JPS63141590A - Method for inserting genetic substance in monocot or growth part thereof - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention describes a novel method for inserting genetic material into monocots or their growing parts using suitable transfer microorganisms. The present invention relates to the expression of the inserted genetic material in growing parts and to the transgenic plants obtained by the above method.

[Conventional technology]

In view of the rapid growth of the world's population and the associated increasing demands on food and raw materials, increasing the yield of useful plants and increasing the extraction of plant reserves, advances in the field of food and medicine are essential. , is one of the most urgent challenges of biological and biotechnological research. In this regard, for example, the following should be stated as substantive objectives: improving the resistance of useful plants to unfavorable soil conditions or to diseases and pests, plant protection agents such as insecticides, herbicides, fungicides. and improving the resistance to bactericides, and advantageously altering the vegetative anastomosis or yield of plants, etc.

Such desirable effects are generally due to the introduction or increased formation of stimulants, important proteins or toxins.
and can result from intervention in the control systems of plant metabolism. Influencing the genotype of a plant is carried out, for example, by transferring new genes into the whole plant or into plant cells.

Many of the most important cultivated plants from an agricultural economic point of view belong to the class Monocot and the Poaceae family, which includes our most important types of cereals, such as wheat, barley, rye, oats, maize, rice and millet, among others. Special mention should be made of plants.

[Problem that the invention seeks to solve]

In monocotyledonous plants, the biggest problem with using recombinant DNA technology is that with the help of which transformation frequencies can be achieved sufficiently high for practical applications and the ability to specifically introduce into the plant genome. There is a lack of suitable transfer microorganisms that can thus be used as adjuncts for such insertions.

Agrobacterium teumefaciens (Agrob)
acterium tumefaciens, hereafter A.

tumefaciens), one of the most used transfer microorganisms for inserting genetic material into plants, is particularly suitable for the genetic manipulation of many dicotyledonous plants, but the monocotyledonous representatives, especially It has not hitherto been possible to achieve correspondingly satisfactory results with monocotyledonous cultivated plants from the monocotyledons, and hitherto only a few selected families of plants have produced A. tumefacie.
It is only known that genetic manipulation can be used, at least in theory, in response to infection in ns. However, these families are insignificant, uncultivable groups that, from the point of view of agricultural economics, are at most important as model plants [References: 1
), 2), 3), 4) See the list of references below, the same applies hereafter].

Recently developed transformation methods are based on direct insertion of foreign DNA into plant protoplasts, such as "direct gene transfer J (5), 6).

7), 8), 9)) or M.R.I.R.9 Ize: Jie, Kushin (10), 11)] is a problem in many plant species, especially in the grass family, where the capacity is limited. It must be considered that regeneration from plant protoplasts is
Until now, there are still virtually unresolved issues.

However, the grass family includes cultivated plants that are most important from an agricultural economic point of view, such as grained wheat, barley, rye, oats, maize, rice, millet, and are of particular economic importance; Regardless of the above-mentioned limitations, the development of methods by which the representative cisternae of the Poaceae family can be directly genetically modified must be regarded as a matter of urgency.

[Means for solving problems]

Surprisingly, within the scope of the invention by simple means,
I was able to solve this problem.

Contrary to all expectations, during the studies carried out within the scope of the present invention, by using suitable culture and application methods, plants of the monocotyledonous group were also exposed to certain transfer microorganisms, such as A.

It has surprisingly been found that important representatives of the monocotyledonous group, especially cultivated plants belonging to the Poaceae family, can also be transformed by the transfer microorganisms using P. tumefaciens in a specifically guided manner. I understand.

Particular attention has been paid to the regional expansion of the host range of A. tumefaciens to include grasses, thereby allowing direct and specifically targeted genetic manipulation even in representative species of this family.

Plants transformed in this method can be identified by appropriate evaluation methods. Plant pathogenic viruses such as maiz
Streak virus (Maize 5treak
The use of Virus (MSV) has proved particularly suitable for this purpose, whereby successful transformation can be very effectively confirmed by the symptoms of the disease that appear.

In one of its aspects, the invention therefore provides a transfer microorganism that contains genetic material in transportable form and that is capable of inserting the genetic material into monocots or viable parts thereof. be used for the infection of monocot plants by using suitable culture and application methods that allow the induction of toxic gene action, and infecting the monocots or their viable parts with said transfer microorganism. The present invention relates to a method for inserting genetic material into monocotyledonous plants or their growing parts.

Within the scope of the above method, transfer microorganism koji such as A
, ttunefaciens is advantageously grown in a stirred liquid culture for 30 to 60 hours at a temperature of 15 to 40° C. in one of the nutrient media commonly used for culturing microorganisms. The preferred growth temperature is between 24 and 2
It is 9℃. One or more subculture steps are then carried out, preferably in the same medium, advantageously at a dilution ratio of 1:20, each step lasting from 15 to 30 hours, preferably from 18 to 20 hours. In these cases, the culture temperature is between 15 and 40°C, preferably between 24 and 29°C.

When using thermophilic microorganisms, the growth temperature is clearly 40
The temperature may be ℃ or higher.

Other cultivation methods suitable for the propagation of transfer microorganisms carried out within the scope of the invention are obviously also possible.

For example, for culturing the transferred microorganisms, solid media may also be used, which media may be prepared using, for example, agarose or alginate or any other suitable solidifying agent.

To prepare the inoculation solution, the cells are centrifuged and resuspended in a suitable inoculation medium, for example 1/20 part by volume (12) of MSSP medium, to a certain concentration suitable for inoculation.

Infection methods can be carried out by contacting the plant material with said transferred microorganisms, for example by incubation with protoplasts, by wounding the tissues of the whole plant or parts of it, or in particular by injection of a microbial suspension directly into the plant. Starting according to the invention.

Particularly preferred is injection of the inoculum solution in the area of the growth zone, preferably in the area of the stem and leaf sheath of the plant.

Within the scope of the present invention, the frequency of transformation of inoculated plants will depend to a crucial extent on the site of application to the plant, but also on the growth stage of the particular plant tested, as well as on other factors. Even more surprisingly it became possible to show that it also depends on particular factors.

An important part of the invention therefore relates to a more complex differentiation of the sites of application to the plants and the particularly targeted application of the transforming microorganism-containing inoculum solution to precisely defined sites on the plants, so that Significantly increases the frequency of transformation of inoculated plants. Furthermore, the frequency of transformation is
It can also be increased by appropriate selection of the time of application with respect to the developmental stage of the plants to be inoculated.

The invention also particularly relates to a novel method for inserting genetic material into monocot plants or their growing parts, i.e. the method is capable of inserting said genetic material into monocot plants or their growing parts. , and is characterized in that the transfer microorganism containing the genetic material to be inserted in transportable form is inoculated in the form of a microbial suspension into the meristem region of said plants or their growing parts.

In order to ensure a clear and consistent interpretation of the detailed description and claims of the invention and the claims, definitions within the scope of the invention are set forth below.

Transfer microorganism: a microorganism that transfers part of its own DNA to plant material (e.g. A, tume-fac
ens).

T-replicons: can be transferred in whole or in part into plant cells with the aid of genes localized in the replicon itself or in other replicons present in the same microorganism (e.g. A,
tumefaciens Ti-plasmid) replicon (13)).

In products, DNA is added to plant material with the aid of microbial action.
) DNA sequence that results in transfer.

CarboDNA: DNA artificially inserted into a DNA vector Genomic DNA: DNA derived from the genome of an organ cmDNA: A copy of mRNA produced by reverse transcriptase.

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

Heterologous gene or DNA: a DNA sequence that encodes a particular product or biological function and originates from a different species than the one into which the gene is inserted; the DNA sequence is also referred to as a foreign gene or foreign DNA .

Homologous gene or DNA: A DNA sequence that encodes a particular product or biological function and originates from the same species into which the gene is inserted.

Plant cell culture: Culture of plant units such as protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of growth.

Plants: Any photosynthetically active member of the plant kingdom characterized by an Il nucleus, genetic material organized in the form of chromosomes, capsular cytoplasmic organ 2, and the propensity to undergo meiosis.

