Basic Requirements for a Stevia Tissue Culture Laboratory

A lot of you might be curious about how you can set up your own tissue culture laboratory. What are the requirements? What equipment do you need to have? Where you need to be careful while planning a setup?

Setting up your own tissue culture lab at home can initially seem challenging, but with to proper planning and preparation (and diligence), you can be well on your way to propagating many cultures. DIY laboratories can be set up in a variety of ways, as long as some fundamental basics are in place. The first thing you will need is the proper space to set up your lab. This could be a dedicated room, sectioned off area of a room, or small surface area, depending on the amount of plants you wish to propagate and the space you have at your disposal.

To construct your lab, there are a few things that you will require depending on your purpose and level of business. The primary focus of tissue culturing is “aseptic condition” which should be maintained at all levels of culturing processes. Here, a list of basic requirements of tissue culture labs is explained. Wherever you plan to design your lab, these are four areas that you need to assign for purposes explained below.

GLASSWARE WASHING AND STORAGE 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.

Space should also be available to set up drying ovens, washing machines, plastic or steel buckets for soaking labware, acid or deter­gent baths, pipette washers, driers and cleaning brushes. For storage of washed and dried labware, the laboratory should be provided with dustproof cupboards or storage cabinets.

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MEDIA PREPARATION AREA

This part is the central section of the laboratory where most of the activities are performed i.e., media preparation and sterilisation of media and glassware’s needed for culture. There should be sufficient working bench as well as storage space. Benches, suitable for comfortable working while standing (85 to 90 cm) and deep enough (60 cm) to hold equipment listed below are essential. Their tops should be covered with moulded plastic laminate or stainless steel surfaces that can tolerate frequent cleanings.

The essential equipment needed for media preparation and sterilization are as follows -

  1. Refrigerator/freezer-- This is needed to store chemicals and stock solutions. A 180L household refrigerator is often good enough for a small tissue culture lab.

  2. Purified water source -- Bottled water can be purchased inexpensively and placed in the media preparation area. Larger businesses may find it economical to obtain distillation or deionization devices. A good RO water filter is often sufficient.

  3. Balances -- Two digital balances, one with a maximum capacity of 500g and 0.5g precision and another with a maximum capacity of 100g and precision of 0.1 mg (analytical balance) are required.

  4. Hot plate/stirrer -- At least one hot plate with an automatic stirrer is needed to make semi-solid media.

  5. pH meter--This is needed to measure media pH. Some laboratories use pH indicator paper, however this method is considerably less accurate and could severely affect the results.

  6. Vacuum filtration device – A filtration device comprising of a filtration flask and a funnel assembly for holding a sintered or membrane filter fitted with a oil free diaphragm type vacuum pump is required for quick filtration of liquid media and filter sterilization of some media components.

  7. Autoclave--An autoclave or pressure cooker is a vital part of a micropropagation laboratory. High pressure heat is needed to sterilize media, water, glassware, and utensils.

 

Other equipment--A variety of small equipment is necessary for tissue culture laboratories. Small electrical heater, gas fired Bunsen burners, hot air oven are the most important accessory equipment for a tissue culture lab. . Microwave ovens are convenient for defrosting frozen stocks and heating agar media. Dissecting microscopes are useful to have in the laboratory for meristeming, dissecting floral and shoot apices, and observing plant culture growth. Automatic media dispensers are helpful when pipetting large volumes of media.

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RO Water Filter

Refrigerator

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Rough Balance

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Analytical Balance

Hot plate

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TRANSFER AREA

This place is required for inoculating the surface sterilized explants to the sterilized media, and transfer of growing plant material from one medium to another medium. This needs to be the cleanest area of the facility and practically free from any airborne contaminants and micro-organisms.

It is preferable to have a separate room for aseptic transfer. Special laboratory clothes should be worn in this area to minimize airborne microbial load in the air within the room . Laminar flow hoods are to 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 facilitate cleaning and minimize dust accumulation.

 

A laminar air flow hood is the essential equipment for the transfer room. It is a enclosed bench designed to prevent contamination of biological samples. In the hood, air, filtered through High Efficiency Particulate Air Filter (HEPA Filter) is continuously fed as a laminar stream. This filtered air stream ensures a contamination free environment within the hood. Surface sterilized explants are inoculated in sterile media and all other aseptic transfers are done within the laminar air flow hood.  

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Laminar flow hoods may have a UV-C germicidal lamp to sterilize the interior work surfaces before usage. experiment. Germicidal lamps are usually kept on for fifteen minutes to sterilize the interior before the cabinet is used.

