MISCELLANEOUS REPORTS 40 ISSN 0253-6749

ORGANIZING A PLANT MICROPROPAGATION LABORATORY

Maria Ioannou

AGRICULTURAL RESEARCH INSTITUTE MINISTRY OF AGRICULTURE AND NATURAL RESOURCES

NICOSIA CYPRUS

JULY 1990

ORGANIZING A PLANT MICROPROPAGATION LABORATORY

Maria Ioannou

SUMMARY

Plant micropropagation enables rapid clonal multiplication of plants which is of major im­portance in horticultural practice, especially for cultivars difficult to propagate with conven­tional methods. In Cyprus, this method appears to have good prospects for the commercial production of ornamental plants. For the initiation and successful operation of a commercial plant micro propagation unit, apart from the necessary scientific and technical expertise, a properly organized tissue culture laboratory is essential. Such a laboratory should normally be divided into three main parts, i.e. the media and plant preparation area, the transfer area, and the culture incubation area (growth room). It must also be furnished with general pur­pose and specialized tissue culture facilities and equipment and must have an adequate supply of chemicals, glassware and tools, which are listed. Major manufacturers of tissue culture equipment are also listed, to facilitate the aquisition of necessary facilities by prospective plant micropropagators. Finally, a brief reference is made to the greenhouse and related nur­sery facilities required for the hardening of micropropagated plants during their transfer to soil.

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INfRODUCTION

Micropropagation (propagation in vitro) is pres­ently the most important commercial application of plant tissue culture. The main characteristic of this technique is rapid clonal multiplication which results in the production of a large number of uniform plants from a limited number of mother stock plants. In addition, such techniques enable the clo­nal multiplication of species or cultivars which are difficult or impossible to propagate with convention­al methods. Other important advantages of micro­propagation include the relatively small space re­quired for the early stages of multiplication and the fact that the propagation material is maintained un­der aseptic conditions, which reduce the risk of in­fection by pathogens. Thus, plants produced through micropropagation are usually of superior quality and of better health state compared to those produced with conventional methods. Major draw­backs of micropropagation include the high initial cost for the necessary laboratory facilities and the need for specialized scientific and technical person­nel.

In Cyprus, a prospective area of tissue culture application appears to be the commercial produc­tion of ornamental plants. The general principles and the basic methodology for the micropropagation of ornamentals and other horticultural crops were described in a previous report (Ioannou, 1989), in which formulations of major culture media were also given. The objective of this paper is to pro­vide interested horticulturists and nurserymen with basic technical information concerning the correct design, equipment and organization of a plant mi­cropropagation unit. Major laboratory facilities and tissue culture equipment needed are briefly described while the numerous chemicals, glassware, tools and other minor laboratory supplies for plant tissue cul­ture are listed in the form of appendices. A list of major commercial suppliers of tissue culture equip­ment is also given in order to help prospective plant micropropagators acquire the necessary facili­ties.

DESIGN AND BASIC FACILITIES OF THE LABORATORY

The most important factors of a plant tissue cul­ture laboratory are efficiency of operation, order

and cleanliness. These factors depend greatly upon the standard of work of the personnel involved but are also closely related to the correct design of the laboratory (pennel, 1982). An example plan of a properly designed micropropagation laboratory is given in Fig. 1. Such a unit is divided into three main parts: a) media and plant preparation area; b) transfer area; and c) culture incubation area (growth room).

The size of the laboratory depends upon the esti­mated quantity of plant material to be handled and the staff available to work. For a large or medium­size unit the three parts mentioned above are de­signed in separate rooms, as in Fig. 1. In case of a small unit, however, a single laboratory area can also suffice since most essential equipment, like in­cubators, inoculation hoods and others, are available in compact forms which can be properly arranged in the same room.

Wether small or large, any micropropagation unit should be SUitably furnished with common laborato­ry facilities, including adequate benching space and storage cabinets. Since all essential tissue culture equipment are electrically operated, the laboratory must be furnished with an adequate number of elec­trical outlets. Their position and distribution in the laboratory should be such as to enable proper ar­rangement and efficient operation of electrical equipment, including large, floorstanding units (e.g. autoclave, refrigerators, incubators, etc.) and small­er, bench-mounted pieces (e.g. stereomicroscope, balances, pH meter, magnetic stirrer etc.). A sink supplied with hot and cold water is also indispensa­ble, especially in the media and plant preparation area in which all cleaning and other kitchen work is to be carried out.

Irrespective of size and design, any tissue culture laboratory must be furnished with an effective air­conditioning/heating system, to maintain room tem­perature within the range 18-27°C, throughout the year. Such temperature control is essential for the efficient operation of the laboratory, as temperature extremes seriously affect not only the efficiency and quality of human work but also the survival and quality of plant material processed, the shelf-life of chemicals and the durability and proper operation of the various equipment.

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LABORATORY EQUIPMENT AND SUPPLIES Apart from furniture and other general-purpose

facilities, a plant micropropagation unit must be also furnished with certain specialized facilities and equipment, and must have an adequate supply of the necessary chemicals, glassware, tools and other laboratory items, as discussed in the following sec­tions. Complete lists of the required equipment and supplies are given in Appendices 1, 2 and 3.

A. Media and plant preparation area

This part of the micropropagation laboratory is mainly for the preparation and sterilization of cul­ture media and for the preparation of plant materi­al to be used as source of explants. In addition, all general-purpose kitchen work, especially the clean­ing of culture vessels and other laboratory glass­ware, takes place in this room. To satisfy these needs, the room should be supplied with hot and cold water and should be equipped with the follow­ing laboratory facilities (Appendix 1): Laboratory autoclave. Autoclaving or steam sterilization under pressure is the standard technique for sterilizing tis­sue culture media or other liquids (e.g. distilled wa­ter) in sealed containers. This involves exposure of such containers to pressurized steam at 1.05 kg/ern" (121 0c) for 10-20 minutes, the exposure time de­pending on the volume of the medium in the cul­ture vessel (Street, 1977). Various types of laborato­ry autoclaves are commercially available of which the most common ones are steam-heated, i.e. for their operation require a supply of ready steam. Since availabilit y of a source of ready steam is high­ly unlikely under Cyprus conditions, it is important to select an electrically-heated model, which produc­es its own pressurized steam if supplied with dis­tilled water. Both the "horizontal" and the "verti­cal" loading types are equally suitable, provided they comply to the basic safety requirements for la­boratory autoclaves (e.g, BS 2646 or equivalent European or American standards). A working ca­pacity of about 30 litres can satisfy the require­ments of most medium-size micropropagation units. For small laboratories, however, smaller portable models (electrically or gas-heated) or even large pressure cookers may suffice. More information concerning proper steam sterilization of culture me­dia, using either an autoclave or a pressure cooker, can be found elsewhere (Ioannou, 1989).

Sterilizing oven. This equipment is necessary for the dry-heat sterilization of laboratory glassware (petri dishes, pipettes, etc.) and other heat-resistant laboratory articles. The usual treatment for this type of sterilization is about 180°C for 4-5 hours (Dodds and Roberts, 1982). Higher temperatures may sometimes be used for shorter exposure peri­ods. The unit to be purchased should feature a temperature range up to 250-300°C and a fan­assisted circulation system, to maintain uniform temperature throughout the oven. Other important features include a safety cut-out switch and possibili­ty for programmable operation for up to 5 hours. A working capacity of 150-200 litres and a power rating of 2-3 KW are considered satisfactory for a medium-size laboratory. Precision and analytical balances. One top­loading precision balance and one analytical balance, are required in the micropropagation laboratory. The first is a general-purpose unit suitable for weighing loads of up to 3 kg, usually with a preci­sion of about 0.01 g. The analytical balance is suit­able for a much lower weighing load (usually up to 200 g) but enables a much higher precision (usually 0.1 mg). It is therefore necessary for weighing out very small quantities, as in the case of microele­ments, vitamins, hormones and other ingredients which are used at very low concentrations in culture media. Electronic models, with easily read luminous display are more convenient to operate and should be preferred. Other essential equipment. In addition to steriliz­ing and weighing facilities, a series of other labora­tory equipment are also essential for operations car­ried out in the media and plant preparation area of a plant micropropagation unit. These include the following (Appendix 1): a) One or more refrigerators for cold storage (about

