AU611652B2 - Process for the genetic modification of monocotyledonous plants - Google Patents

Description

TO: THE COMMISSIONER OF PATENTS OUR REF: 41563 S&F CODE: 52760 5845/3 9%A 6 52 S F Ref: 41563 FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE: Class Int Class Complete Specification Lodged: Accepted: Published: Priority: Related Art: Name and Address of Applicant: Ciba-Geigy AG Klybeckstrasse 141 4002 Basle

SWITZERLAND'

Lubrizol Genetics, Inc.

3375 Mitchell Lane Boulder Colorado 80301-2244 UNITED STATES OF AMERICA Address for Service: Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Complete Specification for the invention entitled: Process for the Genetic Modification of Monocotyledonous Plants The following statement is a full description of this invention, including the best method of performing it known to me/us 5845/4 LUOrPOrdl IK[1 dIIU III 3WILLrI I.IIU Uii u dulle, Io uy kuU"-U I J I bI

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Q,0 5-16159/1+2/=/ZFM Process for the genetic modification of monocotyledonous 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 L 1: I TO: THE COMMISSIONER OF PATENTS

AUSTRALIA

-2 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, o 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 suffic° ciently high for practical application can be achieved and which could thus be used as auxiliaries for a specifically directed insertion into the plant genome.

Agrobacterium tumefaciens, for example, one of the mostnn used transfer microorganisms for inserting genetic material into plants, is excellently suitable for genetic manipulation cf 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.

DeCleene M, Phytopath. Z, 113: 81-89, 1985; 2) Hernalsteens JP et al., EMBO J, 3: 3039-41, 1984; r2 I

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3 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" Hain et al., 1985; 6) Paszkowski et al., 1984; 7) Potrykus et al., 1985 b, c, d; 8) L6rz et al., 1985, 9) Fromm et al., 1985) or microinjection (10) Steinbiss and Stabel, 1983; 11) 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, Agrobacterium tumefaciens, that is to say, now also important representatives from the monocotyledon group, especially cultivated plants belonging to the Gramineae 4 family, are accessible to infection by the said transfer microorganism.

Attention is drawn especially to the broadening of the host spectrum of Agrobacterium 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 provides a process for inserting genetic material, as herein defined, into monocotyledonous plants or viable parts thereof, which process comprises growing a transfer microorganism of the genus Agrobacterium 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 in culture media known per se; optionally carrying out one or more sub-culturing steps; separating the grown Agrobacteria and resuspending them in a suitable inoculation solution; introducing the Agrobacteria prepared according to steps to into the meristematic regions of the said monocotyledonous plants or of viable parts thereof.

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 150 to 40 0 C over a period of from 30 to 60 hours in a stirred liquid culture. The preferred growing temperature is from 240 to 29"C. There then follow one or more sub-cuLlturiing 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 150 to 40 0

C,

'TC/993v TCW/993v il preferably from 24 to 29°C.

If thermophilic microorganisms are used, the growing temperature may be distinctly higher than 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 medi&, 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 -6plant, 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 Agrobacterium tumefaciens).

T-replicon: A replicon [1 3 )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 Agrobacterium 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.

t 7 7 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 gene(s) 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 qene(s) 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 Splant 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 membraneencapsulated 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.

8 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.

The application describes a plasmid comprising a viral DNA which, if desired, contains incorporated Cargo-DNA, 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.

The application also describes the plasmid pEAP 37 or pEAP 40 as herein defined.

The application further describes the plasmid pMSV 109 as herein defined.

The application describes a transfer-microorganism of the genus Agrobacterium comprising a plasmid of the invention.

The application also describes the transformed Escherichia coli strain DH1 (pEAP 37), a sample of which has been deposited under the deposit number DSM 4147, as herein defined, and all derivatives and mutants thereof that still possess the characteristic properties of the transformed strain.

The application further describes the transformed Escherichia coli strain DH1 (pEAP 40), a sample of which has been deposited under the deposit number DSM 4148, as herein defined, and all derivatives and mutants thereof that still possess the characteristic properties of the transformed strain.

