Download BSc.-I Medical - DAV College Jalandhar

Department of Botany, DAV College, Jalandhar (PB.)
BOTANY
Lab Manual
BSc.-I Medical
Semester II
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Department of Botany, DAV College, Jalandhar (PB.)
Syllabus
1. To study cell structure from onion leaf peels; demonstration of standing and mounting
methods.
2. Comparative study of cell structure in onion cells, Hydrilla and Spirogyra. Study of cyclosis
in Tradescantia Staminal Cells.
3. Study of plastids to examine pigment distribution in plants (e.g. Cassia, Lycopersicon and
Capsicum).
4. Examination of electron micrographs of eukaryotic cells with special reference to organelles.
5. Study of electron micrographs of viruses, bacteria, cyanobacteria and eukaryotic cells for
comparative cellular organization.
6. Examination of various stages of mitosis and mitosis using appropriate plants material
(e.g. onion root tips, onion flower buds).
7. Preparation of karyotypes from dividing root tip cells and pollen grains.
8. Cytological examination of special types of chromosomes: bar body, lampbrush and polytene
chromosomes.
9. Working out the laws of inheritance using seed mixtures.
10. Working out the mode of inheritance of linked genes from test cross and/or F2 data.
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Department of Botany, DAV College, Jalandhar (PB.)
AIM 1. Demonstration of Staining and Mounting Methods
A. Staining is a technique used in microscopy to enhance contrast in a microscopic image.
Stains and dyes are frequently used to highlight structures in microbes for viewing, often with
the aid of different microscopes. Staining may be single or double.
1. Single Stain: Safranin or fast green is used to stain filaments of lagae, fungi, sections of
bryophytes, spores of pteridophytes, pollen grains of angiosperms etc. Aniline blue or safranin is
usually used for single staining methods.
Procedure: - Keep material in watch glass
- Few drops of stain are added to the water to another watch glass to dilute the stain.
- The diluted stain is added to the material.
- After the stain is taken up by sample, the extra stain is washed with water.
- Washing is repeated till the stain stops coming out.
- The stained material is now ready for mounting.
2. Combined Stain: In this technique, two or more stains are used. Generally contrasting colours
are used. The following combinations are commonly employed.
•
Haematoxylin and Safranin
•
Safranin and fast green
•
Safranin and Aniline blue
•
Safranin and Crystal violet
•
Crystal violet and Erythrosine
Procedure: - The section is first stained with principle stain.
- Section is passed through a graded series of alcohol for degradation.
- Section is transferred to watch glass containing required amount of alcohol and water.
- Watch glass should be covered by larger watch glass to avoid alcohol evaporation.
- Now the counter stain is employed for few seconds.
- The section is transferred to absolute alcohol for 5-7 min for complete dehydration.
- Section is finally immersed in xylene. If dehydration is not complete, pure xylene
turns white or turbid.
- Now the section is ready for mounting.
B. Mounting is necessary for proper positioning of objects for clear view. Lactophenol, glycerin
and glycerine jelly are used for temporary mounting while Canada balsam is used for permanent
mounting.
Mounting Media: Following are some of the common media:
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Department of Botany, DAV College, Jalandhar (PB.)
- Canada Balsam: It is resin obtained from conifer. It is most suited for permanent slide
preparation.
- Lactophenol: It is a mixture of equal parts of phenol crystals, lactic acids, glycerine and
distilled water.
- Glycerine: Pure glycerine diluted to 15-25% is widely used for semipermanent and temporary
preparations.
- Glycerine Jelly: Jelly is used for mounting. It is made up of gelatin.
Many other mounting media like cedar oil, balsam, venetiane, turpentine, synthetic resin
etc are also used.
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Department of Botany, DAV College, Jalandhar (PB.)
AIM 2. Comparative study of cell structure in onion cells, Hydrilla and Spirogyra.
Requirement: Onion cells, Hydrilla, Spirogyra, Tradescantia flowers, watch glass, forecep,
slide, coverslip, microscope.
Procedure:
1. A thick onion scale was taken out and teared it from concave
side so as to get transparent thin and membrane onion peel.
2. Placed the peel in watch glass containing water and 2-3 drops of
methylene blue.
3.Cut a small portion of peel and placed it on cleansed glass slide
with a drop of glycerine.
