Plant Tissue Culture

1.Introduction to Plant Tissue Culture: Definition, types of culture and historical development, purposes of cell, tissue and organ culture, cellular totipotency, cell and tissue growth
process, characteristics and measurement method, laboratory organization: Lab design, small and large equipment with their functions.

2.Laboratory Organization and aseptic Techniques: Lab, facilities, design, operation and management, aseptic technique for plant tissues, chemicals,
instruments, glass wares, personal hygiene and laboratory safety management etc..

3. Laboratory Equipments and Sterilization: Major equipment, minor equipment‟s,

sterilization types, procedure etc.

4. Culture Media and Plant growth regulators: Components, composition, functions of

components, preparation of media. Solidification, media selection and maintenance of media.

5 Aseptic techniques: Plant tissues, chemicals, instruments, glassware‟s and personal hygiene.

6. Micropropagation: Definition, direct and indirect method of different plant, factors of shoot and root multiplication.

7. Protoplast Culture: Isolation, purification and culture of protoplast, development and application of somatic hybrids and cybrids.

8. Production of disease free plants: Methods of virus elimination, virus indexing, eradication of pathogens other than virus, application and limitations.

9. Somatic embryo genesis and suspension culture: Initiation of somatic embryo: callus and suspension culture, maintenance of callus and suspension culture,
production and management of somatic embryo and its application, plant formation from somatic embryo.

10. Culture of Anther/pollen, Ovule, Embryo, Endosperm and Their Uses: Rice, wheat, barley, maize, brinjal.

11. Somaclonal Variation: Production and selection of somaclonal and gametoclonal variation, utilization of somaclone and gametoclone in agriculture, in vitro selection of
disease resistant and stress tolerant plants.

12. In-vitro Conservation of Plant Materials: methods and factors affecting in vitroconservation, maintenance of frozen
culture.

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Previous year Question

2018 2017 2016 2015 2014 2013

1.Introduction to Plant Tissue Culture: Definition, types of culture and historical development, purposes of cell, tissue and organ culture, cellular totipotency, cell and tissue growth
process, characteristics and measurement method, laboratory organization: Lab design, small and large equipment with their functions.

1. What is tissue culture ? State the importance of plant tissue culture in twenty first century .

Tissue culture is a technique that involves the cultivation of plant cells or tissues in a laboratory setting,
under controlled conditions. It is also known as plant cell culture or micropropagation.
Plant tissue culture has become increasingly important in the twenty-first century due to its many
applications in plant biotechnology. Some of the significant importance of plant tissue culture are:

1. Mass propagation of plants: Plant tissue culture can produce large numbers of plants from a
single explant, which is particularly useful for commercial production of crops, such as fruits,
vegetables, and ornamental plants.

2. Disease-free plants: Plant tissue culture can produce plants that are free of diseases, viruses, and
other pathogens that are commonly found in the field.
3. Genetic modification: Plant tissue culture allows for the genetic modification of plants, which
has led to the development of crops that are more resistant to pests, diseases, and
environmental stresses, such as drought and salinity.
4. Conservation of rare and endangered species: Plant tissue culture has become a valuable tool for
conserving rare and endangered plant species, by allowing for their propagation and
preservation.

5. Production of secondary metabolites: Plant tissue culture can produce secondary metabolites,
such as alkaloids, flavonoids, and terpenoids, which have various applications in
pharmaceuticals, cosmetics, and food industries.

Overall, plant tissue culture has become an essential tool in plant biotechnology, allowing for the
efficient production of plants with desirable traits, and the preservation of rare and endangered plant
species.

2. Mention the achievements of tissue culture .

And Q-1
Tissue culture has had many significant achievements since its inception, including:
1. Mass propagation of plants: Tissue culture has enabled the production of large numbers
of plants from a single explant, which is particularly useful for commercial production of
crops, such as fruits, vegetables, and ornamental plants.
2. Production of disease-free plants: Tissue culture has made it possible to produce plants
that are free of diseases, viruses, and other pathogens that are commonly found in the
field.
3. Genetic modification of plants: Tissue culture has facilitated the genetic modification of
plants, which has led to the development of crops that are more resistant to pests,
diseases, and environmental stresses, such as drought and salinity.

4. Conservation of rare and endangered species: Tissue culture has become a valuable tool
for conserving rare and endangered plant species, by allowing for their propagation and
preservation.
 5. Production of secondary metabolites: Tissue culture can produce secondary metabolites,
such as alkaloids, flavonoids, and terpenoids, which have various applications in
pharmaceuticals, cosmetics, and food industries.

6. Cloning of plants: Tissue culture has enabled the cloning of plants, which has many
applications in agriculture, horticulture, and forestry.
Overall, tissue culture has had a profound impact on plant biotechnology and has enabled many
advancements in the field of agriculture, medicine, and industry.

3. Plant cells are totipotent " Justify the statement .

The statement "plant cells are totipotent" is justified because they have the ability to
differentiate into any type of cell in the plant, given the appropriate signals and conditions. This
unique feature, known as totipotency, has been exploited in tissue culture, where individual
cells or small tissue fragments are grown under specific conditions to produce new plants. This
ability has many applications in agriculture, horticulture, and forestry, and has led to the rapid
production of large numbers of genetically identical plants.

4. What do you mean by callus ? Write the process of callus formation through tissue culture .
Callus is a mass of undifferentiated cells that is formed when plant cells or tissues are cultured
in vitro under specific conditions. Callus is typically characterized by its rapid growth and lack of
organization, and it is an essential intermediate in the regeneration of plants from tissue
culture.
The process of callus formation through tissue culture involves the following steps:
1. Explant preparation: A small piece of plant tissue, called an explant, is taken from the parent
plant and sterilized to remove any surface contaminants.
2. Culture initiation: The explant is placed in a culture medium that contains nutrients, plant
growth regulators, and other compounds that promote cell growth and division.
3. Callus induction: The cells from the explant start to divide and form a mass of undifferentiated
cells, known as callus.
4. Subculture: The callus is transferred to a fresh culture medium containing the same nutrients
and growth regulators to promote its further growth and development.
 5. Differentiation: The callus can be induced to differentiate into specific types of cells, such as
roots, shoots, or somatic embryos, depending on the specific conditions and culture medium
used.
6. Regeneration: The differentiated cells are stimulated to develop into a complete plant, which can
then be transferred to soil for further growth and development.

In summary, callus formation is an important step in tissue culture, and it provides a platform
for the regeneration of plants from individual cells or small tissue fragments. The process of
callus formation involves the induction of undifferentiated cells from the explant, followed by
subculture, differentiation, and regeneration, leading to the development of a complete plant.

5. Mention the salient feature of plant tissue culture with its limitations
Salient features of plant tissue culture:
1. Aseptic culture conditions: Plant tissue culture requires aseptic conditions to prevent
contamination from bacteria, fungi, and other microorganisms.
2. Mass propagation: Tissue culture can produce large numbers of plants from a single explant,
allowing for the rapid propagation of rare or endangered plant species.
3. Genetic modification: Tissue culture can be used to genetically modify plants, allowing for the
development of crops that are more resistant to pests, diseases, and environmental stresses.
4. Disease-free plants: Tissue culture can produce plants that are free from pathogens and
diseases, which is important for the production of high-quality crops.

5. Cloning: Tissue culture allows for the cloning of plants, which is useful for the production of
plants with desirable traits.

Limitations of plant tissue culture:

1. High cost: Plant tissue culture requires expensive equipment and specialized facilities, making it
costly to set up and maintain.
2. Genetic instability: Plants regenerated from tissue culture may have genetic instability, resulting
in phenotypic variations.
3. Epigenetic changes: Tissue culture can cause epigenetic changes that may affect gene expression
and phenotypic traits.
4. Somatic mutations: Somatic mutations can occur during tissue culture, leading to genetic
variations that can affect the quality of the plant material produced.
5. Environmental factors: The success of tissue culture can be affected by environmental factors,
such as temperature, light, and humidity, which can vary widely depending on the specific plant
species being cultured.

Overall, plant tissue culture has many advantages and has revolutionized plant biotechnology,
but it also has several limitations that need to be taken into consideration when using this
technology.
6 . Show the classification of tissue culture
as-7
Tissue culture can be classified based on several different criteria, including the type of tissue
being cultured, the purpose of the culture, and the stage of development of the cultured tissue.
Here are some common classifications of tissue culture:

1. Based on the type of tissue being cultured:

a. Cell culture: Involves the growth of individual cells in culture, without the
presence of a supporting tissue matrix.
b. Organ culture: Involves the growth of whole organs or sections of organs, such as
roots, stems, or leaves, in culture.
c. Embryo culture: Involves the growth of embryos or embryonic tissues in culture.

2. Based on the purpose of the culture:

a. Basic research: Tissue culture is used to study cell biology, genetics, and other
basic biological processes.
b. Applied research: Tissue culture is used to develop new plant varieties, study
plant metabolism, and produce valuable compounds, such as pharmaceuticals or
fragrances.
c. Industrial applications: Tissue culture is used for large-scale production of plants
for commercial purposes, such as crop improvement or horticulture.

3. Based on the stage of development of the cultured tissue:

a. Callus culture: Involves the growth of a mass of undifferentiated cells, known as

callus, which can be used to regenerate whole plants.
b. Suspension culture: Involves the growth of cells in a liquid medium, without the
presence of a solid matrix.
c. Organogenesis: Involves the differentiation of callus into specific organs, such as
roots, shoots, or somatic embryos.
d. Regeneration: Involves the development of whole plants from differentiated cells
or tissues.
Overall, tissue culture is a versatile technology that can be used for a wide range of applications,
and its classification is based on the specific purpose and tissue being cultured.
7. Briefly discuss the application of tissue culture in industry
Q-1
Plant tissue culture has numerous applications in industry, especially in agriculture, horticulture,
forestry, and pharmaceuticals. Here are some of the applications of tissue culture in industry:
1. Mass production of plants: Tissue culture can be used to produce large numbers of
plants rapidly and economically. This is particularly useful for producing rare or
endangered plant species, or for developing new plant varieties with desirable traits.
2. Crop improvement: Tissue culture can be used to develop new plant varieties with
improved traits, such as disease resistance, increased yield, and enhanced nutritional
content. This has led to the development of high-yielding crops that can meet the
increasing demand for food.
3. Horticulture: Tissue culture has revolutionized horticulture by allowing for the
production of high-quality plants with uniform characteristics. This has resulted in
improved productivity, reduced costs, and increased profitability for growers.
4. Forestry: Tissue culture can be used to propagate trees for reforestation and
afforestation programs. It also provides a means for preserving endangered tree species
and conserving genetic resources.
5. Pharmaceutical production: Tissue culture can be used to produce valuable compounds,
such as alkaloids, flavonoids, and essential oils, for the pharmaceutical industry. This
allows for the production of high-quality, standardized plant-based medicines.
6. Bioremediation: Tissue culture can be used for bioremediation, which involves the use of
plants to clean up environmental pollutants. This technology has the potential to
address some of the most pressing environmental challenges facing the world today.
Overall, tissue culture has a wide range of applications in industry, and its use is expected to
increase in the coming years as new technologies and applications are developed.

2.Laboratory Organization and aseptic Techniques: Lab, facilities, design, operation and management, aseptic technique for plant tissues, chemicals,
instruments, glass wares, personal hygiene and laboratory safety management etc.

8. Specify the basic requirements to design a laboratory for tissue culture experiment . Design a
plant tissue culture laboratory considering the number of basic requirements .
Designing a laboratory for tissue culture experiments requires careful consideration of various
factors to ensure optimal conditions for cell growth, sterility, and efficient workflow. Here are
some basic requirements to consider:
1. Clean and Sterile Environment: The laboratory should be designed to maintain a clean and
sterile environment to prevent contamination of cell cultures. This includes proper air filtration
systems, controlled airflow, and maintaining positive pressure to keep contaminants out.
2. Physical Layout: The laboratory should be spacious enough to accommodate all necessary
equipment, workstations, storage areas, and safety measures. Adequate space between
workstations is essential to prevent cross-contamination. Separate areas should be designated
for different stages of tissue culture, such as preparation, culturing, storage, and sterilization.
3. Workstations and Equipment: Dedicated workstations should be designed for different tasks,
including media preparation, inoculation, subculture, and analysis. Each workstation should be
equipped with laminar flow hoods or biosafety cabinets to provide a sterile working area.
4. Temperature and Humidity Control: Tissue culture requires precise temperature and humidity
control. The laboratory should have temperature-controlled rooms or incubators to maintain
optimal growth conditions. Humidity control is important to prevent excessive evaporation from
culture vessels.
5. Lighting: Adequate lighting is crucial for performing delicate tissue culture procedures and
visual observation of cell cultures. The lighting should be adjustable to provide the appropriate
intensity and spectral quality for different stages of tissue culture.
6. Sterilization and Disposal: A designated area should be available for autoclaving and
sterilizing glassware, media, and other materials. Proper waste disposal facilities should be in
place to handle contaminated materials safely.
7. Storage Facilities: The laboratory should have sufficient storage space for cell lines, culture
media, chemicals, reagents, and equipment. Storage areas should be organized, labeled, and
temperature-controlled for the stability of materials.
8. Safety Measures: Safety should be prioritized in the laboratory design. This includes the
provision of personal protective equipment (PPE) for lab personnel, emergency shower and
eyewash stations, fire safety equipment, and adherence to local safety regulations and
guidelines.
9. Utilities: The laboratory should have access to utilities such as electricity, water, and gas for
the operation of equipment, autoclaves, and media preparation.
10. Monitoring and Control Systems: Monitoring systems should be in place to track
environmental conditions, such as temperature, humidity, and CO2 levels in incubators or
growth chambers. These systems help maintain stable conditions and provide early warning of
any deviations.
It is essential to consult with experts in laboratory design and comply with local regulations and
safety standards when designing a tissue culture laboratory. The specific requirements may vary
based on the scale of the operation, the type of tissue culture work being conducted, and the
resources available.

9. What are the personal safety measures of a scientist during working period in the laboratory
1. Wear appropriate clothing and personal protective equipment (PPE). This includes long
pants, closed-toe shoes, a lab coat, safety glasses, gloves, and a face shield if necessary.
2. Familiarize yourself with the lab's emergency procedures. Know where the fire
extinguishers, eyewash stations, and first aid kits are located.
3. Know your symbols and label all hazardous materials. Be familiar with the different
safety symbols used in the lab and make sure that all hazardous materials are properly
labeled.
4. Keep the lab organized and clean. This will help to prevent accidents and injuries.
5. Follow protocol when handling hazardous materials. Always follow the proper
procedures when handling hazardous materials, such as wearing the appropriate PPE
and working in a well-ventilated area.
6. Be aware of your surroundings. Pay attention to what you are doing and be aware of the
potential hazards in the lab.
7. Do not work alone. If possible, work with a partner so that someone can help you in case
of an accident.
8. Report any hazards or safety concerns to your supervisor immediately.
10. Give an account of safety measures that should be taken at the time of lab closing .
a Here are some safety measures that should be taken at the time of lab closing:
1. Turn off all equipment. This includes all lights, computers, and laboratory equipment.
2. Put away all chemicals and hazardous materials. This includes returning them to their
proper storage containers and properly labeling them.
3. Clean up any spills. This includes using the proper cleaning materials and disposing of
the waste properly.
4. Dispose of waste properly. This includes hazardous waste, biohazardous waste, and
regular waste.
5. Check for leaks. This includes checking all hoses, pipes, and containers for any leaks.
6. Make sure all doors and windows are closed and locked. This includes the main
entrance, fire exits, and any other doors or windows that lead to the lab.
7. Report any hazards to your supervisor. This includes any spills, leaks, or other hazards
that you have found.
By following these safety measures, you can help to prevent accidents and injuries in the
laboratory.
11. How can you combat with polyphenolic compound
Polyphenolic compounds are a type of antioxidant that can be found in many foods, including
fruits, vegetables, and tea. Antioxidants help to protect cells from damage caused by free
radicals, which are unstable molecules that can contribute to the development of chronic
diseases such as cancer and heart disease.
There are a number of ways to combat with polyphenolic compound. One way is to eat a diet
rich in fruits, vegetables, and tea. These foods are all good sources of polyphenolic compounds.
Another way to combat with polyphenolic compound is to take a dietary supplement that
contains polyphenolic compounds.
Here are some examples of foods that are high in polyphenolic compounds:
1. Fruits: Berries, apples, grapes, oranges, and tomatoes
2. Vegetables: Broccoli, kale, spinach, and onions
3. Tea: Green tea, black tea, and oolong tea

3. Laboratory Equipments and Sterilization: Major equipment, minor equipment‟s, sterilization types,
procedure etc.

12. * Write the name of 4 major equipments with their function used in the plant tissue culture
laboratory .
4 major equipments used in the plant tissue culture laboratory, along with their functions:
1. Incubator: An incubator is used to provide a controlled environment for plant growth. It
typically maintains a temperature of 25-28 degrees Celsius and a humidity of 60-70%.
2. Laminar flow hood: A laminar flow hood is used to create a sterile environment for plant
tissue culture. It has a stream of filtered air that flows across the work surface, which
helps to prevent contamination.
3. Autoclave: An autoclave is used to sterilize plant tissue culture media and equipment. It
uses steam under pressure to kill bacteria, fungi, and viruses.
4. Microscope: A microscope is used to observe plant cells and tissues. It can be used to
identify cells, measure cell size, and observe cell division.