The application describes the transformed Escherichia coli strain 3M 83 Rec Aa sample of which has been deposited under the deposit number TCH/993 al| f

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a rc 8a NCIB 12547, as herein defined, and all derivatives and mutants thereof that still possess the characteristic properties of the transformed strain.

The invention also provides a monocotyledonous plant including viable parts thereof containing in a majority of its somatic and/or its germ cells the genetic material introduced by Agrobacterium-mediated gene transfer.

The invention further provides seeds of plants and the progeny thereof.

The invention also provides the use of the transfer microorganism of the invention in plant protection for "immunising" monocotyledonous plants against undesired virus attack.

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.

A9 T Wf993V 4 1~ -9- 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 node 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 conta-n, 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.

hit,- 10 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 microorganismcontaining 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 microorganismcontaining inoculation solution is carried out from 1 to 3 days after germination in the immediate vicinity of, or directly into, the coleoptil ar node of the seedling.

The introduction of the transforming microorganismcontaining 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 separating the grown Agrobacteri and resuspending them n a suitable inoculation solution; introducing the AgroaCt prepared according to steps to into the meristematic regions of the said monocotyledonous k plants or of viable parts thereof. /2

II

11 also varies as a function of the plant species concerned and its stage of development and ranges from 1 to 20 41, an application volume of from 5 to 10 il 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 expcnditure 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 101 0 organisms per ml of inoculation solution. An inoculation concentration of from 107 to 109 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 transformation decreases. The efficiency of the Aqrobacterium.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 13 contain a T-replicon especially bacteria, preferably soil bacteria and, of these, especially those of the genus Agrobacterium.

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 Agrobacterium.

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 Agrobacterium to the genome of the plant cell irrespective of whether the T-DNA region and the virregion are present on the same vector or on different vectors within the same Agrobacterium 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 Agrobacterium into plant cells which is characterised in that the vir-region and the T-DNA region 5845/4 12 concentration of the bacterial suspension, and the number of inoculations carried out per infection.

In a further specific embodinent of the present invention, the application of the transforming microorganism-containing solution is carried out directly into the coleoptilarnode 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 coleoptilarnode. 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 I I I I I.p 14 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 noninfected 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 15 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 infccting 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 Tiplasmid or an Ri-plasmid of an Agrobacterium 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 k ii important representatives from the monocotyledon group, especially cultivated plants belonging to the Gramineae 16 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 Tiplasmid or an Ri-plasmid of an Agrobacterium, 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 LI _i TCNW/993v 4, 17 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 inoculation solution at precisely defined sites on the I I -18 Aqrobacterium tumefaciens or Agrobacterium rhizogenes); 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 Agrobacterium tumefaciens (RifR) C58 (pTi C58; pEAP 200) and also Agrobacterium tumefaciens C58 (pTiC58; pEAP37), C58 (pTiC58; pEAP29), C58 (pTiC58; 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 Agrobacterium 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 i I Carqo-DNA: DNA artificially inserted into a DNA vector.

19 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, Agrobacterium tumefaciens or Aarobacterium 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 descenr.LU""r ii i ii ;ii S_ "wH, dants; b) prevent an and c) if desired copies that ma that are depos: transcription, methods of real integrated cop! d) Furthermor ("superinfecti viral genomes a) lead to thi since the nuclear DNI replacing causing th assist con of the hos the improv Thus, this inv i embodiments of the 20 infection by overinfecting viruses; ,act as a source for other virus y also contain selected Cargo-DNA and ited, via transcription, reverse homologous recombination or other rranging genetic material, from the y.

e, possibly a second infection on") of plant material that contains incorporated into the nuclear DNA may e development of better viral vectors, expression of viral genes from the A may offer the possibility of viral DNA by foreign DNA in the virus e second infection; and siderably in the better understanding t-parasite relationships and thus in ed protectability of the plants.

ention includes a plurality of broad concept.

