industrial plant and animal biotechnology

7/25/2019 Industrial Plant and Animal Biotechnology

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PGPCAS/DBT/Lab in Industrial, Plant & Animal Biotechnology

Department of Biotechnology, PGP College of Arts & Science, Namakkal 1

Lab in Industrial, Plant and Animal

Biotechnology

Students Manual


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INDEX

S.NO DATE DESCRIPTIONPAGE

NO REMARKS

1 PREPARATION OF TISSUE CULTURE MEDIUM

2 PLANT TISSUE CULTURE- AN INTRODUCTION

3 MICROPROPAGATION

4 CALLUS INDUCTION

5 DIRECT ORGANOGENESISSHOOT TIP CULTURE

6 EMBRYO CULTURE

7 ISOLATION OF PROTOPLASTS

8 ENZYMATIC METHOD

9 ANTHER CULTURE

10 CELL SUSPENSION CULTURE

11INTRODUCTION FOR ANIMAL CELL CULTURE

LABORATORY

12 STERILIZATION TECHNIQUES

13 PREPARATION OF MEDIA

14 PRIMARY CELL CULTURE

15 CELL COUNTING AND VIABILITY

16 STAINING OF ANIMAL CELLS

17 CULTURE OF VIRUS IN CHICK EMBRYO

18 CELL CULTURE TERMNOLOGY


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Ex.No: Date:

PREPARATION OF TISSUE CULTURE MEDIUM

INTRODUCTION:

The basic nutritional requirements of cultured plant cells as well as plants are very

similar. However, the nutritional composition varies according to the cells, tissues, organs

and protoplasts and also with respect to particular plant species. The appropriate composition

of the medium largely determines the success of the culture. A wide variety of salt mixtures

have been reported in various media.

A nutrient medium is defined by its mineral salt composition, carbon source,

vitamins, growth regulators and other organic supplements. When referring to a particular

medium, the intention is to identify only the salt composition unless otherwise specified. Any

number and concentration of amino acids, vitamins, growth regulators and organic

supplements can be added in an infinite variety of compositions to a given salt composition in

order to achieve the desired results.

UNITS FOR SOLUTION PREPARATION

The concentration of a particular substance in the media can be expressed in various units

that are as follows :

Units in weight

It is represented as milligram per litre (mg/l) 10-6

= 1.0 mg/l or 1 part per million (ppm) 10-7

= 0.1 mg/l.

10-8

= 0.001 mg/l or 1 g/l.

Molar concentration

A molar solution (M) contains the same number of grams of substance as is given by

molecular weight in total volume of one litre.

1 molar (M) = the molecular weight in g/l

1 mM = the molecular weight in mg/l or 10

-3

M

1 M = the molecular weight in g/l or 10-6

M or 10-3

mM.

Conversion from milli molar (mM) to mg/l

For example, molecular weight of auxin 2,4-D = 221.0 1M 2,4-D

solution consists of 221.0 g per litre


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1 mM 2,4-D solution consists of 0.221 g per litre = 221.0 mg per litre 1 M 2,4-

D solution consists of 0.000221 g/l = 0.221 mg/l

Conversion from mg/l to mM

The molecular weight of CaCl2- 2H2O

= 40.08 + 2 x 35.453 + 4 x 1.008 + 2 x 16 = 147.018

(the atomic weights of Ca, Cl, H and O being 40.08, 35.453, 1.008 and 16.0 respectively).

If, 440 mg/l of CaCl2- 2H2O is to be converted into mM; then

The number of mM CaCl2. 2H2O =

No. of mg CaCl2- 2H2O

Molecular weight of CaCl2. 2H2O

=

440

=2.99 mM

147.019

Thus, 440 mg/l CaCl2- 2H2O = 2.99 mM

REQUIREMENTS

Glassware / plasticware / minor items

1. Aluminum foil

2.

Beakers of different sizes from 50ml to 2000ml

Chemicals of Analar grade, depending upon the medium

Conical flasks (with wide mouth) of different capacities

3. (100ml, 150ml, 250ml, 11, 2.5l)

Culture tubes (25mm x 150 mm)

Funnels

4. Glass markers

5.

Graduated cylinders of various capacities6.

Measuring cylinders of various capacities

7. Non-absorbent cotton and muslin/cheese cloth for cotton plugs

8. Petri dishes of different sizes (glass or sterilized plastic)

9. Pipettes (different capacities from 1ml to 10ml)

10. Sterile filtration assembly


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11. Wrapping paper (brown sheet)

MEDIA COMPOSITION

A number of basic media are listed in Table 1. The salt composition of Murashige and Skoog

(1962) nutrient medium, referred to as MS medium, is very widely used in different culture

systems as it gives satisfactory results. But it must be remembered that it is not always the

best medium. Generally, in all the media, the nutritional milieu consists of inorganic

nutrients, carbon and energy sources, vitamins, growth regulators, and complex organic

supplements. It is desirable to choose a composition according to the knowledge of the

physiology of species vis-a-vis mineral nutrition.

INORGANIC NUTRIENTS

Mineral elements are very important in the life of a plant. Besides, C,H,N, and O, 12 other

elements are known to be essential for plant growth. According to the recommendations of

the International Association for Plant Physiology, the elements required by plants in

concentration greater than 0.5 mmol/l are referred to as macroelemetns or major elements and

those required in concentration less than the prescribed amount are microelements of minor

elements. A variety of salts supply the needed macro and micronutrients that are the same as

those required by the normal plant.

Major salts : The salts of potassium (K), nitrogen (N), calcium (Ca), magnesium (Mg),

phosphorus (P) and sulphur (S) are required in macro or millimole quantities. Nitrogen is

generally used as nitrate or ammonium salts, sulphur as sulphates and phosphorus as

phosphate.

Minor salts : The salts of iron (Fe), manganese (Mn), boron (B), copper (Cu), zinc (Zn),

iodine (I), molybdenum (Mo) and cobalt (Co) are required in micromolar concentrations and

are considered to be minor salts. These salts are essential for the growth of tissues and are

required in trace quantities.

To achieve the maximum growth rate, the optimum concentration of each nutrient can vary

considerably. The mineral composition of a culture medium is defined precisely by the

equilibrium of the concentrations of differention in a solution. When mineral salts are

dissolve in water, they undergo dissociation and ionization. The active factor in the mediums

is the ions of different types rather than the compounds. Therefore, a useful comparison

between the two media can be made by looking into the total concentrations of different types


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of ons in them. To choose a mineral composition and then compare their differnet ionic

balances, one uses ionic concentrations expressed in milli equivalents per litre (Table 2). Any

success with a medium is in all probability due to the fact that the ratios as well as

concentrations most nearly match the optimum requirements for the cells or tissues for

growth and/or differentiation.

Cultured tissues have specific needs vis-a-vis the following ions: K+, NO3

-, NH4

+, Ca

++,

Mg++

. Phosphorus is often carried in low concentrations. The ions K+, NO3

-, and NH4

+

have a profound influence on the growth of tissues.

Tabel 1 : Composition of plant tissue culture media (values expressed as mg per

litre)

ConstituentWhite Murashige & Gamborg's Chu

(1963) Skoog (1962) - MS (1968) - B6 (1978) N6KCl 65 - - -

MgSO4. 7H2O 720 370 250 185

NaH2PO4. H2O 16.5 - 150 -

CaCl2. 2H2O - 440 150 166

KNO3 80 1900 2500 2830

Na2SO4 200 - - -

NH4NO3 - 1650 - -

KH2PO4 - 170 - 400

Ca(NO3)2. 4H2O 300 - - -

(NH4)2SO4 - - 134 463

FeSO4. 7H2O - 27.8 - 27.8

MnSO4. 4H2O 7 22.3 - -

MnSO4. H2O - - 10 3.3

Kl 0.75 0.83 0.75 0.8

CoCl2. 6H2O - 0.025 0.025 -

ZnSO4. 7H2O 3 8.6 2 1.5

CuSO4. 5H2O - 0.025 0.025 -H3BO3 1.5 6.2 3 1.6

Na2MoO4.2H2O - 0.25 0.25 -

Fe2(SO4)3 2.5 - - -

EDTA disodium salt - 37.3 - 37.3

EDTA-Na ferric salt - - 43 -

m-inositol - 100 100 -


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Thiamine HCl 0.1 0.1 1.0 1.0

Pyridoxine HCl 0.1 0.5 1.0 0.5

Nicotinic acid 0.5 0.5 1.0 0.5

Glycine 3 2 - -

Cysteine 1.0 - 10 -

Sucrose 20,000 30,000 20,000 30,000

*In place of FeSO4 . 7H2O (27.8 mg and EDTA - Disodium salt (37.3 mg), EDTA-

Naferric salt (40) mg can be added.