12.1Specify the explant sterilization method followed in the tissue culture.

There are many different explant sterilization methods that can be used in tissue culture. The
most common methods include:
1. Sodium hypochlorite: This is the most commonly used method for explant sterilization.
Sodium hypochlorite is a strong disinfectant that can kill a wide range of
microorganisms. It is typically used at a concentration of 0.5-1.0%. The explant is first
rinsed in 70% ethanol to remove any surface debris. It is then placed in a solution of
sodium hypochlorite for 10-20 minutes. After the sterilization period, the explant is
rinsed thoroughly with sterile water.
2. Ethyl alcohol: Ethyl alcohol is another commonly used disinfectant. It is not as strong as
sodium hypochlorite, but it is still effective at killing most microorganisms. Ethyl alcohol
is typically used at a concentration of 70%. The explant is first rinsed in 70% ethanol to
remove any surface debris. It is then left in the ethanol solution for 1-2 minutes. After
the sterilization period, the explant is rinsed thoroughly with sterile water.
3. Other disinfectants: Other disinfectants, such as hydrogen peroxide, mercuric chloride,
and silver nitrate, can also be used for explant sterilization. However, these disinfectants
are not as commonly used as sodium hypochlorite or ethyl alcohol.
12.2. Briefly describe the sterilization procedure of explant dents c suface c .
12.3 Why is filter sterilization done ?
Filter sterilization is done to remove microorganisms from liquids, gases, or powders that cannot
be sterilized by other methods, such as heat or radiation. It is a cold sterilization method, which
means that it does not use heat or chemicals to kill microorganisms. Instead, it uses a filter with
very small pores to physically remove the microorganisms from the liquid, gas, or powder.
Filter sterilization is often used for heat-sensitive materials, such as pharmaceuticals, biological
products, and medical devices. It is also used for materials that would be damaged by heat or
radiation, such as enzymes and proteins.
Here are some of the advantages of filter sterilization:
1. It is a cold sterilization method, which means that it does not use heat or chemicals to
kill microorganisms.
2. It is suitable for heat-sensitive materials.
3. It is suitable for materials that would be damaged by heat or radiation.
4. It is a very effective method of sterilization.
12.3 What is HEPA filter ? Why is it used ?
A HEPA filter (High Efficiency Particulate Air) is a type of air filter that can remove 99.97% of
airborne particles that are 0.3 microns in size or larger. This includes dust, pollen, mold,
bacteria, and viruses. HEPA filters are used in a variety of settings, including hospitals, schools,
homes, and offices.
HEPA filters are used to improve indoor air quality by removing harmful particles from the air.
This can help to reduce the risk of respiratory problems, such as asthma and allergies. HEPA
filters can also help to reduce the spread of disease by removing viruses and bacteria from the
air.
some of the benefits of using HEPA filters:
1. Improved indoor air quality: HEPA filters can remove harmful particles from the air, such
as dust, pollen, mold, bacteria, and viruses. This can help to reduce the risk of
respiratory problems, such as asthma and allergies.
2. Reduced risk of disease: HEPA filters can help to reduce the spread of disease by
removing viruses and bacteria from the air. This is especially important in hospitals and
other healthcare settings.
3. Increased comfort: HEPA filters can help to improve comfort by reducing the amount of
dust, pollen, and other particles in the air. This can help to reduce coughing, sneezing,
and other symptoms of allergies and asthma.
4. Increased productivity: HEPA filters can help to improve productivity by reducing the
amount of dust, pollen, and other particles in the air. This can help to reduce fatigue and
improve concentration.

13. State the importance of filter sterilization and moist heat sterilization in tissue culture
experiment .
Filter sterilization and moist heat sterilization are both crucial in tissue culture experiments for
different reasons. Here's the importance of each method:
1. Filter Sterilization:
Filter sterilization, also known as membrane filtration, is important in tissue culture experiments
for the following reasons:
i. Heat-sensitive substances: Many components used in tissue culture media, such
as growth hormones, vitamins, and amino acids, are sensitive to high
temperatures and can be degraded or inactivated by autoclaving. Filter
sterilization allows these heat-sensitive substances to remain intact while
achieving microbial sterilization.
ii. Selective sterilization: Filter sterilization allows for the selective sterilization of
specific components or additives in a solution. It can be used to remove microbial
contaminants while retaining desired molecules or substances that are necessary
for the experiment.
 iii. Small volume sterilization: Filter sterilization is particularly useful when sterilizing
small volumes of liquid, such as nutrient solutions or media supplements, that
may not be suitable for autoclaving. It provides a quick and convenient method
for achieving sterility in these situations.
2. Moist Heat Sterilization:
Moist heat sterilization, commonly performed using an autoclave, is important in tissue culture
experiments for the following reasons:
i. Complete sterilization: Autoclaving with moist heat is a highly effective method for
achieving complete sterilization. It can eliminate a wide range of microorganisms,
including bacteria, fungi, spores, and viruses, from the culture media, equipment,
and glassware used in tissue culture.
ii. Decontamination of solid materials: Autoclaving is essential for sterilizing solid
materials, such as agar, agarose, culture vessels, and tools, which cannot be filter
sterilized. It ensures that the solid materials used in tissue culture are free from
any microbial contamination.
iii. Elimination of persistent contaminants: Some microorganisms, such as bacterial
endospores, are highly resistant to other sterilization methods. Autoclaving with
moist heat can effectively destroy these persistent contaminants, ensuring aseptic
conditions in the tissue culture environment.
In tissue culture experiments, the combination of filter sterilization and moist heat sterilization
provides comprehensive microbial control, allowing for the successful growth and propagation
of plant tissues in a sterile environment while preserving the integrity of heat-sensitive
components.
14. What are the chemicals used for explant sterilization ?
The choice of chemicals used for explant sterilization can vary depending on the specific
requirements of the plant species and the laboratory's protocols. However, the most commonly
used chemicals for explant sterilization in tissue culture include:
1. Sodium Hypochlorite (Bleach): Sodium hypochlorite is a common disinfectant used for
surface sterilization of plant tissues. It is effective in killing a wide range of microorganisms,
including bacteria, fungi, and some viruses. The concentration of bleach used may vary but is
typically in the range of 1-10%.
2. Ethanol or Isopropyl Alcohol: Ethanol or isopropyl alcohol (commonly known as rubbing
alcohol) are often used as an additional step for disinfecting the surface of the explant. They
help to remove any residual contaminants and provide a sterile surface before transferring the
explant to the culture medium. Concentrations of 70-95% are commonly used.
3. Mercuric Chloride (HgCl2): Mercuric chloride is a highly effective disinfectant used in some
tissue culture protocols for surface sterilization of plant material. However, its use is restricted
due to its toxicity and environmental concerns. It is important to handle mercuric chloride with
caution and follow appropriate safety guidelines when using it.
4. Plant Preservatives: In certain cases, plant preservatives such as antibiotics or antifungal
agents may be added to the culture medium itself rather than using them for explant
sterilization. These substances help prevent the growth of contaminants in the culture and
support the growth of the desired plant tissues.
It is worth noting that the specific concentration and exposure time of these chemicals can vary
depending on the plant species, explant type, and laboratory protocols. It is crucial to follow
established protocols and ensure proper safety measures are in place when working with
chemicals for explant sterilization.
Write the utility of laminar air flow used in tissue culture laboratory . State the importance of
dry heat sterilization in tissue culture experiment .
15. Describe the process of steam and dry heat sterilization applied in the field of biotechnology
Sure, here are the processes of steam and dry heat sterilization applied in the field of
biotechnology:
Steam sterilization is a process that uses moist heat to kill microorganisms. It is a very effective
method of sterilization and is often used in biotechnology to sterilize equipment and media.
The steam sterilization process typically involves the following steps:
1. Place the equipment or media to be sterilized in a steam sterilizer.
2. Close the door of the sterilizer and seal it tightly.
3. Set the sterilizer to the correct temperature and pressure.
4. Allow the sterilizer to heat up and reach the desired temperature and pressure.
5. Hold the sterilizer at the desired temperature and pressure for the required amount of
time.
6. Allow the sterilizer to cool down and depressurize.
7. Open the door of the sterilizer and remove the sterilized equipment or media.
Dry heat sterilization is a process that uses dry heat to kill microorganisms. It is not as effective
as steam sterilization, but it can be used to sterilize equipment that cannot be sterilized with
steam, such as plasticware.
The dry heat sterilization process typically involves the following steps:
1. Place the equipment to be sterilized in an oven.
2. Set the oven to the correct temperature.
3. Allow the oven to heat up to the desired temperature.
 4. Hold the oven at the desired temperature for the required amount of time.
5. Allow the oven to cool down.
6. Remove the sterilized equipment from the oven.
Both steam and dry heat sterilization are important methods of sterilization in biotechnology.
The choice of which method to use will depend on the type of equipment or media to be
sterilized and the requirements of the specific application.

4. Culture Media and Plant growth regulators: Components, composition, functions of components,
preparation of media. Solidification, media selection and maintenance of media.

16. Mention the name of five important media with their functions used in tissue culture
experiments .
Sure, here are five important media with their functions used in tissue culture experiments:
1. Murashige and Skoog (MS) medium: MS medium is a general-purpose medium that is
used for the growth of a variety of cells and tissues. It contains a balanced mixture of
nutrients, including carbohydrates, amino acids, vitamins, and minerals.
2. B5 medium: B5 medium is another general-purpose medium that is similar to MS
medium. However, it contains a few additional nutrients, such as myo-inositol and
glutamine, which are important for the growth of some cells and tissues.
3. White's medium: White's medium is a specialized medium that is used for the growth of
plant cells and tissues. It contains a higher concentration of sucrose than MS or B5
medium, which is important for the growth of plant cells.
4. Linsmaier and Skoog (LS) medium: LS medium is a specialized medium that is used for
the growth of mammalian cells and tissues. It contains a higher concentration of amino
acids and vitamins than MS or B5 medium, which is important for the growth of
mammalian cells.
5. Eagle's minimal essential medium (MEM): MEM is a specialized medium that is used for
the growth of cells in culture. It contains a very low concentration of nutrients, which
allows the cells to grow in a more defined environment. This can be useful for studying
the effects of different nutrients on cell growth and differentiation.
These are just a few of the many different media that are used in tissue culture experiments.
The choice of which medium to use will depend on the type of cells or tissues being cultured
and the specific goals of the experiment.
17. Write the composition of MS medium used for tissue culture
Murashige and Skoog (MS) medium is a plant tissue culture medium that is widely used for the
growth and development of plant cells and tissues. It is a modified version of the Knop medium,
which was first developed in the early 1900s. MS medium contains a balanced mixture of
macro- and micronutrients, vitamins, and growth regulators.
The composition of MS medium is as follows:

Macronutrients (in mg/L):

1. Calcium nitrate (Ca(NO3)2) - 440
2. Magnesium sulfate (MgSO4) - 270
3. Potassium chloride (KCl) - 170
4. Sodium nitrate (NaNO3) - 160
5. Potassium phosphate (KH2PO4) - 130
Micronutrients (in μg/L):
1. Iron (FeSO4) - 200
2. Manganese (MnSO4) - 50
3. Zinc (ZnSO4) - 10
4. Copper (CuSO4) - 0.1
5. Boron (H3BO3) - 3
6. Molybdenum (Na2MoO4) - 0.05
Vitamins (in mg/L):
1. Thiamine (B1) - 1.0
2. Nicotinic acid (B3) - 0.5
3. Pyridoxine (B6) - 0.5
4. Biotin - 0.01
5. Folic acid - 0.01
6. Cyanocobalamin (B12) - 0.001
Growth regulators (in μM):
1. Indoleacetic acid (IAA) - 2.0
2. Kinetin - 0.2
17.1. Specify the name and function of six micro nutrients used in plant tissue culture .
Here are six micronutrients used in plant tissue culture, along with their functions:
1. Boron (B): Boron is involved in a variety of metabolic processes, including cell division,
cell wall formation, and flower development. It also helps to transport nutrients and
water within plants.
 2. Manganese (Mn): Manganese is involved in a variety of metabolic processes, including
photosynthesis, respiration, and protein synthesis. It also helps to protect plants from
environmental stress.
3. Iron (Fe): Iron is essential for chlorophyll production, which is necessary for
photosynthesis. It also plays a role in cell division and respiration.

4. Copper (Cu): Copper is essential for the production of chlorophyll and the metabolism of
carbohydrates. It also plays a role in the production of auxins and gibberellins, which are
plant hormones that promote growth.
5. Zinc (Zn): Zinc is essential for cell division, growth, and development. It also plays a role
in the production of auxins, which are plant hormones that promote cell division and
elongation.

6. Molybdenum (Mo): Molybdenum is essential for the metabolism of nitrogen and sulfur.
It also plays a role in the production of chlorophyll and the breakdown of toxins.
17.2. Write the functions of macronutrients used in tissue culture media preparation .
Sure, here are the functions of macronutrients used in tissue culture media preparation:
1. Nitrogen is essential for the synthesis of proteins, nucleic acids, and chlorophyll. It is also
involved in the production of energy and the regulation of cell growth.
2. Magnesium is essential for the synthesis of chlorophyll, proteins, and nucleic acids. It is
also involved in the activation of enzymes.
3. Phosphorus is essential for the synthesis of DNA, RNA, and ATP. It is also involved in the
metabolism of carbohydrates and lipids.
4. Sulfur is essential for the synthesis of proteins, vitamins, and coenzymes. It is also
involved in the regulation of enzyme activity.

5. Potassium is essential for the regulation of water balance, enzyme activity, and cell
signaling.
6. Calcium is essential for cell wall formation, enzyme activity, and the transport of
nutrients and minerals.
17.3 Write the name of inorganic compounds specifying their major groups used in plant tissue
culture
Q-15,16
Sure, here are some of the inorganic compounds used in plant tissue culture, along with their
major groups:
 1. Macronutrients: These are nutrients that are required in large quantities by plants. They
include nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg),
sulfur (S), and iron (Fe).
2. Micronutrients: These are nutrients that are required in small quantities by plants. They
include copper (Cu), manganese (Mn), zinc (Zn), boron (B), molybdenum (Mo), chlorine
(Cl), and nickel (Ni).
3. Other inorganic compounds: These include sugars, amino acids, vitamins, and plant
hormones.
The specific inorganic compounds that are used in plant tissue culture will vary depending on
the type of plant being cultured and the desired outcome. However, the major groups of
inorganic compounds listed above are typically included in all plant tissue culture media.
Here is a more detailed description of each of the major groups of inorganic compounds used in
plant tissue culture:

1. Macronutrients: Macronutrients are essential for plant growth and development. They
are involved in a variety of processes, including photosynthesis, respiration, and cell
division.
2. Micronutrients: Micronutrients are also essential for plant growth and development.
They are involved in a variety of processes, including enzyme activity, chlorophyll
production, and nitrogen metabolism.
3. Other inorganic compounds: Other inorganic compounds, such as sugars, amino acids,
vitamins, and plant hormones, can also be included in plant tissue culture media. These
compounds can help to improve the growth and development of plant cells and tissues.
Plant tissue culture is a powerful tool that can be used to propagate plants, produce new
varieties of plants, and study plant biology. The use of inorganic compounds in plant tissue
culture is essential for the success of this technique.

18. Mention the name of natural products used in tissue culture media " preparation
Several natural products are used in tissue culture media preparation to provide essential
nutrients, growth regulators, and supplements for the growth and development of plant tissues.
Here are some common natural products used in tissue culture media:
1. Agar: Agar is a natural gelling agent derived from seaweed. It is used to solidify tissue culture
media and provide a solid support for plant growth. Agar is widely used in plant tissue culture to
support the growth of callus, shoot, and root cultures.
2. Banana Extract: Banana extract is derived from mashed or blended bananas. It is rich in
vitamins, sugars, minerals, and growth regulators, especially auxins. Banana extract is often
added to tissue culture media to promote root formation and enhance plant growth.

1. Coconut Water: Coconut water is a popular natural product used in tissue culture media. It
contains various growth-promoting compounds such as vitamins, minerals, amino acids, sugars,
and growth regulators like cytokinins. Coconut water is often used as a supplement to promote
cell division and shoot proliferation in tissue culture.

4. Tomato Juice: Tomato juice is obtained from tomatoes and is commonly used as a nutrient
supplement in tissue culture media. It provides vitamins, sugars, amino acids, and organic acids,
which can enhance the growth and development of plant tissues.

