Plant tissue culture involves growing plant cells, tissues, or organs in sterile conditions on a nutrient medium. It allows for mass production of plant clones and regeneration of whole plants from modified plant cells. Key aspects of plant tissue culture include using explants, maintaining sterile conditions, and promoting cell differentiation and regeneration into whole plants using techniques like micropropagation and somatic embryogenesis. Success requires selecting the right tissue culture type and optimizing factors like media, light, and temperature for the specific plant.

1. Basics of Cell and
tissue culture
BY. Muhammad Mukheed Jami

2. • Cell culture is the practice, by which cells are
grown under controlled conditions, generally
outside of their natural environment. ……term
for animal….not for plant.
• Two parts of course…plant cell culture and cell
culture (animal cell culture)
• Right now…Focusing on first part…

3. • Plant tissue culture is a collection of
techniques used to maintain or grow plant
cells, tissues or organs under sterile conditions
on a nutrient culture medium of known
composition. Plant tissue culture is widely
used to produce clones of a plant in a method
known as micropropagation
• Purpose…

4. • The production of exact copies of plants that produce
particularly good flowers, fruits, or have other desirable
traits.
• To quickly produce mature plants.
• The production of multiples of plants in the absence of
seeds or necessary pollinators to produce seeds.
• The regeneration of whole plants from plant cells that have
been genetically modified.
• The production of plants in sterile containers that allows
them to be moved with greatly reduced chances of
transmitting diseases, pests, and pathogens.
• The production of plants from seeds that otherwise have
very low chances of germinating and growing,
i.e.: orchids and Nepenthes.
• To clear particular plants of viral and other infections and
to quickly multiply these plants as 'cleaned stock' for
horticulture and agriculture.

5. Introduction
• There are numerous methods to propagate
plants in tissue culture.
• But the one principle that is constant is
totipotency – all plants and plant parts have
this potential.

6. Why does tissue culture work?
• Totipotency:
– The ability of a cell to differentiate and develop
into a whole plant when given the correct
conditions. This is because every cell has the
genetic potential of the parent plant.

7. Imp. terms
• Explant
– Living tissue transferred
from a plant to an artificial
medium for culture.
– It can be any portion of
the shoot, leaves, roots,
flower or cells from a plant.

8. • In vitro culture
– From Latin- “within the glass” performing an experiment in a
test tube.
– All types of culture including animal cells, in vitro fertilization,
etc.
• Tissue culture
– Inclusive term for growth of cells and tissues in a sterile
environment
• undifferentiated plant cells
• plant callus
• plant tissue

9. • Micropropagation
– The production of whole plants from small sections of a plant,
called an “explant”.
• Apical bud
• Axillary bud
• Meristem
– Usually the method used by commercial tissue culture laboratories is
micropropagation, since a whole plant (including shoots and roots) is
produced, which is genetically identical to the mother plant.
– Nowadays, tissue culture, in vitro culture and micropropagation are
sometimes used interchangeably.

10. Imp. terms
• Motherplant
• Ex vivo
• In vivo
• Competent cell

11. Terminology
• Differentiation:
• The cells derived from root apical meristem
(RAM) and shoot apical meristem (SAM) and
cambium differentiate, mature to perform
specific functions. This act leading to
maturation is termed differentiation. They,
undergo a few or major structural changes
both in their cell walls and protoplasm.
–

12. • Dedifferentiation:
• In plants, the living differentiated cells can
regain the capacity to divide mitotically under
certain conditions. The sum of events, that
bestow this capacity to divide once again, are
termed dedifferentiation. A dedifferentiated
tissue can act as meristem (e.g., vascular
cambium, wound meristem, cork cambium)

14. Subculture
• After a period of time, it becomes necessary, due to
nutrient depletion and medium drying, to transfer organs
and tissues to fresh media.
• In general, callus cultures are subcultured every 4-6
weeks. Theoretically plant cell and tissue cultures may be
maintained indefinitely by serial subculturing.

15. Acclimatization
It is a process of gradual hardening of the cultured plants
from laboratory to field so that the regenerated plants
should adjust to field conditions.
Androgenesis
It is a process of regeneration of plants from pollen
grains. Such plants are haploids.
Auxenic or Aseptic culture
Cultures devoid of foreign or undesired life forms are
called auxenic culture

16. Basal medium
Most of the media contain inorganic salts of major and minor
elements, vitamins and sucrose. A medium with these ingredients is
called basal medium.
Callus
Mass of undifferentiated cells produced in tissue culture is called
callus. The callus is highly vacuolated and unorganised cells.
Clone
A clone is a group of plants produced from a single explant through
asexual reproduction. All the members of a clone have the same
genotype as'" that of the parent. They are identical in genotype.
Phenotypic differences within a clone are due to external factors.

17. Cryobiology
It is a technique of preservation of plant tissues and cells
in liquid nitrogen at196°C. This is highly significant for
storage of germplasm of those crops which do not
produce seeds and reproduce only by vegetative
means. Thus cryobiology is very useful in crop
improvement.
Explant
Small piece of viable tissues isolated from parent plant is
called explant .
Inoculation
Transfer of explant to culture medium is called
inoculation.

