WO2005059146A1 - Avehicle for plant transformation - Google Patents

Abstract

The invention relates to the field of transgenic plants. More specifically the invention relates to a recombinant plastid vehicle which can be used for transformation. In addition said vehicle can be used for the generation of inter-species hybrid plants and for the optimization of plant fitness.

Description

A vehicle for plant transformation

Field of the invention

The invention relates to the field of transgenic plants. More specifically the invention relates to a recombinant plastid vehicle which can be used for transformation. In addition said vehicle can be used for the generation of inter-species hybrid plants and for the optimization of plant fitness.

Background of the invention The most fundamental steps in the evolution of the eukaryotic cell were the endosymbiotic acquisitions of organelles. The association of formerly free-living prokaryotes with the host cell was followed by an intermixing and restructurating of their genomes. The resulting eukaryotic cell is characterized by a compartmentalized genetic system, in which the individual genetic compartments interact at various levels and co-evolve (Blier et al., 2001; Herrmann et al., 2003). Consequently, an exchange of organelles between species can lead to malfunctions in cellular development and differentiation, a phenomenon referred to as nucleo/organelle incompatibility (NOI). The initial characterization of the NOI phenomena dates back to 1929, when Renner described aberrant pigment phenotypes of interspecies hybrids in the higher plant genus Oenothera (Renner, 1929; Renner, 1934; Stubbe, 1989). Similar studies throughout the plant kingdom showed that interspecies hybrids from many genera exhibit chlorophyll deficiencies and defects in chloroplast development (Michaelis, 1954; Hagemann, 1964; Kirk and Tilney-Bassett, 1987). The loss of normal chloroplast development has been interpreted as a failure of its genetic potential to function in a foreign genetic background, in particular as reciprocal crosses yielded very different results. Chlorophyll deficiencies make it unlikely for hybrids to survive in the wild, hence the genetic mechanisms that determine NOI may prevent or limit gene flow between populations resulting in reproductive isolation. Evolvement of reproductive isolation barriers between populations can result in speciation or strengthening of existing species boundaries. Taken together, this suggests an important role of cytoplasmic genomes in evolution of plants (Grun, 1976; Kirk and Tilney-Bassett, 1987; Levin, 2002)

An ideal subject to study nucleo-cytoplasmic interactions are interspecific nucleus/organelle chimeras that can be generated somatically by protoplast fusion. Cybrids resulting from somatic cell fusions often display severe defects in organelle development, reminiscent of hybrid incompatibilities (e.g. (Thanh et al., 1988; Zubko et al., 2001). A particularly striking example has been described in the family of Solaπaceae between the sexually incompatible species Atropa belladonna (deadly nightshade) and Nicotiana tabacum (tobacco). Cells that combine Atropa nuclei with tobacco plastids, Ab(Nt), yield plants with an albino phenotype that are only viable when grown under heterotrophic conditions (Kushnir et al., 1991). Intriguingly, the reciprocal cybrids, Nt(Ab), with the tobacco nucleus and Atropa plastid, develop as photoautotrophic plants that are almost indistinguishable from tobacco wild-type (Kushnir et al., 1987; Peter et al., 1999). The Atropal tobacco cybrids offer unique opportunities to identify the molecular nature of NOI: (i) The albino phenotype is drastic and easy to score; (ii) In contrast to other plants frequently used as models, somatic cell genetics is well established for Solanaceae, bypassing problems caused by sexual incompatibility, hybrid sterility and relieving the need of time-consuming back-crossing schemes in order to exchange organelles between species; (iii) A further advantage in the choice of tobacco is that - unlike other models like Arabidopsis or maize - its plastids are amenable to reverse genetic approaches by targeted transformation (Svab and Maliga, 1993). This is a crucial experimental approach for the validation of the role of species-specific polymorphisms in nucleo-plastid (in)compatible interactions. Different processes have been proposed to contribute to NOI, such as the failure to process organellar proteins by proteases of chimeric genetic origin (Babiychuk et al., 1995), or suboptimal interactions of nuclear and organellar subunits in multiprotein complexes (Herrmann and Possingham, 1980; Rawson and Burton, 2002; Herrmann et al., 2003). Genetic studies suggested a polygenic origin of NOI (Yao and Cohen, 2000), but unambiguous evidence for a causal connection between a specific molecular defect and NOI is still missing. In order to locate differences in plastid genomes potentially responsible for defects in the Atropal tobacco cybrids, the entire circular plastid chromosome from Atropa belladonna has been sequenced and compared with the corresponding molecule from tobacco (Schmitz- Linneweber et al., 2002). The two molecules turned out to be nearly identical. Among the relatively few differences found, the most conspicuous concern are nucleotide positions that are subject to RNA editing in corresponding transcripts (Schmitz-Linneweber et al., 2002). RNA editing is a process that mediates co- or posttranscriptional alterations of individual nucleotides in distinct RNA species. It is found in organelles of taxa including protozoa, molluscs, mammals and vascular plants (reviewed in (Gott and Emeson, 2000). Editing of plastid encoded RNAs takes place by posttranscriptional cytidine to uridine conversions, and in some species also by the reverse process. It generally changes codon identities leading to the restoration of conserved amino acid residues in the corresponding polypeptides (Maier et al., 1996). In comparison with other types of RNA processing in plastids, such as RNA splicing, RNA editing is evolutionarily highly dynamic. Even closely related species can exhibit quite different patterns of editing sites (editotypes; (Sasaki et al., 2003). Trans factors involved are nuclear encoded and include a set of specificity factors that interact with their plastid target sequences in a site-specific manner (Hirose and Sugiura, 2001; Miyamoto et al., 2002). Moreover, each editing specificity factor seems to be evolutionary linked to the presence of the site it acts on (Bock and Koop, 1997; Reed and Hanson, 1997). It appears that, in addition to the pattern of plastid editing sites, the composition of nuclear-encoded editing factors is species-specific as well, with sites and trans factors co-evolving as interacting pairs. The absence of nuclear factors required for processing a distinct plastid editing site has been shown to have fatal consequences for organelle development (Bock et al., 1994). In the present invention we have analyzed the Ab(Nt) and Nt(Ab) cybrids to unravel mechanisms responsible for nucleo/organellar (in)compatibility. We have surprisingly found a crucial role of plastid editing sites in the species-specific co-adaptation of nuclei and plastids. The finding of this molecular cause of compartmental incompatibility in eukaryotic cells can be industrially exploited in several ways. The plastid genome of higher plants is a circular double-stranded DNA molecule of 120-165 kilobases that may be present in 5,000 - 10,000 copies per leaf cell. The plastid genome has become a very attractive target for genetic manipulation compared to the nuclear genome of the plant for several reasons. Since proteins in plastids may be expressed at a very high level, the molecular machinery of a plastid is essentially a bacterial one. Also, a higher degree of containment can be achieved (virtual lack of transgene pollen transmission in most plants) and because integration of heterologous DNA occurs via homologous recombination mechanism. DNA integrates randomly into the nuclear genome of a plant. The location of integration in the plastid genome, on the other hand, may be controlled such as by way of specific flanking sequences. There is no gene silencing or so-called position effects, so the level of expression is much more predictable. The level of expression is also much higher because there are many more DNA copies per plant cell. The risk of gene release into the environment (referred to as "outcrossing") is essentially eliminated because in most plants chloroplasts do not move into pollen. The transformation of plastids seems to be a bottleneck in plant breeding applications and indeed plastid transformation has only been achieved in a limited number of plants (e.g. tobacco and tomato). The present invention solves this problem since a recombinant plastid is provided in which the editing sites are adapted towards an alien nucleus, id est a plant nucleus derived from a plant species which is normally incompatible with a plastid. The resulting vehicle can easily be transformed to a recombinant plant vehicle which can be further used for the transformation of a variety of plant species. Other applications of this transformation vehicle are the generation of inter-species hybrid plants and the optimization of the fitness of plant species.

