Expt No: 14 Date :

PLANT TISSUE CULTURE

LABORATORY ORGANISATION

AIM
To establish a plant tissue culture laboratory in our college.

INTRODUCTION
The importance and application of plant tissue culture in plant science are
vast and varied. Plant tissue culture is considered to be an important tool under
biotechnology. The cells or tissues obtained from any part of the plant like stem,
root, leaf etc are encouraged to produce more cells in culture and there by express
their totipotency. Totipotency is the genetic potential of plant cells to produce the
entire plant.

Various types of cultures are advocated which includes organ culture, callus
culture, cell suspension, meristem culture, embryo culture, anther culture, pollen
culture etc. therefore, to carry out these experiments using tissue culture technique, a
well equipped plant tissue culture laboratory is required.

MATERIALS REQUIRED
Sterile rooms, laminar airflow, autoclave, hot air oven, distillation units, pH
meter, balance, test tubes, glass wares, forceps, scalpels, glass beads, sterilizer, air
conditioner etc.

PROTOCOL
An ideal plant tissue laboratory should have three rooms assigned for special
purposes. The 3 rooms selected to establish their purposes are:
1) Incubation/culture room
2) Inoculation room
3) Preparation room
 1) Incubation/ culture room

a) The culture racks with white fluorescent tube lights were fixed in the
culture room to maintain the sufficient light energy for the tissue
culture raised plants.
b) The incubation on culture room were also air conditioned to maintain
the suitable incubation condition (25±2° C) in the laboratory.
c) A separate provision was also made to create darkness for certain
experimental purposes.

2) Inoculation Room

The inoculation room was equipped with required instruments like the
laminar air flow chamber, hot bead sterilizer, a bunsen burner,
sterilants, glass wares and basic tools required for the purpose. It also
contains UV light for sterilization purposes.
3) Preparation Room

The preparation room was established for preparing media required

for tissue culture purpose. This room was equipped with a washing
area provided with large sink, having provision for running hot and
cold tap water, brushes of various sizes and a mild detergent.
Autoclave, hot air oven, pH meter, and water distillation unit were
also installed in the preparation room.
RESULT:
A full fledged tissue culture laboratory was established in our college.
Expt No: 15 Date :

PREPARATION OF
PLANT TISSUE CULTURE MEDIUM
AIM
To prepare Murashige and Skoog (MS) tissue culture medium.

PRINCIPLE
Artificially prepared nutrient medium is used for growing excised plant tissue
in controlled environment condition. The culture medium is composed of inorganic
salt which are supplied as macro and micro nutrients. Macronutrients are salts needed
in higher amounts like phosphorous, nitrogen, carbon, potassium, magnesium,
sodium etc. Micronutrients are added in little amounts like iron, manganese, boron,
copper, zinc, iodine, molybdate, cobalt etc.

EDTA is used as chelating agent and it ensures the availability at pH level up

to 8.Vitamins play an important role in the growth of tissue and sucrose is a
carbohydrate source. The hormones (or) growth regulator play an important role in
the growth source and differentiation of tissue in vitro. Plant growth regulators are
highly important to determine and enhance the cellular level division and
differentiation of plants. A range of natural and artificial plant growth regulators are
available which are added in media either individually at specific concentration (or)
in combination with other plant regulators for various tissue culture responses. Agar-
agar can be added as gelling agent for the preparation of semisolid media.

MATERIALS REQUIRED
Murashige- skoog salt, beakers, conical flasks, autoclave, weighing balance,
distilled water, pH meter, tissue culture vials, laminar air flow chamber, forceps,
scalpel etc.
COMPOSITION OF MS MEDIA (pH 5.8 ± 0.1)

