeric genes which are capable of being expressed in plant cells, which utilize promoter regions derived from viruses which are capable of infecting plant cells. One such virus comprises the cauliflower mosaic virus (CaMV). Two different promoter regions have been derived from the CaMV genome and ligated to heterologous coding sequences to form chimeric genes. These chimeric genes have been shown to be expressed in plant cells. This invention also relates to plant cells, plant tissue, and differentiated plants which contain and express the chimeric genes of this invention.

This invention involves a cloning or expression vector comprising a gene which encodes 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) polypeptide which, when expressed in a plant cell contains a chloroplast transit peptide which allows the polypeptide, or an enzymatically active portion thereof, to be transported from the cytoplasm of the plant cell into a chloroplast in the plant cell, and confers a substantial degree of glyphosate resistance upon the plant cell and plants regenerated therefrom. The EPSPS coding sequence may be ligated to a strong promoter, such as the 35S promoter from cauliflower mosaic virus, to create a chimeric gene. Such genes can be inserted into plant transformation vectors, and subsequently introduced into plant cells. Plant cells transformed using such genes and plants regenerated therefrom have been shown to exhibit a substantial degree of glyphosate resistance.

Glyphosate-resistant plants

This invention relates to chimeric genes which are capable of being expressed in plant cells. Such genes contain (a) a promoter region derived in a gene which is expressed in plant cells, such as the nopaline synthase gene; (b) a coding or structural sequence which is heterologous with respect to the promoter region; and (c) an appropriate 3' non-translated region. Such genes have been used to create antibiotic-resistant plant cells; they are also useful for creating herbicide-resistant plants, and plants which contain mammalian polypeptides.

A recombinant plasmid which may be used for propagation of cloned cDNAs and also for the in vitro synthesis of RNA which is an exact copy of the natural sequence, wherein the transcript is devoid of vector-derived sequence. The novel vector was generated de novo by genetic engineering procedures using a synthetic double strand oligodeoxyribonucleotide fragment and a larger DNA fragment derived from plasmid pSP64. The vector, plasmid pHST-O, carried by Escherichia coli HB101, deposited with the ATCC on Dec. 30, 1985 and designated ATCC 53381, is distinguished from other vectors containing the SP6 promoter by the following characteristics: it contains a unique site for the restriction endonuclease BglII; the engineered GBlII site overlaps the downstream border of the SP6 promoter sequence; and the presence and positioning of the BglII restriction site permit insertion of cDNA molecules in such a way that transcription by the SP6 RNA polymerase begins exactly at the 5' terminus of the RNA, providing that the 5' terminal nucleotide of the mRNA transcript is a guanosine (G) residue, thus excluding the transcription of nucleotides derived from the vector. The novel vector permits synthesis of RNA molecules which have a defined 5' terminus and which are devoid of vector-derived sequence. The vector has potential use in research in molecular biology and in the in vitro production of RNA and proteins.

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Glendale, MO, United States of America

Robert T Fraley