Brief description of figures and tables Figure 1. Phenotypes of Atropal tobacco cybrids

For details on the genetic composition of plant materials used see Table 1. A - Cybrid plant of genome composition Nt(Ab). B - Albino cybrid plant Ab(Nt), line Abw3. C - Ab(Nt^m) plant, line L3 in which the cytoplasm ically inherited mutation suppresses albino pigment deficiency of the cybrid line Abw3 shown in B. D - Symmetric nuclear hybrid AbNt(Nt), line Ab27. Inset illustrates differences in flower morphology. Tobacco flower is to the left, Atropa to the right, flower of the hybrid, in the middle, has an intermediate morphology. E - Asymmetric nuclear hybrid Ab^Nt(Nt), line Bar103. Both leaf and flower morphology of this plant is highly similar to the one of Atropa. Figure 2. RNA editing in plastids of cybrids and rescued lines RNA editing was assessed by sequencing PCR products derived from plastid cDNA. Asterisks denote editing sites. (A) RNA editing of tobacco-specific sites in the albino Ab(Nt) cybrid in comparison to editing in wild-type tobacco, Nt(Nt), and rescued lines L3 [Ab(N )] and Bar103 [Ab^Nt(Nt)]. As a control, the albino tobacco mutant, Nt(Δ/po) was assayed as well (De Santis- Maciossek et al., 1999). Note that site atpA-264 is mutated from C to T on the DNA level in line L3. (B) RNA editing of Λfropa-specific sites in the green Nt(Ab) cybrid in comparison to Atropa wild-type. (C) RNA editing of site ndhD-293 in tobacco and parental species. Figure 3. Introduction of recombinant atpA constructs into the tobacco plastid chromosome (A) Plasmid vectors with wild-type and mutant atpA fragments, respectively, designed for plastid transformation of tobacco, were generated by introducing the aadA cassette into the Bcl\ site of a PCR fragment amplified from tobacco plastid DNA (indicated by solid bar). Nucleotide positions on the tobacco plastid chromosome (acc.-no Z00044) are indicated. The promoter driving aaoW-transcription is designated 'P', its 3'-regulatory region (Koop et al., 1996). atpA and aadA are transcribed from left to right, the two tRNA genes, trnR and trnG

' from right to left. In the blow up nucleotide differences between the control construct (KA) and the mutant construct (WAT) including the codon exchange are marked by asterisks. (B) Phenotype of transformed tobacco plants: control line KA-7 (left), variegated leaf from mutant line WAT-9 (middle), albino line WAT-9a (right). All plant material was grown aseptically on agar-solidified MS medium including spectinomycin. (C) Correct integration of the aaαfA cassette was tested using primer pairs P1/P2 and P3/P4 (see A) yielding the expected amplification products of 1197 bp and 1850 bp, respectively, which confirms that the construct has been successfully integrated into the plastid chromosome. (D) As test for the presence of point mutations and homoplastomy of WAT transformants, primers EatpAfor and EatpArev were used to amplify the region spanning the atpA codon 264 from a control plant (KA-7) and from various mutant WAT lines. For variegated lines albino (as) and/or green sectors (gs) were analyzed. An aliquot of the PCR product was digested with the enzyme Mnl\, which recognizes the wild-type (WT), but not the mutant sequence and yields two fragments of 113 and 112 bp. M = Marker with size of bands indicated in base pairs. P = pWAT as template of PCR; N = water instead of template. (E) Northern analysis of atpA and aadA transcripts. Total RNAs from leaves of wild-type tobacco, the Ab(Nt) cybrid, WAT plants and KA control plants were fractionated in formaldehyde containing agarose gels, blotted onto Nylon membranes and hybridized with radioactively labeled, strand specific atpA or aadA probes, respectively. RNA species are given on the right. Methylene blue stains of 25S rRNA below the autoradiograms demonstrate even sample loading (F) Western analysis of plastid proteins. Total leaf proteins of leave tissue samples from in vitro grown tobacco, the Ab(Nt) cybrid, WAT plants and KA control plants, were fractionated by SDS PAGE gel electrophoresis and transferred onto nitrocellulose membranes. Specific plastid proteins were detected by probing filters with monospecific, polyclonal antisera as indicated on the right. The antisera employed were elicited against the α-subunit of the plastid ATP synthase (AtpA), reaction center photosystem I subunit PsaC, the photosystem II subunit PsbE and cytochrome f (PetA) of the cytochrome b₆f complex.

Figure 4. Model for postmating reproductive barriers caused by an insufficient editing capacity Schematic presentation of interpopulation crosses between two populations with different editing capacities for locus E/e. In this example, organelles are inherited maternally. The resulting F1 is heterozygous for the editing factor and hence viable. After backcrossing to the male parent or after selfing, genotypes will segregate that are devoid of the editing factor gene, but still carry the editing site. These genotypes will be burdened with defects in plastid proteins. circle = nucleus; double-oval = plastid; E = editing factor present; e = editing factor absent; U = editing site is processed; C = editing site is not processed; T = editing site absent.

Table 1: ^a GC, Genome Composition. "Ab" or "Nf stands for the nuclear genomes of Atropa belladonna and Nicotiana tabacum, respectively. "(Ab)" or "(Nt)" refers to respective plastomes. "(Nt^al )" refers to the tobacco plastome that carries an unidentified mutation with albino phenotype. ^"m" stands for putative mutations induced in genomes. "AbNf corresponds to a hybrid nuclear genome with complete complements of tobacco and Atropa nuclear genomes as found in symmetric nuclear hybrids. ^"Nr indicates partial presence of the tobacco nuclear genome in asymmetric hybrid. Identification numbers, ID, of genetic lines as known in the laboratory. ^c Pigment phenotype of plants is indicated: "green" - green, photoautotrophic plants; "albino" - a class of pigment deficiency comprising white plants. ^d Beside pigment phenotype, phenotypes of other genes were used as additional selectable markers in protoplast fusion experiments. "nptlF- kanamycin resistance gene cassette; "baf

- resistance gene for the herbicide known as BASTA; ^urm1(F "- a mutated version of the 16S chloroplast ribosomal RNA gene, mutation confers resistance to streptomycin; "x"- stands for an unknown albino mutation with maternal (cytoplasmic) inheritance. ^θ Origins of a given line and some of essential information are summarized. For the plant material generated in this study, schematic presentations of screenings followed to obtain indicated genome composition and ID's of actual parental lines used are shown. NMU - N- nitroso-N-methyl urea, γ-rays were used to generate asymmetric nuclear hybrids. ^f Some of genetic lines were described previously, respective references to original publications are given: (1) Kushnir, 1991; (2) Maliga, 1975; (3) Svab, 1986; (4) Kushnir et al.

