this presentation cover the topics of cell biotechnology and plant tissue culture. the basic terms used in plant cell culture are used and then different types of culture media and methods are discussed including cell suspension and callus culture,
Aseptic Techniques and sterile handling in atc labAkshdeep Sharma
This document discusses aseptic techniques and sterile handling in animal cell culture. It explains that all cell culture procedures must be done aseptically under controlled conditions, such as a laminar flow hood. Basic aseptic techniques include cleaning the work area with ethanol and sterilizing all equipment. Materials used in cell culture must be carefully sterilized, with heat-stable components autoclaved and heat-labile materials filtered. Autoclaving is the preferred sterilization method as it is cheaper and more effective than filtration. Safety precautions for cell culture include keeping cultures antibiotic-free and never sharing media between cell lines.
This document discusses three main modes of fermentation: batch culture fermentation, continuous culture fermentation, and fed batch culture fermentation. Batch culture fermentation involves a closed system with limited initial nutrients that microbes pass through lag, log, stationary, deceleration, and death phases. Continuous fermentation maintains exponential growth by continuously adding fresh medium and removing culture. Fed batch culture adds medium without removing culture, initially operating in batch mode before strategies like continuously adding limiting substrates are used to increase biomass concentration while maintaining a constant cell concentration and decreasing growth rate in quasi steady state.
Secondary cell cultures refer to cells that have been subcultured, or transferred, from a primary culture to a new culture vessel. Subculturing provides fresh nutrients and space for continuously growing cell lines. Cell lines can be finite or continuous depending on their lifespan in culture. Characterization of cell lines is important to confirm identity and purity through analysis of biochemical, genetic, and chromosomal parameters such as karyotyping.
This document provides a history and overview of animal cell culture techniques. It discusses the development of cell culture media and reagents used to support cell growth in vitro. It also describes different techniques for culturing mammalian cells, tissues, and organs, including organ culture, explant culture, and cell culture. The goal of animal cell culture is to maintain cells, tissues, or organs outside of their natural environment for research purposes.
The document discusses axenic and synchronous cultures. It defines axenic culture as a pure culture containing only a single type of microorganism, free from contamination. Methods to achieve axenic cultures include isolation, antibiotic treatments, and checking for contamination over time. Synchronous cultures are composed of populations of microorganisms that are at the same stage of their life cycle, allowing study of cellular processes during division. Techniques like temperature shocks, starvation, and filtration are used to generate synchronous cultures from asynchronous populations where cell cycles are random.
Animal Cloning Procedure, Problems and PerspectivesShafqat Khan
Cloning in farm animals has problems and perspectives. Key issues include developmental anomalies in cloned animals, the large offspring syndrome observed in cattle and sheep clones, and safety apprehensions regarding meat and milk from cloned animals. However, cloning also has potential applications for transgenic animal production, creating disease models, bioreactors, and research into xenotransplantation. It allows the propagation of elite livestock and conservation of endangered species. Further optimization is needed to improve cloning efficiency and resolve health issues.
This document discusses screening techniques used to isolate microorganisms of interest from a population. It describes primary screening as an initial process to discard many non-useful microbes while detecting a small percentage that may have industrial applications. Secondary screening further tests the capabilities of these isolated microorganisms to determine their real potential value. Some primary screening techniques mentioned include using crowded plates, detecting organic acid production, and screening for antibiotic production. The document also discusses improving crowded plate techniques and the goals and approaches of secondary screening to evaluate a microorganism's potential for industrial use.
Aseptic Techniques and sterile handling in atc labAkshdeep Sharma
This document discusses aseptic techniques and sterile handling in animal cell culture. It explains that all cell culture procedures must be done aseptically under controlled conditions, such as a laminar flow hood. Basic aseptic techniques include cleaning the work area with ethanol and sterilizing all equipment. Materials used in cell culture must be carefully sterilized, with heat-stable components autoclaved and heat-labile materials filtered. Autoclaving is the preferred sterilization method as it is cheaper and more effective than filtration. Safety precautions for cell culture include keeping cultures antibiotic-free and never sharing media between cell lines.
This document discusses three main modes of fermentation: batch culture fermentation, continuous culture fermentation, and fed batch culture fermentation. Batch culture fermentation involves a closed system with limited initial nutrients that microbes pass through lag, log, stationary, deceleration, and death phases. Continuous fermentation maintains exponential growth by continuously adding fresh medium and removing culture. Fed batch culture adds medium without removing culture, initially operating in batch mode before strategies like continuously adding limiting substrates are used to increase biomass concentration while maintaining a constant cell concentration and decreasing growth rate in quasi steady state.
Secondary cell cultures refer to cells that have been subcultured, or transferred, from a primary culture to a new culture vessel. Subculturing provides fresh nutrients and space for continuously growing cell lines. Cell lines can be finite or continuous depending on their lifespan in culture. Characterization of cell lines is important to confirm identity and purity through analysis of biochemical, genetic, and chromosomal parameters such as karyotyping.
