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Practical manual for Plant Tissue Culture

Basic Techniques of Plant Tissue Culture and Molecular Biology

Fachbuch 2012 56 Seiten




1 Sterilization techniques

2 Preparation of stock solutions

3 Preparation of media ms medium

4 Surfacesterilization of explants

5 Preparation of explant for callusinduction

6 Characterization of callus

7 Sub culturing of the callus on differentiation media

8 Establishment of suspension culture from grown callus

9 Extraction of secondary metabolites from callus

10 Micro-propagation of plant By shoot tip & nodal culture method

11 Anther culture for haploid production

12 Protoplast isolation

13 Preparation of synthetic seeds

14 H airy root induction through Agrobacterium m ediated gene transfer

15 Rapid extraction of plant DNA

16 Development of RAPD map




Sterilization techniques are designed to kill or remove a wide range of micro-organism including protozoa, fungi, bacteria, viruses. Autoclaving, dry heat sterilization and filtration are adequate for most cell and organ culture materials.



The laboratory apparatus designed to use steam under regulated pressure to achieve sterilization is called autoclave.


Water boils at 100°C depending on the vapor pressure of the atmosphere. If the atmospheric pressure is raised the boiling temperature for water will also rise. So if the steam pressure inside a close vessel is increased to 15 lb/inch2. The temperature can go up to 121.6°C. An autoclave is equipped with device that can maintain saturated steam at a designated temperature and pressure for any period of time.


It is essentially a double jacketed vertical cylinder made up of a strong metal and is separated by a case made up of iron sheet. The lid is very heavy and made up of gun metal and is screwed by buffer files screws. It is rendered air tight with interposition of an asbestos washer. The cylinder contains water up to certain level. The articles to be sterilized are placed on a perforated diaphragm serving as a platform situated above the water level. Heating can be done either by gas burner or by electricity. The chamber is furnished with a steam tap at the top and a pressure gauge with a safety valve. The pressure employed is 15 lb/inch for 15-20 minutes under this pressure H2O boils at 121°C under operation it is necessary that air in the chamber is replaced by saturated steam otherwise required by temperature will not be obtained. It is not the pressure that kills the organism but the temperature of steam. The time of operation to achieve sterility depends upon the nature of the material being sterilized, the type of the containers and the volumes. Sterilization by autoclaving kills all the organisms including spores. Death of cells and spores is due to coagulation of proteins.


Sterilization of most type of solid or liquid media, distilled water, normal saline, discarded cultures, contaminated media, aprons, rubber tubes, gloves etc.


Dry heat or hot air sterilization is recommended where it is either undesirable or unlikely that steam under pressure will make direct and complete contact with the materials to be sterilized. The apparatus employed for this type of sterilization may be special electric or gas oven or even the kitchen stove oven.


Sterilization is carried out by dry heat at high temperature. Bacterial cells and spore die due to dehydration.


It consists of a chamber having triple wall. The inner two walls are made up of copper sheet and outer of asbestos. The chamber is filled with several adjustable shelves and a thermometer is interested to record temperature. There are adjustable holes which are kept open during rising the temperature and are again open partially when sterilization completes clean glassware, like petri-dishes, test tube, flasks, pipettes etc. are place inside and the door is closed. Electric heated is put ON and the temperature of chamber is opened only after the temperature comes down to normal.


It is useful device for sterilization of laboratory glassware. It is also used to sterilize substances which can not be sterilized by moist.


Growth substances that are thermolabile such as Zeatine, Gibberilic acid (GA3), Absisic acid (ABA), urea and certain vitamins are sterilized by membrane filtration with pore size 0.22 –0.45 µ. Four types of filters are commonly used i.e. asbestos sintered glass, unglozed porcelain and membrane filters. Membrane filters are made up of cellulose acetate, cellulose nitrate or a mix of both nylon and polysulphone. A common form is a paper like disc about 50mm in diameter and 0.1mm in thickness. Filter setup is wrapped in aluminium foil and autoclaved prior to use. Membrane filters are kept in petridish for autoclaving swinnex model filter is available.


Filtration is done under reduced pressure connected to vacuum desiccators with a safety of flask attachment in between. Here first liquid to be filtered is filled up. These generate a positive pressure inside the lower container. Now the cotton plug at lower end connected to a vacuum pump. This generates a negative pressure inside the container. If some liquid is still left, take an empty syringe filled with air and blow on the lid till it is filtered. The vacuum pump is disconnected only when the positive and negative pressure balance each other.


They are soaked in chromic acid solution for 24 hours. They washed in high pressure set of tap water with brushing followed by several rinses, First with tap water and then with distilled water and finally with double distilled water. Washed glassware then Dried in oven (3 hours at 100°C). Nowadays chromic- sulfuric acid method is replaced by use of detergents in subsequently rinsed with tap water followed by distilled water.

