NO864681L - PROCEDURE FOR DIRECT TRANSMISSION IN PLASTIDES AND MITOCONDRIES. - Google Patents

Abstract

A method of direct entrapment of DNA in plastids and mitochondria of plant protoplasts which is essentially characterized in that in the absence of a pathogen of said DNA in a medium causes penetration of DNA into the protoplasts of the plastids and mitochondria therein where the protoplasts are placed. so long in contact that this penetration is ensured so that it eventually results in plants with improved properties.

Description

The present invention comprises a method for direct gene transfer in plastids and mitochondria, preferably in the chloroplasts of plant protoplasts.

Within the scope of the present invention, the term plants includes all unicellular or multicellular organisms that can carry out photosynthesis.

Plant protoplasts include plant cells whose cell walls have been completely or partially removed by treatment with cell-lytic enzymes.

The chlorine patches form a point of attack for substitutes of different herbicide classes, such as, for example, the triazine herbicides. Only a short time ago, it was found that atrazine, a substitute for triazine herbicide, interacted with a 32 kd polypeptide encoded by a chloroplast gene, the so-called psbA gene (Hirschberg et al., 1984).

The substitution of individual nucleotides within the coding part of the psbA genes, which results in the replacement of a single amino acid in the 32 kd polypeptide, leads to a resistance to atrazine. Such mutations are found in weeds, such as Amaranthus hybridus and Solanum nigrum.

It would now be desirable in connection with genes to be incorporated directly into the plastid and mitochondrial genomes of plants, especially cultivated plants. This will make it possible to add new undesirable properties to such plants. In order to obtain herbicide-resistant plants, it is necessary, for example, to have genes that provide herbicide resistance in herbicide-sensitive chlorine plasters.

Previous efforts mainly concerned a genetic manipulation of the nuclear genome in plant cells to achieve a resistance or tolerance to herbicides that were

active in chloroplasts.

However, these methods easily led to marginal tolerance differences in relation to these herbicides and not to real resistance.

In the past, it was considered impossible to transfer true resistance to herbicides that were active in chloroplasts with the help of direct gene transfer to cultivated plants, as it was previously assumed that a transformation of plastids, such as e.g. chloroplasts was not possible with this method.

A further disadvantage of the inclusion of genes which gave a desired property such as herbicide resistance in the nuclear genome of a plant lay in the possibility of certain plants to cross-pollinate weeds. Such crossings opened up an opportunity to transfer these desired properties from weeds in cultivated plants.

One of the most frequently used methods for the inclusion of genes in nuclear genomes in plants consists in infection of the cells with pathogens, such as e.g. an agrobacterium, which contains the Ti plasmid vector system. (Barton et al., 1983; Chilton et al., 1985). However, these methods have clear disadvantages which are related to the course of the infection. These disadvantages include, firstly, a limited host specificity, and secondly, the necessity to rid the transformed plant cells of the pathogens used for the transformation.

There are also concerns about the release of such pathogens into the environment. (Roberts, 1985).

De Block et al. (1985) mentions the use of agrobacterium Ti-plasmid vector systems for the inclusion of a gene coding for antibiotic resistance in the chloroplast genomes of tobacco protoplasts from which complete cells and finally complete fertile plants could be regenerated. However, the authors found that the gene was unstable in the absence of the antibiotic in question and that it was lost after a short time. However, the preservation of plants under antibiotic selection pressure did not provide any practical application.

Procedures for the direct transformation of plant protoplasts with exposed linear DNA or circular plasmid DNA are in all cases known (Paszkowski et al., 1984, as well as Schilperoort et al., 1983). In these methods, no pathogen is necessary for the course of the infection. Until today, however, these methods have been limited to nuclear transformation.

There is thus a need for a method which allows the direct transfer of useful genes into plant and mitochondrial genomes so that advantageous new properties of plant cells can be transferred without a previous infection of the cells with a pathogen being necessary, and where it is also prevented that these characteristics are transferred to weeds. Furthermore, there is a need for a method which ensures stable gene transfer in plastids and mitochondria from plant cells and whole plants so that the expression of said gene is not lost during development of the cultured plants in the marrow.

An essential component of the present invention thus deals with the description of a method for direct gene introduction into the genome of plastids and mitochondria, preferably chloroplasts without infection of the cells with a pathogen and maintenance of the gene in said genome. A further object of the present invention comprises the production of whole plants containing genetically manipulated plastids and mitochondria and also under conditions under which the characteristic(s) tailored according to recombinant genetic technology remain stable and give expression.

As can be seen from the following description, these and further objectives are achieved by carrying out the method according to the invention for direct inclusion of DNA in plastids and mitochondria of plant protoplasts, whereby said DNA comprises one or more genes and active promoters in plastids and mitochondria and where the method is distinctive in that a pathogen for this DNA is brought into contact with the protoplasts in a medium for so long that the penetration of the gene into the protoplasts and the plastids and mitochondria present therein is ensured.

Figures

Fig. 1 shows a flow diagram of the individual steps for the construction of plasmid pCAT^ and p32CAT. Fig. 2 shows a flow diagram of the individual steps for the construction of the plasmids pUCH1. Fig. 3 shows a flow diagram of the individual steps for the construction of the plasmids pBRCAT.

In the attached figures, the following abbreviations are used:

X = Xhol

S = Narrow

H = Hindlll

B = BamHI

RI = EcoRi

SI = Sali

EQUAL. = Ligation with a T4 DNA Ligase

X/S1 denotes a site where a hybrid restriction cleavage point is produced by linking an XhoI sequence with a SalI sequence which cannot be cleaved by any of the enzymes.

CAT denotes the coding region for chloramphenicol acetyltransferase. CAT^ denotes the promoter-free form of the CAT gene.

In Figures 1 to 3, the psbA gene is represented by a black bar and the associated promoter by a black box.

In Figures 1 to 3, the CAT coding region is drawn with dots.

In the present description of the invention and in the patent claims, the following definitions apply: A "gene" is a DNA sequence that is characterized by a promoter and a transcribing DNA sequence.

The promoter, which in most cases is not transcribed, causes transcription of the subsequent and in some cases also in the surrounding gene sequences of the corresponding RNA. However, RNA cannot be translated into a polypeptide either. If RNA is translated, it is called mRNA. The DNA sequence that gives mRNA as well as mRNA itself is composed of a 5' region that is not translated by a coding region as well as a 3' region that is also not translated. Only the coding region is translated into the corresponding polypeptide.

The "active" parts of a DNA sequence form corresponding regions that are necessary for the function of the DNA sequence. Some examples of active regions of DNA sequences include the RNA polymerase binding region, the initiator signal (TATA box) of the promoter, the ribosome binding region as well as the translation initiation signal of the untranslated 5' region, the coding sequence, transcription stops - signal as well as the polyadenylation signal of the 3' untranslated region. The various spacer parts in which the DNA sequence is not important are not considered active DNA sequences.

A "variant" of a natural DNA sequence forms a modified form of the natural sequence that fulfills a corresponding function. Thereby, it may be a mutant or a synthetic DNA sequence which is essentially homologous with the corresponding natural sequence.

A DNA sequence which is at least 70%, preferably at least 80% and especially at least 90% homologous to the active part of the DNA sequence is thus considered substantially homologous to another DNA sequence. To determine the essential homology, two different nucleotides within a DNA sequence of a coding region are also considered homologous when the opposite substitution of this nucleotide leads to a silent mutation.

A DNA sequence "descends from" a source, such as plastids or mitochondria when this DNA sequence occurs in this source. The present invention also includes variants of such a DNA sequence.

A DNA sequence is "functional" in plastids or mitochondria when it fulfills its expected function and remains in the succeeding plastids and mitochondria.

