Plant Tissue Culture (PTC)
Plant tissue culture is used to describe the in vitro and
aseptic growth of any plant part on a tissue culture medium. This technology
is based on three fundamental principles:
•
The plant part or explant must be selected and isolated from the rest of plant
body.
•
The explant must be maintained in controlled physically (environmental) and
chemically defined (nutrient medium) conditions.
Explant: The tissue taken from a selected plant transferred to a
culture medium often to establish a new plant.
For PTC, the laboratory must have the following facilities:
·
Washing facility for glassware and ovens for drying glassware.
·
Medium preparation room with autoclave, electronic balance and pH
meter.
·
Transfer area sterile room with laminar air-flow bench and a
positive pressure ventilation unit called High Efficiency Particulate Air
(HEPA) filter to maintain aseptic condition.
·
Culture facility: Growing the explant inoculated into culture
tubes at 22-28° C with illumination of light 2400 lux, with a photoperiod of
8-16 hours and a relative humidity of about 60%.
Sterilization is the technique employed to get rid of microbes
such as bacteria and fungi in the culture medium, vessels and explants.
i.
Maintenance of Aseptic Environment: During in vitro tissue
culture maintenance of aseptic environmental condition should be followed,
i.e., sterilization of glassware, forceps, scalpels, and all accessories in wet
steam sterilization by autoclaving at 15 psi (121°C) for 15 to 30 minutes or
dipping in 70% ethanol followed by flaming and cooling.
ii.
Sterilization of culture room: Floor and walls are washed first with
detergent and then with 2% sodium hypochlorite or 95% ethanol. The cabinet of
laminar airflow is sterilized by clearing the work surface with 95% ethanol and
then exposure of UV radiation for 15 minutes.
iii.
Sterilization of Nutrient Media: Culture media are
dispensed in glass containers, plugged with non-absorbent cotton or sealed with
plastic closures and then sterilized using autoclave at 15 psi (121°C) for 15
to 30 minutes. The plant extracts, vitamins, amino acids and hormones are
sterilized by passing through Millipore filter with 0.2 mm pore diameter and
then added to sterilized culture medium inside Laminar Airflow Chamber under
sterile condition.
iv.
Sterilization of Explants: The plant materials to be used for tissue
culture should be surface sterilized by first exposing the material in running
tap water and then treating it in surface sterilization agents like 0.1%
mercuric chloride, 70% ethanol under aseptic condition inside the Laminar Air
Flow Chamber.
The success of tissue culture lies in the composition of the
growth medium, plant growth regulators and culture conditions such as
temperature, pH, light and humidity. No single medium is capable of maintaining
optimum growth of all plant tissues. Suitable nutrient medium as per the
principle of tissue culture is prepared and used.
MS nutrient medium (Murashige and Skoog 1962) is commonly used. It
has carbon sources, with suitable vitamins and hormones. The media formulations
available for plant tissue culture other than MS are B5 medium (Gamborg.et.al
1968), White medium (white 1943), Nitsch’s medium (Nitsch & Nitsch 1969). A
medium may be solid or semisolid or liquid. For solidification, a gelling agent
such as agar is added.
Agar: A complex mucilaginous polysaccharide obtained from marine
algae (sea weeds) used as solidifying agent in media preparation.
Composition of MS (Murashige and
Skoog) Medium
Macronutrients:
Ammonium
nitrate (NH4NO3) 1650.0 mg/l
Potassium
nitrate (KNO3) 1900.0 mg/l
Calcium
chloride (CaCl2 2H2O) 440.0 mg/l
Magnesium
sulphate (MgSO4 6H2O) 370.0 mg/l
Potassium
dihydrogen phosphate (KH2PO4) 170.0 mg/l
Micronutrients:
Manganese
sulphate (MnSO4 4H2O) 22.3 mg/l
Zinc
sulphate (ZnSO4 4H2O) 8.6 mg/l
Boric
acid (H3BO3) 6.2 mg/l
Potassium
iodide (KI) 0.83 mg/l
Minor nutrient:
Sodium
molybdate (Na2 MO4 2H2O) 0.250 mg/l
Cupric
sulphate (CuSO4 5H2O) 0.025 mg/l
Cobaltous
chloride (CoCl2 6H2O) 0.025 mg/l
Iron stock
Na
EDTA 37.25 mg/l
Ferrous
Sulphate (FeSO4 7H2O) 27.85 mg/l
Vitamins
Glycine
2.0 mg/l
Nicotinic
acid 0.5 mg/l
Pyridoxin
HCl 0.5 mg/l
Thaiamine
HCl 0.1 mg/l
Growth Hormones
IAA
1.30 mg/l
Kinetin
0.4–10.0 mg/l
Myo-inositol
100.0 mg/l
Sucrose
30.0 g/l
Solidifying Agent
Agar 8.0 g/l
pH
The pH of medium is normally adjusted between 5.6 to 6.0 for the
best result.