5. Potato Extract: Potato extract is derived from mashed or blended potatoes. It is rich in
carbohydrates, vitamins, and growth regulators like cytokinins. Potato extract is frequently used
in tissue culture media for the culture and proliferation of plant shoots and callus.

6. Yeast Extract: Yeast extract is obtained from the autolysis of yeast cells. It is a rich source of
amino acids, vitamins, and growth factors. Yeast extract is often added to tissue culture media
as a nutrient supplement to support cell growth and metabolism.

These natural products, along with other synthetic components, are carefully combined to
formulate tissue culture media that provide the necessary nutrients, growth regulators, and
supplements for the successful growth and development of plant tissues in vitro. The specific
concentrations and combinations of these natural products may vary depending on the plant
species and the specific requirements of the tissue culture experiment.
19. What is stock solution **? Justify the importance of stock solution preparation during
experiment
*. Why is it prepared in the laboratory ?
A stock solution is a concentrated solution of a chemical that is used to prepare working
solutions of the desired concentration.
A stock solution is a concentrated solution of a substance that is used to prepare a more diluted
solution. Stock solutions are commonly used in laboratories and industries where precise and
accurate measurements are necessary
Stock solutions are prepared for several reasons.
1. One reason is to save time and effort by eliminating the need to measure out small
quantities of a substance for each experiment. Instead, a small amount of the stock
solution can be used to prepare a more dilute solution as needed.\
2. Additionally, preparing a stock solution can reduce errors associated with measuring
small quantities of a substance, as errors in measuring small amounts can be magnified
in the final solution.
3. Finally, stock solutions can be used to ensure consistency and accuracy in experiments,
as they provide a known starting concentration for each trial.

Stock solutions are important in experiments because they allow for the precise and accurate
preparation of solutions of different concentrations. This is important for a number of reasons,
including:
1. Accuracy: Stock solutions can be made with high accuracy, which is important for
experiments that require precise measurements.
2. Conservation of materials: Stock solutions can be made in large quantities and then
diluted to the desired concentration, which saves on the amount of material needed.
3. Ease of use: Stock solutions are easy to use, as they can be simply diluted with water or
another solvent to the desired concentration.
In addition, stock solutions can be stored for long periods of time, which makes them a
convenient and cost-effective way to prepare solutions for experiments.

20. What is myo inositol *** Why is it used as vitamin ? Write the utility of myo - inositol and
amino acid used in media preparation . State the functions of myoinositol in tissue culture
media . 4. *
Myo-inositol, often referred to as simply inositol, is a naturally occurring compound that
belongs to the vitamin B complex group. It is a sugar alcohol with a chemical structure similar to
glucose. Myo-inositol is found in various foods, particularly in fruits, beans, grains, and nuts.
Why is it used as vitamin
Myo-inositol is used as a vitamin in plant tissue culture because it is a growth factor and
structural element of secondary messengers in eukaryotic cells. In plant tissues, it acts as a
principal storage form of phosphorus, making it an effective vitamin in plant tissue culture
media.
Myo-inositol is a type of sugar alcohol that is found in all living cells. It is a precursor to many
important molecules, including phospholipids, which are essential for cell membranes. Myo-
inositol also plays a role in signal transduction, which is how cells communicate with each other.
In tissue culture media, myo-inositol serves several important functions to support the growth
and development of plant tissues. Here are the key functions of myo-inositol in tissue culture
media:
1. Carbon Source: Myo-inositol serves as a carbon source for the growth and metabolism of
plant cells in tissue culture. It provides a readily available carbon skeleton that can be utilized
for energy production, biosynthesis of macromolecules, and various metabolic processes.
2. Osmotic Balance: Myo-inositol acts as an osmolyte and helps maintain osmotic balance
within plant cells. It contributes to the regulation of cellular hydration and prevents osmotic
stress, which is particularly important in tissue culture environments where the water potential
can fluctuate.
3. Hormone Metabolism and Signaling: Myo-inositol is involved in the metabolism and signaling
of plant hormones, especially auxins. It plays a role in the synthesis and metabolism of indole-3-
acetic acid (IAA), the primary auxin involved in cell elongation, differentiation, and
organogenesis. Myo-inositol availability can influence the production and activity of auxins in
tissue culture, thereby affecting growth and development processes.
4. Antioxidant and Reactive Oxygen Species (ROS) Regulation: Myo-inositol exhibits antioxidant
properties and helps protect plant cells from oxidative stress. It can scavenge free radicals and
reduce the harmful effects of reactive oxygen species (ROS) generated during tissue culture
processes, which can otherwise cause cellular damage and inhibit growth.
5. Lipid Metabolism: Myo-inositol is involved in lipid metabolism and the synthesis of
phospholipids, which are essential components of cell membranes. It contributes to membrane
integrity and function, affecting cell growth, division, and differentiation.
6. Callus Induction and Proliferation: Myo-inositol has been found to enhance callus induction
and proliferation in tissue culture. It promotes the formation of undifferentiated cell masses
(callus) from explants and supports their rapid growth and multiplication.
The addition of myo-inositol to tissue culture media helps provide a carbon source, maintain
osmotic balance, regulate hormone metabolism, protect against oxidative stress, support lipid
metabolism, and promote callus formation and proliferation. These functions contribute to the
successful growth and development of plant tissues in vitro and are essential for various tissue
culture applications, including micropropagation, somatic embryogenesis, and cell suspension
cultures.
21.Write the role of 2,4 - D and myoinositol used in the plant tissue culture media preparation .
2,4-Dichlorophenoxyacetic acid (2,4-D) and myo-inositol are commonly used components in
plant tissue culture media and play distinct roles in supporting the growth and development of
plant tissues. Here is a brief description of the roles of 2,4-D and myo-inositol in tissue culture
media preparation:
1. 2,4-Dichlorophenoxyacetic acid (2,4-D):
1. Role: 2,4-D is a synthetic auxin, or plant growth hormone, that is widely used in tissue
culture media for its role in promoting cell division and callus induction.
2. Callus Induction: 2,4-D stimulates the growth of undifferentiated cell masses known as
callus from explants. It promotes the dedifferentiation of plant cells, allowing them to
revert to a more embryonic state and form a mass of cells capable of regeneration.
3. Auxin-Like Effects: 2,4-D mimics the effects of naturally occurring auxins, particularly
indole-3-acetic acid (IAA). It regulates various processes such as cell elongation,
differentiation, and organogenesis. In tissue culture, 2,4-D helps initiate the formation of
callus and provides the necessary growth stimulus for subsequent steps in tissue culture,
such as organogenesis or somatic embryogenesis.

2. Myo-Inositol:
i. Role: Myo-inositol is a naturally occurring sugar alcohol that serves several
functions in tissue culture media.
ii. Carbon Source: Myo-inositol acts as a carbon source for the growth and metabolism
of plant cells in tissue culture. It provides a readily available carbon skeleton that
can be used for energy production and the synthesis of macromolecules.
iii. Osmotic Balance: Myo-inositol acts as an osmolyte and helps maintain osmotic
balance within plant cells. It helps regulate cellular hydration and prevents osmotic
stress, which can be especially important in tissue culture environments.
iv. Hormone Metabolism and Signaling: Myo-inositol is involved in hormone
metabolism and signaling, particularly with regards to auxins. It influences the
synthesis and metabolism of auxins like indole-3-acetic acid (IAA), which are critical
for cell elongation, differentiation, and organogenesis. Myo-inositol availability can
affect the production and activity of auxins in tissue culture, influencing growth and
development processes.

The combination of 2,4-D and myo-inositol in tissue culture media provides an optimal
environment for the initiation of callus formation, subsequent cell proliferation, and the
development of specific plant tissues or organs. These components, along with other nutrients
and growth regulators, help to support the successful growth and regeneration of plant tissues
in vitro.
22. What are PGRs? *Write the name of auxins and cytokinins with their functions used in
tissue culture .
PGRs, or Plant Growth Regulators, are naturally occurring or synthetic compounds that regulate
various physiological processes in plants. They play a crucial role in plant growth, development,
and responses to environmental stimuli. PGRs are widely used in tissue culture to manipulate
and control the growth and differentiation of plant cells and tissues. Two major classes of PGRs
used in tissue culture are auxins and cytokinins.
1. Auxins:
1. Indole-3-acetic acid (IAA): IAA is the most common naturally occurring auxin in
plants. In tissue culture, it is used to induce cell elongation, stimulate root
formation, and promote the growth of callus and shoots.
2. 1-Naphthaleneacetic acid (NAA): NAA is a synthetic auxin commonly used in tissue
culture. It promotes root initiation and development and can also induce callusc
formation.
- Functions: Auxins regulate various processes in tissue culture, including cell division,
elongation, and differentiation. They are involved in root initiation, shoot formation, and
organogenesis. Auxins also influence apical dominance and phototropism in plant tissues.
2. Cytokinins:
1. 6-Benzylaminopurine (BAP): BAP is a synthetic cytokinin widely used in tissue culture.
It promotes cell division and shoot proliferation, enhances axillary bud growth, and
stimulates the production of multiple shoots from explants.
2. Kinetin: Kinetin is a naturally occurring cytokinin. It plays a role in cell division, shoot
development, and the prevention of senescence in plant tissues.
Functions: Cytokinins are critical for promoting cell division and shoot proliferation in tissue
culture. They stimulate the growth of lateral buds, prevent aging and senescence, and influence
the balance between shoot and root development. Cytokinins work synergistically with auxins
to regulate tissue differentiation and morphogenesis.
The balance between auxins and cytokinins is crucial in tissue culture to achieve desired
outcomes, such as callus induction, shoot proliferation, or rooting. The specific choice and
concentration of auxins and cytokinins in tissue culture media depend on the plant species and
the desired response. By manipulating the concentrations and ratios of these PGRs, tissue
culture researchers can control and direct the growth and development of plant tissues for
various applications, including micropropagation, somatic embryogenesis, and genetic
transformation.
23. Give an account on vitamins used in tissue culture media preparation
Vitamins are essential organic compounds that play crucial roles in the growth and development
of plant tissues. They are often added to tissue culture media to provide necessary cofactors
and coenzymes that support various metabolic processes. Here is an account of some
commonly used vitamins in tissue culture media preparation:
1. Thiamine (Vitamin B1): Thiamine is involved in various enzymatic reactions, particularly in
carbohydrate metabolism. It plays a crucial role in energy production and the biosynthesis of
key molecules. Thiamine is added to tissue culture media to support cell growth, division, and
differentiation.
2. Riboflavin (Vitamin B2): Riboflavin is essential for various redox reactions in plants. It acts as a
cofactor for enzymes involved in energy metabolism, photosynthesis, and antioxidant defense
systems. Riboflavin is included in tissue culture media to support cell metabolism and growth.
3. Niacin (Vitamin B3): Niacin is involved in the synthesis of NAD (nicotinamide adenine
dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate), coenzymes that play
crucial roles in cellular metabolism and energy production. Niacin is added to tissue culture
media to support cell growth and metabolism.
4. Pyridoxine (Vitamin B6): Pyridoxine is involved in amino acid metabolism and is required for
the biosynthesis of several important compounds, including nucleic acids, neurotransmitters,
and hormones. Pyridoxine is included in tissue culture media to support cell division, growth,
and differentiation.
5. Biotin (Vitamin B7): Biotin is involved in various metabolic pathways, including fatty acid
synthesis, amino acid metabolism, and carbohydrate metabolism. It serves as a cofactor for
several enzymes involved in these processes. Biotin is added to tissue culture media to support
cell growth and metabolism.
6. Folic Acid (Vitamin B9): Folic acid is crucial for DNA synthesis, cell division, and the
metabolism of amino acids. It plays a key role in the transfer of one-carbon units in various
metabolic reactions. Folic acid is included in tissue culture media to support cell proliferation
and growth.
7. Ascorbic Acid (Vitamin C): Ascorbic acid is a powerful antioxidant that protects cells from
oxidative damage. It is involved in various metabolic processes, including the biosynthesis of
collagen, hormone metabolism, and redox reactions. Ascorbic acid is added to tissue culture
media to prevent oxidative stress and promote cell growth and development.
These vitamins, along with other components such as macronutrients, micronutrients, and
plant growth regulators, are carefully balanced in tissue culture media to provide the necessary
nutritional requirements for the successful growth and development of plant tissues in vitro.
The specific concentrations of vitamins may vary depending on the plant species and the
experimental objectives of the tissue culture study.

5 Aseptic techniques: Plant tissues, chemicals, instruments, glassware‟s and personal hygiene.
Q-8,9,10
6. Micropropagation: Definition, direct and indirect method of different plant, factors of shoot and root multiplication.
27. What is explant ? Write the name of explants used for plant culture.
In plant tissue culture, an explant refers to a small piece of plant tissue or organ that is excised
and used as the starting material for establishing a new culture. The explant serves as the
source of cells or tissues that will be propagated and grown in vitro under controlled conditions.
These cultured explants have the potential to regenerate into whole plants or produce specific
plant parts, such as shoots, roots, or callus.
There are various types of explants used in plant tissue culture, depending on the specific
purpose and objectives of the culture. Some commonly used explants include:
1. Leaf explants: Leaves are often used as explants due to their accessibility and ability to
regenerate shoots and roots. They can be taken from young, healthy leaves of the desired plant
species.
2. Stem explants: Stem segments, including nodes and internodes, can be used as explants.
They are often utilized for shoot regeneration and multiplication, as they possess meristematic
regions capable of cell division and growth.
3. Root explants: Root sections or root tips can be employed as explants for root culture or for
the induction of adventitious shoots or callus formation.
4. Shoot tip or apical meristem explants: The shoot tips or apical meristems, which contain
actively dividing cells, are used to initiate cultures with a high regeneration potential. They are
valuable for obtaining disease-free plant material through meristem culture techniques.
5. Embryo explants: Embryos at different stages of development can be used as explants for the
propagation of plants via somatic embryogenesis or embryo rescue techniques.
6. Seed explants: Whole seeds or embryo-containing seeds can serve as explants for various
purposes, such as the induction of callus, somatic embryogenesis, or germination studies.
7. Floral explants: Different floral parts, such as petals, anthers, ovaries, or ovules, can be
utilized as explants for specific objectives, including somatic embryogenesis, microspore culture,
or in vitro pollination and fertilization.
The choice of explant depends on the specific tissue culture technique, plant species, objectives
of the study, and the desired outcome. Different explants have varying regeneration potentials
and responses to culture conditions, so researchers select the most suitable explants for their
specific experiments or applications.
27.1 Define micropropagation . Mention the stages of micropropagation with its advantages and
limitations
***The main difference between the two techniques is that micropropagation is a specific type
of tissue culture used for mass production of identical plants, while plant tissue culture
encompasses a range of techniques used for different purposes such as genetic transformation,
conservation of endangered species, production of secondary metabolites, and research on
plant development and physiology.***
Micropropagation is a tissue culture technique that involves the aseptic multiplication of plant cells,
tissues, or organs under controlled conditions to produce large numbers of genetically identical plants. It
is also commonly known as in vitro propagation or tissue culture propagation.

Micropropagation, also known as plant tissue culture, is a method of asexual propagation of plants in
vitro. The stages of micropropagation include:

1. Initiation: In this stage, explants, which are small pieces of plant tissue, are taken from the
desired plant and sterilized to eliminate any contaminants. The explants are then placed on a
nutrient-rich agar medium to encourage cell division and callus formation.
2. Multiplication: Once the callus has formed, it is divided into small pieces and transferred to a
fresh nutrient medium. The pieces of callus continue to multiply and form new shoots and roots.
3. Rooting: The newly formed shoots are transferred to a rooting medium, where they develop
roots.
4. Acclimatization: The rooted plantlets are removed from the sterile culture conditions and placed
in a soil-based medium in a controlled environment to acclimate to the natural environment.

Advantages of micropropagation:

1. Rapid propagation: Micropropagation allows for the rapid multiplication of desirable plants in a
short period.
2. Uniformity: All the plants produced through micropropagation are genetically identical,
providing uniformity in plant characteristics.
3. Disease-free propagation: The sterile conditions of tissue culture ensure that the plantlets are
disease-free.
4. Conservation of endangered species: Micropropagation can be used to propagate and conserve
endangered plant species.

Limitations of micropropagation:

1. Cost: Micropropagation can be expensive due to the need for specialized equipment and sterile
conditions.
2. Genetic stability: While micropropagation produces genetically identical plants, there is a risk of
genetic instability and somaclonal variation.
3. Labor-intensive: Micropropagation requires specialized skills and can be labor-intensive,
especially during the initiation stage.
4. High failure rates: There is a high failure rate associated with the process of micropropagation,
especially during the rooting stage.
Overall, micropropagation offers many advantages in the production of desirable plants, but its
limitations must be taken into account for successful implementation.