This document provides a history and overview of animal cell culture techniques. It discusses the development of cell culture media and reagents used to support cell growth in vitro. It also describes different techniques for culturing mammalian cells, tissues, and organs, including organ culture, explant culture, and cell culture. The goal of animal cell culture is to maintain cells, tissues, or organs outside of their natural environment for research purposes.
The document discusses axenic and synchronous cultures. It defines axenic culture as a pure culture containing only a single type of microorganism, free from contamination. Methods to achieve axenic cultures include isolation, antibiotic treatments, and checking for contamination over time. Synchronous cultures are composed of populations of microorganisms that are at the same stage of their life cycle, allowing study of cellular processes during division. Techniques like temperature shocks, starvation, and filtration are used to generate synchronous cultures from asynchronous populations where cell cycles are random.
Animal Cloning Procedure, Problems and PerspectivesShafqat Khan
Cloning in farm animals has problems and perspectives. Key issues include developmental anomalies in cloned animals, the large offspring syndrome observed in cattle and sheep clones, and safety apprehensions regarding meat and milk from cloned animals. However, cloning also has potential applications for transgenic animal production, creating disease models, bioreactors, and research into xenotransplantation. It allows the propagation of elite livestock and conservation of endangered species. Further optimization is needed to improve cloning efficiency and resolve health issues.
This document discusses screening techniques used to isolate microorganisms of interest from a population. It describes primary screening as an initial process to discard many non-useful microbes while detecting a small percentage that may have industrial applications. Secondary screening further tests the capabilities of these isolated microorganisms to determine their real potential value. Some primary screening techniques mentioned include using crowded plates, detecting organic acid production, and screening for antibiotic production. The document also discusses improving crowded plate techniques and the goals and approaches of secondary screening to evaluate a microorganism's potential for industrial use.
This document describes the process of preparing and isolating genomic DNA from bacterial cells. It involves 4 main steps:
1) Growing and harvesting bacterial cells in nutrient broth media. Common media used are M9 and Luria-Bertani broth.
2) Preparing a cell extract by lysing the bacterial cells using enzymes like lysozyme and detergents like SDS.
3) Purifying the DNA from other cell components like proteins and RNA. This is done using phenol-chloroform extraction and protease/RNase digestion. Ion-exchange chromatography can also be used.
4) Concentrating the purified DNA using ethanol precipitation, which causes the long DNA strands to precipitate out of
Cryopreservation is the process of preserving living cells and tissues by cooling them to very low sub-zero temperatures. This stops all biological and chemical processes, halting the living material in a state of suspended animation. There are several key steps in cryopreservation including preculturing materials, adding cryoprotectants, slow or stepwise freezing, storage in liquid nitrogen at -196°C, rapid thawing, and then reculturing. Common cryopreservation methods include slow freezing, vitrification, encapsulation-dehydration, and cryopreservation has many applications for preserving genetic resources like semen, embryos, oocytes, and more.
Primary and established cell line cultureKAUSHAL SAHU
Introduction
Primary Culture
Steps of Primary Culture
Isolation Of Tissue
Dissection And Disaggregation
Types Of Primary Culture
Primary Explants Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
Reference
Direct Gene Transfer method (gene gun method).ShaistaKhan60
Direct gene transfer methods rely on delivering naked DNA directly into plant cells without the use of Agrobacterium. Biolistic or gene gun particle bombardment is a direct gene transfer method where gold or tungsten particles are coated with DNA and shot into plant tissue using a gene gun. The DNA can then integrate into the plant genome. The method involves creating recombinant plasmids with the gene of interest, coating them onto microcarriers, bombarding embryogenic plant callus, selecting transformed cells, and regenerating plants. Transgenic papaya developed using the gene gun method were resistant to papaya ring spot virus.
Single cell culture involves isolating single cells from plant tissue and culturing them on a nutrient medium. There are mechanical and chemical methods for isolation. Cells can be cultured using various techniques like microchamber, microdroplet, or nurse culture techniques. The paper raft nurse culture places isolated cells on nutrient-soaked paper placed on actively growing callus tissue. Single cell culture is important for fundamental studies, mutation analysis, and industrial applications like crop improvement and production of medicinal compounds.
Animal cell culture media typically contain energy sources like glucose, amino acids as nitrogen sources, vitamins, inorganic salts, fatty acids, antibiotics, growth factors, and hormones. Most media also require an incubator to maintain optimal temperature, pH, osmolality, and gaseous environment for cell growth. Cell cultures can be grown adhered to surfaces or in suspension, and may have limited or continuous proliferation. Common applications of animal cell culture include vaccine production, cancer research, pharmaceutical drug production, and studying nerve cell function.
