DD279503A5 - PROCESS FOR INJECTION OF VIRAL DNA IN PLANT MATERIAL - Google Patents

Abstract

The invention relates to a method for introducing viral DNA into plant material using a transfer microorganism containing the viral DNA.

Description

Field of application of the invention

The present invention relates to a novel process for the introduction of viral DNA into plant material and to vegetable products obtainable by this process.

Characteristic of the known technical solutions

It is ready. In many cases, it has been possible to incorporate selected DNA fragments into virus DNA and then to infiltrate the virus into another organism. However, while the meiotic plant viruses are transmitted under natural conditions by insects, which feed on infected and non-infected plants during food intake and thereby cause a newintoctiology of plants, this route is too complicated and too difficult to control for targeted and systematic transmission. For example, for such a method, insect populations raised under controlled conditions would be needed. Furthermore, a scheduled viral infection would be very difficult to achieve, especially for large amounts of plant material. The method of mechanical inoculation of leaves with viruses used in genetic engineering is of limited use, since cloned viral DNA is only infectious in some cases, in many other cases

not yet. Although it is possible to clone and study certain types of viral genomes in bacteria, such as single-stranded DNA viruses obtained by cloning of double-stranded DNA ("Mullineaux, PM" et al., 1984), many in Bacteria Cloned viruses can not be reintroduced into plants or used to infect plant material, but this also includes the use of methods such as in vitro mutagenesis and recombinant DNA technology for basic investigations as well as the use of such viruses excluded as carriers of selected foreign DNA Such problems do not occur when using the method according to the invention described below.

Object of the invention

The aim of the invention is to provide an improved method for the genetic modification of plants.

Explanation of the essence of the invention

The invention has for its object a new technology aufzufinae ^ to improve plants by introducing new genetic material.

In the context of the present invention, the following definitions apply:

Transfer microorganism: a microorganism that converts some of its DNA into plant material

(for example, Agrobacterium tuinefaciens) T replicon: a replicon ('' Jacob, F. et al., (1963), using genes derived from this replicon itself

or on another replicon present in the same microorganism, in

can be transported in its entirety or as a part in plant cells (example / the

Ti plasmid from Agrobacterium tumefaciens) T-DNA border sequences: DNA sequences that in one or more copies with the aid of microbial functions DNA transfer

cause in plant material

Cargo DNS: DNA used artificially in a DNA vector

Plant cell cultures: cultures and plant units such as protoplasts, cell culture cells, cells in

Plant tissues, pollen, pollen tubes, ova, embryo sacs, zygotes and embryos in

Different stages of development Fully transformed plants: plants in which the genome of each cell is transformed in the desired direction.

The present invention relates to a novel process for the introduction of viral DNA into plant material, in particular whole plants or plant cell culture cells, which is generally applicable. The method consists essentially in that one

a) in a T-replicon, such as a Ti plasmid or an Ri-plasmid of an Agrobacterium, viral DNA, for example DNA of Cauliflower Mosaic Virus (CaMV), which optionally incorporates cargo DNA incorporated adjacent to one or a plurality of T-DNA border sequences, wherein the distance between viral DNA and T-DNA border sequence (s) is selected so that the transfer of the viral DNA including possibly existing cargo DNA into plant material,

b) subsequently causing the replicon to be incorporated into a transfer microorganism, the replicon entering the transfer microorganism, and

c) infected with the according to b) modified transfer microorganism plant material.

This procedure ensures that after the microwave functions have been induced, which promote the transfer of the plasmid DNA into plant material, the viral DNA used is also transferred, including the optional cargo DNA. The transformed plant material thus obtained can be regenerated into completely transformed plants.

The method according to the invention thus consists essentially of the following substeps:

a) isolation of viral DNA or its equivalents (see below) from infected plant material, for example of the genus Brassica, and cloning of these DNA into vectors of a suitable bacterium, such as Escherichia coli;

b) construction of a plasmid (= BaP) which contains more than one viral genome or parts of viral genomes which are located in the vicinity of one or more T-DNA border sequences, the distance between viral DNA and T-DNA Border sequence (s) is so selected that the transfer of the viral DNA, including any cargo DNA incorporated therein, into plant material occurs;

c) construction of a vector system by transfer of the plasmid BaP into a transfer microorganism (for example Agrobacterium tumefaciens or Agrobacterium rhizogenes); and

d) Infection of plant material with the vector system mentioned above under c).

