Two parametric cell cycle analyses of plant cell suspension cultures with fragile, isolated nuclei to investigate heterogeneity in growth of batch cultivations

Christiane Haas, Richard Hegner, Karsten Helbig, Kristin Bartels, Thomas Bley, Jost Weber*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Plant cell suspensions are frequently considered to be heterogeneous with respect to growth in terms of progression of the cells through the cell cycle and biomass accumulation. Thus, segregated data of fractions in different cycle phases during cultivation is needed to develop robust production processes. Bromodeoxyuridine (BrdU) incorporation and BrdU-antibodies or 5-ethynyl-2′-deoxyuridine (EdU) click-it chemistry are frequently used to acquire such information. However, their use requires centrifugation steps that cannot be readily applied to sensitive cells, particularly if nuclei have to be extracted from the protective cellular milieu and envelopes for DNA analysis. Therefore, we have established a BrdU-Hoechst stain quenching protocol for analyzing nuclei directly isolated from delicate plant cell suspension cultures. After adding BrdU to test Harpagophytum procumbens cell suspension cultures the cell cycle distribution could be adequately resolved using its incorporation for the following 72h (after which BrdU slowed biomass accumulation). Despite this limitation, the protocol allows resolution of the cell cycle distribution of cultures that cannot be analyzed using commonly applied methods due to the cells' fragility. The presented protocol enabled analysis of cycling heterogeneities in H. procumbens batch cultivations, and thus should facilitate process control of secondary metabolite production from fragile plant in vitro cultures.

Original languageEnglish
JournalBiotechnology and Bioengineering
Volume113
Issue number6
Pages (from-to)1244-1250
Number of pages7
ISSN0006-3592
DOIs
Publication statusPublished - 2016
Externally publishedYes

Keywords

  • Harpagophytum procumbens
  • Hoechst-BrdU-quenching
  • Plant cell suspension and in vitro culture
  • Process monitoring
  • Proliferation activity
  • Two-parametric cell cycle analysis

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