4°C) of stock solutions, growth regulators and sterilized culture media as well as for the tempo­rary storage of plant material used for prepara­tion of explants.

b) A pH meter for measuring and adjusting the pH of culture media.

c) A deionizer or water still to furnish deionized or distilled water needed for the preparation of stock solutions and nutrient media, the operation of the autoclave, the rinsing of glassware, etc. (Butcher and Ingram, 1979).

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d) A magnetic stirrer, to help dissolve various in­gredients in water or other solvents.

e) An adjustable volume dispenser or other suitable device to dispense aliquots of hot medium.

f) A Millipore apparatus for the sterilization of heat-labile ingredients of tissue culture media that cannot withstand autoclaving. Such ingredients, dissolved in distilled water or other suitable sol­vent are vacuum-filtered through a suitable Milli­pore filter which can withhold all bacterial, fun­gal and other contaminants. Such filter-sterilized ingredients are added aseptically to the medium after autoclaving. In addition to the Millipore apparatus and the necessary filters, a source of vacuum (e.g, a small vacuum pump) is essential for this type of sterilization (Dodds and Roberts, 1982).

Laboratory supplies. A large amount of various types of glassware, especially of culture vessels, are usually used in a tissue culture laboratory. Various types of glass containers are used as culture vessels, including test tubes of various sizes, conical flasks, baby food jars, Petri dishes etc. The shape and size of the culture vessels depend on the purpose of cul­ture. In all cases, however, such vessels must be fitted with autoclavable closures, to exclude contam­inants and at the same time allow free gas ex­change. Other glassware commonly needed in a tis­sue culture laboratory include an assortment of beakers, measuring cylinders', graduated pipettes etc.

An itemized list of the most essential glassware, tools and other minor laboratory supplies needed is given in Appendix 2. Chemicals commonly needed for nutrient media preparation include a large num­ber of inorganic salts, suitable carbon sources, gell­ing agents, vitamins, growth regulators, antioxidants and various other chemical supplies (Appendix 3). Tissue cultures are very sensitive to toxic chemical contaminants; therefore, all media should be pre­pared with the purest possible chemicals available, "Analar" or similar grades (Butcher and Ingram, 1979). Information on media preparation has been summarized elsewhere (Ioannou, 1989).

B. Transfer area All aseptic operations, including the excision of ex­plants, the initiation of new cultures and the trans­fer of existing cultures to fresh media, are carried out in this area. Consequently, it is most important

