In this article we will discuss about:- 1. Introduction to Plant Tissue Culture 2. History of Plant Tissue Culture 3. Apparatus Required 4. Techniques.
Introduction to Plant Tissue Culture:
Plant tissue culture is not a separate branch of plant science like taxonomy, cytology, plant physiology etc., rather it is an experimental method 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 anther 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 glassware containing a medium with mineral nutrients, vitamins and phytohormones. Therefore, to carry out experiments using tissue culture techniques, a well-equipped laboratory is first required.
In recent year there has been a phenomenal increase in number of research laboratories using tissue culture techniques to investigate many fundamental and applied aspects of plants. However, the use of these techniques is not confined to research alone. Tissue culture techniques are now being exploited for commercial purposes. Even many horticultural companies are setting up small tissue culture units to multiply plants which are difficult to propagate by conventional methods.
History of Plant Tissue Culture:
The history of in vitro culture of plant tissues goes back to Schwann and Schleiden (1836) who put forward the theory of ‘totipotency’, which states that cells are autonomic and capable of regenerating to give a complete plant. This theory was the foundation of plant cell and tissue cultures. The first attempts by Hyberlandt, the father of plant tissue culture, in 1902 on plant tissue culture techniques failed. However, between 1907 and 1909, Harrison, Burrows and Carrel succeeded in animal and human tissues in vitro.
Although earlier workers have achieved in vitro culture of orchid seeds (seedlings), culturing of roots done successfully by Nobecourt, Gautheret and White independently in 1939. Plant tissue culture lagged behind, animal and human tissue culture because of the late discovery of plant hormones. The first regulator to be discovered, the auxin (IAA) created great opportunities for the in vitro culture of plant tissues. The discovery of the plant regulator kinetin (a cytokinin) in 1955 was a further stimulus in this direction.
A few important milestones in understanding the in vitro propagation of plants are:
1838 – Schleiden and Schwann proposed theory of Totipotency.
1892 – Sacchs observed that plants synthesize organ forming substances which are polarly distributed.
1902 – Haberlandt made first attempt to grow plant tissues in vitro.
1904 – Hanning made an attempt to culture cruciferous embryo.
1909 – Kuster demonstrated fusion of two plant protoplasts but the product failed to survive.
1922 – Knudson could make orchid seed to germinate in vitro without mycorrhiza.
1922 – Robbins could culture root tips.
1925 and 1929 – Laibach demonstrated interspecific crosses through embryo culture.
1934 – White cultured tomato root tips.
1934 – Gautheret demonstrated in vitro culture of the cambium tissue of a few trees and shrubs but failed to sustain since auxin had not yet been discovered.
1936 – La Rue cultured embryos of gymnosperms.
1939 – Gautheret, Nobecourt and White independently succeeded in maintaining growth of callus culture.
1940 – Gautheret studied adventitious shoot formation from cambial tissues through tissue culture.
1941 – Braun could grow Crown-gall tissues.
1948 – Skoog and Tsui demonstrated that formation of adventitious shoots and roots of tobacco is determined by the ratio of auxin/ adenine.
1952 – Morel and Martin could get virus – free Dahlias by meristem culture.
1954 – Muri et al. were the first to get a plant from single cell.
1959 – Gautheret published first hand book on plant tissue culture.
1960 – Kanta for the first time demonstrated fertilization in papaver in test tube.
1964 – Giha and Maheshwari developed haploid plants from pollen grain of Datura.
1970 – Power et al. were successful in protoplast fusion technique.
1971 – Takebe et al. regenerated plants from protoplast.
Apparatus Required for Tissue Culture Work:
The general laboratory for tissue culture techniques should be provided with the following- a washing area with a large sink, running hot and cold water, brushes of various sizes, detergent and a bucket of distilled water for a final rinse of the washed glass materials. A number of plastic buckets are required for soaking the glassware to be washed.
Another separate bucket with lid is also required for disposing off the used or infected media before cleaning. Only this bucket should have to be kept outside the room or cleaning area.
List of apparatus required for tissue culture work:
1. Flasks (100, 250, 500 ml, 1 litre, 5 litre).
2. Volumetric flasks (500 ml, 1 litre, 2 litre, 3 litre).
3. Measuring cylinders (25, 50, 100, 500 ml, 1 litre).
4. Graduated pipettes (1, 2, 5, 10 ml).
5. Culture vials (culture tubes, screw – cap bottles of various size, Petri dishes, nipple flasks etc.) with suitable closure.
6. Plastic or steel buckets to soak glassware for washing.
7. Hot-air oven to dry washed lab-ware.
8. Oven for dry-heat sterilization of glassware.
9. Wire-mesh baskets, to autoclave media in small vials and for drying lab-ware.
10. Water distillation unit or demineralization unit, to obtain high quality water.
11. Plastic carboys (10, 20 litres) to store high quality water.
12. Balance, one to weigh small quantities and the other to weigh comparatively larger quantities.
13. Hot plate-cum-magnetic stirrer, to dissolve chemicals.
14. Vacuum pump, to facilitate filter sterilization.
15. Plastic bottles of different sizes, to store and deep-freeze solutions.
16. Refrigerator, to store chemicals, stock solutions of media, plant materials etc.
17. Deep freeze, to store stock solutions of media for longer periods, certain enzymes, coconut milk etc.
18. Steamer, to dissolve agar and melt media.
19. pH meter, to adjust pH of media and solutions.
