In this article, we will learn about the- What is Plant Tissue Culture? Principles of Plant Tissue Culture, Techniques of Plant Tissue Culture etc.
What is Plant Tissue Culture?
Plant Tissue culture is technology which helps to keep pace with increasing food demand and to provide sufficiently fast and efficient systems for crop improvement.
Plant tissue culture is a collection of techniques used to maintain or grow and multiply plant cells, tissue or organs in-vitro that is under sterile, aseptic controlled conditions on a artificial nutrient culture medium of known composition. The basic principle of tissue culture is the Totipotency. The ability of a living plant cell to form an entire plant is called Totipotency.
What is Cellular Totipotency?
The ability of a single plant cell to divide or differentiate into a mature plant if placed in the appropriate environment is called cellular Totipotency.
Plant tissue culture is invaluable when traditional plant breeding cannot generate plants with desired traits. The culturing or growing isolated protoplasts or cells or tissue or organ on nutrient medium under controlled aseptic conditions to produce complete plant or plant parts is called tissue culture technique.
Explant-
A tissue isolated from intact plant inoculated on medium to initiate the culture is called explant.
Callus:
A mass of undifferentiated and unorganized cells obtained from the explant is called Callus.
Inoculation:
The method of insertion of the explant onto the sterile culture medium aseptically is called inoculation.
Clones:
The genetically identical organisms produced from the original parent organism are described as clones of each other.Requirements of tissue culture technique:
1. Glassware:
Test tube bumper without rim (50ml), petriplates, measuring cylinders, coupling jars, conical flaks, beakers, pipettes, glass rods etc.
2. Instruments/ Equipments:
Hot air oven, autoclave, refrigerator, incubator, digital balance, distillation unit, hot water bath, pH meter, laminar clean air flow carbinet, spirit lamp, large blunt forceps, surgical blade with blade holder, test tube holder, scalpel, needle, bent scissor, plastic trays etc.
3. Chemicals:
Distilled water, liquid detergent, 0.1% aqueous HgCl2, rectified spirit, absolute alcohol, sources of micronutrients and macronutrients, vitamins, growth regulators, sources of carbohydrates and proteins, agar powder, and other relevant chemicals.
4 . Miscellaneous:
Match box, brown paper, aluminum foil, micropipette, muslin cloth, absorbent, and non-absorbent cotton, rubber band, cotton plugs, pH paper etc.
Technique:
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| Techniques for Plant Tissue Culture |
A. Cleaning and sterilization of glassware:
1) Soak the glassware and equipments in detergent solution for about 4 to 5 hours.
2) Wash it thoroughly under running water followed by rinsing with distilled water.
3. Keep it in the hot air oven for about 1-2 hours at 100°C -120°C temperature and store in dust-free containers.
4. Prepare cotton plugs with the help of muslin cloth and absorbent cotton.
5. Plug the test tubes, conical flasks by using cotton plugs and then cover by aluminum foil and finally wrap with the brown paper in bundles.
6. Cover the equipments like needle, forceps, scalpels etc. with brown paper.
7. Autoclave the wrapped glassware and equipments at 15 lb/inch² for 20 min at 121°C.
B) Preparation and Sterilization of the Nutrient Medium:
Nutrient medium is a mixture of all required nutrients required for plant growth in proper concentrations and proportions. Nutrients are the substances that are absorbed, assimilated and utilized in building up of the plant body. The standard nutrient medium used for tissue culture is MS medium i.e. Murashige and Skoog's medium. It contains micronutrients, macronutrients, iron source, vitamins, growth regulators and carbon source in the form of sucrose or D-glucose and distilled water. Agar is an inert substance obtained from red algae. It is used as a solidifying or gelling agent having low melting point.
1. First prepare stock solutions for all the micronutrients, macronutrients, vitamins, and growth regulators needed for preparation of MS medium.
These are stored in refrigerator for at the most 3 months, while only one month for growth regulators.
2. These are then mixed in proper proportions and sequence to prepare one litre MS medium.
a) Preparation of stock solution:
Following stock solutions are to be prepared and stored at low temperature in refrigerator.
1. Macronutrients stock solution:
Ingredient stock (mg)
NH4NO3 16.5 g
KNO3 19 g
CaCl2. 2H2O 4.4 g
MgSO4. 6H2O 3.7 g
KH2PO4 1.7
Dissolve the above chemicals one by one in distilled water and make final volume 1000 ml with distilled water.
Label the solution as stock solution A with date of preparation.
2. Iron stock:
Iron stock stock (mg)
Na2 EDTA 373
FeSO4. 7H2O 278
Dissolve the quantity of Na2 EDTA in little amount of distilled water then add a quantity FeSO4.7H2O. Yellow colour solution is formed. Make final volume 100 ml with distilled water. This stock is also called "Iron Chelate".
Label the solution as Stock solution B with date of preparation and store it in a dark bottle at 0° to 4°C in refrigerator.
3. Micro-nutrients stock:
Micro-nutrients stock stock(mg)
MnSO4. 4H2O 223
ZnSO4. 4H2O 86
H3BO3 62
KI 8.3
Na2MO4. 2H2O 0.25
CuSO4. 5H2O 0.25
COCl2. 6H2O 0.25
Dissolve the following chemicals one by one in distilled water and make final volume 1000 ml with distilled water.
