Method of plant transformation

Details Method of plant transformation Method of plant transformation

1999-09-28

2001-07-24

Guzo, David (Department: 1636)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

Via agrobacterium

C435S320100, C435S252300, C435S252200, C435S410000, C435S468000, C435S469000, C435S430000, C800S278000

Reexamination Certificate

active

06265638

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to methods and compositions for the transformation of plants, more particularly to methods for transformation utilizing Agrobacterium.
BACKGROUND OF THE INVENTION
Agrobacterium, a natural plant pathogen, has been widely used for the transformation of dicotyledonous plants and more recently for transformation of monocotyledonous plants. The advantage of the Agrobacterium-mediated gene transfer system is that it offers the potential to regenerate transgenic cells at relatively high frequencies without a significant reduction in plant regeneration rates. Moreover, the process of DNA transfer to the plant genome is well characterized relative to other DNA delivery methods. DNA transferred via Agrobacterium is less likely to undergo any major rearrangements than is DNA transferred via direct delivery, and it integrates into the plant genome often in single or low copy numbers.
Currently the most commonly used Agrobacterium-mediated gene transfer system is a binary transformation vector system where the Agrobacterium has been engineered to include a disarmed, or nononcogenic, Ti helper plasmid, which encodes the vir functions necessary for DNA transfer, and a much smaller separate plasmid called the binary vector plasmid, which carries the transferred DNA, or the T-DNA region. The T-DNA is defined by sequences at each end, called T-DNA borders, which play an important role in the production of T-DNA and in the transfer process.
Agrobacterium-mediated introduction of multiple genes, or so-called co-transformation, occurs naturally. In nature, the Ti plasmid of both the octopine producing strain, A348, and the
A. rhizogenes
strains have multiple T-DNA regions that transfer independently and integrate into the plant genome.
Co-transformation has been carried out with genetically engineered Agrobacterium strains. Introduction of multiple genes into plants using Agrobacterium-mediated transformation systems may result in linked or unlinked integration of T-DNA regions depending upon the method of co-transformation used. In some instances, a transformed plant may have both linked and unlinked integration of the T-DNA regions when multiple T-DNA regions are transferred. As a result, subsequent generations of transformed plants may retain only subsets of the originally transferred and integrated T-DNA regions. Such segregation of T-DNA regions carrying different genes may be desirable when the T-DNA region or regions that are lost to segregating progeny carry selectable marker genes that are desirable for selecting initial transformed plants, but are undesirable in the progeny of those transformed plants.
Co-transformation in engineered Agrobacterium binary vector systems has thus far been accomplished in three different ways. The first of these methods involves coinfection of a plant with two Agrobacterium strains, each of which has a unique T-DNA carried on identical binary vector plasmids (McNight et al. (1987)
Plant Mol. Biol.
8:439-445) or different binary vector plasmids (DeNeve et al. (1997)
Plant J.
11(1):15-29. Alternatively, co-transformation may be achieved by infection of a plant with a single Agrobacterium strain that has one T-DNA residing on the Ti helper plasmid and a second T-DNA residing on a binary plasmid (de Framond et al. (1986)
Mol. Gen. Genet.
202:125-131). A third method of co-transformation involves infection of a plant with a single Agrobacterium strain having two separate T-DNA regions on a single binary plasmid (Komari et al. (1996)
Plant J.
10(1):165-174). The first two methods favor unlinked integration of T-DNA regions, while the latter method favors linked integration of the T-DNA regions, though both types of integration may occur within one plant.
Given the advantages of Agrobacterium-mediated transformation systems and the need for simultaneous transformation of plants with more than one gene of interest, additional methods for efficient co-transformation using Agrobacterium are needed.
SUMMARY OF THE INVENTION
The present invention is drawn to novel compositions and methods for the efficient co-transformation of a plant. Novel compositions are Agrobacterium strains that have been engineered to comprise at least two binary vector plasmids in addition to a helper plasmid comprising the vir functions. Each of the binary vectors comprises its own T-DNA borders flanking a heterologous nucleotide sequence of interest. The heterologous nucleotide sequence within at least one of the T-DNA border sequences comprises at least one expression cassette expressing a coding sequence for a plant scorable marker gene. The nucleotide sequence of interest may additionally comprise at least one coding sequence of interest operably linked to regulatory regions that are functional within a host plant. Methods of the invention comprise the use of these novel compositions to co-transform a plant. In this manner, heterologous nucleotide sequences of interest residing on different binary vectors can be independently introduced into the plant in a single transformation event and incorporated in the plant's genome in an unlinked manner. The transferred nucleotide sequences will be flanked by their respective T-DNA borders and present in the transformed plant in low copy number.
Transformed plant cells, tissues, plants, and seed are also provided. Such transformed compositions are characterized by the presence of independently integrated T-DNA borders and a low copy number of the transferred nucleotide sequences. The invention encompasses regenerated, fertile transgenic plants, transgenic seeds produced therefrom, and T
1
and subsequent generations.


