CA1340925C - Process for genetic modification of monocotyledonous plants - Google Patents

Abstract

The present invention relates to a novel process for inserting genetic material into monocotyledonous plants or viable parts thereof, which is characterised in that transfer microorganisms that are capable of inserting the said genetic material into monocotyledonous 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 bacterial suspension into a meristematic tissue region of the plant or of a viable part thereof. By suitable selection of the time of application from the point of view of the stage of development of the recipient plant it is possible to increase the transformation frequency even further.

Description

Process for the qenet:ic modification of monocotyledonous plants The present: invention relates to a novel process for inserting genetic matEarial into monocotyledonous plants or viable parts thereof, using suitable transfer micro-organisms, the expression of the inserted genetic material in monocotyledonous plants or viable parts thereof and the transc~enic 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 yield of plants. Such desirable effects could in general be brought about by induction or increased formation of protective substance;, valuable proteins or toxins and by interventions in the regulatory system of plant metab-olism. 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 impor-tant types of cereal such as, for example, wheat, barley, rye, oats, maize, rice, millet, inter alia.
The greateat problem in using recombinant DNA
technology in p:Lants from the monocotyledon group resides in the lack of :suitable transfer microorganisms, with the aid of which tr~~nsfor:mation frequencies that are suffi-ciently high fo~c practical application can be achieved and which could thus lbe used as auxiliaries for a specifically directed insertion into the plant genome.
Acrrobacterium tumefac.iens, for example, one of the most-used transfer microorganisms for inserting genetic material into pT.ants, is excellently suitable for genetic manipulation of numerous dicotyledonous plants, but so far it has not been possible to achieve correspondingly satisfactory re_~ults with representatives of monocotyled-ons, especially monocotyledonous cultivated plants since, from the monocotyledon class, so far only a few selected families are knc>wn that respond to infection with Aarobacterium tumefaciens and thus, at least theoretically, might be open to genetic manipulation.
These families a.re, however, from the point of view of agricultural economic::, insignificant marginal groups which could, at most, be of importance as model plants.
DeCleene M, Phytox~ath. Z, 113: 81-89, 1985;
2~ Hernalsteens JP et al., EMBO J, 3: 3039-41, 1984;

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 proto-plasts, such as, for example, "direct gene transfer"
(5) Hain et al.,, 1985; 6) Paszkowski et al., 1984;
7) Potrykus et al., 1'985 b, c, d; 8) Lorz et al., 1985, Fromm et al.,. 1985) or microinjection (1~~ Steinbiss and Stabel, 1983; 11) Morikawa and Yamada, 1985), must be regarded as problematic inasmuch as the ability of numerous plant :specie:~, especially from the Gramineae group, to become' regenerated from plant potoplasts currently still presents an essentially unsolved problem.
It is precisely i~he Gramineae family, however, which includes the cu7.tivatESd plants that are the most impor-tant from the point oiE view of agricultural economics, such as, for example, wheat, barley, rye, oats, maize, rice, millet, inter a7Lia, 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 h.as 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 tu:mefaciens, that is to say, now also important repres~sntatives from the monocotyledon group, especially cultivated plants belonging to the Gramineae 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 targetedlmanipulation of the genome is possible.
The plants transformed in this manner can be identified by suitable methods of verification. There has proved espe~~ially 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 effi-ciently 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 mono-cotyledonous plants or viable parts thereof, wherein a trans-fer microorganism that contains the genetic material in a transportable f~crm is made usable for infection of monocot-yledons by empl~~ying suitable culturing and application methods that make possible the induction of the virulence gene functions of the transfer mincroorganism, and wherein monocotyledonou~~ plants or viable parts thereof are infected therewith.
Within the scope of this process, the transfer microorganisms such as, for example, Ag~robacterium 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, preferably from 24° t:o 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 microorg<~nism 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 onl~~ to a decisive degree on the application site on the plant, buts 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 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 i:n that transfer microorganisms that are capable of inserting the said genetic material into monocotyledenou,s plants or viable parts thereof and that contain the genetic material to be inserted in a trans-portable form, .are inoculated in the form of a micro-organism 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 Aqrobac-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, cyan be transported entirely or partially into plant cell: (exa:mple: the Ti-plasmid of Agrobac-terium tumefaciens).
T-DNA-border sequences: DNA sequences that in one or more copies effect DN.A transfer into plant material with the aid of microbial functions.
Cargo-DNA: DNA artificially inserted into a DNA vector.

-' - 13409 25 Genomic DNA: DNA derived from the genome of an organism.
c-DNA: Copy of a mRNA produced by reverse transcrip-tase.
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.
Homolo oq-us genet'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 plant tissues, pollen,, pollen tubes, ovules, embryo sacs, zygotes and embryos in various stages of develop-ment.
Plants: Any photosynt:hetically 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.

-$- 1340925 Proto~last: "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 differen-tiated 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 imt~roved 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 sta~~e 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 develognent (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.

