Practical Manual

B.Sc. I

(Botany)

Under

Star college scheme

Of
Department of Biotechnology
Government of india, New delhi

By
Post Graduate Department of Botany
Jankidevi Bajaj College of Science,Wardha

Jamnalal Bajaj Marga, Civil Lines,

Wardha – 442001 (M.S).
website : jbsw.shikshamandal.org
E-mail : [email protected] & [email protected]
Phone No. (07152) 230515
 Contents

Sr.No. Name of Experiment Page No.

1 Simple staining technique 1
2 Gram staining technique 2-3
3 Preparation of Plant Tissue culture media 3-4
4 Embryo culture 4-5
5 Anther culture 5-6
 EXPERIMENT.1
Simple staining techniques

Requirements

24 hours old culture of Bacilus subtillis, Escherichid coli, and Staphylloceccus

oureus. Staining solution of methylene blue,Staining tray, Glass slides, Inoculating
loop, Bunsen burner/ spirit lamp, Blotting papers,etc..

Procedure

1). Take clean glass slides, wash and dry them. 2) Prepare bacterial smears of all
the bacterial cultures following the usual procedure. 3) Keep a slide (heat fixed
smear) on the staining tray and apply about 5 drops of a stain (any one of the
above) for the designated period. 4). Pour off the stain and wash the smear gently
with slowly running tap water. 5) Blot dry the slide using blotting paper (do not
wipe the slide) 6) Repeat steps 1-5 for the other two cultures.

Observations.

1). Examine all the three preparations under oil-immersion objective. 2) Make
drawings for each organism. 3) On the basis of the microscopic observations, write
description of the organisms indicating colour, shape and arrangement of cells in
each.

Results

The bacteria stain a deep blue. The Bacillus is rod shaped with clear areas
(i.e.endospores) within them S.aureus cells are spherical,, occurring singly, in pairs
and irregular clusters, the small rod of E.coli frequently
 EXPERIMENT 2
Gram staining technique

Requirements – 24 hours cultures

Gram staining reagent – Crystal violet, Gram’s iodine solution.

95 percent ethyl alcohol

Safranin
Staining tray/ clothes pin
Wash bottle of distilled water
Droppers
Inoculating loop
Glass slides
Blotting paper/Absorbent paper
Lens paper
Bunsen burner/spirit lamp
Microscope

Procedure
1. Make thin smears of Staphylococcus and Escherichia on separate glass slides.
2. Let the smears air dry.
3. Heat fix the smears.
4. Hold the smears using slide rack or clothes pin.
5. Cover each smear with crystal violet for 30 seconds.
6. Wash each slide with distilled water for a few seconds, using wash bottle.
7. Cover each smear with Gram’s iodine solution for 60 seconds.
8. Wash off the iodine solution with 95 pler cent ethyl alcohol. Add ethyl alcohol
drop by drop, until no more colour flows from the smear. (the gram-positive
bacteria are not affected while all gram- neagative bacteria are completely
decolorized).
9. Wash the slides with distilled water and drain.
10. Apply safranin to smears for 30 seconds (Counter-staining)
11 Wash with distilled water and blot dry with absorbent paper.
12. Let the stained slides air dry
.
Obserations
1. Examine the slides microscopically using oli-immersion objective.
2. Identity the gram reaction of both the cultures and classify them.
3. Make sketches for morphology of the cultures.
4. Describe the morphology and arrangement of the cells.

Results
Those bacteria that appear purple are referred to as Gram-positive, those
appearing pink are described Gram-negative.
In S. aureus the appear dark purple or blue in colour, thus it is a gram-positive
bacterium whereas in E.coli the rods appear pink and is thus a gram-negative
bacterium. To get reliable results, one should use cultures that are 18 to 24 hours
old.

EXPERIMENT 3
Preparation of Plant Tissue culture media

Requirements.
Constituents of the MS medium.
Erlenmeyer flasks (100,250,500 ml, 1 litre capacity)
Measuring cylinders (100,1000 ml capacity)
Pipettes (1,5,10 ml)
Distilled or demineralized water
pH Meter
1.0 N NaOH, HCL or KOH
Autoclave
Procedure
1. Prepare macronutrients solution in 100 ml distilled water.
2. Prepare micronutrients solution in 100 ml distilled water following the stock
solution dilution chart.
3. Add macronutrient and micronutrient solution while stirring into 700 ml distilled
water taken in 1 litre Erlenmeyer flask.
4. Add the other heat stable constituents (e.g. sucrose, vitamins and hormones) and
agar powder (if desired at a concentration of 0.8 – 1.0 %) (vitamins and auxins can
be added after autoclaving for better results).
5. Make the final volume of the medium by the addition of more distilled water.
6. Adjust pH of the medium to 5.7, while stirring, using 0.1 N NaOH or 0.1 N
HCL.
7. If solid medium is desired agar is used, heat the solution while stirring until agar
is dissolved.
8. Pour the medium into the desired culture vessels (15 ml in a 25 x150mm culture
tube and 50 ml n a 250ml flask).
9. Plug the culture vessels with non-absorbent cotton wool wrapped in cheese-
cloth, or with any other suitable closure.
10.Transfer the culture vessels to appropriate baskets covered with aluminium jfoil
to check wetting of plugs duting autoclaving.
11. Transfer the baskets to autoclave.
12. Sterilize the medium by autoclaving at 121 0c (1.05 kg/cm2) (15 psi) for the
time period depending upon the volume of the medium in the vessel (e.g. for
25,50,100,250,500,1000,2000,4000 ml capacity time required is
20,25,28,31,35,40,48 and 63 minutes respectively).
13. The medium is allowed to cool at room temperature.

Observations and Results

The cooled medium is ready for inoculation and should be stored at 4 0 C for
future use.
 EXPERIMENT 4
Embryo culture
\