Use of Laminar Air Flow Cabinets:

They are available in various sizes. A size 1.2m X 1.2m X 7.1m fitted with glass on top upper half work bench with sunmica top end to end a small sink and a door. They are fitted with germicidal U.V. for inside sterilization and cool white fluorescent tubes for uniform illumination. They given an enclosed area in which air circulated there, dust and microbe screening filters over the working surface at uniform rate. These are built to keep the transfer area under positive pressure starting the experiment entire in travel. Surface of the transfer chamber is hand sprayed with 70% ethanol for demission of air borne contaminants the door is slightly closed and UV switched on for about 45 minutes for inside sterilization. At the starting of operation the illuminating tubes are switched ON. The blower is switched and the door is open. This cause positive pressure inside so that air from can’t rush in. Before entry into the transfer room, it would be advisable for the researcher to wear a clean lab coat a sterile cap, use a face mask and rub his hands with ethanol. Change into new canvas shoes.




Preparation of stock solution is the most basic and important steps in the plant tissue culture technology because to prepare any basal medium the ingredients, macronutrients, micronutrients, Iron EDTA and vitamins are common. So, in order to prepare any basal medium it is complicated process of weighing the ingredients of micronutrients, macronutrients, vitamins, iron EDTA each and every time and it is time consuming process. To avoid all the above difficulties it is better to prepare stock solutions of standard concentration and use whenever needed.


Chemical: Mineral salts

Glassware:Amber colour bottles, funnel, beaker, glass rod, filter paper, pipette etc.

Instrument: Autoclave


1. Preparation of Macronutrients stock:

Normally stock solutions of macronutrients are prepared at 20X concentration. Weigh the ingredients as given in the table as dissolve one by one make up to volume 1000ml that is 20X concentrations. For the preparation of 1litre of basal medium 50ml of above solution is required.

2. Preparation of Micronutrients stock:

Normally stock solutions of macronutrients are prepared at 100X concentration. Weigh the elements as given in table and dissolve in 1000ml of distilled water that gives 100X concentrations. To prepare 1 liter basal medium 10ml of micronutrients solution is required.

Iron-EDTA: An iron stock is prepared separately because of the problem of iron solubility. This element requires acidic condition for solubility. Usually the iron stock is prepared in cheated form as the Sodium salt of Ferric-Ethylene Diamine Tetra Acetic Acid (Fe-EDTA).

3. Preparation of Vitamins stock:

Weigh each vitamin as given in the table and dissolve in 1000ml of distilled water that gives 100X concentrations, 1ml is required for preparation of 1litre basal medium.

4. Preparation of Hormone Stock Solution:

Various hormones of auxins (dissolved in 70% alcohol, 1N HCl) and cytokinins (1N NaOH) are prepared at a concentration of 1mg/ml in suitable solvent.


Abbildung in dieser Leseprobe nicht enthalten


1. All stock solution should be clear and transparent, free of dust and without precipitation.
2. Stock solutions should be stored in amber colour bottles.
3. Stock solutions are kept at 4˚C in dark room. These solutions are stored for limited period of times.
4. After using stock solution immediately keep close and don’t expose for longer time.




The success of getting callus and morphogenesis of plant tissues and applications of in vitro methods is largely governed by the better understanding of nutritional requirements of the cultured cells and tissues and tissue composition of the cultured media. Although the basic requirements of the cultured plants are similar to that of whole plants, the nutritional requirement consists of inorganic salts, carbon and energy source, vitamins and phyto-hormones, other components including organic nitrogen compound, organic acids, and complex substances. In practice nutritional components promoting optimal growth of tissue under laboratory conditions may vary with respect to the particular species. Media composition was therefore formulated considering specific requirements of particular system.



Stock solutions, distilled water, sucrose, myo-inositol, agar-agar, PVP


Culture vessels/tubes, funnel, beaker, glass rod, filter paper, pipette, measuring cylinder.


Autoclave, PH meter


Abbildung in dieser Leseprobe nicht enthalten


1. Pipette out the required volume of the solution as given in the table.
2. Weigh the other ingredients as given in the table and add one by one effectively and dissolve.
3. Add filter sterilized growth hormone in desired concentration.
4. Adjust the pH 5.80-5.85 with the help of 0.1N HCl or 0.1 N NaOH.
5. Lastly agar is added to the solution and boil up to the clear solution formation.
6. The medium is allowed to cool for some time until it reaches around 45 0C to 50 0C and then dispense it into a heat sterilize culture tube around 15-20 ml/tube and cover it with cotton plug and wrap with paper.
7. The above culture tubes with media are autoclaved at 15 lbs pressure, 121 0C for 20 minutes.
8. Keep these sterilized tubes in slant position and allowed them to solidify. Now these tubes are ready for further use.
9. The media should be clean and milky white.


