Simple and efficient plastid transformation system for the liverwort Marchantia polymorpha L. suspension-culture cells
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We have established a simple and efficient plastid transformation system for liverwort, Marchantia polymorpha L., suspension-culture cells, which are homogenous, chloroplast-rich and␣rapidly growing. Plasmid pCS31 was constructed to integrate an aadA expression cassette for spectinomycin-resistance into the trnI–trnA intergenic region of the liverwort plastid DNA by homologous recombination. Liverwort suspension-culture cells were bombarded with pCS31-coated gold projectiles and selected on a medium containing spectinomycin. Plastid transformants were reproducibly isolated from the obtained spectinomycin-resistant calli. Selection on a sucrose-free medium greatly improved the efficiency of selection of plastid transformants. Homoplasmic plastid transformant lines were established by␣successive subculturing for 14 weeks or longer on the spectinomycin-containing medium. The plastid transformation system of liverwort suspension-culture cells should facilitate the investigation of the fundamental genetic systems of plastid DNA, such as replication.
KeywordsPlastid transformation Marchantia polymorpha Homoplasmy Bryophyte Suspension-culture
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Plasmid pCT08 was kindly provided by Dr. T. Shikanai. We thank Dr. F. Sato for the use of a biolistic delivery system. S.C. was supported by the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to Kyoto University. This work was supported in part by the grant “Knowledge Cluster Initiative” from MEXT.
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