INDEX SEMESTER- III - Ramniranjan Jhunjhunwala College · 10 Separation of amino acids by circular...

INDEX SEMESTER- III

PRACTICAL: I ( Plant Diversity- II)

Sr.

No.

Experiments Page

No.

Date Sign

Algae

1 Study of stages in life cycle of Sargassum 1

2 Study of range of thallus and economic importance of brown algae

(Phaeophyta)

2-4

Bryophyta

3 Study of stages in life cycle of Anthoceros 5-6

4 Study of stages in life cycle of Funaria 7-8

Angiosperms

5 Study of Plants for anatomy in relation to taxonomy

a) Stomata

b) Trichome

9-10

6 Study of Plants for chemicals in relation to taxonomy

(Chemotaxonomy)

11

7 Study of Plants for Palynology in relation to taxonomy 12

8 Study of Families : Morphological Pecularities and Economic

Importance

1. Leguminaceae- Papilionaceae/Fabaceae 13

2. Leguminaceae- Caesalpinaceae 14

3. Leguminaceae- Mimosaceae 15

4. Asteraceae 16

5. Amaranthaceae 17

6. Palmae 18

Techniques to study Plant Diversity

9 Preparation of herbarium and wet preservation technique. 19

10 Separation of amino acids by circular paper chromatography.

20

11 Separation of carotenoids by thin layer chromatography.

21

12 Horizontal and Vertical Gel Electrophoresis. (Demonstration)

22

PRACTICAL: II ( Form and Function - II)

Sr.

No.

Experiments Page

No.

Date Sign

Cell Biology

1 Study of the Ultrastructure of Cell Organelles by photomicrograph

– Mitochondria, Peroxisomes, Glyoxysomes and Ribosomes.

23-24

2 Estimation of DNA from plant material (One standard & one

unknown; No standard Graph)

25

3 Estimation of RNA from plant material (One standard & one

unknown; No standard Graph)

26

Cytogenetics

4 Study of inheritance patern with reference to plastid inheritance.

27

5 Study of of chromosomal aberrations - ( Laggards; Chromosomal

bridge; Ring Chromosome)

28

6 Mitosis

29

7 Meiosis

30

Molecular Biology

8 DNA sequencing – Sanger’s method.

31

9 Determining the sequence of amino acids in the protein molecule

synthesised from the given m-RNA strand. ( Prokaryotic and

Eukaryotic)

32-33

PRACTICAL: III ( Current Trends in Plant Sciences I)

Sr. No.

Experiments Page No. Date Sign

Pharmacognosy

1 Monograph/ Test:

a) Eclipta alba b) Saraca asoca

34-35

Forestry and Economic Botany

2 Visit to National Park/Botanical Garden 36

3 Sources ,Properties and Uses of

a) Fibres b) Spices and condiments.

37

4 Herbal cosmetics (Face pack/ detanning cream)

38-39

5 Estimation of crude fibre in cereals and their products

40

Industry based on Plant Products

6 Preparation and evaluation of probiotic food

41

7 Evaluation of neutraceutical value of mushroom (Protein Content)

42

Sargassum ---Habit

Male Conceptacle

Female Conceptacle

Sargassum

Classification

Division : Phaeophyta

Class : Phaeophyceae

Sub class : Cyclosporae

Order : Fucales

Family : Sargassaceae

Genus : Sargassum

Thallus Structure

- The plant body is diploid and sporophytic.

- Sporophyte is thalloid, erect, well branched with a hold fast and main axis.

- Height of plant varies from species to species.

- Main axis produces no. of primary and secondary branches.

- Primary branches produce leaf like secondary laterals which are called leaves.

- Leaves are simple, flat, broad and with midrib.

- Their margins may be serrate, dentate or entire with acute apex.

- Lower portion of main axis produces no. of air bladders instead of leaves.

- They help in floating of plant and its respiration.

- From axillary position of some leaves, some long, repeatedly branched, cylindrical or flat

structures are produced called receptacles.

- Receptacles have many flask shaped conceptacles in which sex organs are present.

Sexual Reproduction: (To observe the sex organs, take T.S. of receptacle, mount the section and observe it at

location of conceptacle.)

- Sexual reproduction occurs through production of sex organs inside conceptacles.

- Male and female sex organs are produced in different conceptacles.

- Conceptacles open by small openings called ostioles.

Male sex organ:

- Male sex organs are antheridia which are club shaped or oval and are produced on

paraphyses inside conceptacle

Female sex organ:

- Female sex organs are produced on the walls of conceptacle.

- The oogonium (female sex organ) remains attached to conceptacle wall by means of a

mucilaginous stalk.

- A single egg is developed in an oogonium.

1

Ectocarpus

Dictyota Padina

Range Of Thallus in Brown Algae

The plant body in brown algae is highly complexed and well organized. Unicellular, colonial,

coccoid and unbranched filamentous thalli are absent. Except a few microscopic members of

Ectocarpales, Tilopteridales and Sphacelariales, most of the brown algae are macroscopic and

very large in size. Because of their bulky appearance they have been called ‘Giant Kelps’.

Brown algae show basically three types of thallus organization – Heterotrichous,

Pseudoparenchymatous and Parenchymatous.

1. Heterotrichous :-

- Members of Ectocarpales, Tilopteridales and Sphacelariales show the characteristic

heterotrichous habit.

- This is most primitive habit.

- The plant body is differentiated into a creeping prostrate system and an erect projecting

system.

- In Ectocarpous sp., the erect system is more developed. In Ectocarpus parasiticus erect

system is extremely reduced.

Many of the brown algae, which are devoid of heterotrichous constructions, show

pseudoparenchymatous(haplostichous) or parenchymatous( polystichous) constructions.

2. Parenchymatous : -

- This thallus habit is shown by many members.

- Undifferentiated parenchymatous construction is exhibited by Phloeospora, but in many

members of Dictyo-siphonaceae, Laminariales and Fucales it is well developed and

differentiated.

- Besides the dichotomous ribbon shaped habit of Dictyota, Laminariales attain a highly

elaborate external and internal organization in Dictyota, Macrocystis etc.

- Plant body is divided into holdfast, stipe and frond or lamina.

- Thallus of Padina contains concentric rings.

- The thallus of Dictyota is dichotomously branched.

- Thallus of Sargassum looks like aquatic angiosperm.

- Examples are Sargassum, Dictyota, Padina etc.

2

Fucus

Sargassum

Economic Importance Of Brown Algae

The brown algae provide food and shelter for fish and other marine animals. In different parts of

world, these algae are used as food or in food preparation and as fodder. They are also good

source of carbohydrates, vitamins and minerals. Few examples are as follows–

i) Fucus –

- It is a marine alga commonly known as ‘rockweed’.

- It grows up to 30 cm. Thallus is dark brown, leathery and slimy to touch.

- The frond (lamina) is a repeatedly branched, ribbon like with a distinct midrib.

- Air bladders are present on the surface of of the frond.

- Primary chemical constituents of this organism include mucilage, algin, mannitol,

beta-carotene, zeaxanthin, iodine, bromine, potassium, volatile oils, and other minerals.

- The seaweed was also used as fertilizer for crop land in the same areas in which it was

harvested.

- Fucus to have the highest antioxidant activity from a range of edible seaweeds, possibly

due to the presence of Fucoxanthin.

- Fucus species have been reported to have a direct effect on the metabolism of the human

body which shows potential for controlling weight and cellulite deposits.

ii) Sargassum –

- It is a marine alga commonly known as ‘Gulf Weed’.

- It looks like aquatic angiosperm.

- The thallus is rich in proteins, fats and vitamins.

- It contains dietary fibre and minerals such as Iron, Calcium, Magnesium etc.

- Some are consumed fresh, others are cooked in coconut milk or a little vinegar or lemon

juice.

- It is smoke-dried to preserve it.

- It contains antibacterial fatty acids, antioxidants and is mild diuretic.

- It is used as raw material for biodiesel production.

3

http://en.wikipedia.org/wiki/Mucilage

http://en.wikipedia.org/wiki/Algin

http://en.wikipedia.org/wiki/Mannitol

http://en.wikipedia.org/wiki/Beta-carotene

http://en.wikipedia.org/wiki/Zeaxanthin

http://en.wikipedia.org/wiki/Iodine

http://en.wikipedia.org/wiki/Bromine

http://en.wikipedia.org/wiki/Potassium

http://en.wikipedia.org/wiki/Mineral

Laminaria

Macrocystis

iii) Laminaria –

- It is a marine alga commonly known as ‘Kelp’.

- Lamina or blade is terminal expanded leaf- like borne terminally on stipe.

- The thallus is rich in proteins, fats and vitamins.

- It acts as good laxative and helps in digestion.

- Laminarin lowers the level of lipids in blood.

- Algin is obtained from Laminaria is used in a number of industrial processes. It has the

property of thickening and gelling mixtures.

- Alginic acid helps to treat lead poisoning.

- Young stipe is edible.

iv) Macrocystis –

- Also known as ‘Giant Kelp’ or ‘Giant Bladder Kelp’.

- This genus contains the largest of all the brown algae.

- It is widely distributed in subtropical and temperate oceans.

- The demand for Macrocystis pyrifera is increasing due to the new found uses of these

plants in commercially available products like these below:

- in anti-aging cream.

- cleansing milk, cleansing gel, and as a skin cleanser.

- body polish, facial mask, and makeup remover.

- shampoo and conditioner.

- nutritional and dietary supplements.

- seasonings and spices.

- animal feed

- fertilizers and soil treatments

- Humans harvest this kelp for its rich iodine, potassium, polysaccharides, and other

minerals.

- The primary product obtained from giant kelp is algin for alginates.

- Algin is an emulsifying and binding agent used in the production of many foods and

cosmetics, such as salad dressings, ice cream, toothpaste.

4

Anthoceros

BRYOPHYTA

A. Anthoceros

Classification:

Division : Bryophyta

Class : Anthocerotae or Anthocerotopsida

Order : Anthocerotales

Family : Anthocerotaceae

Genus : Anthoceros

External Morphology: The thallus is dorsiventrally flattened, irregularly lobed and has folded margins. It is yellowish

to dark green in colour and prostrate. The upper surface of the plant is smooth. There is no

distinct mid rib. The scales are absent. Areolae are absent. On the ventral side develop simple,

rhizoids. The tuberculated rhizoids are absent. Also on the lower surface are been small rounded

dark coloured areas. These are Nostoc colonies. Present on the upper surface are horn-like

sphorophytes.

V. S. of Thallus:

Internally, the thallus shows no differentiation of tissues into photosynthetic and storage regions.

The outermost layer is formed of compactly arranged cells forming upper epidermis. Next to the

upper epidermis is storage region formed of parenchymatous cells. Each cell possesses a single

chloroplast with a single pyrenoid in the centre of the chloroplast. Ventral portion shows

mucilage-filled schizogenuous cavities having Nostoc colonies.

In some species there are stomata-like pores or slits called the slime pores which are guarded by

two bean shaped guard cells. Slime pores lead into the large mucilage cavities. The guard cells

do not control the opening of the pores. They are always open. From the lower epidermis a

number of unicellular, simple rhizoids develop.

