DD258827A5 - Method for direct introduction of DNA into the plastids and mitochondria of plant protoplasts - Google Patents

Abstract

The invention relates to a method for the direct introduction of DNA into the plastids and mitochondria of plant photoplasts, which is characterized essentially by the fact that in the absence of a pathogen, said DNA in a medium in which the DNA in the protoplasts and in these Plastoplast and mitochondria are able to penetrate, with the protoplasts so long in contact, that this penetration is guaranteed, so that ultimately result in plants with improved properties.

Description

For this 3 pages drawings'

Field of application of the invention

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

In the context of the present invention, the generic term plants is to be understood as meaning all monocellular or multicellular organisms which are capable of carrying out photosyntheses.

Characteristic of the known state of the art

Plant protoplasts are plant cells whose cell walls have been completely or partially removed by treatment with cellulolytic enzymes.

The ChNoroplasts form a target for representatives of different herbicide classes, such. B. for the triazine herbicides. It has recently been discovered that the atrazine, a member of the triazine herbicides, interacts with a 32 kd polypeptide encoded by a chloroplast gene, the so-called psbA gene (Hirschberg et al., 1984). Substitution of a single nucleotide within the encoded region of the psbA gene, resulting in the replacement of a single amino acid in the 32 kd polypeptide, results in atrazine resistance. Such mutations are found in weeds, such. In Amaranthus hybridus and Solanum nigrum.

It would now be desirable to be able to incorporate genes directly into the plastid and mitochondrial genomes of plants, especially crops. This would make it possible to give such plants new, desirable properties. For example, to produce herbicide-resistant plants, genes that confer herbicide resistance are needed in herbicide-sensitive chloroplasts.

Efforts to date have generally involved genetic manipulation of the plant cell nuclear genome to provide resistance or tolerance to chloroplast-active herbicides. However, this approach leads only to marginal tolerance to these herbicides, but not to actual resistance.

Heretofore, it has been considered impossible to confer real resistance to herbicides which are active in chloroplasts to culture plants by means of direct gene transfer, since it has previously been assumed that transformation of plastids, such as, e.g. As the chloroplast, with this method is not possible.

Another disadvantage with the introduction of genes which have a desirable property, e.g. a herbicide resistance mediate into the nuclear genome of a plant is due to the ability of some plants to cross-pollinate weeds. Such crossings open up a possibility of transferring these desirable properties to crops in weeds.

One of the most commonly used methods for introducing genes into the nuclear genome of plants is by infecting cells with pathogens, such as. An Agrobacterium containing Ti plasmid vector systems. (Barton et al., 1983; Chilton et al., 1985). However, these methods have clear disadvantages that are associated with the infection process.

On the one hand, these disadvantages consist of a limited host specificity and, on the other hand, the necessity of freeing the transformed plant cells from the pathogen used for the transformation.

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

De Block et al. (1985) report the use of an Agrobacterium Ti plasmid vector system for the introduction of an antibiotic resistance-encoding gene into the chloroplast genome of tobacco protoplasts from which whole cells and finally complete, fertile plants could be regenerated. However, the authors found that the gene was unstable in the absence of the corresponding antibiotics and was lost again after a short time. The conservation of plants under antibiotic selection pressure is not a practical application.

Methods for the direct transformation of plant protoplasts with naked linear DNA or circular plasmid DNA are also known (Paszkowski et al., 1984, and Schilperoort et al., 1983). These methods do not require a pathogen for the infection process, but at the present time these methods have remained limited to nuclear transformation.

Object of the invention

The aim of the invention is to provide methods which allow the direct transfer of useful genes into the genome of plastids and mitochondria so that beneficial new properties can be transferred to plant cells without requiring prior infection of the cells with a pathogen and at the same time preventing these properties from being transmitted to weeds.

Explanation of the essence of the invention

The invention has for its object to provide a method that ensures the stable transfer of genes into plastids and mitochondria of plant cells and whole plants, so that the expression of said genes not during the

Development of crops in the field is lost. ,

An essential part of the present invention is thus to provide a method for the direct introduction of genes into the genome of plastids and mitochondria, preferably chloroplasts, without infection of the cells with a pathogen and maintenance of the genes in said genome. Another object of the present invention is to produce whole plants containing genetically manipulated plastids and mitochondria under conditions in which the properties tailored by recombinant gene technology are stably obtained

stay and get to expression. ' "

As will be apparent from the description below, these and other objects are achieved by providing the method of the invention for direct introduction of DNA into the plastids and mitochondria of plant protoplasts, said DNA consisting of one or more genes and promoters active in plastids and mitochondria and characterized in that, in the absence of a pathogen, bringing said DNA into contact with protoplasts in a medium for so long as to assure penetration of the gene into the protoplasts and the plastids and mitochondria therein.

pictures

FIG. 1 shows a flow chart of the individual steps for the construction of the plasmids pCAT and p32CAT.

FIG. 2 shows a flow chart of the individual steps for the construction of the plasmid pUCHI.

FIG. 3 shows a flow chart of the individual steps for the construction of the plasmid pBRCAT.

In the attached figures the following short symbols are used:

X = Xhol

S = smal. , '

H = HindIII

B = BamHI

R1 = EcoRI

LIG. = Ligation using a T4 DNA ligase

X / Sl denotes a location at which joining a Xhol sequence to a Sall sequence results in a hybrid

Restriction interface is created, which can be cut by any of the enzymes. CAT refers to the coding region of chloramphenicol acetyl trarisferase. CAT refers to the promoterless form of the CAT gene.

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

In Figures 1 to 3, the CAT coding region is dotted. In the present invention description and claims the following definitions apply: A "gene" is a DNA sequence characterized by a promoter and a transcribable DNA sequence The promoter, which in most cases is not transcribed, causes the transcription of the The RNA may or may not be translated into a polypeptide, if the RNA is translated, it is referred to as mRNA, the DNA sequence that corresponds to the mRNA, as well as the

mRNA itself is composed of a 5 'region that is not translated, a coding region, as well as a 3' region, which is also not translated. Only the coding region becomes the corresponding polypeptide

translated. ,

The "active" parts of a DNA sequence make up those parts that are responsible for the function of the DNA sequence.

Some examples of active regions of DNA sequences include the RNA polymerase binding site, the initiation signal (TATA box) of the promoter, the ribosome binding site as well as the translation initiation signal of the untranslated 5 'region, the coding sequence, the transcriptional stop signal and the polyadenylation signal of the untranslated 3 'region. The various spacer sections in which the DNA sequence is meaningless are not considered active DNA sequences.

A "variant" of a natural DNA sequence forms a modified form of the natural sequence that performs the same function, which may be a mutant or a synthetic DNA sequence that is substantially homologous to the corresponding natural sequence.

A DNA sequence is considered to be substantially homologous to a second DNA sequence if at least 70%, preferably at least 80%, in particular at least 90% of the active portions of the DNA sequence are homologous. To determine substantial homology, two different nucleotides within a DNA sequence of a coding region are still considered to be homologous if the mutual exchange of these nucleotides results in a silent mutation.

A DNA sequence "is derived from" a source, such as the plastids or mitochondria, when that DNA sequence is present in this source The present invention also encompasses variants of such a DNA sequence.

A DNA sequence is "functional" in plastids or mitochondria when it performs its expected function and remains in the progeny of the plastids and mitochondria.