Embryo culture, can be defined as the in vitro culture either of the polarized
egg, zygote, proembryo or mature embryo.
The applications of embryo culture are:
1. To obtain rare hybrids. In many interspecific and intergeneric crosses
fertilization occurs normally, but poor or abnormal development of endosperm
causes pre-mature death of hybrid embryos. The embryo culture technique is
extensively used to rescue such hybrid embryos from wide crosses which
otherwise would abort.
2. To study morphogenesis and nutritional requirements. It is an ideal
technique for studying morphogenesis and nutritional requirements of the
developing embryo.
3. To overcome seed dormancy and fro shortening the breeding cycle of
deciduous trees. The breeding work of deciduous trees is delayed due to long
dormancy period of their seeds. By growing excised embryo in culture this period
can be reduced.
4. For raid seed viability test. The possibility of breaking seed dormancy by
embryo culture allows the use of this technique for testing the viability of a
particular seed batch rapidly.
5. Propagation of rare plants. The seeds of some wild varieties of the
cultivated plants do not germinate in nature. Seedlings can be obtained by culturing
their excised embryos, eg.seeds of Musa Bulbisiana, a wild relative of commercial
banana.
6. Haploid Production. Through elimination of chromosomes of one of the
parents following distant hybridization, haploids can be produced. For instance in
the cross of Hordeum vulgare x H. bulbosum, the chromosomes of H. bulbosum
are preperentially lost resulting in haploid embryo plants.
 In vivo the embryo follows a definite pattern of cell division and growth. The
first divisions usually lead to the production of a linear filamentous structure. From
the funicular end of this structure, a globular embryo develops and the remainder
forms the suspensor. In dicots, the globular embryo develops into a heart-shaped
embryo and finally into the mature torpedo-shaped embryo with well-defined
radical and cotyledons.
Depending upon the age of the embryo, the stimulus for its continued growth
is thought to be present within its own cells or in the surrounding endosperm. The
fertilized egg and proembryo develop on the nutritional resources of the
endosperm.
In general, full –grown embryos can be successfully cultured on a standeard
mendium containing sugar, mineral salts and vitamins. Younger embryos, in
addition to the above, also require trace elements and other growth-promoting
substances. Knop’s mineral solution was first used for embryo culture but
proembryo were unable to develop on it, eg. Capsella bursapastris.
The basic requirements for a successful culture of embryos is a well-balanced
medium containgin macro-and micro-nutrients and a carbon source.
Supplementing the medium with organic nitrogen like amino acids or casein
hydrolysate can enhance the growth of excised
 EXPERIMENT 5
Anther culture
Name of Expt. – To demonstrate the technique of anther culture.

Requirements – 9 cm. sterile petridishes, 9cm. non sterile petridishes, 2 forceps.

Dissecting needles (sterile) (2) prepared from soft glass rod
Iron needles(2)
Scalpel with a narrow blade (sterile)
Cm-scale
Parafilm
Spirit lamp
Dissection microscope
Microscope slides, coverslips(10 each)
Incubator or refrigeration unit for chilling buds (preset at 70 - 80
C)
Growth chamber equipped with fluorescent lighting (1000-1200
lux)
Plastic pots (50 mm diameter) (10) filled with sterile sand:
soil(1:1)
b. Culture media. Washing solutions, sterilizing agents and other
chemicals
Culture tubes (10) containing 20ml of medum
20-ml liquid medium in petri dishes (10)
Cultural tubes (10) containing 20ml of root-induction medium
B-medium in 5 cm petri dishes (10)
Culture tubes (10) containing 20 ml or R medium
30- ml of mercuric chloride solution (0.01%) with Tween- 20 as a
wetting agent in a petri
Dish
Sodium hypochlorite solution (0.8%)
Ethanol (70%) (for sterilization)
 Ethanol (80%v/v) in a coupling jar to be used as a dip. Sterile
double distilled water
In 250 ml conical flasks (4)
Acetocarmine stain (1%)
Colchicine solution (0.5% w/v)
c. Source tissue.
Flower-buds of Nicotiana tabacum or other plant.

PROCEDURE
1. Anther culture
i) Do not collect the flower-buds from the plants until the laboratory is ready
ii) Collect the flower- buds using a pair of forceps in a non-sterile petri dish and
measure the length of each bud using a cm-scale. Select the buds with a corolla
length of 21-23 mm. pollen in these flower-budswould usually be in the late
uninucleate or early binucleate stage.
iii) Chill the buds at 70 to 8O C for 12 days in a refrigeration unit.
Iv) Surface- sterilize the buds in a petri dish containgin 0.01% solution of HgCl 2
(with Tween-20 added as a wetting agent) for 10 minutes
v) Rinse the buds 3-4 times with sterile double distilled water in a sterile air
cabinet
vi) Carefully tease, oenn the buds and remove the anthers using forcepas and a
dissecting needle.
 Practical Manual
B.Sc. II

(Botany)

Under

Star college scheme

Of
Department of Biotechnology
Government of india, New delhi

By
Post Graduate Department of Botany
Jankidevi Bajaj College of Science,Wardha

Jamnalal Bajaj Marga, Civil Lines,

Wardha – 442001 (M.S).
website : jbsw.shikshamandal.org
E-mail : [email protected] & [email protected]
Phone No. (07152) 230515
 Contents

Sr.No. Name of Experiment Page No.

1 Effect of plant growth regulators 1
Preparation of biofertilisers, Biopesticides and 2-6
2
Bioherbicides
3 Effect of Biopesticides and Bioherbicides on 7
plant
4 Stages in development of embryo sac 8-9
Vegetative propagation through root, stem and 10
5
leaf cuttings
 Experiment No. 1
Effect of plant growth regulators

Experiment 1 A: Effect of gibberellins on plant growth

Object: - To demonstrate the effect of gibberellins on plant growth.

Requirements:- Seedlings of pea (Pisum sativum) and beans (Phaseolus vulgaris),

100 ppm gibberellic acid in distilled water, acid washed sand.
Prepare five solutions of gibberellic acid (GA) of different concentrations
(molecular weight of gibberellic acid is 348) as under:
1. GA 10-1 M (0.01 gm molecular weight of GA dissolved in 1ml ethanol and
diluted with water to 100ml)
2. GA 10-2 M (1ml 10-1 M GA diluted with water to 10ml).
3. GA 10-3 M (1ml 10-2 M GA diluted with water to 10ml).
4. GA 10-4 M (1ml 10-3 M GA diluted with water to 10ml).
5. GA 10-5 M (1ml 10-4 M GA diluted with water to 10ml).

Method and observations :- Use genotypically dwarf as well as tall varieties of

pea and bean plants.
a) Duxarf Pea Seedlings : 1 – 20 seeds of dwarf pea variety are sown in two rows
in acid washed neutral sand under relatively controlled conditions in a greenhouses
and wait for 14 days.
2. After 14 days, measure the height of all the 20 seedlings and the length of their
leaves and internods.
3. Spray the pea plants of one row with 100 ppm gibberellic acid solution and the
other row of pea plants with distilled water containing the same amount of
gibberellic acid solution.
4. For 4 weeks observe the weekly changes in the characters studied earlier.
5. Make a table of all the results and prepare curve from the mean of 10 plants for
the treatment and control separately to show the differences.
b) Bean Plants: 1. Take б-glazed pots containing good garden soil and sow 5 bean
seeds in each pot.
2. Place these pots in greenhouse for 2 to 3 weeks.
3. In each pot, retain two healthy plants and remove the remaining plants.
4. Place one drop of each of the following solutions on the tip of the retained plants
in the pot i)distilled water (control), ii) GA 10-1M, iii) GA 10-2M, iv) GA 10-3 M,
v) GA 10-4 M).
The first solution, i.e. distilled water (control) should contain ethanol, equal
in amount to these used in the preparation of GA solution.
5. Weekly observe the morphological changes related with the growth for 4 weeks
and note them in the from of a table.

