EXERCISE No.
– 1
Requirements For Plant Tissue Culture and Biotechnology Laboratory
Maintenance or growth of plant cell /tissue / organ primordial /whole plant under in
vitro (laboratory or controlled conditions) and aseptic (microorganism free) condition is
known as plant tissue culture. While, molecular manipulation of genes is known as genetic
engineering. Plant biotechnology refers to the combination of tissue culture and genetic
engineering. Plant tissue culture and biotechnological research requires dust free laboratories
well equipped with continuous supply of electricity, adequate water availability and proper
air conditioning facilities for conducting efficient research. Plant biotechnology laboratory
laid out into different sections depending on functions and purpose of the operations as well
as the fund availability. The overall design of laboratory must focus on maintaining aseptic
or sterile conditions. The following building layout and apparatus are required in a standard
plant tissue culture and biotechnological laboratory to conduct errorless research.
[A] PLANT TISSUE CULTURE LABORATORY
(1) Media Preparation Room
All the necessary minerals and other components required for normal plant growth
are provided in tissue culture media for in vitro regeneration. This room is also known as
kitchen of plant tissue culture laboratory. At least two laboratories (size 20’ × 15’ and 20’ ×
20’) are required, one for preparation of solutions, stock solution, culture media, preparation
of explants and the other for sterilization of living and non-living materials.
Suggestions
1. The operating manuals of all the equipments must be kept along with respective
equipments so the user can refer it prior using the instrument.
2. The chemicals, glass wares and plastic wares should be assigned to specific areas.
3. The chemicals should be arranged alphabetically so that they are easy to find out
whenever needed and stored according to its temperature requirement to preserve them
efficiently.
4. Strong acids and bases should be stored separately.
5. Chemicals like chloroform, alcohol, phenol, etc. which are volatile or toxic in nature,
must be stored in a fume hood. Fume hood expels volatilized compound out of
laboratory and prevents relevant hazards.
(2) Inoculation Room OR Transfer Room
Explant is any part of plant utilized for initiation of cultures. Transferring the plant
material (explants) to the culture vessels having tissue culture medium is called inoculation
or transfer. Transfer room of 20’ × 15’ size is required for this purpose. The room area must
have sterilized, clean and dry atmosphere with protection against airborne microorganisms
for routine transfer and manipulation work. It should have an arrangement of UV lights on
the walls. The inoculation chamber consists of the Laminar airflow cabinets which is
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commonly known as laminar hood. It is the most common scientific instrument used as a
bench for aseptic manipulation / transfer of the plant materials in culture vessels. One or more
laminar airflow cabinets can be accommodated.
(3) Culture Room OR Incubation Room OR Growth Room
Cultures are incubated for growth in this room so it is known as culture room or
incubation room or growth room (20’ × 20’). All the favourable environmental conditions
are provided in this room for normal plant growth. The most important aspect is maintenance
of sterilized atmosphere in this room. It is cut off from the outside air and completely isolated.
It should be cleaned regularly to avoid any sort of dust deposition. For placing cultures, this
room should have enough wooden or iron racks. Each rack should have tube lights and bulbs
for providing the required photoperiod. Culture room should have attached dark room for
liquid cultures and other cultures requiring dark conditions.
Suggestions to avoid contamination in culture room
1. Persons should wash and disinfect the hands prior to enter the room.
2. Outside clothes and shoes should not be allowed into the culture room.
3. As far as possible, entry of people should be restricted in this area.
Plant tissue cultures should be incubated under conditions of well-controlled
temperature, illumination (light intensity), photoperiod (light duration), humidity and air
circulation. For this purpose, there should be air conditioners, heaters, humidifiers,
fluorescent lights, lux meter and maximum / minimum thermometers in this room. All these
should be kept under the control of regulators and auto-timers so that desired conditions are
maintained throughout 24 hours. A twenty-four hours automatic timer can be used to control
the lighting duration and temperature. Generally, the controlled conditions of culture room
includes temperature around 25 ± 2°C, light intensity of 2000-3000 lux (luminous flux), light
duration of 16 hrs light / 8 hrs dark regimes per day and relative humidity around 60 to 80 %
but close to 100 % in the culture vessels.