Table 2 : Composition of mineral solutions used in vitro

Ions Gautheret Gamborg Murashige & Skoog White

NO3- 5.49 25.0 39.4 3.34

H2PO4- 0.92 1.1 1.3 0.12

SO4- 1.0 4.0 3.0 8.64

Cl- - 2.0 6.0 0.88

Total anions (mEq/l) 7.41 32.1 49.7 12.98

K+ 2.17 25.0 20.1 1.68

NH4+ - 2.0 20.6 -

Na+ - 1.1 - 2.92

Ca++

4.24 2.0 6.0 2.54

Mg++ 1.0 2.0 3.0 5.84

Total cations (mEq/l) 7.41 32.1 49.7 12.98

Total anions + cations 11.70 60.2 93.3 17.45(10

3M/I)

The role of different elements has been described below:

Nitrogen: Nitrogen is the major component supplied in the form of nitrates or

ammonium salts. Nitrogen is an important part of amino acids, proteins, nucleic acids.

Inorganic nitrogen is utilized in order to synthesize organic molecules. For most

purposes, a nutrient medium should contain from 25 to 60 mM inorganic nitrogen. The

cells may grow on nitrate alone, but often there is a distinct beneficial effect and

requirement for ammonium or another source of reduced nitrogen. Besides, nitrate alone

in the medium drifts the pH towards alkalinity. Adding a small amount of an ammonium

is the range of 25-40 mM and ammonium in the range of 2-20 mM. Nitrate cannot be

simply used to synthesize organic molecules but has to be reduced to ammonia first. The

response to ammonium varies from inhibitory to essential, depending upon the tissue and

the purpose of culture


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e. In case of amounts in excess of 8 mM of ammonium or if grown solely on this source of

nitrogen, then citrate, malate, succinate or another TCA cycle acid should be present. Most

plants prefer nitrate to ammonium, although the opposite is also true in some cases.

Potassium: Potassium is required at concentrations of 2 to 26 mM. This element isgenerally

supplied as the nitrate or as the chloride form and cannot be substituted by sodium. It is a

monovalent cation with high mobility in the plant. Potassium salts have an important function

in the osmotic regulation of the cell. Potassium ion is essential for the activation of many

enzymes. In photosynthesis, K+regulates the ion balance and pH of chloroplasts.

Calcium: Calcium is essential for cation-anion balance by counteracting organic and

inorganic anions. A concentration of 1-3 mM of calcium is usually adequate. Calcium is also

important for cell and root multiplication. Calcium, a component of the cell wall, is largely

bound to the cell wall and membrane. This is because of the large number of Ca++

binding

places on the cell wall and limited mobility of calcium through the membrane into the

cytoplasm. The stability of cell membrane is highly influenced by Ca++

.

Phosphorus: Phosphorus is present in the plant in the form of inorganic phosphate (iP). A

concentration of 1-3 mM phosphate is usual adequate. The high-energy pyrophosphate bond

of phosphorus, when bound to another P atom as in ATP, is very important for the energy

metabolism in the cell. Phosphorous is an essential element in DNA and RNA nucleic acids.

In phospholipids, this element is very important for the energy metabolism of the plant in

form energy-rich phosphate esters.

Magnesium: A concentration of 1-3 magnesium is usually adequate. This element is an

essential component for many enzymes reactions and is very important in photosynthesis.

Magnesium is indispensable for the energy metabolism of the plant because of its importance

in the synthesis of ATP.

Sulphur: A concentration of 1-3 mM sulphate is usually adequate. These have to be reduced

first for the synthesis of sulphur containing compounds such as amino acids, proteins and

enzymes. Sulphur in its non-reduced form is incorporated in sulpholipids and

polysaccharides.

Boron: Boron is required for the synthesis of cell wall as well as in the stabilization of the

constituents of cell wall and cell membrane.


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Chlorine: Chlorine is taken up as a chloride and is very mobile in the plant. The main

functions of the ions are in osmoregulation. Chlorides play a role in photosystem II during

the Hill reaction. Chlorine also regulates the opening and closing of stomata and is thus very

important in the regulation of the osmotic potential of vacuoles as also to turgor-related

processes.

Copper: Copper is taken up by the plant as Cu++

or as a copper chelate complex. Withinthe

cell, copper is mostly part of the enzyme complexes and important in redox reactions

executed by these enzymes. It is useful in photosynthesis.

Cobalt: Cobalt is assumed to be important in nitrogen fixation. In higher plants thefunction

of this element is not very clear.

Manganese: Manganese is taken up by the plant as bivalent unbound Mn++

ions. The

element is strongly bound to several metalloproteins. The ion is involved in the Hill reaction

of photosystem II in which water is split into oxygen and protons.

Molybdenum: Molybdenum is used as a cofactor in many enzymes, including nitrogenase

and nitrate reductase. It is also directly involved in the reduction of N2.

Zinc: Zinc is taken up by the roots as Zn++

. It is neither oxidized nor reduced in theplants. It

is an important component of a number of enzymes, e.g. alcohol dehydrogenase in the

meristem zone of the plant. Zinc is also very important for protein synthesis.Iron: Iron is generally added as a chelate with ethylene diamine tetra acetic acid (EDTA). In

this form, iron remains available up to a pH of 8.0. It is mainly bound to chelators and

complex compounds in plants. Most plants absorb only ferric ions (Fe3+

). The main function

of iron is to form iron chelates and two kinds of proteins: haeme proteins and iron sulphur

proteins. The most well-known haeme proteins are the cytochromes, functioning as

intermediates for electrons required for the reduction of nitrate to nitrite by the enzyme

nitrate reductase in nitrogen assimilation. The second group of iron-binding proteins are theiron sulphur proteins. The iron is bound to a thiol group (-SH) of cystine and/or inorganic

sulphur. Ferridoxin is the most common iron sulphur protein. It functions as a carrier in the

electron transport reaction catalyzed by nitrate reductase, sulphate reductase, the synthesis of

NADP+during photosynthesis and nitrogen reduction by nitrogenase complex. Iron is also


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important in the biosynthesis of chlorophyll.

Carbon and energy source

The standard carbon source without exception is sucrose but plant tissues can utilize a variety

of carbohydrates such as glucose, fructose, lactose, maltose, galactose and starch. In the

cultured tissues or cells, photosynthesis is inhibited and thus carbohydrates are needed for

tissue growth in the medium. Sucrose, at a concentration of 2-5% in the medium, is widely

used. The autoclaving process does cause an alteration in the sugars by hydrolysis but

presents no drawbacks to the growth plan. Most media contain myo-inositol at a

concentration of 100-mg per litre, which improves cell growth.

VITAMINS

Normal plants synthesize the vitamins required for growth and development, but plant cells

in culture have an absolute requirement for vitamin B1(thiamine), vitamin B (nicotinic acid)

and vitamin B6 (pyridoxine). Some media contain pantothenic acid, biotin, folic acid, p-

amino benzoic acid, choline chloride, riboflavine and ascorbic acid. The concentrations are in

the order of one mg/l. Myo-inositol is another vitamin used in the nutrient medium with a

concentration of the order of 10-100 mg/l.

GROWTH REGULATORS

Hormones now referred to as growth regulators are organic compounds that have been

naturally synthesized in higher plants, which influence growth and development. These are

usually active at different sites from where they are produced and are only present and active

in very small quantities. Two main classes of growth regulators of special importance in plant

tissue culture are the auxins and cytokinins, while others are of minor importance, viz.,

gibberellins, abscisic acid, ethylene, etc. Some of the naturally-occurring growth regulators

are indole acetic acid (IAA), an auxin and zeatin and isopentenyl adenine (2 iP) as cytokinins,

while others are synthetic growth regulators. Characteristics of growth regulators have been

shown in Table

Table Characteristics of growth regulators

Name Chemical formula Molecular Solubility

weight

p-Chlorophenoxy acetic C8H7O3Cl 186.6 96% ethanol

acid


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2,4-Dichlorophenoxy C8H6O3Cl 221.0 96% ethanol, heated

acetic acid lightly

Indole-3 acetic acid C10H9NO2 175.2 1N NaOH/96% ethanol

Indole-3 butyric acid C12H13NO2 203.2 1N NaOH/96% ethanol

-Naphthalene acetic C12H10O2 186.2 1N NaOH/96% ethanolacid

-Naphthoxy acetic C12H10O3 202.3 1N NaOH

acid

Adenine C5H5N5.3H2O 189.1 H2O

Adenine sulphate (C5H5N5)2.H2SO4.2H2O 404.4 H2O

Benzyl adenine 6 C12H11N5 225.2 1N NaOH

benzyl amino purine

N-isopentenyladenine C10H13N5 203.3 1N NaOH

(2 iP)

Kinetic C10H9N5O 215.2 1N NaOH

Zeatin C10H13N5O 219.2 1N NaOH/1N HCl,

heated lightly

Gibberellic acid C19H22O6 346.4 Ethanol

Abscisic acid C15H20O4 264.3 1N NaOH

Colchicine C22H25NO6 399.4 H2O

Auxins

Auxin was discovered following experiments on the reaction of coleoptile curvature inGramineae. It owes its name to its effect on the elongation of cells (auxesis). Auxins have

an indole nucleus with the basic formula C10H9O2N.

