Tissue Culture
Tissue culture is the culture and
maintenance of plant cells or organs in sterile, nutritionally and environmentally
supportive conditions (in vitro). Tissue culture produces clones, in which all
product cells have the same genotype (unless affected by mutation during culture).
It has applications in research and commerce. In commercial settings, tissue
culture is primarily used for plant propagation and is often referred to as
micro propagation.
What conditions do plant cells
need to multiply in vitro?
Tissue culture has several
critical requirements:
· Appropriate tissue (some tissues culture better
than others)
· A suitable growth medium containing energy
sources and inorganic salts to supply cell growth needs. This can be liquid or semisolid.
· Aseptic (sterile) conditions, as microorganisms
grow much more quickly than plant and animal
tissue and can overrun a culture.
· Growth regulators - in plants, both auxins &
cytokinins.
· Frequent subculturing to ensure adequate
nutrition and to avoid the build-up of waste metabolites
Appropriate tissue (Explant)
· Cell, tissue or organ of a plant that is used to
start in vitro cultures.
· Axillary buds, meristems Leaves, stems, roots,
hypocotyl…
· Many different explants can be used for tissue
culture, but axillary buds and meristems are most commonly used.
· The explants must be sterilized to remove
microbial contaminants. This is usually done by chemical surface sterilization
of the explants.
· With an agent such as bleach at a concentration
and for a duration that will kill or remove pathogens without injuring the
plant cells.
· Many plants are rich in polyphenolics
· After tissue injury during dissection, such
compounds will be oxidized by polyphenol oxidases
→ tissue turn brown/black
· Phenolic products inhibit enzyme activities and
may kill the explants
Methods to overcome browning:
Adding antioxidants [ascorbic
acid, citric acid, PVP (polyvinylpyrrolidone), dithiothreitol], activated
charcoal or presoaking explants in antioxidant Incubating the initial period of
culturing in reduced light/darkness Frequently transfer into fresh medium.
Nutrition 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.
· 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.
· This is accomplished by culturing on or in a
defined culture medium which is periodically replenished.
· A nutrient medium is defined by its mineral salt
composition, carbon source, vitamins, plant growth regulators and other organic
supplements.
· 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.
Mineral salt
§ NH4NO3 Ammonium nitrate
§ KNO3 Potassium nitrate
§ CaCl2 -2 H2O Calcium chloride (Anhydrous)
§ MgSO4 -7 H2O Magnesium sulfide
(Epsom Salts)
§ KH2PO4 Potassium hypophosphate
§ FeNaEDTA Fe/Na
ethylene-diamine-tetra acetate
§ H3BO3 Boric Acid
§ MnSO4 - 4 H2O Manganese sulfate
§ ZnSO4 - 7 H2O Zinc sulfate
§ KI Potassium iodide
§ Na2MoO4 - 2 H2O Sodium
molybdate
§ CuSO4 - 5 H2O Cupric sulfate
§ CoCl2 - H2O Cobaltous sulfide
Mineral salt composition
v Macroelements: The elements required in
concentration > 0.5 mmol/l
v
The essential macroelements: N, K, P, Ca, S, Mg,
Cl
v
Microelements: The elements required in conc.
< 0.5 mmol/l
v
The essential microelements: Fe, Mn, B, Cu, Zn,
I, Mo, Co
v
The optimum concentration → maximum growth rate
Carbon sources and vitamins
· Sucrose or glucose (sometimes fructose),
concentration 2-5%
· Most media contain myo-inositol, which improves
cell growth
· An absolute requirement for vitamin B1
(thiamine)
· Growth is also improved by the addition of
nicotinic acid and vitamin B6 (pyridoxine)
· Some media contain pantothenic acid, biotin,
folic acid, p-amino benzoic acid, choline chloride, riboflavine and ascorbic
acid (C-vitamin)
Plant growth regulators
Auxins:
· Induces cell division, cell elongation, swelling
of tissues, formation of callus, formation of adventitious roots.