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

Protoplast: A naked plant cell separated from a plant cell or tissue and lacking a cell wall that has the ability to regenerate into cell clones or whole plants.

Plant tissue: A group of plant cells organized into structural and functional units.

Plant Organs: Parts of a plant that are limited and clearly visible to the naked eye, such as roots, graves, leaves or embryos.

Fully transformed plant: A plant in which the genome of each cell has been transformed in the desired manner.

In particular, the present invention relates to an improved method for transforming monocot plants using a transformable strain of Agrobacterium, which method includes the timing of inoculation with respect to the growth stage of the plants to be inoculated, and the growth zone. The site of inoculation in the area of 1. is characterized in that the site of inoculation in the area of 2 is adjusted in such a way as to significantly increase the rate of transformation that can be achieved by comparison with known methods.

According to the invention, with regard to the growth stage of the plants to be infected for applying the transforming microorganism-containing suspension, the preferred time is the period starting from the growth of the plant embryo and ending at the doubling stage. , and throughout the growth and growth (differentiation) phases of the plants thus infected.

A plant that has reached a stage of growth between seed germination and the four-leaf stage,
Particularly suitable for the application according to the invention.

In another embodiment of the invention, the inoculation of the transformation inoculum solution containing the microorganism is carried out on the immature growing embryo after pollination of the ovule and fertilization by the sperm nucleus, but preferably before the formation of the seed coat. .

The distance between the blastoderm and the apical coleoptile is 1 to 2 mm.
Seedlings that are about 3 days old are more preferred.

Particularly preferred, however, are plants at a stage of growth in which the coleoptile tubes are clearly identifiable.

Inoculation of the transformation suspension containing the microorganisms is preferably carried out in areas of the plant containing meristems. These are the parts of the tissue that are active in terms of division and metabolism, and in particular contain the totipotent embryonic cells from which all somatic cells and tissues differentiate and are ultimately the starting point for the growth of embryonic cells.

By using the method according to the invention, transgenic y; (transg
enic) plants, but also plants containing transformed embryonic cells from which transformed ovules and/or pollen can grow, especially during the differentiation of other cells and tissues.

After fertilization with the addition of transformed ovules and/or transformed pollen, seeds are obtained that contain genetically converted embryos and can be used to produce genetically converted plants.

A particularly suitable application site for the introduction of a suspension containing the transforming microorganism into a plant that has already differentiated into stems, roots and leaves is between the roots and the stem, the so-called root collar.
tcollar).

Repeated application of the inoculum solution containing the transforming microorganism to the meristem region of the plant is particularly preferred within the scope of the invention.

In a particular embodiment of the invention, application of the suspension containing the transforming microorganisms is effective on seedlings about 1 to 3 days after germination. Preferred application sites are coleoptiles and plate sections.

Very good transformation results can be achieved by application in the vicinity of the coleoptile tube or, in particular, by direct application to the coleoptile tube.

Therefore, another particularly preferred embodiment of the invention provides that the application of the inoculum solution containing the transforming microorganisms is carried out 1 to 3 days after germination near the coleoptile or directly to the core of the seedlings. Features.

The introduction of the inoculum solution containing the transforming microorganism into the plant can be carried out by methods that can vary widely, for example by artificially wounding the epidermal tissue and instilling the transforming suspension containing the microorganism into the wounded tissue. Alternatively, the transfer microorganism and plant protoplasts may be cultured together.

Injection of the inoculum solution using a hypodermic syringe is preferred, which allows a very precise and specifically directed application to precisely defined areas of the plant.

In general, a hypodermic syringe with an exchangeable needle having a cross-section of 1 to 0.5 mm is used, depending on the requirements and special needs of the plant species concerned and the growth stage at the time of application. used. The application volume also varies depending on the plant species and growth stage involved, ranging from 1 to 20 μm, with 5 to 10 μm being preferred.

Naturally, other suitable aids for the targeted application of the columbar solution to the plants can be used, for example by means of ultra-fine glass cavities, using micromacobrators, to achieve a minimum application rate on the plants. It can be applied to precisely defined tissue areas (eg meristems).

The method of applying the inoculum solution to plants or seedlings may vary as well, but can be easily optimized for different plant species. These optimization tests can be performed within standard optimization programs according to the guidelines of the present invention by those skilled in the art without requiring any additional expense.

In addition to the parameters mentioned above, the concentration and developmental stage of the inoculated transfer microorganism are also important with respect to the efficiency of transformation. The preferred concentration is an inoculum solution ranging from 1 to 1010 cells/d. 107 to 109 pieces/
Particularly preferred is the inoculum solution of d.

Dilution experiments carried out within the scope of the present invention have shown that as the dilution of the inoculum solution increases, the frequency of transformation decreases. D imparted by Agelobacterium to monocots
The frequency of N-transfer is D to the dicotyledonous host plant.
NA) Same order as transfer (see D in results section).

Possible variations within the scope of the method according to the invention result, for example, in the choice of application method, the depth of drilling into the plant tissue, the composition and concentration of the bacterial suspension, and the inoculation carried out for each infection. There are a number of

In another particular embodiment of the invention, the application of the solution containing the transforming microorganism is carried out directly on the tissue of the coleoptile tube after cutting off the coleoptile tip in the region of the coleoptile tube. Most plumules can be removed without adversely affecting other growth of the seedlings.

A preferred method of application in this case also involves the use of a hypodermic syringe, the depth of the perforation being able to be varied within a certain range in relation to the removal of the truncated area of the coleoptile tube. However, direct inoculation into the coleoptile tissue is preferred in all cases.

Application of the inoculum solution can be carried out either to the peripheral parts or especially to the central part of the exposed coleoptile tissue, the area of the meristem being particularly preferred.

If immature embryos are used, apart from the inoculum solution method already described, it is also possible to use a method in which the embryos are first removed from the mother plant in the preparation and then brought into contact with the transfer microorganism in a suitable medium. It is possible (14)).

Suitable transfer microorganisms which can transfer genetic material into monocots and which can be used in the method according to the invention are in particular microorganisms containing T-replicons.

Microorganisms containing T-replicons are understood in particular to be bacteria, preferably soil bacteria, and among these in particular of the species Agrobacterium.

Naturally, only harmless bacterial strains can be used within the scope of the method according to the invention, such as bacterial strains which cannot survive in the natural environment or which pose no environmental problems.

Suitable T-replicons are especially bacterial replicons, such as Agerobacterium replicons, especially Agrobacterium Ti- or Ri-plasmids.

The Ti-plasmid has two regions that are essential for the production of transformed cells.

In dicots, one of these is the transfer DN that transfers into the plant and introduces the mfs.
This is area A. The other region is the virence donor (vir) region, which is essential not only for growth but also for tumor maintenance. The transfer DNA region can be increased in size by incorporating foreign DNA without compromising its ability to transform. By removing the tumor-causing gene, the resulting gene-transformed plant cells remain non-neoplastic, and by incorporating a selective marker, the modified Ti-plasmid transfers the genetic material into suitable plant cells. can be used as a vector for

The vir region is a component of the Agrobacterium incorporation into the genome of a plant cell, regardless of whether the T-DNA region and the vir-region are present on the same vector or on different vectors within the same Agrobacterium cell. T-D of
Achieve transfer of NA area.

Similarly, the vir region on the chromosome is T-D from one vector.
Directs the transfer of NA into plant cells.

Preferred is a system for transferring the T-DNA region from Agrobacterium to a plant cell, characterized in that the vir region and the T-DNA region are on different vectors. Such systems are known as ``primary vector systems'', and vectors containing T-DNA are referred to as ``primary vectors''.

T-DNA-containing vectors that can be transferred into plant cells and that allow detection of transformed cells are suitable for use within the scope of the present invention.

Plant cells or plants transformed according to the invention are
Selection can be made using appropriate phenotypic markers. Examples of such phenotypic markers include, but are not limited to, antibiotic resistance markers such as kanamycin resistance genes, hygromycin resistance genes, or herbicide resistance markers.

Other phenotypic markers are known to those skilled in the art and may similarly be used within the scope of the present invention.

A preferred embodiment of the invention relates to a novel and generally applicable method of genetic modification of monocots by inserting viral DNA or its equivalent into whole plants or their growing parts.

