What is extraction of plant?

•Extraction can be defined as the treatment of the plant (or animal tissues) with solvent, where the medicinally active constituents are dissolved and most of the inert particles remain undissolved.
• The process of extraction developed into a distinct area and contributes mainly to the progress of phytochemistry.
• Extraction processes includes maceration, steam or hydro-distillation, pressing, decoction, infusion, percolation and Soxhlet extraction.
• It is difficult to find a production process in the perfume, cosmetic, pharmaceutical, food, bio fuel, or fine chemicals industries, which does not use extraction processes these days.

Why extraction process of plant materials important?

• To siphon the required chemical components from the plant materials for further separation and characterization.
• To isolate an unknown compound responsible for the particular activity.
• To discover bioactive constituents from plant materials.
• It has great impact on the reliability of a natural medicine.
• Significant for the standardization of herbal/ayurvedic products.

Solvent extraction method

• The efficient determination of biologically active compounds from plant material largely depends on the type of solvent used in the extraction process.
• The ruptured cells are integrated in the extracting solvent and the mixture is kept for some time (half an hour to 24 hr) to let the solvent prick all parts of the ruptured cells.

1. • Maceration:

   • The simplest mode of cold extraction process which is suitable mostly for the thermolabile drugs.
   • In a stoppered container, the powdered plant material is taken and soaked with the solvent for a specified period of time.
   • Agitation is performed until the soluble matter dissolves.

2. • Percolation:

   • It is the process in which movement of mass across a porous material takes place where a percolator is generally used.
   • Percolator is a narrow, cone-shaped vessel open at both ends.
   • The plant (Crude) material is taken in a percolation tube plugged with cotton or fitted with a filter and a stopcock and is moistened with an appropriate amount of solvent(esp. menstruum).
   • It is left to stand for approximately 4 hr in a well closed container.
   • Further menstruum is added to create a shallow layer above the mass, and the mixture is permitted to macerate in the closed percolator (for 24 hr)
   • The liquid contained is allowed to drip slowly by opening the outlet of the percolator.
   • Menstruum is added as desired, until the percolate measures about three-quarters of the desired volume of the final product.
   • The marc is then pressed and the obtained liquid is added to the percolate.
   • Marc is inert fibrous and other insoluble material remaining after extraction.

3. • Digestion:

   • It is a type of maceration in which mild heat (40-60oC) is applied.
   • It is used when moderately elevated temperature is acceptable.
   • It can be modified by mixing the material with the solvent using magnetic stirrer, mechanical stirrer or by shaking occasionally using hand.
   • The extract is filtered and fresh solvent is added after 8-12hrs.
   • The process is repeated until all the desired solutes are extracted.
   • It is used for the tougher plant parts or those that consist poorly soluble substances.

4. • Infusion:

   • The plant material is macerated in boiling water for a short period of time.
   • In this process, chemical compounds or flavours from plant material are siphoned in a solvent as water, oil or alcohol, by permitting the material to stay suspended in the solvent over a period of time.

5.  Decoction:

   • The crude plant material is boiled in a specified volume of water for a given time frame.
   • It is then cooled and strained or filtered.
   • Decoction time varies depending on the uniformity of the parts to extract.
   • It is suitable for extracting water soluble and heat stable constituents.
   • Decoctions are prepared for fresh purpose and shouldn’t be stored for more than 24 hours.
   • Decoction may be used to prepare herbal teas, leaf teas, coffees, tinctures and similar solutions.

Extraction with boiling solvents (Refluxion):

• The plant material is treated with boiling solvent and hence is a hot extraction process.
  The solvent vapor is recycled by a condenser fitted on top of the container, preferentially a round bottomed flask.

1.  Hot Continuous Extraction (Soxhlet):
   • It is named after ‘Franz Ritter von Soxhlet’, a German agricultural chemist.
   • It is the convenient method for the continuous extraction of a solid by a hot solvent.
   • In this process, the transfer of partially soluble components of a solid to the liquid phase takes place using a Soxhlet extractor.
   • The finely ground crude plant material is placed in a thimble, which is made of strong filter paper that allows liquid to pass through and is placed inside the Soxhlet apparatus.
   • The apparatus is then fitted to a round bottomed (RB) flask containing the solvent and to a reflex condenser.
   • The solvent in the RB flask is boiled mildly.
   • The vapor passes up through the side tube, gets condensed by the condenser and falls into the thimble containing the material and slowly fills the Soxhlet.
   • When the solvent reaches the top, it extracts over into the flask, removing portion of the substance which it has siphoned and the process repeats.
   • After extraction, solvent is eliminated by means of a rotary evaporator, resulting extracted compound.
   • The non-soluble portion of the extracted solid stays in thimble and is usually relinquished.
   • Advantages:
   • Large amount of drugs can be extracted with much smaller quantity of solvent.
   • More economical and feasible on medium or large scale.
2. • Steam Distillation:
   • It is used for the separation of essential oil from crude plant material.
   • Simple vaporization is gained by passing steam directly through the material.
   • The steam vaporizes the plant material’s volatile compounds which finally pass through a condensation and collection process.
   • Since water and oil do not mix in collection process, the essential oil floats on top of the water and it is extracted off.
3. • Hydro Distillation:
   • It is used instead of steam distillation.
   • The plant material is soaked in water and boiled by use of heating mantle.
   • Due to the impact of hot water, the essential oil is freed from the oil glands in the plant tissues and passed along with the steam.
   • In a typical glass apparatus known as Clevenger apparatus, the steam oil mixture is condensed and oil is separated from water and the condensed water is recycled.
4. • Enfluerage:
   • It is used for the extraction of delicate fragrances as that of some flowers.
   • The flower petals are spread over a layer of refined fat which picks up the odour of the flowers.
   • The saturated fat is handled with a solvent, usually alcohol in which the fragrant components are soluble.
   • The residual fat dissolved in alcohol may be eliminated by cooling the alcohol extract to 20oC, when fat separates out.
   • The volatile components are then retrieved from alcohol by concentrating the solution at reduced pressure in a rotavapor.

• Supercritical Fluid Extraction (SFE):

• Critical point represents the conditions above which distinct liquid and gas phases do not exist, but a homogenous supercritical fluid state exists.
• Supercritical fluid is obtained by heating above the critical temperature and compressing above the critical pressure and has the properties of a liquid as well as that of a gas.
• Alternative method with reduced use of organic solvents and increased sample throughput.
• Most commonly used supercritical fluid is CO2 .
• The lower viscosities and higher diffusion rates of supercritical fluids compared to liquids amplifies the extraction process.
• It is non inflammable, chemically inert, odour free, easy for disposal and economical with high purity and also can be recycled.
• Other gases such as ethylene, ethane, propylene, propane and nitrous oxide can also be used.
• High pressure carbon dioxide can be applied in place of various traditional organic solvents and steam distillation.
• Above 1100 psi and 31.70C, carbon dioxide reaches in “supercritical region.”
• Under these conditions, it has the solvating power of a liquid and the diffusion property of a gas.
• Carbon dioxide, being a supercritical fluid is a superior solvent for the extraction of a wide variety of natural products.
• This technique has been in use in industry for decaffeination of coffee and the removal of nicotine from tobacco.
• It is a more efficient method for the extraction of taxol and baccatin from the Yew tree.
• The major advantage is the implementation of mild conditions, which avoid the risk of thermal degradation compared to distillation and solvent extraction.

• Ultrasonic Extraction:

• It involves the use of ultrasound with frequencies ranging from 20 kHz to 2000 kHz.
• It increases the porosity of cell walls and produces cavitation.
• Ultrasound aided extraction can be used with mixtures of immiscible solvents such as hexane with methanol/water.
• In cases where the process creates heat, the extraction container is placed in ice bath to prevent heat labile compounds from decomposition.

• Microwave Assisted Extraction:

• Microwaves are electromagnetic radiations with the frequency range 0.3 to 300 GHz.
• It is made up of two oscillating perpendicular fields: electrical field and magnetic field.
• They can be used as information carriers or as energy vectors.
• Ionic conduction and dipole rotation in both the solvent and the sample which efficiently changes microwave energy to thermal energy.
• If the solvent selected has a high dielectric constant it strongly absorbs the microwave energy.
• However, in some cases, only the sample matrix may be heated, so that the solutes are freed in a cold solvent to avoid the degradation of thermolabile compounds.
• Microwave energy is applied to the sample suspended in solvent, with short intervals of cooling time.
• It’s merits include e.g., shorter extraction time, less solvent, higher extraction rate and lower cost.

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• The term vermiculture refers to the cultivation or production of earthworms.
• Vermicomposting is the method by which worms are used to turn organic materials (usually waste) into a humus-like substance known as Vermicast.
•  The term vermicast is also termed as worm castings, worm manure, worm feces and worm humus.
• Vermicompost includes not only worm castings, but also bedding materials and organic waste in different phases of decomposition.
• It also includes worms that are at different stages of development and microorganisms involved  in the composting process.
• In the home garden, earthworm castings often contain between 5 and 11 times more nitrogen, phosphorous, and potassium than the surrounding soil.
• Vermicompost also adds soil with humic acid that helps link soil with minerals and nutrients.
• Chemical fertilizers can rapidly improve the growth and development of plants, but will not develop soil.
• Studies suggest the best growth in plants with 10 to 40 percent vermicompost content in a soil mix.
• Many beneficial microbes are destroyed by the heat of daily compost.
• Vermicompost is aerobic and therefore can have up to 1000 times the normal compost microbial activity.
• Better disease tolerance indicates increased microbial activity in the soil.
• Vermicompost can be generated within 1/3 of the normal compost period.

What are the requirements for the preparation of the vermicomposting?

1. Breeder worms (Earthworms)
2. Feeding materials: Any type of biodegradable waste: vegetable waste, crop residues, weed biomass, dead leaf litter, agro-industry waste, urban and rural biodegradable portion of waste
3. Bedding materials
4. Production systems (a container or pit in a shady place)

What role does earthworm plays in vermicomposting?

• Physical/Mechanical:
  • As the earthworm moves inside the substrate, it helps in the aeration of substrate, as well as it grinds the substrate.
  • This keeps the organic wastes aerated and cooling is aerated.
  • It also saves costs required for mechanical process units for mixing that are used in conventional composting.

• Biochemical:
  • Microbial decomposition of substrate takes place in the intestines of the earthworm.

• Other roles:
  • They maintain aerobic conditions in the mixture and ingests solids from the substrate.
  • A portion of the organic materials is converted to worm biomass and respiration products.
  • The remaining partially stabilised matter is expelled as discrete material i.e. termed as castings.
  • worms and aerobic mesophilic organisms act symbiotically and fastens the decomposition of the organic matter.

Types of vermicomposting worms:

• On the basis of their feeding habits, they are classified as detrivores and geophages.
• Detrivores feed on plant litter or dead roots, and other plant debris or on mammalian dung on or near the soil surface.
• These worms are referred to as humus formers and comprise the epigeic and anecic forms.
• Some examples of detrivorous worms are  Perionyx excavatus, Eisenia fetida, Eudrilus euginae, Lampito mauritii, Polypheretima elongata, Octochaetona serrata and Octochaetona curensis.
• Geophagous worms, feed below the surface and intake greater quantities of organically rich soil.
• It comprise the endogeic earthworms. Metaphire posthuma and Octochaetona thurstoni are two general examples of geophages.
• Epigeics are surface dwellers who feed on the surface of the soil with organic matter.
• Endogeic earthworms spend much of their time mainly in the mineral layer of the soil and burrow.
• For use in the vermicomposting process, the epigeics and anecics were harnessed.
• Eisenia fetida, the tiger or branding worm, is one of the most widely used worldwide.
• Lumbricus rubellus, Eudrilus eugeniae and Perionyx excavatus, an Asian genus, and Eisenia andrei are other suitable species.
• Redworm species in both Eisenia fetida and Lumbricus rubellus are composters, living naturally in soils that contain a lot of organic matter.
• For this reason, they are often used together, with Eisenia fetida on the surface and Lumbricus rubellus farther down, in vermicomposting systems.

Eisenia fetida in Vermicomposting:

• The genus Eisenia is named after a naturalist from Sweden.
• The name of the genus fetida (foul-smelling) derives from the pungent liquid exuded when roughly handled.
• They are are more generally referred to as red Wigglers, brandling worm, panfish worm, trout worm, tiger worm, red Californian earth worm, etc.
• They are identified by the yellow stripes in between the segments.

Process of vermicomposting:

1. Feeding materials:

• Worms can eat dung from animals, agricultural waste, residues from vegetables, waste from the food market, waste from the flower market, agro-industrial waste, waste from the fruit market and all other bio-degradable waste.
• Before being used for vermicompost production, cattle dung should be dried in open sunlight.
• Depending on the feedstock being used, temperature, moisture levels and the density of the worm population, the exact loading rate (at which raw feedstock will be applied to a worm bed) can differ.
• Proper loading rates require no inclusion of new feedstock until the bulk of the feedstock previously introduced has been decomposed.
• A high protein feedstock such as grains, mash, or cottonseed meal is added if worms are not growing.

2. Bedding materials:

• As bedding products, certain agricultural residues may be used, such as plant waste and solid composted manure.
• In general, because of bedding content’s effect on increasing soil pH, which is harmful to worms, the bedding content should maintain moisture, stay loose and aerated, and be low in protein and nitrogen.
• The bedding content should be varied to provide the earthworms with a variety of nutrients and to create richer compost.
• Suitable bedding materials include:
  • 1. coir waste
  • 2. cardboard
  • 3. shredded fall leaves
  • 4.  sawdust
  • 5. chopped straw
  • 6. mulched paper such as newspaper
  • 7.  semi-composted solid manure
• Sieving and shredding:
  • By shredding raw materials into small pieces, decomposition can be accelerated.

