EP1458875A2 - Methods for the transformation of vegetal plastids - Google Patents

Abstract

The invention relates to novel methods for the production of transgenic plants with genetically modified plastids, in addition to transgenic plants produced according to said method.

Description

Process for the transformation of plant plastids

description

The present invention relates to new processes for producing transgenic plants with genetically modified plastids and to the transgenic plants produced using these processes.

The aim of biotechnological work on plants is the production of plants with advantageous new properties, for example to increase agricultural productivity, to increase the quality of food or to produce certain chemicals or pharmaceuticals.

Piastids are organelles within plant cells with their own genome. They have an essential function in photosynthesis as well as amino acid and lipid biosynthesis. The plastid genome consists of a double-stranded, circular DNA with an average size of 120 to 160 kb and is present - for example in leaf cells - with approx. 1900 to 50,000 copies per cell (Palmer (1985) Ann Rev Genet 19: 325- 54). A single plastid has a copy number of approx. 50 to 100. The term plasti u includes chloroplasts, proplastids, etioplasts, chro oplasts, amyloplasts, leucoplasts and elaioplasts (Heifetz P (2000) Biochimie 82: 655-666). The different forms can be converted into one another and all arise from the proplastids. Therefore, all forms of the plastids contain the same genetic information. In the literature, however, green cells which contain chloroplasts as a form of expression are preferably used as the starting material for the transformation of plastids.

It is of great economic interest for plant biotechnology to develop efficient methods for plastid transformation (McFadden G (2001) Plant Physiol. 125: 50-53). The stable transformation of plastids from higher plants is one of the major challenges.

The one often used when inserting into nuclear DNA

The technique of undirected (illegitimate) DNA insertion has the disadvantage in plastid transformation that it is very likely that an essential gene will be struck on the gene-tight plastid genome, which would often be lethal for the plant. A targeted insertion of foreign DNA is therefore advantageous in plastids. Various methods for targeted insertion into the plastid genome have been described. The plastid transformation in green algae was first described (Boynton JE et al. (1988) Science 240: 1534-1538; Blowers AD et al. (1989) Plant Cell 1: 123-132), later in higher plants such as tobacco (Svab Z et al. (1990) Proc Natl Acad Sei USA 87: 8526-8530).

EP-A 0 251 654, US 5,932,479, US 5,451,513, US 5,877,402,

WO 01/64024, WO 00/20611, WO 01/42441, WO 99/10513, WO 97/32977, WO 00/28014, WO 00/39313 describe methods and DNA constructs for transforming plastids of higher plants, the to transforming DNA is incorporated into the piastome (piastid genome) via homologous recombination ("double crossover"). In general, homologous regions of 1000 bp or more are used on each side of the sequence to be inserted. This very quickly leads to large vectors, whose handling is not very comfortable. In addition, the efficiency of the transformation decreases. The efficiency of the homologous recombination decreases with increasing length of the foreign DNA to be integrated. Another disadvantage is the fact that a homologous area must be identified for each plant species that can be used for the process of DNA integration by means of double crossover. WO 99/10513 claims to have identified an intergenic DNA sequence that is sufficiently homologous between the genomes of the chloroplasts of many higher plants and can thus function as a universal target sequence. However, it has not been proven that this vector can be successfully used in species other than tobacco. On the contrary: In WO 01/64024 the same inventor adapts the transformation vector to non-tobacco plant species by using homologous DNA sequences isolated from them. Since only a few recombination events result in all of the methods described above, a selection of the recombinant plastid DNA molecules is necessary.

The plastid DNA of higher plants is available in up to several thousand copies per cell. To achieve a stable integration of the foreign DNA, all copies of the plastid DNA must be changed equally. In the case of plastid transformation, one speaks of having reached the homotransplastomic state. This state is achieved through a so-called "segregation and sorting" process, in which the plants are kept under constant selection pressure. Due to the persistent selection pressure, those plastids in which many copies of the plastid DNA have already been changed are enriched during the division of the cells and plastids. The selection pressure is maintained until the homotransplastoma is reached (Guda C et al. (2000) Plant Cell Reports 19: 257-262). It is a major challenge to modify all copies of the plastid genome in order to obtain homotransplastome plants that have stably incorporated the foreign gene into their plastid genome without the addition of a selection agent for generations (Bogorad L (2000) TIBTECH 18: 257-263). In addition to the constant selection pressure, repeated regeneration of already transformed tissue ensures that the homotransplastomic state is reached (Svab Z and Maliga P (1993) Proc Natl Acad Sei USA 90: 913-917). However, this limits the plant material that is available for plastid transformation. It is therefore proposed to couple the transgene with another gene that is essential for the survival of the plant. Tissue culture techniques and selection processes are usually not universally applicable to all plant species and represent a significant limitation of plastid transformation, which particularly affects the transferability of the method to species other than tobacco (Kota M et al. (1999) Proc Natl Acad Sei USA 96: 1840-1845). A recently published transformation of plastids from tomato is based on modifications in the regeneration and selection scheme (Ruf S et al. (2001) Nature Biotech 19: 870-875), which, however, are costly and time-consuming. Another approach aims to reduce the number of plastids per cell and the number of DNA molecules per plastid so that fewer DNA molecules have to be modified (Bogorad L (2000) TIBTECH 18: 257-263). Overall, the selection and segregation processes are very time consuming.

WO 99/10513 describes a method in which a plastid ORI (origin of replication, origin of replication) is located on the plasmid to be transformed, so as to increase the number of copies of the vector to be transformed which are available for integration into the plastid genome (Guda C et al. (2000) Plant Cell Reports 19: 257-262).

The need to improve the technique of plastid transformation is also mentioned in Heifetz and Tuttle (Heifetz P and Tuttle AM (2001) Curr Opin Plant Biol 4: 157-161). WO 00/32799 teaches how to increase the efficiency of plastid transformation by using plants with enlarged plastids. This results in a large surface of the plastids, through which the DNA to be transformed can penetrate the plastids more easily. The mechanism of DNA integration also takes place here by means of conventional homologous recombination as in the methods described above. Various other methods for the sequence-specific integration of DNA - in particular into the nuclear DNA - have been described. A method is described based on self-splicing introns of group II. Self-splicing introns of group II can insert sequence-specifically - for example into intronless genes. The sequence-specific hydrolysis of the target DNA is catalyzed by an RNA-protein (ribonucleoprotein) complex. The sequence specificity of the endonuclease function is primarily determined by forming base pairs between the RNA

10 fraction of the ribonucleoprotein complex and the target DNA determined. The use of Group II introns as vectors for foreign DNA has been discussed. The target specificity could be changed by modifying certain sequences of a group II intron. Other sequences in group II introns

15 can be inserted without destroying their functions (Yang J et al. (1996) Nature 381: 332-335; Eickbush TH (1999) Curr Biol 9: R11-R14; Matsuura M et al. (1997) Genes Develop 11: 2910-2924; Cousineau B et al. (1998) Cell 94: 451-462). The adaptation to certain target sequences and the determination of the associated

However, 20 rules is complex and has so far only been decrypted in detail for the Ll.ltrB intron (Mohr G et al. (2000) Genes Develop 14: 559-573). In addition, the efficiency of retrohoming was significantly reduced by the modification and not every of the modified introns tested inserted into the desired one

25 target DNA. The disadvantage of the technique is that some positions are fixed in the nucleotide sequence, which limits the selection of the target region in the DNA to be transformed (Guo H et al. (1997) EMBO J 16: 6835-6848). In addition, the efficiency of the retrohoming process seems to be different from that of the wild-type introns.

30 decreases. The efficiency of intron insertion at different

Places on the genes under consideration were of different heights. The aim of the work was to provide an improved method for the targeted insertion of DNA into the nuclear DNA of organisms that do not allow efficient homologous recombination (Guo et al.

35 (2000) Science 289: 452-456). The experiments described have been carried out extrachromosomally both in the prokaryote E. coli and in human cells. The transferability to the chromosomal DNA of higher organisms or the applicability to plastid DNA was neither described nor shown. It was

40 only proposed to try to optimize this system in such a way that it can be inserted into chromosomal DNA of higher eukaryotes. This system is said to be an alternative method in higher eukaryotes that do not show efficient homologous recombination (Guo et al. (2000) Science

45 289: 452-456). This does not apply to plastids of higher plants where homologous recombination occurs - at least for some plastid DNA molecules - can usually be realized without problems.

In addition to tobacco, the plastid transformation in potatoes (Sidorov VA et al. (1999) Plant J 19: 209-216; WO 00/28014), petunia (WO 00/28014), rice (Khan MS and Maliga P (1999) Nature Biotech 17: 910-915; WO 00/07431; US 6,153,813), Arabidopsis (Sikdar SR et al. (1998) Plant Cell Reports 18: 20-24; WO 97/32977) and rapeseed (WO 00/39313) (review article : Bogorad L (2000) TIBTECH 18: 257-263). Transplastome tomato plants have also recently been described (Ruf S et al. (2001) Nature Biotech 19: 870-875).

The generation of sequence-specific double-strand breaks with the help of restriction enzymes in eukaryotic genomes and others. Plants have been described (Puchta H (1999) Methods Mol Biol 113: 447-451).

WO 96/14408 describes the Homing restriction endonuclease I-Scel and various ways of using it. An application for inserting DNA sequences into plastid DNA is not described.

Posfai et al. describe a method for exchanging genes in the prokaryote E. coli (Posfai G et al. (1999) Nucleic Acids Res 27 (22): 4409-4415). This results in an intramolecular recombination between the endogenous and the mutated gene in the E. coli genome, which is induced by a cut with the restriction enzyme I-Scel. Recombinations in E. coli are significantly more efficient and presumably different mechanisms than in the nucleus of higher eukaryotes (for example described in Kuzminov A (1999) Microbiol Mol Biol Rev. 63 (4): 751-813).

"Homing" means the phenomenon that two or more copies of a DNA sequence exist in a compartment, at least one of these two sequences being interrupted by a further DNA sequence, and consequently a copy of the interrupting DNA sequence also not in the interrupted DNA sequence is introduced. The phenomenon usually occurs as intron homing. Here there are two or more alleles of a gene in a compartment, with at least one of these alleles not having an intron. As a result, a copy of the intron is also introduced into the intronless allele.

Introns in plastid genes of higher plants are described (Vogel J et al. (1999) Nucl Acids Res 27: 3866-3874; Jenkins BD et al. (1997) Plant Cell 9: 283-296; Xu MQ et al. (1990) Science 250: 1566-1570). Splicing a species-specific, non-fashionable infected introns with the natural exon areas at an ectopic locus in the plastid genome is also described (Bock R and Maliga P (1995) Nucl Acids Res 23 (13): 2544-2547). So far there have been no attempts to introduce introns of other species, which are also modified in such a way that they have additional genetic information and / or are splicing in a non-natural sequence environment, in plastids of higher plants.

Experiments by Eddy and Gold on the "homing" process in E.coli showed that certain recombination systems are required. The type of host recombination system is a key variable (Eddy SR and Gold L (1992) Proc Natl Acad Sei USA 89: 1544-1547). Therefore, it could not be assumed that the naturally occurring process of "homing" can be arbitrarily transferred from one organism to another, especially if this process does not naturally occur in this organism.

Dürrenberger et al. describe the induction of an intrachromosomal recombination in chloroplasts of the unicellular green algae Chlamydomonas reinhardtii using the I-Crel homing endonuclease (Dürrenberger F et al. (1996) Nucleic Acid Res 24 (17): 3323-3331). The recombination takes place between the endogenous 23S gene and a 23S CDNA which is inserted into the chromosome of an I-Crel deletion strain and which contains an I-Crel cleavage site. Double-strand breaks are induced by "mating" the corresponding transgenic organism with an organism that naturally expresses I-Crel. At the time of the double strand break, the foreign DNA is already inserted into the chromosomal DNA and the recombination takes place intramolecularly and not between two separate molecules.

It has recently been shown that a mobile intron naturally occurring in Chlamydononas reinhardtii, which also encodes a homing endonuclease, can be efficiently transformed into an intronless copy (Odo OW et al. (2001) Mol Cell Biol 21: 3472-3481). The increase in the transformation rate was dependent on the presence of the homing endonuclease. In the discussion, it is generally proposed, without specific instructions, to improve plastid transformation by inducing double-strand breaks. For this purpose, the recognition regions of rarely cutting nucleases should first be introduced in a first step, and the subsequent integration event should then take place at the same locus. More precise information on how to introduce the recognition regions, which nucleases and recognition regions can be used, how the first and second steps are designed, etc. not given. So far, it has only been shown that the introduction of a species-specific intron into plastids of the alga Chlamydomonas worked by means of the homing endonuclease naturally associated with the mobility of the intron. The results were also generated in an algae species. The experiments by Eddy and Gold with E. coli mentioned above, in which no mobile introns of group I are known, as in plastids of higher plants, show that transferability to heterologous systems is not readily possible. The transferability of the observations from the alga Chlamydomonas to higher plants is therefore by no means obvious to the person skilled in the art. On the contrary, there are numerous indications that question the portability:

1. Homing systems are not easily transferable from one system to another (Eddy SR and Gold L (1992) Proc Natl

Acad Sei USA 89: 1544-1547). The transferability to higher plants is all the more questionable since no homing endonucleases are identified in the already sequenced plastid genomes of higher plants (http: //it.egasun.bch.umontreal. Ca / ogmp / projects / other / cp_list .html) were. It can therefore be assumed that the introns found in the plastid genome of higher plants are not mobile and there is naturally no homing mechanism there.

2. Chlamydomonas has only one plastid at a time, while in the

Cells of higher plants have up to 100 plastids per cell.

3. Conventional plastid transformation is several orders of magnitude more efficient in Chlamydomonas than in higher plants, suggesting that these two systems are not directly comparable. With regard to the regeneration of transplastomic algae or transplastomic plants, it could also play a decisive role that the division of the plastids of the algae is synchronized with the cell cycle, while this does not apply to the plastids of the higher plants (Sato N (2001) Trends Plant Science 6 : 151-155).

4. The mechanisms of DNA integration in plastids from Chlamydomonas and higher plants appear to be fundamentally different. It was found, for example, that the intermediate plastid transformation (using homologous areas instead of identical sequences) in Chlamydomonas leads to a significant reduction in the transformation efficiency, but this cannot be observed in tobacco. The same applies to the distance between a molecular marker on the homologous DNA and the heterologous sequence on the Transformation plasmid: The closer the molecular marker is to the edge of the target region for integration by means of double crossover, the less frequently it is transferred from Chlamydomonas during transformation into plastids. Multiple recombination mechanisms were observed in tobacco, but molecular markers close to the edge of the homologous regions were also effectively transferred during the transformation into the plastid genome (Kavanagh TA et al. (1999) Genetics 152: 1111-1122 and references therein).

5. In Chlamydomonas, the plastids of the two parents merge during a cross, even at an interspecific cross. The plastid fusion is a natural process in Chlamydomonas and the DNA of the plastids is mixed and recombined. Therefore, mobile introns make sense in the organelles of these organisms. In contrast, the plastids in most higher plants are inherited uniparental, so that there is no mixing of the plastid DNA, and no recombinations can occur between the plastid DNA of the mother and the father.

No fusion of the plastids was observed even in the plant species in which biparental inheritance of the plastids occurred. It is therefore reasonable to assume that natural plastid fusion in higher plants is excluded (Hagemann R (1992) Plastid genetics in higher plants; in Cell Organelles, publisher: Herrmann RG, Springer Verlag, Vienna, pp. 65-96) and mechanisms like intron homing are not developed or even suppressed.

Increasing the efficiency of homologous recombination within nuclear DNA with the help of rarely cutting endonucleases has been described in various organisms (Puchta H et al. (1993) Nucleic Acids Research. 21 (22): 5034-40; Puchta Het al. ( 1996) Proc Natl Acad Sei USA 93: 5055-5060; Rong YS and Golic KG (2000) Science 28: 2013-2018; Jasin M (1996) Trends Genet 12: 224-229). In contrast to plastids, insertion by means of homologous recombination in the core DNA is problematic and usually takes place by means of random illegitimate integration. This shows that techniques established in the core genome cannot necessarily be transferred to the plastids. In contrast to the situation in the core, plastids of higher plants are almost exclusively and highly efficiently integrated via homologous recombination (Bock R and Hagemann R (2000) Progress in Botany 61: 76-90; Maliga P et al. (1994) Homologous recombination and integration of foreign DNA in plastids of higher plants. In Ho ologous recombination and gene silencing in plants. Paszkowski J, ed. (Kluwer Academic publishers), p.83-93).

The efficiency of homologous recombination for DNA integration into the piastom was generally not seen as a limiting factor and - on the contrary - was classified as uncritical. Current research on optimizing plastid transformation is therefore not aimed at optimizing homologous recombination, but rather, for example, at improved selection markers, improved selection and regeneration techniques etc. Nevertheless, no significant breakthroughs have been achieved so far.

As the processes and problems in plastid transformation described above clearly emphasize, the provision of new processes for the production of homotransplastomeric plants is a long-standing unsolved need in plant biotechnology. Another need is to avoid antibiotic or herbicide selection markers for regulatory and consumer acceptance reasons. No method has yet been described for plastid transformation that can do without such a selection marker.

The task therefore arose to develop new processes which ensure efficient integration of foreign DNA into all copies of the plastid DNA and an efficient selection of appropriate homotransplastomeric plants. This object was surprisingly achieved by providing the integration / selection method according to the invention.

A first subject of the invention relates to a method for integrating a DNA sequence into the plastid DNA of a multicellular plant or cell derived therefrom and for the selection predominantly homotransplastomeric cells or plants, characterized in that

a) the plastid DNA molecules of said multicellular plant or cells derived therefrom contain at least one recognition sequence for the targeted induction of DNA double strand breaks and

b) at least one enzyme suitable for inducing DNA double-strand breaks at the recognition sequence for the targeted induction of DNA double-strand breaks and at least one transformation construct comprising an insertion sequence in at least one plastid of said multicellular plant or brought together from this derived cell, and

c) the induction of DNA double strand breaks occurs at the recognition sequences for the targeted induction of DNA double strand breaks, and

d) the insertion sequence is inserted into the plastid DNA, the functionality of the recognition sequence for the targeted induction of DNA double-strand breaks being deactivated, so that said recognition sequence can no longer be cut by the enzyme suitable for the induction of DNA double-strand breaks, and

e) plants or cells are isolated in which the insertion sequence has been inserted into the plastid DNA molecules.

The system surprisingly enables a significant increase in efficiency in the production of predominantly homotransplastomer plants. Both the efficiency of the insertion into the plastid DNA and the efficiency of the selection process of predominantly homotransplastomeric plants are increased.

The use of the method according to the invention results in a selection pressure for incorporating the insertion sequence into all copies of the plastid DNA. Ideally, the insertion sequence is distributed independently of selection markers such as herbicide or antibiotic resistance. This has clear advantages in terms of approval and / or consumer acceptance. However, using such selection markers can further increase efficiency. The process according to the invention for the production of homoplastomeric plants is clearly superior to that described in the prior art, since it offers a faster, more efficient and therefore less expensive route to homotransplastomic plants. The system also has the advantage that the constructs used for the transformation can be kept small, since, compared to the integration by means of the double crossover, the homologous regions in the plastid transformation vector can be smaller or completely absent. The transformation of plastids has numerous advantages over the transformation of the cell nucleus. These include:

a) While homologous recombination in nuclear DNA is difficult to achieve, in plastid DNA can easily be integrated at a predefined location using double crossover, a form of homologous recombination. Position effects or "genesilencing", which can be found in core transformations due to the illegitimate integration into a non-predefined locus, are thus avoided.

b) Very high levels of expression can be achieved, presumably due to the high copy number of the plastid DNA.

c) The DNA of the plastids is found in higher plants in the

Usually inherited only maternally, so that the introduced foreign DNA is not spread via pollen and cross-breeding can thus be effectively prevented.

d) The prokaryotic nature of the plastids enables the expression of genes in the context of a polycistronic operon structure. It is therefore not necessary to equip each gene to be expressed with its own promoter, etc. This makes it easier to insert many genes at once, for example to insert entire biosynthetic pathways into the plastids.

“Plastid” means the proplastids and all organelles resulting from them, such as chloroplasts, etioplasts, chromoplasts, amyloplasts, leukoplasts, dermatoplasts and elai plasters (Heifetz P (2000) Biochimie 82: 655-666).

"Piastom" means the genome, that is, the entirety of the genetic information, of a plastid.

"Homotransplastoma" means a transplastomic and homoplastic state.

"Transpiastome" means, for example with respect to a plant, cell, tissue, plastid or plastid-DNA, all such forms of the aforementioned that have been obtained by genetic engineering methods and comprise a plastid DNA that has been modified by genetic engineering methods, the modification exemplifying substitutions, May include additions, deletions, inversions or insertions of one or more nucleotide residues. "Heteroplasto" means the presence of a mixed population of different plastid DNAs within a single plastid or within a population of plastids within a plant cell or tissue.

"Homopiastoma" means a uniform population of plastid DNA within a single plastid or within a population of plastids within a plant cell or tissue. Homoplastoma cells, tissues or plants are genetically stable because they only contain one type of plastid DNA i.e. they generally remain homoplastic even when the selection pressure no longer persists. Offspring obtained by selfing are also homoplastic.

In the context of this invention, "predominantly homoplastom" or "predominantly homotransplastom" means all those plants or cells in which the proportion of the desired plastid-modified DNA molecules with regard to a characteristic - for example with the recognition sequence for the targeted induction of DNA double-strand breaks or the inserted insertion sequence - is at least 50%, preferably at least 70%, very particularly preferably at least 90%, most preferably at least 95% of the totality of all plastid DNA molecules in a plant or a tissue, cell or plastid thereof. Predominantly homoplastic or predominantly homotransplastome plants can be converted into homoplastome or homotransplastome plants by further maintaining the selection pressure and, if necessary, repetitive regenerations. However, due to the "homing" process, continuous selection printing is not absolutely necessary. In a particular embodiment, therefore, a predominantly homoplastic or homotransplastome plant is purely homoplastic or homotransplastoma. A plant which is predominantly homoplastom or homotransplastom or purely homoplastom or homotransplastom with respect to a DSB recognition sequence is consequently referred to as a "master plant". The proportion of the desired plastid-modified DNA molecules with regard to a feature can be determined, for example, by Southern analysis - as described by way of example in Example 4 - in the manner known to the person skilled in the art. The ratio between the plastic base DNA molecules and the plastid DNA molecules modified with regard to a feature can be determined by comparing the intensity of the respective bands.

"Multicellular plant or cell derived from this" generally means any cell, tissue, part or propagation material (such as seeds or fruit) of a plant which in its adult state represents or can represent a multicellular organism. Included in the scope of the invention are all genera and species of higher and lower plants in the plant kingdom. Annual, perennial, monocot and dicot plants are preferred. Included are mature plants, seeds, shoots and seedlings, as well as parts derived therefrom, propagation material (for example tubers, seeds or fruits) and cultures, for example cell or callus cultures. Mature plants mean plants at any stage of development beyond the seedling. Seedling means a young, immature plant at an early stage of development.

Plants from the following plant families are preferred: Amarantheaea, Asteraceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae

Rubiaceae, Saxifragaceae, Scrophulariaceae, Solanacea, Sterculiaceae, Tetragoniacea, Theaceae, Umbelliferae.

Preferred monocotyledonous plants are selected in particular from the monocotyledonous crop plants, such as, for example, the family of the Gramineae such as rice, maize, wheat or other types of cereals such as barley, millet, rye, triticale or oats, and sugar cane and all types of grasses.

Preferred dicotyledonous plants are in particular selected from the dicotyledonous crop plants, such as, for example

- Asteraceae such as sunflower, tagetes or calendula and others,

- Compositae, especially the genus Lactuca, especially the species sativa (salad) and others,

- Cruciferae, especially the genus Brassica, especially the species napus (rape), campestris (turnip), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli) and other types of cabbage; and the genus Arabidopsis, especially the species thaliana as well as cress or canola and others,

- Cucurbitaceae such as melon, pumpkin or zucchini and others,

- Leguminosae especially the genus Glycine, especially the type max (soybean) soy, as well as alfalfa, peas, beans or peanuts and others Rubiaceae, preferably of the subclass Lamiidae, such as, for example, Coffea arabica or Coffea liberica (coffee shrub) and others,

- Solanaceae especially the genus Lycopersicon, especially the species esculentum (tomato) and the genus Solanu, especially the species tuberosum (potato) and melongena (eggplant) as well as tobacco or peppers and others,

Sterculiaceae, preferably of the subclass Dilleniidae such as Theobroma cacao (cocoa bush) and others,

Theaceae, preferably of the subclass Dilleniidae, such as, for example, Camellia sinensis or Thea sinensis (tea bush) and others,

- Umbelliferae, especially the genus Daucus (especially the species carota (carrot)) and Apium (especially the species graveolens dulce (Seiarie)) and others; and the genus Capsicum, especially the species annum (pepper) and others,

as well as flax, soybeans, cotton, hemp, flax, cucumber, spinach, carrot, sugar beet and the various types of trees, nuts and wines, in particular banana and kiwi.

Also included are ornamental plants, useful or ornamental trees, flowers, cut flowers, shrubs or lawn. Examples include, but are not limited to, angiosperms, bryophytes such as hepaticae (liverwort) and musci (mosses); Pteridophytes such as ferns, horsetail and lycopods; Gymnosperms like conifers, cycads, ginkgo and gnetals, the families of rosaceae like rose, ericaceae like rhododendrons and azaleas, euphorbiaceae like poinsettias and croton, caryophyllaceae like carnations, solanaceae like petunias, gesneriaceae like that

African violets, balsaminaceae like balsam, orchidaceae like orchids, iridaceae like gladiolus, iris, freesia and crocus, compositae like marigold, geraniaceae like geraniums, liliaceae like the dragon tree, moraceae like ficus, araeeae like philodendron and others.

Most preferred are Arabidopsis thaliana, Nicotiana tabacum, Tagetes and Brassica napus and all genera and species that are used as food or feed, such as the cereals described, or are suitable for the production of oils, such as oilseeds, nuts, soybeans, Sunflower, pumpkin and peanut. "Enzyme suitable for inducing DNA double-strand breaks on the recognition sequence for the targeted induction of DNA double-strand breaks" (as a result of "DSBI enzyme" for "double strand-break inducing enzyme") generally means all those enzymes which are capable of being sequence-specific Generate double-strand breaks in double-stranded DNA. For example, but not restrictive:

1. Restriction endonucleases, preferably type II restriction endonucleases, particularly preferably homing endonucleases as described in detail below.

2. Artificial nucleases as described in detail below, such as, for example, chimeric nucleases, mutated restriction or homing endonucleases or RNA protein particles derived from group II mobile introns.

Both natural and artificially produced DSBI enzymes are suitable. Preferred are all those DSBI enzymes whose recognition sequence is known and which are either in the form of their

Proteins can be obtained (for example by purification) or expressed using their nucleic acid sequence.