4. Put a cover slip over it and observe under the microscope.
Onion Peel Cell
1. A cleansed glass slide was taken.
2. Mount the material on it with a drop of glycerine.
3. Cover it with watch glass and observe it under microscope.
Hydrilla Cell
1. A cleansed glass slide was taken.
2. Mount the material on it with a drop of glycerine.
3. Cover it with watch glass and observe it under microscope.
Spirogyra Cell
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Department of Botany, DAV College, Jalandhar (PB.)
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AIM 3: To study cyclosis from Tradescantia stamina hair. (e-Reource
Reource generated)
Requirements: Tradescantia flowers, Microscope, Cover slip, Glass slide, brush and forceps.
forceps
Theory: Cyclosis is automatic movement of protoplasm of a cell. This movement may be due to
the movement of organelles like chloroplast in response to changing intensity of light. It is
believed that cyclosis occurs due to Sol-gel
Sol
interconversions or microfibrils.
Procedure: 1. Fresh tradescantia flowers were taken .
2. Staminal hairs were removed and kept on clean glass slide with a drop of water.
3. Placed a coverslip over sample and observed under microscope.
Precautions: 1. Flowers should be fresh.
2. There should not be any air bubble.
Filament with stamina hairs
Enlarged view of Staminal Hair
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Department of Botany, DAV College, Jalandhar (PB.)
AIM 4: Examination of electron micrographs of eukaryotic cells with special reference to
organelles.
Plants Eukaryotic Cell
It has a cell wall, plasma membrane, cytoplasm
having
various
vacuole,
organelles
chloroplast,
like
nucleus,
mitochondria,
golgi
bodies, ribosomes and fat granules embedded
in it.
Nucleus
1. It is a specialized double membrane bound
protoplasmic body.
2.It is divided into 5 main parts like nuclear
envelop,
nucleoplsm,
nuclear
matrix,
chromatin and nucleolus.
3. The nuclear membrane is interconnected
with endoplsmic reticulum.
Mitochondria
1. It is double membrane bound power house
of the cell.
2. Its outer membrane is smooth and inner
membrane is highly folded inwards.
3. It contains its own DNA called mtDNA.
4. It is actively involved in oxidative
phosphorelation in aerobic eukaryotes.
Chloroplast
1. It is greenish plastid which possesses
photosynthetic pigments.
2. It takes part in synthesis of food from
inorganic raw material in presence of sun light.
3. It possess stroma and thylakoids.
4. It is a double membrane organelle.
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Department of Botany, DAV College, Jalandhar (PB.)
Endoplasmic reticulum
1. Endoplasmic reticulum is double membrane
bound cell organelle.
2. It contains 3 main components like
cisternae, vesicles and tubules.
3. ER is of two types i.e., rough endoplasmic
reticulum (RER) and smooth endoplasmic
reticulum (SER).
4. RER is bound by ribosomes.
Golgi body
1. It is an important part of endomembrane
systems.
2. It has a cis and trans face.
3. Cis face is formative face and trans face is
productive face.
4. It induces glycosylation of proteins.
Vacuole
1. These are non cytoplasmic spaces present
inside the cytoplasm.
2. They can be sap vacuoles, contractile
vacuoles, food vacuoles and air vacuoles.
3. These vacuoles are membrane bound. The
membrane is called tonoplast.
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Department of Botany, DAV College, Jalandhar (PB.)
AIM 5: Study of electron micrographs of viruses, bacteria, cyanobacteria and eukaryotic
cells for comparative cellular organization.
Bacteriphage
1. A bacteriophage is a virus that infects and
replicates within a bacterium.
2. Bacteriophages are composed
of proteins that encapsulate a DNAor RNA genome.
3. Bacteriophages may have a lytic cycle or
a lysogenic cycle, and a few viruses are capable of
carrying out both.
4. To enter a host cell, bacteriophages attach to
specific receptors on the surface of bacteria.
Bacterial cell
1. Bacteria constitute a large domain of prokaryotic
microorganisms.
2. Bacterial cells are about one-tenth the size of
eukaryotic
cells
and
are
typically
0.5–
5.0 micrometres in length.
3. Bacteria do not have a membrane-bound nucleus,
and their genetic material is typically a single
circular DNA chromosome located
in
the
cytoplasm.
Cyanobacteria
1. Cyanobacteria also known as Cyanophyta, is
a phylum of bacteria that obtain their energy
throughphotosynthesis.
2. Cyanobacteria are a photosynthetic nitrogen fixing
group that survives in wide variety of habitats, soils,
and water.
3. Their thalli vary from unicellular to filamentous.
4. Many cyanobacteria form motile filaments of
cells, called hormogonia.
