A new vector, pFaadAII, for transformation of plastids of Nicotiana tabacum L. has been developed. It harbours a chimeric gene consisting of the aadA coding region from Escherichia coli, the 16S rDNA promoter from tobacco combined with a synthetic ribosome-binding site, a 500-bp fragment containing the 3′ untranslated transcript region (UTR) of the Chlamydomonas rbcL gene and 3.75-kb (5′) and 0.95-kb (3′) tobacco plastome sequences allowing for targeting the foreign sequences to the intergenic region between the rpl32 and trnL genes of the tobacco plastome. The vector thus targets foreign sequences to the small single-copy region of the plastome, which has so far not been modified by transformation. Leaf protoplasts of Nicotiana tabacum L. were treated with polyethylene glycol (PEG) in the presence of the vector. The protocol for PEG treatment aiming at plastome transformation was optimized. Cell lines were cultured in the presence of spectinomycin and streptomycin using a novel and efficient protoplast culture and selection system. Regenerants were characterized by polymerase chain reaction (PCR) analysis, Southern hybridization and reciprocal crossings. The transformation procedure is described in detail and parameters influencing its efficiency are presented. Special effort is placed on analyzing suitable selection conditions. Only a proportion of the cell lines with a resistant phenotype could be confirmed by molecular analysis and/or reciprocal crossings to represent plastome transformants. Integration of the plastome specific aadA. cassette into the nuclear genome accounted for a fraction of the resistant cell lines. Still, as many as 20–40 plastome transformants can be expected from the treatment of 106 protoplasts. Therefore, the improved protocol for PEG-mediated plastome transformation in combination with the new aadA-vector supplies a simple, reproducible and cost-efficient alternative to the biolistic procedure.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
polymerase chain reaction
Bendich AJ (1987) Why do chloroplasts and mitochondria contain so many copies of their genome? Bioessays 6: 279–282
Bock R, Kössel H, Maliga P (1994) Introduction of a heterologous editing site into the tobacco plastid genome: the lack of RNA editing leads to a mutant phenotype. EMBO J 13: 4623–4628
Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM, Jones AR, Randolph-Anderson BL, Robertson D, Klein TM, Shark KB, Sanford JC (1987) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240: 1534–1538
Carrer H, Hockenberry TN, Svab Z, Maliga P (1993) Kanamycin resistance as a selective plastid marker. Mol Gen Genet 241: 49–56
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50: 151–158
Golds T, Maliga P, Koop HU (1993) Stable plastid transformation in PEG-treated protoplasts of Nicotiana tabacum. Bio/Technology 11: 95–97
Goldschmidt-Clermont M (1991) Transgenic expression of amino-glycoside adenine transferase in the chloroplast: a selectable marker for site-directed transformation of Chlamydomonas. Nucleic Acids Res 19: 4083–4089
Gritz L, Davis J (1983) Plasmid-encoded hygromycin-B-resistance: the sequence of hygromycin-B-phosphotransferase-gene and its expression in Escherichia coli and Saccharaomyces cerevisiae. Gene 25: 179–188
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS-fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907
Kanevski I, Maliga P (1994) Relocation of the plastid rbcL gene to the nucleus yields functional ribulose-1,5-bisphosphate carboxylase in tobacco chloroplasts. Proc Natl Acad Sci USA 91: 1969–1973
Koop HU, Kofer W (1995) Plastid transformation by polyethylene glycol treatment of protoplasts and regeneration of transplastomic tobacco plants. In: Potrykus I, Spangenberg G (eds) Gene transfer to plants, pp 75–82. Springer-Verlag Berlin, Heidelberg, New York
