Summer Training ReportOn

“PLANT TISSUE CULTURE”(15th May – 30th June 2010)

Done at

Professional Institute of Technology and Science

Lucknow

By

Puja

B.Tech(Biotech)

5th semester

Amity Institute of Biotechnology

Amity University, Lucknow Campus

ACKNOWLEDGEMENT

I bow in gratitude to Almighty, who enabled me to complete this task and blessed me with

love and light.

I wish to offer my thanks to Dr. A.K Agarwal, (Director) of Professional Institute Of

Technology & Sciences for not only being the hon’ble guide and philosopher but also a

source of immense inspiration to me in completing this task.

I am deeply indebted to Mr. Kuldeep Srivastava (Project Head) for his ever flowing,

invaluable and knowledgeable guidance and assistance throughout the training. I am

extremely grateful to him for his support, help and encouragement during the course of

training and my entire stay here.

I place on record my sincere regards for Vatsala Mam, Neerja Mam and Puspa Mam

for giving me constant intimate support and all possible help throughout the training

program.

I am very thankful to all PITS faculty for providing constant support and technical help

during the training program.

.

I place on record my sincere thanks to my parents for their constant support and

encouragement and also to all my friends for their cooperation and continuous support.

Puja

Introduction

Plant Tissue Culture refers to the technique of growing plant cells, tissues, organs, seeds

or other plant parts in a sterile environment on a nutrient medium. It is propagation of a

plant by using a plant part or single cell or group cell in a test tube under very controlled

and hygienic conditions.

The first commercial use of plant propagation on artificial media was in the

germination and growth of orchid plants in the 1920’s. It was only after the development

of a reliable artificial medium by Murashige & Skoog in 1962 that plant tissue culture

really took off commercially.

Plant research often involves growing new plants in a controlled environment. These may

be plants that we have genetically altered in some way or may be plants of which we need

many copies all exactly alike. These things can be accomplished through tissue culture of

small tissue pieces from the plant of interest. These small pieces may come from a single

mother plant or they may be the result of genetic transformation of single plant cells

which are then encouraged to grow and to ultimately develop into a whole plant. Tissue

culture techniques are often used for commercial production of plants as well as for plant

research.

Tissue culture involves the use of small pieces of plant tissue (explants) which are

cultured in a nutrient medium under sterile conditions. Using the appropriate growing

conditions for each explant type, plants can be induced to rapidly produce new shoots,

and, with the addition of suitable hormones new roots. These plantlets can also be

divided, usually at the shoot stage, to produce large numbers of new plantlets. The new

plants can then be placed in soil and grown in the normal manner. The most important

part of this activity, however, is to maintain as sterile an environment as possible. Even

one fungal spore or bacterial cell that comes into contact with the growth media will

rapidly reproduce and soon completely overwhelm the small plant piece that you are

trying to clone.

.

Advantages of Plant Tissue Culture

 It can create a large number of clones from a single seed or explant.

1. It is easy to select desirable traits directly from the culture setup (in vitro), thereby

decreasing the amount of space required for field trials.

2. The time required is much shortened, no need to wait for the whole life cycle of seed

development.

3. For species that have long generation time, low levels of seed production, or seeds

that do not readily germinate, rapid propagation is possible.

4. It overcomes seasonal restrictions for seed germination.

5. It enables the preservation of pollen and cell collections form which plants may be

propagated (like a seed bank).

6. It allows for the international exchange of sterilized plant materials (eliminating the

need for quarantine.)

7. It helps to eliminate plant diseases through careful stock selection and sterile

techniques.

8. It enables cold storage of large numbers of viable plants in a small space.

9. Very helpful in the genetically modified organism studies

Disadvantages of Plant Tissue Culture

1. If large scale production is being thinking, the costs of the equipments are very

expensive.

2. The procedure is very variable and it depends on the type of the species so sometimes it

needs trial-and-error type of experiments if there is not any review about that species.