Quantity for Quantity for

SN Constituents
1X media(mg/L) 100x media(g/L)
I Ammonium nitrate 1650.00 165
II Potassium nitrate 1900.00 190
Disodium EDTA 37.30 3.730
III
Ferrous sulphate 27.80 2.780
IV Calcium Chloride 440.00 44.00
Potassium Dihydrogen 170.00 17.00
V
Orthophosphate
Boric Acid 6.20 0.620
Sodium Molybdate 0.25 0.025
VI
Cobalt Chloride 0.025 0.0025
Potassium iodide 0.830 0.0830
Manganese sulphate 22.30 2.230
Magnesium sulphate 370.00 37.00
VII
Copper sulphate 0.250 0.025
Zinc sulphate 10.50 1.05
Thiamine-HCl 2.00 0.20
Nicotinic Acid 1.00 0.10
VIII
Pyridoxine HCl 1.00 0.10
Biotin 2.00 0.20
XI Mesoinositol 100 10

ROLE OF PLANT GROWTH REGULATORS ON CELL AND

TISSUE RESPONSES:

PLANT CELLULAR TISSUE

S.
GROWTH LEVEL LEVEL
No.
REGULATOR RESPONSE RESPONSE
Root induction &
1. Auxin Cell division
Callus induction
Cell
2. Cytokinin Shoot induction
differentiation
Root & Shoot
3. Gibberellic Acid Elongation
Elongation
Growth
4. Abscisic Acid Growth inhibition
inhibition

PROCEDURE
 (1) Individual stocks are prepared by mixing the respective chemicals at 100x
concentration.
(2) 10 ml of solution was taken from each stock and mixed together in a 2 litre
conical flask.
(3) 30 gm of sucrose was added
(4) Plant growth regulators must be added in this stage at appropriate quantity
if required.
(5) The solution was made up to 950 ml using double distilled water.
(6) The PH was adjusted to 5.8 using 0.1N NaOH.
(7) The final volume was made up to1L using double distilled water and 0.8%
agar-agar was added for the preparation of semi solid medium.
(8) The mixture was boiled in a microwave oven and dispersed into suitable
culture vials and test tubes.
(9) The vials containing the medium were autoclaved at 121˚C for 20 min,15
lbs pressure.
(10) Sterilized medium from the autoclave was taken out and kept at room
temperature for solidification.
(11) The solidified MS medium was used for all tissue culture experiments.
RESULT:
MS medium was prepared in tissue culture tubes and used for all tissue
culture experiments. There was a good growth and response obtained in cultured
explants and there was no trace of contamination.
Expt. No: 16 Date :
STERILIZATION PROCEDURES FOR
PLANT TISSUE CULTURE

AIM
To sterilize culture room laminar air flow chamber, glass wares, culture
media and explants required for various tissue culture applications.

PRINCIPLE
The culture medium, especially when it contains sugars as carbon source,
sucrose will also support the growth of micro organisms like bacteria, fungi etc. So if
they come in contact with medium either in cellular form or in spore form, the micro
organisms grow faster than the higher plant tissue due to their brief life cycle and
will kill the tissue. The micro organisms may come from glass vials, instruments,
nutrient medium used for culture and even from plant material itself. Therefore, the
surface of plant tissue and all non living articles including nutrient medium should be
sterilized.

MATERIALS REQUIRED
Autoclave, laminar air flow chamber, glass bead sterilizer,70% alcohol,
0.1%HgCl2, sterile distilled water, formaldehyde , KMN o4, bunsen burner, sterile
forceps, cotton, glass vial, medium etc.

PROCEDURE
There are several possible sources of contamination during plant tissue
culture experiments
1. The environment of culture room and transfer area.
2. Culture vessels and instruments used during inoculation and subculture
process.
3. The media and the explants.

It is therefore essential to maintain a completely aseptic environment inside the

tissue culture laboratory.
(1) THE ENVIRONMENT OF THE CULTURE ROOM
The sterilization of the culture room is brought about by fumigation with
Formaldehyde and Potassium permanganate. The excess of the fumigant is removed
by usage of sudden absorbents like Ammonia.

(2) THE ENVIRONMENT OF TRANSFER AREA

It’s very essential that all precautions are taken to prevent the entry of any
contaminant into the culture vials when its mouth is opened either for sub culturing
or for inoculating fresh tissues. To achieve this, all transfer operations are carried
strictly under aseptic condition.

