RRC ID 28110
著者 Ogawa Y, Sakurai N, Oikawa A, Kai K, Morishita Y, Mori K, Moriya K, Fujii F, Aoki K, Suzuki H, Ohta D, Saito K, Shibata D.
タイトル High-throughput cryopreservation of plant cell cultures for functional genomics.
ジャーナル Plant Cell Physiol
Abstract Suspension-cultured cell lines from plant species are useful for genetic engineering. However, maintenance of these lines is laborious, involves routine subculturing and hampers wider use of transgenic lines, especially when many lines are required for a high-throughput functional genomics application. Cryopreservation of these lines may reduce the need for subculturing. Here, we established a simple protocol for cryopreservation of cell lines from five commonly used plant species, Arabidopsis thaliana, Daucus carota, Lotus japonicus, Nicotiana tabacum and Oryza sativa. The LSP solution (2 M glycerol, 0.4 M sucrose and 86.9 mM proline) protected cells from damage during freezing and was only mildly toxic to cells kept at room temperature for at least 2 h. More than 100 samples were processed for freezing simultaneously. Initially, we determined the conditions for cryopreservation using a programmable freezer; we then developed a modified simple protocol that did not require a programmable freezer. In the simple protocol, a thick expanded polystyrene (EPS) container containing the vials with the cell-LSP solution mixtures was kept at -30 °C for 6 h to cool the cells slowly (pre-freezing); samples from the EPS containers were then plunged into liquid nitrogen before long-term storage. Transgenic Arabidopsis cells were subjected to cryopreservation, thawed and then re-grown in culture; transcriptome and metabolome analyses indicated that there was no significant difference in gene expression or metabolism between cryopreserved cells and control cells. The simplicity of the protocol will accelerate the pace of research in functional plant genomics.
巻・号 53(5)
ページ 943-52
公開日 2012-5-1
DOI 10.1093/pcp/pcs038
PII pcs038
PMID 22437846
PMC PMC3345369
MeSH Arabidopsis / cytology Arabidopsis / genetics Arabidopsis / physiology Cell Culture Techniques / methods* Cell Line Cell Survival / drug effects Cryopreservation / methods* Cryoprotective Agents / pharmacology Freezing Gene Expression Regulation, Plant / drug effects Genomics / methods* Glucuronidase / metabolism High-Throughput Screening Assays / methods* Plant Cells / drug effects Plant Cells / metabolism* Proline / pharmacology
IF 4.062
引用数 22
WOS 分野 PLANT SCIENCES CELL BIOLOGY
リソース情報
シロイヌナズナ / 植物培養細胞・遺伝子 rpc00008 rpc00001