By "transformable plant protoplasts" is meant protoplasts which, after a direct gene transfer, contain a plastid or a mitochondrial genome which has the covalently-bonded DNA which does not normally occur in these plastids or mitochondria.

Plant protoplasts, which can be transformed using the method according to the invention, can originate from cultured cells as well as from cells that are built into the plant parts or multicellular plants. However, the transformable plant protoplasts can also originate from unicellular plants such as algae. Some examples of unicellular plants include cyanobacteria (eg Synechococcus spp.) as well as Chlamydomonas and Euglena. Particularly preferred within the present invention, however, are plant protoplasts originating from multicellular plants. According to the following transformation, the protoplasts can

possibly be regenerated from whole plants.

With the help of the present invention, it is thus possible to genetically modify the plastids and mitochondria from any plant protoplast.

Some examples of such plant protoplasts are:

Solanum spp. (potato), Gossypium spp. (cotton), Glycine spp.

(soybean), Petunia spp. (petunia), Daucus spp. (carrot), Citrus spp. (orange, lemon), Lycopersicon spp. (tomato), Brassica spp. (turnip, cabbage, cauliflower, etc.), Beta spp. (turnip), Phaseolus spp. (bean), Helianthus spp. (sunflower), Arachis spp. (peanut), Amaranthus spp. (foxtail), Medicago spp.

(alfalfa), Trifolium spp. (clover), Atropy spp. (sweet vetiver), Hyoscyamus spp. pine).

Transformable plastids are e.g. chromoplasts, amyloplasts, 1eucoplasts, etioplasts, proplastids and chloroplasts. Particularly preferred for the transformation are chloroplasts.

The present invention also includes genes that are functional in the plastids and mitochondria. The genes according to the present invention transfer a desired useful property when they are enclosed in the plastids and mitochondria. Certain examples of such useful properties are: phytotoxin resistance, such as e.g. herbicide or antibiotic resistance, improved photosynthetic effect as well as enzyme activity in cases where a chromogenic substrate is known to the enzyme.

In the present case, herbicide resistance is understood as a resistance in relation to all herbicides that are active in the plastids or mitochondria. For example, numerous herbicides are active in chloroplasts. This herbicide class includes e.g. triazine, urea, sulfonylurea as well as uracil derivatives as well as glyphosate (N-phosphormethylglycine).

Some examples of triazine herbicides include atrazine, ametryn, metribuzin as well as simazine. Some examples of urea herbicides include phenylurea, which e.g. diuron, chloroxouron and fluoromethuron as well as DCMU (dichloromethyl urea). Examples for sulphonylureas are Oust and Glean, and for uracin herbicides bromasil and terbasil. These and other herbicides are described by LeBaron and Gressel (1982).

In order to make a cell resistant to herbicides, it is not necessary that, by direct gene transfer, each of the 50 to 100 chloroplasts in each cell contains a gene coding for herbicide resistance. A direct gene transfer in a small part of the existing chloroplasts is completely sufficient to protect the cells against herbicides.

The possibility of transferring a herbicide resistance e.g. against atrazine to plants is desirable for various reasons. This allows, for example, the use of such herbicides in larger doses for plants which are thus tolerant to this herbicide. With higher herbicide doses, a better weed control effect is thus achieved at the same time.

In addition, however, the herbicide resistance can also be used as a selective marker that is genetically linked with a physiological characteristic that is in itself difficult to select. In order to make use of this possibility, a donor plasmid is constructed which contains a gene which produces herbicide resistance as well as a second gene which adds the second desired characteristic. This can, for example, be about an increased photosynthetic effect. These donor DNA are enclosed in plant protein patches that can be regenerated into a plant cell culture or into whole plants. The resulting plants thus possess both the properties for herbicide resistance and also the other properties mentioned. If these plants are allowed to grow in the presence of the herbicide in question, it is thus possible to select plants that have this aforementioned second property.

In addition, the introduction of a donor DNA that transfers both a herbicide resistance and another, from an agronomic point of view, useful property to plant protoplasts gives this other property stably in the chloroplasts when the plants are grown in the presence of the herbicide.

Regarding instability of foreign genes in chloroplasts, it has previously been shown to De Block et al. (1985) see above.

Particularly preferred is a herbicide resistance to atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine).

For the in vitro identification of cells with transformed plastids or mitochondria, an antibiotic resistance can be used.

An antibiotic resistance is a property that can be used for in vitro identification of cells containing transformed plaques or mitochondria. Genes containing such selected markers are very useful when they have been genetically linked to agronomically useful traits. For this, for example, resistance to chlormaphenicol, kanamycin or, in principle, also resistance to other existing antibiotics are suitable.

A useful property that is primarily suitable as a marker for a selection (Screening) for the identification of plant cells from tissue cultures that contain genetically manipulated plastids or mitochondria is obtained, for example, by including a gene that codes for an enzyme that has a coloring substrate. If the said enzyme is, for example, beta-galatosidase, the plant cells are planted on a tissue culture medium containing the color-giving substrate Xgal (5-chloro-4-bromo-3-indolyl-jSD-galactosidase). Plant cells containing genetically manipulated plastids or mitochondria are thereby stained through the dye indigo blue due to the cleavage of Xgal by fjgalactosidase being released.

The genes, which are suitable for the present invention, can be extracted by methods known per se. With these methods, it concerns e.g. about natural isolation usually only genes occurring outside the plastids or mitochondria or their variants to be included.

Said gene can be any naturally occurring gene or one of its functional variants in plastids or mitochondria. Naturally, existing plastid, mitochondrial or bacterial genes are preferred. Particularly preferred is chloroplast gene.

In order to be sure that said gene is actually located in the plastids or mitochondria and not, for example, in the cell nucleus, one can preferably, but not necessarily, insert a gene in the cell nucleus that is not or at least clearly less functional than in the plastids or mitochondria.

Certain examples of naturally occurring bacterial genes are e.g. such as code for antibiotic resistance or for enzymes with a color-giving substrate. A bacterial gene which in position transfers an antibiotic resistance to plastids or mitochondria is e.g. the chloramphenicol acetyl transferase gene. A bacterial gene that codes for an enzyme with a chromogenic substrate is e.g. lacZ, which encodes £galactosidase. Certain examples of plastid or mitochondrial genes are genes that code for herbicide resistance or for an improved photosynthetic effect. A chloroplast gene that is in a position to transfer herbicide resistance is e.g. the mutated psbA gene, which is described by Hirschberg et al. (1983) or a mutant of the dsbD genes (Rochaix et al., 1984). A chloroplast gene that is in a position to transfer an improved photosynthetic effect is e.g. represented by a modified form of rbcL encodes a modified large subunit of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). A gene that gives the large subunit of Rubisco is easily enclosed in the chloroplasts and activated during photosynthesis. However, it is also possible to contain a modified (mutated, manipulated or heterologous) form of this gene with improved photosynthetic effect [Jordan et al.,

(1981)] A further possibility for obtaining a gene that is functional in plastids or mitochondria consists in constructing a chimeric gene. A chimeric gene is a gene or at least part of a gene, preferably an active part of a gene which is naturally not covalently bound to the other parts. The chimeric gene can either code for a specific RNA transcript or for a polypeptide.

As promoters for chimeric genes, within the scope of the present invention, all promoters that are functional in plastids or mitochondria come into consideration. Promoters for chimeric genes may originate from a naturally occurring plastid or mitochondrial gene or from a gene that does not occur in the plastids or mitochondria. Among the naturally occurring promoters from a plastid or mitochondrial gene, those originating from the psbA, psbD or rbcL gene are particularly preferred. Said promoters can also be variants of natural promoters. A heterologous promoter for the chimeric gene may, according to the invention, be a natural promoter which does not normally occur in plastids or mitochondria.