Sterile transfer is usually done in laminar air flow cabinet. Before the start of
any sterile procedure, the table surface of the cabinet should be swapped with ethanol
and the UV lamp should be put on for 15-20 minutes. Sterilization is done only for
20 minutes after the air flow is put on. The laminar air flow cabinet is equipped with
high efficiency particulate air filters (HEPA) which removes particles larger than
0.3µ and the ultra clean air (free of fungal and bacterial contaminants) flows through
the working area . The air flow doesn’t in any way hamper the use of a spirit lamp or
bunsen burner.

(3) CULTURE VESSELS AND INTRUMENTS

Glass culture vials are mostly sterilized together with media. The glass vials
are sterilized by autoclaving at 121˚C for 15 min at 15 lbs pressure. The objects to be
sterilized are wrapped in Aluminium foil and kept for autoclaving.

The instruments used for aseptic manipulations such as forceps, scalpel, blade
etc are normally sterilized by dipping in 70% alcohol followed by flaming and
cooling. This is done at the start of the transfer work and it is repeated several times
during the operations. Autoclaving is not recommended for metal instruments
because they may rust and become blunt.
(4) THE CULTURE MEDIA
Microbial contaminants are present in the medium right from the start. To
destroy them, the mouth of the container is properly closed using a Bacteria proof
closure and is autoclaved at 121˚C for 15-20 min..Nutritive value of the culture
media may be reduced by prolonged sterilization. It also results in decomposition of
chemicals present in the medium. Some growth factors like gibberllic acid, zeatin,
absicic acid are thermo-labile. When using such compounds, the sterilization is
usually carried out by Membrane filtration technique. For filter sterilization of the
solution, bacterial proof filter membranes of pore size 0.22µ or less are used.

(5) THE EXPLANT

Surface of plants parts carry a wide range of microbial contaminants. Plant
material which is to be cultured should be surface sterilized to remove the surface –
born micro organisms. The explants are cut into suitable size and the surface
sterilization is brought about by treatment with 0.1% HgCl 2. Since these surface
sterilants are toxic to the plant tissues, they are thoroughly washed thrice with sterile
distilled water to remove traces of the sterilant. The explants part exposed to HgCl2
are excised using sterile blades and subsequently used for inoculation.

RESULT:
The culture room, laminar air flow chamber, glass wares, culture media and
explants were sterilized and used for various tissue culture practicals.
Expt. No: 17 Date :

IN VITRO GERMINATION OF SEEDS

AIM
Germination of seeds under in vitro conditions.

PRINCIPLE
Normally, seeds require water and congenialdimatic condition for sprouting.
In vitro techniques can be adopted for the germination of seeds under nutrients and
conditions available in the tissue culture medium. The main application of invitro
seed germination is to produce sterile explants for the further culturing. The plants
which produce abortive or adventive embryos can be germinated easily by this
method. Thus the time period of regeneration can be reduced.

MATERIALS REQUIRED
Seeds, mercuric chloride, sterile distilled water, sterilized Petri plates, scalpel,
blade, forceps, MS medium, culture vials, bunsen burner.

PROCEDURE
(1) COLLECTION OF EXPLANT
Seeds of Cicer arictinum were collected.

(2) PREPARATION OF MEDIA

MS media was prepared by mixing the recommended macro nutrients
followed by sterilization.
(3)STERILIZATION OF SEED
Subjected to surface cleaning using distilled water. Then, the seeds were
rinsed with 0.1% mercuric chloride for 5 minutes, to remove contamination.
Following this, the seeds were washed with sterile distilled water for 3-4 minutes.

(4) CULTURING OF STERILISED SEEDS

The sterilized seeds were transferred to culture vials containing media under
sterilized condition in laminar air flow chamber.
 (5) INCUBATION
The culture was incubated in tissue culture laboratory at 25±2˚C and kept
under fluorescent light (1500 lux).The lux relative humidity was maintained at
85±5%

RESULT:
The moist Cicer arietinum seeds were germinated under in vitro conditions
after 3 weeks.
Expt. No: 18 Date :

CALLUS INDUCTION

AIM
Induction of callus from a cotyledon explants under in vitro conditions.

