Isolation of Plant DNA (Tomato)
Aim: To isolate DNA from tomato pulp
Principle: DNA is a chief components of living things. DNA of the organisms become inactive on heating. The application of alkali and detergent disturb the cell wall or cell membrane. Protoplast contents are expelled out of the cell membrane by slow heating.
Chemicals required:
Sodium chloride (weighted and 10 ml of li) mild liquid dish washing detergent, 20 ml cold ethanol, fresh raw papaya juice or pineapple juice. Ice cubes. Ripe tomato fruits.
Apparatus:.
Mortar and pistle, Glass beaker, Muslin Cloth, glass rod, Glass tubes, Hot water bath, Watmann filter paper, Forceps, knife etc.
Procedure:
3 grams of sodium chloride weighted and 10 ml of liquid detergent is added to sodium chloride to the flask. The volume is made up to 100 ml by adding distilled water. Cut the ripe tomato into 1×1cm pieces, put them into a beaker and incubate at 65c for 15 minutes. Cool the pieces by placing the beaker on ice for 10 minutes. Crash and macerate the pieces gently and filter the solution through the Muslin cloth. Add 20 ml of cold ethanol and allow DNA to precipitate and take out DNA with the help of glass rod.
Results:
DNA is visible as white time stands which should be spooled by glass rod.
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Isolation of DNA
Make up the volume to 100 ml by adding distilled water
Take out 10 ml into a separate beaker
Make up the volume to 100 ml by adding distilled water
Take ripe tomato, cut into 1X1 cm pieces
Put them into beaker, incubate at 650 C for 15 minutes and bring back
to room temperature with ice water
Macerate tomato pieces gently
Filter it with muslin cloth
Add 20 ml of cold ethanol and allow DNA to precipitate and take out DNA with the help of glass rod.
Synthetic Seed Preparation
Aim: To prepare hydrated synthetic seeds invitro.
Materials required: Beaker, petri dish, micropipette , micro tips.
Chemicals required: Sodium alginate (4%), calcium chloride (4%) and distilled water.
Procedure:
1. Embryo is isolated from the viable seeds under aspectic conditions.
2. It was then transferred to sodium alginate solution and incubated for 5 to 10 minutes
3. The embryo is then transferred from sodium alginate to calcium chloride.
4. The heads formed where transferred to a separate plates for storage.
Results:
Hydrated or encapsulated seeds are formed.
Preparation of M.S media
Aim: to prepare medium for culturing of plant tissue and organs. Excised plant tissue and organs cannot synthesize their own food so nutrients must be supplied exogenously through the culture medium. The media are often named after the discoverers name who formulated the media.
Ex: MS medium, B5 medium, white and SH medium, MS media was formulated by Mursige and Skoog (1962) . MS medium is generally used in plant tissues and organ cultures.
Composition of medium:
1. Macronutrients: NH4NO3: 1650mg/l, KNo3:1900mg/l., Cacl-2Ho:440mg/l. MgSo4-7Ho: 370mg/l. KH2Po4: 170 mg/l.
2. Micronutrients: H3Bo3:6.2mg/l. KI:0.83mg/l. ZnSo4-4H2o : 86 mg. CuSo4 5H2o:0.025mg
Feso4 7H2o: 27.25mg. Na2Mno4 : 0.025mg. NaFe EDTA: 37.25mg.
3.Carbon Sources: Source sucrose-30gms
4. Vitamin: nicotinic acid 0.6 mg / litre, Pyridoxine - 0.5mg/l. HCl:0.5mg. Thiamine, Hcl-0.1mg/l
5. Amino acid: Glycine - 2mg/l.
Most of the culture media contain organic salts an iron sources, vitamins, amino acids, growth hormones and carbohydrates supply. In organic salts are supplied into two groups as Macronutrients and Micronutrients. The Salt needle in higher amount are called Macronutrients. They include nitrogen, phosphorus, Sulphur, magnesium calcium and potassium. The other inorganic elements like needle in little amounts are called Micronutrients. Micronutrients are Fe, Mn ,Zn. , B, Cu and Mo.