As the functions of plastid or mitochondrial genes are often similar or identical to the function of bacterial genes, bacterial promoters can also be functional in plastids or mitochondria. Every bacterial promoter which is functional in the plastids or mitochondria can thus also, according to the invention, be inserted as a promoter for the chimeric genes. Some useful bacterial promoters are e.g. such as the neomycin phosphortransferase II gene and the T-DNA genes such as e.g. the nopaline synthase gene to the Ti plasmid.

As a heterologous promoter, every partially or completely synthetically produced promoter that is functional in plastids or mitochondria also comes into consideration.

Partially or completely synthetically produced promoters that resemble their natural models in that they contain 10 nucleotides below the transcriptional amplification a TATAAT-like sequence are also a component of the present invention.

The untranslated 5' region of the chimeric gene of the present invention can be any suitable untranslated 5' region that is functional in the plastids or mitochondria. The untranslated 5' region can, for example, originate from a plastid or mitochondrial gene or from a gene of other origin. A preferred homologous 5' untranslated region originates from the psbA, psbD or rbcL gene. A preferred heterologous 5' untranslated region is derived from bacterial genes. Synthetic 5' untranslated regions which, because of an effective ribosome binding site, resemble their natural models are in any case a constituent of the present invention.

In principle, any suitable coding region which is in a position to transfer a desired property to a plant cell is suitable for use according to the invention as a coding region. Both the coding regions of the above-mentioned naturally occurring gene as well as coding regions from other sources can be used within the present invention for the production of chimeric genes.

Accordingly, the coding regions within the present invention may originate from naturally occurring plastid or mitochondrial genes or from another source and they may also be completely or partially synthetically produced or by fusion of two or more such coding regions while maintaining the reading frame.

Any of these coding sequences encodes a polypeptide that transfers one or more new properties to the cells and plants.

An example of a polypeptide which is encoded via a gene naturally occurring in the chloroplasts of certain plants is the mutated form of the 32 kd polypeptide, which is described by Hirschberg et al. (1983). This polypeptide could be shown to transfer an atrazine resistance to certain weeds. However, the psbA genes, which code for said polypeptide, can also be transferred to useful plants such as cultivated plants using the method according to the invention.

An example of a coding region that does not originate from plastids or mitochondria is the coding region of plant, animal or bacterial gshA and gshB gene or those of the glutathione reductase (gor) gene by which all its useful properties can be transferred when enclosed in plant plastids or mitochondria. gshA and gshB code for enzymes that can catalyze the synthesis of the tripeptide glutathione, which in turn ensures the conjugative detoxification of numerous herbicides (Meister et al., 1983, as well as Rennenberg 1982).

A further example of a coding region that does not originate from plastids or mitochondria is the coding sequence for a glutathione S-transferase gene from plants, animals, insects or bacteria. In many plants [Shimabukuroy et al.

(1971)], which are known to be tolerant to the herbicide atrazine, the presence of glutathione-S-transferase forms the basis for this tolerance. Glutathione-S-transferase detoxifies the phytotoxin by catalyzing the formation of an atrazine-glutathione conjugate. Another coding region useful for the chimeric gene of the present invention encodes an efficient form of Rubisco. Such coding sequences may possibly originate from naturally occurring genes.

The coding region according to the present invention may optionally also include two or more different coding regions. Polypeptides, which are specified via such coding regions, are termed fusion polypeptides.

An example of a fusion polypeptide produces an efficient form of Rubisco. Rubisco has two functions, firstly as a carboxylase and secondly as an oxygenase. The carboxylase function is active during photosynthesis, the oxygenase function, on the other hand, is undesirable. A usable fusion polypeptide, on the other hand, has a part where the carboxylase function is maximal and another part where the oxygenase function is minimal.

The 3' untranslated region of the chimeric gene can either be naturally present in a plastid or mitochondrial gene or it can be of heterologous origin. Preferred is an untranslated 3' region originating from a plastid or mitochondrial gene. The preferred 3' untranslated sequence corresponds to the psbA, psbD or rbcL gene.

The transcribed regions of the gene according to the present invention can specify RNA transcripts and are in some cases also applicable as such.

These transcripts can be tRNA or rRNA. The transcript can also be an RNA that has a sequence that is at least complementary to part of a sequence of another RNA transcript. Such complementary transcripts are known under the term anti-sense sequences, and if they are long enough, they can disrupt the function of the RNA sequences to which they are complementary or they can block the transcription of these RNAs by the corresponding gene, [Compare Pestka et al ., (1984), Melton (1985), Izant et al., (1984), Simons et al. (1984) and Coleman (1984)].

Transcribed regions, which are applicable within the present invention, may optionally originate from naturally transcribed sequences or they may be completely or partially produced synthetically. Antisense sequences can nevertheless originate from at least part of a naturally occurring gene, by reversing the orientation of said part of the naturally occurring gene in relation to its promoter.

The genes, which are intended for use within the scope of the present invention, are introduced or built into a plastid cloning vector in a manner known per se [Maniatis et al. , (1982) ].

For the integration of foreign genes into the genome DNA from plant cells, it is advantageous if the gene is coated with neutral DNA sequences, so-called Carrier DNA. Carrier DNA can consist of two linear DNA chains so that the complete construct to be contained in the plant cells produces a linear DNA molecule. Said construction can, however, for the gene transformation also exhibit a circular structure - plasmid structure. Carrier DNA can be of synthetic origin as well as originating from naturally occurring DNA sequences that can be treated with suitable restriction endonucleases. For this, for example, naturally occurring plasmids that can be opened with one or more restriction endonucleases are also suitable for use as Carrier DNA. An example of such a plasmid is the readily available plasmid pUC8 [described by Messing et al., (1982)]. According to the invention, fragments of naturally occurring plasmids can also be used as Carrier DNA. The construction suitable for the transformation may optionally contain a Ti plasmid or DNA derived from a modified Ti plasmid. A plasmid is regarded as a modified Ti plasmid when it has at least one T-DNA border region. A T-DNA border region is a DNA sequence within the agrobacterium's genome which causes contact between the introduction of another sequence within the genome (T-DNA) in the plant cells with that of the agrobacterium. The DNA boundary region can, for example, be a sequence of a vector such as a Ti or Ri plasmid or a modified Ti or Ri plasmid. In the case of T-DNA, it can also be a sequence that is located on the same vector in the border region or on another vector.

The probability of genetic transformation (the degree of transformation) of a plant cell can be increased by various factors. As is known from experiments with yeast, the number of successful stable gene transformations thus increases:

1) the increase in the number of copies of new genes per cell,

2) when the replication signal is combined with a new gene, and 3) when an integration signal is combined with a new gene whereby the integration signal is understood as a signal that requires the integration of a DNA chain into another DNA chain.

The method according to the invention thus finds a particularly advantageous application when the transferred gene is coupled with a replication signal which is active in plant cells or with an integration signal which is active in plant cells or with a combination of both signals.

Protoplasts, cell culture cells, cells in plant tissue, pollen, pollen sacs, egg cells, embryo sacs or zygotes as well as enbryos in various stages of development are representative examples of plant cells that are suitable as starting material for a transformation. Protoplasts are preferred in that they can be used directly and without further pre-treatment. Isolated plant protoplasts, cells or tissues can be produced using methods known per se or using methods which are analogous to known methods.

Isolated plant protoplasts, which are suitable as starting material for the production of isolated cells and tissues, can also be isolated from all parts of the plant, e.g. from the leaves, embryos, stems, flowers, roots or pollen. Protoplasts from leaves are preferred. However, isolated protoplasts can also be obtained from cell culture. Methods for isolating protoplasts are, for example, described in Gamberg et al. (1975).