Label this solution as Stock solution C with date of preparation.
4. Vitamin Stock:
Vitamin stock stock (mg)
Nicotenic acid 5
Pyredoxin- HCl 5
Thiamine - HCl 1
Glycine 20
Myoinositol 1000
The above chemicals are dissolved one by one in distilled water and make final volume 100ml with distilled water.
Label this solution as stock solution D with date of preparation.
5. Growth regulators:
Cytokinins- kinetin and/ or auxins are added to the medium to the necessary. Dissolve 100mg of kinetin in 100ml of distilled water to prepare stock solution. Similarly prepare stock solution of IAA by dissolving 100 mg in 100ml distilled water. The stock solution are stored in refrigerator for one month only.
b) Preparation of MS medium from stock solution:
1. Take 600 ml distilled water. Add the following stock solutions one after the other with stirring:
- 100 ml of macronutrient stock solutionA.
- 10 ml of Fe-EDTA stock solution B.
- 10 ml of micronutrient stock solution C.
- 10 ml of vitamin stock solution D.
2. Dissolve 30 gm of D-glucose in 200 ml distilled water and mix it with the above solution.
3. Make the final volume one litre by adding distilled water.
4. Add 6-10 gm of agar as solidifying agent.
5. Adjust pH at 5.8 by adding 0.1N NaOH/ 0.1N HCl.
6. Sometimes little quantity of antibiotics can also be mixed with the medium to avoid fungal or bacterial contamination.
c) Sterilization of the culture medium:
The nutrient medium before it's use has to be sterilized.
1. Pour the nutrient medium in pre-sterilized conical flaks properly by using glass funnels. Seal it by using non-absorbent cotton plugs and wrap it with aluminum foils.
2. Autoclave the culture flasks for continuous 20 min under constant pressure of 15 lb/inch². This is called steam sterilization. At this pressure, temperature of steam goes up to 121°C at which all the microbial contaminants present get killed.
3. After cooling the autoclave remove the culture flasks.
4. Add 1 ml of stock solution of growth regulators, if necessary.
5. Dispense the sterilized medium in pre-sterilized test tubes aseptically in the laminar air flow cabinet. Such culture tubes are also cotton pluged.
C) Preparation of an explant:
An explant is a plant material to be used for culturing. The explant can be an embryo/ nodal sector/ bud meristem. The explant has to be sterilized before it's use.
a) selection of plant material:
1) Select the plant parts from healthy, disease-free plants preferably grown under garden conditions.
2) Detach plant parts using a sharp blade.
3) Collect in clean and moist polythene bags or glass containers.
b) Surface sterilization:
1) Wash the explant under running tap water.
2) Rinse it in 70% ethanol for 30-60 seconds.
3) Wash it again with distilled water.
4) Treat it with commercial chlorine water (chlorogen, Kilton and Saaf) containing at least 1% aqueous solution of HgCl2 (mercuric chloride), for 5-10 minutes. Ensure that the explants sink in the solution by agitating the container. The above steps should be followed in dust free room.
5) Handle such material under aseptic conditions for subsequent manipulations.
D) Inoculation of the explant:
Inoculation is the transfer of the explant to the nutrient medium under aseptic conditions. Aseptic manipulations including transfer are carried out in specially designed inoculation hood or laminar clean air flow cabinet. Inoculation hood has a glass or stainless steel floor. It is internally equipped with ultraviolet (UV) light and day-light fluorescent tubes.
1. Wipe the floor of the hood with rectified spirit.
2. Place culture vessels (medium), inoculation equipment, sterile distilled water, sterile Petri dishes, spirit lamp and sterile beakers etc. inside the hood.
3. Close the windows and switch on the UV light for at least 20 minutes. This will kill all the microbes in the cabinet. Five minutes after switching off the UV tube, switch on the fluorescent light tube.
4. Then open the window and switch on the HEPA flow. Clean both the hands and wipe quickly with rectified spirit and allow it to dry. Light the spirit lamp.
5. Wash the plant material with sterile distilled water at least three times under the hood.
6. Dissect out the required explant from plant material in a petri dish. Flame the dissecting instruments before use.
7. Remove the plug of culture vessel over the flame. Quickly transfer the explant onto the medium and replace plug.
8. After the inoculation is over, put off the spirit lamp. Remove the inoculated culture tubes from the cabinet. Wipe the floor of hood with rectified spirit.
9. Label the culture tubes mentioning name of the explant, date of inoculation, medium used and investigator name.
E) Inocubation :
1. Transfer the culture vessels (with explant) to inoculation chamber/ culture room.
2. Arrange the culture vessels on culture or stands or rocks, orbital shakes or rotary shakers as required.
3. Adjust the intensity of light as required and temperature around 25°C.
4. Observe the culture periodically and remove the contaminated cultures if any.
5. Record the changes in the explant and proceed as required for specific experiment.
The inocubation chambers are usually maintained at 25+10°C. Intensity and duration of light is adjusted as per requirement. Day light fluorescent tubes are employed as source of light.