The DSBI enzyme is preferably selected with knowledge of its specific recognition sequence so that it is in addition to the

Target recognition sequence has no other functional recognition regions in the plastid genome. Homing endonucleases are therefore particularly preferred (overview: Beifort M and Roberts RJ (1997) Nucleic Acids Res 25: 3379-3388; Jasin M (1996) Trends Genet 12: 224-228; Internet: http: //rebase.neb .com / rebase / rebase.homing.html; Roberts RJ and Macelis D (2001) Nucleic Acids Res 29: 268-269). Due to their long recognition sequences, these meet this requirement. However, due to the small size of the piastome, it is also conceivable that DSBI enzymes with shorter recognition sequences (for example restriction endonucleases) can be used successfully.

In addition to the preferred embodiment described above, in which only a single recognition sequence for the DSBI enzyme is present in the plastid DNA, cases are conceivable in which further, functionally identical recognition sequences can also be used advantageously. This is particularly the case if the piastome comprises duplicated genes (for example in the form of inverted "repeats"). The integration into all copies should take place here, so that a cut in all copies is also desirable. The sequences coding for such homing endonucleases can be isolated, for example, from the chloroplast genome of Chlamydomonas (Turmel M et al. (1993) J Mol Biol 232: 446-467). They are small (18 to 26 kD), have a "coding usage" in their open reading frame (ORF), which is directly used for nuclear or plastid expression of higher plants (Monnat RJ Jr et al. (1999) Biochem Biophys Res Com 255: 88-93) is suitable.

Further homing endonucleases are listed at the Internet address given above, for example homing endonucleases such as F-Scel, F-Scell, F-Suvl, F-Tevl, F-TevII, I-Amal, I-Anil, I-Ceul, I-CeuAIIP, I-Chul, I-Cmoel, I-Cpal, I-CpaII, I-Crel, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-Csml, I- Cvul, I-CvuAIP, I-DdiII, I-Dirl, I-Dmol, I-HspNIP, I-Llal, I-Msol, I-Naal, I-NanI, I-NclIP, I-NgrIP, I-NitI, I-Njal, I-Nsp236lP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP, I-PobIP, I-Porl, I-PorIIP, I-PpbIP, I- Ppol, I-SPBetalP, I-Scal, I-Scel, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-SexIP, I-SneIP, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp6803I, I-SthPhiJP, I-SthPhiST3P, I-SthPhiS3bP, I-TdeIP, I-Tevl, I-TevII, I-TevIII, I-UarAP, I- UarHGPAlP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI, PI-MtuHIP, PI-MtuHIIP, Pl-PfuI, Pl-PfuII, Pl-Pkol, Pl-PkoII, PI-PspI, PI-Rma43812IP, PI-SPBetalP, Pl-Scel, PI-TfuI, PI-TfuII, PI-Thyl, PI-Tlil, PI-Tlill.

Preferred are the homing endonucleases, the gene sequences of which are already known, such as F-Scel, I-Ceul, I-Chul, I-Dmol, I-Cpal, I-CpaII, I-Crel, I-Csml, F- Tevl, F-TevII, I-Tevl, I-TevII, I-Anil, I-Cvul, I-Llal, I-NanI, I-Msol, I-NitI, I-Njal, I-PakI, I-Porl, I-Ppol, I-Scal, I-Sce I, I-Ssp6803I, Pl-Pkol, Pl-PkoII, PI-PspI, PI-TfuI, PI-Tlil.

Homing endonucleases are particularly preferably used which are naturally, particularly preferably naturally

Organelles occur. Most preferably, the homing endonucleases come from organisms that live at temperatures similar to plants. Of particular interest here are the homing endonucleases identified from yeast and Chlamydomonas species. Of course, it is also conceivable to use homing endonucleases isolated from extremophilic organisms as long as they are active in the plastids of the plant to be transformed. Are very particularly preferred

I-Ceul (Cote MJ and Tur el M (1995) Curr Genet 27: 177-183 .; Gauthier A et al. (1991) Curr Genet 19: 43-47; Marshall (1991) Gene 104: 241-245; GenBank Acc.-No .: Z17234 nucleotides 5102 to 5758),

I-Chul (Cote V et al. (1993) Gene 129: 69-76; GenBank Acc.-No .: L06107, nucleotides 419 to 1075),

I-Cmoel (Drouin M et al. (2000) Nucl Acids Res 28: 4566-4572),

I-Cpal from Chlamydomonas pallidostigmatica (GenBank Acc.-No .: L36830, nucleotides 357 to 815; Turmel M et al. (1995) Nucleic Acids Res 23: 2519-2525; Turmel, M et al. (1995) Mol Biol Evol 12: 533-545; see also SEQ ID NO: 13 or 14)

I-CpaII (Turmel M et al. (1995) Mol Biol Evol 12: 533-545; GenBank Acc.-No .: L39865, nucleotides 719 to 1423),

I-Crel (Wang J et al. (1997) Nucleic Acids Res 25: 3767-3776; Dürrenberger, F and Rochaix JD (1991) EMBO J 10: 3495-3501; GenBank Acc.-No .: X01977, nucleotides 571 bis 1062),

I-Csml (Ma DP et al. (1992) Plant Mol Biol 18: 1001-1004)

I-NanI (Eide M et al. (1999) Eur J Biochem. 259: 281-288; GenBank Acc.-No .: X78280, nucleotides 418 to 1155),

I-NitI (GenBank Acc.-No .: X78277, nucleotides 426 to 1163),

I-Njal (GenBank Acc.-No .: X78279, nucleotides 416 to 1153),

- I-Ppol encodes the extrachromosomal DNA in the core of Physarum polycephalum (Muscarella DE and Vogt VM (1989) Cell 56: 443-454; Lin J and Vogt VM (1998) Mol Cell Biol 18: 5809-5817; GenBank Acc. -No .: M38131, nucleotides 86 to 577). In addition, the longer sequence starting from an alternative start codon, coding for I-Ppol, can also be used. In the sequence according to GenBank Acc. No M38131, this sequence comprises nucleotides 20 to 577. The shorter sequence is preferably used; however, it can be replaced at any point by the longer one. An artificial sequence which codes for the same amino acids as the sequence of the nucleotides 86 to 577 of the sequence is particularly preferably used in the context of this invention. sequence according to GenBank Acc.-No .: M38131 (see also SEQ ID NO: 5, 11, 12, 70 or 71),

I-Pspl (GenBank Acc.-No .: U00707, nucleotides 1839 to 3449),

I-Scal (Monteilhet C et al. (2000) Nucleic Acids Res 28: 1245-1251; GenBank Acc.-No .: X95974, nucleotides 55 to 465)

I-Scel from the mitochondria of baker's yeast (WO 96/14408; US 5,962,327 there Seq ID NO: 1),

Endo Scel (Kawasaki et al. (1991) J Biol Chem 266: 5342-5347, identical to F-Scel; GenBank Acc.-No .: M63839, nucleotides 159 to 1589),

I-SceII (Sarguiel B et al. (1990) Nucleic Acids Res 18: 5659-5665),

I-SceIII (Sarguiel B et al. (1991) Mol Gen Genet. 255: 340-341),

I-Ssp6803l (GenBank Acc.-No .: D64003, nucleotides 35372 to 35824),

I-Tevl (Chu et al. (1990) Proc Natl Acad Sei USA 87: 3574-3578; Bell-Pedersen et al. (1990) Nucleic Acids Resl8: 3763-3770; GenBank Acc. -No.: AF158101, nucleotides 144431 until 143694),

I-TevII (Bell-Pedersen et al. (1990) Nucleic Acids Res 18: 3763-3770; GenBank Acc.-No .: AF158101, nucleotides 45612 to 44836),

I-TevIII (Eddy et al. (1991) Genes Dev. 5: 1032-1041),

Commercially available homing endonucleases such as I-Ceul, I-Scel, I-Ppol, PI-PspI or Pl-Scel are very particularly preferred. Most preferred are I-Scel and I-Ppol. While the gene coding for I-Ppol can be used in its natural form, the gene coding for I-Scel has an editing point. Since the corresponding editing is not carried out in the plastids of higher plants, in contrast to the mitochondria of yeast, an artificial sequence coding for the I-Scel protein must be used for the heterologous expression of this enzyme (US Pat. No. 5,866,361). In addition to the homing endonucleases listed, there are other intron-encoded enzymes that can be found at homologous sites in the genomes of related organisms. These homing endonucleases generally have a similar sequence specificity and are therefore also suitable as a DSBI enzyme for the introduction of a DSB into the piastome at the DSB recognition sequence. In addition to I-Crel, the corresponding sequence can also recognize Sob2593c, Clu2593c, Col2593c, Ciy2593c, Hla2593c, Cag2593c, I-Cvul, I-PakI, Tmu2593c, Msp2593c, I-Msol, Sdu2593c, Mvi2593m, Nol2593m or Aca25. In addition to I-Ceul, corresponding sequences also recognize I-Cecl, I-Cmol, I-Cell, I-CpaIII, I-Cmul, I-Clul, I-SobI or I-Astl. In addition to I-Cpal, a corresponding sequence can also be recognized by Cbrl931c, Cfrl931c, Cmel931c, Cgel931c, Pcrl931c, Mspl931c, Msol931c, Ptul931c, Cvul931m, Mspl931m, Msol931m, Noll931m, Acal931bm or Sn. In addition, there are introns inserted at position 1951 of the 23S rDNA (numbering refers to the homologous position in the 23S rDNA of E. coli). These introns also code for putative homing endonucleases, which can be used as DSBI enzymes for the specific cutting of plastid DNA. These include, for example, Cbrl951c,

Mspl951c, Msol951c, Cvul951m or Acal951m (Lucas P et al. (2001) Nucl Acids Res 29: 960-969).

Most preferred are the homing endonucleases according to the protein sequences described by SEQ ID NO: 5, 12 or 14. When producing corresponding expression cassettes, therefore, nucleic acid sequences are used which code for a protein according to SEQ ID NO: 5, 12 or 14, particularly preferred is the use of the nucleic acid sequences according to SEQ ID NO: 11 or 13 or an expression cassette according to SEQ ID NO: 4.

The enzymes can be purified from their organisms of origin in the manner familiar to the person skilled in the art and / or the nucleic acid sequence coding for them can be cloned. The sequences of various enzymes are stored in the GenBank (see above).

Examples of artificial DSBI enzymes are chimeric nucleases which are composed of an unspecific nuclease domain and a sequence-specific DNA binding domain (eg consisting of zinc fingers) (Smith J et al. (2000) Nucl Acids Res 28 (17): 3361- 3369; Bibikova M et al. (2001) Mol Cell Biol. 21: 289-297). For example, the catalytic domain of the restriction endonuclease FokI was fused to zinc finger binding domains, which defined the specificity of the endonuclease (Chandrasegaran S & Smith J (1999) Biol Chem 380: 841-848; Kim YG & Chandrasegaran S (1994) Proc Natl Acad Sei USA 91: 883-887; Kim YG et al. (1996) Proc Natl Acad Sei USA 93: 1156-1160). The catalytic domain of the ho endonuclease from yeast could also be given a predefined specificity using the technique described, by fusing it to the zinc finger domain of transcription factors (Nahon E & Raveh D (1998) Nucl Acids Res 26: 1233-1239).

As mentioned, zinc finger proteins are particularly suitable as DNA binding domains in the context of chimeric nucleases. These DNA-binding zinc finger domains can be adapted to any DNA sequence. Appropriate methods for producing corresponding zinc finger domains are described and known to the person skilled in the art (Beerli RR et al. (2000) Proc Natl Acad Sei USA 97 (4): 1495-1500; Beerli RR et al. (2000) J Biol Chem 275 (42) : 32617-32627; Segal DJ and Barbas CF 3rd. (2000) Curr Opin Chem Biol 4 (1): 34-39; Kang JS and Kim JS (2000) J Biol Chem 275 (12): 8742-8748; Beerli RR et al. (1998) Proc Natl Acad Sei USA 95 (25): 14628-14633; Kim JS et al. (1997) Proc Natl Acad Sei USA 94 (8): 3616-3620; Klug A (1999) J Mol Biol 293 (2): 215-218; Tsai SY et al. (1998) Adv Drug Deliv Rev 30 (1-3): 23-31; Mapp AK et al. (2000) Proc Natl Acad Sei USA 97 (8): 3930-3935; Sharrocks AD et al. (1997) Int J Biochem Cell Biol 29 (12): 1371-1387; Zhang L et al. (2000) J Biol Chem 275 (43): 33850-33860). Methods for the production and selection of zinc finger DNA binding domains with high sequence specificity are described (WO 96/06166, WO 98/53059, WO 98/53057). By fusing a DNA binding domain obtained in this way to the catalytic domain of an endonuclease (such as, for example, the FokI or Ho endonuclease), it is possible to produce chimeric nucleases of any specificity which can advantageously be used as DSBI enzymes in the context of the present invention.

Artificial DSBI enzymes with changed sequence specificity can also be generated by mutagenesis of known restriction endonucleases or homing endonucleases by methods familiar to the person skilled in the art. In addition to the mutagenesis of homing endonucleases with the aim of achieving an altered substrate specificity, the mutagenesis of maturases is of particular interest. Maturases often have a lot in common with homing endonucleases and can be converted into nucleases if necessary by a few mutations. This was shown for example for the Maturase in the bi2 intron of the baker's yeast. Only two mutations in the Maturase-encoding open reading frame (ORF) were sufficient to confer homing endonuclease activity on this enzyme (Szczepanek & Lazowska (1996) EMBO J 15: 3758-3767). Further artificial nucleases can be generated with the help of mobile group II introns and the proteins encoded by them or parts of these proteins. Many mobile Group II introns form RNA-protein particles with the proteins they encode, which can recognize and cut sequence-specific DNA. The sequence specificity can be adapted to the needs by mutagenesis of certain areas of the intron (see below) (WO 97/10362).

Various methods are known to the person skilled in the art to introduce a DSBI enzyme into plastids or to express them there. Examples include, but are not limited to:

a) Nuclear expression using plastid transit peptides

An expression cassette coding for a DSBI enzyme fusion protein can be constructed in the manner known to the person skilled in the art, inserted into the cell nucleus and - optionally - stably integrated into the chromosomal DNA. For transport into the plastids, the DSBI enzyme is preferably expressed in fusion with a plastid localization sequence (PLS). Methods of specifically transporting proteins that are not per se located in the plastids into the plastids as well as various PLS sequences are described (Klosgen RB and Weil JH (1991) Mol Gen Genet 225 (2): 297-304; Van Breuse- gem F et al. (1998) Plant Mol Biol 38 (3): 491-496). PLS are preferred which, after translocation of the DSBI enzyme into the plastids, are cleaved enzymatically from the DSBI enzyme part. Particularly preferred is the PLS, which is derived from the plastid Nicotiana tabacum transketolase or another transit peptide (e.g. the transit peptide of the small subunit of Rubisco or the ferredoxin NADP oxidoreductase as well as the isopentenyl pyrophosphate isomerase-2) or its functional equivalent. For the nuclear

In principle, all promoters which allow expression in plants are suitable for expression. Examples are given below. Constitutive promoters such as the 35S promoter of the CaMV or the nitrilase-1 promoter of the nitl gene from Arabidopsis (GenBank Acc.-No .: Y07648.2, nucleotides 2456 to 4340; Hillebrand H et al. (1998) Plant Mol Biol 36 (l): 89-99; Hillebrand H et al. (1996) Gene 170 (2): 197-200). Preferred PLS sequences are:

i) the transit peptide of isopentenyl isomerase (IPP) from Arabidopsis (GenBank Acc.-No .: NC_003074; nucleotides 604657 - 604486)

ii) Transit peptides derived from the small subunit (SSU) of ribulose bisphosphate carboxylase (Rubisco ssu) from, for example, pea, corn, sunflower or Arabidopsis.

Arabidopsis thaliana: GenBank Acc.-No .: e.g. AY054581, AY054552;

- Pea, GenBank Acc. -No. : e.g. X00806, nucleotides 1086 to 1256; X04334, X04333 (Hand JM (1989) EMBO J 8 (11): 3195-206). Particularly preferred here: expression cassette and transit peptide (pea, rbcS3A) from vector pJITH7 (Guerineau F (1988) Nucleic Acids Res 16 (23): 11380. The peptide sequence according to SEQ ID NO: 35 is particularly preferred. Most preferred for the The nucleic acid sequence according to SEQ ID NO: 34 is used for the construction of corresponding expression constructs.

Maize, GenBank Acc.-No. : e.g. S42568, S42508

Sunflower, GenBank Acc.-No .: Y00431, nucleotides 301 to 465.

iii) Transit peptides derived from genes of vegetable fat biosynthesis such as the transit peptide of the plastid "acyl carrier protein" (ACP) (eg the Arabidopsis thaliana beta-ketoacyl-ACP synthetase 2; GenBank Acc-No .: AF318307), the stearyl-ACP desaturase , β-ketoacyl-ACP synthase or the acyl-ACP-thioesterase (eg A. thaliana mRNA for acyl- (acyl carrier protein) thioesterase: GenBank Acc. -No.: Z36911).

iv) the transit peptide of GBSSI ("Starch Granule Bound Synthase I")

v) the transit peptide of the LHCP II genes.

The PLS of the plastid transketolase from tobacco (SEQ ID NO: 36) is particularly preferred. In the context of this invention, expression of appropriate fusion proteins can be different PLS nucleic acid cassettes are used in the three reading frames as Kpnl / BamHI fragment (the translation start (ATG codon) is located in the Ncol interface) (pTP09 SEQ ID NO: 37; pTPlO SEQ ID NO: 38; pTPll SEQ ID NO: 39).

Another example of a PLS that can be used advantageously is the transit peptide of the plastidic isopentenyl pyrophosphate isomerase-2 (IPP-2) from Arabidopsis thaliana (SEQ ID NO: 40). The nucleic acid sequences coding for three cassettes (corresponding to the three reading frames) of the PLS of the isopentenyl pyrophosphate isomerase-2 (IPP-2) from Arabidopsis thaliana (EcoRV / Sall cassettes with ATG; IPP-9 SEQ ID NO: 41) can be used very particularly preferably ; IPP-10 SEQ ID NO: 42; IPP-11 SEQ ID NO: 43).

The nucleic acids according to the invention can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural nucleic acid constituents and consist of different heterologous gene segments from different organisms.

The sequence coding for the transit peptide can comprise all or part of the peptide sequence of the original protein. An exact determination of the essential for the transport

Amino acid residues are not required as long as the functionality of the PLS - namely the transport into the plastids - is guaranteed and the function of the DSBI enzyme is not completely destroyed. The following PLS sequences are very particularly preferred:

PLS1: N-MASSSSLTLSQAILSRSVPRHGSASSSQLSPSSLTFSGLKSNPNITTSRRR TPSSAAAAAWRSPAIRASAATETIEKTETAGS-C (SEQ ID NO: 36). Corresponds to the PLS of plastid transketolase from tobacco.

PLS2: N-MSASSLFNLPLIRLRSLALSSSFSSFRFAHRPLSSISPRKLPNFRAFSGTA MTDTKDGSRVDM-C (SEQ ID NO: 40). Corresponds to the PLS of the isopentenyl pyrophosphate isomerase-2 (IPP-2), the last methionine prefers the start

Methionine of the DSBI enzyme is.

The homing endonuclease according to SEQ ID NO: 69 represents a preferred fusion protein from the native I-Ppo I nuclease and that of the IPP plastid localization sequence. Preferred this protein is encoded by the sequence with SEQ ID NO: 68.

In the context of this invention, fusion proteins from PLS and DSBI enzyme are subsumed under the term DSBI enzyme. If a DSBI enzyme is expressed nuclear, the DSBI enzyme is preferably understood to mean a fusion protein composed of PLS and DSBI enzyme. Another object of the invention relates to fusion proteins from DSBI enzymes with plastid localization sequence (PLS)

Sequences and expression cassettes containing a fusion protein from a plastid localization sequence (PLS) and a DSBI enzyme under the control of a promoter functional in the plant cell nucleus. Appropriate suitable promoters are known to the person skilled in the art and are described further below. The expression cassette can contain further elements, such as transcription terminators and / or selection markers.

Expression in plastids

Expression in plastids can also be carried out by directly introducing an expression cassette for the DSBI enzyme into plastids, possibly integrating them into the plastid DNA and expressing the DSBI enzyme. In a preferred embodiment, this expression cassette is contained in the transformation construct comprising the insertion sequence. On the one hand, specific plastid or chromoplast promoters - as described in detail below - can be used. A targeted plastid expression can also be achieved if, for example, a viral, bacterial or bacteriophage promoter is used, the resulting expression cassette is inserted into the plastid DNA and the expression is then induced by the corresponding viral, bacterial or bacteriophage RNA polymerase. The corresponding RNA polymerase can in turn be introduced into the plastids in various ways - preferably by nuclear transformation as a fusion protein with a PLS. Corresponding methods are described (WO 95/16783, WO 97/06250, US 5,925,806). The introduction into plastids is preferably carried out by means of microinjection and particularly preferably by means of particle bombardment. c) Introduction as RNA

The DSBI enzyme can also be introduced by introducing the mRNA coding for the DSBI enzyme - for example generated in vitro - e.g. can be introduced into plastids via microinjection, particle bombardment (bio- listic processes), polyethylene glycol or liposome-mediated transfection. This embodiment is advantageous since no sequences coding for DSBI enzyme remain in the piastome or genome. The RNA encoding the DSBI enzyme is preferably produced by in vitro transcription in the manner known to the person skilled in the art.

d) Introduction as a protein

The DSBI enzyme can be introduced directly into plastids, for example via microinjection, particle bombardment (biolistic method), polyethylene glycol transfection or liposome-mediated transfection. This embodiment is advantageous since no sequences coding for DSBI enzyme remain in the piastome or genome. A corresponding method is described for example by Segal DJ et al. (1995) Proc Natl Acad Sei USA 92: 806-810.

The DSBI enzyme can be used as a fusion protein with the VirE2 or

VirF protein of an agrobacterium and a PLS can be introduced into plant cells. Corresponding methods are described, for example, for Cre recombinase (Vergunst AC et al. (2000) Science 290: 979-982). This embodiment is advantageous since no sequences coding for DSBI enzyme remain in the genome.

Of course, combinations of the options described above are also conceivable.

The expression cassette for the DSBI enzyme is preferably contained on the insertion sequence or the transformation construct.

The DSBI enzyme is preferably introduced or activated simultaneously with or after the insertion sequence has been introduced into the plastids. Expression or activation at the right place and at the right time can be ensured by different approaches: a) Inducible expression

The expression of a DSBI enzyme can be controlled using an inducible promoter, preferably a chemically inducible promoter. For this purpose, for example, the expression cassette coding for the DSBI enzyme can be stably transformed into the plastidic or nuclear DNA of a master plant. If the transformation into the nuclear genome takes place, the subcellular localization must be ensured by suitable PLS transit peptides, as described above. Shortly before or during the transformation with the insertion sequence or the transformation construct, depending on the inducible system selected, the expression of the DSBI enzyme is switched on by application of the corresponding inductor. Various methods or promoters for induced expression are known to the person skilled in the art. Chemical substances or physical stimuli such as increased temperature or wounding etc. can act as a stimulus. Various examples are described below.

b) Inducible activity

The DSBI enzyme can already be present in the plastids of the master plant, if the activity is suitable

Techniques are only induced at the selected point in time, for example by adding chemical substances. Appropriate methods have been described for sequence-specific recombinases (Angrand PO et al. (1998) Nucl Acids Res 26 (13): 3263-3269; Logie C and Stewart AF (1995) Proc Natl Acad Sei USA 92 (13): 5940-5944; Imai T et al. (2001) Proc Natl Acad Sei USA 98 (1): 224-228). In these methods, fusion proteins from the DSBI enzyme and the ligand binding domain of a steroid hormone receptor (e.g. the human androgen receptor, or mutated variants of the human estrogen receptor as described there) are used. Induction can be carried out using ligands such as estradiol, dexamethasone, 4-hydroxytamoxifene or raloxifene.

Some DSBI enzymes are active as dimers (homo- or heterodimers) (I-Crel forms a homodimer; I-Ppol forms homodimer, Flick KE et al. (1998) Nature 394: 96-101). In general, enzymes of the LAGLIDADG family tend to form homodimers if they contain only one LAGLIDADG motif per monomer (Jurica MS & Stoddard BL (1999) Cell Mol Life Sei 55: 1304-1326; I-Ceul may be mentioned as an example). Dimerization can be designed to be inducible, for example by using natural ones Dimerization domains are exchanged for the binding domain of a low molecular weight ligand. The addition of a dimeric ligand then causes the fusion protein to dimerize. Corresponding inducible dimerization processes and the preparation of the dimeric ligands have been described (Amara JF et al. (1997) Proc Natl Acad Sei USA 94 (20): 10618-1623; Muthuswamy SK et al. (1999) Mol Cell Biol 19 (10) : 6845-6857; Schultz LW and Clardy J (1998) Bioorg Med Chem Lett 8 (l): 1-6; Keenan T et al. (1998) Bioorg Med Chem. 6 (8): 1309-1335).

c) Cotransfection

The expression cassette coding for the DSBI enzyme is preferably simultaneously with the insertion sequence into the

Plastids introduced. The expression cassette for the DSBI enzyme and the insertion sequence can be present on one DNA molecule or on two separate ones. The two sequences are preferably present together on a DNA molecule, so that the expression cassette is contained in the transformation construct comprising the insertion sequence.

In a particularly preferred embodiment, after homoplastomic plants have been regenerated, the sequence coding for the DSBI enzyme is removed from the genome of the transformed plastids again. Various methods are known to the person skilled in the art for this purpose, which are described in detail below.

Some of the DSBI enzymes described above (in particular homing endonucleases) can have E. coli recognition sequences in the preferred intermediate host. In addition, since some expression cassettes for expression in plastids are also functional in E. coli, the expression of the DSBI enzyme in E. coli is preferably prevented in various ways known to the person skilled in the art in order to have any adverse effects on E. coli during the aplification of the expression cassette avoid. For example, several consecutive codons rare for E. coli (eg the codons AGA and AGG coding for arginine) can be inserted into the reading frame of the DSBI enzyme. This prevents expression in E. coli, but still allows expression in the plastids due to the different codon usage. Alternatively, promoters can be used which are not active in E. coli but are active in the plastids of higher plants (eg promoters of the nuclear-encoded plastid RNA polymerases [NEP promoters; see below]). A preferred method is the use of promoters that are neither of the plastids nor are recognized by E. coli (eg viral or bacteriophage promoters), which only become functional when the corresponding viral / bacteriophage RNA polymerase is present at the same time. Corresponding methods are known to the person skilled in the art and are described below. It would also be conceivable to destroy the corresponding DSB recognition sequences in E. coli or to use another host which does not have any DSB recognition sequences for the respective DSBI enzyme. Furthermore, it is conceivable and advantageous to have the coding region of the DSBI enzyme for the amplification in E. coli without a promoter. In this case, the sequence coding for the DSBI enzyme is preferably contained on a plasmid which can integrate into the plastid genome of the plant to be transformed. The integration site can be chosen in such a way that the gene coding for the DSBI enzyme comes under the control of a naturally or artificially inserted promoter present in the piastome and therefore the expression of the DSBI enzyme occurs in the plastids. In a further preferred embodiment, it is ensured that the gene coding for the DSBI enzyme can be deleted from the piastome again (see below). In addition, it is possible to establish a link between a promoter and a DSBI enzyme by adding such an in vitro upstream of the open reading frame by means of PCR techniques known to the person skilled in the art. The PCR product can then be used to introduce it into the plant plastids. In addition, non-functional parts of an expression cassette for a DSBI enzyme can be created and amplified in E. coli if they recombine with one another after being introduced into plant plastids (for example by homologous recombination in overlapping regions of the non-functional parts of the expression cassette). and so result in a functional expression cassette.