3. The procedure needs special attention and diligently done observation.

4. There may be error in the identity of the organisms after culture.

5. Infection may continue thorough generations easily if possible precautions are not

taken

6. Decrease genetic variability.

Tissue Culture Media-MURASHIGE & SKOOG MEDIA (M.S. Media)

The appropriate composition of the medium largely determined the success of the

cultures. Plant materials do vary in their nutritional requirements and therefore it is often

necessary to modify the medium to suit a particular tissue.

Initially tissues and organs from a wide variety of plant species were cultured on

the nutrient salt solutions formulated by Gautheret and White. However,these were found

inadequate for sustaining growth of many plant tissues.

This led to the formulation of several basal nutrient media (Hildebrandt et al.,1946;

Burkholderand Nickell, 1949; Heller, 1953; Murashige and Skoog,1962; Gamborg et al.,

1968.).

Culture media used for the in vitro cultivation of plant cells are composed of following

basic components:

(i) inorganic salts;

(ii) vitamins;

(iii) growth regulators;

(iv) carbon source; and

(v) organic supplements.

Inorganic salts: These are divided into two groups- major and minor salts.

Major salts: The salts of potassium, nitrogen, calcium, magnesium, phosphorus and

sulphur constitute the major salts. These elements usually comprise at least 0.1% of the

dry weight of plants.

Nitrogen is most commonly supplied as a mixture of nitrate ions (from the KNO3) and

ammonium ions (from the NH4NO3). Theoretically, there is an advantage in supplying

nitrogen in the form of ammonium ions, as nitrogen must be in the reduced form to be

incorporated into macromolecules.

Nitrate ions therefore need to be reduced before incorporation. However, at high

concentrations, ammonium ions can be toxic to plant cell cultures and uptake of

ammonium ions from the medium causes acidification of the medium. In order to use

ammonium ions as the sole nitrogen source, the medium needs to be buffered. High

concentrations of ammonium ions can also cause culture

Minor salts: The salts of iron, zinc, manganese, boron, copper, cobalt, molybdenum,

iodine, etc. make up the minor salts. These salts are essential for the growth of tissues and

are required in trace quantities.

Iron is usually added as iron sulphate, although iron citrate can also be used.

Ethylenediaminetetraacetic acid (EDTA) is usually used in conjunction with the iron

sulphate. The EDTA complexes with the iron so as to allow the slow and continuous

release of iron into the medium. Uncomplexed iron can precipitate out of the medium as

ferric oxide.

Element Function

Nitrogen Component of proteins, nucleic acids and some coenzymes Element

required in greatest amount

Potassium Regulates osmotic potential, principal inorganic cation

Calcium Cell wall synthesis, membrane function, cell signaling

Magnesium Enzyme cofactor, component of chlorophyll

Phosphorus Component of nucleic acids, energy transfer, component of intermediates

in respiration and photosynthesis

Sulphur Component of some amino acids (methionine, cysteine) and some

cofactors

Chlorine Required for photosynthesis

Iron Electron transfer as a component of cytochromes

Manganese Enzyme cofactor

Cobalt Component of some vitamins

Copper Enzyme cofactor, electron-transfer reactions

Zinc Enzyme cofactor, chlorophyll biosynthesis

Molybdenu

m

Enzyme cofactor, component of nitrate reductase

Table: Some of the elements important for plant nutrition and their physiological function.

Vitamins: The B-vitamins play an important role in the growth of tissues. Thiamine,

nicotinic acid and pyridoxine are generally incorporated in all media, although

pantothenic acid, folic acid, biotin, riboflavin, etc. have also been used. However, other

vitamins are often added to plant cell culture media for historical reasons.

Amino acids are also commonly included in the organic supplement. The most frequently

used is glycine (arginine, asparagine, aspartic acid, alanine, glutamic acid, glutamine and

proline are also used), but in many cases its inclusion is not essential. Amino acids

provide a source of reduced nitrogen and, like ammonium ions, uptake causes

acidification of the medium. Casein hydrolysate can be used as a relatively cheap source

of a mix of amino acids.

Growth regulators: Growth as well as differentiation of tissues in vitro is controlled by

various growth regulators auxins, cytokinins, gibberellins, ethylene, abscisic acid, etc.