28. Write the differences between micropropagation and plant regeneration .

Feature Micropropagation Plant Regeneration

1. Definition A type of plant tissue culture Any process by which a plant
that uses small pieces of plant is able to regrow from a
tissue to produce clones of damaged or destroyed part.
the original plant.
2. Purpose To produce clones of plants To help plants recover from
that are difficult to grow from damage caused by pests,
seed or that are rare or diseases, or environmental
endangered. factors.
3. Techniques Uses plant tissue culture Can be induced naturally or
techniques to induce callus through tissue culture
formation and organogenesis. techniques.
4. Applications Used to propagate plants for Used to propagate rare or
commercial purposes, such as endangered plants, produce
for the ornamental plant plants with desired traits, and
industry or for the production help plants recover from
of food crops. damage.
Micropropagation and plant regeneration are two tissue culture techniques that involve the
multiplication of plant cells, tissues, or organs under controlled conditions. Although they have
some similarities, there are also several differences between the two techniques:

1. Definition: Micropropagation involves the in vitro multiplication of plant material to

produce large numbers of genetically identical plants, while plant regeneration involves
the in vitro development of whole plants from single cells or small tissue explants.
1. Purpose: The primary purpose of micropropagation is to produce large numbers of
identical plants for commercial or research purposes, while the main purpose of plant
regeneration is to produce genetically modified plants or to regenerate plants from
cells or tissues that have been damaged or lost.
2. Starting material: In micropropagation, the starting material is usually a whole plant, a
shoot, or a piece of a shoot, while in plant regeneration, the starting material can be a
single cell or a small tissue explant.
3. Multiplication rate: Micropropagation typically involves a high multiplication rate,
with numerous plantlets produced from a single explant, while plant regeneration
usually involves a lower multiplication rate, with only a few plantlets produced from a
single cell or explant.
 4. Developmental stages: Micropropagation involves the multiplication and rooting of
the cultured plant material, while plant regeneration involves the induction and
development of shoots and roots from single cells or tissue explants.
5. Applications: Micropropagation is primarily used for the mass production of plants for
commercial purposes, while plant regeneration is used for the genetic transformation
of plants and the production of transgenic plants with desirable traits.

Overall, micropropagation and plant regeneration are both important tissue culture techniques
with different applications and methods of operation.
29. Write the steps involved in micropropagation .
Q-28.1
Micropropagation is a tissue culture technique that involves the aseptic multiplication of plant
cells, tissues, or organs under controlled conditions to produce large numbers of genetically
identical plants. The process of micropropagation typically involves the following steps:
1. Selection of the plant material: The first step in micropropagation is to select the plant
material that will be used for propagation. This may involve selecting specific plant
cultivars or varieties that have desirable traits.

2. Surface sterilization: The selected plant material is then subjected to surface sterilization
to remove any contaminants that may be present on the surface of the plant tissue. This
is typically done using a series of washes in disinfectants such as bleach or ethanol.
3. Culture initiation: Once the plant material is sterilized, it is placed on an appropriate
culture medium that provides the necessary nutrients and growth factors for the plant
material to grow. The culture medium typically consists of a mixture of inorganic salts,
vitamins, sugars, and plant growth regulators.
4. Multiplication: After the culture is initiated, the plant material is allowed to grow and
multiply in vitro. This is typically achieved through the repeated subculture of the plant
material onto fresh culture media, which stimulates the proliferation of new shoots or
other plant structures.
5. Rooting: Once sufficient multiplication has been achieved, the plantlets are induced to
root. This is typically done by transferring the plantlets to a rooting medium that
contains higher levels of auxin plant growth regulators.
6. Acclimatization: Finally, the rooted plantlets are removed from the culture vessel and
transferred to a soil mixture for acclimatization to normal environmental conditions. This
may involve gradually increasing the light and humidity levels to prepare the plantlets
for life outside the laboratory.
Overall, the process of micropropagation requires careful attention to the sterile techniques
involved in plant tissue culture and the optimization of culture conditions to promote the
proliferation and growth of the cultured plant material.
30. Define synthetic seed What do you mean by encapsulated seeds ? ? Write the
characteristics and types of synthetic seed .
Ps-L-8
Artificial seed or synthetic or encapsulated seeds are somatic embryo covered with a protective
gel.
Synthetic seed is a novel technology in plant tissue culture that involves encapsulating somatic
embryos, shoot buds, or aggregates of cells in a protective coating material to produce a seed-
like structure. This technology offers a number of advantages over traditional methods of
propagation, including the potential for mass propagation, storage, and transport of plant
materials.
The main characteristics of synthetic seeds are as follows:
1. Encapsulation: Synthetic seeds are characterized by their encapsulation of somatic
embryos or other plant material in a protective coating material. This coating material
may be a gel, alginate, or other substance that can protect the plant material from
external stresses.
2. Similarity to natural seed: Synthetic seeds resemble natural seeds in size, shape, and
appearance, and can be treated in a similar manner to natural seeds during sowing and
germination.
3. Germination ability: Synthetic seeds have the potential to germinate and grow into
mature plants under appropriate conditions, making them a valuable tool for mass
propagation of genetically identical plant material.
There are two main types of synthetic seeds:
1. Artificial seed: Artificial seed is produced by encapsulating somatic embryos or other
plant material in a protective coating material that contains nutrients and growth
regulators to support growth and development.
2. Encapsulated somatic embryos: Encapsulated somatic embryos are produced by
encapsulating somatic embryos in a protective coating material that provides support
and protection during germination and early growth.
In both cases, the main benefits of synthetic seed technology are the potential for mass
propagation, storage, and transport of plant material, as well as the ability to produce
genetically identical plants with desirable traits.
31. ***Briefly discuss the steps of synthetic seed production procedure . Write the advantages
of synthetic seeds over somatic embryos for propagation
PS- L-8
The production of synthetic seeds involves the encapsulation of somatic embryos or other plant
material in a protective coating material. The following are the general steps involved in
synthetic seed production
1. Culture initiation: The plant material is first initiated in vitro on an appropriate culture
medium. This may involve the culture of somatic embryos, shoot buds, or other plant
aggregates.
2. Encapsulation: Once the plant material has been cultured, it is encapsulated in a
protective coating material that can provide support and protection during germination
and early growth. This may involve the use of alginate, gelatin, or other substances that
can form a gel-like matrix around the plant material.
3. Maturation: After encapsulation, the synthetic seeds are matured on an appropriate
culture medium that provides the necessary nutrients and growth regulators to support
the development of the enclosed plant material.
4. Drying: Once the synthetic seeds have matured, they are dried and stored until they are
ready to be used for propagation.
The advantages of synthetic seeds over somatic embryos for propagation include:
1. Ease of handling: Synthetic seeds are easier to handle and transport than somatic
embryos, as they are encapsulated in a protective coating that can prevent damage
during transport.
2. Longer shelf life: Synthetic seeds can be stored for longer periods of time than somatic
embryos, which can be more sensitive to environmental stresses.
3. Ease of storage: Synthetic seeds can be stored at room temperature, while somatic
embryos require specialized storage conditions.
4. Uniformity: Synthetic seeds produce uniform plants, as each seed contains a genetically
identical plant embryo.
5. Reduced risk of contamination: Synthetic seeds reduce the risk of contamination during
propagation, as the encapsulation process can help to prevent contamination by
pathogens and other microorganisms.
Overall, encapsulation of matured somatic embryos is a relatively simple and straightforward
process that can be used to produce large numbers of synthetic seeds for propagation.
32. How do you encounter browning of medium during micropropagation
Browning of the medium during micropropagation is a common problem that can occur due to the
release of phenolic compounds from the plant tissues. These phenolic compounds can react with the
medium components and cause discoloration, which can negatively impact the growth of the cultured
plant material.
To prevent browning of the medium during micropropagation, various approaches can be used,
including:

1. Using activated charcoal: Activated charcoal can adsorb the phenolic compounds released from
the plant tissues and prevent their oxidation, thus reducing browning of the medium.
2. Using antioxidants: Adding antioxidants, such as ascorbic acid or sodium metabisulfite, to the
medium can help to scavenge the reactive oxygen species generated during browning reactions
and prevent discoloration.
3. Using different culture media: Different culture media formulations can have different effects on
browning, and selecting a suitable medium can help to reduce the occurrence of browning.

Overall, the prevention of browning in the medium during micropropagation is crucial for maintaining
healthy, proliferating plant cultures, and several approaches can be used to mitigate this problem.

Here are some key differences between zygotic embryos and somatic embryos:
1. Origin: Zygotic embryos are formed by fertilization of the egg cell and the sperm cell,
while somatic embryos are formed from somatic cells.
2. Ploidy: Zygotic embryos are usually diploid, while somatic embryos can be haploid,
diploid, or polyploid depending on the type of cells used.
3. Genetic variation: Zygotic embryos are the result of sexual reproduction and thus have
genetic variation due to the mixing of genetic material from two different parents, while
somatic embryos are produced asexually and have the same genetic makeup as the
parent plant.
4. Developmental potential: Zygotic embryos have the ability to develop into a mature
plant, while somatic embryos may or may not have the same developmental potential.
In summary, somatic embryogenesis is a process by which somatic cells are induced to
differentiate into embryos, resulting in the formation of somatic embryos. Zygotic embryos, on
the other hand, are formed by the fertilization of the egg cell and the sperm cell and have
different characteristics compared to somatic embryos.

. SEED

33. Briefly paraphrase artificial seed production through plant tissue culture .
Q-31
34. Write the production method of stress résistant plant in the plant biotechnology laboratory
GEB , SUST .
35. Write down the scopes . objectives and applications of encapsulated seed in agriculture . .
Scopes, Objectives, and Applications of Encapsulated Seeds in Agriculture:
Scopes:
1. Propagation: Encapsulated seeds offer a means to efficiently propagate plants and
produce a large number of identical plantlets. This method allows for the rapid
multiplication of desirable plant varieties with consistent traits.
2. Germplasm Preservation: Encapsulated seeds provide a means to preserve valuable
germplasm for long-term storage and conservation. The encapsulation process helps
maintain the viability and genetic integrity of plant material, enabling the preservation
of rare, endangered, or genetically modified plant varieties.
3. Seed Banking: Encapsulated seeds can be used for seed banking, allowing for the long-
term storage of diverse plant species. This contributes to the conservation of plant
biodiversity and serves as a valuable resource for future research, breeding programs,
and restoration efforts.
Objectives:
1. Storage and Transportability: Encapsulated seeds are designed to withstand storage and
transportation, providing a convenient method to store and distribute plant material.
They offer improved shelf-life compared to traditional seeds or plant tissues, ensuring
that valuable genetic resources remain viable and accessible over extended periods.
2. Protection and Germination Enhancement: The primary objective of encapsulation is to
provide a protective barrier around the enclosed plant material. The coating shields the
seeds from desiccation, physical damage, microbial contamination, and other adverse
environmental conditions. Additionally, encapsulation matrices can be formulated to
enhance germination rates and promote early seedling development.
Applications:
1. Plant Breeding: Encapsulated seeds find applications in plant breeding programs for
clonal propagation, allowing for the mass production of genetically superior individuals.
This facilitates the rapid dissemination of improved varieties, including disease-resistant,
high-yielding, or agronomically desirable plants.
2. Crop Improvement: Encapsulated seeds offer a tool for the propagation and distribution
of elite cultivars, enabling farmers to access high-quality planting material for improved
crop production and productivity. They also aid in the dissemination of new varieties
with desirable traits, such as drought tolerance, nutrient efficiency, or pest resistance.
3. Genetic Engineering and Biotechnology: Encapsulated seeds play a crucial role in the
field of genetic engineering and biotechnology. They enable the delivery and expression
of transgenes, facilitating the production of genetically modified plants with novel traits,
such as herbicide resistance or enhanced nutritional content.
4. Ecological Restoration and Conservation: Encapsulated seeds have applications in
ecological restoration projects, where they can be used to propagate native or
 endangered plant species for habitat restoration and conservation efforts. They provide
a means to reintroduce or establish plant populations in degraded or threatened
ecosystems.
In summary, encapsulated seeds offer diverse scopes and objectives in agriculture,
encompassing propagation, germplasm preservation, storage, protection, and enhancement of
plant material. Their applications range from plant breeding and crop improvement to genetic
engineering, ecological restoration, and conservation, contributing to the advancement and
sustainable management of agricultural systems.

7. Protoplast Culture: Isolation, purification and culture of protoplast, development and application of somatic hybrids
and cybrids.

36.What is protoplast culture ? Define hybrids and cybrids .

Protoplast culture refers to a technique in plant tissue culture where the cell wall is
enzymatically removed from plant cells, resulting in individual naked cells called protoplasts.
These protoplasts can be cultured in a suitable nutrient medium to regenerate into whole plants
or used for various other purposes, such as fusion with other protoplasts or genetic
modification.
In genetics, hybrids are the offspring resulting from the crossbreeding of two genetically distinct
individuals or varieties of the same species.
Cybrids, on the other hand, are hybrids that are formed through the fusion of protoplasts from
different species or even different genera.
36.1. What is protoplast ?*** Shortly state about different methods of protoplast isolation .
Define somatic hybridization.
A protoplast is a single cell with its surrounding cell wall removed. Protoplasts can be isolated
from plant, fungal, or bacterial cells. They are often used in research to study cell biology,
genetics, and biotechnology.
Somatic hybridization is a technique used to create a hybrid cell by fusing two or more somatic
cells. Somatic cells are any cells in a multicellular organism that are not reproductive cells. This
technique can be used to create new varieties of plants and animals with desired traits.

AS-p-21

Sure. There are two main methods for protoplast isolation: mechanical and enzymatic.
1. Mechanical method
 The mechanical method is the oldest method for protoplast isolation. It involves
physically removing the cell wall from plant cells using a sharp blade or a microscalpel.
This method is not very efficient and can damage the protoplasts, making them difficult
to culture.
1. Enzymatic method
The enzymatic method is the most common method for protoplast isolation. It involves
using enzymes to digest the cell wall of plant cells. The enzymes used for protoplast
isolation are typically a mixture of cellulase, hemicellulase, and pectinase. These
enzymes break down the different components of the cell wall, leaving the protoplasts
intact.
There are two main types of enzymatic protoplast isolation methods: sequential and
simultaneous.
Sequential method
The sequential method involves using two different enzymes to digest the cell wall. In the first
step, pectinase is used to break down the middle lamella, which is the layer of material that
holds cells together. In the second step, cellulase is used to break down the cellulose
microfibrils, which are the main component of the cell wall.
Simultaneous method
The simultaneous method involves using a mixture of enzymes to digest the cell wall. This
method is faster than the sequential method and can produce a higher yield of protoplasts.
Once protoplasts have been isolated, they need to be cultured in a special medium that
provides them with the nutrients they need to survive and grow. Protoplasts can be cultured in
liquid medium or on solid medium. Liquid medium is often used for short-term culture, while
solid medium is often used for long-term culture.
Protoplast culture is a valuable tool for plant biotechnology. It can be used to generate new
plants with desired traits, such as resistance to herbicides or diseases. It can also be used to
study plant cell biology and development.

37.Describe the drug sensitivity method for the selection of somatic hybrid cell .
38. Describe a biochemical method for the selection of somatic hybrid cell .

37. *How do you assess protoplast viability ?

There are several methods for assessing protoplast viability. Some of the most common methods include:

2. Evans blue dye exclusion assay: This assay is based on the fact that dead protoplasts will take up
Evans blue dye, while live protoplasts will not. To perform this assay, protoplasts are incubated
with Evans blue dye for a certain period of time. The number of protoplasts that have taken up
the dye is then counted. The percentage of live protoplasts is calculated by dividing the number
of protoplasts that have not taken up the dye by the total number of protoplasts.
3. Fluorescein diacetate (FDA) assay: This assay is based on the fact that live protoplasts will take
up fluorescein diacetate (FDA), which is a fluorescent dye. To perform this assay, protoplasts are
incubated with FDA for a certain period of time. The protoplasts are then examined under a
fluorescent microscope. The percentage of live protoplasts is calculated by dividing the number
of protoplasts that are fluorescent by the total number of protoplasts.
4. Cell counting: This method is based on the fact that live protoplasts will be able to divide and
grow, while dead protoplasts will not. To perform this assay, protoplasts are counted at different
time points. The percentage of live protoplasts is calculated by dividing the number of
protoplasts that have divided by the total number of protoplasts.
The choice of method for assessing protoplast viability will depend on the specific needs of the researcher. The Evans blue dye exclusion assay
and the FDA assay are both relatively simple and quick to perform. The cell counting method is more time-consuming, but it can provide more
accurate information about the viability of the protoplasts.