Temperature: The real temperature of tissues inside culture vessels can be 2 to 4°C greater
than that of the culture room. Thus, the room temperature should be regulated at 2°C lower
than actual requirement of cultures. It is advisable to maintain a temperature of 20 ± 2°C for
temperate species and 25 ± 2°C for tropical species.
Light: Light requirements for the cultures can be divided into different parameters: (a) Light
intensity (b) Duration of light and (c) Quality of light. Photoperiod, light quality and intensity
may be set according to the types of culture. Carbohydrates are present in the media hence,
the process of photosynthesis is not important at initial stage of cultures. However,
photosynthesis is always going on slowly in the tissues.
(a) Light intensity: Unit used for measuring light intensity are Lux, Foot candles, Joule
per square meter (J/m2), Watts per square meter (W/m2) and Watts per square centimeter
(W/cm2). In tissue culture rooms, light intensities ranges from 5 to 25 W/cm2 (1000 to 5000
lux), with the most commonly used 10 to15 W/cm2 (2000 to 3000 lux).
(b) Duration of light: It is the quantity of light energy received which equals to intensity
x light duration. Most of the tissue culture rooms have a light duration of 16 to 18 hours/day
and dark duration of 8 to 6 hours.
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(c) Quality of light: Spectral quality (wavelength) of light received by in vitro cultures
is very important. Generally, commercial white fluorescent tubes are adequate for plant tissue
culture work.
Plant Growth Chamber provides all the facilities which are present in a culture room.
It has UV lights for time to time sterilization, fluorescent lights, arrangements of timers for
the maintenance or control of required humidity, temperature and timer to maintain the
required photoperiod etc.
(4) Observation Room
Plant tissue culture laboratory should also have space (10’ × 10’) for the observation
of cultures. Usually a separate space is provided attached with culture room itself. This
requires various kinds of microscopes like Binocular stereomicroscope, Inverted microscope
and Compound microscope with camera for detailed study of cultures or contamination or
growth. The area should be neat and clean with sufficient light arrangements.
(5) Dark Room
A small dark room (10’ × 10’) is required for photography equipped with cameras.
(6) Washing Room
A single span (20’ × 15’) washing room is required for cleaning glass wares or plastic
wares. This room should include the following facilities.
➢ How to wash the glass wares or reusable plastic wares?
1. Cleaning of glassware involves acid soaking followed by thorough washing with tap
water and subsequent rinsing with distilled water. However, due to corrosive nature
of acids, the glasswares should be soaked in a 2 % detergent for 16 hrs followed by
cleaning with help of a brush and scrub, washing with 60-70°C hot tap water to
remove the last traces of detergent and finally rinsed with double distilled water.
2. The glassware is first air dried and then kept in an oven for two hours at 160°C for
dry sterilization.
3. The clean glasswears are stored in completely clean, dust free wall racks / cupboards
present in the washing room.
4. The reusable plastic wares should be washed with mild detergent followed by rinsing
first with 60-70°C hot tap water and then with distilled water.
(7) Green house
Tissue culture regenerated plantlets are first transferred to green house for hardening
or acclimatization before actual field transfer. Green house (40’ × 20’) with controlled
temperature and relative humidity is required to grow regenerated plants up to maturity
especially during the off seasons. Green house requires cooling system, misting system etc
for hardening of the plantlets. Artificial lighting system is also necessary for adjustment of
photoperiod.
[B] GENETIC ENGINEERING LABORATORY
(8) Molecular Biology Laboratory
At least two laboratories (size 20’ × 15’ and 20’ × 20’) are required for molecular
biology research. One lab is needed for routine preparation work and one for sophisticated
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instrumentation. The following items/instruments are essential in this laboratory.
(9) Genetic Transformation Laboratory
A laboratory of a single (20’ × 15’) with double door entry is required for genetic
transformation work using Agrobacterium and particle gun methods. The following
items/instruments are essential in this laboratory.
