Throughout this application various publications are referenced by citations within parentheses the complete cites for which may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
Transgenic plants are widely used to study nuclear gene function and regulation and to improve agronomically important crop plants (Benfey and Chua, 1989; Weising, et al., 1988). Routine application of the transgenic technology is made feasible by the alternative methods developed for the transformation of the nuclear genome of higher plants. However, the transgenic technology has not been applied yet to the genomes of the cytoplasmic organelles, plastids and mitochondria of higher plants.
The size of plastid DNA (ptDNA) in higher plants is in the range of 120 kb to 160 kb (Palmer, 1985), and encodes the genes involved in plastid maintenance and photosynthesis. By now three plastid genomes have been sequenced, including that of Nicotania tabacum (Shinozaki et al. 1986). In addition to photosynthesis, plastids serve as the compartment for amino acid biosynthesis and lipid biosynthesis (Boyer et al., 1989). Most if not all the genes involved are encoded by the nucleus. The enzymes encoded by nuclear genes are synthesized on cytoplasmic ribosomes, and are subsequently transported into the plastids. Expression and accumulation of nuclear gene products and of plastid gene products is coordinated (Gruissem, 1989; Zurawski and Clegg, 1987).
Formation of stable transformed plastid genomes requires integration of the transforming DNA by recombination. That recombination is a mechanism contributing to the evolution of plastid genome is evident when comparing genomes of different plant species. Also, continued recombination through the plastid inverted repeat has been described (Palmer 1985). It has also been shown that intergenomic plastid recombination occurs in heteroplastidic cells obtained by protoplast fusion (Medgyesy et al. 1985; Thanh and Medgyesy, 1989), and is extensive (Fejes et al., 1990).
Introduction and stable integration of exogenous DNA has been reported recently in the plastid genome of a unicellular alga, Chlamydomonas reinhardtii (Boynton et al. 1988; Blowers et al. 1989). Initial success in transforming the plastid genome of Chlamydomonas by Boynton et al. (1988) was made possible by the development of a microprojectile DNA delivery system based on a particle gun that delivers DNA-coated tungsten microprojectiles into the cell. In addition, a powerful selection scheme was applied that relied on complementing nonphotosynthetic deletion mutants. Subsequently, Blowers et al. (1989) have shown that the Chlamydomonas plastid genome can be expanded by integrating the coding sequence of an E. coli enzyme, neomycin phosphotransferase. Transformation of the psbA gene encoding the D1 reaction center polypeptide of photosystem II, and of the 16s rRNA gene conferring resistance to streptomycin and spectinomycin was reported by Boynton et al., 1990. The psbA and rRNA genes in Chlamydomonas are located in the repeated region. In the transformed clones the repeated regions were identical indicating that transformation was followed by copy correction. The frequency of transformation was as high as 1.4.times.10.sup.-4. Reducing the number of chloroplast genomes five to seven fold by growing the recipient cells on 5-fluorodeoxyuridine prior to transformation increased the frequency of transformation 20 to 280 fold (Boynton et al., 1990).
In higher plant chloroplasts, only transient expression of introduced DNA has been claimed. DNA uptake and transient expression by isolated cucumber etioplasts of the large and small subunits of ribulose bisphosphate carboxylase/oxygenase of Anacystis nidulans, or of the E. coli enzyme, chloramphenicol acetyltransferase (CAT), has been reported (Daniell and McFadden 1987). The 5' end of the psbA (pea), and rbcL (maize) plastid genes were fused with the CAT gene. Transient expression of the constructs in chloroplasts of cultured tobacco cells has been claimed by Daniell, et al. (1990) after biolistic delivery. Some of the vectors contained replication origins from ptDNA. CAT activity was sustained longer when the replicon origins were present. CAT activity, however, was not shown to be localized in chloroplasts. CAT activity, therefore, may be the result of expression in the nucleus, since plastid gene promoters are known to support transcription initiation in the nucleus (Cornelissen and Vandewiele, 1989). European Application No. 87305573.5, filed Jun. 23, 1987, by M. C. Cannon and F. C. Cannon, describes a method for producing a plant whose cells express a desired gene by inserting the desired gene into plastids of a plant cell. However, this application does not suggest a method for stably transforming plastids using nonlethal selection. Transformation of plastids in higher plants was claimed after Agrobacterium-mediated transformation of N. tabacum (DeBlock et al. 1985). A CAT gene was engineered for expression in the nucleus, and transgenic clones were selected for resistance to 10 .mu.g/ml chloramphenicol. The authors claim that there was fortuitous integration of the CAT gene into the ptDNA and expression from a plastid promoter. The line was reportedly unstable, and the authors' claims have not been confirmed.