Result ;- Application of different concentrations of gibberlic acid show much

quicker growth of stem and leaves of pea and bean plants.

Experiment 1 B: Influence of IAA on apical dominance

Object : To demonstrate the influence of IAA on apical dominance.

Requirements : Kidney bean plants (3) or any other potted plants, blade or razor,
IAA-Ianolin paste (1%).

Method :- 1. Take 3 kidney bean plants of same age and almost identical growth.
2. Remove the apical bud of two plants with blade and leave one plant undisturbed.
3. Apply 1% IAA-lanolin paste at the tip of one of the decapped plant and plain
lanolin at the tip of another decapped plant. Observe after about two weeks.

Results : (a) All lateral buds remain inhibited by the shoot tip in the control plant
(A). Lateral buds begin to grow in the plant in which plain lanolin was applied (B).
Lateral bud growth remains inhibited in the plant on which IAA-lanolin paste was
applied. This indicates the influence of IAA on apical dominance.

Experiment 1 C: Effect of Kinetin on cell division.

Object : To show the effect of kinetin on cell division.

Requirements: Freshly cultured tissues of carrot or tobacco in the culture medium,

kinetin, petridishes, alcohol, distilled water, beakers, pipettes.
Method : 1 Culture the tissues of carrot or tobacco in six petri-dishes containing
culture medium as discussed earlier in the chapter of Tissue Culture.
2. Prepare various concentrations ( 1 ppm to 5 ppm) of kinetin.
3. Pour 5ml each of 1 ppm kinetin in one petridish. 2 ppm kinetin in 2 nd petri-dish,
3 ppm kinetin in 3rd petri-dish, 4 ppm kinetin in 4th petridish and 5 ppm kinetin in
5th petri-dish. Keep the 6th petri-dish as control and add in it only distilled water
instead of kinetin. Observe after about a weak.

Observation and Results:

 Experiment No. 2

Preparation of Biofertilisers, Biopesticides

and Bioherbicides

Blue green algae in open air, shallow culture. Each farmer can prepare his
own algal biofertiliser as follows. It has been recommended that trays (6’+3’+9’)
of galvanized iron, sheet or brick and morter or pits lined with polythene be
prepared. A mix of 10kg soil and 200gm of superphosphate is added to trays and
water filled in a trays to the level of 2-6 inch. It is necessary to keep the ph of the
soil to neutral and Tolypothrix, Nostoc, Anaebaena, and plectonema is sprinkled is
over the water in tray within a week time, a thick allgal slum is formed over the
surface. The water is allowed todry at this stage and the dried algal flakes are
collected from the trays and stored in polythene bags and applied at the rate of
10kg/ha in the field, one week after rice transplantation.

Rhizobium:

Rhizobium production is based on batch culture technology. For the growth of

rhizobia, good aeration is required and an aeration rate of 5 litres air/hour is
adequate. Aeration requirement varies from species to species. For fast growing
rhizobia, aeration at a rate of 100 liters air/hr for 201 of broth culture for 3 days at
300C has been found suitable. Slow growers may need more air depending upon
the species. The stuitable temperature for their growth is 28-300 C.
The following steps are required in production.
 Isolation
 Authentication and strain selection
 Preparation of mother culture
 Mass production under non-sterile system or sterile system.

Isolation
Suitable culture media are needed for isolation of the required strain. A number of
media composition for Rhizobium production hae been recmmmended by different
scientists/manufacturers. Rhizobium is heterotrophic bacteria and can readily
utilize mono and disaccharides. Mannitol is the traditional carbon and energy
source used for small volume cultiavation of all thizobium although some slow
growers prefer pentose e.g. arabinose. Most fast growers utilize disaccharides (e.g.
sucrose). However, efficient utilization of carbon source depends on aeration,
method of sterlisation and particular carbon source.
Some production units do not use calcium carbonate in order to avoid
confusion regarding source of turbidity (whether due to bacterial growth of
CaCO3). Few reputed orgnisations are using the following composition: 10 ml
glycerol, 0.4 g yeast extract, 0.5 g. K2HPO4, 0.2 g MgSO4. 7H2O and 0.1 g NaCl
per litre of broth and getting successful results.

Process:
Rhizobium forms nodule on the root of legumies. Nodule is the site of nitrogen
fixation and Rhizobium is isolated from these as follows.
 Wash roots thoroughly
 Select pink and healthy nodules
 Collect nodule from plants without damaging it
 Immerse undamaged nodule in 95% ethanol for 5-10 seconds.

Experiment : Preparation of Biopesticides

The neem tree is indigenous to India. Indians have revered the neem tree for a
very long time. To Millions of Indian, neem has miraculous power. Indian farmers
have kept away insects with different neem extracts. The tree is considered so
invaluable that it is found in every part of the country, every roadside, every field
and almost every house. India has shared its “free tree” and knowledge of its
utilization with the world community. The freedom of diverse species to exist and
the freedom of people to exchange knowledge about them are best symbolized in
the neem.
After the introduction of chemical agriculture, the use of such neem-based
extracts and other products has diminished to a large extent. Farmers have been
made more and more dependent on chemical inputs and have lost confidence in
their age-old methods. However, the current crop of pests has developed resistance
to a wide range of pesticides available. Farmers are thus caught in a vicious circle
the moment they start using chemicals.

The Centre for Indian Knowledge Systems (CIKS) has attempted to identify
the technologies that farmers were using for pest control before chemical
agriculture came into the picture. It has tested these technologies in farmers fields
and developed them in such a way that farers can be totally selfreliant wtith these
technologies. They need no longer be dependent on the pesticide companies. The
preparation of these products is extremely simple, as is their application. In terms
of efficiency, these products are very good and, in certain cases, are even more
efficient than the commercially available products.

Preparation of extracts
Neem kernel extract:

For 5 liters of water, 1 kg of green neem leaf is required. Since the quantity of
leaves required for the preparation of this extract is quite high (nearly 80 kg are
required for 1 hectare), this can be used for nursery and kitchen gardens. The
leaves are soaked overnight in water. The next day, they are ground and the extract
is filtered. The extract is suited for use against leaf eating caterpillars, grubs,
locusts and grasshoppers. To the extract, emulsifier is also added.

