The following points highlight the four main aspects required for plant tissue culture. The aspects are: 1. Aseptic Condition 2. Aeration 3. Equipment 4. Nutrient Medium.
Aspect # 1. Aseptic Condition:
Tissue culture should be done in completely aseptic condition. Care should be taken so that the tissues, equipment, culture media and the room should be completely free from microorganisms. For sterilisation of plant materials, glass equipment, other instruments, culture media etc. several methods are used. These are dry heat, wet heat, ultrafiltration and chemicals. Dry heat is used to sterilise equipment in an incubator.
Wet heat sterilization is done in an autoclave at 120°C at 15 lb pressure for 15 minutes. Liquid media, which are unstable at high temperature are sterilized by ultrafiltration. Chemicals, such as, alcohol is used to sterilize working area and instruments.
(i) Sterilisation of Materials:
The tissue to be cultured may carry microorganisms. To prevent such contamination it is surface sterilized chemically.
The chemical agents to be used and the time of treatment depends on the material to be sterilized. The sterilising agent should be easily removable, because retention of such agents is harmful.
Some of the commonly used sterilising agents are:
(a) 9—10% calcium hypochlorite (Ca [OCl2]) is usually used for 5—30 minutes;
(b) 2% sodium hypochlorite solution (NaOCl) is used for 5—30 minutes. Hypochlorite solutions (Na or Ca) are used at about pH6. After sterilising in these solutions the materials are thoroughly washed in double distilled water;
(c) 10—12% hydrogen peroxide (H202) solution is used for 5—15 minutes;
(d) 1—2% bromine water is used for 2—10 minutes.
Some other sterilising agents are:
1% chlorine water, mercuric chloride, silver nitrate, antibiotics etc. If the tissue is sufficiently hard then it can be sterilised by washing in absolute alcohol. The process of sterilisation differs according to the material to be sterilised.
A short account of the method of sterilisation in various plant materials is discussed below:
The seeds are submerged in absolute alcohol for 10 minutes followed by washing in sterile distilled water. Then the seeds are submerged in 10% calcium hypochlorite solution for 20—30 minutes or treated with 1 % bromine water for 5 minutes.
The time required depends upon the rigidity of the seed coat. After repeated (3—5 times) washing in sterile distilled water the seeds are allowed to germinate in sterile water or on sterile filter paper.
Fruits are washed with absolute alcohol and then submerged in 2% hypochlorite solution for 10 minutes. Fruits are repeatedly washed in sterile distilled water. By dissecting such fruits seeds are obtained, from which sterile seedlings can be grown.
Pieces of stem are washed in running tap water, rinsed in absolute alcohol and then immersed in 2% sodium hypochlorite solution for 15’—30 minutes. The end of the Stem pieces are cut off. The stem piece is either planted vertically on solid agar medium or it is dissected out and the required tissue is taken for culture.
Very young leaves are selected and their surfaces are wiped with absolute alcohol. The leaves are then immersed in 0.1% mercuric chloride for 1 minute. After repeated washing in sterile distilled water the leaves are dried with sterile tissue paper.
(e) Storage Organ:
The material is washed in running tap water and then submerged in 2% sodium hypochlorite solution for 20—30 minutes. After repeated washing in distilled water it is dried with sterile tissue paper. The storage organ is dissected out and internal tissue is taken for culture.
For plant materials having cutin, suberin or epidermal appendages a small amount of detergent (0.05%), such as, teepol or tween 80 is added to the hypochlorite solution. If the tissue is contaminated, as is evident from localized internal discoloration, then it should be discarded.
(ii) Sterilisation of Equipment:
Glasswares (Pyrex or similar borosilicate glass) are generally cleaned with chromic acid-sulphuric acid mixture followed by thorough washing in running water for at least 5 minutes. This is followed by rinsing in distilled water and finally with double distilled water. Cleaned glassware’s should be stored in a dust free place and washed with double distilled water before use.
Glasswares can also be cleaned with hydrochloric acid or nitric acid or detergent. Then the equipment’s are washed thoroughly with sterile distilled water. Due to high corrosive nature of chromic acid-sulphuric acid mixture, in some laboratories detergents are used for cleaning glassware’s.
