Plant Tissue Culture: Laboratory Organization | Biotechnology

In this article we will discuss about:- laboratory organization of plant tissue culture

Introduction:

Plant tissue culture is not a separate branch of plant science like taxonomy, cytology, plant physiology etc.

Rather it is a collection of ex­perimental methods of growing large number of isolated cells or tissues under sterile and con­trolled conditions.

The cells or tissues are ob­tained from any part of the plant like stem, root, leaf etc. which are encouraged to produce more cells in culture and to express their totipotency (i.e. their genetic ability to produce more plants). Cells or tissues are grown in different types of glass vials containing a medium with mineral nutrients, vitamins and phytohormones. Therefore, to carry out the experiments using tissue culture techniques, a well-equipped laboratory is first required.

In recent years there has been a large in­crease in the number of research laboratories us­ing tissue culture techniques to investigate many fundamental and applied aspects of higher plants. However, the use of these techniques is not confined to research alone.

Tissue culture techniques are being exploited by many com­mercial laboratories. Even many horticultural companies are setting up small units to multiply plants which are difficult to propagate by con­ventional means. The general organization of a tissue culture laboratory and the basic tech­niques will be discussed under different subhead­ings.

Laboratory Design and Development:

The size of tissue culture lab and the amount and type of equipment used depend upon the nature of the work to be undertaken and the funds available. A standard tissue culture laboratory should provide facilities for:

•  washing and storage of glassware, plasticware

•  preparation, sterilization and storage of nutrient media

•  aseptic manipulation of plant material

•  maintenance of cultures under controlled temperature, light and humidity

•  observation of cultures, data collection and photographic facility

•  acclimatization of in vitro developed plants. The overall design must focus on maintaining aseptic conditions.

At least three separate rooms should be available one for washing up, storage and media preparation (the media preparation room); a second room, containing laminar-air-flow or clean air cabinets for dissection of plant tissues and subculturing (dissection room or sterilization room); and the third room to incubate cultures (culture room). This culture room should contain a culture observation table provided with binoculars or stereozoom microscope and an adequate light source. Additionally, a green house facility is required for hardening-off in vitro plantlets. For a commercial set-up, a more elaborate set-up is required.

1.1.  Media preparation room

The washing area in the media room should be provided with brushes of various sizes and shapes, a large sink, preferably lead-lined to resist acids and alkalis, and running hot and cold water. It should also have large plastic buckets to soak the labware to be washed in detergent, hot-air oven to dry washed labware and a dust-proof cupboard to store them. If the preparation of the medium and washing of the labware are done in the same room, a temporary partition can be constructed between the two areas to guard any interference in the two activities. A continuous supply of water is essential for media preparation and washing of labware. A water distillation unit of around 2 litre/h, a Milli-Q water purification systems needs to be installed.

1.2.  Culture room

The room for maintaining cultures should be maintained at temperature 25 ±2°C, controlled by air conditioners and heaters attached to a temperature controller are used. For higher or lower temperature treatments, special incubators with built-in fluorescent light can be used outside the culture room. Cultures are generally grown in diffuse light from cool, white, fluorescent tubes. Lights can be controlled with automatic time clocks. Generally, a 16-hour day and 8-hour nights are used. The culture room requires specially designed shelving to store cultures. Some laboratories have shelves along the walls, others have them fitted onto angle-iron frames placed in a convenient position. Shelves can be made of rigid wire mesh, wood or any building material that can be kept clean and dust-free. Insulation between the shelf lights and the shelf above will ensure an even temperature around the cultures. While flasks, jars and petridishes can be placed directly on the shelf or trays of suitable sizes, culture tubes require some sort of support. Metallic wire racks or polypropylene racks, each with a holding capacity of 18-24 tubes, are suitable for the purpose.

1.3.  Dissection room or sterilization room

This area should have restricted entry, which is needed to ensure the sterile conditions required for the transfer operations. For sterile transfer operations, the laminar-air-flow cabinets are used. Temperature control is essential in this room as the heat is produced continuously from the flames of burners in the hoods. The room should be constructed in a way to minimize the dust particles and for easy cleaning. Several precautions can be taken including the removal of shoes before entering the area.

The laminar horizontal flow sterile transfer cabinets are available in various sizes from many commercial sources. They should be designed with horizontal air flow from the back to the front, and equipped with gas cocks if gas burners are to be used. Electrical outlets are needed for use of electric sterilizers and microscopes, and if weighing is to be done in the hoods. A stainless steel working platform is most durable, easy to keep clean and to prevent the unwanted damage due to accidental fire. Sometimes it is fitted with Ultraviolet light to maintain sterility inside the cabinet. UV light is a source of ozone, which can be mutagenic, therefore, utmost care is to be taken while using this. Although UV light is not necessary, a short exposure time of 3-5 min to cabinet is fine sometimes. Work can be started after 10-15 min of switching on the air flow, and one can work uninterrupted for long hours.

