Tissue culture is rapidly becoming a commercial method for propagating
new cultivars, rare species, and difficult-to-propagate plants. From a
few research laboratories several years ago, a whole new industry is emerging.
Currently, the demand for micropropagated plants is greater than the supply
with some plants. Some growers specialize in only the micropropagation
of plantlets, leaving the growing-on to others; many growers are integrating
a tissue culture laboratory into their overall operation.
In designing any laboratory, big or small, certain elements are essential for a successful operation. The correct design of a laboratory will not only help maintain asepsis, but it will also achieve a high standard of work.
Careful planning is an important first step when considering the size
and location of a laboratory. It is recommended that visits be make to
several other facilities to view their arrangement and operation. A small
lab should be set up first until the proper techniques and markets are developed.
A convenient location for a small lab is a room or part of the basement of a house, a garage, a remodeled office or a room in the headhouse. The minimum area required for media preparation, transfer and primary growth shelves is about 150 sq ft. Walls may have to be installed to separate different areas.
A good location includes the following:
Cleanliness is the major consideration when designing a plant tissue
culture laboratory. Most companies are not aware of their losses from contamination,
but estimates run from less than 1% up to 50%. When you consider the high
value of the product, no losses from contamination are acceptable. Routine
cleaning and aseptic procedures can decrease your losses to less than 1%.
Laboratories should have easy to wash walls and floors. Acrylic or urethane
epoxy wall paints Can be used; cement floors can be painted with an epoxy
or urethane floor enamel or have an inlaid linoleum installed. High efficiency
particulate air (HEPA) filters or regular furnace filters can be installed
over air intakes to the laboratory or on furnaces. If possible, an enclosed
entrance should precede the laboratory; sticky mats can be laid there to
help collect dirt from the outside, or shoes can be removed.
The traffic pattern and work flow in a laboratory must be considered in order to maximize cleanliness. The cleanest rooms or areas are the culture room, i.e. primary growth room, and the aseptic transfer area. It is best to design these rooms so they are not entered directly from the outside of a building. The media preparation area, glassware washing area, or storage area should be located outside these rooms. The primary growth room and aseptic transfer room should be enclosed with doors leading to each. Traffic through these areas can be minimized by installing pass-through windows. Ideally, the media preparation area would lead to the sterilization area, which would lead to the aseptic transfer room and eventually the primary growth room.
Unusual requirements for electricity and fire safety dictate that power installation be done by professional electricians. Most wiring will require 110 volts. Temperature and fire alarms are to be connected directly to telephone lines to give fast warnings of problems. An emergency generator should be available to operate essential equipment during power outages.
The glassware washing area should be located near the sterilization and
media preparation areas. When culture vessels are removed from the growth
area, they are often autoclaved to kill contaminants or to soften semi-solid
media. The vessels can be easily moved to the washing area if the autoclave
or pressure cooker is nearby. Locate the glassware storage area close to
the wash area to expedite storage; these areas also need to be accessible
to the media preparation area.
The glassware area should be equipped with at least one large sink; two sinks are preferable. Adequate work space is required on both sides of the sink; this space will be used for glassware soaking tubs and drainage trays. Plastic netting can be placed on surfaces near the sink to reduce glassware breakage and enhance water drainage. The pipes leading from the sink can be PVC to resist damage from acids and alkalis. Both hot and cold water should be available with water distillation and/or deionization devices nearby. Mobile drying racks can be stored nearby and lined with cheesecloth to prevent water dripping and loss of small objects. Locate ovens or hot air cabinets (75 C) close to the glassware washing and storage area. Dust-proof cabinets, low enough to allow easy access, can be used in the storage area.
The water source and glassware storage area should be convenient to the
media preparation area. Benches, suitable for comfortable working while
standing (34 to 36in.) and deep enough (24 in.) to hold equipment listed
below are essential. Their tops should be made with molded plastic laminate
surfaces that can tolerate frequent cleanings.
