NZ222435A - Genetic modification of monocotyledonous plants - Google Patents

Description

r > i M > • / _ /, v,' V " f '• - "f Specific 'fA :a*ion f-i'.c-d: •••••••»•««» ? ■ ' t Pi *1- '!•- |26 /..v. ' r1 i- ' < AP ( 22 2 4 35 Patents Form No. 5 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION PROCESS FOR THE GENETIC MODIFICATION OF MONOCOTYLEDONOUS PLANTS I t i: vT/We, CIBA-GEIGY AG/ Klybeckstrasse 141, 4002, Basle, Switzerland, a Swiss corporation, and LUBRIZOL GENETICS, INC. (LGI), 3375 Mitchell Lane, Boulder, Colorado 80301-2244, U.S.A., hereby declare the invention, for which I/Ve pray that a patent may be granted to vt€/\is, and the method by which it is to be performed, to be particularly described in and by the following statement: (followed fa page la) - 1 a~ 2 2 2 4 -16159/l+2/=/ZFM Process for the genetic modification of monocotvledonous plants The present invention relates to a novel process for inserting genetic material into monocotyledonous plants or viable parts thereof, using suitable transfer microorganisms, the expression of the inserted genetic material in monocotyledonous plants or viable parts thereof and the transgenic plant products obtainable in accordance with this process.

In view of the rapid increase in world population and the associated greater need for foodstuffs 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 unfavourable soil conditions or to diseases and pests, increasing resistance to plant-protecting agents such as insecticides, herbicides, fungicides and bactericides, and beneficially modifying the nutrient content or the (followed by page 2) 22 2 4 3 5 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.

Many of the most important cultivated plants from the point of view of agricultural economics belong to the monocotyledon class, and special mention should be made of the Gramineae family, which includes our most important types of cereal such as, for example, wheat, barley, rye, oats, maize, rice, millet, inter alia.

The greatest problem in using recombinant DNA technology in plants from the monocotyledon group resides in the lack of suitable transfer microorganisms, with the aid of which transformation frequencies that are sufficiently high for practical application can be achieved and which could thus be used as auxiliaries for a specifically directed insertion into the plant genome. Aarobacterium tumefaciens. for example, one of the most-used transfer microorganisms for inserting genetic material into plants, is excellently suitable for genetic manipulation of numerous dicotyledonous plants, but so far it has not been possible to achieve correspondingly satisfactory results with representatives of monocotyledons, especially monocotyledonous cultivated plants since, from the monocotyledon class, so far only a few selected families are known that respond to infection with Agrobacterium tumefaciens and thus, at least theoretically, might be open to genetic manipulation. These families are, however, from the point of view of agricultural economics, insignificant marginal groups which could, at most, be of importance as model plants. [x) DeCleene M, Phvtopath. Z. 113: 81-89, 1985; 2) Hernalsteens JP et al., EMBO J. 3: 3039-41, 1984; 222435 3) Hooykaas-Van Slogteren GMS et al., Nature 311; 763-764, 1984; 4) Graves ACF and Goldman SL, J. Bacteriol.. 169(4): 1745-1746, 1987].

Recently developed transformation processes based on a direct insertion of exogenic DNA into plant protoplasts, such as, for example, "direct gene transfer" (5) Hain et al., 1985; 6) Paszkowski et aJL. , 1984; 7) Potrykus et al., 1985 b, c, d; 8) Lorz et al., 1985, Fromm et al., 1985) or microinjection (^0) Steinbiss and Stabel, 1983; 13-) Morikawa and Yamada, 1985), must be regarded as problematic inasmuch as the ability of numerous plant species, especially from the Gramineae group, to become regenerated from plant potoplasts currently still presents an essentially unsolved problem.

It is precisely the Gramineae family, however, which includes the cultivated plants that are the most important from the point of view of agricultural economics, such as, for example, wheat, barley, rye, oats, maize, rice, millet, inter alia, which are of particular economic interest, so that the development of processes that make it possible, irrespective of the above-mentioned limitations, also to make Gramineae representatives open to direct genetic modification must be regarded as an urgent problem.

Surprisingly it has now been possible to solve this problem within the scope of the present invention by simple measures. Contrary to all expectations, in the course of the investigations carried out within the scope of this invention it has surprisingly been shown that by using suitable culturing and application methods it is now also possible for plants from the monocotyledon group to be transformed in a specifically directed manner using certain transfer microorganisms such as, for example, Aqrobacterium tumefaciens. that is to say, now also important representatives from the monocotyledon group, especially cultivated plants belonging to the Gramineae 22 2 4 35 family, are accessible to infection by the said transfer microorganism.

Attention is drawn especially to the broadening of the host spectrum of Agrobacteriuni tumefaciens to include Gramineae. by means of which even in representatives of this family a direct and specifically targeted manipulation of the genome is possible.

The plants transformed in this manner can be identified by suitable methods of verification. There has proved especially suitable for this the use of virus genomes of plant-pathogenic viruses, such as, for example, Maize Streak Virus (MSV), by means of which successful transformations can be verified very efficiently by way of the disease symptoms that appear.

In one of its aspects the present invention therefore relates to a process for inserting genetic material into monocotyledonous plants or viable parts thereof, wherein a transfer microorganism that contains the genetic material in a transportable form is made usable for infection of monocotyledons by employing suitable culturing and application methods that make possible the induction of the virulence gene functions of the transfer mincroorganism, and wherein monocotyledonous plants or viable parts thereof are infected therewith.

Within the scope of this process, the transfer microorganisms such as, for example, Agrobacterium tumefaciens. are advantageously grown in one of the nutrient media normally used for culturing microorganisms at a temperature of from 15° to 40°C over a period of from 30 to 60 hours (h) in a stirred liquid culture. The preferred growing temperature is from 24° to 29°C.

There then follow one or more sub-culturing steps, preferably in the same medium, advantageously in a dilution ratio of 1:20, each of which lasts for a period of from 15 to 30 h, preferably from 18 to 20 h. In these cases, too, the culture temperature is from 15° to 40°C, ? ? ? 4 3 5 i-tw luyi • preferably from 24* to 29°C.

If thermophilic microorganisms are used, the growing temperature may be distinctly higher than 40°C.

Obviously, it is also possible for other culturing measures suitable for growing the transfer microorganisms to be carried out within the scope of this invention.

For example, it is also possible to use solid culture media for culturing the transfer microorganisms, which media, for example, can be produced using agarose or alginate or any other suitable solidifying agent.

To prepare the inoculation solution, the cells are centrifuged off and resuspended in a concentration, suitable for infection, in a suitable inoculation medium, for example in 1/20th part volume of an MSSP medium [12) Stachel et al., Nature, 318. 624-629, 1985]. The infection process is commenced in accordance with the invention by bringing the afore-described transfer microorganism into contact with the plant material, for example by incubation with protoplasts, by wounding whole plants or portions of tissue or, especially, by injection of the microorganism suspension directly into the plant.

Injection of the inoculation solution in the region of the growth zones, preferably those of the plant stem and the leaf sheaths, is especially preferred.

Within the scope of the present invention, it has furthermore surprisingly been possible to show that the frequency of transformation of the inoculated plants depends not only to a decisive degree on the application site on the plant, but also very especially on the stage of development of the particular plant being tested, as well as on other parameters.

An important part of the present invention therefore relates to a more sophisticated differentiation of the application site on the plant and thus to the specifically directed application of the transforming microorganism-containing inoculation solution at precisely defined sites on the 22 2 4 plant, resulting in a significant increase in the frequency of transformation of the inoculated plants. Furthermore, the frequency of transformation can be even further increased by suitable selection of the time of application as regards the stage of development of the recipient plant.

The present invention thus also relates especially to a novel process for inserting genetic material into monocotyledenous plants or viable parts thereof, which is characterised in that transfer microorganisms that are capable of inserting the said genetic material into monocotyledenous plants or viable parts thereof and that contain the genetic material to be inserted in a transportable form, are inoculated in the form of a microorganism suspension into a meristematic tissue region of the plants or of a viable part thereof.

To ensure a clear and uniform understanding of the description and the claims and also of the scope the said claims are to have, the following are given as definitions within the scope of the present invention. Transfer-microorganism; Microorganism that can convert a part of its DNA into plant material (for example Agrobac-terium tumefaciens).

T-replicon: A replicon [13)Jacob F et al., 1963] that, with the aid of genes that are located on this replicon itself or on another replicon present in the same microorganism, can be transported entirely or partially into plant cells (example: the Ti-plasmid of Agrobac-terium tumefaciens).

T-DNA-border sequences; DNA sequences that in one or more copies effect DNA transfer into plant material with the aid of microbial functions.

Cargo-DNA: DNA artificially inserted into a DNA vector. 22 2 4 3 5 Genomic DNA: DNA derived from the genome of an organism. c-DNA: Copy of a mRNA produced by reverse transcriptase.

Synthetic DNA: A DNA sequence that codes for a specific product or products or for a biological function and that is produced by synthetic means.

Heterologous qenefs) or DNA: A DNA sequence that codes for a specific product or products or for a biological function and that originates from a species different from that into which the said gene is to be inserted; the said DNA sequence is also referred to as a foreign gene or foreign DNA.