(1987). Table 2

U's resulting from posttranscriptional C-to-U editing are given in bold capital letters; codons and "wrong" amino acid residues resulting from non-editing of respective transcripts are shaded, the non-edited nucleotides are given in bold lower case letters; codons and "correct" amino acid residues rescued by point mutation or editing are given in black background, those with increased editing relative to Ab(Nt) plants in dark-grey background; * partially edited

Aims and detailed description of the invention

In the present invention we have demonstrated that nucleo-cytoplasmic substitution lines have a common problem with editing species-specific RNA editing sites in plastids. Analysis of asymmetric nuclear hybrids shows that these problems arise due to the lack in an alien nuclear genome of factors that are required for the editing of individual plastid transcripts. We have further demonstrated that RNA editing defects can have deleterious effects on plant growth and development, thus lowering organism fitness. The invention also shows that evolutionary evolvement of the RNA editing machinery in higher plants contributes for post-zygotic isolation barriers between species and that manipulation of the plastid editing sites can break these barriers. In our experiments we have investigated the problem of nuclear organelle incompatibility (NOI) by the formation of cybrids between Atropa and tobacco. Specific editing defects were detected in the reciprocal Nt(Ab) and Ab(Nt) cybrids. In a first embodiment the present invention provides a method to produce a plant transformation vehicle comprising a) isolating the cDNA's encoded by a plant chloroplast genome, b) comparing the sequence of said cDNA's with the genomic DNA of said chloroplast and identifying the editing sites, c) mutating said editing sites in at least one gene into the corresponding sequence identified in the cDNA encoded by said gene. In another embodiment the invention provides a transformation vehicle obtainable by the method of the invention. A transformation vehicle as understood herein is a mutated plant chloroplast. The mutation is preferentially introduced by homologous recombination. In a particular embodiment said mutation has been introduced by random mutagenesis in the plastid. Random mutagenesis methods comprise UV-mutagenesis and chemical mutagenesis methods known in the art. Thus the transformation vehicle of the present invention comprises at least one mutation. In our experiments we have observed that strategies to rescue the albino Ab(Nt) cybrid by mutagenesis (or by introgression of tobacco nuclear material) always affected a specific editing site in the atpA gene. Furthermore, replacement of the codon in which this editing site resides by a non-editable version in a transplastomic approach yielded albino plants. This shows that the failure to edit atpA mRNA is the major cause of the albino phenotype in Ab(Nt) cybrids. In a preferred embodiment the transformation vehicle comprises a mutation in the atpA gene whereby said mutation means an adaptation of the editing site into the atpA gene for an alien host plant or in other words whereby said mutation means adaptation into a site that does not need editing anymore. Beside the site in atpA, six additional sites located in genes ndhD, rps14, rpl20 and ndhB are negatively affected in albino Ab(Nt) cybrids. Thus in yet another embodiment a transformation vehicle comprises adapted editing sites (or in other words mutated editing sites) in the genes comprising from the list atpA, ndhD, rps14, φl20 and ndhB.

In another embodiment a transformation vehicle is derived from the plant family Solanaceae. In a specific embodiment said transformation vehicle is derived from Nicotiana tabacum. In another particular embodiment the transformation vehicle is a recombinant transformation vehicle.

The term "recombinant vehicle" as used herein refers to a plant transformation vehicle of the invention further comprising sequences containing a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding polynucleotide sequence in said plastid. Plant plastids are known to utilize promoters and 3' sequence elements stabilizing resulting transcripts. In a specific embodiment the recombinant vehicle of the invention comprises an expression cassette. Expression cassettes are generally DNA constructs preferably including (5' to 3' in the direction of transcription): a promoter region, a gene encoding for a desired protein product operatively linked with the translation initiation region, and a 3' stabilizing sequence element. It is understood that all of these regions should be capable of operating in plant plastids to be transformed. The promoter region comprising the transcription initiation region, which preferably includes the RNA polymerase binding site, and the 3' stabilizing sequence may be native to the biological cell to be transformed or may be derived from an alternative source, where the region is functional in the biological cell. The genes encoding for proteins of interest may be expressed in a plastid under the control of a promoter that directs constitutive expression or regulated expression. Regulated expression comprises temporally or spatially regulated expression and any other form of inducible or repressible expression. Temporally means that the expression is induced at a certain time point, for instance, when a certain growth rate of the plant cell culture is obtained (e.g. the promoter is induced only in the stationary phase or at a certain stage of development). Spatially means that the promoter is only active in specific organs, tissues, or cells (e.g. only in roots, leaves, epidermis, guard cells or the like). Other examples of regulated expression comprise promoters whose activity is induced or repressed by adding chemical or physical stimuli to the plant cell. Alternatively, genes encoding proteins of interest are placed under the control of a constitutive promoter. A constitutive promoter directs expression in a wide range of cells under a wide range of conditions.

The term "plant" as used herein refers to vascular plants (e.g. gymnosperms and angiosperms).

These plants may include, but not limited to, plants or plant cells of agronomically important crops, such as tomato, tobacco, diverse herbs such as oregano, basilicum and mint. It may also be applied to plants that produce valuable compounds, e.g. useful as for instance pharmaceuticals.

Plastids are a family of closely related organelles that in one form or another are present in all living plant cells. All plastids share certain features. For example, they have their own small genome and are enclosed by an envelope composed of a double membrane. All plastids develop from protoplastids, relatively small organelles present in meristematic cells. Plastids develop according to the requirements of each differentiated cell. For instance, if the leaf is grown in darkness, the protoplastids develop into etioplasts that contain a yellow chlorophyll precursor called protochlorophyll. If, on the other hand, the leaves are grown in light, the etioplasts further develop into chloroplasts by converting protochlorophyll to chlorophyll. Still other examples of plastids are chromoplasts, leucoplasts and amyloplasts. According to one aspect of the present invention, a process of transformation of plants is provided whereby the recombinant vehicle of the invention is introduced into a non-native environment In a first step a recombinant vehicle is constructed (e.g. a recombinant chloroplast). Thereto a plant comprising the vehicle of the present invention and in which chloroplast transformation can be achieved is selected (e.g. chloroplast transformation has been described for tomato and tobacco). "Transformed" is used herein to mean genetically modified by the incorporation of nucleic acid of interest (e.g., encoding a protein) into plant plastids. In general, the nucleic acid is DNA exogenous to the donor plant (i.e., the plant from which the plastid originates), the recipient and/or the ultimate recipient. By "exogenous", it is meant that the nucleic acid is not normally found in the plant that is to be transformed or not normally found at the copy number that is being introduced. In preferred embodiments, the nucleic acid is exogenous to the plant. Transfer of plastids from one plant species to another can be carried out by fusing of protoplasts derived from the plant species in which chloroplast transformation can take place and a recipient plant cell. Plastid transfer can be also be accomplished by sexual hybridization wherein pollen provides the non-native nuclear environment.

The preferred method for the introduction of the nucleic acid of interest in plastids is by particle gun (biolistics) or PEG-mediated gene transfer. See, Daniell, Methods Enzymol. 217:536-556 (1993); Ye, et al., Plant Mol. Biol. 75 :809-819 (1990) ; Daniell, et al., Proc. Natl. Acad. Sci. U.S.A. 87:88-92 (1990); Daniel, et al., Plant Ceil Reports 9:615-619 (1991) and Svab, et al. Proc. Natl. Acad. Sci. U.S.A. 90:913-917 (1993). The nucleic acid includes a marker in order for the identification of transformants. Selection schemes include spectinomycin resistance due to a mutation in plastid 16S ribosomal RNA genes or conferred by the expression of an engineered bacterial aadA (Svab, et al., Proc. Natl. Acad. Sci. U.S.A. 87:8526-8530 (1990); Svab & Maliga, Proc. Natl. Acad. Sci. USA 90:913-917 (1993) and resistance to kanamycin based on expression of neomycin phosphotransferase (Huang et al. (2002) Mol. Gen. Genet. 268,19-27). The marker gene does not have to be physically linked to the DNA e.g., encoding the protein of interest; it may be delivered via another vector. See, Carrer, et al., BIOTECHNOLOGY 13: 791-794 (1995). It is preferred but not essential that the nucleic acid is integrated into the plastid genome. To facilitate efficient and targeted integration of the nucleic acid of interest into the plastid genome, the nucleic acid is flanked by DNA sequences present in the target plastid. Specific sequences are exemplified in the examples. See also International Patent Publications WO 00/28014 ("Plastid Transformation of Solanaceous Plants") and WO 00/39313 ("Plastid Transformation of Brassica").

