Pioneer Hi-bred International, Inc.

Other

Iowa State University Research Foundation, Inc.

Boyce Thompson Institute for Plant Research

Pioneer Hi-bred Int'l Inc.

Christopher L Baszczynski

Laura Ann Tagliani

Benjamin A Bowen

David J Peterson

William James Gordon-Kamm

Xueni Guan

Leszek Alexander Lyznik

Tong Zhu

Pramod B Mahajan

Zuo-Yu Zhao

Aragula Gururaj Rao

Gary A Sandahl

Bruce J Drummond

Grace Marie St Clair

John H Duesing

Terry EuClaire Meyer

Jinrui Shi

John A McElver

Peter B Kipp

Julian Adin Mann, Iii

Roberto Barreiro

Gregory D May

Lesek Alexander Lyznik

Argula Gururaj Rao

Ashli J Armstrong

Compositions for the targeted integration of nucleotide sequences into a plant are provided. Plants and plant cells having multiple transfer cassettes stacked at precise locations in the genome of a plant or plant cell are provided. In specific embodiments, the plant or plant cell comprises at least two transfer cassettes. The first transfer cassette comprises a first nucleotide sequence of interest flanked by a first and a second recombination site, wherein the first and the second recombination sites are non-identical. The second transfer cassette comprises a second nucleotide sequence of interest flanked by the second recombination site and a third recombination site, wherein the third recombination site is non-identical to the first and said second recombination site. The second recombination site is shared between the first and the second transfer cassette, such that the genome of the plant cell comprises in the following order the first recombination site, the first nucleotide sequence of interest, the second recombination site, the second nucleotide sequence of interest, and the third recombination site.

Compositions for the targeted insertion of a nucleotide sequence of interest into the genome of a plant

Methods for the targeted integration of nucleotide sequences into a plant are provided. Transfer cassettes comprising nucleotide sequences of interest flanked by non-identical recombination sites are used to transform a plant comprising a target site. The target site contains at least a set of non-identical recombination sites corresponding to those on the transfer cassette. Exchange of the nucleotide sequences flanked by the recombination sites is effected by a recombinase.

Compositions and methods for the targeted insertion of a nucleotide sequence of interest into the genome of a plant

Compositions and methods for genetic modification of plants

The present device enables measurement of the susceptibility of corn plants to root lodging. The device is used to push over a corn stalk and the force used to push over the stalk, and the vibration of the stalk caused by the push are recorded. As material breaks in the stalk, an accelerometer, measures stalk vibration response to the breaking events; the data is then recorded to allow quantitative measurements of the susceptibility of corn plants to root lodging. This allows meaningful comparisons of various hybrids at early stages of hybrid evaluation and advancement.

Correlating push force and stalk vibration to a plant'S susceptibility to root lodging

Methods and compositions for the stacking of multiple nucleotide sequences at precise locations in the genome of a plant or plant cell are provided. Specifically, transfer cassettes comprising nucleotide sequences of interest flanked by non-identical recombination sites are introduced into a plant comprising a target site. The target site contains at least a set of non-identical recombination sites corresponding to those on the transfer cassette. Exchange of the nucleotide sequences flanked by the recombination sites is effected by a recombinase. The transfer cassettes and target sites are designed so as to allow for the stacking or ordering of nucleotide sequences at precise locations in the plant genome.

Compositions and methods to stack multiple nucleotide sequences of interest in the genome of a plant

The invention relates to methods and compositions for site-specific recombinase-mediated mobilization of viral replicons and associated DNAs of interest from T-DNA. The methods of the invention comprise-mediated transfer of T-DNA to a plant cell, wherein the T-DNA contains a viral replicon flanked by directly repeated target sites for a site-specific recombinase and optionally a DNA of interest linked to the viral replicon. The DNA of interest may also contain a non-identical target site for the recombinase. An expression cassette for the site-specific recombinase is present on the T-DNA or the plant genome, or is transiently introduced into the plant cell. Expression of the site-specific recombinase in the plant cell results in excision of the viral replicon and the associated DNA of interest. The viral replicon and DNA of interest are then replicated to high copy number in the host plant cell.