In a further embodiment of the present invention, the inoculation of the microorganism-containing trans-forming inoculation solution is carried out on the immature developing.embryo after pollination and fer-tilisation of tile 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 inocul<~tion of the microorganism-containing transforming su:~pension 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 mei;.abolism and that contain, especially, omnipotent embr~~onic 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 tile process according to the invention, therefore, it i:~ possible to obtain not only transgenic plants with transformed somatic cells, but also espec-ially plants that contain transformed germ cells from which, in the course of further cell and tissue differen-tiation, transformed ovules and/or pollen can develop.
After fert_~lisation with participation of trans-formed ovules and/or 'transformed pollen, seeds are obtained that contain transgenic embryos and that can be used to produce transgenic plants.
A particularly sositable application site for the insertion of thE~ tran:~forming microorganism-containing suspension into plant:lets already differentiated into stem, root and 7~eaves is the boundary area between root and stem, the so-called root collar.

A repeated application of the transforming micro-organism-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 embodi-ment 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 coleoptil ar node of the seedling.
The introduction of the transforming microorganism-containing inocvulation 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 hypo-dermic 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 <~ 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 develop-ment at the tinca of application. The volume applied also varies as a funcaion of the plant species concerned and its stage of development and ranges from 1 to 20 ~1, an application volume. of from 5 to 10 ~,1 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 procedvure 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 eff:icienc.y of the transformation. The preferred concentration ranges from 105 to 1010 organisms per ml of inoculation solution. An inoculation con-centration of from 10~~ to 109 organisms/ml is especially preferred.
Dilution eoperim~ents carried out within the scope of this invention have slhown 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 variaticms 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 concentration of then bacterial suspension, and the number of inoc:ulations carried out per infection.
In a further specific embodiment of the present invention, they application of the transforming micro-organism-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 th.e 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 u~;e 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 coleoptil~'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 inocula-tion 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., 1!84; 15) Pareddy DR, et al., 1987, Planta, 170: 141-143, 1987).
Suitable i~ransf~er microorganisms that are capable of transferring genetic material to monocotyledonous plants and can be used in tlhe process according to the invention are especially microorganisms that contain a T-replicon.
There are to be understood by microorganisms that 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 inven-tion.
A suitable T-replicon is especially a bacterial replicon, such ;as a replicon of Aqrobacterium, especially a Ti- or Ri-pla;smid of an Aqrobacterium.
Ti-plasmid;s have two regions that are essential for the production of transformed cells. In dicotyledonous plants one of these, the transfer-DNA region, is trans-ferred to the plant and leads to the induction of tumours. The ovther, 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 abi:Lity 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 <~ vector for the transfer of genetic material into a suitalble plant cell.
The vir-region effects the transfer of the T-DNA
region of A_grob~~cteri~um 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 i:he same Actrobacterium cell. A vir-region on a chromosome likewise induces the transfer of the T-DNA from a ve<aor into a plant cell.
Preferred is a system for transferring a T-DNA
region from an ~~ robacterium into plant cells which is characterised in that the vir-region and the T-DNA region ->l4- 1340925 lie on different vecltors. Such a system is known as a "binary vector system" and the vector containing the T-DNA is callecL a "b:inary vector" .
Any T-DNA--containing vector that is transferable into plant cel7.s and that allows detection of transformed cells is suitable fo:r use within the scope of this invention.
Plant cel7.s 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 marl;ers, which are not, however, to be construed as limiting, include antibiotic-resistance , markers such a::, 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 preferrE:d embodiment of the present invention relates to a novel, generally applicable process for the genetic modification of plants from the monocotyledon group by insertion o:E viral DNA or its equivalents into whole plants ox- viable parts thereof.
There has already been some success in incorporating selected DNA fragments into viral DNA and then inserting these fragment: with the virus into another organism.
Whereas under natural conditions most plant viruses are transferred by inseci:.s which feed on infected and non-infected plant:> and as a result cause new infection of plants, this meathod :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 condi-tions. Furthez-more, 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 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 construc-tions 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 possibl~s 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 'r-replicon such as, for example, a Ti-plasmid or an R:i-plasmid of an Actrobacterium 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 se-quence (s) being chosen su ch that the viral DNA, including any Cargo-DNA that ma:y be present, is transferred into plant material.