Glasswares are dipped overnight in strong detergent solution and then washed thoroughly with hot water followed by a wash with cold sterile distilled water.
Glass, metal or silicon rubber equipment’s can be sterilised in an incubator by keeping them at 180°C for one hour. The equipment’s are wrapped in aluminium foil before placing in an incubator. After sterilisation sufficient time is allowed for cooling. According to Collins and Lyre (’84) sterilisation can be done at 160°C for 45 mins., at 170°C for 18 mins., at 180°C for 7½ minutes and at 190°C for 1½ minutes.
All equipment can be sterilised in an autoclave at 15 lb pressure, at a temperature of 120°C for 20—30 minutes. For wrapping during autoclaving un-waxed papers are used. Aluminium foil is unsuitable as it is impermeable to steam. Paper products after autoclaving are kept in a drying oven at a temperature above 60°G to remove moisture. In an autoclave demineralized water is used.
All metal (stainless steel or nickel plated) instruments (such as, scalpel, scissor, forceps etc.) used for inoculation are dipped in alcohol, then flamed and cooled before use. The instruments may also be sterilised by exposing to UV light.
Culture apparatus and equipment should be wrapped in celophane paper or aluminium foil to keep the outer surfaces sterile during transport from the autoclave to the aseptic area. Discarded and contaminated cultures are autoclaved for a short period to kill the contaminants and liquify the agar. Glasswares are then cleaned.
(iii) Sterilisation of the Culture Medium:
The culture medium may contain micro-organisms from the beginning. Sugar containing nutrient media favour rapid growth of various micro-organisms, which grow faster than the cultured tissue and ultimately destroy the tissue. Slow growing micro-organisms also interfere proper growth of the tissue by producing toxins or stimulants.
To prevent growth of the micro-organisms the medium can be sterilised in an autoclave at 15 lb pressure at 120°C for 15 minutes. Sterilisation time increases with increase in the liquid volume. 75 c.c. of liquid per vial requires a period of 20 minutes, 250—500 c.c. vial needs a period of 25 minutes and 1000 c.c./vial requires 30 minutes of autoclaving. The sterile medium should be used within 14 days of sterilisation.
Substances in pure aqueous solution may be stable when autoclaved; but they may be chemically modified in presence of some other substance of the medium during sterilisation. Such substances are separately autoclaved and after sterilisation added together.
Some of the chemicals used in tissue culture degrade at high temperature during autoclaving, e.g. gibberellins, vitamins, 1, 3 or 9 substituted purines. Sucrose on autoclaving is partially degraded to d-glucose and d-fructose, which may have some inhibitory effect.
Such substances are sterilised by ultrafiltration, that is, by passing through a membrane filtration unit attached to a graduated syringe. The sterilised liquid is added directly to the autoclaved medium.
Ultra filters may be of different types. These are:
(a) Durapore membrane filter consisting of fluorocarbon polymer, it is most suitable for membrane filtration;
(b) Nucleopore filter consisting of polyethelene film and having pores of definite diameter;
(c) Disposable filter of overlapping cellulose fibres;
(d) Fluorcpore filter consisting of thin teflon is used to filter non-aqueous solution, such as, solvent dimethyl sulphoxide. Pores of an ultra filter are usually 0.4 µm in diameter. A pore of 0.22 µm only can remove all micro-organisms.
Antibiotics, such as, penicillin, streptomycin etc. are sometimes added to the nutrient medium to prevent the growth of the micro-organisms. Potassium benzyl penicillin in 100 fig /ml concentration prevents the growth of gram positive bacteria.
Streptomycin sulphate in 100 µg/ml concentration prevents the growth of gram negative bacteria. Gentamycin in 100 µg/ml concentration is supposed to prevent the growth of gram positive and gram negative bacteria. But, antibiotics are not very effective in plant tissue and cell culture.
Antibiotics are not recommended for sterilisation of plant tissue culture because:
(a) No antibiotic is effective against all contaminating micro-organisms;
(b) Degradation products of antibiotics might be metabolised by plant tissues causing unpredictable results;
(c) Antibiotics may inhibit growth as recorded in tobacco callus growth.