that this,part of the laboratory is free from contam­inating microorganisms, especially bacteria and fun­gi. Thus, the transfer area should preferably occupy a completely separate room which should be kept clean and free of dust. The number of people working in this room should be at a minimum and the door should be kept closed to avoid drafts. Floors should never be vacuum-cleaned but, instead, they should be mopped with clean water every day to avoid formation of dust in the air. The person­nel should wear clean laboratory gowns and surgical caps and masks and should thoroughly wash their hands before undertaking any sectioning or transfer work (Dodds and Roberts, 1982). The proper steril­ization of culture media, glassware and instruments used, the effective disinfestation of plant material, and the exclusion of mites and thrips are also im­portant measures against contamination (Ioannou, 1989). Laminar flow cabinet. The maintenance of aseptic conditions in the transfer room can be greatly im­proved through the use of a laminar flow cabinet (inoculation hood). This allows a gentle flow of ul­trafiltered sterile air to pass across the working area, thus preventing entrance of air-borne contami­nants. With this unit it is possible to carry out aseptic manipulations of tissue cultures even in a small, multipurpose laboratory, where a completely separate and isolated transfer room is not avail­able. In such a case the laminar flow cabinet should be sited in an infrequently used corner of the main laboratory, to avoid air currents as much as possible (Dodds and Roberts, 1982). Various types of lami­nar flow cabinets are commercially available. Mod­els with horizontal rather than vertical air flow are more suitable for tissue culture work and should be preferred. The unit must also be equipped with a series of prefilters, to withhold large dust particles, and a high efficiency particulate air (REPA) filter, to withhold small dust particles, fungal or bacterial spores, and other microscopic air-borne contami­nants. The availability of UV light is highly desirable as this enables the elimination of UV-sensitive mi­croorganisms which might exist in the cabinet be­fore starting a particular aseptic operation. Both floor-standing and bench-mounted models are equal­ly suitable. The unit size will be determined by the overall size and design of the laboratory and the number of people available to work at the same time.

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Other essential equipment. Other equipment needed in the transfer area include one or more stereoscopic microscopes and an array of surgical instruments, glassware and minor laboratory supplies (Appendix 1). The stereomicroscopers), placed in­side the laminar flow cabinet, is necessary for sec­tioning work with plant tissues, preparation and handling of minute explants and various aseptic ma­nipulations of tissue cultures. It must preferably feature continuous (ZOOM) magnification of up to 50 or lOOX and possibility for either top or bottom light attachment. Surgical instruments needed include an assortment of forceps, scalpel handles and surgi­cal blades, dissecting needles, transfer needles, etc. (Appendix 2).

C. Incubation area (growth room) Tissue cultures prepared in the transfer area of the laboratory are transported to the incubation area for growth and development under controlled envi­ronmental conditions. Such conditions can be ob­tained by including in the laboratory building one or more separate, walk-in growth rooms. These are small, windowless and well-insulated rooms, equipped with control systems for temperature, light, humidity and ventilation, and furnished with a series of shelves for the incubation of cultures. One such room with three shelf units, each divided in up to 6 levels, about 0.5 m apart, is usually needed for the incubation requirements of a medium-size mi­cropropaganon unit. Light control. Cultures under incubation need to be exposed to defined light conditions, in terms of quality, intensity and duration. Cool white (Gro Lux) lamps with significant emission in the blue and red regions are the most suitable light sources. In case such lamps are not available, common fluores­cent light can also be used instead. The lamps are fixed on the undersurface of the incubation shelves as shown in Fig.2. To satisfy the illumination re­quirements of different culture stages it is suggested that light intensity approximates 1,000 lux on the two upper shelf levels, 3,000 lux on the two middle ones, and 10,000 lux on the two lower shelves. To avoid overheating of cultures, the shelves with high­intensity illumination should be doubled and well­insulated. Temperature control. The system should maintain constant temperature in the growth room at any de­

sired setting in the range from 18°C to 30oe, with maximum temperature fluctuation I-2°C, The tem­perature should also be as uniform as possible throughout the room, with maximum variation <b1­2°C. To achieve these conditions, the system should include separate cooling and heating units, controlled by separate, high-accuracy thermostats. A third thermostat should provide for safety cut-off in case of failure of the heating system. Humidity and ventilation control. A relative hu­midity of about 60% is considered satisfactory for the incubation area. In case of extremely dry envi­ronment, the use of an electric humidifier is recom­mended. A ventilation system consisting of one ex­haust fan and an interval timer for programmable intermittent operation should also be installed. Other equipment. One or more gyratory platform shakers may be needed for the incubation of agitat­ed liqutd cultures (Ioannou, 1989). This can be ac­commodated on the floor or on a bench inside the growth room. For convenient operation of the var­ious environmental control systems of the growth room from outside, all necessary controls and indi­cator lamps should be provided in the form of an easily accessible control panel installed outside the room. A dial thermometer and a weekly-recording, distance hygrothermograph should also be installed outside the room for continuous monitoring of tern­perature and relative humidity in the incubation area.

