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9. Sterilization ofplant material
9.1.Introduction
In principle there are 4 sourcesofinfections: the plant (internal as well external), the nutrient medium (insufficiently sterilized), the air, and the research worker (inaccurate work). The most importantofthese is the plant itself, and plant material should be well sterilized before being iso- lated in vitro. Before beginning sterilization, any remaining soil or dead parts etc. should be removed from the plants (or partsofthe plants). This should be followed by washing in waterifthe external contamination is very bad (e.g. potatoesorrhizomes grown in the soil). Peeling results in the lossof the outer most layer (lossofthe potato skin or the dry dirty layersofa bulb). After these steps, sterilization is begun, usually as follows: (^) the organ is dipped into 70% alcohol for a few second (96% alcohol is too strong, resulting in excessive dehydration) to eliminate air bubbles, then sterilized for a 10-30 minutes in 1% NaClO containing a few dropsof Tween 20 or 80, and rinsed (to remove hypochlorite) in sterile tap water (usually 3 times, for 2,5,and 15 min respectively). After these steps work can begin on cutting the plant pieces, in sterile conditions (the laminar air-flow cabinet), and using sterile instruments (placed in 96% alcohol and then flamed). If, despite' good' chemical sterilizationofthe plant material, infections later occur, the causeofthese are probably:
- So-called internal infections, which are dealt with in Section 9.4.
- Imprecise work (unwashed hands; table-top not sterilized with 96% alcohol; non-sterile forceps or scalpels; not sufficiently sterilized Petri dishes, paper and/or nutrient media; dirty laboratory coats, etc.). The use of face masks, covering the hair, and using sterile gloves can all contribute to a lower number of (^) infections.
- A defective laminar air-flow cabinet. This cabinet should be tested every year by the factory, with a so-called particle counter. The front filters should be periodically renewed.
- The alcohol in which the instruments are placed prior to flaming is contaminated. It is advisable to renew this alcohol regularly.
- The tubes, etc. containing nutrient media are not sterile on the out- side. After sterilizing nutrient media, they should be stored under sterile conditions.
- The floors are not regularly washed and disinfected. When entering the inoculation room plastic covers should be placed over the shoes, or the soles should be dipped in sterilizing fluid.
- The room containing the laminar air-flow cabinets is not sterile. This can be avoided by blowing in sterile air, and by irradiating with UV light during the night.
- Too many non-essential visitors are allowed into the inoculation room, infecting the floor and the air.
- Infections often occur when isolating shoot-tips of rosette plants, because these are difficult to sterilize. To avoid this the shoots are often sterilized in stages: the shoot is washed, sterilized, a few leaves removed, sterilized again, etc. Generally speaking the chances of infection with shoot-tips is smaller if they are isolated when small. Preferably a meris:em with only a few leaf primordia should be iso- lated; the disadvantage of this is that the chances of growth in vitro are smaller.
- If the growth rooms are not kept clean (the floors not regularly cleaned and disinfected), infections can result, in some cases due to mites which can carry, especially, fungal infections with them. Since these mites can easily pass through cotton plugs and plastic film, the seriousness of this form of infection should not be underestimated. The eradication of mites is best carried out, over a few weeks with Vapona insecticide strips (dichlorovos) from Shell (Bonga and Dur- zan, 1982).
9.2. Chemical sterilization
Chemical sterilization (eradication of micro-organisms with the aid of chemicals) can be realized by:
- Alcohol (ethanol); 70% alcohol is used for plant material since 96% alcohol dehydrates too much. When sterilizing tables and instruments 96% alcohol must be used, since 70% alcohol leaves a layer of water
The choiceofsterilization time and the concentrationofthe bleach can be made depending on the particular conditions. They strongly depend on whether the surface layersofthe explant being sterilized are to remain (gentle sterilization) or are to be cut off before inoculation (vigorous ster- ilization). It is sometimes possible that sterilization is accomplished after only 5 min treatment with 1% NaClO, and in other cases 30 min might be necessary. Lengthy sterilization can result in detrimental effects on the explant, and the correct timing and concentrationofbleach should be decided for each individual experimental material. To give an ideaof some ofthe concentrations and times used a fewexamples are given below:
Anthurium andreanum leaves: 30 min 1% NaClO Hyacinthus scale tissue: 15 min. 1% NaClO Rhododendron stems: 20 min 1% NaClO Gerbera petioles: 15 min 1% NaClO Freesia flower buds: 20 min 1% NaClO Strelitzia leaves: 45 min l% NaClO Tulipa seeds: 30 min 2% NaClO Phaseolus stems: 10 min 1% NaClO Shoot tipsof Nephrolepis: 5 min 1% NaClO (Soede, 1979).