Sexual reproduction:

Sex organs are embedded in gametophytic tissue.

V.S. of thallus showing antheridia:

The antheridia are present in the antheridial cavity or chamber. The roof of the chamber is of

two layers. Each antheridial cavity contains 1 to 4 antheridia. The secondary antheridia may

develop from the buds formed on the stalks of primary antheridia.

Antheridium:

The male sex organ (antheridium) consists of a stalk and a body. The body has a single jacket

layer on its outer side which encloses numerous androcytes.

5

The androcytes metamorphose into biflagellate antherozoids. The antheridia are enclosed in

antheridial cavities or chambers having a two-celled thick roof.

V.S. of thallus showing archegonia:

The archegonia are embedded in the thallus, only the cover cells project beyond the surface of

the thallus. The archegonia are in direct contact with vegetative cells.

Archegonium:

The female sex organ (archegonium) consists of four to six neck canal cells, a ventral canal cell

and an egg. There is no sterile jacket layer, but four cover cells form the lid cells. The lid cells

project above the surface of the thallus

Sporophyte:

The upper surface of the thallus shows elongated, erect bodies called sporophytes. The involucre

or annulus is present at the base of the sporophyte.

Take L.S. of sporophyte, mount the second or the third section as the first section may show

only the wall of the sporophyte.

L.S. of sporophyte :

The sporophyte is differentiated into foot and capsule. The Seta is replaced by intermidiate zone

formed of meristematic tissues. The foot is parenchymatous and is embedded in gametophytic

tissue. A capsule can be differentiated into central sterile columella formed or sixteen rows of

cells. It gives mechanical support and helps in conduction. Sporogenous tissue is cylindrical and

surrounds the columella. It possesses spores at various stages of development; the terminal

region has mature spores and elaters, central region has sporocytes and elaters and the basal

zone has sporogenous tissue. Elaters are simple of branched and multicellular. Wall of the

capsule is formed of four to six layers of cells. The outermost layer of this wall is epidermis

which possesses stomata. Inner layers form the photosynthetic region with cells possessing

chloroplasts. The sporophyte of Anthoceros is thus a partial parasite.

.

6

Gametophyte with Gametophyte

sporophyte

Antheridial Head Archegonial Head and Archegonium

Funaria

Systematic Position :

Division : Bryophyta

Calss : Musci (Bryopsida)

Sub Class : Eubrya (Bryidae)

Order : Funariales

Family : Funariacea

Genus : Funaria

External Morphology:

Gametophyte of Funaria consists of two stages, the juvenile stage called protonema and the

adult stage called gametophore. The gametophore can be differentiated into three regions; (1)

rhizoids (2) cylindrical stem and (3) sessile leaves.

Rhizoids: They arise from the base of the stem. They are slender, multicellular and branched.

The multicellular rhizoids are one-cell thick and possess oblique septa. Functions of rhizoid are

fixation and absorption.

Stem (cauloid): The stem of moss is cylindrical and branched. The branches are extra axillary

and develop below the leaves.

Leaves (phylloides):They are spirally arranged on stem and branches, and are sessile. Leaves

are crowded at the apex forming bud-like structure called the head. The leaf is thin, flat and one

cell in thickness except the mid rib.

Sex organs of Funaria:

L.S. of Antheridial head

It shows club-shaped antheridia (male sex organs) intermingled with sterile, colourless

paraphyses. The terminal cell of paraphysis is larger than the rest of the cells. The green foliage

leaves surrounding the antheridia and paraphysis are called perigonial leaves. Each antheridium

is club-shaped consisting of stalk and body. The outermost sterile layer of body is called jacket-

layer which encloses androcytes.

L.S. of Archegonial head

It shows flask-shaped archegonia, the female reproductive organs, intermingled with sterile

paraphysis. The archegonia with paraphysis are surrounded by green foliage leaves called

perichaetial leaves. The flasked shaped archegonium has basal round venter and a terminal neck.

The stalk of the archegonium is short and massive. The venter has ventral canal cell and an egg

while neck possesses neck canal cells

7

External morphology of sporophyte :

The sporophyte is seen at the apex of the female branch. It consists of foot, seta and capsule.

Sporophyte is a partial parasite

Foot: It is multicellular, embedded part of the sporophyte. It is dagger-like and helps sporophyte

in attaching to the gametophyte.

Seta: It is a long, slender, stalk-like structure which elevates the capsule above the gametophore.

Capsule: It is pear-shaped body present at the tip of the seta. Capsule at its distal end bears the

remenant of archegonial neck called calyptra.

L.S. of Capsule

The capsule shows three distinct regions apophysis, theca and operculum.

Apophysis: It is a basal region of the capsule. The central cells are elongated, colourless and

form conducting region. The conducting region is surrounded by parenchymatous cells

containing chloroplasts. The chlorophyllous tissue is surrounded by single layered epidermis

having stomata.

Theca: It is the middle region of the capsule, between apophysis and operculum. The central

region of theca consists of sterile cells and is called columella. The upper part of columella

projects into the operculum. Surrounding the columella is a spore sac containing many spores.

The spore-sac is surrounded by an air chamber. The air chamber is traversed by a number of

trabeculae. The trabeculae connect the innermost layer of capsule wall to the outer wall of the

spore-sac. Epidermis, the outermost layer of the capsule wall is continuous with that of the

apophysis but stomata are absent.

Operculum: It consists of three parts operculum, peristome and annulus. Operculum is the lid

of the capsule and present at the apex of the capsule. Peristome lies below the operculum and

consists of peristomal teeth. Peristomal teeth are in two rings, each ring consisting of 16 teeth.

The teeth of outer ring show bars of lignosuberin. The teeth of inner ring are smaller in size and

without lignosuberin bars. The peristomal teeth are hygroscopic and help in spore dispersal.

8

Stomata

Observations:-

ANGIOSPERMS

Anatomy in relation to Taxonomy

Exp 1: Study of Stomata

Study of stomata has gained increasing importance as a taxonomic character. In

angiosperms there are four main types of stomata with respect to the arrangement of the

surrounding subsidiary cells. These are-

anomocytic (without subsidiary cells),

anisocytic (with unequal subsidiary cells),

paracytic (with two parallel subsidiary cells) and

diacytic (with two subsidiary cells at right angles to the guard cells).

These types are characteristic of certain plant families.

Aim :- To study stomata from different genera of family Rubiaceae.

Requirements:- Different plants of family Rubiaceae, slides, coverslips, blade, glycerine

and compound microscope.

Protocol :-

Take a peel of the lower surface of the leaves and mount in glycerine. Place a coverslip

and observe under the microscope.

Conclusion -

9

Trichomes

Observations :-

2. Study of Trichomes

Tichomes are unicellular or muliticellular epidermal outgrowth meant for various

functions like protection, reduction in transpiration, trapping insects.The type of

trichomes on the leaf and stem surface is also a taxonomic character and is a valuable tool

in plant classification.

Aim:- To study trichomes from the leaf surface of cohort Malvales.

Requirements:- Different plants of cohort Malvales, slides, coverslips, blade, glycerine

and compound microscope.

Protocol:-

Using a blade, scrape the surface of the leaves (upper or lower) and mount the scraped

material with a drop of glycerine. Place a coverslip and observe under the microscope.

Conclusion -

10

Phytochemistry in relation to Taxonomy (Chemotaxonomy)

The phytochemical characters of plants often find a wide application in taxonomic

delimitations. The occurrence and distribution of various types of chemical substances

present in plants, mainly secondary metabolites, form the taxonomic evidence. Secondary

metabolites like phenolic compounds, flavonoids, alkaloids terpenes etc. are some of the

group of chemical compounds.

Requirements: Plants containing phenolic compounds, alcohol, FeCl3, Methylene chloride/ Dichloro

methane (CH2Cl2), pyridine, NaOH, Dil. HCl, 10 % lead acetate, test tubes, pipettes.

Hydro-alcoholic plant extract – Hot extract the phenolic/ flavonoid compounds from

the plant material / powder in 50% alcohol. Cool and use as plant extract.

Test for Phenols :-

1) Test for Water- soluble Phenols:

2,3ml hydro-alcoholic plant extract + 1 to 2 drops of 1% aqueous FeCl3 solution gives a

red/blue/green/purple colour indicating presence of water soluble phenols.

2) Test for Water-insoluble Phenols:

2-3ml hydro alcoholic plant extract + 0.5ml of Dichloro methane (CH2Cl2) + 3-5 drops of

1% FeCl3 in Dichloro methane + a drop of pyridine, stir. If the mixture turns coloured,

phenols are present.

3ArOH + FeCl3 → Fe (OAr)3

Aromatic phenols

The ferric chloride forms a complex with the phenol, provided they are both very dilute,

and the complex acts as a colour test for phenols.

Test for Flavonoids :-

1) Ferric chloride test – Test solution + few drops of FeCl3 solution → blackish red

colour indicating presence of flavonoids.

2) Alkaline reagent test :- Test solution + NaOH solution shows increase in the intensity

of yellow colour which could become colourless on addition of a few drops of dilute HCl,

indicating presence of flavonoids.

3) Lead acetate test :- Test solution + a few drops of lead acetate (10%) solution gives

yellow precipitate indicating presence of flavonoids.

11

Observations:

Pollen grains of all three species exhibited following characteristics-

1. Shape- Spheroidal

2. Exine ornamentation- Heterobrochate, Reticulate, Tagillate, Sexine, Pilate

3. Excrescenses- Present- Spinate: Spines with broad base and blunt tips.

4. Aperture- Pantoporate

Palynology in relation to Taxanomy

Aim: To study pollen morphology in relation to Taxonomy.

Requirements: Pollen from Different Malvaceae members like Hibiscus rosasinensis (wild

variety), Hibiscus rosasinensis (cultivated variety), Hibiscus esculentis, Benzene, Castor oil,

slides, coverslips, microscope.

Principle: Taxopalynology is a branch where palynological study is used in taxonomy to bring

out affinities between different families or to ascertain dispute position of any genus or to

identify different species of the same genus. Pollen morphology structures with respect to size,

shape, exine ornamentation, excrescenses and apertures are used to identify different pollen and

derive taxopalynological conclusions.

Simple method like Benzene-Castor oil method can be used for this purpose. Acetolysis is the

method for pollen slide preparation to be used in long term.

Procedure:

1. Take flower from different members of Family Malvaceae like Hibiscus rosasinensis (wild

variety), Hibiscus rosasinensis (cultivated variety), Hibiscus esculentis on different slides.

2. Tap gently to remove anther wall. Pinhead can be used for this purpose.

3. Add 1-2 drops of benzene. White precipitate is formed on the surface.

4. Continue to add Benzene till no precipitate is seen indicating that total extraction of waxy

deposits from pollen surface making ornamentation clear and visible.

5. Mount in castor oil. Observe under low power and then high power of microscope.

6. Record observations for pollen morphology with respect to shape, exine ornamentation,

excrescenses and aperture.

7. Comment on the degree of similarity or dissimilarity between species in terms of palynology

and conclude.