The transfer of the new genes in the plant cells is direct, i.e. without previous infection of the cells with a pathogen such as a plant pathogenic bacterium, virus or fungus and without the transfer of DNA via insects or fungi that are in a position to infect plants with DNA-transmitting pathogens. This direct gene transfer is achieved by bringing the gene-containing DNA into contact with the plant protoplasts and the plastids and mitochondria contained therein in a medium for such a period of time that the introduction of the gene into the protoplasts and into the plastids and mitochondria contained therein is achieved.

The transformation frequency can thereby be increased by combining this step with different gene transfer techniques. Examples of such techniques include the treatment with poly-L-ornithine or poly-L-lysine, Liposome fusion, DNA-protein complex formation, change of 1ading conditions in the protoplast membrane, fusion with microbial protoplasts or calcium phosphate precipitation, as well as especially by treatment with certain polyhydric alcohols, e.g. polyethylene glycol, further by heat shock treatment and electroporation as well as by a combination of the three latter techniques.

As useful media according to the invention, all media that allow the penetration of the DNA carrying the gene into protoplasts as well as into plastids or

mitochondria within these protoplasts.

Suitable media for joint incubation of the foreign gene with the receptor protoplasts are preferably osmotically stabilized culture media developed for protoplast cultures.

Many, later obtained carbon media differ in certain components or groups of components. The composition of all these media is, however, in accordance with the principle that they all have a group of organic ions, a concentration of approx. 10 mg/litre to approx. 100 mg/liter (so-called macroelements such as nitrate, phosphate, sulfate, calcium, magnesium, iron, etc.), and a further group of organic ions with a maximum concentration of a few mg/liter (so-called trace elements such as cobalt, zinc, copper, manganese , etc.) a number of vitamins (e.g. inositol, folic acid, thiamine), an energy and carbon source, for example sucrose or glucose and growth regulators in the form of natural or synthetic phytohormones of the auxin and cytokinin class in a concentration of 0, 01 to 10 mg/litre.

These culture media are also osmotically stabilized by the addition of sugar alcohols (e.g. mannitol), sugar (e.g. glucose) or salt ions (e.g. CaCl^) as well as being adjusted to a pH value in the range from 5.6 to 6.5.

A more detailed description of conventional cultural media can be found e.g. in Kobliz et al., (1974).

A particularly suitable medium for the direct transformation of protoplasts contains a polyhydric alcohol which is in a position to alter the protoplast membrane and which proves useful in producing the cell fusion. The preferred polyhydric alcohol is polyethylene glycol. Polyhydric alcohols with longer chains, for example polypropylene glycol (425 to 4000 g/mol), polyvinyl alcohol or polyhydric alcohols whose hydroxyl groups are partially or completely alkylated can also be used. Polyhydroxylated detergents, which are common in agricultural use and which are tolerated by plants, are in all cases prepared from suitable polyhydric alcohols: such detergents are e.g. described in the following publication: "Mc Cutcheons's Detergents and Emulsifiers Annual"

MC Publishing Corp., Ridgewood, New Jersey, (1981);

Stache, H., "Tensid-Taschenbuch",

Carl Hanser Verlag, Munich/Vienna, (1981).

The polyhydric alcohols preferred for this purpose are polyethylene glycols with a molecular weight between 1000 and 10000 g/mol, preferably between 3000 and 8000 g/mol.

The transformation frequency for the direct gene transfer can be increased by the following method described in detail.

During the polyethylene glycol treatment, a protoplastic suspension of the culture medium is then added. The DNA, which contains the gene and which can be present as linear DNA or in the form of a circular plasmid, is finally added to the present mixture of polyethylene glycol and culture medium. As an alternative to this method, the protoplasts as well as the gene-containing DNA can also be present in the culture medium and then polyethylene glycol can be added.

Within the scope of the present invention, electroporation as well as heat shock treatment prove to be particularly advantageous methods.

Neumann et al. (1982) mentions that with electroporation, the protoplasts are subsequently transferred into an osmotic medium, e.g. in a mannitol/magnesium suspension and this protoplast suspension is finally brought into an electroporator chamber between two electrodes. When discharging a capacitor above the suspension, the protoplasts are for a short moment supplied with a short electrical impulse of high voltage, which leads to a polarization of the protoplast membrane and an opening of the pores in the membrane.

During the heat treatment, the protoplasts are suspended in an osmotic medium, e.g. a solution of mannitol/calcium chloride. Finally, the suspension is heated in small containers, e.g. in a centrifuge tube, preferably in a water bath. The duration of the heating depends on the previously selected temperature. Typically, these are temperatures in the range from 40°C to 80°C and are maintained for a duration of 1 second to an hour. The best results are obtained at a temperature in the range from 40°C to 50°C for 4 to 6 minutes, especially at 45°C and 5 minutes. The suspension is finally cooled to room temperature or below. It can also be shown that the transformation frequency is increased by inactivation of extracellular nuclease. The inactivation can be achieved by using divalent cations which are tolerated by plants, such as magnesium or calcium. The inactivation of more efficient when the transformation is carried out at a high pH value whereby the optimal pH range is between 9 and 10.5.

Surprisingly, the selective use of these different methods leads to a significant increase in the transformation frequency, which has long been a goal within genetic engineering.

The lower the transformation frequency in gene transformation experiments, the more difficult and time-consuming it is to find the small cloned cells that originate from the transformed cells compared to the large number of non-transformed clones. With only a small transformation frequency, it is practically impossible to use conventional screening methods and thus the gene used has a selective marker (eg resistance to a specific substance). Small transformation frequencies thus require very careful work and time when using genes without the marker function.

When bringing together the foreign gene and receptor protoplasts when using other measures such as e.g. polyethylene glycol treatment, electroporation and heat shock treatment, it is possible in connection with a method to achieve a significant improvement in the transformation frequency by a single method step in a different order.

A combination of two or three of the techniques selected below has proven beneficial: polyethylene glycol treatment, heat shock treatment and electroporation, whereby particularly good results are achieved when these techniques are used in a liquid when introducing the foreign gene and the protoplasts. The preferred method consists of a heat shock treatment before polyethylene glycol treatment and possibly subsequent electroporation. In general, the additional electroporation leads to a further increase in the transformation frequency and in many cases the results obtained by heat shock treatment and polyethylene glycol treatment cannot be significantly improved by even an additional electroporation.

You can also add divalent cations that are tolerated by plants and/or combine a transformation at a pH value between pH 9 and 10.5 with the individual measures described above that change the transformation frequency, namely polyethylene glycol treatment, heat shock treatment and electroporation.

Method according to the invention thus makes it possible to achieve high transformation frequencies without having to use pathogens, such as viruses and agrobacteria or natural or modified Ti plasmids for the transformation.

A preferred embodiment of the method according to the invention is, for example, characterized by the fact that the protoplasts are transferred in mannitol solution and finally that the thus obtained protoplast suspension is mixed with an aqueous DNA solution containing the gene. The protoplasts are then incubated in this mixture for 5 minutes at 45°C and then cooled for 10 seconds to 0°C. At the end of the incubation, so much polyethylene glycol (MG 3000 to 8000) is added to this mixture that a PEG concentration of one to 25% is achieved, preferably approx. 8%. After careful and thorough mixing, treatment follows in the electroporator. The protoplast suspension is then diluted with culture medium and can finally be stored for regeneration of their cell walls in culture medium.

The method according to the invention is suitable for the transformation of all plant cells, especially for cells of the systematic group angiosperm and gymnosperm.

Under gymnosperma, in the first line, plants of the class Coniferae are of interest.

Under angiosperma are under deciduous trees and shrubs the plants of the following families of special interest: Solanaceae, Cruciferae, Compositae, Liliaceae, Vitaceae, Chenopodiaceae, Rutaceae, Bromeliaceae, Rubiaceae, Theaceae, Musaceae or Gramineae and within the order Leguminosae the family of Papilionaceae.