By "transformable plant protoplasts" is meant protoplasts containing, after direct gene transfer, a plastid or mitochondrial genome having covalently-linked DNA not normally present in these plastids or mitochondria.

Plant protoplasts which can be transformed by means of the method according to the invention can be derived from cultured cells, as well as from cells which are present in plant parts or multicellular plants. The transformable plant protoplasts can also be of unicellular plants such. B. algae derived. Some examples of unicellular plants include the cyanobacteria (e.g. Synechococcus spp.) As well as Chlamydomonas and Euglena. However, particularly preferred within the scope of the present invention are plant protoplasts derived from multicellular plants. After the transformation has taken place, the protoplasts may be regenerated to whole plants.

With the aid of the present invention, it is now possible to selectively genetically modify the plastids and mitochondria of any plant protoplasts.

Some examples of such plant protoplasts are:

Solanum spp. (Potato), Gossypium spp. (Wool), 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. (Nightshade), Hyoscyamus spp. (Henbane), Digitalis spp. (Thimble), Catharanthus spp. (Evergreen), Pisum spp. (Pea), and Pinus spp. (Pine).

Transformable plastids are z. Chromoplasts, amyloplasts, leucoplasts, etioplasts, proplastides and chloroplasts.

Particularly preferred for the transformation are the chloroplasts.

The present invention further relates to genes that are functional in the plastids and mitochondria. The genes of the present invention confer a desired useful property by their confinement to the plastids and mitochondria. Some examples of such useful properties are: phytotoxin resistance, such as As herbicide or antibiotic resistance, improved photosynthetic efficiency and enzyme activity in cases where a chromogenic substrate for the enzyme is known.

In the present case, herbicide resistance is understood to be a resistance to all herbicides which are active in the plastids or mitochondria. For example, many herbicides are active in chloroplasts. These herbicide classes include

z. As the triazine, urea, sulfonylurea and uracil derivatives, as well as the glyphosate (N-phosphomethylglycine). *

Some examples of triazine herbicides include atrazine, ametryn, metribuzin and simazine. Some examples of urea herbicides include the phenyl ureas, such as. As diuron, chloroxuron and fluorometuron and the DCMU (dichloromethylurea). Examples of sulfonylureas are Oust and Glean, as well as uracil herbicides Bromacfl and Terbacil. These and other herbicides are described in LeBaron and Gressel (1982).

In order to make a cell resistant to herbicides, it is not necessary to introduce a gene coding for herbicide resistance into each of the 50 to 100 chloroposes of this cell by direct gene transfer. Direct gene transfer into a small fraction of the available chloroplasts is quite sufficient to protect the cells from herbicides.

The possibility of herbicide resistance z. B. against atrazine to transfer to plants is desirable for various reasons. It allows, for example, the application of this herbicide in higher doses to plants which are then tolerant of said herbicide. At the same time, the higher herbicide doses will result in better efficiency in weed control.

In addition, however, herbicide resistance can also be used as a selective marker that is genetically coupled to a physiological property that is difficult to select per se. To exploit this possibility, a donor plasmid is constructed that contains a gene that provides herbicidal resistance and a second gene that mediates the other desirable trait. This may, for example, be an increased photosynthetic efficiency. This donor DNA is introduced into plant protoplasts, which can be regenerated into a plant cell culture or whole plants. The resulting plants then have both the properties of herbicide resistance and the said second property. If these plants are allowed to grow in the presence of the corresponding herbicide, it is possible to select plants having said second property.

In addition, the introduction of a donor DNA conferring both herbicide resistance and a second agronomically useful property on plant chloroplasts allows this second property to be stably maintained in the chloroplasts when grown in the presence of the herbicide ,

De Blocketal already has the instability of foreign genes in chloroplasts. (1985) (see above). Particularly preferred is a herbicide resistance to atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine). For in vitro identification of cells with transformed plastids or mitochondria, antibiotic resistance may be used.

Antibiotic resistance is a property that can be used for the in vitro identification of cells containing transformed plastids or mitochondria. Genes that carry these selectable markers are extremely useful when genetically linked to agronomically beneficial properties. For example, resistance to chloramphenicol, kanamycin or, in principle, resistance to any other antibiotics are suitable for this purpose.

A useful trait, which is primarily useful as a marker for screening plant cells from tissue cultures containing genetically engineered plastids or mitochondria, is obtained, for example, in U.S. Pat. By introducing a gene encoding an enzyme having a coloring substrate. For example, when said enzyme is beta-galactosidase, the plant cells are plated on a tissue culture medium containing the coloring substrate Xgal (5-chloro-4-bromo-3-indolyl-β-D-galactoside) , Plant cells containing genetically manipulated plastids or mitochondria are stained by the dye indigo blue, as this is released due to the cleavage of Xgal by ß-galactosidase.

The genes which are suitable for the present invention can be obtained by methods known per se. These methods are, for. As the isolation of natural, usually occurring only outside of the plastids or mitochondria genes or variants that are to be introduced.

Said gene may be any naturally occurring gene or one of its variants operable in the plastids or mitochondria. Preferred are naturally occurring platinum, mitochondrial or bacterial genes. Particularly preferred are chloroplast genes.

To be sure that said gene is actually in the plastids or mitochondria and not in the cell nucleus, one can advantageously, but not necessarily, not use a nucleus in the nucleus or at least significantly less than in the plastids or mitochondria functional gene.

Some examples of naturally occurring bacterial genes are e.g. For example, those that code for antibiotic resistance or for enzymes with a coloring substrate. A bacterial gene capable of conferring antibiotic resistance on plastids or mitochondria is e.g. B. the chloramphenicol acetyl transferase gene. A bacterial gene encoding a chromogenic substrate enzyme is known e.g. B. lacZ, which encodes the β-galactosidase. Some examples of plastid or mitochondrial genes are genes that encode herbicide resistance or improved photosynthetic efficiency. A chloroplast gene capable of conferring herbicide resistance is e.g. For example, the mutated psbA gene described by Hirschberg et al. (1983) or a mutant of the psbD gene (Rochaix et al., 1984). A chloroplast gene capable of conferring improved photosynthetic efficiency is e.g. Represented by a modified form of rbcL coding for a modified large subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco). A gene that expresses the large subunit of Rubisco is already contained in the chloroplasts and involved in photosynthesis. However, it is also possible to introduce a modified (mutant, manipulated or heterologous) form of this gene with improved photosynthetic efficiency [Jordan et al., (1981)]. Another way to obtain a gene that is functional in the plastids or mitochondria is to construct a chimeric gene. A chimeric gene is a gene or at least part of a gene, preferably an active part of a gene that is not naturally covalently bound to the other parts. The chimeric gene can either specify an RNA transcript or encode a polypeptide.

Suitable promoters of the chimeric gene in the context of the present invention are all promoters which are functional in plastids or mitochondria. Promoters for chimeric genes can be 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 plastids or mitochondrial genes, particular preference is given to those derived from psbA, psbD and rbcL genes. Said promoters may also be variants of natural promoters. According to the invention, a heterologous promoter of the chimeric gene may be a natural promoter which does not normally occur in plastids or mitochondria.