Some tissues are grow on simple media containing sucrose and inorganic salt. such a medium is called Minimal or Basal medium with vitamins, amino acids and growth regulators such a medium is referred to a synthetic medium. The most commonly used amino acid is glycine. The addition of growth hormones like auxin and cytokinins in different promotes the initiation of organs (organogenesis). The auxins(include cell division in) that are commonly used to cytokinins are 6-BAP, Zeatene and 2IPA(2 Iso peptenyl adenine), gibberellins, acetic acid are used in cultural medium.
The ratio of amine and cytokinins in the culture medium is very important with respect to morphogenesis for embryogenesis callus initiation the requisite ratio of amines to cytokinesis is high , white. The reverse leads to shoot proliferation.
Some plant tissues grows in the presence of natural plant products such as coconut milk, ceasin by drolysote, yeast extract, watermelon juice, malt extracts, ripe tomato extract, orange juice etc... Diphenyl urea, a growth factor found in coconut milk, exhibits cytokinins like responses, so as source of cytokinin 10-15% v/v coconut milk is added to medium. Yeast extract is good source of organic nitrogen and vitamin ceasin hydrolysate contain all the common AA. Fruit juices contribute a no. of essential nutrients and vitamins.
Preparation of nutrient medium:
Various components of the medium are dissolved in distilled water. The pH of the medium is adjusted around 5.8. The pH of the medium is likely to given a hard medium while lower pH results in solidification of the agar. The medium can be made semi solid with the addition of agar (58%). A medium without agar remains in liquid form and its prepared for callus culture and liquid medium used for cell suspension culture.
Aseptic condition :
The culture medium, especially when it contains sucrose will support the growth of microorganisms like bacteria fungi etc. If they come into contact with the medium either in cellular or spore form.
The Microorganisms grow faster then the higher tissues and culture medium should necessarily should have aseptic environment.
There are three major sources of contamination of medium. a. Medium b. Glass ware c. Plant tissues.
A. Sterilization of medium:
The Microorganisms maybe present in the medium right from the beginning. To destroy such microorganisms the culture vials containing medium must be sterilized. It can be done with the help of an autoclave or by filter sterilization. The pressure should not exceed 20 atms. Higher pressure lead to destruction of the medium and vitamins .As plant extracts and hormones are thermolabile. They decompose during autoclaving. So the solution of their compounds require filter sterilization. In this process solution are filter through bacterial filters.
B. Sterilization of culture vessels instruments:
Glass culture vessels, metal instruments are wrapped with brown paper later they are sterilized by exposure to dry air(160-180*c) for 24 hours in hot air oven. Metal instruments such as forceps, scalpels, needle and spatula are sterilized by dipping in 95% ethanol followed by drying and cooling. This technique is called flame sterilization.
Proper plugging:
The entry of microorganisms into the vials of the medium can be prevented by plugging them with sterilized cotton.
Demonstration of Micropropagation
Aim: Demonstration of Micropropagation using axillary buds or shoot meristems as Explant.
Apparatus : Culture tubes with plugs, forceps, surgical blade, knife, cotton, spirit lamp, laminar air flow cabinet and autoclave
Chemicals: Ms Medium, Benzyl aminopurine (BAP), kinetine, Indole acetic acid (IAA), Mercuric chloride, 70% Alcohol.
Plant Material: Axillary buds, or Shoot meristems.
Procedure: Axillary buds or shoot meristems of healthy plants are selected. They are cleaned with water and dried well with blotting papers.
They are sterilized with 0.1% HgCl2 for 3-4 mins, so that the spores if any on the explant are completely sterilized. Explants thus sterilized are inoculated into the culture tubes which are readily filled with MS medium supplemented with suitable auxins and cytokinins.
Observation and Results:
Shoot formation from explant on the above medium is observed. these are dissected or separated and culture in separate medium. when required they are transferred to rooting medium enriched with IAA or IBA, so that rhizogenesis is initiated and roots are formed. Then are transferred to green house.