Particularly preferred are members of the Solanaceae, Cruciferae and Gramineae families.

Of particular interest are plants of the family Nicotiana, Petunia, Hyoscyamus, Brassica and Lolium, and e.g. Nicotiana tabacum, Nicotiana plumbaginifoli a, Petunia hybrida, Hyoscymaus muticus, Brassica napus, Brassica rapa and Lolium mulitiflurum.

Plastids and mitochondria from each plant which are regenerable from protoplasts can in any case be easily transformed using the method according to the invention. Until today, it was not possible to manipulate plastids and mitochondria from members of the family Graminae (grasses) and also plant species, e.g. maize, wheat, rice, barley, oats, millet, rye and millet (Sorghum) genetically. The present invention now allows the genetic transformation of plastids and mitochondria from graminae cells and finally grain cells using direct gene transformation. In a similar way, it is possible to carry out the transformation of plastids and mitochondria in any other suitable cultivated plant such as e.g. plants from the family Solanum, Nicotiana, Brassica, Beta, Pisum, Phaseolus, Glycine, Helianthus, Allium, Triticum, Hordeum, Avena, Setaria, Oryza, Cydonia, Pyrus, Malus, Rubus, Fragaria, Prunus, Arachis, Secale, Panicum Saccharum, Coffea, Camellia, Musa, Pineapple, Vitis, Sorghum, Helianthus or Citrus.

The incorporation of transformed genes into plastids or mitochondria can be detected using common methods, e.g. by genetic crossing experiment or with molecular biological detection tests, especially the detection test according to Southern for plastid and mitochondria DNA as well as enzyme activity tests.

The detection method according to Southern can, for example, be carried out as follows: DNA that is isolated from plastids or mitochondria from transformed cells or protoplasts is post-treated with restriction enzymes, subjected to electrophoresis in a 1% agarose gel and then transferred to a nitrocellulose membrane [Southern et al., ( 1975)] and hybridized with an in vitro labeled DNA whose existence is to be determined. DNA can be labeled with specific activity in vitro

8 8

between 5 x 10 and 10 x 10 c.p.m./microgram by means of "nick translation" [Rigby et al., (1977)]. The filter is finally washed three times for one hour with an aqueous solution of a 0.03 molar sodium citrate solution and a 0.3 molar sodium chloride solution at a temperature of 65°C. The hybridized DNA is detected by several days of auto-radiography on an X-ray film.

The cells, which have been transformed with the desired gene, are isolated using methods known per se. These methods retain the selection and screening. The selection of nuclear genes is described by Fraley et al., (1983), Herrera-Estrella et al., (1983), and Bevan et al., (1983). A screening can according to £galactosidase [Helmer et al.,

(1984)], Nopalin synthase or Octopin synthase [Wostmeyer et al., (1984), DeGreve et al., (1982)] or atrazine resistance.

Before the present invention, it was not shown that the plastid or the mitochondria could be transformed by direct gene transfer. Consequently, it was previously not possible to functionally introduce usable genes in the form of isolated DNA into these organelles. Genes are considered functional in the genome of plastids or mitochondria when they are incorporated for replication and expression. Plasmids and mitochondria of any plant that are regenerable from protoplasts can nevertheless be transformed by means of the method according to the invention. Until today, it was previously not possible to manipulate plastids and mitochondria from members of the family Gramina (grasses) nor cereals, e.g. maize, wheat, rice, barley, oats, millet, rye and millet. The present invention now allows the genetic transformation of plastids and mitochondria from graminae cells and finally said grain cells using direct gene transformation. In a similar way, it is possible, it is possible to carry out transformation of plastids and mitochondria from other similar cultivated plants such as e.g. plants from the family Solanum, Nicotiana, Brassica, Beta, Pisum, Phaseolus, Glycine, Helianthus, Allium, Triticum, Hordeum, Avena, Setaria, Oryza, Cydonia, Pyrus, Malus, Rubus, Fragaria, Prunus, Arachis, Secale, Panicum, Saccharum, Coffea, Camellia, Musa, Pineapple, Vitis, Sorghum, Helianthus or Citrus.

The inclusion of transformed genes in plastids or mitochondria can be detected using known methods, e.g. by genetic crossing experiment or with molecular biological detection tests, especially the detection tests according to Southern for plastid and mitochondria DNA as well as enzyme activity tests.

An advantage of introducing genes into plastids or mitochondria by direct gene transfer lies in the possibility of simultaneous inactivation of unwanted genes that are easily present in the plastid or mitochondrial genome. This is thus possible because the inclusion of foreign genes in the plastid or the mitochondrial genome when using direct gene transfer does not necessarily proceed via a homologous recombination. Thus, for example, a donor DNA molecule that has an atrazine-resistant form of psbA is not necessarily integrated at the place where the corresponding atrazine-sensitive gene is located. Since, on the other hand, the plastid and the mitochondrial genome are relatively small, it is thus possible to select the transformed plant cells and thus find in these the donor DNA which happens to be embedded in some desired function of the recipient genome. The direct gene transfer in the plastid and the mitochondrial genome thus also opens up a way of inactivating genes that was previously not feasible. By means of a corresponding screening, it is thus possible, for example, to find transformed plant cells in which the atrazine-sensitive psbA gene has been inactivated by a donor DNA that has an atrazine-resistance gene.

A further advantage of direct gene transfer in the genome from plastids and mitochondria lies in the fact that, in contrast to nuclear genomes, this genome is inherited maternally through the cytoplasm and is thus usually not transmitted through pollen in a sexual way. Thereby, the possibility of transferring genes that convey desirable properties to cultivated plants is greatly reduced from cultivated plants to weeds.

Plant cells in cell cultures which are transformed using the method according to the invention can optionally be used for the production of polypeptides which are coded for by the contained gene. Such synthetic products include, for example, the 32 kd polypeptide expressed from psbA and further chloramphenicol acetyltransferase, glutathione S-transferase and the large subunit of Rubisco.

The plant cells, which contain the enclosed genes, can in many cases also be regenerated into multicellular plants that develop the gene and thus have the desired new properties. Any suitable plant which is regenerable from plant cells and which in turn is derived from protoplasts can be produced by the method of the invention.

Examples of corresponding whole plants are: Solanum spp.

(potato), Petunia spp. (petunia), Daucus spp. (carrot), Lycopersicon spp. (tomato), Brassica spp. (turnip, cabbage, cauliflower, etc.), Medicago spp. (alfalfa), Trifolium spp.

(clover), Zitruns, Atropa, Hyosxyamus, Salpiglossis, Arabidopsis, Digitalis, Cichorium, Gossipium, Glycine, Geranium, Anthirrhinum and Aspargus. The plants are regenerated using methods known per se, see Evans and Bravo,

(1983), Dale (1983); and plants can, for example, be regenerated from any suitable offshoot such as cells, calli, tissues, organs, buds, cuttings, shoots, roots, seedlings, somatic embryos and others.

The present invention further relates to protoplasts, plant cells, cell aggregates, plants and especially the grains from plants which have been produced using the method according to the invention and in addition as long as the formed seeds contain the built-in genes and thereby the resulting desired properties. It also includes all subsequent plants that have been produced using the method according to the invention, both sexually and vegetatively.

Sexual offspring can be obtained by self- or cross-pollination whereby the offspring also contain all crossing and fusion products as defined in the previous section and transformed plant material provided that this offspring exhibits the characteristic introduced via transformation. Seeds and progeny with herbicide resistance or tolerant parents that have unstable herbicide resistance or tolerance can maintain their herbicide resistance or tolerance as long as they grow in the presence of herbicide. Preferred herbicide is atrazine. The present invention thus allows the production of genetically manipulated plants which are in a position to survive a treatment with herbicides such as e.g. with atrazine in concentrations lethal to sensitive plants.