Homologous gene(si or DNA: A DNA sequence that codes for a specific product or products or for a biological function and that originates from the same species as that into which the said gene is to be inserted.

Plant cell cultures: Cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos in various stages of development.

Plants: Any photosynthetically active member of the Planta kingdom that is characterised by a membrane-encapsulated nucleus, genetic material organised in the form of chromosomes, membrane-encapsulated cytoplasmatic organelles and the ability to carry out meiosis.

Plant cell: Structural and physiological unit of the plant, consisting of a protoplast and a cell wall.

Protoplast: "naked plant cell" without a cell wall isolated from plant cells or tissues, with the ability to regenerate to a cell clone or a whole plant.

Plant tissue; A group of plant cells that are organised in the form of a structural and functional unit.

Plant organ: A defined and clearly visible differentiated part of a plant such as, for example, a root, stem, leaf or embryo.

Fully transformed plants; Plants in which the genome of each cell has been transformed in the desired manner.

In particular, the present invention relates to an improved process for transforming monocotyledonous plants using strains of Agrobacterium that are capable of carrying out the said transformation, which process is characterised in that the time of inoculation as regards the stage of development of the recipient plant, and the site of inoculation in the region of the growth zones, are so coordinated that there is a significant increase in the rates of transformation that can be achieved by comparison with known processes.

According to the invention, the preferred time as regards the stage of development of the recipient plant for applying the transforming microorganism-containing suspension extends over a period that commences with the development of the plant embryo and ends with the flowering stage, and thus with the growth and development (differentiation) phase of the recipient plant.

Plants that have reached the stage of development extending between seed germination and the 4-leaf stage are especially suitable for the application of the process according to the invention. 222 In a further embodiment of the present invention, the inoculation of the microorganism-containing transforming inoculation solution is carried out on the immature developing embryo after pollination and fertilisation of the ovules by the sperm nucleus, but preferably before the seed coat has developed.

More especially preferred are 1- to 3-day-old seedlings in which the distance between the scutellar node and the apical coleoptile tip is from 1 to 2 cm. Plants that are at a stage of development that renders possible a clear identification of the coleoptilar ncde are, however, especially suitable.

The inoculation of the microorganism-containing transforming suspension is carried out preferably in regions of the plant that contain meristematic tissue. These are portions of tissue that are active as regards division and metabolism and that contain, especially, omnipotent embryonic cells from which all of the somatic cells and tissues differentiate and which are thus ultimately also the starting point for the development of the germ cells.

By using the process according to the invention, therefore, it is possible to obtain not only transgenic plants with transformed somatic cells, but also especially plants that contain transformed germ cells from which, in the course of further cell and tissue differentiation, transformed ovules and/or pollen can develop.

After fertilisation with participation of transformed ovules and/or transformed pollen, seeds are obtained that contain transgenic embryos and that can.be used to produce transgenic plants.

A particularly suitable application site for the insertion of the transforming microorganism-containing suspension into plantlets already differentiated into stem, root and leaves is the boundary area between root and stem, the so-called root collar. 22 2 4 3 5 A repeated application of the transforming microorganism-containing inoculation solution into the meristematic tissue regions of the plant is especially preferred within the scope of this invention.

In a special embodiment of the present invention, the application of the transforming microorganism-containing suspension is effected on the seedling approximately from 1 to 3 days after germination. Preferred application sites are the coleoptile and coleorhiza areas.

Very good transformation results can be achieved by application in the immediate vicinity of, or especially by application directly into, the coleoptilar node.

Accordingly, a further especially preferred embodiment of the present invention is characterised in that the application of the transforming microorganism-containing inoculation solution is carried out from 1 to 3 days after germination in the immediate vicinity of, or directly into, the coleoptilar node of the seedling.

The introduction of the transforming microorganism-containing inoculation solution into the plant can be carried out by a wide variety of methods, for example by artificially wounding the epidermal tissue and rubbing the microorganism-containing transforming suspension into the wounded tissue, or by incubating the transfer microorganism and the plant protoplasts together.

Injection of the inoculation solution using a hypodermic syringe is preferred, by means of which a very accurately located and thus specifically directed application at precisely defined sites on the plant can be effected.

As a rule, hypodermic syringes with exchangeable needles having a cross-section of from 0.1 to 0.5 mm are used, adapted to the requirements and special demands of the plant species concerned and to its stage of development at the time of application. The volume applied m 222435 also varies as a function of the plant species concerned and its stage of development and ranges from 1 to 20 /xl, an application volume of from 5 to 10 jxl being preferred. ^ Obviously, it is also possible to use other suitable aids for the targeted application of the inoculation solution into the plant, such as, for example, very finely drawn glass capillaries, by means of which, using micromanipulators, the smallest application quantities " can be applied into accurately defined tissue regions of the plant (such as, for example, the meristem).

The procedure for applying the inoculation solution to the plant or seedling may likewise vary, but can easily be optimised for different species of plant.

These optimising tests can be carried out, without appreciable expenditure by any person skilled in the art, within the limits of a standard optimising programme in accordance with the guidelines of the present invention.

In addition to the parameters already mentioned, the concentration and the growth phase of the inoculated transfer microorganisms are also of significance as regards the efficiency of the transformation. The preferred concentration ranges from 105 to 1010 organisms per ml of inoculation solution. An inoculation con- J -1 Q centration of from 10 to 10 organisms/ml is especially preferred.

Dilution experiments carried out within the scope of this invention have shown that as dilution of the inoculation solution increases the frequency of transfor-^ mation decreases. The efficiency of the Agrobacterium- imparted DNA transfer to monocotyledonous plants is of the same order as the DNA transfer to dicotyledonous host plants (Results section, Point D).

Possible variations within the scope of the process according to the invention consequently reside in, for example, the choice of application method, the depth of puncture into the plant tissue, the composition and 22 2 4 concentration of the bacterial suspension, and the number of inoculations carried out per infection.

In a further specific embodiment of the present invention, the application of the transforming micro-organism-containing solution is carried out directly into the coleoptilar node tissue after decapitating the tip of the coleoptile in the region of the coleoptilar node. The majority of the plumule can be removed without the further development of the seedling being adversely affected.

A preferred method of application in this case, too, includes the use of hypodermic syringes, it being possible for the depth of puncture to be varied within specific limits as a function of the removal of the decapitated region of the coleoptilar node. However, inoculation directly into the coleoptilar node tissue is in any case preferred.

Application of the inoculation solution can be effected either in the peripheral tissue areas or, especially, in the central part of the exposed coleoptilar node tissue, the areas of meristematic tissue being especially preferred.

If using immature embryos, apart from the inoculation techniques already mentioned it is also possible to use a process in which the embryo is first of all, in preparation, removed from the mother plant and then brought into contact with the transfer microorganism in a suitable culture medium (14) Culture and Somatic Cell Genetics of Plants, Vol. 1, ed. IK Vasil, Academic Press, Inc., 1984; 15) Pareddy DR, et al.. 1987, Planta, 170: 141-143, 1987).

Suitable transfer microorganisms that are capable of transferring genetic material to monocotyledonous plants and can be used in the process according to the invention are especially microorganisms that contain a T-replicon.

There are to be understood by microorganisms that m 222435 contain a T-replicon especially bacteria, preferably soil bacteria and, of these, especially those of the genus Aqrobacterium.

Obviously, only strains of bacteria that are harmless, that is to say, for example, strains of bacteria that are not viable in a natural environment or that do not cause any ecological problems, can be used within the scope of the process according to the invention.

A suitable T-replicon is especially a bacterial replicon, such as a replicon of Aqrobacterium. especially a Ti- or Ri-plasmid of an Aqrobacterium.

Ti-plasmids have two regions that are essential for the production of transformed cells. In dicotyledonous plants one of these, the transfer-DNA region, is transferred to the plant and leads to the induction of tumours. The other, the virulence-conferring (vir) region, is essential only for the development but not for the maintenance of the tumours. The transfer-DNA region can be increased in size by incorporating foreign DNA without its ability to be transferred being impaired. By removing the tumour-causing genes, as a result of which the transgenic plant cells remain non-tumorous, and by incorporating a selective marker, the modified Ti-plasmid can be used as a vector for the transfer of genetic material into a suitable plant cell.

The vir-region effects the transfer of the T-DNA region of Aqrobacterium to the genome of the plant cell irrespective of whether the T-DNA region and the vir-region are present on the same vector or on different vectors within the same Aqrobacterium cell. A vir-region on a chromosome likewise induces the transfer of the T-DNA from a vector into a plant cell.

Preferred is a system for transferring a T-DNA region from an Aqrobacterium into plant cells which is characterised in that the vir-region and the T-DNA region 222435 lie on different vectors. Such a system is known as a "binary vector system" and the vector containing the T-DNA is called a "binary vector".

Any T-DNA-containing vector that is transferable into plant cells and that allows detection of transformed cells is suitable for use within the scope of this invention.

Plant cells or plants that have been transformed in accordance with the present invention can be selected by means of a suitable phenotypic marker. Examples of such phenotypic markers, which are not, however, to be construed as limiting, include antibiotic-resistance markers such as, for example, kanamycin-resistance genes and hygromycin-resistance genes, or herbicide-resistance markers. Other phenotypic markers are known to the person skilled in the art and can likewise be used within the scope of this invention.