The nucleic acid of interest varies widely and embraces any DNA encoding proteins whose expression in plants would be valuable from some standpoint. The invention can for example be used for the introduction new traits in a transplastomic plant that require very high levels of expression. The transplastomic plants produced in accordance with the present invention contain thousands of copies of the DNA in each cell, leading to extraordinary levels of gene expression. The DNAs and proteins fall into the broad categories of crop protection, crop improvement, production of specialty compounds including specific chemicals, neutraceuticals and other products associated with food quality such as modified starch, oils and protein compositions, that, in total, require the expression of a coordinate set of genes and thus a specialized transformation system in order to have the plant exhibit the trait of interest. The exogenous genes can be useful for modifying the input requirements of a plant such as their response to the environment, their ability to protect themselves form pests, protection from exobiotic agents, or which alter other traits such as overall yield, production of nutritionally balanced protein, better quality starch, high quality or quantity of oil or vitamin levels. The genes may also allow the plants to perform functions they normally do not perform such as the production of pharmaceuticals such as proteins (e.g., growth hormones such as somatotropins), antigens and small molecules. For example, the literature has reported the genetic engineering of plastids with transgenes that impart resistance to insects and herbicides and cytoplasmic male sterility. See, McBride, et al., Bio/Technology 13:362-365 (1995) (transplastomic tobacco leaves containing "unprecedented" 3-5% of crylAC protein); Kota, et al., Proc. Natl. Acad. Sci. USA 96:1840-1845 (1999) (reporting over expression of Bt Cry2Aa2 protein in chloroplasts to reduce insect resistance); McBride & Maliga, PCT patent, WO 95/24492; McBride & Stalker, PCT patent WO 95/24493; Daniell, et al., Nature/Biotechnology 16:345-348 (1998); Blowers, et al. PCT patent WO 99/05265; Maliga, PCT patent WO 95/25787.

Once the transformed plastids have been transferred into or formed within the cells of the ultimate recipient plant, transplastomic plants are regenerated from cells that express the selectable marker gene. In preferred embodiments, the plants are regenerated from homoplastomic cells. Homoplastomy may be achieved in accordance with standard techniques such as selective elimination of wild-type plastome copies during repeated cell divisions on a selective medium. Copy number of the DNA (i.e., the introduced sequences) is indicative of whether homoplastomy has been achieved. See, Daniell, era/., (1998), supra, Kanevski, et al., Plant Physiol. 779:133-141 (1999) (obtaining homoplastomic spectinomycin resistant plants by a repeated cycle of shoot regeneration from leaves on the same selective medium and then rooting the shoots on antibiotic-free Murashige-Skoog agar). Once homoplastomic recipient cells are obtained, the transformed plastids are transferred to a cell of the donor plant. The homoplastomic nature of the recipient facilitates the selection following transfer. Preferably, the transfer is conducted via fusing protoplasts derived from the respective cells. Fertile plants may be regenerated from the protoplasts in accordance with standard techniques. Obtaining various plant parts such as roots, shoots, leaves and stems, and deriving seed from the plants are likewise accomplished using standard procedures.

In another embodiment the transformation vehicle of the present invention is used to generate inter-species hybrids comprising a) transforming a transformation vehicle derived from plant species 1 to plant species 2, b) crossing the species generated in a) with plant species 1 and c) obtaining a progeny of inter-species hybrids consisting of species 1 and 2. In a particular embodiment said transformation vehicle is recombinant. In yet another particular embodiment said transformation vehicle is derived from Nicotiana tabacum. In yet another particular embodiment said plant species 1 and 2 belong to the same family. In a particular embodiment said plant family is the Solanaceae. In a specific embodiment the transformation vehicle of the present invention can be used for optimizing the fitness of plant species. Thereto an adapted (recombinant) transformation vehicle of the invention is transformed to a plant. We here show that not only a complete block in editing but also the observed reduction of editing efficiencies, that results in partially edited transcript pools, can cause a developmental disorder. We demonstrate that a single amino acid replacement in AtpA results in an albino phenotype of plants. Both, a significant impact on plant fitness and evolutionary instability are the major requirements for a trait causing NOI. Interestingly, the same could be said of a factor that contributes to the reproductive isolation of species, because loci with these characteristics can cause defects in interpopulation hybrids. Postzygotic isolating mechanisms consist of

hybrid sterility, F₁ hybrid lethality and F₂ hybrid breakdown (Dobzhansky, 1970). As illustrated in Figure 4, interbreeding of populations with different sets of editing factors and likewise different sets of editing sites will lead to F₂- genotypes that are burdened with editing defects and hence decrease the overall reproductive success of hybrids. In addition, even in the F^ editing defects may be encountered in case the allele capable of producing the correct editing factor becomes silenced or lost.

Examples The recombinant DNA and molecular cloning techniques applied in the below examples are all standard methods well known in the art and are e.g. described by Sambrook et al. (1989) Molecular cloning: A laboratory manual, second edition, Cold Spring Harbor Laboratory Press. Methods for plant cell culture and manipulation applied in the below examples are methods described in or derived from methods described in Nagata et al. (1992) Int. Rev. Cytol. 132, 1.

