In Vitro Cell.Dev.Biol.—Plant DOI 10.1007/s11627-011-9418-8
PROTOCOLS/METHODS
Micropropagation of gerbera using chlorine dioxide (ClO2) to sterilize the culture medium Jean Carlos Cardoso & Jaime A. Teixeira da Silva
Received: 16 March 2011 / Accepted: 2 November 2011 / Editor: J. Finer # The Society for In Vitro Biology 2011
Abstract One of the alternatives to autoclaving culture media is chemical sterilization, which may cause fewer changes to the chemical composition of the media. In this study, the effect of chemical sterilization by inclusion of chlorine dioxide (ClO2) in the culture medium on the in vitro development of gerbera (Gerbera jamesonii) cv. AL101, cultured at different stages of micropropagation, was evaluated. The following five concentrations of ClO2 were tested: 0%, 0.0025%, 0.0050%, 0.0075%, and 0.010%. Autoclaved medium was used as the control. ClO2 in the culture medium reduced contamination at rates comparable to autoclaving when tested at three stages of the culture process: in vitro establishment, multiplication, and rooting. Plantlets grown in culture media sterilized with ClO2 showed similar or better development than those grown in autoclaved culture medium. Use of 0.0025% ClO2 to sterilize the culture medium resulted in better plantlet development than autoclaved medium, regardless of the stage of micropropagation. Keywords Gerbera jamesonii . Culture medium . Micropropagation . Sterilization . Development
J. C. Cardoso (*) Lab of Plant Biotechnology, Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, C.P. 96, 13416-000 Piracicaba, Sao Paulo, Brazil e-mail:
[email protected] J. A. Teixeira da Silva Department of Horticultural Science, Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Miki-cho, Ikenobe 2393, Kagawa-ken 761-0795, Japan
Introduction Micropropagation has been used to accelerate plant production and generate virus-free plantlets (Kozai et al. 1997). The development of technologies for low-cost tissue culture is a priority in agriculture, horticulture, forestry, and floriculture in many developing countries (IAEA-TECDOC 2004), and it would be useful to reduce plant production costs associated with this technology (Brink et al. 1998). The process of autoclaving, which uses high temperature (121°C) and pressure (1 kg cm−2) for 15 to 30 min to eliminate contaminating microorganisms from the culture medium (Torres et al. 1998), increases the production costs of plantlets and can lead to the degradation of culture media components, changing its composition (Pan and van Staden 1999). As a result, chemical alternatives to sterilization such as sodium hypochlorite (NaOCl) (Teixeira et al. 2006) and hydrogen peroxide (Yanagawa et al. 1995) have been tested. However, technical difficulties can be associated with their practical use, which have not allowed their commercial application for in vitro plantlet production or for standard tissue culture research on a wide scale. Liquid chlorine dioxide (ClO2) is a potent antibacterial agent, fungicide and viricide (Srebernich 2007) and is stable in solutions at pH ranging from 3.0 to 9.0, with higher effectiveness than a disinfectant at neutral pH (Huang et al. 1997). It is effective in the presence of organic matter, is stable at very low concentrations (