It is possible to use as Cargo-DNA either homologous or heterologous genes) or DNA as well as synthetic genes) or DNA in accordance with the definition given within the scope of the present invention.
The coding DNA sequence can be constructed ex-clusively 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 gen~omic DNA, or alternatively both the cDNA
and the genomic DNA may originate from different genes.
In any case, ho~~aever, both the genomic DNA and/or the cDNA may each b~~ 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 differE~nt sections of DNA sequence can be linked to one another i.o foam a complete coding DNA sequence by methods known ~~r 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 con-sists, essentia7_ly, o:E the following:
a) inserting viral D1JA, for example DNA of Maize-Streak Virus (MSV) which, if desired, contains incorporated Cargo-DNA, into a T-rEaplicon, such as, for example, a Ti-plasmid or an Ri.-plasmid of an Actrobacterium, in the vicinity of one or more T-DNA border sequences, the distance between the viral DNA and the T-DNA border se-quences) being chosen such that the viral DNA, including any Cargo DNA that many 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 on~~ viral genome or alternatively portions of viral genome;s 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 sequences) being cho-sen such that the viral DNA, including any Cargo-DNA that may be incorpor<~ted 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 -F
-1$- 1340925 Agrobacterium tumefac:iens or Agrobacterium rhizo~enes);
d) infecting whole monocotyledonous plants or viable parts thereof with th.e 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 Actrobacterium tumefaciens (RifR) C58 (pTi C58; pEAP 200) and also Acrrobacterium 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 t~umefaciens C58 (pTiC58; pEAP37), C58 (pTiC58; pEAP2~9), C58 (pTiC58; pEAP40) and also C58 (pTiC58, ~ISV 10~~) .
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 ex~~mple Gemini viruses, such as Maize Streak Virus (MSV));
- natural viral DNA (for example CaMV);
- cDNA copies o:E viral RNA or viroid RNA (for example of Tobacco-Mosaics virus or Cadang-Cadang viroid);
- any lethal or viable mutants of viruses;
- cloned DNA under the influence of viral replication and/or expres:~ion signals;
- cloned DNA under the influence of eucaryotic replica-tion and/or expression signals;
- portions of viral D1NA;
- equivalents oi= the above-listed types of DNA in tandem form and 13409 z~
- equivalents of the above-listed types of DNA with incorporated Cargo-DNA.
The applic,~tion of the process according to the invention described in the example of the afore-described vector system h;ss, 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 Agrobac-terium rhizoctenes, to monocotyledons.
- The possibility of systemic infection of whole plants by using viral I~NA or equivalents thereof.
- Rendering in:Eectious 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 ex<~mple, 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 simplificai~ion of inoculation by avoiding DNA
purification, and drastic reduction of the amount of inoculum necessary fo:r inoculation.
- The possibil:Lty of transforming cells, tissues and whole plants, w:Lth 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 incor-porated 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 int:egratE~d virus genomes can a) be tran:cferred by sexual means to the descen-r dams;
b) prevent an infection by overinfecting viruses;
and 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 i~he expression of viral genes from the nuclear DNA :may offer the possibility of replacing viral DNA by foreign DNA in the virus causing the aecond infection; and Q) assist considerably in the better understanding of the host-parasite relationships and thus in the improved protectability of the plants.
Thus, this invenition includes a plurality of embodiments of t:he broad concept.
Using the above-described process, Cargo-DNA
incorporated into the virus genome can also be trans-ported into plant matcarial in which it proliferates. The ~ropagationin plants of the viruses, and thus also of the foreign gene transport=ed by them, is in particular most advantageous if t:he plants are to be propagated asexually or are: to bea protected against harmful in-fluences 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 r 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, with~~ut this implying any limitation, for example DNA of Caulimo viruses, including Cauliflower Mosaic Virus (C~~MV), 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 'ilirus (CLV), Curly Top Virus (CTV), Maize Streak Virus (MSV), Tomato Golden Mosaic Virus (TGMV) and Wheat Dwarf Virus (WDV).
Representaltives from the Caulimo viruses group, but especially Cauliflower Mosaic Viruses, are especially suitable for use within the scope of the process accord-ing to the invention, since owing to their genome structure (doub:le-stranded DNA) they are directly accessible to gESnetic manipulation.
All experiments ao far and the associated observa-tions argue than also representatives of the Gemini viruses, the genome of which is constructed from single-stranded (ss) DtJA, ca:n be used as vectors for transfer-ring genetic mai=erial. In addition, it is also known that in the course of the development cycle of the Gemini viruses double-:>tranded ds-DNA is formed in infected plants and that this ds-DNA is infectious [1~) Kegami M
et al., Proc. N~itl. Acad. Sci.. USA, 78: 4102, 1981].
Consequent7.y, alao representatives from the Gemini viruses group that foam ds-DNA in the course of their development cyc7_e and are thus accessible to direct r genetic manipulation are suitable within the scope of the present invention as carriers of foreign genetic mat-erial.
The present invention also relates to the use of Gemini viruses as markers, since successful gene trans-fers 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, pl~~nt tissue cultures or cell culture cells, with viral DNA ;end 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 from the transformation, and also to all hybridis-ation 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 viab:Le parts of those plants, especially pollen, ovules, zygotes, embryos or any other reproduc-tive material emerging from transformed ~xerm-line cells.