REFERENCES:
patent: 5538878 (1996-07-01), Thomas et al.
Bevan, Michael, Binary Agrobacterium Vectors for Plant Transformation, Nucleic Acids Research, 1984, pp. 8711-8721, vol. 12, No. 22, IRL Press Limited, Oxford, England.
Watson et al., New Cloning Vehicles for Transformation of Higher Plants, EMBO Journal, 1985, pp. 277-284, vol. 4, No. 2, IRL Press Limited, Oxford, England.
Depicker et al., Frequencies of Simulataneous Transformation With Different T-DNAs and Their Relevance to the Agrobacterium/Plant Cell Interaction, Biol. Gen. Genet, pp. 477-484, vol. 201, MGG, Springer-Verlag.
Simpson et al., A Disarmed Binary Vector fromAgrobacterium TumefaciensFunctions inAgrobacterium TumefaciensFunctions in Agrobacterium Rhizogenes, Plant Molecular Biology, 1986, pp. 403-415, vol. 6, Martinus Nijhoff Publishers, Dordrecht—Printed in the Netherlands.
de Framond et al., Two Unlinked T-DNAs Can Transform The Same Tobacco Plant Cell and Segregate in the F1 Generation, Biol. Gen. Ganet, 1986, pp. 125-131, vol. 202, MGG Springer-Verlag.
McKnight et al., Segregation of Genes Transferred to One Plant Cell From Two Separate Agrobacterium Strains, Patent Molecular Biology, 1987, pp. 439-445, vol. 8, Martinus Nijhoff Publishers, Dordrecht—Printed in the Netherlands.
Hooykmass, Paul, J.J., Agrobacterium Molecular Genetics, Pllant Molecular Biology Manual, 1988, pp.1-13, vol. A4, Kluwer Academic Publishers, Dordrechtp—Printed in Belgium.
An et al., Binary Vectors, Plant Molecular Biology Manual, 1988, pp. 1-19, vol. A3, Kluwer Academic Publishers, Dordrecht—Printed in Belgium.
De Block et al., Two T-DNA's Co-Transformed intoBrassica Napusby a DoubleAgrobacterium TumefaciensInfection are Mainly Integrated at the Same Locus, Theor. Appl. Genet, 1991, pp. 257-263, vol. 82, Plant Genetic Systems, N.V., Jozef Plateaustraat 22, 900 Gent, Belgium.
Walkerpeach et al., Agrobacterium-Mediated Gene Transfer to Plant Cells: Cointegrate and Binary Vector Systems, Plant Molecular Biology Manual, 1994, pp. 1-19, vol. B1, 1994 Kluwer Academic Publishers, Belgium.
Frishch et al., Complete Sequence of the Biary Vector Bin 19, Plant Molecular Biology, 1995, pp. 405-409, vol. 27, 1995 Kluwer Academic Publishers, Belgium.
Komari et al., Vectors Carrying Two Separate T-DNAs for Co-Transformation of Higher Plants Mediated byAgrobacterium Tumefaciensand Segregation of Transformants Free from Selection

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