caa a22 4500 477101909 CHVBK 20180405111544.0 cr unu---uuuuu 170330e19960601xx s 000 0 eng 10.1007/BF00196559 doi (NATIONALLICENCE)springer-10.1007/BF00196559 Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation [Elektronische Daten] [Hans-Ulrich Koop, Klaus Steinmüller, Hiltrud Wagner, Christine Rößler, Christian Eibl, Lydia Sacher] 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. Springer-Verlag, 1996 Nicotiana nationallicence Plastid transformation nationallicence Polyethylene glycol nationallicence Protoplast nationallicence PCR : polymerase chain reaction nationallicence PEG : polyethylene glycol nationallicence WT : wild type nationallicence Koop Hans-Ulrich Botanisches Institut der Universität München, Menzinger Strasse 67, D-80638, München, Germany aut Steinmüller Klaus Botanisches Institut, Universität Düsseldorf, Universitätsstrasse 1, D-40225, Düsseldorf, Germany aut Wagner Hiltrud Botanisches Institut der Universität München, Menzinger Strasse 67, D-80638, München, Germany aut Rößler Christine Botanisches Institut der Universität München, Menzinger Strasse 67, D-80638, München, Germany aut Eibl Christian Botanisches Institut der Universität München, Menzinger Strasse 67, D-80638, München, Germany aut Sacher Lydia Botanisches Institut der Universität München, Menzinger Strasse 67, D-80638, München, Germany aut Planta Springer-Verlag 199/2(1996-06-01), 193-201 0032-0935 199:2<193 1996 199 425 https://doi.org/10.1007/BF00196559 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00196559 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Koop Hans-Ulrich Botanisches Institut der Universität München, Menzinger Strasse 67, D-80638, München, Germany aut NATIONALLICENCE 700 1- Steinmüller Klaus Botanisches Institut, Universität Düsseldorf, Universitätsstrasse 1, D-40225, Düsseldorf, Germany aut NATIONALLICENCE 700 1- Wagner Hiltrud Botanisches Institut der Universität München, Menzinger Strasse 67, D-80638, München, Germany aut NATIONALLICENCE 700 1- Rößler Christine Botanisches Institut der Universität München, Menzinger Strasse 67, D-80638, München, Germany aut NATIONALLICENCE 700 1- Eibl Christian Botanisches Institut der Universität München, Menzinger Strasse 67, D-80638, München, Germany aut NATIONALLICENCE 700 1- Sacher Lydia Botanisches Institut der Universität München, Menzinger Strasse 67, D-80638, München, Germany aut NATIONALLICENCE 773 0- Planta Springer-Verlag 199/2(1996-06-01), 193-201 0032-0935 199:2<193 1996 199 425 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer