Stable transformation of petunia plastids

Plastid transformation results in stably expressed foreign genes, which for most Angiosperms are largely excluded from sperm cells, thereby greatly reducing the risk of foreign gene spread through pollen. Prior to this work, fertile plastid transformants were restricted to tobacco, tomato and Lesquerella . Application of plastid engineering in the important floriculture industry requires the development of stable plastid transformation in a major ornamental plant species such as Petunia hybrida. Here we describe the successful isolation of fertile and stable plastid transformants in a commercial cultivar of P. hybrida (var. Pink Wave). Plastid targeting regions from tobacco were used to integrate aad A and gusA between the acc D and rbc L genes of P. hybrida plastid DNA following particle bombardment of leaves. For three spectinomycin and streptomycin resistant lines, DNA blot analysis confirmed transgene integration into plastid DNA and homoplasmy. Maternal inheritance and homoplasmy resulted in 100 transmission of spectinomycin resistance to progeny after selfing. Plastid transformants expressed the gusA gene uniformly within leaves and to comparable levels in all three lines. Insertion of trait genes in place of gusA coding sequences enables immediate applications of our plastid transformation vector. Establishment of plastid transformation in P. hybrida facilitates a safe and reliable use of this important ornamental plant for research and plant biotechnology.These two authors contributed equally to this work.     

Chloroplast transformation in plants: polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems

Nicotiana plumbaginifolia protoplasts were directly transformed by PEG treatment with a cloned 16S rRNA gene isolated from a double antibiotic-resistant Nicotiana tabacum plastid mutant. Putative plastid transformants were selected in cell culture by their spectinomycin resistance and identified by their unselected streptomycin resistance. Alternatively, cell lines were selected in the presence of both antibiotics. The cell line (and its regenerated plants) selected solely for spectinomycin resistance demonstrated an extensive segregation of streptomycin resistance in subsequent tests, while the double-selected line showed stable resistance for both antibiotics. The resistance markers were inherited maternally. In the putative plastid transformants the origin of the resistance mutations was identified by the absence of an Aat ll site, missing in the donor N. tabacum plastid gene (spectinomycin resistance site) but present in that of wild-type N. plumbaginifolia , and a sequence analysis of the particular nucleotide changes in both resistance sites. Restriction enzyme analysis of total plastid DNA (ptDNA), and the recloning and full sequencing of the fragment introduced, investigated in one of the plastid transformants, showed no DNA rearrangements accompanied with the integration process. Sequence analysis indicated a targeted, homologous integration of the DNA fragment introduced but an unexpectedly complete homology of the parental ptDNA sequences in this region prevented the location of borders. Although the frequency of plastid transformant colonies (2 × 10⁻⁵) should still be improved, this method for stable chloroplast DNA transformation is comparable with or more efficient than the particle bombardment techniques.     

Simple and efficient plastid transformation system for the liverwort <Emphasis Type="Italic">Marchantia polymorpha</Emphasis> L. suspension-culture cells

We have established a simple and efficient plastid transformation system for liverwort, Marchantia polymorpha L., suspension-culture cells, which are homogenous, chloroplast-rich and␣rapidly growing. Plasmid pCS31 was constructed to integrate an aadA expression cassette for spectinomycin-resistance into the trnI–trnA intergenic region of the liverwort plastid DNA by homologous recombination. Liverwort suspension-culture cells were bombarded with pCS31-coated gold projectiles and selected on a medium containing spectinomycin. Plastid transformants were reproducibly isolated from the obtained spectinomycin-resistant calli. Selection on a sucrose-free medium greatly improved the efficiency of selection of plastid transformants. Homoplasmic plastid transformant lines were established by␣successive subculturing for 14 weeks or longer on the spectinomycin-containing medium. The plastid transformation system of liverwort suspension-culture cells should facilitate the investigation of the fundamental genetic systems of plastid DNA, such as replication.     