20. Autoclave or domestic pressure cookers, for steam sterilization of media and apparatus.
21. Heat-regulated hot plate, domestic pressure cooker for steam sterilization.
22. Membrane filters and their holders to filter sterilize solutions.
23. Hypodermic syringes, for filter sterilization of solution.
24. Trolley with suitable trays, to transport cultures, media and apparatus.
25. Laminar air-flow cabinet, for aseptic manipulations.
26. Spirit lamp or Bunsen burner.
27. Atomizer, to spray spirit in the inoculation chamber.
28. Screw-cap bottles, to sterilize plant material.
29. Instruments stand, to keep sterilized instruments during aseptic manipulations.
30. Large forceps with blunt ends, for inoculation and subcultures.
31. Forceps with fine-tips, to peel leaves.
32. Fine needles, for dissection.
33. Scalpels, for shredding of the tissues.
34. Spatula, to subculture friable tissues.
35. Cork-borer, for excising tissue cylinders of standardized size.
36. Binocular microscope, for dissecting out microscopic explants.
37. Air-conditioners, to maintain temperature of the culture room.
38. Shaker, to grow suspension cultures.
39. Stainless steel or Teflon sieves of various pore size to separate cell clumps of various dimensions.
40. Low speed bench centrifuge, to sediment cells for determining cell-packed volume and cleaning of protoplasts.
41. Haemocytometer, for cell counting.
42. Cavity slides, for hanging – drop cultures.
43. Ordinary microscope slides and cover-glass, to make microscopic preparations of cells and tissues.
44. Compound microscope, to observe cells and tissues.
Techniques of Plant Tissue Culture:
In plant tissue cultures several techniques are being adopted. The details of the procedure are described below:
i. Preparation of Medium:
Plant cells or organs or tissues are cultured in vitro on a suitable nutrient medium which is known as culture medium (table 15.1).
A culture medium is composed of inorganic salts, vitamins, amino acids, growth substances and a carbohydrate supply. Inorganic salts are supplied in two groups as macro salts and micro salts. The salts needed in higher amounts are called macro-salts and they include nitrogen, phosphorous, sulphur, magnesium, calcium and potassium. The other essential inorganic salts needed in trace amounts are called micronutrients. Nitrogen is mostly provided in two forms as nitrates and as ammonium compounds.
When nitrate is used along, the pH of the medium drifts towards alkalinity, but adding ammonium compounds together with nitrate, checks the drift of pH. In most media, iron is added in the form of sodium ethylene-amine tetra-acetate (Fe-EDTA) facilitating the gradual release of iron into the culture medium. Vitamins used in culture media are meso-inositol, nicotinic acid, pyridoxine, thiamine etc. Carbohydrate is supplied usually as sucrose. The most commonly used amino acid is glycine.
In addition, auxins and cytokinins or their synthetic counterparts are added either singly or in combination to initiate and maintain cell division. The concentration and ratio of hormones may vary from plant to plant. The auxins that are commonly used in tissue culture medium are IAA (indole-3-Acetic acid), 2,4-D (2,4- dichlorophenoxy acetic acid), BTOA (2-benz-thiozyl acetic acid), NAA (x-naphthalene acetic acid) and IBA (3-indole butyric acid). The cytokinins are kinetin (6-furfuryl-amino purine), 6-BAP (6, Benzyl amino purine) Zeatin and 2 IPA (2, isopentenyl adenine). Gibberellic acid is rarely used in the medium.
The inorganic or organic chemicals used in the preparation of media should be analytical grades i.e., AR or GR (Guaranteed reagent).
Some of the plant tissues are grown in the presence of complex additives such as coconut milk, casein hydrolysate, yeast extract, ripe tomato extract, orange juice etc. Yeast extract is a good source of organic nitrogen and vitamins. Casein hydrolysate is obtained from milk by removing the cream and acidifying the skimmed milk which causes casein to precipitate without any decomposition.
It contains all the common amino acids. Potato extract, ripe tomato extract, orange juice and water melon juice contribute a number of essential nutrients and vitamins. Diphenyl urea, a growth factor found in coconut milk, exhibits cytokinin – like responses. So, as a source of cytokinin, 10-15% V/V coconut milk is added to the medium.
It is not possible to weigh and mix all the constituents just before the preparation of medium. So it is convenient to prepare the concentrate stock-solutions of macrosalts, microsalts, vitamins, amino acids, hormones etc., all stock solutions should be stored in a refrigerator for a limited period.
ii. Sterilization of Nutrient Media:
Before placing on a medium, the seeds, parts of plants, organs and tissues etc., must be sterilized i.e., make free from all microorganisms.