Conclusion:

12

ANGIOSPERM FAMILIES :

Leguminosae : Papilionaceae

Classification

Division : Spermatophyta (Seed bearing plants)

Sub-division : Angiospermae(Ovules enclosed by ovary wall)

Class : Dicotyledoneae (Reticulate venation in leaves, tetra- or pentamerous

symmetry of flowers)

Sub-class : Polypetalae ( petals free)

Series : Calyciflorae ( flowers perigynous, calyx gamosepalous )

Cohort : Rosales (carpels 1 or many apocarpous, marginal placentation )

Family : Leguminosae

Sub-family : Papilionaceae

Genus : Clitoria

Species : ternatea

Distinguishing characters

Habit : A twining herb.

Leaves : Leaves imparipinnate, stipules linear, leaflets 5-7, elliptic, oblong, reticulate

venation.

Inflorescence : Flowers axillary, solitary.

Flower : Bracteate, bracteolate, pedicellate, zygomorphic, bisexual, hypogynous,

complete.

Calyx :- Sepals 5, gamosepalous, unequal.

Corolla : Petals 5, polypetalous, unequal, papilionaceous, largest posterior petal is called

standard or vexillum, 2 lateral petals smaller are called wings or alae and 2 anterior still

smaller petals forming boat shaped keel or carina. Aestivation vexillary. Blue or white.

Androecium : Stamens 10, diadelphous, 9 stamens united and 1 free.

Gynoecium : Monocarpellary, ovary superior, unilocular, manu ovules on marginal

placentation, style bent, stigma hairy.

Fruit : a legume.

Plants of economic importance 1) Pisum sativum – peas, seeds rich in protein

2) Cajanus cajan – Tur

3) Vigna catjang – Chavli

4) Dolichos lablab – Vaal

5) Trigonella foenum graecum – fenugreek- methi, seeds and leaves are edible

6) Dalbergia sissoo – Shisam wood used in furniture making.

7) Butea frondosa – flame of the forest, Palash.

13

Leguminosae : Caesalpinaceae

Classification









Sub-family : Caesalpinaceae

Genus : Caesalpinia

Species : pulcherrima

Distinguishing characters Habit : A small shrub.

Leaves : Alternate, stipulate, bipinnately compound, large.

Inflorescence : Erect raceme.

Flower : Bracteate, pedicellate, zygomorphic, bisexual, complete, hypogynous.

Calyx : Sepals 5, unequal, coloured, free or connate at the base, imbricate, one large boat shaped

.

Corolla : Petals 5, unequal, 4 large 1 small, small petal opposite the large sepal, petals clawed.

Androecium : Stamens 10, free, unequal, exerted, anthers versatile.

Gynoecium : Monocarpellary, ovary superior, unilocular, many ovules on marginal placenta,

stlye long, stigma capitate.

Fruit : Legume.

Plants of economic importance

1) Delonix regia – Gulmohur, cultivated in gardens for shade and beautiful flowers.

2) Tamarindus indica – tamarind, imli fruits sour used as condiment, carminative & laxative.

3) Bauhinia racemosa – apta, kanchan, sacred plant with beautiful flowers, cultivated in

gardens.

4) Cassia angustifolia – senna, leaves used for relieving constipation, loss of appetite.

5) Cassia fistula, C. javanica, C.renigera, C.auriculata are grown as avenue trees.

14

Leguminosae : Mimosaceae

Classification









Sub-family : Mimosaceae

Genus : Acacia

Species : auriculiformis

Distinguishing characters

Habit : medium sized tree.

Leaves : petioles are modified to phyllodes which are flat sickle shaped with parallel convergent

venation. Leaf lamina is not developed.

Inflorescence : long, erect spikes

Flower : sessile, actinomorphic, hypogynous or perigynous, bisexual, complete.

Calyx : Sepals 5, gamosepalous, sepals equal.

Corolla : Petals 5, polypetalous, equal, recurved.

Androecium : Stamens many free, colored.

Gynoecium : monocarpellary, ovary superior, single ovule on marginal placenta, style long,

stigma minute.

Fruit : legume or lomentum.

Plants of economic importance

1) Acacia Arabica – babool, young stems used as toothbrush. Wood used in making cart

wheels, yields gum Arabic.

2) Acacia catechu – khair, kaath, used as an astringent.

3) Pithecolobium dulce – vilayati chincg, fruits edible sweet.

4) Mimosa pudica – touch-me-not, leaves sensitive to touch.

5) Adenanthera pavonia – red sandle wood tree, ratangunj, red seeds.

15

Asteraceae

Classification


Sub-division : Angiospermae (Ovules enclosed by ovary wall)



Sub-class : Gamopetalae ( petals united, stamens epipetalous)

Series : Inferae (flowers epigynous, ovary inferior)

Cohort : Asterales ( inflorescence capitulum or head )

Family : Asteraceae ( Compositae)

Genus : Helianthus

Species : annus

Distinguishing characters Habit : Herbs

Leaves: simple, alternate, exstipulate, margin serrate or lobed.

Inflorescence: capitulum

Florets: 2 types of florets are present on the inflorescence.

Ray florets: Situated towards periphery of capitulum, zygomorphic, unisexual, incomplete,

epigynous.

Calyx: sepals modified into 2 scaly segments.

Corolla: petals 5, united to form strap shaped or ligulate corolla.

Androecium: absent

Gynoecium: bicarpellary, syncarpous, ovary inferior, one celled, ovule is absent style single,

stigma bifid.

Disc florets: Situated in the centre of the inflorescence, actinomorphic, bisexual,

fertile, epigynous, bracteate, centripetal arrangement.

Calyx: sepals modified into 2 scaly segments.

Corolla: petals 5, gamopetalous, tubular.

Androecium: stamens 5, epipetalous, syngenesious-anthers united but filaments free

Gynoecium: bicarpellary, syncarpous, ovary inferior, one celled, single ovule on

basal placentation, style single, stigma bifid.

Fruit: cypsela

Plants of economic importance: 1) Chrysanthemum indicum (Shevanti)

2) Tridax procumbens – common herbaceous weeds

3) Eclipta alba (Maaka) – (Bhringaraj) – extract of leaves mixed with coconut oil keeps

hair black. It is a good tonic for spleen also cures leucoderma.

4) Helianthus annus – (sunflower) seeds yield edible sunflower oil used in cooking.

5) Carthamus tinctorius – (safflower or kardai) seeds yield edible oil, flowers are tonic

for liver and also yield red dye.

6)Dahlia, Zinnia, Tagetes, Gallardia, Cosmos, Gerbera etc. are annual flowering ornamentals

16

Amaranthaceae

Classification:

Division – Spermatophyta (seed bearing plants)

Sub-division – Angiospermae (ovules enclosed in the ovary)

Class - Dicotyledoneae (Reticulate venation, tetra or pentamerous

symmetry)

Sub-class – Apetalae/ Monochlamydeae (flowers often unisexual with or without

perianth, perianth with only one whorl, usually corolla absent )

Series – Curvembryeae ( flowers regular, ovary superior, embryo curved)

Order – Chenopodiales ( mostly herbs, ovary superior, monocarpellary with one ovule)

Family – Amaranthaceae

Genus – Gomphrena

Species – globosa

Distinguishing characters:

Habit – small herb, hairy with swollen pinkish nodes.

Leaves – simple, opposite, exstipulate, sessile.

Inflorescence – globose cyme.

Flower – Actinomorphic, bisexual, presence of dry bracts and bracteoles, bracts green,

bracteoles scarious (thin, dry, membranous), hypogynous.

Perianth – Tepals – 5, in a single whorl, free or connate, scarious, brightly coloured.

Androecium – Stamens - 5, united to form stamina tube, stamens opposite the tepals.

Gynoecium – bicarpellary, syncarpous, one celled , superior, single ovule on basal placenta,

style slender, stigma bifid.

Plants of economic importance:

1. Gomphrena globosa – Gentleman’s button, ornamental plant.

2. Amaranthus paniculatus-seeds edible– (Rajgira), leaves used for purifying bloo

3. Amaranthus polygamous – Chavli, lal math, leaves used as vegetable.

4. Celosia cristata – Ornamental plant– cock’s comb, in yellow, orange & red in

colour

5. Celosia argentea – common weed after rains, seeds useful in diarrhea.

6. Achyranthus aspera – Aghada, common weed, useful in dropsy, piles,

boils and colic. Also used as a cure for cough, hydrophobia.

17

Palmae

Classification:

Division: Spermatophyta (seed bearing plants)

Sub-division: Angiospermae (ovules enclosed in the ovary)

Class: Monocotyledoneae(leaves with parallel venation, trimerous symmetry)

Series: Calycinae ( unbranched trees with palmate or pinnately compound leaves,

inflorescence compound spadix, flowers cyclic, hypogynous, 3 carpels )

Family: Palmae

Genus Caryota

Species urens

Distinguishing characters:

Habit – Tree with an unbranched stems.

Leaves – Large bipinnately compound forming a crown at the apex with well developed

petiole, spirally arranged , leaflets, triangular, irregularly cut upper margin

prolonged into fish-tailed process.

Inflorescence – spadix, very large drooping and freely hanging covered by 2- 3 spathes.

Male Flower Flower – Actinomorphic, sessile, hypogynous.

Perianth – Tepals 6 in two whorls of 3 each, outer small and inner large, green, leathery,

hard (calycine), valvate aestivation.

Androecium – Stamens many, filaments hort and white, anthers versatile.

Gynoecium – ovary rudimentary ( pistillode).

Female Flower

Flower – Actinomorphic,sessile, hypogynous and small.

Perianth – Tepals 6 in two whorls of 3 each, outer small and inner large, green, leathery,

hard (calycine), valvate aestivation.

Androecium – Staminodes, usually 3 present.

Gynoecium – tricarpellary, syncarpous, superior, 3 celled but only one ovule, stigma

sessile. Plants of economic importance:

1) Cocos nucifera – coconut ( Kalpvriksha) – leaves used for thatching, making baskets,

hats & brooms. Coconut water is the liquid endosperm, very nutritious, also used in

tissue culture. The solid endosperm is eaten raw or cooked, drier coconut yields oil

which is edible. The fibrous husk ( mesocarp ) is used in making coir which is used in

making mats and rugs etc.

2) Borassus flabellifer - Taad, sap is tapped and fermented (taadi) or consumed fresh

(nira), stem used for making poles, pillars, rafters.

3) Phoenix dactylifera – dates (khajur)

4) Areca catechu –(supari)- grown commercially in India, used as masticatory & chewed

along with Paan, powered nut is useful in checking diarrhea & urinary disorders. An

alkaloid , arecoline is used in ophthalmic treatment.

5) Metroxylon rumphii – stem pith yields starch from which sago (sabudana) is made.

6) Calamus rotang - (cane) – valued for the stem, used in furniture making, housing,

walking sticks etc.

18

HERBARIUM AND WET PRESERVATION TECHNIQUE

Aim: To study hebarium and wet preservation technique.

