Plant Tissue Culture Protocol

PTC Methods
Basic steps
• Only little variation in the methods of plant cell,
tissue and organ culture, but the basic steps are
almost same.
(1) Preparation of suitable nutrient medium.
(2) Selection of explants.
(3) Sterilisation of explants.
(4) Inoculation (transfer)
(5) Incubations
(6) Regeneration
(7) Hardening
(8) Plantlet Transfer
Preparation of suitable medium
• Suitable nutrient medium as per objective of
the culture is prepared and transferred into
suitable containers
• Eg. Flasks, Petriplates, culture tubes etc.,
• Autoclaved at 15 psi (pound per inch square)
for 30 minutes.
• Hormones and vitamins are sterilised using
millipore filter.
Selection of explants
• Explants are any excised part of plant to be
used in tissue culture.
• Eg., axillary, bud, leaf and stem segments, root
tip, shoot tip, anther, ovary, endosperm etc.,
• Always young and health parts of the plants
are selected as explant.
Sterilisation of Explants
• Sterilisation is the complete eradication of
microorganisms present on the surface of any
material.
• The explants are sterilised by disinfectants (eg.
Sodium hypochlorite, mercuric chloride)
• Washed aseptically for 6-10 times with
sterilised distilled water.
Explant excision
Inoculation (Transfer)
• The sterile explant is inoculated on the surface
of solidified nutrient medium under aseptic
conditions.
• The cabinet of laminar airflow provides the
sterile conditions.
Incubations
• The cultures are incubated in the growth
chamber or tissue culture room at 25±2°C, 50-
60% relative humidity and 16 hours of
photoperiod (light and dark created
artificially).
• After defined period callus develops on the
medium or shoots/roots developing from
explant.
Regeneration
• Plantlets regenerate after transferring a
portion of callus onto another medium and
induction of roots and shoots or directly from
explants.
Hardening
• Hardening is the gradual exposure of plantlets
for acclimatisation to environmental
conditions.
Hardening process( Acclimatization)
Plantlet transfer
• After hardening process plantlets are
transferred to green house or field conditions.
Types of cultures
(1) Explant culture.
(2) Callus culture.
(3) Organ culture.
(4) Cell or suspension culture.
(5) Protoplast culture.
Explant culture
• There are variety of types of seeds plants viz.,
trees, herbs, grasses which exhibit the basic
morphological units i.e., root, stem and leaves.
• Parenchyma is the most adaptable of all types
of tissues.
• They are capable of cell division and growth.
• Development of tissue is characterized by cell
division, cell elongation and cell
differentiation.
• Young plants parts are used.
• Parenchyma of stems, rhizomes, tubers, roots
is easily accessible and will generally respond
quickly to culture conditions in vitro.
• Explant cultures are generally used for
induction of callus or regeneration of plant.
Callus formation / culture
• Callus develops by infection of microorganisms
from wounds due to stimulation by endogenous
growth hormones, the auxins and cytokinins.
• It has been artificially developed by adopting
tissue culture techniques.
• Explant, a 2-5 mm sterile segment, excised from
a stem, tuber or root is transferred into nutrient
medium incubated at 25-28°C in a artificial day
light regime.
• Nutrient medium supplemented with auxins
induces cell division.
• The upper surface of explant is covered by
callus.
• A callus is an amorphous mass of loosely
arranged thin walled parenchyma cells
developing from proliferating cells of the
parent tissue.
• When callus has grown on nutrient medium
after a long time it becomes essential to
subculture within 28 days on a fresh medium.
Organogenesis
• Root, shoot and leaves (not embryos) are the
organs that are induced in plant tissue culture.
• Organogenesis starts with stimulation caused
by the chemicals of medium, substances
carried over from the original explants and
endogenous compounds produced by the
culture.
• Skoog (1944) indicated that the organogenesis
can be chemically controlled.
• He observed root initiation (rhizogenesis) and
shoot inhibition (caulogenesis) after addition
of auxin to the medium.
• Generally excised root is cultured in liquid
medium.
• It has several advantages over solid medium.
• (i) nutritional requirements, (ii) infection by
Rhizobium and nodulation (iii) physiological
activities.
Micropropagation
• “… the art and science of multiplying plants
in vitro
It implies
• - regeneration
• - multiplication
• - uniformity
Starting material for
Micropropagation (Explant)
• Propagation from plant Tip bud
cells, tissues or organs
Leaf
under aseptic conditions
in synthetic medium in
vitro.
• Many different explants Axillary
bud
can be used for
micropropagation, but Internode
axillary buds and
meristems are most Root
commonly used
Stages of Micropropagation
• Stage I - Selection & preparation of the mother

plant – sterilization of the plant tissue takes
place Initiation of culture – explant placed into
growth media
• Stage II – Multiplication – explant transferred to
shoot media; shoots can be constantly divided.
• Stage III - Rooting– explant transferred to root
media
• Stage IV - Transfer to soil– explant returned to
soil; hardened off
STAGE I - Sterilization STAGE II: Shoot Production
STAGE III:
Pretransplant STAGE IV: Transfer to Natural
(rooting) Environment
Micropropagation
Cell (suspension culture)
• Cell suspension is prepared by transferring a
fragment of callus (about 500 mg) to the liquid
medium (500 ml) and agitating them
aseptically to make them cells free.
• It is difficult to have suspension of single cell.
• Suspension cell includes, single cell, cell
aggregates, residual inoculum and dead cells.
• King and street (1977) described the
techniques of cell separation by changing the
nutritional composition of medium.
• No standard technique for separation of cells
from callus has been recommended.
Benefits of cell culture
(1) The suspension can be pipetted.
(2) They are less heterogenous and cell
differentiation is less pronounced.
(3) They can be subcultured in volumes up to 1,500
litres.
(4) They can be subjected to more stringent
environmental controls.
(5) They are can be manipulated for production of
natural products by feeding precursors.

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What are the basic steps involved in plant tissue culture?

What is the purpose of sterilizing explants?

PTC Methods

• Stage I - Selection & preparation of the mother

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