Remarks: The advantage of using neem leaf extract is that it is available

throughout the year. There is no need to boil the extract since boiling reduces the
azadirachtin content. Hence the cold extract is more effective. Some farmers prefer
to soak the leaves for about one week, but this creates a foul smell.

Neem cake extract:

A hundred grams of neem cake are required for 1 litre of water. The neem cake
is put in a muslin pouch and soaked in water overnight. It is then filtered and an
emulsifier is added at the rate of 1 millilitre for 1 litre of water, after which it is
ready for spraying.

Neem oil spray:

Thirty milliliters of neem oil are added to the emulsifier and stirred well to
ensure that the oil and water can mix well. After this, 1 litre of water is added and
stirred well. It is very essential to add the emulsifier with the oil before adding
water. It should be used immediately, otherwise oil droplets will start floating. A
knapsack sprayer is better for neem oil spraying than a hand sprayer.

Pongam, aloe and neem extract:

One kilogram of pounded pongam cake, 1 kg of pounded neem cake and 250 g
of pounded poison nut tree seeds are put in a muslin pouch and soaked overnight in
water. In the morning, the pouch is squeezed and the extract is taken out. This is
mixed with ½ litre of aloe Vera leaf juice. To this, 15 litres of water are added.
This is again mixed with 2-3 litres of cow’s urine. Before spraying. 1 litre of this
mixture is diluted with 10 litres of water. For an acre, 60-100 litres of spray are
used. This is effective in the control of pests of cotton and crossandra.

Custard apple, neem, chilli extract:.

Five hundred millilitres of water are added to 2 kg of ground custard apple

leaves and stirred. This is filtered to get the extract and the filtrate is kept aside.
Separately, 500 g of dry fruits of chilli are soaked in water over night. The next
day, this is ground and the solution filtered to get the extract. One kilogram of
crushed neem fruits is soaked in 2 litres of water overnight and the extract is
filtered. All the three filtrates are subsequently mixed with 50-60 litres of water,
filtered again and sprayed over the crops.

Note: For all the above extracts. 250 milliliters of khadi soap solution should
be added as an emulsifier before spraying.

General remarks about spraying:.

a) Spraying should be undertaken in the morning or late in the evening. Under hot
conditions, the frequency of spraying should be increased in winter, spraying once
in 10 days and every day in the rainy season is recommenede.
b) Insects lay eggs on the underside of the leaves. Hence it is important to spray
under the leaves also.

c) while using a power sprayer, the quantity of water used should be halved.

d) It is better to use low concentrations of extracts frequently.

e) As a general guideline, it can be said that each acre of land to be protected can
be sprayed with 60 liters of ready-to-use solution (not the concentrate). Of course,
the volume may have to be varied depending on the exact conditions prevailing,
such as the intensity of the pest attack.

Biological effects of neem on insects:

The action of neem products as pest control agents can be manifested at

different levels and in different ways. This is a very important point to be noted
since the farmer would be used to the “knock-out” effect of chemical pesticides.
Neem extracts do not exhibit this type of effect on pests but affect them in several
other ways.
 Experiment 3
Effect of Biopesticides and Bioherbicides on plant
Nitrogen fixation which is required for the growth of higher plants is hindered
by pesticides in soil. The insecticides DDT,Methyl parathion and especially
pentachlorophenol have been shown to interfere with legume rhizobium chemical
signaling result in reduced nitrogen fixtion and thus reduced nitrogen crop yield.
Root nodule formation in these plant saves. Pesticides have some direct harmful
effect on plant including poor root hair development shoot yellowing and reduced
plant growth.

Weed killers (herbicides) can save considerable labor in the yard and garden.
Some of these kill plants selectively, so the manager can control weeds but not
injure desirable plants. Others are not selective and may kill all plants in an area.
They must be applied directly to weeds carefully to avoid damaging nearby plants.

Like many powerful tools, weed killers cause serious harm if used incorrectly.

When using an herbicide, or any pesticide read the label. The label will state
whether protective clothing needs to be worn when applying the weed killer.

Make sure that any equipment (such as a sprayer) used is free of other pesticide
before adding a new one. It is important always to shower after applying
herbicides. Wash clothes separaterly that have been worn when spraying. Failure to
follow label directions can result in damage to other plants. Or to animals or
people. It is also illegal.

Different herbicides work on different types of plants, on different components

of plant metabolism, and at different times in plant growth cycles. Some are
translocated (taken up into the plant’s vascular system), while others affect the
plant on contact. Some pose a risk of moving as a vapor to other sites, while others
remain in the soil for long periods, injuring crops planted in those locations at a
later time. In either case non-larget plants can be damaged.

It can be difficult to determine whether a plant has been damaged by herbicides

or other causes, such as insects or disease. Careful examination of the plant is
essential before making any decision about plant damage.
Different herbicides affect different plantsystems, resulting in a range of symptoms
from discolored or distorted leaves and stem to a lack of seedling emergence.
When landscape plants come into contact with herbicides. Major problems can
ensue. Effects may be mistaken for indications of insect infestation,disease,
nutritional deficiency or environmental disorder.
 Experiment 4
Stages in development of embryo sac
L.s. of ovule showing binucleate embryo sac.

Study the slide of longitudinal section of ovule howing binucleate embryo sac.

Observations.

The slide shows following characters.

The ovule is attached to the placenta by a stalk called funicle. The ovule is made of
intefuments and the nucellus. Outermost part of the ovule is made of two
integuments. Inner to the integuments lies nucellus. A few layers below the
nucellar epidermis, binucleate embryo sac is situated. At the top of the sac, three
degenerating megaspores can still be scen. The embryo sac has two nuclei, one at
each pole, separated by a large vacuole.

L.s. of ovule showing 4 nucleate embryo sac

Study the slide of longitudinal section of ovule showing 4 nucleate embryo sac.

Observations.

The slide shows following characters.

1). The ovule consists of stalk and the body. 2). The body fo the oule is made of
integuments and the nucellus. 3). The outer covering of the ovule is made of two
integuments. 4). Nucellus is situated inner ot the integuments. 5). A few layers
below the nucellar epidermis, 4 nucleate embryo sac is present. 6). The embryo sac
shows four nuclei, out of which two are located at the micropylar end and the rest
two at the chalazal end. 7). The nuclei at two ends are separated by large vacuole
in the centre.

L.S. of ovule showing 8-nucleate poygonum type of embryo sac

Study the slide of longitudinal section of ovule showing 8-nucleate Poygonum

type of embryo sac.
Observations

Following characters are observed.