HERBARIUM

A collection of dried plant specimens, mounted on sheets is known as herbarium. Freshly-

picked specimens are dried and pasted on mounting paper of regulation-sized herbarium sheets.

The purpose of such a collection is to study the vegetation of a locality and maintain its record.

Preparation of herbarium sheets

1. Equipments - On excursion, for collection of plants, several items are required like

trowel - to collect the entire plant, if possible, vasculum - to bring few numbers of plants

to the lab. Along with this, news papers or blotting papers are required to press the

plants on the field itself to avoid loss of natural colour of some of the plants.

2. Collection – Collected plants are placed in the collecting sheets. The most practical size

is 16.5 x 23 inches and when folded, 16.5 x 11.5 inches. A specimen collected should

represent the root, stem, leaves and flowers. The plants are placed between the sheets or

newspapers. Herbaceous plants, 2 feet or less higher, may be collected entire. The most

desirable is to collect a branch, about one foot high, containing leaves and flowers.

After a specimen has been collected and placed in a collecting sheet, it is kept in a plant

press. This collecting sheet is placed in between blotting papers, one on either side.

While on collection, it is important to note the date, locality, habitat, height, method of

branching, colour of reproductive parts, common name etc. This should be noted

separately in a field-book.

3. Pressing - The collecting sheets should be transferred to a heavy laboratory press.

The press is securely tightened. The press should be placed in a warm, well-aired

place to dry. After 24 hours, the press is taken out and opened. The old newspapers

and blotting. Sheets are replaced by new unused ones. At least such 3 – 4 changes

are given at an interval of 2 – 3 days. An average specimen takes about a week for

complete drying. Sometimes to hasten the process of drying, plant press may be

placed near an appropriate heat source.

4. Mounting – The specimen are ready for mounting once they are completely dry. The

standard size of the sheet is 16.5 x 11.5 inches. The paper should be of good weight and

not thin and flexible. The quality should be so that it does not turn yellow even with a

considerable lapse of time.

To mount the specimen, one of the following methods can be used.

a) The gum is spread on a glass plate and the specimen is laid on it. As soon as all the

parts come in contact with the gum, it is lifted and then placed in position on a

mounting sheet.

b) The specimen is inverted and painted with gum by a brush and then transferred to

a mounting sheet.

c) The specimen is placed on a herbarium sheet and small strips of gummed tape

or cellulose tape are pasted at suitable places, so that most of the part remains loose.

After mounting the specimen, a label is pasted in the right hand lower corner of the sheet.

This carries information regarding the botanical name of the plant, common name, date,

collector’s name, place of collection etc.

5. Arrangements of sheets – The sheets are finally arranged in accordance with a standard

classification system (preferably Bentham and Hooker’s classification for Angiosperms).

The sheets are arranged into groups according to species, genera, families, classes,

orders, series and sub-divisions etc. Each group is placed in a separate envelope, slightly

larger than the herbarium sheets.

6. Care of sheets – Herbarium sheets are often attacked by museum pests, fungi etc. To

guard them against specimens, these are fumed with carbon bisulphide, 3 – 4 times a

year. Mounted specimens may also be treated with mercuric bichloride or copper

sulphate. To prevent them from being attacked, powdered naphthalene balls or

gamaxene powder should also be spread or dust from time to time.

WET PRESERVATION –

While on a collection trip, local or outstation, following things are to be carried along.

(1) Containers - For packing the collected material, preferably unbreakable plastic

containers or polythene bags.

(2) Preservatives - Formalin-Acetic-Alcohol (FAA) or Alcohol 70 % or Alcohol 90 %,

and/or Formalin 4 %-10 %.

(3) Other requirements - Scalpel, knife, blade, forceps, pencil, paper, a hand lens, a bag

or vasculum for keeping plants or a plant press with many newspapers or blotting

papers.

After collecting the plant, it should be immediately killed and preserved or pressed to avoid its

rotting and dehydration. On return to the laboratory, collected material should be transferred to

new and suitable containers with fresh preservative.

A few plants e.g. filamentous algae, fungi, reproductive parts of bryophytes, fertile parts of

pteridophytes and different parts of gymnosperms, if collected in large quantities, are preserved

in containers. Even if large quantities of such plants are available, one plant with fertile parts

should be preserved in the form of a herbarium sheet, while others should be packed in a

container.

Every tube should be labeled. It is important to write the name of the specimen, place and date

of collection. The place of collection and date should also be written on a small piece of white

card paper with a pencil, on the spot and inserted in the container. On return to the

laboratory, the material is identified with the help of standard books. A label bearing the name

of the division and class to which the material belongs, the name of the material, date and place

of collection and also the name of student should be pasted on the container. All the containers

should be of uniform size as far as possible.

Generally for cryptogamic and gymnospermic material, 4 % formalin or formaldehyde is

preferred. But occasionally after a period lapse, because of formalin the specimen may get

bleached and lose their original colours. Some of the fungi can grow even in this condition and

contaminate the preservation. To avoid this 1 % copper sulphate solution is used. When

specimens are preserved (anthers, root tips etc) for cytological studies, they are pretreated with

fixative agents like FAA and then preserved in 70 % alcohol. For VAM studies roots are

preserved in 70 % alcohol before staining where as after staining procedure, they are preserved

in lacto phenol.

19

Observations –

1. Distance traveled by solvent - __________________ cm.

2. Distance traveled by first spot (solute) - ___________ cm.

3. Distance traveled by second spot (solute) - _________ cm

Calculations –

Rf = Distance traveled by solute

Distance traveled by the solvent

CHROMATOGRAPHIC SEPARATION OF AMINO ACIDS

Aim – To separate aminoacids by circular paper chromatography.

Requirements - Chromatography paper strips, Solvent system- butanol: acetic acid: water

(40:11:25), capillaries, chromatography chamber, amino acids sample, ninhydrin spray reagent.

Principle - Cellulose in the form of paper sheet makes an ideal support medium where water is

adsorbed between the cellulose fibres and forms a stationary hydrophilic phase. Butanol serves

as the mobile phase. Amino acids from the mixture separate on the basis of differences of size,

mass, solubility and affinity to the solvent.

Ninhydrin reacts with alpha amino acids to give a purple color and with imino acids to give a

yellow colour.

Procedure –

a. Take a circular Whatman no 1 filter paper, mark the centre and make a slit to attach a wick.

b. Load the known amino acid solutions and the sample at different spots equidistant from the

centre.

c. Place the loaded circular disc on the petridish containing solvent system on the wick.

d. Cover the entire system with a glass jar. Make it airtight by using petroleum jelly on the rims

of glass jar.

e. Allow the solvent to run across the circular disc upto 3/4th

of the diameter.

f. Remove the sheet and mark the solvent front. Develop the chromatogram by spraying spray

reagent.

g. Let it dry, mark the spots and calculate Rf value.

Result –

20

Observations –

1. Distance traveled by solvent - __________________ cm.

2. Distance traveled by first spot (solute) - ___________ cm.

3. Distance traveled by second spot (solute) - _________ cm

Calculations –

solventby travelledDistance

soluteby travelledDistancefR

SEPARATION OF CAROTENOIDS BY TLC

Aim - To separate carotenoids by thin layer chromatography from the plant material.

Requirements – Slides, chromatography chamber, pestle and mortar, muslin cloth, glass rod,

separating funnel, Acetone, petroleum ether, benzene, 5% sodium sulphate solution, methanolic

KOH, silica gel powder, distilled water, solvent system – Petroleum ether: benzene (1: 9),

Tomato fruit / Carrot root.

Principle – Carotenoids are tetraterpenoid (C40) compounds existing as hydrocarbons

(carotenes) or oxygenated derivatives (xanthophylls) giving characteristic colour to plant parts,

flowers and fruits. The total carotenoids are extracted and separated on the basis of differential

absorption between the stationary phase and mobile solvent phase. The solutes compete with the

solvent for surface sites of the absorbent depending on the distribution co-efficient on the

surface of the absorbent.

Procedure – (Extraction of carotenoids) a. Weigh 5 – 10 gm of tomato pulp or carrot tissue and extract it with 10 ml acetone in pestle

and mortar.

b. Filter it and take the filtrate in a separating funnel containing 20 ml petroleum ether.

c. Add 20 ml of 5% sodium sulphate solution and shake it gently. Two separate layers are

obtained.

d. Collect the upper petroleum ether layer in a brown bottle containing 10 gm anhydrous sodium

sulphate. Keep it aside for 30 minutes.

e. Decant the extract in separating funnel and add 60% methanolic KOH, distilled water and

more ether. Two layers are obtained.

f. The upper layer containing yellow carotenoid pigments is collected for separation.

Separation of carotenoids –

a. Prepare TLC plates with silica gel and acetone, keep at 100 – 1200 C for 1 hour.

b. Load the carotenoid extract on activated TLC plate and place it in pre saturated

chromatographic chamber. Keep it covered.

c. Allow the solvent to run 2/3rd

of the plate, remove the plate and mark the solvent front.

d. Let it dry and mark the spots of carotenoids to calculate the Rf value.

Result – Rf of solute/s

21

Horizontal Gel Electrophoresis apparatus

Vertical Gel Electrophoresis Apparatus

GEL ELECTROPHORESIS

Electrophoresis is a technique for separation of components on the basis of charge and mass

ratio. Agarose gel electrophoresis is the standard method for the separation, identification and

purification of DNA and RNA fragments. The location of DNA within the gel can be

determined directly by staining with low concentrations of intercalating fluorescent dye

‘Ethidium bromide’ under U.V. light.

HORIZONTAL GEL ELECTROPHORESIS

The apparatus consists of buffer tank, gel casting unit, comb and lid. Agarose gels are cast by

melting the agarose in the presence of suitable buffer. DNA samples are mixed with glycerol

and bromophenol blue (tracking dye) is loaded in wells specially made in the gel with the help

of the comb. When electric field is applied across the gel, the DNA, which is negatively charged

at neutral pH, migrates towards the anode. TAE (Tris- Acetate EDTA) buffer is the commonly

used buffer for the DNA samples. DNA fragments migrate at different rate under the influence

of electric field (due to their mass difference) and separate as bands.

VERTICAL GEL ELECTROPHORESIS

The apparatus consists of buffer tank, glass plates, spacers, comb, clips, clamps, petroleum

jelly, lid, silica plate etc. After cleaning and drying the glass plates and spacers, assemble them

properly. Hold the assembly together with the help of bulldog clips. White petroleum jelly or

2% melted agar is then applied around the edges of the spacers to hold them in place and seal

the chamber between glass plate and silica plate. Prepare a sufficient volume of gel mixture

( agarose or polyacrylamide gel) and pour the gel solution in the chamber between silica plate

and glass plate and place the comb in the upper end of gel and allow the gel to set (30-60 min.)

After setting, remove the comb carefully. Then install the gel after removing the clips, agar etc.

in electrophoresis apparatus. Fill it with electrode buffer. Prepare sample for electrophoresis.

Mark the well properly with permanent marker for correct and easy sample loading in sample

well. Sample is loaded with the help of micropipettes. Cover the apparatus properly with lid.