39. State the PEG mediated protoplast fusion techniques . Shortly narrate PEG mediated
protoplast 2 + fusion with its advantages .
Ap-p27
#A heterokaryon is a cell that contains multiple nuclei, each with a different genetic makeup.
This can occur naturally, such as in the mycelium of fungi, or it can be artificially induced, such
as in the production of hybridomas.
PEG-mediated protoplast fusion is a technique used in genetic engineering and plant breeding
to combine the genetic material of two different protoplasts, which are plant cells with their cell
walls removed. This fusion process involves the use of polyethylene glycol (PEG), a substance
that helps the protoplasts adhere and merge together.
The procedure begins by isolating protoplasts from two different plant sources. The protoplasts
are then mixed together in a solution containing PEG. The PEG acts as a fusogen, promoting the
adhesion of the protoplasts and facilitating their fusion. Through this process, the plasma
membranes of the protoplasts merge, allowing the exchange of genetic material.
PEG-mediated protoplast fusion offers several advantages in plant genetic engineering. First, it
enables the combination of genetic traits from different plant species or varieties that may be
difficult or impossible to crossbreed using conventional methods. This allows for the transfer of
desired traits, such as disease resistance or increased yield, between plants that would not
naturally interbreed.
Furthermore, this technique offers a rapid means of generating genetic diversity and creating
new plant hybrids. It bypasses the lengthy process of traditional breeding methods, which often
involve multiple generations and strict selection criteria. Protoplast fusion can result in the
direct production of novel hybrid plants with desirable characteristics.
Additionally, PEG-mediated protoplast fusion allows for the incorporation of genes from
different species, including those from unrelated organisms. This opens up the possibility of
introducing beneficial traits from non-plant sources, such as genes encoding enzymes or
proteins with specific functions. It enables the creation of genetically modified plants that
exhibit improved agronomic traits, increased nutritional value, or enhanced tolerance to biotic
and abiotic stresses.
In summary, PEG-mediated protoplast fusion is a valuable tool in plant genetic engineering,
enabling the combination of genetic material from different plant sources, facilitating the
generation of novel hybrids, and allowing the incorporation of genes from unrelated organisms.
This technique provides a means to accelerate the development of new plant varieties with
desired traits, ultimately contributing to the advancement of agriculture and crop improvement.
40 . Briefly discuss the process of electrofusion method of protoplast
As-p28
41. What do you mean by symmetric and asymmetric hybrids ?
Symmetric and asymmetric hybrids are two types of hybrids that can be formed when two cells
with different genetic makeups fuse.
In a symmetric hybrid, the two cells contribute an equal number of chromosomes to the hybrid
cell. This means that the hybrid cell has a complete set of chromosomes from each parent cell.
Symmetric hybrids are usually fertile and can reproduce.
In an asymmetric hybrid, one cell contributes more chromosomes to the hybrid cell than the
other cell. This means that the hybrid cell has an incomplete set of chromosomes from one
parent cell. Asymmetric hybrids are usually sterile and cannot reproduce.

Here is a table that summarizes the key differences between symmetric and asymmetric
hybrids:

Characteristic Symmetric Hybrid Asymmetric Hybrid

Number of chromosomes Complete set from each Incomplete set from one
parent cell parent cell
Fertility Fertile Sterile
42. Briefly cite the biochemical methods of selecting somatic hybrid cells .
Biochemical methods are commonly employed to select and identify somatic hybrid cells, which
are hybrid cells formed by fusing protoplasts from different plant species or varieties. Here are
some of the biochemical methods used for the selection of somatic hybrid cells:

1. Enzyme Isozyme Analysis: Isozyme analysis involves studying the variation in enzyme
patterns among different plant species or varieties. By comparing the isozyme profiles of
parental lines and potential somatic hybrids, specific enzyme bands or patterns can be
used as markers to identify the presence of hybrid cells.

2. DNA Hybridization: DNA hybridization techniques, such as Southern blotting or DNA-DNA

hybridization, can be used to detect and identify hybrid cells. Probes specific to the
nuclear or chloroplast DNA of one parent species are labeled and used to hybridize with
the DNA of the putative somatic hybrids. The presence of hybridization signals indicates
the presence of hybrid cells.

3. Polymerase Chain Reaction (PCR): PCR can be used to amplify specific DNA sequences
from parental lines and potential somatic hybrids. Primers specific to certain genetic
markers or sequences from each parent are used, and the resulting PCR products are
analyzed to determine the presence of hybrid cells. PCR-based techniques like Random
Amplified Polymorphic DNA (RAPD) or Simple Sequence Repeats (SSR) analysis are
commonly employed.

4. Protein Analysis: Protein analysis techniques, such as SDS-PAGE (Sodium Dodecyl Sulfate-
Polyacrylamide Gel Electrophoresis), can be used to compare protein patterns between
parental lines and putative somatic hybrids. Differences in protein bands or patterns can
indicate the presence of hybrid cells.

5. HPLC (High-Performance Liquid Chromatography): HPLC can be used to analyze the

presence and quantity of specific metabolites or biochemical compounds in parental lines
and potential somatic hybrids. Differences in metabolite profiles or concentrations can
serve as markers to identify hybrid cells.

43.Write the concepts of cybrids with production process -polten ent

A cybrid is a cell that has been created by fusing a cell with a nucleus from one organism with a
cell with cytoplasm from another organism. The nucleus contains the cell's genetic material,
while the cytoplasm contains all of the other cellular components, including mitochondria.
Cybrids can be produced in a number of ways, but the most common method is to use a
technique called electrofusion. In electrofusion, the two cells are placed in a solution and then
subjected to an electric field. The electric field causes the cell membranes to fuse, creating a
single cell with the nucleus from one organism and the cytoplasm from the other organism
Cybrids are often created in order to study the effects of different genetic or environmental
factors on cell function. For example, cybrids can be used to study the effects of mitochondrial
mutations on cell death or disease. Cybrids can also be used to create cells that are resistant to
certain types of cancer treatments.
44. . Describe in brief the method of somatic hybridization . Give a diagrammatic representation
including hybridization and cybridization process . 7
Immg
Here is a brief overview of the method of somatic hybridization:

Somatic hybridization is a technique used to create a hybrid cell by fusing two different somatic
cells (cells that are not reproductive cells). This can be done using a variety of methods,
including:
Chemical fusion: This method uses chemicals to weaken the cell membranes of the two cells,
making them more likely to fuse.
Electrofusion: This method uses an electric field to force the cell membranes of the two cells to
fuse.
Virus-mediated fusion: This method uses a virus to deliver genes into the cells, which can then
fuse together.
Once the two cells have fused, they form a hybrid cell with the genetic material of both cells.
This hybrid cell can then be cultured to grow into a hybrid organism.

45. Write the methodology and application of cybridization

Sc Methodology of Cybridization:
1. Isolation of Protoplasts: Protoplasts are isolated from the desired parental species or
genera by enzymatic digestion of the cell walls. This involves treating plant tissues with
 cell wall-degrading enzymes, such as cellulases and pectinases, to release the
protoplasts.
2 . Cytoplasmic Donor Selection: A cytoplasmic donor plant is chosen for its desirable
cytoplasmic traits, such as enhanced disease resistance, higher yield potential, or altered
metabolic pathways. The cytoplasmic donor should possess the desired mitochondrial
and/or chloroplast DNA.
3. Protoplast Fusion: The isolated protoplasts from the nuclear donor and cytoplasmic
donor are mixed together. Fusion is induced using methods like chemical fusogens (e.g.,
PEG), electrical stimulation, or specialized fusion-inducing agents. These techniques
promote the fusion of the protoplast plasma membranes, resulting in hybrid cybrids.
4. Regeneration of Cybrids: The fused protoplasts are cultured in a nutrient-rich medium
to support their growth and development. The medium contains essential nutrients,
growth regulators (e.g., auxins and cytokinins), and sometimes antibiotics or selective
agents to eliminate unfused protoplasts. The cultured protoplasts divide and
differentiate, leading to the development of mature cybrids.
5. Screening and Selection: The regenerated cybrids are screened and selected based on
the desired traits. This can involve molecular techniques, such as PCR or DNA
sequencing, to confirm the presence of the desired cytoplasmic DNA. Phenotypic
assessments are also conducted to evaluate the expression of specific traits associated
with the cytoplasmic genome.
Applications of Cybridization:
1. Cytoplasmic Male Sterility (CMS): Cybridization is commonly used in hybrid seed production.
By transferring cytoplasmic male sterility (CMS) traits from a donor plant to a nuclear male-
fertile line, it is possible to produce hybrid seeds without manual emasculation. This simplifies
the process and increases the efficiency of hybrid seed production.
2. Enhancement of Disease Resistance: Cybridization allows for the incorporation of cytoplasmic
traits associated with disease resistance. By combining the nuclear genome of a desired crop
plant with the cytoplasmic genome of a wild relative or another resistant species, plants with
improved resistance to pathogens can be developed.
3. Improvement of Yield and Quality Traits: Cybridization offers the opportunity to combine the
nuclear and cytoplasmic genomes from different parental species or genera to enhance yield
potential and improve quality traits. For example, cybrids can be created to improve
photosynthetic efficiency, nutrient uptake, or stress tolerance, leading to increased productivity
and crop quality.
4. Altered Metabolic Pathways: By transferring specific cytoplasmic genes or organelles,
cybridization can result in altered metabolic pathways in plants. This can be exploited for the
production of secondary metabolites, such as pharmaceuticals or industrial chemicals, or for
improving nutritional profiles in crops.
5. Fundamental Research: Cybridization is also used as a tool for fundamental research in plant
biology. By studying the interaction between nuclear and cytoplasmic genomes, researchers can
gain insights into organelle function, gene expression, and the mechanisms underlying
cytoplasmic inheritance.
Overall, cybridization has diverse applications in plant breeding and research. It allows for the
combination of desirable cytoplasmic traits with nuclear genomes, leading to the development
of improved crop varieties with enhanced traits, disease resistance, and increased productivity.
Here are some of the applications of cybridization:

1. Crop improvement: Cybridization can be used to improve crop yields, disease resistance,
and tolerance to environmental stress. For example, cybrid wheat plants have been
created that are more resistant to drought and pests.
2. Animal breeding: Cybridization can be used to improve animal breeding by creating
animals with desired traits, such as increased milk production or improved growth rate.
For example, cybrid dairy cows have been created that produce more milk.
3. Biomedical research: Cybridization can be used to study the effects of different genes on
cell function. For example, cybrid cells can be used to study the effects of genes involved
in cancer.
46 .Siquattle dis What are the applications of somatie hybridization ?
Somatic hybridization, also known as somatic cell fusion, is a technique used in plant breeding
to create hybrid plants by fusing protoplasts from different plant species or varieties. The
resulting somatic hybrids possess a combination of traits from both parental plants. The
applications of somatic hybridization are as follows:
1. Disease Resistance: Somatic hybridization can be employed to transfer disease
resistance traits from wild or resistant plant species to commercially valuable crops. By
fusing protoplasts from a susceptible crop plant with those from a resistant species, the
resulting somatic hybrids can exhibit improved resistance to various pathogens, such as
viruses, bacteria, and fungi.
2. Stress Tolerance: Somatic hybridization allows for the introduction of stress tolerance
traits into crop plants. By fusing protoplasts from plants that are adapted to specific
environmental conditions, such as drought, salinity, or extreme temperatures, somatic
hybrids with enhanced tolerance to these stresses can be developed.
3. Improved Yield and Quality Traits: Somatic hybridization can contribute to the
improvement of yield-related traits in crops. By combining the traits of different parental
 plants, such as increased biomass production, enhanced photosynthetic efficiency, or
altered hormonal responses, somatic hybrids can exhibit improved yield potential.
Additionally, somatic hybridization can be utilized to enhance the nutritional quality of
crops by introducing genes responsible for increased vitamin or mineral content.
4. Crop Improvement and Diversification: Somatic hybridization enables the creation of
novel hybrid plants that would not occur through conventional breeding methods. It
provides a means to combine genetic material from distantly related species, expanding
the genetic diversity available for crop improvement. This can lead to the development
of new crop varieties with unique traits, such as novel flavors, colors, or growth habits.
5. Heterosis (Hybrid Vigor): Somatic hybrids can exhibit heterosis, also known as hybrid
vigor, which is the phenomenon of increased vigor, growth, and productivity compared
to their parental lines. By combining complementary traits from different parental
plants, somatic hybrids can demonstrate enhanced growth, yield, and adaptability,
making them valuable for commercial crop production.
6. Fundamental Research: Somatic hybridization serves as a tool for studying various
aspects of plant biology and genetics. It allows researchers to investigate gene
expression patterns, organelle interactions, and nuclear-cytoplasmic interactions.
Somatic hybrids provide insights into genetic and molecular processes, contributing to a
deeper understanding of plant development and evolution.
Overall, somatic hybridization has broad applications in crop improvement, disease resistance,
stress tolerance, yield enhancement, and fundamental research. It offers a valuable approach
for developing improved crop varieties with desirable traits, contributing to sustainable
agriculture and food security.
47. Mention the causes of variations of the chromosomal number in somatic hybrids .
There are a number of factors that can cause variations in the chromosomal number of somatic
hybrids. These include:
1. Multiple fusions: When two or more protoplasts fuse, the resulting hybrid may have a
higher or lower chromosome number than either parent plant.
2. Chromosome loss: During cell division, chromosomes can be lost or damaged. This can
lead to a decrease in the chromosome number of the hybrid.
3. Chromosome duplication: During cell division, chromosomes can be duplicated. This can
lead to an increase in the chromosome number of the hybrid.
4. Chromosome rearrangement: The chromosomes in the hybrid can be rearranged. This
can lead to changes in the genes that are present in the hybrid.
48. State the culture method of protoplast.
R
49. What is sphaeroplast how can you purify protoplast .
A sphaeroplast is a type of cell that has been partially stripped of its cell wall, resulting in a
spherical shape.
Protoplasts, on the other hand, are plant cells that have had their cell walls enzymatically
removed, leaving behind the plasma membrane and other cellular components. Protoplasts can
be purified from plant tissues using the following steps:

1. Isolation of Protoplasts: Plant tissues, such as leaves or stem segments, are treated with
enzymes, such as cellulases and pectinases, to break down the cell wall and release the
protoplasts. The specific enzyme composition and incubation conditions depend on the
plant species and tissue type.
2. Filtration and Centrifugation: After the enzymatic digestion, the resulting cell suspension
is filtered through a fine mesh or nylon filter to remove any undigested cell debris or
large particles. The filtrate is then centrifuged at a low speed to pellet the protoplasts
while leaving behind the lighter components.

3. Washing and Resuspension: The protoplast pellet is gently resuspended in a suitable

washing buffer, typically a balanced salt solution, to remove any remaining enzymes, cell
debris, or contaminants. This washing step is usually repeated several times to ensure a
clean preparation of protoplasts.

4. Density Gradient Centrifugation (Optional): In some cases, density gradient

centrifugation can be used to further purify the protoplasts. A density gradient medium,
such as Percoll or Ficoll, is prepared with a range of densities. The protoplast suspension
is carefully layered on top of the density gradient and centrifuged. As a result,
protoplasts with different densities separate into distinct bands, allowing for the
collection of highly purified protoplasts from a specific band.

5. Viability Assessment: The purified protoplasts can be evaluated for viability using various
techniques, such as staining with vital dyes (e.g., fluorescein diacetate or propidium
iodide) or conducting plating assays to assess cell division and regeneration potential.
8. Production of disease free plants: Methods of virus elimination, virus indexing, eradication of pathogens other than virus, application and limitations.

50.What do you mean by disease free plant production?