The use of vector systems, as described above under c), as well as new vector systems, such as bacteria of the strain Agrobacterium tumefaciens A136 (pTiBo542, pEAP18 :: Ca305) and Agrobacterium tumefaciens A136 (pTiBo 542, pEA 1) further aspects of the present invention.

Particularly suitable as a T-replicon is a bacterial replicon, such as a replicon of Agrobacterium, in particular a Ti or Ri plasmid of an Agrobacterium.

Microorganisms which contain o-T replicon include, in particular, bacteria, preferably soil bacteria, and of these, in particular, those of the genus Agrobacterium.

Viral DNA and its equivalents are preferably to be understood as the following types of DNA:

natural virus DNA (for example CaMV);

double-stranded DNA forms of single-stranded DNA viruses (for example gemini viruses);

- cDNA copies of viral RNA or viroid RNA (for example, tobacco mosaic virus or cadang-cadang viroid);

any lethal or viable mutants of viruses;

Monoded DNA under the influence of viral replication, and / or expression signals;

- parts of virus DNA;

- equivalents of the above listed types of DNA in tandem form and

- Equivalents of the above-listed DNS types with built-in cargo DNS.

As a vegetable material both whole plants as well as plant parts come into consideration. As plant parts, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, oocytes, embryo sacs or zygotes in different stages of development are mentioned. Preferred are whole plants and cell culture cells.

The method according to the invention is suitable for viral infection of all plants, of the systematic units Gymnospermae and Angiospermae (including ornamental plants) the latter being emphasized.

Among the angiospermae are in addition to deciduous trees and shrubs of particular interest plants by families Solanaceae, Cruciferae, Malvaceae, Compositae, Liliaceae, Vitaceae, Chenopodiaceae, Rutaceae, Cucurbitaceae, Bromiliaceae, Rubiaceae, Theaceae, Musaceae or Graminsae and the order Leguminosae and here especially the family Papilionaceae.

Preference is given to representatives of the families Solanaceae, Cruciferae and Graminoae.

The crops with high crop yields such as corn, rice, wheat, barley, rye, oats or millet are particularly worth mentioning.

Target crops are for example those of plants of the genera Solanum, Nicotiana, Gossypium (cotton), Brassica (rape), Beta, Pisum, Phaseolus, Glycine, Helianthus, Allium, Triticum (wheat), Hordeum (barley), Avena (oats), Setaria , Sorghum (millet), Oryza (rice), Zea (corn), Cydonia, Pyrus, Malus, Rubus, Fragaria, Prunus, Arachis, Seeale, Panicum, Saccharum, Coffea, Camellia, Musa, Pineapple, Vitis, Citrus and Persea ( Avocado).

The infection process is carried out mainly by combining the previously described transfer microorganism with protoplasts or by injury of whole plants or tissue pieces and subsequent infection.

The use of the method according to the invention using the vector system described above provides numerous advantages over previously used methods:

- To achieve the infectivity of viruses that could not be artificially induced to be infected (for example, Maize streak virus), bypassing the natural vectors such as insects;

Possibility of manipulation of virus DNA in a bacterial system such as E. coli;

- extension of the host range of viruses;

Simplification of inoculation by avoiding DNA purification and drastically reducing the amount of inoculum required for inoculation.

Under the control of bacterially encoded functions, the T-DNA, including the selected viral DNA, can be incorporated into the host genome. Since a whole plant can be regenerated after transformation with bacteria from a single cell, viral DNA can be introduced into the nuclear genome of all cells of a plant. Such integrated virus genomes can

- transmitted sexually to offspring;

- prevents an infection with over-infecting viruses; and

- Where appropriate, serve as a source for further, optionally also selected cargo DNA-containing virus copies which settle on the integrated copy via transcription, Revers-Transkriptic.i, homologous recombination or other methods of rearrangement of genetic material.

