Tissue culture refers to the growth and maintenance of a plant in nutrient
medium in vitro. The term “plant tissue culture” is used for culturing of
unorganized tissues or callus.
It is now used as a blanket term for protoplast, cell, tissue, organ, or whole
plant culture under aseptic conditions. The methodology of tissue culture
consists of separation of the cells, tissues, and organs of a plant called
“explants”, and growing them aseptically on a nutrient medium under controlled
conditions of temperature and light. The explants give rise to an unorganized,
proliferative mass of differentiated cells called callus, which later produces
Plant tissue culture techniques are now being used as powerful tools for
the study of various kinds of basic problems not only in plant physiology, cell
biology, and genetics but also in agriculture, forestry, horticulture, and industry.
An important contribution made by this technique is the revelation of a unique
capacity of plant cells that is the cellular totipotency, which means that all
leaving cells in a plant body can potentially give rise to a whole plant.
Plant tissue culture technology can be divided into 5 classes based on the
type of materials used.
- Callus culture: Culture of callus on agar medium produced from explants.
- Cell culture: Culture of cell in liquid media, usually aerated by agitation.
- Organ culture: Aseptic culture of embryos, anthers, roots, shoot and ovaries,
- Meristem culture: Aseptic culture of shoots meristem or explant tissue in
- Protoplast culture: The aseptic isolation and culture of plant protoplast from
cultured cell or plant tissue.
Preparation of the Media
- Hot air oven
- Magnetic stirrer
- pH meter
- Distillation unit
- Spirit lamp
- Forceps with blunt end (to inoculate and subculture) forceps with fine tips
(to dissect leaves)
- Culture bottles
- Conical flasks
- Measuring cylinders
- Screw cap bottles.
The best-suited media is Mushige and Skoogs Media, or MS Media. It has the
Preparation of Stock Solution
- Stock A—major elements
- Stock B—minor elements
- Stock C—iron elements
- Stock D—vitamin stores
- Sucrose (as carbon source) and agar (as solidifying agent).
The stocks A, B, C, and D are prepared by weighing the respective chemicals
written in the following chart and dividing them in the minimum amount of
distilled water. The final volume in each stock is made up to 1000 mL by
adding distilled water. While preparing Stock C, slight heating is done to
dissolve sodium ethylene diamino tetra acetate and ferrous sulfate in water.
These stock solutions are stored in a refrigerator.
Preparation of Growth Regulators
|Composition of Stock Solutions
|1. Ammonium nitrate (NH4NO3)
|2. Potassium nitrate (KNO3)
|3. Magnesium sulfate (MgSO4, 7H2O)
|4. Potassium dihydrogen phosphate (KH2PO4)
|5. Calcium chloride (CaCl2, 7H2O)
|1. Boric acid (H3BO3)
|2. Manganese sulfate (MnSO4, 4H2O)
|3. Zinc sulfate (ZnSO4, 7H2O)
|4. Potassium iodide (KI)
|5. Sodium molybdate (Na2MoO4, 5H2O)
|6. Cobalt chloride (CoCl2, 6H2O)
|7. Cupric sulfate (CuSO4, 5H2O)
|1. Disodium ethylene diamino tetra acetate (Na2EDTA)
|2. Ferric sulfate (FeSO4, 2H2O)
|2. Nicotinic acid
|3. Thiamine hydrochloride
| 4. Pyridoxine hydrochloride
| 5. Sucrose
| 6. Dyco-bacto agar
- Auxins: The different types of auxins used are as follows:
- Indole acetic acid-IAA
- Indole-3-Butyric acid-IBA
- Naphthalene acetic acid-NAA
- 2,4-Dichloro phenoxy acetic acid-2,4-D.
The known quantities of these hormones are first dissolved in 5 mL of
NaOH or KOH and the final volume is made up by adding distilled water.
The required quantities of hormones are well dissolved in 5 mL of 0.1 N
HCl and the final volume is made by water.
- 6-Benzyl amino purine-BAP and
- 6-perfuryl amino purine (kinetin).
The liquid endosperm from the coconut is collected. It is filtered through clean
cloth and a known quantity of coconut milk is added to the medium before
The medium is prepared depending upon the quantity required to prepare
1 liter of the medium. The quantity of the stock required is 100 mL for Stock
A, 1 mL for Stock B, 10 mL for Stock C, and 1 mL for Stock D added to the
boiling water. Known amounts of sucrose (2%) and agar (0.8%) are added.
Then, the stock solution that is the boiling solution is made up to 1000 mL by
adding distilled water. PH is adjusted to 5.6–5.8.
- 25 mL of the media are dispensed into culture bottles, and then the mouths
of the bottles are closed with lid.
- The culture bottles are then transferred to the autoclave.
- The instrument necessary for the inoculation, and distilled water, are also
kept inside the autoclave.
- The media is autoclaved at 15 inch/inch2 pressure and temperature of
121°C for 15 minutes.
- It is allowed to cool and the autoclaved media is removed. The culture
vessels with media are allowed to solidify before they are transferred to the
inoculation chamber for inoculation.
A collection of explant materials, pieces of seedlings, swelling of buds, stems,
leaves, immature embryos, and anthers is assembled from a suitable plant
Sterilization of Plant Material
The surface of plant parts gets a wide range of microbial contaminants like
fungi and bacteria. To avoid the microbial growth in a culture, the explants
must be surface-sterilized in disinfectant solutions before planting to the medium.
The procedure is as follows:
- The explants are washed with tap water.
- They are washed thoroughly by keeping them under running tap water for
an hour. Thorough washing of the material is necessary because it removes
- After washing, the material is treated with freshly prepared saturated
chlorine water for 2 minutes.
- It is then rinsed with sterile distilled water.
- The material is surface-sterilized with freshly prepared 0.1% mercuric
chloride solution for 1 minute, followed by 4–5 times with sterilized distilled
water to remove all traces of contamination.
The surface-sterilized explants are transferred to an inoculation chamber where
aseptic conditions are maintained. The chamber is cleaned with ethanol and
subjected to UV radiation for 1 hour. The outer surfaces of the culture bottles
are also cleaned with ethanol before placing them in the chamber. The materials
are placed on the sterilized petri plates.
Flame sterilization of the forceps is done by dipping them in ethanol and
holding them into the spirit lamp. With the help of the flamed forceps, the
explants are inoculated onto the media. Flaming of outlets of the bottles is done
and the caps are replaced tightly.
Incubation of Cultures
The culture bottles are transferred to the incubator, where controlled conditions
of temperature and light are maintained. The temperature inside the culture
room is maintained at around 25°C. The culture is exposed to 15 hours of light
and 5 hours of darkness. Fluorescent tubes are used as light source. The cultures
are allowed to grow and periodic observations are made. Subculturing is done
once in 6 weeks.