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Literature review
misting will facilitate contamination by wetting the foliage (KANE, 2004). Placing the
plant material in a less humid and dry environment a few weeks prior to taking
explant material can reduce contamination of cultures (SMITH, 2000b).
Plant materials growing in soil (roots, tubers, bulbs) or near the soil surface (stolons,
rhizomes, orchid protocorms, etc.) are often harder to clean and disinfect than aerial
plant material (SMITH, 2000b). Explants are much easier to clean if the plant has
been growing in an artificial medium, such as washed sand or perlite (KYTE &
KLEYN, 1996). After cutting the explant from the source plant, it should be placed in
a plastic bag containing a moist paper towel and kept refrigerated until culture
initiation (KYTE & KLEYN, 1996).
2.9.2 Explant preparation
Explants require surface-disinfection before they can be placed in culture on the
nutrient agar for in vitro culture (SMITH, 2000b). Explants are washed in sterile water
and rinsed in ethanol and the surface is sterilised using chemicals with a chlorine
base (AHLOOWALIA et al., 2002). There are a number of products used for surface
disinfection, the most commonly used is commercial chlorine bleach (SMITH, 2000b).
For soft, herbaceous material a calcium or sodium hypochlorite based solution is
often used at a concentration of 1-3% (AHLOOWALIA et al., 2002). Before surface-
disinfection any remaining soil or dead parts should be removed from the explant
(PIERIK, 1997). An inexpensive and ready-made alternative is a 5-7% solution of
Domestos® (a toilet disinfectant by Lever Bros. Ltd., UK), which contains 10.5%
sodium hypochlorite, 0.3% sodium carbonate, 10.0% sodium chloride and 0.5%
sodium hydroxide and a patented thickener (AHLOOWALIA et al., 2002). Explants
are washed in sterile water before and after sterilization (AHLOOWALIA et al., 2002).
A general procedure for preparing the explant involves washing the explant in warm,
soapy water after which it is rinsed in tap water (PIERIK, 1997; SMITH, 2000b). The
explant is then rinsed in a freshly made chlorine bleach solution (PIERIK, 1997). One
to 2 drops of wetting agent should be added to every 100 ml of bleach solution. The
explant is then rinsed in sterile water three to five times.
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