"Recognition sequence for the targeted induction of DNA double-strand breaks" (as a result of "DSB recognition sequence" for double-strand break recognition sequence) generally means those sequences which, under the conditions in the plastids of the plant cell or plant used in each case, the detection and cleavage by a DSBI enzyme allow. DSB recognition sequences for homing endonucleases which are naturally encoded in mitochondria or nucleus of other organisms are particularly preferred. DSB recognition sequences of the homing endonucleases derived from plastids (for example from green algae) can also be used. The DSB recognition sequence is preferably singular in the plastid DNA, ie a double-strand break is only generated at the point predefined in this way. However, cases are also conceivable in which there is more than one DSB recognition sequence in the piastom. This is particularly the case if the DSB- Recognition sequence is localized in duplicated genes (eg in inverted "repeats"). In the latter case, there is more than one identical DSB recognition sequence, but their context is identical, so that a targeted insertion also takes place here. It is even preferred that the integration be made in all copies so that a cut in all copies is also required. DSB recognition sequences that occur more than once in a piastom but are localized in the same plastomic context (eg in "repeats" or gene duplications) are subsumed within the scope of this invention under the term singular DSB recognition sequences.

Preferably, the plant used or the cell derived from it is predominantly homoplastomic or homotransplastomic with respect to the DSB recognition sequence, i.e. that the majority of the plastid DNA molecules contained in a plastid have this DSB recognition sequence. Such plants are also referred to as master plants in the context of this invention.

In principle, two types of DSB recognition sequences can be used:

a) Natural, endogenous DSB recognition sequences

As could be shown in the context of this invention, the piastomes of higher plants contain different sequences which can act as recognition sequences for DSBI enzymes (for example homing endonucleases), even if no such endonucleases could be detected for higher plants. Such DSB recognition sequences can be identified by screening the plastid DNA sequence using the known DSB recognition sequences (for example those described in Table 2). The plastid genome of various plants is known (http: //megasun.bch.umontreal. Ca / ogmp / projects / other / cp_list.html). The sequences of the piastomes are reported by

Arabidopsis thaliana (Sato S et al. (1999) DNA Res. 6 (5): 283-290) (GenBank Acc. -No.: AP000423; NCBI Acc.-No. NC_000932)

Epifagus virginiana (Beechdrops; Wolfe KH et al. (1992) J Mol Evol 35 (4): 304-317; NCBI Acc.-No .: NC_001568; GenBank Acc.-No.: M81884) Lotus japonicus (Kato T et al. (2000) DNA Res 7 (6): 323-330; NCBI Acc.-No .: NC_002694; GenBank Acc. -No.: AP002983)

Oryza sativa (rice) (Hiratsuka J et al. (1989) Mol Gen Genet 217 (2-3): 185-194; NCBI Acc.-No .: NC_001320; GenBank Acc.-No: X15901),

Marchantia polymorpha (Liverwort; Ohyama K et al. (1988) J Mol Biol 203 (2): 281-298; Yamano Y et al. (1984) Nucl Acids Res 12 (11): 4621-4624; GenBank Acc.-No .: X04465 and

Y00686; NCBI Acc.-No .: NC_001319)

Nicotiana tabacum (tobacco) (GenBank Acc.-No .: Z00044 and S54304; NCBI Acc.-No .: NC_001879; Shinozaki K et al. (1986) EMBO J 5: 2043-2049)

- Oenothera elata ssp. hookeri (Monterey evening primrose; GenBank Acc.-No .: AJ271079; NCBI Acc.-No .: NC_002693; Hupfer H et al. (2000) Mol Gen Genet 263 (4): 581-585)

Medicago truncatula (Gen Bank Acc.-No .: AC093544)

Pinus thunbergii (black pine; Tsudzuki J et al. (1994) Curr Genet 26 (2): 153-158; NCBI Acc.-No.: NC_001631; GenBank Acc.-No.: D17510)

Spinacia oleracea (GenBank Acc.-No .: AJ400848 J01442 M12028 M16873 M16878 M27308 M55297 X00795 X00797 X01724 X04131 X04185 X05916 X06871)

Triticum aestivum (wheat; GenBank Acc.-No .: AB042240; NCBI Acc.-No .: NC_002762) and

Zea mays (GenBank Acc.-No .: X86563; NCBI Acc.-No .: NC_001666)

In addition, other piastomes can be sequenced to identify DSB recognition sites there as well. It is generally sufficient to isolate highly conserved regions from the piastome by means of PCR methods familiar to the person skilled in the art and only to sequence them.

Furthermore, natural, endogenous DSB recognition sites can be determined experimentally, for example by isolating the plastid DNA (e.g. according to Mariac P et al. (2000)

BioTechniques 28: 110-113), which amplified fragments of the plastid genome to be examined by means of PCR or synthetic Uses fragments and performs a restriction analysis with the respective DSBI enzyme. This is preferably carried out under conditions which are present in the plastid of a higher plant.

The endogenous DSB recognition sequences for natural homing endonucleases identified in the context of this invention and described in Table 1 are furthermore in the conserved regions of the piastome, so that - in particular taking into account the given variability of the corresponding homing endonucleases with regard to their respective recognition sequences - It can be assumed that these recognition sequences occur almost universally in all plastomes of higher plants. Behind the positions given in Table 1 is the sequence and the reverse complement, since all recognition regions shown in Table 1 are located in the "inverted repeats" of the plastid genome. Of the homing endonucleases listed in Table 1, I-Cpal, I-Ceul, I-Chul, I-CpaII and I-Crel are particularly preferred.

The recognition sequences identified in this way can be used for the insertion of foreign DNA by generating a double-strand break by introducing the corresponding DSBI enzyme. If the DSB recognition sequence is located in a highly conserved region within a gene of the organelle genome, the foreign DNA is preferably inserted in the form of a self-splicing intron, which enables the mRNA of the affected gene to be reconstituted (see below).

Methods are also known to the person skilled in the art according to which any endogenous sequence can act as a recognition sequence for chimeric, mutated or artificial endonucleases by their DNA recognition region - for example by modifying a zinc finger domain fused to an endonuclease domain or modifying the RNA Sequence of a group II intron-RNA-protein complexes - is specifically changed (see; WO 96/06166, Bibikova M et al. (2001) Mol Cell Biol 21: 289-297).

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In addition, there are unique interfaces of restriction endonucleases in the plastid genome. However, these are usually located in less highly conserved regions and are therefore not necessarily universally usable in all plant species. Examples include:

a) The enzyme Sfil has a unique recognition site in the Arabidopsis plastid genome (GenBank Acc.-No .: AP000423) at position 40846-40858 with the sequence GGCCTTTATGGCC.

b) In the plastid genome of maize (GenBank Acc.-No .: X86563) there is a unique interface for the enzyme Ascl at position 42130-42137 with the sequence GGCGCGCC.

c) In the plastid genome of rice (GenBank Acc.-No .: X159019) there is a unique interface for the enzyme Sgfl at position 77309-77316 with the sequence GCGATCGC and for the enzyme Ascl at position 39776-39783 with the sequence GGCGCGCC ,

d) In the plastid plastid genome (Accession Z00044) there is a unique interface for the enzyme Sfil at position 42475-42487 with the sequence GGCCTTTATGGCC, for the enzyme Sgrl at position 78522-78529 with the sequence CACCGGCG, and for the enzyme Pmel at position 120895-120902 with the sequence GTTTAAAC.

e) In the plastid genome of wheat (Accession AB042240) there is a unique interface for the enzyme Pmel at the

Position 59331-59338 with the sequence GTTTAAAC, for the enzymes Narl, KanI, Ehel and Bbel a unique interface at position 41438-41443 with the recognition sequence GGCGCC, and for the enzyme Sfil a recognition region at position 112656-112668 with the sequence

GGCCCAGGGGGCC.

All of these plants with endogenous, natural DSB recognition sequences represent quasi naturally occurring "master plants". The DSB recognition sequence is naturally homoplastic in them. This eliminates the need to introduce and select artificial DSB recognition sequences. Artificially introduced DSB recognition sequences

The person skilled in the art is aware that the recognition region for a rare cutting enzyme does not have to be natural in a master plant. In principle, any recognition sequence of any DSBI enzyme can be inserted at any location on the plastid DNA. The preparation is preferably carried out using a construct for inserting the DSB recognition sequence (as a result of the DSBE construct). The DSBE construct preferably comprises a selection marker in order to facilitate the selection of transplastomeric plants with the successfully inserted DSB recognition sequence, which is required to generate corresponding master plants. Various selection markers are known to the person skilled in the art which enable a selection of plastids (see below). Preferred are aadA, nptll or BADH, with aadA being particularly preferred. The selection takes place, for example, with the aid of the "segregation and sorting" process known to the person skilled in the art (described by way of example in Example 4). The selection marker is preferably constructed so that a subsequent deletion from the piastome is made possible. Corresponding methods are known to the person skilled in the art and are described below.

A plant homotransplastome which is homotransplastome with respect to the inserted DSB recognition sequence and which has a DSB recognition sequence in all or the predominant number of the plastids of the plant under consideration is thus preferably first produced. Such plants can advantageously be used as master plants.

In addition to the selection marker, the DSBE construct can contain further sequences. These can contain, for example, further regulatory elements for the expression of the insertion sequences to be introduced as a result. In a preferred embodiment, the selection marker introduced in the context of the construct for inserting the DSB recognition sequence is deleted after the homoplastic master plant has been obtained by methods known to the person skilled in the art (see below).

In a preferred embodiment, the DSBE construct comprises, in order to enable a site-specific insertion on at least one, preferably on both sides of the DSB recognition sequence, further flanking sequences which are of sufficient length and homology to corresponding target sequences in the piastome in order to use a site-specific insertion to ensure homologous recombination. Due to the large number of DSBI enzymes described in the prior art with defined recognition sequences, it is possible and preferred to produce master plants which have incorporated several different singular DSB recognition sequences into their plastid genome.

As an example, but not by way of limitation, the recognition sequences for the respective DSBI enzymes listed are mentioned in Table 2 below.

Table 2: Detection sequences and organism of origin of DSBI enzymes (" ^Λ " indicates the interface of the DSBI enzyme within a detection sequence.)

Deviations (degenerations) of the recognition sequence are also included, which nevertheless enable recognition and cleavage by the respective DSBI enzyme. Such deviations - also in connection with different framework conditions such as calcium or magnesium concentration - have been described (Argast GM et al. (1998) J Mol Biol 280: 345-353). Core sequences of these recognition sequences are also included. It is known that the inner parts of the recognition sequences are also sufficient for an induced double-strand break and that the outer parts are not necessarily relevant, but can help to determine the efficiency of the cleavage. For example, an 18bp "core" sequence can be defined for I-Scel. In this respect, the term DSB recognition sequence also encompasses all essentially identical recognition sequences. Essentially identical recognition sequences mean those recognition sequences which, although they deviate from the recognition sequence found to be optimal for the particular enzyme, still allow cleavage by the same.

Different locations of the localization (in the case of already existing endogenous DSB recognition sequences) or integration (in the case of artificially generated DSB recognition sequences) are possible for the DSB recognition sequence. Examples include: a) Localization (integration) in a transcriptionally silent region

Localization (integration) of the DSB recognition sequence in a transcriptionally silent region of the plastid genome (intergenic region) is the preferred embodiment. A disruption of the plastid functions can be largely excluded. It should be noted here that appropriate regulatory elements such as promoters etc. may have to be incorporated for expression.

b) Localization (integration) in a transcriptionally active, but non-coding (intercistronic) region

This localization (integration) has the advantage that the insertion sequence to be introduced is ultimately encoded in a plastid operon and the promoter (s) or terminator (s) do not have to be introduced separately, but the endogenous ones present at this locus can be used ( but do not have to). In such a case, only ribosome binding sites should be present at a suitable distance upstream of the coding region of the foreign genes to be introduced.

However, it is also conceivable that an intergenic region is not completely transcriptionally silent because, for example, the transcription of an adjacent gene or operon is terminated only inefficiently.

c) Localization (integration) in a transcriptionally active coding region.

The localization (integration) of the DSB recognition sequence at a non-coding locus described under a) and b) has the advantage that the insertion of the foreign DNA has a high probability of not influencing the function of the plastid genome. However, non-coding areas are less conserved than coding areas. In order to have a method which is as universal as possible and which works in many plant species, in a particularly preferred embodiment the DSB recognition sequence (and consequently the insertion sequence) is localized in the coding sequence of an existing gene. The destruction of the gene function by the introduction of the DSB recognition sequence (in the case of an artificially generated DSB recognition sequence) or the introduction of the insertion sequence is prevented in an inventive manner in that the DSB recognition sequence or the insertion sequence in one preferred variant of this embodiment is introduced i frame of an intron. In this way, the complete, coding mRNA is generated again by splicing the pre-RNA of the gene at the integration site.

DSB recognition sequences not naturally occurring in the plastid DNA can be introduced into the plastid DNA in various ways. Examples include:

a) Integration using a double crossover

The integration into the plastid genome is preferably carried out with the aid of the above-described methods (double crossover) which are generally known to the person skilled in the art.

b) Integration using natural, endogenous DSB recognition sequences

c) Integration using recombinases and corresponding recognition sequences.

Even if the effort for inserting an artificial DSB recognition sequence into the plastid DNA is relatively high and in the case of a) corresponds to that for the plastid transformation process currently described in the prior art, this effort only has to be carried out once. The homotransplastome master plant obtained can then be used for any number of different subsequent transformations using the method according to the invention, which enables a considerable increase in the transformation efficiency: instead of having to go through the conventional selection process for a homotransplastome plant every time, it only has to be done once here will be realized.

"Deactivating the functionality" of a DSB recognition sequence means that by inserting the insertion sequence at or near the position of the double strand break, the DSB recognition sequence is destroyed, that is to say that the corresponding DSBI enzyme no longer recognizes the region and accordingly no longer has a double strand break there induced.

Construction of the transformation construct with the insertion sequence

Using one of the master plants described above or cells derived therefrom, which contain a natural and / or an artificially generated DSB recognition sequence in the piastome, the insertion sequence is converted into said DSB recognition sequence inserted as part of a transformation. This happens with the simultaneous presence of a DSBI enzyme, which recognizes one of the DSB recognition sequences in the piastom.

In its simplest form, the transformation construct consists solely of the insertion sequence itself, for example an expression cassette, which is intended to ensure the expression of a specific gene in the plastids. The sequence-specific induction of double-strand breaks is sufficient to ensure the placement of this insertion sequence at this position and thus to deactivate the DSB recognition sequence.

In a preferred embodiment, the insertion sequence comprises at least one nucleic acid sequence to be expressed. In order to ensure expression (transcription and / or translation), depending on the embodiment and the place of insertion, these are to be provided with regulatory elements. If the insertion takes place in a transcriptionally active locus, no promoter sequences are required, as described above. In any case, the sequences to be expressed are advantageously provided with ribosome binding sites at a suitable distance upstream of the open reading frame or already have such inherently. However, these regulatory sequences or parts thereof can naturally also be present in the piastome or already in the first step, i.e. when generating a non-natural master plant together with that of the DSB recognition sequence are introduced into the plastid DNA.

An increase in the insertion efficiency and accuracy can be brought about by the fact that the insertion sequence contained in the transformation construct and the DSB recognition sequence are flanked by homologous sequence regions which ensure homologous recombination as a result of the induced double-strand break. In a preferred embodiment, the insertion sequence comprises flanking homology sequences A 'or B', the sequence to be inserted into the plastid DNA lying between A 'and B'. The DSB recognition sequence is flanked by homology sequences A and B, the DSB recognition sequence being between A and B. A and B may be of natural origin, or may have been introduced as part of the insertion of non-natural DSB recognition sequences. A and A 'or B and B' are of sufficient length and sufficient homology to one another to ensure homologous recombination between A and A 'or B and B'. In a further embodiment, the DSB recognition sequence is flanked only by a homology sequence A which has sufficient homology to a sequence A 'flanking the insertion sequence on one side.

With regard to the homology sequences, “sufficient length” preferably means sequences of a length of at least 20 base pairs, preferably at least 50 base pairs, particularly preferably of at least 100 base pairs, very particularly preferably of at least 250 base pairs, most preferably of at least 500 base pairs.

"Adequate homology" in relation to the homology sequences A and A 'or B and B' preferably means sequences which have a homology within these homology sequences of at least 70%, preferably 80%, preferably at least 90%, particularly preferably at least 95% particularly preferably at least 99%, most preferably 100% over a length of at least 20 base pairs, preferably at least 50 base pairs, particularly preferably of at least 100 base pairs, very particularly preferably of at least 250 base pairs, most preferably of at least 500 base pairs.

Homology between two nucleic acids is understood to mean the identity of the nucleic acid sequence over the entire entire length of the sequence, which can be determined by comparison using the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA) with the following parameters is calculated:

Gap Weight: 12 Length Weight: 4

Average Match: 2,912 Average Mismatch: -2, 003

Since the homologous recombination is promoted by the induced double-strand break, the requirements for the length and homology of the sequences are significantly lower than, for example, in the case of a conventional homologous recombination. The homologous areas can also be significantly smaller than 250 bp. The use of homology sequences has the advantage that if A 'and B' are different, or only one homology sequence A 'is used, the insertion sequence can be inserted into the plastid DNA in a directed manner.

The transformation construct preferably comprises or

Insertion sequence a selection marker that enables a selection of transplastomer plastids (see below), particularly preferred aadA, BADH or a "binding type" marker. The selection marker is preferably constructed so that a subsequent deletion from the piastome is made possible. Corresponding methods are known to the person skilled in the art and are described below.

The insertion sequence or the transformation construct preferably has the structure and sequence of an intron. As a rule, naturally occurring introns are modified so that they meet the requirements of the method according to the invention. Such artificial introns are particularly preferred if the insertion into a transcriptionally active or even coding region is to take place, for example using a natural, endogenous DSB recognition sequence. The insertion is preferably carried out in such a way that the inserted sequence is completely removed by splicing the pre-mRNA. The spliced RNA (i.e. the artificial intron) then represents the mRNA, for example for translation on its encoded proteins. This method has further advantages:

- The introns used have a pronounced secondary folding, so that a relatively stable RNA results. As a result, the genes of interest encoded in the intron can be expressed particularly highly, as has been demonstrated, for example, in E. coli (Chan KYW et al. (1988) Gene 73: 295-304).

- If the intron is integrated into a gene, the transcription of the intron is subject to the regulatory control of the gene into which the intron has been integrated. Therefore, all regulatory elements upstream or downstream of the genes or of the gene of interest in the intron can be saved. This allows the constructs to be kept correspondingly small and it is certain that a transcription actually works in the species under consideration. The use of heterologous regulatory elements entails a residual risk that they are not functional in the plastids of the plant species in question. Due to the sequence duplication, the use of homologous sequences can lead to spontaneous recombination events with the endogenous sequences and thus to instability of the organelle genome. Due to the possibility of being able to largely dispense with the introduction of regulatory elements - for example by coding the gene of interest in an intron, which is inserted into a transcriptionally active region of the piastome, in this embodiment the method according to the invention can additionally increase the insertion and spreadability of the Many other disadvantages of conventional plastid transformation can be avoided.

In addition, all introns can be used if the corresponding factors that mediate the splicing are simultaneously expressed in the plastids or imported into them. The factors supporting the splicing are preferably coded in the intron itself. In this embodiment, group II introns are particularly preferred which themselves encode at least one of the factors necessary for the splicing. This includes the Ll.ltrB intron from Lactococcus. Also preferred are introns that occur naturally in plastids of higher plants, especially introns of group II, very particularly preferably introns that code for a protein, most preferably introns of the trnK genes of the plastid genome. In the latter case, the introns from the trnK genes of the plastids from the Arabidopsis, maize and tobacco species are particularly preferred.

Preferred are introns with a self-splicing activity that does not depend on other protein factors, introns that use general factors for splicing that are universal and therefore also present in plastids, and introns that code themselves for factors that are used for splicing are necessary .. These introns include, for example

a) the intron of Group I from Tetahymena (GenBank Acc.-No .: X54512; Kruger K et al. (1982) Cell 31: 147-157; Roman J and Woodson SA (1998) Proc Natl Acad Sei USA 95: 2134 -2139)

b) the group II rll intron from Seenedesmus obliquus (GenBank Acc.-No .: X17375.2 nucleotides 28831 to 29438; Holländer V and Kück U (1999) Nucl Acids Res 27: 2339-2344; Herdenberger F et al. (1994) Nucl Acids Res 22: 2869-2875; Kück U et al. (1990) Nucl Acids Res 18: 2691-2697).

c) the Ll.LtrB intron (GenBank Acc.-No .: U50902 nucleotides 2854 to 5345)

d) the trnK intron from Arabidopsis (GenBank Acc.-No .: AP000423 neucleotides complementary 1752 to 4310)

e) the trnK intron from maize (GenBank Acc.-No .: X86563 nucleotides complementary 1421 to 3909)

f) the trnK intron from tobacco (GenBank Acc.-No .: Z00044 nucleotides complementary 1752 to 4310) Both intrinsic and introns naturally occurring in the plastids of the respective plant can be used. In order to avoid instabilities caused by sequence duplication, introns of a different species - for example trnK introns of a different species - are preferred. In a preferred embodiment, introns naturally occurring in the plastids of the respective plant are modified such that they can still fulfill their function, but the sequence homology is less than 95%, preferably 80%, particularly preferably 70% of the sequence of the starting intron ,

In a further preferred embodiment, a factor which causes the intron under consideration to be spliced is available in trans, i.e. is not encoded in the intron itself. If this factor is not naturally present in the plastid under consideration, but is only introduced into it, this can be done in various ways known to the person skilled in the art. For example, the introduction of a corresponding coding and expressible sequence into the piastome or the introduction into the nuclear DNA may be mentioned, in which case the factor is preferably fused to a PLS.

Introns which naturally encode a DSB enzyme (in particular a homing endonuclease) are particularly preferred. The intron Cp.LSU2 from Chlamydomonas pallidostigmatica, which encodes the enzyme I-Cpal (Turmel M et al. (1995) Mol Biol Evol 12: 533-545), is particularly preferred. Group II introns from the yeast mitochondria are also preferred.

In a preferred embodiment, the intron sequence is adapted to the insertion site so that they can splice at this locus. The adaptation can affect the internal guide sequence (IGS) for group I introns or the exon binding sequence (EBS) I or / and II for group II introns.

In the case of the trnK intron from maize, it should be noted that the protein encoded by the trnK intron, which also includes the Maturase function, is probably not functional in its naturally encoded form without editing. It was shown that the corresponding mRNA is edited (His420Tyr) in barley plastids (Vogel J et al. (1997) J Mol Biol 270: 179-187). Tyrosine at position 420 of the matK protein is highly conserved. A codon coding for His was also found in the coding DNA in the corresponding position in the monocots rice and maize. It can therefore be assumed that - as in barley - the matK transcript is also edited in these plants. However, since other plant species may edit this RNA cannot guarantee, in a preferred embodiment the matK gene in the trnK intron from maize is already modified by an appropriate His / Tyr exchange at the DNA level, so that RNA editing is no longer necessary. For example, the sequence CATTATCATAGTGGAT of the maize trnK intron can be mutated to CATTATTATAGTGGAT.

In the case of group I introns, the splice point is determined by mating the IGS with the exon of the corresponding transcript located 5 'and / or 3' to the intron (Lambowitz AM & Beifort M (1993) Annu Rev Biochem 62: 587-622). Using techniques known to the person skilled in the art, such as PCR or the synthetic generation of nucleotide sequences, the IGS of any group I introns can be adapted accordingly such that splicing takes place at the predefined insertion point within the DSB recognition region. The modified IGS is designed such that it can - at least partially - base pair with the sequences of the transcript 5 'and 3' of the insertion site. The intron CpLSU2 from C. pallidostigmatica, which codes for the homing endonuclease I-Cpal, is preferably used. If this is used in connection with the expression of the DSBI enzyme I-Cpal, which results in an insertion of the DNA to be transformed into the 23SrDNA of the plastid genome of higher plants, no adaptation of the intron is necessary. The insertion takes place at a locus in the plastid genome of higher plants, which is homologous to that where the intron is naturally located in C. pallidostigamtica. This intron is therefore already designed in such a way that pairings with the 5 'and 3' exon can be made and correct splicing takes place in this nucleotide environment. Group I intron from Tetrahymena thermophila is also preferred. There it interrupts the 26S rRNA coding region as a 413 bp "Intervening Sequence" (IVS) (Accession V01416 J01235 nucleotides 53 to 465). The naturally found IGS with the sequence 5'-ggaggg-3 '(Waring RB et al. 1985 Cell 40: 371-380; Been, MD & Cech, TR 1986 Cell 47: 207-216) can be obtained by techniques known to those skilled in the art be adapted to the new insertion point. If, for example, integration into the DSB recognition site of the I-Cpal enzyme at the point marked with ^Λ is desired (cggtcct ^Λ aaggtagcgaaattc), the mutated, adapted IGS can have the following sequence, for example: 5 '-gggacc-3'.

In group II introns that are mobile, other activities in addition to the Maturase are often coded in the protein portion of the ribonucleoprotein complex. However, these are not absolutely necessary for the described method and can therefore be deleted. They are even preferred to be deleted, since this makes the corresponding construct smaller and easier to handle is. Various possibilities are known to the person skilled in the art as to how the corresponding activities can be removed from the protein portion. This can be done, for example, by creating a synthetic gene that only covers the desired areas, or by using suitable PCR methods.