A preferred embodiment of the present invention relates to a novel, generally applicable process for the genetic modification of plants from the monocotyledon group by insertion of viral DNA or its equivalents into whole plants or viable parts thereof.

There has already been some success in incorporating selected DNA fragments into viral DNA and then inserting these fragments with the virus into another organism. Whereas under natural conditions most plant viruses are transferred by insects which feed on infected and non-infected plants and as a result cause new infection of plants, this method is too expensive and too difficult to control for a directed and systematic transfer. For example, for such a method it would be necessary for insect populations to be reared under controlled conditions. Furthermore, especially for large quantities of plant material, a systematic virus infection would be very difficult to achieve.

The method of mechanical inoculation of leaves with 22 2 4 35 viruses that is used in gene technology can be applied in practice only to a limited extent since cloned viral DNA is infectious only in some cases whilst in many others it is not. Although it is possible to clone and study certain types of virus genomes in bacteria such as, for example, single-stranded DNA viruses that are obtained by cloning double-stranded DNA [16) Mullineaux PM et al., (1984)], many viruses cloned into bacteria cannot be reinserted into the plants or used for infecting plant material. This therefore also precludes the use of methods such as in vitro mutagenesis and recombinant DNA technology for basic investigations and the use of such viruses as carriers of selected foreign DNA.

Such problems do not arise when using the process according to the invention described hereinafter.

Especially preferred in this process are constructions that contain one or more viral replicons or parts of a viral replicon incorporated in a manner that allows release and replication of the viral replicon in the plant cell independently of the chromosomal DNA.

This arrangement of the viral replicon therefore renders possible a release of infectious viral DNA based on an intramolecular recombination via transcription, reverse transcription or other methods of rearranging genetic material.

Especially preferred within the scope of the present invention is a T-replicon such as, for example, a Ti-plasmid or an Ri-plasmid of an Aqrobacterium that contains, adjacent to one or more T-DNA border sequences, viral DNA, for example DNA of Maize-Streak Virus (MSV), which, if desired, contains incorporated Cargo-DNA, the distance between viral DNA and the T-DNA border sequence (s) being chosen such that the viral DNA, including any Cargo-DNA that may be present, is transferred into plant material.

It is possible to use as Cargo-DNA either homologous 222 4 35 or heterologous gene(s) or DNA as well as synthetic gene(s) or DNA in accordance with the definition given within the scope of the present invention.

The coding DNA sequence can be constructed exclusively from genomic DNA, from cDNA or from synthetic DNA. Another possibility is the construction of a hybrid DNA sequence consisting of both cDNA and genomic DNA and/or synthetic DNA.

In that case the cDNA may originate from the same gene as the genomic DNA, or alternatively both the cDNA and the genomic DNA may originate from different genes. In any case, however, both the genomic DNA and/or the cDNA may each be prepared individually from the same or from different genes.

If the DNA sequence contains portions of more than one gene, these genes may originate from one and the same organism, from several organisms that belong to more than one strain, one variety or one species of the same genus, or from organisms that belong to more than one genus of the same or of another taxonomic unit (kingdom).

The different sections of DNA sequence can be linked to one another to form a complete coding DNA sequence by methods known per se. Suitable methods include, for example, the in vivo recombination of DNA sequences that have homologous sections and the .in vitro linking of restriction fragments.

The process according to the invention thus consists, essentially, of the following: a) inserting viral DNA, for example DNA of Maize-Streak Virus (MSV) which, if desired, contains incorporated Cargo-DNA, into a T-replicon, such as, for example, a Ti-plasmid or an Ri-plasmid of an Aqrobacterium. in the vicinity of one or more T-DNA border sequences, the distance between the viral DNA and the T-DNA border sequence (s) being chosen such that the viral DNA, including > 22 2 4 35 any Cargo DNA that may be present, is transferred into plant material, b) subsequently causing the replicon to be taken up into a transfer microorganism, the replicon passing into the transfer microorganism, c) infecting plants from the monocotyledon group or viable parts thereof with the transfer microorganism modified in accordance with b).

This process ensures that, after the microbial functions that promote the transfer of the plasmid-DNA into the plant have been induced, also the viral DNA inserted, including any Cargo-DNA that may be present, is transferred.

The process according to the invention thus consists essentially of the following steps: a) Isolating viral DNA or its equivalents (see further below) from infected plants, for example those of the genus Zea. and cloning this DNA in vectors of a suitable bacterium such as, for example, Escherichia coli; b) constructing a plasmid (=BaP) that contains one or more than one viral genome or alternatively portions of viral genomes that are located in the vicinity of one or more T-DNA border sequences, the distance between the viral DNA and the T-DNA border sequence (s) being chosen such that the viral DNA, including any Cargo-DNA that may be incorporated therein, is transferred into whole plants or viable parts thereof; c) constructing a vector system by transferring the plasmid BaP into a transfer microorganism (for example 22 2 4 35 Aqrobacterium tumefaciens or Aqrobacterium rhizoqenes); d) infecting whole monocotyledonous plants or viable parts thereof with the vector system described above under c).

The present invention also relates to the use of vector systems such as those described above under c), and novel vector systems such as, for example, bacteria of the strain Aqrobacterium tumefaciens (Rif^) C58 (pTi C58; pEAP 200) and also Aqrobacterium tumefaciens C58 (pTiC58; pEAP37), C58 (pTiC58; pEAP29), C58 (pTiC58; pEAP40) and also C58 (pTiC58,MSV 109) for the controlled transformation of monocotyledonous plants or viable parts thereof.

Most especially preferred are bacteria of the strain Aqrobacterium tumefaciens C58 (pTiC58; pEAP37), C58 (pTiC58; pEAP29), C58 (pTiC58; pEAP40) and also C58 (pTiC58, MSV 109).

Within the scope of the present invention there are to be understood by viral DNA and its equivalents especially the following types of DNA: - double-stranded DNA forms of single-stranded DNA viruses (for example Gemini viruses, such as Maize Streak Virus (MSV)); - natural viral DNA (for example CaMV); - cDNA copies of viral RNA or viroid RNA (for example of Tobacco-Mosaic virus or Cadang-Cadang viroid); - any lethal or viable mutants of viruses; - cloned DNA under the influence of viral replication and/or expression signals; - cloned DNA under the influence of eucaryotic replication and/or expression signals; - portions of viral DNA; - equivalents of the above-listed types of DNA in tandem form and 222435 - equivalents of the above-listed types of DNA with incorporated Cargo-DNA.

The application of the process according to the invention described in the example of the afore-described vector system has, for example, the following important advantages: A broadening of the range of hosts of normally dicotyledon-specific transfer microorganisms such as, for example, Aqrobacterium tumefaciens or Aqrobacterium rhizogenes. to monocotyledons.

- The possibility of systemic infection of whole plants by using viral DNA or equivalents thereof.

- Rendering infectious viruses that hitherto could not artificially be caused to infect (for example Maize Streak Virus), whilst avoiding the use of natural vectors such as, for example, insects.

- The possibility of manipulating viral DNA in a bacterial system such as, for example, E. coli.

- A broadening of the range of hosts of viruses.

- A simplification of inoculation by avoiding DNA purification, and drastic reduction of the amount of inoculum necessary for inoculation.

- The possibility of transforming cells, tissues and whole plants, with the consequence that limitations that might occur as a result of difficulties in regenerating whole plants from protoplasts are overcome.

- Under the control of bacterially coded functions, T-DNA, including the selected viral DNA, can be incorporated into the host genome. Since, in many cases, after transformation with bacteria a whole plant can be regenerated from a single cell, viral DNA can be introduced into the nuclear genome of all cells of a plant. Such integrated virus genomes can a) be transferred by sexual means to the descen- 22 2 4 35 dants; b) prevent an infection by overinfecting viruses; and ( Is c) if desired, act as a source for other virus copies that may also contain selected Cargo-DNA and that are deposited, via transcription, reverse transcription, homologous recombination or other methods of rearranging genetic material, from the integrated copy. d) Furthermore, possibly a second infection ("superinfection") of plant material that contains viral genomes incorporated into the nuclear DNA may a) lead to the development of better viral vectors, since the expression of viral genes from the nuclear DNA may offer the possibility of replacing viral DNA by foreign DNA in the virus causing the second infection; and /?) assist considerably in the better understanding of the host-parasite relationships and thus in the improved protectability of the plants.

Thus, this invention includes a plurality of embodiments of the broad concept.

Using the above-described process, Cargo-DNA incorporated into the virus genome can also be transported into plant material in which it proliferates. The propagation in plants of the viruses, and thus also of the foreign gene transported by them, is in particular most advantageous if the plants are to be propagated asexually or are to be protected against harmful influences directly and in the shortest possible time; for example by the introduction of a resistance gene into the plants.

The process according to the invention is especially suitable for inserting selected genes, and thus a desired property, into plant material and also into fully grown 22 2 4 plants, and increasing these therein.