A common feature of auxins is their property to induce cell division and cell elongation.

The stimulation of division of cells of cambial origin resulted in initial successes with in

vitro cultures. This effect leads to a number of cells, which further result in the formation

of callus. Auxin has a clear rhizogenic action, i.e. induction of

adventitious roots. It often inhibits adventitious and auxillary shoot formation. At low auxin

concentration, adventitious root formation predominates, whereas at high auxin

concentration, root formation fails to occur and callus formation takes place. All the plants

synthesize auxin that is modulated according to the stage of development. Auxin is present in

sufficient concentration in the growing shoot tips or flowering tips of plants to ensure cell

multiplication and elongation. Auxin circulates from the top towards the base of the organs


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with a polarity strongly marked in young organs. The compounds most frequently used and

highly effective are 2,4-dichlorophenoxy acetic acid (2,4-D), naphthalene acetic acid (NAA),

indole acetic acid (IAA), indole butyric acid (IBA). Other auxins in use are 2,4,5-

trichlorophenoxy acetic acid (2,4,5-T), p-chlorophenoxy aceticacid (pCPA) and pichoram (4-

amino-3,5,6-trichloropicolinic acid).

Cytokinins

Cytokinins were discovered during in vitro culture studies. Coconut milk was known to have

a favourable effect on cellular multiplication and bud formation. Researchers discovered that

an active compound of a purine nature causes such an effect. This enabled Skoog in 1956 to

isolate an active substance from denatured RNA, which he called kinetin. Thus, cytokinins

are derivatives of adenine and have an important role in shoot induction. Cytokinins also

have a clear effect on cell division. Often used to stimulate growth and development, they

usually promote cell division if added together with an auxin. Auxins favour DNA

duplication and cytokinins enable the separation of chromosomes. Cytokinins have a clear

role in organogenesis where they stimulate bud formation. They are antagonistic to

rhizogenesis. At higher concentrations (1 to 10 mg/l), adventitious shoot formation is induced

but root formation is generally inhibited. Cytokinins promote axillary shoot formation by

decreasing apical dominance. The most frequently used compounds are kinetic, benzyl

adenine (BA) or 6-benzyl amino purine (BAP), zeatin, and isopentenyladenine (2 iP). Zeatin

and 2 iP are natural cytokinins. Zeatin, the first endogenous cytokinin, was identified in 1963

in immature embryos of maize, while 2 iP was discovered a little later from plants attacked

by the bacterium

Cornyebacterium fascines.

Stock solutions of IAA and kinetic are stored in amber bottles or bottles covered with a black

paper and kept in the dark since they are unstable in light.

Other Growth Regulators

Gibberellins, although found in all plants and fungi, are unevenly distributed. The sites of

synthesis are very young, unopened leaves, active buds, root tips and embryos. There are

various gibberellin compounds with a similar gibbane nucleus but differ in the quality and

position of the substituents of the nucleus. Gibberellic acid (GA3) is the most widely used

compound.


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Gibberellins are normally used in plant regeneration. GA3is essential for meristem culture of

some species. In general, gibberellins induce elongation of internodes and the growth of

meristems or buds in vitro. In its absence, the culture appears globular, due to the

accumulation of nodes. Gibberellins usually inhibit adventitious root as well as shootformation. During organogenesis, gibberellins are antagonistic. They seem to oppose the

phenomenon of dedifferentiation. Thus, in in vitro cultures, they cannot be used for this

purpose, but can be utilized for explants already organized (meristems, apices, buds).

Abscisic acid is an important growth regulator for induction of embryogenesis. Abscisic acid

was identified in 1965 and since then, has been found in all plants. This is a growth inhibitor,

which seems to be synthesized when a plant is under difficult conditions. It has a favourable

effect on abscission.

Ethylene is a gaseous compound identified a long time ago, but its functions as a growth

regulator were not evident. Ethylene is produced by cultured cells, but its role in cell and

organ culture is not known. It also influences fruit maturation, acceleration of the process of

leaf or fruit abscission, flowering induction and tuberization. The practical use of ethylene,

which is difficult in a gaseous state, made great progress after the discovery of 2-

chloroethane phosphoric acid. This product, when applied in a powder form, penetrates the

tissue where it liberates ethylene.

ORGANIC SUPPLEMENTS

Certain complex substances are also added in the media which supply organic nitrogen,

carbon or vitamins. Organic nitrogen in the form of casein hydrolysate (0.2-1 g/l) or certain

amino acids such as glutamine and asparagine, nucleotide as adenine are included in the

medium L-glutamine (upto 8 mM, i.e. 150 mg/l) may replace the casein hydrolysate. The

amino acids, when added, should be used with caution, since they can be inhibitory. The

other amino acids included in the media in mg/l include: glycine (2), aspargine (100),

tyrosine (100), arginine (10), cysteine (10), and aspartic acid, glutamic

acid and proline, etc. Only L-isomers are used, while D-isomers have proved to be

ineffective. Adenine or adenine sulphate (2-120 mg/l) is added to agar media for

morphogenesis. Addition of TCA cycle acids such as citrate, malate, succinate or fumarate

permits the growth of plant cells on ammonium as the sole nitrogen source. A variety of

extracts, viz., protein hydrolysate, yeast extract, malt extract, coconut milk, orange and


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tomato juices have also been tested. With the exception of protein hydrolysate and coconut

milk, most of the others are used as a last resort. Coconut milk is commonly used at 2-15%

(v/v). The present trend is, however, towards fully defined media and the use of complex

mixtures is losing favour.

Antibrowning compounds: Many plants are rich in polyphenolic compounds. After tissue

injury during dissection, such compounds will be oxidized by polyphenol oxidases and the

tissue will turn brown or black. The oxidation products are known to inhibit enzyme activity,

kill the explants, and darken the tissues and culture media, a process which severely affects

the establishment of explants. Activated charcoal at concentrations of 0.2 to 3.0% (w/v) is

used where phenol-like compounds are a problem for in vitro growth of cultures. It can

adsorb toxic brown/black pigments and also stabilize pH. Besides activated charcoal,

polyvinylpyrolidone (250-1000 mg/l), citric acid and ascorbic acid (100 mg/l each), thiourea

or L-cysteine are also used to prevent oxidation of phenols.

Some of the procedures used by various workers to combat this problem of browning are: (i)

Adding antioxidants to culture medium, viz, ascorbic acid, citric acid, polyvinylpyrolidone

(PVP), dithiothreitol, bovine serum albumin, etc. (ii) Pre-soaking explants in antioxidant

before inoculating into the culture medium; (iii) incubating the initial period of primary

cultures in reduced light or darkness because it is known that phenolic oxidation products are

formed under illumination; and (iv) Frequently transferring explants into fresh medium

whenever browning of the medium is observed.

GELLING AGENTS

Agar, the most popular solidifying agent, is a seaweed derivative. Plant tissue culturists often

use Difco Bacto agar at a concentration of 0.6 to 1.0% (w/v), although other forms of agar

(agarose, phytoagar, flow agar, etc.) are also gaining popularity. Solubilized agar forms a gel

that can bind water and adsorb compounds. The higher the agar concentration, the stronger is

the water bound. With higher concentrations, the medium becomes hard and does not allow

the diffusion of nutrients into the tissues. Thus in vitro growth may be adversely affected if

the agar concentration is too high. Besides agar, the following alternatives can be used.

i. Alginate can be used for plant protoplast culture.

ii. Gelrite at 0.2% can be used for solidification of media. Gelrite gels are remarkably

clear in comparison to those formed by agar. Gelrite requires both a heating cycle and the


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presence of divalent cations (Mg++

or Ca++

) for gelation to take place.

iii. Synthetic polymer biogel P200 (polyacrylamide pellets) or a starch polymer can be

used.

iv.

Agargel: A mixture of agar and synthetic gel has been developed by Sigma Company.It has the properties of both synthetic gel and agar.