Group II self-splicing introns have a conserved structure and generally consist of 6 different domains. Domain I contains the exon binding sites (EBS1 and EBS2), which interact with the 5 'from during the splicing process

Exon intron located. In addition, there is an interaction between the "5 region" (immediately 5 'of EBS1) and the "δ'region" at the 3 'exon (Lambowitz AM & Beifort M (1993) Annu Rev Biochem 62: 587-622; Michel F & Ferat JL (1995) Annu Rev Biochem 64: 435-461). These sequences can be adapted in each case by techniques known to the person skilled in the art, such as synthetic creation of the introns or suitable PCR methods, in such a way that a correct choice of the splice sites at the insertion location selected in the DSB recognition region is ensured. This is done by modifying the regions mentioned so that base pairings with the corresponding sequences upstream (intron binding sequences, IBS) and downstream (δ ') of the artificial insertion sequence can be entered into. If, for example, cggtcctaaggt ^Λ agcgaaattc is selected as the insertion site ( ^Λ ) for the Ll.LtrB intron in the I-Cpal recognition region, the δ and EBS1 region can, for example, the sequence TCGCTACCTTAG (natural sequence: TTATGGTTGTG), the EBS2, for example, the sequence GACCG (natural Sequence: ATGTG). If one chooses the trnK intron from Arabidopsis thaliana, the δ and EBS1 region can be assumed if the same insertion point as for the

Ll.LtrB intron indicated, for example, assume the sequence CGCTACCTTAGG (natural sequence: AATGTTAAAAA).

If the DSB recognition sequence is a natural, endogenous recognition sequence of a homing endonuclease, a selected intron is preferably inserted at the location of the DSB recognition region at which the intron naturally also belongs to the homing endonuclease under consideration.

The artificial insertion site of an intron in the DSB recognition site is preferably selected such that 5 'and 3' of the inserted intron, as many bases as possible correspond to those at the natural insertion site of the intron under consideration and the DSB recognition sequence is no longer functional after the insertion of the intron , This corresponds very particularly preferably in each case directly upstream or downstream of the Insertion site of the intron located at the natural insertion site.

In a particularly preferred embodiment, the intron is flanked by homology sequences in order to enable a directed insertion. As described above, the homology sequences are homologous to the sequences flanking the DSB recognition sequence and thus enable exact insertion.

Another object of the invention therefore relates to DNA constructs comprising at least one nucleic acid sequence and intron sequence elements which are capable of ensuring the deletion of the ribonucleic acid fragment coding for said nucleic acid sequence in a ribonucleic acid sequence derived from said DNA construct, wherein said Nucleic acid sequence is heterologous with respect to said intron sequence elements.

In a preferred embodiment, the nucleic acid sequence is flanked at least by a splice acceptor sequence and a splice donor sequence.

In a further embodiment, the DNA construct at the 5 'and 3' ends comprises sequences Hl or H2 which are of sufficient length and homology to plastid sequences Hl 'or H2' to enable a homologous recombination between Hl and Hl ' or H2 and H2 'and thus an insertion of the sequence flanked by Hl and H2 into the piastome.

The invention further relates to a transgenic plastid DNA comprising at least one nucleic acid sequence and intron sequence elements which are capable of ensuring in a ribonucleic acid sequence derived from said transgenic plastid DNA the deletion of the ribonucleic acid fragment coding for said nucleic acid sequence, the nucleic acid sequence being said Relative to said intron sequence elements is heterologous. In a preferred embodiment, the nucleic acid sequence is flanked at least by a splice acceptor sequence and a splice donor sequence.

The insertion sequence or the transformation construct can be cloned into a standard vector such as pBluescript or pUClδ to build up a transformation vector. In a preferred embodiment, the insertion sequence or transformation construct is applied as a linear or linearized DNA molecule. Preferably only the part of the transformation vector is applied which comprises the insertion sequence or the transformation construct with possibly homology sequences, selection markers and / or the expression cassette for the DSBI enzyme. If homology sequences are completely or partially dispensed with, the linearized DNA molecule is preferably obtained by digestion with restriction endonucleases which generate single-stranded DNA overhangs at one or both ends which are compatible with those which the DSBI enzyme produces in the plastid DNA.

In a preferred embodiment, the transformation vector can comprise elements (for example a plastid ORI origin of replication, origin of replication) which enable it to replicate autonomously in the plastid before integration into the plastid DNA or to exist stably as an extrachromosomal DNA molecule in the plastids , Corresponding methods are known to the person skilled in the art (US 5,693,507; US 5,932,479; WO 99/10513). This method is preferred because it increases the number of copies of the insertion sequences available for integration in the plastid.

One of the constructs described above can be introduced into the plastids of a corresponding master plant using one of the methods described. Microinjection and particle bombardment are preferred.

Cloning, expression, selection and transformation procedures

"Expression cassette" means - for example in relation to the expression cassette for the DSBI enzyme - such constructions in which the DNA to be expressed is functionally linked to at least one genetic control element that enables or regulates its expression (i.e. transcription and or translation). For example, the expression can be stable or transient, constitutive or inducible. Various direct (e.g. transfection, particle bombardment, microinjection) or indirect methods (e.g. agrobacterial infection, virus infection) listed below are available to the specialist for the introduction, which are listed below.

A functional link is generally understood to mean an arrangement in which a genetic control sequence can perform its function in relation to a nucleic acid sequence - for example coding for a DSBI enzyme. Function can, for example, control the expression, ie transcription and / or translation, of the nucleic acid sequence - for example coding for a DSBI enzyme - mean. Control includes, for example, the initiation, increase, control or suppression of expression, ie transcription and possibly translation. The control, in turn, can take place in a tissue-specific or time-specific manner. It can also be inducible, for example, by certain chemicals, stress, pathogens, etc.

A functional link is understood to mean, for example, the sequential arrangement of a promoter, the nucleic acid sequence to be expressed - for example coding for a DSBI enzyme

- and if necessary. further regulatory elements such as a terminator such that each of the regulatory elements can fulfill its function in the expression of the nucleic acid sequence - for example coding for a DSBI enzyme. It is not absolutely necessary that the functional link is already given on the transformation constructs. The functional linkage can also result from the insertion into the core or plastid DNA, with the regulatory elements already being present in the core or plastid DNA. In this regard, the regulatory elements can be natural or in an upstream step - for example when an artificial DSB recognition sequence is introduced

- be introduced.

This does not necessarily require a direct link in the chemical sense. Genetic control sequences, such as, for example, enhancer sequences, can also perform their function on the target sequence from more distant positions or even from other DNA molecules. Arrangements are preferred in which the nucleic acid sequence to be expressed - for example coding for a DSBI enzyme - is positioned behind a sequence which acts as a promoter, so that both sequences are covalently linked to one another. The distance between the promoter sequence and the nucleic acid sequence - for example coding for a DSBI enzyme - is preferably less than 200 base pairs, particularly preferably less than 100 base pairs, very particularly preferably less than 50 base pairs.

Various ways are known to the person skilled in the art in order to arrive at one of the transformation constructs according to the invention, these vectors or one of the expression cassettes. The production can be carried out using common recombination and cloning techniques, as described, for example, in Maniatis T, Fritsch EF and Sambrook J, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in Silhavy TJ, Berman ML and Enquist LW, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987). The direct fusion of a nucleic acid sequence functioning as a promoter with a nucleotide sequence to be expressed is preferred - for example coding for a DSBI enzyme.

The term "genetic control sequences" is to be understood broadly and means all those sequences which have an influence on the formation or the function of an expression cassette or transformation vector. Genetic control sequences ensure transcription and, if necessary, translation in the cell nucleus (or cytoplasm) or plastids. The expression cassettes according to the invention preferably comprise a promoter 5 'upstream of the respective nucleic acid sequence to be expressed and a terminator sequence 3' downstream as an additional genetic control sequence, and optionally further customary regulatory elements, in each case functionally linked to the nucleic acid sequence to be expressed.

Genetic control sequences are described, for example, in "Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990)" or "Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton , Florida, eds. Glick and Thompson, Chapter 7, 89-108 "and the quotations cited therein.

Examples of such control sequences are sequences to which inducers or repressors bind and thus regulate the expression of the nucleic acid. In addition to these new control sequences or instead of these sequences, the natural regulation of these sequences may still be present before the actual structural genes and may have been genetically modified so that the natural regulation has been switched off and the expression of the genes increased. However, the expression cassette can also have a simpler structure, ie no additional regulation signals are inserted in front of the genes mentioned above and the natural promoter with its regulation is not removed. Instead, the natural control sequence is mutated so that regulation no longer takes place and gene expression is increased. These modified promoters can also be placed in front of the natural genes to increase activity. Depending on the host organism or starting organism described in more detail below, which is converted into a genetically modified or transgenic organism by introducing the expression cassettes or vectors, different control sequences are suitable.

In principle, all promoters which can control the expression of genes, in particular foreign genes, in plants are suitable for nuclear expression (for example a viral / bacteriophage RNA polymerase or a DSBI enzyme with a plastid transit peptide).

Promoters which allow constitutive expression in plants are suitable (Benfey et al. (1989) EMBO J. 8: 2195-2202). In particular, a vegetable one is preferably used

Promoter or a promoter derived from a plant virus. The promoter of the 35S transcript of the cauliflower mosaic virus (Franck et al. (1980) Cell 21: 285-294; Odell et al. (1985) Nature 313: 810-812; Shewmaker et al. (1985) Virology 140: 281-288; Gardner et al. 1986, Plant Mol. Biol. 6, 221-228) or the 19S CaMV promoter (US 5,352,605 and WO 84/02913). As is known, this promoter contains different recognition sequences for transcriptional effectors, which in their entirety lead to a largely permanent and constitutive expression of the introduced gene (Benfey et al.

(1989) EMBO J 8: 2195-2202). Another suitable constitutive promoter is the "Rubisco small subunit (SSU)" promoter (US 4,962,028). Another example of a suitable promoter is the LeguminB promoter (GenBank Acc.-No .: X03677). Further preferred constitutive promoters are, for example, the promoter of nopaline synthase from Agrobacterium, the TR double promoter, the OCS (octopine synthase) promoter from Agrobacterium, the ubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29: 637- 649), the promoters of the vacuolar ATPase subunits, the FBPaseP promoter (WO 98/18940) or the promoter of a proline-rich protein from wheat (WO 91/13991). Further suitable and preferred constitutive promoters within the scope of this invention are the SuperPromotor (Ni M et al. (1995) Plant J 7: 661-676; US 5,955,646) and the nitrilase-1 promoter of the nitl gene from Arabidopsis (GenBank Acc. No .: Y07648.2, nucleotides 2456 to 4340; Hillebrand H et al. (1998) Plant Mol Biol 36 (l): 89-99; Hillebrand H et al. (1996) Gene 170 (2): 197-200).

Preferred are inducible, particularly preferably chemically inducible promoters (Aoyama T and Chua NH (1997) Plant J

11: 605-612; Caddick MX et al. (1998) Nat. Biotechnol 16: 177-180; Rewiew: Gatz (1997) Annu Rev Plant Physiol Plant Mol Biol 48: 89-108) by which expression can be controlled at a particular point in time. Examples include the PRP1 promoter (Ward et al. (1993) Plant Mol Biol 22: 361-366), a promoter induced by salicylic acid (WO 95/19443), a promoter induced by benzenesulfonamide (EP-A-0388186) , a tetracycline-inducible promoter (Gatz et al. (1992) Plant J 2: 397-404), an abscisic acid-inducible promoter (EP-A 335 528), an ethanol-inducible promoter (Salter MG et al. ( 1998) Plant J. 16: 127-132), the heavy metal-inducible metallothionein I promoter (Amini S et al. (1986) Mol Cell Biol 6: 2305-2316), the steroid-inducible MMTV LTR promoter (Izant JG et al (1985) Science 229: 345-352) and a cyclohexanone-inducible promoter (WO 93/21334). The inducible expression of a PLS / DSBI enzyme fusion protein in the core is particularly preferred. Inducible promoters also include those that can be regulated by certain repressor proteins (eg tet, lac). Corresponding repressor proteins can be translocated into the plastids in fusion with PLS and there regulate the expression of certain genes under the control of appropriate promoters. The repressor binds in the plastids to an artificial repressor binding site inserted into the piastom and can thus suppress the expression of the gene located downstream (cf. W095 / 25787). In this way, for example, the expression of a plastid-encoded DSBI enzyme can be induced if necessary or suppressed until the moment when expression is desired.

Furthermore, promoters are preferred which are induced by biotic or abiotic stress, such as, for example, the pathogen-inducible promoter of the PRPl gene (Ward et al.,

Plant Mol Biol 1993, 22: 361-366), the heat-inducible hsp70 promoter or the hsp80 promoter from tomato (US 5,187,267), the cold-inducible alpha-amylase promoter from the potato (WO 96/12814) or the wound-induced pinll promoter (EP-A 0 375 091).

In a particularly preferred embodiment, especially the nucleic acid coding for the DSBI enzyme is expressed under the control of an inducible promoter. In this way a controlled, controllable expression is achieved and any

Problems caused by constitutive expression of a DSBI enzyme avoided.

Advantageous control sequences for the expression cassettes or vectors according to the invention include viral, bacteriophage or bacterial promoters such as cos, tac, trp, tet, phoA, tat, lpp, lac, laclq, T7, T5, T3 -, gal-, trc-, ara-, SP6-, λ-PR or in the λ-PL promoter. These are preferably used in combination with the expression of the corresponding RNA polymerase.

Expression in plastids can be realized using plastid promoters and / or transcription regulatory elements. The RNA polymerase promoter (WO 97/06250) or those in WO 00/07431, US 5,877,402, WO 97/06250, WO 98/5559,5 WO 99/46394, WO 01/42441 and Promoters described in WO 01/07590. These include the rpo B promoter element, the atpB promoter element, the clpP promoter element (see also WO 99/46394) or the 16S rDNA promoter element. A polycistronic "operon" can also be assigned to the promoter (EP-A 1 076 095; WO 00/20611). Systems are also described in which a non-vegetable (eg viral) RNA polymerase is imported into the plastid using plastid transit peptides and specifically induces the expression of transgenic sequences there, which are under the control of the recognition sequences of the RNA polymerase and previously into the plastid one DNA were inserted (WO 95/16783; US 5,925,806; US 5,575,198).

In addition to the promoters mentioned, it would also be preferable to use:

a) the PrbcL promoter (SEQ ID NO: 44)

b) the Prpslδ promoter (SEQ ID NO: 50)

c) the Prrnl6 promoter (SEQ ID NO: 46)

In a particularly preferred embodiment, NEP promoters are used. These are promoters that are functional in plastids and are recognized by the nuclear-encoded, plastid RNA polymerases (NEP). Preferred are: Prrn-62; Pycf2-1577; PatpB-289; Prps2-152; Prpsl6-107; Pycfl-41; PatpI-207; PclpP-511; PclpP-173 and PaccD-129 (WO 97/06250; Hajdukiewicz PTJ et al (1997) EMBO J 16: 4041-4048).

The following are particularly preferred:

a) The PaccD-129 promoter of the accD gene from tobacco (WO 97/06250; SEQ ID NO: 47)

b) The PclpP-53 promoter of the clpP gene as a highly active NEP promoter in chloroplasts (WO 97/06250; SEQ ID NO: 48) c) The Prrn-62 promoter of the rrn gene (SEQ ID NO: 49)

d) The Prpsl6-107 promoter of the rpsl6 gene (SEQ ID NO: 45):

e) The PatpB / E-290 promoter of the atpB / E gene from tobacco (Kapoor S et al. (1997) Plant J 11: 327-337) (SEQ ID NO: 51)

f) The PrpoB-345 promoter of the rpoB gene (Liere K & Maliga P (1999) EMBO J 18: 249-257) (SEQ ID NO: 52)

In general, all promoters belonging to class III can be used in this preferred embodiment (Hajdukiewicz PTJ et al (1997) EMBO J 16: 4041-4048) as well as all fragments of the class II promoters which control the transcription initiation by the NEP. Such promoters or promoter parts are not particularly highly conserved. The consensus near the transcription initiation site of NEP promoters is given: ATAGAATAAA (Hajdukiewicz PTJ et al (1997) EMBO J 16: 4041-4048).

Normally, genes are surrounded by regulatory sequences that come from the plastids of the organism to be transformed. This creates sequence duplications that can lead to instabilities due to spontaneous, intrachromosomal homologous recombination events (Heifetz PB (2000) Biochimie 82 (6-7): 655-666). To solve this problem, it has been proposed to use heterologous regulatory sequences or to use endogenous regulatory units endogenously in the plastid genome (WO 99/46394; WO 01/42441). A reduction in homology by mutagenesis of the endogenous promoter sequence is also described (WO 01/07590).

In principle, all natural promoters with their regulatory sequences such as those mentioned above can be used for the method according to the invention. Promoters isolated from prokaryotes are particularly preferred. Promoters isolated from Synechocystis or E. coli are very particularly preferred. In addition, synthetic promoters can also be used advantageously, for example a synthetic promoter derived from the E. coli consensus sequence for σ70 promoters:

5'-TTGACA Nι ₆ _ ₁₉ TATAAT N ₃ CAT -3 ',

where N stands for any nucleotide (ie A, G, C or T). It is obvious to the person skilled in the art that individual to few base exchanges are also possible in the specified, conserved regions without destroying the function of the promoter. The variable design of these synthetic promoters through use different sequence sequences makes it possible to create a large number of promoters which do not have extensive homologies, which increases the stability of the expression cassettes in the piastom in particular in the event that several promoters are required. The following particularly preferred promoter sequences, which are derived from the above-mentioned consensus sequence, may be mentioned by way of example but not by way of limitation:

a) 5 '-TTGACATTCACTCTTCAATTATCTATAATGATACA-3' (SEQ ID NO: 53) b) 5 '-TTGACAATTTTCCTCTGAATTATATAATTAACAT-3' (SEQ ID NO: 72)

It is obvious to the person skilled in the art that these synthetic promoters can control the expression of any genes. For example, they can be used to drive the expression of a selection marker - also in order to be able to select from transplastome plants under regenerative conditions with the aid of the said selection system. Selection markers are listed below as examples. In addition, such synthetic promoters can be linked to any gene, for example to genes coding for antibodies, antigens or enzymes. The expression cassettes consisting of such promoters preferably also contain 5 'untranslated regions (or ribosome binding sites) or 3' non-coding regions described in more detail below.

The invention further relates to expression cassettes containing a nucleic acid sequence coding for a DSBI enzyme under the control of a promoter which is functional in plant plastids, for example one of the promoters described above. The expression cassette can contain further elements, such as, for example, transcription terminators and / or selection markers.

Genetic control sequences also include further promoters, promoter elements or minimal promoters that can modify the expression-controlling properties. Genetic control sequences also include the 5 'untranslated region (5'-UTR) or the 3' non-coding region (3'-UTR) of genes (Eibl C (1999) Plant J 19: 1-13). It has been shown that these can play a significant role in regulating gene expression in plastids of higher plants. At its core, genetic control elements such as 5'-UTR, introns or 3'UTR can also have a function in gene expression. For example, it has been shown that 5 'untranslated sequences can increase the transient expression of heterologous genes. You can further promote tissue specificity (Rouster J et al., Plant J. 1998, 15: 435-440.).

The following are preferably used as 5 'UTRs and 3'UTRs in plastids:

a) 5'psbA (from tobacco) (SEQ ID NO: 54)

b) 5'rbcL including 5 'portions from the coding region of the rbcL gene (from tobacco) (SEQ ID NO: 55); the sequence described with SEQ ID NO: 55 was mutated relative to the native sequence in order to introduce a Pagl or Ncol interface.

c) 5'rbcLs (SEQ ID NO: 56); the sequence described with SEQ ID NO: 56 was mutated to a by the native sequence

Pagl interface.

d) 3'psbA-1 from Synechocystis (SEQ ID NO: 57)

e) 3'psbA from tobacco (SEQ ID NO: 58)

f) 3'rbcL from tobacco (SEQ ID NO: 59)

Genetic control sequences, especially for expression in plastids, in particular also include ribosome binding sequences for initiating translation. These are usually included in the 5 'UTRs. This is particularly preferred if the nucleic acid sequence to be expressed does not provide appropriate sequences or if these are compatible with the expression system. Particularly preferred is the use of a synthetic ribosome binding site (RBS) with the sequence 5 '-GGAGG (N) ₃ _ι ₀ ATG-3', preferably 5 '-GGAGG (N) ₅ ATG-3' (SEQ ID NO: 60) preferably 5 '-GGAGGATCTCATG-3' (SEQ ID NO: 61).

The expression cassette can advantageously contain one or more so-called "enhancer sequences" functionally linked to the promoter, which enable increased transgenic expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminators, can also be inserted at the 3 'end of the nucleic acid sequences to be expressed transgenically. The nucleic acid sequences to be expressed transgenically can be contained in one or more copies in the gene construct. Furthermore, it is possible to insert a so-called downstream box after the start codon, which generally increases expression (translation enhancer WO 00/07431; WO 01/21782).

As genetic control sequences especially Polyadenylation signals suitable for core transformation are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) of the Ti plasmid pTiACHS (Gielen et al., EMBO J. 3 (1984), 835 ff) or functional equivalents thereof. Examples of particularly suitable terminator sequences are the OCS (octopine synthase) terminator and the NOS (nopalin synthase) terminator.

The transformation vectors and insertion sequences according to the invention can comprise further nucleic acid sequences. Such nucleic acid sequences can preferably represent expression cassettes. The following may be mentioned as examples, but not restrictive, for the DNA sequences to be expressed in the expression constructs:

1. Selection marker

"Selection marker" means all those nucleic acid or protein sequences whose expression (i.e. transcription and possibly translation) gives a cell, tissue or organism a different phenotype than an untransformed one. Selection markers include, for example, those nucleic acid or protein sequences whose expression of a cell, tissue or organism is an advantage (positive selection marker) or disadvantage

(negative selection marker) against cells that do not express this nucleic acid or protein. Positive selection markers work, for example, by detoxifying a substance that has an inhibitory effect on the cell (e.g. antibiotic / herbicide resistance), or by forming a substance that enables the plant to improve regeneration or increase growth under the selected conditions (for example nutritive markers, hormone-producing markers such as ipt; see below). Another form of positive selection marker includes mutated proteins or RNAs that are not sensitive to a selective agent (for example, 16S rRNA mutants that are insensitive to spectinomycin). Negative selection markers work, for example, by catalyzing the formation of a toxic substance in the transformed cells (for example the codA gene). Furthermore, selection markers can also include reporter proteins, insofar as these are suitable, transformed from non-transformed cells, tissues or organs (for example by coloring or another detectable phenotype).

The following selection markers are exemplary and not restrictive:

1.1 Positive selection markers:

The selectable marker inserted into the cell nucleus or the plastids with the expression cassette gives the successfully transformed cells resistance to a biocide (for example a herbicide such as phosphinothricin, glyphosate or bromoxynil), a metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456) or an antibiotic, such as, for example, tetracycline, ampicillin, kanamycin, G 418, neomycin, bleomycin or hygromycin. The selection marker allows the selection of the transformed cells from untransformed (McCormick et al., Plant Cell Reports 5 (1986), 81-84; Dix PJ & Kavanagh TA (1995) Euphytica 85: 29-34).

Particularly preferred selection markers are those which confer resistance to herbicides. Examples of selection markers are:

- DNA sequences coding for phosphinothricin acetyltransferases (PAT), which acetylate the free amino group of the glutamine synthase inhibitor phosphinothricin (PPT) and thus achieve detoxification of the PPT (de Block et al. 1987, EMBO J. 6, 2513-2518) ( also called Bialophos® resistance gene (bar). The bar gene coding for a phosphinothricin acetyltransferase (PAT) can be isolated from, for example, Streptomyces hygroscopicus or S. viridochromogenes. Corresponding sequences are known to the person skilled in the art (from Streptomyces hygroscopicus GenBank Acc.-No .: X17220 and X05822, from Streptomyces viridochromogenes GenBank

Acc.-No .: M 22827 and X65195; US 5,489,520). Furthermore, synthetic genes are described, for example, for expression in plastids. A synthetic Pat gene is described in Becker et al. (1994) The Plant J. 5: 299-307. The genes confer resistance to the herbicide bialaphos or glufosinate and are common markers in transgenic plants (Vickers JE et al. (1996). Plant Mol Miol Reporter 14: 363-368; Thompson CJ et al. (1987) EMBO J 6: 2519 -2523).

- 5 ~ enolpyruvylshikimate-3-phosphate synthase genes (EPSP synthase genes) which confer resistance to glyphosate (N- (phosphonomethyDglycine). The unselective herbicide glyphosate has 5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS) as a molecular target. This has a key function in the biosynthesis of aromatic amino acids in microbes and plants, but not in mammals (Steinrucken HC et al. (1980) Biochem. Biophys. Res. Commun. 94: 1207-1212; Levin JG and. Sprinson DB (1964) J. Biol. Chem. 239: 1142-1150; Cole DJ (1985) Mode of action of glyphosate a literature analysis, pp. 48-74. In: Grossbard E and Atkinson D (eds.). The herbicide glyphosate. Buttersworths, Boston.). Glyphosate-tolerant EPSPS variants are preferably used as selection markers (Padgette SR et al. (1996). New weed control opportunities: development of soybeans with a Roundup Ready ™ gene. In: Herbicide Resistant Crops (Duke, SO, ed.), Pp 53-84 CRC Press, Boea Raton, FL; Saroha MK and Malik VS (1998) J Plant Biochemistry and Biotechnology 7: 65-72). The EPSPS gene of the Agrobacterium sp. strain CP4 has a natural tolerance to glyphosate, which can be transferred to corresponding transgenic plants. The CP4 EPSPS gene was derived from Agrobacterium sp. strain CP4 cloned (Padgette SR et al. (1995) Crop Science 35 (5): 1451-1461). Sequences of 5-enolpyrvylshikimate-3-phosphate synthases that are glyphosate tolerant, as described, for example, in US 5,510,471; US 5,776,760; US 5,864,425; US 5,633,435; US 5,627,061; US 5,463,175; EP 0 218 571 are both described in the patents and deposited in the GenBank. Further sequences are described under GenBank Accession X63374. The aroA gene is also preferred (M10947 S. typhimurium aroA locus 5-enolpyruvylshikimate-3-phosphate synthase (aroA protein) gene).

the gox gene (glyphosate oxidoreductase) coding for the glyphosate® degrading enzyme. GOX (for example the glyphosate oxidoreductase from Achromobacter sp.) Catalyzes the cleavage of a C-N bond in the glyphosate, which is thus converted to aminomethylphosphonic acid (AMPA) and glyoxylate. GOX can thereby confer resistance to glyphosate (Padgette SR et al. (1996) J Nutr. 1996 Mar; 126 (3): 702-16; Shah D et al. (1986) Science 233: 478-481).

the deh gene (coding for a dehalogenase that inactivates Dalapon®), (GenBank Acc. No .: AX022822, AX022820 and W099 / 27116)

bxn genes which code for bromoxynil-degrading nitrilase enzymes. For example, the nitrilase from Klebsiella ozanenae. Sequences are in the Genbank, for example, under Acc.-No: E01313 (DNA encoding bromoxynil specific nitrilase) and J03196 (K. pneumoniae bromoxynil-specific nitrilase (bxn) genes, complete cds).