3. Blending:

• To achieve a near optimal C/N ratio of 30:1-40:1, carbonaceous substances such as sawdust, paper and straw can be combined with nitrogen-rich products such as sewage sludge, biogas slurry, and fish scraps.
• Good quality compost, rich in main and micro nutrients, is produced by a varied mixture of substances.

4. Pre-composting/Half digestion:

• In order to avoid worm systems from feeling so much sun, manure feedstocks and bedding should be pre-composted.
• When introduced into the worm systems, fresh manures produce a lot of energy that transfers into additional heat.
• Strong heat in the beds of worms can be deadly.
• The bedding and feeding materials are then combined, watered and allowed to ferment for approximately two to three weeks.
• It is necessary to hold the raw materials in piles to allow the temperature to exceed 50-55^o C.
• The substance is overturned 3 to 4 times during this phase to get the temperature down and to aid in uniform decomposition.
• It is passed to the vermicompost production method as the material becomes very fragile, and worms are inserted into it ranging from a few days to a few weeks old.

5. Moisture, temperature and pH:

• 50-60 percent is the optimal moisture level for maintaining aerobic conditions.
• The temperature should be within 25-30^o C of the stacks.
• The activity of microflora and earthworms can be decreased by higher or lower temperatures.
• The height of bed can help to regulate the increase in temperature.
• The raw material’s pH should not be greater than 6.5 to 7.

Methods of vermicomposting:

• A 1 meter by 1 meter by 0.3 meter container carries about 30-40 kg of bedding and feeding materials.
• It is possible to prepare a vermiculture bed or worm bed (3 cm) by putting dust or husk or coir waste or sugar cane garbage in the bottom of the tub/container.
• The culture bed can be spread with a sheet of fine sand (3 cm) followed by a layer of garden soil (3 cm).
• A15-20 cm sheet of organic waste material (pre-composted/half digested) can be spread on the worm bed. 
• It is sprinkled with rock phosphate powder (to increase the content of phosphate) if required.
• Soil or cow dung is used to cover the organic layer with (sprinkle cow dung slurry).
• The selected earthworms are released through the cracks created (1000-1500).
• In order to prevent birds from eating the earthworms, cover the ring with wire mesh or gunny sack.
• Water is sprinkled to maintain adequate humidity and temperature regularly/daily.

Harvesting of vermicompost:

• In about 3 months, the vermicompost is ready (may vary depending on organic waste used as substrate).
• It will be black, granular, lightweight and humus-rich.
• Vermicompost harvesting requires manual isolation of worms from the castings
•  Watering is stopped for two to three days before emptying the beds to facilitate the removal of the worms from the compost.
• The worms will be pushed to the bottom of the bed by this.
• For new culture beds, the worms are collected.
• To retrieve the cocoons, young worms, and unconsumed organic waste, the gathered vermicompost is dried and passed through a 3 mm sieve.
• For seeding the new culture beds, cocoons and young worms are used.

Storage and packing of vermicompost:

• The harvested vermicompost should be stored in dark, cool location.
• It should have moisture of at least 40 percent.
• Sunlight should not fall on the content being composted.
• At the point of sale, packaging can be done.
• Periodic sprinkling of water can be done to retain the level of moisture and also to maintain a beneficial microbial population if it is kept in an open location.
• Vermicompost may be preserved for a duration of one year without loss of quality if the moisture level is kept at 40%.

What are the precautions to be taken while preparing vermicompost?

• In vermicompost preparation, only plant-based materials such as hay, leaves or vegetable peelings can be used.
• Animal products such as eggshells, beef, bone, chicken droppings, etc, are not appropriate for vermicompost preparation.
• For the rearing of earthworms, toxic plant species like tobacco are not appropriate.
• It is necessary to protect earthworms from birds, termites, ants and rats.
• During the process, sufficient moisture should be maintained.
• The earthworms could die by either stagnant water or lack of moisture.
• The vermicompost should be withdrawn from the bed at periodic intervals after completion of the process and replaced by fresh waste materials.

Vermicompost production system:

1. Beds and/or Bins:

• The most popular small-scale device is bins.
• Bins may be built with a drain hole at the bottom of many materials, such as wooden crates, cardboard buckets or other reusable containers.
• The bottom of the tub is designed to slope like a structure to drain the vermicompost unit from the waste water.
• Bins can measure between 8 and 12 inches deep.
• The size would depend on the available amount of feedstock and bedding.
• As a rule of thumb,  at least 1 square foot of surface area per pound of feedstock should be provided.

2. Pits below the ground:

• There are 1 m deep and 1.5 m wide pits made for vermicomposting.
• As required, the length varies.

3. Windrows (Heap above the ground):

• Windrows are longitudinal stacks of either covered or exposed feedstocks placed at ground level.
• The windrow is initiated by extending a layer 12 to 18 inches deep of organic materials.
• Next, at a rate of up to one pound per square foot, redworms may be added.
•  2 to 3 inches a week on top of the existing pile is layered to apply feedstock to the windrow.
• For ease of management, the pile should not reach 3 feet in height.
• Harvest worms using the light technique.

4. The wedge system:

• This system is a modification of the windrow method by adding feedstock at a 45 degree angle to the current windrow.
• The redworms would move towards the “fresh” pile by creating a “wedge” next to the actual windrow being used.
• Organic materials are applied to this new pile until a height of 3 feet is achieved, then a new wedge starts.
• Worms can pass across the piles laterally.
• We would finally be able to harvest the first pile and subsequent piles after 2-3 months.

5. Continuous flow reactors:

• These are systems with elevated beds that have side walls and mesh bottoms that are either 2 inches by 4 inches or 2 inches by 2 inches with openings.
• To preventbedding from slipping through, place a material such as newspaper on the mesh.
• About 12 inches of bedding is spread on top of the newspaper.
• Redworms are placed at a rate of 1/2 to 1 pound per square foot of surface area on top of the bedding.
• Feedstocks are then inserted on top of the bedding in layers.
• By scraping a thin layer just above the mesh to allow material to fall into the catchment chamber, Vermicompost can be harvested.
• Such systems work better under cover.

The post Vermicomposting appeared first on Online Biology Notes.

• Semen is also termed as seminal fluid. It is the fluid that is released from the male reproductive tract and comprises of sperm cells that can fertilize the female’s eggs.
• Among the cells, sperm are unique in form and function.
• Mature sperms are the terminal cells that are incapable to go further division or differentiation.
• One of the standard methods for evaluation of the fertility of breeding males is by examination of semen.

Evaluation of semen of bull and fertility of bull:

• For the evaluation of semen of bull, physical examination and a vaccination/ health certification is required.
• Generally, the minimal standards required for a classification of a specimen of bull semen are:
  • a. over 500 million sperm per ml.
  • b. >50% of motile sperm leads to forward progression.
  • c. Normal morphology is confirmed by >80%of spermatozoa.
• We can conclude that the bull is sterile only if the motile sperm are completely absent and the reproductive system has been properly examined.

Appearance and volume of bull semen:

• Bull semen should have a comparatively uniform and opaque appearance.
• Opaque appearance is the indicator of high sperm cell concentration.
• However, translucent samples consist of few sperm.
• The sample should be devoid of contaminants such as hair, dirt etc.
• Semen which is curd like in appearance comprising of chunks of material are avoided i.e. it suggests infection.
• Sometimes, presence of riboflavin results in yellow appearance of semen which is usually considered harmless.
• Repeated ejaculation leads in lower average volume.
• If the two ejaculates are extracted consecutively, the second generally has the lower volume.

Measurement of Sperm concentration:

• The measurement of sperm concentration can be done by using colorimeter, hemocytometer, or spectrophotometer.
• A hemocytometer is a microscopic slide with accurate scored chambers.
• The number of sperm per chamber are then counted.
• It is more tedious job but has greater preciseness.
• In contrast to that, spectrophotometer and colorimeter have a merit of being fast and accurate.
• The calibration of machine is done at 550nm.
• For the dilution of the ejaculate, 2.9% sodium citrate and 5ml of 10% formalin per litre.
• A standard curve that measures concentration versus 0.5% increment of light transmittance provides a range that is required to measure concentration.
• However, in presence of contamination, photometers are not accurate.
• The results can confound if the cloudy extenders are added prior to the estimation of concentration.
• The concentration of sperm ranges from 2X 10^8 sperm/ml in young bulls to 1.8 X 10^9 sperm/ml in mature bulls.

Measurement of Sperm motility:

• The measurement of the sperm motility requires subjective estimation of the viability of the spermatozoa and the effectiveness of motility.
• Light microscopic analysis is widely used.
• Sperm motility assessment is performed with raw and prolonged semen.
• Raw semen assessment is an indicator of sperm performance in its own accessory gland fluid.
• Greater sperm concentration will hinder the calculation of motility in the raw form, making it hard to distinguish individual patterns of motility.
• An aliquot of semen should be extended in a good quality extender (i.e. concentration of 25X 10⁶ sperm/ml) to overcome this restriction.
• The motility of sperm is highly prone to environmental factors (as such excessive heat or cold).
•  Hence, the semen needs to be shielded from harmful agents or circumstances, prior to study.

Process of sperm motility assessment:

• On a glass slide, a drop of extended semen is placed and smeared with other slide.
• Then, it is observed under microscope with a built-in-stage warmer and phase-contrast optics.
• To estimate sperm motility, magnification of 200X or 400X is usually used.
• The parameters of motility consists of:
  • a. Percentage of motile sperm (normal is 70%-90% motile).
  • b. Percentage of increasingly motile sperm.
  • c. velocity of sperm (based on an arbitraty scale on between 0-4).
  • d. Longevity of sperm motility in raw semen (at room temperature 20^oC to 25^oC) and in extended semen (at room temperature, or refrigerated temperature 4^oC to 6^oC).
• Several patterns of motility are observed.
• In diluted semen, general patterns of sperm motility appear in a long semiarc pattern.

Several factors that affect sperm motility:

• The extender might alter motility slightly, generally by enhancing velocity measures.
• A high percentage of sperm may show circular motility pattern, which generally resolves after 5-10 minutes in the extender.
• If the sperm are swimming in a tight circular motion, it indicates that they could have been in presence of cold shock.
• There is correlation between motility patterns and infertility or subfertility of males.
• For unbiased evaluation of sperm motility, various techniques have been developed.
• Some of the procedures are time-lapse photomicrographs, frame-by-frame playback videomicrography, spectrophotometry, and computerized analysis.

Abnormalities in morphology of sperm:

• Each semen sample comprises of few abnormal sperm cells.
• The abnormalities in morphology of sperm is directly related to the fertility of livestock.
• One of the causes for the abnormality is heat stress.
• Heat stress results in large number of damaged sperm.
• Long exposure of high ambient temperature in combination of high humidity may cause sterility in male for upto 6 weeks.
• During the recovery duration, high number of abnormal sperm are observed in ejaculates.
• The effects of heat stress can be reduced by supplying sufficient shade and clean cool water.
• In the condition, when abnormal sperm cells exceed 20%, it results in infertility.
• The microscopic magnification is determined by the type of abnormality being evaluated.
• The classification of morphologic abnormalities are as:
  • 1. Primary
  • 2. Secondary
  • 3. Tertiary
• Primary abnormalities are related with sperm heads and acrosome.
• Secondary abnormalities is defined by the presence of the droplet on the midpiece of the tail.
• Other tail defects are shown by tertiary abnormalities.
• The use of eosin-nigrosin stain analyzes the sperm morphology.
• Wright’s and William’s stains can also be used.
• By the use of high microscopic magnification, the examination of stained slides are done.
• Among 150 spermatozoa examined with abnormal sperm were classified into 5 categories:
  • a. tailless
  • b. abnormal heads
  • c. abnormal tail formations.
  • d. formation of abnormal tail with a proximal cytoplasmic droplet.
  • e. Formation of abnormal tail with a distal droplet.

Evaluation of quality of semen:

• The technique for sperm quality evaluation from frozen semen are linked with motility and intact acrosome.

Procedure:

• In a 95^oF water bath, two straws (0.5ml-0.25ml) are thawed.
• After 45 seconds, one straw is removed and a paper towel is used to dry it. Note: Drying is essential as water can be harmful to sperm.
• Towards the end of the cotton plug, the contents are shaken.
• The other end of straw is cut off and the semen is released into a small clean disposable test tube by cutting a small opening just below the cotton plug.
• On a warm side with a cover slip, a small drop of semen is placed.
• Microscopic analysis is used to perform initial motility readings.
• After incubation of the second straw for 3h at 95^oF, a microscope with interference optics (1000X) and oil immersion is used to analyze the % of intact acrosomes and abnormalities of the sperm.
• The anterior two-thirds portion of the sperm head is covered by the acrosome.
• The acrosome comprises of the enzymes that facilitate the capacitated sperm to penetrate the egg.
• After the 3hrs of incubation, there is direct correlation with the percent intact acrosomes, and fertility.

