Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system.

Incorporation of a selectable marker gene during transformation is essential to obtain transformed plastids. However, once transformation is accomplished, having the marker gene becomes undesirable. Here we report on adapting the P1 bacteriophage CRE-lox site-specific recombination system for the elimination of marker genes from the plastid genome. The system was tested by the elimination of a negative selectable marker, codA, which is flanked by two directly oriented lox sites (>codA>). Highly efficient elimination of >codA> was triggered by introduction of a nuclear-encoded plastid-targeted CRE by Agrobacterium transformation or via pollen. Excision of >codA> in tissue culture cells was frequently accompanied by a large deletion of a plastid genome segment which includes the tRNA-ValUAC gene. However, the large deletions were absent when cre was introduced by pollination. Thus pollination is our preferred protocol for the introduction of cre. Removal of the >codA> coding region occurred at a dramatic speed, in striking contrast to the slow and gradual build-up of transgenic copies during plastid transformation. The nuclear cre gene could subsequently be removed by segregation in the seed progeny. The modified CRE-lox system described here will be a highly efficient tool to obtain marker-free transplastomic plants.

Identification of a functional respiratory complex in chloroplasts through analysis of tobacco mutants containing disrupted plastid ndh genes.

The plastid genomes of several plants contain homologues, termed ndh genes, of genes encoding subunits of the NADH:ubiquinone oxidoreductase or complex I of mitochondria and eubacteria. The functional significance of the Ndh proteins in higher plants is uncertain. We show here that tobacco chloroplasts contain a protein complex of 550 kDa consisting of at least three of the ndh gene products: NdhI, NdhJ and NdhK. We have constructed mutant tobacco plants with disrupted ndhC, ndhK and ndhJ plastid genes, indicating that the Ndh complex is dispensible for plant growth under optimal growth conditions. Chlorophyll fluorescence analysis shows that in vivo the Ndh complex catalyses the post-illumination reduction of the plastoquinone pool and in the light optimizes the induction of photosynthesis under conditions of water stress. We conclude that the Ndh complex catalyses the reduction of the plastoquinone pool using stromal reductant and so acts as a respiratory complex. Overall, our data are compatible with the participation of the Ndh complex in cyclic electron flow around the photosystem I complex in the light and possibly in a chloroplast respiratory chain in the dark.

Targeted deletion of sprA from the tobacco plastid genome indicates that the encoded small RNA is not essential for pre-16S rRNA maturation in plastids.

The small plastid RNA (spRNA) which includes a segment that is complementary to the pre-16S rRNA has been suggested to facilitate maturation of pre-16S rRNA in tobacco. To investigate the function of spRNA, the gene encoding it (sprA) was removed from the plastid genome using targeted gene deletion. We report here that deletion of sprA does not significantly affect pre-16S rRNA maturation, nor does it cause any obvious phenotype. Although the spRNA still may be involved in rRNA maturation, it is non-essential under normal growth conditions.

Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco.

The Bacillus thuringiensis (Bt) crystal toxins are safe biological insecticides, but have short persistance and are poorly effective against pests that feed inside plant tissues. Production of effective levels of these proteins in plants has required resynthesis of the genes encoding them. We report that amplification of an unmodified crylA(c) coding sequence in chloroplasts up to approximately 10,000 copies per cell resulted in the accumulation of an unprecedented 3-5% of the soluble protein in tobacco leaves as protoxin. The plants were extremely toxic to larvae of Heliothis virescens, Helicoverpa zea, and Spodoptera exigua. Since the plastid transgenes are not transmitted by pollen, this report has implications for containment of Bt genes in crop plants. Furthermore, accumulation of insecticidal protein at a high level will facilitate improvement in the management of Bt resistant insect populations.

Efficient targeting of foreign genes into the tobacco plastid genome.

The pPRV plasmids are vectors for targeted insertion of foreign genes into the tobacco plastid genome (ptDNA). The vectors are based on the pUC119 plasmid which replicates in E. coli but not in plastids. The spectinomycin resistance (aadA) gene and a multiple cloning site (MCS) are flanked by 1.8-kb and 1.2-kb ptDNA sequences. Biolistic delivery of vector DNA, followed by spectinomycin selection, yields plastid transformants at a reproducible frequency, approximately 1 transplastomic line per bombarded sample. The selected aadA gene and linked non-selectable genes cloned into the MCS are incorporated into the ptDNA by two homologous recombination events via the flanking ptDNA sequences. The transplastomes thus generated are stable, and are maternally transmitted to the seed progeny. The pPRV vector series targets insertions between the divergently transcribed trnV gene and the rps12/7 operon. The lack of readthrough transcription of appropriately oriented transgenes makes the vectors an ideal choice for the study of transgene promoter activity.