The process according to the invention can also be used in the field of plant protection for "immunising" plants against attack by a virus by means of a transfer microorganism as described above, by transforming the plants with a weakened non-pathogenic or only slightly pathogenic virus, which has the result of protecting the plants from undesired further virus infections.

It is possible to employ as the viral DNA that can be used within the scope of the process according to the invention, without this implying any limitation, for example DNA of Caulimo viruses, including Cauliflower Mosaic Virus (CaMV), and also DNA of representatives of the Gemini viruses, such as, for example, Bean Golden Mosaic Virus (BGMV), Chloris Striate Mosaic Virus (CSMV), Cassava Latent Virus (CLV), Curly Top Virus (CTV), Maize Streak Virus (MSV), Tomato Golden Mosaic Virus (TGMV) and Wheat Dwarf Virus (WDV).

Representatives from the Caulimo viruses group, but especially Cauliflower Mosaic Viruses, are especially suitable for use within the scope of the process according to the invention, since owing to their genome structure (double-stranded DNA) they are directly accessible to genetic manipulation.

All experiments so far and the associated observations argue that also representatives of the Gemini viruses, the genome of which is constructed from single-stranded (ss) DNA, can be used as vectors for transferring genetic material. In addition, it is also known that in the course of the development cycle of the Gemini viruses double-stranded ds-DNA is formed in infected plants and that this ds-DNA is infectious [17^ Kegami M et al. . Proc. Natl. Acad. Sci. . USA. 78.: 4102, 1981].

Consequently, also representatives from the Gemini viruses group that form ds-DNA in the course of their development cycle and are thus accessible to direct genetic manipulation are suitable within the scope of the present invention as carriers of foreign genetic material .

The present invention also relates to the use of Gemini viruses as markers, since successful gene transfers to plants using viruses can be recognised very easily in the usually macroscopically visible symptoms of infection, for example by way of yellow dots or streaks, at the base of newly formed leaves when using MSV.

The range of hosts of the Gemini viruses includes a whole series of economically important cultivated plants such as maize, wheat, tobacco, tomatoes, beans, and numerous tropical plants.

Of particular commercial importance is the range of hosts of Maize Streak Virus, which includes numerous monocotyledonous cultivated plants and cereals such as, for example, maize, rice, wheat, millet, sorghum and various African grasses.

The process according to the invention is especially suitable for infecting whole plants from the class of Monocotvledone or viable parts of those plants, such as, for example, plant tissue cultures or cell culture cells, with viral DNA and equivalents thereof. This invention therefore also relates to the transformed protoplasts, plant cells, cell clones, cell aggregates, plants and seeds and progeny thereof resulting from the process according to the invention, that have the novel property resulting ftcmthe transformation, and also to all hybridisation and fusion products of the transformed plant material that have the novel properties produced by the transformation.

The present invention also relates to transformed whole plants and viable parts of those plants, especially pollen, ovules, zygotes, embryos or any other reproductive material emerging from transformed germ-line cells.

Oi n L I n L 4 35 The present invention furthermore includes also completely transformed plants that have been regenerated from viable parts of transformed monocotyledonous plants. ^ Monocotyledonous plants that are suitable for the use according to the invention include, for example, species from the following families: Alliaceae. Amarvl-lidaceae. Asparaqaceae. Bromeliaceae. Gramineae. Lilia-ceae, Musaceae. Orchidaceae or Palmae.

Especially preferred are representatives from the Gramineae family, such as, for example, plants that are grown over a large area and produce high yields. The following may be mentioned as examples: maize, rice, wheat, barley, rye, oats and millet.

Other target crops for the application of the process according to the invention are, for example, plants of the following genera: Allium. Avena. Hordeum. Orvzae. Panicum. Saccharum. Secale. Setaria. Sorghum. Triticum. Zea. Musa, Cocos. Phoenix and Elaeis.

Successful transformation by transferring MSV-DNA to the test plant concerned can be verified in a manner known per se. for example in the light of disease symptoms, and also by molecular biological investigations including, especially, the "Southern blot" analysis.

The extracted DNA is first of all treated with restriction enzymes, then subjected to electrophoresis in 1% agarose gel, transferred to a nitrocellulose membrane [22) Southern, E. M., J. Mol. Biol. 98., 503-517 (1975) ] and hybridised (DNA-specific activities of from ^ 5 x 10s to 10 x 108 c.p.m./jig) with the DNA to be detected, which has previously been subjected to a nick-translation [23) Rigby, W.J., Dieckmann, M., Rhodes, C. and P. Berg, J. Mol. Biol. 113. 237-251]. The filters are washed three times for one hour each time with an aqueous solution of 0.03M sodium citrate and 0.3M sodium chloride at 65°C. The hybridised DNA is made visible by blackening an X-ray film for from 24 to 48 22? 4 35 pair fla» ■ hours.

To illustrate the rather general description, and for a better understanding of the present invention, reference will now be made to specific Examples, which are not of a limiting nature unless there is a specific indication to the contrary.

Non-limiting Examples: Example 1: Construction of a vector with dimeric MSV genome MSV-genomes can be isolated from naturally occurring infected maize plants in accordance with 16) Mullineaux PM et al., EMBO J. 3: 3063-3068, 1984, virion ss DNA acting as a matrix for the in vitro synthesis of double-stranded MSV-DNA using Klenow-polymerase I and an endogenous primer [ls) Donson J et al., EMBO J. 3: 3069-3073, 1984].

Another possibility consists of the isolation of double-stranded MSV-DNA ("supercoiled MSV-DNA) directly from infected leaf material. Double-stranded MSV-DNA is formed as an intermediate during virus replication. It is referred to as "replicative form DNA" or "RF-DNA".

The MSV-genomes are cloned by incorporating the RF-DNA or the in vitro synthesised DNA into a pUC9 vector linearised by BamHI [19) Vieira T and Messing T, Gene 19: 259-268, 1982]. The lac complementation test is used to identify the recombinant phages.

The next stage in the procedure is first of all to excise the cloned MSV-DNA at the single BamHI restriction incision site. The resulting linearised DNA fragment is then isolated by gel-electrophoretic separation of the DNA mixture [20) Maniatis et al., (1982)].

In virus strains having two or more BamHI restriction sites, either the MSV-genome is partially digested or another suitable restriction site is sought that 22 2 4 llMI appears only once in the MSV-genome. This applies also to the case where there is no BamHI restriction site in the MSV-genome. "*> There then follows the splicing of the BamHI fragment in tandem arrangement into the Bglll restriction site of the plasmid pGA471 [21) An G et al., EMBO J.. 4: 277-284, 1985], which site is located between the T-DNA border sequences. This so-called tandem-cloning can be controlled by way of the respective concentrations of vector and insert. The insert should be present in the ligation solution in excess. The preferred concentration ratio is in this case 10:1 (insert:vector).

The plasmid pGA471 is a so-called shuttle vector, which is stably replicated both in E.coli and in Aqrobacterium tumefaciens in the presence of tetracycline.

This vector possesses, in addition to the ColEl-replication origin lying between the T-DNA border sequences, a further broad host range replication origin» that makes it possible for the plasmid to be received in Aqrobacterium tumefaciens.

This replication origin originates from the plasmid pTJS75, a plasmid with a broad range of hosts and a tetracycline-resistance gene, a derivative of RK2 [21) An w G et al., EMBO J. 4: 277-284, 1985].

Other characteristic properties of the plasmid pGA4 71 are: 1) The possession between the T-DNA border sequences of ■ various restriction sites that render possible w incorporation of foreign DNA? 2) a cos-region of the bacteriophage which permits cloning of large DNA fragments (25-35 kb); 3) a chimar marker gene, composed of the control sequences of the nopalin synthase gene (nos) and a DNA sequence coding for neomycin phosphotransferase, and also 4) a bom-incision site, which renders possible transfer of the plasmid from E.coli into Agrobacterium tume- 22 2 4 35 faciens.

The above-mentioned incubation solution containing the vector and the MSV-DNA to be spliced in, preferably in a concentration ratio of 1:10, is used for the transformation of the E.coli strain DH1 [22J Hanahan D and Meselson M, Gene. 10: 63-67, 1980]. The selection is effected on the basis of the tetracycline resistance of the transformed clones and hybridisation experiments using radioactively labelled MSV-DNA.

A selection of positive clones is then examined for the presence of MSV-genomes in tandem arrangement. For this purpose the plasmid DNA is isolated from the positive clones according to methods known per se [20) Maniatis et ai., (1982)] and then subjected to restriction analysis.

One of the "tandem clones" is selected and is transferred from E. coli DH1 to Aqrobacterium tumefaciens (RifR) C58 (pTiC5 8).

The transfer is carried out by "triparental mating", as described in detail in 23) Rogers SG et al. (1986). In this case, rifampicin (100 /xg/ml) and tetracycline (5 Hg/ml) are used for the selection. The successful transfer of the dimeric MSV-genome is tested by Southern hybridisation f24) Dhaese P et al.., Nucleic Acids Res. 7.: 1837-1849, 1979]. •p Aqrobacterium tumefaciens (Rif ) C58 (pTiC58) t25) Holsters et al., Plasmid. 3./ 212, 1980] contains a wild-type Ti-plasmid with intact virulence functions, rendering possible the transfer of the shuttle vector into the plant cell.