1. RNA editing defects of species-specific sites in both reciprocal Atropal tobacco cybrids Cybrid plants that possess the nuclear genome of Atropa and the plastid genome of tobacco, Ab(Nt), exhibit a pigment deficiency of the albino type, although both genomes are bona fide wild-type (Table 1, Figure 1B). Cells with the reciprocal combination of genomes, Nt(Ab), however produce plants that are green, with no apparent defect in plant development (Table 1 , Figure 1A). The plastid genomes of tobacco and Atropa are more than 96% identical and share gene content and organization. Expression signals known from the well-studied tobacco plastid chromosome, notably promoters, Shine-Dalgarno sequences, transcriptional or translational enhancers, are all fully conserved in Atropa (Schmitz-Linneweber et al., 2002). However, inspection of the editotypes (34 and 32 editing sites in tobacco and Atropa, respectively) revealed five tobacco-specific and three Λfropa-specific editing sites (Table 2; Schmitz-Linneweber et al., 2002). To check whether these species-specific sites are edited on the heterologous nuclear background in cybrids, cDNAs for the sites in question were sequenced (Figure 2 and summarized in Table 2). It turned out that in the albino Ab(Nt) cybrid, tobacco-specific sites atpA-264, ndhD-200 and ndhD-225 were not edited, whereas site psbE- 72 was edited completely and φs14-50 partially (Figure 2A). This editing failure in the albino cybrid is probably not a consequence of impaired chloroplast development, since the five tobacco-specific sites are edited in the albino tobacco mutant disrupted in the plastid rpoB gene (De Santis-Maciossek et al., 1999). Remarkably, sites φs14-27 and φ/20-103 of the tobacco plastid chromosome, although present in Atropa, exhibit only weak editing in the albino Ab(Nt) cybrid (Figure 2A). The sequence environments of both sites are almost identical between Atropa and tobacco. That of site φs14-27 shows only a single base substitution occurring 4 base pairs (bp) downstream of the editing site (G in tobacco; A in Atropa), whereas the next upstream substitution is located 72 bp away. For ηpl20, the coding sequence is identical between both species with a only base substitution found 233 bp upstream of site φ/20-103. While immediate upstream sequences are known to function as a target for the trans machinery (e.g. Chaudhuri and Maliga, 1996; Bock et al., 1997; Miyamoto et al., 2002), these findings provide compelling in vivo evidence that more distantly located sequences may play a role as well and that even downstream located residues may be involved in specific interaction with the editing machinery. As expected, editing of sites ndhB-Λ96 and ndhB-204, which has been shown to be selectively inhibited in bleached tobacco seedlings (Karcher and Bock, 1998) was found significantly reduced in the albino Ab(Nt) cybrid (data not shown). All other sites common between both species were edited (data not shown), confirming that the Ab(Nt) cybrid is not a global editing mutant. In view of the role of plastid RNA editing in restoring codons for phylogenetically highly conserved amino acid residues, the failure to edit three of the five tobacco-specific sites plus the strong decrease in editing of one tobacco- specific (φsf -50) and two common sites (φs14-27 and φ/20-103) is expected to have deleterious consequences for the function of the corresponding polypeptides in the Ab(Nt) cybrid. In the reciprocal green Nt(Ab) cybrid, of the three Λfropa-specific sites, ndhD-293 and nof 7 \-189 were edited efficiently, whereas φoβ-809 remained unedited (Figure 2B). This shows that, concerning editing, even the green cybrid exhibits NOI, although to a much lower extent. This is in accordance with previous data that also indicated subtle defects in this cybrid (Babiychuk et al., 1995; Peter et al., 1999). It is expected that among the various loci involved in NOI, some exert a stronger, others a weaker effect on the plant. Regarding editing defects, it remains to be determined, how strongly each singular editing defect influences the phenotype. The editing defect in rpoB obviously has no dramatic consequence for the function of the corresponding RNA polymerase subunit since φoβ knock-out mutants exhibit an albino phenotype (Allison et al., 1996; De Santis-Maciossek et al., 1999) in contrast to the green Nt(Ab) cybrids. Still, the resulting amino acid exchange in the protein could compromise RNA polymerase function in a more subtle manner, in particular as the concerned leucine codon is conserved in higher plants (data not shown). Shared editing sites φs14-27 and φ/20-103 are fully edited in Nt(Ab) plants, whereas wild-type Atropa edits these sites only partially (Figure 2B). This indicates that the Atropa editing machinery is less efficient than the tobacco one for these sites and explains in part, why editing of these two sites is reduced in Ab(Nt) cybrids. 2. Heterologous editing of plastid RNAs in cybrids

The outlined data established that the tobacco nuclear genome is able to provide the appropriate trans factors for two of three tfropa-specific sites (nofM-189; ndhD-293), whereas the Atropa nucleus is capable of processing only one of the five tobacco-specific sites completely (psόE-72) and a second one partially (φs 4-50). This capacity of nuclear genomes to edit heterologous plastid editing sites is unexpected, since most attempts to edit introduced foreign sites had failed (Bock et al., 1994; Reed and Hanson, 1997). The only instance of heterologous editing reported concerned the homologue o Atropa ndhA- 9 from spinach that is edited in the tobacco nuclear background (Schmitz-Linneweber et al., 2001). Tobacco is allotetraploid with Nicotiana sylvestris and Nicotiana tomentosiformis as parental species and N. sylvestris being the donor of its plastids. It was shown that N. tomentosiformis, the male progenitor, possesses and processes nd7?A-189 and hence probably bequeathed the respective editing capability to its descendant. For ndhD-293, the same scenario seems to apply: again, N. tomentosiformis edits the ndhD mRNA at that position (Figure 2C). The various instances of heterologous editing in the cybrids show that both plant genomes harbour more editing factors than needed to serve their respective plastid transcriptomes. This could indicate that the genes for at least some nuclear editing factors are phylogenetically more stable than anticipated so far. The question remains, whether the perseverance of these factors was a chance event or whether there is a selection for their presence even without the known target site. A factor could for instance be selectively stabilized, because its involvement in processing another site as well (Chateigner-Boutin and Hanson, 2002; Chateigner-Boutin and Hanson, 2003). In any case," the allotetraploid nature of the tobacco nucleus obviously reduces NOI in Nt(Ab) cybrids with respect to RNA editing to a single site (Table 2).

3. Genetic suppression of the albino phenotype of Ab(Nt) cybrids correlates with a loss of the tobacco-specific editing site in the plastid gene atpA

If the failure to edit individual sites in the Ab(Nt) cybrid is contributing to the observed defect in chloroplast development, removal of editing sites should at least partially relieve these problems. One experimental approach to test this possibility was to screen for cytoplasmaticly inherited genetic suppressors and to analyze their editotype. N-nitroso-N-methyl-urea (NMU) is known to induce mutations in chloroplast DNA at high frequency (Hagemann, 1982). This alkylating mutagen was used in a genetic suppressor selection scheme that relied on a selection for green tissue sectors in otherwise white, albino leaves. Three independent green Ab^m(Nf) lines (the "m" note stands for "mutated"), designated AG1, AG2 and AG7, were obtained after mutagenesis of leaf explants from two different Ab(Nt) cybrid lines (Table 1). As compared with albino plants used for mutagenesis, all of the putative suppressor mutants were impaired in normal growth and leaf development, presumably because of a heavy mutational load of the nuclear genome. The reversions from the albino to a green phenotype were stable. No albino shoots or sectors of albino tissue in leaves were ever observed over an extended period of micropropagation, implying that the Ab^m(Nt^m) lines were homoplastomic. Thus, chemical mutagenesis with NMU allowed to isolate genetic suppressors of NOI in Ab(Nt) cybrids. To determine whether genetic suppressor mutation(s) are cytoplasmically inherited, plastids from the randomly chosen Ab^m(Nt^m) line AG7 were first transferred into tobacco cells, and then back to Atropa. The albino cytoplasmic tobacco mutant A15 (Table 1) served as a recipient of the AG7 plastids. In a protoplast fusion experiment, numerous green calli that regenerated tobacco-like shoots were selected. Tobacco plants of the presumed Nt(Nf) genotype were healthy, fertile and after backcrossing with wild-type tobacco produced only green progeny. This established that putative mutation(s) in the plastome of the Ab^m(Nt ) line AG7 do not affect its ability to cooperate with the tobacco nuclear genome in chloroplast development and supports the previous assertion that the poor growth of Ab^m(Nt^m) plants is due to nuclear mutations. Next, plastids from one of the Nt(Nt^m) lines (L3/7) were combined with the nuclear genome of the original Ab(Nt) cybrid albino line Abw3. The resulting plants of the expected genotype Ab(Nf¹) were green, photoautotrophic, and displayed normal vegetative development and the morphology of Atropa, but were male sterile (Figure 1C). Male sterility is a common phenotype for cybrids obtained by protoplast fusions. It is caused by rearrangements in mitochondrial genomes (Hanson, 1991) and was not analyzed further as least relevant to the presented study. Upon pollination with wild-type Atropa, normally looking >A.ropa-like green progeny plants were obtained. Analysis of the Ab(Nt^m) plants in regard of chromosome complement and species-specific nuclear markers, such as isoenzyme types of peroxidases and amylases and tubulin genes, did not reveal the presence of tobacco nuclear genes (data not shown). Taken together, the outlined plastid transfer experiments strongly indicated that the suppressor mutation in the Ab^m(Nt^m) line AG7 is cytoplasmically inherited and most likely borne by the tobacco plastid chromosome. DNA sequence analysis of RNA editing sites in plastomes of the three Ab^m(Nt^m) lines AG1, AG2 and AG7 revealed that, of all the sites, which are not or only partially edited in the Ab(Nt) plants, only the atpA editing site was reversed from C to T in the tobacco plastid chromosome, whereas the other sites remained unaffected (Table 2). Examination of RNA editing in these lines and the line L3 [Ab(Nf")j revealed that the extent of the processing of the other sites was not altered in comparison to the original albino Ab(Nt) cybrid (Fig. 2A). The occurrence of a C to T base change at the tobacco-specific atpA editing site in tobacco plastid chromosomes from plants of three independent lineages suggested that this mutational change may cause the genetic suppression of NOI. 4. Tobacco nuclear genes suppress the albino phenotype of Ab(Ntt plants, which correlates with a normal editing of atpA messenger RNA