I

Using the above-described process, Cargo-DNA incorporated into the virus genome can also be transported into plant material in which it proliferates. The propagationin 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 j The application describes the transformed Escherichia coli strain JM 83 Rec A a sample of which has been deposited under the deposit number TCN/993 21 21 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 2ightly 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 singlestranded (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

IH

TN993v 22 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 Monocotyledone 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 also provides the transformed protoplasts, plant cells, cell aggregates, plants and seeds and descendants thereof that have the novel property produced by the transformation and that result from the process of the invention.

This invention also provides all fusion products with the transformed plant material that have the novel properties produced by the transformation.

The invention also provides monocotyledonous plants or viable parts of monocotyledonous plants that have been transformed in accordance with the process of the invention.

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.

The present invention further provides a completely transformed plant that has been regenerated from viable parts of monocotyledonous plants, TCW/993v Kl suspension into plantlets already differentiated into stem, root and leaves is the boundary area between root and stem, the so-called root collar.

-4 23 Monocotyledonous plants that are suitable for the use according to the invention include, for example, species from the following families: Alliaceae, Amaryllidaceae, Asparagaceae, Bromeliaceae, Gramineae, Liliaceae, 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, Oryzae, 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, J. Mol. Biol. 98, 503-517 ?0 (1975)] and hybridised (DNA-specific activities of from 5 x 108 to x 108 with the DNA to be detected, which has previously been subjected to a nick-translation E 23 Rigby, 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 0 C. The hybridised DNA is made visible by blackening an X-ray film for from 24 to 48 TCW/993v the plant species concerned and to its stage of development at the time of application. The volume applied 24hours.

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 Klenowpolymerase I and an endogenous primer [18) 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 BaiHI 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 example, the choice of application method, the depth of puncture into the plant tissue, the composition and 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 BglII restriction site of the plasmid pGA471 [21) An G et al., EMBO 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 Agrobacterium tumefaciens in the presence of tetracycline.

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

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

Other characteristic properties of the plasmid pGA471 are: 1) The possession between the T-DNA border sequences of various restriction sites that render possible incorporation of foreign DNA; 2) a cos-region of the bacteriophage X, 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- 1 characterised in that the vir-region and the T-DNA region 26 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 [22) 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 Maniatis et al., (1982)] and then subjected to restriction analysis.

One of the "tandem clones" is selected and is transferred from E. coli DH1 to Agrobacterium tumefaciens (Rif

R

C58 (pTiC58).

The transfer is carried out by "triparental mating", as described in detail in 23) Rogers SG et al. (1986).

In this case, rifampicin (100 gg/ml) and tetracycline pg/ml) are used for the selection. The successful transfer of the dimeric MSV-genome is tested by Southern hybridisation [24) Dhaese P et al., Nucleic Acids Res. 7: 1837-1849, 1979].

Agrobacterium tumefaciens (RifR) C58 (pTiC58) 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 Agrobacterium strain transformed in the manner described above has been given the following strain name: Agrobacterium tumefaciens (Rif

R

C58 (pTiC58; pEAP 200).

Example 2: Construction of a control vector To construct a control vector without T-DNA border

VI

L-I a contain a T-replicon.

There are to be understood by microorganisms that

A

-27 sequences, the plasmid pRK252 KanIII, 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 abovedescribed BamHI fragment (see page 25) in tandem arrangement, with the aid of a SalI/BamHI adaptor, into the SalI restriction site of the plasmid pRK252 KanIII.

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

The transformed Agrobacterium strain has been given the following strain name: Aqrobacterium tumefaciens (Rif

R

C53 (pTiC58; pEA 21).

Example 3: Construction of the bacterial vector pEAP By exchanging a 0.65 kb Hind III-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 III-Sal I restriction site of pHC 79 is rendered possible by adding Hind III-Sal I linker sequences.

A 2.9 kb Sal I-Bst EII fragment that contains a gene coding for kanamycin-resistance in plants 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 vety airricult to achieve.

The method of mechanical inoculation of leaves with I n' 28 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 pCIB 10 was developed and made available by Mary-Dell Chilton, CIBA-GEIGY Biotechnology Facility, Research Triangle Park, Raleigh

U.S.A..