Temperature
The cultures should be incubated normally at constant temperature
of 25°C ± 2°C for optimal growth.
Humidity and Light Intensity
The cultures require 50-60% relative humidity and 16 hours of
photoperiod by the illumination of cool white fluorescent tubes of
approximately 1000 lux.
Aeration
Aeration to the culture can be provided by shaking the flasks or
tubes of liquid culture on automatic shaker or aeration of the medium by
passing with filter-sterilized air.
Explant of 1-2 cm sterile segment selected from leaf, stem, tuber or root is inoculated (transferring the explants to sterile glass tube containing nutrient medium) in the MS nutrient medium supplemented with auxins and incubated at 25°C ± 2°C in an alternate light and dark period of 12 hours to induce cell division and soon the upper surface of explant develops into callus. Callus is a mass of unorganized growth of plant cells or tissues in in vitro culture medium.
The callus cells undergoes differentiation and produces somatic
embryos, known as Embryoids. The embryoids are sub-cultured to
produce plantlets.
The plantlets developed in vitro require a hardening period and so are transferred to greenhouse or hardening chamber and then to normal environmental conditions.
Hardening is the gradual exposure of in vitro developed
plantlets in humid chambers in diffused light for acclimatization so as to
enable them to grow under normal field conditions.
Based on the explants some other plant tissue culture types are
1. Organ culture
2. Meristem culture
3. Protoplast culture
4. Cell culture.
The culture of embryos, anthers, ovaries, roots, shoots or other
organs of plants on culture media.
The culture of any plant meristematic tissue on culture media.
Protoplasts are cells without a cell wall, but bounded by a cell
membrane or plasma membrane. Using protoplasts, it is possible to regenerate
whole plants from single cells and also develop somatic hybrids. The steps
involved in protoplast culture.
i. Isolation of protoplast: Small bits of plant tissue like leaf
tissue are used for isolation of protoplast. The leaf tissue is immersed in
0.5% Macrozyme and 2% Onozuka cellulase enzymes dissolved in 13% sorbitol or
mannitol at pH 5.4. It is then incubated over-night at 25°C. After a gentle
teasing of cells, protoplasts are obtained, and these are then transferred to
20% sucrose solution to retain their viability. They are then centrifuged to
get pure protoplasts as different from debris of cell walls.
ii. Fusion of protoplast: It is done through the
use of a suitable fusogen. This is normally PEG (Polyethylene Glycol). The
isolated protoplast are incubated in 25 to 30% concentration of PEG with Ca++
ions and the protoplast shows agglutination (the formation of clumps of cells)
and fusion.
iii. Culture of protoplast: MS liquid medium is used
with some modification in droplet, plating or micro-drop array techniques.
Protoplast viability is tested with fluorescein diacetate before the culture.
The cultures are incubated in continuous light 1000-2000 lux at 25°C. The cell
wall formation occurs within 24-48 hours and the first division of new cells
occurs between 2-7 days of culture.
iv. Selection of somatic hybrid cells: The fusion
product of protoplasts without nucleus of different cells is called a cybrid.
Following this nuclear fusion happen. This process is called somatic
hybridization.
The growing of cells including the culture of single cells or
small aggregates of cells in vitro in liquid medium is known as cell suspension
culture. The cell suspension is prepared by transferring a portion of callus to
the liquid medium and agitated using rotary shaker instrument. The cells are
separated from the callus tissue and used for cell suspension culture.
Cell suspension culture can be useful for the production of
secondary metabolites like alkaloids, flavonoids, terpenoids, phenolic
compounds and recombinant proteins. Secondary metabolites are chemical
compounds that are not required by the plant for normal growth and development
but are produced in the plant as ‘byproducts’ of cell metabolism. For Example:
Biosynthesis and isolation of indole alkaloids from Catharanthus roseus plant
cell culture.
The process of
production of secondary metabolites can be scaled up and automated using
bio-reactors for commercial production. Many strategies such as
biotransformation, elicitation and immobilization have been used to make cell
suspension cultures more efficient in the production of secondary metabolites.
Few examples of industrially important plant secondary metabolites are listed
below in the table:
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