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Department of Botany, DAV College, Jalandhar (PB.)
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AIM 6: Examination of various
vario stages of mitosis and meiosis using appropriate plants
material (e.g. onion root tips, onion flower buds).
Requirements: Onion root tips, onion flower bulbs, slides, watch glass, distilled water,
acetocarmine/acetooricine, burner, forecep, needle, coverslip.
A. Mitosis
Interphase
1. This stage is prior to actual mitotic cycle.
2. This cell appears to be inactive.
3. Nuclear membrane and nucleolus are very
distinct.
4. Chromosome appear double stranded.
stran
Early Prophase
1. Nuclear membrane appears distinct.
2. Nucleolus is also clear.
3. Chromosome become clear and shortened.
Late Prophase
1. Nucleolus and nuclear membrane partially
disappear.
2. Chromosomes are condensed.
3. Spindle fibres also begin to appear.
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Department of Botany, DAV College, Jalandhar (PB.)
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Metaphase
1.
Nuclear
membrane
and
nucleolus
disappears.
2. Centromere get arranged on equatorial plate
and are attached to spindle fibres.
3. Spindle is made up of fibres only.
Anaphase
1. Chromatids are pulled toward opposite
poles.
2. Each chromosome appear in characteristic
shape.
Telophase
1. Chromosomes are present at both ends.
2.Chromosomes
increase
in
l;ength
and
become thread like.
3. Nuclear membrane and nucleolus reappear.
4. Spindle fibres appear to deassemble.
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Department of Botany, DAV College, Jalandhar (PB.)
Cytokinesis
1. Cytoplasm divides into two. It results in two
daughter cells.
2. Cell plate formation begins at the centre of
plant cell and progress towards the periphery.
Interphase
Prophase
Metaphase
Anaphase
Telophase & Cytokinesis
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Department of Botany, DAV College, Jalandhar (PB.)
B. Meiosis
Leptotene (Prophase I)
1. Nuclear membrane and nucleolus are
intact.
2. Chromosomes are long thread like.
3. This stage is also called bouquet stage.
4. All the chromosomes finally move towards
one part of the nucleus.
Zygotene (Prophase I)
1. Nucleolus and nuclear membrane are still
very clear.
2. Synapsis of homologous chromosomes
occur.
3. Synaptonemal complex is formed.
Prophase (I)
Pachytene (Prophase I)
1. Nucleus and nuclear membrane are
distinct.
2. Chromosomes are thickened, coiled and
thread like.
3. Crossing over occurs.
Diplotene (Prophase I)
1. The nucleolus and nuclear membrane is
disappearing.
2. Homologous chromosomes are still intact.
3. Chromosomes are shortened and
thickened.
Diakinesis (Prophase I)
1. Nuclear membrane and nucleolus
completely disappear.
2. Chromosomes start separating from
centromere.
3. The chromosomes appear circular due to
contraction.
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Department of Botany, DAV College, Jalandhar (PB.)
Metaphase (I)
1. Nuclear membrane and nucleolus completely
absent.
2. Spindle is distinct.
3. Bivalents are arranged on equatorial plate.
Anaphase (I)
1. Nuclear membrane and nucleolus completely
absent.
2. Chromosomes separate out of the pair.
3. Results in haploid set of chromosomes at each
pole.
Telophase (I)
1. Nuclear membrane and nucleolus reappear.
2. There are two nuclei on each pole.
Meiosis (II)
Prophase (II)
1. Nuclear membrane and nucleolus are distinct in
early stages.
2. Chromosome are short and thick.
3. Each chromosome is made up of two
chromatids.
Metaphase (II)
1. Nuclear membrane and nucleolus are absent.
2. Soindle is quite visible.
3. Chromosomes lie at equater.
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Department of Botany, DAV College, Jalandhar (PB.)
Anaphase (II)
1. Nuclear membrane and nucleolus are absent.
2. Spindle fibres contract and chromosomes are
pulled to opposite poles.
3. Chromatids show characteristic shape.
Telophase (II)
1. Chromosomes occur in group at end of parent
cell.
2.
Nuclear
membrane
and
nucleolus
start
reapperaring.
3. Four haploid daughter cells are formed.
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Department of Botany, DAV College, Jalandhar (PB.)
AIM 7: Preparation of karyotypes from dividing root tip cells and pollen grains.
Procedure: 1.Karyotype refers to the phenotypic appearance of the chromosome at somatic
metaphase. Ideogram is the diagrammatic representation of karyotype.