Example 5: Manufacture of the bacterial vector pEAP A 1.6 mer of the MSV-genome [BglII-BamHI fragment (0.6 mer) BamHI-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 Ag 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, mM sodium acetate, 2 mM EDTA), eluted from the latter and spliced into the single BamHI restriction site of the It is possible to use as Cargo-DNA either homologous 29 binary T-DNA vector pBinl9 (26) Bevan, 1984).

For the ligation, a 100-fold molar excess of the 2.7 kb MSV fragment (of the "insert") in relation to the vector pBinl9, and a high T 4 -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 0 C for a period of 16 hours in the presence of 5 units of T 4 -DNA ligase in a total volume of 10 Al. Half of this ligation mixture is transformed into competent E.coli JM83 rec A cells, and plated out onto "Luria Broth" (LB)-agar (20) Maniatis et al. 1982) supplemented with 50 ug/ml of kanamycin sulphate and 40 ig/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 cc-tains the dimeric MSVinsert in tandem arrangement (pMSV109) is selected for the conjugation into Agrobacterium tumefaciens C58NalR (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 .g/ml of kanamycin sulphate and Ag/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 (pEA 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 precursor-plasmid of pBIN19 (tb) 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)-resisquence(s) being chosen such that the viral DNA, including 30 tance of the control vector.

Example 8: Introduction of the plasmids pEAP 25, pEAP 37 and pEA 2 into Agrobacterium 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 Agrobacterium tumefaciens. The plasmids pEA 2, pEAP 37 and pEAP 40 are transferred via "triparental mating" into Agrobacterium tumefaciens (23) Rogers, S.G. et al., 1986). The recipient strains used are two Agrobacterium 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 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 Agrobacterium 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-BglII 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 Since the plasmid pEAP 25 does not replicate in Aqrobacterium tumefaciens, the selection -f the exconjugants on rifampicin, kanamycin and carbenicillin yields the new Agrobacterium strain Agrobacterium tumefaciens C58 (pTiC58, pEAP 29) in which the plasmid pEAP 25 has been integrated into the binary vector pEAP 18 by homologous recombination.

plasmid BaP into a transfer microorganism (for example 31 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 Aqrobacterium 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 40 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 microorganisms for the purposes of patent procedure. A declaration regarding the viability of the deposited samples was prepared by the said International Depositories.

uaki -La -c ranaem form and i i 32 microorganisms deposition date deposition number date of the viability certificate pEAP 37 16 June 1987 DSM 4147 19 June 1987 (Escherichia coli DH1 transformed with pEAP 37 plasmid-DNA) pEAP 40 16 June 1987 DSM 4148 19 June 1987 (Escherichia coli DH1 transformed with pEAP plasmid-DNA) pMSV 109 23. Sept. 1987 NCIB 12547 24 Sept. 1987 (Escherichia coli JM 83 Rec transformed with pMSV lo9 plasmid-DNA) Limitations on the availability of the said microorganisms have not been requested by the depositor.

Example 9: Culturing the Agrobacterium strains (RifR) C58 (pTiC58; pEAP 200), (RifR)C58 (pTiC58, pEA21), C58 (pTiC58, pEA 2) and C58 (pTiC58, pEAP 37), C58 DTiC58.

pEAp 29); C58 (pTiC58, pEAP 40) and also C58 (pTiC58, (rPTiC58, pMSV109) and the manufacture of the inoculation solution Before inoculation, the Agrobacteria strains are plated out onto YEB medium [Bacto beef extract 5 g/l, Bacto yeast extract 1 g/l, peptone 5 g/l, sucrose 5 g/1 MgSO 4 2 mM, pH which has been augmented beforehand with 100 pg/ml of rifampicin and 25 Ag/ml of kanamycin or Mg/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 a) be transferred by sexual means to the descen- 33 Erle-.ieyer 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 10 9 /ml.

The bacteria cells are harvested by centrifuging and are then resuspended in an equivalent volume of a 10 mM MgSO 4 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 MgSO 4 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 j iiv> iii l jLti eapeclally 7 suitable for inserting selected genes, and thus a desired property, into plant material and also into fully grown i Ili 34 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-Nage-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 gl or 100 pl) 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-day-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 pL 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).