Homeologous plastid DNA transformation in tobacco is mediated by multiple recombination events.

Efficient plastid transformation has been achieved in Nicotiana tabacum using cloned plastid DNA of Solanum nigrum carrying mutations conferring spectinomycin and streptomycin resistance. The use of the incompletely homologous (homeologous) Solanum plastid DNA as donor resulted in a Nicotiana plastid transformation frequency comparable with that of other experiments where completely homologous plastid DNA was introduced. Physical mapping and nucleotide sequence analysis of the targeted plastid DNA region in the transformants demonstrated efficient site-specific integration of the 7.8-kb Solanum plastid DNA and the exclusion of the vector DNA. The integration of the cloned Solanum plastid DNA into the Nicotiana plastid genome involved multiple recombination events as revealed by the presence of discontinuous tracts of Solanum-specific sequences that were interspersed between Nicotiana-specific markers. Marked position effects resulted in very frequent cointegration of the nonselected peripheral donor markers located adjacent to the vector DNA. Data presented here on the efficiency and features of homeologous plastid DNA recombination are consistent with the existence of an active RecA-mediated, but a diminished mismatch, recombination/repair system in higher-plant plastids.     

Long regions of homologous DNA are incorporated into the tobacco plastid genome by transformation.

We investigated the size of flanking DNA incorporated into the tobacco plastid genome alongside a selectable antibiotic resistance mutation. The results showed that integration of a long uninterrupted region of homologous DNA, rather than of small fragments as previously thought, is the more likely event in plastid transformation of land plants. Transforming plasmid pJS75 contains a 6.2-kb DNA fragment from the inverted repeat region of the tobacco plastid genome. A spectinomycin resistance mutation is encoded in the gene of the 16S rRNA and, 3.2 kb away, a streptomycin resistance mutation is encoded in exon II of the ribosomal protein gene rps12. Transplastomic lines were obtained after introduction of pJS75 DNA into leaf cells by the biolistic process and selection for the spectinomycin resistance marker. Homologous replacement of resident wild-type sequences resulted in integration of all, or almost all, of the 6.2-kb plastid DNA sequence from pJS75. Plasmid pJS75, which contains engineered cloning sites between two selectable markers, can be used as a plastid insertion vector.     

Streptomycin and lincomycin resistances are selective plastid markers in cultured Nicotiana cells

Summary Resistance to streptomycin and lincomycin in plant cell culture is used as a color marker: resistant cells are green whereas sensitive cells are white on the selective medium. Streptomycin and lincomycin at appropriate concentrations do not kill sensitive Nicotiana cells. The selective value of plastid ribosomal DNA mutations, conferring resistance to streptomycin and lincomycin, was investigated by growing heteroplastidic cells on a selective medium. The heteroplastidic cells were obtained by protoplast fusion, and contained a mixed population of streptomycin resistant plastids from the N. tabacum line Nt-SR1-Kan2, and lincomycin resistant plastids from the N. plumbaginifolia line Np-LR400-Hyg1. Clones derived from protoplast fusion were selected by kanamycin and hygromycin resistance, transgenic nuclear markers. Somatic hybrids were then grown on a selective streptomycin or lincomycin medium, or in the absence of either drug to a 50 to 100 mg size callus. Southern analysis of a polymorphic region of plastid DNA (ptDNA) revealed that somatic hybrids grown on streptomycin contained almost exclusively ptDNA from the streptomycin resistant parent, somatic hybrids grown on lincomycin contained almost exclusively ptDNA from the lincomycin resistant parent whereas somatic hybrids grown in the absence of either drug contained mixed parental plastids. Sensitive ptDNA was below detection level in most clones on selective medium, but could be recovered upon subsequent culture in the presence of the appropriate drug. The drugs streptomycin and lincomycin provide a powerful selection pressure that should facilitate recovery of plastid transformants.     