Ancillary tests to evaluate quality of sperm

a. Electron microscopy:

• In light microscopy, limited magnification is found, and hence the evalution of sperm morphology is limited.
• By the use of scanning and/or transmission electron microscopy, abnormalities in sperm structure can be detected precisely.
• These two microscopic techniques provide details of high-resolution and allow closer examination of sperm morphology.
• Scanning Electron microscopy  observes the 3D visualization of the entire sperm.
• Transmission electron microscopy allows to view cross-section of sperm revealing ultra-structural detail.

b. Sperm chromatin structure assay (SCSA):

• The procedure termed as flow cytometry is employed for the evaluation of structural integrity of the sperm chromatin.
• It does so by measuring the relative amounts of double stranded and single stranded DNA in sperm populations.
• SCSA has been considered fruitful for the identification of some forms of subfertility in bulls and stallions.
• In a few minutes, SCSA can screen 5,000 to 10,000 sperm.

c. Other fluorescent probes:

• The investigation of new fluorescent probes is being carried on for the evaluation of their reliability for sperm function tests by the use of microscope or flow cytometer.
• These assays can provide detailed information on specific sperm features such as plasma membrane integrity, acrosomal integrity, and mitochondrial integrity, and mitochondrial potential.
• Hence, these assays can prove to be very useful accessory tests.

d. Antisperm antibody assay:

• The maturation of sperm completes in the adluminal compartment of the seminiferous tubules in order to circumvent immunologic attack.
• If the sperm escapes from the adluminal compartments of the seminiferous tubules, it triggers an immune response following the formation of antisperm antibodies.
• For this immunologic attack of sperm, the precipitating factors include lacerations, neoplasms, biopsies, trauma, or degenerative changes of the testes.
• Antisperm antibodies hinder the fertilization process in case of humans, however, the mechanism of action stays unresolved.
• Antisperm antibodies can play as a factor to affect the fertility of stallions.
• Primarily, the autoantibody response to sperm is of the IgG type in serum and of IgA type in the seminal plasma.
• For the presence of auto antibodies, evaluation of serum, seminal plasma, and sperms of stallions is lately being investigated.
• The best predictive fertility index is provided by the tests that evaluate the actual binding of antibodies to sperm.

e. Biochemical analysis of seminal plasma/ Secretions of female reproductive tract:

• On post-thaw motility of cryo-preserved sperm, the protein concentration, specific protein composition of seminal plasma, electrolyte concentration do not supply abundant predictive information.
• Osteopontin and lipocalin-type prostaglandin D synthase are the 2 proteins of epididymal origin that serve as markers of bull fertility potential.
• The accessory glands secrete deoxyribose-1 like enzyme and 2 tissue inhibitor of metalloproteinases at ejaculation and bind to sperm as they traverse the male reproductive tract, and are also linked with greater bull fertility.
• The components of the female reproductive tract secretions such as glycosaminoglycans bind to bull sperm and cause acrosome reactions invitro.
• There is a high correlation between % acrosome reacted sperm triggered by glycosaminoglycans and non-return days.

The post Semen analysis and bull fertility appeared first on Online Biology Notes.

• There are various equipments used in animal cell culture and the basic equipments required to carry out the animal cell culture are enlisted as follows:

List of Basic Equipments needed in animal cell culture lab:

1. Sterile Work Area/Cell culture hood (i.e., laminar-flow hood or biosafety cabinet)
2. Incubator (humid CO2 incubator recommended)
3. Water bath
4. Centrifuge
5. Refrigerator and freezer (–20°C)
6. Cell counter (e.g. Automated Cell Counter or hemocytometer)
7. Inverted microscope
8. Liquid nitrogen (N₂) freezer or cryostorage container
9. Sterilizer (i.e., autoclave)

1. Sterile work area required for cell culture:

• In order to maintain clean cell culture work, it is necessary to prepare a separate room or space if possible.
• This room should be devoid of traffic, and if possible it should be equipped with an air flow cabinet that provides filtered air surrounding to the work surface.
• A HEPA (High Efficiency Particle Air Filter) filtered air is appropriate but is not economical.
• The laboratory must be especially designated for clean culture work and it should be strictly restricted to culture the primary animal tissue and micro-organisms in or near the cell culture laboratory.
• The laboratory coats should be placed at the entry gate of the laboratory and should not be used outside the lab.
• A laminar flow hood (i.e. biosafety cabinet) is supposed to the simplest and the most cost effective way to supply aseptic conditions.
• While permitting the containment of infectious splashes or aerosols produced by many microbiological processes, the laminar flow hood provides an aseptic work area.
• In order to meet the diversified research and clinical needs, Three kinds of laminar flow hoods, have been designated as Class I, II and III.
• When used with proper microbiological techniques, Class I laminar flow hoods supplies essential levels of protection to laboratory workers and to the environment, but they do not protect cultures  from contamination. 
• They are identical to chemical fume hoods in design and air flow characteristics.
• For work that involves BSL-1, 2, and 3 materials, Class II laminar flow hoods are designed and they also allow an aseptic environment essential for cell culture experiments. 
•  In order to handle exclusively harmful materials (e.g., primate-derived cultures, virally infected cultures, radioisotopes, carcinogenic or toxic reagents) a Class II biosafety cabinet should be employed.
• Class III biosafety cabinets are gas-tight, and they supply the highest achievable level of protection to personnel and the environment. 
• A Class III biosafety cabinet is needed for work that involves known human pathogens and other BSL-4 materials.
• Air-Flow Characteristics of Cell Culture Hoods:
  • The working environment is protected by the laminar flow hoods from dust and other airborne contaminants by retaining a constant, unidirectional flow of HEPA-filtered air over the work area. 
  • The flow can be both horizontal, blowing parallel to the work surface, and it can be vertical, blowing onto the working surface from the top of the cabinet.
• Clean Benches:
  • Vertical laminar flow or horizontal laminar flow “clean benches” are not biosafety cabinets.
  • These pieces of equipment discharge HEPA-filtered air from the back of the cabinet across the work surface toward the personnel, and the user might be exposed to potentially hazardous materials.
  • These devices only aids product protection.

2. Incubator:

• An incubator will be needed in order to supply the suitable temperature environment for cell growth at 30-40⁰ C.
• Depending on the type of cells being cultured, the  incubation temperature will vary.
• An incubator that has been designated to permit CO2 to be supplied from a main supply or gas cylinder is needed in order to maintain an atmosphere of between 2-5% CO2 is maintained in the incubator.
• In the medium, the concentration of CO2 is kept in the equilibrium with sodium bicarbonate.
• In general, several cell lines can be retained in an atmosphere of 5% CO2: 95% air at 99% relative humidity.
• Dry incubators are relatively cost-effective, but the cell cultures are needed to be incubated in sealed flasks to avoid evaporation.
• In a dry incubator, if the water dish is placed, it can supply some humidity however, they do not provide appropriate control of atmospheric conditions in the incubator.
• Humid CO₂ incubators are relatively expensive, however it allows superior control of culture conditions.
• They can be used to incubate cells that are cultured in petri-dishes or multiwell plates that needs a regulated atmosphere of high humidity and increased CO2 tension.

3. Refrigerators and freezer (-20 °C) for specimen storage:

• Both refrigerators and freezer are very essential for storage of liquid media at 2–8°C and for enzymes (e.g. trypsin) and some media components (e.g., glutamine and serum) at –5°C to –20°C.
•  To store medium and buffers, a refrigerator or cold room is needed. 
• A freezer is required for keeping pre-aliquoted stocks of serum, nutrients and antibiotics. 
• Cryogenic Storage
  • There is high possibility for genetic instability in cell lines of continuous culture as their passage number increases, hence, it is necessary to prepare working stocks of the cells and preserve in cryogenic storage.
  • It is to be noted that the cells should not be stored in 20^oC or -80^oC freezers as their viability reduces when they are not stored at these temperatures.
  • Liquid nitrogen freezers permit storage in the vapor phase just above the liquid at temperature between -140^oC and -180^oC, or submerged in the liquid at a temperature below -196^oC.
  • The possibility of leaky vials or ampules exploding during removal is highly reduced by use of vapor phase storage, however, the liquid phase systems generally have longer static holding times, and are thus, more cost-effective.

4. Microscopes:

• In order to examine the cultures in flasks and dishes, a simple inverted microscope is needed.
• The morphological changes in cultures should be recognized as they are the first indicators for the identification of deterioration of a culture.
• Although, a microscope of very high quality will be needed for chromosome analysis or autoradiography work, a very simple light microscope with X100 magnification will suffice for routine cell counts in a hemocytometer.

5. Tissue culture ware:

• A diverse tissue culture plasticware is found, the most general being specially treated polystyrene. 
• Even if all tissue culture plasticware should support cell growth maximally, it is necessary to make sure that the new supplier facilitates the growth of cultures.
• Cells can be kept in petri dishes or flasks (25 cm2 or 75 cm2) , that have added the benefit that the flasks can be gassed and then sealed so that a CO2 incubator should not be used.
• This is especially useful in case if incubators fail.

6. Washing up and sterilizing facilities:

• Glassware such as pipettes should be immersed in a suitable detergent, then passed through a strict washing procedure with thorough soaking in distilled water prior to drying and sterilizing.
• Pipettes are often stuffed with non-absorbent cotton wool before being placed in sterilization containers.
• Glassware such as pipettes, conical flasks, beakers (covered with foil of aluminum) is sterilized for one hour in a hot air oven at 160 ° C.
• All other equipment, like automatic pipette tips and bottles (lids loosely attached) are sterilized by autoclaving at 121 °C for 20 min.

7. Water still or reverse osmosis apparatus:

• For preparation of media, and rinsing glassware, a double distilled or reverse osmosis water supply is required.
• The pH of the double distilled water should be checked regularly, as this can vary in some instances.
• Variations in the quality of water used may account for variations in outcomes, so it is necessary to use water from one source.
• Water is sterilized for 20 minutes at 121 °C by autoclaving.

8. Filter sterilization

• Media which can not be autoclaved must be sterilized through a membrane filter of 0.22 μm pore size.
• These can be obtained in different designs to filter a wide range of volumes.
• They can be bought as sterile disposable filters, or they can be sterilized in appropriate filter holders by autoclaving.

9. Centrifuge

• Periodically, to increase the concentration of cells or to wash off a reagent, cell suspensions require centrifugation.
• For most purposes, a small bench-top centrifuge, preferably with proportionally controlled braking, is enough.
• Refrigeration is not necessary, although, set at room temperature, it can be used to prevent overheating of cell samples.
• At 80 to 100 g, cells sediment satisfactorily; higher g may cause damage and encourage pellet agglutination.

Other expanded equipments and additional supplies:

• Other additional equipments and supplies needed in animal tissue culture lab are:
  • Aspiration pump (peristaltic or vacuum)
  • pH meter
  • Roller racks (for scaling up monolayer cultures)
  • Confocal microscope
  • Flow cytometer
  • Cell culture vessels (e.g., flasks, Petri dishes, roller bottles, multiwell plates)
  • Pipettes and pipettors
  • Syringes and needles
  • Waste containers
  • Media, sera, and reagents
  • Cells
  • Cell cubes

1. Aspiration pump:

• An aspirator is a form of ejector-jet pump, that creates vacuum by means of the Venturi effect.
• Fluid (liquid or gaseous) passes through a tube in an aspirator that gets narrower and then expands in the cross-sectional area and thus volume.
• The pressure of the fluid diminishes as the tube narrows.
• The most popular type of aspirator is the cheap and simple water aspirator.
• It is used in laboratories for chemistry and biology and consists of a tee fitting connected to a tap and has a hose barb on one side.
• The water flow passes through the tee’s straight portion, which at the intersection where the hose barb is attached has a restriction.
• To this barb, the vacuum hose should be attached.
• Although historically popular for low-strength vacuums used in chemical bench work, they use a lot of water, and depending on what the vacuum is being used for, i.e. removal of solvents, by mixing these potentially dangerous solvents into the water stream, they can breach environmental protection laws such as RCRA, then rinsing them down a drain which often directly leads to the municipal sewer.
• The intensity of the vacuum generated is restricted by the vapor pressure of the liquid (for water, 3.2 kPa or 0.46 psi or 32 mbar at 25^o C or 77 F) if a liquid is being used as the working fluid.
• This restriction does not exist if a gas is used.
• The industrial steam ejector (also named the ‘steam jet ejector’, ‘steam aspirator’, or ‘steam jet aspirator’) uses steam as a working fluid.

2. pH meter:

• PH meter is an electrical instrument for calculating the activity of hydrogen ions (acidity or alkalinity) in the solution.
• A pH meter comprises necessarily of a voltmeter connected to a pH-responsive electrode and a reference (unvarying) electrode.
• The pH-responsive electrode is normally glass, and a mercury-mercurous chloride (calomel) electrode is usually the reference, although sometimes a silver-silver chloride electrode is used.
• The two electrodes act like a battery when they are submerged in a solution.
• The electrical potential (charge) of the glass electrode is directly related to the hydrogen ion activity in the solution (59.2 millivolts per pH unit at 25^o C [77 °F]) and the potential difference between the glass and reference electrodes is determined by the voltmeter.

3. Confocal microscope:

• Confocal microscopy is a specialized type of standard fluorescence microscopy (also termed widefield fluorescence microscopy) that produces high-resolution images of material stained with fluorescent probes using specific optical components.

4. Flow cytometer:

• Flow cytometry is a method for cell analysis that was first used in the 1950s to determine the volume of cells in a fluid stream that circulated quickly as they flowed in front of a viewing aperture.
• Fluidics, optics and electronics are the three principal components of a flow cytometer.
• The transport of the sample from the sample tube to the flow cell is the function of the fluidic system of the flow cytometer.
• The sample is either sorted (in the case of cell sorters) or transported to waste after it has been through the flow cell (and past the laser).
• The optical system components include excitation light source, lenses, and filters used to capture and move light around the instrument and the photocurrent-generating detection system.
• The brains of the flow cytometer are the electronics.
• Here the photocurrent from the detector is digitized and analyzed to be saved for subsequent analysis.

