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Semester 2: M.Sc. Biotechnology Syllabus 2023-2024
Introduction of plant tissue culture, composition of media, Micropropagation, somatic embryogenesis, haploid and triploid production, protoplast isolation and fusion, hybrid and cybrid, synthetic seed production
Introduction to Plant Tissue Culture
Overview of Plant Tissue Culture
Plant tissue culture is a technique used to grow plant cells, tissues, or organs in a controlled environment. This method is essential for the propagation of plants, disease elimination, and genetic modification.
Composition of Culture Media
Culture media is vital for the growth and development of plant cells in vitro. It generally contains macronutrients, micronutrients, vitamins, hormones, and agar as a solidifying agent.
Micropropagation
Micropropagation involves propagating plants from small tissue samples under sterile conditions. This technique allows for rapid multiplication of plants and ensures uniformity and disease-free plants.
Somatic Embryogenesis
Somatic embryogenesis is the process whereby somatic cells develop into embryos. This technique can be used for clonal propagation and the production of genetically uniform plants.
Haploid and Triploid Production
Haploid production involves generating plants with half the number of chromosomes, leading to potential breeding applications. Triploid plants, possessing three sets of chromosomes, often exhibit increased vigor and sterility.
Protoplast Isolation and Fusion
Protoplasts are plant cells without their cell walls, isolated for genetic engineering and fusion. Cell fusion techniques allow for the creation of hybrid cells and the introduction of new traits.
Hybrid and Cybrid Production
Hybrid production combines genetic material from two different species, while cybrids contain cytoplasmic components from one species and nuclear material from another. Both techniques expand genetic diversity.
Synthetic Seed Production
Synthetic seeds are artificially encapsulated somatic embryos or other tissue that can germinate into a plant. This technology allows for the storage and transport of plant material and facilitates propagation.
Plant Transformation Direct transformation by electroporation and particle gun bombardment. Agrobacterium, Ti plasmid vector. Theory and techniques for the development of new genetic traits, conferring resistance to biotic and abiotic stresses. Plant engineering towards the development of enriched food products, plant growth regulators Molecular Marker aided breeding RFLP maps, RAPD markers, QTL, Map based cloning and Molecular marker assisted selection
Plant Transformation
Direct Transformation Techniques
Direct transformation methods include electroporation and particle gun bombardment. Electroporation involves applying an electrical field to plant cells or tissues to increase the permeability of the cell membrane, allowing the introduction of DNA. Particle gun bombardment, or biolistic transformation, uses high-velocity microprojectiles coated with DNA to penetrate cell walls and membranes, delivering genetic material directly into the cells.
Agrobacterium-Mediated Transformation
Agrobacterium tumefaciens is a soil bacterium commonly used for plant transformation. It transfers T-DNA from its Ti plasmid into the plant genome. The genes included in the T-DNA can confer desired traits such as pest resistance. The effectiveness of this method is due to the bacterium's natural ability to transfer DNA, making it a widely used tool in genetic engineering.
Development of New Genetic Traits
Genetic engineering techniques enable the development of new traits in plants. These traits can provide resistance against biotic stresses like pathogens and pests, as well as abiotic stresses such as drought and salinity. Through targeted modifications and the introduction of specific genes, plants can be tailored to thrive under adverse environmental conditions.
Plant Engineering for Nutritional Enrichment
Plant biotechnology is also focused on enhancing the nutritional value of food products. Genetic modifications can lead to the development of crops with increased levels of essential vitamins and minerals, improving food security and public health. Techniques involve the alteration of metabolic pathways to boost the accumulation of beneficial compounds.
Molecular Marker-Aided Breeding
Molecular markers are essential tools for plant breeding, aiding in the selection of desired traits. Techniques such as RFLP (Restriction Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA), and QTL (Quantitative Trait Loci) mapping facilitate the identification of genetic variation, allowing breeders to develop superior varieties through marker-assisted selection.
Map-Based Cloning and Molecular Marker Selection
Map-based cloning involves the identification and isolation of genes linked to traits of interest using molecular markers. This technique is supported by the construction of detailed genetic maps. Molecular marker-assisted selection enhances the efficiency of breeding programs by allowing the early selection of desirable traits in breeding populations.
