TOOLS AND TECHNIQUES USED FOR PLANT TISSUE CULTURE:

Different types of tools are used in tissue culture lab which can be describe as follows;

1. pH and pH meter
2. Autoclave
3. Plant growth chamber
4. Laminar air flow bench
5. Colorimeter
6. Centrifugation
7. Chromatography
8. Thermometer
9. Hygrometer
10. Methods of sterilization

 pH AND pH METER:

The hydrogen ion concentration of most solutions is extremely low. In 1909, Sorenson
introduced the term pH as a convenient way of expressing hydrogen ion concentration. pH of
a solution is strictly defined as the negative logarithm of the hydrogen ion activity. But in
practice usually hydrogen ion concentration is taken.

pH = – log10 (H+) = 7

WORKING OF pH METER:

An approximate idea of the pH of a solution can be obtained using indicators. These are
organic compounds of natural or synthetic origin whose Colour is dependent upon the pH of
the solution. Indicators are usually weak acids, which dissociate in solution. A standard pH
meter has two electrodes, one glass electrode for measuring pH and the other calomel
reference electrode. Reference electrode is filled with saturated KCI solution.

Indicator = Indicator– + H+

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  AUTOCLAVE:

Autoclave is used to sterilize medium, glassware and tools for the purpose of plant tissue
culture. The same equipment is used in hospitals to sterilize gauge, cotton, tools and linen,
etc. Sterilization of material is carried out by increasing moist heat (121 °C) due to increased
pressure inside the vessel (15-22 psi, pounds per square inch or 1.02 to 1.5 kg/cm 2) for 15
minutes for routine sterilization. Moist heat kills the microorganism and makes the material
free from microbes.

WORKING:

An autoclave is a pressure chamber that is used to sterilize equipment and supplies. When
these items are placed inside the autoclave they are exposed to high temperature steam
(usually around 132 degrees Celsius or 270 degrees Fahrenheit) for about twenty minutes.
These times vary based on the amount and physical size of the equipment that needs
sterilized. This hot steam will kill germs that simple detergent or boiling water could not.

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  PLANT GROWTH CHAMBER:

Plant growth chambers can be constructed in a suitable sized room or can be purchased as
commercially available equipment. Thermal insulation of walls increases the efficiency of the
cooling system.

Essentially plant growth chamber has three environmental control systems:

 LIGHT:

Light is fixed in the roof of equipment or in shelves. Light is provided by commercially

available light sources like cool white fluorescent tubes and incandescent lamps in a ratio of
3:1 and usually a light intensity of 2000-2500 lux (about 200-250 candles or 30 µ mol m -2 s1)
is used.

 TEMPERATURE:

Usually temperature of 22-28 °C is used for growing plant tissue culture. Temperatures
should be measured in a constructed growth chamber at different levels and places, viz., light
racks, central and corners to have a correct temperature setting.

 HUMIDITY:
Humidity inside the growth chamber is provided by humidifier (a mist generating system)
and controlled by humidistat. Usually 60% RH (relative humidity) is used to maintain healthy
growth. Low RH may cause early drying of medium while high humidity may cause fungal
growth in the environment.

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  LAMINAR AIR FLOW BENCH:

Laminar air flow (LAF) bench is the main working table for aseptic manipulations related to
plant tissue culture. This is equipment fitted with High Efficiency Particulate Air (HEPA)
Filters, which allow air to pass but retain all the particles and micro-organisms. These HEPA
Filters have a very small pore size (0.3 µm) with 99.97-99.99% efficiency.

WORKING:

Air blown by a blower is passed through these filters, thus, always generally a positive
pressure of air is maintained from inside to outside. This positive pressure does not allow any
particle to enter in the working area of LAF bench. Air pressure inside the instrument is
measured by a manometer and pressure of more than 12 bars shows choking of filters and at
this point filters should be replaced. Equipment is fitted with UV light and visible light
source.

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  COLORIMETER:
The most commonly used method for determining the concentration of biochemical
compounds is colorimetry. It uses the property of light such that when white light passes
through a coloured solution, some wavelengths are absorbed more than others. Hyaline
solution can be made coloured by specific reactions with suitable reagents. These reactions
are generally very sensitive to determine quantities of material in the region of millimole per
litre concentration.

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  CENTRIFUGATION:

A centrifuge is an instrument which produces centrifugal force by rotating the samples

around a central axis with the help of an electric motor. Centrifuges can be categorized as the
clinical type (5-10,000 rpm), refrigerated high-speed centrifuges (10,000-20,000 rpm) and
ultra -centrifuges (20,000 to 80,000 rpm).