PRINCIPLE
Callus tissue means an unorganized proliferate mass of cells produced from
isolated plant cells, tissues (or) organs when grown aseptically on artificial nutrient
medium. Any part of the plant like shoot-apex, bud, cambium, anther, pollen, fruit
etc can be used as explants.
The callus tissues can be used to produce embryoids which directly develop
into plantlets, eventually giving rise to a whole viable plant.
MATERIALS REQUIRED
Explants, MS medium with plant growth regulators, mercuric chloride, test
tubes, blade, sterile distilled water, sterilants, laminar air flow chamber,etc.
PROCEDURE
Preparation and sterilization of explants
1. The fruit wall was cut opened using sterile blade and forceps.
2. The explants were washed in running water.
3. The explants were immersed in a beaker containing Mercuric chloride,
transferred to the laminar air flow and then washed with sterile distilled
water.
4. The above process was repeated thrice and the explants were kept in a clean
petridish.
5. The seed coat was removed with a knife and the cotyledons were obtained.
6. The explants were carefully taken with the forceps and placed on the center
of the medium in a test tube
7. The test tubes were tightly plugged with cotton in order to maintain sterile
conditions.
8. Then, the tubes were transferred into the tissue culture laboratory and
incubated at 25°C under white fluorescent light [1500 lux] and 85±5%
 relative humidity.
9. Careful observations were made daily.
RESULT:
The tubes with explants showed the induction of callus after 1 week. Callus
formation in different plant growth regulators supplemented with MS medium is
tabulated in the table.
Expt. No: 19 Date :
EMBRYO CULTURE
AIM
In vitro isolation of zygotic embryo and culturing in suitable culture medium.
PRINCIPLE
For the natural growth of zygotic embryo, nutrients are provided by the
cotyledons during seed germination. However, naked embryo culture may be
required for certain cases where natural germination is difficult or not possible.
Under in vitro conditions, the nutrients for zygotic embryo to develop are provided
by the nutrients present in the medium.
MATERIALS REQUIRED
Explants, beaker, petri plates, sterile blade, forceps, culture vials, 0.1%
mercuric chloride [HgCl2], sterile distilled water, MS medium, laminar air flow
chamber, etc.,
PROCEDURE

1. Young seeds were taken and washed with tap water.

2. Seeds were sterilized by rinsing in mercuric chloride [HgCl 2] 0.1% for 5
minutes.
3. These seeds were washed thrice with sterile distilled water to remove
mercuric chloride traces from the explants.
4. Seed coat was then removed and embryo was excised out under sterile
conditions.
5. These excised embryos were transferred to culture vials containing MS
media.
6. Culture vials were incubated at 25ºC under white fluorescent light [1500
lux] and 85±5% relative humidity was maintained in the tissue- culture
laboratory.
RESULT:
Embryos were germinated after one week of incubation in MS media.
Expt. No: 20 Date :

SOMATIC EMBRYOGENESIS

AIM
In vitro production of somatic embryos from stem hits.

PRINCIPLE
Somatic embryogenesis is the process in which single cells or group of cells
initiating the developmental pathway that leads to reproducible regeneration of non-
zygotic embryos capable of germination to form complete plants.
MATERIALS REQUIRED
Explants, culture tubes, petri plates, conical flask, 70% alcohol, distilled
water, sterile blade, forceps, culture vials, laminar flow chamber, MS media with
explant growth regulators.
PROCEDURE
1. The MS media was prepared with different concentration of plant growth
regulators.
2. The explant was taken to the laminar air flow-chamber and cut into small
pieces using sterile blade.
3. The stem pieces used as explant were then carefully placed at the centre of
the culture tube containing media.
4. The tubes were tightly capped.
5. The culture tube was transferred to the culture chamber and incubated at 25ºC
under white fluorescent light.
6. Observations were made daily and the changes were noted.
RESULT:
At the end of the fourth week, embryogenic callus were obtained at the cut
end.
Expt. No: 21 Date :

ISOLATION AND REGENERATION OF

PROTOPLASTS

AIM
To isolate the protoplast from Catharanthus roseus leaf explant and culturing
them for regeneration.