A Laminar-air-flow cabinet has small motor to blow air which first passes through a coarse filter, where it loses large particles, and subsequently through a fine filter known as ‘high efficiency particulate air (HEPA). The HEPA filters remove particles larger than 0.3 µm, and the ultraclean air flows through the working area. The velocity of the ultra clean air is about 27 ± 3 m min-1 which is adequate for preventing the contamination of the working area as long as the flow is on. The flow of the air does not in any way hamper the use of a spirit lamp or a Bunsen burner. 

1.4.  Greenhouse

The greenhouse facility is required to grow parent pants and to acclimatize in vitro raised plantlets. The size and facility inside the green house vary with the requirement and depends on the funds available with the laboratory. However, minimum facilities for maintaining humidity by fogging, misting or a fan and pad system, reduced light, cooling system for summers and heating system for winters must be provided. It would be desirable to have a potting room adjacent to this facility.

1.5. Equipments and apparatus

1.4.1. Media preparation area

•  benches at a height suitable to work while standing

•  pH meter is used to determine the pH of various media used for tissue culture. pH indicator paper can also be used for the purpose but it is less accurate. The standard media pH is maintained at 5.8.

•  hot-plate-cum-magnetic stirrer for dissolving chemicals and during media preparation

•  an autoclave or domestic pressure cooker is crucial instrument for a tissue culture laboratory. High pressure heat is needed to sterilize media, water, labware, forceps, needles etc. Certain spores from fungi and bacteria can only be killed at a temperature of 121°C and 15 pounds per square inch (psi) for 15-20 min. A caution should be taken while opening the door of autoclave and it should be open when the pressure drops to zero. Opening the door immediately can lead to a rapid change in the temperature, resulting in breakage of glassware and steam burning of operator.

•  plastic carboys for storing distilled water required for media preparation and final washing of labware.

•  balances near dry corner of the media room. High quality microbalance are required to weigh smallest of the quantities. Additionally a top pan balance is required for less sensitive quantities.

•  hot-air oven to keep autoclaved medium warm before pouring into vessels. It is also used for the dry heat sterilization of clean glassware like, Petridishes, culture tubes, pipettes etc. Typical sterilizing conditions are 160-170 °C/1hr.

•  Dishwasher for cleaning glass pipettes in running water

1.4.2. Storage area

•  a deep freezer (-20°C to -80°C) / refrigerator for storage of enzyme solutions, stock solutions plant materials and all temperature-sensitive chemicals.

•  microwave oven to melt agar solidified media

• Upright and inverted light microscope with camera attachment for recording the morphogenic responses from various explants, calli, cells and protoplasts. Inverted microscope gives the clear views of cultures settled at the bottom of Petridishes.

1.4.3. Dissection room

•  laminar-air-flow cabinet within which tissue culture work can be carried out under sterilized environment

•  glass bead sterilizer where temperature of beads is raised to 250°C in 15-20 min with 15 s cut off. Here the sterilization of instruments is effecting by pushing them into the beads for 5-7 s. This is much safer compare to the Bunsen burner heating of instruments like, forceps, needles, scalpels etc.

•  binocular microscope to observe surface details and morphogenic responses of cultures and their possible contamination.

•  low speed table-top centrifuge to sediment cells or protoplasts

1.4.4. Culture room

•  air (or heating / cooling system) to maintain 25±2 °C temperature

•  racks for holding test-tubes

•  lights to provide diffuse light and to maintain photoperiod

•  shakers with various sized clamps for different sized flasks to grow cells in liquid medium

•  thermostat and time clock for lights

•  wall cabinets for dark incubation of cultures

1.4.5. Other apparatus

•  beakers (100 mL, 250 mL, 1 L, 5 L)

•  measuring cylinders (5 mL, 10 mL, 25 mL, 50 mL, 100 mL, 500 mL, 1L, 2 L, 5 L)

•  graduated pipettes and teats

•  reagent bottles for storing liquid chemicals and stock solutions (glass or plastic)

•  culture tubes and flasks (glass or polypropylene or disposable)

•  plastic baskets

•  filter membrane, preferably nylon, of sizes 0.22 µm and 0.45 µm, holders and hypodermic syringes (for solutions requiring filter sterilization)

•  large forceps (blunt and fine points) and scalpels for dissecting and subculturing plant material.

•  Scalpel handles (no. 3) and blades (no. 11)

•  Chemicals and reagents for preparing culture media

•  Disposable gloves and masks.

•  Micropipettes of maximum volume size 5000 µL, 1000 µL, 500 µL, 250 µL, 100 µL