The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation.

The new pPZP Agrobacterium binary vectors are versatile, relatively small, stable and are fully sequenced. The vectors utilize the pTiT37 T-DNA border regions, the pBR322 bom site for mobilization from Escherichia coli to Agrobacterium, and the ColE1 and pVS1 plasmid origins for replication in E. coli and in Agrobacterium, respectively. Bacterial marker genes in the vectors confer resistance to chloramphenicol (pPZP100 series) or spectinomycin (pPZP200 series), allowing their use in Agrobacterium strains with different drug resistance markers. Plant marker genes in the binary vectors confer resistance to kanamycin or to gentamycin, and are adjacent to the left border (LB) of the transferred region. A lacZ alpha-peptide, with the pUC18 multiple cloning site (MCS), lies between the plant marker gene and the right border (RB). Since the RB is transferred first, drug resistance is obtained only if the passenger gene is present in the transgenic plants.

Plastid engineering in land plants: a conservative genome is open to change.

We have developed efficient transformation protocols to modify each of the 500-10,000 plastid genome copies in a tobacco cell. The transforming DNA is introduced on the surface of microscopic tungsten particles by the biolistic process. Selection for transplastomes is by spectinomycin resistance based on expression of aminoglycoside-3"-adenyltransferase from a chimeric aadA gene in the transforming DNA. Manipulations that are now feasible include replacement of endogenous plastid genes with DNA sequences modified in vitro, targeted gene disruption, and insertion of reporter genes into the plastid genome. Alternative methods for plastid genome manipulations may be developed utilizing an extrachromosomal element which was identified during the transformation studies. Introduction of foreign genes under control of plastid gene expression elements results in duplication of endogenous regulatory sequences. A sensitive somatic assay to detect deletions via such direct repeats confirmed that these sequence duplications do not result in significant genome instability. The ability to transform plastids will facilitate the study of plastid gene regulation, and the application of genetic engineering to crop improvement.

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 micrograms/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 micrograms/ml). Incorporation of kan into the plastid genome led to its amplification to a high copy number, about 10,000 per leaf cell, and accumulation of NPTII to about 1% of total cellular protein.

High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene.

We report here a 100-fold increased frequency of plastid transformation in tobacco by selection for a chimeric aadA gene encoding aminoglycoside 3"-adenylyltransferase, as compared with that obtained with mutant 16S rRNA genes. Expression of aadA confers resistance to spectinomycin and streptomycin. In transforming plasmid pZS197, a chimeric aadA is cloned between rbcL and open reading frame ORF512 plastid gene sequences. Selection was for spectinomycin resistance after biolistic delivery of pZS197 DNA into leaf cells. DNA gel-blot analysis confirmed incorporation of the chimeric aadA gene into the plastid genome by two homologous recombination events via the flanking plastid gene sequences. The chimeric gene became homoplasmic in the recipient cells and is uniformly transmitted to the maternal seed progeny. The ability to transform routinely plastids of land plants opens the way to manipulate the process of photosynthesis and to incorporate novel genes into the plastid genome of crops.

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

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 micrograms/ml), SPC23 seedlings are resistant to low levels (50 micrograms/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.

Stable transformation of plastids in higher plants.

Stable genetic transformation of the plastid genome is reported in a higher plant, Nicotiana tabacum. Plastid transformation was obtained after bombardment of leaves with tungsten particles coated with pZS148 plasmid DNA. Plasmid pZS148 (9.6 kilobases) contains a 3.7-kilobase plastid DNA fragment encoding the 16S rRNA. In the 16S rRNA-encoding DNA (rDNA) a spectinomycin resistance mutation is flanked on the 5' side by a streptomycin resistance mutation and on the 3' side by a Pst I site generated by ligating an oligonucleotide in the intergenic region. Transgenic lines were selected by spectinomycin resistance and distinguished from spontaneous mutants by the flanking, cotransformed streptomycin resistance and Pst I markers. Regenerated plants are homoplasmic for the spectinomycin resistance and the Pst I markers and heteroplasmic for the unselected streptomycin resistance trait. Transgenic plastid traits are transmitted to the seed progeny. The transgenic plastid genomes are products of a multistep process, involving DNA recombination, copy correction, and sorting out of plastid DNA copies.

