Tissue Culture

Tissue Culture Techniques: An Introduction

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

· 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…

·  Shoot induction, cell division
·  BAP, Kinetin, zeatin, 2iP…

·  Plant regeneration, elongation of internodes

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.


§  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.    


· 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

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