PRINCIPLE
Mesophyll tissue is the most frequent choice for isolation of protoplasts
because of high yield and uniformity of protoplast.
Experimentally produced protoplasts are known as “isolated protoplast”.
Isolated protoplast is plant cells surrounded by plasma membrane which is
potentially capable of cell wall regeneration, cell division, growth and plant
regeneration in culture.
The cells are stabilized by plasmolysis and the protoplasts are released from
cell wall either mechanically or enzymatically, intact tissue can be incubated with
pectinase which will dissolve the middle lamella between the cells and separate
them. Subsequent treatment with cellulase will digest the cellulosic cell wall. These
two enzymatic treatment can be done either simultaneously [direct technique] or
separately [sequential technique].
MATERIALS REQUIRED
EXPLANT: Leaf tissue
REAGENTS: CPW media, Mannitol, cellulose (Hi-media), pectinase (Hi-
media) and MS media.
OTHER MATERIALS: Membrane filtration unit, glass syringe, glass slide,
microscope, glass vials, centrifuge tubes.
CPW MEDIA
KH2PO4 - 27.2 mg/l
KNO3 - 101.0 mg/l
CaCl2.2H2O - 1480.0 mg/l
MgSO4.7H2O - 246.0 mg/l
 KI - 0.16 mg/l
CuSO4.5H2O - 0.25 mg/l
1) WASH SOLUTION (pH-5.8)
CPW Medium
Mannitol - 13 %
2) ENZYME MIXTURE (pH-5.8)
CPW Medium
Mannitol - 13 %
Pectinase - 2%
Cellulase - 2%
3) FLOATING MEDIA (pH-5.8)
CPW Medium
Mannitol - 13 %
Sucrose - 22 %
4) CULTURE MEDIA (pH-5.8)
MS Media
Mannitol -9%
Sucrose -1%
PROCEDURE
1. Young fully expanded leaves from the upper parts of plants growing in a
green house are detached and leaves are washed with tap water.

2. Surface sterilization of leaves was first done by immersing in 0.1%

mercuric chloride for 5 minutes followed by a distilled water wash.

3. The sterilant was poured off and leaves were washed aseptically with
sterile double distilled water for 3-4 times.

4. Lower epidermis of the leaves were peeled off as completely as possible

and cut into small pieces for better enzyme contact.

5. The peeled off leaf bits were incubated in 10 ml enzyme mixture in a

glass vial at 25ºC for 16-18 hours.

6. The digested leaf pieces were gently agitated and squeezed with sterile
 forceps to facilitate the release of protoplast.

7. The protoplast suspension was then allowed to pass through 100 mesh
sieve.

8. The filtrate was transferred to centrifuge tube and spinned at 2000 rpm for
5 minutes.

9. The filtrate or supernatant was discarded and the pellet was suspended in
fresh wash solution.

10. The centrifugation was repeated thrice.

11. The washed protoplasts were purified using floating medium by

centrifugation.

12. Viable protoplasts were collected from the surface and resuspended in
culture media.

13. The protoplasts were observed under microscope to check the viability
and yield.

14. The protoplasts were allowed to be cultured in a petriplate by micro

droplet techniques and suspension culture.

RESULT:
Intact live protoplasts were observed under light microscope. This can be
used for further regeneration or fusion purpose. Regeneration of protoplast is
in progress.
Expt. No: 22 Date :

PROTOPLAST FUSION

AIM
To induce fusion among isolated Catharanthus roseus protoplasts.
PRINCIPLE
In vitro fusion of plant protoplast derived either from somatic cell of same
plant or from two genetically different plants is called somatic hybridization. It
overcomes the species barrier of sexual hybridization.
Polyethylene glycol (PEG) is the most commonly employed fusagen in order
to obtain high frequency of heterokaryon viability, it is essential to use PEG with low
carbonyl content. The fused protoplasts are grown in vitro conditions with an aim to
obtained hybrid plants. Hybrids can be identified based on their pigmentation,
nuclear staining or cytoplasmic markers.
MATERIALS REQUIRED
Isolated protoplasts, 30% PEG, 50% MS media with 9% mannitol, sterile
tips, beakers, glass slides etc.
PROCEDURE
1. A solution of 30% PEG and 50% PEG was prepared in MS medium with 9%
mannitol separately and sterilized

2. Suspension of protoplast of Catharanthus roseus was prepared in sterile MS

media containing 9% mannitol.

