What are the steps for plant tissue culture?
Basic
steps involved in plant tissue
culture:
Preparation
of suitable nutrient medium:
MS medium,
White’s medium, B5 medium are few culture media. Auxins, cytokines and
Gibberellins are the most commonly used growth hormones in culture medium
prepared for tissue culture.
Agar: It
solidifies the medium and acts as a substratum. It doesn’t have any nutritive importance. It is
obtained from sea weeds and is used to provide solid surface for growth.
All these
important nutrients are incorporated in the culture medium and transferred into
suitable containers and then sterilized.
Selection
of explant:
Explants
are any excised part of the plant to be used in tissue culture like axillary buds,
leaf and stem segments, root tip, shoot tip, anther, ovary and endosperm.
Always
young and healthy parts of plants are selected as explants.
Sterilization
of the explant:
Explants
are sterilized by using different types of disinfectants like sodium hypochlorite
(2%), silver nitrate (1%) calcium hypochlorite (9-10%), bromine water (1-2%),
mercuric chloride (0.1-1%), hydrogen peroxide etc. and different types of
antibiotics.
Inoculation
of explant:
The
sterile explant is inoculated on the surface of the solidified nutrient medium
under aseptic (free from contamination) condition.
Laminar
air flow can be used for this inoculation process.
Incubation:
Incubation
is done at 25+/- 2 0C, with a relative humidity of 50-60% with 16 hours of
photoperiod.
After
defined period of incubation, an unorganized and undifferentiated (no root and
shoot) mass of cells called callus is obtained from each explant. In some
cases, root and shoot directly develop from the explant after incubation.
Transfer
of callus (subculture)
and regeneration:
The callus
obtained from incubation of explant is transferred into another medium and
reincubated.
Plantlet
regenerates from the callus with the induction of roots and shoot directly
from the callus.
Hardening
or acclimatization:
Hardening
is the gradual exposure of the plantlets for acclimatization
or adjustment to the environmental conditions.
Transfer
of plantlets to the field:
After the
hardening process, plantlets are transferred to either the green house or the
field conditions outside.
These
plantlets will grow into independent and healthy plants.
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