1). The ovule shows stalk and the body. 2). The body consists of integuments,
nucellus and the embryo sac. 3). There are two integuments which form the
outermost covering of the ovule. 4). A small amount of nucellus is present between
the integuments and the embryo sac. 5). Embryo sac is present deep into the tissue
of nucellus. 6). Orgnised 8-nucleate Polygonumtype embryo sac has an egg
apparatus, two polar nuclei and three antipodals. 7). Egg apparatus is situated at the
micropylar end. It consists of centrally placed egg cell with two synergids, one on
each side of the egg. 8). An egg cell has a large vacuole towards its micropylar end
while synergids have a small vacuole toward its chalazal end. 9). Each synergid
has a beak like structure on its lateral side and filiform apparatus at its micropylar
end. 10). Two polar nuclei are located in the centre of the embryo sac. These later
fuse to form the secondary nucleus. 11). Three antipodal cells are located at the
chalazal enc. These degenerate soon, either before or just after fertilization. 12).
Since this embryo sac develops from a single megaspore, it is known as
monosporic, 8-nucleate Polygonum type embryo sac.
 Experiment 5
Vegetative propogation through root, stem
and leaf cutting
Types of cutting.

Theoretically, all plants with primary meristems are capable of propagation by

means of cutting. Cuttings are generally made from the vegetative parts like root,
stem or leaf and even from modified plant part like tubers, rhizomes and bulbs.
They may, thus be root cuttings. Stem cuttings, tuber cuttings and so on depending
on the part of the organ from which these are made.

Root cuttings: Root cuttings are used for propagation of either succulents
where they are capable of producing both the root and shoot primordial or of plants
which produce suckers freely under natural conditions. The method has proved
auccessful in some plants which could not be raised successfully by other methods.
Some of the common plants which are propagated through root cuttings include
Cyrilla, Prunus, Albiaaia, Aesculus, Taraxaceum , Chaenomeles , Cladrastis,
Clethra, Clerodendron, Rhus, Iles, Loelteutria, Maackla, Rhododendron, Syringla
and Robinia.

Stem Cutting: Propagation by stem cuttings is widely practiced by foresters,

horticulturists and others. the shoot is cut into segmens of suitable size which are
then planted for rooting under suitable conditions. On the basis of the type of wood
which is used for making cuttings, they may be hardwood, semihardwood,
softwood and hervaceous cuttings.

i) Hardwood cutting:- In general, hardwood cutting are used for propagating

deciduous trees. They have also been used for propagating plants like olive, citrus
and some gymnosperms. Many ornamental plants and furit trees are propagated by
hardwood cuttings 10+11 Cuttings are made from the vigorous branches when the
tree is still in dormant state and are planted as soon as the active season sets in.

ii) Semi hardwood cutting: The cuttings made from the wood of evergreen
broadleaved species or form partially mature wood of deciduous plants are semi-
hardwood and are used for propagating many ornamental and fruit plants. Cuttings
are usually made from the upper parts of branches although the lower parts can
also root. Cuttings in which some of the upper leaves are retained are then planted
for rooting under conditions of minimum water lo: this is achieved by planting
them under mist sprays.

iii) Softwood cuttings: these are the cuttins which are made from the saft green
succulent new growth and are used for propagating many ornamental woody
shrubs. Such cutting root easily and quickly if careis take to retain leaves and they
are planted under conditions of minimum water los in green houses under constant
mist sprays. The most suitable material maing cuttings is the lateral or side
branches of the stock plants. Head back the main shoot is helpful as it causes the
production of more shoots f te cuttings to be made from. Some of the fruit and
other plants are propagated through softwood cuttings 12-14

Iv) Herbaceous cuttings: Cuttings made mostly from greenhouse plar are
herbaceous, soft, tender and succulent and require special attention. The root easily
under favourable conditions which are similar to those needed of softwood
cuttings. Cuttings of most of the floriculture crops belong to category.

Leaf cuttings: In general, plants possessing thick and fleshy leaves may be
propagated by leaf cuttings. Thin textured leaves are not suitable for the purpose as
they dry quickly. The leaf cuttings when planted suitably, develop both
adventitious roots as well as shoots. A new poant is thus, establishe. The cutting
itself does not become a part of the new plant. Bryophyllui Sansevieria and
Begonia are the common plants theat are propagated through leaf cuttings .
 Practical Manual
B.Sc. III

(Botany)

Under

Star college scheme

Of
Department of Biotechnology
Government of india, New delhi

By
Post Graduate Department of Botany
Jankidevi Bajaj College of Science,Wardha

Jamnalal Bajaj Marga, Civil Lines,

Wardha – 442001 (M.S).
website : jbsw.shikshamandal.org
E-mail : [email protected] & [email protected]
Phone No. (07152) 230515
 Contents

Sr.No. Name of Experiment Page No.

Demonstration of the technique of
1 1–2
micropropogation using different explants.
2 Demonstration of the root and shoot formation. 3
Isolation of protoplast from different tissues using
3 4–7
commercially available enzymes.
4 Demonstration of the protoplast production. 8 – 10
Estimation of Anthocyanin pigment from
5 different plants materials and preparation of 11 – 13
absorption spectra.
 EXPERIMENT 1
Micropropogation

Aim :- Demonstration of the technique of micropropogation using different

explants

Requirements :- The stock plants, incubator, laminar flow cabinet, growth cabinet
or glass house, 70% ethanol, manoxol(or any other surfactant), distilled water, 2%
chloros, nutrient media MS or B5 (powdered and prepared), compost (3:2, peat :
sand), 50mm and 85mm diameter sterile petri dishes, 250ml sterile jars (or conical
flask), propagation trays, seed trays, plastic bags, glass sheets, forceps, blades,
scissor, needles, scalpel, hand gloves, laboratory sealing film, pots, etc.

Procedure :- the following is a representative schedule for a typical

micropropogation. However, the procedure would require modifications and
adjustments depending upon the material used and the laboratory facilities
available.

1. Cut off the shoot tip with a clean and sharp blade.
2. Put the severed apical meristems (or axillary buds) in distilled water.
3. Conduct the next few steps in laminar flow cabinet or under perfect aseptic
conditions.
4. Transfer upto 20 axillary buds in a sterile test tube.
5. Fill the test tube with 70% ethanol. Allow ethanol to remain in the test tube
as such for about 2 minutes.
6. Pour alcohol and replace it with 2% chloros solution. Shake the test tube
vigorously (about 120 strokes per minute) for about 10 minutes.
7. Decant the chloros solution and fill the test tube with sterile distilled water.
8. Rinse the material for 3 to 4 times. Store it in distilled water (but not for
more than 2 hours).
9. Empty the contents of the tube into a sterile petri dish.
10.Pour MS (MS I) medium in a sterilized petri dish. Place up to 4 buds (50mm
petri dish) or 10 buds (85mm petri dish).
11.Take care to ensure that each bud is stuck firmly in the medium but is not
buried.
12.Seal the petri dish with laboratory sealing film. Make 2-4 fine holes in the
film with sterilized needle.
13.Place the petri dish in growth room or incubator. Allow it to remain for a
period of about 1 week to 2 months depending upon the material used.
14.If multiplication is desired, cut internodes and transfer apical cuttings to
sterile jars containing medium MS 2 or MS 4.
15.The basal portion of the cutting should be firmly placed in the medium
without burying it.
16.If plants are desired to be rooted and transferred to compost, instead of steps
14 and 15 follow the procedure given below.
17.Cut off apex with 2 or 3 nodes (about 5mm in length).
18.Transfer about 12 plants per 250ml jar containing culture medium MS 4.
19.Allow them to grow for about 3 to 5 days till approximately 5mm long roots
develop.