· Inhibits adventitious and axillary shoot
formation
-
2,4-D, NAA, IAA, IBA, pCPA…
Cytokinins:
· Shoot induction, cell division
· BAP, Kinetin, zeatin, 2iP…
Gibberellins:
· Plant regeneration, elongation of internodes
- GA3…
Abscisic acid:
· Induction of embryogenesis
- ABA
Organic supplements
· N in the form of amino acids (glutamine,
asparagine) and nucleotides (adenine)
· Organic acids: TCA cycle acids (citrate,
malate, succinate, fumarate), pyruvate
· 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. Also, to prevent oxidation of phenols PVP (polyvinylpyrrolidone), citric acid,
ascorbic acid, thiourea and L-cysteine are used.
Different Techniques of Plant
Tissue Culture:
• Callus and Cell culture
• Somatic embryogenesis
• Haploid culture
• Protoplast culture
• Micro propagation
• Organogenesis
• Soma clonal variation
• In vitro Mutagenesis
This includes:
ØCell culture
ØTissue culture
ØOrgan culture
Cellular
totipotency and plant regeneration
§ The
capacity of a cell (or a group of cells) to give rise to an entire organism.
§ Unlike
an animal cell, a plant cell, even one that highly maturated and
differentiated, retains the ability to change a meristematic state and
differentiate into a whole plant if it has retained an intact membrane system
and a viable nucleus.
§ An
excised piece of differentiated tissue or organ (Explant) → dedifferentiation →
callus (heterogenous) → re differentiation (whole plant) = cellular totipotency.
§ Two
hormones affect explants’ differentiation:
§ ↑
Auxin ↓Cytokinin = Root development
§ ↑
Cytokinin ↓Auxin = Shoot development
§ Auxin
= Cytokinin = Callus development
Callus culture
§ Un-differentiated
mass of cells.
§ Callus
is formed at the peripheral surfaces as a result of wounding and hormones.
§ Genotype,
composition of nutrient medium, and physical growth factors are important for
callus formation.
Organogenesis
§ Process
of differentiation by which plant organs are formed (roots, shoot, buds, stem
etc.).
§ Plant
development through organogenesis is the formation of organs either de novo
(from callus) or adventitious (from the explants) in origin.
Why we need plant tissue culture?
§ Create
a large number of clones
§ New
genes can be introduced in to the plants which boost crop yield resistance to
pests and infections
§ Time
required is much shortened & Decrease the amount of space required for
field trials
§ Rapid
propagation is possible.
§ Overcomes seasonal restrictions for seed germination
§ Preserve pollen and cell collections
§ Helps
to eliminate plant diseases
§ Reduces the
storage space
§ The
production of exact copies of plants that produce particularly good flowers,
fruits, or have other desirable traits
E.g.: Tea crop plantation
Rubber crop plantation
§ The
production of plants from seeds that other wise have very low chances of
germinating.
E.g.
: Orchids, Nepenthes
§ If
there is plant with partially infected tissue, it is possible to produce a new plant without infection.
Applications
· Widely used in forestry
· Pharmaceuticals
e.g.Ginseng and taxol
· Selection
of crops with advantageous characters e.g. herbicide
resistance/tolerance
· To cross distantly related species
· Production of dihaploids from haploids
· Produce large numbers of identical individuals
Basic requirements of a tissue
culture laboratory
ü Preparation
area
ü Transfer
area
ü Culture
area
ü Acclimatization
area
Preparation area
ü Running
water facility
ü Drainage
system
ü Equipments
for sterilization (Autoclave etc.)
ü Distilled
water supply
ü Necessary
chemicals and glass-ware
Transfer area
ü Laminar
air flow bench
ü Disinfectants
ü Other
sanitary aids (glows, masks etc.)
Culture area
ü Incubators
ü Shakers
ü Controlled
light, temperature RH etc.
Acclimatization area
ü Facility
to provide different shade levels