GREENHOUSE FACILITIES Plants produced in vitro through micropropagation are eventually transferred to soil for growth in vivo under normal greenhouse conditions. Consequently, the micropropagatton unit must be supplemented With adequate greenhouse and related nursery facili­ties to enable proper care of plants during the final stage of propagation, i.e. after they have been transferred to soil. The facilities required for this purpose are basically identical to those of a conven­tional plant propagation unit. However, it must be pointed out that for successful establishment in soil, micropropagated plants require a weaning process (acclimatization or hardening) during which they gradually adjust to normal greenhouse conditions. For this purpose, the newly potted ex vitro plantlets need to be placed for some time in a special green­house compartment, which should be equipped with

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light regulation (shading) system, heating and cool­ing system enabling effective temperature control throughout the year, and intermittent mist facility for humidity control. In this compartment plants adjust gradually to higher light intensity, fluctuating temperatures and lower relative humidity, thus avoiding excessive loss of water and dessication, whtch is the most frequent cause of poor plant sur­vival in soil. Protection against infection by patho­gens is also very important during the hardening pe­riod and until plants adjust to non-sterile condi­tions. For this purpose, ex vitro plantlets are usual­ly transplanted to soil mixes which have been steril­ized by steam or dry heat. The availability of such soil sterilization facilities is therefore very impor­tant. Facilities for insect exclusion (double doors, in­sect-proof nets on doors and windows etc.) are also important as a means for pest control and for pre­venting infection by insect-borne virus and virus­like pathogens.

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REFERENCES Butcher, D.N., and D.S. Ingram. 1979. Plant Tissue

Culture. Edward Arnold, London. 67 p.

Dodds, J.H., and L.W. Roberts. 1982. Experiments in Plant Tissue Culture. University Press, Cam­bridge. 178p.

Ioannou, Maria. 1989. Agricultural applications of plant tissue culture: rapid clonal propagation of horticultural crops. Miscellaneous Reports 37, Agricultural Research Institute, Nicosia, Cyprus. 11p.

Pennel, D. 1982. Introduction to the Microprops­gation of Horticultural Crops. Ministry of Agriculture, Fisheries and Food, London. 47 p.

Street, H.E. 1977. Plant Tissue and Cell Culture. Blackwell Scientific Publications, Oxford. 614 p.

TRANSFER ROOM

PREPARATION ,--­

ROOM

CULTURE ROOM

Fig. 1. Example plan of micropropagation laboratory

10, 000 LUX

~ I

Fig. 2. Culture shelves in growth room

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Appendix 1. Essential facilities and equipment required for a plant rnicropropa­gation laboratory

General-purpose Facilities Benches and storage cabinets Sink supplied with hot and cold water Air-condltioning/heating system Electrical outlets

Media and Plant Preparation Area Deionizer or water still Autoclave (or large pressure cooker/s) Sterilizing oven Refrigerators pH meter Analytical balance Precision balance Magnetic stirrer Vacuum pump Millipore apparatus Adjustable volume dispenser Glassware, tools and other laboratory supplies (Appendix 2) Chemicals for media preparation (Appendix 3)

Transfer Area Laminar flow cabinet Stereomicroscope Surgical instruments, glassware and other laboratory supplies (Appendix 2).