The Aqrobacterium strain transformed in the manner described above has been given the following strain name: p Aqrobacterium tumefaciens (Rif ) C58 (pTiC58; pEAP 200).

Example 2: Construction of a control vector To construct a control vector without T-DNA border 22 2 4 sequences, the plasmid pRK252 Kanlll, a derivative of the plasmid pRK [26) Bevan M, Nucleic acid Res.. 12: 204-207, (1984)] is used, which contains no T-DNA border sequences.

The incorporation of the dimeric MSV-genome into the control vector is carried out by splicing the above-described BamHI fragment (see page 25) in tandem arrangement, with the aid of a Sall/BamHI adaptor, into the Sail restriction site of the plasmid pRK252 Kanlll.

The transfer of the control vector into Aqrobacterium tumefaciens (RifR) C58 (pTiC58) is carried out by "triparental mating", as described in detail in 23) Rogers SG et al., (1986).

The transformed Aqrobacterium strain has been given the following strain name: Aqrobacterium tumefaciens (Rif ) I (pTiC58; pEA 21).

Example 3: Construction of the bacterial vector pEAP 25 By exchanging a 0.65 kb Hind Ill-Sal I fragment in the cosmid pHC 79, a derivative of the E.coli plasmid pBR322 (27) Hohn and Collins, 1980), for a 1.2 kb fragment from the transposon Tn 903 (28) Grindley et al.. 1980), which carries a Kanamycin-resistance gene, the hybrid cosmid p22Gl is formed. The integration of the 1.2 kb fragment into the Hind Ill-Sal I restriction site of pHC 79 is rendered possible by adding Hind Ill-Sal I linker sequences.

A 2.9 kb Sal I-Bst EII fragment that contains a gene coding for kanamycin-resistance in plants (6) Paszkowski et al.. 1984) is excised from the plasmid pCaMV6Km and exchanged for a 2.4 kb Sal I-Bst EII fragment from P22G1.

The final construction of pEAP 25 is carried but by integration of the plasmid pB6 previously cut with Sal I into the Sal I incision site of pEAP 1. Plasmid pB6 was developed and made available by J. Davies of the John Innes Institute, Norwich, England. This plasmid has 22 2 4 since been published in 29) N. Grimsley et al., 1987, under the name pMSV 12.

Plasmid pB6 contains a dimeric MSV-genome that has previously been cloned in the plasmid pACYC184 (30) Chang and Cohen, 1978).

Example 4: Construction of the bacterial vector pEAP 37 The bacterial vector pEAP 37 is constructed by inserting the plasmid pB6, which has previously been cut with Sal I, into the Sal I restriction site of the plasmid pCIB 10. The plasmid pClB 10 was developed and made available by Mary-Dell Chilton, CIBA-GEIGY Biotechnology Facility, Research Triangle Park, Raleigh N.C., U.S.A..

Example 5: Manufacture of the bacterial vector pEAP 40 A 1.6 mer of the MSV-genome [Bglll-BamHI fragment (0.6 mer) + BaxnHI-BamHI-fragment, (monomer)] is spliced into the BamHI restriction sites of the plasmid pTZ19R, which is described in 31) Mead et al. (1986). The resulting plasmid, called p3547, which contains a 1.6 mer of the MSV-genome, is cut with EcoRI and then spliced into the EcoRI site of the plasmid pCIB200 (32) Rothstein et al., 1987). By means of these steps the MSV sequences are placed between the T-DNA border sequences of pCIB200.

Example 6; Construction of the bacterial vector pMSV 109 mg of the plasmid pMSV12, the construction of which has already been described in Example 3, are digested for a period of 2 hours at a temperature of 37°C with BamHI in a buffer solution (20) Maniatis et al., 1982). The 2.7 kb DNA fragment resulting from this enzymatic digestion is, after electrophoretic separation of the sample in a 1% agarose-TAE gel (40 mM tris-HCl, 20 mM sodium acetate, 2 mM EDTA), eluted from the latter and spliced into the single BamHI restriction site of the 22 2 4 35 binary T-DNA vector pBinl9 (26) Bevan, 1984).

For the ligation, a lOO-fold molar excess of the 2.7 kb MSV fragment (of the "insert") in relation to the vector pBinl9, and a high T4-DNA ligase concentration, are used in order to ensure a high rate of incorporation of the dimeric MSV-DNA into the vector. In detail, the concentrations used are 625 ng of pMSV DNA and 25 ng of pBinl9 DNA, which are ligated at a temperature of 10°C for a period of 16 hours in the presence of 5 units of T4-DNA ligase in a total volume of 10 fil. Half of this ligation mixture is a transformed into competent E.coli JM83 rec cells, and plated out onto "Luria Broth" (LB)-agar (20) Maniatis et al., 1982) supplemented with 50 ug/ml of kanamycin sulphate and 40 jig/ml of 5-dibromo-4-chloro-3-indolylgalactoside (X-gal), and incubated overnight at 37"C.

White colonies that contain the MSV-insert are selected and a clone that contains the dimeric MSV- insert in tandem arrangement (pMSV109) is selected for R the conjugation into Aqrobacterium tumefaciens C58Nal (33) Hepburn et al., 1985), which is carried out in accordance with a process described by Ditta et al., 1980. The selection of exconjugants is carried out on LB-agar containing 50 /ig/ml of kanamycin sulphate and 50 /xg/ml of nalidixic acid. The selected colony, which in the inoculation experiments described hereinafter initiates an infection in maize, is catalogued as pMSV 114.

Example 7; Construction of a control vector fpEA 2) without T-DNA border sequences To construct the control vector pEA 2, the Sal I restriction site of the plasmid pRK 252/kmIII, of a precur-sor-plasmid of pBIN19 (^"J Bevan, 1984), is linked with the Sal I cut plasmid pB6.

The selection of pEA 2 is carried out on the basis of the kanamycin (KmR)- and chloramphenicol (CmR)-resis- 222435 tance of the control vector.

Example 8; Introduction of the plasmids pEAP 25. pEAP 37 and pEA 2 into Aqrobacterium tumefaciens The plasmid pEAP 25 is cloned in bacteria of the strain Escherichia coli GJ23 (pGJ28, R64rdll) (33) van Haute et al., 1983). This E.coli strain renders possible the transfer by conjugation of plasmids that have a bom incision site into Aqrobacterium tumefaciens. The plasmids pEA 2, pEAP 37 and pEAP 40 are transferred via "triparental mating" into Aqrobacterium tumefaciens (23) Rogers, S.G. et al., 1986). The recipient strains used are two Aqrobacterium tumefaciens strains: 1) C58 (pTiC58) for the binary vectors pEAP 40, pEA 2 and pEAP 37 2) C58 (pTiC58), pEAP 18) for the plasmid pEAP 25.

Wild-type strains of Aqrobacterium tumefaciens can be obtained from the "Culture Collection of the Laboratory of Microbiology, Microbiology Department of the University of Gent". pEAP 25: The Aqrobacterium strain C58 (pTiC58, pEAP 18) acts as a recipient strain for the plasmid pEAP 25. pEAP 18 is a binary vector that is constructed by replacing the 6.7 kb EcoRI-BamHI fragment of the plasmid pGA472 (21) An, G. et al., 1985) by the 2.6 kb EcoRI-Bglll fragment of the plasmid pHC79 (27) Hohn, B. et al., 1980) which contains, between T-DNA border sequences, a region for homologous recombination in the plasmid pEAP 25.

Since the plasmid pEAP 25 does not replicate in Aqrobacterium tumefaciens. the selection of the exconjugants on rifampicin, kanamycin and carbenicillin yields the new Aqrobacterium strain Aqrobacterium tumefaciens C58 (pTiC58, pEAP 29) in which the plasmid pEAP 25 has been integrated into the binary vector pEAP 18 by homologous recombination. of 22 2 4 3 5 pEAP 37. pEAP 40; The mobilisation of the plasmide pEAP37 and pEAP40 from E.coli into Aqrobacterium tumefaciens via "triparental mating" results in the construction of a binary vector system.

PEA 2: The control plasmid pEA 2 is inserted into the Agrobacteriuro strain C58 (pTiC58) where it establishes itself in the trans-position to the Ti plasmid already present there.

The plasmids newly constructed in the manner described above are tested by way of DNA isolation and restriction mapping.

The plasmids used within the scope of the present invention, pEAP 37, pEAP 4 0 and pMSV 109, were deposited at the "Deutsche Sammlung von Mikroorganismen" (DSM), in Gottingen, Federal Republic of Germany and "The National Collection of Industrial Bacteria" (NCIB), Torry Research Station, P.O. Box 31, 135 Abbey Road, Aberdeen, both recognised as International Depositories in accordance with the requirements of the Budapest Treaty on the international recognition of the deposit of micro-organisms for the purposes of patent procedure. A declaration regarding the viability of the deposited samples was prepared by the said International Depositories. 222435 microorganisms deposition date deposition number ■ date of the viability certificate pEAP 37 (Escherichia coli DH1 transformed with pEAP 37 plasmid-DNA) 16 June 1987 DSM 4147 19 June 1987 pEAP 40 (Escherichia coli DH1 transformed with pEAP 40 plasmid-DNA) 16 June 1987 DSM- 4148 • 19 June 1987 pMSV 109 (Escherichia coli JM 83 Rec^ transformed with pMSV lo9 plasmid-DNA) 23. Sept. 1987 NCIB 12547 24 Sept. 1987 Limitations on the availability of the said microorganisms have not been requested by the depositor.