Sometimes the surfaceofa wound may be coated in paraffin to prevent the penetration by the sterilizing fluid (into a stem), and /or to prevent bleeding (e.g. in the Euphorbiaceae).
9.3. Apparently sterile cultures
It should be borne in mind that seeming sterile cultures are not in fact always sterile.Ifthe centreofinfection is in the inner tissuesofthe plant, then the infection often only becomes apparent when the siteofinfection is cut open (during subculture), and makes contact with the medium. Contamination often only becomes evident after a few subcultures have been made. Poor growth and/or chlorosis can be an indication of an internal infection, e.g. bacteria such as Erwinia carotovora (Knauss and Miller, 1978). Apparently sterile cultures can also arise due to the fact that growth may take place on a relatively poor medium, on which micro-organisms seldom if ever develop. Infections only become evident if inoculation takes place onto a richer medium, or if mutation of the micro-organism takes place, allowing it to develop on a poorer me- dium.
To be certain that a culture is sterile, a shoot tip can be cut longitudi- nally, and placed with the cut surface on a rich medium. This can be prepared by the addition of 2-3% tryptone or peptone (a mixture of amino acids and vitamins), and within a few days there is usually an explosion of microbial growth (Knauss, 1976). This technique is not always sufficient since there is no good detection medium for many of
the endogenous bacteria.
Internal contamination (see Section 9.4) in plants is often in the form
of rod bacteria (particularly Bacillus licheniformis and/or Bacillus subtil-
is), which have been given the name of white ghost in the U.S.A. These saphrophytic bacteria are also often encountered in microbiological lab- oratories. They are well known because their spores are able to tolerate unfavourable periods (heat, drought, cold, UV-radiation, presence of sterilizing fluids). Sterilization of the air, floor and the plant material itself may be needed to rid the plants and the laboratory of the 'white ghost'.
9.4. Internal infections
Internal infections, which can be a considerable problem, are caused by micro-organisms present inside the plant itself, and cannot be eliminated by external sterilization. In principle there are two ways of combating this problem: meristem culture (since most of the micro-organisms are not present in the meristem), or by the addition of antibiotics to the nutrient media. Since meristem culture is very complicated and the addi- tion of antibiotics to the media is largely ineffective, easily the best solu- tion is the use of internally sterile plants! The addition of antibiotics often leads to phytotoxic phenomena: such high concentrations of antibiotics are necessary that the growth and development of the higher plant is also inhibited. The use of antibiotics can also lead to the selection of a resistant micro-organism. When used most antibiotics are added to the medium by filter sterili- zation. Stichel (1959) and Montant (1957) described the use of penicillin and achromycin, and other workers have used tetracyclin and 8-hydroxy- quinoline. Staritsky et al. (1983) compared the eftfects of different anti- biotics (oxytetracycline, streptomycin, chloromycetin, penicillin-G, ri- fampicin, and gentamycin) on the culture of Cryptocoryne and Cinchona which were internally infected with bacteria. They concluded that only in the case of rifampicin were the bacteria inhibited, and at the same time there was no effect of the antibiotic on the growth and development of
10. Isolation, inoculation and subculturing
10.1. Introduction
It is obvious that inoculation and subculturing should also be carried out under the same sterile conditions as isolation. In a professional laborato- ry these take place in the laminar air-flow cabinet, but if there is not enough laminar air-flow space available (e.g. during practical courses), cutting etc., can be carried out between sterile filter paper. It is advisable to wear a clean laboratory coat during preparations, and to wash your hands as well as washing the table top with 96% alcohol. Instruments such as scalpels, forceps, inoculation needles, etc. must be previously sterilized by immersing in 96% alcohol followed by flaming. Alternatively they can be put in a beaker containing glass balls at a tem-
perature of 250 oc. This method (factory: S. Keller, Lyssachstrasse 83,
CH 3400, Burgdorf, Switserland), has the great advantage that there is no need for flaming which can be a fire hazard. Sterilization of instruments without the use of alcohol can also be accomplished with the so-called Bacti-Cinerator, which has been developed in the U.S.A. This apparatus is electrically heated to very high temperatures, and it is only necessary to put the instruments inside for 5 seconds for them to be sterilized. As with the heated glass balls, care should be taken that the instruments are allowed time to cool before use. If the alcohol is used for sterilization of instruments then it should be regularly changed since bacteria are sometimes able to survive emersion in alcohol, and the alcohol becomes contaminated with pieces of plant material and agar, etc. Ex plants, can in principle, be cut in two different ways (Fig. 10.1) : on a glass plate sterilized with 96% alcohol (this has the disadvantage that the knives quickly become blunt), or on (between) sterile filter paper (the knives stay sharp longer). It is very easy to work with two stacks of sterile papers: one for use in cutting the explants (regularly replaced) and one
Fig. 10.1. Preparation of bulb scale explants from a hyacinth bulb. Scales (below left) are first sterilized, before explants (below right) are cut in the laminar air-flow cabinet.