F

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, Amaryl-lidaceae, Asparaqacea.e, 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, Oryzae, Panicum, Saccharum, Secale, Setaria, Sorghum, Triticum, Zea, 1!riusa, 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 DN.A is first of all treated with restriction enz~,Tmes, 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 hyb~.idised (DNA-specific activities of from x 108 to l0 x 108 c.p.m./~,g) 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. Mo:l. 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 blacl~;ening an X-ray film for from 24 to 48 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
en~ome MSV-genome~s can be isolated from naturally occur-ring infected m~~ize 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 doable-stranded MSV-DNA using Klenow-polymerase I and an endogenous primer [18) Donson J et al., EMBO J, 3: 3069-3073, 1984].
Another po:~sibility consists of the isolation of double-stranded MSV-DNA ("supercoiled MSV-DNA) directly from infected lE:af material. Double-stranded MSV-DNA is formed as an ini~ermed.iate 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 v~~tro s!~nthesised 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 rE~sulting linearised DNA fragment is then isolated by gel-ealectrophoretic separation of the DNA mixture [20) Maniatis et al., (1982)].
In virus strains having two or more BamHI restric-tion sites, either the MSV-genome is partially digested or another suitable restriction site is sought that 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 tan~3em arrangement into the BglII 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 soluti~~n in excess. The preferred concentration ratio is in thia case 10:1 (insert: vector).
The plasmid pGA471 is a so-called shuttle vector, which is stably replicated both in E.coli and in Agrobac-terium tumefacinns 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 Agrobacterium tumefaciens.
This replication origin originates from the plasmid pTJS75, a plasm:id with a broad range of hosts and a tetracycline-re:~istance gene, a derivative of RK2 [21) An G et al., EMBO ~T, 4: 277-284, 1985].
Other characteristic properties of the plasmid pGA471 are:
1) The possession between the T-DNA border sequences of various restrici~ion sates that render possible incorporation o~E foreign DNA;
2) a cos-region of tlhe bacteriophage ~, which permits cloning of largE~ DNA :fragments (25-35 kb);
3) a chimar marker gene, composed of the control sequences of then nopa:lin 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 i:rom E.coli into Aqrobacterium tume-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 [20) Maniatis e~t al., (1982)] and then subjected to restriction ana:Lysis.
One of the "tandem clones" is selected and is transferred from E. coli DH1 to Agrobacterium tumefaciens (RifR) C58 (pTic~58) .
The transfer is carried out by "triparental mating", as described in detail in 23) Rogers SG et al. (1986).
In this case, r:ifampicin (100 ~,g/ml) and tetracycline (5 ug/ml) are used for t:he selection. The successful transfer of the dimeric MSV-genome is tested by Southern hybridisation [''4) Dh;aese P et al., Nucleic Acids Res. 7:
1837-1849, 1979;).
Actrobacter:ium tumefaciens (RifR) C58 (pTiC58) [25) Holsters ei_ 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 A robacaerium strain transformed in the manner described above has been given the following strain name:
Aqrobacterium tumefac:iens (RifR) C58 (pTiC58; pEAP 200).
Example 2: Con:atruct:ion of a control vector To constructs a control vector without T-DNA border a sequences, the plasmi.d pRK252 KanIII, a derivative of the plasmid pRK [26) Beva.n M, Nucleic acid Res., 12: 204-207, (1984)] is used, which contains no T-DNA border se-quences.
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 arrange-ment, with the aid of a SalI/BamHI adaptor, into the SalI
restriction sits°_ of the plasmid pRK252 KanIII.
The transfer of the control vector into Actrobac-terium tumefaci~ens (RifR) C58 (pTiC58) is carried out by "triparental mating", as described in detail in 23) Rogers SG e~t al., (1986).
The transf~~rmed Aqrobacterium strain has been given the following strain name: Agrobacterium tumefaciens (RifR) C5.3 (pTiC58; pEA 2:L ) .