Mobilization of viral genomes from T-DNA using site-specific recombination systems

Methods to find optimal integration sites within a plant genome are provided. More particularly, a plant is transformed with a target site having an expression cassette comprising a nucleotide sequence operably linked to a promoter active in the plant. The target site is flanked by non-identical recombination sites. Transformed protoplast, tissues, or whole plants can be tested to determine the levels of activity of the inserted gene. By comparison of cellular activities of the gene in different insertion sites, preferred integration sites may be found wherein the gene is expressed at high or acceptable levels. These plants can then be utilized with subsequent retargeting techniques to replace the nucleotide sequence with other genes or nucleotide sequences of interest contained in a transfer cassette.

Compositions and methods for locating preferred integration sites within the genome of a plant

Compositions comprising non-identical recombination sites are provided. Compositions include plants, plant parts and plant cells comprising transfer cassettes comprising nucleotide sequences of interest flanked by non-identical recombination sites.

Compositions comprising non-identical recombination sites

Compositions and methods to reduce the complexity of transgene integration into the genome of a plant

The invention relates to methods and compositions for site-specific recombinase-mediated mobilization of viral replicons and associated DNAs of interest from T-DNA. The methods of the invention comprise-mediated transfer of T-DNA to a plant cell, wherein the T-DNA contains a viral replicon flanked by directly repeated target sites for a site-specific recombinase and optionally a DNA of interest linked to the viral replicon. The DNA of interest may also contain a non-identical target site for the recombinase. An expression cassette for the site-specific recombinase is present on the T-DNA or the plant genome, or is transiently introduced into the plant cell. Expression of the site-specific recombinase in the plant cell results in excision of the viral replicon and the associated DNA of interest. The viral replicon and DNA of interest are then replicated to high copy number in the host plant cell. The compositions of the invention comprise nucleic acids, such as T-DNAs containing a viral DNA flanked by directly repeated target sites for a site-specific recombinase. The nucleic acids of the invention may additionally contain expression cassettes encoding the cognate site-specific recombinase for the target sites flanking the viral genome. The compositions of the invention further comprisecontaining the nucleic acids of the invention.

Compositions and methods for introducing a DNA of interest into a genomic target site are provided. In particular, the methods and compositions involve the use of a combination of target sites for two site specific recombinases and expression of a chimeric recombinase with dual target site specificity. Thus, the compositions comprise novel site-specific recombinases with specificities to multiple target sites, and nucleotide sequences and expression cassettes encoding these recombinases or target sites. The methods involve transforming a eukaryotic cell having target sites for the novel recombinase with a DNA of interest that is flanked by corresponding target sites. Expression of the recombinase results in integration of the DNA of interest into the genome of the cell. The compositions and methods of the invention have use in the construction of stably transformed eukaryotic cells, and in particular, plant cells. The methods result in the efficient targeted genomic integration of DNA by site-specific recombination.

Method for the integration of foreign DNA into eukaryotic genomes

The invention relates to methods and compositions for site-specific recombinase-mediated mobilization of viral replicons and associated DNAs of interest from T-DNA. The methods of the invention comprise-mediated transfer of T-DNA to a plant cell, wherein the T-DNA contains a viral replicon flanked by directly repeated target sites for a site-specific recombinase and optionally a DNA of interest linked to the viral replicon. The DNA of interest may also contain a non-identical target site for the recombinase. An expression cassette for the site-specific recombinase is present on the T-DNA or the plant genome, or is transiently introduced into the plant cell. Expression of the site-specific recombinase in the plant cell results in excision of the viral replicon and the associated DNA of interest. The viral replicon and DNA of interest are then replicated to high copy number in the host plant cell. The compositions of the invention comprise nucleic acids, such as T-DNAs containing a viral DNA flanked by directly repeated target sites for a site-specific recombinase. The nucleic acids of the invention may additionally contain expression cassettes encoding the cognate site-specific recombinase for the target sites flanking the viral genome. The compositions of the invention further comprisecontaining the nucleic acids of the invention. The compositions and methods of the invention have use in increasing the efficiency of agroinfection, providing high copy numbers of a DNA of interest for transient expression or for integration into a plant chromosome, and in simplifying the construction and stable maintenance of vectors for agroinfection and transformation.

Growing community of inventors

Urbandale, IA, United States of America

Christopher L Baszczynski