The other way of micropropagation is through callus culture. Once the callus is formed on the suitable medium, this callus is cut and transferred to suitable medium containing auxins and cytokinins. They regenerate multiple shoots. These shoots are dissected and transferred to medium containing rooting hormones. Within two or three weeks plantlets are formed. Then they are transferred to pots .
Anther Culture
Aim:
To isolate and incubate anthers for haploid production.
Principle:
Haploid refers to those plant which possess a gametophytic number of chromosomes in there sporophyte. Haploid maybe grouped into two board categories
A. Monoploids which possess half number of chromosomes from a polyploidy species.
B. Polyploids which process half the number of chromosomes from a polyploidy species.
Haploid production through anther culture is called as androgenesis, while gynogenesis is the production of haploid plants from ovary or ovule culture when the female gametes or gametophyte is trigger to sporophyte development.
Materials required:
Anthers from hibiscus, MS medium, Growth factors, 70% of ethanol, 2% of Mercuric chloride, Meso inositol, Scissors, Petri dishes, Scalpels, Forceps,
Procedure:
1. Flower bud of hibiscus were collected
2. The Flower buds are surface sterilized by immersing in 70% ethanol for 60 seconds followed by immersing in Mercuric chloride.
3. The buds are washed out 4-5 times with sterilized distilled water.
4. The buds are transferred to a sterile petri dish.
5. The buds are split open using a blade and the anthers are removed without damage and the filaments were removed.
6. The anther were placed horizontally on the MS medium supplemented with the different concentration of plant growth regulators or Meso inositol.
7. The petri plates were sealed and incubated in dark at 28°c
8. The Petri plates/dish were examined for the generation of anthers.
Results:
The anther under went generation leading to the formation of haploid plantlets.
Demonstration of PCR
The polymerase chain reaction (PCR) is a laboratory techniques for a DNA replication that allows all target DNA sequence to be selectively amplified. PCR Can use the smallest sample of the DNA to cloned to amplify it to millions of copies in just few hours. Discovered in 1985 by Karry Mullis,PCR has become both and essential or routine tool in most biological laboratories.
Principle:
The PCR involves the primer mediated amplification of DNA. PCR is based on using the ability of DNA polymerase to synthesize new stand of DNA. Complementary to the offered template strand primer is needed because DNA polymers can add a nucleotide only into a pre existing 3'- OH group. After adding the first nucleotide DNA polymerase can then elongate. it's 3 end by adding more nucleotides to generate an extended region of double standard DNA.
Component of PCR::
The PCR reaction requires the following components
1. DNA template:
The double standard DNA of interest separated from the sample.
2. DNA polymerase:
Usually a thermostable taq polymerare that does not rapidly denature at high temperature (98°) can function at a temperature optimum at about 76°c.
3.Oligonucleotide primers:
Short pieces of single standard DNA (often 20-30bp) which are complementary to the 3' end of sequence and anti sense strands of the largest sequences.
4. Deoxy nucleotide triphosphate:
Single units of the bases A,T,G and C (dATP, dTTP, dGTP, dCTP) provide the energy for polymerisation and the building blocks for DNA synthesis.
5. Buffer system:
Includes magnesium and potassium to provide the optimal condition for DNA denaturation and renaturation. Also important for polymerase activity, stability and fidelity.
Procedure of PCR::
All the PCR components are mixed together and taken through series of a major cyclic reaction and conducted in an automated self contaminated thermo cycle medium.
1. Denaturation:
This step involves heating of the mixture to 94°c for 15-30 sec. During this double standard DNA is denatured to single strand due to breakage in hydrogen bonds.
2. Annealing:
The Reaction temperature is rapidly lowered to 54- 60°c for 20-40sec. This allow the primers to bind to their complementary sequences in template DNA.
3. Elongation:
Also known as extension this step usually occurs at 72 -80°c.