R Example 9: Culturing the Aqrobacterium strains (Rif ) C58 (ffTiC58; pEAP 200) , (PdfR)C58 foTiC58', pEA21) , C58 fpTiC58. PEA 2) and C58 fpTiC58. PEAP 37). C58 PTiC58. pEAp 29); C58 rpTiC58. pEAP 40) and also C58 (pTiC58, (tfPiC58 , PMSV109) and the manufacture of the inoculation solution Before inoculation, the Agrobacteria strains are plated out onto YEB medium [Bacto beef extract 5 g/1, Bacto yeast extract 1 g/1, peptone 5 g/1, sucrose 5 g/1 MgS04 2 mM, pH 7.2), which has been augmented beforehand with 100 /xg/ml of rifampicin and 25 /ig/ml of kanamycin or 50 fig/ml of nalidixic acid and solidified with 1.5 % agar. After a culturing period of 48 h at a temperature of 28°C, a single colony is used to inoculate a liquid culture. The inoculation is carried out in 100 ml 222435 Erlenmeyer flasks in a liquid YEB medium that has been augmented with antibiotics in the afore-mentioned concentration. Culturing is carried out at a temperature of 28'C on a stirring machine at a speed of 200 r.p.m. The culturing period is 24 h.

Then, a second sub-culturing process is carried out in liquid medium at a dilution ratio of 1:20 under otherwise identical conditions. The incubation period is in this case 20 h.

These steps lead to a population density of living agrobacteria of approximately 109/ral.

The bacteria cells are harvested by centrifuging and are then resuspended in an equivalent volume of a 10 mM MgS04 solution that does not contain any antibiotics.

This suspension is referred to as an undiluted strain solution in the following procedure. When preparing a series of dilutions, 10 mM MgS04 solution is again used as diluent.

Example 10: Sterilisation and germination of maize seeds For the inoculation experiments plants of the varieties Golden Cross Bantam, B 73, North Star and/or Black Mexican Sweetcorn are used, all of which can be successfully agroinfected.

For the following experiments, as a rule 3-day-old, previously sterilised seedlings are used. The sterilisation of the seedlings comprises the following process steps: 1. Sterilisation of the seeds in a 0.7 % w/v calcium hypochlorite solution (250 ml solution/100 seeds). The seeds and solution are thoroughly mixed using a magnetic stirrer.

After 20 minutes the sterilisation solution is decanted. 2. The seeds treated in this manner are then washed 3 22 2 4 3 5 times with distilled water (250 ml dist. water/100 seeds) for 30 minutes each time.

The seeds sterilised in this manner are then introduced into seed chambers that have also already been sterilised. The seed chambers are petri dishes which each contain 3 sterile Macherey-NageJ^^round filters having a diameter of 8.5 cm and also approximately 10 ml of sterile water. seeds are introduced into each of these seed chambers and incubated in the dark for approximately 3 days at a temperature of 28"C.

For the subsequent inoculation experiments, only seedlings in which the distance between scutellar node and the apical coleoptile tip is 1-2 cm are used. In any case, however, it must be ensured that the coleoptile node is clearly identifiable.

Example 11: Inoculation of the maize seedlings Hamilton hypodermic syringes (A 50 fil or 100 fil) fitted with exchangeable needles 0.4 mm in diameter are used to introduce the inoculation solution described under point 3. into the maize seedlings.

The inoculation solution is taken up into the hypodermic syringe in such a manner that no air bubbles are formed. 11.1. Inoculation of 10-dav-old maize plants The inoculation of the bacteria-containing suspension into 10-day-old maize plants is carried out by various methods and at different sites on the plant. 1. Application of 20 fil of bacterial suspension to one of the upper leaves and rubbing the suspension into the leaf with the aid of carborundum powder until the entire leaf appears wet (position A in diagram 1). 22 2 4 35 2. Injection of 10 /xl of the bacterial suspension using a 100 /xl Hamilton hypodermic syringe into the central part of the plant a) exactly above the ligula of the primary leaf (position B in diagram 1) b) 1 cm below the ligula of the primary leaf (position C in diagram 1) c) at the base of the plant in the so-called root collar, a meristematic tissue from which adventitious roots later develop (position D in diagram 1) • 11.2. Inoculation of 3-dav-old maize seedlings The inoculation of the bacterial suspension into 3-day-old maize seedlings is carried out by injection into the seedling using a 100 /xl Hamilton hypodermic syringe. 1. Injection of the bacterial suspension into the coleoptilar node by introducing the hypodermic needle through the coleoptile, starting from the apical coleoptile tip and passing into the region of the coleoptilar node (position E in diagram 2). 2. Injection of the bacterial suspension directly into the coleoptile, 2 mm below the apical coleoptile tip (position F in diagram 2). 3. Injection of the bacterial suspension directly into the coleoptile, 2 mm above the coleoptile node (position G in diagram 2). 4. Injection of the bacterial suspension directly into the coleoptilar node (position H in diagram 2). 22 2 4 35 . Injection of the bacterial suspension directly into the coleoptile, 2 mm below the coleoptilar node (position I in diagram 2). 6. Injection of the bacterial suspension directly into the scutellar node (position J in diagram 2). 7. Injection of the bacterial suspension into the scutellar node by introducing the hypodermic needle through the primary root, starting from the root tip and passing into the region of the scutellar node (position K in diagram 2). 11.3. Decapitation of the coleoptile in the region of the coleoptilar node 3-day-old maize seedlings are decapitated at various points in the region of the coleoptilar node (see diagram 3) . 1. directly at the level of the coleoptilar node 2. 1 mm above the coleoptilar node 3. 2 mm above the coleoptilar node 4. 5 mm above the coleoptilar node.

The decapitated seedlings are then planted in moist earth and cultivated in accordance with the conditions given under point 6.

The actual inoculation experiments with Aqrobacterium are carried out on seedlings in which the coleoptile tips have, in preparation, been removed 2 mm above the coleoptilar node.

Example 12: Cultivating the treated maize plants and maize seedlings Directly after the inoculation treatment the maize seedlings are planted in moist earth and cultivated in the same manner as the 10-day-old maize plants at a 222435 temperature of 22°C ±2°C with permanent lighting with white light (Phillips 400 W/G/92/2) at 3000-5000 lux.

The plants are then examined daily for the presence of symptoms of a virus infection, which is characterised by the appearance of yellow dots and/or streaks at the base of newly formed leaves.

Example 13: DNA extraction from infected, symptomatic maize plants Approximately 400 mg (fresh weight) of young leaf tissue is first of all homogenised in a mortar on ice, with the addition of 0.5 ml-1.0 ml STEN (15% sucrose, 50 mM tris-HCl, 50 mM Na3 EDTA, 0.25M NaCl, pH 8) and sand (~50 mg) to assist the tissue digestion. The homogenisate is then transferred into a small centrifug-ation tube (1.5 ml) and centrifuged for 5 minutes at a temperature of 4°C in a table centrifuge at maximum speed. The supernatant is discarded and the pellet is resuspended in 0.5 ml of ice-cold SET (15% sucrose, 50 mM Na3 EDTA, 50 mM tris-HCl, pH 8) while stirring first of all with a sterile toothed rod, and then briefly using a vortex mixer (5 seconds) . Subsequently, 10 /Ltl of a 20% SDS solution and 100 /itl of proteinase K (20 mg/ml) are added and mixed in and the whole is then heated in the small tube for 10 minutes at 68°C. After the addition of 3M sodium acetate (1/10 volume) the lysate is extracted twice with phenol/chloroform (3:1). The DNA is then ^ precipitated by the addition of 2 parts by volume of ^ ethanol and stored overnight at -20°C. Centrifugation (10 min.) in a table centrifuge at maximum speed yields a DNA-containing pellet which is subsequently dissolved in 40 til of TE buffer (40 mM tris-HCl, 1 mM Na3 EDTA, pH 8) .

Aliquots of this DNA solution are used for the "Southern blot" experiments (36) Southern EM, J.Mol. Biol.. 98: 503-517, 1975). 22 2 4 Example 14; "Southern blot" analysis The extracted DNA is first of all treated with restriction enzymes and then subjected to electrophoresis in 1% agarose gel, transferred onto a nitrocellulose membrane [22) Southern, E.M., J. Mol. Biol. 98, 503-517 (1975)] and hybridised (DNA-specific activities of 5 x 108 to 10 x 108 c.p.m.//xg) with the DNA to be detected, which has previously been subjected to a nick-translation [23) Rigby, W.J., Dieckmann, M., Rhodes, C. and P. Berg, J.Mol.Biol. 113. 237-251)]. The filters are washed three times for an hour each time with an aqueous solution of 0.03M sodium citrate and 0.3M sodium chloride at 65°C. The hybridised DNA is made visible by blackening an X-ray film for from 24 to 48 hours.