Liquid media with a support can also be used instead of solid media. Such media include

i. Liquid medium without agar using clean foam plastic, glasswool or rockwool as

support.

ii. Filter paper-bridge, which is hung in a liquid medium.

iii. Growth on a liquid medium containing glass beads.

iv. Viscose sponge underneath the filter paper as a carrier for a liquid medium instead of

agar.

pH

pH determines many important aspects of the structure and activity of biological

macromolecules. pH is the negative logarithm of the concentration of hydrogen ions. Nutrient

medium pH ranges from 5.0 to 6.0 for suitable in vitro growth of explant. pH higher than 7.0

and lower than 4.5 generally stops the growth and development. The pH of the medium

changes during autoclaving. It generally falls by 0.3 to 0.5 units after autoclaving. If the pH

falls appreciably during plant tissue culture (the medium becomes liquid), then a fresh

medium should be prepared. One should know that a starting pH of 6.0 could often fall to 5.5

or even lower during growth. pH higher than 6.0 gives a fairly hard medium and a pH below

5.0 does not allow satisfactory gelling of the agar.

General methodology for preparation of medium (MS Medium)

Preparation of stock solutions: Since it is a time - consuming and tediousprocess to weigh

the necessary products each time a medium is prepared, concentrated solutions of the desired

composition of a medium are used which one dilutes adequately. These concentrated

solutions are called stock solutions. Simple stock solutions comprise only one constituent at a

time. Complex stock solutions comprise several chemicals. Stock solutions of macro and

micronutrients, vitamins and growth regulators are prepared in distilled or high purity

demineralized water. Chemicals should be of the highest grade.

i. Macronutrient stock solution(s): Usually, the stock solution of macronutrients is


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prepared as 10x. Dissolve all the macronutrients one by one except (CaCl2for macronutrient

stock solution. The stock solution of CaCl2should be prepared separately. Another way is to

dissolve the different macronutrients one after the other and CaCl2 is dissolved separately

and later added to the rest of the stock solution in order to avoid precipitation.

ii. Micronutrient stock solution: A stock solution of all the micronutrientswith 100x is

generally prepared. Since copper and cobalt are required in very small quantities, it is

preferable to first make a separate stock solution of these two salts (100x) and then an

appropriate volume can be pipetted and put into the main micronutrient stock solution. These

nutrient solutions can be dispensed in plastic bags with zipper seals and stored frozen (e.g.

10x macronutrient solution is dispensed into a bag containing 100ml of solution to prepare 1

litre medium).

iii. Iron-EDTA: Iron EDTA should be added fresh. If stock solution (100x) is prepared,

then it should be stored after autoclaving in an amber bottle or a bottle covered with an

aluminium foil.

iv. Vitamins and growth regulators stock solutions: These are simple stocksolutions.

All the growth regulators are not soluble in water. Solubility of different growth regulators is

given in Table 4.3. The compound should be dissolved in a few ml of solvent and then water

is slowly added to make the requisite volume. Concentrations of compounds can be taken as

mg/l or in molarity.

Concentration in mg/l - It is preferable to dissolve 50 mg / 100 ml to give aconcentration of

0.5 mg/ml or 100 mg/100 ml in order to give a concentration of 1 mg/ml.

Concentration in mM - The growth regulator solutions can be prepared as 1 mM for100ml.

If a culture medium is to contain 10 M of the growth regulator (e.g. 2,4-D M.W. = 221.0),

then

1M = 221 g/l

1 mM = 221 mg/l or 0.221 mg/ml

The amount in 100 ml stock solution = 0.221 x 100ml = 22.1

mg 10 M = 2210 g or 2.210 mg

The required volume of stock solution to be added = 10ml of this stock (22.1 / 10 = 2.210

mg).


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1. Media chart is prepared as shown in Table 5. In a sequence, different components are

added into a beaker according to the list : nutrients, iron-EDTA, vitamins, myo-inositol,

growth regulators (if thermostable and autoclavable), organic supplements, sucrose etc., by

using the correctly sized graduated cylinders or pipettes or balance.

2. Water is added to just below the final volume (e.g. 800 ml volume for one litre medium)

3. pH of the medium is adjusted to the required value (e.g. pH 5.8 for MS) by adding

dropwise while stirring 1N KOH or 1N HCl.

4. Required quantity of agar or any other gelling agent is added while the medium is being

stirred.

5. The solution to brought to the final volume, i,e 1 Litre and heated with continuous

stirring until all the agar is dissolved and the solution becomes transparent.

6.

The medium is dispensed in glass or polypropylene vessels and plugged with cotton

plugs.

7. Culture medium is sterilized in an autoclave for 20 min at 121oC at 15 psi (105 kPa).

8. If the medium contains heat-labile substances :F

a. steps 1- 5 are followed except for the addition of heat labile substances.

b. Culture medium is sterilized as such in a big Erienmeyes flask without dispensing in

vessels in an autoclave for 20-25 min at 121oC at 15 psi (105 kPa).

c.

the thermolabile compound solutions are filter sterilized using millipore or any other

filter assembly using 0.22 m filter.

d. After autoclaving, the medium is kept in a laminar airflow hood and allowed to cool to a

temperature of around 50oC. The requisite quantity of the compound is added to the medium

with the help of micropipettes while the mediuim is being stirred.

e. the medium is dispensed into sterile containers (generally sterile petri dishes) under the

hood of laminar airflow, provided the neck of the Erlenmeyer flask is passed over a flame

before the medium is poured from it.

1 Medium is allowed to cool and solidify in a laminar airflow hood.

2. Prepation of the commercial medium:

1. The commercial medium which is obtained contains the nutritional components, agar

and sucrose. But it is devoid of CaCl2and the growth factors. These components have to


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be prepared as stock solution and added in the medium during preparation.

2. The obtained medium should be disolved in 1L of water, if the amount of the the total

content is mentioned it should be weighed and dissolved in the appropriate amount of

water and should be heated for complete dissolving of agar. The medium while heating

should be strried continuously inorder to prevent charing of agar.

3. After dissolving it the required amount is dispensed into culture tubes and kept for

autoclaving.

4. After sterilization the medium is cooled to the room temperature.

5. Medium is allowed to cool and solidity in a laminar airflow hood.

STORAGE OF MEDIA

After cooling, the media containers are stored preferably at 4-10oC but that is not absolutely

necessary. Medium should be used after 3-4 days of preparation, so that it medium is not

properly sterilized, contamination will start to appear.

Table 4 Preparation of stock solutions of Murashige and Skoog (MS) medium

Concentration inConcentration in Volume to be

Constituent the stock solution taken/litre of

MS medium (mg/l) (mg/l) medium

Macronutrients (10x)Stock solution I

NH4NO3 1650 16500 100 ml

KNO3 1900 19000MgSO4. 7H2O 370 3700

KH2PO4 170 1700

Macronutrient (10x) Stock solution II

CaCl22H2O 440 4400 100 ml

Micronutrients (100x) Stock solution III

H3BO3 6.2 620 10 ml

MnSO4. 4H2O 22.3 2230

ZnSO4. 7H2O 8.6 860

Kl 0.83 83

Na2MoO4.2H2O 0.25 25

CuSO45H2O 0.025 2.5

CoCl2. 6H2O 0.025 2.5

Iron source


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Fe EDTA Na salt 40 Added fresh

Vitamins

Nicotinic acid 0.5 50 mg/100 ml 1 ml

Thiamine HCl 0.1 50 mg/100 ml 0.2 ml

Pyridoxine HCl 0.5 50 mg/100 ml 1 mlMyo-inositol 100 Added fresh

Others

Glycine 2.0 50 mg/100 ml 4 ml

Sucrose 30,000 Added fresh

Agar 8000 Added fresh

pH 5.8

Nutrientmedium chart for preparation of culture medium

Quantity required

Stock

Quantity

for volume of

Constituents solution medium underRemarksrequired for 1 L(conc.) preparation

(e.g. 500ml)

Macro stock 10x 100ml 50 ml

solution I

Macro stock 10x 100 ml 50 ml

solution II (CaCl2)

Micro stock 100x 10 ml 5 ml

solution III

Iron-EDTA Na Added fresh 40 mg 20 mgsalt

Vitamins

Nicotinic acid 50 mg/100 ml 0.5 mg/l = 1 ml 0.5 ml

Thiamine HCl 50 mg/100 ml 0.1 mg/1 = 0.2ml 0.1 ml

Pyridoxine HCl 50 mg/100 ml 0.5 mg/l = 1ml 0.5 ml

Myo-inositol Added fresh 100 mg 50 mg

Others

Glycine 50 mg/100 ml 2 mg/l = 4ml 2.0 ml

Growthregulators

Sucrose Added fresh 30 g 15 g

Agar Added fresh 8 g 4 g

pH


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Ex.No: Date:

PLANT TISSUE CULTURE- AN INTRODUCTION

Plant tissue culture is the science or art of growing plant cells, tissues or organs isolated from the

mother plant, on artificial media. The culture initiation and plant regeneration are still

accomplished ampirically by varying conditions until the desired response is obtained. Scheiden

and Schwanns suggestion that the totipotency of cells was the foundation of the plant cell and

the tissue culture. However it was Haberlandt who look the first real steps in in vitro cultures.