Mutations conferring lincomycin, spectinomycin, and streptomycin resistance in Solanum nigrum are located in three different chloroplast genes

A number of Solanum nigrum mutants resistant to the antibiotics spectinomycin, streptomycin and lincomycin have been isolated from regenerating leaf strips after mutagenesis with nitroso-methylurea. Selection of streptomycin- and spectinomycin-resistant mutants has been described earlier. Lincomycin-resistant mutants show resistance to higher levels of the antibiotic than used in the initial selection, and in the most resistant mutant (Ll7A1) maternal inheritance of the trait was demonstrated. The lincomycin-resistant mutant L17A1 and a streptomycin plus spectinomycin resistant double mutant (StSpl) were chosen for detailed molecular characterisation. Regions of the plastid DNA, within the genes encoding 16S and 23S rRNA and rps12 (3) were sequenced. For spectinomycin and lincomycin resistance, base changes identical to those in similar Nicotiana mutants were identified. Streptomycin resistance is associated with an A C change at codon 87 of rps 12 (converting a lysine into a glutamine), three codons upstream from a mutation earlier reported for Nicotiana. This site has not previously been implicated in streptomycin resistance mutations of higher plants, but has been found in Escherichia coli. The value of these mutants for studies on plastid genetics is discussed.     

Stable chloroplast transformation in cabbage (<Emphasis Type="Italic">Brassica oleracea </Emphasis>L. var. <Emphasis Type="Italic">capitata </Emphasis>L.) by particle bombardment

The objectives of this research were first to isolate plastid gene sequences from cabbage (Brassica oleracea L. var. capitata L.), and to establish the chloroplast transformation technology of Brassica. A universal transformation vector (pASCC201) for Brassica chloroplast was constructed with trnV–rrn16S (left) and trnI–trnA–rrn23S (right) of the IR_A region as a recombination site for the transformed gene. In transforming plasmid pASCC201, a chimeric aadA gene was cloned between the rrn16S and rrn23S plastid gene borders. Expression of aadA confers resistance to spectinomycin and streptomycin antibiotics. The uidA gene was also inserted into the pASCC201 and transferred into the leaf cells of cabbage via particle gun mediated transformation. Regenerated plantlets were selected by 200 mg/l spectinomycin and streptomycin. After antibiotic selection, the regeneration percentage of the two cabbage cultivars was about 2.7–3.3%. The results of PCR testing and Southern blot analysis confirmed that the uidA and aadA genes were present in the chloroplast genome via homologously recombined. Northern blot hybridizations, immunoblotting and GUS histochemical assays indicated that the uidA gene were stable integrated into the chloroplast genome. Foreign protein was accumulated at 3.2–5.2% of the total soluble protein in transgenic mature leaves. These results suggest that the expression of a variety of foreign genes in the chloroplast genome will be a powerful tool for use in future studies.     

Transfer of transformed <Emphasis Type="Italic">Lesquerella fendleri</Emphasis> (Gray) Wats. chloroplasts into <Emphasis Type="Italic">Orychophragmus violaceus</Emphasis> (L.) O.E. Schulz by protoplast fusion

Asymmetric intergeneric hybrid plants were obtained through protoplast fusion between Orychophragmus violaceus (L.) O.E. Schulz and Lesquerella fendleri (Gray) Wats. The latter carried chloroplasts transformed with the fused aadA16gfp gene construct, conferring streptomycin–spectinomycin resistance and UV-induced green fluorescence. The somatic hybrids were selected using the properties of spectinomycin-induced plastid defects in “albino” O. violaceus plants (chloroplast recipient) combined with the γ-irradiation-induced inactivation of nuclei in plastid donor L. fendleri. The morphology and esterase isozyme pattern of the hybrid plant as well as the results of the PCR analysis of internal transcribed spacer of nuclear ribosomal DNA proved that the regenerated hybrids carried O. violaceus nuclei, while PCR amplification of the atpB– rbcL spacer and aadA16gfp gene fragments confirmed the presence of the transformed L. fendleri chloroplasts in these plants. Expression of the fused aadA16gfp gene construct was confirmed by sodium dodecylsulfate–polyacrylamide gel electrophoresis analysis and the resistance of the obtained plants to both streptomycin and spectinomycin.     