Results; A) Inoculation of 10-dav-old maize plants Table 1 shows the results of inoculation experiments on 10-day-old maize plants described under point 10.1. The inoculation is carried out using pEAP 37 DNA.

Table 1 inoculation number of plants with symptoms/ site number of inoculated plants PEAP 37 pMSV 109 pEAP 200 pEAP 25 A 0/46 C <2%) - - - B 0/44 «2%) - C 3/46 (6.5%) + + D 42/68 (62%) 26/65 ++ ++ (40%, The results in Table 1 show clearly that the preferred site of application on the plant is located in the region of the root collar, where 62% and 40% of the 22 2 4 35 treated plants exhibit symptoms of infection, whilst the number of plants exhibiting symptoms of infection after being inoculated at the other inoculation sites on the plant (A, B, C) is 0 or negligibly small.

B) Inoculation of 3-dav-old maize seedlings Table 2 shows the results of inoculation experiments on 3-day-old maize seedlings described under point 10.2.

The inoculation is carried out using pEAP 37 and pEAp 40 DNA.

Table 2 inoculation site number of plants with symptoms/ number of inoculated plants pEAP 37 1 pEAP 40 E 21/27 (78%) | F 0/20 (<5% ) | G 3/19 (16%) | H /30 (83%) | 51/58 (88%) I 8/51 (16%) | J 1/20 ( 5%) | K 2/12 (17%) | 1 As the results in Table 2 show, the preferred site of application on the maize seedling is in the region of the coleoptile node, direct and indirect application of the bacterial suspension directly into the coleoptile node, with 83% and 88% or 78% of the plants becoming infected, being clearly preferred by comparison with with all other application sites investigated. Whether the suspension is injected directly into the coleoptile node laterally, or is injected indirectly through the coleoptile, is clearly of no significance. 2 2 2 4 3 5 C) Decapitation of 3-dav-old maize seedlincrs Table 3 shows the number of surviving seedlings 2 weeks after decapitation of the coleoptile at various sites in the region of the coleoptile node.

Table 3 decapitation site number of surviving seedlings/ number of decapitated seedlings 1 0/7 | 2 /8 3 CO N CO 4 8/8 It can be seen that the plumule can be removed up to 2 mm above the coleoptile node without any impairment of the viability of the seedlings treated in this manner being observed. Even removal of the plumule only 1 mm above the coleoptile node still results in approximately 60% of cases in completely viable plantlets.

Table 4 shows the results of inoculation experiments on seedlings decapitated 2 mm above the coleoptile node. The inoculation is carried out using pEAP 37 DNA.

Table 4 inoculation site number of plants with symptoms/ number of inoculated plants 1 L 48/49 (98%) j M 14/44 (32%) | „ , , The results in Table 4 show clearly that position L on the decapitated seedling, that is to say the meris-tematic tissue region, is distinctly preferred to 22 2 4 3 5 position M, which covers the peripheral area of tissue.

D) Dilution experiments The bacterial suspension described under point 9 is diluted in YEB medium without the addition of antibiotics and applied into the coleoptilar node in the concentrations indicated below. dilution estimated number of number of plants bacteria remaining in with symptoms/ the inoculation site number of inocu lated plants undiluted 2 X io6 84/102 (82%) "1 2 X 105 42/55 (76%) H O 1 CO 2 X o H 34/54 (62%) io~3 2 X io3 19/56 (34%) ~4 0 0/10 «10%) io'5 0 0/10 (<10 %) Assuming that the number of copies of the binary vector that contains the MSV sequences is approximately 10 and that the bacteria do not increase further in the inoculation site, 104 bacteria contain approximately 400 fg (4 x 1013 g) of MSV-DNA.

This means that Aqrobacterium transfers its DNA to maize with an efficiency comparable to that with which it transfers its DNA to dicotyledonous host plants.

E) Aqrobacterium host range Apart from maize, it was possible to ascertain other representatives from the Gramineae class that are accessible to infection by Aqrobacterium.

The results of inoculation experiments with these Gramineae species are shown in Table 5: 222435 Table 5 Gramineae species number of plants with symptoms/ number of inoculated plants barley (Maris Otter) wheat (Maris Butler) wheat (normal) paring oats (Saladin) Panicum milaceum Digitaria sanguinalis Lolium temulentum 1/15 ( 6%) 1/40 ( 2%) 1/25 ( 4%) 1/25 ( 4%) 3/8 (35%) 2/10 (20%) 1/25 ( 4%) Some of the less effective results are possibly attributable to technical difficulties arising in the course of inoculation, since the plants are in some cases very small and therefore have only small stem diameters, which makes a specifically targeted injection of the inoculation solution difficult.

This apart, the results above show that, besides maize, it is possible to transform a number of other representatives from the Gramineae group by means of Aqrobacterium.

F) Aqrobacterium strains In addition to the Aqrobacterium tumefaciens strain C58 routinely used in the inoculation experiments with maize, other A. tumefaciens and A. rhizoqenes strains were also tested. It was also possible using the following Aqrobacterium strains listed in Table 6 to detect transfer of MSV-DNA to maize: 22 2 4 3 5 Table 6 Agrobacterium strain number of plants with symptoms/ number of inoculated plants pMSV 109 *1 pEAP 37 *2 A. tumefaciens T 37 LBA 4301 (pTiC58) A 6 A. rhizogenes R 1000 LBA 9402 2626 3/6 (50%) 6/6 (100%) 21/23 (91%) 15/21 ( 71%) 0/8 (<1%) 2/37 ( 5%) 17/22 (81%) /20 (75%) 7/12 (51%) *1 The inoculation experiments with pMSV 109 were carried out on 10-day-old maize plants *2 The inoculation experiments with pEAP 37 were carried out on 3-day-old maize seedlings.

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Claims (68)

WHAT WE CLAIM IS:- - 46 -

1. A process for inserting genetic material into monocotyledonous plants or viable parts thereof, which material is heterologous both with respect to the transfer microorganism and the plant or its viable parts, characterised in that a transfer microorganism that contains the genetic material in a transportable form and that is capable of inserting the said genetic material into monocotyledonous plants or viable parts thereof is made usable for infection of monocotyledons by employing suitable culturing and application methods that make possible the induction of the virulence gene functions of the transfer microorganism, and that monocotyledonous plants or viable parts thereof are infected with the said transfer microorganism.

2. A process according to claim 1, characterised in that the said transfer microorganisms are grown in culture media known per se, one or more sub-culturing steps if necessary being carried out, the transfer microorganisms are centrifuged off and then resuspended in a suitable medium, and the resulting inoculation solution is introduced into a growth zone of a monocotyledonous plant.

3. A process according to claim 1 or 2, characterised in that the said transfer microorganism is grown in an agitated culture over a period of from 30 to 60 hours (h) at an incubation temperature of from 15* to 40*C in a ■41* o\ medium suitable for culturing transfer microorganisms, //x //V •"* \\ and then, if necessary, one or more sub-culturing steps (!z °!1 are carried out, each of these sub-culturing steps lasting for a period of from 15 to 30 hours and being | 2 0 MAR 1990^ carried out at a temperature of from 15° to 40"C.

4. A process according to claim 3, characterised in that the incubation period for the culturing of the transfer microorganism and for the sub-culturing steps that may be - 47 - 222435 necessary is from 40 to 50 hours.

5. A process according to claim 3, characterised in that the incubation temperature for the culturing of the transfer microorganism and for the sub-culturing steps that may be necessary is from 24# to 29°C.

6. A process according to any one of claims 1 to 3, characterised in that a culture medium solidified with agarose or alginate or any other suitable solidifying agent is used.

7. A process according to claim 1, characterised in that the said transfer microorganisms are inoculated in the form of a microorganism suspension into a meri-stematic tissue region of the plant or viable parts thereof.

8. A process according to claim 7, characterised in that the said microorganism suspension is inoculated repeatedly.

9. A process according to claim 1, characterised in that the time of inoculation - as regards the stage of development and the stage of differentiation of the plant - and the inoculation site on the plant are so coordinated that there is a significant increase in the frequency of transformation.

10. A process according to claim 9, characterised in that the inoculation of the transforming microorganism suspension is carried out on a plant that is at a stage of development between the beginning of the development of the plant embryo and the beginning of flower formation - 48 - 222435

11. A process according to claim 10, characterised in that the said recipient plant is at a stage of development between seed germination and the 4-leaf stage.

12. A process according to claim 11, characterised .in that the plant is in the first, second or third day of the germination phase, the distance between the scutellar node and the apical coleoptile tip being from 1 to 2 cm.

13. A process according to claim 7, characterised in that the inoculation of the transforming microorganism suspension is carried out by injection into a meri-stematic tissue region of the plant.

14. A process according to claim 13, characterised in that the transforming microorganism suspension is injected in the region of the root collar of plantlets that are already differentiated into root, stem and leaves.

15. A process according to claim 13, characterised in that the transforming microorganism suspension is injected into the seedling in the immediate vicinity of the coleoptilar node.

16. A process according to claim 15, characterised in that the transforming microorganism suspension is injected directly into the coleoptile node or within an area of approximately 2 mm around the coleoptilar node.