To test whether plastids that possess the tobacco plastid chromosome will differentiate into functional chloroplasts on a hybrid tobacco + Atropa nuclear background, symmetric hybrids were generated. Editotype analysis of the AbNt(Nt) hybrid line Ab27 (Figure 1D, Table 1) revealed the rescue of editing of all sites compromised in Ab(Nt) cybrids (data not shown). The tobacco nuclear genome obviously encodes the enzymatic activity(ies) required for the processing of these editing sites. To investigate, whether all of the editing events restored in the symmetric hybrid are necessary to maintain chloroplast development, we earned out a random introduction of tobacco nuclear material into the Ab(Nt) cybrid by generating a radiation hybrid panel (Gleba et al., 1988). Further, this approach also allows estimating the number of tobacco nuclear genes required for editing of the sites in question. To generate the radiation hybrid panel, the nuclear genome of donor cells is fragmented in vivo by ionizing radiation before protoplast fusion (Menczel et al., 1982). Such pre-treatment usually result in elimination of the donor nuclear genome after the first divisions of heterokaryocytes. To impose a positive selective pressure for the recovery of the so-called asymmetric hybrids that carry tobacco chromosomal fragments we used the transgenic tobacco line BarD, which is resistant to an herbicide known as BASTA. BarD was generated by .Agroόacfera/m-mediated genetic transformation with a 35S::bar chimerical gene. Leaf protoplasts of BarD tobacco plants were irradiated with γ rays and fused with protoplasts from the Ab(Nt) albino cybrid Abw3. BASTA-resistant green colonies were selected and regenerating shoots were screened for the absence of tobacco-specific morphological traits. Shoots from the thus selected clone Bar103 developed into Airopa-like green plants of the expected genotype Ab^Nt(Nt). Pollination of flowers of Bar103 plants with wild-type Atropa pollen yielded twelve plants in the progeny that all were purely albino. Most probably, the tobacco nuclear genome fragments were not transmitted to the F1 progeny plants due to their loss during meiosis, further suggesting that the tobacco plastome in Bar103 has not been altered by γ irradiation or propagation in tissue culture, as it is still incompatible with the Atropa nuclear genome. Analysis of RNA editing in the Ab^Nt(Nt) line Bar103 revealed that editing of tobacco-specific sites atpA-264 and φs14-50 was complete, as in tobacco wild-type (Figure 2A). In addition, editing of site ndhD-225 is restored partially, while all other sites were not edited differently from Ab(Nt) plants. This strongly suggests that the introgressed tobacco nuclear genetic material contained genes for several, but not all editing factors that function in plastids of the Ab^Nt(Nt) asymmetric nuclear hybrid. At least three tobacco nuclear loci will be required for the effective processing of all editing sites compromised in Ab(Nt) cybrids as indicated by the complete editing, partial editing or non-editing of individual sites in Ab^Nt(Nt) plants. Editing of sites ndhD-200, and full editing of φs14-27 and φ/20-103, is obviously not required for chloroplast differentiation. Confirming its dependence on active chloroplast metabolism, editing of sites ndhB-^96 and ndhB-204 was restored in the Bar103 plants (data not shown). Intriguingly, similar to the situation in the mutated Ab(Nt) lines, atpA editing seems to go in hand with a regain in photoautotrophy, again concordant with the hypothesis that the failure of editing in atpA mRNA contributes significantly to the albino phenotype of Ab(Nt) plants.

5. A transplastomic substitution of the edited atpA codon leads to albino plants

In order to determine whether editing at the atpA site affects plant development, the wild-type atpA gene, which encodes the α subunit of the thylakoid-located ATP synthase, was replaced by a mutated version in a transplastomic approach. It was not possible to mimic the atpA editing defect in tobacco by the genetic change that will result in L264P amino acid replacement in a nascent AtpA polypeptide, because all praline codons would be changed to codons specifying leucine by C-to-U editing at the second codon position. Therefore, the respective praline codon was replaced by a tryptophane codon (Figure 3A), since this amino acid residue, comparably to praline, does not sterically fit into the α subunit polypeptide, when modeled into the available 3D structure of the spinach ATP synthase (PDB 1 FXO; Groth and Pohl, 2001 ). A plastid transformation vector pWAT was constructed, which carries a mutated version of the atpA gene linked to a selectable marker, the aadA cassette conferring resistance to spectinomycin and streptomycin (Figure 3A). The control vector pKA was identical to pWAT with the exception that it carried the wild-type atpA gene. Both plasmids were introduced into tobacco plastids by microparticle bombardment. Transformed spectinomycin resistant calli were selected, and, after shoot induction, the correct integration of the transgene was verified by PCR analysis (Figure 3C). In this way, more than 40 transformed lines were retrieved. Remarkably, almost one-fourth of all WAT-lines did show variegation, whereas all KA control lines remained green and indistinguishable from wild-type. White sectors were excised and used to regenerate completely white plants (Figure 3B). In this way, altogether 8 independent lines were isolated. The presence of the mutation was analyzed by RFLP analysis of a PCR product spanning the mutated region (Figure 3D). Apparently, white lines were homoplastomic for the mutation as no signal corresponding to the wild-type restriction fragment could be detected, whereas green lines appeared heteroplastomic. Southern analysis confirmed the homoplastomic nature of the transgenic lines transformed with pKA (data not shown). In higher plant plastids atpA is encoded as part of the rps2latpllHIFIA operon which is transcribed from multiple promoters and undergoes complex transcript processing (Miyagi et al., 1998). Accumulation of atpA containing messages in the albino WAT plants was reduced to less than 20% of the wild type level but was normal in KA control lines as assayed by Northern hybridization (Figure 3E). A comparable reduction in atpA mRNA accumulation has been shown for non-photosynthetic tobacco cells and has been attributed to differential development specific transcription and transcript processing (Miyagi et al., 1998). Accordingly, significantly decreased atpA transcript levels were also observed in the albino Ab(Nt) cybrid (Figure 3E). The reduction in atpA transcript accumulation is therefore probably consequence of and not the primary cause for the albino phenotype in WAT plants. Western analysis demonstrated that the amount of AtpA polypeptide in both, the albino WAT mutant and the albino Ab(Nt) cybrid, was reduced to nearly undetectable amounts (Figure 3F). The levels of other thylakoid complex subunits like PetA of the cytochrome b₆f complex, and PsaC and PsbE of photosystem I and II, respectively, were also drastically reduced in both WAT and Ab(Nt) plants (Figure 3F). The expression of the aaofA-cassette conferring spectinomycin-resistance in WAT plants shows that plastid translation is still functional, in contrast to other albinotic tissues like that from the albostrians line of barley (Hess et al., 1994). The observed loss of AtpA could be due to the reduction in atpA message or result from the rapid degradation of the mutant AtpA polypeptide. Loss of AtpA could directly cause albinism, although no such effect has been described so far. Alternatively, the editing factor binding to the mutant atpA message is immobilized on the RNA and hence is not available for a second function. It has been shown that some RNA editing factors are potentially involved in processing more than one site (Chateigner-Boutin and Hanson, 2002; Chateigner-Boutin and Hanson, 2003). If the atpA- factor processed a site important for the function of a protein involved in translation or transcription, its immobilization on the mutant atpA RNA would impact gene expression, which can cause albinism (Allison et al., 1996; Han et al., 1992). There are eleven editing sites in genes coding for ribosomal subunits or in genes coding for subunits of the plastid RNA polymerase in the tobacco plastid chromosome (Tsudzuki et al., 2001). All eleven sites (including φs14-27 and φ/20-103) were processed in WAT plants (data not shown). This makes it unlikely that a secondary editing defect resulting from immobilization of the atpA factor causes albinism. In conclusion, the replacement of the proline codon 264 - de facto a leucine codon after editing in wild-type - by a tryptophane codon leads to a pronounced defect in chloroplast development, i.e. albinism. Hence, the atpA editing defect in Ab(Nt) cybrids can alone explain the observed cybrid albino phenotype, and therefore likely is a decisive factor in NOI in this system.