Large quantities of distilled water is needed for tissue culture. Distilled water should not be stored for a long time and the reservoir should be frequently cleaned. Water may be sterilised in an autoclave for an hour in a pyrex glass flask provided with a non-absorbent plug fitted to its mouth.
Mouths of the culture tubes and flasks are flamed during sterilisation. During combustion process ethylene is produced and this gas may enter in high concentration in the culture vials. Ethylene being a plant hormone may influence the growth and morphogenesis of plant tissue culture.
(iv) Sterilisation of the Environment:
All sterilisation, inoculation etc. should be done under aseptic conditions in a clean room free from dust and convection current which may carry spores of microorganisms. Culture may be best done in an enclosed chamber (Fig. 1) under filtered air pressure in a well-lighted, sterile and suitably ventilated condition.
The inoculation chamber is sterilised with ultraviolet ray before inoculation. The UV germicidal lamp is turned on about two hours before using the chamber and should be turned off before using the chamber. The chamber is kept free from microorganisms by continuous flow of filter sterilised air.
The table to be used for inoculation is wiped with ethyl alcohol or isopropanol (70% or 80%). The neck of the culture vial is quickly flamed and its plug is rapidly removed. The tissue is inserted and the plug is immediately replaced.
Hands should be washed thoroughly with soap and hot .water for several minutes and the skin is wiped with paper towels. Hands and fingers may be dipped in ethanol before certain operations, such as, removing seed coats by hands. Discarded cultures should be immediately removed. All unnecessary glass-wares, instruments, aluminium foils etc. which have been used should be removed,
Aspect # 2. Aeration:
Proper aeration of the tissue in the culture medium is essential. Those tissues, which are cultured on semi-solid medium do not require any special method for aeration. But those tissues, which are cultured in liquid medium remain submerged and require special device for aeration.
(a) For proper aeration in liquid medium filter paper bridge may be used. The filter paper is placed in such a manner so that its two ends are dipped in a medium and the middle horizontal portion on which the tissue is placed remains above the level of the medium.
(b) By passing filter sterilised air through the medium and by stirring the medium proper aeration is possible.
Usually proper supply of oxygen is provided by keeping the culture vial with liquid medium on an automatic shaker. This method will also help in the separation of the tissue into individual cells or small groups of cells. The mouths of the culture vials are closed with non-absorbent cotton covered in cheese cloth. This allows proper aeration but prevents the entry of micro-organisms.
Aspect # 3. Equipment:
Various equipment are necessary for tissue culture.
(i) Culture vessels—Glasswares used for tissue culture should be of borosilicate glass (pyrex glass), because soda glass may hamper the growth of the tissue. Pyrex glass containers can be easily sterilised and their mouths can be flamed during transfer processes.
Various types of culture vessels, such as, test tubes, conical flasks, bottles etc. are used. Usually 100 ml conical flask or large test tubes (25 X150 mm) are used. Sometimes special flat tubes are used so that the culture is easily visible. Watch glasses were earlier used for organ culture.
(ii) In addition to culture vessels, different kinds of glassware’s are required for preparing the nutrient media and culture tissue. These include measuring cylinders, beakers, funnels, petridishes, graduated pipette, conical flasks etc.
Petridishes made of pyrex glass or plastic are used for culturing or to hold the material during transferring.
Petridishes are sterilised in an autoclave for 30 minutes or more by placing them within metal containers or wrapped paper bags.
(iii) The mouth of the culture vial may be closed with non-absorbent cotton plug, screw cap back-elite cover, metal or plastic cover, polyurethene foam or polypropylene plug.
The exposed part of the plug and the rim may be covered with aluminium, foil. This will prevent the plug from becoming wet when autoclaved. Wet and contaminated plugs are immediately discarded.
The mouth of the culture vials may be directly covered with sterile square soft aluminium foil (0.25 mm thick). Sides of the foil are pressed neatly to the sides of the test tubes or flasks by downward movement. These foils can be easily removed and replaced again and again. After one culture aluminium foils are discarded.