Apply electric field for separation. Then continue the separation until the bromophenol blue (

tracking dye) reaches the bottom of the gel. After the run is complete, carefully remove the gel

from between the plates.

22

A Cell ( Internal 3 D View)

Mitochondrion- Ultrastructure Peroxisome- Photorespiratory cycle

P II – CELL BIOLOGY

CELL ORGANELLES

The cell is regarded as a structural and functional unit of living organisms. A typical eukaryotic

cell has a well defined nucleus surrounded by a nuclear envelope. The cells are characterized by

a complex structural organization in which various cell organelles carry out different functions.

Mitochondria:

- Mitochondria are present in all plant and animal cells.

- They may be oval, rod shaped, club shaped etc.

- Oval mitochondria range from 0.5 -1.0 µ in diameter and length may be twice the

diameter.

- It is surrounded by two membranes.

- The outer membrane is stretched and the inner membrane shows invagination known as

Cristae.

- The inner membrane is associated with small stalked particles referred to as F1 particles

or elementary particles.

- Each F1 particle shows a base piece, a stalk and a head piece.

- The mitochondria is filled with a jelly like matrix which also contains ribosomes,

mitochondrial DNA, sugars and salts.

- Matrix is site for Kreb’s cycle and inner membrane for the ETS. Mitochondrion is called

the power house of the cell.

Microbodies :

- In addition to lysosomes the cytoplasm contains other membrane bound vesicles called

microbodies.

- Two common microbodies are Peroxisome and Glyoxysome.

- Microbodies contain catalase and oxidase enzymes.

Peroxisome

- It is having potential peroxidase activity.

- It is oblate or spherical organelle containing granular and amorphous matrix.

- It is bounded by single unit membrane which arises from Endoplasmic Reticulum.

- The membrane permits the substrates like amino acids, α – hydroxyl acids and uric acid.

- The peroxisome oxidizes a variety of substrates in to two step reaction.

- In first step, substrates (like uric acid, amino acids and lactic acid) are oxidized by

molecular Oxygen to form H2O2 by oxidase enzymes. H2O2 is destructive to cells and has

to be removed.

- In second step, H2O2 is broken down into water molecule and the process is catalized by

Catalase enzymes.

- Peroxisome involves in photorespiration process occurring in C3 plants.

23

Glyoxysome ( showing Glyoxylate cycle)

Ribosome

Glyoxysome

- It is oblate or spherical organelle containing granular and amorphous matrix.

- It is bounded by single unit membrane which arises from Endoplasmic Reticulum.

- In Glyoxysomes, the glyoxylate cycle converts two acetyl co.A into succinic acid i.e. four

Carbon compound for gluconeogenesis.

- Glyoxylate cycle enzymes have been found in the Protozoa, some Fungi and the

Metazoa.

- Glyoxysomes also contain the enzymes for β – oxidation of fatty acids.

- Seeds rich in fat convert storage fats to carbohydrates during germination.

- Conversion of storage fat to carbohydrates in germinating seed via glyoxylate cycle

involves three organelles – Oleosome, Glyoxysome and mitochondria.

- The four carbon organic acid – Succinic acid which form inside glyoxysome converts

into hexose sugar inside mitochondria.

Ribosomes :

- Palade , 1955, revealed the presence of dense granules in cytoplasm. Later on it is termed

as Ribosomes.

- These granules may be found in the free state (either monosomes or associated with m-

RNA i.e. polysome) or attached to membranes of organelles.

- It is made up of protein and RNA and having small and large two subunits.

- Ribosomes are of two basic types, 70S and 80S ribosome.

- The ‘S’ refers to Svedberg Unit. This is a Sedimentation Coefficient and it is not additive.

- 70S ribosomes are relatively smaller and found in prokaryotes e.g. bacteria. The 70S

ribosome consists of a large 50S subunit and a small 30S subunit.

- 80S ribosomes are found in eukaryotes e.g. algae, fungi, higher plants and animals. The

80S ribosome consists of a large 60S subunit and a small 40S subunit.

- Ribosomes found in mitochondria and chloroplasts of eukaryotes are closer to

prokaryotic ribosomes.

- Metal ions, mainly Magnesium ion, play an important role in holding the two subunits

together.

- Ribosomes take part in protein synthesis.

24

Observation Table –

Sr. No Amount of extract

(ml)

Saline citrate

buffer

(ml)

Diphenylamine

reagent (ml)

O. D.

1 Standard DNA – 0.1

ml.

0.9 4.0 X

2 0.5 ml. plant sample 0.5 4.0 Y

0.1 ml. standard DNA contains 0.05 mg DNA

Calculations –

(X)___ ∆ O.D. = __ 0.05mg DNA (purine).

(Y) ___ ∆ O.D = Y x 0.05 / X = (Z) mg DNA (purine).

0.5 ml of RM contain _____ Z mg DNA

10 ml RM will have 20 X Z mg = _____ mg DNA (purine).

100 gm of plant material contains = _______ (A) mg DNA (purine).

Since only the deoxyribose of the purine nucleotide reacts, the value obtained represents only

half of the total deoxyribose present.

Therfore the total DNA content = (A) X 2 = _____ mg DNA %

QUANTITATIVE ESTIMATION OF PLANT GENOMIC DNA.

Aim – To estimate DNA from the plant material

Requirements – Pestle and mortar, test tubes, pipettes, colorimeter, centrifuge, water

bath,extraction buffer ( preparation- 50 mM Tris- HCl pH- 8.0 + 50 mM EDTA + 250 mM

NaCl + 15% Sucrose), ethanol, Isopropanol, 5 M potassium acetate solution, standard DNA –

0.5 mg / ml, freshly prepared diphenylamine reagent ( 5 gm. Diphenylamine in 500 ml. glacial

acetic acid + 13. 75 ml. conc. H2SO4), saline citrate buffer (o.15 M NaCl + 0.15 M sodium

citrate- pH – 7),, radical of germinating seeds.

Principle – The nucleic acids are usually present in the cell in combination with proteins and are

called nucleoproteins. During isolation this complex breaks and the nucleic acids are

precipitated with alchohol. The deoxyribose of DNA in presence of acid forms hydroxylevulinic

aldehyde which reacts with diphenylamine to give a blue colouration. In DNA, only the

deoxyribose of the purine nucleotide reacts, so the value obtained represents only half of the

total deoxyribose present. This reaction is given by deoxypentoses and is not specific for DNA.

The absorption maxima is read at 600nm.

Procedure –

I. Isolation of DNA – (not to be performed by students)

1. 2 g of radical is homogenized with 15 ml of chilled Saline extraction buffer and filter

with muslin cloth.

2. To 15 ml of filtrate add 5 ml. potassium acetate solution and keep in ice for 20 minutes.

3. Centrifuge at 4000 rpm for 20 minutes.

4. Take the supernatant and add 2/3 vol of isopropanol and refrigerate for 30 minutes.

5. Centrifuge and collect the pellets and solubilize in 10 ml. saline citrate buffer.

II. Estimation of DNA –

1. Prepare a standard tube with 0.1 ml. standard DNA solution and 0.9 ml. saline citrate

buffer.

2. Take 0.5 ml. sample + 0.5 ml saline citrate buffer.

3. Add 4 ml of diphenylamine reagent to each of the test tube.

4. Place both the test tubes in boiling water bath for 10 minutes.

5. After cooling measure O. D. at 600 nm.

Result – 100 g plant material contains ______ mg of DNA.

25

Observation Table –

Sr. No Amount of extract

(ml)

D.W. (ml) Bial’s reagent (ml) O. D.

1 Standard RNA –

1 ml.

1 2 X

2 1.0 ml. plant sample 1.0 2 Y

Calculations –

OD of standard = X

OD of plant sample = Y

X OD = 5 µg ribose

Y OD = ? (Z µg )

1 ml sample contains Z µg ribose

10 ml. sample contains = 10× Z / 1 µg

= A µg

10 ml. sample made from 1 gm. Plant material. = _______A µg

Then 100 gm. Plant material contains = ? _A X 100 = _________ µg of Ribose

10

QUANTITATIVE ESTIMATION OF PLANT RNA.

Aim – To estimate RNA from the plant material

Requirements –

Glassware and apparatus – Pestle and mortar, test tubes, pipettes, colorimeter, centrifuge,

water bath etc.

- Standard RNA (standard ribose – 5 µg/ml.)

- Bial’s reagent

- Onion

Principle – Orcinol or Bial reaction is specific for pentose sugars. The ribose sugar of RNA

when heated with con. HCl forms furfuraldehyde (furfural). Orcinol reacts with the furfural in

the presence of ferric chloride as a catalyst to give a green colour. Since ribose sugar of only

purine nucleotides reacts, the value obtained represents only half of the total nucleotides.

Procedure –

- Prepare a standard test tube with 1 ml. of standard ribose solution + 1 ml. distilled water

+ 2 ml. Bial’s reagent.

- Crush 1 gm. Onion in distilled water, filter and centrifuge. Make the volume of filtrate to

10 ml.

- Prepare the reaction mixture tube with 1 ml. sample extract + 1 ml. distilled water + 2 ml.

Bial’s reagent.

- Keep both the test tubes in boiling water bath for 15 minutes, after which cool them to

room temperature and measure the O. D. at 660 nm.

Result – 100 g plant material (Cauliflower head) contains ______ µg of RNA.

26

CYTOGENETICS

STUDY OF INHERITANCE PATTERN WITH REFERENCE TO PLASTID

INHERITANCE

Inheritance due to factors present in the cytoplasm (non- nuclear) and does not follow

Mendelian inheritance is called cytoplasmic inheritance. They show maternal effects.

Example: Inheritance of plastid transmission.

In certain variety of Mirabilis jalapa (4 o’clock plant), leaves show green, white or mixed

(variegated) colour pattern. In various crosses conducted for the above said trait it was observed

that the progeny inherits the character of the female parent plant irrespective of the male parent

plant. This trait is due to the type of plastids inherited through the cytoplasm.

The phenotype of the progeny depends upon a factor present within the cytoplasm of the egg.

The plant leaves show green colour due to chloroplast, white colour due to leucoplasts while

variegated variety shows mixed plastids of both types in the cytoplasm of the egg.

27

Study of Chromosomal aberrations

Laggard Chromosome : Due to break in DNA molecule, the chromosome fragments are

formed without centrosomes ( acentric). Such fragments are not included in anaphase

segregration or do not get attached to spindle fibres. Hence, they are left out when other

chromosomes reach to opposite poles and are enclosed in daughter nuclei. Such lagging behind

chromosomes are destroyed by cytoplasmic enzymes and hence are lost gradually. These

chromosome fragments are called as laggard chromosomes or lagging chromosomes. They can

be observed at the beginning of the mitotic telophase or very alte anaphase. Such laggards cause

few abnormalities since the parental chromosome from which these laggards are formed will be

showing ‘deletion’ syndromes.

Chromosomal bridge: Chromatin bridge is a mitotic event that forms when teleomeres of

sister chromatids fuse together and fail to completely segregate into their respective daughter

cells. Such phenomenon is commonly observed during anaphase and called as chromosomal

bridge. After the formation of individual daughter cells, the DNA bridge connecting

homologous chromosome remains fixed and can be observed in next interphase stage.