EXAMPLES:

Certain procedural measures of the following examples can be read in general form from Maniatis et al., (1983). The enzymes can be obtained from New England Biolabs and, with the exception of the special deviations in the text, are used according to the manufacturer's guidelines.

Example 1: Construction of pCAT^ (see Fig. 1)

1) The vector plasmid pMON 187 osm has a Km<r>gene of Tn 903 is cleaved with HindIII and BamHI. 2) A promoter-free CAT gene is isolated as a gel-purified HindIII/BamHI fragment of the plasmid pSOV [Gorman et al.,

(1982)]. 3) The joining of the fragments from step 2) with the fragment from step 1) gives a plasmid which is transformed into E. coli HB101. The selection follows o Ap IT. A Km<S> colony has the plasmid with the one in fig. 1 indicated structure for pCAT°.

Example 2: Alternative construction of pCAT°

Example 2 can be repeated with a different plasmid than pMON 187.

A suitable plasmid having the Km<r>gene of Tn 903 can be constructed as follows:

A 1.2 kb Avall fragment, which contains the Km gene

from Tn 903, is isolated from the plasmid pA02 [Oca et al.

(1982)]. The ends from the Avall fragments are filled in with the help of Klenow polymerase and are spliced together with BamHI linker with the smooth end of the fragment. The DNA is finally treated with the restriction enzymes TaqI and BamHI and inserted into the plasmid pBR327, which was previously treated with ClaI and BamHI. The recombinant material which

TC

containing the Tn903 fragment transfers Km to E.coli.

Example 3: Construction of p32CAT (see Fig. 1)

The following work steps are carried out to splice the psbA promoter with the promoter-free CAT^ gene.

4) pCAT° is digested with Smal and HindIII.

5) A gel-purified 161 bp Smal/HindIII fragment containing the psbA promoter is isolated from plasmid pAH484. The recombinant plasmid pAH484 contains the 3.68 kb EcoRI fragment of chloroplast DNA of (atrazine-) herbicide-resistant Amaranthus hydrids that was cloned into pBR322.

[Hirschberg and Mclntosch, (1983)].

6) The fragment isolated in step 5 is linked with the large fragment that appears in step 4.

let

ling leads to p32CAT, which transfers Cm to transformed E.coli cells.

Example 4: Construction of plasmid pUCH1, a vector with a psbA gene and a chimeric psbA/CAT gene construct (see fig. 2).

The plasmid pUCH1 is constructed in 5 steps in the pUC8 vector.

(Steps 7 to 11):

7) (Fig. 1) A 3.6 kb EcoRI fragment (gel-purified) containing the complete psbA gene is isolated from the above-described plasmid pAH484. 8) pUC8 (Fig. 2) is linearized at its only EcoRI site. 9) The linearized pUC8 fragment from step 8) is joined with the fragment described from step 7). The transformation of E.coli gives Ap r colonies that have the desired incorporated fragment whereby one of these colonies is designated pUC8-32. 10) A gel-purified 1.8 kb XhoI/BamHI fragment was isolated from p32CAT. 11) The resulting plasmid from step 9), pUC8-32, was partially digested with SalI and completely with BamHI. The splicing with the fragment resulting from step 10) and selection for Cm of subsequent transformation of E.coli leads to an E.coli colony containing pUCH1.

Example 5 Construction of pBRCAT containing a

chimeric psbA/ CAT gene.

The construction of pBRCAT is achieved by splicing the following three DNA fragments. a) the EcoRI/HindIII (promoter) fragment (approx. 660 bp) from the psbA genes in plasmid pAH484 (step 12); b) the HindIII/BamHI fragment of pSVO with a promoterless CAT gene (step 2); and c) the EcoRI/BamHI digested pBR322 as vector (step 13).

14) The splicing leads to a plasmid pBRCAT which transfers

Cm r to E.coli host cells in which this plasmid is contained upon transformation.

Example 6: Containment of donor DNA in plant protoplasts

Tobacco protoplasts with a population concentration of 2x10 per ml was suspended in 1 ml of a K^ medium [see Z.Pflanzenphysiologie 78, 453-455 (1976), Mutation Research 81, 165-175 (1981)] containing 0.1 mg/liter 2,4 dichlorophenoxyacetic acid, 1.0 mg/liter 1-naphthylacetic acid and 0.2 mg/liter 6-benzylaminopurine. The protoplasts were obtained from an enzyme suspension by flotation in 0.6 M sucrose at a pH of 5.8 and then sedimentation for 5 minutes at 100 g in 0.17 M calcium chloride at pH 5.8. To this suspension, 0.5 ml of 40% polyethylene glycol (PEG) with a molecular weight of 6000 in modified (after autoclaving in an adjusted pH value of 5.8) F medium [Nature 296, 72-74,

(1982)], and also added 65 µl of an aqueous solution containing 15 µg of donor DNA (p32CAT, pUCH1 or pBRCAT) and 50 µg of calf hour DNA. This mixture was cultured for 30 min at 26°C whereby the solution was gently stirred and finally diluted stepwise with F medium. The protoplasts were isolated by centrifugation (5 minutes at 100 g) and finally resuspended in 30 ml of fresh K^ medium. Further incubation followed at 24°C in the dark for 10 ml portions in Petri dishes with a cross section of 10 cm.

After three days, the culture medium was diluted in each Petri dish with 0.3 volume units of fresh K^ medium and incubated for a further 4 days at 24°C and 3000 lux. After a total of 7 days, the clones that had developed into protoplasts were enclosed in a 1% agarose solid culture medium containing 10 mg/liter chloramphenicol and cultivated at 34°C in the dark according to the "bead type culture method" [Plant Cell Reports, 2 , 244-247 (1983)]. The culture medium was replaced by a fresh medium every 5 days in the same way. After 3 to 4 weeks of uninterrupted cultivation in chloramphenicol-containing culture medium, the resistant plants of 2-3 mm in cross-section were transferred to an agar-fixed LS culture medium [Physiol. Plant. 18, 100-127 (1965)] which contained 0.05 mg/liter 2,4-dichlorophenoxyacetic acid, 2 mg/liter 1-naphthylacetic acid, 0.1 mg/liter 6-benzylaminopurine, 0.1 mg/liter kinetin and 10 mg/litre chlormaphenicol. Chloramphenicol resistant Nicotiana tobacco Petit Havana SRI plants were obtained by shoot induction on LS medium with 10 ml/liter chloramphenicol and 0.2 mg/liter 6-benzylaminopurine and finally root induction on T medium [Science 163, 85-87, (1969 )]

The selection of chloramphenicol resistant shoots and plants essentially followed that of De Block et al.,

(1984) stated procedure.

Example 7: Screening of atrazine-resistant chloroplast transformants

a) sprouts

An atrazine toxicity curve was prepared for non-transformed sprouts in a concentration range of 1 to 10 (ummol atrazine and different light intensities. Similarly, sprouts derived from chloroplast transformants and control sprouts were sown on nutrient agar plates with the atrazine concentration and light intensity as complete bleached out the chlorophyll from the control tissue Resistance to atrazine was visually tested based on the green coloration of the resistant tissue obtained.

b) Whole plants

Plants, which were obtained from chloroplast transformants, were

by the following method examined for atrazine resistance: Chlorophyll fluorescence induction in leaves. If the electron transport at the reducing site of photosystem II is inhibited, as for example with atrazine, then the radiation energy absorbed by chlorophyll is re-emitted as fluorescence. This fluoride

essence can be measured on the leaf surface. The fluorescence measurement was carried out on small leaves that were stretched horizontally over a transparent Lucite window as described in Nalkin et al.,

(1981). [Lucite stands for polymerized resin of methyl methacrylate.] The exciting light originates from a projector powered by direct current which, when passed through a filter, results in bands of actinic light of 500 to 600 nm with an intensity of approx. 10 nE x cm s . The duration of the exciting light is approx. 3 ms. The exciting light is directed at right angles to the leaf surface and the fluorescence from this surface is collected by means of a flexible light guide system and then, after passing through a cut-off filter (660 nm), transferred to a red-sensitive photomultiplier. The fluorescence passage was recorded on an oscilloscope and filmed directly.

c) Light modulated flotation

Excised leaf pieces are allowed to float on a phosphate buffer

containing detergent, this remains on the surface as long as photosynthesis continues, as this ensures a high O^/CO^ ratio in the intercellular spaces.

