Document Detail


Synchronization, transformation, and cryopreservation of suspension-cultured cells.
MedLine Citation:
PMID:  16739567     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We have recently described the selection of rapidly dividing Arabidopsis cell suspension cultures MM1 and MM2d that provide a powerful platform for plant cell-cycle research. Here we provide detailed protocols and procedures to achieve high levels of synchronization, either by starving the cell cultures of sucrose or by applying the toxin aphidicolin. Cell-cycle activity during cell-cycle reentry (starvation-induced synchrony) or further cell-cycle progression (aphidicolin-induced synchrony) can be conveniently followed by using various validation procedures, such as determination of labeling index and metaphase/anaphase index or flow cytometry. We also describe a procedure that allows clonal transformed cell-suspension lines to be produced using Agrobacterium-mediated transformation, and an optimized and straightforward method for the cryopreservation and recovery of both parental and transformed lines which is applicable both to Arabidopsis and the tobacco BY2 cell lines. Cell-cycle synchronization capacity of the parental lines is maintained after both transformation and recovery from cryopreservation. The techniques described here require no specialized equipment and are suitable for routine laboratory use, greatly facilitating the handling and maintenance of cell cultures. The ability to store easily large numbers of transformed lines opens the possibility of using Arabidopsis cell suspension cultures for future high-throughput cell-cycle analysis.
Authors:
Margit Menges; James A H Murray
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Methods in molecular biology (Clifton, N.J.)     Volume:  323     ISSN:  1064-3745     ISO Abbreviation:  Methods Mol. Biol.     Publication Date:  2006  
Date Detail:
Created Date:  2006-06-02     Completed Date:  2006-06-23     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  9214969     Medline TA:  Methods Mol Biol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  45-61     Citation Subset:  IM    
Affiliation:
Institute of Biotechnology, University of Cambridge, UK.
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MeSH Terms
Descriptor/Qualifier:
Antimetabolites / pharmacology
Aphidicolin / chemistry
Arabidopsis / cytology*
Botany / methods*
Bromodeoxyuridine / pharmacology
Cell Culture Techniques / methods*
Cell Cycle
Cells, Cultured
Cryopreservation
Plants, Genetically Modified
Rhizobium / metabolism
Sucrose / metabolism
Time Factors
Transgenes
Chemical
Reg. No./Substance:
0/Antimetabolites; 38966-21-1/Aphidicolin; 57-50-1/Sucrose; 59-14-3/Bromodeoxyuridine

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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