Since the functions of plastid or mitochondrial genes are often similar or identical to the functions of bacterial genes, bacterial promoters in plastids or mitochondria may also be functional. Any bacterial promoter which is functional in plastids or mitochondria can therefore also be used according to the invention as a promoter of the chimeric gene. Some useful bacterial promoters are e.g. Such as the neomycin phosphotransferase II gene and the T-DNA genes, such as. For example, the nopaline synthase gene of the Ti plasmid.

Also suitable as a heterologous promoter is any partially or completely synthetically produced promoter which is functional in plastids or mitochondria.

Partially or fully synthetically produced promoters which resemble their natural counterparts by having a TATAAT-like sequence 10 nucleotides below the transcriptional stem are also part of the present invention.

The untranslated 5 'region of the chimeric gene of the present invention may be any untranslated 5' region that is functional in the plastids or mitochondria. The untranslated 5 'region may be derived, for example, from plastid or mitochondrial genes or from genes of other origins. A preferred homologous untranslated 5 'region is from the psbA, psbD or rbcL genes. A preferred heterologous untranslated 5 'region is derived from bacterial genes. Synthetic untranslated 5 'regions that resemble their natural counterparts due to an effective ribosome binding site are also part of the present invention. In principle, any coding region capable of conferring a desirable trait on a plant cell is suitable for use as the coding region of the invention. Both the coding regions of the above-mentioned naturally occurring genes and coding regions from other sources can be used in the context of the present invention for the production of a chimeric gene. .

Thus, in the context of the present invention, the coding regions may be derived from naturally occurring plastid or mitochondrial genes or from another source; They can also be complete or partial

be produced synthetically or by fusion of two or more such coding regions while maintaining the reading frame.

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

An example of a polypeptide determined by a gene naturally occurring in the chloroplast of certain plants is the mutated form of the 32 kd polypeptide described by Hirschberg et al. (1983). It has been shown that this polypeptide transfers atrazine resistance to certain weeds. However, the psbA gene which encodes said polypeptide may also be restricted to natinal plants, such as. B. crops are transferred using the method according to the invention.

An example of a non-plastid or mitochondrially encoded region is the coding region of the plant, animal or bacterial gshA and gshB genes, or the glutathione reductase gene (gor), all of which can confer useful properties thereto, when introduced into plant plastids or mitochondria. gshA and gshB encode enzymes that catalyze the synthesis of the tripeptide glutathione, which in turn is responsible for the conjugative detoxification of many herbicides (Meister et al., 1983, and Rennenberg, 1982). Another example of a non-plastid or mitochondrial coding region is the coding portion of a glutathione S-transferase gene from plants, animals, insects or bacteria. In some plants {Shimabukuroy et al., [1971]) known to be tolerant of the herbicide atrazine, the presence of 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 a more efficient form of Rubisco. Such coding portions may optionally be derived from naturally occurring genes. Optionally, the coding region of the present invention may also be derived from two or more differently coding portions. Polypeptides specified by such coding regions are referred to as fusion polypeptides.

An example of a fusion polypeptide is a more efficient form of Rubisco. Rubisco has two functions, one being carboxylase and the other being oxygenase. The carboxylase function is effective in the course of photosynthesis, but the oxygenase function is undesirable. A useful fusion polypeptide accordingly has a proportion of the _:

Carboxylase function maximizes and a second part that minimizes oxygenase function.

The untranslated 3 'region of the chimeric gene may either be naturally present in a plastid or mitochondrial gene, or it may be of heterologous origin. Preferred is a 3'-untranslated region derived from a plastid or mitochondrial gene. The preferred untranslated 3 'portions are those of the psbA, psbD or rbcL genes.

The transcribed regions of the genes of the present invention may specify RNA transcripts and in some cases are useful as such.

These transcripts may be tRNA or rRNA. The transcript may also be an RNA having a sequence that is complementary to at least a portion of a sequence of another RNA transcript. Such complementary sequences, known as anti-sense sequences, if long enough, may interfere with the function of RNA sequences to which they are complementary, or they may transcribe that RNA from the corresponding one Block gene; (See Pestka et al., [1984]; Melton [1985]; Izant et al., [1984]; Simons et al., [1984] and Colemann, [1984]). ,

Transcribed regions useful in the present invention may optionally be derived from naturally transcribed portions, or they may be wholly or partially synthetically produced. Anti-sense sequences can also be derived from at least part of a naturally occurring gene by reversing the orientation of said part of the naturally occurring gene with respect to its promoter. The genes suitable for use in the present invention are incorporated into a plasmid cloning vector by methods known per se (Maniatis et al., [1982]).

For the integration of foreign genes into the genomic DNA of plant cells, it is advantageous if the genes are flanked by neutral DNA sequences, so-called carrier DNA. The carrier DNA can consist of two linear DNA strands so that the entire construct to be introduced into the plant cell is a linear DNA molecule. However, said structure may also have a circular structure-plasmid structure for the gene transformation. The carrier DNA may be of synthetic origin as well as derived from naturally occurring DNA sequences treated with appropriate restriction endonucleases. Thus, for example, naturally occurring plasmids 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 in Messing et al., [1982]). Fragments of naturally occurring plasmids can also be used as carrier DNA according to the invention. The construction intended for transformation may optionally contain DNA derived from a Ti plasmid or from a modified Ti plasmid. A plasmid is considered to be a modified Ti plasmid if it has at least one T-DNA border region. A T-DNA border region is a DNA sequence within an Agrobacterium genome that causes the incorporation of another sequence within the genome (T-DNA) into the plant cells that the Agrobacterium contacts. For example, the T-DNA border region may be a sequence of a vector, such as a vector. A Ti or Ri plasmid or a modified Ti or Ri plasmid. The T-DNA may also be a sequence located on the same vector as the border region or on another vector. The likelihood of genetic transformation (transformation rate) of a plant cell can be increased by several factors. As we know from experiments with yeast, the number of successful stable gene transformations increases accordingly:

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

2) when a replication signal is combined with a new gene and

3) when an integration signal is combined with a new gene, an integration signal being a signal that promotes the integration of one strand of DNA into another strand of DNA.

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

Protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, oocytes, embryonic sacs or zygotes, as well as embryos at different stages of development are representative examples of plant cells suitable as starting material for transformation. Preference is given to protoplasts, since they can be used directly without further pretreatment. Isolated plant protoplasts, cells or tissues can be obtained by methods known per se or by methods analogous to known methods. Isolated plant protoplasts, which are suitable as starting material for the isolation of isolated cells and tissues, can be obtained from all parts of the plant, eg. As the leaves, embryos, stems, flowers, roots or pollen isolate. Preference is given to protoplasts from leaves. However, isolated protoplasts can also be obtained from cell cultures. Methods for the isolation of protoplasts are described, for example, in Gamborg et al. (1975). The transfer of the new gene into the plant cell takes place directly, i. without prior infection of the cell with a pathogen such as a plant pathogenic bacterium, virus or fungus and without transfer of DNA by insects or fungi capable of infecting plants with DNA-transmitting pathogens. This direct gene transfer is achieved by contacting the DNA containing the gene with the plant protoplasts and the plastids and mitochondria contained therein in a medium for a period of time sufficient to allow the gene to penetrate into the protoplasts and into the plastids therein and mitochondria is sufficient. The transformation frequency 1 <ann can be increased by combining this step with various gene transfer techniques. Examples of such techniques include treatment with poly-L-ornithine or poly-L-lysine, liposome fusion, DNA-protein complexation, alteration of charge ratios on the protoplast membrane, fusion with microwave protoplasts or calcium phosphate precipitation, and especially treatment with certain polyhydric Alcohols such. For example, polyethylene glycol, further by heat shock treatment and electroporation and by a combination of these last three techniques.

