Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1995-06-07
2001-03-13
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S278000, C800S288000, C800S292000, C435S419000, C435S421000, C435S468000, C435S470000
Reexamination Certificate
active
06201169
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel process for transforming hereditary material of plants and to the plant products obtained by said process.
Plants having novel and/or improved properties can be produced by introducing new genetic information into plant material.
BACKGROUND OF THE INVENTION
In view of the rapid rise in world population and the concomitant increase in the need for food and raw materials, increasing the yield of useful plants as well as the increased extraction of plant storage substances, and in particular advances in the field of nutrition and medicine, are among the most urgent tasks of biological research. In this connection, the following essential aspects may be mentioned by way of example: strengthening the resistance of useful plants to unfavourable soil or climatic conditions as well as to disease and pests; increasing resistance to plant protective agents such as insecticides, herbicides, fungicides and bactericides; and a useful change in the nutrient content or of the harvest yield of plants. Such desirable effects could be produced generally by induction or increased formation of protective substances, valuable proteins or toxins. A corresponding influence on the hereditary material of plants can be brought about, for example, by inserting a specific foreign gene into plant cells without utilising conventional breeding methods.
The transfer of novel DNA sequences into plant cells using genetically manipulated plant infecting bacteria has beer described in the literature in a number of publications, for example Nature, Vol. 303, 209-213 (1983); Nature, Vol. 304, 184-187 (1983); Scientific American 248(6), 50-59 (1983); EMBO-Journal 2(6), 987-995 (1983); Science 222, 476-482 (1983); Science 223, 247-248 (1984); or Proc. Natl. Acad. Sci. USA 80, 4803-4807 (1983). In these publications, the natural properties of these bacteria for infecting plants were utilised to insert new genetic material into plant cells. So far such insertion has been made using preferably
Agrobacterium tumefaciens
itself or the Ti plasmid thereof, and also cauliflower mosaic virus.
SUMMARY OF THE INVENTION
In contradistinction thereto, the novel process of this invention makes possible the direct transfer of a gene without the use of biological vectors, in particular, without the T-DNA border regions of the Ti-plasmid. Pathogens have been used as vectors in the known processes. As the process of this invention is performed without pathogens, the limitations imposed by the host specificity of pathogens also do not apply. The development of the plants on which the novel process of transformation is carried out is not impaired by said process.
In addition to the process for transforming hereditary material of plants, the present invention also relates to the products obtainable by said process, in particular protoplasts and plant material derived therefrom, for example cells and tissues, in particular complete plants that have been regenerated from said protoplasts and the genetically identical progeny thereof.
DESCRIPTION OF THE FIGURES
FIG. 1
depicts the construction of the pABDI and pABDII plasmids.
FIG. 2
depicts the construction of the pCaMV6Km plasmid.
DETAILED DESCRIPTION OF THE INVENTION
Within the scope of the present invention, the following definitions apply:
gene: structural gene with flanking expression signals
structural gene: protein-coding DNA sequence
expression signals: promoter signal and termination signal plant expression
signal: expression signal that functions in plants
promoter signal: signal that initiates transcription
termination signal: signal that terminates transcription
enhancer signal: signal that promotes transcription
replication signal: signal that make s possible DNA replication
integration signal: DNA sequence that promotes the integration of the gene into genomic DNA
hybrid gene: gene constructed from heterologous DNA sequences, i.e. DNA sequences of different origin that may be natural as well as synthetic DNA sequences
carrier DNA: a neutral (i.e. not participating in the function of the gene) DNA sequence flanking the gene
isolated gene: DNA sequence coding for a single protein and separated from the original DNA
NPT II gene: neomycin 3′-phosphotransferase gene, type II, of transposon Tn 5 [Rothstein, S. J. and W. S. Retznikoff, Cell 23, 191-199 (1981)]
genomic DNA: DNA of the plant genome (total or part thereof).
The present invention is concerned with a novel process for the transformation of hereditary material of plants, which process comprises transferring a gene direct into plant cells without the aid of natural systems for infecting plants, more specifically without the T-DNA border regions of the Ti-plasmid. Such transformation is accordingly a vector-free transformation. In this vector-free transformation, the foreign gene for insertion is under the control of plant expression signals. The vector-free transformation of plant genes is preferably carried out by introducing a foreign gene for insertion into plant cells together with plant protoplasts acting as recipients (receptor protoplasts) into a suitable solution and leaving them until the gene has been taken up by the protoplasts.
As protoplasts it is preferred to use those of a single plant species or of a systematic unit which is a suborder of a species.
The foreign gene and the protoplasts are conveniently left in the solution for a period of time ranging from several seconds to several hours, preferably from 10 to 60 minutes, most preferably for 30 minutes.
The process of this invention is susceptible of broad application. Thus it is possible to transfer any structural genes of plant origin, for example the zein gene [Weinand, U., et al., Mol. Gen. Genet. 182, 440-444 (1981)], of animal origin, for example the TPA gene [tissue-type plasminogen activator gene; Pennica, D., et al., Nature 301, 214-221 (1983)], of microbial origin, for example the NPT II gene, or also of synthetic origin, for example the insulin gene [Stepien, P., et al., Gene 24, 289-297 (1983)], into hereditary material of plants, provided that the structural genes are flanked by expression signals which are expressed in plants and which expression signals may be of plant, animal, microbial or synthetic origin.
The transferred genes, consisting of structural gene and flanking expression signals, may be naturally occurring genes as well as hybrid genes. In the process of this invention, it is preferred to use those genes whose expression signals are of animal or, in particular, of plant or synthetic origin. Exemplary of such genes are:
a) complete genes of plants consisting of the structural gene with its natural expression signals;
b) completely synthetic genes consisting of a structural gene of synthetic origin, flanked by expression signals of synthetic origin;
c) structural genes of plant origin, flanked by plant expression signals, with the structures and expression signals originating from various plant species;
d) structural genes of plant origin, flanked by expression signals of synthetic origin;
e) structural genes of animal, microbial or synthetic origin, flanked by expression signals of plant origin; or
f) structural genes of animal or microbial origin, flanked by expression signals of synthetic origin.
Most preferred are structural genes of bacterial origin, flanked by expression signals of plant origin, in particular those originating from plant viruses. Particularly suitable expression signals for use in the process of this invention are the expression signals of gene VI of cauliflower mosaic virus.
The hybrid genes are prepared by microbiological techniques which are known per se, retaining the reading frame of the coding for the proteins to be produced by the plant cell. Such techniques are known and are described e.g. in the following publications: “Molecular Cloning”, Maniatis, T., Fritsch, E. F. and J. Sambrook, Cold Spring Harbor Laboratory, 1982, and “Recombinant DNA Techniques”, Rodriguez, R. L. and R. C. Tait,
Hohn Barbara
Hohn Thomas
Mandak Vaclav
Paszkowski Jerzy
Potrykus Ingo
Fox David T.
Lebel Edouard G.
Morton Marcia
Novartis Finance Corporation
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