WORKING:

A centrifuge is a laboratory device that is used for the separation of fluids, gas or liquid,
based on density. Separation is achieved by spinning a vessel containing material at high
speed; the centrifugal force pushes heavier materials to the outside of the vessel.

 CHROMATOGRAPHY:

Chromatography (meaning ‘colored writing’) is a technique to separate molecules on the

basis of differences in size, shape, mass, charge and adsorption properties. The term
chromatography was used by the Russian botanist Tswett to describe the separation of plant
pigments on a column of alumina. There are different types of chromatography but they all
involve interactions between these components: the mixture to be separated, a solid phase,
and a solvent.

WORKING:

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Chromatography is actually a way of separating out a mixture of chemicals, which are in gas
or liquid form, by letting them creep slowly past another substance, which is typically a
liquid or solid. ... As the mobile phase moves, it separates out into its components on the
stationary phase.

 THERMOMETER:

The thermometer is a device that measures temperature or temperature gradient using a

variety of different principles; it comes from the Greek roots thermo, heat, and meter, to
measure. A thermometer has two important elements: the temperature sensor (e.g., the bulb
on a mercury thermometer) in which some physical change occurs with temperature, plus
some means of converting this physical change into a value (e.g., the scale on a mercury
thermometer).

WORKING:

Temperature is measured by maximum-minimum thermometer or a continuous rotary dram

chart type thermometer. The U- shaped maximum-minimum thermometer is commonly used
for determining diurnal maximum and minimum range of temperature in the culture room.

During the dark period, temperature remains slightly (1-2 °C) lower than light period (all
bulbs and tube lights of the culture room increase the temperature). Therefore, chokes of the
tube lights are fitted outside the culture roo

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  HYGROMETER:

Hygrometers are instruments used for measuring humidity.

WORKING:

A simple form of a hygrometer is specifically known as a “psychrometer” and consists of two

thermometers, one of which includes a dry bulb and the other of which includes a bulb that is
kept wet to measure wet-bulb temperature. Evaporation from the wet bulb lowers the
temperature, so that the wet-bulb thermometer usually shows a lower temperature than that of
the dry-bulb thermometer, which measures dry-bulb temperature.

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  METHODS OF STERILIZAION:

ASEPSIS:

Plant tissue culture requires contamination free environment, tools and cultures or strict
maintenance of germ free system in all the operations, known as asepsis. Particularly in
commercial production units, the contamination of one batch of the cultures may result in
heavy financial losses or even loss of a culture strain. Therefore strict control measures are
enforced to maintain the entry of the personnel and living materials. The basic rules and
practices of asepsis are followed in all the tissue culture laboratories.

Plant tissue culture media are rich in nutrients and very suitable for the growth of microbes
also. These microorganisms grow faster, consume nutrients rapidly and suppress the growth
of plant tissues (by over growth). This saprophytic unwanted growth of microbes is formed as
contamination. There are many ways by which these microbes suppress the growth of plant
cells and tissues, e.g., release of enzymes and toxins (which inhibit the growth of plant cells),
selective absorption of specific nutrients, and some tissue infection.

Suitable sized plant material is sterilized as follows:

1. Clean the working area with ethanol, and start the air flow of the laminar air flow bench.

2. Sterilized petridishes, distilled water, scalpel, filter paper sheets (suitable sized and
autoclaved) alcohol, disinfectant (mercuric chloride 0.01-0.1% aqueous solution, or 20%
sodium hydrochloride), forceps, bead sterilizer or beaker or coupling jar, sprit lamp or gas
burner are collected on Laminar flow bench. Put on the UV light of the bench and put it off
after 30 minutes.

3. Bring the plant material to be sterilized on the bench and prepare pieces for sterilization.

4. Clean the working area and hands with alcohol, put on mask and cap, and light the sprit
lamp.

5. Keep 3-4 petridishes in a line; add disinfectant in 1st plate and autoclaved distilled water in
subsequent plates.

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6. Place plant pieces in 1st plate and immerse the material with the help of forceps for 5-10
minutes depending upon the disinfectant used.

7. Transfer material from 1st to 2nd plate, rinse gently and pass to 3rd and 4th plate, one by
one with thorough rinsing.

8. Finally, drain the distilled water or place the material in a fresh petridish or filter paper and
prepare suitable sized explants

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