Next generation synthetic vectors for transformation of the plastid genome of higher plants

Plastid transformation vectors are E. coli plasmids carrying a plastid marker gene for selection, adjacent cloning sites and flanking plastid DNA to target insertions in the plastid genome by homologous recombination. We report here on a family of next generation plastid vectors carrying synthetic DNA vector arms targeting insertions in the rbcL-accD intergenic region of the tobacco (Nicotiana tabacum) plastid genome. The pSS22 plasmid carries only synthetic vector arms from which the undesirable restriction sites have been removed by point mutations. The pSS24 vector carries a c-Myc tagged spectinomycin resistance (aadA) marker gene whereas in vector pSS30 aadA is flanked with loxP sequences for post-transformation marker excision. The synthetic vectors will enable direct manipulation of passenger genes in the transformation vector targeting insertions in the rbcL-accD intergenic region that contains many commonly used restriction sites. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Stability of Soybean Recombinant Plastome Over Six Generations

The stability of a plastid transgene has been evaluated in soybean transformants over six generations. These transformants had integrated the aadA selection cassette in the intergenic region between the rps12/7 and trnV genes. Three independent homoplasmic T0 transformation events were selected and ten plants from each event propagated to generation T5 in the absence of selection pressure. No transgene rearrangement nor wild-type plastome were detected in generation T5 by Southern blot analysis. All tested progenies were uniformly resistant to spectinomycin. Therefore, soybean transformants of generations T0 and T5 appear to be genetically and phenotypically identical.     

Mutations conferring lincomycin,spectinomycin, and streptomycin resistance in Solanum nigrum are located in three different chloroplast genes

A number of Solanum nigrum mutants resistant to the antibiotics spectinomycin, streptomycin and lincomycin have been isolated from regenerating leaf strips after mutagenesis with nitroso-methylurea. Selection of streptomycin- and spectinomycin-resistant mutants has been described earlier. Lincomycin-resistant mutants show resistance to higher levels of the antibiotic than used in the initial selection, and in the most resistant mutant (Ll7A1) maternal inheritance of the trait was demonstrated. The lincomycin-resistant mutant L17A1 and a streptomycin plus spectinomycin resistant double mutant (StSpl) were chosen for detailed molecular characterisation. Regions of the plastid DNA, within the genes encoding 16S and 23S rRNA and rps12 (3′) were sequenced. For spectinomycin and lincomycin resistance, base changes identical to those in similar Nicotiana mutants were identified. Streptomycin resistance is associated with an A → C change at codon 87 of rps 12 (converting a lysine into a glutamine), three codons upstream from a mutation earlier reported for Nicotiana. This site has not previously been implicated in streptomycin resistance mutations of higher plants, but has been found in Escherichia coli. The value of these mutants for studies on plastid genetics is discussed.     

Spontaneous spectinomycin resistance mutations of the chloroplast <Emphasis Type="Italic">rrn16</Emphasis> gene in <Emphasis Type="Italic">Daucus carota</Emphasis> callus lines

Bioballistic transformation of carrot (Daucus carota L.) callus cultures with a plasmid containing the aadA (aminoglycoside 3′-adenyltransferase) gene and subsequent selection of transformants on a selective medium containing spectinomycin (100–500 mg/l) yielded ten callus lines resistant to this antibiotic. PCR analysis did not detect exogenous DNA in the genomes of spectinomycin-resistant calluses. Resistance proved to be due to spontaneous mutations that occurred in two different regions of the chloroplast rrn16 gene, which codes for the 16S rRNA. Six lines displayed the G > T or G > C transverions in position 1012 of the rrn16 gene, and three lines had the A > G transition in position 1138 of the gene. Chloroplast mutations arising during passages of callus cultures in the presence of spectinomycin were described in D. carota for the first time. The cause of spectinomycin resistance was not identified in one line. The mutations observed in the D. carota plastid genome occurred in the region that is involved in the formation of a double-stranded region at the 3′ end of the 16S rRNA and coincided in positions with the nucleotide substitutions found in spectinomycin-resistant plants of tobacco Nicotiana tabacum L. and bladderpod Lesquerella fendleri L.     

Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants.

Plastid transformation in higher plants is accomplished through a gradual process, during which all the 300-10,000 plastid genome copies are uniformly altered. Antibiotic resistance genes incorporated in the plastid genome facilitate maintenance of transplastomes during this process. Given the high number of plastid genome copies in a cell, transformation unavoidably yields chimeric tissues, which requires the identification of transplastomic cells in order to regenerate plants. In the chimeric tissue, however, antibiotic resistance is not cell autonomous: transplastomic and wild-type sectors both have a resistant phenotype because of phenotypic masking by the transgenic cells. We report a system of marker genes for plastid transformation, termed FLARE-S, which is obtained by translationally fusing aminoglycoside 3"-adenyltransferase with the Aequorea victoria green fluorescent protein. 3"-adenyltransferase (FLARE-S) confers resistance to both spectinomycin and streptomycin. The utility of FLARE-S is shown by tracking segregation of individual transformed and wild-type plastids in tobacco and rice plants after bombardment with FLARE-S vector DNA and selection for spectinomycin and streptomycin resistance, respectively. This method facilitates the extension of plastid transformation to nongreen plastids in embryogenic cells of cereal crops.     

Persistence of unselected transgenic DNA during a plastid transformation and segregation approach to herbicide resistance

The use of a nonlethal selection scheme, most often using the aadA gene that confers resistance to spectinomycin and streptomycin, has been considered critical for recovery of plastid transformation events. In this study, the plastid-lethal markers, glyphosate or phosphinothricin herbicides, were used to develop a selection scheme for plastids that circumvents the need for integration of an antibiotic resistance marker. The effect of selective agents on tobacco (Nicotiana tabacum) mesophyll chloroplasts was first examined by transmission electron microscopy. We found that at concentrations typically used for selection of nuclear transformants, herbicides caused rapid disintegration of plastid membranes, whereas antibiotics had no apparent effect. To overcome this apparent herbicide lethality to plastids, a "transformation segregation" scheme was developed that used two independent transformation vectors for a cotransformation approach and two different selective agents in a phased selection scheme. One transformation vector carried an antibiotic resistance (aadA) marker used for early nonlethal selection, and the other transformation vector carried the herbicide (CP4 or bar) resistance marker for use in a subsequent lethal selection phase. Because the two markers were carried on separate plasmids and were targeted to different locations on the plastid genome, we reasoned that segregation of the two markers in some transplastomic lines could occur. We report here a plastid cotransformation frequency of 50% to 64%, with a high frequency (20%) of these giving rise to transformation segregants containing exclusively the initially nonselected herbicide resistance marker. Our studies indicate a high degree of persistence of unselected transforming DNA, providing useful insights into plastid chromosome dynamics.     

Mutation proximal to the tRNA binding region of the Nicotiana plastid 16S rRNA confers resistance to spectinomycin.

Summary Nicotiana tabacum lines carrying maternally inherited resistance to spectinomycin were obtained by selection for green callus in cultures bleached by spectinomycin. Two levels of resistance was found. SPC1 and SPC2 seedlings are resistant to high levels (500 g/ml), SPC23 seedlings are resistant to low levels (50 g/ml) of spectinomycin. Lines SPC2 and SPC23 are derivatives of the SR1 streptomycin-resistant plastome mutant. Spectinomycin resistance is due to mutations in the plastid 16S ribosomal RNA: SPC1, an A to C change at position 1138; SPC2, a C to U change at position 1139; SPC23, a G to A change at position 1333. Mutations similar to those in the SPC1 and SPC2 lines have been previously described, and disrupt a conserved 16S ribosomal RNA stem structure. The mutation in the SPC23 line is the first reported case of a mutation close to the region of the 16S rRNA involved in the formation of the initiation complex. The new mutants provide markers for selecting plastid transformants.     