Auxins: Indole acetic acid, indole butyric acid, naphthalene acetic acid, 2,4-

dichlorophenoxyacetic acid are the frequently used auxins. These are generally used at O.

I to 10 mg/litre concentrations in plant tissue culture media. Naphthalene acetic acid and

2,4dicchlorophenoxy acetic acid are thermostable and do not lose their activity on

autoclaving. Whereas, indole acetic acid is thermolabile and loses most of its activity

upon autoclaving. Hence, it is sterilized by filtration.

Table: Commonly used auxins, their abbreviation and chemical name

Cytokinins: Cytokinins have a profound effect on cell division and cell differentiation.

Kinetin, zeatin and 6-benzylaminopurine, the commonly used cytokinins, are used in 0.1-

10 mg/litre concentration. Cytokinins promote cell division. Naturally occurring

cytokinins are a large group of structurally related (they are purine derivatives)

compounds. Of the naturally occurring cytokinins, two have some use in plant tissue

culture media. These are zeatin and 2iP (2-isopentyl adenine). Their use is not widespread

as they are expensive (particularly zeatin) and relatively unstable. The synthetic

analogues, kinetin and BAP (benzylaminopurine), are therefore used more frequently.

Non-purine-based chemicals, such as substituted phenylureas, are also used as cytokinins

in plant cell culture media. These substituted phenylureas can also substitute for auxin in

some culture systems.

Table: Commonly used cytokinins, their abbreviation and chemical name

Auxins and cytokinins are the most widely used plant growth regulators in plant tissue

culture and are usually used together, the ratio of the auxin to the cytokinin determining

the type of culture established or regenerated. A high auxin to cytokinin ratio generally

favours root formation, whereas a high cytokinin to auxin ratio favours shoot formation.

An intermediate ratio favours callus production.

Fig: The effect of different ratios of auxin to cytokinin on the growth and morphogenesis

of callus. High auxin to cytokinin ratios promote root development, low ratios promote

shoot development. Intermediate ratios promote continued growth of the callus without

differentiation.

Others: Gibberellic acid, ethylene releasing compounds and abscisic acid are also

incorporated in the media.

Carbon source: Plant tissues in culture can utilize a variety of carbohydrates- sucrose,

glucose, fructose, starch, and maltose. Sucrose, at 2 - 5% concentrations in the nutrient

media, however, remains the most widely used carbohydrate source.

Organic supplements: Complex substances such as yeast-extract, malt extract and casein

hydrolysate are also added (0.1- 1% w/v). Among various plant extracts, liquid

endosperm of immature coconut (coconut water) is widely used (5 - 20% v/v).

Gelling Agents: Agar has long been used to solidify media for plant tissue culture. The

type of agar or gelling agent used can influence the growth of the tissue in culture. Both

purity and cost of the gelling agent are important factors in any research or production

operation. Many companies have expanded its line of gelling agents to allow greater

selection in choosing the plant cell culture tested gelling agent for your particular

requirement.

Composition of M.S. Media

MAJOR M.S. SALTSSl.No.

NAME OF SALT FOR 10x QUANTITY in g/l1- NH4NO3 1650 mg/l 16.5

2- KNo3 1900 mg/l 19.0

3- CaCl2.2H2O 440 mg/l 4.4

4- MgSo4.7H2O 370 mg/l 3.7

5- KH2Po4 170 mg/l 1.7

MINOR M.S. SALT Sl. No.

NAME OF SALTFOR 100x

QUANTITY in g/l

1-KI

0.83 mg/l 0.083

2- H3BO3 6.20 mg/l 0.620

3- MnSO4.4H2O 22.30 mg/l 2.23

4- ZnSO4.H2O 8.60 mg/l 0.86

5- Na2MoO4.2H2O 0.25 mg/l 0.025

6- CuSO4.5H2O 0.025 mg/l 0.0025

7- CoCl2.6H2O 0.025 mg/l 0.0025

IRON SOURCESl.No.

NAME OF SALTFOR 100x

QUANTITY in g/l

1- Na2.EDTA 3790 mg/l 3.73

2- FeSo4.7H2O 2780 mg/l 2.78

VITAMINSSl.No.