Sterilization can be carried out as follows:
1. Physical destruction of microorganisms by dry hot air, steam or irradiation (UV light or gamma irradiation).
2. Chemical destruction of microorganisms by using microbicides (ethylene oxide, alcohol, hypochlorite etc.) or antibiotics.
3. Physical removal of microorganisms through bacterial filtration or washing.
Generally the medium is sterilized with the help of autoclave or large pressure cooker, less often by filtration and seldom by irradiation. The sterilized nutrient medium, glassware etc., should be stored in a sterile cupboard or metal box which has previously been disinfected.
Autoclave:
Usually nutrient media are sterilized with the help of an autoclave. In this, media are exposed to pressurized steam resulting in the elevation of boiling point of water up to 121°C which is sufficient to destroy all microorganisms. An autoclave has a temperature range of 115-135°C. Good sterilization relies on- time, pressure, temperature and volume of the object to be sterilized.
Advantages of an autoclave are- Speed, simplicity, the additional destruction of viruses and no adsorption (this occurs with filter sterilization).
Disadvantages of an autoclave are- Change in pH, components can separate out and chemical reactions can occur resulting in a loss of activity of media constituents.
Some of the guidelines to use an autoclave are:
1. Test tubes and flasks containing 20-50 ml nutrient media must be kept for 20 minutes at 121°C.
2. Flasks containing 50-500 ml nutrient media must be kept for 25 minutes at 121°C.
3. Flasks containing 500-5000 ml nutrient media must be kept for 35 minutes at 121°C.
4. Empty test tubes, flasks and filter paper must be kept for 30 minutes at 130°C.
It must be realized that the heat penetration is very important in an autoclave and large volumes must in principle be sterilized for longer periods. Material that can be dry sterilized (such as test tubes, empty flasks and petridishes, paper, instruments etc.) require 2-3 hours exposure to dry sterilization at 160°C.
The nutrient media and empty object such as glass, paper etc., should be sterilized separately. The same holds true for large and small flasks.
During and after autoclaving the following points should be taken into account:
1. The pH of the media is lowered by 0.3-0.5 units.
2. Autoclaving at too high temperature can burnt sugars, which may then be toxic.
3. Autoclaving for too long can precipitate salts and at the same time depolymerize the agar.
4. Care should be taken to use the correct duration of pressure and temperature (effective temperature).
5. It must be realized that volatile substances can be destroyed by the use of an autoclave (e.g. ether, ethylene).
6. If nutrient media slopes are needed (e.g. for embryo culture and culture of orchid seeds) the test tubes must be placed on a slope to set after autoclaving (at a temperature of 45-50°C).
7. It is recommended that de-ionized water is used in the autoclave as tap water usually contains too much calcium which gets precipitated and autoclave gets corroded.
(i) Sterilization by Irradiation:
Sterilization of nutrient media through irradiation (via gamma rays) is hardly ever-used in the growth of tissue cultures because it is extremely expensive when compared with the usual method of autoclaving. Although gamma ray sterilization is as effective as autoclaving, tissue culture growth is significantly less on media sterilized by this method. When sterilizing plastic containers, boxes, tubes etc., use of an autoclave is not possible gamma ray sterilization is used.
(ii) The Laminar Air-Flow Cabinet (Fig. 15.1):
The preparation and cutting of explants, the cutting of calluses etc., is done on a sterilized glass plate or between sterile filter paper, it is necessary to carry out these tasks in a laminar airflow cabinet (also called an inoculation cabinet). Research laboratories and commercial tissue culture laboratories always use laminar air flow cabinets to limit the possibilities of contamination.
A laminar air-flow cabinet is one in which the air is sucked from outside, first being filtered through very fine filters before reaching the table top of the inoculation cabinet. This filtering system ensures that the air flow over the table is (this flow being laminar giving the cabinet its name) completely sterile.
Since there is a continuous air flow through the inoculation cabinet, it is practically impossible that anything can pass from outside room into cabinet itself. When not in use the air flow cabinet can be closed from the outside air with a plastic cover. The air flow can be regulated and fluorescent tubes, for illumination are fixed in the roof of the cabinet. A gas supply is needed on the table top for use in flaming. However, flaming may be substituted by a spirit lamp or so called dry sterilization process.
In modern laboratories the laminar air-flow cabinet is built in a special (clean) isolation room, which is kept sterile and dust free by the use of filters. The room is vacuumed so that non- sterile air from outside cannot enter.
The filters of the laminar air-flow cabinet should be regularly vacuumed and replaced annually. The floor of the room containing cabinet should be decontaminated every day and only clean indoor shoes and clean laboratory coats should be used. Visitors from outside are particularly a source of infection.
(iii) Sterilization by Filtration:
All the particles, microorganisms and viruses which are bigger than the pore diameter of the filter used in a medium are removed. The greatest advantage of this method of sterilization is that thermolabile substances (broken down during autoclaving) can be sterilized without any harm. Disadvantages can be adsorption of substance into the filter. Sometimes virus particles pass through the filter, this procedure is time consuming and not as simple as autoclaving.
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