The document discusses the key stages in downstream processing as part of bio manufacturing or biosynthesis of products. It describes how downstream processing involves removing cells and impurities from fermentation broth to produce the final product. The main stages discussed are removal of insolubles, product isolation, product purification, and product polishing. Key operations at each stage include filtration, centrifugation, precipitation, crystallization, and lyophilization.
The document discusses upstream processing in biomanufacturing. Upstream processing involves growing cells in bioreactors to produce target proteins for pharmaceuticals. Key aspects of upstream processing include media preparation and sterilization, inoculum development, and cell culture in bioreactors. The main goal of upstream processing is to provide optimal environmental conditions for cell growth and protein production before downstream processing separates and purifies the target proteins.
The document discusses the essential components of plant tissue culture media, including macro and micro nutrients, organic nutrients like vitamins and carbohydrates, and plant growth regulators. It explains that the success of tissue culture depends on using the right type of culture media, which must contain nutrients, carbon sources, and other components to support in vitro plant growth. The various roles and forms of important media components like nitrogen, calcium, iron, and cytokinins are also outlined.
This document provides an overview of media formulation for fermentation and bioprocessing. It discusses the types of media, including complex and synthetic media. The key requirements for formulated media are then outlined, including carbon sources, oxygen sources, water, nitrogen sources, minerals, growth factors, and antifoams. Specific examples are given for each requirement. The document emphasizes that media formulation is essential for successful laboratory experiments and manufacturing processes.
Application of Microbial Biotechnology in Food Technology, Critical Review an...Zohaib HUSSAIN
1. Microbial fermentation is used to produce many important fermented foods like beers, wines, cheeses and more. Microbes add flavors and end products to foods through fermentation.
2. Nisin is an antibacterial peptide produced by Lactococcus lactis that is used as a food preservative. It is effective against many gram-positive bacteria and is considered generally safe for use in foods.
3. Lactobacillus sakei is used in meat fermentation and preservation. It produces compounds that form biofilms on meat surfaces to exclude other microbes and keeps meat safe during low-temperature storage.
This document summarizes two main types of fermentation processes: solid state fermentation and submerged fermentation. Solid state fermentation occurs without free water and uses natural raw materials like grains as the carbon source to cultivate microorganisms. Submerged fermentation uses a liquid substrate and is best for microbes that require high moisture. Both methods have various applications, with solid state fermentation used for producing enzymes, biopesticides, and in bioremediation, while submerged fermentation is common in industrial manufacturing.
The document discusses totipotency in plant cells. Totipotency refers to the ability of single plant cells to regenerate into a whole plant through cell differentiation and tissue culture techniques. The document outlines various tissue culture systems used to study totipotency, including callus culture, suspension culture, single cell culture, and protoplast culture. Factors that influence a cell's ability to express totipotency, such as the explant source and culture conditions, are also discussed.
Micromanipulation involves manipulating individual cells, sperm, or embryos under a microscope using microtools to improve fertilization and pregnancy rates. It can be performed physically using instruments like micromanipulators that carry microtools, or optically using lasers. Key applications of micromanipulation include IVF, stem cell research, transgenics, and basic biological research like studying protein interactions.
Protoplast fusion involves removing the cell walls of plant cells through enzymatic or mechanical means to create naked protoplasts. These protoplasts can then be fused using chemicals, electricity, or other methods. This allows the cytoplasms and sometimes nuclei of different plant cells to merge, creating hybrid cells. Successful fusion can generate hybrid plants through regeneration of cell walls and tissues. Protoplast fusion overcomes sexual incompatibility and is used to introduce traits like disease resistance between species. It remains a technically challenging process with limitations like genetic instability and uncertain expression of transferred traits.
Culture techniq and type of animal cell culturePankaj Nerkar
A primary culture refers to the initial culture of cells directly taken from an organism before the first subculture. A cell line refers to the propagation of cells after the first subculture. Primary cultures contain a variety of differentiated cell types and require higher cell quantities due to lower survival rates. Tissues are disaggregated into single cells using mechanical or enzymatic techniques for primary culture. Organ cultures involve culturing whole organs or tissues to preserve their structure and function in vitro. Various techniques like plasma clot, raft, and grid methods are used to culture different organ explants.
This document discusses protoplast isolation, culture, and fusion. It defines protoplasts as plant, fungal, or bacterial cells without cell walls. Protoplasts can be isolated from plant tissue through either mechanical or enzymatic methods, with enzymatic isolation being more common. The document outlines steps for protoplast isolation, purification, and assessing viability. It also discusses protoplast culture techniques and methods for inducing protoplast fusion, including electrofusion and PEG treatment. Applications of protoplast techniques in plant research include genetic engineering and crop breeding.
This document discusses methylases, which are enzymes that add methyl groups to DNA. Specifically:
- Methylases transfer methyl groups from S-adenosylmethionine to adenine or cytosine bases within their recognition sequence on DNA. This methylation protects the DNA from restriction endonucleases.