NAME OF SALTFOR 100x QUANTITY in g/l

1- Myo-inositol 100 mg/l 10.0

2- Nicotinic Acid 50 mg/l 0.05

3- Pyridoxin HCl 50 mg/l 0.05

4- Thiamic HCl 10 mg/l 0.01

5- Glycine 20 mg/l 0.2

SUCROSE

Quantity 30 g/l

pH 5.8

AGAR8 g/l

Preparation of Plant Tissue culture media-Stock Solution:

Stock solutions of major salts, minor salts and vitamins are prepared to be used in the

preparation of media are stored in a refrigerator. For preparing one litre of the medium:

1. Transfer appropriate amounts of stock solutions of salts( Stock A and B), iron source

(Stock-C) and vitamins (Stock-D) to a one-litre flask

2. Add required hormones from hormone stocks. IAA and GA3 can not be added at this

stage.

3. Add a carbohydrate source such as sucrose, glucose or fructose (2 - 5%),adjust the

volume nearly 100ml with dd water.

4. Adjust the pH 5.8 using a pH meter.

5. Make the volume to 1 litre with double distilled water.

6. Add powdered agar 8 g for 1000 ml for making the medium semisolid.

7. Cover the flask with paper or aluminium foil and boil for 15-20 minutes.

8. Dispense the medium into culture tubes or flasks as the case may be.

9. Autoclave tubes or flasks containing medium at 121° for 20 min .

Sterilization

Sterilization of glassware

Cleaning

It is important to use clean glassware for the growth of tissues in vitro. In laboratory,

these include:

Boil all glassware in washing-soda solution (l0%) for 1 hr

Rinse thoroughly with tap water and leave in hydrochloric acid (1 N) for 2 hr

Remove traces of acid by thorough washing with tap water

Rinse glassware with double-distilled water.

Allow glassware to dry overnight at room temperature.

Sterilization Techniques

The objective of sterilization is to make media, glassware and instruments free from

microorganisms. It is accomplished by wet heat, dry heat or filtration.

1. Wet-heat sterilization

Plug glassware such as conical flasks, test-tubes, etc. with non-absorbent

cotton

Wrap petri dishes with wrapping paper or aluminum foil

Place forceps and scalpels in test-tubes. Plug the tubes with nonabsorbent

cotton and cover with brown paper

Plug the mouth end of graduated pipettes (1,2,5 and 10 m1) with cotton.

Wrap them individually in wrapping paper or aluminium foil

Autoclave glassware and instruments a1 121°C for 1 hr

2. Dry-heal sterilization

Plug glassware such as conical flasks, test-tubes, etc. with non-absorbent cotton. The final

step involves placing the instruments in an oven at 140°- 160°C for 2 hI. .

3. Filter sterilization

Amino acids, vitamins, phytohormones, etc. may get destroyed during autoclaving, such

chemicals are, therefore, sterilized by filtration through a Millipore-filtration assembly

using filter membranes of 0.45 or 0.22 μm porosity

Plug 500 or 1,000 ml receiver flask with cotton

Assemble Millipore filtration unit with bacteriological membrane filter (0.45

or 0.22 Jim)

Wrap filtration unit with wrapping paper

Autoclave receiver flask and filtration unit at 121°C for 1 hr (do not sterilize in dry

heat as membrane filters get damaged)

Fix filtration unit to the receiver flask in a sterile cabinet

Pour solution to be sterilized into the filtration unit.

Apply slight air pressure to commence filtration (do not exceed air pressure by

7.03 kg/em 2) .

Transfer under aseptic conditions, to sterile flasks.

Using a sterile pipette and filter sterilized solution to the autoclaved medium,

shake well and dispense into sterile culture tubes or flasks under aseptic

conditions.

Media Sterilization

Plant tissue culture media are generally sterilized by autoclaving at 121 °C and 1.05

kg/cm2 (15-20 psi). The time required for sterilization depends upon the volume of

medium in the vessel. It is advisable to dispense medium in small aliquots whenever

possible as many media components are broken down on prolonged exposure to heat.