Example 3: Construction of the bacterial vector pEAP 25 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 tra:nsposon Tn 903 (28) Grindley et al., 1980), which carries .a Kanamycin-resistance gene, the hybrid cosmid p;?2G1 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 sequence:a.
A 2.9 kb Sal I-B;st EII fragment that contains a gene coding for k anamycin-:resistance in plants (6) Paszkowski et al., 1984) is excised from the plasmid pCaMV6Km and exchanged for a 2.4 kh Sal I-Bst EII fragment from P22G1.
The final construction of pEAP 25 is carried out by integration of t:he plasmid pB6 previously cut with Sal I
into the Sal I »ncision site of pEAP 1. Plasmid pB6 was developed and made av<~ilable by J. Davies of the John Innes Institute, Norwich, England. This plasmid has r since been published in 29) N. Grimsley et al., 1987, under the name pMSV 1.2.
Plasmid pB6 contains a dimeric MSV-genome that has previously been cloned in the plasmid pACYC184 (30) Chang and Cohen, 1978).
Example 4: Construcaion 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 N.C., U.S.A..
Example 5: Manufacture of the bacterial vector pEAP 40 A 1.6 mer ~~f the MSV-genome [BglII-BamHI fragment (0.6 mer) + Bam:HI-BamHI-fragment, (monomer)] is spliced into the BamHI :restriction sites of the plasmid pTZl9R, which is descrilbed 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: Con:~truction of the bacterial vector pMSV 109 ~g of the plasmid pMSVl2, the construction of which has already been described in Example 3, are digested for a ~~eriod of 2 hours at a temperature of 37°C
with BamHI in a buffer solution (20) Maniatis et al., 1982). The 2.7 kb DN,A fragment resulting from this enzymatic digesl~ion ins, after electrophoretic separation of the sample in a 1~ agarose-TAE gel (40 mM tris-HC1, 20 mM sodium accatate, 2 mM EDTA), eluted from the latter and spliced into the ;single BamHI restriction site of the 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, arid a high T4-DNA ligase concentra-tion, are used in ordler to ensure a high rate of incor-poration 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 ~,1. Half of this ligation mixture is transformed into competent E.coli JM83 recA cells, and plated out onto "Luria Broth" (LB)-agar (20) Maniatis et al . , 1982 ) supplemented. with 50- ,ucyml of kanamycin sulphate and 40 ~Cg/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 the conjugation into Agrobacterium tumefaciens C58Na1R
(33) Hepburn et al., 1985), which is carried out in accordance with a process described by Ditta et al., 1980. The sele~~tion of exconjugants is carried out on LB-agar containing 50 ~,g/ml of kanamycin sulphate and 50 ~g/ml of nalidi:Kic acid. The selected colony, which in the inoculation experiments described hereinafter initiates an in:Eection in maize, is catalogued as pMSV 114.
Example 7: Construction of a control vector ~~pEA 2) without T-DNA border seguences To constru~~t the control vector pEA 2, the Sal I
restriction site of t:he plasmid pRK 252/kmIII, of a precur-sor-plasmid of ~?BIN19 (~b~ Bevan, 1984), is linked with the Sal I cut p:Lasmid pB6.
The selection of pEA 2 is carried out on the basis of the kanamycin (KmR)- and chloramphenicol (CmR)-resis--3~- 1340925 tance of the control vector.
Example 8: Cntroduction of the nlasmids pEAP 25 nEAP 37 and t~EA 2 ini~o Agrobacterium tumefaciens The plasmid p:EAP 25 is cloned in bacteria of the strain Escherichia coli GJ23 (pGJ28, R64rd11) (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 Acrrobacterium tumefaciens. The plasmids pEA 2, pEAP 37 and pEAP 40 are transferred via "triparental mating" into AQrobacterium tumefaciens (23) Rogers, S.G. Eat 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 (pTiC~58), pEAP 18) for the plasmid pEAP 25.
Wild-type strains of Actrobacterium tumefaciens can be obtained from the "Culture Collection of the Laborat-ory of Microbiology, Microbiology Department of the University of Ghent".
pEAP 25: The A robacterium 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. e~t al., 1985) by the 2.6 kb EcoRI-BglII
fragment of tlZe 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 pla:~mid p:EAP 25 does not replicate in Acxrobac_-terium tumefac:iens, the selection of the exconjugants on rifampicin, kanamyc.in and carbenicillin yields the new Acrrobacterium strain Acrrobacterium tumefaciens C58 (pTiC58, pEAP 29) in which the plasmid pEAP 25 has been integrated into the binary vector pEAP 18 by homologous recombination.