Disease-free plant production refers to the cultivation and propagation of plants that are free
from pathogens and diseases. It involves implementing various measures and techniques to
prevent the introduction, spread, or persistence of harmful pathogens that can negatively
impact plant health and productivity.
The goal of disease-free plant production is to ensure that plants are healthy, vigorous, and free
from any known pathogens that could cause diseases.
This can be done through a variety of methods, including:
1. Meristem culture: This method involves isolating a small piece of tissue from the tip of a
plant's shoot (the meristem) and growing it in a sterile environment. The meristem is
typically free of diseases, so the plants that are grown from it will also be disease-free.
2. Soilless culture: This method involves growing plants in a sterile environment without
soil. This helps to prevent the spread of diseases that are transmitted through the soil.
3. Chemical treatment: Plants can be treated with chemicals to kill diseases. However, this
can be harmful to the environment and can also leave residues on the plants.
4. Genetic engineering: Plants can be genetically engineered to be resistant to diseases.
This is a relatively new technology, but it has the potential to significantly reduce the
incidence of plant diseases.
51. * *Write the reasons for escape of meristem from virus .
There are a few reasons why meristems may escape virus infection.
1. Vascular system: Viruses typically move through a plant's vascular system, which is
absent in meristems. This means that viruses cannot easily reach meristem cells.
2. High metabolite activity: Meristem cells are actively dividing, which requires a high level
of metabolism. This high metabolic activity may make it difficult for viruses to replicate
in meristem cells.
3. Antiviral defense mechanisms: Meristem cells may have antiviral defense mechanisms
that help to protect them from infection. These mechanisms may include the production
of antiviral proteins or the upregulation of genes that are involved in antiviral responses.
52. *Describe the production methods of virus - free plants in an ideal plant tissue culture
laboratory .
Virus-free plant production is essential for maintaining healthy crops and preventing the spread
of plant viruses. There are several methods for producing virus-free plants, including meristem
culture, shoot-tip culture, and thermotherapy.
1. One method is meristem culture, also known as shoot apical meristem (SAM) culture.
This method involves the aseptic excision of the apical meristem, which is the growing
tip of the plant shoot. The meristem is then placed on a nutrient-rich agar medium
containing plant growth regulators and antibiotics to prevent contamination. The
 meristem cells divide and differentiate, producing a virus-free plantlet that can be
transferred to soil.
2. Another method is shoot-tip culture, which involves the aseptic excision of a portion of
the shoot tip. The shoot tip is then placed on a nutrient-rich agar medium containing
plant growth regulators and antibiotics to prevent contamination. The shoot tip cells
divide and differentiate, producing a virus-free plantlet that can be transferred to soil.
3. Thermotherapy is another method used to produce virus-free plants. This method
involves exposing the infected plant tissue to elevated temperatures, which can either
kill the virus or inhibit its replication. The infected plant material is then cultured in vitro
to produce virus-free plantlets.
Overall, virus-free plant production is important for maintaining healthy crops and preventing
the spread of plant viruses. Meristem culture, shoot-tip culture, and thermotherapy are
effective methods for producing virus-free plants.
53. How would you use PCR technique for detecting virus in virus free plant production ? d .
Why is virus indexing important in in vitro culture system ?
PCR (polymerase chain reaction) is a molecular technique used to amplify and detect specific
DNA sequences, including those present in plant viruses. PCR can be used to detect the
presence of viruses in virus-free plant production by detecting viral DNA or RNA in plant tissue
samples.
To use PCR for virus detection in virus-free plant production, the following steps can be taken:
1. Collect plant tissue samples: Collect plant tissue samples from the plants that are
suspected to be virus-free. The samples should be collected aseptically and handled
carefully to prevent contamination.
2. Extract nucleic acids: Isolate DNA or RNA from the plant tissue samples using a
commercial kit or a standard protocol. This involves breaking open the cells and
separating the nucleic acids from other cellular components.
3. Design primers: Design PCR primers that are specific to the target virus. The primers
should be designed based on the viral genome sequence and should be able to amplify a
specific region of the viral genome.
4. Perform PCR amplification: Perform PCR amplification using the extracted nucleic acids
and the designed primers. The PCR reaction will amplify the target viral DNA or RNA if
present in the sample.
5. Analyze PCR products: Analyze the PCR products by gel electrophoresis to confirm the
presence or absence of the target virus.
By using PCR for virus detection in virus-free plant production, it is possible to detect viruses
that are not visible through visual inspection. This can help ensure the production of truly virus-
free plants and prevent the spread of plant viruses.
54. Write the methods of virus elimination through tissue culture .
Q-52
55. Write down the advantages and limitations of disease resistant plant in agriculture .
Advantages of Disease-Resistant Plants in Agriculture:

1. Increased Crop Yield: Disease-resistant plants exhibit improved resistance to various

pathogens, including viruses, bacteria, fungi, and nematodes. By reducing the impact of
diseases, resistant plants can achieve higher crop yields compared to susceptible counterparts.
This leads to increased productivity and improved food security.

2. Reduced Dependency on Chemical Pesticides: Disease-resistant plants can significantly

reduce the reliance on chemical pesticides for disease control. This has several benefits,
including lower production costs, decreased environmental pollution, and reduced risks to
human health and biodiversity.

3. Enhanced Crop Quality: Diseases can negatively impact crop quality, leading to reduced
market value and consumer acceptance. Disease-resistant plants can maintain better quality
attributes, such as improved color, texture, flavor, and nutritional content. This enhances the
marketability and economic value of the harvested crops.

4. Sustainable Agriculture: Disease-resistant plants play a crucial role in promoting sustainable

agricultural practices. By minimizing disease incidence and severity, these plants contribute to
resource conservation, including water, energy, and soil health. They also help maintain
ecological balance by reducing the need for extensive pesticide use.

5. Long-Term Stability: Incorporating disease resistance into crop varieties provides long-term
stability and durability against specific pathogens. Unlike chemical control methods, which may
face challenges due to the development of resistance in pathogens, disease-resistant plants
offer a sustainable and stable solution for disease management.

6. Reduced Crop Losses: Plant diseases can cause significant crop losses, leading to economic
hardship for farmers and food scarcity. Disease-resistant plants can mitigate these losses by
reducing disease incidence and severity, thereby ensuring a more reliable and consistent food
supply.

Limitations of Disease-Resistant Plants in Agriculture:

1. Limited Spectrum of Resistance: Disease resistance in plants is often specific to certain

pathogens or strains. Developing resistance against a broad range of pathogens can be
challenging. Therefore, disease-resistant plants may still be susceptible to other diseases
that are not targeted by their specific resistance traits.

2. Potential for Pathogen Adaptation: Continuous cultivation of disease-resistant plants

can exert selective pressure on pathogens, potentially leading to the emergence of new
strains or races that can overcome the plant's resistance mechanisms. This necessitates
ongoing research and development efforts to stay ahead of evolving pathogen
populations.

3. Genetic Uniformity: In some cases, disease-resistant plants are derived from a limited
number of genetic sources or possess a narrow genetic base. This can result in genetic
uniformity, making the plant population susceptible to new diseases or susceptible to
changes in the pathogen population.

4. Time and Resources for Development: Developing disease-resistant plant varieties

requires significant time, expertise, and resources. It involves extensive breeding
programs, field trials, and rigorous testing to ensure the stability, efficacy, and safety of
the resistant traits. The development process can be time-consuming and costly.

5. Compatibility with Other Traits: Disease resistance traits may not always be
compatible with other desirable traits, such as high yield, desirable agronomic
characteristics, or market preferences. It is important to carefully consider and balance
multiple traits during the breeding process to ensure that disease resistance does not
compromise other important traits.
While disease-resistant plants offer numerous advantages, it is important to address the
limitations and challenges associated with their development and deployment. By combining
disease resistance with other breeding objectives and implementing integrated pest
management strategies, sustainable and resilient crop production systems can be achieved.
56. Briefly describe the ways to eliminate viruses in laboratory
lhlhjkl In the laboratory, there are several methods used to eliminate viruses and ensure a virus-
free environment. These methods aim to either inactivate or remove viruses from samples,
surfaces, or laboratory equipment. Here are some commonly employed techniques:

1. Heat Treatment: Viruses can be heat-inactivated by subjecting them to high

temperatures. The specific temperature and duration required for effective inactivation
vary depending on the virus. Typically, incubating the samples at temperatures above
60-70°C for a specific period of time can achieve virus inactivation. Autoclaving, which
uses high-pressure steam, is another method for heat inactivation of viruses.

2. Chemical Inactivation: Various chemicals can be used to inactivate viruses. Commonly

used agents include aldehydes (e.g., formaldehyde), oxidizing agents (e.g., bleach or
hydrogen peroxide), phenols, and detergents. These chemicals disrupt the viral
structure, rendering them non-infectious. Proper safety protocols should be followed
when working with these chemicals.

3. Ultraviolet (UV) Irradiation: UV light in the germicidal range (UV-C) can effectively
inactivate viruses by damaging their genetic material. UV irradiation can be performed
using UV lamps or specific UV chambers. The duration and intensity of exposure should
be carefully controlled to ensure effective virus inactivation.

4. Filtration: Virus elimination through filtration involves passing the sample or solution
through filters with pore sizes small enough to trap and remove viruses. Filters with pore
sizes of 20-100 nanometers (nm) or smaller, such as membrane filters or ultrafiltration
membranes, can effectively remove viruses from liquid samples.

5. Chemical Disinfection: Disinfectants specifically formulated to target viruses can be

used to decontaminate surfaces and equipment. These disinfectants are designed to
 inactivate or destroy viral particles on contact. Examples include quaternary ammonium
compounds, chlorine-based disinfectants, and alcohol-based disinfectants.

6. Molecular Techniques: Molecular methods, such as polymerase chain reaction (PCR)

or other nucleic acid amplification techniques, can be employed to detect and eliminate
viral nucleic acids. By targeting and amplifying specific viral genetic material, these
techniques allow for the identification and removal of viral contaminants from samples
or cultures.

It is crucial to choose the appropriate virus elimination method based on the specific virus, the
type of sample or material being treated, and the intended downstream application.
Additionally, strict adherence to biosafety protocols and regulations is necessary to ensure the
safety of laboratory personnel and prevent the accidental release of infectious agents.
57. What do you mean by thermotherapy.*How do you detect banana bunchy top virus using
molecular techniques .
Thermotherapy refers to a treatment method that utilizes controlled heat to combat or manage
diseases in plants or organisms. It involves subjecting plants or plant parts to specific
temperature ranges for a predetermined duration to achieve therapeutic effects.
Thermotherapy has applications in plant disease management, particularly for controlling viral
infections.
In the context of plant diseases, thermotherapy is primarily used to eliminate or reduce viral
infections in infected plant material. The treatment is typically carried out in controlled
environments, such as growth chambers or greenhouses, where temperature and humidity can
be carefully regulated.
There are several molecular techniques that can be used to detect banana bunchy top virus
(BBTV) infection. These include:

1. Polymerase chain reaction (PCR): PCR is a technique that amplifies specific DNA
sequences. In the case of BBTV, PCR primers can be designed to target specific
sequences within the BBTV genome. This allows for the detection of even very low levels
of virus infection.
2. Loop-mediated isothermal amplification (LAMP): LAMP is a rapid and sensitive method
for detecting DNA that is similar to PCR, but it can be performed isothermally (at a
constant temperature). This makes it a more convenient and cost-effective option for
some applications.
 3. Reverse transcription PCR (RT-PCR): RT-PCR is a variation of PCR that is used to detect
RNA viruses. In the case of BBTV, RT-PCR is used to first convert the virus RNA into DNA,
which can then be amplified using PCR.
4. Immunohistochemistry (IHC): IHC is a method that uses antibodies to detect specific
proteins. In the case of BBTV, antibodies can be used to detect the virus protein in
infected plant tissue.
The choice of which molecular technique to use will depend on a number of factors, including
the availability of equipment and reagents, the desired level of sensitivity, and the cost.
58. What is virus indexing* ? How is it done ?
Virus indexing is a method of detecting viruses in plants. It is done by grafting a piece of tissue
from a plant suspected of being infected with a virus to a healthy plant called an indicator plant.
If the indicator plant develops symptoms of the virus, then the original plant is also infected.
Virus indexing is a technique used to detect and identify the presence of viruses in plants. It
involves the systematic testing of plant material to determine if it is infected with specific
viruses or virus groups. Virus indexing is an important tool in plant disease diagnosis, quarantine
measures, and ensuring the production of virus-free planting material.
Virus indexing is done by collecting plant material, extracting virus particles or viral nucleic acids
from the sample, and inoculating the extracted material onto indicator plants. The indicator
plants are monitored for the development of characteristic symptoms associated with viral
infection. Confirmation and identification of the virus can be done using serological assays or
molecular techniques.
59. Briefly describe two methods of virus indexing .
Sure. Here are two methods of virus indexing:
1. Biological indexing: This method involves grafting or budding a plant sample onto a
healthy plant. If the healthy plant becomes infected, then the original plant sample was
infected with a virus. This method is relatively slow and can be unreliable, but it is still
widely used because it is relatively inexpensive.
2. Molecular indexing: This method uses techniques such as polymerase chain reaction
(PCR) or enzyme-linked immunosorbent assay (ELISA) to detect the presence of viruses
in plant samples. These methods are much faster and more reliable than biological
indexing, but they can be more expensive.

Method Advantages Disadvantages

Biological indexing Inexpensive Slow, unreliable
Molecular indexing Fast, reliable Expensive
Here is a table that summarizes the advantages and disadvantages of each method:
60.Briefly discuss a low - cost virus indexing method for the management of viral pathogen .

9. Somatic embryo genesis and suspension culture: Initiation of somatic embryo: callus and suspension culture, maintenance of callus and suspension culture,
production and management of somatic embryo and its application, plant formation from somatic embryo.

61.* What do you mean by somatic embryogenesis ? *Differentiate between somatic embryo
and zygotic embryos .
R Somatic embryogenesis is a process in which plant cells are induced to form embryos without
fertilization. The resulting embryos can develop into whole plants.

62. What is suspension culture ? Write down the steps involve of suspension culture .
embryogenesis
Somatic embryogenesis is a process by which plants can be regenerated from somatic cells,
such as cells from leaves, stems, or roots. Somatic embryos are morphologically similar to
zygotic embryos, which are embryos that develop from fertilized eggs.

The steps involved in suspension culture and somatic embryogenesis are as follows:

1. Inoculation: The first step is to inoculate the cells into the liquid medium. The cells can
be inoculated from a solid culture or from a single cell suspension.
2. Growth: The cells will then grow and divide in the liquid medium. The growth rate of the
cells will depend on the type of plant, the composition of the medium, and the
environmental conditions.
3. Induction of embryogenesis: Once the cells have reached a certain density, they can be
induced to undergo somatic embryogenesis. This can be done by changing the
composition of the medium or by adding plant growth regulators.
4. Embryogenesis: The cells will then begin to form embryos. The embryos will grow and
develop into plantlets.
5. Plantlet regeneration: Once the embryos have reached a certain size, they can be
transferred to solid medium and allowed to regenerate into plants.
63. . What are the types of suspension culture ? State the maintenance of suspension culture .
Suspension cultures are a type of plant cell or tissue culture where cells are grown in a liquid medium
without attachment to a solid surface. There are two main types of suspension cultures:
 1. Batch Suspension Culture: In a batch suspension culture, cells are initially inoculated into
a fixed volume of liquid medium in a culture vessel. The cells proliferate and grow in the
medium until the available nutrients are depleted or waste products accumulate,
limiting further growth. Batch cultures require regular monitoring and periodic
subculturing to maintain cell viability and prevent cell death due to nutrient exhaustion
or toxic metabolite accumulation.
2. Continuous Suspension Culture: Continuous suspension cultures, also known as
chemostat cultures, are designed to maintain cells in a continuous state of growth. In
this system, fresh medium is continuously supplied to the culture vessel, while an equal
volume of spent medium containing cells and metabolites is removed. This allows for a
steady-state condition where cells can grow continuously without nutrient depletion or
accumulation of inhibitory metabolites. Continuous cultures require careful monitoring
of nutrient levels, growth rates, and cell density to maintain optimal conditions.

Maintenance of Suspension Cultures:

To maintain suspension cultures, certain steps and considerations need to be followed:

1. Subculturing: Regular subculturing is necessary to prevent cell overcrowding and maintain

optimal growth conditions. Cells are transferred to fresh medium to dilute out accumulated
waste products, provide fresh nutrients, and maintain an appropriate cell density.

2. Nutrient Supply: Suspension cultures require a balanced nutrient supply to support cell
growth. The medium should contain essential nutrients such as sugars, amino acids, vitamins,
and minerals. The medium composition may vary depending on the specific cell or tissue type
being cultured.

3. pH and Osmolality Control: The pH of the medium should be monitored and maintained
within an appropriate range to support cell growth. Osmolality, which affects the osmotic
balance of the medium, should also be controlled to ensure cell health and prevent osmotic
stress.

4. Agitation: Suspension cultures require continuous agitation or stirring to keep the cells
suspended and prevent settling. This promotes efficient nutrient and gas exchange and helps
maintain a homogeneous culture.
5. Sterility: Maintaining sterility is crucial to prevent contamination and maintain the integrity of
the suspension culture. Strict aseptic techniques should be followed during media preparation,
subculturing, and handling of the cultures.

6. Monitoring and Analysis: Regular monitoring of cell viability, growth rates, cell density, and
metabolite levels is important for assessing culture health and making necessary adjustments to
the growth conditions.