Furthermore, a secondary infection ("superinfection") of plant material containing parts of viral genomes incorporated into the nuclear DNA may possibly occur,

a) lead to the development of better viral vectors, since the expression of virulent genes from the nuclear DNA may offer the possibility of replacing viral DNA with foreign DNA in the secondary infection virus; and

b) contribute significantly to a better understanding of the host-parasite relationships and thus to better protectability of the plants.

The method of the invention can also be used in crop protection in order to "immunize" plants against virus attack by a transfer microorganism as described above, by transforming the plants with a weakened, non-pathogenic or only weakly pathogenic virus.

Using the method described above, cargo DNA incorporated into the virus genome can also be transported into plant matter and spread therein. The propagation of the viruses and thus also of the foreign gene transported through them in plants is extremely advantageous, in particular, when the plants are intended to be axsexually propagated or are to be protected directly against harmful influences in a very short time (for example by introducing a resistance gene into the plants). ,

The method according to the invention is particularly suitable for introducing selected genes into plant material, for example adult plants, and for propagating them therein.

DNA transfer into plant material can be accomplished using any of the known systems, such as the binary vector system described by ³¹ An, G. et al., 1985. This vector system can be improved by, for example, incorporating sequences for homologous recombination of the DNA to be transferred into the plant.

Ausführtingsbeispiel

The invention will be explained in more detail below by way of example.

In the following example, in which as virus CaMV, as a cloning bacterium t. coli and used as the vehicle bacterium Agrobacterium tumefaciens, construction and use of a suitable vector system are further illustrated. The example can be carried out in an analogous manner with Agrobacterium thizogenes.

Preparation of Bacterial Vector pCa305 Containing 1.3 CaMV Genomes (Figure 1)

ii) In a plasmid pHC79 C "Hohn, B. et al., 1980), the small Be ^r calibration between the EcoRI site and the Clal interface by the EcoRI-Glal Gragment which (from the plasmid pMON 30 Ninem precursor from pMON 1? 0; ^5I Fraley, RT et al., 1983) and substituted for spectinomycin / streptomycin resistance In the plasmid thus obtained, the 2.5 kb region between the Sail and BstEII cleavage sites is replaced by the 3, Fragment which has been ^excised from CaMV ( ^6I Hohn, T. et al., 1982) at the Sail and BstEII cleavage sites. In the plasmid pCa292 thus obtained, a complete CaMV CM-4184 is transformed into the remaining Sall site Genome I ⁷¹ Howarth, AJ et al., 1981), thus obtaining the plasmid pCa305, which contains 1.3 genomes of CaMV in tandem-like arrangement (Figure 1) infectious cloned virus DNA on the recipient's plant not by lysing the agrobacte rien cells, the plasmid pGV1106 ( ^8I Leemans, J. et al., 1982), which has a broad host range, is cut open with the enzyme EcoRI and the single SphI site of pCa305 inserted. The resulting plasmid pEAI is introduced into Agrobacterium tumefaciens and arrives there for independent replication. In FIG. 1,

Ap ": ampicillin resistance

Sp ^R / Sm ^R : spectinomycin / streptomycin resistance

ori: replication origin

bom: mobilization origin

BstEII, Sphl, Sail: Restriction Sites

I-Vll: Open Reading Frame of the Cauliflower Mosaic Virus

kb: kilo bases

Introduction of plasmids pCa305 and pEAI in Agrobacterium tumefaciens

The plasmids pCa305 and pEAI are each transformed into bacteria of the strain Escherichia coli GJ 23 (pGJ28, R64drd11) ( ^Dl van Haute, E. et al., 1983). This E. coli strain allows the conjugal transfer of plasmids having a bom-interface into Agrobacterium tumefaciens. As a recipient, two different strains of Agrobacterium are used, both derived from Agrobacterium tumefaciens A136 and containing the wild-type Ti plasmid pTiBo542 (' ^ol Hood, EE et al., 1984).