pEAP 37, pEAP 40: The mobilisation of the plasmide pEAP37 and pEAP40 from E.coli into Agrobacterium tumefaciens via "triparental mating" results in the construction of a binary vector system.
Ep A 2: The control plasmid pEA 2 is inserted into the Aarobacterium 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 des-cribed 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 micro-organisms for the purposes of patent procedure. A
declaration regarding the viability of the deposited samples was prepared by the said International Deposit-ories.

MicroorganismsDepositionDeposition Date of the viability Date Number certificate pEAP 37 16 June DSM 4147 19 June 1987 (Escherichia coli DH1 transformed with pEAP 37 plasmid-DNA) pEAP 40 16 June DSM 4148 19 June 1987 (Escherichia coli DH1 transformed with pEAP 40 plasmid-DNA) pMSV 109 23 Sept. NCIB 12547 24 Sept. 1987 (Escherichia 1987 coli JM 83Re~

transformed with pMSV 109 plasmid-DNA) Limitations on the availability of the said microorganisms have not been requested by the depositor.
Example 9: _C'.ulturin~ the Agrobacterium strains (Rif C58 (~pTiC58: pEAP
200y, i(Rif )C58 l~Ti('.58;~EA21). C58 (~TiC58. pEA 2) and C58 (~TiC58, pEAP
37).
C58 pTiC58, pEAP ~:9 ~ 58 (pTiC58, pEAP 40) and also C58 lpTiC58. pMSV1091 and the manufacture ~~f the inoculation solution Before inoculation, the, Agrobacteria strains are plated out onto YEB medium [Bacto beef extract S ;,/1, Bacto yeast extract 1/g/1, peptone 5/g/l, sucrose 5/g/l, MgS04 2mM, pH 7.2), which has been augmented beforehand with 100 ~g/ml of rifampicin and 25 ~g/ml of kanamycin or 50 ~g/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 earned out in 100 ml ..,-Erlenmeyer flasks in a liquid YEB medium that has been augmented with antibiotics in the afore-mentioned con-centration. 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/ml.
The bacteria cells are harvested by centrifuging and are then resusp~=_nded 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 d:iluent.
Example 10: StE~rilis,ation and ctermination of maize seeds For the inoculation experiments plants of the varieties Golden Cross Bantam, B 73, North Star and/or Black Mexican Sweetco:rn are used, all of which can be successfully agroinfected.
For the fo7Llowing experiments, as a rule 3-day-old, previously sterilised seedlings are used. The sterilisa-tion of the seedlings comprises the following process steps:
1. Sterilisation of ithe 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 s 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 aterile Macherey-Nage~ round filters having a diameter of 8.5 cm and also approximately 10 ml of sterile water.
20 seeds a:re 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 seedlincts Hamilton h~Tpodermic syringes (A 50 ~,1 or 100 ~1) fitted with exchangeable needles 0.4 mm in diameter are used to introduce the inoculation solution described under point 3. :into tlhe maize seedlings.
The inoculation aolution is taken up into the hypo-dermic syringe :in suclh a manner that no air bubbles are formed.
11.1. Inoculation of 10-day-old maize plants The inoculation of the bacteria-containing sus-pension into 10--day-o:Ld maize plants is carried out by various methods and ait different sites on the plant.
1. Application of 20 ~1 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: (pos:ition A in diagram 1) .

J
1349 ~5 2. Injection o~f 10 Nil of the bacterial suspension using a 100 ~,1 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 l.igula of the primary leaf (position C in diagram 1) c) at the base of t:he plant in the so-called root collar, a merist:ematic tissue from which adven-titious 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 ~cl Hamilton hypodermic syringe.
1. Injection ~~f the bacterial suspension into the coleoptilax-node by introducing the hypodermic needle through the coleoptile, starting from the apical col~~optile tip and passing into the region of the coleop~til~ node (position E in diagram 2).
2. Injection of the bacterial suspension directly into the coleop~~ile, 2 mm below the apical coleoptile tip (position 1~ in diagram 2).
3. Injection of the bacterial suspension directly into the coleopi~ile, 2 mm above the coleoptile node (position (z in diagram 2).
4. Injection of the bacterial suspension directly into the coleopt:ils'~ node (position H in diagram 2) .

5. 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 th~~ 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 reaion 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 i=he co:leoptihr node 3. 2 mm above i~he co:leoptilar node 4. 5 mm above i~he co:leoptilar node.
The decapii:ated ;seedlings are then planted in moist earth and culti~rated .in accordance with the conditions given under point 6.
The actual inocu:Lation experiments with Aarobac-terium are carried oust on seedlings in which the coleop-tile tips have, in preparation, been removed 2 mm above the coleoptil~' node.
Example 12: Cu7.tivatina the treated maize plants and maize seEadlinas 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 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-HC1, 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 ~E°C in a table centrifuge at maximum speed. The sups:rnatant is discarded and the pellet is resuspended in 0.5 ml of ice-cold SET (15% sucrose, 50 mM
Na3 EDTA, 50 mM tris-HC1, pH 8) while stirring first of all with a sterile toothed rod, and then briefly using a vortex mixer (5 seconds). Subsequently, 10 ~,1 of a 20%
SDS solution andl 100 ~cl of proteinase K (20 mg/ml) are added and mixed in and the whole is then heated in the small tube for 1.0 minutes at 68°C. After the addition of 3M sodium acetate (1/7_0 volume) the lysate is extracted twice with phena~l/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 ~,1 of TE buffer (4Ci mM tris-HC1, 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).

-3$- 1340925 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 x 108 to 10 x 108 c.p.m./~g) with the DNA to be detected, which has previously been subjected to a nick-translation [231 Rigby, W.J., Dieckmann, M., Rhodes, C.
and P. Berg, J.lKol.Biol. 113, 237-251)]. The filters are washed three times for an hour each time with an aqueous solution of 0.0:3M sodium citrate and 0.3M sodium chloride at 65°C. The h~~rbridised DNA is made visible by blacken-ing an X-ray fi:Lm 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 lams pEAIP 37 IpMSV 109 ~pEAP 200 IpEAP 25 A ~ 0/46 C <2%) ~ - I - I -B ~ 0/44 C<2%) ~ - ~ - I -C ~ 3/46 (6.5%)~ - ~ + ~ +
D ~ 42/68 (62%)~ 26/65 ~ ++ ~ ++
(40%) i i i i i i The result~~ in Table 1 show clearly that the preferred site of app7Lication on the plant is located in the region of the root, collar, where 62% and 40% of the 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 t:he 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 number of plants with symptoms/
site number of inoculated plants pEAP 37 I pEAP 40 E ~ 21/27 (78%) ~ __-0/20 (<5% ) ~ ___ G ~ 3/19 (16%) ~ _-_ H ~ 25/30 (83%) ~ 51/58 (88%) I ( 8/51 (16%) ( ---1/20 ( 5%) ~ ---2/12 (17%) ~ _-_ 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 clear:Ly preferred by comparison with with all other application sites investigated. Whether the suspension is injeacted directly into the coleoptile node laterally, or is injected indirectly through the coleoptile, is clearly of no significance.