for lying the sterile instruments on or between. To work efficiently a number of forceps, scalpels, etc. should be available.
10.2. Isolation
After sterilization and rinsing, the explant is laid on the sterile filter paper or glass plate using sterile forceps. If the cut surfaces have been in contact with bleach, the effected parts are first removed using a sterile scalpel. Sterilized seeds (if no embryos need to be isolated) can be directly ino- culated without any further treatment. It if often necessary to cut out a standard amount or volume of tissue, and to make this more easy to realize, graph paper (coated in plastic) is available. Other necessary pieces of apparatus are the cork borer, the cutting apparatus developed by Bouriquet (19 52), and scales for sterile weighing on aluminium foil. When cutting explants it should be borne in mind that the volume made available can have important consequences (see also Chapter 12): the amount of food reserve, the cut surface area (ethylene production).
air-flow cabinet) should be avoided, since this can result in ethylene pen-
etration into the test tube or flask (Hughes, 1981).
The method of inoculating on solid media strongly depends on the experimental material. Seeds are usually placed on rather than in the medium, which results in oxygen deficiency. This also applies to meris- tems, which are inoculated on the medium using an inoculation needle (dampened with sterile agar), or a piece of razor blade mounted on an inoculation needle holder. Explants (e.g. a piece ofpith tissue) are usually pushed half-way into the agar. Care should be taken not to push shoot tips far into the agar (oxygen deficiency will result). Explants retain their polarity after inoculation: the physiological upper-side remaining the upper side, etc. It is very important when regenerating organs to know how the inoculation has been carried out: polar (straight up, with the basal side of the explant in the medium), or apolar (upside down, with
Fig. 10.3. Soon after isolation a shoot of Pelargonium hybr. has excreted a halo of black substances into the culture medium.
the basal side above the medium). Adventitious roots are mainly formed on the basal side of the explant (Fig. 10.2), which results in better adven- titious root formation with apolar inoculation, as expected from the bet- ter oxygen availability. If the original plant material is orthotropic or plagiotropic then these usually remain so in vitro. For the formation of axillary shoots on isolated shoot tips, it is sometimes best to lie the shoots horizontally on the medium, which promotes the formation of side shoots (Frett and Smagula, 1983).
As has been mentioned earlier shortly after in vitro isolation a brown/black halo of pigment can exudate in the agar (Fig. 10.3). Ways of overcoming this problem can be found in Section 6.4.9.
10.4. Subculturing
Subculturing can be necessary for a number of reasons:
- The nutrient medium is exhausted (deficiency phenomena).
- The nutrient medium dries out (resulting in too high salt and sugar concentrations).
- Growth has filled the tube or flask.
- The material is needed for further propagation.
- Brown and/or black colouring appears in the agar: plant tissues some- times give off toxic substances during the first few weeks, which dif- fuses into the agar or liquid medium.
- It is needed to give the isolated material a different growth and devel- opment pattern, on a known nutrient medium.
- The medium has become liquid due to a lowering of the pH by the plant.
Subculturing is carried out as follows :
- The tube or flask is externally sterilized with 96% alcohol (on a cotton wad).
- Any aluminium foil or film and then the cotton wad (or steristop) are removed from the tube or flask in the laminar air-flow cabinet.
- The explant or callus clump is taken out and put in a sterile Petri dish or on (between) sterile filter paper.
- After any cutting out the material is inoculated onto a new nutrient medium. When cutting pieces out, strong homogeneous (not necrotic etc.) material is selected.