Suitable media for use in the invention are all media; which allows the DNA carrying the gene to penetrate into the protoplasts as well as into the plastids or mitochondria within these protoplasts. Suitable media for the co-incubation of alienation with the receptor protoplasts are preferably osmotically stabilized culture media, as have been developed for protoplast cultures.

Many cultural media now available differ in individual components or groups of components. However, the composition of all these media is in accordance with the principle that they all have a group of inorganic ions in a concentration range of about 10mg / liter to several hundred mg / liter (so-called macroelements such as nitrates, phosphates, sulfates, calcium, magnesium, Iron, etc.); another group of inorganic ions with a maximum concentration of several mg / liter (so-called trace elements such as cobalt, zinc, copper, manganese, etc.); a number of vitamins (eg, 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 range of 0.01 to 10 mg / liter.

These culture media are additionally stabilized osmotically by the addition of sugar alcohols (eg mannitol), sugars (eg glucose) or salt ions (eg CaCl ₂ ) and to a pH in the range of 5, 6 to 6.5 set. A more detailed description of conventional culture media can be found, for example, in Kobliz et al. (1974). A particularly suitable medium for the direct transformation of protoplasts contains a polyhydric alcohol which is capable of altering the protoplast membrane and which proves to be useful in mediating 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. The polyhydroxylated detergents commonly used in agriculture and tolerated by the plants are also suitable polyhydric alcohols. Such detergents are described, for example, in the following publication:

"McCutcheon's Detergents and Emulsifiers Annual" MC Publishing Corp., Ridgewood, New Jersey, (1981); Stäche, H., "Tensid-Taschenbuch", Carl Hanser Verlag, Munich / Vienna, (1981).

The polyhydric alcohols preferred herein are polyethylene glycols having a molecular weight between .1,000 and 10,000 g / mol, preferably between 3,000 and 8,000 g / mol.

The transformation frequency of direct gene transfer can be significantly increased by the methods described in detail below.

In the polyethylene glycol treatment, a protoplast suspension is first added to the culture medium. The DNA containing the gene, which may be present as linear DNA or in the form of a circular plasmid, is then added to the initially introduced mixture of polyethylene glycol and culture medium. As an alternative to this procedure, the protoplasts and the DNA containing the gene may also be initially introduced in the culture medium and then the polyethylene glycol first added.

In the context of the present invention, the electroporation and the heat shock treatment also proved to be particularly advantageous method measures.

Neumann et al. (1982) report that in electroporation, protoplasts are first converted to an osmoticum, e.g. in a mannitol / magnesium suspension and this protoplast suspension is then introduced into an electroporator chamber between two electrodes. By discharging a capacitor over the suspension, the protoplasts are momentarily energized with an electrical pulse of high voltage, resulting in polarization of the protoplast membrane and opening of pores in the membrane.

In the heat treatment, the protoplasts are suspended in an osmoticum, e.g. B. a solution of mannitol / calcium chloride. Subsequently, the suspension in small containers z. B. in centrifuge tubes, preferably in a water bath heated. The duration of the heating process depends on the preselected temperature. In general, the temperatures range from 40 ° C to 80 ⁰ C and maintained for a period of 1 second up to one hour. The best results are obtained at a temperature in the range of 4O ⁰ C to 50 ⁰ C to 4 to 6 minutes, in particular at 45 ⁰ C and 5 minutes. The suspension is then cooled to room temperature or less. It can also be shown that the transformation frequency is increased by inactivating extracellular nucleases. Inactivation can be achieved by using divalent cations tolerated by plants, such as magnesium or calcium. Inactivation is more effective when the transformation is performed at high pH, with the optimum pH range between 9 and 10.5.

Surprisingly, the selective use of these different methods leads to a significant increase in the transformation frequency, which represents a long-sought goal in the field of genetic engineering. The lower the transformation frequency in the gene transformation experiments, the more difficult and time consuming it is to find out the few cloned cells derived from transformed cells among the large number of untransformed clones. With only a low transformation frequency, it is virtually impossible to use conventional screening methods unless the gene used has a selective marker (eg, resistance to a particular substance). Low transformation frequencies therefore require a considerable amount of work and time when using genes without marker functions.

By bringing foreign genes and receptor protoplasts together prior to the application of other means, e.g. polyethylene glycol treatment, electroporation, and heat shock treatment, can achieve a significant improvement in transformation frequency as compared to a procedure in which the individual process steps are performed in a different order.

A combination of two or three of the techniques enumerated below has been found to be advantageous: polyethylene glycol treatment, heat shock treatment and electroporation, with particularly good results being obtained when these techniques are applied after introduction of the foreign gene and protoplasts in a liquid. The preferred procedure is a heat shock treatment prior to the polyethylene glycol treatment and optionally subsequent electroporation. In general, the additional electroporation leads to a further increase in the transformation frequency; However, in some cases, the results achieved by heat shock treatment and polyethylene glycol treatment can not be significantly improved by additional electroporation.

It is also possible to use divalent cations which are tolerated by plants and / or combine a transformation at pH values between pH 9 and 10.5 with the individual transformation frequency-changing measures already described above, namely with the polyethylene glycol treatment. heat shock treatment and electroporation.

The inventive method thus makes it possible to achieve high transformation frequencies without pathogens such. As viruses and Agrobacterium or natural or modified Ti plasmids must be used for the transformation. An advantageous embodiment of the process according to the invention is characterized, for example, by the fact that the protoplasts are converted into a mannitol solution and then the resulting protoplast suspension is mixed with an aqueous DNA solution which contains the gene. The protoplasts are then incubated in this mixture for 5 minutes at 45 ° C and then cooled to 0 ° C for 10 seconds. Following the incubation, this mixture is added as much polyethylene glycol (MG3000 to 8000) that a PEG concentration of 1 to 25%, preferably by 8%, is reached. After careful and thorough mixing, the treatment then takes place in the electroporator. The protoplast suspension is then diluted with culture medium and may eventually remain in the culture medium to regenerate its cell walls.

The method according to the invention is suitable for the transformation of all plant cells, in particular for cells from the systematic groups of angiosperms and gymnosperms.

Among the Gymnospermae are primarily plants from the class Coniferae of interest.

Among the angiospermae, in addition to deciduous trees and shrubs, plants of the following families of particular 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 families of Solanaceae, Cruciferae and Gramineae.