There is evidence that medium exposed to temperatures in excess of 121 °C may not

properly gel or may result in poor cell growth. Times may vary due to differences several

medium components are considered thermolabile and should not be autoclaved. Stock

solutions of the heat labile components are prepared and filter sterilized through a 0.22

μm filter into a sterile container. The filtered solution is aseptically added to the culture

medium, which has been autoclaved and allowed to cool to approximately 35-45 °C..

Sterilization of plant material

Before inoculating the medium with the explant, it is necessary to surface sterilile it.

There are many sterilizing agents such as calcium hypochlorite, chlorine water bleaching

powder, mercuric chloride, hydrogen peroxide and ethylene oxide. The concentration of

the sterilizing agent and the duration of treatment varies with the plant material. There

should he no damage to the explants in the process of surface sterilization The plant

material should then be thoroughly washed with sterile distilled water before transferring

it to the nutrient medium.

Sterilization of Seeds

Select 15 - 20 healthy seeds and wash them thoroughly with water

Now add 2 - 3 drops of liquid detergent in 100 ml of water and shake well for 5

min

Pour off the detergent solution and wash thoroughly to remove any traces of the

detergent

Rinse seeds in 50 ml of 70% ethanol for I min

Wash thoroughly 3 - 4 times with single distilled water

Transfer seeds to a sterile 25D-ml flask and add mercuric chloride solution (0.1%)

Treat for 20 min, shake the flask occasionally. All the operations thereafter should

be carried out under sterile conditions

Transfer the flask to a sterile cabinet, decant the mercuric chloride solution and

wash the seeds thoroughly with sterile water to remove any traces of mercuric

chloride.

Sterilization of Leaf Explants

Collect the younger leaves.

Dip in 70% alcohol for 10 seconds followed by quick rinsing in sterile water.

Using sterile forceps, place segments in a sterile petri dish and cut it from corners.

Transfer I - 2 leaf segments to each tube containing 20 ml of semisolid medium.

Sterilization of Stem/ root explants

The long shoots are excised from the tree and brought to the laboratory.

The defoliated shoots are cut in to 3-4 cm in size.

The shoots are first washed under running tap water and than kept in a solution

containing 0.1 % Carbendazime (Bavestin) + 25 mg/l Rifampicin and two drop of

Tween-20 for one hour.

After thorough washing the explants are surface sterilized with 0.1 % HgCl2 for 6

minutes aseptically followed by 5- 6 washing with sterile distilled water.

Sterilization procedures may be enhanced by:

1. Placing the material in a 70% ethyl alcohol solution prior to treatment with another

disinfectant solution. The use of a two-step (two-source) sterilization procedure has

proven beneficial with certain species.

2. Using a wetting agent, such as Tween 20 or 80, can be added to the disinfectants to

reduce surface tension and allow better surface contact.

3. Conducting the sterilization process under vacuum. This results in the removal of air

bubbles and provides a more efficient sterilization process.

Inoculation

In inoculation first seeds or explants are sterilized properly and then transferred carefully

to the bottle containing the prepared M.S. Media.

This whole process of inoculation is done inside laminar chamber to avoid any kind of

contamination which can cause hindrance in germination of that seed or callus formation

from explants.

Procedure

1. Take few seeds and wash with 70% ethanol for 30 secs with continuous shaking.

2. Drain the excess and add 0.1% mercuric chloride for 2-5 minutes (treatment with

mercuric chloride depends upon size of seed, bigger seeds require more time).

3. Drain excess and then wash with distilled water (approx 5 times).this is done to

remove mercuric chloride which will hamper seed germination if present.

4. Dry the seeds in sterilized blotting paper. Transfer the healthy seeds carefully to M.S

media.

Photograph showing the inoculated seeds

Incubation

In incubation all the bottles or test tubes containing media with seeds or explants to be

grown are kept inside a chamber with sufficient light and air and this chamber should be

free from any kind of contamination to provide a healthy growth to the germinating seeds

and developing callus.

The exposure of seeds and explants to light and air causes desired growth in them which

can be seen with time. This time of growth varies with the variety of seeds and explants

chosen. Some can show growth in a week or some can grow in months.