3. 10 µl of the suspension of protoplast was taken on a glass slide and mixed

gently and the protoplast was allowed to settle at the bottom.

4. 20 µl of 30% PEG and 50% PEG, respectively, were added around the
periphery of the protoplast mixture in the slide and incubated at room
temperature for 30 minutes.

5. The slides were then observed under the microscope.

RESULT
The fused protoplast or heterokaryons were observed under the microscope.
50% PEG solution induced more hybridization than 30% PEG.
Expt. No: 23 Date :

ARTIFICIAL SEED PRODUCTION

AIM
To produce artificial seeds.
PRINCIPLE
In nature, true seeds are products of fertilized ovules, consisting of zygotic
embryo enclosed by a protective coat developed from integuments.
In a seed, the zygotic embryo grows into seedling by seed germination, while
a somatic embryo gives rise into a plant only under aseptic conditions. Somatic
embryos are not enclosed by a seed coat and will not survive if sown into a field
directly due to microbial contamination and desiccation.
Tissue culturist try to develop technique by which isolated somatic embryo
could be encapsulated by a protective gel like substance, in order to aid their survival
and to prevent desiccation. Such encapsulated embryo could be used as artificial
seeds.
MATERIALS REQUIRED
Somatic embryo, 2% sodium alginate, 0.7% calcium chloride, agar medium,
glass beaker, micropipettes, forceps etc.
PROCEDURE
1. Somatic embryogenesis was induced from the callus culture of the explant.
2. The somatic embryos were allowed to complete development.
3. Isolated embryos were mixed with sodium alginate and dropped into 0.7%
w/v calcium chloride solution, using a wide mouth pipette tips
4. Surface complexation began immediately and drops were completed within
30 minutes.
5. Encapsulated embryos were placed aseptically on agar medium with minimal
nutrients.
RESULT
1. Proper synthetic seeds were prepared by encapsulating the embryos.
2. Uniform germination of somatic embryos was observed.
Expt. No: 24 Date :

MERISTEM CULTURE
AIM
Production of plantlets at in vitro condition by meristem culture technique.
PRINCIPLE
The simplest type of meristem culture is the stimulation of apical bud growth.
Apical buds are used for meristem culture. The meristem is a dome of actively
dividing cells about 0.25 mm long. Shoot elongation and further rooting are required
for production of new plantlets.
MATERIALS REQUIRED
Culture tubes containing nutrient media [MS media], sterile Petri plates,
knife, sterile distilled water, mercuric chloride [0.1%], MS medium with PGR [BAP,
IAA, NAA, GA3] and shoot tip explants.
PROCEDURE
1. Carefully selected explants were washed thoroughly in running tap water.
2. The apical buds were excised using a clean sharp blade and stored in distilled
water.
3. The tools were sterilized by immersing them in 70% alcohol and then flame
sterilization was done.
4. Explants were sterilized in 0.1% mercuric chloride for 5 minutes and washed
thrice with sterile distilled water.
5. The cut ends which were exposed to mercuric chloride were excised in sterile
environment.
6. The explants were inoculated in MS media, supplemented with different plant
growth regulators.
7. The culture tubes were kept in incubation room.
8. Observations were made daily for the determination of good shoot
proliferation in medium.
RESULT
Proliferation of apical buds was observed in the cultured explants after 3-4
weeks of incubation in tissue culture media.
Expt. No: 25 Date :
MICROPROPAGATION

