Plant tissue culture generally refers to in vitro culture of all plant parts under sterile conditions. Basic facilities are must in the laboratory where tissue culture technology is to be performed. This includes general cleaning and media preparation, sterilization, storage, sterile transfer, observation / data acquisition, and environmentally controlled incubator or culture room areas. The most important work area is the Cultural Transfer Office, where core activities take place. Plant tissue culture should be cultured for a specified period of time under well-controlled temperature, humidity, airflow, and light quality. Plant culture techniques require a variety of organic and inorganic chemicals to prepare the culture medium. The growing room is an equally important area where plant cultures are maintained under controlled environmental conditions for optimal growth. After in vitro culture of plants they are transferred to the pots. The potted plants are immediately transferred to the greenhouse or growth cabinets and are maintained under proper conditions of light, temperature, and humidity.
Facilities in tissue culture labs are:
- Area for general washing, media preparation, sterilization, and storage
- Aseptic transfer area
- Incubators and culture rooms
- Lab should be free from dust, smoke, molds, spores, and chemicals.
- Plant pathology lab should be far from the laboratory.
Large sinks should be there in the lab including draining boards, racks, dryers, automated dishwashers, storage cabinets, and access to demineralized water, distilled water, and double-distilled water. Guidelines for the washing area:
- Glassware should be washed and dried out after the use without allowing Media or agar to dry.
- Glassware with corrosive chemicals should be separated from rest of the glassware and contaminated glassware should be autoclaved before washing.
- Immediately after completion of an experiment the contents of containers should be discarded.
Requirements of media preparation in the room:
- Refrigerator/freezer and high quality water
- Hot plate/stirrer, pH meter, balances, vacuum pump
- Double-distillation assembly and autoclave
- Equipment like dissecting microscopes, microwave, water baths, laboratory washers, ovens, automatic media (dispensers are helpful when pipetting large volumes of media) should be there
- Bunsen burners.
- Type 2 reagent grade water should be used for preparation of the media. Type 2 water is free from pyrogens, gases, and organic matter, and has electrical conductivity less than 1.0 umho/cm
In culture room light, temperature, and humidity are maintained and are varied according to the size of the room. Plant cultures are kept in the growth room under controlled environmental conditions to achieve optimal growth. Temperature (temperature should be constant throughout the culture room, i.e., no hot or cold spots; 13 and 30°C, with a temperature fluctuation of less than 0.3°C; always with a temperature alert alarm system to monitor temperature fluctuations), humidity (range of 20–98 percent controllable to 3%, air circulation), and light quality (fluorescent lighting to reach 10,000 lu) are all important factors to consider when incubating tissue cultures. For a 24-hour period, both light and temperature should be programmable. Protoplast cultures, low-density cell suspension cultures, and anther cultures are especially sensitive to cultural conditions in the environment. Plant cultures can also be shifted to another room if one room's cooling or lighting fails. This prevents cultures from being lost. To avoid contamination, the number of doors in the growth chamber should be kept to a minimum. Except when natural light is employed, there is no need for windows in the growth room. External light can interfere with the photoperiod and temperature of the growth room when artificial lighting is employed. The culture containers can be put on either fixed or mobile shelves, depending on the quantity of available space and cost. Mobile shelves have the benefit of allowing access to cultures on both sides of the shelf. The shelf height should not exceed 2 metres. To place and retrieve cultures from high shelving, step-up stools are required, which can be unsafe and time consuming. The lights positioned on the shelves are usually the dominant source of illumination in the growth chamber. Overhead light sources can be reduced because they will only be used during the dark cycle. The traditional downward illumination may not provide homogeneous light to plant cultures. Uneven heat distribution is caused by lights that are directly attached to the racks. This results in a high level of humidity within the culture containers, which might lead to hyperhydricity. Sideways illumination is an option that uses fewer lights and produces more uniform lighting. The following are some of the most important needs for a culture room.
Laminar Air Flow
A laminar flow hood is another form of transfer area. A dust filter forces air into the unit, which is then passed through a HEPA filter. After that, the air is directed either downward (vertical flow unit) or outward (horizontal flow unit) across the working surface. Nonfiltered air and particle matter do not settle on the working surface because of the steady flow of bacteria-free filtered air.
Safety rules in the lab:
- In the lab eating, smoking, and drinking are strictly prohibited.
- Toxic chemicals should be discarded with properly with precautions.
- First aid kits and fire extinguishers should be equipped in the labs.
- It is necessary to use autoclaved Pipettes, tips, Pasteur pipettes, and other items in the lab to prevent contamination.