(10) Transgenic Glass House
A transgenic glass house (20’ × 15’) with very good temperature and light control
facility is required to grow transgenic plants up to the stage of maturity and further
maintenance.
(11) Laboratory for Radioactive Work
In southern and northern blotting techniques, radio labeled DNA or RNA probes are
used to hybridize with the target gene. Probe is a known oligonucleotide sequence which is
labeled and utilized as bait in hybridization assay to find out complementary sequence from
a DNA sample or mixed pool of DNA samples. As probes were usually labeled with
radioactive elements generally heavy isotops of hydrogen, sulphur and phosphorous, such
hybridization experiments should be carried out in specialized laboratory (20’ × 20’) with
necessary safety measures and radioactive disposal facilities under the permission and
guidance of Bhabha Atomic Research Center (BARC), Trombay. However, recent approach
is utilization of fluorescent labels which are safe considering health hazards.
(12) Cold Room
A small cold room (10’ × 10’) is maintained at 4°C is required for doing DNA/protein
work.
(13) Store Room
A store of about 20’ × 15’ is needed for storing chemicals, apparatus, glasswares,
plastic wares, stationery etc.
(14) Generator Room
A generator room of about 10’ × 10’ is needed for supplying uninterrupted power
supply in case of non-availability of power in plant tissue culture and biotechnology
laboratories.
Equipment’s, Glassware’s and Safety Measures for Plant Biotechnology Laboratory
There are certain general purpose apparatus in the biotechnology laboratory, which are
discussed below:
1. Autoclave 17. Water Bath
2. Hot Air Oven 18. Centrifuge Machine
3. Tissue Lyser 19. Electrophoresis Unit
4. Microwave Oven 20. UV Transilluminator
5. Hybridization Oven 21. Laminar Air Flow Cabinet
6. Bio-Spectrometer 22. Microscope
7. PCR Thermocycler 23. Deep Freeze -20 ℃
8. Real Time PCR 24. Deep Freeze -80 ℃
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9. Gel documentation System 25. Fume Hood
10. Micro-centrifuges 26. Vortex Mixer
11. Orbital Incubator Shaker 27. Micropipettes
12. Electronic Weighing Balance 28. pH meter
Autoclave
An autoclave is a large pressure cooker. It is a steam sterilization unit.
Principle: It operates with the principle of steam under pressure as the sterilizing agent. High
pressures enable steam to reach high temperature, thus increasing its heat content and killing
power. Most of the heating power of steam comes from its latent heat of vaporization (the amount
of heat required to convert boiling water to steam).
Standard temperatures/pressures employed are 115oC/10 p.s.i., 121oC/15 p.s.i., and 132oC/27
p.s.i. (psi=pounds per square inch).
Do not autoclave items containing solvents, volatile or corrosive chemicals (phenol,
trichloroacetic acid, ether, chloroform, etc.) or any radioactive materials.
Hot Air Oven
It is a dry heat sterilization unit. A dry heat cabinet is easy to install and has relatively low
operating costs; it is nontoxic and does not harm the environment and it is noncorrosive for
metal and sharp instruments.
Principle: Sterilization by dry heat is accomplished through conduction. The heat is absorbed
by the outside surface of the equipment, and then passes towards the centre of it, layer by layer.
The entire system will eventually reach the temperature required for sterilization. Dry heat does
most of the damage by oxidizing molecules. The temperature is maintained for almost an hour
to kill the most difficult of the resistant spores. The most common time-temperature
relationships for sterilization with hot air sterilizers are: 170oC (340 oF) for 60 minutes, 160 oC
(320 oF) for 120 minutes, and 150 oC (300 oF) for 150 minutes or longer depending upon the
volume.
pH Meter
A pH meter is used to measure the acidity and alkalinity of a solution, or a pH meter is an
electronic device used for measuring the pH of any solution. pH Meter Calibration is required
at regular intervals or as per the defined frequency to maintain its accuracy.
Electronic Weighing Balance
Principle & Working: Electronic weighing balance accurately measures the weight. Calibrate
the balance by internal calibration. Place the weighing boat and tare the weight. Wait till it
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becomes zero. Chemical should be weighed slowly according to the need.
