Plant Tissue Culture

Introduction
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 shoots.

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.

1. Callus culture: Culture of callus on agar medium produced from explants.
2. Cell culture: Culture of cell in liquid media, usually aerated by agitation.
3. Organ culture: Aseptic culture of embryos, anthers, roots, shoot and ovaries, etc.
4. Meristem culture: Aseptic culture of shoots meristem or explant tissue in nutrient media.
5. Protoplast culture: The aseptic isolation and culture of plant protoplast from cultured cell or plant tissue.

Materials

• Balance
• Autoclave
• Hot air oven
• Magnetic stirrer
• Refrigerator
• Heater
• pH meter
• Distillation unit
• Microscope
• Spirit lamp
• Forceps with blunt end (to inoculate and subculture) forceps with fine tips
  (to dissect leaves)
• Needles
• Culture bottles
• Racks
• Conical flasks
• Beakers
• Measuring cylinders
• Scalpels
• Screw cap bottles.

Preparation of the Media
The best-suited media is Mushige and Skoogs Media, or MS Media. It has the following ingredients:

1. Stock A—major elements
2. Stock B—minor elements
3. Stock C—iron elements
4. Stock D—vitamin stores
5. Sucrose (as carbon source) and agar (as solidifying agent).

Preparation of Stock Solution
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.

Composition of Stock Solutions
Stock A
Composition	mg/liter
1. Ammonium nitrate (NH4NO3)	1650.00
2. Potassium nitrate (KNO3)	1900.00
3. Magnesium sulfate (MgSO4, 7H2O)	370
4. Potassium dihydrogen phosphate (KH2PO4)	170
5. Calcium chloride (CaCl2, 7H2O)	440
Stock B
Composition	mg/liter
1. Boric acid (H3BO3)	6.200
2. Manganese sulfate (MnSO4, 4H2O)	2.300
3. Zinc sulfate (ZnSO4, 7H2O)	8.600
4. Potassium iodide (KI)	0.830
5. Sodium molybdate (Na2MoO4, 5H2O)	0.250
6. Cobalt chloride (CoCl2, 6H2O)	0.250
7. Cupric sulfate (CuSO4, 5H2O)	0.025
Stock C
Composition	mg/liter
1. Disodium ethylene diamino tetra acetate (Na2EDTA)	37.3
2. Ferric sulfate (FeSO4, 2H2O)	27.8
Stock D
Composition	mg/liter
1. Glycine	2.00
2. Nicotinic acid	0.50
3. Thiamine hydrochloride	0.50
4. Pyridoxine hydrochloride	0.50
5. Sucrose	20 gm/liter
6. Dyco-bacto agar	8 gm/liter

Preparation of Growth Regulators

• Auxins: The different types of auxins used are as follows:
  1. Indole acetic acid-IAA
  2. Indole-3-Butyric acid-IBA
  3. Naphthalene acetic acid-NAA
  4. 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.
• Cytokinins:
  
  1. 6-Benzyl amino purine-BAP and
  2. 6-perfuryl amino purine (kinetin).
  The required quantities of hormones are well dissolved in 5 mL of 0.1 N HCl and the final volume is made by water.

Coconut Milk
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 autoclaving.

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.

1. 25 mL of the media are dispensed into culture bottles, and then the mouths of the bottles are closed with lid.
2. The culture bottles are then transferred to the autoclave.
3. The instrument necessary for the inoculation, and distilled water, are also kept inside the autoclave.
4. The media is autoclaved at 15 inch/inch² pressure and temperature of 121°C for 15 minutes.
5. 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.

Procedure
A collection of explant materials, pieces of seedlings, swelling of buds, stems, leaves, immature embryos, and anthers is assembled from a suitable plant material.


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:

1. The explants are washed with tap water.
2. They are washed thoroughly by keeping them under running tap water for an hour. Thorough washing of the material is necessary because it removes superficial contaminants.
3. After washing, the material is treated with freshly prepared saturated chlorine water for 2 minutes.
4. It is then rinsed with sterile distilled water.
5. 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.

Inoculation
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.