There are several differences between the Chlamydomonas system and higher plants that maybe relevant for successful transformation of plastids. Two of these are discussed below. The number of plastids, and the number of plastid genomes per cell is much lower in Chlamydomonas than in Nicotiana. Chlamydomonas contains a single plastid. Every plastid carries up to 80 identical plastid genomes (Harris, 1989). Nicotiana tabacum cells contain a variable number of plastids, about 100 in leaf cells, and 12 to 14 in meristematic cells and dedifferentiated tissue culture cells (Thomas and Rose, 1983). In a study with cultured cells the number of plastid genome copies was estimated to be 3,000 to 12,000 per cell (Cannon, et al., 1985; Yasuda, et al., 1988). Clearly, there are many more plastid genome copies in Nicotiana than in Chlamydomonas. Another important difference is that Chlamydomonas cells are grown photoautotrophically which allows a stringent selection for photosynthetic ability, that is, functional plastids. Photoheterotrophic culture in higher plants, however, reduces the stringency of selection for functional plastids, a requirement for transformation with all the proven methods including Agrobacterium-mediated transformation (Weising et al. 1988), electroporation (Fromm et al 1986), calcium phosphate coprecipitation (Krens et al. 1982) and transformation by high-velocity microprojectiles (Klein et al. 1988a), and polyethylene glycol treatment (Negrutiu, et al, 1987).
Given the large number of plastid genomes in plant cells (Possingham and Lawrence, 1983; Palmer, 1985) the ability to select for the transformed genome in culture is a key element in achieving plastid transformation. Available markers are reviewed below. Since most of the selectable plastome markers have been developed through cell culture, it is not surprising that most plastome markers are available in Nicotania tabacum and Nicotiana plumbaginifolia, two species that are easy to grow in cell culture and to subsequently regenerate into plants. Resistance to inhibitors of protein synthesis, conferred by mutation in the plastid 16S rRNA and 23S rRNA genes, are the most readily available markers. The list of markers includes resistance to streptomycin (Maliga et al. 1973; Etzold et al. 1987; Fromm et al. 1989), spectinomycin (Fromm et al. 1987) and lincomycin (Cseplo and Maliga 1984; Cseplo et al. 1988) which are the equivalent rRNA gene mutation used for transformation in Chlamydomonas (Harris et al. 1989). These mutants have been characterized genetically, after plant regeneration, and at the DNA sequence level. Higher plant cells in photoheterotrophic culture respond to these drugs by bleaching and retarded growth, but not cell death. Bleaching in cell culture is not lethal because culture medium containing sucrose dispenses with the requirement for photosynthesis. Differentiation in culture of resistant mutants from the sensitive parental type is based on greening and faster growth. Chlamydomonas cells are grown photoautotrophically, hence the same markers are lethal. In more detail, mutant line SR1 (Maliga et al. 1973; Etzold et al. 1987), SPC1 and SPC2 (unpublished) carry mutations in the 16S rDNA that confer resistance to streptomycin, spectinomycin, and both drugs, respectively. In the SR1 line a change of C to A in the 16S rDNA at position 860 confers resistance to streptomycin (Etzold et al. 1987). The SR1 line is sensitive to spectinomycin. In the SPC1 line mutation A1138 to C in the 16S rDNA confers resistance to spectinomycin. This line is sensitive to streptomycin. The SPC2 line is a derivative of the SR1 streptomycin resistant line, and was selected for spectinomycin resistance. A second rDNA mutation, a change of C to U at position 1139, confers resistance to spectinomycin. The line is resistant to high levels of streptomycin and spectinomycin (500 ug each) and resistance to both drugs is simultaneously expressed (unpublished results of Zora Svab). Mutations similar to those in the SPC1 and SPC2 lines are known in Chlamydomonas (Harris et al. 1989). Plastome mutants resistant to triazine herbicides, have also been obtained in cultured Nicotiana cells. Triazine herbicides inhibit photosynthesis by interruption of electron flow at the acceptor of photosystem II. Selection was feasible in cultures when lowering the concentration of sucrose in the medium made cellular proliferation partially dependent on photosynthesis (photomixotrophic cultures; Cseplo, et al., 1985; Sato, et al., 1988). Selection for resistance to this class of herbicides is also a nonlethal color selection. The resistant clones are identified by their green color (Cseplo, et al., 1985). A mutation in two of the lines was localized to the psbA gene (Pay et al. 1988; Sato et al. 1988). Similar mutant have been found in higher plants under field conditions (Maliga, et al. 1990), and isolated in Chlamydomonas (Erickson, et al., 1985). Naturally occurring resistance to tentoxin is also plastome-encoded (Durbin and Uchytil 1977). Pigment deficiency caused by plastome mutation is frequent. It does not appear to be a useful marker in culture. Pigment mutation in combination with antibiotic resistance mutations, however, proved important in recovering a recombinant plastid genome (Medgyesy et al. 1985).
The present invention provides a method for stable transformation of the plastids of higher plants. Others have attempted to obtain stable plastid transformation in higher plants, but without success.