The document discusses the environmental conditions required for successful plant tissue culture. It outlines five key areas: 1) laboratory design and management, 2) environmental conditions, 3) sterilization techniques, 4) conditions in the inoculation area, and 5) conditions inside growth chambers. Specific requirements include maintaining temperature between 24-26°C, controlling air movement, using sterile culture vessels and media, and employing sterilization methods like autoclaving. The inoculation area must be inside a laminar flow hood with positive pressure and UV sterilization. Growth chambers need temperature control, lighting of 3000-8000 lux for 16 hours, and 40-60% humidity. Proper environmental protocols are necessary for contaminant-free culture and

1. ENVIRONMENTAL CONDITIONS FOR PLANT
TISSUE CULTURE LABORATORY
S. No. CONTENT
1 Laboratory Design and
Management
2 Environmental Conditions
3 Sterilization Techniques
4 Conditions required in
inoculation area
5 Environment in growth
chamber
Sandhya Upadhyay

2. MANDATORIES BEFORE PRACTICISING PLANT TISSUE
CULTURE
Plants should be diagnosed before
culturing in tissue culture laboratories
Plant should remain its genetic
fidelity during sub culturing.
Mother plant should be of high
quality.

3. Laboratory Design and Management
• There are 3 major factor
that depend to design any
tissue culture laboratory.
1. Physical environment
conditions in culture room
and the culture vessel.
2. Preparation and
sterilization of plant
material.
3. Culture media
composition.

4. PHYSICAL ENVIRONMENT
• Temperature
• Air movement
• Physical boundary of culture
vessels
• Physical characteristics of culture
media
• Humidity
• Light
CHEMICAL ENVIRONMENT
• Purified water
• pH
• Macro and micro nutrient
• Organic chemicals

5. PHYSICAL ENVIRONMENT
To maintain physical environmental
factors followings are required:-
1. AC to maintain temperature.
2. Pressure module to maintain
fresh air and air curtains to
avoid air movement from inside
of the culture lab to outside or
visa-versa.
3. Narrow mouth culture bottles.
4. Laminar Air Flow Hood for
performing inoculation.
5. Operators should wash their
exposed body area and wear
safety clothes while performing
inoculation.

6. CHEMICAL ENVIRONMENT
• PURIFIED WATER:- Water used in plant tissue culture should meet
the standard for type 2 reagent grade water (be free of gasses ,
organic matter, and have an electric conductivity less than 1.0.
1. Deionization Treatment followed by 1 to 2 distillation process.
2. Use of Activated carbon
3. Membrane filtration
4. Reverse Osmosis
• Media should of pH around 5.7 to 6.8
• Components of media should be sterilized by autoclave that are
non- temperature sensitive.

7. Air curtain Autoclave

8. Sterilization and its techniques
Sterilization is the process of making something free from bacteria or other
living microorganisms.
1. Sterilization can be done by either dry or moist heat.
- Dry heat technique requires longer exposure time (1.5 to 3 hours) and higher
temperatures (170°C for 30 minutes and160°C for 60 minutes), mainly used for
glassware.
Various available methods of dry heat sterilization are; hot air oven,
incineration, flaming (wire loop) etc.
2. Wet heat sterilization is usually required for sterilization of media and the
other components used in tissue culture practices like forceps, scalpel, tissue
paper etc.
3. Chemical sterilization is also important for maintenance of laboratory-
-used for sterilizing contamination (Chromic acid- 20gm dichromate + 300ml of
concentrated sulphuric acid)
-for explant sterilization ( ethanol, mercuric chloride, detergents, antibiotics
sodium hypo chloride etc.)
-for sterilizing laboratory fumigation should be perform by formalin,
formaldehyde + potassium per magnet or by using UV lights.

9. Conditions for transfer area
• Sub-culturing process should be
performed inside laminar flow
hood (LFH) as of its most desirable
environment due to the presence
UV light and positive pressure
ventilation unit ( by HEPA filters).
• Before culturing the LFH is
sterilised by UV light for 15 to 20
minutes and wipe with 70%
ethanol.
• Temperature should be maintain
at 24 ±2 °C.
• Operator working on LFH should
wear lab coats and masks.
• Mouth of open culture bottles
should be at the front of air filters
when inoculating.
• Always use sterilised instruments
for culturing.
• Don’t talk while working on LFH

10. Conditions inside growth chambers
Temperature
should be at 24
± 2 °C.
To maintain this
temp.
requirement of
air conditioner is
essential.
Light Intensity
should be at
3000 to 8,000
lux for 16/8
hours time
period.
Humidity
should
maintain at
40 to 60% .