RESUMO
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 (106 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 plastids 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 plastids in Nicotiana tabacum using an optimized protoplast culture protocol. PEG-mediated plastid transformation has the potential to be developed into a high-throughput, automated pipeline.
Assuntos
DNA de Plantas/genética , Resistência a Medicamentos , Nicotiana/genética , Plantas Geneticamente Modificadas/genética , Plastídeos/genética , Polietilenoglicóis/farmacologia , Transformação Genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Nicotiana/efeitos dos fármacos , Nicotiana/crescimento & desenvolvimentoRESUMO
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.