Incubation Area (Growth Room) Culture shelves Gyratory shaker(s) Artificial illumination system Temperature control system Humidifier and ventilation control system Dial thermometer Distance thermograph

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Appendix 2. Glassware, tools and other supplies required for plant micropropagation laboratory

Culture tubes with caps Measuring cylinders Flasks, conical and volumetric Beakers Culture jars (or baby food jars) Petri dishes Glass rods Funnels Pipettes Dropping bottles Alcohol burner (or Bunsen burner) Coplin jars Laboratory scoops and microspatulas Asbestos gloves Wash bottles Brushes for test tubes and flasks Test tube racks Test tube baskets Forcep racks Scalpel handles and surgical blades Alarm clock (to time disinfestation) Dissecting needles Garden pruner Forceps Filter paper Cheesecloth Paper towels Aluminium foil Millipore filters Scalpel blades Gro Lux lamps Disposable gloves Cotton wool Chlorine bleach Detergent concentrate Sponges

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Appendix 3. Chemicals needed for nutrient media preparation Inorganic salts

Ammonium nitrate Potassium nitrate Calcium chloride, dihydrate Magnesium sulfate, heptahydrate Potassium phosphate, monobasic Boric acid Manganous sulfate, monohydrate Cupric sulfate, pentahydrate Sodium molybdate, dihydrate Zinc sulfate, heptahydrate Cobalt chloride, heptahydrate Potassium iodide Potassium hydroxide Hydrochloric acid 36.5-38% pH 7 buffer pH 4 buffer Ferrous sulfate, heptahydrate Disodium EDTA

Hormones, vitamins and other growth regulators Indoleacetic acid (IAA) Indolebutyric acid (IBA) Naphthaleneacetic acid (NAA) Kinetin (kn) N6-isopentenyladenine (2iP) N6-benzyladenine (BA or BAP) Adenine sulfate, dihydrate Inositol Thiamine. HCl Nicotinic acid Pyridoxine. HCl

Other chemical supplies

Sucrose (and/or white table sugar) Glucose Gelrite or agar Ascorbic acid Citric acid Activated charcoal Ethanol 95% Tween 20

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Appendix 4. Some major commercial suppliers of tissue culture equipment Baird and Tatlock (London) Ltd., P.O. Box 1, Freshwater Road Chadwell Heath, Romford

RMI IHA, U.K. (general equipment and supplies). Bellco Glass, Inc., 340 Edrudo Road, Vineland, NJ 08360, U.S.A. (general equipment and sup­

plies). . A. Gallenkamp & Co. Ltd., P.O. Box 290, Technico House, Christopher Street, London

EC2P 2ER, England (general equipment and supplies). Flow Laboratories Ltd., P.O. Box 17, Second Avenue, Industrial Estate, Irvine, Ayrshire,

Scotland, KAI28ND, U.K. (general equipment and supplies). Sigma Chemical Company, Fancy Road, Pool, Dorset BH17 7NH, U.K. (biochemicals). BDH Chemicals Ltd., Poole, Dorset BH 12 4NN, U.K. (chemicals). May and Baker Ltd., Laboratory chemicals, Liverpool Roard, Barton Moss, Eccles, Manches­

ter M30 7RT, U.K. (chemicals). Gibco Biocult Ltd., 3 Washington Road, Abbotsinch Industrial Estate, Paisley, Scotland PA3

4EP, U.K. (premixed powders).

John Bass Ltd., Bassaire Building Duncan Road, Swanwick, Southampton, Hampshire S03 7ZS, England (Laminar flow cabinets).

Hepair Manufacturing Ltd., Station Road, Thatcham Newbury, Berks, U.K. (Laminar flow cabinets).

MiIIipore Corporation, Bedford, Massachusetts 01730, U.S.A. (membrane filters and apparati). GTE Sylvania, 60 Boxton Street, Salem, Massachusetts U.S.A. (Gro Lux lumps). Magenta Corporation, Laboratory Products Division, 4149 W. Montrose Avenue, Chicago, il­

linois 60641, U.S.A. (plastic culture vessels).

Sterilin Ltd., 43-45 Broad Street, Teddington, Middlesex, TWll 8QZ, U.K. (sterile disposable containers).

Swann - Morton Ltd., Penn Works, Owlerton Green, Sheffield S6 2BJ, England (surgical in­struments).

The Paragon Razor Company, Sheffield S8 OUJ, England (scalpel blades).

P.LO.38/90-600

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