Neomycin phosphotransferases confer resistance to antibiotics (aminoglycosides) such as neomycin, G418, hygromycin, paromomycin or kanamycin by reducing their inhibitory effect through a phosphorylation reaction. The nptll gene is particularly preferred. Sequences can be obtained from GenBank (AF080390 mini transposon mTn5-GNm; AF080389 mini transposon mTn5-Nm, complete sequence). In addition, the gene is already part of numerous expression vectors and can be isolated from them using methods familiar to the person skilled in the art (such as, for example, polymerase chain reaction) (AF234316 pCAMBIA-2301; AF234315 pCAMBIA-2300, AF234314 pCAMBIA-2201). The NPTII gene codes for an aminoglycoside 3 'O-phosphotransferase from E. coli, Tn5 (GenBank Acc.-No: U00004 position 1401-2300; Beck et al. (1982) Gene 19 327-336). In addition, the aphA-6 gene from Acine-to acter baumannii, coding for an aminoglycoside phosphotransferase, can also be used as a selection marker (Huang et al. (2002) Mol Genet Genomics 268: 19-27)

the DOG ^R l gene. The gene D0G ^R 1 was isolated from the yeast Saccharomyces cerevisiae (EP 0 807 836). It encodes a 2-deoxyglucose-6-phosphate phosphatase that confers resistance to 2-DOG (Randez-Gil et al. 1995, Yeast 11, 1233-1240; Sanz et al. (1994) Yeast 10: 1195-1202, Sequence: GenBank Acc.-No .: NC001140 Chromosome VIII, Saccharomyces cervisiae Position 194799-194056).

Sulfonylurea and imidazolinone inactivating acetolactate synthases which confer resistance to imidazolinone / sulfonylurea herbicides. The active ingredients imazamethabenz-methyl, imazamox, imazapyr, imazaquin, imazethapyr may be mentioned as examples of imidazolinon herbicides. Examples of sulfonylurea herbicides are amidosulforon, azimsulfuron, chlorimuronethyl, chlorosulfuron, cinosulfuron, imazosulforon, oxasulforon, prosulforon, rimsulforon, sulfosulforon. Numerous other active substances of the classes mentioned are known to the person skilled in the art. Nucleic acid sequences such as those under GenBank Acc-No are suitable. : X51514 filed sequence for the Arabidopsis thaliana Csr 1.2 gene (EC 4.1.3.18) (Sathasivan K et al. (1990) Nucleic Acids Res. 18 (8): 2188). Acetolaetatesynthasen, which confer resistance to imidazolinone herbicides are also described under GenBank Acc.-No .:

a) AB049823 Oryza sativa ALS mRNA for acetolaetate synthase, complete eds, herbicide resistant biotype

b) AF094326 Bassia scoparia herbicide resistant acetolaetate synthase precursor (ALS) gene, complete eds

c) X07645 Tobacco acetolaetate synthase gene, ALS SuRB (EC 4.1.3.18)

d) X07644 Tobacco acetolaetate synthase gene, ALS SuRA (EC 4.1.3.18)

e) A19547 synthetic nucleotide mutant acetolaetate synthase

f) A19546 synthetic nucleotide mutant acetolaetate synthase

g) A19545 Synthetic nucleotide mutant acetolaetate synthase

h) 105376 Sequence 5 from Patent EP 0257993

i) 105373 Sequence 2 from Patent EP 0257993

j) AL133315

Hygromycin phosphotransferases (X74325 P. pseudomallei gene for hygromyein phosphotransferase) which confer resistance to the antibiotic hygromyein. The gene is part of numerous expression vectors and can be isolated from them using methods known to the person skilled in the art (such as, for example, polymerase chain reaction) (AF294981 pINDEX4; AF234301 pCAMBIA-1380; AF234300 pCAM-BIA-1304; AF234299 pCAMBIA-1303; AF234298 pCAMBIA-130; AF354046 pCAMBIA-1305.; AF354045 pCAMBIA-1305.1)

Resistance genes against

a) chloramphenicol (chloramphenicol acetyl transferase),

b) Tetracycline, various resistance genes are described, e.g. X65876 S. ordonez genes class D tetA and tetR for tetraeycline resistance and repressor proteins X51366 Bacillus cereus plasmid pBC16 tetraeycline resistance gene. In addition, the gene is already part of numerous expression vectors and can be used can be isolated from these by processes familiar to the person skilled in the art (such as, for example, polymerase chain reaction)

c) Streptomycin, various resistance genes are described e.g. with the GenBank Acc.-No. : AJ278607 Corynebacterium acetoacidophilum ant gene for streptomycin adenylyl transferase.

d) Zeocin, the corresponding resistance gene is part of numerous cloning vectors (e.g. L36849 cloning vector pZEO) and can be isolated from these using methods familiar to the person skilled in the art (such as, for example, polymerase chain reaction).

e) Ampicillin (β-lactamase gene; Datta N, Richmond MH.

(1966) Biochem J. 98 (l): 204-9; Heffron F et al (1975) J. Bacteriol 122: 250-256; the Amp gene was first cloned to produce the E. coli vector pBR322; Bolivar F et al. (1977) Gene 2: 95-114). The sequence is part of numerous cloning vectors and can be isolated from these using methods familiar to the person skilled in the art (such as, for example, polymerase chain reaction).

- Genes such as isopentenyl transferase from Agrobacterium tumefaciens (strain: P022) (Genbank Acc.-No.: AB025109). The ipt gene is a key enzyme in cytokinin biosynthesis. Its overexpression facilitates the regeneration of plants (e.g. selection on cytokinin-free medium). The procedure for using the ipt gene is described (Ebinuma H et al. (2000) Proc Natl Acad Sei USA 94: 2117-2121; Ebinuma, H et al. (2000) Selection of Marker-free transgenic plants using the oncogenes (ipt , rol A, B, C) of Agrobacterium as selectable markers, In Molecular Biology of Woody Plants. Kluwer Academic Publishers).

Various other positive selection markers which give the transformed plants a growth advantage over non-transformed ones, and methods for their use are described, inter alia, in EP-A 0 601 092. Examples include β-glucuronidase (in conjunction with, for example, cytokinin glucuronide), Mannose-6-phosphate isomerase (in connection with mannose), UDP-galactose-4-epimerase (in connection with eg galactose), with mannose-6-phosphate isomerase being particularly preferred in connection with mannose. For a selection marker which is functional in plastids, preference is given in particular to those which are resistant to spectinomycin, streptomycin, kanamycin, lincomycin, gentamycin, hygromyein, methotrexate, bleomycin, phleoycin, blasticidin, sulfonamide, phosphinotricin, chlorosulfuron, bromoxymil, glyoxysil , 2,4-Datrazine, 4-methyltryptophan, nitrate, S-aminoethyl-L-cysteine, lysine / threonine, aminoethyl cysteine or betaine aldehyde. The genes aadA, nptll, BADH, FLARE-S (a fusion of aadA and GFP, described by Khan MS & Maliga P, 1999 Nature Biotech 17: 910-915) are particularly preferred.

The aadA gene is primarily described as a selection marker which is functional in plastids (Svab Z and Maliga P (1993) Proc Natl Acad Sei USA 90: 913-917). Modified 16S rDNA, the nptll gene (kanamycin resistance) and the bar gene (phosphinothricin resistance) are also described. Due to the preference of the selection marker aadA, this is preferably "recycled" i.e. deleted after use from the genome or piastome (Fischer N et al. (1996) Mol Gen Genet 251: 373-380; Corneille S et al. (2001) Plant J 27: 171- 178), so that aadA in further Transformations of a previously transplastomic plant can be used again as a selection marker. Another possible selection marker is betaine aldehyde dehydrogenase (BADH) from spinach (Daniell H et al. (2001) Trends Plant Science 6: 237-239; Daniell H et al. (2001) Curr Genet 39: 109-116 ; WO 01/64023; WO 01/64024; WO 01/64850). Lethal-acting agents such as glyphosate can also be used in conjunction with corresponding detoxifying or resistant enzymes (WO 01/81605).

Binding type markers can also be used. The use of the DBS recognition sequence of the homing endonuclease I-Cpal preferred as an insertion site in the gene of the 23S rRNA is surrounded by at least the 3 * end of the insertion sequence (preferably an artificial intron) with homologous sequences of the target region. So sequences of the 23SrDNA are included in the transformation vector. At one point (position 2073 or 2074 of the 23SrRNA from tobacco, sequence: AAAGACCCTATGAAG) point mutations can be introduced (e.g. sequence: (≥GAGACCCTATGAAG) which confer resistance to lincomycin derived from such a mutated 23SrDNA (Cseplö A et al. ( 1988) Mol Gen Genet 214: 295-299) Further point mutations include those in the 16S rRNA of tobacco which confer resistance to spectinomycin (mutation underlined):

a) 5 '-GGAAGGTGAGGATGC.-3'("native" here: A) Other mutations confer resistance to strepomycin:

b) 5 '-GAATGAAACTA-3' ("native" here: C)

5 1.2 Negative selection markers

For example, negative selection markers enable the selection of organisms with successfully deleted sequences that comprise the marker gene (Koprek T et al. (1999) The Plant Journal 10 19 (6): 719-726). For example, sequences coding for selection markers or for DSBI enzymes can be deleted again from the genome / piastome after successful use of the method according to the invention.

15 In the case of negative selection, for example, the negative selection marker introduced into the plant converts a compound which otherwise has no adverse effect on the plant into a compound with an adverse effect. Also suitable are genes that have an adverse effect per se, such as

20 Example TK thymidine kinase (TK) and Diphtheria Toxin A fragment (DT-A), the codA gene product coding for a cytosine deaminase (Gleave AP et al. (1999) Plant Mol Biol 40 (2): 223-35; Perera RJ et al. (1993) Plant Mol Biol 23 (4): 793-799; Stougaard J (1993) Plant J 3: 755-761), the cytochrome P450 gene (Koprek et al. (1999) 5 Plant J. 16: 719 -726), genes coding for a haloalkane dehalogenase (Naested H (1999) Plant J. 18: 571-576), the iaaH gene (Sundaresan V et al. (1995) Genes & Development 9: 1797-1810) or the tms2 gene (Fedoroff NV & Smith DL (1993) Plant J 3: 273-289).

30 The concentrations of antibiotics, herbicides, biocides or toxins used for the selection must be adapted to the respective test conditions or organisms. Examples of plants to be mentioned are kanamycin (Km) 50 to 100 mg / L, hygromyein B 40 mg / L, phosphinothricin (Ppt) 6 to 20 5 mg / L, sepctinomycin (Spec) 15 to 500 mg / L.

Furthermore, functional analogs of the nucleic acids mentioned can be expressed coding for selection markers. Functional analogs here means all the sequences which essentially have the same function, ie are capable of selecting transformed organisms. The functional analogue can differ in other characteristics. For example, it may have a higher or lower activity, or it may have other functionalities. 5 Functional analogs furthermore mean sequences which code for fusion proteins consisting of one of the preferred selection markers and another protein, for example another preferred selection marker, one of the reporter proteins mentioned below or a PLS. A merger of the GFP is an example

(green fluorescent protein) and the aadA gene

(Sidorov VA et al. (1999) Plant J 19: 209-216).

2. Reporter genes

Reporter genes code for easily quantifiable proteins, which use their own color or enzyme activity to ensure an assessment of the transformation efficiency, the expression site or time or the identification of transgenic plants. Genes coding for reporter proteins are very particularly preferred (see also Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13 (1): 29-44) such as

"Green fluorescence protein" (GFP) (Chui WL et al., Curr Biol 1996, 6: 325-330; Leffel SM et al., Biotechniques. 23 (5): 912-8, 1997; Sheen et al. (1995 ) Plant Journal 8 (5): 777-784; Haseloff et al. (1997) Proc Natl Acad Sei USA 94 (6): 2122-2127; Reichel et al. (1996) Proc Natl Acad Sei USA 93 (12): 5888-5893; Tian et al. (1997) Plant Cell Rep 16: 267-271; WO 97/41228).

chloramphenicol,

Luciferase (Millar et al., Plant Mol Biol Rep 1992 10: 324-414; Ow et al. (1986) Science, 234: 856-859); allows bioluminescence detection.

- β-galactosidase, encoded for an enzyme for which various chromogenic substrates are available.

β-glucuronidase (GUS) (Jefferson et al., EMBO J. 1987, 6, 3901-3907) or the uidA gene, which encodes an enzyme for various chromogenic substrates.

- R-Locus gene product: protein that regulates the production of anthocyanin pigments (red coloring) in plant tissue and thus enables a direct analysis of the promoter activity without the addition of additional auxiliaries or chromogenic substrates (Dellaporta et al., In: Chromosome Structure and Function : Impact of New Concepts, 18th Stadler Genetics Symposium, 11: 263-282, 1988). β-lactamase (Sutcliffe (1978) Proc Natl Acad Sei USA 75: 3737-3741), enzyme for various chromogenic substrates (eg PADAC, a chromogenic cephalosporin).

- xylE gene product (Zukowsky et al. (1983) Proc Natl Acad Sei USA 80: 1101-1105), catechol dioxygenase, which can convert chromogenic catechols.

Alpha amylase (Ikuta et al. (1990) Bio / technol. 8: 241-242).

Tyrosinase (Katz et al. (1983) J Gen Microbiol 129: 2703-2714), enzyme that oxidizes tyrosine to DOPA and dopaquinone, which consequently form the easily detectable melanin.

- Aequorin (Prasher et al. (1985) Biochem Biophys Res Commun 126 (3): 1259-1268), can be used in calcium-sensitive bioluminescence detection.

The selection marker or the reporter gene is preferably encoded on the transformation construct, particularly preferably on the insertion sequence. However, it can also be coded on an independent transformation construct, which is introduced into the core or the plastids of a plant cell in a co-transformation with the transformation construct of interest.

The transformation vectors and insertion sequences according to the invention can contain further functional elements. The concept of further functional elements is to be understood broadly. Preference is given to all those elements which have an influence on the production, multiplication, function, use or value of the insert sequences, transformation constructs or vectors used in the process according to the invention. Examples of the other functional elements, however, are not restrictive:

i. Replication origins (ORI; origin of DNA replication), which ensure an increase in the expression cassettes or vectors according to the invention in, for example, E. coli or also in plastids. Examples of E. coli ORI be mentioned are the pBR322 ori, the P15A ori (Sambrook et al .: Molecular Cloning. A Laboratory Manual, ed ^nd 2nd Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) or the colEl- ORI, for example, from pBLUESCRIPT. Plastid ORIs are described in US 5,693,507, US 5,932,479 or WO 99/10513. ii. Multiple cloning regions (MCS) allow and facilitate the insertion of one or more nucleic acid sequences.

iii. Sequences that enable homologous recombination or insertion into the genome or piastome of a host organism.

iv. Elements for example “border sequences” which enable an agrobacterium-mediated transfer in plant cells for the transfer and integration into the plant genome, such as for example the right or left border of the T-DNA or the vir region.

The introduction of an insertion sequence or an expression construct for a DSBI enzyme can advantageously be implemented using vectors into which these constructs or cassettes are inserted. Vectors can be, for example, plasmids, cosmids, phages, viruses, retroviruses or also agrobacteria.

In an advantageous embodiment, the expression cassette is introduced by means of plasmid vectors. Preferred vectors are those which enable stable integration of the expression cassette into the host genome or plastome.

The production of a transformed organism or a transformed cell requires that the corresponding DNA is introduced into the corresponding host cell or into the plastids thereof. A large number of methods are available for this process, which is referred to as transformation (see also Keown et al. (1990) Methods in Enzymology 185: 527-537). For example, the DNA can be introduced directly by microinjection, electroporation or by bombardment with DNA-coated microparticles. The cell can also be chemically permeabilized, for example with polyethylene glycol, so that the DNA can get into the cell by diffusion. The transformation can also be carried out by fusion with other DNA-containing units such as minicells, cells, lysosomes or liposomes. Also worth mentioning are transfection using calcium phosphate, DEAE-dextran or cationic lipids, transduction, infection, and the incubation drier

Embryos in DNA-containing solution, sonication and the transformation of intact cells or tissue by micro or macro injection into tissue or embryos, tissue electroporation or the vacuum infiltration of seeds. Such processes are familiar to the person skilled in the art. In the case of injection or electroporation of DNA into plant cells, there are no special requirements for the plasmid used. Simple plasmids like that of pUC series can be used. If complete plants are to be regenerated from the transformed cells, it is useful to have an additional selectable marker gene on the plasmid. The processes for regenerating plants from plant tissues or plant cells are also described.

There are various ways of introducing the DNA into the plastids. It is only crucial for the present invention that the DNA is introduced into the plastids. However, the present invention is not limited to a specific method. Any method which allows the DNA to be transformed to be introduced into the plastids of a higher plant is suitable. The stable transformation of plastids is a process familiar to the person skilled in the art and has been described for higher plants (inter alia in Svab Z and Maliga P (1993) Proc Natl Acad Sei USA 90 (3): 913-917). The methods are based, for example, on a transformation using a “particle gun” and an insertion into the plastid genome by homologous recombination under selection pressure. Further methods are described in US 5,877,402. In EP-A 0 251 654 the DNA is introduced by means of Agrobacterium tumefaciens (see De Block M et al. (1985) EMBO J 4: 1367-1372; Venkateswarlu K and Nazar RN (1991) Bio / Technology 9: 1103-1105 ). It has also been shown that DNA can be introduced into isolated chloroplasts by electroporation and thus transient expression can be achieved (To KY et al. (1996) Plant J 10: 737-743). A transformation by means of a direct transfer of the DNA into plastids of protoplasts is preferred, for example using PEG (polyethylene glycol) (Koop HU et al. (1996) Planta 199: 193-201; Kofer W et al. (1998) In Vitro Cell Dev Biol Plant 34: 303-309; Dix PJ and Kavanagh TA (1995) Euphytica. 85: 29-34; EP-A 0 223 247). Biolistic transformation methods are most preferred. The DNA to be transformed is e.g. Gold or tungsten particles applied. These particles are then accelerated to the explant to be transformed (Dix PJ and Kavanagh TA (1995) Euphytica. 85: 29-34; EP-A 0 223 247). Then, in the manner familiar to the person skilled in the art, transplastome plants are regenerated under suitable pressure on a suitable medium. Corresponding methods are described (e.g. US 5,451,513; US 5,877,402;

Svab Z et al. (1990) Proc Natl Acad Sei USA 87: 8526-8530; Svab Z and Maliga P (1993) Proc Natl Acad Sei USA 90: 913-917). In addition, the DNA can be introduced into the plastids by means of microinjection. A particular method of microinjection has recently been described (Knoblauch M et al. (1999) Nature Biotech 17: 906-909; van Bei AJE et al. (2001) Curr Opin Biotechnol 12: 144-149). This method is for the present invention particularly preferred. It is also possible to introduce the plastids from one species into another species by means of protoplast fusion, to transform them there and then to convert them back to the original species by means of protoplast fusion (WO 01/70939).

In addition to these "direct" transformation techniques, a transformation can also be carried out by bacterial infection using Agrobacterium tumefaciens or Agrobacterium rhizogenes

10 (Horsch RB (1986) Proc Natl Acad Sei USA 83 (8): 2571-2575; Fraley et al. (1983) Proc Natl Acad Sei USA 80: 4803-4807; Bevans et al. (1983) Nature 304: 184 -187). The expression cassette, for example for the DSBI enzyme, is preferably integrated into special plasmids, either into an intermediate vector (English: shuttle

15 or intermediate vector) or a binary vector. Binary vectors are preferably used. Binary vectors can replicate in both E. coli and Agrobacterium and can be transformed directly into Agrobacterium (Holsters et al. (1978) Mol Gen Genet 163: 181-187). Different binary vectors

20 are known and some are commercially available, for example pBIN19 (Clontech Laboratories, Inc. USA; Bevan et al. (1984) Nucl Acids Res 12: 8711). The selection marker gene allows selection of transformed agrobacteria and is, for example, the nptll gene which confers resistance to kanamycin.

25 The binary plasmid can be transferred into the agrobacterial strain, for example, by electroporation or other transformation methods (Mozo & Hooykaas 1991, Plant Mol. Biol. 16, 917-918). The coculture of the plant explants with the agrobacterial strain usually takes place for two to three days.

30 The agrobacterium acting as the host organism in this case should already contain a plasmid with the vir region. Many strains of Agrobacterium tumefaciens are able to transfer genetic material, e.g. the strains EHA101 [pEHAl01] (Hood EE et al. (1996) J Bacteriol

35 168 (3): 1291-1301), EHA105 [pEHA105] (Hood et al. (1993) Transgenic Research 2: 208-218), LBA4404 [pAL4404] (Hoekema et al. (1983) Nature 303: 179-181 ), C58Cl [pMP90] (Koncz and Schell (1986) Mol Gen Genet 204: 383-396) and C58C1 [pGV2260] (Deblaere et al. (1985) Nucl Acids Res 13: 4777-4788).

40

For the transfer of the DNA to the plant cell, plant explants with Agrobacterium tumefaciens or Agrobacterium rhizogenes are co-cultivated. Starting from infected plant material (e.g. leaf, root or stem parts, however

45 also protoplasts or suspensions of plant cells) can transplant whole plants using a suitable medium, for example antibiotics or biocides for selection. formed cells can be regenerated. A co-transformed selection marker allows the selection of transformed cells from untransformed ones (McCormick et al. (1986) Plant Cell Reports 5: 81-84). The plants obtained can be grown, grown and crossed in a conventional manner. Two or more generations should be cultivated to ensure that genomic integration is stable and inheritable. The methods mentioned are described for example in Jenes B et al. (1993) Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by Kung SD and Wu R, Academic Press, p.128-143 and in Potrykus (1991) Ann Rev Plant Physiol Plant Molec Biol 42: 205-225).

The Agrobacterium-mediated transformation is best suited for dicotyledonous plant cells, whereas the direct transformation techniques are suitable for every cell type.

The Agrobacterium-mediated transformation is particularly preferably used for the core transformation, the direct transformation techniques are particularly preferably used for the plastid transformation.

Once a predominantly homotransplastoma plant cell has been produced by the method according to the invention, a complete plant can be obtained using methods known to the person skilled in the art. This is based, for example, on callus cultures. The formation of shoots and roots can be induced in a known manner from these still undifferentiated cell masses. The sprouts obtained can be planted out and grown.

deletion method

In the described methods according to the invention, it is advantageous at various stages to remove certain previously introduced sequences (for example for selection markers and / or DSBI enzymes) from the piastome or genome of the plant or cell. So it is advantageous but not absolutely necessary to remove the selection marker, which was introduced, for example, when inserting a non-natural DBS recognition sequence, from the master plant, because then in a subsequent transformation (for example with the insertion sequence) again the same selection marker can be used. A deletion is particularly advantageous since the selection marker is no longer absolutely necessary after the selection phase and is therefore superfluous. The deletion also increases consumer acceptance and is desirable when considering approval technology. In addition, the protein synthesis apparatus of the plastid is not unnecessarily burdened by the synthesis of the marker protein, which has a potentially advantageous effect on the properties of the corresponding plant.

Various methods are known to the person skilled in the art for the deletion of sequences. However, excision by means of recombinases should not be mentioned as a limitation.

10 Various sequence-specific recombination systems are described, such as the Cre / lox system of Bacteriophagen Pl (Dale EC and Ow DW (1991) Proc Natl Acad Sei USA 88: 10558-10562; Russell SH et al. (1992) Mol Gen Genet 234: 49 -59; Osborne BI et al. (1995) Plant J. 7, 687-701), the FLP / FRT system of the yeast (Kilby

15 NJ et al. (1995) Plant J 8: 637-652; Lyznik LA et al. (1996) Nucleic Acids Res 24: 3784-3789), the Mu phage gin recombinase, the E. coli pin recombinase or the R / RS system of the pSRI plasmid (Onouchi H et al. (1995) Mol Gen Genet 247: 653-660; Sugita K et al. (2000) Plant J 22: 461-469). These procedures are

20 not only for the deletion of DNA sequences from the nuclear genome but also from the piastome (Corneille et al. (2001) Plant J 27: 171-178; Hajdukieicz et al. (2001) Plant J 27: 161-170) , Examples of other recombinases which can be used are: PhiC31 (Kuhstoss & Rao (1991) J Mol Biol

25 222: 897-908), TP901 (Christiansen et al. (1996) J Bacteriol 178: 5164-5173), xisF from Anabaena (Ramaswamy et al. (1997) Mol Microbiol 23: 1241-1249), integrase from phage PhiLC3 (Lillehaug et al. (1997) Gene 188: 129-136) or the recombinase encodes the sre gene of the R4 phage (Matsuura et al. (1996) J Bacteriol

30 178: 3374-3376).

In a preferred embodiment, however, the deletion is implemented by intrachromosomal recombination on the basis of sequence duplications which have been introduced accordingly. The latter can

35 through the targeted introduction of double strand breaks near the

Sequence duplications can be increased in efficiency (cf. FIG. 8). For this purpose, the sequence to be deleted is flanked on both sides by homology sequences H1 and H2, which are of sufficient length and homology to recombine with one another.

The recombination is induced by induction of at least one sequence-specific double-strand break near one of the two homology sequences of another DSB recognition sequence (which, however, is preferably different from the first). This DSB recognition sequence is preferred between the two

45 homology sequences localized. To induce the double-strand break, a second DSBI enzyme is preferably expressed or introduced, which is different from the first. Especially this method is preferably used for the deletion of selection markers from the piastome.

Another object of the invention relates to the transplastomic, predominantly homoplastic, plants produced by the process according to the invention, and parts thereof, such as leaves, roots, seeds, fruits, tubers, pollen or cell cultures, callus, etc. - derived from such.

Another object of the invention relates to the plants used in the method according to the invention, which contain an expression cassette according to the invention for a DSBI enzyme or a fusion protein of PLS and DSBI enzyme. The expression cassette for the fusion protein composed of PLS and DSBI enzyme is - particularly preferably - under the control of a promoter which is functional in the plant cell nucleus and is stably integrated into the nuclear DNA. The expression cassette coding for a DSBI enzyme is preferably present stably integrated into the piastome under the control of a promoter active in plant plastids. Also included are parts of the same, such as leaves, roots, seeds, tubers, fruits, pollen or cell cultures, callus, etc. - derived from the aforementioned plants.

Genetically modified plants according to the invention that can be consumed by humans and animals can also be used, for example, directly or after preparation known per se as food or feed.

Another object of the invention relates to the use of the transplastomic, predominantly homoplastic, plants according to the invention described above and the cells, cell cultures, parts derived therefrom - such as roots, leaves, etc. in transgenic plant organisms - and transgenic propagation material such as seeds or fruits, for the production of food or feed, pharmaceuticals or fine chemicals.