In this step the polymerase enzyme sequentially adds bases to the 3'OH primer extending the DNA sequences in the 5'-3'direction. Under optimal conditions DNA polymerase will add about 1000 bp/min.
With one cycle a single segment of double standard DNA template is amplified into two separate pieces of double standard DNA. There two pieces are then available for amplification in the next cycle. As the cycle are repeated more and more copies are generated and the number of copies of the template is increased exponentially.....
Polio
Polio, or poliomyelitis,
is a crippling and potentially deadly disease. It is caused by the poliovirus.
The virus spreads from person to person and can invade an infected person’s
brain and spinal cord, causing paralysis (can’t move parts of the body).
Polio can be prevented
with vaccine. Inactivated polio vaccine (IPV) is the only polio vaccine that
has been given in the United States since 2000. It is given by shot in the arm
or leg, depending on the person’s age. Oral polio vaccine (OPV) is used in
other countries.
Centers for Disease Control and Prevention (CDC) recommends that
children get four doses of polio vaccine. They should get one dose at each of
the following ages:
·
2 months old
·
4 months old
·
6 through 18 months old
·
4 through 6 years old
Almost all children (99
out of 100) who get all the recommended doses of polio vaccine will be
protected from polio.
The first polio vaccine was available in the United States in 1955.
Hepatitis
B
Hepatitis B is a liver disease caused by a virus.
There are 2 types of hepatitis B:
- Acute (short-term) hepatitis B
- Chronic (long-term) hepatitis B
Many children who get acute hepatitis B don’t have any symptoms, but most adults do. Symptoms may include:
- Fever
- Feeling tired
- Upset stomach and throwing up
- Not feeling hungry
- Dark pee or clay-colored poop
- Pain in the muscles, joints, and stomach
- Jaundice (yellow skin or eyes)
There are 2 vaccines that protect against hepatitis B:
- The hepatitis
B vaccine protects infants, children, and adults from hepatitis B
- The hepatitis A and B combination vaccine protects adults from both hepatitis B and hepatitis A
- Getting vaccinated is the best way to prevent hepatitis B.
Varicella vaccine
al It is also known as chickenpox vaccine,
i It is s a vaccine that protects against chickenpox.
One dose of vaccine prevents
95% of moderate disease and 100% of severe disease. Two doses of vaccine are
more effective than one.
Antibiotics
m Antibiotics are substances produced by various microorganisms or a similar product produced wholly (synthetic) or partially (semi-synthetic) by chemical synthesis and which in low concentration inhibits the growth of or kills the microorganisms.
Penicillin
1. The first antibiotic discovered was penicillin.
2. It was discovered by Alexander Fleming in 1928. He noticed that some bacteria he left in a petridish had been killed by naturally ocurring penicillin mould.
3. Ernst Chain and Howard Fleming developed the process for mass production of penicillin for medical use
3. Uses:
Skin and soft tissue infections
Diptheria and Tetanus
Intra abdominal infection
Ear and Lung infection
Respiratory Tract infection
Urinary Tract infection
Dental Infection
Syphilis, gonorrhoea
Streptomycin
1. Streptomycin is a broad-spectrum antibiotic which is used as the primary treatment of tuberculosis, a disease which mainly affects the respiratory system.
2. Also used for
certain other infectious diseases which affect the brain, the respiratory
system, and the inner tissues of the heart, especially the valves.
3. It is the first aminoglycoside antibiotic which was isolated form the Actinomycetes griseus.
4. Discovered in 1943 by Selman Abraham Wakesman.
Amoxicillin
Amoxicillin is
an antibiotic often used for the treatment of a number of bacterial infections.
Uses
Amoxicillin is used to treat a wide variety
of bacterial
infections. This medication is a penicillin-type antibiotic. It works by stopping the growth of bacteria.
T This antibiotic treats only bacterial
infections. It will not work for viral
infections (such as common cold, flu). Unnecessary use or misuse of any antibiotic can lead to its decreased
effectiveness.
