RESUMO
We describe a simple and efficient plastid transformation method for the liverwort, Marchantia polymorpha L. Use of rapidly proliferating cells such as sporelings, which are immature thalli developing from spores, as targets made plastid transformation by particle bombardment efficient. Selection on a sucrose-free medium and linearization of the transformation vector significantly improved the recovery rate of plastid transformants. With the method described here, a few plastid transformants are obtained from a single bombardment of sporelings. Homoplasmic transformants of thalli are obtained immediately after primary selection.
Assuntos
Engenharia Genética/métodos , Marchantia/genética , Plantas Geneticamente Modificadas/genética , Plastídeos/genética , Esporos/genética , Transformação Genética , Marchantia/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Esporos/crescimento & desenvolvimentoRESUMO
The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.
Assuntos
Evolução Biológica , Embriófitas/genética , Genoma de Planta , Marchantia/genética , Adaptação Biológica , Embriófitas/fisiologia , Regulação da Expressão Gênica de Plantas , Marchantia/fisiologia , Anotação de Sequência Molecular , Transdução de Sinais , Transcrição GênicaRESUMO
We describe simple and efficient plastid transformation methods for suspension-cultured cells and sporelings of the liverwort, Marchantia polymorpha L. Use of rapidly proliferating cells such as suspension-cultured cells and sporelings, which are immature thalli developing from spores, as targets made plastid transformation by particle bombardment efficient. Selection on a sucrose-free medium and linearization of the transformation vector significantly improved the recovery rate of plastid transformants. With the methods described here, a few plastid transformants are obtained from a single bombardment of sporelings, while more efficient plastid transformation is expected in suspension-cultured cells, ~60 transformants from a single bombardment. Homoplasmic transformants of thalli are obtained immediately after primary selection, whereas homoplasmic transformants from suspension-cultured cells are obtained after 12-16 weeks of repeated subculture.
Assuntos
Biolística/métodos , Cloroplastos/genética , Marchantia/genética , Transformação Genética , Antibacterianos/farmacologia , Técnicas de Cultura de Células , Células Cultivadas , DNA de Plantas/genética , Resistência a Medicamentos/genética , Vetores Genéticos/genética , Recombinação Homóloga , Nucleotidiltransferases/genética , Plantas Geneticamente Modificadas/genética , Plasmídeos/genética , Espectinomicina/farmacologia , Esporos/crescimento & desenvolvimentoRESUMO
Agrobacterium-mediated transformation has not been practical in pteridophytes, bryophytes and algae to date, although it is commonly used in model plants including Arabidopsis and rice. Here we present a rapid Agrobacterium-mediated transformation system for the haploid liverwort Marchantia polymorpha L. using immature thalli developed from spores. Hundreds of hygromycin-resistant plants per sporangium were obtained by co-cultivation of immature thalli with Agrobacterium carrying the binary vector that contains a reporter, the beta-glucuronidase (GUS) gene with an intron, and a selection marker, the hygromycin phosphotransferase (hpt) gene. In this system, individual gemmae, which arise asexually from single initial cells, were analyzed as isogenic transformants. GUS activity staining showed that all hygromycin-resistant plants examined expressed the GUS transgene in planta. DNA analyses verified random integration of 1-5 copies of the intact T-DNA between the right and the left borders into the M. polymorpha genome. The efficient and rapid Agrobacterium-mediated transformation of M. polymorpha should provide molecular techniques to facilitate comparative genomics, taking advantage of this unique model plant that retains many features of the common ancestor of land plants.
Assuntos
Haploidia , Marchantia/genética , Marchantia/microbiologia , Modelos Biológicos , Rhizobium/metabolismo , Transformação Genética , Sequência de Bases , Southern Blotting , Cinamatos/farmacologia , DNA Bacteriano/metabolismo , DNA de Plantas/genética , Resistência Microbiana a Medicamentos , Genoma de Planta/genética , Genótipo , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Marchantia/efeitos dos fármacos , Dados de Sequência Molecular , Mutagênese Insercional/efeitos dos fármacos , Rhizobium/efeitos dos fármacos , Análise de Sequência de DNA , Transformação Genética/efeitos dos fármacosRESUMO
The liverwort, Marchantia polymorpha L., belongs to a group of basal land plants and is an emerging model for plant biology. We established a procedure to prepare sporangia of M. polymorpha under laboratory conditions by promoting its transition to reproductive development by far-red light irradiation. Here we report an improved direct transformation system of M. polymorpha using immature thalli developing from spores. Hygromycin-resistant transformants were obtained on selective media by transformation with a plasmid carrying the hygromycin-phosphotransferase gene (hpt) conferring hygromycin resistance in 4 weeks. The aminoglycoside-3''-adenyltransferase gene (aadA) conferring spectinomycin resistance was also successfully used as an additional selectable marker for nuclear transformation of M. polymorpha. The availability of the aadA gene in addition to the hpt gene should make M. polymorpha a versatile host for genetic manipulation. DNA gel-blot analyses indicated that transformed thalli carried a variable number of copies of the transgene integrated into the genome. Although the previous system using thalli grown from gemmae required a two-step selection in liquid and solid media for 8 weeks, the system reported here using thalli developing from spores allows generation of transformants in half the time by direct selection on solid media, facilitating genetic analyses in this model plant.
Assuntos
Engenharia Genética/métodos , Marchantia/genética , Plantas Geneticamente Modificadas/genética , Transformação Genética , Biolística , Cinamatos/farmacologia , DNA de Plantas/genética , Genes Reporter , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Marchantia/efeitos dos fármacos , Nucleotidiltransferases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plasmídeos , Espectinomicina/farmacologia , Esporos/efeitos dos fármacos , Esporos/genética , Técnicas de Cultura de TecidosRESUMO
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.