McBride KE, Schaaf DJ, Daley M, Stalker DM (1994) Controlled expression of plastid transgenes in plants based on a nuclear DNA-encoded and plastid-targeted T7 RNA polymerase. Proc Natl Acad Sci USA 91: 7301–7305
McBride KE, Svab Z, Schaaf DJ, Hogan PS, Stalker DM, Maliga P (1995) Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco. Bio/Technology 13: 362–365
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–479
Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8: 4321–4325
Negrutiu I, Shillito R, Potrykus I, Biasini G, Sala F (1987) Hybrid genes in the analysis of transformation conditions. I: Setting up a simple method for direct gene transfer in plant protoplasts. Plant Mol Biol 8: 363–373
Negrutiu I, Dewulf J, Pietrzak M, Bottermann J, Rietveld E, Wurzer-Figurelli EM, Ye D, Jacobs M (1990) Hybrid genes in the analysis of transformation conditions. II: Transient expression vs. stable transformation analysis of parameters influencing gene expression levels and transformation efficiency. Physiol Plant 79: 197–205
O'Neill C, Horvath GV, Horvath E, Dix PJ, Medgyesy P (1993) Chloroplast transformation in plants: polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems. Plant J 3: 729–738
Prentki P, Krisch HM (1984) In vitro insertional mutangenesis with a selectable DNA fragment. Gene 29: 303–313
Schröder M, Dixelius C, Rahlen L, Glimelius C (1994) Transformation of Brassica napus by using the aadA gene as selectable marker and inheritance studies on the marker genes. Physiol Plant 92: 37–46
Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Zaita N, Chungwongse J, Obokata J, Yamaguchi-Shinozaki K, Ohto C, Torazawa K, Mend BY, Sugita M, Deno H, Komoyashira T, Yamada K, Kusuda J, Tkawa F, Kato A, Tohdoh N, Shimada H, Sugiura M (1986) The complete nucleotide sequence of the tobacco chloroplast genome: Its gene organization and expression. EMBO J 5: 2043–2049
Spörlein B, Streubel M, Dahlfeld G, Westhoff P, Koop HU (1991) PEG-mediated plastid transformation: A new system for transient gene expression assays in chloroplasts. Theor Appl Genet 82: 717–722
Staub J, Maliga P (1993) Accumulation of D1 polypeptide in tobacco plastids is regulated via the untranslated region of the psbA mRNA. EMBO J 12: 601–606
Staub J, Maliga P (1994) Extrachromosomal elements in tobacco plastids. Proc Natl Acad Sci USA 91: 7468–7472
Staub J, Maliga P (1995) Expression of a chimeric uidA gene indicates that polycistronic mRNAs are efficiently translated in tobacco plastids. Plant J 7: 845–848
Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA. gene. Proc Natl Acad Sci USA 90: 913–917
Svab Z, Hajdukiewicz P, Maliga P (1990a) Stable transformation of plastids in higher plants. Proc Natl Acad Sci USA 87: 8526–8530
Svab Z, Harper EC, Jones JDG, Maliga P (1990b) Aminoglycoside-3′-adenyltransferase confers resistance to spectinomycin and streptomycin in Nicotiana tabacum. Plant Mol Biol 14: 197–205
Töpfer R, Matzeit V, Gronenborn B, Schell J, Steinbiss HH (1987) A set of plast expression vectors for transcriptional and translational fusions. Nucleic Acids Res 15: 5890
Zoubenko OV, Allison LA, Svab Z, Maliga P (1994) Efficient targeting of foreign genes into the tobacco plastid genome. Nucleic Acids Res 22: 3819–3824
The work was supported by the Deutsche Forschungsgemeinschaft (SFB #184). The authors wish to express their gratitude to M. Goldschmidt-Clermont for supplying plasmid pUC-atpX-AAD, A. Blowers for pNtcPs1, and Z. Svab and P. Maliga (Waksman Institute, Piscataway, N.Y., USA) for pZS197, P. Dörfel and W. Kofer (Botanisches Institut, Universität München, Germany) contributed valuable suggestions.
About this article
Cite this article
Koop, H., Steinmüller, K., Wagner, H. et al. Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation. Planta 199, 193–201 (1996). https://doi.org/10.1007/BF00196559
- Plastid transformation
- Polyethylene glycol