Abstract
BackgroundThe repeated weekly subculture of plant cell suspension is labour intensive and increases the risk of variation from parental cells lines. Most of the procedures to preserve cultures are based on controlled freezing/thawing and storage in liquid nitrogen. However, cells viability after unfreezing is uncertain. The long-term storage and regeneration of plant cell cultures remains a priority.ResultsSycamore (Acer pseudoplatanus) and Arabidopsis cell were preserved over six months as suspensions cultures in a phosphate-free nutrient medium at 5°C. The cell recovery monitored via gas exchange measurements and metabolic profiling using in vitro and in vivo 13C- and 31P-NMR took a couple of hours, and cell growth restarted without appreciable delay. No measurable cell death was observed.ConclusionWe provide a simple method to preserve physiologically homogenous plant cell cultures without subculture over several months. The protocol based on the blockage of cell growth and low culture temperature is robust for heterotrophic and semi-autotrophic cells and should be adjustable to cell lines other than those utilised in this study. It requires no specialized equipment and is suitable for routine laboratory use.
Highlights
The repeated weekly subculture of plant cell suspension is labour intensive and increases the risk of variation from parental cells lines
A plateau corresponding to a cell fresh weight (FW) of 150 ± 15 g l-1 of culture is reached after two weeks
If cells are subcultured at this stage, a 2-d lag phase attributed to the exhaustion of sucrose supply and to the beginning of the related process of autophagy is observed
Summary
The repeated weekly subculture of plant cell suspension is labour intensive and increases the risk of variation from parental cells lines. Suspension culture of isolated plant cells is an invaluable tool for providing the material for high-throughput studies such as metabolic analyses, production of secondary plant products, and herbicide discovery. It enables easy experimentation on physiologically and biochemically homogenous population of cells. Different methods for cultivating large quantities of plant cells in liquid nutrient medium (NM) have been described for a long time [1,2,3,4] These methods are based on the subculture of cell suspensions having reached their growth plateau when most of the nutrients initially added to NM, carbohydrates, are metabolised.
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