There is a variety of equipment available for micropropagation laboratories; this equipment is generally located in the media preparation area. The equipment budget will determine the type and amount purchased. All laboratories need the following basics:
Temperature, relative humidity, lighting units, and shelves need to be
considered in the culture room. All of these environmental considerations
will vary depending on the size of the growth room, its location, and the
type of plants grown within it. For example, a small primary growth room
located in a cool, North American climate, can be placed in an unheated
or minimally heated basement. The ballasts from the fluorescent lights
do not need to be separated; rather they can be used as a heating source.
Excess heat can be blown out of the growth room and used to heat other
parts of the basement or building. In this case, solid wood shelves with
air spaces located between shelves are recommended to prevent the cultures
on shelves above lights from becoming over-heated. A larger growth room
located in an above-ground location may need to have remote ballast and/or
a heat pump installed. Shelves in a larger growth room could then be glass
or expanded metal.
Temperature is the primary concern in culture rooms; it affects decisions on lights, relative humidity, and shelving. Generally, temperatures are kept 76 +/-2 F. Heating can be accomplished by traditional heating systems supplemented with heat from light ballasts or space heaters. Cooling the room is usually a greater problem than heating; cooler temperatures can be obtained by installing heat pumps, air conditioners, or exhaust fans. Using outside windows to cool culture rooms invites contamination problems in the summer and humidity problems in the winter.
Some plant cultures can be kept in complete darkness; however, most culture rooms are lighted at 1 klux (approximately 100ft-c) with some going up to 5 to 10 klux. The plant species being micropropagated will determine the intensity used. The developmental stage of the plants will also help determine if wide spectrum or cool white fluorescent lights are used. Rooting has been shown to increase with far-red light; therefore, wide spectrum lights should be used during stage III and cool-white lights can be used during Stages I and II. Automatic timers are needed to maintain desired photoperiods. Reflectors can be placed over bulbs to direct their light. Heat generated by the lights may cause condensation and temperature problems. In addition to using procedures previously mentioned, small fans with or without polyethylene tubes attached, can be placed at the ends of shelves to increase air flow and decrease heat accumulation.
Relative humidity (RH) is difficult to control inside growing vessels, but fluctuations in the culture room may have a deleterious effect. Cultures can dry out if the room's RH is less than 50%; humidifiers can be used to correct this problem. If the RH becomes too high, a dehumidifier is recommended.
Shelving within primary growth rooms can vary depending upon the situation and the plants grown. Wood is recommended for inexpensive, easy-to-build shelves. The wood for shelves should be exterior particleboard or plywood and should be painted white to reflect the room's light. Expanded metal is more expensive than wood, but provides better air circulation; wire mesh of 1/4 or 1/2 in. hardware cloth can be used but tends to sag under load. Tempered glass is sometimes used for shelves to increase light penetration, but it is more prone to breaking. Air spaces, 2 to 4 in., between the lights and shelves will decrease bottom heat on upper shelves and condensation in culture vessels. A room that is 8 ft high will accommodate 5 shelves, each 18 in. apart, when the bottom shelf is 4 in. off the floor. The top and bottom shelves may be difficult to work.
In addition to the primary growth room, the aseptic transfer area needs
to be as clean as possible. It is preferable to have a separate room for
aseptic transfer; this decreases spore circulation and allows personnel
to leave shoes outside the room. Special laboratory shoes and coats should
be worn in this area. Laminar flow hoods or still-air boxes can be placed
in this room and used for all aseptic work. Ultraviolet (UV) lights are
sometimes installed in transfer areas to disinfect the room; these lights
should only be used when people and plant material are not in the room.
Safety switches can be installed to shut off the UV lights when regular
room lights are turned on. Surfaces inside the aseptic transfer area should
be smooth to minimize the amount of dust that settles. Several electric
outlets are to be installed to accommodate balances, flow hoods, bacti-cinerators,