a) As a recipient of the plasmid pCa305 the strain Agrobacterium tumefaciens A136 (pTiBo542.pEA18) is used. PEAP18 is a binary vector constructed by expressing the 6.7kb EcoRI-BamHI fragment of plasmid pGA472 ( ^3I An, G., et al., 1985) through the 2.6kb EcoRI-BglII fragment of the plasmid pHC79 ( ⁴ 'Hohn, B. et al., 1980), which contains a region for homologous recombination in the plasmid pCA305 between T-DNA border sequences. Since the plasmid pCa305 does not replicate in Agrobacterium tumefaciens, the selection of the exconjugants suf rifampicin, spectinomycin and streptomycin results in the new Agrobacterium strain Agrobacterium tumefaciens A136 (pTiBo542, pEAP18 :: pCa305), in which the plasmid pCa305 is transformed into the binary vector pEAPI8 homologous recombination has been integrated ('' Leemans, J. et al., 1981);

b) As the recipient of the plasmid pEA1 the strain Agrobacterium tumefaciens A136 (pTiBo542) is used. The uptake of the plasmid pEA1, which independently replicates in Agrobacterium tumefaciens, leads to the new strain Agrobacterium tumefaciens A136 ipTiBo542, pEA1). The exconjugants are selected for rifampicin (selective for Agrobacterium fumefaciens), kanamycin, spectinomycin and streptomycin.

The plasmids of the strains obtained according to a) and b) as described above are checked by DNA isolation and restriction mapping.

Transfer of Agrobacterium Strains Containing Plasmids pCa305 and pEA1 to Brassica rapa Plants Agrobacteria of the strains Agrobacterium tumefaciens A136 (pTiBo542, pEAP18 :: pCa305) or Agrobacterium tumefaciens A136 (pTiBo542, pEAI) are incubated for 40 hours in YEB medium (1 liter Water containing 5 g each bacto-bovine extract, peptone and sucrose, 1 g Bacto-yeast extract, and 2mM MgSO ₄ ), which onthält the suitable for selecting the plasmids antibiotics spectinomycin and / or streptomycin cultured. Subsequently, the agrobacteria are enriched by centrifuging and resuspending in 1/100 volume of YEB (based on the previously used amount of YEB). From this suspension are used: for inoculation of leaves 10μΙ, for leaf stalks 20μΙ. For inoculation, three weeks old Brassica rapa cv Just Right plants are used, either injuring three leaves per plant by rubbing with CeMt, or drilling a hole through a tuft of leaves near the soil surface with a sterile toothpick. The site of injury is then charged with the above-indicated amount of Agrobacterium suspension.

Results

While inoculation with the control strain Agrobacterium tumefaciens A136 (pTiBo542, pEA1) does not result in viral infection, a systematic infection with CaMV is usually achieved in the test strain Agrobacterium tumefaciens A136 (pTiBo542, pEAP18 :: pCa305) in a period of about 3 weeks. Since both strains contain 1.3 CaMV genomes in the same arrangement, it results from the different infectivity that the infestation of the plants in the test strain is a consequence of the escape of the virus located between the T-DNA border sequences from the plasmid in that the transmission of the virus to the plants is promoted by bacterially encoded functions, and that the infection of the plants is not attributable to the lysing of the Agrobacterium cells with release of the DNA.

Claims (31)