There has already been some success in integrating selected DNA fragments into viral DNA and then inserting the fragment together with the virus into another organism. On the other hand, under natural conditions many plant viruses are transferred by insects that feed on infected and non-infected plants and result in infection of new plants, but this method does not control direct and planned transfer. It's too expensive and too difficult to do. For example, such a method would require a large number of insects reared under controlled conditions. Moreover, especially due to the large number of plants, planned virus infection is extremely difficult to carry out.

The methods of mechanically infecting leaves with viruses used in genetic engineering are only infective in the presence of cloned viral DNA, but are only practical to a limited extent because in many cases this is not possible. Can be applied. Although it is possible to clone and study specific types of viral genomes within bacteria, such as single-stranded DNA viruses (16) obtained by cloning double-stranded DNA, Many viruses that have been infected cannot be reinserted into plants or used to infect plant material.

Therefore, this also applies to the use of methods such as in vitro mutated and recombinant DNA for basic research,
and prevent the use of such viruses as carriers of selected foreign DNA.

Such problems do not arise when using the method according to the invention described below.

Constructs comprising one or more viral replicons or portions of viral replicons inserted in a manner that allows release and replication of the viral replicon in the plant separate from the chromosomal DNA are particularly preferred in the present method.

Therefore, this arrangement of the viral replicon allows for the release of infectious viral DNA based on intramolecular recombination via transcription, reverse transcription or other methods of rearranging the genetic material.

Within the scope of the invention, a T-replicon, e.g. a cell flanking one or more T-DNA border sequences, a virus D
NAFor example, D of Mize Streak Virus (MSV)
Contains NA, as desired) Contains cargo-DNA, contains viral DNA,! : Ti-plasmid or Ri-plasmid of Agrobacterium, with the distance to the T-DNA border sequence chosen such that the viral DNA, including the optional cargo DNA, is transferred into the plant material.

As cargo-DNA, either homologous or heterologous genes or DNA as well as synthetic DNA may be used according to the definition given within the scope of the present invention.

The encoding DNA sequence is derived from genomic DNA by c, p
It can be composed only of NA or synthetic NA. Another possibility is to consist of a hybrid DNA sequence consisting of both cDNA and genomic DNA and/or synthetic DNA.

In that case, the cDNA may be derived from the same gene as the genomic DNA, or one of the cDNA and the genomic DNA may be derived from a different gene.

However, in either case both genomic DNA and/or cDNA may each be produced separately from the same or different genes.

If the DNA sequence contains more than one segment, these genes may be derived from more than one species and the same organ, or from several organs belonging to more than one strain. It may be derived from a return tube belonging to the same or different indicator unit (kingdom).

The different parts of the DNA sequence are joined to each other to form the complete coding DNA sequence in a manner known per se.

Suitable methods include, for example, in vivo recombination of DNA sequences with homologous parts and in vitro ligation of restriction fragments.

The method according to the invention comprises: a) Viral DNA containing optionally incorporated cargo-DNA, such as Myzu Streak Virus (MSV).
), T-replicons such as Agrobacterium Ti
- in the vicinity of one or more T-DNA border sequences in the plasmid or Ri-plasmid, the viral DNA
b) the distance between A and said T-DNA border sequence is selected such that the viral DNA, including the optional cargo DNA, is transferred into the plant material; b) subsequently said T-replicon is transferring the replicon into a suitable transfer microorganism such that it is absorbed into the transfer microorganism, and producing a modified transfer microorganism according to b).

It becomes substantial because it infects monocot plants.

This method involves the introduction of inserted viral DNA, including cargo-DNA that may be present, after the action of microorganisms that promote the transfer of plasmid DNA into plants has been induced.
is also transferred.

The method according to the invention comprises: a) viral DNA or its equivalent (see below);
The DN is isolated from infected plant material, e.g. Triticum spp., and in a vector in a suitable bacterium, e.g.
A) and b) the cloned virus D.
Viral DNA and T-D
of one or more T-DNA border sequences, the distance from which the T-DNA border sequences are selected such that the viral DNA, including the cargo-DNA that may be incorporated into them, is transferred into the plant material. 1 localized nearby
C) forming a plasmid (Bap) comprising one or more viral genomes or parts of viral genomes and cargo-DNA optionally contained therein; C) transferring said plasmid Bap to a suitable transfer microorganism (e.g.
tumefaciens or A.

rhizogenes) to form a vector system which can be used in plants; d) with the thus modified vector system, substantially consisting of infecting whole monocot plants or their growing parts; .

The present invention provides vector systems, such as the vector systems of C) above, and new vector systems, such as A, tum, for the controlled transformation of monocotyledonous plants or their growing parts.
efacien5j strain (Rif) C58 (pTiC5
8; pEAP2o0) and A, tume-facie
ns” C58 ('pTiC58; pEAP37),
C58 ('pTiC58; pEAP29), C3B (
pTiC58; pEAP 40 ) and C58 (pT
ic58. MSV109) bacteria.

A, tumefacien5t: C58(('pT
iC58; pEAP37), C3B('9TiC58
: pEAP29), C58 (pTiC58; pE
AP4 o ) and C3B (:pTiC58, MSV
109) strains of bacteria are highly preferred.

Within the scope of the invention, viral DNA and its equivalents F
i is particularly understood as the following types of DNA: Single-stranded D
Double-stranded DNA bodies of NA (e.g. dieminiviruses, e.g. Miz Streak Virus (MSV)), cDNA copies of natural viral DNA (e.g. CaMV), viral RNAI and viroid RNAs (e.g. tobacco mosaic virus or Kadan-kadan). Viroid (('adang -Cad an gvir
(oid)) - Cloned DNA under the influence of lethal or viable mutant virus replication signals and/or expression signals - Cloned DNA under the influence of eukaryotic replication and/or expression signals - Viral DNA - equivalents of the above type of tandem DNA, and - equivalents of the above type of DNA with inserted cargo-DNA.

The application of the method according to the invention as described in the example of vector systems above has, for example, the following important advantages: Transfer microorganisms specific for common dicots, such as A. tumefaciens or A. rhiz
Expanding the host range of o-genes to monocots.

- Possibility of systemic infection of the entire plant by viral DNA or their equivalents.

- Make viruses that could not be infected artificially (e.g. MSV) infectious and avoid the use of natural vectors, e.g. insects.

- Possibility to manipulate viral DNA in bacterial systems such as E. coli.

・Expanding the range of hosts for the virus.

- Avoids DNA purification and simplifies inoculation by significantly reducing the amount required for inoculation.

- The possibility of transforming cells, tissues and whole plants, with the result that the limitations resulting from the difficulty of regenerating whole plants from pro)doplasts are overcome.

- T-DNA containing selected viral DNA can be inserted into the host genome under the control of bacterially encoded functions. Thus, in many cases, after transformation with bacteria, whole plants can be regenerated from single cells and the viral DNA can be introduced into the nuclear genome of all cells of the plant.

Such fused viral genomes can be a) passed on to progeny by sexual means, b) prevent infection by superinfecting viruses, and C) optionally contain selected cargoD
may contain NA and act from the fused replica via transcription, reverse transcription, homologous recombination, or other methods of rearranging genetic material as a source of other deposited viral copies. obtain.

d) A second infection (superinfection) of the plant material containing the viral genome further inserted into the nuclear DNA is likely due to α) expression of the viral DNA from the nuclear DNA, thus causing the foreign DNA within the virus to cause a second infection. Virus DN by
A) would lead to better virus spectrope evolution as it could offer the possibility of transposing A. It can be quite supportive in its action.

The invention thus includes multiple embodiments of the broad concept.

Using the methods described above, the cargo DNA inserted into the viral genome can also be transferred to the plant material where it is propagated. The multiplication within plants of viruses and foreign genes transferred by them is particularly advantageous if plants are to be propagated asexually or to be protected against harmful effects directly and for the shortest possible period of time. ; for example, by introducing a resistance gene into the plant.

The method according to the invention is particularly suitable for inserting and increasing selected genes and desired characteristics into plant material and also into fully grown plants.