Photograph showing the incubation of explants

Results and Discussion

After sterilization of seeds with 0.1% HgCl2 followed by rinsing with sterile water we

inoculation of seeds into the MS media the seeds started germination. The germination of

seeds proved that they were viable. There was no contamination in the culture bottles, this

showed that the media as well as the explants was sterilized properly and the germination

of plants shows that they were getting the nutrients.

Micropropagation

The in vitro regeneration of plants from organs, tissues , cells or protoplasts and the true

to type propagation of a selected genotype using in vitro culture technique

A variety of plants species can be conveniently propagated through the techniques of cell,

tissue or organ culture. This is particularly described as clonal propagation or micro

propagation

Explants for micro propagation

• apical shoots

• axillary buds

• adventitious shoots

• bulbs

• leaf size ( Begonia sp)

Stages in micropropagation

In vitro clonal propagation is a complicated process requiring many steps or stages.

Murashige proposed four distinct stages that can be adopted for overall production

technology of clones commercially. Stages I to III are followed under in vitro conditions,

whereas stage IV is accomplished in a green house environment

Stage 0 – Preparative stage

This step involves selection of mother plant to provide a suitable explant of good quality

for initiation of aseptic cultures. Selection of suitable explant is essential to the success of

in vitro propagation wherever possible mother plants should be grown under protected

environment like glasshouse, greenhouse etc. while taking explant from the field age of

the mother plant, yield potential and healthiness should be considered.

Stage 1: Initiation of aseptic culture

This step is meant for obtaining an aseptic culture of the plant which is under

investigation. The culture may be initiated from shoot tips, buds, stem root explants,

flowers, sterile plantlets, callus etc. At this stage it is absolutely essential that the explant

to be cultured should be totally free from microbial infection and that a high percentage of

the explants survive and show rapid growth.

Stage II: Shoots proliferation stage

This stage aims at obtaining a rapid increase in the number of shoots or asexual embryos

which can ultimately be used to provide the large number of plants. This cycle generally

lasts for 4 – 10 weeks The cytokines in the media stimulates pre existing shoot buds

present in the explants ( apical meristems in shoot tips, and axillary buds in nodal

Explants) to develop into shoots. After sometime (4-6 weeks) the axillary branching in a

culture reaches the maximum. The individual shoots are then excised and sub cultured

onto fresh medium to initiate a new cycle of multiplication by axillary branching with a

final yield of 510 – 612 plants in one year.

Stage III: Root development

This stage involves preparation of shoots for rooting. The proliferated shoots are

transferred to a rooting medium. Sometimes shoots are directly established in soil as

micro cuttings to develop roots.

Stage IV: Hardening

An important and critical stage in this process is when the plantlets under aseptic

conditions of laboratories have to be shifted to glasshouses or greenhouse. The sterile

plants are hardened or acclimatized to their new surroundings.

Advantages of micropropagation 1) Rapid multiplication of superior clones and maintenance of uniformity

2) Multiplication of disease free plants

3) Multiplication of sexually derived sterile hybrids

4) Supplies planting material irrespective of season

5) Facilitates storage of a large scale

6) Eases the transportation of planting material

7)Stocks of germplasm can be maintained for many years

8) Multiplication of cloning dioecious species

9) Requires minimum growing space in commercial nurseries

10)In case of forest trees mature elite trees can be identified and rapidly cloned by this

technique

11) In ornamentals tissue culture plants give better growth , more flowers,

12) Very valuable when limited tissue is available as explant

Limitations High investment cost

Requirement of more skill

High electricity consumption

Presence of traces of somaclonal variation

Possibility of loss of rare genetic material when there is a severe contamination

Suitable techniques of micropropagation are not available for many valuable

species

Vitrification may be a problem in some species

Procedure:

Sterilization

All glassware’s like Petri plates, flasks, beakers; instruments like forceps, scissors,

scalpel; M.S media; distilled water; blotting paper to be properly autoclaved.