- The methylase and restriction enzyme of a bacterial species together form the restriction-modification system, with the methylase protecting the host DNA.
- Methylases are of interest because methylation of some restriction enzyme recognition sites protects the DNA from being cleaved by that enzyme. This allows study of DNA isolated from strains expressing common methylases like Dam or Dcm.
This document discusses plant cell culture production in bioreactors. It describes various bioreactor types including mechanically agitated, bubble column, and airlift bioreactors. Mechanically agitated bioreactors employ impellers but can cause high shear stress on plant cells. Bubble column and airlift bioreactors have no moving parts, providing mixing through rising gas and liquid circulation with less shear stress, but have lower oxygen transfer rates than mechanically agitated bioreactors. The document compares these bioreactor types for oxygen transfer ability and low shear on plant cells.
Plant tissue culture involves growing plant cells, tissues or organs in an artificial nutrient medium under sterile conditions. Some key points:
1. It allows for the rapid mass propagation of plants through micropropagation and the production of genetically uniform plants.
2. It facilitates the production of disease-free plants through culture of meristems and shoot tips.
3. It enables genetic modification of plants through techniques like protoplast fusion, anther culture and recombinant DNA technology.
This document describes the process of preparing and isolating genomic DNA from bacterial cells. It involves 4 main steps:
1) Growing and harvesting bacterial cells in nutrient broth media. Common media used are M9 and Luria-Bertani broth.
2) Preparing a cell extract by lysing the bacterial cells using enzymes like lysozyme and detergents like SDS.
3) Purifying the DNA from other cell components like proteins and RNA. This is done using phenol-chloroform extraction and protease/RNase digestion. Ion-exchange chromatography can also be used.
4) Concentrating the purified DNA using ethanol precipitation, which causes the long DNA strands to precipitate out of
Cryopreservation is the process of preserving living cells and tissues by cooling them to very low sub-zero temperatures. This stops all biological and chemical processes, halting the living material in a state of suspended animation. There are several key steps in cryopreservation including preculturing materials, adding cryoprotectants, slow or stepwise freezing, storage in liquid nitrogen at -196°C, rapid thawing, and then reculturing. Common cryopreservation methods include slow freezing, vitrification, encapsulation-dehydration, and cryopreservation has many applications for preserving genetic resources like semen, embryos, oocytes, and more.
Primary and established cell line cultureKAUSHAL SAHU
Introduction
Primary Culture
Steps of Primary Culture
Isolation Of Tissue
Dissection And Disaggregation
Types Of Primary Culture
Primary Explants Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
Reference
Direct Gene Transfer method (gene gun method).ShaistaKhan60
Direct gene transfer methods rely on delivering naked DNA directly into plant cells without the use of Agrobacterium. Biolistic or gene gun particle bombardment is a direct gene transfer method where gold or tungsten particles are coated with DNA and shot into plant tissue using a gene gun. The DNA can then integrate into the plant genome. The method involves creating recombinant plasmids with the gene of interest, coating them onto microcarriers, bombarding embryogenic plant callus, selecting transformed cells, and regenerating plants. Transgenic papaya developed using the gene gun method were resistant to papaya ring spot virus.
Single cell culture involves isolating single cells from plant tissue and culturing them on a nutrient medium. There are mechanical and chemical methods for isolation. Cells can be cultured using various techniques like microchamber, microdroplet, or nurse culture techniques. The paper raft nurse culture places isolated cells on nutrient-soaked paper placed on actively growing callus tissue. Single cell culture is important for fundamental studies, mutation analysis, and industrial applications like crop improvement and production of medicinal compounds.
Animal cell culture media typically contain energy sources like glucose, amino acids as nitrogen sources, vitamins, inorganic salts, fatty acids, antibiotics, growth factors, and hormones. Most media also require an incubator to maintain optimal temperature, pH, osmolality, and gaseous environment for cell growth. Cell cultures can be grown adhered to surfaces or in suspension, and may have limited or continuous proliferation. Common applications of animal cell culture include vaccine production, cancer research, pharmaceutical drug production, and studying nerve cell function.
The document discusses the key stages in downstream processing as part of bio manufacturing or biosynthesis of products. It describes how downstream processing involves removing cells and impurities from fermentation broth to produce the final product. The main stages discussed are removal of insolubles, product isolation, product purification, and product polishing. Key operations at each stage include filtration, centrifugation, precipitation, crystallization, and lyophilization.
The document discusses upstream processing in biomanufacturing. Upstream processing involves growing cells in bioreactors to produce target proteins for pharmaceuticals. Key aspects of upstream processing include media preparation and sterilization, inoculum development, and cell culture in bioreactors. The main goal of upstream processing is to provide optimal environmental conditions for cell growth and protein production before downstream processing separates and purifies the target proteins.