The method according to the invention involves the use of a transferred microorganism as described above to incubate plants with a weakened non-disease R1 or slightly pathogenic virus, i.e. a virus which has the effect of protecting the plant from infection with other undesirable viruses. They can also be used in the field of plant protection to "immunize" plants against attack by viruses by transformation.

Viral DNA that can be used within the scope of the method according to the invention
For example, cauliflower mosaic virus (CaM
DNA of Kauridama virus including V), typical dieminiviruses such as Bean Golden Mosaic Virus (BGMV), Cloris Streato
Mosaic virus (C8MV), Kasapa latent virus (CLV), Kaley top virus (C
TV), Myce Streak Virus (MSV), Tomato Golden Mosaic Virus (TGMV), and Wheat Dwarf Virus (WDV) DNA
can also be used, including but not limited to.

Representatives of the caulimovirus group, in particular cauliflower mosaic virus, are particularly suitable for use within the scope of the present invention. This is because these viruses have a genome structure (double-stranded DNA') that is amenable to direct genetic manipulation.
This is for the sake of

All experiments and related observations to date indicate that the genome, which is typical of dieminiviruses and is composed of single-stranded (kjj) DNA, can also be used as a vector for transforming genetic material. show. Furthermore, during the dieminivirus life cycle, double-stranded ds-DNA is formed within the infected plant, and this ds-DNA
is also known to be infectious (17)).

Representative dieminiviruses that form ds-DNA in their life cycle and are thus available for genetic manipulation are also suitable within the scope of the present invention as carriers of foreign genes.

Successful gene transfer into plants using M. spp. virus is usually accompanied by visible signs of infection, such as yellow dots or streaks at the base of newly formed leaves when using M.S.V. The present invention also relates to the use of dieminiviruses as markers.

The host range of dieminiviruses includes a range of economically important cultivated plants, such as corn, wheat, tobacco, tomato, soybean, and various tropical plants.

The host range of Mize streak virus is of particular commercial importance, including a variety of monocotyledonous plants and cereals;
Examples include corn, rice, wheat, millet, sorghum and various African grasses.

The method according to the invention is particularly suitable for infecting whole monocotyledonous plants or growing parts of these plants, such as plant tissue cultures or cell cultures with viral DNA and their equivalents. The invention therefore also relates to transformed protoplasts, plant cells, cell clones, cell aggregates, plants and seeds, and their progeny obtained by the method of the invention, which are obtained from transformation. It has novel properties and also relates to all hybrids and fusion products of transformed plant material having novel properties obtained by transformation.

The invention also relates to transformed whole plants and growing parts of these plants, in particular pollen, ovules, zygotes, embryos or other regenerated materials arising from transformed germline cells.

The invention further includes fully transformed plants regenerated from growing parts of transformed monocots.

Monocotyledonous plants which are suitable for use according to the invention include, for example, species of the following families: Alideaceae, Amaryllidaceae, Asparagaceae, Pinetaceae, Poaceae, Liliaceae, Papilinae, Orchidaceae or Icicaceae. Department.

Representatives of the Poaceae family are particularly preferred, eg plants that grow over large areas and that produce high yields.

Examples include corn, rice, wheat, barley, rye,
Oats and millet may be mentioned.

Other target crops for the application of the method according to the invention are, for example, plants of the following genera: Allium, Oat, Barley, Oryza, Panicum, Sugarcane, Lamium, Setaria, Sorghum, Triticum, Maize, Bacteria, Cocos, Phoenix and Ellaeis.

Transfer-Mss-D to the relevant test plants
Successful transformation with NA can be confirmed in a manner known per se, for example by considering disease symptoms and by molecular biological tests, including in particular 6 Southern blot analysis.

The extracted DNA was first treated with restriction enzymes, then subjected to electrophoresis on a 1% agarose gel, transferred to a nitrocellulose membrane (39): and a detection plate previously exposed to nick translation (31). hybridize with the desired DNA (5X108~10X10" c, p,
m/pi DNA-specific activity L) The filter was incubated at 65°C for 3 times for 1 hour each with 0.05M sodium citrate and CL3.
Wash with an aqueous solution of M sodium chloride. Hybridized DNA is visualized by darkening the X-ray film for 24-48 hours.

Although reference has been made to specific examples for purposes of general description and for a better understanding of the invention,
It is not intended to limit the nature of the invention unless specifically indicated.

[Examples and effects of the invention]

The invention is not limited to the examples below.

Example 1: Preparation of vectors with dimeric MSV genomes The MSV-genomes were prepared from naturally occurring infected maize plants using Klenow-polymerase I and free-length primers according to 16). Pirion ss DNA as a matrix for in vitro synthesis of double-stranded MSV-DNA
(18) ) Can be separated by 't' action.

Another possibility is that direct two-z chain MsV from infected leaf material
-Consists of separation of DNA (@Superase/CMSV-DNA-). Double-stranded MSV-DNA is formed as an intermediate during viral replication. It is °replicative DN
A" or "RF-DNA".

The IS - genome is cloned by inserting RF-DNA or in vitro synthesized RNA into the pUC9 vector linearized with BamHI (19)).

1 (ac complementation test is used to identify recombinant phages.

The next step in production is to first cut the cloned MSV-DNA at one BamHI restriction site. The generated linearized DNA fragments are then applied to a gel of the DNA mixture.
separated by electrophoretic fractionation (20)).

In virus strains with two or more BamHI restriction sites, the MSV-genome is partially digested, or another suitable restriction site that appears only once in the MSV-genome is sought.

This applies even if there is no 1cBamHI restriction site within the MSV-genome.

Next is BamHIlf in tandem arrangement? 1 piece of rasmid
The Bgl I of GA471 [tin rises within the tfyl restriction site (21)), which is localized between the T-DNA border sequences. This so-called tandem-cloning can be controlled by vectors and typical methods of enrichment of inserts. The insert is present in excess ligation solution. The preferred concentration ratio in this case is 10:
1 (insert: vector).

Plasmid pGA4;M is a so-called shuttle vector, and in the presence of tetracycline, A and
It is stably replicated in both tumefaciensc.

This vector has C
o1E1 In addition to the origin of replication, the plasmid has A, t
It has a broad host range origin of replication that allows it to be accommodated within the umefacien sigma. This origin of replication is obtained from plasmid pTJ S75, a plasmid carrying a wide range of host and tetracycline resistance genes, a derivative of PK2 (21)).

Other features of plasmid pGA471 are as follows: 1) Possession of viral restriction sites that allow insertion of foreign DNA between the T-DNA border sequences 2) Cloning of large DNA fragments (25-35 kb) cos-region of the bacteriophage.

3) A chimar (chimar?) consisting of the control sequence of the navalin synthase gene (nos) and the DNA sequence encoding neomycin phosphotransferase.
-7! J - gene, oyohi 4) bom - cleavage site that allows transfer of glasmid from E. coli to A, tumefacient)'.

The above vector and MSV-DNA to be spliced
a culture solution containing t, preferably at a concentration of 1:10;
used for the transformation of E. coli strain DH1 in [22]
). Selection is based on the tetracycline resistance of the transformed clones and on hybridization experiments with radiolabeled M8V'-DNA.

Selected positive clones are then tested for the presence of the MSV-genome in tandem configuration. For this purpose,
Grasmid DNA'i is isolated from positive clones using a known method (20)), and then restriction analysis is performed.

One of the "tandem clones" is selected and E,
coli DHI to A, tumefacienf/
,! , (Rif)C58 (Moromi 1C58) is transferred.

Transfer u, ') Repair Rental Mating (
triparental mating) 2 by the method detailed in 25). In this case, 7ambicin (100μg7tst) and tetracycline (5μgAt) are used for selection. Successful transfer of the dimeric MSv-genome is tested by Southern hybridization (24)).

A, tumefaciens・: (RifRI C5
8 (pTiC58) (25)) contains a wild-type Ti-1 lasmid with complete viral functionality, allowing transfer of the shuttle vector into plant cells.

The Agelobacterium strain transformed by the above method has the following legal name: A, tumefacien et al. (R
ifR)C58(pTic58:pEAP200).

Example 2: Production of control vector To produce a control vector without T-DNA border sequences, plasmid pRK252 without T-DNA border sequences was used.
Kan I, a derivative of plasmid protein (26))
use.