If, on the other hand, the leaf pieces are allowed to float in the dark or if photosynthesis-inhibiting herbicides are added to the medium, they very quickly lose their buoyancy and sink. An assay method for atrazine resistance is described by Hensley (1981). Leaf pieces are added to small tubes of atrazine-containing solution and placed under vacuum. The leaf pieces are quickly absorbed by the solution and sink to the bottom of the solution. The vacuum is finally removed and a bicarbonate solution is added and the tubes exposed to light. If the photosynthesis is not inhibited by atrazine, then the photosynthesis produces a buoyancy by produced oxygen within the tissue and the leaf pieces float towards the surface. Atrazine-sensitive pieces, on the other hand, remain on the bottom.

Example 8: Transformation of cells of Brassica rapa

(compare Just Right)

Brassica rapa protoplasts are grown with a suitable osmotic medium and suspended at a population density of 5x10^ per ml in a culture medium prepared according to the description in [Protoplast 83, Proceedings Experientia Supplementum, Birkhauser Verlag, Basel, Vol 45 (1983), 44-45]. 40% polyethylene glycol (PEG) with a molecular weight of 6000 dissolved in modified F medium (pH 5.8) (compare example 1b) is mixed with the protoplast suspension to a final concentration of 13% PEG. Finally, a solution consisting of 50 micrograms of endonuclease Sal I digested plasmid for pBRCAT or p32CAT and 60 µm of water is quickly added to this mixture. With gentle stirring, this mixture is incubated for 30 minutes at 20° to 25°C. Then becomes 3x2

mm modified F medium (total 6 ml) and 2 x 2 mm culture medium (total 4 ml) added at 5 minute intervals. The protoplast suspension is transferred to Petri dishes with a 10 cm cross-section and additional culture medium is added to a total volume of 20 ml. This protoplast suspension is incubated for 45 minutes at 26°C in the dark.

The protoplasts are isolated by 5-minute sedimentation and taken up in a subsequent liquid and later fixed by agarose gel culture medium and cultivated according to the "bead type culture method" [Plant Cell Reports, 2, 244-247 (1983)]. After four days in the development stage to the first cell division, chloramphenicol is added in a concentration of 10 mg/litre to the culture. The liquid culture medium, which surrounds the agarose segments, is added every four days with fresh chlorine-amphenicol-containing nutrient solution. After four weeks, the chloramphenicol-resistant clones are isolated and finally further cultured, adding chloramphenicol-containing nutrient solution (10 mg/litre) weekly.

Example 9: Transformation of protoplasts

The starting point is Lolium multiflorum protoplasts with a concentration of 2x10 per ml in a 0.4 molar mannitol solution with pH 5.8. To this suspension are added successively 0.5 ml of 40% polyethylene glycol (PEG) with a molecular weight of 6000 in modified (pH 5.8) F medium [Nature 296, 72-74, (1982)] and 65 µl of a aqueous solution with 50 µg of the plasmids p32CAT or pBRCAT. This mixture is incubated for 30 minutes at 26°C and gentle stirring and finally diluted with F-medium. This happens according to the description in [Nature 296, (1982)]. The protoplasts are isolated by centrifugation (5 minutes at 100 g) and taken up in 4 ml CC culture medium [Potrykus, Harms and Lorz, Callus Formation from Cell Culture Protoplasts of Corn (Zea Mays L.), Theor. Appl. The gene. 54, 209-214 (1979)] and incubated at 24°C in the dark. After 14 days, the developed cell cultures are transferred into a corresponding medium which, however, now contains chloramphenicol (10 mg/litre). Resistance colonies are transferred to an agar medium - the same medium as above 10 mg/litre chloramphenicol without osmotic agent, and finally after they have reached a size of several grams of fresh weight per colony analyzed with regard to the maintenance of the bacterial genes and the biological activity of the genes.

Example 10: Transformation of cultured cells from Nicotiana tabacum by transfer of p32CAT, pBRCAT or pUCH1 using electroporation

Protoplasts are obtained by sedimentation of 50 ml of a 10 g phase suspension culture of a nitrate reductase defective variant from Nicotiana tabcum, cell strain nia-115 [Muller, A.J. and R. Grafe, Mol. Gen. The gene. 161, 67-76 (1978)] and resuspended in 20 ml of an enzyme solution [2% Cellulase Onozuka R-10, 1% Macerozume R019 and 0.5% Driselase (from der Chemischen Fabrik Schweizerhalle, Basel) in a washing solution (0.3 M mannitol, 0.04 M calcium chloride and 0.5% 2-(N-morpholine)ethanesulfonic acid) adjusted to pH 5.6 with KOH] and incubated for 3 hours on a rotary shaker at 24°C. The protoplasts were finally separated by filtration through a sieve with a mesh width of 100 µm of undiluted tissue. The corresponding volume was added to a 0.6 M sucrose solution and the resulting suspension centrifuged for 10 minutes at 100 g. The protoplasts floating on the surface are collected and washed 3 times by sedimentation in the washing solution.

The transformation is carried out using electroporation. The chamber of a Dialog "Porator" (from Dialog Gmbh Harffstr. 32, 4000 Diisseldorf, Germany) is sterilized by washing with 70% alcohol and then with 100% alcohol and dried by a stream of sterile air by a laminar air-flow blast . The protoplasts are suspended in a concentration of 1x10^ per ml in a 0.4 M mannitol solution and adjusted to a resistance of 1.4 KOhm with magnesium chloride. Finally, pBRCAT, pUCH1 or p32CAT is added at a concentration of 10 µg/ml. 0.38 ml samples of this protoplast suspension are added 3 times at intervals of 10 seconds with a voltage of 1000 or 2000 Volts. The protoplasts are finally grown in a concentration of 1x10^ per

ml in 3 ml of AA-CH medium [AA Medium from Glimelium, K. et al., Physiol. Plant. 44, 273-277 (1978)] which was modified both by increasing the inositol concentration to 100 mg/liter and the sucrose concentration to 34 g/liter and by adding 0.05 ml/liter 2-(3-methyl- 2-butenyl)adenine and due to its 0.6% agarose content [Sea Plaque, FMC Corp., Marine Colloids Division, P.O. Box 308, Rockland, Maine 04841, USA], After one week, the agarose layer containing the protoplasts is transferred into 30 ml of a liquid AA-CH medium containing 10 mg/liter chloramphenicol. After three weeks, during which the corresponding half of the medium is replaced weekly by fresh medium with the same composition, the transformed cell colonies can be examined visually. 4 weeks after the transfer in the chloramphenicol-containing medium, these colonies are transferred to an AA medium [Glimelium, K. et al., Physiol. Plant. 44, 273-277 (1978)] with 0.8% agar containing 10 mg/liter chloramphenicol for further cultivation and examination.

Analogous investigations with protoplasts from Brassica rapa and Lolium multiflorum likewise lead to similar trans-

formation.

Example 11: Transformation of chloroplasts of Nicotiana tabacum cells by transfer of donor DNA using electroporation

Preparation of the electroporator and protoplasts follows according to Example 10 or Example 6.