!,1 1- *i u-ne course or tnelr development cycle and are thus accessible to direct 35 2. Injection of 10 Al of the bacterial suspension using a 100 Al 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-day-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 Al Hamilton hypodermic syringe.

1. Injection of the bacterial suspension into the coleoptilarnode by introducing the hypodermic needle through the coleoptile, startin 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).

36 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 coleoptiar 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 Agrobacterium 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

-II

TCW/993v 37 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, mM tris-HCl, 50 mM Na 3 EDTA, 0.25M ,'aCl, pH 8) and sand (~50 mg) to assist the tissue digestion. The homogenisate is then transferred into a small centrifugation 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 Na 3 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 gl of a SDS solution and 100 gl of proteinase K (20 mg/ml) are jadded and mixed in and the whole is then heated in the smnll 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 The DNA is then precipitated by the addition of 2 parts by volume of ethanol and stored overnight at -20*C. Centrifugation min.) in a table centrifuge at maximum speed yields a DNA-containing pellet which is subsequently dissolved in l1 of TE buffer (40 mM tris-HCl, 1 mM Na 3 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).

I Lion si-es, eirner tne Msv-genome is partially digested or another suitable restriction site is sought that 38 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, J. Mol. Biol. 98, 503-517 (1975)] and hybridised (DNA-specific activities of x 108 to 10 x 108 c.p.m./gg) with the DNA to be detected, which has previously been subjected to a nicktranslation [23) Rigby, Dieckmann, 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-day-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 lants pEAP 37 pMSV 109 pEAP 200 pEAP A 0/46 B 0/44 C 3/46 D 42/68 26/65 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 of the plasmid from E.coli into Agrobacterium tume- 39 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 B, C) is Q or negligibly small.

B) Inoculation of 3-day-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

DNA.

Table 2 inoculation site

E

F

G

H

I

J

K

number of plants with symptoms/ number of inoculated plants pEAP 37 pEAP 21/27 0/20 3/19 25/30 8/51 1/20 2/12 (78%) (16%) (83%) (16%) (17%)

I

51/58 (88%) 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.

I 40 C) Decapitation of 3-day-old maize seedlings 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 number of surviving seedlings/ site number of decapitated seedlings S1 0/7 I 2 5/8 3 8/8 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 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 L 48/49 (98%) M 14/44 (32%) The results in Table 4 show clearly that position L on the decapitated seedling, that is to say the meristematic tissue region, is distinctly preferred to 41 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 coleoptiar node in the concentrations indicated below.

dilution estimated number of number of plants bacteria remaining in with symptoms/ the inoculation site number of inoculated plants undiluted 2 x 106 84/102 (82%) 1 2 x 105 42/55 (76%) -2 2 2 x 104 34/54 (62%) -3 3 2 x 103 19/56 (34%) 4 0 0/10 10-5 0 /10 0 0/10 Assuming that the number of copies of the binary vector that contains the MSV sequences is approximately 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) Agrobacterium host range Apart from maize, it was possible to ascertain other representatives from the Gramineae class that are accessible to infection by Agrobacterium.

The results of inoculation experiments with these Gramineae species are shown in Table Table Gramineae species barley (Maris Otte wheat (Maris Butle wheat (normal) apring oats (Saladi Panicum milaceum Digitaria sanguina Lolium temulentum Some of the les possibly attributabl in the course of ino some cases very smal diameters, which mak of the inoculation s This apart, the maize, it is possibl representatives from Agrobacterium.

42 number of plants with symptoms/ number of inoculated plants r) 1/15 6%) r) 1/40 2%) 1/25 4%) n) 1/25 4%) 3/8 lis 2/10 1/25 4%) s effective results are e to technical difficulties arising culation, since the plants are in 1 and therefore have only small stem es a specifically targeted injection olution difficult.

results above show that, besides e to transform a number of other the Gramineae group by means of F) Agrobacterium strains In addition to the Agrobacterium tumefaciens strain C58 routinely used in the inoculation experiments with maize, other A. tumefaciens and A. rhizogenes 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: 43 Table 6 Agrobacterium number of plants with symptoms/ strain number of inoculated plants pMSV 109 *1 pEAP 37 *2 A. tumefaciens T 37 3/6 6/6 (100%) LBA 4301 (pTiC58) 21/23 15/21 71%) A 6 0/8 2/37 A. rhizogenes R 1000 17/22 LBA 9402 15/20 2626 7/12 *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.