Of particular interest are plants of the genus Nicotiana, Petunia, Hyoscyamus, Brassica and Lolium, for example Nicotiana tabacum, Nicotiana plumbaginifolia, Petunia hybrida, Hyoscyamus muticus, Brassica napus, Brassica rapa and Lolium multiflorum;

Plastids and mitochondria of all those plants which can be regenerated from protoplasts can also be successfully transformed by means of the method according to the invention. To date, it has not been possible to isolate plastids and mitochondria from members of the Gramineae family (grasses), as well as crops such as cereals. Corn, wheat, rice, barley, oats, millet, rye and sorghum include genetically manipulating. The present invention now permits the genetic transformation of plastids and mitochondria of Gramineae cells, including the cereal cells, using direct gene transformation. In the same way, it is possible to carry out the transformation of plastids and mitochondria of any other crop, such. B. plants of the genus Solanum, Nicotiana, Brassica, Beta, Pisum, Phaseolus, Glycine, Helianthus, Allium, Triticum, Hordeum, Avena, Setaria, Oryza, Cydonia, Pyrus, Malus, Rubus, Fragaria, Prunus, Arachis, Seeale, Panicum Saccharum, Coffea, Camellia, Musa, Pineapple, Vitis, Sorghum, Helianthus or Citrus.

The incorporation of transformed genes into plastids or mitochondria can be detected by conventional methods, eg. As by genetic crossing experiments or molecular biological detection methods, in particular the detection method according to Southern for plastid and mitochondrial DNA and the enzyme activity test.

The detection method according to Southern can be carried out, for example, as follows: the DNA isolated from plastids or mitochondria of transformed cells or protoplasts is subjected to electrophoresis after treatment with restriction enzymes 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. The DNA can be labeled in vitro at a specific activity between 5χ 10 ^{a and} 10χ 10 ⁸ cpm / microgram by nick translation (Rigby et al., 1977) .The filters are then washed three times for one hour with a aqueous solution of a 0.03 M sodium citrate solution and a 0.3 M sodium chloride solution at a temperature of 65 ° C. The hybridized DNA is visualized by one to several days of autoradiography on an X-ray film.

The cells transformed with the desired gene are isolated by methods known per se. These methods include selection and screening. The selection of nuclear genes is described in Fraley et al., (1983); Herrera-Estrella et al., (1983); and Bevan et al., (1983). Screening may be for β-galactosidase (Helmer et al., [1984]), nopaline synthase or octopine synthase (Wostemeyer et al., [1984]; DeGreve et al., 1982) or for atrazine resistance. Prior to this invention, it was not known that plastids or mitochondria could be transformed by direct gene transfer. Accordingly, it has previously not been possible to operably introduce useful genes in the form of isolated DNA into these organelles. Genes are considered to be operably incorporated into the genome of plastids or mitochondria when incorporated into replication and expression. Plasmids and mitochondria of all those plants that are regenerable from protoplasts can also be successfully transformed by the method of the invention. To date, it has not been possible to date to detect plastids and mitochondria from members of the Gramineae family (grasses), which also have cereals, such as, e.g. Corn, wheat, rice, barley, oats, millet, rye and sorghum include genetically manipulating. The present invention now permits the genetic transformation of plastids and mitochondria of Gramineae cells, including the said cereal cells, using direct gene transformation. In the same way it is possible to carry out the transformation of plastids and mitochondria of any other crop, such. B. plants of the genus Solanum, Nicotiana, Brassica, Beta, Pisum, Phaseolus, Glycine, Helianthus, Allium, Triticum, Hordeum, Avena, Setaria, Oryza, Cydonia, Pyrus, Malus, Rubus, Fragaria, Prunus, Arachis, Secaie, Panicum , Saccharum, Coffea, Camellia, Musa, Pineapple, Vitis, Sorghum, Helianthus or Citrus. The incorporation of transformed genes into plastids or mitochondria can be detected by conventional methods, eg. As by genetic crossing experiments or molecular biological detection methods, in particular the detection method according to Southern for plastid and mitochondrial DNA and the enzyme activity test.

An advantage of introducing genes into plastids or mitochondria by direct gene transfer is the possibility of simultaneous inactivation of unwanted genes already present in the plastid or mitochondrial genome. This is possible because the incorporation of foreign genes into the plastid or mitochondrial genome does not necessarily proceed via homologous recombination when direct gene transfer is used. For example, a donor DNA molecule carrying an atrazine-resistant form of psbA will not necessarily be integrated at the site where the corresponding atrazine-sensitive gene is located. On the other hand, since the plastid and mitochondrial genomes are relatively small, it is quite possible to select the transformed plant cells and find out those in which the donor DNA was accidentally incorporated into any desired function of the recipient genome. Direct gene transfer into plastid and mitochondrial genomes also opens the way to inactivation of genes, which was previously not feasible. With the aid of appropriate screening, it is thus possible, for example, to find transformed plant cells in which the atrazine-sensitive psbA gene is inactivated by the incorporation of a donor DNA carrying an atrazine resistance gene. Another advantage of direct gene transfer into the genomes of plastids and mitochondria lies in the fact that, unlike nuclear genomes, these genomes are inherited maternally by the cytoplasm and are therefore usually not transmitted by pollen to their sexual offspring. Therefore, the possibility of transferring genes that confer desirable traits on crops is greatly reduced by crop plants on weeds. Plant cells in cell cultures transformed by the method of the invention may optionally be used for the production of polypeptides encoded by the inserted genes. Such synthesis products include, for example, the 32 kd polypeptide expressed by psbA, chloramphenicol acetyltransferase, glutathione S-transferase, and the large Rubisco subunit.

The plant cells containing the incorporated genes can in some cases also be regenerated into multicellular plants which express the gene and therefore possess the desired new property. Any plant which can be regenerated from plant cells, which in turn are derived from protoplasts, can be produced by means of the method according to the invention. , ·

Examples of such 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), Citrus, Atropa, Hyosxyamus, Salpiglossis, Arabidopsis, Digitalis, Cichorium, Gossipium, Glycine, Geranium, Anthirrhinum and Asparagus. The plants are regenerated by methods known per se; see Evans and Bravo, (1983); Dale, (1983); For example, plants may be regenerated from any suitable offshoot, such as cells, cilia, tissues, organs, buds, cuttings, shoots, rootlets, plantlets, somatic embryos, and others.

The present invention further relates to protoplasts, plant cells, cell aggregates, plants, and more particularly to the seeds of plants produced by the method of the invention, as long as the seeds formed contain the incorporated gene and thus the desired property resulting therefrom. It also relates to all progeny of plants produced by the method of the invention, both sexual and vegetative progeny.

Sexual progeny can be obtained by self or cross pollination, with progeny also including all cross and fusion products with the transformed plant material defined in the preceding paragraph, provided that these progeny have the property introduced by the transformation. Seeds and progeny of herbicidally resistant or tolerant parents having unstable herbicide resistance or tolerance may maintain their herbicide resistance or tolerance as long as they grow in the presence of the herbicides. A preferred herbicide is the atrazine. The present invention thus allows the production of genetically engineered plants that are capable of treatment with herbicides such. With atrazine, in concentrations which are lethal to sensitive plants.

embodiment

The invention is explained in more detail below with reference to some examples.

Examples:

Individual procedures of the following examples can be found in general terms in Maniatis et al., (1983). The enzymes may be obtained from New England Biolabs and, except as specifically stated in the text, should be used in accordance with the manufacturer's guidelines.

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

1) The vector plasmid pMON 187 carrying a Km ^r gene of Tn 903 is digested 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 assembly of the fragment of step 2) with the fragment of step 1) yields a plasmid which is transformed into E. coli HB101 '. The selection is made on Ap ^r . One Km ^s colony has the plasmid with the structure for pCAT "given in FIG.