Insertion of the dimeric MSV-S No into the control vector
This is done by splicing the 13amHI fragment described above in tandem arrangement into the 5alI control site of plasmid pRK252Kanl with the aid of the tI/BamHI adapter.

Control vector tD this cien5 ・(R+if R
) Transfer into C58 (pTiC58) is
23) by “Tori Bear Rental Mating” described in detail in 23).

The transformed Agrobacterium strain has the following legal name: A, tumefacien5 (RifR)
csa(pTiC58: pEA21).

Example 3: Preparation of bacterial vector pEAP 25 Cosmid pHc 79, E, cli plasmid p
B.R.

α65 kb Hind in a derivative of 322 (27))
Hybrid cosmid 922G1 is formed by exchanging with l-8al [fragment E or a 1.2 kb fragment from transfozoy Tn 905 carrying the kanamycin resistance gene [28]]. pHC79 Hindl
-Addition of the 1.2 kb fragment to the 5alI control site
This is possible by adding an nd-8al linking sequence.

Contains a gene encoding kanamycin resistance in plants 2
．． The 9 kbsal I-BstEI fragment [6)] was cut from the plasmid pcaMV6 JGn, and the 9
From 22Gl (D 2.4 kb OSal I-Bs
tE [Exchange to fragment.

The final production of pEAP 25 is carried out by joining plasmid pB6, previously cut with Sal I, into the Sal I cleavage site of pEAPl. Plasmid pBA was obtained from J. Davies of John, Norwich, UK.
Developed and available by Innes In5titute. This plasmid (29) has since been published under the name pMSv12.

Plasmid pB6 is plasmid pACY0184 (3
0)) contains the dimeric MSV-genome previously cloned into.

Example 4: Preparation of bacterial vector pEAP37 Bacterial vector pEAP37 was prepared by inserting plasmid pB6, previously cut with 8al, into the Sall restriction site of plasmid pcIB10.

Plasmid pcIB10 was developed and made available by Marie Del Chilton, Civer-Geigy Biotechnology Facility, Research Triangle Park, Raleigh N,C, USA.

Example 5: Production of bacterial vector pEAP 40 1.6m MSv-genoA (Bgl I-Bam
HI fragment (α6mev) + BamHI-BamH
I fragment, (monomer)] into the BamHI restriction site of the plasmid 1) TZ19R described in 31).

The generated plasmid is called p3547, which is 1.6
contains the MSV-genome of meV and converts it into an EcoR
I then cut the plasmid pc'lB200 (32
)) into the EcoRI site. These steps, Msv sequence t, pcIB200 (7) T-D
Located between NA border arrays.

Example 6: Preparation of bacterial vector pMsv 109 The construct already described in Example 3, plasmid pMsV1
2-5 μg are digested with 13amHI in buffer for 2 hours at a temperature of 37° C. (20)). The 2.7 kb DNA fragments resulting from this enzymatic digestion were transferred to a 100% agarose-TA solution.
After electrophoretic separation of the samples in a B-gel (40mM Lis-HCI, 20mM sodium acetate, 2mM EDTA), the latter was eluted and the binary T-DNA vector pBin19 (26)) was eluted with a single BamHI cutting site. Take a chair inside the curfew area.

100 for vector pBiv+19 for ligation.
A two-fold molar excess of the 2.7 kb MSV fragment, and high agility (D T4-DNA on IJ agar, is used to ensure a high rate of insertion of dimeric MSV-DNA into the vector. , the concentration used was pMsVDNA6
25 ng and pain 19 DNA 25 ng
and T4-DN in a total volume of 10 μl at 10°C for 16 hours.
Ligate in the presence of 5 units of A ligase. Half of this M solution was added to competent E, coli JMB 5
transfected into recA cells and kanamycin sulfurini) 50μν'ILI,! : 5-Schiff"como-4-chloro-5-indolylgalactoside (X-ga
l) °Luliapros' (LB) supplemented at 40 μg/d
-7 gar (20)) and plate out on 37
The cells were incubated overnight at .

Select white colonies containing the λl5V-insert, /i
:/ Te A configuration (pMsV 109) A clone containing the Nakanoji body MSV f
Selection was made by conjugating to sC58Nal''[55)3, and this was done by the method described by Ditta et al.
L containing 1@/ILt and nalidixic acid 50μm
B - Performed on agar. The selected colonies are those that initiate infection of maize as described below, and phts
Classified as v114.

To produce control vector pEA2, pBIN19
Plasmid pRK 2 of the precursor plasmid [26)]
52/kml (D Sal [restriction site 8al
ligated with the cut plasmid pB6.

Selection of pEA2 was performed using the control vector Kananisin (I).
m′)- and chloramphenicol (Gm R)
-'# Do it based on gender.

Grasmid pEAP25ft, strain E, Cal i G
J 23 (pGJ28, R6a rd 11 ) (3
5) Clone into the bacteria of ). This E, C
o11 strain has 1 in A, tume-facience]
Transfer is enabled by conjugation of a plasmid with an Om cleavage site. Plasmi)' 9gA2, p
EAP37 O and pEAP40i, A via “Tribe Rental Mating”.

turnefaciens (23))
. The susceptibility methods used are two A, tumefac
iens strains: 1) binary vector pEAP40. pEA2 and pEA
C58 (pTiC58)2) 7' for P57
Rasmi)' pEAP25 Notame (7) C58 (pT
iC58, pEAP18) A, tumefaciens (7) wild type strain is “
fj A/f Year Collection of Laboratory Op Microbiology, Microbiology
Department Op The University Op Gent (Culture Co11ection of t
he Laboratory of Microbiol
ogy, Microbiology Department
ment of the [Juniversity of the
Gent)'.

pEAP25: Agrobacterium strain csa (p
'riC58, pEAP183 is plasmid pEAP
Acts as a 25 susceptibility method. pEAP 18 is a 6.7 Kb Ec of plasmid pGA472 [21)]
o 几I-BatnHI fragment was added to plasmid PHC7q.
The binary vector was replaced with the 2.6 kb EcoI-Bgl fragment of [27)) and contains the homologous recombination region in Grasmid pEAP25 between the T-DNA border sequences. Plasmid pEAP25 is A, tumef
Nonconjugative selection on rifampicin, kanamycin and carbenicillin resulted in a novel 7 Globacterium strain A, tumefac
ienscss (pTic58.pEAP29) was obtained,
Therein, plasmid pEAP25 has been inserted into the original vector pEAP18 for homologous recombination.

pEA'P57, pEAPa O: E, coli
pEAP 37 and pEAP
40 (D ” A, turnefaciens heno flow through hair Lz talmating,
This results in the production of a binary vector system.

pEA2: The control plasmid pEA2 is inserted into the Agrobacterium strain csa (pTi058) in trans position relative to the already existing T1-1 lasmid.

Grasmid newly produced by the above method is tested by the method of DN separation and restriction mapping.

Grasmid used within the scope of the invention, pEAP37
, pEAP40 Oyohi pMsV1091d, 1 Toichie Sammlung, Göttingen, West Germany.
Phono Microorganisme (Devtscbe S
ammlung von Mikroorganism
en ) ” (D8M) and Aberdeen 135 Abbey Road, P-O Box 31, Tory Research Station 1 The National Collection/of
Industrial Bacteria (The National
Co11,ection of IndustryVia
l Bacteria)'(NCIB);
Both were admitted for the purposes of the present invention as international deposits in accordance with the requirements of the Petabes Treaty on the International Agreement on the Deposit of Microorganisms. Certification as to the viability of the deposited samples was made by the international depositary institution.

No restrictions regarding the use of said microorganisms are required by the depositor.

Prior to inoculation, Agrobacterium strains were grown in YEB medium [Bactogifex 5 g/l] enriched with 100 μV of rifamubicin and 25 μV of kanamycin or 50 μV of nalidixic acid and solidified with 1.5% agar before use. ! , Bakto yeast extract 1g/l!
, peptone 577/l.