20.Remove each plantlet carefully without disturbing the roots. Also try to
remove from roots as much as agar is possible.
21.Fill polythene bags or small pots with compost. Water the bags or pots as the
case may be.
22.Transfer the plantlets to damp compost.
23.Maintain high humid conditions for about 12 to24 hours.
24.Transfer the polythene bags or pots to glass house. Leave them in shade
away from the direct sunlight.
25.Once the plants show signs of maturity (about 70mm in height), transfer
them to larger pots or nursery beds.
Nutrient Media Useful in Micropropogation by Axillary Bud Culture (amount of
ingredients –gl-1 or mgl-1)

Chemicals Medium 1 Medium 2 Medium 3 Medium 4

(M&S) (M&S) (BS) (M&S)
NH4NO3 1.65g 1.65g - 1.65g
KNO3 1.9g 1.9g 2.5g 1.9g
CaCl2.2H2O 0.44g 0.44g 0.15g 0.44g
MgSO4.7H2O 0.37g 0.37g 0.25g 0.37g
KH2PO4 0.17g 0.17g - 0.17g
(NH4)2SO4 - - 13.4g -
NaH2PO4.H2O - - 0.15g -
FeNa, EDTA 36.7mg 36.7mg 40.0mg 36.7mg
H3BO3 6.2mg 6.2mg 3.0mg 6.3mg
MnSO4.4H2O 22.3mg 22.3mg 10.0mg 22.3mg
ZnSO4.7H2O 8.6mg 8.6mg 2.0mg 8.6mg
KI 0.83mg 0.83mg 0.75mg 0.83mg
Na2MoO4.2H20 0.25mg 0.245mg 0.25mg 0.025mg
CuSO4.5H2O 0.025mg 0.025mg 0.025mg 0.025mg
CoCl2.6H2O 0.025mg 0.025mg 0.025mg 025mg
Nicotinic acid 0.50mg 0.50mg 1.0mg 0.50mg
Thiamine HCL 0.10mg 0.10mg 10.0mg 0.10mg
Pyridoxine 0.50mg 0.50mg 1.0mg 0.50mg
HCL
Glycine 2.0mg 2.0mg - 2.0mg
Sucrose 20.0g 20.0g 20.0g 20.0g
Inositol 0.1g 0.1g 0.1g -
Glutamine 0.1g 0.1g 0.1g -
BAP (6- 0.25mg 0.25mg 0.25mg 0.25mg
benzyl-amino
purine)
GA3 0.1mg - - -
(gibberellic
acid)
pH 5.64 5.64 5.64 5.64
Agar (Difco 8.0g 8.0g 8.0g 8.0g
Bacto)
 EXPERIMENT 2
Root and shoot formation
Aim :- Demonstration of the root and shoot formation

Requirements :- Controlled temperature growth room, dissecting microscope,

laminar air flow cabinet, autoclave, hydrochloric acid, Murashige and Skoog’s
basal medium, naphthalene acetic acid (NAA), 6-benzyl aminopurine (BAP),
kinetin, sodium hydroxide, etc.

Procedure :-

The following is a tentative schedule for a typical plant tissue culture. The exact
procedure would, however, differ depending upon the material to be used and the
laboratory facilities available.

1. Cut off the shoot tip or any desired part with a clean and sharp blade.
2. Place the severed part in a petridish containing distilled water.
3. Conduct the next few steps in laminar air flow cabinet or under perfect
aseptic conditions.
4. Cut portion (explant) is dipped into 5% teepol (or any other surface sterilizer
) for 5 to 10 minutes for surface sterilization. Wash the explants in sterilized
distilled water.
5. The explants is further surface sterilized by immersing in 70% ethanol (V/V)
for 40 to 60 seconds followed by 5% sodium hypochlorite (V/V) for 20-30
minutes.
6. The explants is finally washed at least three times in sterilized distilled
water.
7. A thin solid section of the explants is now cut by a sterilized scalpel and
placed in sterilized petri dish. 5% sodium hypochlorite solution is now
added to the petri dish. The section is allowed to remain in this solution for
about 5 – 10 minutes.
8. The explants is now repeatedly washed in sterile distilled water.
9. The explants is then placed in another clean and sterilized petri dish.
10.Prepare sterilized conical flasks with nutrient medium and keep them
plugged with cotton.
11.The cambium is carefully removed from the sections under total aseptic
conditions.
12.The separated cambial tissue is now transferred to the conical flasks
containing medium.
13.The flask marked for induction of root should contain low concentration of
cytokinin and relatively high concentration of auxins in the medium.
14.The process of incubation involves removal of cotton plug, insertion of
explants and replugging under completely aseptic conditions.
15.Allow atleast 4-6 weeks for the callus growth to take place.
16.Callus tissue can now be removed for sub-culture.
 EXPERIMENT 3
Isolation of protoplast

Aim :-Isolation of protoplast from different tissues using commercially available

enzymes

Three different tissues are used and therefore, requirements and

methods for each one of them are described separately.

EXPERIMENT 3 A
Isolation of protoplasts from mesophyll cells of tobacco by
simultaneous method
Requirements :- Tobacco leaves, 70%ethanol, 0.5% sodium hypochlorite solution,
distilled water, 600m mol1-1 mannitol- CPW solution, (CPW – cell protoplast
washing medium contains (mg1-1): KH2PO4 (27.2), KNO3(101), CaCl2. 2H2O
(1480), MgSO4. 7H2O (246), KI (0.16), CuSO4.5H2O (0.025), pH (5.8), enzyme
4% cellulose SS, 0.4% macero-enzyme SS, sucrose, parafilm, forceps, petridishes,
Pasteur pipette, 60-80 mµ mesh, centrifuge tubes, etc.