The document discusses the essential components of plant tissue culture media, including macro and micro nutrients, organic nutrients like vitamins and carbohydrates, and plant growth regulators. It explains that the success of tissue culture depends on using the right type of culture media, which must contain nutrients, carbon sources, and other components to support in vitro plant growth. The various roles and forms of important media components like nitrogen, calcium, iron, and cytokinins are also outlined.
This document provides an overview of media formulation for fermentation and bioprocessing. It discusses the types of media, including complex and synthetic media. The key requirements for formulated media are then outlined, including carbon sources, oxygen sources, water, nitrogen sources, minerals, growth factors, and antifoams. Specific examples are given for each requirement. The document emphasizes that media formulation is essential for successful laboratory experiments and manufacturing processes.
Application of Microbial Biotechnology in Food Technology, Critical Review an...Zohaib HUSSAIN
1. Microbial fermentation is used to produce many important fermented foods like beers, wines, cheeses and more. Microbes add flavors and end products to foods through fermentation.
2. Nisin is an antibacterial peptide produced by Lactococcus lactis that is used as a food preservative. It is effective against many gram-positive bacteria and is considered generally safe for use in foods.
3. Lactobacillus sakei is used in meat fermentation and preservation. It produces compounds that form biofilms on meat surfaces to exclude other microbes and keeps meat safe during low-temperature storage.
This document summarizes two main types of fermentation processes: solid state fermentation and submerged fermentation. Solid state fermentation occurs without free water and uses natural raw materials like grains as the carbon source to cultivate microorganisms. Submerged fermentation uses a liquid substrate and is best for microbes that require high moisture. Both methods have various applications, with solid state fermentation used for producing enzymes, biopesticides, and in bioremediation, while submerged fermentation is common in industrial manufacturing.
The document discusses totipotency in plant cells. Totipotency refers to the ability of single plant cells to regenerate into a whole plant through cell differentiation and tissue culture techniques. The document outlines various tissue culture systems used to study totipotency, including callus culture, suspension culture, single cell culture, and protoplast culture. Factors that influence a cell's ability to express totipotency, such as the explant source and culture conditions, are also discussed.
Micromanipulation involves manipulating individual cells, sperm, or embryos under a microscope using microtools to improve fertilization and pregnancy rates. It can be performed physically using instruments like micromanipulators that carry microtools, or optically using lasers. Key applications of micromanipulation include IVF, stem cell research, transgenics, and basic biological research like studying protein interactions.
Protoplast fusion involves removing the cell walls of plant cells through enzymatic or mechanical means to create naked protoplasts. These protoplasts can then be fused using chemicals, electricity, or other methods. This allows the cytoplasms and sometimes nuclei of different plant cells to merge, creating hybrid cells. Successful fusion can generate hybrid plants through regeneration of cell walls and tissues. Protoplast fusion overcomes sexual incompatibility and is used to introduce traits like disease resistance between species. It remains a technically challenging process with limitations like genetic instability and uncertain expression of transferred traits.
Culture techniq and type of animal cell culturePankaj Nerkar
A primary culture refers to the initial culture of cells directly taken from an organism before the first subculture. A cell line refers to the propagation of cells after the first subculture. Primary cultures contain a variety of differentiated cell types and require higher cell quantities due to lower survival rates. Tissues are disaggregated into single cells using mechanical or enzymatic techniques for primary culture. Organ cultures involve culturing whole organs or tissues to preserve their structure and function in vitro. Various techniques like plasma clot, raft, and grid methods are used to culture different organ explants.
This document discusses protoplast isolation, culture, and fusion. It defines protoplasts as plant, fungal, or bacterial cells without cell walls. Protoplasts can be isolated from plant tissue through either mechanical or enzymatic methods, with enzymatic isolation being more common. The document outlines steps for protoplast isolation, purification, and assessing viability. It also discusses protoplast culture techniques and methods for inducing protoplast fusion, including electrofusion and PEG treatment. Applications of protoplast techniques in plant research include genetic engineering and crop breeding.
This document discusses methylases, which are enzymes that add methyl groups to DNA. Specifically:
- Methylases transfer methyl groups from S-adenosylmethionine to adenine or cytosine bases within their recognition sequence on DNA. This methylation protects the DNA from restriction endonucleases.
- The methylase and restriction enzyme of a bacterial species together form the restriction-modification system, with the methylase protecting the host DNA.
- Methylases are of interest because methylation of some restriction enzyme recognition sites protects the DNA from being cleaved by that enzyme. This allows study of DNA isolated from strains expressing common methylases like Dam or Dcm.
This document discusses plant cell culture production in bioreactors. It describes various bioreactor types including mechanically agitated, bubble column, and airlift bioreactors. Mechanically agitated bioreactors employ impellers but can cause high shear stress on plant cells. Bubble column and airlift bioreactors have no moving parts, providing mixing through rising gas and liquid circulation with less shear stress, but have lower oxygen transfer rates than mechanically agitated bioreactors. The document compares these bioreactor types for oxygen transfer ability and low shear on plant cells.