Chromatin bridges are earliest and most readily visible when chromosomes are well stained.

Occurrence of chromatin bridges have been used as diagnostic marker for cancer

(tumourogenesis) in humans, since frequent changes in chromosomal structure and number may

be the basis of cancer development.

Ring Chromosome : Chromosomes are often considered as static containers for genetic

information. Chromatin information is known to play an important role for regulation of gene

expression. The topography (properties like shape, size etc) plays vital role in gene expression.

However a change in topology from linear structure to circular structure totally disrupts the

normal sequence of chromosomal segregation. Formation of ring chromosomes is due to two

reasons: a) By two DNA breaks, one in each arm of the same chromosome followed by fusion

of the proximal broken ends which results in formation of ring chromosome. b) By fusion of

dysfunctional telomeres from the same chromosome. It takes place due to deattachment of

protective proteins from the chromosome ends.

Formation of ring chromosome brings about some abnormalities (genetic syndromes) such as

abnormality due to deletion of terminal portion of chromosomes. It may also result into partial

trisomy since ring can form with two normal homologoues or corresponding chromosomes. In

most of the cases, occurrence of ring chromosome proves to be fatal in the early age because

somatic cells are prevented to proliferate normally.

28

MITOSIS

Aim : To study mitosis in the given planrt material.

Requirement : Root tips, 10 % HCl, Acetocarmine stain, slides, coverslips.

Procedure : Take a root tip and wash in water. Transfer the root tips in a watch – glass. Treat

the root tips with 1 % HCl to hydrolyse the cell wall and to separate the cells from each other.

After 10 minutes drain off HCl and wash the root tips with water repeatedly for 4 – 5 times.

Transfer the root tips on slide and add 2 – 3 drops of acetocarmine stain. After 5 min. place a

coverslip on the root tip and press it gently so that the cells spread evenly. Blot the excess stain

and observe under low power and then under high power to see the different stages of mitosis.

Principle : Mitosis is an equational division in which each cell divides to form two genetically

identical cells. Mitosis mainly occurs during growth. Mitosis produces daughter cells with same

number of chromosomes as the parent cell. Various stages of chromosomal separation can be

observed.

i) Prophase: Spirally coiled thread – like chromosomes appear and nuclear membrane

dissolves.

ii) Metaphase: Chromosomes are arranged on the equatorial plane. Each chromosome

consists of two chromatids.

iii) Anaphase: Chromatids separate and move towards opposite poles.

iv) Telophase: Chromosomes reach at the poles. Two daughter nuclei are formed by

construction of nuclear membrane. Telophase is immediately followed by the process

of cytokinesis in which cytoplasmic division takes place resulting in formation of two

daughter cells.

29

MEIOSIS

Aim: To study meiosis by smear technique from Tradescantia buds .

Principle : Pollen mother cells isolated from the anthers of the immature flower bud appear

spherical in shape. These are found to be undergoing division and appear to be in various stages

of meiosis. Acetocarmine is a nuclear stain. The acetic acid component makes the nucleus

enlarge while carmine component stains the chromosomes red in colour.

Requirements : Tradescantia flower buds, Acetocarmine, forcep, needle, glass slide, cover slip,

dissecting and compound microscope.

Theory : During microsporogenesis, the pollen mother cells undergo meiotic division

(reductional division) in which the chromosome number is reduced to half (n). Meiosis consists

of Meiosis I (reductional division) and Meiosis II ( equational division). The stages of Meiosis I

are Prophase I, Metaphase I, Anaphase I, Telophase I which is followed by Meiosis II consisting

of Prophase II, Metaphase II, Anaphase II and Telophase II.

Prophase I is of longest duration and is further sub divided into following 5 sub stages:

a) Leptotene or Leptonema : Chromatin condenses to form individual thread like chromonema.

b) Zygotene or Zygonema : Pairing of homologous chromosomes (synapsis) occurs.

c) Pachytene or Pachynema : Crossing over occurs between non-sister chromatids of

homologous chromosomes. Cross over regions are called chiasma. Exchange of genes

(recombinations) occur.

d) Diplotene or Diplonema : The chiasma opens out due to repulsion between homologous

chromosomes.

e) Diakinesis : Chromatin spindle starts appearing. Nuclear membrane and nucleolus

disappears. Homologous chromosomes are seen attached to the spindle fibre.

During Metaphase I, paired homologous chromosomes are found attached to and at the centre of

the spindle apparatus. During Anaphase I, homologous chromosomes separate and move

towards opposite poles resulting in reduction of number of chromosomes at poles to half.

During Telophase I, Chromosomes unwinds and forms chromatin, Nuclear membrane and

nucleolus reappear. Meiosis II is similar to Mitosis. During Anaphase II, sister chromatids

separate due to division of centromere and move towards opposite poles.

Procedure:

30

Take a clean dry slide

Isolate out the smallest (2nd or 3rd from base) flower bud onto the slide

Dissect open the flower bud and isolate the anthers (appearing transluscent) with the

help of a needle under the dissecting microscope

Tap the anthers to liberate the pollen mother cells

Discard away the anther wall cells.

Add a drop of acetocarmine onto the smear

Wait for 10 mins ( ensure that the slide does not dry)

Place a coverslip slowly and press it gently. Blot away the excess stain

Observe under the low power and then under high power of a compound microscope

AUTORADIOGRAM FOR DNA SEQUENCING

DNA SEQUENCING – SANGER’S METHOD

Aim - To read the DNA sequence from the given autoradiogram.

Principle - DNA sequencing can be carried out by two methods.

1. Maxam and Gilbert’s chemical degradation method

2. Sanger and Coulson’s enzymatic method.

Sanger and Coulson’s enzymatic method

Theory - In this method, a single stranded DNA is used as a template for DNA synthesis. 2′ 3′

Dideoxynucleotides (ddNTP) are incorporated leading to termination of synthesis. These

ddNTPs are used as triphosphates and they can be incorporated into the growing chain. The

respective ddNTPs terminate the synthesis since they can not form a phosphodiester bond with

the incoming nucleotide.

Procedure – This method consists of following steps:

1. Four reaction tubes are set up each containing single stranded DNA sample to be

sequenced cloned in M13 phage.

2. The DNA synthesis is terminated by the incorporation of ddNTP to terminate any one of

the four bases.

3. The reaction mixtures are then electrophoresed.

4. It is expose to X ray photographic plates which gives the autoradiogram.

Reading autoradiogram – The autoradiogram is always read from the anode to the cathode.

The sequence is that of the nascent strand which is in 5’ 3’ direction. The complemetary

strand can be deduced from this in 3’ 5’ direction.

Conclusion:

31

AMINO ACID REFERENCE DICTIONARY

Phe– Phenalamine Leu – Leucine Ser – Serine Tyr – Tyrosine

Cys – Cysteine Trp – Tryptophan Pro – Proline His – Histidine

Gln – Glutamine Arg – Arginine Ile – Isoleucine Thr – Threonine

Asn – Asperagine Lys – Lysine Ser – Serine Arg - Arginine

Val – Valine Ala – Alanine Asp– Aspartic acid Glu – Glutamine

Gly – Glycine Met – Methionine

(Initiator)

UAA /UGA – Ochre

(Stop)

UAG – Amber

(Stop)

AMINO ACIDS SEQUENCING IN THE PROTEIN MOLECULE SYNTHESISED FROM THE

GIVEN m – RNA STRAND

Aim - To determine the sequence of amino acid in the protein molecule synthesized from the

given m – RNA strand. .

Theory – Proteins are polymers of amino acids linked by polypeptide bonds. It starts from the

amino group which begins as N- terminal and ends with either C- terminal or the carboxy end.

Protein exhibit the phenotypic character of an organism. It is synthesized as a result of

transcription and translation.

Transcription – This involves the synthesis of complementary single stranded m – RNA from

the double stranded DNA template. The RNA based sequences are read in 5’ 3’ direction.

Translation – It involves the formation of protein molecules. It can be divided into three steps

namely initiation, elongation and termination of polypeptide chain.

Reading m – RNA strands of PROKARYOTES – The given m – RNA strand does not show

7 methylated C cap (7 mc) at the 5’ end. Therefore it is inferred that it is prokaryotic

m – RNA. In the prokaryotes, 30S ribosomal unit always gets attached to the ribosome binding

site called “The Leader sequence” or “Shine – Delgarno sequence (SD)”. It is a purine rich

sequence (5’ – AGGAGGU – 3’). It is 8 – 13 bases upstream of initiation codon AUG or GUG.

After the identification of SD, AUG or GUG is identified as initiating codon both of which

could code for formylated Methionine (f - met). The m- RNA is now divided into codons of 3

nucleotides each. The amino acid sequence is read with the help of genetic code dictionary. The

m – RNA of prokaryotes is said to be polycistronic and hence more than 2 polypeptide chains

can be synthesized.

Reading m – RNA strands of EUKARYOTES – The eukaryotic strands are always having 7

mc cap at the 5’ end. The end of the tail is always made up of poly A base. The initiating codon

AUG or GUG is identified and the amino acid sequence is read till a terminating codon is

reached. The m – RNA of eukaryotes is monocistronic therefore only one type of polypeptide

chain can be syntheised.

32

mRNA Strands

Prokaryotes Eukaryotes

Diagram of Eclipta

PRACTICAL - III

PHARMACOGNOSY AND PHYTOCHEMISTRY

Aim: To study pharmacological properties of plants.

(A) Eclipta alba

Bhringraj consists of the dried entire plant of Eclipta alba (L.) Hassk. (Fam:

Asteraceae/Compositae).

Chemical constituents: The plant contains alkaloids, ecliptine. The roots contain a nematicidal

compound called thiophenes. The aerial parts contain β- sitosterol. Also contains not less than

0.1 % of wedelolcatone and resins.

Description: A greenish brown colour when completely dry.

Macroscopic characters:

Root: Well developed, a number of secondary branches arise from main root.

Stem: Herbaceous, occasionally rooting at nodes, cylindrical, or flat, rough due to opposed

white hairs, node distinct, greenish, and occassionly brownish.

Leaf: Simple, opposite, sessile to sub-sessile, with hair on both surfaces.

Infloresence: Head or Capitulum

Flower: Presence of Ray and Disc florets

Uses: Extract added in coconut oil used for promoting hair growth. It prevents premature

graying of the hair.

Chemical tests:

Tests Observation Inference / Result

Extract + drop of conc. H2SO4 Greenish colour Alkaloids present

Extract + Dragendroff’s reagent Dark orange red colour Alkaloids present

Extract + Mayer’s reagent White colured ppt Alkaloids present

34

Diagram of Saraca

(B) Saraca (Ashoka) bark

Botanical source: It consists of dried stem bark of Saraca asoca (Rose.) De. Willd, (Family

Leguminosae). The bark is collected in spring from mature, wild or cultivated trees, found in

Central and Eastern Himalayas, Western Ghats and Deccan.