By following these maintenance procedures, suspension cultures can be sustained for an

extended period, allowing for the production of desired metabolites, studying cellular
processes, or scaling up for industrial applications.
64. Why and how encapsulation of matured somatic embryos is done ?
Encapsulation of matured somatic embryos is done to produce synthetic seeds. Synthetic seeds
are a novel technology in plant tissue culture that involves encapsulating somatic embryos,
shoot buds, or aggregates of cells in a protective coating material to produce a seed-like
structure.
Encapsulation involves the use of a gel-like matrix material, such as sodium alginate or gelatin,
to encapsulate the matured somatic embryos. The encapsulation material provides support and
protection to the somatic embryos during the germination and early growth stages. The
encapsulated embryos are then matured on an appropriate culture medium that provides the
necessary nutrients and growth regulators to support their development.
The advantages of encapsulating matured somatic embryos to produce synthetic seeds include:
1. Mass propagation: Synthetic seeds can be produced in large quantities, allowing for
mass propagation of desirable plant material.
2. Easy handling and storage: Synthetic seeds are easier to handle and store than somatic
embryos, as they can be stored at room temperature and do not require specialized
storage conditions.
3. Reduced risk of contamination: Encapsulation can help to reduce the risk of
contamination by pathogens and other microorganisms during propagation.
4. Uniformity: Synthetic seeds produce uniform plants, as each seed contains a genetically
identical plant embryo.
5. Transportability: Synthetic seeds can be transported over long distances without
damage, allowing for the distribution of plant material to different locations.
Encapsulation of matured somatic embryos is typically done using a gel-like matrix material,
such as sodium alginate or gelatin. The following steps are involved in encapsulation of matured
somatic embryos:
1. Preparation of the encapsulation material: The encapsulation material, such as sodium
alginate or gelatin, is prepared by dissolving it in an appropriate solvent to form a gel-like
matrix.
2. Preparation of the somatic embryos: The somatic embryos are matured on an
appropriate culture medium until they are ready for encapsulation.
3. Coating the somatic embryos: The somatic embryos are gently mixed with the
encapsulation material to coat them evenly.
4. Dropping the coated embryos into a solution: The coated somatic embryos are then
carefully dropped into a solution containing a divalent cation, such as calcium chloride or
barium chloride. The divalent cation reacts with the encapsulation material, causing it to
form a gel-like coating around the somatic embryos.
5. Hardening of the gel coat: The coated somatic embryos are then left in the solution
containing the divalent cation for a specific period of time, allowing the gel coat to
harden.
6. Washing: After the gel coat has hardened, the coated somatic embryos are washed with
an appropriate solution to remove any excess encapsulation material and divalent
cation.
7. Storage: The encapsulated somatic embryos, or synthetic seeds, can then be stored in an
appropriate container until they are ready to be used for propagation.
65. Define somatic embryogenesis . Compare zygotic embryos and somatic embryos
Somatic embryogenesis is the process by which somatic cells, which are any cells of a plant
except for the germ cells, are induced to undergo embryogenesis, resulting in the formation of
somatic embryos. Somatic embryogenesis can be induced in vitro by culturing plant cells or
tissues in appropriate media containing growth regulators and other supplements.
Zygotic embryos are formed by the fertilization of the egg cell by the sperm cell, resulting in the
formation of an embryo that develops into a mature plant. On the other hand, somatic embryos
are formed by the direct differentiation of somatic cells into embryos, without the need for
fertilization.

10. Culture of Anther/pollen, Ovule, Embryo, Endosperm and Their Uses: Rice, wheat,
barley, maize, brinjal.

65. Define anther culture . What are the factors affecting androgenesis ?
Anther culture is a technique used in plant tissue culture that involves the culture of anthers,
the male reproductive organs of plants, in vitro in order to induce androgenesis, the
development of haploid male gametes, which can be used for the production of haploid plants.
Factors affecting androgenesis in anther culture include:
1. Genotype: The ability of anthers to produce androgenic embryos is genotype-
dependent, with some genotypes being more responsive than others.
2. Stage of anther development: The stage of anther development at the time of culture is
important, as anthers at certain stages are more responsive to culture than others.
3. Culture medium: The composition of the culture medium, including the types and
concentrations of plant growth regulators and other supplements, can affect
androgenesis in anther culture.
4. Temperature: The temperature at which anthers are cultured can affect androgenesis,
with optimal temperatures varying depending on the genotype and stage of anther
development.
5. Light: The presence or absence of light during anther culture can also affect
androgenesis, with some genotypes showing better responses under dark conditions
while others require light.
6. pH: The pH of the culture medium can affect androgenesis in anther culture, with
optimal pH values varying depending on the genotype and stage of anther development.
In summary, anther culture is a technique used for inducing androgenesis in plants, which can
be used for the production of haploid plants. Factors affecting androgenesis include genotype,
stage of anther development, culture medium composition, temperature, light, and pH.
66. Specify the process of haploid plant production through anther culture
*** 1) Selection of explants(eg. Flower bud)
2) preparation of explant
3) disinfection of bud
4) selected buds are pretreated
5) surface sterlization
6) inoculation
7) transfer to culture room
8) transplanted to small pots in greenhouse
***
Haploid plant production through anther culture involves the following steps:
 1. Selection of appropriate donor plant: Plants with high androgenic potential are selected as
donors for anther culture.
2. Preparation of explants: Anthers are carefully excised from the selected donor plants and surface
sterilized to prevent contamination.
3. Inoculation of explants: The anthers are cultured on a nutrient-rich medium containing plant
growth regulators that induce androgenesis.
4. Induction of embryogenic callus: Under optimal conditions, the anthers start to divide and form
embryogenic callus, which contains androgenic embryos.
5. Selection and regeneration of haploid plants: The androgenic embryos are screened for haploidy
using chromosome counting or molecular markers. Haploid embryos are then cultured on a
suitable medium to allow for plant regeneration.
6. Rooting and acclimatization: The regenerated haploid plants are transferred to a rooting medium
to promote root development, and then gradually acclimatized to the external environment
before being transferred to the field.

Overall, the production of haploid plants through anther culture is a complex process that
requires careful selection of donor plants, preparation of explants, induction of
androgenesis, screening for haploidy, and regeneration of haploid plants. The success of the
process depends on a variety of factors, including the genotype of the donor plant, culture
conditions, and the expertise of the researcher.
67. . Give a diagrammatic representation of anther culture for producing haploid plant

68. What is anther and pollen culture? What are the advantages of pollen culture over anther
culture ?
Anther and pollen culture are two types of in vitro culture techniques used for plant breeding
and genetic improvement.
Anther culture involves the culture of anthers, which are the male reproductive structures
found in flowers, on a nutrient-rich medium in aseptic conditions. This method allows for the
development of haploid plants or callus tissues that can be used for genetic analysis, mutation
breeding, and somatic cell hybridization.
Pollen culture, on the other hand, involves the culture of pollen grains, which are the male
gametophytes of plants, in a nutrient medium. This method is used to produce haploid plants,
which can be used for breeding programs, genetic engineering, and the study of plant genetics.
Advantages of pollen culture over anther culture include:
Here is a table comparing the advantages of pollen culture over anther culture:

Feature Pollen culture Anther culture

Simpler Pollen culture is a simpler technique than Anther culture is a more complex
anther culture. It is easier to isolate technique than pollen culture. It
 pollen grains than anthers, and pollen requires the use of hormones to
culture does not require the use of induce embryogenesis, and it can be
hormones. difficult to isolate anthers from the
plant without damaging them.
More Pollen culture is more efficient than Anther culture is less efficient than
efficient anther culture. It is possible to produce pollen culture. It is possible to
more haploid plants from a single pollen produce fewer haploid plants from a
culture than from a single anther culture. single anther culture than from a
single pollen culture.
Can be used Pollen culture can be used with difficult- Anther culture can be used with
with to-grow species. Anther culture is not as most species of flowering plants.
difficult-to- effective with difficult-to-grow species, as However, pollen culture may be
grow the anthers may not develop properly. more effective with difficult-to-grow
species species.
1. Higher success rate: Pollen culture has a higher success rate compared to anther culture
since the pollen grains are more resilient and can tolerate a wider range of culture
conditions.
2. Shorter culture time: Pollen grains require a shorter culture time compared to anthers,
which can take several weeks to develop haploid plants.
3. Greater genetic variability: Pollen culture produces a wider range of genetic variability
compared to anther culture, which is limited to the genetic makeup of the donor plant.

In summary, both anther and pollen culture are important techniques for plant breeding and
genetic improvement. However, pollen culture has certain advantages over anther culture in
terms of success rate, culture time, and genetic variability.
69. Why haploid are useful in plant breeding ?
Haploids are useful in plant breeding for several reasons:

1. Rapid development of homozygous lines: Haploids carry only one set of chromosomes
and are therefore homozygous for all genes. This allows for the rapid development of
homozygous lines in a single generation, which would otherwise take several
generations to achieve through conventional breeding methods.

2. Creation of new genetic variability: Haploids can be induced to double their

chromosome number, resulting in the creation of new genetic variability that can be
used for plant breeding.
 3. Facilitation of genetic analysis: Haploids allow for the easier and more efficient analysis
of genetic traits and gene mapping, as they carry only one set of chromosomes.

4. Production of hybrid seeds: Haploids can be used to produce hybrid seeds through the
process of double haploid breeding, which results in the production of homozygous lines
that are identical to the original hybrid.

In summary, haploids offer a valuable tool for plant breeders to rapidly develop homozygous
lines, create new genetic variability, facilitate genetic analysis, and produce hybrid seeds..
70. Define pollen bank with its advantages and disadvantages
A pollen bank is a collection of pollen grains from a variety of plant species that are stored
under controlled conditions for later use in plant breeding programs. The advantages and
disadvantages of a pollen bank are:

Advantages:
1. Genetic diversity: A pollen bank provides a source of genetic diversity that can be used
in plant breeding programs to create new varieties with desirable traits.
2. Conservation of genetic resources: By storing pollen from rare and endangered plant
species, a pollen bank can help to conserve genetic resources for future generations.
3. Efficient use of resources: A pollen bank allows plant breeders to efficiently use their
resources by providing a source of pollen for use in controlled pollination experiments.
Disadvantages:
1. Maintenance costs: A pollen bank requires a significant amount of resources to maintain
the controlled conditions necessary for long-term storage of pollen grains.
2. Limited shelf life: Pollen grains stored in a pollen bank have a limited shelf life and may
lose viability over time, reducing their usefulness in plant breeding programs.
3. Risk of contamination: There is a risk of contamination of stored pollen grains, which
could compromise the genetic integrity of the pollen bank and the breeding programs
that rely on it.
In summary, a pollen bank provides a valuable source of genetic diversity and helps to conserve
genetic resources, but requires significant resources to maintain and has limitations due to the
limited shelf life and risk of contamination of stored pollen grains.

70. Briefly describe the " Bulbosum " method of doubled haploid production .
The Bulbosum method is a technique used for the production of doubled haploid plants in
which anthers from the donor plant are cultured in vitro to produce haploid embryos, which are
then treated with a chemical agent to double their chromosome number and create
homozygous, doubled haploid plants.

The process involves the following steps:

1. Anthers are collected from the donor plant at the appropriate developmental stage.
2. The anthers are sterilized and cultured in vitro on a nutrient medium containing plant
growth regulators and other necessary nutrients.
3. Haploid embryos are produced from the anthers through a process called androgenesis.
4. The haploid embryos are treated with a chemical agent such as colchicine, which
disrupts the process of cell division and causes the chromosomes to double.
5. The doubled haploid embryos are then cultured in vitro to produce plantlets that are
homozygous for all their genes.
6. The doubled haploid plantlets can be transplanted to soil and grown into mature plants.
The Bulbosum method has several advantages over other techniques for doubled haploid
production, including the ability to produce homozygous plants in a single generation, the
ability to generate large numbers of doubled haploid plants quickly, and the ability to use a wide
variety of plant species. However, it also has some limitations, such as the high sensitivity of the
haploid embryos to environmental factors and the difficulty of inducing androgenesis in some
plant species.
71. Write down the application and limitations of anther and pollen culture in plant kingdom .
Anther and pollen culture are techniques used for the production of haploid and doubled
haploid plants, which have several applications in plant breeding and biotechnology. However,
these techniques also have some limitations that must be considered.

Applications of anther and pollen culture:

 1. Production of haploid and doubled haploid plants: Anther and pollen culture can be
used to produce haploid and doubled haploid plants, which can be used in plant
breeding to create new varieties with desirable traits.
2. Production of homozygous lines: Doubled haploid plants produced through anther and
pollen culture are homozygous for all their genes, which makes them ideal for creating
pure breeding lines.
3. Genetic transformation: Anther and pollen culture can also be used as a tool for genetic
transformation, where genes of interest can be introduced into haploid cells to create
transgenic plants.

Limitations of anther and pollen culture:

1. Genetic variability: Anther and pollen culture can result in genetic variability due to
mutations or chromosome aberrations, which can be a disadvantage when creating pure
breeding lines.
2. Limited success rates: Anther and pollen culture can have low success rates, especially in
some plant species where androgenesis is difficult to induce.
3. Limited applicability: Anther and pollen culture can only be applied to plant species that
produce viable pollen and have a high rate of androgenesis.

4. Time and resource-intensive: Anther and pollen culture require specialized equipment
and techniques, and can be time and resource-intensive, which can limit their use in
large-scale plant breeding programs.

5. Susceptibility to environmental factors: Haploid cells are highly sensitive to

environmental factors such as temperature, light, and nutrient conditions, which can
affect the success of anther and pollen culture.
72. Define in vitro androgenesis . Write down the techniques involve of in vitro androgenesis .
In vitro androgenesis refers to the process of inducing haploid or doubled haploid plant
production from cultured cells of the male gametophyte (pollen or anther) through tissue
culture techniques.
There are two main techniques involved in in vitro androgenesis:
1. Anther culture: Anthers from flower buds are excised and cultured on a suitable nutrient
medium under controlled environmental conditions. The anthers undergo
dedifferentiation and form callus, which is then induced to produce haploid or doubled
haploid plants.
 2. Pollen culture: Pollen grains are isolated from anthers and cultured in a suitable nutrient
medium. The cultured pollen undergoes haploid or doubled haploid plant production by
the induction of embryogenesis or organogenesis.

These techniques have immense potential in plant breeding, especially in the development of
new cultivars with desirable traits. They can also be used for the production of genetically
uniform planting material for agricultural and horticultural purposes. However, these
techniques also have limitations, such as low efficiency and genotype dependency, which
restrict their widespread use.
73. What do you mean by embryo culture ? Describe in brief the methods involve of in vitro
embryo culture .
Embryo culture is a tissue culture technique used for the in vitro growth and development of
plant embryos. It involves the isolation and culture of immature or mature embryos in a
nutrient medium under controlled environmental conditions.

There are mainly two methods involved in in vitro embryo culture:

1. Embryo rescue: Embryo rescue is a technique used for the recovery of abnormal or
immature embryos from crosses that are otherwise unviable. The technique involves the
isolation of embryos at a critical stage of development, usually before or after
fertilization, and their culture in a suitable nutrient medium. The embryos are then
grown to maturity, and the resulting plants are screened for desirable traits.

2. Somatic embryogenesis: Somatic embryogenesis is a technique used for the in vitro

induction of somatic embryos from cultured plant cells or tissues. The technique
involves the regeneration of embryos from callus tissue, which is then transferred to a
suitable nutrient medium for further growth and development. The resulting embryos
can then be germinated to produce genetically uniform plants.

Embryo culture has several applications in plant breeding, such as the development of new
cultivars and hybrids, the rescue of hybrid embryos that would otherwise be lost, and the
production of genetically uniform planting material. However, the technique is also limited by
low efficiency and the difficulty of controlling the developmental stages of the embryos.
74. What does it mean by gametoclonal variation ?
Gametoclonal variation is a type of genetic variation that arises from the culture of isolated
gametes or haploid cells (e.g., microspores or megaspores) in vitro, followed by their
development into multicellular structures, such as calli or plants. The resulting plants can exhibit
genetic variation due to the occurrence of somatic mutations during cell division, as well as the
recombination of chromosomes during meiosis in haploid cells.
75. Briefly narrate the pollen culture technique with flow diagram . Illustrate the chromosome
diplodization techniques .

76. Write the trouble shooting in plant tissue culture

. Some common problems that can occur during plant tissue culture, and their potential
solutions, include:
1. Contamination: This can be caused by bacteria, fungi, or other microorganisms, and can
be prevented by maintaining sterile conditions, using proper disinfection techniques,
and avoiding overcrowding of cultures.
2. Callus browning: This can be caused by excess exposure to light, poor nutrient balance,
or the accumulation of phenolic compounds, and can be prevented by adjusting culture
conditions, using different media formulations, or adding activated charcoal to the
medium.
3. Slow growth: This can be caused by inadequate nutrient supply, low light levels, or
genetic factors, and can be remedied by adjusting nutrient concentrations, increasing
light intensity, or selecting faster-growing genotypes.
4. Low frequency of regeneration: This can be caused by poor explant quality, inadequate
growth conditions, or genetic factors, and can be addressed by using healthier explants,
optimizing culture conditions, or screening for high-regeneration genotypes.
5. Genotypic variation: This can arise from genetic drift or somaclonal variation, and can be
mitigated by using clonal propagation techniques or screening for stable genotypes.
6. Technical errors: These can include mistakes in media preparation, contamination of
equipment, or improper handling of cultures, and can be minimized through rigorous
quality control procedures and staff training.
Overall, effective troubleshooting in plant tissue culture requires careful attention to detail,
good record-keeping, and a willingness to experiment with different variables to find optimal
conditions for each culture.

11. Somaclonal Variation: Production and selection of somaclonal and gametoclonal variation, utilization of somaclone and gametoclone in agriculture, in vitro selection of
disease resistant and stress tolerant plants.