Fine chemicals means enzymes such as the industrial enzymes mentioned below, vitamins such as tocopherols and tocotrienols (e.g. vitamin E) and vitamin B2, amino acids such as methionine, lysine or glutamate, carbohydrates such as starch, amylose, amylopectin or sucrose, fatty acids such as saturated , unsaturated and polyunsaturated fatty acids, natural and synthetic flavors, aromas such as linalool, menthol, borneon (camphor), pinene, limonene or geraniol and dyes such as retinoids (e.g. vitamin A), flavonoids (e.g. quercetin, rutin, tangeretin , Nobiletin) or Carotenoids (e.g. ß-carotene, lycopene, astaxanthin). The production of tocopherols and tocotrienols and carotenoids is particularly preferred. The transformed host organisms and the isolation from the host organisms or from the growth medium are cultivated using methods known to those skilled in the art. The production of pharmaceuticals such as antibodies or vaccines has been described (Hood EE, Jilka JM. (1999) Curr Opin Biotechnol. 10 (4): 382-386; Ma JK and Vine ND (1999) Curr Top Microbiol Immunol .236 : 275-92).

The method according to the invention is particularly suitable for the production of industrial enzymes in the context of so-called "phytofarming". Examples, however, are not restrictive for the industrial enzymes: lipases, esterases, proteases, nitrilases, acylases, epoxyhydrolases, amidases, phosphatases, xylanases, alcohol dehydrogenases, amylases, glucosidases, galactosidases, pullulanases, endocellulases, glucancleas, nuclitaminases, cellenase, cellulase, cellulase, cellulase, cellulase, cellulase, monolitase, cellulase, cellulase, cellulase, cellulase, cellulase, cellulase, cellulase, cellulase, cellulase, cellulase, cellulase, cellulase, cellulase, cellulase, cellulase, cellulase, cellulase, and cellulase type , Lysozymes and laccases.

Embodiments which are particularly preferred in the context of the invention are described in more detail below in the context of the explanations for the figures.

sequences

1. SEQ ID NO: 1 pCB42-94 base vector for plastid transformation.

2. SEQ ID NO: 2

Nucleic acid sequence inserted into the multiple cloning site of pCB42-94 (SEQ ID NO: 1). Resulting vector: PCB199-3.

3. SEQ ID NO: 3

Nucleic acid sequence inserted into the multiple cloning site of pCB42-94 (SEQ ID NO: 1). Resulting vector: pCB401-20

4. SEQ ID NO: 4

PCB289-13 expression cassette for plastid expression of the I-Ppol homing endonuclease.

5. SEQ ID NO: 5 amino acid sequence of the I-Ppol homing endonuclease encoded by the pCB289-13 expression cassette. 6. SEQ ID NO: 6

Xhol / Bglll fragment used to create the vector pCB304-25.

5 7. SEQ ID NO: 7

Nucleic acid sequence inserted into the multiple cloning site of pGEMTeasy. Resulting vector: pCB220-17

8th . SEQ ID NO: 8

10 Inserted into the multiple cloning site of pBluescript

Nucleic acid sequence. Resulting vector: pCB270-l

9. SEQ ID NO: 9

Sequence from vector pCB315-l: LacZ gene with inserted intron 15 for the detection of the splicing.

10. SEQ ID NO: 10

Ll.LtrB intron from vector pCB345-34.

20 11. SEQ ID NO: 11

Synthetic sequence of the homing endonuclease I-Ppol (ORF: 16 to 507)

12. SEQ ID NO: 12

25 Protein sequence of the homing endonuclease I-Ppol

13. SEQ ID NO: 13

Nucleic acid sequence of the homing endonuclease I-Cpal from Chlamydomonas pallidostigmatica (changes to the published sequence at position 69. An Ncol interface was introduced at the ATG. (ORF: 4 to 462)

14. SEQ ID NO: 14

Protein sequence of the homing endonuclease I-Cpal 35

15. SEQ ID NO: 15

Sequence containing the CpLSU2 intron

16. SEQ ID NO: 16: oligonucleotide primer pl9 40 5 '-TAAGGCCCTCGGTAGCAACGG-3'

17. SEQ ID NO: 17: oligonucleotide primer p20 5 '-GGGGTACCAAATCCAACTAG-3'

45 18. SEQ ID NO: 18: Oligonucleotide primer p21: 5 '-GGAGCTCGCTCCCCCGCCGTCGTTC-3' 19. SEQ ID NO: 19: oligonucleotide primer p22 5 '-GATGCATGATGACTTGACGGCATCCTC-3'

20. SEQ ID NO: 20: oligonucleotide primer pl90 5 5 '-GTCGACAGATCTTTAA-3'

21. SEQ ID NO: 21: oligonucleotide primer pl91 5 '-AGATCTGTCGACTTAA-3'

10 22. SEQ ID NO: 22: oligonucleotide primer pl99 5 '-GATCTCCAGTTAACTGGGGTAC-3'

23. SEQ ID NO: 23: oligonucleotide primer p200 5 '-CCCAGTTAACTGGA-3'

15

24. SEQ ID NO: 24: oligonucleotide primer p218 5 '-TTAAGCCAGTTAACTGGGCGGAGCT-3'

25. SEQ ID NO: 25: oligonucleotide primer p219 20 5 '-CCGCCCAGTTAACTGGC-3'

26. SEQ ID NO: 26: oligonucleotide primer p276 5 '-TCGAGAAGATCAGCCTGTTATCCCTAGAGTAACT-3'

25 27. SEQ ID NO: 27: Oligonucleotide primer p277 5 '-CTAGAGTTACTCTAGGGATAACAGGCTGATCTTC-3'

28. SEQ ID NO: 28: oligonucleotide primer p91 5 '-AGAAGACGATCCTAAGG-3'

30

29. SEQ ID NO: 29: oligonucleotide primer p92 5 '-TGAAGACTTGACAAGGAATTTCGC-3'

30. SEQ ID NO: 30: oligonucleotide primer pl02

35 5 '-AGAAGACGATCCTAAATAGCAATATTTACCTTTG ^ SÄQCAAAAGTTATCAGGCATG-3

31. SEQ ID NO: 31: oligonucleotide primer pl03

5 'TGAAGACTTGACAAGGAATTTCGCTACCTTCGAGTACTCCAAAACTAATC-3'

40 32. SEQ ID NO: 32: Oligonucleotide Prime p207 5 '-GAGAAGACATTCCTAACACATCCATAACGTGCG-3'

33. SEQ ID NO: 33: Oligonucleotide prime p208 5 '-TGAAGACTTGACATTTGATATGGTGAAGTAGG-3' 45 34. SEQ ID NO: 34

Nucleic acid sequence coding for the transit peptide of the small subunit (SSU) of ribulose bisphosphate carboxylase (Rubisco ssu) from pea 5

35. SEQ ID NO: 35

Transit peptide of the small subunit (SSU) of pea ribulose bisphosphate carboxylase (Rubisco ssu)

10 36. SEQ ID NO: 36

Transition peptide of tobacco plastid transketolase.

37. SEQ ID NO: 37

Nucleic acid sequence coding for the transit peptide of the 15 tobacco plastid transketolase (reading frame 1; pTP09)

38. SEQ ID NO: 38

Nucleic acid sequence coding for the transit peptide of tobacco plastid transketolase (reading frame 2; pTPlO) 20

39. SEQ ID NO: 39

Nucleic acid sequence coding for the transit peptide of tobacco plastid transketolase (reading frame 3; pTPll)

25 40. SEQ ID NO: 40

Arabidopsis thaliana plastid isopentenyl pyrophosphate isomerase-2 (IPP-2) transit peptide.

41. SEQ ID NO: 41

30 nucleic acid sequence coding for the transit peptide of plastid isopentenyl pyrophosphate isomerase-2 (IPP-2) from Arabidopsis thaliana (reading frame 1; IPP-9)

42. SEQ ID NO: 42

35 nucleic acid sequence coding for the transit peptide of the plastid isopentenyl pyrophosphate isomerase-2 (IPP-2) from Arabidopsis thaliana (reading frame 2; IPP-10)

43. SEQ ID NO: 43

40 nucleic acid sequence coding for the transit peptide of plastid isopentenyl pyrophosphate isomerase-2 (IPP-2) from Arabidopsis thaliana (reading frame 3; IPP-11)

44. SEQ ID NO: 44

45 nucleic acid sequence coding for the PrbcL promoter from tobacco. 45. SEQ ID NO: 45

Nucleic acid sequence coding for the tobacco Prpsl6-107 promoter.

5 46. SEQ ID NO: 46

Nucleic acid sequence coding for the Prrnl6 promoter from tobacco.

47. SEQ ID NO: 47

10 nucleic acid sequence coding for the PaccD-129 promoter from tobacco.

48. SEQ ID NO: 48

Nucleic acid sequence coding for the PclpP-53 promoter from 15 tobacco.

49. SEQ ID NO: 49

Nucleic acid sequence coding for the Prrn-62 promoter from tobacco. 20

50. SEQ ID NO: 50

Nucleic acid sequence coding for the Prpsl6 promoter from tobacco.

25 51. SEQ ID NO: 51

Nucleic acid sequence coding for the PatpB / E-290 promoter from tobacco.

52. SEQ ID NO: 52

30 nucleic acid sequence coding for the PrpoB-345 promoter from tobacco.

53. SEQ ID NO: 53

Nucleic acid sequence coding for a promoter derived from the consensus sequence of the σ70 promoters from E. coli.

54. SEQ ID NO: 54

Nucleic acid sequence coding for the 5 'untranslated region of the psbA gene from tobacco (5'psbA) 40

55. SEQ ID NO: 55

Nucleic acid sequence coding for the 5 'untranslated region including 5' parts from the coding region of the rbcL gene from tobacco (5'rbcL). 45 56. SEQ ID NO: 56

Nucleic acid sequence coding for the 5 'untranslated region of the rbcLs gene from tobacco.

5 57. SEQ ID NO: 57

Nucleic acid sequence coding for the 3 'untranslated region of the psbA-1 gene from Synechocystis (3'psbA-1)

58. SEQ ID NO: 58

10 nucleic acid sequence coding for the 3 'untranslated region of the psbA gene from tobacco (3'psbA)

59. SEQ ID NO: 59

Nucleic acid sequence coding for the 3 'untranslated 15 region of the rbcL gene from tobacco (3'rbcL)

60. SEQ ID NO: 60

Nucleic acid sequence coding for synthetic ribosome binding parts (RBS) 20

61. SEQ ID NO: 61

Nucleic acid sequence coding for synthetic ribosome binding site (RBS)

25 62. SEQ ID NO: 62

Complete insert of the vector pCB304-25

63. SEQ ID NO: 63

Bglll / Muni fragment of the vector pCB320-192. 30

64. SEQ ID NO: 64: oligonucleotide primer p93 5 '-AAAGATCTCCTCACAAAGGGGGTCG-3'

65. SEQ ID NO: 65: oligonucleotide primer p97 35 5 '-TCGAAGACTTAGGACCGTTATAG-3'

66. SEQ ID NO: 66: oligonucleotide primer p98 5 '-AGGAAGACCTTGTCGGGTAAGTTCCG-3'

40 67. SEQ ID NO: 67: oligonucleotide primer p95: 5 '-CTCAATTGGGGTCTCTCTGTCCAGGTGCAGG-3'

68. SEQ ID NO: 68: Nucleic acid sequence coding for fusion proteins from the native I-Ppo-I nuclease and that of the 45 IPP plastid localization sequence (ORF for I-Ppol: 181-672; IPP transit peptide: 1-180; native sequence from 1-172). 69. SEQ ID NO: 69: fusion proteins from the native I-Ppo-I nuclease and that from the IPP plastid localization sequence.

70. SEQ ID NO: 70: Nucleic acid sequence coding for long version of the I-Ppol homing endonuclease.

71. SEQ ID NO: 71: amino acid sequence coding for long version of the I-Ppol homing endonuclease.

10 72. SEQ ID NO: 72: Nucleic acid sequence coding for a

Promoter sequence derived from the consensus sequence of the σ70 promoters from E. coli.

15 73. SEQ ID NO: 73: Nucleic acid sequence coding for the artificial intron TetIVS2a.

74. SEQ ID NO: 74: insert of the vector pCB459-l

20 75. SEQ ID NO: 75: insert of the vector pCB478-3

76. SEQ ID NO: 76: insert of the vector pCB492-25

77. SEQ ID NO: 77: oligonucleotide primer p396

25 5 '-TAGTAAATGACAATTTTCCTCTGAATTATATAATTAACATGGCGACTGTTTACCAAAA AC-3

78. SEQ ID NO: 78: oligonucleotide primer p95

5 '-CTCAATTGGGGTCTCTCTGTCCAGGTGCAGG-3' 30

79. SEQ ID NO: 79: Nucleic acid sequence coding for PCR product

Prom-TetIVS2a-Cpa

80. SEQ ID NO: 80: insert of the vector pCB435-45 35

81. SEQ ID NO: 81: Nucleic acid sequence coding for probe for

Southern blot analysis (directed against portions of the 16SrDNA).

40 82. SEQ ID NO: 82: Nucleic acid sequence coding for probe for

Southern blot analysis (directed against portions of the 23SrDNA).

83. SEQ ID NO: 83: insert of the vector pCB456-2 45

84. SEQ ID NO: 84: insert of the vector pCB528-2, from Kpnl to Sacl pictures

In the context of the method according to the invention, the embodiments illustrated in the following figures are particularly preferred. The following abbreviations generally apply to the illustrations:

A, A 'pair of homologous sequences A and A'

A / A 'Result of a homologous recombination between A and A' and / or an exchange between A and A 'due to repair synthesis.

B, B 'pair of homologous sequences B and B'

B / B 'Result of a homologous recombination between B and B' and / or an exchange between B and B 'due to repair synthesis.

Hl, H2: pair of homologous sequences Hl and H2

Hl / 2: sequence as a result of the homologous recombination from Hl and H2 DS functional DSB recognition sequence nDS non-functional "half page" of a DSB recognition sequence E: DSBI enzyme P: promoter I: further nucleic acid sequence (gene of interest) S, S ' Positive selection marker NS Negative selection marker

IS intron sequences. The intron is marked overall as a box. The box contains all the elements required for a functional intron.

As already described above, A / A 'or B / B' is the result of a homologous recombination and / or an exchange caused by repair synthesis. The resulting sequence can in turn be the starting sequence for further homologous recombinations or repair syntheses. To simplify this sequence (A / A 'or B / B') is referred to again as A or B in subsequent steps.

1. Fig. 1: Introduction of a DSB recognition sequence into the piastom by means of double "cross-over"

In a particularly preferred embodiment 1, a DSBE construct is first introduced into plastids of a higher plant. In this embodiment, the DSBE construct is preferably equipped with homologous target regions and with an expressible selection marker (promoter - 5'UTR - selection marker - 3'UTR) and in this embodiment preferably contains one Recognition region for a DSBI enzyme which preferably does not have a natural recognition sequence in the plastid genome of the (non-transformed) plant under consideration. The DSBE construct can - optionally - already encode other genes of interest. Mainly homoplastic master plants are produced (Fig. 1).

2. Fig. 2A-E: Introduction of an insertion sequence with a

Expression cassette for a DSBI enzyme and possibly selection markers and other genes of interest

Explants of the master plants produced by embodiment 1 are used for a further transformation with a transformation construct according to the invention. The transformation construct according to the invention preferably has regions on both sides (FIGS. 2A, 2B) or on one side (FIGS. 2C, 2D) of the insertion sequence that are homologous to the sequences surrounding the insertion site of the DSBE construct. The insertion then takes place via homologous recombination (e.g. cross-over) or via repair synthesis.

The sequences to be inserted are particularly preferably flanked (inwardly after the homology sequences) by parts of the DSB recognition sequence (nDS) which correspond to the parts resulting from a cut with the DSBI enzyme (FIG. 2B). The insertion sequence thus contains sequences which correspond directly to the ends resulting from a cut in the piastome and thus ensure particularly efficient installation.

If the transformation construct or the insertion sequence has no such homologous regions, the insertion sequence is preferably provided at these ends with overhanging ends which are also produced by the DSBI enzyme after cutting the piastome of the master plant (FIG. 2E).

If there is only one homology sequence, an nDS sequence (as described for FIG. 2B) adjoins this in a particularly preferred embodiment, while the other side of the insertion sequence is provided with overhanging ends which correspond to the DSBI enzyme in the piastom correspond to the master plant produced (Fig. 2D).

Optionally, the insertion sequence codes for a further, expressible selection marker, which differs functionally from that of the DSBE construct, possibly one or more expressible genes of interest and the expressible DSBI enzyme, which the introduced by the DSBE construct Recognition sequence at the insertion site in the plastid genome of the master plant. The insertion sequence of the transformation construct is inserted at one point in such a way that said recognition region is no longer functional after the insertion.

3. Fig.3: Introduction of a DSB recognition sequence in the

Piastom by means of double "cross-over" into a transcriptionally active area

In a further, particularly preferred embodiment 2, a DSBE construct is first introduced into plastids of a higher plant. In this embodiment, the DSBE construct is preferably equipped with homologous target regions and with an expressible selection marker, an endogenous promoter of the piastome being used for this purpose, and additionally preferably contains a recognition region for a DSBI enzyme, which preferably does not have a natural recognition sequence in the plastid genome of the ( non-transformed) considered plant. The DSBE construct can already code for genes of interest. Mainly homoplastic master plants are produced (Fig. 3).

4. Fig.4A-E: Introduction of an insertion sequence with a cassette coding for a DSBI enzyme and possibly selection markers and other genes of interest

Explants of the master plants produced by embodiment 2 are used for a further transformation with a transformation construct according to the invention. The transformation construct according to the invention preferably has on both sides

(Fig. 4A, 4B) or one-sided (Fig. 4C, 4D) of the insertion sequence areas that are homologous to the sequences surrounding the insertion site of the DSBE construct. The insertion then takes place via homologous recombination (e.g. cross-over) or a repair synthesis. Those to be inserted are particularly preferred

Sequences - inwardly following the homology sequences - flanked by parts of the DSB recognition sequence (nDS) which correspond to the parts resulting from a cut with the DSBI enzyme (FIG. 4B). The insertion sequence thus contains sequences which correspond directly to the ends resulting from a cut in the piastome and thus ensure particularly efficient installation.

If the transformation construct or the insertion sequence has no such homologous regions, the insertion sequence at these ends is preferably provided with overhanging ends, which also from the DSBI enzyme after cutting the piastome of the master plant (Fig. 4E).

If there is only one homology sequence, an nDS sequence (as described for FIG. 4B) adjoins this in a particularly preferred embodiment, while the other side of the insertion sequence is provided with overhanging ends which correspond to the DSBI enzyme in the piastom correspond to the master plant produced (Fig. 4D).

Optionally, the insertion sequence codes for a further, expressible selection marker, which differs functionally from that of the DSBE construct, possibly one or more expressible genes of interest and the expressible DSBI enzyme, which the introduced by the DSBE construct

Recognition sequence at the insertion site in the plastid genome of the master plant. The insertion sequence of the transformation construct is inserted at one point in such a way that said recognition region is no longer functional after the insertion.

5. Fig. 5A-E: Introduction of an insertion sequence with a cassette coding for a DSBI enzyme and possibly selection markers and other genes of interest using natural, endogenous DSB recognition sequences

In a further, very particularly preferred embodiment 3, a transformation construct according to the invention contains an expressible DSBI enzyme which has an endogenous, natural recognition sequence in the piastome of the plant under consideration.

Explants of these natural master plants are used for a transformation with a transformation construct according to the invention. The transformation construct according to the invention preferably has regions on both sides (FIGS. 5A, 5B) or on one side (FIGS. 5C, 5D) of the insertion sequence which are homologous to the sequences surrounding the insertion site of the DSBE construct. The insertion then takes place via homologous recombination (e.g. cross-over) or repair synthesis.

The sequences to be inserted are particularly preferably flanked (inwardly following the homology sequences) by parts of the DSB recognition sequence (nDS) which correspond to the parts resulting from a cut with the DSBI enzyme

(Fig. 5B). The insertion sequence thus contains sequences which immediately end the ends resulting from a cut in the piastome. bar appropriate and thus ensure a particularly efficient installation.

If the transformation construct or the insertion sequence has no such homologous regions, the insertion sequence is preferably provided at these ends with overhanging ends, which are also produced by the DSBI enzyme after cutting the piastome of the master plant (FIG. 5E).

If there is only one homology sequence, an nDS sequence (as described for FIG. 5B) adjoins this in a particularly preferred embodiment, while the other side of the insertion sequence is provided with overhanging ends which correspond to that of the DSBI enzyme in the piastom of the master plant produced (Fig. 5D).

The insertion sequence of the transformation construct is preferably inserted at one point such that said recognition region is no longer functional after the insertion. The insertion sequence preferably codes for an expressible selection marker (S '), one or more genes of interest and for the expressible DSBI enzyme. The selection marker is optional.

6. Fig. 6A-E: Introduction of an insertion sequence with a cassette coding for genes of interest and possibly selection markers and introduction of a DSBI enzyme into trans

In further preferred embodiments 4, the DSBI enzyme is not encoded by the transformation construct, but rather is either expressed in trans (in plastids or as PLS fusion protein in the core) or in the form of RNA or as protein in the plastids. The DSBI enzyme recognizes either an artificially introduced (Fig. 6A, 6B) or natural (Fig. 6C, 6D) DSB recognition sequence. This embodiment is particularly preferred when the transformation construct does not comprise any promoter elements and the encoded genes are only expressed after insertion into the piastome using plastidic, endogenous promoters.

As in the exemplary embodiments already described above, the transformation construct preferably has regions on both sides (FIGS. 6A, 6B) or one side (not shown) of the insertion sequence which are homologous to the sequences surrounding the insertion site of the DSBE construct. The insertion takes place then via homologous recombination (eg cross-over) or repair synthesis.

The sequences to be inserted are particularly preferably flanked (inwardly after the homology sequences) by parts of the DSB recognition sequence (nDS) which correspond to the parts resulting from a cut with the DSBI enzyme (FIGS. 6B, 6D). The insertion sequence thus contains sequences which correspond directly to the ends resulting from a cut in the piastome and thus ensure particularly efficient installation.

If the transformation construct or the insertion sequence has no such homologous regions, the insertion sequence is preferably provided at these ends with overhanging ends, which is also produced by the DSBI enzyme after cutting the piastome of the master plant (FIG. 6E). The transformation construct can additionally comprise a sequence coding for a DSBI enzyme. However, the expression takes place only after successful insertion into the piastome, so that the provision of a first amount of functional RNA or protein of a DSBI enzyme is desirable.

If there is only one homology sequence, an nDS sequence (as described for FIGS. 6B, 6D) adjoins this in a particularly preferred embodiment, while the other side of the insertion sequence is provided with overhanging ends which correspond to that of the DSBI enzyme correspond in the piastome to the master plant (not shown).

The insertion sequence of the transformation construct is preferably inserted at one point such that said recognition region is no longer functional after the insertion.

7. Fig. 7A-E: Introduction of an insertion sequence comprising one

Intron sequence with a cassette coding for genes of interest and possibly selection markers or DSBI enzymes

In a further, very particularly preferred embodiment 5, the gene of interest (as well as optionally a selection marker S 'and / or the DSBI enzyme) is encoded within an intron which is functional at the selected insertion site, ie can splice from the transcript formed there , The transformation construct according to the invention preferably has regions on both sides (FIGS. 7A, 7B) or on one side (not shown) of the insertion sequence which are homologous to the sequences surrounding the insertion site of the DSBE construct. The insertion then takes place via homologous recombination (eg cross-over) or repair synthesis.

If the transformation construct or the insertion sequence has no such homologous regions, the insertion sequence is preferably provided at these ends with overhanging ends, which is also generated by the DSBI enzyme after cutting the piastome of the master plant (not shown).

The expression can be controlled by means of a promoter (FIG. 7B) contained on the transformation construct or an enogenous, plastidic promoter (FIG. 7A). In the first case, the DSBI enzyme is preferably contained on the transformation construct (FIG. 7B), while in the latter case it is expressed (at least in parallel) either in trans (in plastids or as a PLS fusion protein in the nucleus) or in the form of RNA or is transfected into the plastids as protein (FIG. 7A).

The insertion sequence of the transformation construct is preferably inserted at one point such that said recognition region is no longer functional after the insertion. The transformation construct can additionally optionally comprise a sequence coding for a DSBI enzyme. However, the expression takes place only after successful insertion into the piastome, so that the provision of a first amount of functional RNA or protein of a DSBI enzyme is desirable.

8. Fig. 8: Deletion of sequences by means of intramolecular homologous recombination induced by sequence-specific double strand breaks

In all of the above-described embodiments, sequences — for example coding for selection markers or DSBI enzymes — are preferably flanked by homology sequences Hl and H2 which are of sufficient length and homology to recombine with one another. The recombination is induced by induction of at least one double-strand break between the two homology sequences located in the DSB recognition sequence. A DSBI enzyme is preferably transiently expressed or introduced to induce the double-strand break (FIG. 8). The person skilled in the art is aware that the order of the expressed genes in an operon is interchangeable and can therefore vary in the embodiment described above. If only one homology sequence is used to insert the insertion sequence, it can also be located on the 5 '(as shown in the figures) or 3' side of the double-strand break. In principle, the DSBI enzyme can be expressed on the transformation construct and / or separately (in the core or plastids) or introduced in another way - for example by transfection with RNA or protein in plastids.

9. Fig. 9: Southern analysis of predominantly homotransplastomas

plants

Wild-type and predominantly homotransplastome master plants were analyzed for the modification (introduction of a DSB recognition sequence) (cf. Example 4). The modification detected a band of 1750 bp (lanes 2, 3, and 4 corresponding to lines CB199NTH-4, -6, and -8), while a band of 3100 bp was detected in the unmodified wild type plant (lane 1) ,

10. Fig. 10: Modifying the IGS of the Tetrahymena LSU Intron.

Uppercase letters show the sequence of the intron, while lowercase letters show the sequence of the surrounding exons. Shown are the flanking exon sequences, the 5 * and 3 ^λ part of the intron or intronderivative and the sequence comprising the IGS. Bars between the bases indicate possible base pairings that can be formed to initiate the splicing process.

A: The sequence sections of the naturally occurring Tetrahymena LSU intron in its natural exon environment are shown.

B: The sequence segments of the Tetrahymena LSU intron derivative (TetIVS2a) produced in the context of this invention are shown in the predefined exon environment, as can be found in the CpLSU5 intron within the DSB recognition sequence of the DSBI enzyme I-Cpal. Letters in bold represent the mutations compared to the natural sequence. 11. Fig. 11:

A: Southern analysis with total DNA cut with BamHI from the tobacco lines CB255 + 435NTH-16b, -16c, -19 and -20. An area of the 16SrDNA was used as a probe. The bands which represent piastom copies which correspond to the wild type (WT) (approx. 3.2 kb band detected) and which carry the transgene (TG) (approx. 2.3 kb band detected) are indicated by arrows.