-4~- 1340925 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 2 ~ 5/8 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 coleo~ptile 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/I
number of inoculated plants L ( 48/49 (98%) 14/44 (32%) t i The result:a in Table 4 show clearly that position L
on the decapitai~ed seedling, that is to say the meris-tematic tissue region, is distinctly preferred to 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 concentra-tions indicated below.
dilution estimated number of number of plants bacteria remaining in with symptoms/
t:he inoculation site number of inocu-lated plants undiluted 2 x 106 84/102 (82%) 1 2 x 105 42/55 (76%) 10 2 2 x 104 34/54 (62%) 10 3 2 x 103 19/56 (34%) 10 4 0 0/10 (QQ%) 10 5 0 0/10 (ap%) Assuming that the number of copies of the binary vector that conitains 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 MS'V-DNA.
This means that ,~larobacterium transfers its DNA to maize with an e~Eficie;ncy comparable to that with which it transfers its DtJA to dicotyledonous host plants.
E) Aarobacterium host range Apart from maize, it was possible to ascertain other representatives from ithe Gramineae class that are accessible to infection by Aarobacterium.
The results of inoculation experiments with these Gramineae speciea are shown in Table 5:

Table 5 Gramineae species number of plants with symptoms/

number of inoculated plants barley (Maris Otter) 1/15 ( 6%) wheat (Maris Butler) 1/40 ( 2%) wheat (normal) 1/25 ( 4%) sing oats (Saladin) 1/25 ( 4%) Panicum milaceum 3/8 (35%) Digitaria sanguinalis 2/10 (20%) Lolium temulentum 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 Actrobacterium .
F) Agrobacterium strains In addition to the Acrrobacterium 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 Agrob<~cterium strains listed in Table 6 to detect transfer of MS'V-DNA to maize:

Table 6 Aqrobacterium number of plants with symptoms/

strain number of inoculated plants pMSV 109 *1 pEAP 37 *2 A. tumefacien:~

_ T 37 3/6 (50%) 6/6 (100%) LBA 4301 (pTiC58) 21/23 (91%) 15/21 ( 71%) A 6 0/8 (<1%) 2/37 ( 5%) A. rhizo enes R 1000 17/22 (81%) ---LBA 9402 15/20 (75%) ---2626 7/ 12 (51%) ---*1 The inoculation e:~periments with pMSV 109 were carried out on 10-day-old maize plants *2 The inoculation e:~cperiments with pEAP 37 were carried out on 3-day-old maize seedlings.
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Claims (51)

1. A process for inserting genetic material into monocotyledonous plants of the family Gramineae or viable parts thereof, which process comprises (a) integrating the genetic material to be introduced into a T-replicon of a transfer microorganism of the genus Agrobacterium in the vicinity of one or more T-DNA
border sequences, the distance between the genetic material to be integrated and the T-DNA
border sequence(s) being chosen such that the said genetic material is transferred into plant material;
(b) causing the T-replicon to be taken up in a transfer microorganism of the genus Agrobacterium, the replicon passing; into the said transfer microorganism and growing the said transfer microorganism in a culture medium commonly used for the cultivation of Agrobacteria;
(c) separating the grown Agrobacteria and resuspending them in an inoculation solution;
(d) infecting the said monocotyledonous plants of the family Gramineae or viable parts thereof with the Agrobacteria prepared according to steps (b) to (c).

2. A process according to claim 1, wherein one or more sub-culturing steps are carried out prior to the preparation of the inoculation solution.

3. A process according to claim 1, wherein 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 and optionally one or more sub-culturing steps lasting for a period of from 15 to 30 hours are carried out at a temperature of from 15° to 40°C.

4. A process according to claim 3, wherein the incubation period for the culturing of the transfer microorganism and optionally for the sub-culturing steps is from 40 to 50 hours.

5. A process according to claim 3, wherein the incubation temperature for the culturing of the transfer microorganism and optionally for the sub-culturing steps is from 24° to 29°C.

6. A process according to any one of claims 1 to 3, wherein a culture medium solidified with agarose or alginate is used.

7. A process according to claim 1, wherein the said microorganism suspension is inoculated repeatedly.

8. A process according to claim 1, wherein 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.

9. A process according to claim 1, wherein the said recipient plant is at a stage of development between seed germination and the 4-leaf stage.

10. A process according to claim 9, wherein the plant is in the first, second or thins day of the germination phase, the distance between the scutellar node and the apical coleoptile tip being approximately from 1 to 3 cm.

11. A process according to claim 1, wherein the inoculation of the transforming microorganism suspension its carried out by injection into a meristematic tissue region of the plant.

12. A process according to claim 11, wherein the transforming microorganism suspension is injected in the region of the root collar of plantlets that are already differentiated into root, stem and leaves.

13. A process according to claim 11, wherein the transforming microorganism suspension is injected into the seedling in the immediate vicinity of the coleoptilar node.