17. A process according to claim 13, characterised in that the transforming microorganism suspension is injected directly into a meristematic tissue region of the coleoptile after decapitation of the coleoptile tip. O N -V. ' Oi 2 0 MAR 1990 y . O//' t 222435 49

18. A process according to claim 17, characterised in that the coleoptile tip is decapitated in an area that is from 1 to 5 mm above the coleoptilar node.

19. A process according to claim 1, characterised in that the said genetic material is viral DNA which may, if desired, contain incorporated Cargo-DNA.

20. A process according to claim 19 for inserting viral DNA which, if desired, contains incorporated Cargo-DNA, into plants from the Monocotvledone class, characterised in that a) viral DNA which, if desired, contains incorporated Cargo-DNA, is integrated into a T-replicon in the vicinity of one or more T-DNA border sequences, the distance between the viral DNA and the T-DNA border sequence (s) being chosen such that the viral DNA, including any Cargo-DNA that may be present, is transferred into plant material, b) subsequently, the T-replicon is caused to be taken up in a suitable transfer microorganism, the replicon passing into the transfer microorganism, c) plants from the Monocotvledone class are infected with the transfer microorganism modified in accordance with b).

21. A process according to claim 19, characterised in that a) viral DNA or its equivalents is (are) isolated from infected plant material and cloned with the aid of suitable vectors in a host organism; - 50 - 222435 b) the cloned viral DNA or parts thereof as well as any Cargo-DNA that may be incorporated therein is(are) used to construct a bacterial plasmid (=BaP) that contains more than one viral genome or portions of viral genomes as well as any Cargo-DNA that may be incorporated therein, which are located in the vicinity of one or more T-DNA border sequences, the distance between the viral DNA and the T-DNA border sequences being chosen such that the viral DNA, including any Cargo-DNA that may be incorporated therein, is transferred into plant material; c) the plasmid BaP is transferred into a suitable transfer microorganism in order to construct a vector system that can be used for plants, d) plants from the Monocotvledone class or viable parts thereof are infected with the so-modified vector system.

22. A process according to any one of claims 19 to 21, characterised in that double-stranded DNA forms of single-stranded DNA viruses are used as viral DNA.

23. A process according to claim 22, characterised in that DNA of Gemini viruses is used as the viral DNA.

24. A process according to claim 23, characterised in that DNA of Maize Streak Virus (MSV) , Bean Golden Mosaic (■Jj Virus (BGMV) , Chloris Striate Mosaic Virus (CSMV) , Cassava Latent Virus (CLV), Curly Top Virus (CTV), Tomato Golden Mosaic Virus (TGMV) or Wheat Dwarf Virus (WDV) is used as the viral DNA.

25. A process according to any one of claims 19 to 21, characterised in that the viral DNA used is natural viral DNA. £ N c> 2 OMAR 1990', \ J, .. O J; 1 . C p ] - 51 - 2224 35

26. A process according to claim 25, characterised in that DNA of Cauliflower Mosaic Virus is used as the viral DNA.

27. A process according to any one of claims 19 to 21, characterised in that cDNA copies of viral RNA are used as the viral DNA.

28. A process according to any one of claims 19 to 21, characterised in that cDNA copies of viroid RNA are used as the viral DNA.

29. A process according to any one of claims 19 to 28, characterised in that DNA of lethal or viable mutants of viruses are used as the viral DNA.

30. A process according to claim 19, characterised in that cloned DNA that is under the control of viral replication signals is used as the viral DNA.

31. A process according to claim 19, characterised that cloned DNA that is under the control of viral f[*f <" expression signals is used as the viral DNA. \ t2 OMAR 1990

32. A process according to claim 19, characterised in that cloned DNA that is under the control of viral replication and expression signals is used as the viral DNA.

33. A process according to claim 19, characterised in that cloned DNA that is under the control of eucaryotic replication and expression signals is used as the viral DNA.

34. A process according to claim 19, characterised in that portions of viral DNA are used as the viral DNA. 222-135 - 52 -

35. A process according to claim 19, characterised in that the viral DNA is used in tandem form.

36. A process according to any one of claims 19 to 34, characterised in that the viral DNA or equivalents thereof is(are) used in tandem form.

37. A process according to any one of claims 19 to 3 6, characterised in that viral DNA or equivalents thereof with incorporated Cargo-DNA is(are) used.

38. A process according to claim 20 or 21, characterised in that a bacterial T-replicon is used.

39. A process according to claim 38 , characterised in that a T-replicon of a bacterium of the genus Aqrobacterium is used.

40. A process according to any one of claims 38 or 39, characterised in that the T-replicon used is a Ti-plasmid or an Ri-plasmid from a bacterium of the genus Agrobacterium.

41. A process according to claim 1, characterised in that a bacterium is used as the microorganism that accommodates the T-replicon according to any one of claims 38 to 40.

42. A process according to claim 41, characterised in that a soil bacterium is used as the microorganism acco^^^rj^^ modating the T-replicon. if**- t- 0 MAR 199017!'

43. A process according to claim 42, characterised in\ ^ ' I; 5 222«Z5 - 53 - that a bacterium of the genus Agrobacterium is used as the microorganism accommodating the T-replicon.

44. A process according to claim 37, characterised in that the said Cargo-DNA consists of genomic DNA, of cDNA or of synthetic DNA.

45. A process according to claim 37, characterised in that the said Cargo-DNA is composed of genomic as well as of cDNA and/or synthetic DNA.

46. A process according to claim 37, characterised in that the said Cargo-DNA is composed of gene fragments of several organisms that belong to various genera.

47. A process according to claim 37, characterised in that the said Cargo-DNA is composed of gene fragments of more than one strain, one variety or one species of the same organism.

48. A process according to claim 37, characterised in that the said Cargo-DNA is composed of portions of more than one gene of the same organisms.

49. A process according to claim 1, characterised in that there are used as viable parts of monocotyledonous plants plant tissue cultures or cell culture cells.

50. A process according to claim 49, characterised in that there are used as plants or viable parts thereof 0 plants from one of the following families: Alliaceae. jLj Amarvllidaceae. Asparaaaceae. Bromel iaceae. Gramineae. Q HAR Liliaceae. Musaceae. Orchidaceae or Palmae. V '• C M ^

51. A process according to claim 50, characterised in that there are used as plants or viable parts of these ! ' ' 222435 - 54 - plants those from the Gramineae family.

52. A process according to claim 51, characterised in that there are used as plants or viable parts of plants maize, rice, wheat, barley, rye, oats or millet.

53. A process according to claim 49, characterised in that there are used as plants or viable parts of plants those from the following genera: Allium. Avena. Hordeum, Orvzae. Panicum. Saccharum. Secale. Setaria. Sorghum. Triticum. Zea. Musa. Cocos. Phoenix. Elaeis or parts of those plants.

54. A process according to claim 1, characterised in that protoplasts are incubated together with the transfer microorganism.

55. A transfer microorganism or relevant parts thereof that can be used in the process according to claim 1, which transfer microorganism is characterised in that it contains the genetic material to be transferred in a transportable form and is capable of inserting the said genetic material into monocotyledonous plants or viable parts thereof.

56. The plasmid pEAP 37 or pEAP 40 or a transfer micro-^Ffj>\, organism containing one of those plasmids. f/^ '>Sv "v\ O

57. The plasmid pMSV 109 or a transfer microorganism4^ 2 0 MAR £990 jj containing that plasmid. ''

58. The transformed Escherichia coli strain DH1 (PEAP 37), a sample of which has been deposited under the - 55 - deposit number DSM 4147, and all derivatives and mutants thereof that still possess the characteristic properties of the transformed strain.

59. The transformed Escherichia coli strain DH1 (pEAP 40), a sample of which has been deposited under the deposit number DSM 4148, and all derivatives and mutants thereof that still possess the characteristic properties of the transformed strain. 6o.

The transformed Escherichia coli strain jm 83 Rec* A a sample of which has been deposited under the deposit number NCIB 1254 7, and all derivatives and mutants thereof that still possess the characteristic properties of the transformed strain.

61. Monocotyledonous plants and the progeny thereof, manufactured in accordance with one of the processes of any one of claims 1 to 48, characterised in that a majority of the somatic cells and/or of the germ cells of the said plants and their descendants is transformed.

62. Seeds of plants and the progeny thereof according to claim 61.

63. Monocotyledonous plants or viable parts of monocotyledonous plants that have been transformed in accordance with the process described in any one of claims 1 to 48.

64. Transformed parts of monocotyledonous plants according to claim 63, characterised in that those parts are seeds, pollen, ovules, zygotes, embryos or other reproductive material originating from transformed Nr germ-line cells. 2 OMAR -se- ss.

A completely transformed plant that has been regenerated from viable parts of monocotyledonous plants according to any one of claim 4 9 or 64.

66. The transformed protoplasts, plant cells, cell aggregates, plants and seeds and descendants thereof that result from the process claimed in any one of claims 1 to 48 .

67. All hybridisation and fusion products with the transformed plant material defined in any one of claims 61 to 66.

68. The use of the transfer microorganism according to claim 1 in plant protection for "immunising" plants against undesired virus attack. CIBA-GEIGY AG and LUBRIZOL GENETICS INC