Example 2: Alternative Construction of pCAT

Example 2 can be repeated with a plasmid other than pMON 187. A suitable plasmid having the Km ^r gene of Tn 903 can be constructed as follows:

A 1.2kbAvall fragment containing the Km ^r gene of Tn 903 is isolated from the plasmid pA02 (Oca et al., [1982]). The ends of the Avail fragment are filled in using Klenow polymerase and BamHI linkers are linked to the blunt ends of the fragments. The DNA is then treated with the restriction enzymes Tagl and BamHI and spliced into the plasmid pBR327, which was previously digested with CIaI and BamHI. Recombinants possessing the Tn903 fragment confer Km ^r on E. coli.

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

The following procedures are performed to link the psbA promoter to the promoterless CAT ° gene.

4) pCAT ⁰ is digested with SmaI and HindIII

5) A gel-purified 161 bp SmaI / HindIII fragment containing the psbA promoter is isolated from the plasmid pAH484. The recombinant plasmid pAH484 contains the 3.68 kb EcoRI fragment of chloroplast DNA from (atrazine) herbicide-resistant Amaranthus hybridus cloned into pBR322. (Hirschberg and McIntosch, [1983]).

6) The fragment isolated in step 5 is linked to the larger fragment resulting from step 4. The linkage leads to p32CAT, which transmits Cm 'to transformed E. coli cells.

Example 4: Construction of plasmid puCHI, a vector with a psbA gene and a chimeric psbA / CAT gene construction (see Fig. 2)

The plasmid pUCHI is constructed in 5 steps in the pUC8 vector. (Steps 7 to 11):

7) (Figure 1) A 3.6kb EcoRI fragment (gel-purified) containing the complete psbA gene is isolated from plasmid pAH484 described above.

8) pUC8 (Fig. 2) is linearized at its unique EcoRI site.

9) The linearized pUC8 fragment from step 8) is linked to the fragment described in step 7). The transformation of E. coli gives Ap ^r colonies possessing the desired, incorporated fragment, one of these colonies being designated pUC8-32.

10) A gel-purified 1.8 kb Xhol / BamHI fragment was isolated from p32CAT.

11) The resulting plasmid from step 9), pUC8-32, is partially digested with Sail and completely digested with BamHI. The linkage with the fragment resulting from step 10) and selection for Cm ^r after transformation of E. coli leads to an E. coli colony containing pUCHI.

Example 5: Construction of pBRCAT containing a chimeric psbA / CAT gene

The construction of pBRCAT is achieved by linking the following three DNA fragments:

a) the EcoRI / HindIII (promoter) fragment (about 660 bp) from the psbA gene in plasmid pAH484 (step 12);

b) the HindIII / BamHI fragment of pSVO with a promoter-free CAT gene (step 2;) and

c) EcoRi / BamHI digested pBR322 as a vector (step 13).

14) The linkage leads to a plasmid, pBRCAT, which transmits Cm ^r to E.coli host cells, into which this plasmid is introduced by transformation.

Example 6: Introduction of donor DNA into plant protoplasts

Tobacco protoplasts with a population density of 2 × 10 ⁶ per ml are dissolved in 1 ml of a medium (compare Z. Pflanzenphysiologie 78, 453-45 [1976]; Mutation Research 81, 165-175 [1981]) containing 0.1 mg / Liter of 2,4-dichlorophenoxyacetic acid containing 1.0 mg / liter of 1-naphthylacetic acid and 0.2 mg / liter of 6-benzylaminopurine. Protoplasts are obtained from an enzyme suspension by flotation to 0.6 M sucrose at pH 5.8 and subsequent sedimentation for 5 minutes at 100 g in 0.17 M calcium chloride at pH 5.8. 0.5 ml of 40% strength polyethylene glycol (PEG) having a molecular weight of 6000 in modified (after autoclaving back to a pH of 5.8) are added to this suspension in succession. F-Medium (Nature 296, 72-74) 1982]) and 65 microliters of aqueous solution containing 15 micrograms of donor DNA (p32CAT, pUCHI, or pBRCAT) and 50 micrograms of calf thymus DNA. This mixture is cultured for 30 minutes at 26 ° C, with the solution occasionally agitated and then gradually diluted with F medium. The protoplasts are isolated by centrifugation (5 minutes at 100g) and then resuspended in 30ml fresh K ₃ medium. Further incubation is carried out at 24 ° C. in the dark in 10 ml portions in Petri dishes with a cross section of 10 cm. After three days, the culture medium in each Petri dish is diluted with 0.3 volume of fresh K ₃ medium and incubated for a further 4 days at 24 ° C and 3000 lux. After a total of 7 days, the clones arising from the protoplasts

have developed, embedded in a solidified culture medium with 1% Ägarose containing 10mg / Lite'r chloramphenicol and at 24 ⁰ C in darkness after the "bead type culture method" (Plant Cell Reports, 2, 244-247 [1983] The culture medium is replaced every 5 days by a fresh medium of the same species After 3 to 4 weeks of continuous culture in chloramphenicol-containing culture medium, the resistant cells of 2 to 3 mm in diameter are placed on agar-solidified LS culture medium. Medium (Physiol Plant, 18, 100-127 [1965]) containing O, 05 mg / liter 2,4-dichlorophenoxyacetic acid, 2 mg / liter of 1-naphthylacetic acid, 0.1 mg / liter of 6-benzylaminopurine, 0.1 mg / liter of kinetin chloramphenicol-resistant Nicotiana tabacum Petit Havana SRI plants are obtained by sprout induction on LS medium with 10 ml / liter chloramphenicol and 0.2 mg / liter 6-benzylaminopurine and subsequent rooting on T-medium (Science 163, 85-8 7, [1969]).

The selection of chloramphenicol-resistant cells and plants is essentially in accordance with the information provided by De Blocketal, (1984).

Example 7: Screening for atrazine-resistant chloroplast transformants

a) KaIIi

An atrazine toxicity curve is generated for untransformed kaIIi in a concentration range of 1 to lomicromole atrazine and various light intensities. Chimeric transformant-derived kaIIi and control kaIIi are applied to nutrient agar plates, treated with atrazine concentrations and light intensities that completely bleach the chlorophyll out of the control tissues. Resistance to atrazine is visually examined due to the persistent greening of resistant tissues.

b) Whole plants

Plants obtained from chloroplast transformants are tested for atrazine resistance by the following methods:

Chlorophyll fluorescence induction in leaves. If the electron transport on the reducing side of Photosystem II inhibited, such as by atrazine, then the chlorophyll absorbed radiation energy is re-emitted as fluorescence. This fluorescence can be measured on the leaf surface. Fluorescence measurement is made on leaf discs stretched horizontally across a transparent lucite window as described by Nalkin et al. (1981). (Lucite stands for polymerized resin of methyl methacrylate.) The excitation light comes from a de-driven projector, which, after passage of a filter, in a band of actinic light of 500 to 600nm with intensities around 1OnE χ cm ~ ² xs ~ ¹ results. The duration of the excitation light is 3 ms. The excitation light is oriented perpendicular to the sheet surface and the fluorescence from the same surface is collected by means of a flexible light guide system and then transferred to a red-sensitive photomultiplier after passage of a cut-off filter (600 nm). The fluorescence passages are recorded on an oscilloscope and directly filmed.

c) light-modulated flotation

Letting cut-out pieces of leaf float on a phosphate-buffered detergent, they remain on the surface as long as the photosynthesis stops, which ensures a high O ₂ ZCO ₂ ratio in the intercellular spaces. On the other hand, if the leaf pieces are allowed to float in the dark or if the medium containing photosynthesis inhibits herbicides, they quickly lose their buoyancy and go under. An assay method for atrazine resistance is described in Hensley (1981). Leaf pieces are placed in tubes containing atrazine-containing solution and placed under vacuum. The leaf pieces are quickly penetrated by the solution and sink to the bottom of the solution. The vacuum is then released, a bicarbonate solution is added and the tubes exposed to light. If photosynthesis is not inhibited by atrazine, the photosynthetically produced oxygen within the tissue restores buoyancy and the leaf fragments swim to the surface. Atrazine sensitive pieces, however, remain on the ground.

