著者

Masaki Odahara Most Tanziman Ara Remi Nakagawa Yoko Horii Shougo Ishio Shinjiro Ogita Keiji Numata

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.4, pp.263-271, 2023-12-25 (Released:2023-12-25)

参考文献数

34

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The plastid is a promising target for the production of valuable biomolecules via genetic engineering. We recently developed a plastid-specific gene delivery system for leaves or seedlings using KH-AtOEP34, a functional peptide composed of the polycationic DNA-binding peptide KH and the Arabidopsis thaliana plastid-targeting peptide OEP34. Here, we established a liquid culture system for inducing multiple shoots in the model plants A. thaliana and Nicotiana benthamiana and the crop plant strawberry (Fragaria×ananassa) and tested the use of these plant materials for peptide-mediated gene delivery to plastids. Our liquid culture system efficiently induced multiple shoots that were enriched in meristems. Using these meristems, we performed KH-AtOEP34-mediated gene delivery to plastids and tested the delivery and integration of a cassette composed of the spectinomycin resistance gene aadA, the GFP reporter gene, and sequences homologous to plastid DNA. Genotyping PCR revealed the integration of the cassette DNA into plastid DNA several days after delivery in all three plants. Confocal laser scanning microscopy and immunoblotting confirmed the presence of plasmid-derived GFP in the plastids of meristems, indicating that the plasmid DNA was successfully integrated into plastid DNA and that the cassette was expressed. These results suggest the meristems developed in our liquid culture system are applicable to peptide-mediated delivery of exogeneous DNA into plastids. The multiple shoots generated in our liquid novel culture system represent promising materials for in planta peptide-mediated plastid transformation in combination with spectinomycin selection.

著者

Kazusato Oikawa Takuto Imai Yutaka Kodama Keiji Numata

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.38, no.2, pp.257-262, 2021-06-25 (Released:2021-06-25)

参考文献数

15

被引用文献数

2

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Mitochondria-selective fluorescent probes such as MitoTracker are often used for mitochondria imaging in various plants. Although some of the probes are reported to induce mitochondria dysfunction in animal cells, the effect on plant cells remains to be determined. In the present study, we applied quantitative methods to analyze mitochondrial movement, speed frequency, and speed-angle changes, based on trajectory analysis of mitochondria in mesophyll protoplast cells of Arabidopsis thaliana expressing the mitochondria-localized fluorescent protein. Using the quantitative method, we assessed whether MitoTracker Red (FM and CMXRos) induce mitochondria dysfunction in A. thaliana. Although both the fluorescent probes well-stained mitochondria, the CMXRos probe, not the FM probe, gave a severe effect on mitochondrial movement at the low concentration (10 nM), indicating a MitoTracker-induced mitochondria dysfunction in A. thaliana. These results revealed that our quantitative method based on mitochondrial movement can be used to determine the appropriate concentrations of mitochondria-selective fluorescent probes in plants.