Autoclave

Vacuum Filtration Set

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Small Modular Cleanroom for Aseptic Transfer

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Aseptic transfer in Laminar Air Flow Hood

Laminar Air Flow Hood

GROWTH AREA

After inoculation of the explants in the media, the culture vessel is transferred in the growth area. The growing plants remain here until they are ready for transplantation in pots. The growth area is generally a clean dust free place provided with racks/shelves. The racks/shelves are fitted with fluorescent lamps with correct light intensity – so that the growing plant parts and plants get enough light for their photosynthesis. The temperature and humidity of this area is also precisely controlled by air conditioning and humidification. The fluorescent lamps are turned on and off by an automatic timer to provide correct photo-period (light – dark cycle) according to the requirement of the plants.  

  

Growth racks can be made with metallic frame and metal/glass/laminated composit shelves. If metallic/laminated composit sheets are used as shelves, they should be painted white. The shelves’ dimensions may vary. However, the optimal shelf dimension is 900mm x 300mm. The gap between the shelves should be 350mm to allow good illumination.   The gap between the shelves and the wall should be at least 100 for facilitating good air circulation. The walls of the growth area are to be preferably painted with epoxy paint. Just like the transfer room, a very good sanitation and hygiene is to be maintained in the growth area.

The light intensity on the surface of the shelves should be between 2500 – 3500 lux. The ambient temperature in the area should be 25°C ± 2°C.

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TRANSPLANTATION AREA

Due to very high humidity inside the culture vessel and artificial conditions of development, the plantlets are tender and are therefore are not ready for coping up with the filed conditions.

After growing plants in tissue culture media in laboratory, those plants are to be adapted to the open air field condition. As the first step for this adaptation process, those plants are taken out from nutrient media and washed thoroughly with water. Then those plants are grown in netted plastic pots filled with liquid nutrient medium and kept in under high humidity and temperature controlled condition for few weeks. This process is called primary hardening. 

 

The next step of the acclimatization process comprises transplanting of the primary hardened plants in small pots filled with either soil or specific potting mix. Then the plants are grown in partial shade and then gradually exposed to full sunlight over a period of time. This is called secondary hardening. This operation is done in a glass house of a poly green house annexed to the tissue culture laboratory. Partial shading can be achieved by using agro-shed net coverings on the glass/poly house. This growth area should be provided with active humidification system of misting, and a temperature control system. Constant temperature and humidity monitoring systeme are also an essential pre-requisite for this growth and acclimatization area.  

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BASIC TOOLS REQUIRED FOR PLANT TISSUE CULTURE

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Scalpels

#7L (length 20.5 cm)

#7 (length 16.5 cm)

#3 (length 12.5 cm)

#4L (length 22.0 cm)

#4 (length 13.5 cm)

Forceps

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Standard –  straight tip –  23 cm
Standard –  straight tip –  25 cm
Waugh –  straight tip –  20 cm
Brophy –  straight tip –  30 cm
Brophy –  curved tip –  30 cm
Potts Smith –  straight tip –  25 cm
Potts Smith –  curved tip –  20 cm
Potts Smith –  curved tip –  25 cm

Scissors

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Doyen operating 18cm Straight
Metzenbaum 20 cm Straight
Metzenbaum 20 cm curved
Mayo 20 cm Straight
Angular dissection 15 cm

GLASSWARE AND PLASTICWARE REQUIRED FOR PLANT TISSUE CULTURE

Beakers

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Short form with spout

1000 ml

500 ml

250 ml

100 ml

Conical flasks

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Erlenmeyer type, with rim

1000 ml

500 ml

250 ml

Measuring cylinders

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Class B, with spout

1000 ml

500 ml

250 ml

100 ml

10 ml

Petri dish

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Re-usable borosilicate glass or disposable polystyrene 

100 mm x 15 mm

150 mm x 20 mm

Glass PTC vessel

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With autoclavable polypropylene or polycarbonate screw cap

250, 350 and 650 ml

Phytocon vessel

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Autoclavable polypropylene with vented closure

237 ml

473 ml

946 ml

Pipettes

Class B, Graduated

25 ml

10 ml

1 ml

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Reagent bottles

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With plastic cap, narrow mouth, with pouring ring

1000 ml

500 ml

100 ml

Culture tube

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Glass with screw cap,

autoclave-able polycarbonate cap, 24 mm dia

100 mm length

145 mm length

Plantcon vessel

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Presterilized, disposable, plastic growth containers

Bottom: 9.5 x 9.5 x 3.5 cm Cover: 9.5 x 9.5 x 7.0 cm

Wash bottle

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HDPE squeeze type wash bottle, narrow mouth

500 ml

1000 ml

Glass drying tray

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Borosilicate glass

320 mm x 180 mm x 51 mm