Plant Tissue

Culture
 Plant Tissue Culture
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.
Plants have the ability to reproduce asexually, so this
natural ability is the basis of plant tissue culture.

All plants have TOTIPOTENCY which means they each

retain a latent capacity to produce a whole plant

Totipotency is a special characteristic of young tissues or

meristems to develop into an entire plant if suitable
stimulated
The term describes all kinds of in vitro plant cultures
under control conditions which lead to cell multiplication or
regeneration of organs or whole plants

It is the source to obtain plants from the cells which would
not normally take part in the plant reproduction.

Plant tissue culture is used to produce large number of

plants from small pieces of the parent plant in shorter
periods of time.
Why do Plant Tissue Culture?

• The objective of plant tissue culture

A more recent advance is the use of
plant tissue culture along with genetic
modification using bacterial vectors and
gene guns to create genetically
engineered plants
 Where does the New growth come
from in plants?

• Embryo Culture
• Organ Culture
1. Shoot tip culture
2. Root culture
3. Leaf culture
4. Anther culture
• Callus culture
• Cell suspension
• Protoplast culture
What is needed for Plant Tissue
Culture?

•Appropriate tissue
•Nutrient growth medium
•Aseptic environment
•Growth regulators
•Frequent sub culturing
What is needed for Plant Tissue Culture
Appropriate tissue
Selection of the plant tissue (explant) from a healthy vigorous
‘mother plant’
Usually, the younger, less differentiated
the explant, the better for tissue culture

The explants must be sterilized to remove

microbial contaminants.

Surface contaminants - principally microbial saprophytes that

are eliminated by surface disinfestation
Internal contaminants - principally pathogens that are
eliminated by thermotherapy (35-40 C) and culture of explants
free of organisms or by antibiotics
 Common Plant Tissue Disinfectants

Conc. of Active
Agent Ingredient Phytotoxicity Time (min)
• Na hypochlorite
(Laundry Bleach) 0.25-1% Moderate 5-20
• Ca hypochlorite 9-10% Moderate 5-20
• H2O2 3-10% High 5-20
• Alcohol(ethanol or
isopropanol) 70% High <30 sec
These disinfestants can be used in combination and the effectiveness of
these solutions is enhanced by using a wetting agent such as a detergent
 Nutrient Medium
• When an explant is isolated, it is no longer able to receive nutrients or
hormones from the plant, and these must be provided to allow growth in
vitro.

• A nutrient medium is defined by its mineral salt composition, carbon

source, vitamins, plant growth regulators and other organic supplements

• The composition of the nutrient medium is for the most part similar,
although the exact components and quantities will vary for different species
and purpose of culture.

• Types and amounts of hormones vary greatly. In addition, the culture must
be provided with the ability to excrete the waste products of cell metabolism.

• pH determines many important aspects of the structure and activity of

biological macromolecules. Optimum pH of 5.0-6.0 tends to fall during
autoclaving and growth.
Medium constituents
• Mineral salts
• Carbohydrates
• Vitamins
• Water
• Growth hormones
• Solidifying agents
• Other supplements
Mineral Salts

Macroelements: The elements required in

concentration > 0.5 mmol/L. (N, K, P, Ca, S, Mg,
Cl)

Microelements: The elements required in conc.

< 0.5 mmol/L (Fe, Mn, B, Cu, Zn, I, Mo, Co)

The optimum concentration → maximum

growth rate
 Mineral Salts Function of Nutrients in Plant Growth

MINERAL SALT FUNCTION

• Nitrogen Component of proteins, nucleic acids and some coenzymes. Element required
in greatest amount
• Potassium Regulates osmotic potential, principal inorganic cation
• Calcium Cell wall synthesis, membrane function, cell signalling
• Magnesium Enzyme cofactor, component of chlorophyll
• Phosphorus Component of nucleic acids, energy transfer, component of
intermediates in respiration and photosynthesis
• Sulphur Component of some amino acids (methionine, cysteine) and some cofactors
• Chlorine Required for photosynthesis
• Iron Electron transfer as a component of cytochromes
• Manganese Enzyme cofactor
• Cobalt Component of some vitamins
• Copper Enzyme cofactor, electron-transfer reactions
• Zinc Enzyme cofactor, chlorophyll biosynthesis
• Molybdenum Enzyme cofactor, component of nitrate reductase
Carbohydrates
• Plants in culture usually cannot meet
their needs for fixed carbon. Usually
added as sucrose at 2-3% w/v

• For large scale cultures, cheaper

sources of sugars (corn syrup) may be
used
 Vitamins
• A wide range of vitamins are available and
may be used.
• Generally, the smaller the explant, the more
exact amount of vitamin are required.
• A vitamin cocktail is often used (Nicotinic
acid, Glycine, Thiamine, pyridoxine).
• Inositol usually has to be supplied at much
higher concentration (100mg/l)
 Plant hormones
• Auxins induces cell division, cell elongation, swelling of
tissues, formation of callus, formation of adventitious roots.-
inhibits adventitious and axillary shoot formation (IAA, NAA,
IBA, 2,4-D)
• Cytokinins shoot induction, cell division (Zeatin, BAP,
Kinetin).
• Gibberellic acids: plant regeneration, elongation of internodes
(GA3)
• Abscisic Acid: induction of embryogenesis (ABA)
• Generally, the ratio of these two hormones can determine
plant development:

↑ Cytokinin ↓Auxin = Shoot development

↑Auxin ↓Cytokinin = Root development
Auxin = Cytokinin = Callus development
Polyphenolics in Tissue Culture
Many plants are rich in polyphenolics, after tissue injury during
dissection, such compounds will be oxidized by polyphenol oxidases,
tissue turn brown/black

