Business Opportunities in Agriculture: 150 Field Interviews (Book)

Tissue culture information

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sanjunaik15

New Member
Dear All,

I am from Goa.I want to set up a tissue culture plant. I am very much interested in tissue culture. Can anyone please guide me for the initial setup, like where i will get the materials, information, How to start , whom to contact etc.

Thanks in advance..

Sanjay
 

Business Opportunities in Agriculture: 150 Field Interviews (Book)

Ashwini

New Member
Hi sanjay
Any laboratory, in which tissue culture techniques are performed, regardless of the
specific purpose, must contain a number of basic facilities. These usually include the
following:

A general washing area

A media preparation, sterilization, and storage area

An aseptic transfer area

Environmentally controlled incubators or culture rooms

An observation/data collection area.

Washing Area
The washing area should contain large sinks, some lead-lined to resist acids and alkalis,
draining boards, and racks, and have access to demineralized water, distilled water, and
double-distilled water. Space for drying ovens or racks, automated dishwashers, acid
baths, pipette washers and driers, and storage cabinets should also be available in the
washing area.
Media Preparation Area
The media preparation area should have ample storage space for the chemicals, culture
vessels and closures, and glassware required for media preparation and dispensing.
Bench space for hot plates/stirrers, pH meters, balances, water baths, and media-
dispensing equipment should be available. Other necessary equipment may include air
and vacuum sources, distilled and double-distilled water, Bunsen burners with a gas
source, refrigerators and freezers for storing stock solutions and chemicals, a microwave
or a convection oven, and an autoclave or domestic pressure cooker for sterilizing media,
glassware, and instruments.
In preparing culture media, analytical grade chemicals should be used and good
weighing habits practiced. To insure accuracy, and exact step-by-step routine should be
developed for media preparation and a complete checklist required of all media preparers
even for the simplest media.
The water used in preparing media must be of the utmost purity and highest quality.
Tap water is unsuitable because it may contain cations (ammonium, calcium, iron,
magnesium, sodium, etc.), anions (bicarbonates, chlorides, fluorides, phosphates, etc.),
microorganisms (algae, fungi, bacterial), gases (oxygen, carbon dioxide, nitrogen), and
particulate matter (silt, oils, organic matter, etc.) Water used for plant tissue culture
should meet, at a minimum, the standards for type II reagent grade water, i.e., be free of
pyrogens, gases, and organic matter and have an electrical conductivity less than 1.0
μmho/cm.
The most common and preferred method of purifying water to type II standards is a
deionization treatment followed by one or two glass distillations. The deionization
treatment removes most ionic impurities, and the distillation process removes large
1
organic molecules, microorganisms, and pyrogens. Three other methods that will
produce type II purity water are absorption filtration, which uses activated carbon to
remove organic contaminants and free chlorine; membrane filtration, which removes
particulate matter and most bacterial contamination; and reverse osmosis, which removes
approximately 9% of the bacterial, organic, and particulate matter as well as about 90%
of the ionized impurities.
Transfer Area
Under very clean and dry conditions, tissue culture techniques can be successfully
performed on an open laboratory bench. However, it is advisable that a laminar flow
hood or sterile transfer room be utilized for making transfers. Within the transfer area
there should be a source of electricity, gas, compressed air, and vacuum.
The most desirable arrangement is a small dust-free room equipped with an overhead
ultraviolet light and a positive-pressure ventilation unit. The ventilation should be
equipped with a high-efficiency particulate air (HEPA) filter. A 0.3-μm HEPA filter of
99.97-99.99% efficiency works well. All surfaces in the room should be designed and
constructed in such a manner that dust and microorganisms do not accumulate and the
surfaces can be thoroughly cleaned and disinfected. A room of such design is
particularly useful if large numbers of cultures are being manipulated or large pieces of
equipment are being utilized.
Another type of transfer area is a laminar flow hood. Air is forced into the unit through
a dust filter then passed through a HEPA filter. The air is then either directed downward
(vertical flow unit) or outward (horizontal flow unit) over the working surface. The
constant flow of bacteria-free filtered air prevents nonfiltered air and particulate matter
from settling on the working surface.
The simplest type of transfer area suitable for tissue culture work is an enclosed plastic
box commonly called a glove box. This type of culture hood is sterilized by an
ultraviolet light and wiped down periodically with 95% ethyl alcohol when in use. This
type of unit is used when relatively few transfers are required.
Culture Room
All types of tissue cultures should be incubated under conditions of well-controlled
temperature, humidity, air circulation, and light quality and duration. These
environmental factors may influence the growth and differentiation process directly
during culture or indirectly by affecting their response in subsequent generations.
Protoplast cultures, low-density cell suspension cultures, and anther cultures are
particularly sensitive to environmental cultural condition.
Typically, the culture room for growth of plant tissue cultures should have a temperature
between 15° and 30° C, with a temperature fluctuation of less than ±0.5°C; however, a
wider range in temperature may be required for specific experiments. It is also
recommended that the room have an alarm system to indicate when the temperature has
reached preset high or low temperature limits, as well as continuous temperature recorder
to monitor temperature fluctuations. The temperature should be constant throughout the
entire culture room (i.e., no hot or cold spots). The culture room should have enough
fluorescent lighting to reach the 10,000 lux; the lighting should be adjustable in terms of
quantity and photoperiod duration. Both light and temperature should be programmable
for a 24-hr period. The culture room should have fairly uniform forced-air ventilation,
and a humidity range of 20-98% controllable to ±3 percent. Many incubators, large
growth chambers, and walk-in environmental chambers meet these specifications.