For the transformation, protoplasts of Nicotiana tabacum are resuspended in a concentration of 1.6x10^ per ml in a mannitol solution [0.4 M, buffered with 0.5% w/v 2-(N-morpholine)ethanesulfonic acid, pH 5.6]. The resistance of the protoplast suspension is measured in the porator chamber (0.38 ml) and adjusted with a magnesium chloride solution to a value between 1 and 1.2 KOhm. 0.5 ml samples are taken out and come in capped plastic tubes (5 ml volume) in which 40 µl of water with 8 µg of donor DNA and 20 µg of calf thymus DNA were previously added as well as 0.25 ml of a polyethylene glycol solution (24% w/v in 0.4 M mannitol). 9 minutes after addition of DNA, 0.38 ml samples are added to the electroporator chamber. 10 minutes after the addition of DNA, the protoplast suspension is fed into the chamber with three pulses (1000-2000 Volts) at an interval of 10 seconds. The treated samples are added to Petri dishes with a 6 cm cross section and kept for 10 minutes at 20°C. Finally, 3 ml of K^ medium at 0.7% w/v "Sea Plaque Agarose" is added to each Petri dish and the contents of the dishes are carefully mixed. After solidification, the contents of each dish of the cultures were kept for one day at 24°C in the dark and 6 days in the light. The protoplast-containing agarose is then cut into 4 parts and added to a liquid medium. The protoplasts are finally cultivated according to the "bead type culturing method". Newly formed plant tissue, which is obtained by selection of the transformed material with chloramphenicol and plants that are regenerated from there, contains the CAT enzyme (chloramphenicol acetyl transferase) as a product of the CAT gene. The electroporation leads to a 5 to 10-fold increase in the transformation frequency compared to the method without electroporation. Analogous investigations with Brassica rapa cv. Just Right and Lolium multifluroum likewise gave an increase in the transformation frequency in a similar order of magnitude.

Example 12: Transformation of cells of Nicotiana tabacum by transfer of the CAT gene using heat shock treatment

Protoplasts that were isolated from leaves or from Nicotiana tabacum cell cultures are, as described in examples 6 and 10, recovered and corresponding to previous examples transferred to an osmotically stabilized medium.

The protoplast suspensions are kept for 5 minutes at 45°C and then cooled for 10 seconds with ice at 0°C and finally the plasmids pBRCAT, pUCH1 or p32CAT are added as described in Examples 6 and 10.

The heat shock treatment increases the transformation frequency by a factor of 10 or more, compared to a transformation carried out without this treatment.

Example 13: Transformation of plant cells of different

origin by transmission of the CAT genes,

as in the first step protoplasts and genes are brought together and finally a combined treatment is carried out

Plant proto-patch: Nicotiana tabacum cv. Petit Havana SRI (A); Brassica rapa cv. Just Right (B) and Lolium multiflorum (C) were isolated and transferred according to example 11 to an osmotically stabilized medium. The protoplast suspensions are mixed with the plasmids pUCH1, p32CAT or pBRCAT according to examples 6 to 9, but without simultaneous treatment with polyethylene glycol. The protoplast suspensions are then subjected to a heat shock treatment according to example 12 and finally to a polyethylene glycol according to examples 6 to 9 and finally subjected to an electroporation according to example 11. The transformation frequency in this method is between 10 and 10 and can, however, be increased to 1 according to the chosen conditions % to 2%.

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Claims (36)

1. Method for direct inclusion of DNA in plastids and mitochondria of plant protoplasts whereby said DNA comprises one or more genes and in the plastids and mitochondria active promoters, characterized in that in the absence of a pathogen of said DNA in a medium in which this DNA is able to draw into the protoplasts and the plastids and mitochondria contained therein and bring this into contact with the protoplasts for such a long time that this penetration is ensured. 2. Method according to claim 1, characterized in that this is allowed to continue until regeneration of transformed plant cells by the transformed protoplasts. 3. Method according to claim 1 or 2, characterized in that the method is allowed to proceed to regeneration of transformed plants. 4. Method according to claim 1, characterized in that it concerns linear DNA in the enclosed DNA. 5. Method according to claim 1, characterized in that circular DNA is involved in the enclosed DNA. 6. Method according to claim 5, characterized in that the circular DNA contains 1 or more T-DNA border regions. 7. Method according to claim 1, characterized in that it concerns a DNA of the enclosed DNA which transfers a herbicide resistance to the plastids or mitochondria. 8. Method according to claim 7, characterized in that the herbicide is atrazine. 9. Method according to claim 7, characterized in that the enclosed DNA, in addition to the herbicide resistance, transmits a second property useful for agriculture. 10. Method according to claim 1, characterized in that the contained DNA contains a selectable marker and a gene which transmits an agriculturally significant property. 11. Method according to claim 10, characterized in that the marker gene confers antibiotic resistance. 12. Method according to claim 11, characterized in that the antibiotic resistance is chloramphenicol or kanamycin resistance. 13. Method according to claim 10, characterized in that the agriculturally significant property is a herbicide resistance. 14. Method according to claim 13, characterized in that the herbicide resistance is atrazine resistance. 15. Method according to claim 1, characterized in that the enclosed gene is a chimeric gene. 16. Method according to claim 1, characterized in that the contained DNA contains a replication signal. 17. Method according to claim 1, characterized in that the enclosed DNA contains an integration signal. 18. Method according to claim 1, characterized in that the method is carried out with protoplasts from leaves. 19. Method according to claim 1, characterized in that an osmotically stabilized culture medium suitable for protoplasts is added. 20. Method according to claim 1, characterized in that a medium containing a plant-tolerable divalent cation is used. 21. Method according to claim 20, characterized by the action being magnesium or calcium. 22. Method according to claim 1, characterized in that the medium contains a polyhydric alcohol which changes the cell membrane and causes cell fusion. 23. Method according to claim 22, characterized in that the polyhydric alcohol is polyethylene glycol, polypropylene glycol or polyvinyl glycol. 24. Method according to claim 23, characterized in that the polyhydric alcohol is polyethylene glycol (PEG). 25. Method according to claim 24, characterized in that the polyethylene glycol has a molecular weight between 100 and 10,000. 26. Method according to claim 1, characterized in that the DNA and the protoplasts are subjected to a heat shock treatment. 27. Method according to claim 1, characterized in that the DNA and the protoplasts are subjected to electroporation. 28. Method according to claim 1, characterized in that the introduction of DNA into the protoplasts is restored by a combination of two measures selected from polyethylene glycol treatment, heat shock treatment and electroporation. 29. Method according to claim 28, characterized in that the gene transfer is carried out in such a way that, in order to contain the foreign gene, one adds said gene and the protoplasts in a solution and subjects the resulting suspension, first to a heat shock treatment and then to a polyethylene glycol treatment. 30. Method according to claim 1, characterized in that the gene transfer is carried out in such a way that, in order to contain the foreign gene, one adds said gene and the protoplasts to a solution and subjects the resulting suspension, first to a heat shock treatment and then to a polyethylene glycol treatment and finally an electropora-s ion. 31. Method according to claim 1, characterized in that the medium used contains a polyhydric alcohol which changes the protoplasmic membrane and causes cell fusion and where the DNA suspended in this medium together with the protoplasts is subjected to electroporation and/or heat shock treatment. 32. Method according to claim 1, characterized in that additional cellular nucleases are additionally inactivated. 33. Method according to claim 1, characterized in that protoplasts from plants of the family Gramineae, Solamaceae or Gruciferae are inserted. 34. Method according to claim 33, characterized in that protoplasts of plant cells from the plants of the Gramineae family are inserted. 35. Method according to claim 34, characterized in that the Gramineae are grains. 36. Method according to claim 35, characterized in that the grain is corn, wheat, rice, barley, oats, millet, rye or millet.