Bibliography 1) Decleene M, Phytopath. 113, pp 81-89, (1985) 2) Hernalsteens JP et al., EMBO J 3, pp 3039-3041, (1984) 3) Hooykaas-Van Slogteren GMS et al., Nature 311, pp 763-764 4) Graves AFC and Goldman SL, J. Bacteriol., 169(4): 1745-1746, 1987 Hain R et al., Mol. Gen. Genet., 199: 161-168, 1985 6) Paszkowski J et al., EMBO J, 3: 2717-2722, 1984 7) Potrykus I et al., (1985a) "Direct gene transfer to protoplasts: an efficient and generally applicable method for stable alterations of plant genoms", In: Freeling M. Plant Genetics Alan R. Liss Inc., New York, pp 181-199 8) L6rz H et al., Mol. Gen. Genet., 119: 178-182, 1985 9) Fromm M et al., Proc. Natl. Acad. Sci., USA, 82: 5824-5828, 1985 recombination.

44 Steinbiss HH and Stabel P, Protoplasma 116: 223-227; 1983 11) Morikawa H and Yamaday, Plant Cell Physiol., 26: 229-236, 1985 12) Stachel et al., Nature, 318: 624-629, 1985 13) Jacob F et al., Cold Spring Harbor Symp. 28, 329, (1963) 14) Cell Culture and Somatic Cell Genetics of Plants, Vol. 1, ed. IK Vasil, Academic Press, 1984 Pareddy DR, et al., Planta 170; pp 141-143, Leemans, J. et al., Gene 19, pp 361-364, (1982) 16) Mullineaux PM et al., EMBO J, 3(13): 3063-3068, 1984 17) Kegami M et al., Proc. Natl. Acad. Sci. USA, 78, pp 4102 (1981) 18) Donson J et al., EMBO J, 3(13): 3069-3073, 1984 19) Vieira T and Messing T, Gene, 19: 259-268, 1982 Maniatis T et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, (1982) 21) An G et al., EMBO J: 277-284, 1985 22) Hanahan D and Meselson M, Gene, 10: 63-67, 1980 23) Rogers SG et al, Methods in Enzymology, 118: 630-633, 1986 24) Dhaese P et al., Nucl. Acids Res., 7: 1837-1849, 1979 Holsters et al., Plasmid, 3: 212, 1980 26) Bevan Nucl. Acids Res., 12: 204-207, 1984 27) Hohn, B. and Collins, Gene 11, pp 291-298 (1980) 28) Grindley et al., Proc. Natl. Acad. Sci. USA, 77, pp 7176-7180, (1980) 29) Grimsley NH, et al., Nature, 325(8), pp 177-179, (1987) Chang ACY and Cohen SN, J. Bacteriol. 134, pp. 1141-1156, (1978) 31) Mead DA, et al., Protein Engineering 1, pp 67-74, (1986) 32) Rothstein SJ, et al., Gene, 53, pp 153-161, (1987) 45 33) Hepburn, 34) Ditta G, et al. Proc. Natl. Acad. Sci., US, 77(12), pp 7347-7351, (1980) van Haute E et al., EMBO J. 2, No. 3 pp 411-417, (1983), 36) Southern EM, J. Mol. Biol.. 98: 503-517, (1975).