Since then many investigate have studied plant regeneration under in vitro conditions and have

obtained further understanding of cell totipotency.

Tissue culture is a collective term commonly used to describe all kinds of in vitroplant cultures.

The tissue cultures can be cultures of an organized tissue, viz., callus cultures, suspension or cell

cultures, protoplat cultures, pollen cultures etc., or cultures of organized structure like meristem

cultures, shoot and root cultures, embryo cultures, inflorescence cultures, ovule cultures, etc..

Plant tissue culture has direct commercial applications as well as value in basic research into cell

biology, genetics and biochemistry.

Prepartion of glassware:

Cleaning:

It is important to use clean glassware for the growth of tissue in vitro. In our laboratory these

include:

1. Boil all glassware in washing soda solution (10%) for 1 hour.

2. Rinse thoroughly with tap water and leave in HCl (1N) for 2 hours.

3. Remove traces of acid by thorough washing with tap water.

4. Rinse glassware with double-distilled water.

5. Allow glassware to dry overnight at room temperature.

Sterilization:

The objective of sterilization is to make media, glassware and instruments free frommicroorganisms. It is accomplished by wet heat, dry heat or filteration.

1)Wet-heat sterilization:

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


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2. Wrap petriplates dishes with wrapping paper or aluminium foil.

3. Place forceps and scalpels in test tubes. Plug the tubes with non-absorbent cotton and cover

with brown paper.

4. Plug the mouth end of graduated pipettes (1, 2, 5 and 10ml) with cotton. wrap them

individually in wrapping paper or aluminium foil.

5. Autoclave glassware and instruments at 1210C for 1 hour.

2)Dry-heat sterilization:

Steps 1-4 are similar as wet-heat sterilization. But the final step involves placing the instruments

in an oven at 1400-160

0C for 2 hours.

3)Filter sterilization:

Aminoacids, vitamins, phytohormones,etc.. may get destroyed during autoclaving. So such as

chemicals are therefore sterilized by filtration through a Seitz or Millipore filtration assemblyusing filter membranes of 0.45 or 0.22m porosity.

1. Plug 500 or 1000ml receiver flask with cotton.

2. Assemble Millipore filtration unit with bacteriological membrane filter (0.45 or 0.22 m).

3. Wrap filtration until with wrapping paper

4. Autoclave receiver flask and filtration unit at 121C for 1 hour (do not sterilize in dry heat as

membrane filters get damaged)

5. Fix filtration unit to the receiver flask in a sterile cabinet.

6. Pour solution to be sterilized into the filtration.

7. Apply slight air pressure to commence filtration (don not exceed air pressure by 7.03

Kg/cm2).

8. Transfer under aseptic conditions to sterile flasks.

9. Using a sterile pipette and filter sterilized solution to the autoclaved medium, shake well and

dispense into sterile cultures tubes or flasks under aseptic conditions.

Preparation of media:

The appropriate composition of the medium largely determines 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 cultures on the nutrient salt solutions formulated by Gautheret and White. However,


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these were found inadequate for sustaining growth of many plant tissues. This led to the

formation of several basal nutrient media (Hildebrandt et al., 1946; Burkholder & Nickel,1949;

Heller,1953; Murashige and Skoog,1962; Gamborg et al., 1968). In general, the medium

contains 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, phosphorous and sulphur

constitute the major salts. Nitrogen is generally used as nitrate or ammonium salts, sulphur as

sulphates and phosphorus as phosphates.

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.

Vitamins:

The B-vitamins play an important role in 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.

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-dichlorophenoxy acetic acid

are the frequently used auxins. These are generally used at 0.1 to 10mg/l concentration in plant

tissue culture media. Naphthalene acetic acid and 2,4-dichlorophenoxy acetic acid are

thermostable and do not lose their activity on autoclaving. Whereas, Indole acetic acid is

thermolabile and loses most of their activity on autoclaving. Hence, it is sterilized by filtration.

Cytokinins:Cytokinins have a profound effect on cell division and cell differentiation. Kinetin, zeatin and 6-

benzylamino purine, the commonly used cytokinins, are used in 0.1-10mg/l concentration.

Others:


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Gibberellic acid, ethylene releasing compounds and abscisic acid are also incorporated in the

media.

Carbon sources:

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 substance such as yeast extract, malt-extract and casein hydrolysates are also added

(0.1- 1% w/v).

Among various plant extracts, liquid endosperm of immature (coconut water) is widely used (5-

20%v/v)

General methodology for media preparation:

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 1 litre of the medium:

Transfer appropriate amounts of stock solution of salts to 1 litre flasks.

1. Add vitamins, auxins, cytokinins and inorganic supplements.

2. Add a carbohydrate source such as sucrose (2-5%)

3. Adjust the pH to 5.6-5.8 using a pH meter.

4. Make up the volume to 1 litre with double distilled water.

5.

Add powdered agar 8g/l for making the medium semisolid.

6. Cover the flask with paper of aluminium foil and keep in a steamer or water-bath at 1000C for

dissolving the agar.

7. Shake the flask well for the uniform distribution of agar.

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

9. Autoclave tubes or flasks containing medium at 1210C for 20 minutes.

10.Again steam the medium for 20 minutes on the following day (i.e the day after it is

prepared).

Surface sterilization of plant material:

Before inoculating the medium with the explants, it is necessary to surface sterilize it. There are

many sterilizing agents such as calcium hypochorite, chlorine water, bleaching powder, mercuric


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chloride, hydrogen peroxide and ethylene oxide. The concentration of the sterilizing agent and

the duration of treatment varies with the plant material. These should be 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.

a)

Sterilization of seeds:

1. About 15-20 healthy seeds were selected and wash them thoroughly with water.

2. Now added 2-3 drops of liquid detergent 100ml of water and shake well for 5 minutes.

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

4. Rinse seeds in 50ml of 70% ethanol for 1 minutes.

5. Wash thoroughly 3-4 times with single distilled water.

6. Transfer seeds in a sterile 250ml flask and add mercuric chloride solution (0.1%).

7. Treat for 20 minutes, shake the flask occasionally. All the operations thereafter should be

carried out under sterile conditions.

8. 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.

b) Sterilization of leaves:

1. Collect the apical portion about 20cm long from 6-8 month old plants.

2. Remove the outer green sheathing leaves with a scalpel till the inner thin and white leaves are

exposed.

3.

Dip 2-3 cm long segments of this portion in 70%alcohol for 1 minute. Then follow the steps 1-

8 as in sterilization of seeds.

4. Using sterile forceps, place segments in a sterile petridish and remove 1-2 outer leaves with

scalpel.

5. Cut the inner leaves into 5mm segments.

6. Transfer 1-2 leaf segments to each tube containing 20ml of semisolid medium.


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25

Ex.No: Date:

MICROPROPAGATION

Tissue culture is particularly useful for multiplication of plants which are slow growing

(turmeric, ginger, cardamom); cross- pollinated (coconut, teak, eucaluptus, cashew, mango and

those which show wide variation in the progeny); male-sterile lines (cotton, sorghum, pearl-

millet); newly produced varieties (normally vegetatively propagated); and for multiplication of

virus free plants by meristem cultures (sugarcane, potatoes, tapioca, etc..).

Tissue culture is now being commonly used for clonal propagation of a large number

of horticultural plants. Crop plants and also for forest trees (Murashige;1974; Conger,1981).

The success of clonal multiplication in higher plants depends generally on 3 main stages:

STAGE 1: ESTABLISHMENT OF AN ASEPTIC CULTURE

The explants taken from the plant has first to be made free of microorganisms which would

outgrow the plant tissue when placed on a nutrient medium. This would result in the death of the

explants. These surface contaminants, e.g. bacteria, fungi and yeast are removed by surface

sterilization prior to culture, but without killing the plant tissue.

STAGE 2: MULTIPLICATION

The surface sterilized material when inoculated on sterile nutrient media and incubated at

252C with a definite photoperiod and light intensity grows to form large nuber of shoots.

STAGE 3: ROOTING AND HARDENING OF PLANTS

The shoots obtained are carefully excised and transferred to a rooting medium,

preferably a liquid medium,containing an auxin and supported on a filterpaper platform in

order to obtain rooting in these shoots.