Plant tissue culture involves growing plant cells, tissues or organs in an artificial nutrient medium under sterile conditions. Some key points:
1. It allows for the rapid mass propagation of plants through micropropagation and the production of genetically uniform plants.
2. It facilitates the production of disease-free plants through culture of meristems and shoot tips.
3. It enables genetic modification of plants through techniques like protoplast fusion, anther culture and recombinant DNA technology.
Secondary metabolites are organic compounds produced by plants and organisms that are not essential for growth or reproduction. They play important roles in defense against herbivores and pathogens. Approximately 1500 new secondary metabolite molecules are identified from plants each year, around 30% of which show some biological activity. Secondary metabolites have many applications in medicine, food, cosmetics, and other industries. Plant tissue culture is used to produce many important secondary metabolites in a controlled environment, as production from native plants can be limited by environmental and geographical factors. Common production methods include cell suspension cultures, hairy root cultures, and immobilized cell cultures. Factors like media composition, temperature, pH, and elicitors can influence metabolite yield. An example is the production
This document provides information about plant tissue culture techniques. It discusses that plant tissue culture involves culturing small explant tissues in a sterile nutrient medium under controlled conditions. With the addition of hormones, new shoots and roots can be induced to grow from explants. Tissue culture is used for micropropagation to produce clones of plants. Single plant cells also have the ability to regenerate into whole plants given the right conditions. The document outlines the composition of solid and liquid culture media, different tissue culture techniques, types of culture, advantages, applications and nutritional requirements for plant tissue culture.
It gives the general knowledge about plant tissue culture. As this topic is an important aspects of plant biotechnology, it will remind a brief idea about why it is necessary.
Plant tissue culture is a collection of techniques used to grow plant cells, tissues or organs under sterile conditions. It allows for the mass production of clones of plants with desirable traits. The key aspects of plant tissue culture are maintaining sterile conditions on a nutrient medium, and providing proper aeration. Common types of plant tissue culture include callus culture, single cell culture, root tip culture, shoot tip culture, and anther culture. Plant tissue culture has many applications for plant conservation, breeding, and production of secondary metabolites.
Dr. Rehab Al Mousa. Plant Tissue CultureRehab Moussa
Plant tissue culture is a technique for growing plant cells, tissues or organs in vitro on artificial nutrient media under sterile conditions. It allows for the clonal propagation of plants as well as applications in plant breeding such as haploid production, somatic hybridization and genetic modification. Some challenges include contamination, hyperhydricity, phenolic exudation, shoot tip necrosis and somaclonal variation. Tissue culture has many uses in micropropagation, plant breeding, germplasm preservation, plant physiology and production of secondary metabolites.
This document contains protocols for various plant tissue culture techniques. It discusses the introduction to plant tissue culture, sterilization techniques used, and then outlines 8 specific protocols: 1) tissue culture media preparation, 2) explant preparation and surface sterilization, 3) embryo culture, 4) culture of anther for haploid production, 5) meristem culture, 6) meristem tip culture for virus-free plants, 7) induction of somatic embryogenesis, and 8) protoplast isolation, culture, and regeneration. The goal of these protocols is to describe the principles and procedures of different plant tissue culture methods.
Mass multiplication procedure for tissue culture and PTC requirementDr. Deepak Sharma
This presentation include basic Micropropagation protocol: Application and advantages of mass multiplication. Beside this the requirement of tissue culture are there (Nutrient, gelling agent, energy source, vitamins and PGRs) are also included.
This document provides an outline and overview of organogenesis in agriculture biotechnology. It defines organogenesis as the development of organs like flowers, roots, and shoots directly from an explant or callus culture. There are two main methods of organogenesis - direct organogenesis from an explant and indirect organogenesis through callus culture. The process involves two steps, caulogenesis which is the induction of adventitious shoot buds, and rhizogenesis which is the induction of adventitious roots. Organogenesis is regulated by plant growth hormones like auxins and cytokinins, and is affected by factors like growth regulator concentration, light intensity, temperature, and physical state of the culture medium.
Regeneration of plants and application of plant tissue culture SuruchiDahiya
Plant tissue culture is a collection of techniques used to grow plant cells, tissues, or organs in sterile conditions. It relies on the fact that many plant cells can regenerate a whole plant. There are several types of tissue culture including callus culture, organ culture, and suspension culture. Plant tissue culture has many applications including micropropagation, production of pharmaceuticals, and genetic engineering of plants. It is a valuable tool for producing disease-free plants and increasing crop yields.