Claims (28)

1. 2. The seeds treated in this manner are then washed 3 46 The claims defining the invention are as follows: 1. A process for inserting genetic material, as hereinbefore defined, into monocotyledonous plants or viable parts thereof, which process comprises growing a transfer microorganism of the genus Agrobacterium 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 in culture media known per se; optionally carrying out one or more sub-culturing steps; separating the grown Agrobacteria and resuspending them in a suitable inoculation solution; introducing the Agrobacteria prepared according to steps to into the meristematic regions of the said monocotyledonous plants or of viable parts thereof. 2. A process according to claim 1 characterised in that the said transfer microorganism is grown in an agitated culture over a period of from 30 to 60 hours at an incubation temperature of from 15° to 40°C in a medium suitable for culturing transfer microorganisms, and then, if necessary, one or more sub-culturing steps are carried out, each of these sub-culturing steps lasting for a period of from 15 to 30 hours and being carried out at a temperature of from 150 to 400C.
2. 3. A process according to claim 1, characterised in that the incubation period for the culturing of the transfer microorganism and for the sub-culturing steps that may be necessary is from 40 to 50 hours. TCW/993v LU1LJJ u ne entlre leaf appears wet (position A in diagram i). 47
3. 4. A process according to claim l, 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. A process according to any one of claims 1 or 2, characterised in that a culture medium solidified with agarose or alginate or any other suitable solidifying agent is used.
4. 6. 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 of viable parts thereof by wounding the plant or a portion of tissue and rubbing in the microorganism suspension, or by directly injecting the microorganism suspension.
5. 7. A process according to claim 6, characterised in that the said microorganism suspension is inoculated repeatedly.
6. 8. 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.
7. 9. A process according to claim 8, 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 beginnin:g of flower forma- tion. i/0 !L W the coleoptilar node (position H in diagram 2). -48- A process according to claim 9, characterised in that the said recipient plant is at a stage of develop- ment between seed germination and the 4-leaf stage.
8. 11. A process according to claim 10, 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 approximately from 1 to 3 cm.
9. 12. A process according to claim 6, characterised in that the inoculation of the transforming microorganism suspension is carried out by injection into a meri- stematic tissue region of the plant.
10. 13. A process according to claim 12, 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.
11. 14. A process according to claim 12, characterised in that the transforming microorganism suspension is injected into the seedling in the immediate vicinity of the coleoptilar node. A process according to claim 14, 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.
12. 16. A process according to claim 12, characterised in that the transforming microorganism suspension is injected directly into a meristematic tissue region of the coleoptile after decapitation of the coleoptile tip. A r are pauLdeuL ini mu isjLu ear-rn ana cultivated in the same manner as the 10-day-old maize plants at a 49
13. 17. A process according to claim 16, characterised in that the coleoptile tip is decapitated in an area that is from 1 to 5 mm above the coleoptilarnode.
14. 18. A process according to claim 1, characterised in that the said genetic material is viral DNA which may, if desired, contain incorporated Cargo-DNA.
15. 19. A process according to claim 18 for inserting viral DNA which, if desired, contains incorporated Cargo-DNA, into plants from the Monocotyledone 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 se- quence(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 of the genus Agrobacterium, the replicon passing into the said transfer microorganism, c) plants from the Monocotyledone class are infected with the transfer microorganism of the genus Agrobacterium modified in accordance with b) by wounding the plant or a portion of tissue and rubbing in the microorganism suspension, or by directly injecting the microorganism suspension. a a 1! n) I 50 A process according to claim 18, 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; 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 of the genus Aqrobacterium in order to construct a vector system that can be used for plants. d) plants from the Monocotyledone class or viable parts thereof are infected with the so-modified vector system.
16. 21. A process according to any one of claims 18 to characterised in that double-stranded DNA forms of single-stranded DNA viruses are used as viral DNA.
17. 22. A process according to claim 21, characterised in that DNA of Gemini viruses is used as the viral DNA. A 1 i I I 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