These plants which have rooted and have developed secondary roots with root hairs

can be transferred to pots containing soil:vermiculite mixture (1:1). This mixture is

preautoclaved for 1 hour at 15 psi and steamed for 3 days successively and cooled. The

potted plants can be transferred to the field where the first new leaf emerges.

MULTIPLICATION BY SUBCULTURE AT STAGE 2However, excised shoot tips can be inoculated on the same medium used in stage 2 instead

of the rooting media. By regular repetition of this subculture procedure, high rates of

multiplication can be achieved.

Vegetative multiplication of plants depends on various factors as nutrient medium, agar


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26

concentration, photoperiod and light intensity, hydrogen ion concentration, size and source of the

explants (Conger,1983).

Explants source:

Requirements:

a)

Equipments

Conical flasks (100ml capacity) Test tubes (25mm*150mm) Petridishes (80mm diameter)

Pair of forceps and scalpel (15 cm long), Environmental growth cabinets adjusted to 252C with

18hr photoperiod and 1500lux intensity and 152 and 600 lux light intensity, Shaker with

120rpm and 1000 lux light intensity.

b) Culture media, washing solutions, sterilizing agents, Glass distilled water, Sterile glass distilled

water0.5%HgCl solutionDetergent, Medium

c)Source tissue

Procedure

a. Sterilization of glassware

b. Preparation and sterilization of media

c. Explants collection:

1. Select a twig (60-90 cm long, 10-15mm wide) from mature elite trees and cut, making sure

that the twig contains many young axillary buds. The length is important in selecting twig

that do not wither before being brought to the laboratory.

2.

Bring the twigs containing axillary bud to the laboratory, remove the leaves and cut them

into small pieces of about 5-8 cm.

3. Transfer the buds to a sterile 250ml conical flask and surface sterilize the explants.

d. Culture of buds:

1. Keep sterile petridishes, scalpel, forceps and medium inside a sterile cabinet along with the

flask containing surface-sterilized explants.

2. Transfer these explants into sterile petri dishes with the help of a pair of sterile forceps and

cut these explants into small pieces of 10-15 mm each containing atleast one axillarybud.3. Inoculate 2 pieces to each tube containing medium.

4. Incubate the tubes in an environment growth cabinet at 152 and 500 lux light intensity for

72 hours.

5. Transfer the cultures after 72hr to another incubator maintained at 252C with 16hr


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27

photoperiod and 1500lux intensity.

6. After 25 days, the young buds start sprouting.

7. When the sprouts are 10-15mm long, transfer them to liquid medium in 100 ml

Erlenmeyer flasks.

8.

Incubate the flasks on a rotatory shaker at 120 rpm and 500 lux light intensity.

9. Observe the formation of multiple shoots after 10-15 days.

e. Multiplication by subculture:

1. Transfer the multiple shoots from the flask to a sterile petridish aseptically.

2. Incubate the cultures in an environmental growth cabinet at 252C and at 1000 lux light

intensity (12 hr photo periods) and observe the cultures regularly.

3. Observe the explants produces multiple shoots within 15 days.

4. Separate these shoots again aseptically and transfer the tubes containing medium for

shoot formation.

f. Transfer of plants to pots:

1. Remove the rooted plantlet from the tube and wash the roots gently with tap water to

remove any traces of medium.

2. Transfer the plantlets to soil: vermiculite (1:1) sterile mixture in a pot.

3. Irrigate with about 20 ml of tap water.

4. Keep the pots in a growth cabinet at 252C and at 1000 lux light intensity and water

them.

5. Transfer the plants to the field after 8 days of hardening in which 70-80% plants survive.

Result:

S.No. Number of bottles

inoculated

Number of explants

inoculated per bottle

Number of explants

developed per bottle

1.


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28

Ex.No: Date:

CALLUS INDUCTION

Callus is an actively-dividing non-organized mass of undifferentiated and

differentiated cells often developing either from injury (Wounding) or in tissue culture in

the presence of growth regulators. Explants from both mature and immature organs can

be induced to form callus. However, explants with mitotically active cells (young,

juvenile cells) are generally good for callus initiation. Callus is produced on explants in

vitro from peripheral layers as a result of wounding and in response to growth regulators,

either endogenous or exogenously supplied in the medium. The season of the year, donor

conditions of the plant, the age and physiological state of the parent plant contribute to

the success of organogenesis in cell cultures.

Growth regulator concentration in the culture medium is critical for morphogenesis.

Auxin, at a moderate to high concentration, is the primary hormone used to produce

callus. In some species, a high concentration of auxin and a low concentration of

cytokinin in the medium promotes abundant cell proliferation with the formation of

callus. Callus may be serially subcultured and grown for extended periods, but its

composition and structure may change with time as certain cells are favoured by the

medium and come to dominate the culture.

Callus tissue from different plants species may be different in structure and growth habit:

white or coloured, soft (watery) or hard, friable (easy to separate in to cells) or compact.

The callus growth within a plant species is dependent on various factors such as the

original position of the explant within the plant, and the growth conditions.

Although the callus remains unorganized, with increasing growth, some kinds of

specialized cells may be formed again. Such differentiation can appear to take place at

random, but may be associated with centers of morphogenesis, which can give rise to

organs such as roots, shoots and embryos.

AIM:

To induce callus from the explants of Phaseolus mungo(Green Gram)

Reagents and other requirements

1. Culture tubes or conical flasks containing media

2. Sterile Petri dishes


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29

3. Scalpel, blades, forceps and steel dissecting needles

4. Sterile distilled water

5. Alcohol

6. Detergent (Tween 20, Teepol, etc.)

7. SterilantsHgCl2, Sodium. Hypochlorite

8.Nutrition medium reagents MS basic salts and

vitamins Growth regulators2, 4-D

Plant materialGreen gram

Media

Seed Germination: MS Medium

Callus Induction: MS + 2, 4-D (2mg/lL)

I.

Seed Germination

1. The seeds washed by submerging in water with a few drops of detergent in a

beaker with vigorous shaking.

2. The seeds were submerge in 70% alcohol for 40 s after which the alcohol was

decanted.

3. The seeds were transfer to a flask containing 20% commercial sodium

hypochlorite solution and left there for 20 min for surface sterilization. Later they

were rinsed thrice with sterile distilled water.

4. 2-3 seeds were placed on the surface of MS medium and incubated at 25oC for 16

h photoperiod with 250 E/m2/ s light intensity for 2 weeks.

5. Observe regularly for germination. If need be, transfer the individual plantlets to

half MS medium.

II.Callus Induction

1. The leaves were removed from in vitrogerminated seeds 2 weeks were cut into

pieces and placed on the MS mediu.As a control measure, some explants should beinoculated on MS medium without hormones.

2. The cultures were incubated in dark at 25oC. Callus started appearing within 2

weeks and good callus growth can be observed in 3-4 weeks.

3. Callus can be subcultured after the 4th

week on fresh medium with the same


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30

composition.

Result:

The undifferentiated mass of cells was formed from the inoculated leaf explant.

Callus induction from the explant of Brassica


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31

Ex.No: Date:

DIRECT ORGANOGENESISSHOOT TIP CULTURE

Plant production through organogenesis can be achieved by two modes: (i)

Organogenesis through callus formation with de novo origin; and (ii) Emergence of

adventitious organs directly from the explant. In this exercise, organogenesis through

callus formation has been discussed.

By varying the growth regulator levels, i.e. lowering the auxin and increasing the

cytokinin concentration is traditionally performed to induce shoots from the explant. The

next phase involves the induction of roots from the shoots developed. IAA or IBA auxins,

either alone or in combination with a low concentration of cytokinin, are important in the

induction of root primordia. Thus organ formation is determined by quantitative

interaction, i.e. ratios rather than absolute concentrations of substances participating in

growth and development.

Aim :

To perform regeneration of the plant from shoot tip ofBougainvillea

Material Required :

1. Bougainvillea shoot tips

2. M.S Medium

3. Growth regulators: Auxin2,4 D (0.5mg/l)

4. CytokininBAP (1.5mg/l)

Procedure:

1. M.S medium was amended with required concentration of growth hormones and

sterilized

2. Shoot tips ofBougainvilleawere obtained from 5 year old plants growing outside

in the lawn campus. One cm long shoot tips of Bougainvillea

3.

The apical portions of the plant were collected and the shoot tip was carefully cut

using a sterile surgical blade.

4. It is washed under running tap water for 30 mins to remove dust particles and then

surface sterilized in alcohol for 30s followed by sodium hypochloride for 3 mins and

rinsed thoroughly in sterile distilled water.


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32

5. The explant was then air dried and inoculated in the medium and incubated at

25oC for 16 h photoperiod with 250 E/m

2/ s light intensity for 2 weeks.

Result:

Shoot proliferation was observed after 2 weeks from the explants.