5. Cell culture vessels:

• In order to shield cultures from the external environment while retaining the correct internal environment, culture vessels provide a contamination barrier.
• The vessels have an effective and consistent cell attachment substrate for anchorage-dependent cells.
• Simple access to cultures and optically transparent viewing surfaces are more features of vessels.
• All cultural vessels were originally glass.
• In comparison to plastic, glass drawbacks include heavy weight, cost, labor-intensive cleaning, and poor microscopic viewing.
• Surface treatment techniques were developed for polystyrene in the 1960s, enabling plastic vessels to replace glass for most applications of cell culture.
• a. Glass flasks:
  • In the 1920s, the first glass flasks were developed by Alexis Carrel.
  • The more conventional straight neck rectangular (also hexagonal) glass T-flasks were created by Harry Earle in the 1940s.
  • Today, with a number of growing areas, a variety of shapes, with several different neck designs, plastic flasks are available.

b. Cell culture dishes:

• The best economy and access to the surface of growth are provided by cell culture dishes.
• Hence, they are regarded as the vessels of choice for cloning or other manipulations such as scraping that need the direct access to cell monolayer.
• They should be used with incubators that regulate CO2 and humidity.
• The majority of manufacturers provide dishes in four sizes: 35 mm, 60 mm, 100 mm and 150 mm.
• These are nominal diameters and may not be the growth surface’s true diameter.
• Cell culture dishes are accessible for growing anchorage-dependent cells with either specially treated surfaces, or untreated (native) surfaces for growing suspension cultures where attachment is not required.

c. Roller bottles:

• To cultivate large numbers of anchorage-dependent cells, the roller bottle was created.
• Today, they suppy a more cost-effective means of growing large volumes of cells using basically the same culture techniques as with flasks, but with substantially less labor.
• In addition to the conventional smooth wall design, roller bottles are available with small ridges that roughly double the available surface area for developing cells without raising the bottle dimensions.

d. Pipettes and pipettors:

• The serological pipette is a relatively common laboratory instrument used for transferring milliliter volumes of liquid.
• In order to calculate the volume of liquid being aspirated or dispensed, serological pipettes usually have gradations along their sides.
• These instruments are most commonly used with a pipette dispenser, which enhances the liquid transfer through the development of a partial vacuum.
• Depending on the amount of volume you want to pass, the same pipette dispenser can be used with a number of serological pipette sizes.
• Serological pipettes are usually sterilizable and reusable, either plastic, sterile, and disposable or glass.
• For the transfer of fluids, all serological pipettes involve the use of a pipette dispenser.
• This primitive liquid transfer method is not recommended, as it may lead to liquid entering the oral cavity and some severe adverse side effects may be caused.
• One type of dispenser, the pipette bulb supplies the least amount of accuracy and is usually used with glass serological pipettes in order to transfer non-specific volumes of liquid.
• The pipette pump is often used for glass pipettes and enables the liquid volume to be controlled more precisely.
• For dispensing repeated volumes of solution, pipette pumps are especially useful.
• The most popular pipette dispenser type is the pipet-aid.
• It consists of several primary components: the nose cone is where the pipette is attached and where the filter is also located, which protects the inside of the pipet-aid from fluid and retains sterility.
• Two triggers can be found on the pipet-aid handle; the top trigger for aspirating liquids is depressed, the bottom for dispensing.
• Pipet-aids are also fitted with settings that monitor the speed at which fluid is dispensed.
• For example, the instrument can be fixed to dispense liquid using pressurized air, in a blow-out setting, and with no force, in a gravity setting.
• Although there are cords in some pipet-aids, most are battery operated.
• Some pipet-aids have a stand attached to the handle that allows the pipet-aid to rest on its side without removing the pipette.

The post Equipment and materials used in animal cell culture appeared first on Online Biology Notes.

• Cell culture is the technique where cells are allowed to grow under controlled conditions, usually outside of their natural environment.
• Likewise, animal cell culture is a technique in which the cells are removed and are allowed to grow in a favorable artificial environment.
• The removal of tissue can either take place from tissue directly or from disaggregation by enzymatic or mechanical means before culture, or they may be originated from a cell line or cell strain that has been established earlier.
• Even if the basic mammalian/animal cell culture techniques are similar to those that are applied to bacteria, fungi, and yeast, there are however some characteristic differences.
• Usually, mammalian cells are found to be more delicate and more susceptible to mechanical damage.
• They have lesser growth rates and need more complex culture media along with special substrates.

Steps to culture animal cells:

• Harvest cells
• Isolation of the cells with the use of appropriate enzymes.
• In a culture dish with appropriate growth media, the cells are placed.
• The culture dish is now kept in incubator for the culture of cells.
• Cells can be sub-cultured in order to fix the problem or to get the pure culture.
• Now, cells are ready to be manipulated or modified for lab procedures.

Media Composition for animal cell culture:

• The basic components for the animal cell culture are enlisted as bellows:
• Sources of Energy: Glucose, Fructose, Amino acids
• Nitrogen sources: Amino acids
• Vitamins: Usually, water soluble vitamins B & C.
• Inorganic salts: Na+, K+, Ca²⁺, Mg²⁺
• Fat and Fat soluble components: Fatty acids, cholesterols
• Antibiotics
• Growth factors and hormones.
• Oxygen and CO₂ concentration.
• Physical environment: The physical environment consists of the optimum pH, temperature, osmolality and gaseous environment, supporting surface and protecting the cells from chemical, physical, and mechanical stresses.
• CO₂ incubators are employed and designed to resemble the environmental conditions of the living cells.
• For visualizing cell cultures in vitro, an inverted microscope is used.
• Low speed centrifuges are needed for most animal cell cultures.
• Cryopreservation is the storage of cells using liquid nitrogen at a very low temperature (-180 °C to -196 °C).
• DMSO is a cryo-preservative molecule that prevents cells from being harmed.
• For the culture of animal cells, serum is necessary and contains growth factors that promote cell proliferation.
• Even if animal cell culture media differ in their complexity, most include:
  • Amino acids:   0.1-0.2 mM
  • Vitamins:  1 microM
  •  Salts (NaCl) :  150 mM
  • KCl : 4-6 mM 
  • CaCl₂ :  1 mM
  • Glucose : 5-10 mM

Animal Culture media:

• In animal tissue culture, 2 types of culture media are used:
  • Natural media
  • Artificial media
• The type of medium relies basically on the type of cells to be cultured and its objectives.

1. Natural media:

• These media include the naturally occurring biological fluids and are of the following three types:
  • Clots
  • Biological fluids
  • Tissue extract

i) Clots:

• Plasma clots are the most commonly used clots and has been employed for a long time.
• In the present time, plasma is commercially found in liquid state that can be prepared in the laboratory.

ii) Biological Fluids:

• Several biological fluids can be employed as culture media such as amniotic fluid, pleural and ascetic fluids, hemolymph of insects, aqueous humoral from eye, serum etc.
• Among them, serum is the mostly preferred.

iii) Tissue Extracts:

• The most commonly used tissue extract is chick embryo extract, however, bovine embryo extract is also used.
• In the culture media, the extracts from spleen, liver, bone marrow and leucocytes were also used.
• The substitution for tissue extract can be a mixture of amino acids and certain other organic compounds.

2. Artificial media:

• For the following purposes, various artificial media have been employed:
• Immediate survival (a balanced salt solution with specified pH and adequate osmotic pressure)
• Prolonged survival (a balanced salt solution in addition with serum, or appropriate formulation of organic compounds.
• Indefinite growth
• Specialized functions
• Artificial media may be classified  into following types:
  • Serum containing media
  • Serum-free media
  • Protein free media
  • Chemically defined media

• Serum:
  • Serum is the yellowish liquid and is a transparent content that remains left over after the removal of fibrin and cells from the blood.
  • 2-10% of serum is often contained by normal growth media.
  • The most commonly employed supplement in animal cell culture is fetal bovine serum (FBS).
  • It supplies the basic nutrients for cells.
  • It also contains several hormones and various growth factor.
  • In addition to it, it also acts as a buffer.
• Serum containing media:
  • In animal cell culture media, fetal bovine serum is the most common supplement.
  • In order to supply an optimal culture medium, it is employed as an economical supplement.
  • Serum supplies carriers water-insoluble nutrients, protease inhibitors, hormones and growth factors and binds and neutralizes the toxic moieties.
• Serum free media:
  • In case of immunological studies, presence of serum in media can result to serious misinterpretations.
  • In general, these media are specifically designed to promote the culture of a single type of cell and incorporate specified amounts of purified growth factors, lipoproteins and other proteins normally supplied by the serum.
  • As the components of these media are known, thus it is referred to as ‘defined culture media’
• Chemically defined media:
  • These media contain ultra-pure inorganic and organic ingredients free of contaminants and may also contain pure protein additives, such as growth factors.
  • Their constituents are produced by genetic modification in bacteria or yeast with the addition of vitamins, cholesterol, particular amino acids, and fatty acids.
• Protein free media:
  • Protein-free media is devoid of any protein and only include non-protein constituents.
  • Usage of protein-free media enhances superior cell growth and protein expression in contrast to serum-supplemented media and enables downstream purification of any expressed product.

Development of animal culture media:

• Early efforts to culture cells, such as chick embryo extract, plasma, serum, and lymph, were conducted in natural media based on tissue extracts and body fluids.
• Demand for greater quantities of a medium of more reliable quality has led to the emergence of chemically defined media.
• However, Eagle’s Basal medium and subsequently Eagle’s Minimal Essential Medium (MEM) were broadly accepted, variously supplemented with human, horse or calf serum, protein hydrolysates, and embryo extract.
• In order to choose a medium, the minimum criteria required usually involve:
  • The medium should supply the cells with all of the nutrients.
  • Keep the physiological pH about 7.0 with ample buffering.
  •  The medium should be sterile, and isotonic to the cells.
  • The balanced salt solution that was initially used to establish a physiological pH and osmolarity needed to sustain cells in vitro was the basis for the cell culture media.
  • Various components (glucose, amino acids, vitamins, growth factors, antibiotics etc have been added to promote cell growth and proliferation, and several media have been created.

Physicochemical properties of culture media

• In order to promote good growth and proliferation of the cultured cells, the culture medium is required to possess certain physicochemical properties (pH, O2, CO2, buffering, osmolarity, viscosity, temperature etc.).
• pH:
  • Most cells can develop in the 7.0-7.4 pH range, but there are minor differences depending on the cell type.
  • Phenol red is usually used as an indicator.
  • It becomes orange at pH 7.0, yellow at pH 6.5, lemon yellow below pH 6.5, more pink at pH 7.6, and purple at pH 7.8. It is red at pH 7.4.
•  CO₂, bicarbonate and buffering:
  • The medium’s carbon dioxide is in a dissolved state, the concentration of which depends on the atmospheric CO₂ tension and temperature.
  • As seen below, CO2 in the medium occurs as carbonic acid (H2CO3), and as bicarbonate (HCO3-) and H+ ions.                                    

                             CO₂ + H₂O ↔ H₂CO₃ ↔ H⁺ + HCO₃^– 

• The CO2, HCO3-and pH concentrations are interrelated, as is obvious from the above equation.
• The pH would be decreased by increasing atmospheric CO2, rendering the medium acidic.
• Bicarbonate ions are neutralized by the addition of sodium bicarbonate (as a part of bicarbonate buffer).