14. A process according to claim 13, wherein the transforming microorganism suspension is injected directly into the coleoptile node or within an area of approximately 2 mm above or below the coleoptilar node.

15. A process according to claim 11, wherein the transforming microorganism suspension is injected directly into a meristematic tissue region of the coleoptile after decapitation of the coleoptile tip in an area that is firm 1 to 5 mm above the coleoptilar node.

16. A process according to claim 1., wherein the said genetic material is viral DNA which optionally may contain incorporated Cargo-DNA.

17. A process according to claim 16, wherein a) viral DNA or its equivalents is (are) isolated from infected plant material and cloned 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 constrict 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 transfer microorganism of the genus Agrobacterium in order to construct a vector system that can be used for plants, d) monocotyledonous plants of the family Gramineae or viable parts thereof are infected with the so-modified vector system.

18. A process according to any one of claims 16 or 17, wherein double-stranded DNA
forms of single-stranded DNA viruses are used as viral DNA.

19. A process according to claim 18, wherein DNA of Gemini viruses is used as the viral DNA.

20. A process according to claim 19, wherein 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.

21. A process according to any one of claims 16 or 17, wherein the viral DNA
used is natural viral DNA.

22. A process according to claim 21, wherein DNA of Cauliflower Mosaic Virus is used as the viral DNA.

23. A process according to any one of claims 16 or 17, wherein cDNA copies of viral RNA are used as the viral DNA.

24. A process according to any one of claims 16 or 17, wherein cDNA copies of viroid RNA are used as the viral DNA.

25. A process according to any one of claims 16, 17, 19, 20, or 22, wherein DNA of lethal or viable mutants of viruses are used as the viral DNA.

26. A process according to claim 16, wherein cloned DNA that is under the control of viral replication signals is used as the viral DNA.

27. A process according to claim 16, wherein cloned DNA that is under the control of viral expression signals is used as the vial DNA.

28. A process according to claim 16, wherein cloned DNA that is under the control of viral replication and expression signals is used as the viral DNA.

29. A process according to claim 16, wherein cloned DNA that is under the control of eucaryotic replication and expression signals is used as the viral DNA.

30. A process according to claim 16, wherein portions of viral DNA are used as the viral DNA.

31. A process according to claim 16, wherein the viral DNA is used in tandem form.

32. A process according to any one .of claims 16, 17, 19, 20, 22 and 26 to 31, wherein the viral DNA or equivalents thereof is (are) used in tandem form.

33. A process according to any one of claims 16, 17, 19, 20, 22 and 26 to 31, wherein viral DNA with incorporated Cargo-DNA. is used.

34. A process according to any one of claims 16, 17, 19, 20, 22 and 26 to 31, wherein viral DNA or equivalents thereof with incorporated Cargo-DNA is (are) used.

35. A process according to any one of claims 16 or 17, wherein the T-replicon used is a Ti-plasmid or an Ri-plasmid from a bacterium of the genus Agrobacterium.

36. A process according to claim 1, wherein a bacterium is used as a transfer microorganism that accommodates a T-replicon which is a Ti-plasmid or an Ri-plasmid from a bacterium of the genus Agrobacterium.

37. A process according to claim 16, wherein the said Cargo-DNA consists of genomic DNA, of cDNA or of synthetic DNA.

38. A process according to claim 16, wherein the said Cargo-DNA is composed of genomic as well as of cDNA and/or synthetic DNA.

39. A process according to claim 16, wherein the said Cargo-DNA is composed of gene fragments of several organisms that belong to various genera.

40. A process according to claim 16, wherein the said Cargo-DNA is composed of gene fragments of more than one strain, one variety or one species of the same organism.

41. A process according to claim 16, wherein the said Cargo-DNA is composed of portions of more than one gene of the same organisms.

42. A process according to claim 1, wherein there are used as viable parts of said monocotyledonous plants of the family Gramineae plant tissue cultures or cell culture cells.

43. A process according to claims 1, wherein there are used as plants or viable parts of plants maize, rice, wheat, barley, rye, oats or millet.

44. A process according to claim 1, wherein there are used as plants or viable parts of plants those from the following genera: Avena, Hordeum, Oryzae, Panicum, Saccharum, Secale, Setaria, Sorghum, Digitaria, Lolium, Triticum, Zea or pans of those plants.

45. A process according to claim 1, wherein protoplasts are incubated together with the transfer microorganism.

46. Use of a transfer microorganism of the genus Agrobacterium or relevant parts thereof in the process according to claim 1.

47. The plasmid pEAP 37 or pEAP 40 or a transfer microorganism of the genus Agrobacterium containing one of those plasmids.

48. The plasmid pMSV 109 or a transfer microorganism of the genus Agrobacterium containing that plasmid.

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

50. 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.

51. The transformed Escherichia coli strain JM 83RecA a sample of which has been deposited under the deposit number NCIB 12547, and all derivatives and mutants thereof that still possess the characteristic properties of the transformed strain.