Macroscopic characters:

Bark: Channeled, externally dark circular lenticels, sometimes cracked, internally reddish-

brown, taste astringent.

Chemical constituents: It mainly contains condensed tannins, catechol, sterol, haematoxylin,

organic calcium compound.

Uses: Uterine tonic and sedative. Shows oxytocic activity.

Chemical tests:

Tests Observation Inference / Result

Aqueous extract + 5 % Ferric

chloride solution

Dark colouration / blue

black colour

Tannins present

Fresh aqueous extract + lime water Brown colour on

standing for 3 minutes

red precipitate is formed

Tannins present

1 % aqueous extract + Ferric

ammonium sulphate

Dark green colour, on

addition of NaOH

Changes to purple

(because of tannins)

Tannins present

35

Report making for the visit to forest /garden

SOURCES, PROPERTIES AND USES OF FIBRES & SPICES & CONDIMENTS

A) PLANTS YIELDING FIBRES –

Fibres are elongated cellular strucutures which may parenchymatous or sclerenchymatous

in natue. They can be obtained from root, stem, leaves, fruits and seeds. They provide

mechanical strength to the respective organs.

1. Cotton (Kapas):

Botanical name: Gossipium hirsutum

Family: Malvaceae

Source: upper surface of the seed coat

The fibres are of two kinds: viz. Lint and Fuzz. Lint fibres are long hairs which are more

commercially important. Fuzz are short hairs and commercially not important. The fibres are

chiefly composed of cellulose.

Uses:

In textile industry, stuffing, rubber tyres, fabrics, carpets, blankets, cordage, cellulose industry,

paper making, rayon etc.

2. Jute:

Botanical Name: Corchorus capsularis

Family: Tiliaceae

Source: Stem

It is a textile fibre, soft fibre or bastfibre, achived from the secondary phloem of the stem.

Uses:

This fibre is utilized in various purposes i.e. in preparation of gunny bags, twines, carpets,

inferior cloth, curtains, packing cloth, ropes etc.

B) PLANTS USEFUL AS SPICES AND CONDIMENT –

Spices and condiments are plant products used to improve the taste, flavor and aroma of

food. They also have medicinal properties.

1. Cardamom (Elaichi):

Botanical name: Elettaria cardamomum

Family: Zingiberaceae

Source: dried fruits

Uses:

1. Used in curry powder.

2. Flaouring sweetmeats, pastries, cakes etc.

3. Medicinally used in treatment of clod and cough, as carminative, in tea.

4. Eating a cardamom once daily with a tablespoon of honey improves eye-sight, strengthens the

nervous system and keeps one healthy.

2. Saffron (Kesar):

Botanical name: Crocus sativus

Family: Iridaceae

Source: Dried stigma of flowers

Uses:

1. Used as colouring, flavouring and garnishing agent in sweetmeats.

2. Used in treatment of fevers and enlargement of the liver and spleen.

3. Helps in urinary, digestive and uterine troubles.

37

PREPARATION OF ANY HERBAL COSMETICS

Aim: To prepare herbal cosmetics (Face pack/ De-tanning cream)

Principle: Acne, black head, pimples, dark circle are common among youngsters and person

who suffers from it. According to Ayruveda, skin problems are normally due to impurities in

blood. Accumulated toxins in the blood during improper food and lifestyle are causing skin

related diseases. Various herbs, medicines are described in Ayurveda for blood purification.

Herbs like Manjistha, Lodhra, Chandana, Haridra etc. are good example of blood purifier. The

herbal paste which is applied on face to treat acne, pimple, scars, marks and pigments are known

as “Mukhalepa” in ayurveda. The process of smearing this herbal mix on face is known as

“Mukhlepana”. This beauty therapy is popular as facial. The smooth powder which is used for

facial application is “face pack.” A good herbal face pack must supply necessary nutrients to

skin. It should penetrate the subcutaneous tissues in order to deliver the required nutrients.

Different types of skin need different types of herbal face packs.

Benefits of Applying Face Pack:

1. Nourishes the skin, by supplying nutrients to skin.

2. Helps to reduce, acne, pimple, scars and marks depending on its herbal ingredients.

3. Usually remove dead cells of skin.

4. Provides a smoothing and reflexing effect on skin.

5. Restore the lost shine and glow of skin in short span of time.

6. Brings glow to skin, improve skin texture and complexion.

7. Prevents premature aging of skin.

8. Effectively controls wrinkles, fine lines and sagging of skin.

9. Effectively combats with harmful effects of pollution and harsh climates.

10. Makes the skin look young and healthy.

Materials and Methods:

For preparation of facepack following plant materials are taken,

1. Multani Mitti (Calcium bentonite): It removes all impurities and dead skin cells.

Makes the skin radiant and excellent for aggravated and irritated skin.

Its cooling action soothes the skin, relives the inflammation caused due to aggravated pitta.

It removes the dirt and dead skin cells accumulated and replace with fresh, radiant and glowing

skin.

2. Manjistha (Rubia cordifolia): It holds the reputation of a very good skincare herb.

Used externally and internally, it helps one to gain luster and glow of the skin and aids to

remove pimples, frecckes and discolouration. Its paste should be applied in various skin

disorders like itching, black spots on the face, pimples, leucoderma etc. according to Charaka,

Manjistha is varnya (improving the complexion), jvarahara (febrifuge) and visaghna (detoxifier).

3. Haridra (Curcuma longa): Haridra has anti-inflammatory and anti-allergic activity. It is best

blood purifier and helos in wound healing. It possess best blood purification action so it is used

in all disease with blood impurities origin. Haridra is rejuvinatior of skin and revitalizes skin;

delays the signs of aging like wrinkles.

4. Chandan (Santalum album): Chandan (Red Sandalwood powder) has curative value in skin

allergies. Chandan powder has cooling and soothing action, protects the skin against the impact

of environmental pollution and keep the skin cool, fair and healthy. Sandalwood is helpful

ayurvedic herb with antimicrobial properties is used for healing various skin problems and

removes scars.

5. Lodhra (Symplocos racemosa): It nourishes the skin and benefits in acne, wrinkles and other

health issues related with skin. It lightens skin colour, reduces skin irritation and benefits for

acne, wrinkles and other skin related issues. Lodhra is useful in skin diseases requiring

purification of the skin. Lodhra is shavaro (making the body and skin very attractive) and

galavastatha (helping the whole body to maintain its shape).

Formulation of face pack

FACE PACK: For normal skin

Requirement: Multanimitti (50gms), Musta (10gms), Turmeric, Chandana, Jeshthimadhu

(5gms each)

Procedure:

Mix all above powders thoroughly and store in dry air tight container

Make a paste of 2-3 spoonful of above mixture with rose water

Apply on the facial skin

Wash after drying

Remarks: Herbs like turmeric and chandana improves the distribution of melanin. Musta is

useful in pruritus, also it improves the complexion of the skin

FACE PACK: for oily skin

Requirement:Masoor dal (lentil powder 50 gms), Musta, Arjuna, Lodhra, (10 gms each),

Turmeric, Triphala, Neem, Shrish (5 gms each)

Procedure:


Take 2-3 spoonful of above mixture and make a paste with skimmed milk

Apply on oily facial skin

Wash after drying

Remarks: All above herbs are skin rejuvenator. Turmeric, neem and shirish are blood purifier.

Arjuna is used against pimples and black spots.

FACE PACK: for dry skin

Requirement: Shatavari, Jesthimadhu, Musta (10gms each), Sariva, Khus, Turmeric (5gms

each)

Procedure:


Take 2-3 spoonful of above mixture and make a paste with shatdhautaghrita or rose water

Apply on dry facial skin

Wash after drying.

Remarks: Shatavari and Jesthimadhu remove dryness of the skin. The other herbs improve the

complexion of the skin.

Evaluation of Face Pack:

Shinoda Test: Extract + Magnesium turnings + Conc. HCl Red colour indicates the presence

of Flavonoids

38

Skin De- tanning Cream

Aim: To prepare de-tanning cream.

Theory: The skin colour is a function of size, number and the distribution of melanin cells. The

melanin cells of dark skin have thicker, longer and branched dendrites. Tyrosinase is rate

limiting enzyme for this biosynthetic pathway. Melanin cells are produced from the black

pigment due to the increasing tyrosinase activity. Many acting compounds are isolated from

natural herbs and their extracts are used as potential antioxidants in cosmetics. Some of the most

recent are Vit C, Kojic acid, Litoric extract, Burnet root extract, Sterculia extract and Mulberry.

The extracts of Glycyrrhiza (containing Glycirrhzin) and Papaya (Containing Papain and

flavonoids) are rich in natural antioxidants. These antioxidants are responsible for skin

whitening, skin depigmenting, skin lightening, anti aging, anti-erythemic, emollient, anti acne

and photo protection effects.

Preparation of De-tanning night cream:

2 spoon Aloe vera gel + 1/ 2 spoon olive oil + 2-3 threads of Saffron + 1 spoon Shea butter + 1/

2 spoon sandal powder.

All these materials are added and heated in a double boiler. After melting of bees was, mixing is

done to avoid lump formation. The mixture is cooled and refrigerated.

Shinoda Test: Extract + Magnesium turnings + Conc. HCl Red colour indicates the presence

of Flavonoids

39

Observation:

1. Weight of crucible with residue before ignition (W1):

2. Weight of crucible after ignition (W2):

Calculation:

% crude fibre in ground sample = ____W1 – W2___ X 100

Weight of the sample

Estimation of Crude fibre in Cereals and their products

Aim: To find out the amount of crude fibre in a given food sample.

Theory: Crude fibre consisits largely of cellulose and lignin (97%) plus some mineral matter. It

represents only 60% to 80% of the cellulose and 4% to 6 of the lignin. The crude fibre content is

commonly used as a measue of the nutritive value of poultry and livestock feeds and also in the

analysis of various foods and food products to detect adulteration, quality and quantity.

Principle: During the acid and subsequent alkali treatment, oxidative hydrolytic degradation of

the native cellulose and considerable degratdation of lignin occur. The residue obtained after

final filteration is weighed, incinerated, cooled and weighed again. The loss in weight gives the

crude fiber content.

Requirements: a) Sulphuric acid solution (0.255 N) 1.25g conc. H2SO4 diluted to 100ml.

b) Sodium hydroxide solution (0.313 N): 1.25g NaOH in 100ml d.w.

Procedure:

1. Extract 2.0g of groud material with ether or petroleum ether to remove fat (Initial boiling

temp 35to 38oC and final temp 52

oC). If fat content is below 1% extraction may be omitted.

2. After extraction with ether boil 2.0g of dried material ( Sample 1- Whole wheat flour and

Sample 2- Refined wheat flour (Maida) ) with 10ml of H2SO4 for 15min with porcelein chips.