Shoot tip culture Bougainvilla


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33

Ex.No: Date:

EMBRYO CULTURE

Aim:

To isolate embryos of Cicer aertinumand perform in vitroculture

Requirement:

1. Sterilants - alcohol, HgCl2, sodium hypochlorite

2. Nutrition medium reagents - MS basic salts and vitamins

3. Growth regulatorsusually not required for embryogenesis

4. Plant Material- Embryo of Cicer auritinum

5. Culture tubes containing media

6.

Sterile Petri dishes

7. Scalpel, blades, forceps knives and steel-dissecting needles

8. Sterile distilled water

Procedure:

1. The seeds were washed by submerging them in water with a few drops of

detergent in a beaker and shake them by hand.

2. The embryo was teased and collected without any damage

3.

It was washed with distilled water and then treated with 70% alcohol for 30

seconds.

4. This was followed by rinsing completely with distilled water and then transferred

to 20% sodium hypochlorite, where it was left for 0 minutes.

5. Then the embryo was thoroughly rinsed with distilled water for 3 times and dried

using the autoclaved tissue paper and inoculated in the culture tubes containing the

MS medium.

6.

The culture tubes were incubated at 25

o

C under 16 h photoperiod for 2 to 3weeks.

Result:

The plant was developed from inoculated embryo.


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34

Induction of plant let from the embryo of Cicer aerti num


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35

Ex.No: Date:

ISOLATION OF PROTOPLASTS

Protoplast is the living material of the cell where as an isolated protoplast is the cell from

which the cell wall is removed. In plant breeding programme many desirable

combination of characters could not be transmitted through the conventional method of

genetic manipulation. higher plants that could lead to the genetic process involving fusion

between the subsequent developments of a product to a hybrid plant is known as somatic

hybridization.

Plant protoplasts can be isolated from cells by two methods:

1. Mechanical method

2.

Enzymatic method.

MECHANICAL METHOD

Aim:

To isolate protoplast by mechanical method

Principle:

Protoplast can be isolated from almost all plant parts: roots, leaves, fruits, tuber, root

nodules, pollen mother cell etc. Protoplast isolated by mechanical is a crude and tedious

procedure.

Cells are plasmolysed causing the protoplast to shrink from the cell wall. The protoplast

obtained from this method is then cultured on suitable culture medium. The principle

deficiency of this approach is that the protoplast released is few in number. Mechanical

isolation was that of only historical event now.

Materials Required:

1. Plant leavesDuranta repens

2. Mortar and pestle

3.

Phosphate buffer pH-7.0

4. 0.3 M sorbitol

5. 0.3 M mannitol

6. Glass slides

7. Microscope.


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36

Procedure:

1. Young leaves were obtained from plants growing out doors and initially washed

with tap water to remove any dust particles.

2. The leaves were washed with phosphate buffer and homogenized gently with the

mortar and pestle.

3. The crude protoplast suspension was centrifuged at very low 50-100 rpm for 10

minutes.

4. The supernatant containing intact protoplast was carefully pipetted out and the

pellet containing cell debris and other cell organelles were discarded.

5. Small volume of supernatant was placed in the slides and covered with coverslip.

6.

The slide was observed in light microscope to find out viable protoplast

Result:

The spherical shaped protoplasts were observed using the microscope.


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37

Ex.No: Date:

ENZYMATIC METHOD

AIM:

To isolate protoplasts by enzymatic method

PRINCIPLE:

Protoplasts are isolated by treating tissues with a mixture of cell wall degrading enzyme

in solution, which contain osmotic stabilizer. A most suitable source of protoplasts is

mesophyll tissue from fully expanded leaves of young plants or new shoots. The release

of protoplast is very much dependent on the nature and composition of enzymes used to

digest the cell wall. There are three primary components of the cell wall which have been

identified as cellulose, hemicellulase and pectin substance. Pectinase (macrozyme)

mainly degrades the middle lamella while cellulose and hemicellulase degrades the

cellulose and hemicellulosic components of the cell wall. During this enzymatic

treatment, the protoplast obtained should be stabilized because the mechanical barrier of

the cell wall which offered support has been broken. For this reason an osmoticm is

added which prevents the protoplast from bursting.

MATERIALS REQUIRED:

1. Young leaves

2. 70% ethanol

3. 2% cellulose

4. 13% mannitol

5. 0.5% macrozyme

6. CPW salt solution:

KH2PO4 - 27.2mg/l

KNO3 - 101mg/l

CaCl2 - 1480mg/l

MgSo4 - 246mg/l

KI - 0.16mg/l

CaSo4 - 0,026mg/l

pH - 5.8.


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PROCEDURE:

1. The young leaves were collected and washed in sterile distilled water thrice.

2. The leaves were cut into small bits.

3.

Then the leaves were kept immersed in 13% mannitol for 1 h for pre-plasmolysis.

4. Mannitol was removed after incubation ant sterilized enzyme mixture (Cellulase +

macerozyme) was added and incubated at 25C in a shaker for 12 h

5. The filtrate was centrifuged at 100g for 5 min to sediment the protoplast.

6. The supernatant was removed and the protoplast pellet was suspended in 10ml of

CPW +21% sucrose solution.

7. The mixture was centrifuged at 100g for 5 min. The viable protoplast will float to

the surface of the sucrose solution.

8. The supernatant was collected and viewed under microscope.

9. The protoplasts were visualized in microscope.

RESULT:

Protoplasts were isolated by enzymatic method and viewed under the microscope


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Ex.No: Date:

ANTHER CULTURE

AIM:

To isolate and inoculate anthers for haploid production.

PRINCIPLE:

Haploids refer to those plants which possess a gametophytic number of chromosomes in

their sporophytes. Haploids may be grouped into two broad categories:

(a) monoploids which possess half the number of chromosomes from a diploid

species.

(b)

Polyhaploids which possess half the number of chromosomes from a polyploidy

species.

Haploid production through anther culture has been referred to as androgenesis while

gynogenesis is the production of haploid plants from ovary or ovule culture where the

female gamete or gametophyte is triggered to sporophytic development.

MATERIALS REQUIRED:-

1. Anthers fromHibiscus

2. MS medium

3. growth factors

4. 70% ethanol

5. 2% mercuric chloride

6. Meso inositol

7. Scissors

8. Scalples

9. Petriplates

10.

Forceps.

PROCEDURE:

1. Flower buds ofHibiscuswere collected.

2. The flower buds are surface sterilized by immersing in 70% ethanol for 60 sec

followed by immersing in 2% sodium hypochloride solution for 1 min or in mercuric


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chloride.

3. The buds were washed four or five times with sterile distilled water.

5. The buds were transferred to a sterile Petridish.

6.

The buds were split open using a blade and the anthers were removed without

damage and the filaments were removed.

7. The anthers were placed horizontally on the MS medium supplemented with

different concentration of plant growth regulators or mesoinositol.

8. The Petriplates were sealed and incubated in dark at 28C.

9. The Petriplates were examined for the germination of anthers.

RESULT:

The anther underwent germination leading to the formation of haploid plantlets.

Anthers inoculated on the MS medium


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Ex.No: Date:

CELL SUSPENSION CULTURE

INTRODUCTION:

A cell suspension culture consists of cell aggregates dispersed and growing in a moving liquid

media. It is normally initiated by transferring pieces of undifferentiated and friable callus to a

liquid medium, which is continuously agitated by a suitable device. Suspension cultures can

also be started from sterile seedlings or imbibed embryos or leaves by the mechanical method.

Leaves or the other tissues (e.g. hypocotyl, cotyledon, etc.) can be gently grinded or soft

tissues can be broken up in a hand-operated glass homogenizer. This homogenate, containing

intact living cells, dead cells and cell debris is cleared by filtration and centrifugation and then

transferred to moving liquid medium.

For general purpose, the objective with cell suspension cultures is to achieve rapid growth

rates, along with uniform cells, all the cells being viable. Cells should be subcultured at weekly

intervals or less if they are to be used for experimental purposes. Optimally, suspension

cultures passage length is 1-2 weeks, but the exact time and the dilution required must be

determined for each cell line. Dilutions of 1:4 after 1 week or 1:10 after 2 weeks are commonly

used. It is recommended that a small sample should be withdrawn and the cell density

determined before sub culturing.

Equipments:

Shaker

Centrifuge

Microscope

Haemocytometer

Reagents and other requirements

Sterilantsalcohol, HgCl2, sodium hypochlorite

Nutrient medium reagents -MS basic salts and vitamins

Growth regulators - 2, 4-D

Seedlings ofPhaseolus mungo


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Media

MS + (1.5mg/l) 2, 4-D

PROCEDURE

1. Seeds are germinated in vitro.

2. Seedlings are collected when the cotyledons are fully expanded and the epicotyl is

beginning to emerge (2-weeks). Each seedling is place on a sterile slide/Petri dish and

crush the expalnt gently.