B: Schematic representation of transplastomic tobacco plants which resulted from the insertion of the insertion sequence from pCB435-45; as well as the bands to be expected from a corresponding Southern analysis (cf. A).

C: Southern analysis with total DNA cut with HindIII from the tobacco lines CB255 + 435NTH-16b, -16c, -19 and -20. A region of the 23SrDNA was used as a probe. The bands which represent piastom copies which correspond to the wild type (WT) (approx. 1.1 kb band detected) and which carry the transgene (TG) (approx. 1.5 kb band detected) are indicated by arrows.

D: Schematic representation of transplastomic tobacco plants which resulted from the insertion of the insertion sequence from pCB255-l; as well as the bands to be expected from a corresponding Southern analysis (cf. C).

12. Fig. 12:

A: Wild-type and predominantly homotransplastome master plants were analyzed for the modification (introduction of one of the I-Ppol DSB recognition sequences) in a Southern (see Example 14.2). As a result of the modification, a band of approximately 1.7 kb was detected in the DNA treated here with EcoRI (TG) (lanes 1 and 4 according to lines CB456NTH-1 and -15), while a band was found in the unmodified wild type plant (WT) of about 3.1 kb was detected (lane 6). (wt - unmodified wild type plant; wild type - shows the expected fragment size in unmodified wild type plants; transgenic - shows the expected fragment size in plants CB456NTH)

B: Schematic representation of the EcoRI fragment which was to be expected in A by hybridization with the probe (probe) in a modified plant CB456NTH. (trnV - gene coding for a tRNA-Val; rrnl6 - gene coding for the 16S rRNA; aadA gene coding for a selection marker; 3 'psbA (Synec) - non-coding region downstream of the psbA-1 gene from Synechocystis, here incorporated into the expression cassette for the selection marker aadA; Psynth. - synthetic promoter derived from the consensus sequence for E. coli σ70 promoters; DSB-E: DSB recognition sequence).

Examples

General methods:

The chemical synthesis of oligonucleotides can be carried out, for example, in a known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897). The cloning steps carried out in the context of the present invention, such as e.g. Restriction cleavages, agorose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of bacteria, multiplication of phages and sequence analysis of recombinant DNA are carried out as in Sambrook et al , (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. The sequencing of recombinant DNA molecules is carried out with a laser fluorescence DNA sequencer ALF-Express (Pharmacia, Upsala, Sweden) according to the method of Sanger (Sanger et al. (1977) Proc Natl Acad Sei USA 74: 5463-5467) ..

Example 1: Creating a base vector for plastid transformation

First, the selected target regions were cloned from the Piastom of the tobacco variety SRI using PCR. The left target region was amplified with the primers pl9 and p20.

pl9: 5 '-TAAGGCCCTCGGTAGCAACGG-3' (SEQ ID NO: 16)

p20: 5 '-GGGGTACCAAATCCAACTAG-3' (SEQ ID NO: 17)

Primers p21 and p22 were used to amplify the right target region, with the latter primer also introducing spectinomycin resistance into the amplified part of the 16SrDNA in addition to the SRI resistance (binding type marker).

p21: 5 '-GGAGCTCGCTCCCCCGCCGTCGTTC-3' (SEQ ID NO: 18) p22: 5 '-GATGCATGATGACTTGACGGCATCCTC-3' (SEQ ID NO: 19)

The two approved regions were cloned and sequenced in pBluescript and pZeroBlunt, respectively. The left and right target regions were then cloned into the backbone of the pUC19 vector. The interfaces Eco0109l and PvuII of the vector were used for this. A multiple cloning site from the pBluescript (from Kpnl to Sacl) was cloned between the left and right target regions. This is located in the base vector for plastid transformation between the two plastid-encoded genes trnV and rrnlδ. This base vector for plastid transformation was given the designation pCB42-94 (SEQ ID NO: 1). The vector contains the following sequence elements:

a) Position complementary bp 55-1405: right target region with the partial gene of 16SrRNA (complementary bp 56 to 1322). In the latter there are mutations for streptomycin resistance (SRI, position bp 346) and spectinomycin resistance (SPC1, position bp 68). b) Position complementary bp 2374 to 1510: left target region including 0RF131 (bp 1729 to 2124) and trnV gene (complementary bp 1613 to 1542).

c) Position bp 1404 to 1511: multiple cloning site

d) Position bp 2629 to 3417: ampicilin resistance in the vector backbone

Example 2: Creating a vector (pCB199-3) for the introduction of a non-naturally occurring one

Detection region for the homing endonucleases I-Ppol in the piastome of tobacco

Various elements were successively cloned into the multiple cloning site of the base vector pCB42-94 (SEQ ID NO: 1) for the plastid transformation:

a) for recognition region (mutated, does not contain an Xbal interface; complementary 1307-1354)

b) Expression cassette for the expression of the marker gene aadA consisting of:

i) Promoter of the gene for 16SrRNA (complementary 1191-1281) ii) 5 'untranslated region of the tobacco rbcL gene (complementary 1167-1184) including mutated 5' portions of the rbcL gene (duplication of 6 AS of the rbcL gene, partially mutated, as a result of the cloning strategy, so that a fusion coding for a total of 12 amino acids (complementary 1131-1166) with the following element, the aadA gene, was created)

iii) aadA gene (complementary 336-1130)

iv) the 3 'region of the psbA gene (complementary 232-323)

c) Core recognition region for the homing endonuclease I-Ppol (complementary 176-190).

Instead of the multiple cloning site in the base vector for plastid transformation, this vector with the designation pCB199-3 contains the elements listed within the framework of the nucleic acid sequence with SEQ ID NO: 2. The sequence is given which replaces the complete MCS from Kpnl to Sacl. However, due to the cloning strategy, there is no longer a Kpnl interface in the sequence given.

Example 3: Creating another vector (pCB401-20) for the introduction of a non-naturally occurring one

Detection region for the homing endonucleases I-Ppol in the piastome of tobacco

In contrast to the vector pCB199-3 described in Example 2, the vector described here contains no promoter and 3'UTR, which was linked directly to the selection marker aadA. Rather, the expression of the aadA gene is controlled starting from the promoter of the trnV gene, which is located in the plastid genome or in the left target region upstream of the aadA gene. The goal in creating this vector was

Avoid sequence duplications by exploiting regulatory areas from the tobacco plastid genome. For this purpose, various elements were successively cloned into the multiple cloning site of the base vector pCB42-94 for the plastid transformation:

a) Ribosome binding parts (complementary bp 1033 to 1050)

b) aadA gene (complementary bp 238 to 1032) c) Core recognition region for the homing endonucleases I-Ppol (complementary bp 176 to 190)

The vector obtained in this way also conferred spectinomycin resistance in E. coli. Instead of the multiple cloning site in the base vector pCB42-94 (SEQ ID NO: 1) for the plastid transformation, this vector with the designation pCB401-20 contains the elements listed within the framework of the nucleic acid sequence with the SEQ ID NO: 3. Here, too, the whole is the MCS replacing sequence (from Sacl to Kpnl) given.

Example 4: Creation of predominantly homoplastomic tobacco master plants which contain a non-natural DSB recognition sequence

The plasmid pCB199-3 was introduced into the plastids of tobacco (Nicotiana tabacum cv. Petit Havana) as described below. The regenerated plants were named CB199NTH. Independent lines have been given different end numbers (e.g. CB199NTH-4).

Analogously, the vector pCB401-20 is introduced into the plastids of tobacco. The resulting plants are designated CB401NTH accordingly.

First, leaf disks with a diameter of 2.0 to 2.5 cm were cut out from plants grown in vitro using a sterile cork borer and the top of each leaf was placed on a petri dish with bombardment medium [MS salts (Sigma-Aldrich): 4.3 g / L; Sucrose: 30.0 g / L, phytoagar (Duchefa, P1003): 0.6% (w / v); pH 5.8; after autoclaving, 1.0 mg / L thiamine (Duchefa, T0614) and 0.1 g / L yo-innositol (Duchefa, 10609) were added]. The underside of the leaf facing away from the agar was then bombarded with the particle gun. For this purpose, the plasmid DNA to be transformed (purified from E. coli using Nucleobond AX100 Macherey & Nagel) was first applied to 0.6 μm gold particles according to the following protocol ("coating"). First, 30 mg of gold powder (BioRad) was taken up in ethanol. 60 μl of the gold suspension were transferred to a new Eppendorf tube and the gold particles were sedimented by centrifugation (for 10 seconds). The gold particles were washed twice in 200 μl sterile water and, after a further centrifugation step, taken up in 55 μl water. With constant mixing (vortexing), the following was quickly added:

5 μl plasmid DNA (1 μg / μl) 50 μl 2.5 M CaCl ₂ 20 ul 0.1 M spermidine

The suspension was then vortexed for a further 3 minutes and then centrifuged briefly. The sedimented gold-DNA complexes were washed once or twice in 200 μl ethanol and, after a further centrifugation step, finally taken up in 63 μl ethanol. 3.5 μl (corresponding to 100 μg gold) of this suspension were applied to a macro carrier per shot.

According to the manufacturer's instructions, the particle cannon (BioRad, PDSlOOOHe) was prepared and the leaf explants were bombarded with the gold-DNA complexes at a distance of 10 cm. The following parameters were used: Vacuum: 27 inch Hg, pressure 1100 psi. After the bombardment, the explants are incubated for 2 days in climatic chambers (24 ° C, 16 h light, 8 h dark) and then divided into segments of approximately 0.5 cm2 using a scalpel. These segments were then placed on regeneration medium [bombardment medium plus 1 mg / L 6-benzylaminopurine (BAP, Duchefa, B0904) and 0.1 mg / L naphthylacetic acid (NAA, Duchefa, N0903)] plus 500 mg / L spectinomycin (Duchefa, S0188) transferred and incubated for 10 to 14 days under the above-mentioned conditions in a climatic chamber. At the end of this time, the leaf segments were transferred to fresh regeneration medium plus 500 mg / L spectomycin. This process was repeated until green shoots formed on the explants. The shoots were separated with a scalpel and grown on growth medium (like bombardment medium, but 10 g / L sucrose instead of 30 g / L sucrose) plus 500 mg / L spectinomycin.

Optionally, in order to obtain predominantly homoplastic plants, explants can again be cut off from the regenerated plants and placed on regeneration medium with 1000 mg / L spectinomycin. Regenerating shoots are placed in jars with growth medium plus 500 to 1000 mg / L spectinomycin. After rooting, the plants are transferred to the greenhouse and grown there until they reach seed maturity.

In the case of transformation with the plasmid pCB199-3, 8 plants were obtained which showed resistance to spectinomycin. Using PCR and Southern analyzes, it was demonstrated that three of these lines (lines CB199NTH-4, -6 and -8) actually have incorporated the aadA gene into the plastid genome.

To analyze the transplastomic plants according to Southern

Total DNA from leaves of transformed and non-transformed plants using the GenElute Plant Geno ic DNA Kit (Sigma) isolated. The DNA was taken up in 200 ul eluate. 86 μl of this were each mixed with 10 μl lOx restriction buffer and 40 U restriction endonuclease and incubated for 4 to 8 h at the temperature recommended for the restriction enzyme. The DNA was then precipitated with ethanol in a manner known to the person skilled in the art and then taken up in 20 μl of water. The samples were then separated on an agarose gel according to methods known to the person skilled in the art, the DNA in the gel was denatured and transferred to the nylon membrane by means of capillary blot.

A corresponding probe for radioactive hybridization was created using the HighPrime (Röche) system. The membrane was first covered with HybPuffer (1% (w / v)) bovine seum albumin for 1 h at 65 ° C; 7% (w / v) SDS; 1 M EDTA; 0.5 M sodium phosphate buffer, pH 7.2) prehybridized. The heat denatured probe was then added and hybridization was carried out at 65 ° C. overnight. The blots were then washed as follows: one rinse with 2 x SSPE / 0.1% SDS; for 15 min. wash at 65 ° C with 2 x SSPE / 0.1% SDS; for 15 min. wash at 65 ° C with 1 x SSPE / 0.1% SDS and if necessary the last step was repeated again (20 x SSPE is 3 M NaCl; 0.2 M NaH ₂ P0 ₄ ; 0.5 M EDTA; pH 7.4).

The hybridization was then analyzed using a phospho-imager (Molecular Imager FX, BioRad).

For example, total DNA from various plants cut with PstI and regenerated after transformation with pCBl99-3 was labeled as radioactive with the aadA gene

Probe (793bp PstI \ NcoI fragment from pCBl99-3) hybridized. It was found that the lines CB199NTH-4, -6 and -8 had actually incorporated the aadA gene into the DNA. Furthermore, total DNA of CB199NTH-4, -6 and -8 was cut with EcoRI and Xhol using a radioactively labeled probe

(1082 bp Bspl20I / SacI fragment from pCB199-3) which hybridizes with part of the 16S rDNA. While, as expected, a band of approximately 3100 bp was detected in the wild type (untransformed plant), a band at 1750 bp was predominantly detected in the transplastomic lines, which results from the insertion of the insertion sequence from pCBl99-3 into the piastom (FIG . 9). The plants obtained can be understood as predominantly homotransplastoma. Example 5: Creating transformation vectors that can be used to transform the plastids of master plants CB199NTH using the artificial homing process

5.1 Cloning the Homing Endonuclease I-Ppol

The homing endonuclease I-Ppol was generated from 26 synthetically generated oligonucleotides by means of PCR based on the method of Stemmer WPC et al. (1995) Gene 164: 49-53 (SEQ ID NO: 11). The underlying sequence was derived from the published sequence (Accession No. M38131 nucleotides 86 to 577). Some mutations were introduced to remove recognition parts for restriction endonucleases from the gene, but none of them caused an altered amino acid sequence. The following elements were then successively combined in a pBluescript KS (Stratagene) vector backbone in order to create an I-Ppol expression cassette. The sequence is flanked by the interfaces Kpnl and Sacl.

a) Positions 21 to 111: Prrn promoter

b) Position 118 to 135: 5 'untranslated region of the rbcL gene followed by 18 bp coding for 6 amino acids of the rbcL protein (bp 136-152)

c) Positions 154 to 645: nucleic acid sequence coding for I-Ppol.

d) Position 688-779: 3 'untranslated region of the psbA gene.

The resulting plasmid was named pCB289-13. Despite the expected expression of the enzyme I-Ppol in E. coli, no impairment of growth was found. From the Kpnl to the Sacl interface, the sequence described by SEQ ID NO: 4 resulted (vector backbone is unchanged that of the pBluescript KS).

5.2 Creation of a transformation vector for artificial homing with homologous regions flanking the insertion sequence on both sides

I) without I-Ppol in the insertion sequence

Areas around the I-Ppol recognition region from pCBl99-3 were cut out with PstI and Sacl and alloyed into the PstI and Sacl interfaces of the pBluescript. Subsequently, from this Vector unnecessary interfaces were removed by linearizing them with PstI and Bspl20I and recircularizing the vector after treatment with the Klenow fragment. With the help of commercially available enzyme I-Ppol (PROMEGA GmbH, Mannheim, Germany), the corresponding recognition region was cut in the vector obtained and further interfaces were introduced there using the synthetic oligos pl90 and pl91.

Oligo pl90: 5 '-GTCGACAGATCTTTAA-3' (SEQ ID NO: 20)

Oligo pl91: 5 '-AGATCTGTCGACTTAA-3' (SEQ ID NO: 21)

An expression cassette as a Bglll / Xhol fragment (SEQ ID NO: 6) consisting of the following elements was inserted into the Sall and Bglll interfaces thus introduced:

a) Prpsl6 promoter (complementary 1033-1139)

b) 5'rbcL (complementary 1007-1024) with 18 bp coding for the 6 AS (complementary 989-1006)

c) nptll gene (complementary 185-988)

d) 3'rbcL (complementary 6-143)

The resulting vector was named pCB304-25 and also conferred E. coli cells kanamycin resistance. This vector is no longer linearized by commercially available I-Ppol. The entire insert of the vector pCB304-25 (basic structure pBluescript; from Sacl to Kpnl correspondingly replacing the MCS) is described by SEQ ID NO: 62 and thus comprises the following elements:

a) Position bp 19 to 110: 3 'psbA from tobacco

b) Position bp 149 to 160: non-functional "half side" of the I-Ppol recognition sequence

c) Position bp 171 to 277: Prpslδ promoter

d) position bp 286 to 303: 5'rbcL sequence followed by lδbp coding for the first 6 amino acids of the rbcL protein (bp 304-321)

e) Position bp 322 to 1125: nptll f) Position bp 1167 to 1304: 3'rbcL

g) Position bp 1310 to 1319: non-functional "half-side" of the I-Ppol recognition sequence

II) With I-Ppol in the insertion sequence

With the help of the BamHI and EcoRI interfaces, a BglII / Muni fragment which encoded a 5 'psbA-I-Ppol fusion was additionally introduced into the vector pCB304-25. The resulting vector pCB320-192, under the control of the Prpsl6 promoter, thus expressed the nptll gene and the I-Ppol homing endonuclease. The Bgl II / Mun I fragment is reproduced by SEQ ID NO: 63 and comprises the following elements:

a) Position bp 6 to 82: 5 'psbA

b) Position bp 83 to 574: I-Ppol

5.3 Creation of a transformation vector for artificial

Homing with a homologous region flanking the insertion sequence on one side

From the vector pCB320-192, the elements upstream of the Prpsl6 promoter, which are homologous to those in the master plants CB199NTH, were removed by restriction with Kpnl and Bglll. In their place, a BstXI interface was introduced there using synthetic oligonucleotides pl99 and p200.

pl99 5 '-GATCTCCAGTTAACTGGGGTAC-3' (SEQ ID NO: 22)

p200 5 '-CCCAGTTAACTGGA-3' (SEQ ID NO: 23)

By cutting with BstXI, one can now create DNA ends that are compatible with those that result from restriction with I-Ppol. The vector obtained was designated pCB322-l.

For example, with the enzymes BstXI and Sacl can be made from this

Vector a fragment can be obtained, which on one side has a DNA compatible end that was cut with I-Ppol at its core recognition region, and on the other side

Homology to piastome sequences of master plants CB199NTH. 5.4 Creation of a transformation vector for artificial

Homing without homologous areas around the insertion sequence

The remaining part, which is homologous to recombinant plastid sequences of the master plants CB199NTH, was removed from the vector pCB322-1 with Sacl and BspTI. At the same time, by inserting synthetic oligonucleotides p218 and p219, a BstXI interface was generated here which, after cutting with BstXI, generated DNA ends which are compatible with DNA cut with I-Ppol. The resulting vector was labeled pCB347-33.

p218 5 '-TTAAGCCAGTTAACTGGGCGGAGCT-3' (SEQ ID NO: 24)

p219 5 '-CCGCCCAGTTAACTGGC-3' (SEQ ID NO: 25)

With the enzyme BstXI, a fragment can be isolated from this vector which has DNA ends on both sides which are compatible with the DNA ends which the I-Ppol enzyme generates at its core recognition region.

Example 6: Exploitation of the master plants CB199NTH for plastid transformation by means of DSB induction

6.1 Use of the vectors created under 5.1 and 5.2 to transform the master plants CB199NTH by using the DSBI enzyme I-Ppol

The plasmid pCB304-24 was applied to the gold particles simultaneously with the plasmid pCB289-13 as described in Example 4 and shot in explants of the master plants CB199NTH treated according to the statements in Example 4. In deviation from the descriptions in Example 4, incubation is first carried out for 10 days on the regeneration medium without antibiotic, later kanamycin is used in a concentration of 50 mg / L (in contrast to the 500 mg / L spectinomycin given in Example 4).

The plasmid pCB320-192 was applied to gold particles as described in Example 4. After washing with ethanol, 20 U of commercially available I-Ppol enzyme (Promega) were added. The further treatment is carried out as described above.

Furthermore, in another approach, 0.5 μg of a transcript generated in vitro using the T7 polymerase was applied to the gold particles simultaneously with the plasmid pCB320-192. Hindlll served as a template for in vitro transcription linearized DNA of plasmid pCB289-13. The transcript thus created codes for I-Ppol. After the covering, the explants of the master plants are further treated as described above.

6.2 Use of the vectors created under 5.1 and 5.3 to transform the master plants CB199NTH by using the DSBI enzyme I-Ppol and only one-sided homologous regions

A fragment excised from plasmid pCB322-1 with BstXI and Sacl was eluted from an agarose gel. This fragment was then brought onto gold particles simultaneously with 1 μg in vitro transcript of plasmid pCB289-13 (see Example 6.1) linearized with HindIII. After covering the explants of the master plants CB199NTH, the further treatment is carried out as described in Example 6.1.

6.3 Use of the vectors created under 5.1 and 5.4 to transform the master plants CB199NTH by using the

DSBI enzyme I-Ppol without homologous regions

The insertion sequence was excised from plasmid pCB347-33 using BstXI and eluted from an agarose gel. This fragment was applied to gold particles simultaneously with 1 μg in vitro transcript of the plasmid pCB289-13 linearized with HindIII. Covering and subsequent treatment is carried out as described in Example 6.1.

Example 7: Identification of naturally occurring, endogenous recognition regions for homing endonucleases in plastomes of different plant species

Although no homing endonucleases are known in the plastids of higher plants, known piastome sequences were examined for the presence of recognition regions for homing endonucleases. This was done with the help of the computer program SeqMan II (DNASTAR Inc.). The recognition regions identified in this way are summarized in Table 1.

Based on computer analysis, it was not possible to decide whether I-Scel has a recognition region in the plastid genome or not. The region that can most likely act as a recognition region was created synthetically and integrated as oligonucleotides p276 and p277 in the Xbal and Xhol interface of the pBluescript. The resulting plasmid pCB414-1 was then commercialized using available enzyme I-Scel (Röche) analyzed for the presence of a functional interface. In fact, the plasmid was linearized by I-Scel. From this it can be concluded that I-Scel expressed in plastids also recognizes and cuts this sequence. Another endogenous DSB recognition sequence for a DSBI enzyme has thus been identified.

p276 5 '-TCGAGAAGATCAGCCTGTTATCCCTAGAGTAACT-3' (SEQ ID NO: 26)

p277 5 '-CTAGAGTTACTCTAGGGATAACAGGCTGATCTTC-3' (SEQ ID NO: 27)

Example 8: Cloning of homologous regions from the tobacco plastome which flank the endogenous recognition region for the homing endonuclease I-Cpal

Upstream and downstream of the I-Cpal recognition region, DNA fragments from the 23S rDNA of the tobacco piastome were amplified by means of PCR using the primers p93 and p97 or p98 and p95.

p93: AAAGATCTCCTCACAAAGGGGGTCG (SEQ ID NO: 64)

p97: TCGAAGACTTAGGACCGTTATAG (SEQ ID NO: 65)

p98: AGGAAGACCTTGTCGGGTAAGTTCCG (SEQ ID NO: 66)

p95: CTCAATTGGGGTCTCTCTGTCCAGGTGCAGG (SEQ ID NO: 67)

The fragments thus obtained were used to construct the vector pCB270-1. The fragment from BssHII to BssHII of the pBlueScript vector (SEQ ID NO: 8) is indicated, the 5 'end of the sequence indicated being found at the BssHII interface which is closer to the 3' end of the lacZ gene.

Two Bpil interfaces were introduced between the DNA fragments located upstream and downstream of the I-Cpal recognition regions. Bpil creates overhanging ends that are outside of their detection region. This procedure ensured that the vector pCB270-l can be used equally for the subsequent integration of different introns. For this purpose, simply overhanging ends are generated on the introns to be cloned, which are compatible with the ends generated by Bpil in the vector pCB270-1. In addition, the corresponding nucleotides that are missing between the two fragments of the 23S rDNA in the vector pCB270-1 are added to the introns. The selected areas are so highly conserved that no new regions need to be amplified from other plant species. Also located in the downstream of the I-Cpal detection region A sequence mutation was inserted into the 23S rDNA fragment via the PCR strategy, as was also found in lincomycin-resistant mutants. The sequence of the vector pCB270-1 inserted into the pBluescript vector is reproduced under SEQ ID NO: 8. The sequence includes the following elements:

Fragment of the 23SrDNA upstream of the I-Cpal recognition region: nucleotides 37 to 194

- Fragment of the 23SrDNA upstream of the I-Cpal recognition region: nucleotides 237 to 359

- Point mutation for lincomycin resistance: 352 (native is A here)

The vector pCB234-l has the same structure as the vector pCB270-l, it only has a recognition region for the restriction endonucleases Xhol and Sacl only downstream of the sequence given below.

Example 9: Cloning of the CpLSU2 intron including the homing endonuclease I-Cpal

The CpLSU2 intron (SE ID NO: 15).

p91 5 '-AGAAGACGATCCTAAGG-3' (SEQ ID NO: 28)

p92 5 '-TGAAGACTTGACAAGGAATTTCGC-3' (SEQ ID NO: 29)

The choice of oligonucleotides was such that, as described above, cloning into the Bpil sites of the

Vector pCB234-l was possible. The sequence comprises the following elements:

Position 9-17 - portion of tobacco 23S rDNA missing from pCB234-l

CpLSU2 intron: position 18-893

I-Cpal ORF: position 377-835 Position 894-909 - proportion of tobacco 23S rDNA that is missing in pCB234-l

This fragment, which comprises the CpLSU2 intron, was cloned into the backbone of the vector pGEMTeasy (Promega) (vector pCB141-3). The entire fragment was excised from this vector with Bpil and cloned into the vector pCB234-1 linearized with Bpil. The resulting vector was named pCB254-2.

Example 10: Nuclear LSU rRNA intron from Tetrahymena thermophila

10.1 Cloning the nuclear LSU rRNA intron from Tetrahymena thermophila

The LSU rRNA intron was amplified from the organism Tetrahymena thermophila by means of PCR. The oligonucleotides pl02 and pl03 were again chosen in such a way that the nucleotides of the tobacco 23S rDNA which were missing in pCB234-1 were added to the intron to be amplified.

pl02 (SEQ ID NO: 30):

5 '-AGAAGACGATCCTAAATAGCAATATTTACCTTTGGGACCAAAAGTTATCAGGCATG-3'

pl03 (SEQ ID NO: 31):

5 'TGAAGACTTGACAAGGAATTTCGCTACCTTCGAGTACTCCAAAACTAATC-3'

In addition, the choice of the oligonucleotide pl02 mutated the internal guide sequence (underlined in pl02) so that the intron is spliced from the tobacco at the desired position in the 23SrDNA. The sequence given under SEQ ID NO: 7 - the PCR fragment from Bpil to Bpil interface is given - was cloned into the backbone of the vector pGEMTeasy. The vector obtained was designated pCB220-17. The sequence includes the following elements:

Position 9-12 - proportion of tobacco 23S rDNA that is absent in pCB234-l

- LSU intron: position 13-425

Position 426-446 - portion of tobacco 23S rDNA that is absent in pCB234-l

The Tetrahymena LSU intron, including the attached flanking sequences, was excised from the vector pCB220-17 with Bpil and into the Bpil cleavage sites of the vector pCB234-1 inserted. The resulting product was designated pCB255-l.