Sucrose s I/l, Mg 8042mM,
Plate out on pH 7.2). After 48 hours of incubation at 28°C, single colonies are used to inoculate liquid cultures. Inoculation is carried out in a 100 d Erlenmeyer flask containing liquid YEB medium supplemented with 1 antibiotic at the concentration described above. Cultivation is carried out at 28° C. with stirring M at 20 Or, plm speed. The culture time is 24 hours.

A second subculture step is carried out in liquid medium at a dilution ratio of 1:20, other conditions being the same. The culture time in this case is 20 hours.

These steps lead to a population concentration of viable Agrobacterium of 10 6 /l/ml.

Bacterial cells are collected by centrifugation and then suspended in an equal volume of antibiotic-free 10 rnM Mg8 (J4 solution).

This suspension is referred to as the undiluted method solution in the following steps. When preparing a dilution series, 1o mM
Mg5O, solution is used again as diluent.

1i[11J10:) Used for sterilization and germination of corn seeds, Golden Cross Pantum, B75, Nome, all of which can be successfully infected with pesticides.

For the experiments described below, pre-sterilized seedlings, generally at the three-leaf stage, are used. Sterilization of seedlings consists of the following steps: 1. Sterilization of seeds in 07% WΔ calcium perchlorate salt (
2so*solution/100 pieces). Thoroughly mix the particles and the solution using a magnetic stirrer. After 20 minutes, decant the sterilizing solution.

Z Seeds treated in this way were then soaked with distilled water three times each time.
Wash for 0 minutes (250d distilled water/100 seeds).

The seeds sterilized in this way are then introduced into an already sterilized seed chamber. The seed chamber is a veterinary vessel containing 5 sterile Macherey-Nagel round filters each with a diameter of 5 m and approximately 10 ml of sterile water.

20 seeds are introduced into each of these seed chambers and incubated at 28° C. for about 3 days in the dark.

For subsequent inoculation experiments, only seedlings with a distance of 1-2 mm between the platen node and the apical coleoptile tip are used. However, in all cases it must be ensured that the coleoptile tubes can be clearly distinguished.

Example 11: Inoculation of Corn Seedlings A Hamilton hypodermic syringe (50
μl or 100 μz) h,′: used to introduce the inoculum solution described in 3 below into corn seedlings.

Place the inoculum solution into the hypodermic syringe in such a way that no air bubbles form.

11.1. 10th day corn plant concubine 10
The application of the bacteria-containing suspension to day-old corn plants is carried out by various methods and at different sites on the plants.

1. 20 μl of bacterial suspension! Apply to one leaf of Kanagami B111 and rub the suspension into the leaf using carporundum powder until the entire leaf is moistened (injection site in Figure 1).

Z Central part of the plant a) Just above the tongue of the first leaf (point B in Figure 1) b) 1 cm below the tongue of the second leaf (point C in Figure 1) C) The so-called root Apply 100μ to the base of the collar plant, the meristem where adventitious roots will later grow (f in Figure 1).
Prepare the bacterial suspension using a Hamilton hypodermic syringe of 1
Inject 0 μl.

11.2. ! Inoculation of S5-day corn seedlings Inoculation of the bacterial suspension to 3-day corn seedlings
This is done by injecting into the seedlings using a 100 μl Halmitone hypodermic syringe.

1. Inject the bacterial suspension into the coleoptile node by introducing a hypodermic syringe through the coleoptile starting from the apical coleoptile tip and ending in the coleoptile node area (site E in Figure 2). .

2. Inject the bacterial suspension directly into the cotyledon pulp 211 m below the apical coleoptile apex (point F in Figure 2).

5. Inject the bacterial suspension directly into the cotyledon sperm of the upper 21 coleoptile nodes (site C in Figure 2).

4. Inject the bacterial suspension directly into the coleoptile nodes ((Second
H site in the figure).

1. Inject the bacterial suspension directly into the cotyledon spermatozoon 2 cm below the coleoptile node (injection site in Figure 2).

& Inject the bacterial suspension directly into the blastocyst (site J in Figure 2).

Z Inject the bacterial suspension into the blastoderm by introducing a hypodermic syringe through the first root starting at the tip of the root and ending in the area of the blastoderm (site in Figure 2). .

11.5 Cutting of the coleoptile within the area of the coleoptile node Three-day-old maize seedlings are cut off at various points within the area of the coleoptile node (see Figure 3).

1. Directly at the height of the coleoptile node 2. Im above the coleoptile node 5. 2rm above the coleoptile node 4. 5rtys above the coleoptile node Cultivate according to established conditions.

Actual inoculation experiments with Agrobacterium are carried out on seedlings whose coleoptile tips have been removed 2 m above the coleoptile nodes at the time of preparation.

Immediately after the inoculation treatment, the corn plants were planted in moist soil in the same manner as the 100-day corn plants.
22℃±2℃, 3000-5000 lux white light (Philips 400W/G/9/12) -Give your feet-
C.Cultivate.

The plants are then observed daily for signs of viral infection, characterized by the appearance of yellow dots and/or streaks at the base of newly formed leaves.

Approximately 400 η of young leaf tissue (fresh amount) was first mixed with 5TEN (15% sucrose, 5%
0mM) Rimata-HeI 50mM Na5ED
TA, (L25 MNacl, p) (a)
Add L5-1.0 td and sand (~50 vq) and homogenize in a motor on ice. The homogenate is then transferred to a small centrifuge tube (1,5d) and homogenized at maximum speed in a tabletop centrifuge. Centrifuge at 4°C for 5 minutes.

The supernatant was discarded, and the precipitate was mixed first with a sterile toothbrush, then briefly (5 seconds) using a Portex mixer in a water-cooled 5ET (15% C/O, 50
mM Na, EDTA, 50 mM Tris-HC
L pH 8) Resuspend in α5d. Continue to be 20%
10 μl of SDS solution and proteinase K (2(ly
/d) Add 100 μl, mix and the whole is then heated to 68° C. for 10 minutes in a small tube. After adding 3M sodium acetate (1/10 volume), the digest is extracted twice with phenol/chloroform (3:1). D.N.
Af: Then add 2 parts by volume of ethanol to precipitate and store at -20°C overnight. Centrifugation at maximum speed in a tabletop centrifuge (for 10 min) yields a DNA-containing precipitate, which is then diluted with TE buffer (40mM HCl,
1 mM Na, EDTA% pH 8) 40 tt
Dissolve in l.

Perform 1 Southern plot experiment (36) on the fractionated DNA solution.
) used for

<a: 1 Southern Prod 1 Analysis The extracted DNA was first treated with restriction enzymes, then electrophoresed in agarose gel, transferred onto nitrocellulose [3G)], and the pre-nicks detected Exposed to transrage sodium [1)))L) Hybridized with NA (5X 1 as-1o x 1o'
c, p, m, /py DNA % heteroactivity).

The filter was washed with α03M sodium citrate aqueous solution for 1 hour each at 65°C. No Ibrid DN
A is visualized by darkening the X-ray film for 24-48 hours.

Results: A) 100-day corn plant contact (Table 1 shows 1
[The results of the inoculation experiment on 100-day-old corn plants described in Ll are shown.

Inoculation was performed using pEAP 37 DNA.

1'']L The results in Table 1 clearly show that the preferred site of application to plants is localized in the area of the root collar, with 62% and 40% of the treated plants showing signs of infection. , whereas the number of plants showing signs of infection after inoculation of the other negative seed sites (A, B, C) on the plants is zero or negligibly small.

B) Table 2 shows the inoculation of 3-day-old corn seedlings.
1 shows the results of an experiment in which 3-day-old corn seedlings were inoculated as described in 1α2.

ms is pEAP37 and pEAP40 DNA
I used k.

Table 2 The results in Table 2 clearly show that the preferred site of application to maize seedlings is the coleoptile region, and that bacterial suspensions can be applied both directly and indirectly to the coleoptile. Upon application, 83 trees and 88% or 7896 plants were infected, indicating a clear preference compared to other application sites tested. It is clearly not important whether the suspension is injected directly through the whole cotyledon mNK side or indirectly through the coleoptile.

c) Cutting of 5-day-old corn seedlings Table 3:
The number of seedlings that survived 2 weeks after cutting of the coleoptile at different sites in the coleoptile region is shown.

