Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – Via agrobacterium
Reexamination Certificate
2000-05-19
2002-11-19
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
Via agrobacterium
C800S278000, C800S260000, C800S314000, C435S469000, C435S419000, C435S427000, C435S430000, C435S430100
Reexamination Certificate
active
06483013
ABSTRACT:
BACKGROUND OF THE INVENTION
(i) Field of the invention
This invention relates to the field of plant transformation, particularly to an improved method for Agrobacterium mediated transformation of cotton.
(ii) Description of the Related Art
Cotton is an important crop, grown primarily for its lint, which provides much of the high quality fiber for the textile industry. Cotton seed is also a valuable commodity, as it provides a source for oil, meal and seed hulls.
About 90 % of cotton grown worldwide is
Gossypium hirsutum
L., whereas
Gossypium barbadense
accounts for about 8%.
Improvement of various traits such as resistance to pests, stress or disease, fiber quality, reduced gossypol content, has been obtained by conventional breeding techniques, and further improvement of these and other traits can be achieved using molecular and genetic techniques.
Several methods are available for the transformation of cotton including Agrobacterium mediated transformation of cotton explants, such as isolated cotyledons, isolated hypocotyls and isolated hypocotyl transition zones, as well as direct gene transfer by microprojectile bombardment of meristimatic cotton tissues.
Firoozabady et al. (1987, Plant Molecular Biology 10:105-116) described transformation of cotton cotyledon tissues by
Agrobacterium tumefaciens
and regeneration of transgenic plants.
Umbeck et al (1987, Bio/Technology 5: 263-266) reported results on cotton transformation via Agrobacterium using hypocotyls as explants.
PCT publication (“WO”) 89/05344, (“U.S.”) U.S. Pat. Nos. 5,244,802 and 5,583,036 provide methods for regeneration of cotton by tissue and suspension culture starting with explants which are the hypocotyl, cotyledon or immature embryos. Also taught are method to transform cotton and improve cotton by selective growth.
WO 89/12102 and U.S. Pat. No. 5,164,310 relate to a novel method for transforming and rapidly regenerating plant tissue. The method employs target tissues which are the shoot apices, thereby expanding the species range for transformation and reducing the risk of somaclonal variation.
WO 98/15622 provides a method for the in vitro regeneration of fertile Gossypium plants in which cells from the transition region tissue of seedlings is excised and cultured. A method for the production of transgenic Gossypium plants capable of transmitting a foreign gene to progeny is also described in which cells derived from the transition region tissue of seedlings are targeted for transformation.
WO 97/12512 provides a method for regenerating cotton plants from explant tissue which allows the generation of embryogenic callus from a cotton tissue explant, such as a hypocotyl, which is not cultivated on callus initiation media having exogenous plant hormones. The method can be utilized in the Agrobacterium-mediated transformation of cotton plants.
U.S. Pat. Nos. 5,004,863 and 5,159,135 and European patent publication “EP”0 270 355 all disclose a method to achieve genetic transformation of cotton plants and lines, by subjecting immature cotton tissues such as hypocotyl segments in vitro to Agrobacterium mediated genetic transformation.
WO 97/43430 relates to a versatile method of rapidly regenerating cotton plants from explants of apical and/or nodal meristematic tissues which can be coupled with well known methods of transformation such as Agrobacterium-mediated transformation for the rapid production of genetically-engineered cotton varieties of agronomic importance.
WO 92/15675 and EP 0 531 506 describe a method for the particle-mediated transformation of cotton permitting the direct genetic engineering of elite cotton lines without the need for tissue culture or callus proliferation, involving excision of the embryonic axes from germinating seeds and blasting particles carrying foreign genes into the embryonic axes.
Finer and Mc Mullen (1990, Plant Cell Reports, 8: 586-589) described transformation of cotton via particle bombardment of embryogenic suspension cultures.
McCabe and Martinell, (1993, Bio/Technology 11: 596-598) have described transformation of elite cotton cultivars via particle bombardment of cotton meristematic tissue from excised embryonic axes.
Hansen et al (1994, Proc. Natl. Acad. Sci. 91: 7603-7607) have described an Agrobacterium strain comprising a constitutive virg mutant (virGN54D) leading to enhanced transformation efficiencies of isolated cotton cotyledons.
Veluthambi et al. (1989, Journal of Bacteriology 171: 3696-3703) described a markedly increased transformation of cotton shoot tips by the simultaneous addition of acetosyringone and nopaline at the time of infection.
The cotton Agrobacterium-mediated transformation methods characterizing the art to date suffer from the major drawback, as indicated in Murray et al. (1993, in Transgenic Plants Vol 2, Academic Press Inc. p153-168) that the time required to regenerate a transgenic plant from an explant which has been co-cultivated with Agrobacterium cells is extremely long.
Moreover, an additional problem arises from the fact that all described Agrobacterium mediated transformation methods of cotton require the generation of embryogenic callus from the explant containing the transformed cells, as an intermediate step in the regeneration of transgenic cotton plants. Since not all transformed cells of the explant necessarily are competent for the initiation of embryogenic callus, a loss of regeneration of a number of potentially transgenic cells, which consequently results in a low transformation efficiency, is observed with these methods. An increase in starting numbers of putative transformed cotton explants to compensate for this loss, results in an huge increase of the amount of labor to be invested in obtaining regenerated transgenic cotton plants.
WO 89/05344 has suggested in general terms that embryogenic callus may be used as appropriate starting material for Agrobacterium mediated transformation of cotton callus. Nevertheless, during the past decade no reports were made of successful production of transgenic cotton plants using co-cultivation of embryogenic callus with Agrobacterium.
To some extent, this problem has been alleviated by techniques in microprojectile bombardment of meristematic tissue which result in a larger number of transgenic cotton lines. Nonetheless, the generation of germline transformed cotton plants, which are capable of passing on the transgene to their progeny, is infrequent.
The art is thus deficient in providing an efficient method, both in terms of required time and transformation efficiency, for the generation of transgenic cotton plants which are capable of transferring the foreign incorporated DNA to their progeny plants.
SUMMARY AND OBJECTS OF THE INVENTION
In view of the foregoing shortcomings associated with prior art cotton transformation processes as well as other disadvantages not specifically mentioned above, it should be apparent that there still exists a need in the art for a method of transforming cotton which achieves the heretofore elusive combination of a high transformation efficiency in a relatively short period of time. It is, therefore, a primary objective of the present invention to fulfill that need by providing a method for producing a transgenic cotton plant, preferably a fertile transgenic plant, by Agrobacterium-mediated transformation, comprising co-cultivating Agrobacterium cells comprising a DNA fragment of interest operably linked to at least one T-DNA border with cotton embryogenic callus in the presence of a plant phenolic compound.
It is another object of the present invention to provide a process for producing a transgenic cotton plant which eliminates the step of generating embryogenic callus from a cotton explant comprising the transformed cells, thereby both reducing the potential loss of transformed cotton lines from transformed plant cells which are not capable of generating embryogenic callus and enhancing transformation frequencies.
In a first aspect, the present invention relates to a method for producing a transgenic cotton plant comprising incubation
De Sonville Anne
Reynaerts Arlette
Bayer BioScience N.V.
Fox David T.
Hunton & Williams
Kubelik Anne
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