                              NaHCO₃ ↔ Na+ + HCO₃^–

•  The recommended bicarbonate concentration and CO2 tension for the appropriate pH are found in commercially available media.
• In recent years HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer being used in the culture media as it is considered more efficient than the bicarbonate buffer.
• However, due to the low cost, less toxicity and nutritional value to the medium, bicarbonate buffer is favored.
• In contrast to it, HEPES is expensive and along with it, it is toxic to the cells.
• The existence of pyruvate in the medium results in the excessive endogenous production of CO2 by the cells.
• This is good since the reliance on the exogenous supply of CO₂ and HCO^–₃ would be lower.
• In such a scenario, high amino acid concentrations can be used for buffering.
• In summary, cultures in open vessels need to be incubated in the CO₂ atmosphere, the concentration of which is in equilibrium with that of sodium bicarbonate in the medium.
• Oxygen:
  • A large majority of in vivo cells rely on the availability of O₂ for aerobic respiration, which is made possible by hemoglobin’s continuous supply of O₂ to the tissues.
  • The cultured cells mainly depend on the dissolved O₂ in the medium that may be toxic at high concentration due to the production of free radicals.
  • It is also completely necessary to supply appropriate amounts of O₂ in order to comply with the cellular requirements, preventing toxic effects.
  • The introduction into the medium of free radical scavengers such as glutathione,2-mercaptoethanol (β-mercaptoethanol) or dithiothreitol erases the toxicity of O₂.
  • To decrease O₂ toxicity, the addition of selenium to the medium is often recommended.
  • It is because selenium is a cofactor for the synthesis of glutathione.
  • In general, as compared with in vivo cells, glycolysis occurring in cultured cells is more anaerobic.
  • As the rate of diffusion of O₂ is affected by the depth of the culture medium, it is recommended to keep the depth of the medium in the range of 2-5mm.
• Temperature:
  • In particular, the optimum temperature for a provided cell culture is reliant on the body temperature of the organism, acting as the source of the cells.
  • Therefore the optimum temperature for cells obtained from humans and warm blooded animals is 37 ° C.
  • Higher temperatures cannot be handled by in vitro cells and most of them die if the temperature goes beyond 40 ° C.
  • For reproducible outcomes, it is therefore completely important to maintain a steady temperature (± 0.5°C).
  • If the cells are collected from birds, the optimum temperature for culture is slightly higher (38.5°C).
  • The culture temperature can be about 15-25 ° C for cold blooded animals (poikilotherms) that do not control their body heat.
  • In addition to directly affecting cell growth, temperature also influences the solubility of CO2, i.e. higher temperatures increase solubility.
• Osmolality:
  • Most cultured cells have a reasonably wide osmotic pressure tolerance.
  • This is comparable to human plasma osmolality (290mOsm/kg).
  • Once an osmolality is appointed for a culture medium, it should be sustained at that level (with an allowance of ± 10 mOsm/kg).
  • Osmolality is typically determined by the depression of the medium’s freezing point or vapor pressure elevation.
  • The instrument osmometer is used in the laboratory for osmolality measurement.
  • Osmolality is influenced if acids, bases, medications, etc are applied to the medium.
  • If we make up the medium ourselves, the measurement of osmolality is a useful quality control step, as it helps to protect against errors in weighing, dilution, and the like.
  • Osmolality is usually measured by depression of the freezing point or elevation of the vapor pressure, of the medium.
• Viscosity:
  • A culture medium’s viscosity is largely affected by the serum content and will have little impact on cell growth in most cases.
  • When a cell suspension is agitated (e.g. when a suspension culture is stirred), or when cells are dissociated after trypsinization, or in low serum concentrations/absence, viscosity becomes especially important.
  • Increasing the viscosity of the medium with carboxymethylcellulose (CMC) or polyvinylpyrrolidone (PVP) will minimize any cell damage that occurs under these conditions.
•  Surface Tension and Foaming:
  • The consequences of foaming have not been clearly identified, but the rate of denaturation of protein can increase, as might the risk of contamination if the foam enters the culture vessel’s neck.
  • If a film from a foam or spill gets into the capillary space between the lid and the base of a Petri dish or between a slack cap and the neck of a flask, foaming can also restrict gaseous diffusion.
  • In suspension cultures in stirrer vessels or bioreactors, foaming can occur when 5 percent of CO2 in air is bubbled via serum containing medium.
  • The addition of 0.01 percent to 0.1 percent silicone antifoam (Dow Chemical) or Pluronic F68 (Sigma) helps avoid foaming in this situation by minimizing surface tension and can also protect cells from bubble shear stress.

The post Animal cell culture appeared first on Online Biology Notes.

• Epigenetics is a process involving changes in the function and expression of the genes.
• The basis for epigenetics is changes in chromosomal structures and not the alterations in DNA sequence of the chromosomes.
• Hence, epigenetics refers to any heritable phenotypic changes.
• Over all, these are not due to genome sequence changes, such as mutations, etc.
• These changes are affected and controlled by a variety of external influences, such as environmental stresses or internal ones, such as the cell’s own innate developmental program.
• It tends to occur in all Eukaryotes.
• The study of the epigenetics of the cell helps to clarify how newly developed cells of an organism that possess identical genome or DNA sequence goes through differentiation to give various cell types and maintain a particular physiology and morphology.
• For instance, it clarifies why and how leaf epidermis cells and root cells, vary so much even if they possess same DNA sequence in their genome.
• Thus, epigenetics serves as a bridge between the phenotype and genotype of a cell.
•  Like all other eukaryotes plants also rely on the action of epigenetic regulatory mechanisms for their ability to function.
• The study of epigenetics and its benefits may allow breeders to not only recognize the nature of plants they are breeding but also to develop strategies that would inculcate desirable characteristics in their plants such as; exhibit resistance towards environmental stresses or to adapt to some particular conditions.

Modes of epigenetic variations in plants:

• There are set of mechanisms and factors responsible for regulating the process of epigenetics and are listed below.
• They affect the functioning of the genome but not of the DNA sequence.
• When these regulatory changes take place, they are often inherited from generation to generation.
  • DNA methylation
  • Histone modifications
  • RNA interference

1. DNA methylation in plants:

• DNA methylation is one of the covalent modifications found in DNA.
• In methylation, the incorporation of methyl (-CH3) groups takes place at 5C’ position of cytosine dNTP ring that yields 5 methyl-cytosine.
• The addition of methyl groups is performed by two enzymes namely methyltransferases and demethylases.
• Three types of methylations are found in plants on the basis of nature of the plant i.e. CG, CHH, CHG.
• The most common methylation is CG methylation whereas CHG methylation is the least frequent.
• The requirement of methylation of transposons sequences by plants is necessary as it might result in lethal phenotypes when not limited by methylation.
• Their exons also manifest higher level of methylation, however, promoter regions of genes exhibit less methylation and hence they are easily acted upon by transcription factors.
• This process also ensures the presence of a methylated sequence of CpG genes, which is responsible for maintaining chromosomal stability by controlling chromatin content condensation and also maintaining gene silencing patterns.
• This mechanism and that of histone modification together decide which portion of chromatin is to be condensed and rendered inaccessible as a heterochromatin segment of chromosome.

2. Histone modifications in plants:

• In DNA packaging, histone proteins play a very significant role.
• In order to obtain condensed chromosomal configuration, the DNA segments wound around these octamer proteins to yield nucleosomes.
• As histones can stabilize the negative charge of DNA molecule, they are essential for DNA binding.
• The His-DNA interactions are affected by the post translational modifications like phosphorylation, acetylation, biotinylation, ubiquitation and methylation of specific amino acids at histone tails and these are responsible for determining the extent of condensation at that region.
•  Hence, the chromatin state differs along with a particular modification and with the aid of certain particular internal or external signaling factors, it goes through transcriptional activity.
• For instance, upon the methylation of histone H3K4, transcription factors are expressed whereas methylation of H3K9 suppresses it.

3. RNA interference in plants:

• In a number of processes, such as transgene silencing, post-transcriptional processing of mRNAs, combating viral invasions, development of unexpressive heterochromatin, silencing of transposable components, etc., small interference RNAs (siRNA) play a crucial role.
• Research carried out on the Arabidopsis plant shows how these siRNAs serve as mobile markers that cause epigenetic variations.
• By inducing epigenetic variations, they not only affect the normal plant development system but also aid those plants to adapt to invitro conditions and the changing environments.
• Several transcriptional factors, repressor proteins, mRNAs and microRNAs have also been found to function and assist in the display of these epigenetic processes and cause phenotypic variation to occur in the plant.
• Together, they all allow greater polymorphism to occur, which generally brings new phenotypes.

Role of epigenetics in plants:

• In order to function, plants depend on epigenetics.
• This was first observed by Gassner, who found that it was important to provide the plant with a cold phase for its growth.
• After this, some others found that photo-periodism is also very important for the growth of plants.
• They also illustrated how some epigenetic modifications trigger flowering during the vernalization process.
• Biotechnology progress has helped scientists to illustrate the underlying epigenetic mechanisms that govern flowering, germination, fruit ripening, vernalization of fruiting and photoperiodism etc, as well as to research the configurational changes that genome undergoes during cell differentiation.
• By conducting epigenetic research on all aspects of plant development, such as identification of complex characteristics of novel crops through DNA methylation, processes controlling plant flower timing, plant resistance to certain microbes, their activity of virus resistance through gene silencing, etc., gaining an understanding of all this, applicable to a particular type of plant, will help us optimize genomic activity of this crop in a field or cultured in vitro.
• Researchers have also been assisted in the study of the processes of natural selection and other adaptive responses of plants to their environment by assessing the heritable epigenetic marks of a plant.

Environment induced epigenetics:

• Physiological changes in plants are also brought about by the external factors such as the stresses of the environment and its conditions and permit them to respond to these conditions.
• In response to external stimuli, plants exhibit modification in their chromatin material.
• For example, in response to several environmental stress, DNA methylation takes place.
• In order to introduce stable epigenetic modifications, this stress stimuli must be perpetuated by cell divisions.
• The alterations are brought by abiotic stresses such as draught, UV radiation, high saline concentrations, excessive heat.
• The alterations include silencing of reporter transgenes and endogenous loci in specific plant types.
• However, these alterations are short-lived in their expression.
• The previously suppressed silencing process are restored by the return of optimal conditions.
• The other examples are listed as follows:
  • In tobacco plant, the exposure to stresses of cold, and aluminum salts results in DNA methylation.
  • The hypomethylation occurs in Cannabis sativa upon exposure to heavy metal stress.
  • To the rice varieties that ae sensitive to draught, the hyper and hypo-methylation of DNA takes place.

Application of Epigenetics in plant biotechnology:

1. Somaclonal variations:
   • For researchers who want to implement soma clonal variations through in vitro plant culture, research on epigenetic modifications has proved to be of high importance.
   • Through the somatic embryo-genic plant tissue culture technique, researchers have identified soma-clonal variations as the cells attempt to adapt to their in vitro conditions.
   • Such differences arise due to genome rearrangement and not due to any changes in the sequence of DNA.
2. Chromosomal mutations with genotypic variations:
   • It also contributes phenotypic variants or chromosomal mutations in plants with beneficial characteristics.
   • These genotypic variations also lead to aneuploidy and polyploidy, which is used by plant biotechnologists to achieve high quality yields from plants such as cotton and onion.
   • In addition, for inbreeding, vegetative and seed propagation, such variants are useful. Especially gene methylation occurs in these variants.
3. Transgenic plants:
   • Epigenetics is beneficial for in vivo propagated plants.
   • In addition to generating and controlling transgenic plants with desirable characteristics, the implementation of epigenetic modifications can also produce and control natural processes such as flowering, fruiting, germination, dormancy, etc.
   • One such effective way of provoking temporary inactivation or activation of certain genes without the induction of any genetic mutation is chromosome methylation.
   • Over time scientists have been successful in producing transgenic crops that can withstand stress conditions through these alterations.
   • They produce sustainable crops on a large scale and by decreasing greenhouse effects, contribute to environmental conservation.

Noble plant phenotypes produced by Epigenetics:

Following noble phenotypes have been produced through epigenetics:

1. Enhancement in nutritional value:
   • Improvement in nutritional value; Scientists have been able to express certain genes during specific crop development using RNA interference technology.
   • Up until now, this technique has been used to reduce gossypol lines in cotton plants.
   • Furthermore, it has been used to reduce the content of caffeine in coffee plants, to increase the development of amylase in wheat crops and to improve the quality of tomatoes.
2. Dosage compensation:
   • Research into epigenetics allowed scientists to understand the role of the dosage of the parental plant genome in the development of endosperm or seeds.
3. Tolerance against stress conditions: 
   • Under environmental stress, modifications can down regulate certain genes. For example, salinity, intense cold, heat and drought etc.
   • For example, inactivation of genes through epigenetics provide certain resistance abilities to rice crops incourse of their reproductive and vegetative stages.
4. Virus resistance improvement: 
   • Using RNA interference, Researchers have developed  BBrMV ( Banana Bract Mosaic virus) and resistant banana varieties

What are the disadvantages and limitations of epigenetics mechanisms?

• The following are the drawbacks of epigenetic processes during in vitro callus tissue cultivation:
  • They can provide variants that are genetically unstable.
  • Extensive trials are required for the application of such variant crops
  • Unwanted characteristics can be expressed phenotypically
  • It can produce a dangerous genetic mutation.
  • In addition, transgenic crops produced using epigenetics may contain protein content that is modified.
  • Some are phenotypically unobvious and in some cases, may cause an allergic reaction and spread this allergic epidemic.
  • Phenotypic as well as genotypic changes arise from epigenetic variations.
  • Despite being heritable, when transmitted over many generations, there is still disagreement with the Mendalian epigenetic model. It is however clear that epigenetics regulate the growth of a plant rather than inherited characteristics.

What are the future aspects of epigenetics in plants?

• The further study of epigenetics and its understanding can be implemented for wide areas. Such as:
  • Plant generation in a new habitat.
  • Crop manipulation for generating biofuels
  • Reducing the development time of crops
  • Induction of plants, regardless of seasonal changes, to produce fruits or flowers throughout the year.
  • These can generate heterosis and cross-breeding that provides better quality for plants than their parent plants.
  • Magnify stress adaptation in them.
  • These may produce heterosis and cross breeding that give plants with quality better than that of their parent plants.
• It can appear to be an efficient way to eradicate hunger and food crop production with extra nutritional values in nations facing severe environmental stresses such as Ethopia, Ghana, etc.
• It therefore intends to bring cost-effective crops to combat global food demand with characteristics that can guarantee a healthy environment in less time.
• In the foreseeable future, epigenetics can even be used to modify weed genomes in such a way as to either obstruct their growth among desirable plantlets or incorporate characteristics to render them beneficial to humanity.

The post Epigenetics and its roles in plants biotechnology appeared first on Online Biology Notes.

