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Plant Tissue Culture and its


Nasir Hussain
Tissue Culture
• Production of microbe-free plant material in
an in vitro conditions from any part of the
plant in an aseptic (sterile) environment, such
as sterilized nutrient medium.
• It is non conventional method of vegetative
propagation for any crop specie.
• Conventionally vegetative propagation take
place through cuttings, budding, grafting and
layering etc.
Tissue Culture Techniques
• Micropropagation .
• Production of pathogen free plants.
• Meristem Culture.
• Somatic embryogenesis.
• Organ Culture.
• Callus Culture.
• Embryo culture.
• Anther and pollen grain Culture
• Protoplast culture etc.
Advantages of Tissue Culture
• Elimination of viruses from infected plants
• Rapid multiplication of clones (elite)
• Vegetative propagation of difficult to
propagate species
• All the year round propagation of clones
• True to type plantlet production
• Rapid multiplication of seedlings (in cases
where seed is hard to get)
Applications of Tissue Culture
• Organ, meristem, • Micropropagation,
shoot. disease elimination,
germplasm preservation.
• Undifferentiated • In vitro selection,
(callus suspension). secondary metabolites
• Embryo rescue. • Wide hybridization
• Somatic • Artificial seeds, genetic
embryogenesis. engineering.
Applications of Tissue Culture
• Anther, microspore • Homozygous line
culture. production.
• Protoplast • Gene pool
Tissue culture methods for virus
Thermotherapy, chemotherapy and meristem
tip culture.
• Incubation of plants or of cultures at
relatively high temperatures (32° to 40°C)
either alone or
• in combination with the excision and culture
of very small stem tips is the only method of
ridding infected cultivars of their viruses.
• Antivirus (Ribavirin).
Tissue culture methods for virus
• Excise and culture the apical meristem of an infected
plant because this region is most likely to be free of
• It involves a very delicate dissection and such small
explants are difficult to grow in culture.
• A very small stem tip which has, in addition to the
apical meristem, one or two leaf primordia. It is
much easier to grow in culture.
• High magnification stereomicroscope is also needed
to excise the meristems (0.1-0.5 mm).
Tissue culture methods for virus
• However, some viruses invade the
apical meristem and, in these cases, it
is necessary to apply heat treatments
to the plant. In the past, this has meant
heat treatment of the whole plant
followed by stem tip culture. An easier
method involves the application of the
heat treatment to aseptic cultures.

• Meristem tip refers to the

region of shoot apex lying
distal to the youngest leaf
primordial, whereas shoot
apex includes Meristem
tips plus a few sub
adjacent leaf primordial.

• Meristem tip culture is a

method in which shoot
meristem (Meristem tip) is
cultured in vitro with a few leaf
• ELISA (Enzyme-Linked
Immunosorbent Assay )
protein based.
• PCR nucleic acid base.
• Clonal multiplication of virus
free stock
• Micropropagation is the production
of whole plants from small sections
of plant such as a shoot tip, node,
meristem, embryo, or even a seed
• Plant tissue culture is basically the
same thing, except that it implies the
use of callus tissue generated from
plant cells cultured in-vitro.
• Why does micropropagation work?
– Plant cells have the ability to reproduce
the whole plant from single cells. This is
called totipotency.
– Totipotency is the ability of a single cell to
express the full genome in the cells to
which it gives rise by cell division.
• What part of the plant can be used for
– Any part of a plant can be used.
– Meristems, shoot and root tips, leaf
tissue, anthers, embryos, flowers,
virtually all parts of a plant can be
• The only limitation is that each plant is
propagated differently and not every
plant will respond the same way.
• Each genus, species and variety may
require a different tissue which will
obtain the best results.
Tip bud
Starting material for



Micropropagation Methods
• Micro cuttings
• Small sections of plant
tissue are cut from the
mother plant
• Placed into media
• Grown out or
subdivided again to
produce more plants
Micropropagation Methods
• Shoot Tip Culture (less 10 mm)
• Meristem culture (0.1-0.5 mm)
Surface sterilize, wash & culture.
Higher cytokinins to over come
apical dominance.
Adventitious bud production.
sub-culture frequently.
Basic in vitro propagation ...
Micropropagation Stages

• The Basics of Micropropagation

• The Four stages
– Stage one – Explant establishment or initiation
– Stage two – Multiplication
– Stage three – Rooting
– Stage four – Acclimatization or hardening off
Benefits for Micropropagation ...

♣ Rapid multiplication of clones

♣ Difficult species ?
♣ Genetic uniformity ?
♣ Aseptic conditions
♣ Micro- stock plants
♣ Controlled environment
Other applications ...

♣ in vitro micro-grafting
♣ Genetic conservation
♣ Plant improvement
♣ Experimental system