6. Restoration of Compatibility by Removal of Editing Site afpA-264

Next we tested, whether a P264L codon substitution in tobacco atpA would restore compatibility between the nightshade nuclear genome and the tobacco plastome. Direct plastome transformation of the albino Ab(Nt) cybrid was not feasible due to technical difficulties. In an alternative approach, the tobacco plastid chromosome was genetically manipulated in tobacco and compatibility of the engineered plastomes with the nightshade nucleus was assessed in a second step by interspecific plastid transfer experiments. We constructed transplastomic tobacco CAT lines, genomic composition Nt(Nt^CAT), with a single C to T point mutation leading to a P264L codon substitution. Control KA lines, ttM¹^), were generated, which had only the aadA cassette but no point mutation. Transplastomic plants were used as donors of plastids in protoplast fusion experiments with A. belladonna as plastid recipient. Using lines CAT6 and CAT10 as plastid donors, thirty-two cybrid Ab(Nt^CAT) lines were obtained from four different protoplast fusion experiments. These cybrids were all green, photoautotrophic, and essentially indistinguishable from Ab(N ) plants. In contrast, sixteen Ab(Nt^KA7) cybrid plants were albino like the original Ab(Nt) cybrid. We also used CAT8 as a plastid donor, a line heteroplasmic for the mutation. In this somatic cell hybridisation experiment, both green and albino nightshade cybrids were recovered. Sequence and restriction analysis showed that only the green cybrids contained the C to T point mutation. Taken together, these data prove that nucleo/plastid compatibility between the nightshade nuclear genome and the tobacco plastid genome can be directly restored by a single point mutation.

7. Plant inter-species crosses In the present example we have investigated whether the identified genetic mechanism regarding NOI can influence the barriers for sexual incompatibility between plant species. Wild type Nicotiana tabacum and Atropa belladonna are sexually incompatible species. In reciprocal inter-species pollinations, no initiation of seed development was ever observed. In fact, nightshade flowers pollinated with tobacco pollen usually abscise three to four days after pollination. Tobacco flowers pollinated with nightshade pollen remain attached to the inflorescence stems, which is also true for emasculated but not pollinated flowers. This is probably because tobacco does not have an active abscission mechanism of aging organs, including leaves. However, there is no growth of capsules and ovules on placenta turn brown and necrotic in a few days after pollination of tobacco flowers with nightshade pollen. In a further step we looked at seed formation when as pollen acceptors used were either tobacco or nightshade plants that carry tobacco plastid chromosome with a mutation that suppresses nucleo-cytoplasmic incompatibility between these two species, lines L3 and L3/7, respectively (Table 1). Surprisingly, in both reciprocal crosses the fruits began to enlarge, there was no abscission of fruits. When open, fruits showed the enlargement (growth) of several (10 to 50) ovules per placenta. We did not observe the development of normal seeds in either of crosses, yet planting of developing ovules from a cross N. tabacum L3/7 x A. belladonna onto synthetic plant growth medium resulted in a germination of one of ovules into a plant that by all morphological criteria represented a sexual hybrid between tobacco and nightshade. This experiment shows that alterations in cytoplasmic genomes can affect sexual incompatibility. In a next step we will investigate if the genetic interplay around the editing of the atpA transcript has an impact on hybrid formation and growth. Thereto we have generated trans- plastomic lines of tobacco plants that carry an atpA gene in which the editing site is eliminated by site directed mutagenesis. The outcome of the interspecies crosses between these plants, which have wild type tobacco mitochondriome and no other plastome mutations (except an edited atpA gene), and nightshade will be important to investigate the rale of the atpA gene in sexual hybridization barriers.

Materials and Methods

1.Plant material

Genotypes of wild-type and cybrid plants of Atropa belladonna L. and Nicotiana tabacum L. var. Petit Havana were as described (Kushnir et al., 1991). In plastid transfer experiments, the recipient lines carried genetically marked plastomes, N. tabacum A15 (Svab and Maliga, 1986) and A. belladonna Abw3 (Kushnir et al., 1991). To introduce a selectable marker for the preparation of radiation hybrids, the glufosinate-ammonium (PESTANAL®, Riedel-de Haen GmbH, Germany), also known as commercially available herbicide BASTA, resistant transgenic SR1 tobacco line BarD was generated by standard leaf-disk transformation procedure using Agrobacterium tumefaciens CIRifr (pGV2260; pGSFR890)(De Block et al., 1987). Plants were grown aseptically with 8h/16h dark/light cycles at 0.5 - 1 Wm^"2 (Osram L85 W/25 Universal White fluorescent lamps) on a synthetic MS medium (Murashige and Skoog, 1962) that was supplemented with sucrose (30 g/l) and solidified with 0.6% agar at 25°C.

2.Chemical mutagenesis of leave explants

Chemical mutagenesis of somatic cells was done essentially as described (McCabe et al., 1989). Two separate experiments were carried out with the albino cybrid lines Ab and Abw3, which possess the plastome of N. tabacum var. Gatersleben and of N. tabacum var. Petit Havana, respectively (Kushnir et al., 1991 ). Leaves were cut into pieces of approximately 0.5 x 0.5 cm. Approximately 400 leaf explants were incubated for 1 hour in liquid MS medium supplemented with 5 mM of N-nitroso-N-methylurea (NMU). They were then thoroughly washed with sterile water and cultured in petri dishes containing agar-solidified MS medium supplemented with 30 g/l sucrose, 0.5 mg/l 6-benzylaminopurine (Sigma) and 0.1 mg/l α- naphthalene acetic acid (Sigma) to induce somatic organogenesis. Regenerating shoots were scored for the appearance of green leaf sectors that were used to establish stable lines of putative revertants by the induction of de novo shoot organogenesis followed by micropropagation.