• Plant hormones are also termed as phytohormones (named by Thieman), growth factors, growth regulators, growth substances etc.
• Phytohormone is an organic substance, naturally produced in higher plants that regulate plant physiological process such as affecting growth and other functions remote from its place of production and active in very minute amounts.
• They can be either natural or synthetic, stimulatory or inhibitory in nature.
• They act at a distance from the place where they are formed.
• Three types of phytohormones are mostly recognized. They are:
  • Auxin
  • Gibberellin
  • Cytokinin

1. Auxin:

• An auxin is an organic compound responsible for promoting the growth of plants along the longitudinal axis when applied in low concentrations to shoots of the plants.
• Auxin is specifically concerned with cell enlargement or the growth of the shoots.
• Auxin is identical to Indole 3-Acetic Acid (C₁₀H₉O₂N, IAA), i.e. natural true auxin.
• The precursor of Indole 3-Acetic Acid is tryptophan and zinc play a role in its biosynthesis.
• Auxin exhibits polar movement i.e.
• Basipetal movement (from apex to base) in case of shoots.
• Acropetal movement (from root tip to shoot) in case of roots.
• Bioassay test: Bioassay is termed as the functional test of substance in living plants.
• The common bioassay tests of auxin are Avena coleoptile test and root growth inhibition test.

What are the physiological roles of auxin in plants?

• Besides the cell enlargement and growth, auxin (both natural and synthetic) are responsible for various other growth processes. They are:
• Cell elongation:
  • The cell elongation occurs only in the presence of auxin and the rate of elongation is directly proportional to the amount of auxin supplied, given no other factors are limiting.
  • However, relatively high concentrations of auxin show inhibitory effects on this phase of growth.
  • Auxin promotes the elongation of roots at its low concentrations, the growth of roots is retarded at higher concentrations.
  • Flowers need higher concentration of auxin for their growth.
  • Auxin also induces the elongation of coleoptiles and stems by cell enlargement.
  • Auxins are responsible for the elongation of petiole, mid rib and major lateral veins of the leaves.
  •  Hence, adenine aids in enlargement in detached leaves of radish and pea. Similarly, coumarin has been shown to promote expansion of leaves in some plants.
• Cambial activity:
  • During the spring season, the trees manifest growth by developing buds that later on open and elongation of young stems take place.
  • Auxin activates this resumed growth by cambial cells
  • The growth moves basipetally in the stems from developing buds.
• Callus formation and galls:
  • The auxins activate cell division.
  • When 1% IAA in lanolin paste is applied to a de-bladed petiole of a bean plant, prolific division of parenchyma cells occurs.
  • A swelling or callus tissue is formed at the point of application of auxin.
  • The amount of callus tissue formed is directly proportional to the concentration of IAA applied.
• Apical dominance:
  • Apical dominance is the major function of auxin.
  • The growth of lateral buds is suppressed until apical bud is present in the plants.
  • This inhibitory effect of terminal bud upon the growth of lateral buds is termed as apical dominance.
  • Skoog and Thimann (1934) first reported the relation of apical dominance
    with the auxin supply.
  • When agar block containing auxin b or IAA was kept on the decapitated shoot of broad bean (Vicia faba), the lateral buds, as might be expected, resulted in the usual suppression of growth.
  • But when the same decapitated shoot was re-headed with an agar block containing no auxin, these lateral buds resumed growth.
  • When NAA was used as auxin in field-grown tobacco plants, similar results were obtained.
  • Evidence of apical dominance has been practically used in solving the potato storage problem.
  • Potatoes, stored for some time, sprout and become sweet in taste, causing the grower to lose financially as its consumers hate the sweet taste.
  • But by inhibiting the growth of buds or ‘eyes’ by spraying potatoes with auxins such as indole butyric acid ( IBA) and NAA, sprouting (or in other words, prolonging dormancy) can stop sprouting; the effect lasts for as long as 3 years.
• Rooting of stem cuttings (Formation of adventitious roots):
  • It is a common observation that when the lower end is dipped in an acceptable rooting medium, the appearance of buds on a cutting promotes the growth of roots.
  • In accelerating root formation, developing buds are efficient.
  • The initiation of roots on the cuttings are often favoured by young leaves.
  • These findings contributed to the idea that the auxins synthesized in the buds and young leaves favour the root formation and are later translocated to the basal part of the cut.
  • IAA, NAA, 2,4-D, naphthalene acetamide (NAd) etc are the auxins most widely used for this function.
  • Auxin-induced rooting is also of considerable horticultural benefit as it allows cuttings to propagate those plants.
• Delay (or inhibition) of abscission of leaves:
  • By adding auxins on  the surface of  the lamina or on the cut surface of a debladed petiole, abscission of  the leaves may  be delayed or hindered.
  • Laibach (1933), who demonstrated that the extract of orchid pollinia is capable of preventing leaf dropping, first noted the regulating actions of auxins on abscission.
  • Since then, sufficient work in this direction has been carried out.
  • The delaying effect of IAA on the abscission of different plant organs has been shown conclusively by Addicott and Lynch (1955).
  • As for the abscission process, it has been proposed that the basipetal migration of a hormone from the blade to the base of the petiole retards the leaf drop.
  • Leaf blade removal removes the hormone supply to the abscission zone and thus causes the drop of the leaf.
• Flowering:
  • Auxins play role in modifying flowering by following ways:
  • Producing early flowering
  • Inducing flowering
  • Preventing or delaying flowering
• Fruiting:
  • Auxins play significant role in fruiting by altering it in one of the following ways :
  • Fruit setting:
  • The changes in the ovary leading to the development of the fruit is termed as fruit set.
  • These changes are generally induced after pollination and fertilization.
  • The development of fruit without fertilization is termed as parthenocarpy.
  • It is a common characteristics in plants and hence occurs frequently.
  • The parthenocarpy can be induced artificially by the aid of auxin.
  • For example, Yasuda (1934) demonstrated it by application of pollen extracts to cucumber flowers.
  • It was also observed that ovaries of many plants (orange, lemon, grape, banana, tomato etc.) could be induced to develop into seedless fruits by application of IAA in lanolin paste to their stigmas.
  • The various other auxins used for parthenocarpy are IPA, IBA, α-NAA, phenoxyacetic acid (POA), α- naphthoxyacetic acid (NOA) etc.
  •  Fruit thinning:
  • The trees, in many cases, bear a large number of fruits.
  • It leads to the inability of the trees to grow an average number of new flower buds.
  • Therefore, such trees must grow fruit either in alternate years (alternate bearing) or if yearly, the number of fruits is significantly reduced.
  • Clearly, these trees need thinning.
  • For the first time, fruit thinning was achieved in apples when naphthalene acetic acid added to flowers failed to set the fruits and actually caused a decrease in the set of fruits.
  • It is interesting to note that the only effective auxin that induces fruit thinning seems to be naphthalene acetic acid.
  • However, a-2,4,5-trichlorophenoxyacetic acid for thinning of pears and p-chlorophenoxyacetic acid for thinning of grapes are other examples of auxins used for fruit thinning.
  • Control of premature fruit dropping:
  • The development of an abscission sheet causes the falling of unripe fruits in many fruit trees causes significant losses in yield to the gardeners.
  • In several cases, such as apples, the problem has now been successfully overcome by the application of auxins.
  •  Auxins prevent the formation of the abscission layer and thus check the drop of the fruits before harvesting.
  • With 2,4-D and 2,4,5-trichlorophenoxyacetic acid as auxins, regulation has also been induced in citrus fruits (like oranges and lemons).
  • Improving the quality of fruits:
  • The different processes such as colouring, softening, sweetening and ripening are all involved in improving the fruit ‘s quality.
  • In apples, where the use of 2,4, 5-trichlorophenoxyacetic acid has significantly increased red pigments, the auxin effects on fruit colouration are most noticeable.
  • 2,4-D accelerated the ripening process when added to bananas as the auxin stimulates the conversion of starch into sugars.
  • Sugar accumulation has been reported in sugarcane by injecting 2,4-D, IBA or maleic hydrazide.
• Increase in respiration:
  • Auxins enhances the respiration process. It was first identified by James Bonner in 1953.
  • A direct relation between growth due to auxin treatment and rate of respiration has been found i.e., greater the growth, higher is the respiration.
  • Auxins are used to control the growth of weeds in the crop fields.
  • 2,4-D is sprayed for the weeds in the crop fields that acts as weed killer.
  • Graminaceous weeds are destroyed by 2,4-dichloropropionic acid.
• Increased resistance to frost damage:
  • When parsnip is treated by 2,4,5-T, the tops resist damage by frost.
  • In apricot fruits, the application of 2,4,5-T before the onset of frost caused less damage than the untreated fruits.
• Great weapon of war:
  • When auxins are applied in higher concentrations on enemy crop fields by means of air, it causes devastation of land and form the basis for biological warfare.

2. Gibberellins:

• E. Kurosawa, first discovered gibberellin from a fungus called Gibberella fujikoroi in the year 1926.
• A gibberellin is abbreviated as GA, for gibberellic acid.
• Gibberellin may be referred as a compound which is active in gibberellin bioassays and possesses a gibbane ring skeleton.
• However, there are other compounds (like kaurene) that are active in some of the assays but lack a gibbane ring. Such compounds have been termed gibberellin-like rather than gibberellins.
• Brian isolated pure sample of a single gibberellin and termed as gibberellic acid.
• The structure for gibberellic acid was given by Cross et al in 1961.
• More than 100 types of gibberellin are known, among them GA₃ is most common.
• Gas are common in all groups of plants, however it acts as a flowering hormone in angiosperms only.
• All gibberellin possess gibbane ring. Gibbane ring consists of 4 isoprene units (hence, 2 terpenes, di-terpenes).
• The 5 carbon compound isopentenyl pyrophosphate is the precursor of gibberellin.
• Bioassay test: Gibberellins are synthesized via the mevalonic acid (MVA) pathway.
• The biosynthesis of GA3 from MVA takes place by 18 or more steps or intermediates and about 15 associated compounds.

What are major Physiological effects of Gibberellin in plants?

• Genetic dwarfism:
  • In some plants, the mutation of a single gene causes dwarfism.
  • Such individuals are termed as ‘single gene dwarfs’.
  • In these plants dwarfism is due to shortening of internodes rather than reduction in number of internodes.
  • The use of gibberellins on such dwarfs causes them to elongate to the point of being indistinguishable from common tall plants.
  • Hence, gibberellin A3 treatment  has been used to overcome genetic dwarfism successfully in many single gene dwarf mutants like Pisum sativum, Vicia faba and Phaseolus multiflorus.
  • Gibberellin also induces leaf expansion.
• Bolting and flowering:
  • Rosette plants are marked by the prolific growth of leaves and the delayed growth of internodes.
  • But there is striking elongation in the internode before the reproductive process, so that the plant reaches 5 to 6 times the initial height.
  • The treatment of these ‘rosette’ plants with gibberellins stimulates bolting (or shoot elongation) and flowering under conditions that would normally preserve the rosette shape.
  • It is also possible to distinguish shoot elongation from flowering by controlling the amount of gibberellin applied.
  • The plant can bolt but not flower with low gibberellin dosages.
  • GA₃ hastens the flowering and flower yield in many plants such as Coriandrum sativum (coriander).
  • Gibberellin controls flowering in long day plants.
• Light inhibited stem growth:
  • The dark-grown plants showed better stem growth in comparison to light grown plants.
  • This inhibitory effect of light on stem elongation could be reversed by the use of gibberellins in plants as such Pisum sativum.
  • This clearly indicates that the gibberellin is the limiting factor in stem elongation.
• Parthenocarpy:
  • Gibberellins induce parthenocarpy more efficiently than auxins.
  • It has been found in plants such as Cucumis sativa (cucumber), Zepyranthes sp., Solanum melongena (brinjal).
• Breaking dormancy of seeds:
  • In the light sensitive seeds (lettuce, tobacco), the germination is retarded in dark.
  • The application of GA₃ allows the germination of seeds in dark as well.
• Breaking dormancy of buds:
  • Because of very low temperature, the buds produced in winter stays dormant till the next spring in temperate areas.
  • Gibberellin treatment overcomes the dormancy in such cases and replaces the light requirement for breaking dormancy.
  • It breaks dormancy in potato tubers as well.
• Role in abscission:
  • The abscission has been enhanced in explants of bean and Coleus by the GA₃ treatments.
• Stimulation of enzyme activity in cereal endosperm:
  • It was demonstrated that the exogenous application of gibberellins stimulated amylase activity in isolated barley endosperm.
  • It was also found that the treatment of isolated aleurone layer of endosperm with GA could release enzymes, amylase and proteinase.
• Sex expression:
  • Gibberellins show the capability to alter the sex of the flowers.
  • It promotes the production of male flowers in cucurbitis, Cannabis etc.
  • Also, the antheridia have been induced to form in many fern gametophyte
•  Juvenility:
  • Most of the plants manifests two different stages of growth i.e. a juvenile stage and an adult stage.
  • The application of gibberellin helps to determine if a specific part of plant is juvenile or not.

3. Cytokinins:

• Cytokinins are alos named as kinetins because of their absolute power to enhance cell division in the presence of an auxin.
• First naturally occurring cytokinin was recognized from young maize grain by Letham and termed as zeatin.
• Fox (1969) has defined cytokinins as chemicals composed of one hydrophilic adenine group of high specificity and one lipophilic group without specificity.
• Chemically, kinetin (C10H9ON5) is 6-furfurylaminopurine.
• Cytokinins occur in higher plants, diatoms, red and brown algae, mosses.
• These occur widely in embryo sac, roots during seedling stage, flowers, developing fruits, cambial tissue and endosperm.
• The richest source of kinins are fruits and endosperm.
• Bioassay test: Callus pith cell division, chlorophyll retention test, soybean and radish cotyledon cell division are the main bioassay tests.

