Plant and Agricultural Biotechnology (BTN218)

Plant in vitro culture (tissue/cell culture)

Sterile Techniques: Clean Equipment
• Successful tissue culture requires the maintenance of a sterile environment
• All tissue culture work is done in a laminar flow hood
• The laminar flow hood filters air with a dust filter and a high-efficiency particulate air (HEPA) filter
• It is important to keep the hood clean, which can be done by wiping it with 70% alcohol
• The instruments used should also be dipped in 70% ethanol and sterilized using flame or glass
beads
• Hands should be disinfected with ethanol before handling cultures in order to avoid
contamination.
• It is imperative to maintain axenic conditions throughout the life of cultures: from explant to the
production of whole plants
• Especially problematic are fungal contaminants that are propagated by spores that might blow into
a hood from an environmental source
• Therefore, it is important to work away from the unsterile edge of a laminar flow hood
• Culture rooms or chambers must be maintained as clean as possible to control any airborne
contaminants.
 Plant and Agricultural Biotechnology (BTN218)
Plant in vitro culture (tissue/cell culture)
Sterile Techniques: Clean Equipment
 Plant and Agricultural Biotechnology (BTN218)
Plant in vitro culture (tissue/cell culture)
Sterile Techniques: Surface sterilization of explants
• Plant tissues inherently have various bacteria and fungi on their surfaces
• It is important that the explant be devoid of any surface contaminants prior to tissue culture since
contaminants can grow in the culture medium, rendering the culture nonsterile
• In addition, they compete with the plant tissue for nutrition, thus depriving the plant tissue of nutrients
• The surface sterilants chosen for an experiment typically depend on the type of explant and also plant
species
• Explants are commonly surface-sterilized using sodium hypochlorite (household bleach) at 0.3 – 0.6%,
ethanol (70 – 80%), and fungicides when using field-grown tissues
• The time of sterilization is dependent on the type of tissue; for example, leaf tissue will require a shorter
sterilization time than will seeds with a tough seed coat
• Wetting agents such as Tween added to the sterilant can improve surface contact with the tissue
• Overexposing tissues to decontaminating chemicals can also kill tissues, so there is a balancing act
between sterilizing explants and killing the explants themselves
• Internal contamination can be controlled to a certain extent by frequent transfer to fresh medium or by
the use of a low concentration of antibiotics in the medium
 Plant and Agricultural Biotechnology (BTN218)
Plant in vitro culture (tissue/cell culture)
Sterile Techniques: Culture Conditions and Culture Vessels
• Cultures are grown in walk-in growth
rooms or growth chambers
• Humidity, light, and temperature have to
be controlled for proper growth of cultures
• A 16-h light photoperiod is optimal for
tissue cultures, and a temperature of 22–
25˚C is used in most laboratories
• Light is supplied by cool white fluorescent
lamps, but some cultures are also
incubated in the dark
• A relative humidity of 50–60% is
maintained in the growth chambers
• Cultures can be grown in various kinds of
vessels such as petri plates, test tubes,
“Magenta boxes,” bottles, and flasks