Table 5 shows that seedlings can be removed up to 211 tll above the coleoptile without any observed loss of viability of the seedlings treated with this method. By removing only the upper fin of the coleoptile, approximately 60% of the plants are completely viable.

Table 4 shows the results of inoculation experiments with seedlings from which the upper 2 ffim of the coleoptile were removed. Inoculation is pEAP37DNA
It was done using .

Table 4 The results in Table 4 clearly show that the part of the cut seedling, ie the meristem area, is also clearly preferable to the M part, which occupies the peripheral part of the tissue.

D) Dilution experiment The bacterial suspension described in Example 9 above is diluted in YEB medium without addition of antibiotics and applied within the coleoptile nodes at the following concentrations.

Non-selective 2 X 106 84/10
2 (82%) 10” 2X 10!
42/55 (76%) 10”
2X 104 54/ 54 (62%)1
0″″3 2X103 19
156 (34%) 10-4 0
0/10 (<10 easy) 10
-5 0 0/10
(<10%) Assuming that the replication factor of a binary vector containing MSV sequences is approximately 10, and that bacteria do not multiply further at the inoculation site, 104 bacteria will be approximately 400 fg (4X 10'g) (7) MSV
- Contains DNA.

This means that Agrobacterium transfers Agrobacterium (its DNA = k) to maize with an efficiency comparable to that of transfer to dicot host plants.

E) Agrobacterium host range In addition to maize, it was confirmed that other representative plants of the Poaceae family are susceptible to infection by Agrobacterium.

The full results of inoculation experiments with these Poaceae species are shown in Table 5: Some of the results showing lower efficacy than in Table 5 can clearly be attributed to technical difficulties encountered during inoculation, but This is because the plants, if any, are very small and therefore have a small stem diameter which makes it difficult to inject the inoculum solution into a specific location.

Apart from this, the above results show that besides maize, many representative plants of the Poaceae family can be transformed by Agrobacterium.

F) A, t consistently used in experiments in which Agrobacterium strains were inoculated into maize.
In addition to the C58 strain of A. umefaciens, other A.
.

tumefaciens and A, rlxizoge
nes strains were also tested. It was also possible to detect the transfer of MSV-DNA into maize using Agrobacterium a listed in Table 6 below: Skewer 1
Inoculation experiments with pMsVloq were performed on 10-day old maize plants.

-2 Inoculation experiments with pEAP 57 were performed on 3-day-old corn seedlings.

List of references 1) Ecleene M)
, Phytopath, Z.

Sat, pp. 81-89, (1985) 2) Hernalsteens G.I.P.
alteensJP) et al., EMBOJ 3, 303
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S.) et al., Nature 311.

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SL), J, Bacteriol.

169 (4): pp. 1745-1746, 198
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(1985a) @Direct gene trans
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Page 17, (19AS) Figure 1 is an explanatory diagram showing the inoculation site of a bacterial suspension to a 10-day-old corn plant in an example of the present invention. An explanatory diagram showing the inoculation site of a bacterial suspension on a 3-day-old corn seedling. FIG. It is an explanatory diagram showing a part.

Patent applicant Civer Geigy Muff life 1〉Ikame”ゝ′
'J''7''
Ben two! Shiichiwo 1'/li' and 2 others Figure 1 Figure 2 Figure 3 1mm

Claims (69)