10.2 Indirect detection of the splicing activity of the Tetrahymena LSU intron in E. coli

For indirect proof that the modified intron can actually splice in the predetermined environment within the I-Cpal interface, the modified Tetrahymena intron from pCB220-17 together with a portion that surrounds the I-Cpal recognition region from the tobacco 23SrDNA was so in the lacZ gene of the pBluescript clones that, if this intron is spliced into E. coli (strain XLl-Blue), a functional lacZ peptide can be formed. Its expression can be detected in suitable strains by methods familiar to those skilled in the art by converting the substance 5-bromo-4-chloro-3-indolyl-βD-galactopyranoside (X-Gal) in the medium to a blue dye. The corresponding vector was designated pCB315-l. The lacZ gene including the intron in the vector pCB315-l is described by SEQ ID NO: 9. The vector backbone is identical to the pBluescript. The sequence comprises the following elements:

lacZ-5 'Share: complementary (789-765)

- multiple cloning site from the pBluescript: complementary (764-692)

23SRDNA fragment upstream and including the I-Cpal recognition region: complementary (691-682)

modified Tetrahymena intron complementary (681-269)

23SRDNA fragment downstream and including the I-Cpal recognition region: complementary (268-244)

multiple cloning site from the pBluescript: complementary (243-168)

lacZ-5 'Share: complementary (167-1)

As a control, a plasmid was created which corresponds to pCB315-l, only that this plasmid (pCB305-l) lacked the element for the modified Tetrahymena intron. pCB305-l thus served as a positive control to show that the lacZ is still functional with the in-frame nucleotides from the 23SrDNA from the tobacco plastome. This reflects the situation after correct splicing of the Tetrahymena intron. XII-Blue competent cells were transformed with the plasmids pCB315-l and pCB305-l using a method familiar to the person skilled in the art. A single colony was each on LB (Bactotrypton: 10 g / L, yeast extract: 5 g / L, NaCl: 10 g / L, pH7.5) plates, the 15g / L Bacto agar, 40 μg / L ampiciline, 75 μg / L IPTG (isopropyl-ßü-thiogalactopyranoside) and 80 ug / L X-Gal contained, incubated overnight at 37 ° C. In fact, both clones stained blue, suggesting that the modified Tetrahymena intron was spliced in the non-natural environment of the 23SrDNA tobacco in the heterologous organism E. coli.

10.3: Insertion of further sequences into the Tetrahymena LSU intron

In addition to the experiments in Example 10.2, it was investigated whether additional elements can be incorporated into the modified Tetrahymena intron without destroying the splicing activity. For this purpose, pCB315-l was linearized with BglII and the overhanging ends were filled in using the Klenow fragment. An Xhol-Sacl fragment, as occurs in pCB199-3, was then cloned into this vector, also after treatment with the Klenow fragment. This cloning thus inserted a 229 bp fragment into the modified intron. This fragment contains an I-Ppol recognition region. Regardless of the orientation with which the 229bp fragment was inserted into the Tetrahymena intron, a blue color could be detected in the test as described in Example 10.2. This suggests that the Tetrahymena intron can both splice at the desired region in the 23S rDNA, as well as take up additional genetic information and still maintain splice activity.

10.4 Transformation of a natural master plant and

Destruction of the endogenous I-Cpal recognition region with the modified Tetrahymena Intron

According to the principle described in Example 8, the modified Tetrahymena intron was excised from the vector pCB220-17 with Bpil and cloned into the vector pCB234-1 linearized with Bpil. The resulting vector was named pCB255-l. pCB255-l is applied to gold particles simultaneously with in vitro transcript from pCB262-5 (linearized with Sall, use of T7 polymerase) by the method described in Example 4. These gold particles are then shot analogously to the process described in Example 4 in tobacco plants, Petit Havana variety. If necessary, the explants can be selected for lincomycin (250 to 500 mg / L). Example 11: Ll.LtrB intron from Lactocococcus lactis

The Ll. LtrB intron, including a few bases of the flanking exon sequences from Lactococcus lactis, was amplified by PCR using the primers p207 and p208. The PCR product was cloned into the vector pCR2.1TA (Invitrogen) (pCB345-34) and sequenced (SEQ ID NO: 10). Few deviations from the published sequence were found.

p207 5 '-GAGAAGACATTCCTAACACATCCATAACGTGCG-3' (SEQ ID NO: 32)

p208 5 '-TGAAGACTTGACATTTGATATGGTGAAGTAGG-3' (SEQ ID NO: 33)

The cloned fragment in pCB345-34 (from the EcoRI to the EcoRI site of the pCR2. ITA vector) is represented by SEQ ID NO: 10. The rest of the vector is identical to the backbone of pCR2.lTA. The sequence includes the following elements:

Proportion of the natural 5'exon: complementary (2540-2527)

Intron Ll.LtrB: complementary (2526-35)

ORF in the intron: complementary (1953-154)

- Proportion of the natural 3 'exon: complementary (34-28)

Example 12: Creation of a further derivative of the Tetrahymena LSU intron and incorporation of a foreign gene into this intron derivative, as well as transformation into natural master plants

According to a preferred embodiment of this invention, an artificial intron was created which can be built into the plastid genome exactly at the point at which the natural intron is also associated with the DSB recognition sequence under consideration. In the present example, the Tetrahymena LSU intron has been modified such that it may be spliced at the position marked with ^"Λ" position in the identified within the scope of this invention recognition site for the DSBI enzyme I-Cpal in the plastid genome of higher plants: CGATCCTAAGGT ^Λ AGCGAAATTCA.

The gene coding for I-Cpal including an RBS was then inserted into the intron. A splicing-active intron carrying a foreign gene was thus created, which can be incorporated into the plastids of a natural master plant in the middle of an essential gene (coding for the 23S rRNA) by means of the process found within the scope of this invention. 12.1: Creating another Tetrahymena LSU intron derivative

In order to obtain a functional intronderivative at a predefined insertion site, the internal guide sequence (IGS) 5 must be adapted in such a way that it can base pair with the 5 ^X and 3 ^λ exon. FIG. 10 illustrates how this adaptation was carried out in the present example in order to create a Tetrahymena intron derivative which can splice 10 at the natural insertion site of the CpLSU5 intron within the I-Cpal recognition region. Analogously, an adaptation to any insertion point can be carried out. The intron created in this example was designated TetIVS2a and is described by SEQ ID NO: 73.

15 12.2: Indirect detection of the splice activity of the TetIVS2a intron in E. coli

Analogously to example 10.2, the TetIVS2a was incorporated into the lacZ gene of the pBluescript. After corresponding incubation of E. coli 20 XLl-blue cells, which contained the plasmid pCB459-l, a blue color indicated the splicing activity of the TetIVS2a intron at the desired position.

Components of the insert from plasmid pCB459-l (SEQ ID NO: 74; 25 backbone corresponds to pBluescript)

lacZ-3 ^λ portion including parts of the multiple cloning site from the pBluescript (complementary item 1-254) - sequence from the I-Cpal recognition region (complementary item.

30 254-265)

TetIVS2a (complementary item 266-678)

Sequence from the I-Cpal recognition region (complementary pos.

679-687) lacZ-5 'portion including portions of multiple clones

35 from the pBluescript (complementary item 688-791)

12.3: Introduction of further genetic information into the TetIVS2a intron and detection of the splicing activity in E. coli 0

In the context of this example, the gene coding for the DSBI enzyme I-Cpal is introduced into the TetIVS2a without the latter losing its splicing activity at said position within the I-Cpal recognition region. For this purpose, a Bell interface was first inserted into sequence section 5 of TetIVS2a, which corresponds to loop L8 in the Tetrahymena LSU intron, using PCR. Then a non-functional one became in this Bell interface Derivative of the gene coding for I-Cpal incorporated. Since the expression of I-Cpal in E. coli is toxic, a non-functional gene had to be used for the splicing test in E. coli, which gene had previously been generated by linearizing the corresponding gene at the EcoRI interface, the protruding ends were smoothed by Klenow treatment and then the gene sections were ligated together again. This created a grid shift in the reading frame of the gene. By incorporating said intron with the non-functional gene into the lacZ gene of pBluescript, we obtained the plasmid pCB478-3 and, similarly to Example 12.2, by means of blue staining of corresponding colonies, we were also able to splice this intron in E. coli at the desired position within to prove the I-Cpal Erkenunngss elle. Since the functional gene coding for I-Cpal only differs by 4 bases from the non-functional gene used in pCB478-3, it can be assumed that the intron will continue to be used after the functional I-Cpal gene has been inserted instead of the non-functional one has the desired splicing activity.

Components of the insert from plasmid pCB478-3 (SEQ ID NO: 75; backbone corresponds to pBluescript)

lacZ-3 ^l portion including parts of the multiple cloning site from the pBluescript (complementary item 1-265) sequence from the I-Cpal recognition region (complementary item 256-265)

TetIVS2a (complementary item 266-1178), contains a non-functional gene for I-Cpal (complementary item 399-861) and an RBS upstream of the non-functional I-Cpal gene (complementary 866-870)

Sequence from the I-Cpal recognition region (complementary item 1179-1187) lacZ-5 'portion including parts of the multiple cloning site from the pBluescript (complementary item 1179-1291)

12.4: Transformation of a self-propagating, artificial intron in a natural master plant

After it could be shown that the TetIVS2a intron can splice at the desired position and can simultaneously take up additional genetic information without losing this splicing activity, a construct was created which should make it possible to use the I-Cpal gene as a foreign gene using the in the frame to incorporate the method described in the invention into the plastid genome without using a selection marker. For this, the vector pCB492-25 was first created, which contains an insert with the following includes elements (SEQ ID NO: 76; backbone corresponds to that of the pBluescript; sequence is given from BssHII to BssHII in pBluescript, the BssHII interface given here at the 5 'end, that in the pBluescript being the closer to the 3' end of the lacZ Gene is localized):

23SrDNA fragment upstream and including the I-Cpal recognition region (pos. 37-203)

TetIVS2a (Item 204-1112) with inserted gene coding for I-Cpal (Item 521-979) and RBS (Item 512-516)

23SrDNA fragment downstream and including the I-Cpal recognition region (pos. 1113-1247)

In order to ensure expression of the I-Cpal directly after introduction into plastids of natural master plants, a promoter was added in vitro upstream of the said intron-Cpa construct by means of PCR. The primers p396 and p95 as well as pCB492-25 were used as a matrix.

p396 (SEQ ID NO: 77):

5 '-TAGTAAATGACAATTTTCCTCTGAATTATATAATTAACATGGCGACTGTTTACCAAAAAC-3

p95 (SEQ ID NO: 78): 5 '-CTCAATTGGGGTCTCTCTGTCCAGGTGCAGG-3'

The resulting PCR product was named Prom-tetIVS2a-Cpa, is described by SEQ ID NO: 79 and comprised the following elements:

synthetic promoter (items 8-40) - 23SrDNA from tobacco upstream and including portions of the I-Cpal recognition region (items 41-207)

TetIVS2a (pos. 208-1116) comprising gene coding for I-Cpal

(Item 525-983) and RBS (item 516-520)

23SrDNA from tobacco downstream and including parts of the I-Cpal recognition region (pos. 1117-1243)

The plasmid pCB492-25 was applied to the gold particles at the same time as the PCR product Prom-TetIVS2a-Cpa as described in Example 4 and then shot at wild-type tobacco. The expression of the I-Cpal enzyme is said to result in a double-strand break in the 23SrDNA, which is repaired by the PCR product introduced or by the insertion sequence of the plasmid pCB492-25. This inactivates the naturally existing I-Cpal recognition region in the already transformed piastom copies. Plants were regenerated without any selection pressure and these are regenerated using PCR tested for the presence of the insertion sequence in the piastome.

Example 13: Distribution of the modified Tetrahymena LSU intron from pCB255-l in a natural master plant

Expression of the DSBI enzyme I-Cpal in trans

This example shows how a DSBI enzyme can be expressed in the plastids of master plants in order to efficiently distribute an insertion sequence in the copies of the master plant.

13.1: Creation of a vector for plastid transformation which allows the expression of the homing endonuclease I-Cpal in plastids

First, a vector was created that codes for the selection marker aadA and the DSBI enzyme I-Cpal. Since the expression of the I-Cpal enzyme in E. coli is lethal, the accD promoter was chosen to enable expression of this enzyme in the plastids, but to prevent expression in E. coli. This vector could be generated and amplified in a conventional manner with E. coli as the host organism. The resulting vector was designated pCB435-45 and comprised an insert according to SEQ ID NO: 80 with the following elements:

Right target region (as in pCB42-94, see above; complementary

Item 66-1403)

Promoter PaccD (item 1422-1478) - RBS (item 1500-1504)

Gen coding for I-Cpal (Pos. 1509-1967)

Expression cassette for the marker gene aadA consisting of: the 3 * region of the psbA gene (complementary item 2065-1974) aadA gene (complementary item 2872-2078) - 5 'untranslated regions of the tobacco rbcL gene (complementary

Item 2890-2873), partially mutated

Promoter of the gene for the 16SrRNA (complementary item 2987 to

2897)

Left target region (as in pCB42-94, see above; complementary item 3863-3007)

Parts of the pBluescript (including "origin of replication"; items 3864-4746 and 1-65) 13.2 Co-transformation of pCB435-45 and pCB255-l into natural master plants

According to the statements in Example 4, the plasmids pCB435-45 and pCB255-l were simultaneously applied to gold particles and then introduced into plastids of tobacco leaves by means of the particle gun. Analogously to Example 4, transpiastome plants were selected on regeneration medium plus 500 mg / L spectinomycin. As soon as plantlets had formed, they were transferred to growth medium plus 500 mg / L spectinomycin and leaf material was harvested. This leaf material was analyzed by means of ether analysis using the Dig-Easy Hyb® (Röche Diagnostics; Mannheim) with regard to the incorporation of the two plasmids into the plastid genome. In order to determine the proportion of piastom copies which were transgenic with respect to the insertion sequence from pCB435-45, a probe was used which had a sequence according to SEQ ID NO: 81 (directed against parts of the 16SrDNA).

In order to determine the proportion of piastom copies which were transgenic with regard to the insertion sequence from pCB255-1, a probe was used which had a sequence according to SEQ ID NO: 82 (directed against parts of the 23SrDNA).

11 shows that 2 lines (CB255 + 435NTH-19 and -20) were identified in this experiment which are transgenic both with regard to the insertion sequence from pCB435-45 and that from pCB255-1. Furthermore, it could be shown that, surprisingly, the insertion sequence from pCB255-l (modified Tetrahymena LSU intron) was already distributed in more copies of the plastid genome than the insertion sequence from pCB435-45 - although selected for the event of insertion of the insertion sequence from pCB435-45 (aadA marker gene is in pCB435-45). The effectiveness of the method described in the context of this invention - namely the insertion and rapid distribution of an insertion sequence in the plastid genome, without being selected for the presence thereof, through the use of DSBI enzymes and corresponding recognition sites - could thus be said in this example for Lines are detected. Example 14: Generation of further master plants with a DSB recognition region not naturally occurring in plastids, and transformation of these plants using the DSBI enzyme I-Ppol

14.1: Creation of a further vector (pCB456-2) for the introduction of a non-naturally occurring recognition region for the homing endonuclease I-Ppol into the piastome of tobacco

The aim of this approach (analogously to example 3) was to create a further vector for plastid transformation which does not have extensive homologies to sequences in the plastid genome.

The selection marker aadA in this plasmid is under the control of a synthetic promoter, which is derived from the consensus sequence for E. coli σ70 promoters. A region downstream of the 3 'psbA-1 gene from Synechocystis was used as the 3 ^v end. In contrast to the vector pCB199-3 already described, the DSB recognition sequence was introduced into the molecule directly downstream of the aadA gene, but upstream of the 3'psbA-1 sequence from Synechocystis. This enables an operon to be created after insertion of an insertion sequence using a DSBI enzyme. The genes on the insertion sequence can then - optionally - be inserted without a promoter on the insertion sequence. After the insertion, corresponding genes in the insertion sequence then also come under the control of the synthetic promoter upstream of the aadA gene in the master plant. This allows - optionally - to create an operon structure consisting of the aadA and the genes introduced below in the piastom.

To create the plasmid pCB456-2, various elements were successively cloned into the base vector pCB42-94:

- Synthetic promoter (complementary bp 1226-1260) ribosome binding site (complementary bp 1214-1218) aadA gene (complementary bp 414-1208)

Core recognition region for the homing endonuclease I-Ppol (complementary bp 331-345)

3 "psbA-l from Synechocystis (complementary bp 19-155)

The vector obtained in this way also conferred spectinomycin resistance in E. coli. Instead of the multiple cloning site in the base vector pCB42-94 for plastid transformation, this vector with the designation pCB456-2 contains the elements listed within the framework of the nucleic acid sequence with SEQ ID NO: 83. Here too the entire MCS-replacing sequence (from Sacl to Kpnl) is given.

14.2: Creation of predominantly homotransplastomic master plants that contain a non-natural DSB recognition sequence

Analogously to pCBl99-3 in Example 4, the vector pCB456-2 was introduced into the plastids of tobacco. Deviating from the description in Example 4, however, the shoots obtained were cultivated on growth medium which contained 30 g / L sucrose (instead of the 10 g / L given in Example 4). The resulting plants were named CB456NTH. Southern hybridization was used to identify 2 lines (CB456NTH-1 and -15, see FIG. 12) from the spectinomycin-resistant plants which were obtained after the transformation and which had inserted the insertion sequence from pCB456-2 into their piastom. In the Southern experiment, a probe was used which was directed against a fragment of the 16SrDNA (cf. Example 13.2 above). This probe was suitable for detecting a fragment of approximately 3.1 kb from DNA which had been digested with EcoRI and which corresponds to the wild type. In contrast, an approximately 1.7 kb fragment was detected when the insertion sequence from pCB456-2 had been inserted into the corresponding piastom copies.

14.3: Creation of a transformation vector for artificial homing in the master plants CB456NTH

First, an operon structure consisting of the elements RBS - nptll (coding for an enzyme which confers kanamycin resistance) - RBS - I-Ppol (coding for a DSBI enzyme) was created. This cassette was surrounded with BstXI cleavage sites which, after exposure to the enzyme, generate BstXI DNA ends which are compatible with the DNA ends generated by the enzyme I-Ppol. The resulting vector (backbone corresponds to that of the pBluescript) was designated pCB528-2 and contains an insert according to SEQ ID NO: 84 with the following elements:

RBS (item 28-32) - nptll (item 27-840) RBS (item 849-853) gene coding for I-Ppol (item 859-1350)

The 1360 bp fragment from pCB528-2 was then cut out with BstXI and ligated into the I-Ppol site in vector pCB456-2. From the clones obtained after transformation of the ligation products into E. coli, such who had kanamycin resistance. This ensured that said insert in the corresponding clone was inserted into the vector in such a way that the nptII and I-Ppol cassette were in the same orientation as the aadA cassette. This was also verified by the restriction analysis method known to the person skilled in the art. The corresponding vector was named pCB535-ll.

14.4: Transformation of pCB535-ll in master plants CB456NTH Analogously to pCB456-2 in Example 14.2, pCB535-ll was applied to gold particles and then introduced into plastids of the master plant CB456NTH-1 by means of a particle gun.

Some of the explants were incubated on regeneration medium without any selection pressure. The resulting plants were transferred to growth medium (again without selection pressure). The plants are then analyzed by PCR for the presence of the RBS-nptll-RBS-I-Ppol cassette.

Another part of the explants was incubated on regeneration medium plus 15 or 30 mg / L kanamycin. After 2 weeks each, the plants are transferred to fresh regeneration medium and the kanamycin concentration is increased successively to 50 or 80 mg / L.

Claims

claims

1. Method for integrating a DNA sequence into the plastid DNA of a multicellular plant or derived therefrom

Cell and for the selection predominantly homotransplastomer cells or plants, characterized in that

a) the plastid DNA molecules of said multicellular plant at least one recognition sequence for targeted

Induction of DNA double strand breaks included and

b) at least one enzyme suitable for inducing DNA double-strand breaks on the recognition sequence for the targeted induction of DNA double-strand breaks and at least one transformation construct comprising an insertion sequence in at least one plastid of said multicellular plant or cell derived therefrom are brought together, and

c) the induction of DNA double-strand breaks occurs at the recognition sequences for the targeted induction of DNA double-strand breaks, and

d) the insertion sequence is inserted into the plastid DNA, the functionality of the recognition sequence for the targeted induction of DNA double-strand breaks being deactivated, so that said recognition sequence can no longer be cut by the enzyme suitable for the induction of DNA double-strand breaks, and

e) plants or cells are isolated in which the insertion sequence has been inserted into the plastid DNA molecules.

2. The method according to claim 1, characterized in that the recognition sequence for the targeted induction of DNA double-strand breaks and the insertion sequence are flanked at least on one side by sequences which have a sufficient length and sufficient homology to ensure homologous recombination with one another.

3. The method according to any one of claims 1 or 2, characterized in that the transformation construct includes at least one of the elements selected from the group consisting of i) Expression cassette for an enzyme, suitable for inducing DNA double-strand breaks on the recognition sequence for the targeted induction of DNA double-strand breaks

ii) Positive selection markers

iii) negative selection markers

iv) reporter genes

v) Origins of replication

vi) Multiple cloning regions

vii) "Border" sequences for Agrobacterium transfection

viii) sequences which enable homologous recombination or insertion into the genome of a host organism

4. The method according to any one of claims 1 to 3, characterized in that an enzyme suitable for the induction of DNA double-strand breaks on the recognition sequence for the targeted induction of DNA double-strand breaks is selected from the group consisting of restriction endonucleases, homing endonucleases, chimeric nucleases, mutated restriction or homing endonucleases and RNA protein particles derived from group II mobile introns, as well as fusion proteins of the aforementioned proteins with plastid localization sequences.

5. The method according to any one of claims 1 to 4, characterized in that the enzyme is selected from the group of homing endonucleases consisting of F-Scel, F suitable for inducing DNA double-strand breaks on the recognition sequence for the targeted induction of DNA double-strand breaks -Scell, F-Suvl, F-Tevl, F-TevII, I-Amal, I-Anil, I-Ceul, I-CeuAIIP, I-Chul, I-Cmoel, I-Cpal, I-CpaII, I-Crel , I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-Csml, I-Cvul, I-CvuAIP, I-DdiII, I-Dirl, I-Dmol, I-HspNIP, I-Llal, I -Msol, I-Naal, I-NanI, I-NclIP, I-NgrIP, I-NitI, I-Njal, I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI , I-PgrIP, I-PobIP, I-Porl, I-PorIIP, I-PpbIP, I-Ppol, I-SPBetaIP, I-Scal, I-Scel, I-SceII, I-SceIII, I-SceIV, I -SceV, I-SceVI, I-SceVII, I-SexIP, I-SneIP, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp6803I, I-SthPhiJP, I-SthPhiST3P, I-SthPhiS3bP , I-TdeIP, I-Tevl, I-TevII, I-TevlII, I-UarAP, I-UarHGPAlP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI, PI-MtuHIP, PI-MtuHIIP, Pl-PfuI, Pl-PfuII, Pl-Pkol, Pl- PkoII, PI-PspI, PI-Rma43812lP, PI-SPBetalP, Pl-Scel, PI-TfuI, PI-TfuII, PI-Thyl, PI-Tlil, PI-Tlill 5 and fusion proteins of the above with plastid localization sequences.

6. The method according to any one of claims 1 to 5, characterized in that the enzyme is suitable for inducing DNA double-strand breaks on the recognition sequence for targeted

Induction of DNA double-strand breaks is selected from the group of homing endonucleases consisting of the enzymes according to SEQ ID NO: 5, 12, 14 and 71.

15 7. The method according to any one of claims 1 to 6, characterized in that the enzyme is suitable for the induction of DNA double-strand breaks on the recognition sequence for the targeted induction of DNA double-strand breaks is expressed using an expression cassette which one for said

20 Enzyme encoding nucleic acid sequence includes.

8. Expression cassette containing an enzyme suitable for

Induction of DNA double-strand breaks on the recognition sequence for the targeted induction of DNA double-strand breaks 25 according to the definition of one of claims 4 to 6 under the control of a promoter functional in plant plastids.

9. Expression cassette containing a fusion protein from a plastid translocation sequence and an enzyme 30 suitable for inducing DNA double-strand breaks at the recognition sequence for the targeted induction of DNA double-strand breaks according to the definition of one of claims 4 to 6 under the control of a promoter functional in the plant cell nucleus.

35 10. Multi-cell plant obtained by a method according to any one of claims 1 to 7.

11. Multi-cell plant containing an expression cassette according to claim 8 inserted into the plastid DNA.

40

12. Multi-cell plant containing an expression cassette according to claim 9 inserted into the nuclear DNA.

13. Multi-cell plant according to one of claims 10 or 12 selected from the group consisting of Arabidopsis thaliana,

Tobacco, tagetes, wheat, rye, barley, oats, rapeseed, corn, Potato, sugar beet, soy, sunflower, pumpkin or peanut.

14. Cell cultures, organs, tissues, parts or transgenic propagation material derived from a multicellular plant according to claims 10 to 13.

15. Use of an organism according to any one of claims 10 to 13 or cell cultures, organs, tissues, parts or transgenic reproductive material derived from it as claimed in claim 14 as food, feed or seed or for the production of pharmaceuticals or fine chemicals.

16. DNA construct comprising at least one nucleic acid sequence and intron sequence elements which are capable of ensuring the deletion of the ribonucleic acid fragment coding for said nucleic acid sequence in a ribonucleic acid sequence derived from said DNA construct, said nucleic acid sequence being ent in relation to said intron sequence ,

17. DNA construct according to claim 16, wherein the nucleic acid sequence is flanked at least by a splice acceptor sequence and a splice donor sequence.

18. DNA construct according to claim 16 or 17, wherein the DNA construct comprises at the 5 'and 3' end sequences Hl or H2, which have a sufficient length and homology to plastid sequences Hl 'or H2' to to ensure a homologous recombination between Hl and Hl 'or H2 and H2' and thus an insertion of the sequence flanked by Hl and H2 into the piastome.

19. Transgenic plastid DNA comprising at least one nucleic acid sequence and intron sequence elements which are capable of ensuring the deletion of the ribonucleic acid fragment coding for said nucleic acid sequence in a ribonucleic acid sequence derived from said transgenic plastid DNA, said nucleic acid sequence with respect to said nucleic acid sequence is.

20. The transgenic plastid DNA according to claim 19, wherein the nucleic acid sequence is flanked at least by a splice acceptor sequence and a splice donor sequence.

21. A transgenic plant comprising a transgenic plastid DNA according to claim 20.