An Introduction about animal cell culture, Planning and Construction of Lab layout, Equipments - Laminar-flow hood, CO2 Incubators, Inverted microscope, Cryostorage containers, Aseptic concepts and Cell culture vessel. Preparation of Media- defined media and supplements, Types of cell culture media Physical and chemical property of Medium, Balanced salts, Antibiotics, growth supplements Fetal bovine serum Serum free media primary and established culture organ culture tissue culture
An Introduction to Animal Cell Culture
Overview of Animal Cell Culture
Animal cell culture involves the maintenance and growth of animal cells in a controlled environment outside their natural habitat. This technique is essential for research, biotechnology, and pharmaceutical development.
Planning and Construction of Lab Layout
Proper planning of laboratory space is crucial for effective animal cell culture. This includes separation of clean and contaminated areas, efficient workflow design, and adherence to safety regulations.
Essential Equipment
Laminar-Flow Hood
A laminar-flow hood provides a sterile environment by filtering air to prevent contamination during cell culture procedures.
CO2 Incubators
These incubators provide a controlled environment with precise CO2 and temperature regulation, essential for maintaining cell viability.
Inverted Microscope
This type of microscope is used to observe cultured cells without detaching them from their growth surface.
Cryostorage Containers
Cryostorage containers are used for the long-term preservation of cell lines at sub-zero temperatures.
Aseptic Concepts
Understanding aseptic techniques is vital to prevent contamination during cell culture processes.
Cell Culture Vessel
Various types of vessels, such as flasks and plates, are used to grow and maintain cell cultures.
Preparation of Media
Defined Media and Supplements
Defined media contain known quantities of essential nutrients, while supplements like growth factors enhance cell growth.
Types of Cell Culture Media
Cell culture media can be broadly categorized into basic media, serum-containing media, and serum-free media.
Physical and Chemical Properties of Medium
Medium properties such as pH, osmolarity, and viscosity affect cell growth and function.
Balanced Salts
Balanced salts in media help maintain osmotic balance and provide essential ions for cell metabolism.
Antibiotics
Incorporating antibiotics helps prevent bacterial contamination during cell culture.
Growth Supplements
Growth supplements like fetal bovine serum provide critical growth factors and hormones.
Fetal Bovine Serum
Fetal bovine serum is a common supplement used in cell culture for its rich nutrient profile.
Serum-Free Media
These media formulations eliminate the variability associated with serum and are used for specific applications.
Types of Cell Cultures
Primary Culture
Primary culture involves isolating cells directly from tissues and has a limited lifespan.
Established Culture
Established cultures are derived from primary cultures and can be continuously propagated.
Organ Culture
Organ culture refers to maintaining intact organs in vitro to study physiological functions.
Tissue Culture
Tissue culture focuses on the culture of specific tissues for various research applications.
Disaggregation of tissue and primary culture cell separation, Slide and coverslip cultures, flask culture, test tube culture techniques, cell synchronization, cryo preservation. Scaling up of animal cell culture, cell line and cloning micromanipulation and cloning, somatic cell cloning. Karyotyping measuring parameters for growth, measurement of cell death, apoptosis and its determination, cytotoxicity assays
Disaggregation of tissue and primary culture cell separation, Slide and coverslip cultures, flask culture, test tube culture techniques, cell synchronization, cryo preservation, scaling up of animal cell culture, cell line and cloning micromanipulation and cloning, somatic cell cloning, karyotyping measuring parameters for growth, measurement of cell death, apoptosis and its determination, cytotoxicity assays
Disaggregation of Tissue and Primary Culture Cell Separation
This process involves breaking down tissue into individual cells for culture. Techniques include enzymatic digestion using proteolytic enzymes like trypsin or collagenase, mechanical methods using tissue grinders or homogenizers, and the use of chelating agents to detach cells from the matrix. Proper disaggregation is crucial for obtaining viable cells.
Slide and Coverslip Cultures
These techniques involve culturing cells on glass slides or coverslips. They are useful for examining cell morphology and behavior under a microscope. Slides can be pre-treated with extracellular matrix proteins to enhance cell adhesion.
Flask Culture Techniques
Culturing cells in flasks allows for larger-scale growth. There are different types of flasks, including T-flasks and multi-well plates, which facilitate studying cell interactions, drug responses, and proliferation. The culture medium must be regularly changed, and the growth conditions monitored.
Test Tube Culture Techniques
This method is beneficial for small-scale cultures and for growing bacteria, yeast, or specific cell types. The focus is on maintaining sterility and suitable growth conditions. Test tube cultures can be excellent for preliminary experiments and scale-up before moving to flasks.