Laminar Air Flow Chamber
It provides clean air to the working area, a constant flow of air out of the work area to prevent
room air from entering. The air flowing out from the hood suspends and removes contaminants
introduced into the work area.
Principle: The most important part of a laminar flow hood is a high efficiency bacteria-
retentive filter. Room air is taken into the unit and passed through a pre-filter to remove gross
contaminants. The air is then compressed and channeled up behind and through the HEPA filter
(High Efficiency Particulate Air filter) in a laminar flow fashion; that is the purified air flows
out over the entire work surface in parallel lines at a uniform velocity. The HEPA filter removes
nearly all of the bacteria from the air.
Working: Switch on. Let the blower and light on. Wipe with 70% spirit in a vertical manner.
Place all the materials required to work in the hood. All should be Spirit sterilized before
placing in the LAF. Switch on the UV light for 45 minutes. Let the blower run continuously
for 15 minutes. When this time has passed repeat the wipe out of the sterile area with an alcohol
soaked piece of absorbent cotton. For cutting explants use a Petri dish (made of glass) or sterile
steel plate which should be cleaned with an alcohol soaked piece of absorbent cotton.
Orbital Shaker
This is mainly used to provide for gentle and intensive mixing of biological and chemical
compounds in a laboratory.
Working: It is a table-top laboratory instrument applicable for extracting, dissolving slow-
reacting samples; cultivation of cells; extraction of mineral oil of soil, of tissue culture for
analytical diagnostics; de-aeration of tested biodegradable materials and samples; rotating
closed containers for dialysis.
Centrifuges
A centrifuge is a device for separating particles from a solution according to their size, shape,
density, viscosity and rotor speed. In biology, the particles are usually cells, subcellular
organelles, viruses, large molecules such as proteins and nucleic acids.
In a centrifugal field, the gravitational acceleration (g) is replaced by the centrifugal force.
Ultracentrifugation is carried out at speed faster than 20,000 rpm. Super speed ultracentrifugation
is at speeds between 10,000 and 20,000 rpm. Low-speed centrifugation is at speeds below 10,000
rpm.
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Magnetic Stirrer with Hot Plate
It is used to dissolve certain slowly dissolving chemicals and for mixing the solution. It is used
in many biological labs. A magnetic stirrer is a laboratory device consisting of either a rotating
magnet or stationary electromagnets creating a rotating magnetic field. This device is used
to cause a stir bar immersed in a liquid to spin very quickly, agitating or mixing the liquid.
A magnetic stirrer often includes a provision for heating the liquid. They are preferred over
gear- driven motorized stirrers because they are quieter, more efficient, and have no moving
external parts to break or wear out (other than the simple bar magnet itself).
Thermocycler (PCR Machine)
A device which carries out polymerase chain reaction.
Principle & Working: The polymerase chain reaction (PCR) is an enzymatic process that allows
for the detection of specific genes within an environmental DNA sample. PCR utilizes short,
user defined DNA sequences called oligonucleotide primers, the sequence of which are
complementary to target regions of genes known to encode for specific functions (e.g.
contaminant degradation). In brief, the DNA sample is denatured to produce single stranded
DNA, called template DNA, to which the oligonucleotide primers can bind. The enzyme DNA
polymerase then adds nucleotide bases to the end of each primer, using the template DNA as a
guide to extend the primer thereby producing new double stranded DNA. This process is
repeated for a number of cycles to enrich the DNA sample for the desired genes targeted by the
oligonucleotide primers. Since each cycle of PCR involves creating two new double stranded
DNAs from each DNA molecule present, the amount of DNA theoretically doubles with every
cycle of PCR.
Agarose Gel Electrophoresis Unit
Agarose gel electrophoresis is the horizontal gel electrophoresis which is the easiest and most
popular way of separating and analyzing DNA. It separates the DNA fragments based on their
molecular weight.