3. Centrifuge for 5 mins at 2000 rpm and decant.

4. Wash the pellet with 10 ml boiling water and Centrifuge (2000 rpm for 5 mins).

5. Decant and boil the residue with 10 ml of NaOH solution for 20min.

6. Centrifuge at 2000 rpm for 5 mins and decant.

7. Wash the pellet 10 ml of boiling 1.25% H2SO4, and centrifuge ( 2000 rpm , 5 mins). Decant.

8. Wash the pellet with 10 ml water. Centrifuge ( 2000 rpm, 5 mins). Decant.

9. Mix pellet with 10 ml alcohol and transfer contents to a clean dry crucible/ evaporating dish.

10. Dry the residue in air or hot plate. Weigh the crucible (W2).

11. Ignite the crucible with residue for 20 min at 6000C in a muffle furnace.

12. Cool in a dessicator and reweigh (W2).

Result: a) The % crude fibe in sample 1 ( Whole wheat flour) is ___________

b) The % crude fibe in sample 2 ( Refined wheat flour) is ___________

Conclusion :

40

Observation Table:

Evaluation of Probiotic Food

Aim: To evaluate the probiotic food for presence of live microorganisms.

Requirements: Methylene blue, Curd, Probiotic curd, Raw milk, Test tube stand with test tubes

(3), Labels

Principle:

Methylene Blue Dye Reduction Test, commonly known as MBRT test is used as a quick method

to assess the microbiological quality of milk and curd. This test is based on the fact that the blue

colour of the dye solution added to the milk get decolourized when the oxygen present in the

milk or curd get exhausted due to microbial activity. The sooner the decolourization, more

inferior is the bacteriological quality of milk or curd. This test is widely used at the dairy

reception dock, processing units and milk chilling centres where it is followed as

acceptance/rejection criteria for the raw and processed milk.

Principle: MBRT test may be utilized for grading of milk which may be useful for the milk processor to

take a decision on further processing of milk. As per BIS 1479 (Part 3): 1977 criterion for

grading of raw milk based on MBRT is as below:

5 hrs and above Very good

3 to 4 hrs Good

1 to 2 hrs Fair

Less than ½ hrs Poor

Procedure:

1. The given milk and two curd samples are checked for their quality by using MBRT test.

2. Take 5ml of milk and two curd samples in three separate test tubes

3. Add 0.5ml of methylene blue to each test tube and shake the contents.

4. Note the time for decolourization to occur.

Results –

41

Observation Table:

Calculation: From standard tube, ‘A’ ΔO.D. ≡ 0.02 mg of BSA

Therefore, ‘X’ ΔO.D. ≡ ‘X’ x 0.02 = ‘B’ mg of BSA

A

i.e. 0.1 ml of Plant extract contains ‘B’ mg of BSA

Therefore, 50 ml of Plant extract contains ‘B’ x 50 = ‘C’ mg of BSA

0.1

But, 50 ml of Plant extract was prepared from 5 g plant material

i.e 5 g plant material contains ‘C’ mg of BSA

Therefore, 100 g plant material contains ‘C’ x 100 = ‘Z’ mg of BSA

5

Sr. No.

Volume of Std

BSA solution

(ml)

D.W

(ml)

Conc. Of

BSA

(mg)

O.D ΔO.D.

Blank

- 1.0 0

Standard

0.2 ml 0.8 0.02

(A)

R.M. 0.1 ml of Plant

extract 0.9 ?

(X)

Determination of total protein

Aim: To compare and analyse the neutraceutical content in terms of total protein estimation

from the given plant sample/s.

Principle: The Folin- Ciocalteau reagent contains phosphomolybdic acid and tungstate. The

aromatic aminoacids- tyrosine and tryptophan present in the proteins react with these and

produce a blue colour. The colour developed by the biuret reaction of the proteins with alkaline

cupric tartarate is also measured by the Lowry’s method.

Requirements:

Chemicals-

1. Reagent A : 200mg NaOH and 1 gm. Na2CO3 . Dissolved separately in small amount of

distilled water and make volume 50 ml. with distilled water.

2. Reagent B: 120mg. CuSO4 and 250 mg Sodium tartarate dissolved separately in small

amount of distilled and make vol. 25 ml. with distilled water.

3. Reagent C : Mix 50 ml of Reagent A with 1 ml of Reagent B just prior to use.

4. Folin- Coicalteau reagent: Reagent to be diluted with equal volume of water just before

use.

5. Standard working Protein solution: 0.1mg/ ml Bovine Serum Albumin (BSA).

6. D.W.

7. Phosphate- NaOH buffer ( Phosphate buffer pH 7 + 0.1 N NaOH)

Glasswares and other equipments:

Test tube stand with test tubes, pipettes, boiling and cold water bath, incubator, colorimeter.

Procedure For the determination of protein content from fresh mushroom, 5 g is taken with 50 ml phosphate-

NaOH buffer and homogenized using mortar and pestle.

1. Centrifuge at 6000 rpm.

2. Supernatent is collected and final volume is made to 50 ml with NaOH.

Total protein content is measured according to the method of Lowry et al. (1951).

Prepare three tubes, as given in the table.

To all tubes, add 5 ml of Reagent C

Mix well and allow tubes to stand for 15 mins.

Add 0.5 ml of Folin- Coicalteau reagent to all tubes. Mix thoroughly

and allow to stand for 20 mins.

.

Read O.D. at 660 nm.

RESULT : The amount of protein in the plant material is __(Z)__ mg % BSA

42

PRACTICAL MODEL PAPER

SEMESTER - III, S.Y.B.Sc. BOTANY

TIME - PAPER – I Total Marks – 50

Q.1. a) Identify, Classify and describe specimen ‘A’ . Sketch neat and labeled diagram. (06)

b) Identify, Classify and describe specimen ‘B’ . Sketch neat and labeled diagram. (06)

Q.2. a) Assign the specimen ‘C’ to its family giving reasons. Give the distinguishing

characters and floral formula. Sketch the L.S. of flower and T.S. of ovary. (08)

b) Identify and study the anatomy (types of Trichome / Stomata) / Perform tests for

flavanoids and phenolics of Specimen ‘D’, in relation to Taxonomy. (04)

Q.3. To Separate given material ‘E’ by any appropriate chromatography technique . (10)

Q.4. Identify and describe the specimen/ slide/ photograph - ‘F’ and ‘G’. (08)

Q.5. Journal. (04)

Q.6. Submission of Herbarium. (04)

------------------------

KEY :

A. – Sargassum

B. –Anthoceros / Funaria

C. - Any Angiospermic Family as per syllabus.

D. – Trichome ( Malvales) / Stomata ( Rubiaceae) / Chemical Tests ( Flavanoids and Phenolics)

E. – Amino acids by Circular Chromatography/ Carotenoids by TLC. ( Internal Evaluation)

F. Algae – economic importance / range of thallus in Phaeophyta / Bryophyta - Anthoceros / Funaria

G. Horizontal/ Vertical Gel electrophoresis / Dry or Wet Preservation technique.

------------------------------------------------------------------------------------------------------------------------------

SEMESTER - III, , S.Y.B.Sc. BOTANY

TIME - PAPER – II Total Marks – 50

Q.1. Make a Squash / Smear preparation of Specimen ‘A’. Draw and Comment on your

observations and show the slide to the examiner. (10)

Q.2. To estimate DNA/ RNA from the given Sample ‘B’. (10)

Q.3. To determine the sequence of bases in the DNA strand by Sanger’s method from the

given data ‘C’.

OR

To determine the sequence of amino acids in the polypeptide synthesized from the given

m-RNA strand ‘C’. (10)

Q.4. Identify and describe the specimen/ photograph - ‘D’ , ‘E’ and ‘F’. (15)*

Q.5. Journal #. (05)

----------------------

KEY :

A. – Mitosis / Meiosis.

B.- Onion- DNA/ RNA estimation. ( Internal Evaluation) .

C. – DNA sequence / Amino Acid sequence.

D*. - Cell organelles ( Mitochondrion/ Peroxisome/ Glyoxysome/ Ribosomes)

E*. - Plastid inheritance.

F*. – Chromosomal aberrations- ( Laggards/ Chromosomal ring/ Chromosomal bridge)

* 3 marks each for identification. Remaining 06 marks for DBT Star activity submission (MCQ

Test) .

Q5. # Submission of Rough journal.

_________________________________________________________________________________

SEMESTER - III, , S.Y.B.Sc. BOTANY

TIME - PAPER – III Total Marks – 50

Q.1. To study the macroscopic characters and identify the active constituents present in

Specimen ‘A’ by performing suitable chemical tests. (12)

Q.2. a). To prepare face pack/ detanning cream from Specimen ‘B’.* (06)

b) Submission of Biodiversity field Report. (06)

Q.3. To estimate of crude fibre from Specimen ‘ C’.

OR

To evaluate the neutraceutical value from Specimen ‘C’. (12)

Q.4. Identify and describe the specimen - ‘D’, and ‘E’. (10)

Q.5. Journal#. (04)

KEY :

A. – Alkaloids / Glycosides.

B. –* Probiotic analysis from given food sample ‘B’(any two) and interprete

C. Crude fibres from Aata (wheat flour) and Maida (Refined flour) / Protein estimation from

mushroom. ( Internal Evaluation)

D. Fibres: Jute/ Cotton

E. Spices & Condiments: Cardamon/ Saffron

Q5. # Active disciplined participation in Biodiversity Excursion

Common Taxonomic Characters/ Terms to be included for all families

Habit : Tree/ Shrub/ Herb.

Leaves: Simple/ Compound, Alternate/ Opposite/ Whorled, Petiolate/ Sessile, Stipulate/ Exstipulate,

Reticulate/ Parallel venation.

Inflorescence : Racemose/ Cymose, Type of Racemose or Cymose, Axillary/ Terminal.

Flower : Actinomorphic/ Zygomorphic/ Irregular, Bisexual/ Unisexual, If unisexual, its type, Complete/

Incomplete, Pedicellate/ Sessile, Bracteate/ Ebracteate, Bracteolate/ Ebracteolate, Hypogynous/

Perigynous/ Epigynous, Tri-/ Tetra-/ Pentamerous symmetry.

Calyx: Number of sepals, Polysepalous/ Gamosepalous, Type of aestivation.

Corolla: Number of petals, Polypetalous/ Gamopetalous, Type of aestivation.

[ Perianth: Number of Tepals, Polyphyllous/ Gamophyllous, Type of aestivation]

Androecium: Number of stamens, Free or fused from each other, Free or fused with other whorls,

Inserted/ Exerted, Structure of filament, Structure of anther and its attachment to filament.

Gynoecium: Number of Carpels or Pistils, Apocarpous/ Syncarpous, Ovary superior/ Ovary inferior,

Number of chambers or locules, Type of placentation, Number and structure of style,

Number and structure of stigma.

Fruit: If present, describe the type of fruit.

Taxonomic families

Division Spermatophyta

Sub division Angiospermae

Class Dicotyledoneae Monocotyledoneae

Sub- Class Polypetalae Gamopetalae Monochlamydae

Series Calyciflorae Inferae Curvembryeae Calycinae

Cohort Rosales Asterales Chenopodiales

Family Leguminosae Asteraceae Amaranthaceae Palmae

Sub- family Papilionaceae Caesalpinaceae Mimosaceae

INDEX SEMESTER- III - Ramniranjan Jhunjhunwala College · 10 Separation of amino acids by circular...

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