There are only 2 experiment for term 1
EXPERIMENT -1
To isolate DNA from available plant materials such as spinach,, green pea seed, papaya etc.
AIM
Deoxyribonucleic acid (DNA) acts as the genetic material in most of the organisms. It is a long polymer deoxyribonucleotides. A nucleotide has three components:
THEORY
• Purines: Adenine and Guanine
• Pyrimidines Cytosine and Thymine
2. Pentose deoxyribose sugar
1. Nitrogen base:
Nitrogen base is linked to pentose sugar through N-Glycosidic linkage and forms a nucleoside. Phosphate group linked to 5-OH of a nucleoside through phosphoester linkage and forms a nucleotide. Two nucleotides are linke through 3-5 Phosphodiester linkage to form a dinucleotide. A polynucleotide chain has free phosphate group at 5'e of deoxyribose sugar and a free 3-OH group at the other end.
Pentose sugar and phosphate components of DNA are readily soluble in water. The phosphate groups outside the D carry negative charges, which are attracted and neutralised by cations such as sodium.
DNA is made up of two polynucleotide chains, where the backbone is constituted by sugar-phosphate and the base project inside. Both polynucleotide chains have anti-parallel polarity.
These two strands are held together by hydrogen bonds. Both chains are coiled in right-handed fashion. The salts added during DNA isolation shield the negative phosphate ends of DNA which allows the ends to come closer so that the DNA can precipitate out of a cold alcohol solution. The detergent causes the cell membrane to breakdown by dissolving the lipids and proteins of the cell and disrupting the bonds that hold the cell membrane together. The detergent then forms complexes with these lipids and proteins and precipitates them out of the solution.
MATERIAL REQUIRED
Plant tissue knife/razor, mortar-pestle, centrifuge tubes, pipettes, centrifuge machines, ice-bath, water bath and thermometers.
PROCEDURE
1.Weigh about 03 g of the plant tissue (spinach, green pea seeds or papaya).
2. Chop it into small pieces and make a homogeneous paste of it using a pestle.
3. Immediately transfer the tissue to a 1.5 ml micro-centrifuge tube.
4 After this, add 300 ul EBA, 900 μL EBB and 100 µL SDS to it.
5. Vortex the tube in centrifuge at 15000 rpm for about 10 minutes, Incubate it at 65" C for another 10 minutes.
6 Place the tube on ice and add 410 ut of cold potassium acetate to it. Mix by inverting the tube gently a few number of times and place it back on ice for another 3-5 minutes.
7. Centrifuge it again at 13000 rpm for 15 minutes in a refrigerated centrifuge. A pellet will be observed. & Transfer 1 mL of the supernatant to a new micro-centrifuge tube of 1.5 mL. Add 540 μL of ice-cold abso isopropanol to it and incubate it on ice for another 20 minutes.
OBSERVATION
The addition of ethanol causes precipitation of DNA. The DNA strands can be clearly viewed
suspended in the solution of the vial as white-coloured translucent threads.
RESULT
The DNA of the given plant aterial is hence isolated under experimental limits.
PRECAUTIONS
1. Wash the plant material thoroughly with distilled water to remove any dust particle present on it.
2 The amount of leaf sample should weigh between 0.3 g to 0.6 g.
3. The chemicals used for the isolation of DNA should be manufactured by standard pharmaceuticals. 4. Use clean and sterilised apparatus for the experiment.
EXPERIMENT-2
To study the germination of pollen grains under the microscope..
AIM
To study the germination of pollen grains under the microscope.
MATERIAL REQUIRED
Beakers, glass rod, dropper, aniline blue stain, sucrose (10 g), boric acid (10 mg), calcium nitrate (30 mg), magnesium sulphate (30 mg), distilled water, a flower of Tradescantia/Balsam/Jasmine/Vinca/Hibiscus/Petunia for pollen grains. glycerine, glass slides, coverslips and microscope.
THEORY
In flowering plants, a pollen represents the male gametophyte. They can be seen as powdery yellow coloured dust in the anthers of a flower. A pollen grain has two walls. The outer hard wall is made up of sporopollenin and is called the exine. The inner thin wall is made up of cellulose and pectin and is called intine. The exine has prominent apertures called germ pores.
Pollination is the process of transfer of pollen grains from the anthers of a flower to the stigma of the same or another flower of the same species. In case of flowering plants, the ovul is contained within a hollow organ called the pistil and the pollen is deposited on the pistil's receptive surface, know Several pollen .
The germination of a pollen grain begins by the absorption of water and nutrients from the medium or is triggere by the chemicals on the stigma. Upon germination, the intine of the pollen grain, along with its nuclei, emerges ou through one of the germ pores in the exine as pollen tube. The tube nucleus descends to the tip of the tube. The tub The growing grows through the style and reaches the ovule inside the ovary.
PROCEDURE
A) In Vitro Pollen Germination
1. Preparation of culture medium: Prepare 10% sucrose solution by dissolving 10 g of sucrose in 100 mL of water in a beaker. Also add 10 mg boric acid, 30 mg magnesium sulphate and 30 mg calcium nitrate in it. Stir the contents with a glass rod. This is known as nutrient solution or culture medium.
2. Take a clean glass slide. Put 2-3 drops of above nutrient solution in the centre of the slide.
3. Take a mature flower. Break the fresh anther and dust a few pollen grains on the glass slide in the nutrient solution.
4. Observe the slide under a dissecting microscope and note the time of appearance of the pollen tubes.
5. After a few minutes, observe the slide under low magnification of a compound microscope and note down the size of pollen tubes.
6. View the slide after every 10 minutes and record your observations.
7. Similarly observe the pollen germination in different flowers .
(B) In Vivo Pollen Germination
1. Take out the pistil from a flower, say China Rose. Boil it in water for 5-10 minutes.
2. Place the isolated pistil in a watch glass and stain it with aniline blue.
3. Mount the stained material in glycerine. Observe under a compound microscope.
4. While viewing through the microscope, carefully take out a germinating pollen with the help of a needle, mount it in glycerine on another slide and observe under the compound microscope.
5. Repeat the process with pollens of different flowers and record your observations.
OBSERVATION
1. Several pollen grains were found to show germination and the pollen tubes were observed coming out of them. 2. Initially, the pollen grains are found to contain a single nucleus. The exine layer is also visible.
3. Later, a small outgrowth is seen coming out of the pollen grains.
4. The outgrowths continue to grow as pollen tubes and each contains two nuclei.
5. The growing pollen tubes inside the style are later found to contain three nuclei, i.e., two male gametes and one tube nucleus.
RESULT
1. Pollen grains germinate in sucrose solution, which acts as a nutrient medium. Pollen grains absorb water and their exine ruptures.
2. Sever pollen grains and pollen tubes of different lengths were observed and studied germinating over stigma. 3. Although pollen grains of many species germinate in this nutrient medium, the time taken and the percentage of germination varies in different species.
PRECAUTIONS
1. Only a few pollens should be dusted on the slide.
2. Pollens dusted must be dipped in the nutrient solution.
3. Place the coverslip carefully without letting the air bubbles to enter.
4. Do not allow the nutrient medium on the slide to dry away.
VIDEO
(pending)
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