Principle: Here DNA molecules are separated on the basis of charge by applying an electric
field to the electrophoretic apparatus. Shorter molecules migrate more easily and move faster
than longer molecules through the pores of the gel and this process is called sieving. The gel
might be used to look at the DNA in order to quantify it or to isolate a particular band. The
DNA can be visualized in the gel by the addition of Ethidium bromide through gel
documentation unit.
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UV- Spectrophotometer
To measure the chemical constituents of the sample and to get spectrometric analysis of the
given samples.
Principle: If a beam of light of intensity falls on a cell containing the sample, the emergent
radiation intensity I is less intense than the incident radiation, since a portion of it is absorbed
by the sample. The absorption is different at different wavelengths and is characteristic of the
sample. This characteristic quantity is called the absorbance and may be calculated from the
Beer-Lambert principle. The Beer-Lambert law states that in a sample, each successive portion
along the path of the incident radiation, containing an equal number of absorbing molecules
absorbs an equal fraction of the radiation that traverses it.
Water Bath
A water bath is an instrument used in the laboratory for carrying out biochemical,
agglutination, inactivation and biomedical tests and industrial incubation procedures.
Principle: It is a system for the control of temperature in which a vessel containing the
material to be heated is set into or over one containing water and receiving the heat directly.
Laboratory Safety Measures
Working in the laboratory may involve some degree of hazard. The students must be
clear on the task at hand, understand use of the equipments, reagents and biological materials
and must be clear about the procedures for conducting the experiments. Safety issues may be
divided into four areas:
(I) General rules (II) Physical hazards (III) Chemical hazards (IV) Biological hazards
I. General Rules
(1) Keep your belongings and other materials in the storage area away from the
laboratory benches.
(2) Use protective clothing and shoes in the laboratory.
(3) Use disposable gloves while handling microorganisms, enzymes, nucleic acids and
other biochemical reagents.
(4) UV protective goggles are required while using UV-trans-illuminator.
(5) Food, beverages, chewing gum etc are never permitted in the laboratory as reagentsor
microbes could accidentally be introduced into the mouth or skin.
(6) Each person must wash his or her hands before leaving the laboratory.
(7) Avoid mouth pipetting. Use pipettor / automatic micropipettes for all the solution
transfer.
II. Physical Hazards
In biotechnology laboratories, the major physical hazards are posed by fire,
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autoclaved and hot solutions, broken glass wares and sharp instruments, electrical
equipments, power supplier and UV radiations.
(A) Fire
➢ Know the location of the master shut off controls for gas and electricity.
➢ Super heated agarose solutions or media can boil over causing burns. Care must be
taken while removing them from the hot plate. Use tongs and heat proof gloves.
➢ Never leave a flaming Bunsen burner unattended.
(B) Autoclave
➢ Use heat proof gloves to remove media from the autoclave.
➢ When autoclave door is opened, steam may escape, causing burns. The autoclave
should be opened only after sufficient working.
(C) Electrical equipments
➢ Never plug or disconnect any equipment into or from a power supply unless the
power is off.
➢ Electrical equipment should not be used in or near water.
(D) UV-Trans-illuminator
➢ UV radiation can damage the skin and eyes.
➢ Use UV trans-illuminator with a UV- blocking lid.
➢ Use a UV- blocking face mask or goggles.
III. Chemicals Hazards
➢ Before use of any chemical reagent, one must know about its proper use.
➢ Never pipette anything by mouth.
➢ Use disposable gloves, lab coats and chemical splash goggles while using hazardous
chemicals.
➢ Avoid contact with skin or inhalation of chemicals like acryl amide (neurotic),
phenol (corrosive), chloroform, lysozyme etc.
➢ Use gloves while handling ethidium bromide (highly carcinogenic) and other
carcinogenic chemicals.
IV. Biological Hazards
➢ Most biological hazards arise from working with cultures of micro-organisms.
➢ Avoid all fingers to mouth and fingers to eye contact.
➢ Do not put pens into your mouth.
➢ Wash your hands thoroughly with anti-microbial soap before leaving lab.
➢ Never remove reagents, media or cultures directly from the laboratory.
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