REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named “BCS12-2004-WO1_ST25.TXT”, created on Apr. 19, 2013, and having a size of 54 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the field of agronomy. More particularly, the invention provides methods and means to introduce a targeted modification, including insertion, deletion or substitution, at a precisely localized nucleotide sequence in the genome of a plant cell or plant via bacterium-mediated transformation, such asAgrobacterium. The modifications are triggered in a first step by induction of a double stranded break at a recognition nucleotide sequence using a double stranded DNA break inducing enzyme encoded by a T-DNA which has been introduced into the plant cell, while a co-introduced T-DNA comprising a repair DNA molecule is subsequently used as a template for repair of the double stranded break. The frequency of correctly targeted genome modification is increased by co-introduction of the two T-DNA molecules by a single bacterium cell.

BACKGROUND

Agrobacteriummediated DNA transfer is the standard method of transformation for most plants, including crop plants. The advantages of theAgrobacteriummethod over other methods include the high efficiency of transformation, the transfer of pieces of DNA with defined ends, the transfer of relatively large segments of DNA, and the absence of a requirement for protoplast culture techniques (Komari et al., 1996 Plant J. 10: 165-174).

When more than one construct is to be transformed, direct DNA delivery methods such as particle bombardment or electroporation may be more efficient because of a higher frequency of cotransformation compared to the simultaneous transformation with more than oneAgrobacteriumstrains. However, a disadvantage of these direct delivery methods is that they can lead to more complex transgene integration patterns, making the identification of single copy transformants lengthy and laborious process.

In order to be able to introduce a foreign DNA at a predetermined site, so-called gene targeting, it is required to transform plants cells or tissues with two constructs, one comprising a gene encoding an enzyme that induces a double stranded DNA break (DSB) at a specific target site and one comprising a DNA of interest for repair of the break. This process may also be more efficient when plant cells are transformed with the repair DNA and the DNA encoding the double stranded DNA break inducing (DSBI) enzyme simultaneously using direct DNA delivery methods, than when using twoAgrobacteriumstrains.

The frequency of DSB induction and subsequent repair using DNA of interest viaAgrobacterium-mediated transformation can be improved by co-delivery of the repair DNA and DSBI enzyme encoding gene on the same T-DNA using a singleAgrobacteriumstrain. However, this can lead to co-integration of the DSBI enzyme encoding gene at the site of double strand break induction, which is undesirable. This co-integration can be avoided by constructing the T-DNA vector in a way that that the DNA to be introduced into the genome is flanked by regions having homology to the genomic target site, thereby directing insertion via homologous recombination, but whereby the DSBI gene is located outside of these homology regions, but cloning procedures become more complicated because additional elements need to be included in one construct.

Wright et al. (2005, plant J, 44:693-705) discloses Zinc finger nuclease (ZNF) induced chromosomal break repair at an engineered target locus via homologous recombination in tobacco protoplast by simultaneous electroporation of two linearized plasmids containing the ZNF expression construct and donor DNA respectively.

Shukla et al. (2009, Nature 459: 437-441), U.S. Ser. No. 08/018,2332 and U.S. Ser. No. 10/019,9389 describe targeted insertion into an endogenous locus in maize by codelivery of designed ZNF expression constructs with donor plasmids containing homology arms into maize embryogenic cell cultures via Whiskers and particle bombardment.

Lloyd et al. (2005, Proc Natl Acad Sci, 102: 2232-2237) and Zhang et al. (2009, Proc Natl Acad Sci, 107: 12028-1203) and U.S. Ser. No. 10/007,1083 describe an inducible DSBI enzyme encoding gene that has been stably transformed into the plants genome for use in targeted mutagenesis.

Cai et al. (2009, Plant Mol Biol, 69: 699-709) and US2011008833 describe homology mediated targeted insertion into engineered and endogenous tobacco loci usingAgrobacterium, via co-culture of twoAgrobacteriumstrains of which one harbors a donor DNA and the other a designed ZNF expression construct, but also by co-culturing with a singleAgrobacteriumstrain containing a Ti plasmid harboring both the ZNF and donor construct within the same T-DNA.

Komari et al. (1996, Plant J 10: 165-174) and U.S. Pat. No. 5,731,179 discloses super-binary vectors for producing transformants free from selection markers.

Thus, in view of the advantages of theAgrobacteriumsystem for plant transformation, the need remains for more efficient methods for the co-delivery of the repair DNA molecule and the DSBI enzyme encoding chimeric gene usingAgrobacterium. This problem is solved hereinafter, in the detailed description, examples and claims.

