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Establishment of rapidly proliferating rice cell suspension culture and its characterization by fluorescence-activated cell sorting analysis

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Abstract

A rapidly growing and fine-textured cell line, NB2P, was established from Japonica rice cultivar Nipponbare and characterized in this study. Addition of casein enzymatic hydrolysate (2 g/L) and pectinase (0.005%) to the suspension medium resulted in a 2-fold-increased rate of cell growth and reduced aggregation. Remarkably, the medium and conditions described here resulted in growth leading to a 9-fold increase in fresh weight 7 d after subculture. High-quality, well-dispersed nuclei were obtained from this NB2P cell culture. Fluorescence-activated cell sorting (FACS) analysis of the isolated nuclei showed a clear separation of each cell cycle phase in both small- and large-scale preparations. On the basis of representative data from the nuclei fraction in the G1 phase, purity of the sorted and recovered nuclei was higher than 98%. The studies described here demonstrate that NB2P culture can be a powerful tool for studying many important plant processes, including DNA replication and cell cycle-related pathways.

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Abbreviations

2,4-D:

2,4-dichlorophenoxyacetic acid

DAPI:

4′,6-diamidino-2-phenylindole

FACS:

fluorescence-activated cell sorting

MES:

2-(N-morpholino)-ethanesulfonic acid

PI:

propidium iodide

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Authors and Affiliations

  1. Department of Botany, North Carolina State University, 27695, Raleigh, NC, USA

    Tae-Jin Lee, Randall W. Shultz & William F. Thompson

  2. Department of Molecular and Structural Biochemistry, North Carolina State University, 27695, Raleigh, NC, USA

    Linda Hanley-Bowdoin

Authors
  1. Tae-Jin Lee
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  2. Randall W. Shultz
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  3. Linda Hanley-Bowdoin
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  4. William F. Thompson
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Correspondence to William F. Thompson.

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Lee, TJ., Shultz, R.W., Hanley-Bowdoin, L. et al. Establishment of rapidly proliferating rice cell suspension culture and its characterization by fluorescence-activated cell sorting analysis. Plant Mol Biol Rep 22, 259–267 (2004). https://doi.org/10.1007/BF02773136

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