Procedure :-

1. Select fully grown leaves from 7-8 week old plants.

2. Surface – sterilize the leaves by first immersing in 70% ethanol for about 30
seconds followed by rinsing in 0.4-0.5% sodium hypochlorite solution for about
30 minutes.
3. Wash the leaves thoroughly with sterile distilled water to remove all the
traces of hypochlorite.
4. Peel off the lower epidermis with fine forceps. Cut them into pieces.
5. Place the peeled leaf pieces on a thin layer of 600m mol1-1 mannitol- CPW
solution in such a way that the peeled surface is in contact with the solution.
 Stock solutions for Murashige and Skoog’s (MS) medium

Constituents Amount (mg-1)

Stock solution I
NH4NO3 33000
KNO3 38000
CaCl2.2H20 8800
MgS04.7H20 7400
KH2P04 3400
Stock solution II
KI 166
H3Bo3 1240
MnS04.4H20 4460
ZnS04.7H20 1720
Na2Mo04.2H20 50
CuS04.5H20 5
CoCl2.6H20 5
Stock solution IIIb
FeS04.7H20 5560
Na2.EDTA.2H20 7460
Stock solution IV
Inositol 20000
Nicotinic acid 100
Pyridoxine HCL 100
Thiamine HCL 100
Glycine 400

To prepare i litre of medium take 50ml of stock 1,5ml of stock II, 5ml of stock III
and 5ml of stock IV.
b
Dissolve FeSO4. 7H2O and Na2.EDTA. 2H2O separately in 450ml distilled water
by heating and constant stirring. Mix the two solutions, adjust the pH to 5.5 and
add distilled water to make up the final volume to one litre.

6. After about 30 minutes replace the mannitol – CPW solution by filter-

sterilized solution of enzyme containing 4% cellulose SS, 600 m mol1-1
mannitol and CPW salts.
7. Seal the petri dish with parafilm and incubate it in the dark at 24-260C for
16-18 hours.
8. Gently squeeze the leaf pieces with a Pasteur pipette to liberate the
protoplasts.
9. Remove the large debris by filtering through 60-80µm mesh.
10.Transfer the filtrate to a screw-cap centrifuge tube and sediment the
protoplasts at 100g for 3 minutes.
11.Remove the supernatant and transfer the sediment on the top of 860m mol1-
1 sucrose solution (prepared in CPW) in a screw-cap centrifuge tube and
centrifuge it at 100g for 10 minutes.
12.Collect the green protoplast band from the top of the sucrose solution and
transfer it to another centrifuge tube.
13.Add the protoplast culture medium to suspend the protoplasts and centrifuge
at 100g for 3 minutes. Repeat these washings for at least three times.
14.After the final washing add sufficient amount of culture medium to achieve
protoplast density of 0.5 x 105 to 1 x 105 ml-1.
15.Plate the protoplasts as small (100-150µl) droplets or a thin layer in petri
dishes.

EXPERIMENT 3 B
Isolation of protoplasts from mesophyll cells of Cereals

Requirements:- Wheat/Rice seedlings, 0.1% zephirin, 10% ethanol,

washing solution (600m mol1-1 sorbitol solution containing 10m mol-1
CaCl2), 0.5% macerozyme, 1% hemicelluloses, 2% cellulysin, 600m mol1 -1
sorbitol, pH 5.4, nylon mesh (pore size 0.7mm- 0.05mm), petri dishes, test
tubes, etc.
Procedure:-
1. Take primary leaves from 5-6 days old seedlings by cutting at the base of
the leaf. Discard the apical 0.5cm region.
 2. Surface – sterilize the leaves in 0.1% Zephirin – 10% ethanol solution for
5 minutes.
3. Wash twice with washing solution (600m mol1-1 sorbitol solution
containing 10m mol1-1 CaCl2).
4. Cut the leaves into 1 – 2mm wide transverse sections and transfer them to
the enzyme solution (0.5% maceroenzyme, 1% hemi-cellulose, 2%
cellulysin, 600m mol1-1 sorbitol, pH 5.4) with a ratio of 10ml enzyme
solution for each gram of leaf tissue.
5. Infiltrate the leaves under partial vaccum for 3 – 5 minutes.
6. Incubate the leaves in the dark at 23 +-20C, on a shaker with 80 strokes
per minute.
7. After 2 hours filter the leaf digest through two layers of nylon mesh (first
mesh of pore size 0.7mm and the second of 0.05mm).
8. Transfer the filtrate to centrifuge tubes and spin at 50g for 90 seconds.
9. Remove the supernatant and wash the pellet thrice with washing medium.
10.Suspend the protoplast in nutrient medium and culture them.

EXPERIMENT 3 C
Isolation of protoplasts from root nodules

[III] Isolation of protoplasts from root nodules of Trifolium sp.

Requirements :- Roots of Trifolium, protoplast dilution buffer (PDB), 4%

cellulysin, 2% macerase, 1% driselase, 50µm nylon mesh, Pasteur pipette, 30%
sucrose solution

Procedure:-

1. Cut the nodules (1.5mm in length) from aseptically grown plants.

2. Wash thrice with protoplast dilution buffer.
3. Cut the nodules into four pieces and wash again in PDB.
4. Transfer the nodule pieces to the enzyme solution containing 4% cellulysin,
2% macerase, 1% driselase in PDB, pH 5.8.
5. After incubation of about 3 – 4 hours at 230C in the dark dissociate the
partially digested nodues by passing through the orifice of Pasteur pipette
and continue incubation for a further 90 minutes.
6. Sieve through a 50µm nylon mesh and wash the digested tissue twice in
PDB by centrifuging at 200g for 10 minutes.
7. Transfer the suspension onto a 30% sucrose solution and centrifuge at 100g
for 10 minutes.
8. Collect the intact protoplast from the top of the sucrose solution.
 EXPERIMENT 4
Protoplast production
Aim :- Demonstration of protoplast production

Growth and Division of Protoplast

For the growth and nuclear division, the regeneration of cell wall is not seems to be
a prerequisite in protoplast culture. In Convolvulus sp. Protoplast undergo one or
two nuclear division prior to cytokinesis. The same was observed in Haplopappus
sp.

It has been observed that for the rapid growth ofprotoplasts frequent subculturing
in mannitol free medium is necessary. Protoplasts do not grow in large colonies on
high osmotic medium. Its growth gradually shows a downward trend which
ultimately inhibited altogether. The colonies, therefore, should be picked up alng
with small pieces of agar and then transferred on the top of another medium to
avoid any damage to protoplast by rough handling with the forceps.

Protoplast culture : Regeneration of Cell Wall

In culture, protoplasts start developing a wall around itself within few hours and it
takes only few days to complete the process. Wall materials are progressively
deposited. Cellulose is deposited either between the plasmalemma and the
multilamellar wall material or directly on the plamalemma. The nature of
biosynthesis of the cell wall depends on the plant material and the system of
protoplast culture.

