Micropropagacao de aloe vera

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Boisson et al. Plant Methods 2012, 8:4 http://www.plantmethods.com/content/8/1/4

PLANT METHODS

METHODOLOGY

Open Access

A simple and efficient method for the long-term preservation of plant cell suspension cultures
Anne-Marie Boisson, Elisabeth Gout, Richard Bligny* and Corinne Rivasseau*
Abstract
Background: The repeated weekly subculture of plant cell suspension is labourintensive 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. Results: Sycamore (Acer pseudoplatanus) and Arabidopsis cell were preservedover 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. Conclusion: We provide a simple method to preserve physiologically homogenousplant 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. Keywords: Plant cell suspension, Acerpseudoplatanus, Arabidopsis thaliana, cell preservation, in vitro and in vivo NMR spectroscopy, low temperature, phosphate starvation

Background 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 onphysiologically 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 [1-4]. These methods are based on the subculture of cell suspensions having reached their growth plateau when most of the nutrients initially added to NM, particularly carbohydrates, are metabolised. It leads tomore or less homogenous cell populations and usually induces a growth delay (lag phase) following subculture [5]. It has been shown that obtaining homogenous cell suspension cultures requires sophisticated apparatus such as chemostats that optimize NM and cell growth [6]. Alternatively, subcultures every one or two
* Correspondence: rbligny@cea.fr; corinne.rivasseau@cea.fr Commissariat àl’Energie Atomique, institut de Recherche en Technologies et Sciences pour le Vivant, Laboratoire de Physiologie Cellulaire Végétale, Unité Mixte de Recherche 5168 CNRS, UJF, INRA, CEA, F-38054 Grenoble, France

days also yields homogenous cell populations [7], but involve much handling and maintenance that is therefore difficult to perform over long periods of time. For this reason, alternativeprocedures to preserve newly optimized cell suspension cultures, ideally for indefinite periods, have been proposed. Apart from the maintenance of cell callus on solid media which lead to appreciable delays to initiate homogenous cell suspension cultures, most of the procedures are based on controlled freezing/ thawing and storage in liquid nitrogen [8-12]. However, the viability of the cells afterunfreezing is generally low and long lag phases before full recovery of cell culture growth are always mentioned by authors. The highest viability (up to 90%) was observed by Menges and Murray [13] after cryopreservation of Arabidopsis and tobacco cells in the presence of DMSO and sorbitol. Nevertheless, even in this case, it takes at least one week for cells to recover normal post-thaw growth and...
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