Plants grows in their natural environment interact continuously with various biotic and abiotic factors. Among them, biotic factors such as bacteria, fungi and virus might present within the tissues and could be most important factor for losses during initiation of culture (Omamor et al., 2007).
The doubling time of mentioned microbes is between 20-30 minutes, on the other hand plant cells divides after 20-24 hours. Moreover, cell wall and cell membrane compositions are also relatively same for both microbes and different plant species. The number and types of such microorganisms varies with respect to plant stage, weather and species. The most versatile part of aseptic inoculation is to optimize decontamination treatment for different plant species due to their varied cell wall compositions. The sensitivity of tissue towards different sterilants, time of contact and concentration affects the success of decontamination treatments (Goswami & Handique, 2013). These potential biotic factors compete for the available resources during tissue culture and growth of these microbes will lead to signal transduction in plants tissue. The plant tissue produces certain metabolites in response to these microbes. As a result, the explant gets necrosis and culture mortality (Kane, 2003).
These contaminants are not seen all the time during initiation stage; some contaminant appears after several sub culture cycle. Sometimes sequential sterilization gave better response than single sterilization step. Scientist around the world tries lot to eliminate contamination using various chemical treatments. However, on an average 10-15% losses were observed during micropropagation after every subculture cycle (Goswami & Handique, 2013). During aseptic inoculation process, the explants should not lose their meristematic nature and contaminants must be killed. Previous studies reported use of various sterilization agents to decontaminate the tissues.
There are various sterilants like mercuric chloride, sodium chlorite, sodium hypochlorite, sodium benzoate, calcium hypochlorite, ethanol (or isopropyl alcohol), hydrogen peroxide, lactic acid, silver nitrate (Oyebanji, et al., 2009) routinely used in the micropropagation laboratory. The laundry bleach contains sodium hypochlorite as principle compound and having strong bactericidal activity even at very low concentration. During the preparation of sterilants in water, it dissociate into highly active and toxic HoCl salt. Calcium hypochlorite is mild in its effect and alternative to sodium hypochlorite for decontaminating explants (Oyebanji, et al., 2009).
Similarly, mercury chloride is the most common, widely used and extensively reported in various literatures, frequently utilized to alleviate microbial load in tissue culture (Kanwar, 2009; Lal et al., 2009). Ethanol could be potent sterilizer with phytotoxic at prolonged exposure. Above all factors requires lot of efforts, time, money and material which if not properly handled can lead to complete wipe of the cultures present in the laboratory(Webster et al., 2003).