(iv) Alcohol lamps—Small spirit lamps can be used to sterilize and flame the instruments or the mouth of the culture vials.
(v) Sterilising agents—various sterilising agents are used in tissue culture:
(a) Ethyl alcohol is used to wipe the surfaces of materials, working areas, to rinse hands or gloves, to dip instruments.
(b) Calcium or sodium hypochlorite is used to wipe surfaces of working areas and to sterilize equipment. It is very useful to disinfect surfaces of plant tissues and seeds without causing injury.
(c) Bromine or chlorine water is used to sterilize plant materials.
(d) Aqueous or alcoholic mercuric chloride (0.1%) is useful to disinfect working surfaces and materials.
(e) Sterile distilled water is used to rinse the surface of the material after disinfection. For preparing the media distilled, redistilled and deionized water is used.
(vi) Forceps, scalpels etc. are required for excising the explant and transferring the tissue to the culture vials.
(vii) Rubber gloves, aprons etc. are used to maintain aseptic conditions.
(viii) Incubator is required to sterilize equipment.
(ix) Autoclave—All nutrient media and equipment are sterilised in an autoclave.
(x) Inoculating chamber—All aseptic transfers should be done in an inoculation room under completely aseptic condition. If inoculation room is not available then a small inoculation chamber can be used. The inoculation chamber can be made from metal sheets and glass. Inner surface of the chamber is sterilised by wiping it with alcohol. Sterile air is allowed to pass through the chamber.
(xi) A refrigerator in the media room is required for storing chemicals and stock solutions.
(xii) A sensitive balance is necessary for weighing the chemicals.
(xiii) A magnetic stirrer, a pH meter or pH indicator papers etc. are needed.
(xiv) A centrifuge is required.
(xv) Labels and marking pencils are needed.
(xvi) Hand lens, dissecting microscope, compound microscope with micro photographic accessories are required.
(xvii) Shelves are required to keep the media and reagents.
Aspect # 4. Nutrient Media:
Various nutrient media have been used for tissue culture. In the beginning different organic media, such as, plasma, tissue extract, coconut milk etc. were used. Later, various defined media were used. Nutrient media required for culture vary with the kind of plant and the purpose of producing the culture. When starting the work specific medium for that tissue is worked out.
Most of the media contain some inorganic salts of major and minor elements, vitamins and sucrose. Such a medium is called a basal medium. If required the medium is solidified with 0.5-1% agar. The pH of the culture medium is maintained between 5.6-5.8 (slightly acidic). The pH is adjusted by adding a drop of hydrochloric acid or by adding a few drops of caustic potash.
Various substances used in different culture media are:
i. Inorganic Substances:
Some inorganic ions used in culture media are sodium, potassium, calcium, magnesium, chloride, phosphate, sulphate, bicarbonate etc. Micronutrients, such as, manganese, boron, zinc, copper, molybdenum etc. are usually added to the culture media.
(i) Nitrogen is usually added in the form of nitrate or ammonium salt or a mixture of both.
(ii) Potassium is added as KCI or KNO3 or KH2PO4. It is the most abundant cation. High concentration of potassium favours embryogenesis.
(iii) Calcium is added in the form of CaCl2, 2HgO or Ca (NO3)2, 4H2O or anhydrous form of either.
(iv) Magnesium and sulpher are added in the form of magnesium sulphate (MgSO4, 7H2O). Phosphorus is obtained from NaH2PO4, H2O or KH2PO4.
(v) Iron is essential and added to the medium as inorganic iron or chelated iron (such as, NaFeEDTA—i.e., ferric sodium ethylene diamine tatra-acetate). The presence of NaFeEDTA ensures that iron is available over a wide pH range in the medium.
Each inorganic material is made as stock solution having 10 or 100 times the concentration actually required. The solution is autoclaved and stored in a freeze.
ii. Organic Substances:
(i) Sugar—2-4% sucrose (20,000-30,000 mg/l) is usually used in a medium. In some cases glucose is used. Sucrose is thermolabile, so it is good to sterilize glucose try filtration.