18. In vitro
Cells/tissues removed from the intact organism and
grown in controlled condition in laboratory.
Micropropagation (clone propagation)
It is a process of production of clones similar to asexual
reproduction. Plantlets are produced from shoot
tips/axillary buds. Here, small amount of explant
produce millions of clonal parts in a year directly.
Organogenesis
Process of differentiation of callus initially into embryo
like structure (embryoids) and then showing organ like
roots/shoots

19. Protoplast
Animal, plant or fungal cell from which the entire cell wall
has been removed.
Regeneration
Production of entire plant from explant is called
regeneration.
Shoot tip culture
Culture of shoot tip (shoot apical meristem) along with
one or more leaf primordia or mature leaves is called
shoot tip culture.

20. Somaclone
Plants derived from somatic cell culture.
Somaclonal variation
Somaclonal variations are heritable variations for both
qualitative and quantitative traits produced in plants
regenerated from cell culture and tissue cultures.
Variation caused during plant tissue culture is called
somaclonal variation. Somaclonal variations arise as
a result of chromosome structural changes, gene
mutations, gene amplification, transposable
elements etc.

21. Somatic embryogenesis
Process of production of embryo from somatic cells is called
somatic embryogenesis and such embryo is called
embryoid.
Stock plant
The plant from which explant is taken is called stock plant.
Sub-culture
Callus produced and cultured again for production of big mass
of callus is called sub-culture.

22. Suspension culture
A type of liquid culture in which cells or cell
aggregates grow and multiply.
Transplant stage
Process of transfer of regenerated plants from
test tube (i.e. in vitro) to the soil is called
transplant stage

23. Introduction
• Type of cells
– Meristematic cells
• undifferentiated cells at shoot and root tips.
• greatest potential to produce cells that will become
shoots or roots.
– Parenchyma cells
• thin-walled cells that make up the bulk of most non-
woody structures.
• can be induced to divide and differentiate.

24. Types of tissue culture
1. Organized culture:
– The culture of whole or parts of a plant. The characteristics and
organizational structure of a plant or organ is maintained.
– Axillary bud culture
– Terminal bud culture
– Seed culture
– Embryo culture
– Ovary culture
– Pollen culture

25. Types of tissue culture
2. Unorganized culture:
• Callus culture
• Cell suspension culture
• Organogenesis
• Somatic embryogenesis
• Protoplast culture

26. Types of tissue culture
• The type of tissue culture techniques applied is
dependent on the type of explant and what one wants to
achieve.
• Techniques mentioned above is mainly used for
propagation and multiplication.
• Other techniques include:
– Micrografting – to eliminate viruses, to root microcuttings.
– In vitro pollination Used by plant breeders to
– In vitro fertilization create new varieties

27. Advantages
• Mass production of various plant cultivars
– 6 million plants per year from one explant.
– Much higher production rate than other asexual
propagation methods.
• Especially beneficial for:
– Plants in high demand or valuable plants.
– Plants that are slow or difficult to propagate.
– Endangered species.

28. Advantages
• Production of pathogen-free plants
– Maintaining disease-free plants by micropropagation.
• Germplasm preservation
– Germplasm: the DNA of a species
– In the past: seeds
–limited shelf-life
–don’t preserve uniform characteristic (variability)

29. Advantages
• Continuous year round production
– Unaffected by climate
• Propagated in controlled lab conditions
– The ability to change specific conditions to meet
the needs of a particular plant species.
– Mainly, nutrient, light and temperature
requirements.

30. Advantages
• The original plant is not
destroyed in the process
- a factor of considerable
importance to the owner
of a rare or unusual plant.

31. Disadvantages
• Specialized equipment required
– Laminar flow cabinets
– Autoclave
– Water purification systems
– Glassware etc…
• High labor cost is the most limiting factor
– Skilled labor required

32. Disadvantages
• Contamination risks
– Maintenance of aseptic (sterile) environment difficult.
– Rapid spread of contaminants = widespread loss.
• Risk of mutation arising
– Artificial environment induces mutations.
• Responses to tissue culture conditions varies
– Trial and error to determine optimum media or conditions。

33. Factors affecting tissue culture
• The areas in which tissue culture techniques can be used
are very wide.
• The choice of technique is dependent on what one wants
to achieve. It may be mass production, breeding of new
varieties, or producing virus-free plants.
• To be able to successfully propagate plants in vitro,
understanding how and why these factors affect plant
growth in an in vitro environment is crucial.

34. Factors affecting tissue culture
• The in vitro growth and development of a
plant is determined by a number of factors:
– The genetic make-up of the plant
– Source of explants
– Nutrients
– Environmental factors: light, temperature, pH, O2
and CO2 concentrations.

35. Factors affecting tissue culture
• The genetic make-up of the plant.
– The genetic make-up is a decisive factor at every
stage in the plant.
– It determines, for example, if a plant is a
monocotyledon or dicotyledon, or which
temperature is optimal for growth.
– The type of in vitro environment that must be
created in the lab to ensure that growth and
development of the explant takes place, is totally
dependent on the genotype of the plant.

36. Factors affecting tissue culture
• Source of explant
– Young explant vs. old explant
– Usually the younger, less differentiated explant,
the better for tissue culture
– Type of explant – leaf, stem, root, meristem, etc.

37. Factors affecting tissue culture
• Growth medium (Artificial)
– Nutrients
– Plant hormone
– Vitamins
• Environmental factors (Controlled)
– Light intensity
– Photoperiod
– Temperature
– Sterility

38. • Next lecture……
• History of plant cell culture