1. A method for introducing viral DNA into plant Materia !, characterized in that a) inserted into a T-replicon of viral DNA, which if desired contains cargo DNA incorporated, in addition to one or more T-DNA border sequences, wherein the distance between viral DNA and T-DNA border sequence ( en) is selected so that. the transmission of the viral DNA, including, if appropriate, forerunner cargo DNA in vegetable material, b) inducing the uptake of the replicon into a transfer microorganism, the replicon entering the transfer microorganism, and c) infected with the according to b) modified ransfer microorganism plant material. 2. The method according to claim 1, characterized in that one a) isolate viral DNA or its equivalents from: infected plant material and clone it in vectors of a suitable bacterium; b) a bacterial plasmid (= BaP) comprising more than one viral genome or parts of viral 3r genomes located adjacent to one or more T-DNA border sequences, the distance between viral DNA and T-DNA border sequence (s) is selected so that the transfer of the viral DNA, including the optionally incorporated therein cargo DNA into plant material takes place; c) for the construction of a vector system, the plasmid BaP in a transfer Mikroorgankv. transferred, and d) infected plant material with the vector system thus obtained. 3. The method according to claim!, Characterized in that one uses a bacterial T-replicon. 4. The method according to claim 1, characterized in that one uses a T-replicon of a bacterium of the genus Agrobacterium. 5. The method according to claim 1, characterized in that the T-replicon used is a Ti plasmid or a Ri-plasmid from a bacterium of the genus Agrobacterium. 6. The method according to claim!, Characterized in that one uses as a microorganism, which harbors the T-replicon, a bacterium. 7. The method according to claim!, Characterized in that one uses as a microorganism, which houses the T replicon, a soil bacterium. 8. The method according to claim 1, characterized in that one uses as a microorganism harboring the T replicon, a bacterium of the genus Agrobacterium. 9. The method according to claim 1, characterized in that one uses as viral DNA natural virus DNA. 10. The method according to claim 1, characterized in that one uses as viral DNA of Cauliflower mosaic virus. 11. The method according to claim 1, characterized in that one uses as viral DNA double-stranded DNA forms of DNA strand DNA viruses. ! 2. The method according to claim 1, characterized in that as viral DNA, those of gemini Used viruses.
13. The method according to claim 1, characterized in that one uses as viral DNA cDNA copies of virus RNA.
Process according to claim 1, characterized in that viral DNA is cDNA copies of viroid RNA. 15. The method according to claim 1, characterized in that one uses as viral DNA of lethal or viable mutants of viruses. 16. The method according to claim 1, characterized in that one uses as viral DNA under the influence of viral replication signals, cloned DNA. 17. The method according to claim 1, characterized in that one uses as viral DNA under the influence of viral expression signals standing cloned DNA. 18. The method according to claim 1, characterized in that one uses as viral DNA under the influence of viral replication and expression signals standing cloned DNO. 19. The method according to claim 1, characterized in that one uses as viral DNA parts of virus DNA. 20. The method according to claim 1, characterized in that one uses the viral DNA in tandem form. 21. The method according to claim 1, characterized in that one uses the viral DNA or equivalents thereof in tandem form. Method according to claim 1, characterized in that viral DNA with built-in Cargo DNA is used. 23. The method according to claim 1, characterized in that one uses viral DNA or equivalents thereof with incorporated cargo DNA. 24. The method according to claim 1, characterized in that one uses as plant material whole plants or parts of plants. 25. The method according to claim 1, characterized in that one uses as plant material whole plants or plant cell culture cells. 26. The method according to claim 1, characterized in that as plant material plants of the families Solanaceae, Cruciferae, Malvaceae, Compositae, Liliaceae, Vitaceae, Chenopodiaceae, Rutaceae, Cucurbitaceae, Bromeliaceae, Rubiaceae, Theaceae, Musaceae or Gramineae or the order Leguminosae or Used parts of these plants. 27. The method according to claim 1, characterized in that used as a vegetable material plants of the family Papilionaceae or parts of these plants. 28. The method according to claim 1, characterized in that plants of the families Solanaceae, Crucifeiae and Gramineae or parts of these plants are used as plant material. 29. The method according to claim 1, characterized in that one uses as plant material whole plants or parts of corn, rice, wheat, barley, rye, oats or millet. 30. The method according to claim 1, characterized in that as plant material plants of the genera Solanum, Nicotiana, Gossypium, Brassica, Beta, Pisum, Phaseolus, Glycine, Helianthus, Allium, Triticum, Hordeum, Avena, Setaria, Sorghum, Oryza, Zea, Cydonia, Pyrus, Malus, Rubus, Fragaria, Prunus, Arachis, Seeale, Panicum, Saccharum, Coffea, Camellia, Musa Pineapple, Vitis, Persea and Citrus or parts of these plants. 31. The method according to claim 1, characterized in that one carries out the introduction of viral DNA into the plant material using a vector system, said vector system consisting essentially of a bacterium which transfers its DNA into plant cells and viral DNA which is adjacent to one or more T-DNA border sequences, wherein the distance between viral DNA and T-DNA border sequence (s) is selected so that the transfer of the viral DNA takes place in plant cells, and if desired, the viral DNA contains cargo DNA incorporated. 32. The method according to claim 1, characterized in that one brings together protoplasts with the transfer microorganism. 33. The method according to claim 1, characterized in that injuring whole plants cder tissue pieces and infected with the transfer microorganism. 34. A method of transfer microorganism according to claim 1, characterized in that it is used for the immunization of plants against virus attack. For this 1 page drawing