3.Generation of somatic hybrids

Leaf protoplast isolation, fusion and culture were performed as described previously (Kushnir et al., 1991). To generate symmetric nuclear hybrids between Atropa and tobacco that carry a tobacco plastome, leaf protoplasts of the streptomycin resistant tobacco SR1 (Maliga et al., 1975) were fused with leaf protoplasts of the kanamycin resistant Atropa line Ab5 (Kushnir et al., 1991). Calli double resistant to streptomycin (500 μg/ml) and kanamycin (50 μg/ml) were selected and induced to regenerate shoots. These were propagated aseptically on a hormone- free MS medium or transplanted into soil after development of adventitious roots.

In plastid transfer experiments, we relied on a phenomenon of heterokaryon division without fusion of nuclei. This resulted in the segregation of nuclear genomes very early i.e. during the first division(s) of the protoplast fusion product. In all plastid transfer experiments one of the parental lines was albino, thus the difference in pigment phenotype, white-green, was a major screening trait.

To generate asymmetric nuclear hybrids, leaf protoplasts of the tobacco line BarD were irradiated with γ rays from a Co⁶⁰ source and fused with non-irradiated leaf protoplasts of the albino Abw3 line. The ionizing radiation dose used, 500 Gray, completely prevents cell division, i.e. is lethal. Glufosinate ammonium resistant, green calli were selected and induced to regenerate shoots. For further analysis we screened for calli, which regenerated shoots that morphologically resembled Atropa and did not have any of the morphological traits characteristic of tobacco. The morphology of symmetric tobacco + Atropa hybrids served as a reference. Specifically, the counter-selected traits included the absence (i) of abundant trichome development, hirsuteness, a typical tobacco trait; (ii) of characteristic tobacco leaf shape, and (iii) of the morphology of the tobacco root system. The selected Bar103 line displayed Atropa shoot and leaf morphology, and developed greenish adventitious roots, an Atropa trait.

4.Construction of transformation vectors A 2 kbp interval containing an atpA gene fragment was amplified from tobacco plastid DNA using primers HatpAfor and HatpArev and subcloned into TA vector, to yield the recombinant vector designated pSab Identity with the published tobacco atpA was confirmed by sequencing. The aadA expression cassette (Koop et al., 1996) was introduced downstream and in the same transcriptional direction as atpA into the singular Sc/1 site of pSab This clone designated pKA served as template for two Pfu DNA polymerase driven PCRs with primer pairs atpArev-Mu-edatpAfor and atpAfor-Mu-edatpArev, respectively, specifically introducing a TGG tryptophane codon instead of the wild-type CCC proline codon at the atpA codon position 264. A mixture of 20 ng of each purified PCR product served as template for a further PCR with primers HatpAfor and HatpArev. The resulting DNA fragment was digested with BshTI and Λ wa1269l, purified gel-electrophoretically and used to replace the identical sequence in pKA, resulting in plasmid pWAT. The presence of the mutation at atpA codon position 264 and absence of other mutations haphazardly introduced by PCR was confirmed by nucleotide sequence analysis.

5-Plastid transformation and selection for transplastomic lines Leaves of 14-day-old axenically-grown tobacco seedlings were bombarded with plasmid DNA- coated gold particles using a biolistic device (PDS-1000/He system, Bio-Rad, CA). Spectinomycin-resistant shoots were selected on RMOP medium containing 500 mg/l spectinomycin dihydrochloride (Svab and Maliga, 1993). Plastid transformants were identified by PCR using primer pairs P1-P2 and P3-P4, which also test integration polarity into the plastid chromosome. Mnft -digestion of the amplification product derived with primer pair EatpAfor/EatpArev was used to estimate the presence of the mutation as well as the transplastome to wild-type plastid chromosome ratios.

6.Oligonucleotides The following synthetic oligonucleotides (5'-3') were employed for vector construction and analysis of plastid transformants:

HatpAfor: TAGCAAGCTTGGGTACAATAGGCATTGCTC

HatpArev: TAGCAAGCTTAACCCTTTTCCTCAGGATCC

Mu-edatpAfor: TAGCAAGCTTGGGTACAATAGGCATTGCTC Mu-edatpArev: CGGGTCGTCGTATATTATAAGAGTGTGTCGTTCACG

EatpAfor: TTCCAGAATTCACATTACAATACCTTGCTCC

EatpArev: TTCCAGAATTCTTTCCAAAAGGCGTGAATGC

P1: ACGAAATTAGTAATATTATCC

P2: ACTGCGGAGCCGTACAAATG P3: AAGCGGATGTAACTCAATCGG

P4: CGGGTATAGTTTAGTGGTAA

7.lsolation of nucleic acids, RNA analysis, cDNA synthesis, PCR and DNA seguencing

DNA from leaves was isolated as described in (Doyle and Doyle, 1990). Total leaf RNA was isolated using TRIzol reagent (Gibco/BRL). Northern analysis, reverse transcription of RNA primed with hexanucleotide primers using Superscript II (Invitrogen) amplification of cDNA and purification of the products was performed as described (Schmitz-Linneweber et al., 2001). Sequencing reactions were carried out with the DYEnamicTM Terminator cycle sequencing ready reaction kit (Amersham-Pharmacia). Sequencing products were analyzed using an ABI PRISM 377 DNA Sequencer (PE Applied Biosystems).

δ.lmmuno analyses SDS polyacrylamide gel electrophoresis of total leaf proteins was performed as described by Laemmli (Laemmli, 1970). Proteins were electrophoretically transferred onto nitrocellulose filters (Hybond P, Amersham-Pharmacia). The filters were probed with polyclonal antisera raised against the photosystem I subunit PsaC, ATP synthase subunit alpha (AtpA), subunit PsbE of photosystem II and cytochrome f of the cytochrome btf complex. They were developed with goat anti-rabbit serum conjugated with horseradish peroxidase. Peroxidase activity was detected with luminol and H₂O₂ (Sigma).

9.Miscellaneous Species-specific polymorphic markers of nuclear genomes, such as isoenzymes of peroxidases, catalase and malate dehydrogenase, genes encoding plant tubulins were assayed as described (Kushnir et al., 1991). Karyotypes of somatic hybrids were analysed by acetoorceine staining of chromosome spreads from dividing cells in root tips (Gleba et al., 1988). Plastome types were identified by the RFLP analysis of purified chloroplast DNA or by direct DNA sequencing of PCR amplified DNA fragments.

Tables Table 1:

Table 2:

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Claims

Claims

1. A method to produce a plant transformation vehicle comprising a) isolating the cDNA's encoded by a plant chloroplast genome, b) comparing the sequence of said cDNA's with the genomic DNA of said chloroplast and identifying the editing sites, c) mutating said editing sites in at least one gene into the corresponding sequence identified in the cDNA encoded by said gene.

2. A transformation vehicle obtainable by the method of claim 1.

3. A transformation vehicle according to claims 1 and 2 which is derived from Nicotiana tabacum.

4. A transformation vehicle according to claims 1-3 which is a recombinant vehicle.

5. Use of a transformation vehicle according to claims 1-4 to transform plants.

6. Use according to claim 5 wherein said plants belong to the plant family of Solanaceae.

7. Use of a transformation vehicle according to daims 1-4 to generate inter-species hybrids comprising a) transforming a transformation vehicle derived from plant species 1 according to claims 1-4 to plant species 2, b) crossing the species generated in a) with a plant species 1 and c) obtaining a progeny of inter-species hybrids consisting of species 1 and 2.

8. Use according to claim 7 wherein the transformation vehicle is derived from Nicotiana tabacum.

9. Use according to claim 7 wherein said plant species 1 and 2 belong to the same plant family.

10. Use according to claim 9 wherein said plant family is the Solanaceae.