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Nicotiana tabacum: PEG-mediated plastid transformation.
- Source :
-
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2014; Vol. 1132, pp. 165-75. - Publication Year :
- 2014
-
Abstract
- Stable plastid transformation in Nicotiana tabacum has been achieved by using two different methods, the biolistic method, using a particle gun, and the polyethylene glycol (PEG)-mediated transformation. PEG-mediated plastid transformation involves the treatment of isolated protoplasts (plant cells without cell wall) with PEG in the presence of DNA. We have previously shown that in Nicotiana tabacum both methods are equally efficient. The PEG-mediated transformation efficiencies range between 20 and 50 plastid transformants per experiment (10(6) viable treated protoplasts). One advantage of the PEG method is that no expensive equipment such as a particle gun is required. The only crucial points are the handling and the cultivation of protoplasts. Furthermore, markers for the selection of transformed chloroplasts are required. One of the most often used selection markers is the aadA gene which encodes for spectinomycin and streptomycin resistance. Here we describe a simplified and inexpensive protocol for the transformation of chloroplasts in Nicotiana tabacum using an optimized protoplast culture protocol.
- Subjects :
- Biolistics methods
Drug Resistance genetics
Plants, Genetically Modified genetics
Protoplasts cytology
Protoplasts drug effects
Spectinomycin pharmacology
Streptomycin pharmacology
Surface-Active Agents pharmacology
Chloroplasts genetics
Polyethylene Glycols pharmacology
Nicotiana genetics
Transfection methods
Transformation, Genetic drug effects
Subjects
Details
- Language :
- English
- ISSN :
- 1940-6029
- Volume :
- 1132
- Database :
- MEDLINE
- Journal :
- Methods in molecular biology (Clifton, N.J.)
- Publication Type :
- Academic Journal
- Accession number :
- 24599852
- Full Text :
- https://doi.org/10.1007/978-1-62703-995-6_9