Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1996-10-28
2002-04-23
Benzion, Gary (Department: 1638)
Chemistry: molecular biology and microbiology
Vector, per se
C536S023720, C536S023100
Reexamination Certificate
active
06376234
ABSTRACT:
The present invention relates to a novel method of inserting viral DNA, which optionally may contain cargo-DNA, into plants or viable parts thereof, but preferably into plants of the monocotyledon class, and most preferably into plants of the family Gramineae, using suitable transfer microorganisms. Further comprised by the invention are recombinant DNA, plasmid and vector molecules suitably adapted to the specific conditions of the process according to the invention and the transgenic plant products obtainable in accordance with the said process.
In view of the rapid increase in world population and the associated greater need for food-stuffs and raw materials, increasing the yield of useful plants and also increased extraction of plant contents, that is to say progress in the field of foodstuffs and medicines, is one of the most urgent tasks of biological and biotechnological research. In this connection, for example the following should be mentioned as essential aspects: increasing the resistance of useful plants to diseases and pests or to unfavourable soil conditions, increasing resistance to plant-protecting agents such as insecticides, herbicides, fungicides and bactericides, and beneficially modifying the nutrient content or the yield of plants. Such desirable effects could in general be brought about by induction or increased formation of protective substances, valuable proteins or toxins and by interventions in the regulatory system of plant metabolism Influencing the plant genotype appropriately can be effected, for example, by transferring new genes into whole plants or into plant cells.
It has ready proved possible in many cases to insert selected DNA fragments into viral DNA and then, together with the virus, to introduce them into another organism. Although most plant viruses are transmitted under natural conditions by insects that feed on infected and uninfected plants, thereby causing fresh infection of plants, this route is too inconvenient and difficult to control to achieve a selective and systematic transmission of viruses. Thus, for example, specially bred insect populations would be required for such a method under contained conditions. In addition, it would be very difficult to achieve a controlled virus infection, especially of large amounts of plant material.
The mechanical inoculation of leaves with viruses, the method so far employed in genetic engineering, is of only limited applicability, as cloned viral DNA is commonly believed to be non-infectious.
Although it is possible to clone and study in bacteria a variety of types of viral genomes, for example single stranded DNA viruses which are obtained by cloning double-stranded DNA forms [Mullineaux, P. M. et al, 1984], many viruses that are cloned in bacteria cannot be reintroduced into plants or used for infecting plants. The use of methods such as in vitro mutagenesis and recombinant DNA technology are therefore ruled out in basic studies as well as for exploiting such viruses as carriers of selected foreign DNA. Such problems do not arise when using the method of this invention as set forth hereinbelow.
Prior to the present invention there have been only a few reports concerning the introduction of cloned viral DNA into plant cells.
Howell et al (1980), for example, describe infection of turnip plants by cloned CaMV DNA. It is specifically emphasized in the said reference that the cloned viral DNA must be excised from the recombinant plasmid before it is capable of infecting the turnip plants.
Lebeurier et al (1982) demonstrate that a cloned tandem dimer of CaMV DNA with a partial deletion is infectious in a plant assay. The viral genome was inoculated as part of a pBR322 double-stranded DNA plasmid by artificial leaf inoculation. Lebeurier et al do not teach introduction of a tandemly duplicated CaMV genome into the plant cell as part of an Agrobacterium Ti-plasmid using the Agrobacterium transformation system.
Cress et al (1983) demonstrate that dimeric PSTV cDNA is infectious in a plant assay when inoculated by artificial means as part of a recombinant bacterial plasmid. Again, Cress et al do not teach use of the Agrobacterium system as an alternative route for delivering the viral DNA into the plant cell.
Prior to the present invention the only mentioning of viral DNA in connection with the Agrobacterium transformation system can be found in U.S. Pat. No. 4,536,475 [Anderson] and Shewmaker et al (1985).
Anderson [U.S. Pat. No. 4,536,4751] discloses a variety of recombinant plasmid molecules which comprise a bacterial plasmid into which are ligated the border sequences from the T-DNA regions of the Ti-plasmid of
Agrobacterium tumefaciens
. Anderson teach that the CaMV DNA can be employed as a DNA source of an eucaryotic origin of replication, which was considered helpful in increasing the opportunity for integration of the introduced DNA to occur. Accordingly, Anderson disclose CaMV DNA sequences that are situated outside of the the T-DNA and thus have not been assigned for the introduction into the plant cell's genome.
Shewmaker et al (1985), on the other side, describe experiments in which a full-length copy of CaMV is introduced into plant cells using a Ti-plasmid of
Agrobacterium tumefaciens
. However, within the genetic construct used by Shewmaker et al (1985) the full-length viral genome is broken in two places and could thus not give rise to viral infection. Accordingly, by the above experiments Shewmaker et al (1985) were only able to demonstrate that the introduced CaMV genome gave rise to two polyadenylation transcripts. The teaching of Shewmaker et al is thus confined to a showing that the two major promoters of the CaMV genome are supposedly able to function in plant cells.
The above short discussion of the cited references shows that the prior art teaches essentially two different experimental approaches.
The objective of the main approach, which is represented by the Lebeurier et al (1982), the Cress et al (1983), and the Howell et al (1980) reference, is to develop a plant viral transformation system which shall make use of the specific properties of infectious plant virus particles. To achieve this objective, either the cloned viral DNA is excised from the recombinant bacterial plasmid prior to infecting the plant material [Howell et al (1980); Cress et al (1983)], or the whole recombinant plasmid containing duplicated viral copies, which proved able to become recombined out in the plant cell, is introduced into the plant by artificial means [Lebeurier et al (1983)].
However, using the above roughly sketched experimental approach for developing a virus-based plant transformation system would not help to overcome the disadvantages which are involved in a pure viral vector system.
The second experimental approach, which is represented by Shewmaker et al (1983) and by U.S. Pat. No. 4,536,475 [Anderson], relates to studies for establishing novel, improved Ti-plasmid based vector systems, for example by use of plant viral regulatory DNA sequences, such as the two CaMV promoters described in Shewmaker et al (1983), or of the CaMV replication origin as described in U.S. Pat. No. 4,536,475 [Anderson].
Neither Shewmaker et al (1983) nor U.S. Pat. No. 4,536,475 [Anderson] teach the introduction of a complete, intact viral genome, which is capable of giving rise to a functional virus particle in the transformed plant.
Thus, it was one of the main objectives of the instant invention to provide a method for reintroducing cloned viral DNA, that is normally not infectious upon mechanical inoculation of plant material, into plants.
Within the scope of the present invention it was surprisingly found that in order to achieve this object the two principle experimental approaches discussed hereinbefore can be suitably combined. By taking a combination of selected and rather simple measures, parts of which were already known, it is possible to accomplish the transfer of a functional viral DNA to a plant.
This fi
Boulton Margaret Irene
Davies Jeffrey William
Grimsley Nigel Harry
Hohn Barbara
Hohn Thomas
Benzion Gary
Ciba-Geigy
Saliwanchik Lloyd & Saliwanchik
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