The newly built cell wall can be observed either by plasmolyzing the protoplast by
transferring it in a hypertonic solution or by staining the cell wall with calcofluor
white fluorescent stain (0.1%). However, electron microscopic studies and freeze
etching studies have revealed much about the structure and progressive
development of cell wall around the protoplast in culture medium.

Name of some of the families and species in which shoot differentiation of

plant regeneration has been achieved from cultured protoplasts.

Family Species
Compositae Cichorium intybus, Lactuca sativa cultivars (L serriola, L.
saligna), Petasites japonicas, Senecio vulgaris
Cruciferae Arabidopsis thaliana, Brassica compestris (B. Carinata, B.
Juncea, R. Napus, B. Nigra, B. Oleraceae var. capitata),
Sinapix alba
Cucurbitaceae Cucumis sativus
Euphorbiaceae Manihot esculentus
Gramineae Bromus inermis, Oryza sativa, Pennisetum americanum,
Saccharum spp., Triticum aestivum
Leguminosae Glycine argyrea, G. canescens, G. clandestine G.max,
Medicago arborea, M. Difalcata, Psophocarpus
tetragonolobus, Trifolium hybridum, T. Repens
Liliaceae Asparagus officinalis, Hemerocallis sp
Linaceae Linum usitatissimum, L. Strictum, L. Lewissii
Magnoliaceae Liriodendron tulipfera
Ranunculaceae Ranunculus sceleratus
Rutaceae Citrus aurantifolia, C. Grandis, C. Limon
Salicaceae Populus tremula, P. Alba, P. Grandidentata, P. Nigra
Santalaceae Santalum album
Solanaceae Atropa belladonna, Capsicum annum, Datura metel, D.
Innoxia, S. Nigrum, S. Luteum, S. Viarum, S. Xanthocarpum
Ulmaceae Ulmus spp.

1. Observe regularly the regeneration of cell wall, cell division and small callus
formation under inverted microscope.
2. Examine cell wall formation in protplasts with a droplet of 0.1% calcofluor
white R, American Cyanamid, Bound Brooke, N.J. USA, in 0.4m sorbitol
solution on a slide. The cell wall regenerated protoplasts fluoresce.
3. Small cluster of calli are observed after 2-3 weeks of culturing protoplasts.
4. Subculture the cell clusters on a freshly prepeared protoplast culture medium
with or without ½ the initial mannitol and 0.8-1.6% agar.

Determination of Protoplast Plating Efficiency

Calculate the average number of cell colonies per cm2 of the petri dish by
using following formula:

Plating efficiency = X x Area of petri dish x 100

No of protoplast plated/petri dish
X =S1+S2+S3+S4+S5
5

Development of Callus/ Regeneration of Whole Plant

Soon after the formation of wall around the protoplasts, the reconstituted
cells show considerable increase in size and first divisions give rise to small
cell colonies. After 2-3 weeks macroscopic colonies are formed which can
be transferred to an osmotic free medium to develop a callus. The callus may
be induced to undergo organogenic differentiaton or whole plant
regeneration through embryogenesis.
Organogenesis
Organogenesis takes place from a callus and not directly from a single cell.
When isolated protoplasts are put into culture under appropriate conditions
of plant inoculums, medium and environmental factors they go through a set
series of events.
1. Wall regeneration
2. Early mitotic division and callus formation
3. Organogenesis

Embryogenesis
Plant tissue in vitro can induce to form somatic haploid embryos.
Stewards and co-workers first observed the phenomenon of somatic
embryogenesis in carrot. Somatic embryos can be induced in cultural
conditions from three different sources:
1. Vegetative cells of mature plants
2. Reproductive tissues other than the zygote
3. Hyocotyls and cotyledons of embryo
 EXPERIMENT 5
Estimation of Anthocyanin pigment

Aim :- Estimation of Anthocyanin pigment from different plant materials and

preparation of absorption spectra

Requirements :- ethyl alcohol, HCL, acetone, petroleum ether, potassium

hydroxide, petri plates, Impatients balsamia flower, reagent bottle, separating
funnel, colorimeter,

Principle :- The colorimetry or spectrophotometry is based on the principle of

Beer-Lambert law. The coloured samples/solutions absorb complementary
calories.

Absorbance = LCL

Where L – molar extraction coefficient.

C – molar concentration.

L – optical path length.

O.D. = Aborbance = log I0

I

Where, Io – incident light intensity

I – emitted light intensity.

Procedure :- Anthocyanins from Impatients balsamina flowers can also be

separated. Extract is prepared by grinding the material with 1% HCL in 95% ethyl
alcohol. Transfer in a separating funnel, add equal volume of petroleum ether.
Shake the mixture and frequently release the pressure which may develop because
of mixing of the contents. Wash the mixture separately with distilled water to
remove acetone. Discard lower aqueous acetone layer. Collect the upper layer.
This layer contains pigment mixture.

Add 25-30ml of 95% ethyl alcohol in separating funnel, mix by rotation. Allow to
separate two fractions. Collect the lower fraction (B) in a beaker and transfer in
another separating funnel and retain the upper fraction (A)

Lower fraction (B) contains red, purple, blue colour pigment in ethyl alcohol
phase :-

Add 30ml ethyl alcohol. Mix by rotation. Wash by adding 5ml distilled water,
rotate. Discard lower phase. Repeat washing with water 5-6 time, until distinct
layer are observed.

Upper fraction (A) i.e. petroleum ether phase:-

Add 15 ml of freshly prepared 30% ethyl alcohol KOH solution carefully dowm
the inner wall, shake and observe for 10 minutes. Add 30ml of distilled water. It
separates upper and lower fractions.

Preparation of absorption of absorption spectra :-

Use various fractions obtained in the above experiments

1. Acetone extract
2. Petroleum ether extract
3. Various fractions
Use of calorimeter :-

1. Measure absorbance for each filter of the colorimeter. Set zero for each filter
using specific solvent.
2. Note the O.D. for each filter given in the colorimeter manual.
3. Plot the absorbance against wavelength for the filter and find the peak for the
maximum absorbance in a certain wavelength (ƛnm). The O.D. values for
different wavelength will give approximate absorption spectrum for different
pigments.

Observation table:-
Sr.No Wave length Red (O.D.) Purple (O.D.) Blue (O.D.)
(nm)

Plot the absorbance graph (Optical Density) against the wave length for the
filter and find the peak for the maximum obsorbance (ƛnm).
Conclusion:-
The absorption spectrum differs for each pigment because these pigments
absorb light of different wave length in the visible spectrum in between 380nm
to 750nm.