These are the details and for detailed information contact the horticulture department


Regards
Ashwini
 

Business Opportunities in Agriculture: 150 Field Interviews (Book)


Business Opportunities in Agriculture: 150 Field Interviews (Book)

needyou123

New Member
tissue culture

Sir Ashwini,
I noted in your info about tissue culture is short and encouraging. Kindly post the cost and if anyone is partner with me.
Hope this find a resposive message.
Thank youi,
Paul Ponniah.
Please contact..
 
Last edited by a moderator:

Business Opportunities in Agriculture: 150 Field Interviews (Book)

vivek agri

New Member
Hi sanjay
Any laboratory, in which tissue culture techniques are performed, regardless of the
specific purpose, must contain a number of basic facilities. These usually include the
following:

A general washing area

A media preparation, sterilization, and storage area

An aseptic transfer area

Environmentally controlled incubators or culture rooms

An observation/data collection area.

Washing Area
The washing area should contain large sinks, some lead-lined to resist acids and alkalis,
draining boards, and racks, and have access to demineralized water, distilled water, and
double-distilled water. Space for drying ovens or racks, automated dishwashers, acid
baths, pipette washers and driers, and storage cabinets should also be available in the
washing area.
Media Preparation Area
The media preparation area should have ample storage space for the chemicals, culture
vessels and closures, and glassware required for media preparation and dispensing.
Bench space for hot plates/stirrers, pH meters, balances, water baths, and media-
dispensing equipment should be available. Other necessary equipment may include air
and vacuum sources, distilled and double-distilled water, Bunsen burners with a gas
source, refrigerators and freezers for storing stock solutions and chemicals, a microwave
or a convection oven, and an autoclave or domestic pressure cooker for sterilizing media,
glassware, and instruments.
In preparing culture media, analytical grade chemicals should be used and good
weighing habits practiced. To insure accuracy, and exact step-by-step routine should be
developed for media preparation and a complete checklist required of all media preparers
even for the simplest media.
The water used in preparing media must be of the utmost purity and highest quality.
Tap water is unsuitable because it may contain cations (ammonium, calcium, iron,
magnesium, sodium, etc.), anions (bicarbonates, chlorides, fluorides, phosphates, etc.),
microorganisms (algae, fungi, bacterial), gases (oxygen, carbon dioxide, nitrogen), and
particulate matter (silt, oils, organic matter, etc.) Water used for plant tissue culture
should meet, at a minimum, the standards for type II reagent grade water, i.e., be free of
pyrogens, gases, and organic matter and have an electrical conductivity less than 1.0
μmho/cm.
The most common and preferred method of purifying water to type II standards is a
deionization treatment followed by one or two glass distillations. The deionization
treatment removes most ionic impurities, and the distillation process removes large
1
organic molecules, microorganisms, and pyrogens. Three other methods that will
produce type II purity water are absorption filtration, which uses activated carbon to
remove organic contaminants and free chlorine; membrane filtration, which removes
particulate matter and most bacterial contamination; and reverse osmosis, which removes
approximately 9% of the bacterial, organic, and particulate matter as well as about 90%
of the ionized impurities.
Transfer Area
Under very clean and dry conditions, tissue culture techniques can be successfully
performed on an open laboratory bench. However, it is advisable that a laminar flow
hood or sterile transfer room be utilized for making transfers. Within the transfer area
there should be a source of electricity, gas, compressed air, and vacuum.
The most desirable arrangement is a small dust-free room equipped with an overhead
ultraviolet light and a positive-pressure ventilation unit. The ventilation should be
equipped with a high-efficiency particulate air (HEPA) filter. A 0.3-μm HEPA filter of
99.97-99.99% efficiency works well. All surfaces in the room should be designed and
constructed in such a manner that dust and microorganisms do not accumulate and the
surfaces can be thoroughly cleaned and disinfected. A room of such design is
particularly useful if large numbers of cultures are being manipulated or large pieces of
equipment are being utilized.
Another type of transfer area is a laminar flow hood. Air is forced into the unit through
a dust filter then passed through a HEPA filter. The air is then either directed downward
(vertical flow unit) or outward (horizontal flow unit) over the working surface. The
constant flow of bacteria-free filtered air prevents nonfiltered air and particulate matter
from settling on the working surface.
The simplest type of transfer area suitable for tissue culture work is an enclosed plastic
box commonly called a glove box. This type of culture hood is sterilized by an
ultraviolet light and wiped down periodically with 95% ethyl alcohol when in use. This
type of unit is used when relatively few transfers are required.
Culture Room
All types of tissue cultures should be incubated under conditions of well-controlled
temperature, humidity, air circulation, and light quality and duration. These
environmental factors may influence the growth and differentiation process directly
during culture or indirectly by affecting their response in subsequent generations.
Protoplast cultures, low-density cell suspension cultures, and anther cultures are
particularly sensitive to environmental cultural condition.
Typically, the culture room for growth of plant tissue cultures should have a temperature
between 15° and 30° C, with a temperature fluctuation of less than ±0.5°C; however, a
wider range in temperature may be required for specific experiments. It is also
recommended that the room have an alarm system to indicate when the temperature has
reached preset high or low temperature limits, as well as continuous temperature recorder
to monitor temperature fluctuations. The temperature should be constant throughout the
entire culture room (i.e., no hot or cold spots). The culture room should have enough
fluorescent lighting to reach the 10,000 lux; the lighting should be adjustable in terms of
quantity and photoperiod duration. Both light and temperature should be programmable
for a 24-hr period. The culture room should have fairly uniform forced-air ventilation,
and a humidity range of 20-98% controllable to ±3 percent. Many incubators, large
growth chambers, and walk-in environmental chambers meet these specifications.

These are the details and for detailed information contact the horticulture department


Regards
Ashwini
Dear Ashwini

Can i have your contact number.
Thank you
Regards
Vivek
 
Last edited by a moderator:

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