(ii) Vitamins and amino-acids—Vitamins are usually required in traces. Many scientists consider that thiamine (Vit B1 is the only vitamin needed by all plant tissue cultures. Thiamine is added as thiamine hydrochloride.
Nicotinic acid (0.5 mg/1), thiamine (0.1-1 mg/1) and pyrodoxine (0.5 mg/1) are usually added to the medium. Some other materials which may be benificial are inositol (100 mg/1), biotin (0.1 mg/1) and pantothenic acid (0.1 mg/1) or calcium pantothenate (1.0 mg/1) and folic acid (0.5 mg/1).
A stock solution of all these substances at 100 times the required concentration is prepared and stored in a freeze.
Vitamins are thermolabile and are filter sterilised and added with a syringe or pipette to warm unsolidified autoclaved medium.
Plants can prepare necessary nitrogenous substances from simple amino acids such as, glycine and inorganic nitrogenous substances. But the presence of other amino acids is beneficial. Some of these amino acids are—arginine, aspergine, aspertic acid, glutamic acid, glutamine, methionine.
In culture media containing complex nitrogenous substances growth is good. Sometimes organic nitrogen, such as, casein hydrolysate or casamino acid (0.05-0.1%) is added to the medium. Addition of several amino acids to the medium may have an inhibitory effect probably due to competition among various amino acids.
(iii) Hormones—Most callus cultures require a supply of auxin and cytokinin. Auxin stimulates shoot elongation and cytokinin promotes cell division. Auxin and cytokinin balance controls cell division, cell elongation, cell differentiation and organ formation.
Auxins are usually dissolved in a small amount of alcohol before preparing the stock solution. Indole acetic acid (IAA) at 1-50 mg/litre concentration, napthal acetic acid (NAA) at 0.1-10 mg /litre concentration and 2-4 dichloro-phenoxy acetic acid (2-4-D) at 0.05-0.5 mg/litre concentration are generally used.
IAA and NAA are soluble in sodium hydroxide, 2-4-D is soluble in ethanol or dimethyl sulphoxide, which in higher concentration is toxic. For callus initiation 2-4-D is most suitable but it suppresses root and shoot initiation.
IAA is easily destroyed by light and enzyme, so IAA is added at a high concentration to the culture medium. NAA is not easily degraded, thus it is added at low concentration.
In culture cytokinins used are zeatin, kinetin and benzyl adenine. Kinetin at 0.01—10.0 mg/1 concentration is used in culture. Kinetin at 0.1 mg/1 concentration can induce callus formation. A stock solution is prepared by dissolving it in a small amount of hydrochloric acid and then water is added.
Gibberellin is not usually required, but it is needed for apical meristem culture.
Ethylene helps differentiation of tracheary elements and initiation of buds.
(iv) Other organic substances—Sometimes natural extracts are added to supplement the media especially when chemically defined media cannot produce desired results. Some such extracts are —coconut milk, yeast extract, casein hydrolysate, malt extract, fruit juice (such as, tomato juice).
10-15% (by volume) of coconut milk is usually added to the medium. Preparation of coconut milk—Fresh coconut milk is taken out through micropyler region of a ripe coconut. It is filtered and the filtrate is boiled for 10 minutes, the proteins precipitate out. This is cooled, decanted and filtered. This coconut milk is used in culture. Unused coconut milk is stored in a freeze.
In orchid seed culture banana extract is beneficial. Explants of Citrus sp. is stimulated by orange extract.
(v) Charcoal—Activated charcoal is sometimes used in the culture medium.
It has adsorbent property and may serve various purposes, such as:
(a) Removes contaminants from the medium,
(b) Absorbs secondary products secreted by the culture tissue,
(c) Controls the supply of endogenous growth hormone and
(d) Darkens the supporting matrix.
According to Kolenbach and Wernicke (78) presence of activated charcoal stimulates embryogenesis. But in some cases it may inhibit growth and morphogenesis.
Osmotica—The uptake of water by a cell depends upon the osmotic concentration of the cell sap and the external medium. Water availability of a culture medium, depends upon the concentration of agar, carbon source and any external non-metabolite added as an osmoticam. Sugar, such as, mannitol or sorbitol is used as an external osmoticum. In protoplast culture polyethylene glycol (PEG) is used as an osmoticum.