Oxidation products inhibit enzyme activities and may kill the explant
 Prevention:

• Adding antioxidants (ascorbic acid, citric acid, PVP,

dithiothreitol) or presoaking explants in
antioxidant

• Incubating the initial period of culturing in reduced

light/darkness

• Frequently transferring into fresh medium

 Other Supplements
• Complex substances: yeast extract, malt extract, coconut
milk, protein hydrolysate

• Activated charcoal is used where phenol-like compounds are

a problem, absorbing toxic pigments and stabilizing pH
MICRO-PROPAGATION

“ The practice of rapidly multiplying stock plant

material to produce a large number of progeny
plants, using modern plant tissue culture
methods.”

Two general forms of plantlet production.

• Micro cuttings
• Callus production
MICROPROPAGATION - THE STEPS

• Microcutting
 Meristem and shoot tip culture
 Bud culture
• Embryogenesis
 Direct embryogenesis
 Indirect embryogenesis
• Organogenesis
 Direct adventitious organ formation
 Organogenesis via callus formation
 MICROCUTTINGS
Explant: Mother plant

• Size
• Source
• Species dependent
• Physiological age
• Degree of contamination
a) External infection
b) Internal infection
 MICROCUTTINGS
• Establishment of the explant
in the culture medium:

The medium sustains the

plant cells and encourages cell
division. It can be solid or
liquid. Each plant species
(and sometimes the variety
within a species) has
particular medium
requirements that must be
established by trial and error
 MICROCUTTINGS

• Root formation:

The shoots are then transferred

to growing medium with
suitable auxin:cytokinin ratios
MICROCUTTINGS
Shifting of Plants to soil:
• The rooted shoots are
potted up and ‘hardened
off’/acclimatized by
gradually decreasing the
humidity

• This is necessary as many

young tissue culture
plants have no waxy
cuticle to prevent water
loss
 Features of Micropropagation
• Can be recycled to produce unlimited clones
• Routinely used commercially for many ornamental
species, some vegetatively propagated crops

• Easy to manipulate production cycles

• Not limited by field seasons/environmental
influences

• Disease-free plants can be produced

• Has been used to eliminate viruses from donor
plants
 Callus Production
Callus:
•Undifferentiated tissue that develops
on or around an injured or cut plant
surface or in tissue culture.

• It is expanded into a larger mass of

undifferentiated cells.

• Callus is then activated, by

selective use of plant hormones to
dedifferentiate to produce, shoots,
roots and ultimately plantlets
Callus culture-Phases of growth

• Lag phase: cells prepare to divide

• Exponential phase: the rate of cell division is highest

• Linear phase: cell division slows but the rate of cell

expansions increases

• Deceleration phase: rates of cell division and

elongation decreases

• Stationary phase: number and size of cells remain

constant
 Embryogenesis

Embryogenesis is a natural process that produces a plant embryo

from a fertilized ovule by cell division and the differentiation of

undifferentiated cells into tissues and organs. This process
occurs during seed development, when the single-celled zygote
undergoes a programmed pattern of cell division which results in
the formation of mature embryo.

A similar process continues during the plant's life within the

meristems of the stems and roots. Plant cells can be induced to
form embryos in plant tissue culture; these embryos are called
somatic embryos, which are used to generate new plants from
single cells.
Somatic embryogenesis
The first somatic embryo formation was observed on carrot
in 1958 by Steward and Reinert

Somatic embryos are formed from plant cells that are not
normally involved in the development of embryos, i.e.
ordinary plant tissue e.g. meristematic tissues in the stem,
roots etc.

Plant growth regulators in the tissue culture medium can be

manipulated to the induced callus and subsequently
changed to embryogenesis from the callus. The ratio of
different plant growth regulators required to induce callus or
embryo formation varies with the type of plant.
Stages of somatic embryogenesis

• Initiation
• Proliferation
• Pre-maturation
• Maturation
• Germination
Limitations of Somatic
Embryogenesis
• High probability of mutations

• Method is usually difficult

• Chances of losing regenerative capacity with repeated

subcultures

• Induction of embryogenesis is often species dependant

• Embryo dormancy problems

Plant Organogenesis
 Plant Organogenesis
• The process of initiation and development of
a structure that shows natural organ (roots,
shoots, leaves).

• These organs may arise out of pre-existing

meristems or out of differentiated cells.

• This, like embryogenesis, may involve a

callus intermediate but often occurs
without callus.
Applications of PTC
A single explant can be multiplied into
several thousand plants in less than a
year. This allows fast commercial
propagation of new cultivars
Taking an explant does not usually
destroy the mother plant, so rare and
endangered plants can be cloned safely
Once established, a plant tissue culture
line can give a continuous supply of
young plants throughout the year
Applications…
In plants prone to virus diseases, virus
free explants (new meristem tissue is
usually virus free) can be cultivated to
provide virus free plants
Plant ‘tissue banks’ can be frozen, then
regenerated through tissue culture
Plant cultures in approved media are
easier to export than soil-grown plants, as
they are pathogen free and take up little
space (most current plant export is now
done in this manner)
Applications…
Tissue culture allows fast selection for
crop improvement - explants are chosen
from superior plants, then cloned

Tissue culture clones are ‘true to type’ as

compared with seedlings, which show
greater variability
Limitations
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. Protocols not
optimized for all species
Technical expertise in management
positions
It may be too expensive