Cell Synchronization
Methods for synchronizing cell populations are vital for studies in cell cycle dynamics. Techniques include serum starvation, chemical synchronization, and centrifugal elutriation. Synchronization helps in analyzing specific phases of the cell cycle in detail.
Cryo Preservation
Cryopreservation involves storing cells at ultra-low temperatures to maintain viability over extended periods. Common cryoprotectants include DMSO and glycerol. Careful slow-freezing and thawing protocols are critical to prevent ice crystal formation which can damage cells.
Scaling Up of Animal Cell Culture
Scaling up from small laboratory cultures to industrial-scale bioreactors is essential for pharmaceutical and biotechnology applications. Various bioreactor designs accommodate increased cell densities and productivity while ensuring optimal growth conditions.
Cell Line and Cloning Micromanipulation and Cloning
Micromanipulation techniques enable the isolation of single cells for cloning. This process facilitates the development of pure cell lines, which can be important for research and therapeutic purposes. Techniques include cell picking and the use of micropipettes.
Somatic Cell Cloning
Somatic cell cloning involves transferring the nucleus of a somatic cell into an enucleated oocyte. This technique has applications in genetics, agriculture, and medicine, with techniques like SCNT (Somatic Cell Nuclear Transfer) playing a vital role in cloning organisms.
Karyotyping Measuring Parameters for Growth
Karyotyping examines the number and structure of chromosomes in cells. This analysis is critical for identifying genetic abnormalities, studying cell line characteristics, and understanding the genetic basis of diseases.
Measurement of Cell Death, Apoptosis and Its Determination
Quantifying cell death is essential in research and clinical evaluation. Methods include staining techniques (e.g., Annexin V/PI) and assays like TUNEL. Apoptosis pathways can be studied via western blotting or qPCR to assess the expression of proteins involved.
Cytotoxicity Assays
Cytotoxicity assays evaluate the effect of substances on cell viability. Common assays include MTT, LDH, and XTT assays, which measure cell metabolic activity or membrane integrity. These assays are critical in drug development and toxicology studies.
Application of animal cell culture for in vitro testing of drugs, in production of human and animal viral vaccines and pharmaceutical proteins. Culture Scale up and mass production of biologically important compounds. Harvesting of products, purification and assays. Transgenic animals Production and application transgenic animals in livestock improvement, transgenic animals as model for human diseases
Application of animal cell culture for in vitro testing of drugs, production of human and animal viral vaccines and pharmaceutical proteins, culture scale up and mass production of biologically important compounds, harvesting of products, purification and assays, transgenic animals production and application
In Vitro Drug Testing
Animal cell culture provides a controlled environment for testing the efficacy and safety of new drugs. Cells from various animal sources can be cultured to study cellular responses to pharmaceuticals, offering insights into drug mechanisms, toxicity, and metabolic pathways.
Viral Vaccine Production
Animal cell culture techniques are essential in the development of vaccines. Cells are used as hosts for growing viruses, which are then harvested and purified to create vaccines. This method is crucial for producing human and animal vaccines efficiently and safely.
Production of Pharmaceutical Proteins
Animal cells are leveraged for the production of complex proteins, including monoclonal antibodies and therapeutic enzymes. These cells provide the post-translational modifications necessary for protein functionality.
Culture Scale-up and Mass Production
Scaling up animal cell cultures involves optimizing growth conditions to increase yield while maintaining product quality. Techniques such as bioreactor systems are employed to facilitate the mass production of cultured cells and their products.
Harvesting and Purification Techniques
Harvesting refers to the collection of cultured cells or secreted products. Techniques like centrifugation, filtration, and chromatography are utilized for purification to isolate desired biomolecules for further applications.
Transgenic Animals
Transgenic animals are genetically modified to express foreign genes, which can be used for various applications, including enhanced disease resistance and improved agricultural traits. They are also valuable models for studying human diseases and testing therapeutics.
Applications in Livestock Improvement
The use of transgenic animals in agriculture involves enhancing desirable traits such as growth rate, disease resistance, and feed efficiency. This biotechnological approach aims to improve the overall productivity of livestock.
Transgenic Models for Human Diseases
Transgenic animals serve as models for studying human diseases by mimicking specific conditions. These models are crucial in understanding disease mechanisms and developing new treatments.