This document summarizes research on somatic embryogenesis in rice. It describes the process of somatic embryogenesis, including the stages of embryogenesis and factors that affect it. The methodology section outlines the materials and methods used, including collecting rice seeds as explants, sterilizing them, and culturing them on callus induction and embryo germination media with different concentrations of plant growth regulators like 2,4-D, BAP and NAA. The goal is to develop an efficient system for somatic embryogenesis and plant regeneration in rice.
Internship of plant physiology department in universitat deÖzlem Kocaağaoğlu
1) The document discusses plant biotechnology and various plant cell culture techniques used to produce valuable compounds, including callus cultures, suspension cultures, and hairy root cultures. It focuses on using these techniques to study the taxol biosynthesis pathway in Taxus species.
2) Key genes involved in the taxol metabolic pathway are analyzed, including TB506, an unknown enzyme that may catalyze an important step. Experiments are described to test TB506's hydroxylase activity in vitro and in vivo by expressing it in transgenic Nicotiana tabacum plants.
3) The goal is to better understand the taxol biosynthesis pathway to maximize taxol production through these techniques and genetic studies of the pathway's unknown steps.
Callus is an unorganized mass of undifferentiated cells that can be cultured in vitro. It is produced when plant explants are cultured on medium containing auxin and cytokinin hormones under sterile conditions. Callus tissue lacks differentiation and is unable to perform photosynthesis. It can be maintained indefinitely and used for plant regeneration through processes like organogenesis and somatic embryogenesis. Successful callus culture requires aseptic preparation of explants, a nutrient medium with proper hormone balance, and controlled physical conditions for incubation.
Plant regeneration is possible through organogenesis or somatic embryogenesis where differentiated plant cells can become totipotent using hormones. The process involves taking an explant from a plant and culturing it in a nutrient medium under sterile conditions to regenerate a whole new plant. Somaclonal variation may occur during this process, introducing genetic changes into the regenerated plants.
Secondary metabolites are organic compounds produced by plant metabolism that are not essential for growth or reproduction but provide other benefits. They often function in plant defense against herbivores and pathogens. There are several types of plant tissue cultures used to study secondary metabolism, including organized cultures of tissues, disorganized callus cultures, hairy root cultures, and immobilized cell cultures where cells are confined within a matrix.
Role of Phytohormones in Tissue CultureApoorva Ashu
Description about phytohormones and their role in tissue culture, including descriptions about molecular basis of phytohormones with special focus on auxin and cytokinin and their role in calli development, organogenesis and somatic embryogenesis.
This document discusses plant tissue culture, including the types, steps involved, and procedures. It describes the different types of plant tissue culture such as seed culture, embryo culture, and anther culture. The key steps are initiation, multiplication, root formation, shoot formation, and acclimatization. The procedures covered are sterilization of materials, preparation and sterilization of explants, production and proliferation of callus, subculturing, and suspension culture. The document provides details on the composition of culture media and the roles of macronutrients, micronutrients, vitamins, nitrogen supplements, carbon sources, growth regulators, and solidifying agents.
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Histololgy of Female Reproductive System.pptxAyeshaZaid1
Dive into an in-depth exploration of the histological structure of female reproductive system with this comprehensive lecture. Presented by Dr. Ayesha Irfan, Assistant Professor of Anatomy, this presentation covers the Gross anatomy and functional histology of the female reproductive organs. Ideal for students, educators, and anyone interested in medical science, this lecture provides clear explanations, detailed diagrams, and valuable insights into female reproductive system. Enhance your knowledge and understanding of this essential aspect of human biology.
Promoting Wellbeing - Applied Social Psychology - Psychology SuperNotesPsychoTech Services
A proprietary approach developed by bringing together the best of learning theories from Psychology, design principles from the world of visualization, and pedagogical methods from over a decade of training experience, that enables you to: Learn better, faster!
Rasamanikya is a excellent preparation in the field of Rasashastra, it is used in various Kushtha Roga, Shwasa, Vicharchika, Bhagandara, Vatarakta, and Phiranga Roga. In this article Preparation& Comparative analytical profile for both Formulationon i.e Rasamanikya prepared by Kushmanda swarasa & Churnodhaka Shodita Haratala. The study aims to provide insights into the comparative efficacy and analytical aspects of these formulations for enhanced therapeutic outcomes.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of the physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar lead (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
6. Describe the flow of current around the heart during the cardiac cycle
7. Discuss the placement and polarity of the leads of electrocardiograph
8. Describe the normal electrocardiograms recorded from the limb leads and explain the physiological basis of the different records that are obtained
9. Define mean electrical vector (axis) of the heart and give the normal range
10. Define the mean QRS vector
11. Describe the axes of leads (hexagonal reference system)
12. Comprehend the vectorial analysis of the normal ECG
13. Determine the mean electrical axis of the ventricular QRS and appreciate the mean axis deviation
14. Explain the concepts of current of injury, J point, and their significance
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. Chapter 3, Cardiology Explained, https://www.ncbi.nlm.nih.gov/books/NBK2214/
7. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Kat...rightmanforbloodline
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Katzung, Verified Chapters 1 - 66, Complete Newest Version.