Selection of explant:

Appropriate selection of explants is very important for rapid in vitro propagation

The genotype, age and physiological state of the explant respond differently to cultures.

Selection of mother plant

It is necessary to select the mother tree/plant with the care. The explants should be taken

from mother plants possessing superior phenotypes such as disease resistance, stress

tolerance, high yield and product quality. Explants should proliferate readily. Even

materials with strong ability to proliferate should be screened continuously

Surface sterilization of explant

The surface contaminants e.g. bacteria, fungi and yeast have to be removed prior to

culture(otherwise may lead to death of explant or seed) therefore washed with sterilizing

agents like calcium hypochlorite, chlorine water, bleaching powder, mercuric chloride,

hydrogen peroxide and ethylene oxide.

Steps:

Take few seeds and wash with 70% ethanol for 30 secs with continuous shaking

Drain the excess and add 0.1% mercuric chloride for 2-5 minutes (treatment with

mercuric chloride depends upon size of explant, bigger explant require more time).

Drain excess and then wash with distilled water (approx 5 times).this is done to

remove mercuric chloride which will hamper explant germination if present.

Dry the explant in sterilized blotting paper.

Transfer the explant carefully to M.S media

Micropropagation of explants

Results and Discussion

The explants that were sterilized 0.1% HgCl2 were inoculated in the MS media. After 4

days of inoculation, the auxiliary buds started growing, green leaves sprouted. The

auxiliary buds which were dormant under the influence of the Auxin secreted from the

Apical buds. Once the apical buds were removed, these auxiliary started sprouting this

show that the micropropagation was successfull.

Callus Induction and Maintenance

It is an unorganized or undifferentiated mass of proliferative cells produced either in

culture or in nature.

.

Tissues and cells cultured on an agar medium form an unorganized mass of cells i.e.,

callus. Callus formation from explant involves the development of progressively more

random planes of cell division, less frequent specialization of cells and loss of organized

structures. Callus culture is often performed in the dark and the light can encourage

differentiation of the callus.

Micro callus

The initial colony of cells visible but too small to transfer by direct manipulation

recovered from cultured of protoplasts single cells or very small aggregate cells.

Induction and maintenance When freshly cut pieces of surface sterilized plant tissues are grown in agar medium with

appropriate nutrients with suitable proportion of auxin and cytokinin they exhibit

callusing at cut ends, which gradually extends to the entire surface of the tissue. Callus

cultures need to be sub cultured every 3 – 5 weeks in view of cell growth, nutrient

depletion and medium drying. Repeated subculture on an agar medium improves the

friability of the callus

Callus cultures are extremely important in plant biotechnology. Manipulation of the auxin

to cytokinin ratio in the medium can lead to the development of shoots, roots or somatic

embryos from which whole plants can subsequently be produced. Callus cultures can also

be used to initiate cell suspensions, which are used in a variety of ways in plant

regeneration studies.

Applications of callus culture

1. To study nutrition requirement of plants

2. To study cell and organ differentiation and morphogenesis

3. Somaclonal variations and its exploitation

4. Developing cell suspension cultures and protoplasts cultures

5. Genetic transformation using ballistic particle gun technology

6. In the production of secondary metabolism and regulation

Procedure

1. Take the explant and wash with 70% ethanol for 30 secs with continuous shaking.

2. Drain the excess and add 0.1% mercuric chloride for 2-5 minutes (treatment with

mercuric chloride depends upon size of explant, bigger explant require more time).

3. Drain excess and then wash with distilled water (approx 5 times).this is done to

remove mercuric chloride which will hamper explant germination if present.

4. Dry the explant in sterilized blotting paper.

5. Transfer the explant carefully to B.A media.

Photograph showing the regenerated callus

Results and Discussion

The leaf discs were used as explants. These explants were surface sterilized with 0.1% HgCl2 the thoroughly rinsed by sterile water. After rinsing they were dried by sterile blotting pare and then inoculated in the BA media. After 8 days the leaves show the callus formation from the cute margins. In few days the callus was present all over the leaf surface.

This show that the callus starts from the cut ends and then it gradually spreads over the leaf. The De-differentiation of the leaf disc had started.