18. 51- 23. A process according to claim 22, characterised in that DNA of Maize Streak Virus (MSV), Bean Golden Mosaic 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. 24. A process according to any one of claims 18 to characterised in that the viral DNA used is natural viral DNA. A process according to claim 24, characterised in that DNA of Cauliflower Mosaic Virus is used as the viral DNA. 26. A process according to any one of claims 18 to characterised in that cDNA copies of viral RNA are used as the viral DNA. 27. A process according to any one of claims 18 to characterised in that cDNA copies of viroid RNA are used as the viral DNA. 28. A process according to any one of claims 18 to 27, characterised in that DNA of lethal or viable mutants of viruses are used as the viral DNA. 29. A process according to claim 18, characterised in that cloned DNA that is under the control of viral replication signals is used as the viral DNA. A process according to claim 18, characterised in that cloned DNA that is under the control of viral expression signals is used as the viral DNA. A4 ©'YU '-.=aca.Ly uL 11o signiricance. I 52 31. A process according to claim 18, characterised in that cloned DNA that is under the control of viral replication and expression signals is used as the viral DNA. 32. A process according to claim 18, characterised in that clonea DNA that is under the control of eucaryotic replication and expression signals is used as the viral DNA. 33. A process according to claim 18, characterised in that portions of viral DNA are used as the viral DNA. 34. A process according to claim 18, characterised in that the viral DNA is used in tandem form. A process according to any one of claims 18 to 33, characterised in that the viral DNA or equivalents thereof is(are) used in candem form. 36. A process according to any one of claims 18 to characterised in that viral DJA with incorporated Cargo- DNA is used. 37. A process according to any one of claims 18 to characterised in that viral DNA or equivalents thereof with incorporated Cargo-DNA is(are) used. 38. A process according to claim 19 or 20, 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 Agrobac- terium is used. 0OR on the decapitated seedling, that is to say the meris- tematic tissue region, is distinctly preferred to
19. 53- A process according to claim 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 microorganism that accom- modates a T-replicon according to any one of claims 38 to 42. A process according to claim 41, characterised in that a soil bacterium is used as microorganism accom- modating the T-replicon. 43. A process according to claim 42, characterised in that a bacterium of the genus Agrobacterium is used as 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. 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. i uramineae species are shown in Table r 54 4.8. 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. A process according to claim 49, characterised in that there are used as plants or viable parts thereof plants from one of the following families: Alliaceae, Amaryllidaceae, Asparagaceae, Bromeliaceae, Gramineae, Liliaceae, Musaceae, Orchidaceae or Palmae. 51. A process according to claim 50, characterised in that there are used as plants or viable parts of these 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 frcm the following genera: Allium, Avena, Hordeum, Oryzae, Panicum, Saccharum, Secale, Setaria, Sorqhum, Triticum, Zea, Musa, Cocos, Phoenix, Elaeis or parts of those plants.
20. 54. A process according to claim 1, characterised in that protoplasts are incubated together with the transfer microorganism. I 1- -I 55 A monocotyledonous plant including viable parts thereof containing in a majority of its somatic and/or its germ cells the genetic material introduced by Agrobacterium-mediated gene transfer.
21. 56. Parts of a monocotyledonous plant according to claim characterised in that those parts are plant protoplasts, cells, cell aggregates or cell clones.
22. 57. Parts of a monocotyledonous plant according to claim characterized in that those parts are seeds, pollen, ovules, zygotes, embryos or other reproductive material.
23. 58. Seeds of plants and the progeny thereof according to claim
24. 59. 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 49. A completely transformed plant that has been regenerated from viable parts of monocotyledonous plants according to any one of clitims 56 or 57.
25. 61. The transformed protoplasts, plant cells, cell aggregates, plants and seeds and descendants thereof that have the novel property produced by the transformation and that result from the process claimed in any one of claims 1 to 49.
26. 62. All fusion products with the transformed plant material defined in any one of claims 55 to 61 that have the novel properties produced by the transformation.
27. 63. The use of the transfer microorganism according to claim 1 in plant protection for "immunising" monocotyledonous plants against undesired virus attack.
28. 64. A process for inserting genetic material, as hereinbefore defined, into monocotyledonous plants or viable parts thereof which process is substantially as herein described with reference to Example 8 or 11. DATED this TWENTY-FIRST day of MARCH 1991 Ciba-Geigy AG Lubrizol Genetics, Inc. 4 Patent Attorneys for the Applicants SPRUSON FERGUSON TCW/993v