Amoxicillin is also used with other medications to treat stomach/intestinal ulcers caused by the bacteria H. pylori and to prevent the
ulcers from returning.
Bt Cotton
Bt Cotton plants are transgenetically or genetically modified plants.
T They contain a gene that codes for the
production of a protein (Bt toxin) that kills the caterpillar of the Helicoverpa amigera moth.
Bt genes encodes the cry group of endotoxis
cry IAc which is specific to cotton bull worm and kills the infecting pathogen.
Bt Brinjal
Bt Brinjal is a transgenic crop created by inserting a gene cry IAc from the soil bacterium Bacillus thuringiensis.
Bt Brinjal is resistant against lepidopteran insects like Brinjal furit and shoot borer (Leucinodes orbonalis) and fruit borer (Helicoverpa amigera).
It will reduce the level of pesticide residues.
Golden Rice
'Golden Rice” is the name of a rice that has been genetically engineered (genetically modified or GM) to produce beta carotene, which the body can convert into vitamin A.
This beta-carotene gives the rice grains a yellowish colour that inspired its name.
It is intended to produce a fortified food to be grown and consumed in areas with shortage of dietary Vitamin A.
Deficiency of Vitamin A is estimated to kill 670,000 children under the age of 5 and cause an additional 5 lakh cases of irreversible childhood blindness.
Rice is a staple food crop for over half of the world's population, making it an excellent crop for targeting Vitamin deficiencies.
Flavr Savr Tomato
The first commercially grown genetically engineered food granted a license for human consumption
Produced by the Californian company Calgene 1992
- A hot air oven is a laboratory instrument that uses dry heat to sterilize laboratory equipment and other materials.
- We can sterilize Glassware (like petri dishes, flasks, pipettes, and test tubes), Powder (like starch, zinc oxide, and sulfadiazine), Materials that contain oils, Metal equipment (like scalpels, scissors, and blades) by using hot air oven.
- Hot air ovens use extremely high temperatures over several hours to destroy microorganisms and bacterial spores. The ovens use conduction to sterilize items by heating the outside surfaces of the item, which then absorbs the heat and moves it towards the center of the item.
- The temperature range covered by ovens is between 50-250°C.
- The most common time-temperature relationships for sterilization with hot air sterilizers are • 170ᵒC (340ᵒF) for 30 minutes, • 160ᵒC (320ᵒF) for 60 minutes, and • 150ᵒC (300ᵒF) for 150 minutes or longer depending up the volume.
- Sterilizing by dry heat is accomplished by conduction. The heat is absorbed by the outside surface of the item, then passes towards the Centre of the item, layer by layer. The entire item will eventually reach the temperature required for sterilization to take place.
Incubator contain a thermostat which maintains the inside temperature of the incubator. We can monitor this temperature from the outside via the thermometer. By utilizing the heating and no-heating cycles we maintain the inside temperature of the incubator.
Similarly, the incubator also maintains the other parameters such as humidity, airflow, co2 concentration, pH, through different mechanisms which are required for the growth of the organisms.
Electrophoresis Apparatus
Electrophoresis is a laboratory technique used to separate DNA, RNA or protein molecules based on their size and electrical charge.
An electric current is used to move the molecules through a gel or other matrix. Pores in the gel or matrix work like a sieve, allowing smaller molecules to move faster than larger molecules.
Electrophoresis apparatus consists of a gel, which is often made from agar or polyacrylamide, and an electrophoretic chamber (typically a hard plastic box or tank) with a cathode (negative terminal) at one end and an anode (positive terminal) at the opposite end.
The gel, which contains a series of wells at the cathode end, is placed inside the chamber and covered with a buffer solution.
The samples are then loaded into the wells with a pipette. The chamber is connected to a power supply that, when turned on, applies an electric field to the buffer.
The electric field causes negatively charged molecules to migrate through the gel toward the anode.
The molecules’ movement is influenced by the porous gel matrix such that larger, heavier molecules move relatively slowly, whereas smaller, lighter molecules move more quickly.
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