What are Physiological roles of cytokinin in plants?:

• Cell division:
  • In addition to auxins, the kinins are required in right ratio of concentrations for the enormous growth response .
  • When mixture of auxin and cytokinin is added to unspecialized  cells, their differentiation begins.
  •  A high cytokinin to auxin ratio results the formation of shoots, buds and leaves while a low cytokinin to auxin ratio causes root formation.
  • This invitro culture methods allows the rapid production of large number of plants in a small space.
• Cell elongation:
  • Kinetin also enhances cell elongation.
  • It has been demonstrated in tobacco pith cultures, tobacco roots and bean leaf tissues.
• Root growth:
  • Kinetin is responsible for both the stimulation as well as inhibition of root development.
  • When kinetin was applied along with IAA, the root initiation and development in stem callus cultures was stimulated.
  • In lupin seedlings, Kinetins induced increase in dry weight and elongation of the roots.
• Shoot growth:
  • When the balance of IAA and kinetin is maintained, the callus tissue of tobacco can be kept in an undifferentiated state for a long time.
  • When the amount of kinetin is increased, the development of leafy shoots begins.
• Organogenesis:
  • Organogenesis is resulted by cytokinins in several tissue cultures.
  • By changing the relative concentrations of kinetins and auxins, the tobacco pith callus can be directed to develop either buds or roots.
  • High kinetin and low auxin contents causes the production of buds.
  • The roots appear in pith in reverse condition, i.e. high auxin and low kinetin contents.
  • In leaf segments of various plants such as Saintpaulia ionantha, Bryophyllum sp and Begonia sp., the kinins stimulate the production of buds.
  • In addition to the root and shoot differentiation, the cytokinins also bring about other morphogenetic responses.
  • These are :
    (a) maturation of proplastids into plastids
    (b) differentiation of tracheids
    (c) induction of parthenocarpy
    (d) induction of flowering
• Counteraction of apical dominance:
  • Cytokinins are powerful promoters of lateral bud growth.
  • When the culture medium consists of IAA, the growth of lateral buds is inhibited, but the addition of kinetin along with IAA stimulates the growth of lateral buds.
• Breaking dormancy of seeds:
  • Cytokinins show effective role in breaking seed dormancy in lettuce, tobacco, white clover and carpet grass.
  • In such cases, the site of cytokinin action is cotyledon.
  • The seeds of parasites such as Striga asiatica need the host plant for germination. But when treated with kinetin, the seeds germinate even in the absence of their host.
• Delay of senescence (Richmond-Lang effect):
  • The ageing of leaves along with the loss of chlorophyll and the breakdown of proteins is termed as senescence.
  • Richmond and Lang demonstrated that the senescence in the detached leaves of Xanthium could be postponed for several days by kinetin treatment.
  • This effect of kinetin in retarding senescence is termed as Richmond-Lang effect.
• Role in abscission:
  • Depending on the site of application, cytokinins can either accelerate or retard the process of abscission in leaf petioles.
  • It is the common property of cytokinin.
•  Effects on cotyledons:
  • Cytokinins enhances cellular division and expansion in cotyledons.
  • Cytokinins increase the concentration of sugars in cells resulting in endosmosis that causes the expansion of cytokinin treated cells in cotyledons.

Phytohormones: Types and physiological effects in plant growth and development

The post Phytohormones: Types and physiological effects in plant growth and development appeared first on Online Biology Notes.

• It is a distilled alcoholic beverage obtained from fermentation of grains followed by distillation and ageing in white oak barrel for 3-4 years. 
• Primarily, barley is used but other grains such as corn, wheat, oat & rye are also used.
• Whisky may be malt or non-malt.
• The concentration of ethanol in whisky is not less than 40%.
• Whisky is usually colored with the addition of caramel (burned Sugar).
•  Whisky production was first begun on Scotland.
• But some believe that it started on Arabs & Greek.
• Whisky production was supposed to be started first by Irish 1000 years ago.
• But written records suggest that, it was after 1494.
• Microbiology – Improved strains of Saccharomyces Cerevisiae.
• Biosynthesis:   
  • Glucose —–> Pyruvate —–> Acetaldehyde —-> Ethanol

Steps in whiskey making:

• Various steps involved in whisky production are:
  • Selection & Preparation of substrate
  • Malting
  • Mashing
  • Fermentation
  • Distillation
  • Ageing
  • Blending

Step I: Selection and Preparation of substrate:

• Basic substrates used for whisky production are water & grains.
• The water should be free from hazardous material, pesticides, toxic chemicals & heavy metals. 
• But it should have high DO value.
• The water containing lime is considered good for whisky production.
• The basic ingredients for whisky include barley, oat, ray, malt etc. but it must be free from fungal infection.
• Beside those substrate other things needed for whisky production are caramel mix (burnt sugar + others).
• According to substrates used, whisky are of:
  • Straight whisky: No added ingredients i.e. non-blended
  • Blended whisky: Added with different ingredients and sherry (wine)
• Barley—-> malted cooked at 100^oC for 3hrs/150^oC for 1 hr
• Other grains —-> grounded without malting —-> cooked ta 100^oC for 3hrs/150^oC for 1 hr
• Cooking liquify starch and break cellulose wall of grains.

Step II: Malting

• Malting is essential for enzyme activity.
• In Scottish whisky, barley germination heating is provided with hot air which is produced from peat burning.
• Peat is soft carbon rich substance when plant matter decomposed in water.
• It provides characteristics smoky flavor to Scottish whisky.
• But, in other countries, hot air is produced by coal burning.

 Step III: Mashing

• The malted barley is grounded and mixed with cereals and finally subjected to controlled heating with overall temperature 40-85^oC for several days.
• During heating alpha-amylase is converted into beta amylase.
• After mashing it is filtered to obtain wort.
• It is optimized for total sugar content (TSS)= 20-30%.

Step IV: Fermentation

• Mash/wort (TSS=20-30%) —> pH adjusted (3.5-4.5) —-> close fermenter (mostly used)
• Mash/wort (TSS=20-30%) —–> pH adjusted (3.5-4.5) —–> open tank fermenter (require more time, more chance of contamination, more chance of loss of ethanol.

Step V: Distillation

• Now the ethanol is evaporated at 78-80^o C.
• In Scotland, distillation is carried out in copper plant or stain less steel.
• Distillation is carried out to obtain 95% ethanol.
• The ethanol should be free from congeners.
• Congeners are the extra things that produces bad flavors such as aldehyde, ketones & other volatile substances.
•  It is overcome by production of high % or concentrated ethanol.
• Hard/High wine is produced.

Step VI: Maturation/ Ageing

• The high Wine is diluted with addition of water to obtain the solution with alcohol content 50-65%.
•  In the Scotland, the diluted ethanol is kept inside white oak barrel and mouth of the barrel is sealed and kept in cool & wet condition.
• Whereas in American whisky, the ageing is done in warm and dry condition.
•  During ageing, the congeners, esters, sugars, and alcohols react together to form special type of aroma.
• Ageing is conducted minimum for 3-4 years & maximum up to 10-15 years.

Step VII: Blending

• In blending, appropriate proportion of different whisky of previous batch is mixed with the aged whisky.
•  At the same time, the appropriate proportion of Sherry port & other things are also mixed to develop flavors.
• Finally, caramel mix is added in appropriate amount to develop a suitable color.
• Finally, it is checked by testing with master blender.

Production process of Whisky

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• Wine is a kind of undistilled alcoholic beverage mainly prepared from fruit juice. (mainly from grapes).
• The process of preparation of wine is known as vinification and the branch of science that deals with study of wine is known as enology (American) or oenology (British).
• There are different types of wine on different basis.
• Besides fruit and berries, non-toxic plants (flowers) etc can also be used for wine production.
• Since, basic constituent of wine is alcohol, other substrates are also added in it.
• It contains 3-22% of alcohol.

Microbiology: Grape juice (27% sugar) is fermented by various strains of Saccharomyces cerevisiae.

Wine production:

• Wine is basically the transformation of sugars of grapes of yeast under anaerobic condition into ethanol, CO₂ and small amounts of byproducts such as D-glucose.

What are the basic steps of wine making?

• Step I: Harvesting of fruits:
  • Appropriate variety of fruits and berries are harvested.
  • They must contain high amount of fermentable sugars.
  • Grapes usually contain 5-25% total soluble sugar (Total soluble sugar).
• Step II: Crushing and extraction:
  • Thus, obtained fruits are crushed and extracted mechanically.
  • This process releases juice and a little bit pigment.
  • The whole mass is known as Must.
  • For white wine preparation, the skin is removed. The harvested fruits are de-steamed for white wine preparation which is not required for red wine preparation.
  • In case of red wine, the steam gives vegetable aroma due to presence of 2 methoxy-3-isopropyl pyrazine.
  • Color is also extracted from steam.
  • In case of red wine, the Must should be fermented.
• Step III: Optimization:
  • The must is optimized for two parameters, TSS and pH.
  • The TSS is generally optimized between 17-22% and pH in between 3-4, depending on yeast strains to be used.
  • KNS (potassium metabisulphite) may or may not be added at this stage which is an antimicrobial compound against Acetobacter spp. and competitive yeast.
  • It also acts as anti-oxidant and antifungal agent.
• Step IV: Primary fermentation:
  • The optimized Must is inoculated with 2-10% of inoculum and fermentation is carried out under optimum temperature.
  • Red wine preparation= 22-27^oC for 3-5 days
  • White wine preparation= 10-21^oC for 7-14 days
  • During the fermentation, the content is mixed twice a day by punching the floating skin for proper aeration.
  • It also helps in color extraction.
  • This fermentation allows rapid multiplication of yeast cell as well as sugar fermentation to ethanol, when the TSS is decreased nearly about 9-10% then primary fermentation is terminated.
• Step V: Pressing:
  • The skin of must is taken out and pressed in order to release juice and alcohol.
  • The liquid is again transferred into tank.
  • In case of white wine, pressing is carried out before fermentation.
  • During pressing color of fruits and berries is extracted.
• Step VI: Heat and cold sterilization:
  • The main aim of this technique is to remove the tartarate crystals (wine diamonds or wine crystals).
  • In cold sterilization method, the fermented must is cooled to nearly freezing and kept for one to two weeks.
  • During this period, the crystals gets separated or stirred in the wall of fermenter and clear liquid is collected on secondary fermented tank.
  • In heat stabilization technique, it is gently heated in between 50-60^oC for an hour and kept overnight.
  • The proteins get decanted.
  • The clear contents are pumped out and remaining turbid substance adsorbed on to bentonite.
• Step VII: Secondary fermentation:
  • It is carried out in stainless steel or oak barrel or concrete tank lined with plastic.
  • The stabilized, sterilized wine is now kept at 15-20^oC for 3-6 months under strict anaerobic condition usually in case of sweet wine, the fermentation is terminated when sugar content is reduced to 4-6%.
  • During secondary fermentation, aroma is developed.
  • The aroma in wine is categorized into 3 types:
    • Primary aroma —-> contributed by fruits or berries
    • Secondary aroma —–> developed during secondary fermentation
    • Tertiary aroma ——-> developed during bottled ageing
  • The aroma compound may be volatile or non-volatile.
  • It is developed due to chemical reactions among acids (malic acid, citric acid etc), sugars, alcohols and phenolic compounds.
  • The main compound responsible for aroma is methoxyparazine , monoterpenes, nor-isoprenoids, thiols, esters etc. among which ester is the principal one.
  • Esters are produced by reaction between alcohols and acids which is very slow.
  • It takes nearly one year for secondary fermentation
  • Before secondary fermentation malo-lactic fermentation occurs.
  • Malic acid (sharp sour) —–Lactic acid bacteria (LAB)—-> Lactic acid
• Step VIII: Laboratory testing:
  • After secondary fermentation, certain laboratory tests are conducted which includes bricks reading, bricks pH, titrable acidity, residual sugars, free or available sulfur, total sulfur, volatile acidity and alcohol percentage.
• Step IX: Blending and fining:
  • It is the most crucial to produce good quality of wine giving special taste and aroma.
  • In blending process, spices, extracts of aromatic plants, essential oils, fruit juices and other things are added in appropriate proportion.
  • Blending is kept trade secret in winery (wine industry).
  • In fining process, tannins and microscopic particles are removed in order to make clear wine.
  • For this purpose, wine is treated with gelatin, potassium caseinate, egg albumin, lysozymes, skimmed milk powder etc. or it is filtered through membrane filter or diatomaceous earth cellulose filter.
  • Finally, wine is clarified in order to remove pectin which is achieved with the use of pectinase enzyme.
• Step X: Preservation:
  • Pasteurization technique and use of KMS (Potassium metabisulphite) are mainly used for preservation.
  • It kills sugar utilizing micro-organisms.
• Step XI: Bottling:
  • Finally, wine is aseptically filled in bottle and bottle is corked, which is usually made with oak.
  • Finally, the outside cork is sealed.
  • The bottled wine can be directly consumed or preserved.