77. What is somaclonal variation . Write the scopes and objectives of somacional variation in
plant sciences .
Somaclonal variation refers to the genetic variation that arises in plants that have been regenerated from
somatic cells or tissues through in vitro tissue culture techniques. This variation is caused by various
factors such as epigenetic changes, chromosome number changes, and DNA mutations.

The scopes and objectives of somaclonal variation in plant sciences are:

1. Improvement of crop plants: Somaclonal variation provides a way to generate genetic variability
that can be used for crop improvement. By selecting desirable variants, plant breeders can
develop new cultivars with improved traits such as disease resistance, yield, and quality.
2. Study of plant development and genetics: Somaclonal variation can be used to study the genetic
and epigenetic mechanisms that control plant development and gene expression.
3. Production of useful compounds: Somaclonal variation can be used to produce plants with
altered levels of secondary metabolites, such as alkaloids, flavonoids, and terpenoids, which
have various medicinal and industrial applications.
4. Conservation of rare or endangered species: Somaclonal variation can be used to propagate rare
or endangered plant species that cannot be propagated by conventional methods.

However, somaclonal variation can also be a limitation of tissue culture, as it can lead to undesirable
changes in plant morphology, growth, and development, as well as changes in gene expression and
epigenetic modifications. Therefore, careful selection and screening of variants is necessary to ensure
that only desirable variants are used for crop improvement or other applications.

78. Write the scopes and objectives of somaclonal variation in agriculture

Somaclonal variation has various scopes and objectives in agriculture, including:

1. Improvement of crop yield: The main objective of somaclonal variation is to develop new crop
varieties with higher yield potential, disease resistance, and better quality traits.
2. Crop improvement through genetic manipulation: Somaclonal variation can also be used to
develop new crop varieties with specific traits through genetic manipulation.
3. Production of stress-tolerant crops: Somaclonal variation can be used to produce crops that are
tolerant to environmental stress such as drought, high temperature, or salinity.
4. Development of disease-resistant crops: Somaclonal variation can also be used to develop
disease-resistant crops by selecting somaclones that are resistant to a particular disease.
 5. Conservation of plant genetic resources: Somaclonal variation can be used to conserve
endangered plant species by multiplying them through tissue culture techniques.
6. Production of secondary metabolites: Somaclonal variation can be used to produce high-value
secondary metabolites such as alkaloids, flavonoids, and essential oils in plants.

Overall, the scope of somaclonal variation in agriculture is to provide a tool for crop improvement and
genetic manipulation, leading to increased crop productivity, sustainability, and food security.

79. Specify the steps of somaclonal variation with in vitro selection .

The steps involved in somaclonal variation with in vitro selection are as follows:

1. Selection of the initial culture: The culture is initiated from a single cell or tissue
explant.
2. Induction of somaclonal variation: Various methods such as the use of mutagenic
agents or tissue culture conditions that promote genetic instability are used to induce
somaclonal variation.
3. Culture of variant cells: The variant cells are cultured separately from the initial
culture.
4. Screening of variants: The variants are screened for desirable traits such as disease
resistance, increased yield, or improved quality.
5. In vitro selection: The selected variants are cultured in vitro to produce a
homogeneous population of plants with the desired traits.
6. Evaluation: The selected plants are evaluated in the field for their agronomic
performance and compared with the parental line.
7. Release of improved varieties: The selected plants that show superior traits are
released as new varieties.
The main objective of in vitro selection is to identify and isolate somaclonal variants with
improved traits that can be used for crop improvement. This approach can be used to develop
new crop varieties with desirable traits such as disease resistance, increased yield, and
improved quality.
80. Specify the steps of somaclonal variation without in vitro selection .
The steps involved in somaclonal variation without in vitro selection are as follows:
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1. Collection of plant materials: Plant materials are collected from the field or greenhouse.
2. Callus induction: Explants are sterilized and cultured on appropriate media for callus
induction.
 3. Regeneration of plants: Callus is subcultured on appropriate media to induce
regeneration of plants.
4. Screening and selection: The regenerated plants are screened and selected based on the
desired trait.
5. Field evaluation: The selected plants are transferred to the greenhouse or field for
further evaluation.
6. Genetic stability testing: The genetic stability of the selected plants is tested through
molecular and cytological analyses.
7. Commercialization: The selected plants are evaluated for commercial viability and can be
released to farmers for cultivation.
The main advantage of somaclonal variation without in vitro selection is that it is a simpler and
less expensive method compared to in vitro selection. However, the main disadvantage is the
lack of control over the somaclonal variation process, which can result in a low frequency of
desired variants.
81.. Illustrate the reasons of somaclonal variation . (Write the causes of somaclonal variation or
Write the reasons of somaclonal variation .)

Somaclonal variation is caused by various factors that can affect the genetic makeup of plants
during tissue culture. Some of the main causes of somaclonal variation include:

1. Genetic instability: Tissue culture can cause changes in the genetic makeup of plants due
to chromosome abnormalities, mutations, or epigenetic modifications.
2. Cell differentiation: In vitro culture can lead to the differentiation of cells, resulting in a
diverse range of cell types, each with its unique genetic makeup.
3. Stress factors: Stressful environmental conditions during tissue culture, such as high or
low temperature, light intensity, and nutrient availability, can cause genetic variations.
4. Somatic embryogenesis: Plants produced through somatic embryogenesis can exhibit
genetic variation due to the involvement of haploid cells, which can lead to the
development of gametes with different genetic makeup.
5. Contamination: Bacterial, fungal, or viral infections during tissue culture can cause
genetic variations in plants.
82. Specify the mechanism of genetic variations occur during tissue culture .
Ps-l-6,9
Q-83
Tissue culture involves various steps and environmental conditions that can cause genetic
variations in the cells. The mechanism of genetic variation during tissue culture can be
explained by the following factors:
1. Somatic mutation: The cells that are cultured can undergo somatic mutations that can
lead to genetic variation. Somatic mutations are genetic changes that occur in non-
reproductive cells and are not inherited by the offspring.
2. Chromosomal aberrations: During tissue culture, the cells may undergo chromosomal
aberrations such as deletion, duplication, inversion, or translocation of chromosome
segments. These chromosomal changes can result in genetic variation.
3. Epigenetic changes: Epigenetic changes are heritable changes that do not involve
alterations in the DNA sequence. These changes occur due to modifications of DNA or
chromatin structure. Tissue culture conditions such as plant growth regulators, culture
media composition, light intensity, and duration can lead to epigenetic changes.
4. Polyploidization: Polyploidization is a process of acquiring more than two sets of
chromosomes in a cell. Tissue culture can induce polyploidization, resulting in genetic
variation.
5. Horizontal gene transfer: Horizontal gene transfer is a process of transferring genetic
material between organisms that are not parent and offspring. Tissue culture can create
conditions that facilitate the transfer of genetic material between cells.
These mechanisms of genetic variation can lead to somaclonal variation, which can be
beneficial or detrimental, depending on the desired outcome.
83. Briefly mention the mechanism of somaclonal variation .
***
Mechanism of Somaclonal Variations
1. Genetic (Heritable Variations)
• Pre-existing variations in the somatic cells of
explant
• Caused by mutations and other DNA changes
• Occur at high frequency
2. Epigenetic (Non-heritable Variations) or variation
arising due to culture
• Variations generated during tissue culture
• Caused by temporary phenotypic changes
• Occur at low frequency
***

Somaclonal variation is a genetic variation that occurs during plant tissue culture. It is caused by
changes in the genome of somatic cells, which can lead to phenotypic variation in the
regenerated plants. The mechanisms that contribute to somaclonal variation include:
1. Chromosome number variation: During cell division in tissue culture, errors can occur in
chromosome segregation, leading to changes in chromosome number. This can result in
aneuploidy, polyploidy, or chromosomal rearrangements, which can lead to significant
changes in plant morphology, physiology, and fertility.
2. DNA methylation: Changes in DNA methylation patterns can occur during tissue culture,
which can result in altered gene expression and phenotypic variation.
3. Transposable elements: Transposable elements are mobile genetic elements that can
move within the genome, causing mutations and rearrangements. Tissue culture can
activate transposable elements, leading to somaclonal variation.
4. Somatic mutation: Somatic mutations can arise during tissue culture due to DNA
damage caused by stresses such as tissue culture media, subculture, and selection
pressure. These mutations can lead to altered gene expression and phenotypic variation
in the regenerated plants.
The precise mechanism of somaclonal variation is not fully understood, but these factors are
known to contribute to the genetic variability observed in tissue-cultured plants.
84. Mention the schemes of somaclonal variation with in vitro selection
***
Scheme for obtaining somaclonal variation
a) Without invitro selection
b) With invitro selection
Without in vitro selection (Steps)
1. Explant
2. Callus from explant
3. Shoot regeneration
4. Plant
5. Transfer to field
6. Desirable variants observed
7. Agronomic traits
With in vitro selection (Steps)
1. Explant
2. Callus from explant
3. Multiplication of callus
4. Use of toxic materials or antibiotic on callus
5. Shoot regeneration from tolerant calluses
6. Variants from the culture
7. Transfer to field and field testing
8. Screening of desired plant
9. Agronomic traits observation

The schemes of somaclonal variation with in vitro selection are:

1. In vitro selection for herbicide tolerance: The callus or cell culture is subjected to
increasing concentrations of a herbicide, and the surviving cells are selected and
cultured to produce herbicide-tolerant plants.

2. In vitro selection for disease resistance: The callus or cell culture is inoculated with a
pathogen, and the surviving cells that exhibit resistance to the pathogen are selected
and cultured to produce disease-resistant plants.

3. In vitro selection for salt tolerance: The callus or cell culture is grown on a medium
containing increasing concentrations of salt, and the surviving cells that exhibit salt
tolerance are selected and cultured to produce salt-tolerant plants.

4. In vitro selection for drought tolerance: The callus or cell culture is subjected to drought
stress, and the surviving cells that exhibit drought tolerance are selected and cultured to
produce drought-tolerant plants.

The selected cells are then regenerated into plants, and the somaclonal variations that have
occurred during the tissue culture process are evaluated for their usefulness.
85. Write the application of somaclonal variation in the field of plant biotechnology. State the
application of somaclonal variation .
Somaclonal variation has various applications in the field of plant biotechnology, such as:

1. Development of novel plant varieties with desirable traits: Somaclonal variation can lead
to the development of new plant varieties with improved traits, such as disease
resistance, higher yield, and better quality.
2. Genetic improvement of crops: Somaclonal variation can be used to generate genetic
diversity in crops, which can then be used in breeding programs to improve crop
performance.
3. Conservation of endangered species: Somaclonal variation can be used for the
conservation of endangered plant species by producing large numbers of plants from a
limited number of cells.
4. Production of secondary metabolites: Somaclonal variation can be used to enhance the
production of secondary metabolites, such as alkaloids, flavonoids, and terpenoids,
which have pharmaceutical and industrial applications.
5. Study of plant developmental biology: Somaclonal variation can be used as a tool to
study the genetic and epigenetic mechanisms that regulate plant development.

Overall, the application of somaclonal variation can lead to the development of novel plant
varieties with improved traits and enhanced productivity, which can benefit agriculture and
society.
86. Brief about the utility of somaclonal variation
Somaclonal variation has numerous utilities in the field of plant biotechnology, some of which
are:
1. Crop improvement: Somaclonal variation can be used for the development of new plant
varieties with desirable traits, such as disease resistance, improved yield, and enhanced quality.
2. Genetic studies: Somaclonal variation can be used to study the genetic mechanisms
underlying the development of new traits and to identify the genes responsible for these traits.
3. Conservation: Somaclonal variation can be used to conserve endangered plant species by
producing large numbers of plants from a single individual.
4. Mutation breeding: Somaclonal variation can be used to induce mutations in plants that can
lead to the development of new traits, which can then be selected for through in vitro selection
or other means.
5. Production of secondary metabolites: Somaclonal variation can be used to produce
secondary metabolites such as alkaloids, flavonoids, and phenolics in high amounts from in vitro
cultures.
Overall, somaclonal variation is a valuable tool for plant biotechnologists and has many
potential applications in agriculture, horticulture, and forestry.
@@What do you mean by androgenesis Write the importance of chromosome doubling in the
process of androgenesis .
12. In-vitro Conservation of Plant Materials: methods and factors affecting in vitroconservation, maintenance of
frozen culture.

In situ conservation Ex situ conservation

Takes place in the species' natural habitat Takes place in a controlled environment outside of
the species' natural habitat
Aims to protect the species and its habitat in the Can be used to supplement in situ efforts and to
long term help save species that are on the brink of
extinction
Can be more expensive and time-consuming Can be more flexible and can be used to save
species that are not well-suited for in situ
conservation

87 *What is cryopreservation? Briefly describe the basic principle of cryopreservation .

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Cryopreservation is the process of preserving living cells, tissues, organs, or other biological
materials by cooling them to very low temperatures
88. Write the mechanism of cryopreservation .Briefly discuss the different steps of
cryopreservation
89.. What do you mean by cryoprotectants ? Write the importance of using cryoprotectants
during cryopreservation.
Cryoprotectants are substances that are used to protect biological materials from the damage
caused by freezing. They do this by preventing ice crystals from forming, which can damage cells
and tissues. Cryoprotectants are often used in the cryopreservation of cells, tissues, and organs
for transplantation or research. The use of cryoprotectants during cryopreservation is of utmost
importance and offers several key advantages. Here are the primary reasons why
cryoprotectants are essential in the cryopreservation process:
1. Preventing Ice Crystal Formation: Cryoprotectants play a crucial role in preventing the
formation of damaging ice crystals during freezing. By replacing water molecules,
cryoprotectants lower the concentration of freezable water inside the cells or tissues. This
 reduction in freezable water helps minimize ice crystal formation and growth, reducing the
potential for cellular damage.

2. Reducing Osmotic Stress: During freezing, the extracellular solution can become increasingly
concentrated due to the formation of ice crystals. This concentrated solution creates osmotic
stress, which can lead to water loss from the cells and subsequent cell shrinkage or damage.
Cryoprotectants help counterbalance this osmotic stress by maintaining osmotic equilibrium and
preventing excessive water loss from the cells.

3. Preserving Cell Structure and Integrity: Ice crystal formation during freezing can cause physical
damage to cellular structures, membranes, and organelles. Cryoprotectants act as a protective
shield by minimizing ice crystal formation or promoting the formation of amorphous, non-
crystalline structures (vitrification). This preservation of cell structure and integrity helps
maintain cell viability and functionality after thawing.

4. Minimizing Toxic Effects: Cryoprotectants can have additional properties that help protect cells
from freezing-induced damage. Some cryoprotectants have antioxidant properties, which can
reduce oxidative stress and prevent cellular damage caused by reactive oxygen species (ROS)
generated during freezing and thawing. Cryoprotectants can also act as scavengers for ice-
nucleating agents, further reducing the potential for ice crystal formation.

5. Enhancing Post-Thaw Recovery: Cryoprotectants aid in the successful recovery of cells or

tissues after thawing. They provide an osmotically favorable environment for cells during the
removal of cryoprotectants, preventing osmotic shock and minimizing stress during the
rehydration process. This enhances cell viability and facilitates the restoration of normal cellular
functions

By using cryoprotectants, cryopreservation techniques can effectively preserve the viability and
functionality of biological materials during freezing and thawing processes. Cryoprotectants
help prevent ice crystal formation, reduce osmotic stress, preserve cell structure, minimize toxic
effects, and facilitate post-thaw recovery. This enables long-term storage of valuable biological
samples and facilitates various applications in fields such as research, medicine, and
biotechnology.

90. * Write the application of cryoprotectants

Cryoprotectants are chemicals that are used to protect biological materials from damage during
freezing and thawing. They work by preventing the formation of ice crystals, which can damage
cells and other biological structures. Cryoprotectants are used in a variety of applications,
including:

1. Cryopreservation: This is the process of preserving biological materials at very low

temperatures, typically below -130 degrees Celsius. Cryopreservation is used to store
 cells, tissues, and organs for long periods of time, and to make them available for future
use.
2. Cryosurgery: This is a medical procedure that uses freezing to destroy tissue.
Cryoprotectants are used to protect healthy tissue from the freezing process.
3. Food preservation: Cryoprotectants are used to preserve food by preventing the
formation of ice crystals. This can be done by freezing food rapidly, or by adding
cryoprotectants to food before freezing.
4. Plant breeding: Cryoprotectants are used to store plant seeds and pollen for long periods
of time. This allows plant breeders to preserve genetic material from rare or endangered
plants.
5. Space exploration: Cryoprotectants are used to protect biological samples from the
harsh conditions of space travel. This includes exposure to extreme temperatures,
radiation, and vacuum.
91. Give an outline of the protocol for cryopreservation of shoot tip
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92. Write short potes on any FOUR of following : Suspension culture / Gelling agent
Organogenesis Synthetic Seed e ) Amino acid Triploid production LII