Aminoglycoside-3''-adenyltransferase confers resistance to spectinomycin and streptomycin in Nicotiana tabacum.

The bacterial gene aadA encodes the enzyme aminoglycoside-3"-adenyltransferase that confers resistance to spectinomycin and streptomycin in Escherichia coli. Chimeric genes have been constructed for expression in plants, and were introduced into Nicotiana tabacum by Agrobacterium binary transformation vectors. Spectinomycin or streptomycin in selective concentrations prevent greening of N. tabacum calli. Transgenic clones, however, formed green calli on selective media containing spectinomycin, streptomycin, or both drugs. Resistance was inherited as a dominant Mendelian trait in the seed progeny. Resistance conferred by the chimeric aadA gene can be used as a color marker similar to the resistance conferred by the streptomycin phosphotransferase gene to streptomycin.

Stable genetic transformation of intact Nicotiana cells by the particle bombardment process.

We show that the genetic transformation of Nicotiana tabacum can be achieved by bombarding intact cells and tissues with DNA-coated particles. Leaves or suspension culture cells were treated with tungsten microprojectiles carrying plasmid DNA containing a neomycin phosphotransferase gene. Callus harboring the foreign gene was recovered from the bombarded tissue by selection on medium containing kanamycin. Kanamycin-resistant plants have subsequently been regenerated from the callus derived from leaves. Transient expression of an introduced beta-glucuronidase gene was used to assess the efficiency of DNA delivery by microprojectiles. The frequency of cells that were stably transformed with the neomycin phosphotransferase gene was a few percent of the cells that transiently expressed the beta-glucuronidase gene. These results show that gene transfer by high-velocity microprojectiles is a rapid and direct means for transforming intact plant cells and tissues that eliminates the need for production of protoplasts or infection by Agrobacterium.

Improved expression of streptomycin resistance in plants due to a deletion in the streptomycin phosphotransferase coding sequence.

Previous studies have shown that a chimeric streptomycin phosphotransferase (SPT) gene can function as a dominant marker for plant cell transformation. The SPT marker previously described by Jones and co-workers has a limited value since it conferred a useful level of resistance only to a fraction (10%) of Nicotiana plumbaginifolia transgenic lines. Expression of resistance was species specific: no such resistant transformants were found in N. tabacum. In this paper we describe an improved SPT construct that utilizes a mutant Tn5 SPT gene. The mutant gene, SPT*, encodes a protein with a two amino acid deletion close to its COOH-terminus. In N. tabacum cell culture the efficiency of transformation with the improved streptomycin resistance marker was comparable to kanamycin resistance. When the chimeric SPT* gene was introduced linked to a kanamycin resistance gene, streptomycin resistance was expressed in most of the transgenic N. tabacum lines.

Nicotiana tabacum mutants with chloroplast encoded streptomycin resistance and pigment deficiency.

Callus ofNicotiana tabacum SRI, a mutant with maternally inherited streptomycin resistance, was induced from leaf sections. Callus pieces were mutagenised with N-ethyl-N-nitrosourea and inoculated onto a shoot-induction medium on which calli are normally green. White callus sectors were observed in the mutagenised cultures, and white and variegated shoots were regenerated from these sectored calli. The SR1-A10 line regenerated a chimeric shoot with white leaf margins. The chimeric shoot was grafted onto a normal green rootstock, grown into a flowering plant in the greenhouse, and crosses were made. The SRI-A15 line was crossed using flowers formed on albino plants grown in sterile culture. Pigment deficiency was maternally inherited in both lines. Physical mapping of the chloroplast genome of the SR1-A15 mutant by SalI, PstI and BamHI restriction endonucleases did not reveal any difference between the SR1-A15 and the parental SRI chloroplast genomes.

Transient cycloheximide resistance in a tobacco cell line.

Cylcoheximide-resistant cell lines have been recovered from tissue cultures of haploid Nicotiana tabacum at the usual mutant frequency (10(-6)). One such line, CR 4/6 was studied in detail. Resistance is based on the potential of the callus to inactive cycloheximide. The transient nature of the resistance and its association with differentiated cells indicate that the new phenotype is not due to a mutation, but rather to an alteration in gene expression.