1. All stock solution should be clear and transparent, free of dust and without precipitation.
2. Stock solutions should be stored in amber colour bottles.
3. Stock solutions are kept at 4˚C in dark room. These solutions are stored for limited period of times.
4. After using stock solution immediately keep close and don’t expose for longer time.




The first important condition for the successful tissue culture procedures is the maintenance of aseptic condition. Sterilization eliminates microorganism and thus avoids contamination by bacteria and fungi. To maintain an aseptic environment, all culture vessels, media and instruments used in handling tissue, as well as the explant itself is should be surface sterilized. Plant material can be surface sterilized by variety of chemicals. Some commonly used chemicals sterilants are as follows:

1 % sodium hypochlorite ( NaClO) :

It is generally available with 5 % active chlorine content, so 20 % can be used for normal sterilization.

Calcium hypochlorite Ca(ClO)2 :

This comes in the powder form. Generally 100 ml of Ca(ClO)2 is used. The desired weight of hypochlorite is added in to the water, agitated for 10 min, allowed to settle and the clarified filtered supernatant solution is used for sterilization. The filtrate is used immediately because of deliquescent (take up water) nature. Calcium hypochlorite enters the plant tissue slowly as compared to sodium hypochlorite. The standard concentration used is of the order of 4 to 10 % and the soaking time varies from 5 to 30 min.

B romine Water:

1to 2% bromine water solution is used for the sterilization purpose.

Mercuric chloride:

It is dissolved in water to create the solution. Concentration of 0.01 to 0.1 % for 2 to 10 min, depending upon the tissue, is used. Mercuric chloride is an extremely toxic substance for plant, so rinsing must be very thorough at least five times.


70 % alcohol is used for sterilization of plant material by dipping them for a period of 30 sec to 2 min. Generally alcohol alone is not sufficient to kill all the microorganisms and the plant material after alcohol treatment is treated another chemical sterilant.


Cefotaxime antibiotic at 50 mg/L concentration in the nutrient medium is generally used to control bacterial infection.

Explants after treatment with sterilants must be thoroughly rinsed with sterile distilled because retention of such toxic chemicals will seriously affect the establishment of culture.


Reagents & Chemicals:

Tween 20 (liquid detergent) , 0.1% HgCl2 , 70% alcohol , sterile distilled water


Beakers, sterile petri plates, sterile blades, sterile forceps, muslin cloth


Laminar airflow hood, Autoclave


1. Wash leaf with tap water to remove soil and dust particles deposited on surface.
2. Transfer the washed leaf into a glass beaker containing tap water; add few drops of liquid detergent – Tween 20.
3. Cover beaker mouth with muslin cloth with the rubber band and keep under running tap water for 1 hour to remove any waxy/ oily deposition on leaf surface.
4. Wash it trice with distilled water.
5. Transfer the leaf explant into laminar airflow hood for farther work to avoid contamination.
6. Wash the above leaf with sterile distilled water for thrice each washing should be for 3-4 minutes.
7. Treat it with 0.1% HgCl2 solution for 60 sec.
8. After treating it with disinfectant, wash it with sterile distil water for thrice, each washing should be for 3-4 minutes.
9. Wash with 70% alcohol for 30 seconds to remove water from the surface of the leaf.
10.Transfer t he sterile leaf to a sterile petriplate.
11.Cut the leaf into small pieces of about 1x1 cm with sterile blade.
12.Now the explant is ready for inoculation.




Explant is any portion taken from a plant that will be used to initiate in vitro culture. It can be a portion of the shoot or of the leaves or even just some cells. Any part of the plant that is able to regenerate and give rise to a callus can be used as an explant. The selection, preparation and inoculation of explant can be done using specific steps.

Selection of explant

The selection of explant depends on various factors like age of the plant material, region of plant that can be used, etc. In many species explants of various organs vary in their rates of growth and regeneration, while some do not grow at all. The choice of explant material also determines if the plantlets developed via tissue culture are haploid or diploid. Also the risk of microbial contamination is increased with inappropriate explants. Thus it is very important that an appropriate choice of explant be made prior to tissue culture. The specific differences in the regeneration potential of different organs and explants have various explanations. The significant factors include differences in the stage of the cells in the cell cycle, the availability of or ability to transport endogenous growth regulators, and the metabolic capabilities of the cells. The most commonly used tissue explants are the meristematic ends of the plants like the stem tip, auxiliary bud tip and root tip. These tissues have high rates of cell division and either concentrate or produce required growth regulating substances including auxins and cytokinins.



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practical plant tissue culture basic techniques molecular biology



Titel: Practical manual for Plant Tissue Culture