Distillation of wine:

• These are the alcoholic beverages/drinks obtained by the distillation of wine or fermented cereals.
• It may be aged or unaged (i.e. the distilled liquor).
• Distilled liquor is commonly called spirits.
• They consist of more than 40% ethanol.
• There are various types of distilled liquor. The primary types are:
• Whisky (Barley and others) —-> aged
• Brandy (Wine distillation) —–> aged
• Rum (fermented molasses) —–> aged
• Vodka (fermented cereals) —–> not aged
• Gin (distillation of fermented cereals) —–> unaged but flavored

Wine production process

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Following are types of plant tissue culture:

1. Cell or suspension culture
2. Explant culture
3. Callus culture
4. Protoplast culture
5. Embryo culture
6. Anther and pollen culture
7. Ovule culture
8. Ovary culture

1. Cell or suspension culture:

• Cell or suspension culture can be obtained either directly from explant or from callus.
• Pieces of undifferentiated calli are transferred to liquid medium which is continuously agitated to obtain a suspension culture.
• Tissue and cells cultured in a liquid medium produces a suspension of single cells and cell clumps of few to many cells which is called suspension culture.
• Single cells can also be obtained from plant organs (explants) particularly from leaf either by mechanical or enzymatic (pectinase solutions) means.
• Then single cells can be cultured by different techniques.
• Single cell culture methods:
• Filter paper raft nurse tissue technique:
  • Single cells are placed on filter paper (8X8mm) usually which themselves are placed on top of established callus cultures.
  • Single cells will be able to derive nutrient from callus exudates diffusing through the filter papers.
• Microchamber technique:
  • A microchamber can be created either by using a microscopic slide and coverslip or a cavity slide.
  • Single cells are suspended in a condition medium and the drop is placed onto a coverslip which is then inverted into the slide cavity.
  • This method allows microscopic observation that can be sub-cultured on petri dish easily.
• Microdrop method:
  • An especially designed dish (cup rack dish) having a smaller outer chamber to be filled with distilled water to avoid dessications of cells and a larger chamber having several microncells is used.
  • Microdrops of (0.25-0.5) ml are distributed in the micron-cells and the dish is sealed with parafilm.
  • Cell density in the medium is adjusted to give on an average one cell per droplet.
• Bergmann’s plating technique:
  • In this technique, free cells are suspended in a liquid medium and if cell aggregates are present, these are filtered.
  • Culture medium with (0.8-1)% is cooled and maintained at 35^oC in a water bath.
  • Then cells are mixed with the medium and poured in a petri dish.
  • Then, it is allowed to solidify and sealed with parafilm and examined with inverted microscope to mark single cells.
  • When macroscopic colonies develop, they are isolated and cultured separately.

Applications of single cell:

• Induction of somatic embryos and shoots.
• Invitro mutagenesis and selection of mutants.
• Genetic transformation studies.
• Production of secondary metabolites.

2. Explant culture:

• Any excised part of plant which has capacity or capability to develop into a whole plant is called explant.
• Explant should contain parenchyma tissues.
• Parenchyma tissues are versatile and capable of division and growth so far explant culture, the explant we have selected must contain parenchyma tissue.
• Explant should be selected from young and healthy part of plant.
• It can be from stems, rhizomes, tubers, roots, cotyledons, and hyper cotyledons.
• Development of the tissue occur through cell division, cell elongation, and cell differentiation.

3. Callus culture:

• Callus is an amorphous mass of loosely arranged, thin-walled parenchyma cells developing from proliferating cells of the parent tissue.
• It has biological potential to develop normal root, shoot and embryos and ultimately forming a plant.
• Callus culture should be subcultured in every 28 days to prevent nutrient depletion and production of toxic metabolites.
• Factors:
  • Environmental factors
  • Nutrient composition (Concentration of auxins and cytokinins)
  • Explant source

4. Somatic embryogenesis:

• Development of embryo from somatic or vegetative cells is somatic embryogenesis.
• Zygotic embryo:
  • Embryo formed from zygote.
• Non-zygotic embryo:
  • Embryo derived from other parts than zygote.
• Somatic embryo:
  • Embryo derived from somatic or vegetative parts.
• Parthenogenic embryo:
  • Embryo derived from unfertilized egg is known as parthenogenic embryo.
• Androgenic embryo:
  • Embryo derived from pollen.
• In 1959, from the callus culture and suspension culture of carrot (Daucus carota), somatic embryogenesis was done for first time.
• Major nutrient factor for somatic embryogenesis is auxin (high concentration is taken) and smaller amount of cytokinin may also be used.

Characteristics of somatic embryogenesis:

• Are bipolar in nature i.e., it has radicle and plumule end. (Radicle: from where roots are regenerated and plumule end: shoots are regenerated)
• Somatic embryo can be developed from two different cells.
• Dedifferentiation:
  • Dedifferentiation can be defined as reversion process in which mature meristematic step of cell lead to the formation of callus.
  • Mature cell ——–> callus
• Redifferentiation:
  • Component of callus have the ability to grow differentiated cells or plantlet.
  • This process is known as redifferentiation.
• Organogenesis:
  • Organogenesis can be defined as development of organs (shoot, root, leaves) that are induced in plant tissue culture.
  • It can also be known as regeneration.

5. Protoplast culture:

• Protoplast is a cell without cell wall.
• It is important for culture of hybrid plants.
• Hybrid: The fusion of two cells of two different plants.
• Cybrid: The fusion of the cell of nucleated plant and unnucleated cell.
• Protoplast is a cell from which cell wall is removed.
• This can be done either mechanically or enzymatically or by combination of both.
• CaCl₂ is used to stabilize plasma membrane.

Stages of protoplast culture:

I. Isolation of protoplast:

• Protoplast can be isolated from variety of tissue including leaves, root, invitro shoot callus, cell suspension and pollen but most commonly used substances are leaves.
• The process of isolation of protoplast from leaves are as follows:

Steps of protoplast isolation:

• Sterilization of leaf/leaves:
  • Rinse/ wash with clean water to remove dust particles/ soil particles.
  • Dip in 70% ethyl alcohol and 2% solution of sodium hypochlorite for few minutes.
  • Rinse/wash with sterile distilled water few times (3-4 times) to remove sodium hypochlorite.
• Removal of epidermis:
  • Remove lower epidermis gently and cut leaves into small pieces (for early removal of protoplast).
• Enzymatic treatment:
  • This can be performed in two different ways:
  • Direct (One step) method:
    • 0.5% macerozyme or pectinase + 2% cellulase in 13% mannitol or sorbitol at pH 5.4 at (25-30)^oC for few hours.
  • Teased gently and try to isolate protoplast.
  • Sequential (Two step) method:
    • 0.5% macerozyme or pectinase enzyme is taken.
    • Along with it, 0.3% potassium dextran (antioxidant) is added in 10% mannitol or sorbitol solution.
    • Place in water bath maintained at 25^oC and by shaking for 15 minutes.
    • Add fresh enzyme and incubate for an hour.
    • After removing previous enzyme solution, wash with 13% mannitol or sorbitol.
    • Add enzyme B (2% cellulase in 13% sorbitol or mannitol)
    • Incubate for few hours.
• Purification:
  • Filter the solution obtained from enzymatic treatment in 45 μ Nylon mesh and take the filtrate.
  • For further purification, centrifuge filtrate at 100g for 1 minute.
  • Remove supernatant.
  • Wash pellet 3 times (13% mannitol or sorbitol, centrifuge 100g for 1 minute).
  • Resuspend pellet in 20% sucrose solution.
  • Centrifuge at 200g for 1 minute.
  • Pure protoplast form supernatant.
  • Then separate protoplast by using pipette.

II. Culture:

• Make 10⁵ cell (protoplast)/ml solution with mannitol or sorbitol.
• Culture by Bergmann’s culture technique or microchamber technique or microdrop method.
• First callus is obtained which is sub-cultured as plantlet.

Applications of protoplast culture:

• Biochemical and metabolic studies
• Production of hybrid plants (nucleus of two different plants fused)
• Production of cybrid (fusion of nucleated and enucleated cells).
• Genetic manipulation
• To test during sensitivity

Somatic hybridization:

• Production of hybrid plants through the fusion of protoplast of two different plant species or varieties is called somatic hybridization and the hybrid plant obtained is known as somatic hybrid stages involved in somatic hybridization are:
• Isolation of protoplast of two different plant species or varieties.
• Mixing of protoplast by Fusogen treatment (NaNO₃, PEG TR, Ca⁺⁺ treatment, electrofusion).
• Wall regeneration by heterokaryotic cells (having two nucleus from two different species or two different varieties).
• Fusion of nuclei of heterokaryon to produce hybrid cells.
• Plating and production of colonies of hybrid cells.
• Selection of hybrid subculture and plantlet regeneration.

Protoplast fusion (different methods used in protoplast fusion (Induced fusion).

6. Anther and Pollen culture:

• The objectives of anther and pollen culture is:
  • For the production of haploid plants (having ‘n’ number of chromosomes) to produce same type of plants to stable genetic variability.
  • Haploids can be defined as sporophyte with gametophytic chromosome number which have a single complete set of chromosomes that may be useful for the improvement of many crop plants.
  • Anther and pollen or microspore culture are popular methods for the production of haploid plants.
• Parameters for successful anther and pollen cultures:
  • Conditions of growth of donor plant
  • Genotype of donor plant
  • Pre-treatment
  • The developmental stage of anther or pollen
  • The culture medium and the environmental conditions during growth.

i. Anther culture:

• Anther is a male reproductive organ having diploid in chromosome number.
• As a result of microsporogenesis, tetrads of microspores are formed from a single mother spore, they are known as pollen grains after released from tetrads.
• For anther culture, flower buds are brought to laminate air flow chamber and sterilized using appropriate chemical treatment.
• While removing anthers from flower buds, care should be taken to avoid injury because injury may lead to the development of callus which gives the mixture of haploids and diploids.

ii. Pollen culture:

• Pollen culture is preferred over anther culture even though the degree of success is low in this case
• About 50 anthers is placed in 20ml medium squeezed with a glass rod or syringe piston to allow the microspore to squeeze out.
• This solution (suspension of haploids and diploids) is filtrated through nylon sieve which allows only the microspore to pass through.
• The filtrate is centrifuged 3 time for 5 mins each at (500-800) rpm.
• The microspores are inoculated on solid or liquid medium maintained at 25^oC for (16-18) hrs photoperiod.
• The microspores may develop directly into embryoids within 15 days or follow one of the several indirect path to reproduce haploid plantlets.
• In anther culture as well as pollen culture, spontaneous double haploid may also be obtained which does not require colchicine treatment.
• For culture, commonly used medium: MS, White’s medium
  • Sucrose concentration: 2-3%
  • For dhaturo and tobacco: (2-4)% sucrose
  • Cereals: N₆, Potato-2 media
  • Wheat: sucrose concentration, 6%

7. Embryo culture:

• Embryo culture has been done for the production of haploid plants.
• It is used for the recovery of plants from distinct crosses and is useful where embryo fails to develop due to degeneration of embryonic tissues.
• This is extensively used in hybrid barley (Hordeum vulgare X H. bulbosum).

Procedure of embryo culture:

• Pluck or take developed fruit
• Wash it with clean water.
• Surface sterilize with 0.01% tween 20 for 15 minutes.
• Wash with distilled water few times.
• Sterilize with 0.01% mercuric chloride (HgCl₂) for 10-15 minutes.
• Wash with sterile distilled water few times.
• Break seed aseptically and isolate the embryo.
• Inoculate embryo on callus proliferating media.
• After four weeks of inoculation callus appears.
• And after 8 weeks of inoculation, subculture on shoot regenerating medium.
• After 4 weeks, green callus and embryoids starts appearing.
• After 12 weeks shoot appears.
• In some case like Brassica species, embryo can be cultured on very early stage.
• Early stage embryos are heterotrophic and cannot produce required nutrients from the simple tissue culture media. So, they can be cultured in a specialized way.
• Embryo nurse endosperm technique is most commonly used method to culture embryo on very early stage.

8. Ovary culture:

• The objectives of ovary culture is;
  • To overcome pre-fertilization barrier.
  • To overcome post-fertilization barrier (embryo rescue).
• Culture of unfertilized or fertilized ovaries to obtain plant is called ovary culture.
• Ovary culture is also known as gymnogenesis.
• About (0.2-6)% of the cultured ovaries shown gymnogenesis and 1 or 2 rarely upto 8 plantlets originate from each ovary.
• Ovary culture is often used either for invitro pollination and fertilization or for post fertilization barrier (embryo rescue).
• Interspecific hybrid using ovary culture have been successfully obtained in several genera of Brassica (Brassica campestris X B. oleraceae).
• Technique for growing excised ovaries on culture medium was developed in many plant materials including tobacco, tomato, beans etc.
• For interspecific or intergeneric crosses, ovaries are excised at the zygote stage or two-celled pro-embryo space and normal development is completed invitro.
• By addition of different chemicals like IAA, fruit juice, coconut milk to the medium, the development is promoted.
• Limitations of ovary culture:
  • Successful only in less than two dozen species.
  • Success rate is very less (0.2-6)%

Applications of Haploids in plant breeding:

• Homozygous lines for the production of homozygous plant.
• Hybrid sorting in haploid breeding.
  – Selection of recombinant superior gametes.
• Mutation research
• Gametoclonal variation: variation in gametic chromosomal number
• Cytogenic research: Basic chromosome number
• Evolutionary studies
• Genetic studies

Applications of plant tissue culture:

• Production of secondary metabolites. E.g. alkaloids, steroids, phenolics, variety of flavors and perfumes.
• Rapid clonal multiplication or clonal propagation (which also known as micropropagation).
• Production of virus free plant (By thermotherapy, chryotherapy, chemotherapy, and meristem culture).
• Rapid development of homozygous lines by producing haploids (anther culture, ovary culture, etc.)
• Production or recovery of hybrid plants which are difficult to grow in normal conditions. (Embryo rescue, in-vitro pollination).
• Germplasm conservation. (By cryopreservation, and slow growth cultures).
• Genetic modification of plants (somaclonal variation, somatic hybridization and cybridization, gene transfer).
• Creation of genetic maps or genome maps and use of molecular markers.
• Multiplication of the plants of desired size.
• Independent of season so carried out through-out the year.
• Production of synthetic or artificial seeds which are easier to handle, transport and store.

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