Example 8: Transformation of Brassica rapa cells (see Just Right)

Brassica rapa protoplasts are washed with a suitable osmoticum and suspended at a population density of 5 x 10 ⁶ per ml in a culture medium prepared according to the instructions in (Protoplast 83, Proceedings Experientia Supplementum, Birkhäuser Verlag, Basel, Vol. 45 [1983 ], 44-45). 40% polyethylene glycol (PEG) having a molecular weight of 6000 and dissolved in modified F medium (pH 5.8) (see Example 1b) is mixed with the protoplast suspension to a final concentration of 13% PEG. To this mixture is then immediately added a solution consisting of 50 micrograms of the endonuclease Sal I digested plasmid pBRCAT or p32CAT and 60 micrograms of water. With occasional stirring, this mixture is incubated at 2O ⁰ C to 25 ⁰ C for 30 minutes. Then, 3 ml of 2 ml of modified F medium (total 6 ml) and 2 ml of 2 ml culture medium (4 ml total) are added at 5 minute intervals. The protoplast suspension is transferred to Petri dishes with a cross section of 10 cm and supplemented with additional culture medium to a total volume of 20 ml. These protoplast suspensions are incubated for 45 minutes at 26 ^° C. in the dark. The protoplasts are taken up by sedimentation for 5 minutes with 100 g of an initially liquid culture medium, which is later solidified by agarose gel, and cultured by the "bead type culture method" (Plant Cell Reports, 2,244-247 [1983]) Chloramphenicol at a concentration of 10mg / liter is added to the cultures The liquid culture medium surrounding the agarose segments is replaced every four days with fresh chloramphenicol containing broth and after four weeks the chloramphenicol resistant clones are isolated and then further cultivated by weekly feeding with chloramphenicol-containing nutrient solution dOmg / liter).

Example 9: Transformation of protoplasts

It is based on Lolium multiflorum protoplasts with a concentration of 2 · 10 ⁶ per ml in a 0.4 molar Mannitol solution of pH 5.8. To this suspension are successively added 0.5 ml of 40% polyethylene glycol (PEG) having a molecular weight of 6000 in modified (pH 5.8) F medium (Nature 296, 72-74, [1982]) and 65 microliters of an aqueous solution 50 micrograms of the plasmid p32CAT or pBRCAT added. This mixture is incubated for 30 minutes at 26 ° C with occasional stirring and then diluted with F medium. This is done as described in (Nature 296, [1982]). The protoplasts are isolated by centrifugation (5 minutes at 100g) and 4ml CC culture medium (Potrykus, Harms and Lörz,

Callus Formation from Cell Culture Protoplasts of Corn [Zea Mays L.], Theor. Appl. Genet. 54, 209-214 [1979]) and incubated at 24 ° C in the dark. After 14 days, the developing cell cultures are transferred to the same medium but now containing chloramphenicol (10 mg / liter). Resistant colonies are transferred to an agar medium - the same medium as above 10 mg / liter chloramphenicol without osmoticum - and subsequently, having reached a size of several grams fresh weight per colony, in view of the presence of the bacterial gene and the biological Activity of the gene analyzed.

Example 10: Transformation of cultured cells of Nicotiana tabacum by transfer of p32CAT, pBRCAT or pUCHI by means of electroporation

Protoplasts are obtained by sedimentation of 50 ml of a 10 g-phase suspension culture of a nitrate reductase defective variant of Nicotiana tabacum, Zeil strain nia-115 (Müller, AJ and R. Grafe, Mol. Gen. Gen. 161, 67-76. 1978]) and resuspension in 20 ml of an enzyme solution (2% CellulaseOnozuka R-10.1% Macerozyme R019 and 0.5% Driselase [available from Chemische Fabrik Schweizerhalle, Basel] in a washing solution [0.3 M mannitol, 0.04 M calcium chloride and 0.5% 2- (N-morpholino) ethanesulfonic acid] adjusted to pH 5.6 with KOH) and incubation for 3 hours on a rotary shaking machine at 24 ⁰ C. The protoplasts are then removed by filtration through a sieve separated from undigested tissue with a mesh size of 100 microns. The same volume of a 0.6M sucrose solution is added and the resulting suspension centrifuged at 100g for 10 minutes. The protoplasts floating on the surface are collected and washed three times by sedimentation in the washing solution.

The transformation is carried out with the help of electroporation. The chamber of a Dialog® "Porator" (available from Dialog GmbH, Harffstr. 34, 4000 Dusseldorf, Federal Republic of Germany) is sterilized by washing with 79% alcohol followed by 100% alcohol and by a stream of sterile air from a blower The protoplasts are suspended in a concentration of 1 × 10 ⁶ / ml in a 0.4M mannitol solution and adjusted to a resistance of 1.4 kOhm with magnesium chloride, followed by pBRCAT, pUCHI or p32CAT in a concentration of 10 0.38 ml samples of this protoplast suspension are applied three times at intervals of 10 seconds with a voltage of 1000 or 2000 V. The protoplasts are then in a concentration of 1 × 10 ⁶ / ml in 3 ml of an AA -CH medium (AA Medium of Climelius, K. et al., Physiol. Plant. 44, 273-277 [1978]) cultured by both increasing the inositol concentration to 100 mg / And the sucrose concentration to 34 g / liter, as well as by the addition of 0.05 ml / liter of 2- (3-methyl-2-butenyl) adenine was modified and that due to its, content of 0.6% agarose (Sea Plaque , FMC Corp., Marine Colloids Division, PO Box 308, Rockland, Maine 04841, USA). After one week, the agarose layer containing the protoplasts is transferred to 30 ml of a liquid AA-CH medium containing 10 mg / liter chloramphenicol. After three weeks, during which half of the medium is replaced weekly with fresh medium of the same composition, the transformed cell colonies can be visually perceived. Four weeks after transfer to the chloramphenicol-containing medium, these colonies are seeded on an AA medium (Climelius, K. et al., Physiol. Plant., 44, 273-277 [1978]); with 0.8% agar containing 10 mg / liter chloramphenicol for further cultivation and study.

Analogous studies with protoplasts of Brassica rapa and Lolium multiflorum also lead to successful transformations.

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

The preparation of the electroporator and the protoplasts takes place according to Example 10 or Example 6.