3. MS liquid medium is prepared. For experiment purposes, 15 ml medium in a 125 ml

Erlenmeyer flask is taken. Sterilize the opening of a flask with the flame of a burner in the

hood.

4. Pieces of the gently crushed plant material are transferred to sterile petri dish to the

liquid media using pipette.

5. The culture is incubated on a gyratory shaker set at 125 rpm under controlled

conditions of temperature.

6. An aliquot from the flask on a glass slide while maintaining sterile conditions.

CELL COUNT

The cell number in very finely divided suspensions may be counted directly in a

haemocytometer. However, most cultures usually contain aggregates and it is difficult to count

the number of cells in each clump. Thus clumps are generally broken and then the cell number

is counted.

1. Place the haemocytometer and focus the center 25 squares.

2. Place the coverslip and Pipette out 100 l of suspension into a sterile

haemocytometer

3. The cells were counted in 4 corner squares and the center one is counted.

4. The number and the average are counted.RESULT:

Suspension culture containing clumps was observed


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Ex.No: Date:

INTRODUCTION FOR ANIMAL CELL CULTURE LABORATORY

Cell culture is an indispensable technique for understanding the structure and function of cells, in recent

times it has very good implications in biotechnology. Cultured animal cells are commercially used for the

production of interferon, vaccines and clinical materials like growth hormones and urokinase. In the process

of learning the techniques of cell culture and gene transfer you will become familiar with several

terminologies and hypotheses.

Make yourself familiar with the equipments (incubators, centrifuges and microscope etc.,). If you have

problems, approach the faculty members.

Good laboratory work habits will help you a grand success. Follow the following guide lines very strictly.

These will protect you and your experiments.

1. No eating and drinking in the lab.

2.

No storage of food materials in the lab.

3. No mouth pipetting. Use appropriate pipetting aids that are available.

4.

Wear gloves for certain experiments whenever it is necessary.

5. Work cleanly in an organized manner. Wipe your tissue culture hood bench with 70%

ethanol.

6. Most important, label every thing you use with your Reg. number, date and name of the

reagent, buffer or medium.

Handling Methods

Use sterile glassware and pipettes.When you open a glass bottle, before and after use flame the mouth in a flame.

Flame glass pipettes.

Use rubber bulbs to control pipettes. DO NOT MOUTH PIPETTE. This is to protect both you and the

cells from contamination

Prewarm medium and serum to 37C in water bath.

Wipe bottles with filter paper and transfer to the tissue culture hood (Laminar Air Flow).

Open bottles, flame tops, replace caps loosely but so they wont fall off.

Transfer, desired quantity of serum to the medium, or mix in separate (sterile) container.

Using sterile but NOT plugged, Pasteur pipette, aspirate the medium. Use a separate pipette for different

cells.

Using sterile pipette, transfer desired amount of medium and serum to the dish. Do not reuse the pipette.

Return dishes to incubator without shaking to avoid spill-over of medium.

Reflame tops of bottles and close tightly using aluminium foil. Close pipette cans.


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Work only in designated tissue culture area and Wipe surfaces with ethanol before starting.

Work cleanly, mopping spills immediately.

Discard used medium, and especially contaminated plates/TC bottles carefully. Otherwise, they serve as

sources of contamination.

Label bottles and dishes with Reg. No., cell type, date etc. When you open a new bottle of medium or

serum, write the date on it and indicate how much has been removed.

If you add anything to medium, indicate this on the bottle. If there is only a small amount left in a

bottle, discard it.

After you have finished, remove your belongings; discard properly. Eventually, wipe the working

surface with ethanol.

A sterile hood is available for your use. Keep UV, lamp on for at least 10 mins before use. Turn off the

UV lamp while you work.

Sterile glasswareSterile pipetes Autoclaved

Sterile medium

Sterile serum Milliporefiltered (0.45u)


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Ex.No: Date:

STERILIZATION TECHNIQUES

AIM : To prepare the materials required for various cell culture practices in sterile condition.

INTRODUCTION

The term control as used here refers to the reduction in numbers and or activity of the total microbial flora.

The principal reasons for controlling microorganisms and to prevent transmission of disease and infection, to

prevent contamination by or growth of undesirable microorganisms and to prevent deterioration and spoilage

of materials by microorganisms.

Microorganisms can be removed , inhibited or killed by various physical agents, physical processes or

chemical agents. A variety of techniques and agents are available, they act in many different ways and each

has kits own limits of applications.

Steam under pressure: Heat in the form of saturated steam under pressure is the most practical and

dependable agent for sterilization. Steam under pressure provides temperatures above those obtainable by

boiling as shown in Table 22-5. In addition, it has the advantages of rapid heating, penetration, and moisture

in abundance, which facilitates the coagulation of proteins.

TYPES OF STERLISATION TECHNIQUES

AUTOCLAVE

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

autoclave is an essential unit of equipment in every microbiology or cell culture laboratory. Many media,

solutions, discarded cultures, and contaminated materials are routinely sterilized with this apparatus.

Generally, but not always, the autoclave is operated at a pressure of approximately 15lb/in2

(1210

C). The

time of operation to achieve sterility depends on the nature of the material being sterilized, the type of the

container, and the volume. For example, 1000test tubes containing 10ml each of a liquid medium can be

sterilized in 10 to 15 min at 1210C, 10 litres of the same medium contained in a single container would

require 1hr or more at the same temperature to ensure sterilization..

BOILING WATER

Contaminated materials exposed to boiling water cannot be sterilized with certainty. It is true that all

vegetative cells will be destroyed within minutes by exposure to boiling water, but some bacterial spores can

withstand this condition for many hours. The practice of exposing instruments for short periods of time inboiling water is more likely to bring about disinfection(destruction of vegetative cells of disease producing

microorganisms) rather than sterilization. Boiling water cannot be used in the laboratory as a method of

sterilization..

DRY HEAT OR HOT AIR OVEN

Dry hear or hot air sterilization is recommended where it is either undesirable or unlikely that steam under


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pressure will make direct and complete contact with the materials to be sterilized. This is true of certain

items of laboratory glass wares, such as Petri dishes and pipettes, as well as oils, powders and similar

substances. The apparatus employed for this t7ype of sterilization may be a special electric or gas oven or

even the kitchen dry oven. For laboratory glassware, a 2h exposure to a temperature of 1600C is sufficient

for sterilization.

ULTRAVIOLET LIGHT

The ultraviolet portion of spectrum includes all radiation from 150 to 3900 A. Wavelengths around 2650A

have the highest bactericidal efficiency. Although the radiant energy of sunlight partly composed of

ultraviolet light, most of the shorter wavelengths of this type are filtered out by the earths atmosphere.

Consequently, the ultraviolet radiation at the surface of the earth is restricted to the span from about 2670A

to 3900A. From this, we may conclude that sunlight under certain conditions, has microbicidal capacity, but

to a limited degree. An important practical consideration in using this means of destroying microorganisms

is that ultraviolet light has very little ability to penetrate matter. Even as thin layer of glass filters off a large

percentage of the light. Thus, only the microorganisms on the surface of an object where they are exposed

directly to the ultraviolet light are susceptible to destruction.

FILTRATION

For many years a variety of filters have been available to the microbiologist which can remove

microorganisms from liquids or gases. These filters are made of different materials an asbestos pad in the

Seitz filter, diatomaceous earth in the Berkeloid filter, porcelain in the Chamberland-Pasteur filter and

sintered glass disks in other filters.The mean pore diameter in these bacteriological filters ranges from

approximately one to several microfilters; most filters are available in several grades based on the average

size of the pores. However, it should be understand that these filters do not act as mere mechanical sieves;

porosity alone is not the only factor preventing the passage of organisms. Other factors, such as the electric

charge of the filter, the electric charge carried by the organisms, and the nature of the fluid being filtered,

can influence the efficiency of filtration.

In recent years a new type of filter termed the membrane or molecular filter has been developed whose pores

are of a uniform and specific predetermined size. Membrane or molecular filters are composed of

biologically inert cellulose esters. They are prepared as circular membranes of about 150m thickness and

contain million of microscopic pores of very uniform diameter. Filters of this type can be produced with

known porosities ranging from approximately 0.01 to 10m. Membrane filters are used extensively in the

laboratory and in industry to sterilize fluid materials. They have been adapted to microbiological procedures

for the identification and enumeration of microorganisms from water samples and other materials. It is

customary to form the fluid through the filter by applying a negative press

industrial plant and animal biotechnology

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