Sterilization is the technique employed to get rid of microbes such as bacteria and fungi in the culture medium, vessels and explants.
i. Maintenance of Aseptic Environment:
During in vitro tissue culture maintenance of aseptic environmental condition should be followed, i.e., sterilization of glassware, forceps, scalpels, and all accessories in wet steam sterilization by autoclaving at 15 psi (121°C) for 15 to 30 minutes or dipping in 70% ethanol followed by flaming and cooling.
ii. Sterilization of culture room:
Floor and walls are washed first with detergent and then with 2% sodium hypochlorite or 95% ethanol. T he cabinet of laminar airflow is sterilized by clearing the work surface with 95% ethanol and then exposure of UV radiation for 15 minutes.
iii. Sterilization of Nutrient Media:
Culture media are dispensed in glass containers, plugged with non-absorbent cotton or sealed with plastic closures and then sterilized using autoclave at 15 psi (121°C) for 15 to 30 minutes. The plant extracts, vitamins, amino acids and hormones are sterilized by passing through Millipore filter with 0.2 mm pore diameter and then added to sterilized culture medium inside Laminar Airflow Chamber under sterile condition.
iv.Sterlization of Explants
The plant materials to be used for tissue culture should be surface sterilized by first exposing the material in running tap water and then treating it in surface sterilization agents like 0.1% mercuric chloride, 70% ethanol under aseptic condition inside the Laminar Air Flow Chamber.
2. Media Preparation
The success of tissue culture lies in the composition of the growth medium, plant growth regulators and culture conditions such as temperature, pH, light and humidity. No single medium is capable of maintaining optimum growth of all plant tissues. Suitable nutrient medium as per the principle of tissue culture is prepared and used.
MS nutrient medium (Murashige and Skoog 1962) is commonly used. It has carbon sources, with suitable vitamins and hormones. The media formulations available for plant tissue culture other than MS are B5 medium (Gamborg.et.al 1968), White medium (white 1943), Nitsch’s medium (Nitsch & Nitsch 1969). A medium may be solid or semisolid or liquid. For solidification, a gelling agent such as agar is added.
The pH of medium is normally adjusted between 5.6 to 6.0 for the best result.
The cultures should be incubated normally at constant temperature of 25°C ± 2°C for optimal growth.
Humidity and Light Intensity
The cultures require 50-60% relative humidity and 16 hours of photoperiod by the illumination of cool white fluorescent tubes of approximately 1000 lux.
Aeration to the culture can be provided by shaking the flasks or tubes of liquid culture on automatic shaker or aeration of the medium by passing with filter-sterilized air.
4. Induction of Callus
Explant of 1-2 cm sterile segment selected from leaf, stem, tuber or root is inoculated (transferring the explants to sterile glass tube containing nutrient medium) in the MS nutrient medium supplemented with auxins and incubated at 25°C ± 2°C in an alternate light and dark period of 12 hours to induce cell division and soon the upper surface of explant develops into callus. Callus is a mass of unorganized growth of plant cells or tissues in in vitro culture medium.
The callus cells undergoes differentiation and produces somatic embryos, known as Embryoids. The embryoids are sub-cultured to produce plantlets.
The plantlets developed in vitro require a hardening period and so are transferred to greenhouse or hardening chamber and then to normal environmental conditions. Hardening is the gradual exposure of in vitro developed plantlets in humid chambers in diffused light for acclimatization so as to enable them to grow under normal field conditions.