Showing papers by "Pal Maliga published in 1993"

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

Abstract: 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.

Method for stably transforming plastids of multicellular plants

Abstract: This invention provides methods for obtaining a plant cell of a multicellular plant, the plastids of which cell have been stably transformed by a foreign DNA of interest. These methods employ selectable markers some of which are nonlethal, some of which are designed to insert into the plastid genome and some of which are designed to be expressible only from the plastid genome and not from the nuclear genome. This invention also provides cells and multicellular plants the plastids of which have been stably transformed by a foreign DNA of interest.

Kanamycin resistance as a selectable marker for plastid transformation in tobacco

Abstract: 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.

Accumulation of D1 polypeptide in tobacco plastids is regulated via the untranslated region of the psbA mRNA.

Abstract: The plastid psbA mRNA is present in all tissues, while the encoded 32 kDa D1 protein of photosystem II accumulates tissue-specifically and in response to light. To study the regulation of D1 accumulation, a chimeric uidA gene encoding beta-glucuronidase (GUS) under control of the psbA 5'- and 3'-regulatory regions (224 and 393 bp, respectively), was integrated into the tobacco plastid genome. A high level of GUS accumulation in leaves and the lack of GUS in roots, with uidA mRNA present in both tissues, indicated tissue-specific accumulation of the chimeric gene product. Light-regulated accumulation of GUS in seedlings was shown. (i) Light-induced accumulation (100-fold) of GUS in etiolated cotyledons was accompanied by only a modest increase in mRNA levels. (ii) Inhibition of GUS synthesis was observed in cotyledons when light-grown seedlings were transferred to the dark, with no reduction in mRNA levels. Tissue-specific and light-regulated accumulation of GUS indicates that D1 accumulation is controlled via cis-acting regulatory elements in the untranslated region of the psbA mRNA. We propose that in tobacco, control of translation initiation is the primary mechanism regulating D1 protein accumulation.

Stable Plastid Transformation in PEG-treated Protoplasts of Nicotiana tabacum

Abstract: Plastid engineering currently relies on DNA delivery by the biolistic process. We report here stable plastid transformation in tobacco by an alternate direct transformation protocol that is based on polyethylene glycol (PEG) treatment of leaf protoplasts in the presence of the transforming DNA. Clones with transformed plastid genomes were selected by spectinomycin resistance encoded by a mutant 16S ribosomal RNA gene. Incorporation of the transforming DNA into the plastid genome was confirmed by two unselected markers, streptomycin resistance and a novel PstI site that flank the spectinomycin resistance mutation in plasmid pZS148. Our simple and inexpensive protocol eliminates the dependence on the particle gun for chloroplast transformation and should facilitate applications of plastome engineering in crops.

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

Abstract: 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.