[Claims] (1) A transfer microorganism containing genetic material in a transportable form and capable of inserting said genetic material into monocots or viable parts thereof, enabling the induction of toxic gene action in said transfer microorganism. The genetic material is made available for the infection of monocot plants by using suitable culture and application methods, and the monocot plants or their viable parts are infected with said transfer microorganism. A method of insertion into cotyledonous plants or their growing parts. (2) growing the transfer microorganism in a medium known per se, optionally performing one or more subculturing steps, and centrifuging the transfer microorganism;
A method according to claim 1, characterized in that it is then resuspended in a suitable medium and the resulting inoculum solution is introduced into the growing area of the monocot. (3) The transferred microorganism is grown in a medium suitable for culturing the transferred microorganism, stirred for 30 to 60 hours, at a culture temperature of 15 to 40°C, and then optionally one or more subculturing steps. 3. A method according to claim 1 or 2, characterized in that each of these subculturing steps is continued for a period of 15 to 30 hours at a temperature of 15 to 40°C. (4) The method according to claim 3, characterized in that the culture time of the transferred microorganism and the culture time of the optional subculture step are 40 to 50 hours. (5) The method according to claim 3, wherein the culture temperature of the transferred microorganism and the culture temperature of the optional subculture step are 24 to 29°C. (6) Any one of claims 1 to 3, characterized in that a medium solidified with agarose or alginate or any other suitable solidifying agent is used.
The method described in section. (7) The method according to claim 1, characterized in that the transfer microorganism is inoculated in the form of a microbial suspension into the meristem region of the plants or their growing parts. (8) The method according to claim 7, characterized in that the microorganism suspension is repeatedly inoculated. (9) The method according to claim 1, characterized in that the timing of inoculation and the inoculation site of the plant with respect to the developmental and differentiation stages of the plant are adjusted so as to significantly increase the frequency of transformation. (10) The method according to claim 1, characterized in that a plant at a developmental stage between the early stage of plant embryo development and the early stage of flowering is inoculated with a suspension of microorganisms to be transformed. (11) Claim 1, wherein the plant to be infected is at a developmental stage between seed germination and the four-leaf stage.
The method described in item 0. (12) The plant is on the first, second or third day of the germination period, and the distance between the blastoderm and the apical coleoptile tip is about 1 to 3 cm. A method according to claim 11. (13) The method according to claim 7, wherein the inoculation of the microorganism suspension to be transformed is carried out by injection into the meristem region of the plant. (14) A method according to claim 13, characterized in that the microorganism suspension to be transformed is injected into the region of the root collar of the plantlet, which has already differentiated into roots, stems and leaves. (15) Claim 13, characterized in that the microorganism suspension to be transformed is injected into the seedling near the coleoptile node.
The method described in section. (16) The method according to claim 15, characterized in that the microorganism suspension to be transformed is directly injected into the coleoptile node or into a region approximately 2 mm around the coleoptile node. (17) The method according to claim 13, characterized in that the microorganism suspension to be transformed is directly injected into the meristem region of the coleoptile after the tip of the coleoptile has been cut off. (18) Place the tip of the coleoptile 1 to 5 mm above the coleoptile part.
The method according to claim 17, characterized in that the portion is cut off. (19) Genetic material optionally incorporates Cargo-DN
2. The method according to claim 1, wherein the viral DNA may contain A. (20) a) Adding viral DNA, including optionally incorporated cargo-DNA, to the vicinity of one or more T-DNA border sequences in the T-replicon to the viral D
b) the distance between the NA and the T-DNA border sequence is chosen such that the viral DNA, including the cargo-DNA that may be present, is transferred into the plant material; b) subsequently the T-replicon is Transferring the replicon into a suitable transfer microorganism such that it is absorbed into the transfer microorganism, and infecting monocot plants with the transfer microorganism modified according to b). 20. The method according to claim 19, wherein viral DNA containing cargo-DNA is inserted into a monocotyledonous plant. (21) a) viral DNA or its equivalent is isolated from infected plant material and cloned under the action of a suitable vector in a host organ; b) the cloned viral DNA or a portion thereof and its Using the cargo-DNA that may be incorporated into them, the distance between the viral DNA and the T-DNA border sequence can be calculated using the cargo-DNA that may be incorporated into them.
one or more viral genomes, or parts of viral genomes, selected to be transferred into the plant material and localized in the vicinity of one or more T-DNA border sequences; c) transferring said plasmid BaP into a suitable transfer microorganism to form a vector system that can be used in plants; d) thus 20. The method according to claim 19, characterized in that monocot plants or their growing parts are infected with a vector system modified by the vector system. (22) The method according to any one of claims 19 to 21, characterized in that a double-stranded DNA of a single-stranded DNA virus is used as the viral DNA. (23) As viral DNA, D of gemini virus
Claim 22, characterized in that NA is used.
The method described in section. (24) Mize streak as viral DNA
virus (MSV), Bean golden mosaic virus (BGMV), Cloris striate mosaic virus (CSMV), Casava latent virus (CLV), Curly top virus (CTV)
, tomato golden mosaic virus (TGMV)
or Wheat Wolf Virus (WDV) D.
Claim 23, characterized in that NA is used.
The method described in section. (25) The viral DNA used is natural virus D
22. The method according to claim 19, wherein the method is NA. (26) The method according to claim 25, characterized in that cauliflower mosaic virus is used as the viral DNA. (27) As viral DNA, cD of viral RNA
22. A method according to any one of claims 19 to 21, characterized in that an NA replica is used. (28) As viral DNA, viroid RNA c
22. The method according to any one of claims 19 to 21, characterized in that a DNA copy is used. (29) Any one of claims 19 to 21, characterized in that viral DNA of a lethal or viable mutant of the virus is used as the viral DNA.
The method described in section. (30) The method according to claim 19, characterized in that cloned DNA under the control of a viral replication signal is used as the viral DNA. (31) The method according to claim 19, characterized in that cloned DNA under the control of a viral expression signal is used as the viral DNA. (32) Claim 1, characterized in that cloned DNA under the control of viral replication and expression signals is used as the viral DNA.
The method described in Section 9. (33) The method according to claim 19, characterized in that a cloned DNA under the control of eukaryotic replication and expression signals is used as the viral DNA. (34) The method according to claim 19, characterized in that a portion of viral DNA is used as the viral DNA. (35) The method according to claim 19, characterized in that the viral DNA is used in tandem. (36) Claim 19, characterized in that viral DNA or their equivalents are used in tandem.
The method according to any one of items 34 to 34. (37) Virus D with integrated Cargo-DNA
Claim 19, characterized in that NA is used.
The method according to any one of items 36 to 36. (38) Virus D with integrated Cargo-DNA
37. Method according to any one of claims 19 to 36, characterized in that NA or their equivalents are used. (39) The method according to claim 20 or 21, characterized in that a bacterial T-replicon is used. (40) Claim 3, characterized in that a T-replicon of Agrobacterium species is used.
The method described in Section 9. (41) The method according to claim 39 or 40, characterized in that the T-replicon used is a Ti-plasmid or Ri-plasmid of a bacterium of the species Agrobacterium. (42) As a microorganism that provides the T-replicon according to any one of claims 39 to 41,
A method according to claim 1, characterized in that bacteria are used. (43) The method according to claim 42, characterized in that soil bacteria are used as the microorganism that provides the T-replicon. (44) The method according to claim 43, characterized in that bacteria of the species Agrobacterium are used as the microorganism that provides the T-replicon. (45) The method according to claim 38, wherein the cargo-DNA consists of genomic DNA, cDNA, or synthetic DNA. (46) Cargo-DNA is genomic DNA and cDNA
and/or synthetic DNA. (47) The method according to claim 38, wherein the cargo-DNA is composed of gene fragments of various organisms belonging to various genera. (48) The method according to claim 38, wherein the cargo-DNA is composed of gene fragments of one or more strains, one or more varieties, or one or more types of the same organism. (49) Claim 38, characterized in that the cargo-DNA is composed of parts of the same organism of one or more genera.
The method described in section. (50) The method according to claim 1, characterized in that a plant tissue culture or cell culture is used as the growing part of a monocot. (51) A patent claim characterized in that a plant of the Aliaceae, Amaryllidaceae, Asparagaceae, Pineappleaceae, Poaceae, Liliaceae, Musaceae, Orchidaceae, or Coconutaceae family is used as a plant or a growing part thereof. Range 5th
The method described in item 0. (52) Claim 51, characterized in that a plant of the Poaceae family is used as the plant or its growing part.
The method described in section. (53) The method according to claim 52, characterized in that corn, rice, wheat, barley, rye, oats, or millet is used as the plant or its growing part. (54) As plants or growing parts thereof, the genus Allium;
Using plants of the genus Oat, Barley, Rice, Panicum, Sugarcane, Rye, Setaria, Sorghum, Triticum, Maize, Musa, Cocos, Phoenix, Ellaeis or parts of these plants. 51. The method according to claim 50, characterized in that: (55) The method according to claim 1, characterized in that protoplasts are cultured together with the transfer microorganism. (56) Transfer microorganisms containing genetic material in a transportable form and capable of inserting said genetic material into monocots or viable parts thereof, with suitable Transfer microorganisms or theirs, characterized in that they can be used for the infection of monocot plants by using cultivation methods and application methods and are capable of infecting monocot plants or their viable parts. part. (57) Plasmid pEAP37 or pEAP40
, or a transfer microorganism containing one of these plasmids. (58) Plasmid pMSV109 or a transfer microorganism containing the plasmid. (59) The transformed E. coli strain DH1 (pEAP37) deposited under the accession number DSM4147, and all derivatives and mutants thereof that still have the properties of said strain. (60) The transformed E. coli strain DH1 (pEAP40) deposited under accession number DSM4148 and all derivatives and mutants thereof that still have the properties of said strain. (61) The transformed E. coli strain JM83^R^e^e^A deposited under accession number NCIB12547, and all derivatives and mutants thereof which still have the properties of said strain. (62) A transfer microorganism containing genetic material in transportable form and capable of inserting said genetic material into monocots or viable parts thereof, allowing induction of toxic gene action in said transfer microorganism. can be used for the infection of monocot plants by using suitable culture and application methods, and by infecting the monocots or their viable parts with said transfer microorganisms, the bodies of said plants and their progeny are Monocots and their progeny, characterized in that most of their cells and/or embryonic cells have been transformed. (63) A transfer microorganism containing genetic material in a transportable form and capable of inserting said genetic material into monocots or viable parts thereof, enabling the induction of toxic gene action in said transfer microorganism. can be used for the infection of monocot plants by using suitable culture and application methods, and by infecting the monocots or their viable parts with said transfer microorganisms, the bodies of said plants and their progeny are Seeds of monocots and their progeny, characterized in that most of the cells and/or embryonic cells have been transformed. (64) A transfer microorganism containing genetic material in a transportable form and capable of inserting said genetic material into monocots or viable parts thereof, enabling the induction of toxic gene action in said transfer microorganism. characterized in that it can be used for the infection of monocot plants by using suitable culture and application methods and that it has been transformed by infecting the monocot plants or viable parts thereof with said transfer microorganism. monocots or their growing parts. (65) The unit according to claim 64, wherein the transformed part is a seed, pollen, ovule, zygote, embryo or other reproductive organ resulting from transformed germline cells. The growing part of a cotyledonous plant. (66) Transfer microorganisms containing genetic material in transportable form and capable of inserting said genetic material into viable parts of monocotyledonous plants in a suitable manner that enables the induction of toxic gene action in said transfer microorganisms. from a growing part of a monocot plant which has been made available for infection of a monocot plant by using a culture method and an application method and which has been transformed by infecting a viable part of the monocot plant with said transfer microorganism. A fully transformed plant characterized by being regenerated. (67) A transfer microorganism containing genetic material in a transportable form and capable of inserting said genetic material into monocots or viable parts thereof, allowing induction of toxic gene action in said transfer microorganism. can be used for the infection of monocot plants by using suitable culture and application methods, and can be used for the infection of monocots or viable parts thereof with said transfer microorganisms and by transformation. transformed protoplasts with new properties,
Plant cells, cell aggregates, plants and seeds and their progeny. (68) A transfer microorganism containing genetic material in a transportable form and capable of inserting said genetic material into monocots or viable parts thereof, enabling the induction of toxic gene action in said transfer microorganism. can be used for the infection of monocot plants by using suitable culture and application methods, and can be used for the infection of monocots or viable parts thereof with said transfer microorganisms and by transformation. All hybrids and fusions with transformed plant material that have new properties. (69) Transfer microorganisms containing genetic material in a transportable form and capable of inserting said genetic material into monocots or viable parts thereof, which are suitable for the induction of toxic gene action. A transfer microorganism, characterized in that it can be used for the infection of monocot plants by using the cultivation method and the application method, and is capable of infecting monocot plants or their viable parts, is used in plants. A method used to immunize plants against unwanted viral attack in the protection of plants.