Water—Double distilled or demineralized water is used in culture media.
Media matrix—Solid and semisolid culture media usually contain 0.6—1.0% agar. Commercial agar contains various contaminants. Purified agar is available for tissue culture.
For different types of culture various types of culture media may be used. Some of the extensively used culture media are-—White’s medium, Murashige and Skoog’s medium, Nitsch’s medium, Nagata and Takebe’s medium etc.
Knop’s solution is sometimes used for embryo culture. It was first developed by Knop’s in 1865, but later modified by different workers. For callus culture of dicot Murashige and Skoog’s medium is good. For monocot culture Schenk and Hildebrandt’s (’72) medium may be used. It contains 2-4-D kinetin and 2 chlorophenoxy-acetic acid.
Preparation of the stock solution:
Preparation of Murashige and Skoog’s stock solution is described below:
(a) Micronutrient stock—To 400 c.c. of double distilled water each micronutrient salts are added. By adding double distilled water final volume of 1000 c.c. is obtained. This solution is stored in a freeze. 10 c.c. of this solution is added to 1000 c.c. of culture medium.
(b) Iron stock—To 80 c.c. of double distilled water iron salts are added. The final volume of 100 c.c. is made by adding double distilled water. This is stored at room temperature. 5 c.c. of this stock solution is added to 1 litre of the Murashige and Skoog’s culture medium.
(c) Vitamin stock—To 50 c.c. of double distilled water required amount of each vitamin is added. By adding double distilled water final volume of 100 c.c. is obtained. Vitamin solution is sterilised by ultrafiltration and added to the sterilised culture medium while agar is still warm. To 1000 c.c. of MS culture medium 1 c.c. of vitamin stock solution is added. Unused vitamin solution is discarded after one month.
(d) Kinetin stock—Kinetin is weighed and dissolved in a few drops of hydrochloric acid. To it a few c.c. of distilled water is added. The final volume of 100 c.c. is made by adding double distilled water. To 1 litre of culture medium 1 c.c. of this stock solution is added.
Preparation of MS culture medium:
Macronutrients are weighed, dissolved and added to 400 c.c. of double distilled water. To it 10 c.c. micronutrient stock, 5 c.c. of iron stock and 1.c.c. of kinetin stock are added with a pipette. To it 100 mg myo inositol is added. To this medium a mixture of 10 mg of IAA dissolved in a few drops of sodium hydroxide is added. By adding double distilled water the volume of the culture mixture is made up to 800 c.c.
The pH is adjusted to 5.7 by adding few drops of IN NaOH or IN HC1. The final volume is made upto 1000 c.c. by adding double distilled water. 3.0 gm sucrose, 0.8 gm of agar is added to 100 c.c. of MS medium in 250 c.c. flask.
The mouth is closed with an aluminium foil. It is autoclaved and allowed to cool. 1 c.c. of vitamin is added while the agar is still warm and the flask is gently swirled to mix the vitamin with the medium. About 10 c.c. of medium is poured in each culture vial.
The temperature of the medium should be maintained properly. Plant tissues are usually cultured at 25—27°C. In lily and Gladiolus if the tissues are kept at low temperature before culture then regeneration capacity increases.
In animal tissue culture the temperature of the medium should be similar to the body temperature in case of warm blooded animals. In case of cold-blooded animals the temperature of the medium is similar to the temperature of the environment, where the animals live.
The medium to be used for culture depends upon:
(a) The plant species,
(b) The tissue to be cultured and
(c) The purpose of culture.
In different kinds of plant tissue culture various types of media are used:
(1) Solid agar medium is used for different tissue and organ culture. On agar medium tissue grows quickly and usually produces callus.
(2) Static liquid medium is mainly- used for root culture.
(3) Shaking liquid medium is useful for the culture of cell population. Such a medium helps gaseous exchange. In a shaking liquid medium when a tissue is placed and agitated then a free cell culture is obtained.
(4) One drop medium is useful for culture of an isolated cell (clone).