Kanamycin resistance as a selectable marker for plastid transformation in tobacco

We report on a novel chimeric gene that confers kanamycin resistance on tobacco plastids. The kan gene from the bacterial transposon Tn5, encoding neomycin phosphotransferase (NPTII), was placed under control of plastid expression signals and cloned between rbcL and ORF512 plastid gene sequences to target the insertion of the chimeric gene into the plastid genome. Transforming plasmid pTNH32 DNA was introduced into tobacco leaves by the biolistic procedure, and plastid transformants were selected by their resistance to 50 g/ml of kanamycin monosulfate. The regenerated plants uniformly transmitted the transplastome to the maternal progeny. Resistant clones resulting from incorporation of the chimeric gene into the nuclear genome were also obtained. However, most of these could be eliminated by screening for resistance to high levels of kanamycin (500 g/ml). Incorporation of kan into the plastid genome led to its amplification to a high copy number, about 10000 per leaf cell, and accumulation of NPTII to about 1% of total cellular protein.     

Plastid transformation in Arabidopsis thaliana

Plastid transformation is reported in Arabidopsis thaliana following biolistic delivery of transforming DNA into leaf cells. Transforming plasmid pGS31A carries a spectinomycin resistance (aadA) gene flanked by plastid DNA sequences to target its insertion between trnV and the rps12/7 operon. Integration of aadA by two homologous recombination events via the flanking ptDNA sequences and selective amplification of the transplastomes on spectinomycin medium yielded resistant cell lines and regenerated plants in which the plastid genome copies have been uniformly altered. The efficiency of plastid transformation was low: 2 in 201 bombarded leaf samples. None of the 98 plants regenerated from the two lines were fertile. Received: 13 February 1998 / Revision received: 24 April 1998 / Accepted: 5 June 1998     

Plastid Transformation in Lesquerella Fendleri,an Oilseed Brassicacea

A plastid transformation protocol was developed for Lesquerella fendleri, a species with a high capacity for plant regeneration in tissue culture. Transformation vector pZS391B carried an aadA16gfp marker gene conferring streptomycin–spectinomycin resistance and green fluorescence under UV light. Biolistic transformation of 51 Lesquerella leaf samples, followed by spectinomycin selection, yielded two transplastomic clones. The AAD–GFP fusion protein, the marker gene product, was localized to chloroplasts by confocal laser microscopy. Fertile plants and seed progeny were obtained in line Lf-pZS391B-1. In the 51 samples a large number (108) of spontaneous mutants were identified. In five of the lines spectinomycin resistance was localized to a conserved stem structure by sequencing 16S rRNA genes. Success in L. fendleri, a wild oilseed species, extends plastid transformation beyond Arabidopsis thaliana in the Brassicaceae family.     

Generation of fertile transplastomic soybean

We describe here the development of a plastid transformation method for soybean, a leguminous plant of major agronomic interest. Chloroplasts from embryogenic tissue of Glycine max have been successfully transformed by bombardment. The transforming DNA carries a spectinomycin resistance gene (aadA) under the control of tobacco plastid regulatory expression elements, flanked by two adjacent soybean plastome sequences allowing its targeted insertion between the trnV gene and the rps12/7 operon. All generated spectinomycin resistant plants were transplastomic and no remaining wild type plastome copies were detected. No spontaneous mutants were obtained. The transformation efficiency is similar to that of tobacco plastids. All transplastomic T0 plants were fertile and T1 progeny was uniformly spectinomycin resistant, showing the stability of the plastid transgene. This is the first report on the generation of fertile transplastomic soybean.