For transformation, protoplasts of Nicotiana tabacum are resuspended in a concentration of 1.6 x 10 ⁶ VmI in a mannitol solution (0.4 M buffered with 0.5% w / v 2 - (N-morpholine) -ethanesulfonic acid; 5.6). The resistance of the protoplast suspension is measured in the Porator chamber (0.38 ml) and adjusted to a value between 1 and 1.2 kOh with a magnesium chloride solution. 0.5 ml samples are withdrawn and placed in a capped plastic tube (5 ml volume) into which previously 40 microliters of water containing 8 micrograms of donor DNA and 20 micrograms of calf thymus DNA were added, and 0.25 ml of a polyethylene glycol solution ( 24% w / v in 0.4M mannitol). 9 minutes after the addition of DNA, 0.38 ml samples are added to the electroporator chamber. Ten minutes after addition of the DNA, the protoplast suspension in the chamber is charged with three pulses (1000-2000 volts) at a 10 second interval. The treated samples are placed in 6cm diameter petri dishes and held at 20 ° C for 10 minutes. Then, 3 ml of K ₃ medium with 0.7% w / v "Sea Plaque Agarose" is added to each Petri dish and mixed thoroughly the contents of the bowl. After solidification, the content of each dish, the cultures for one day at 24 ⁰ C in The protoplasts containing agarose are then cut into four parts and placed in a liquid medium.The protoplasts are then cultured by the "bead type culturing method". Calli tissue obtained by selecting the transformed material with chloramphenicol and plants regenerated therefrom contain the CAT enzyme (chloramphenicol acetyl transferase) as a product of the CAT gene. Electroporation results in a 5 to 10-fold increase the transformation frequency compared to a method without electroporation. Similar studies with Brassica rapa cv Just Right and Lolium multiflorum also showed an increase in the transformation frequency of the same order of magnitude. -

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

Protoplasts isolated from leaves or Nicotiana tabacum cell cultures are obtained as described in Examples 6 and 10, and converted into an osmotically stabilized medium analogously to the preceding examples. The protoplast suspensions are held at 45 ° C for 5 minutes and then cooled to ⁰ ° C with ice for 10 seconds; then the plasmid pBRCAT, pUCHI or pBRCAT as described in Examples 6 and 10 is added. The heat-shock treatment increases the transformation frequency by a factor of 10 or more compared to a transformation performed without this treatment.

Example 13: Transformation of Plant Cells of Various Origin by Transfer of the CAT Gene by first bringing together protoplasts and genes and then performing a combined treatment

Plant Protoplasts: Nicotianatabacumc.v. Petit Havana SRI (A); Brassicarapac.v. Just Right (B) and Lolium multiflorum (C) are isolated and transferred according to Example 11 to an osmotically stabilized medium. The protoplast suspensions are mixed with the plasmid pUCHI, 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 then subjected to a polyethylene glycol treatment according to Examples 6 bis and finally an electroporation according to Example 11. The transformafion frequency for this procedure is between 10 ~ ³ and 1CT ² , but can be increased to 1% to 2% depending on the selected conditions.

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Claims (36)

Claims: ' A method of directly introducing DNA into the plastids and mitochondria of plant protoplasts, said DNA consisting of one or more genes and promoters active in plastids and mitochondria, characterized in that, in the absence of a pathogen, said DNA in a medium, in which the DNA is able to penetrate into the protoplasts and the plastids and mitochondria therein, as long as it comes into contact with the protoplasts, that this penetration is ensured. 2. The method according to claim 1, characterized in that it can continue to run until the regeneration of transformed plant cells from the transformed protoplasts. 3. The method according to any one of claims 1 or 2, characterized in that it is allowed to continue the process until the regeneration of transformed plants. 4. The method according to claim 1, characterized in that it is the DNA to be injected is linear DNA. 5. The method according to claim 1, characterized in that it is the DNA to be injected is circular DNA. 6. The method according to claim 5, characterized in that the circular DNA contains no or one more T-DNA border regions. 7. The method according to claim 1, characterized in that the DNA to be injected is a DNA which transmits a herbicide resistance in the plastids or mitoehondria. 8. The method according to claim 7, characterized in that said herbicide is atrazine. 9. The method according to claim 7, characterized in that the DNA to be injected in addition to the herbicide resistance transmits a second useful for agriculture property. 10. The method according to claim 1, characterized in that the DNA to be injected contains a selectable marker and a gene which transmits an agriculturally significant property. 11. The method according to claim 10, characterized in that the marker gene generates an antibiotic resistance. 12. The method according to claim 11, characterized in that it is the antibiotic resistance to chloramphenicol or kanamycin resistance. 13. The method according to claim 10, characterized in that it is the agriculturally significant property is a herbicide resistance. 14. The method according to claim 13, characterized in that it is atrazine resistance in the herbicide resistance. 15. The method according to claim 1, characterized in that it is the gene to be inserted is a chimeric gene. 16. The method according to claim 1, characterized in that the DNA to be injected contains a replication signal. 17. The method according to claim 1, characterized in that the DNA to be injected contains an integration signal. 18. The method according to claim 1, characterized in that one carries out the method with leaf-derived protoplasts. 19. The method according to claim 1, characterized in that one uses an osmotically stabilized suitable for protoplasts culture medium. 20. The method according to claim 1, characterized in that one uses a medium containing plant tolerant divalent cations. 21. The method according to claim 20, characterized in that it is the cations to magnesium or calcium cations. 22. The method according to claim 1, characterized in that the medium contains a polyhydric alcohol, which alters the cell membrane and favors the cell fusion. 23. The method according to claim 22, characterized in that it is the polyhydric alcohol is polyethylene glycol, polypropylene glycol or polyvinyl glycol. 24. The method according to claim 23, characterized in that it is the polyhydric alcohol is polyethylene glycol (PEG). 25. The method according to claim 24, characterized in that the polyethylene glycol has a molecular weight between 1,000 and 10,000. 26. The method according to claim 1, characterized in that the DNA and the protoplasts are subjected to a heat shock treatment. 27. The method according to claim 1, characterized in that the DNA and the protoplasts are subjected to electroporation. 28. The method according to claim 1, characterized in that the introduction of the DNA into the protoplasts by combining two measures selected from a polyethylene glycol treatment, heat shock treatment and electroporation takes place. 29. The method according to claim 28, characterized in that the gene transfer is carried out such that for the introduction of the foreign gene said gene and the protoplasts are in a solution and subjecting the resulting suspension first a heat shock treatment and then a polyethylene glycol treatment. 30. The method according to claim 1, characterized in that the gene transfer is carried out such that for the introduction of the foreign gene said gene and the protoplasts are in a solution and the resulting suspension first subjected to a heat shock treatment then a polyethylene glycol treatment, and finally an electroporation. 31. The method according to claim 1, characterized in that the medium used contains a polyhydric alcohol which alters the protoplast membrane and promotes cell fusion and subjecting the DNA suspended in this medium together with the protoplasts of an electroporation and / or a heat shock treatment. 32. The method according to claim 1, characterized in that additionally inactivates the extracellular nucleases. 33. The method according to claim 1, characterized in that one uses protoplasts from plant cells of plants of the families of Gramineae, Solanaceae or Cruciferae. 34. The method according to claim 33, characterized in that one uses protoplasts from plant cells of the plants of the family Gramineaeeinseins. 35. Method according to claim 34, characterized in that the Gramineae are cereals. 36. The method according to claim 35, characterized in that it is the grain to corn, wheat, rice, barley, oats, millet, rye or sorghum.