AIM:
To develop multiple shoots from the single shoot tip and node segments and
their subsequent plantlet development.
MATERIALS REQUIRED:
1. Plant in shade house or field.
2. Culture medium in test tubes or conical flasks.
3. Laminar airflow chamber and spirit lamp.
4. Sterilized tools.
5. Sterilized petriplates, beaker etc.
6. Sterilized double distilled water in flask.
7. 0.1% mercuric chloride solution.
8. Sterilized culture.
PROCEDURE
Preparation of media
Semi solid MS medium supplemented with 3% Sucrose , 0.8% Agar & 2mg/l
BAP was prepared
Preparation of Plant materials (Explant)
1. Nodal & Shoot tip portions having terminal shoot buds and auxiliary buds of
2-3 cm length with stem below and above the node from the mother plants
were collected and were transferred to double distilled water.
2. Few drops of tween twenty or teepol were added and the explants were
washed thoroughly.
3. The buds were then placed in a sterilized Petri plate with double distil water
inside the transfer area.
4. The surrounding leaves having primordial leaf were left and the auxiliary leaf
petioles in nodal section were removed carefully.
5. The Basal region was trimmed off and the final size of the explants was
trimmed to 1-1.5cm in length.
6. The explants were transferred to solution containing 2 drops of tween twenty
in a flask and shaken well.
7. The solution was then decanted and explants were rinsed with water.
 8. Then 70% ethanol was added and the explants were treated for 30sec to
1minute and decanted, followed by sterile water wash.
Then 0.1% mercuric chloride solution was added and shaken well for 2-4minutes.the
surface sterilant was then quickly decanted and was washed with sterile double distil
water repeatedly.
INOCULATION AND INCUBATION:
The following plant species are used for experiment.
Rose, mulberry, syngonium, hibiscus , kalluriki, costus

These explants were transferred aseptically in the surface of agar medium in

culture bottle. The culture bottles with explants were incubated in a controlled
culture room.
OBSERVATION:
Explants were prepared according to the procedure and multiple shoot
induction was observed [table]. The multiple shoot induction time and number of
multiple explants were observed.
Expt. No: 26 Date :

QUALITATIVE ANALYSIS OF ALKALOIDS,

FLAVONOIDS, SAPONINS, TANNINS AND
PHENOLIC COMPOUNDS
AIM
To qualitatively analyze the alkaloids, flavonoids, saponins, tannins and
phenolic compounds present in the given sample.
PLANT COLLECTION AND EXTRACT PREPARATION
Fresh leaves of Neem (Azadirachta indica) were collected from college
campus. The leaves were collected to 25 g and crushed with mortar and pestle until it
formed paste. By adding 30 ml of distilled water filtrate was taken using Whatmann
filter paper.
QUALITATIVE PHYTOCHEMICAL SCREENING
The plant filtrate prepared were analyzed for alkaloids, flavonoids, saponins,
tannins and phenolic compounds.
REAGENTS REQUIRED
Mayer’s reagent: Add 1.5 g of mercuric chloride in 60 ml of distilled water.
Pour this to 5 g of potassium iodide (KI) in 10 ml of distilled water.
TEST FOR ALKALOIDS
About 2 ml of plant filtrate was boiled with 5 ml of 2% HCl in water bath for
5 minutes. Mixture is allowed to cool and then filtered. It is then treated with
Mayer’s reagent and observed for precipitate as turbid formation.
TEST FOR FLAVANOIDS
1ml of plant filtrate was mixed with 2 ml of 10 % lead acetate, a brownish
precipitate indicated a positive test for phenolic flavonoids, while for flavonoids 1 ml
of plant filtrate was mixed with 2ml of dilute NaOH. A golden yellow colour
indicated the presence of flavonoids.
TEST FOR SAPONINS
1 ml of plant filtrate was diluted with 2 ml distilled water and vigorously
shaken and left standing for 10 minutes, the development of foam on surface lasting
for 10 minutes indicated presence of saponins.
TEST FOR TANNINS
 1 ml of filtrate was mixed with 2 ml of FeCl3¬ a dark colour indicated a
positive test for tannins.
TEST FOR PHENOLIC COMPOUNDS
Anthraquinones
1 ml of plant extract was shaken with 10 ml benzene, the mixture was filtered
and 5ml of 10% (v/v) ammonia was added then shaken and observed. A pinkish
solution indicates positive test.
Anthocyanosides
1 ml of plant filtrate was mixed with 5 ml of dilute HCl. A pale pink colour
indicates positive test.
RESULT
Qualitative phytochemical confirmed the presence of three secondary
metabolites namely alkaloids, saponins and tannins.