2. Study the number and morphology of chromosome from well spread out mitotic preparation.
3. Draw well spread out mitotic metaphase plate.
4. Mark the positions of centromere carefully.
5. Measure the arm length of various chromosomes and plot their relative length on graph as
histograms.
6. The chromosomes should be arranged in decreasing order of their total length with the larger
arms towards the bottom.
7. Uniform gaps should be used to indicate primary and secondary constrictions.
8. Compare the karyotypes and idiograms of different species of different varieties of same
species.
Karyotype of Allium cepa
Chromosomes of Vicia faba
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Department of Botany, DAV College, Jalandhar (PB.)
AIM 8: Cytological examination of special types of chromosomes: bar body, lampbrush
and polytene chromosomes.
Polytene Chromosome
1. Polytene chromosomes are over-sized
chromosomes which have developed from
standard chromosomes and are commonly
found in the salivary glands of Drosophila
melanogaster.
2. Cells undergo repeated rounds of DNA
replication without cell division.
3. Polytene cells may also have a metabolic
advantage as multiple copies of genes permits
a high level of gene expression.
4. Polytene chromosomes have characteristic
light and dark banding patterns.
5. Polytene chromosomes have characteristic
light and dark banding patterns.
6. Balbiani ring is a large chromosome puff
.
Lampbrush Chromosome
1. Lampbrush chromosomes are a special
form of chromosome found in the growing
oocytes (immature eggs) of most animals,
except mammals.
2. Chromosomes transform into the
lampbrush form during the diplotene stage of
meioticprophase I due to an active
transcription of many genes.
3. Chromosomes have loops projecting in
pairs.
4. These pairs give lampbrush chromosome
its characteristic appearance.
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Department of Botany, DAV College, Jalandhar (PB.)
Bar Body
1. Rinse your mouth thoroughly with tap
water and followed with distilled water.
2. Scrap the mucosa gently from the deeper
layer of your mouth with the help of
cleanlywashed spoon or spatula.
3. Spread the sample over a small area on a
clean dry slide and allow it for air drying.
4. Add 95% Alcohol and allow it to settle for
5-6 min.
5. Stain the slide with Carbol fuchsin and
allow it to settle for 15-20 min.
6. Wash the slide with distilled water and
immediately observe the slide under the
microscope.
Phorograph of Cheek Cells showing Barr Body
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Department of Botany, DAV College, Jalandhar (PB.)
AIM 9: Working out the laws of inheritance using seed mixtures.
1. Law of Segregation: During gamete formation, the alleles for each gene segregate from each
other so that each gamete carries only one allele for each gene.
Procedure: 1. Count 50 yellow and 50 red buttons and put them in box A.
2. Similarly put count 50 blue and 50 green buttons and put them in box B.
3. Now randomly pick one button from each box, until the boxes are empty. Record each
reading.
4. Find out if the genotypic ration comes out to be 1: 2: 1 and phenotypic ratio as 3:1.
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Department of Botany, DAV College, Jalandhar (PB.)
2. Law of Independent Assortment: Genes for different traits can segregate independently
during the formation of gametes.
Procedure: 1. Count 40 buttons of two different colours and put them in beaker A.
2. Repeat it for beaker B with 40 buttons of two different colours.
3. Now randly draw one capsule from each beaker .
4. Continue this until beakers are empty.
5. Note down the readings and analyse if you get ratio of 9:3:3:1.
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Department of Botany, DAV College, Jalandhar (PB.)
AIM 10: Working out the mode of inheritance of linked genes from test cross and/or F2
data.
Explanation: The coexistence of two or more genes in the same chromosome is called
linkage. If genes are present on same chromosomes, then the test cross ratio comes out to be 1:1
while if the genes are present on different chromosomes the test ratio comes out to be 1:1:1:1.
Procedure: 1.Assume two genes A and B where a and b are recessive alleles.
2. A cross between AABB and aabb would give rise to F1 progeny as AaBb.
3. Depending upon the distance between any 2 genes which is inversely proportional to the
strength of linkage, non cross overs will vary from 50 to 100 %.
4. Thus cross over will vary from 0 – 50% and will never exceed 50%.
5. The easiest way to find out the proportion of non cross overs and cross overs is make a test
cross.
6. From phenotypic ratio of test cross the relative proportion of crossing over and non cross
overs can be easily determined.
Complete Linkage
Incomplete Likage
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