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Katzung, Verified Chapters 1 - 66, Complete Newest Version.
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Katzung, Verified Chapters 1 - 66, Complete Newest Version.
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Katzung, Verified Chapters 1 - 66, Complete Newest Version.
share - Lions, tigers, AI and health misinformation, oh my!.pptxTina Purnat
• Pitfalls and pivots needed to use AI effectively in public health
• Evidence-based strategies to address health misinformation effectively
• Building trust with communities online and offline
• Equipping health professionals to address questions, concerns and health misinformation
• Assessing risk and mitigating harm from adverse health narratives in communities, health workforce and health system
Muktapishti is a traditional Ayurvedic preparation made from Shoditha Mukta (Purified Pearl), is believed to help regulate thyroid function and reduce symptoms of hyperthyroidism due to its cooling and balancing properties. Clinical evidence on its efficacy remains limited, necessitating further research to validate its therapeutic benefits.
Osteoporosis - Definition , Evaluation and Management .pdfJim Jacob Roy
Osteoporosis is an increasing cause of morbidity among the elderly.
In this document , a brief outline of osteoporosis is given , including the risk factors of osteoporosis fractures , the indications for testing bone mineral density and the management of osteoporosis
2. 1. Biotechnology
• Plant Cell Biotechnology
• General terms
2. Plant Cell Culture
3. Development in Plant Cell Culture
4. Recent Approaches for High Production of secondary
metabolites ( Drugs)
CONTENTS:
2
3. The definition of biotechnology usually includes the development of methods by
which biological processes may be controlled such that their rate of production
enables economic industrial production (phytoproduction), or by which living
material is obtained that can be utilized in industry, agriculture and forestry, as well
as in gardening and breeding (plant breeding).
3
PLANT CELL BIOTECHNOLOGY:
BIOTECHNOLOGY:
Plant Cell Biotechnology is broadly concerned with experimental research on
plants and plant like organisms (fungi and cyanobacteria).(Stafford, Morris, &
Fowler, 1986)
4. GENERAL TERMS:
4
Cultures of plant cells taken from their natural
environment and placed under controlled
conditions. Callus a more or less loose
association of cells without visible morphological
differentiation. Cell suspension denotes cultures
consisting of single cells or the smallest cellular
association without differentiation, submersed in
a turbulent medium. Protoplasts are naked cells
of varied origin without cell walls, which are
cultivated in liquid as well as on solid media
Callus, Cell Suspension
and Protoplast: Each cell contains every single attribute
which appears in the differentiated plant.
• Morphological totipotency was
achieved by the regeneration of
completely normal differentiated plants
from callus and protoplasts
• Chemical totipotency was brought by
the examination of anthraquinone
accumulation in the calli of different
parts (root, leaf, shoot, fruit) of Morinda
citrifolia .
Totipotency:
Sterile organs or pieces of tissue used to gain
dedifferentiated cells by proliferation at
sectional planes or wounded areas are called
explants.
These Explants are placed in specific solid
culture media which, because of their
phytohormonal content, encourage cell
proliferation.
Explant:
As a rule, cytokinins and auxins are used as
phytohormones. 2,4-Dichlor-phenoxyacetic acid is
the most powerful, and is therefore called the
dedifferentiation hormone.
Phytohormones: Cytokinins Auxins
6-Benzyl-aminopurine (6-BA) Indole-3-acetic acid (IAA)
6-Furfuryl-aminopurine (kinetin) I-Naphthalene acetic acid (NAA)
2,4-Dichlorphenoxyacetic acid
(2,4-D)
5. Callus Formation: Usually, the ratio between auxin and cytokinin concentrations
determines whether a culture grows in a disorganized fashion as callus or develops
shoots or roots. A particularly effective agent for callus formation is 2,4-di-
chlorophenoxyacetic acid, generally characterized by its great effectiveness. Its effect
is usually increased by the addition of cytokinin.
Organogenesis: By reversing the conditions suitable for callus formation, the
meristematic cells or groups of cells developing in the course of callus formation are
stimulated to organogenesis. In many dicotyledonous callus cultures, bud formation
is generally initiated by a ratio of 100/10, while callus development is favored by the
inverse ratio 10/100. The relevant ratios in tobacco are different, but they do
demonstrate the dependence of the effect on the donor material. However, the
relative effectiveness of different auxins must be kept in mind. In some cases,
culturing without one of the two hormones is sufficient to induce organogenesis.
5
AUXIN TO CYTOKININ RATIO:
7. GENERAL TERMS:
7
• Soma clonal variations can be
obtained by subcultring the callus
into different particular stress
environment either by induced
mutation using mutagens or by
subculturing.
• They are clones but with
phenotypic and genotypic
variations.
Soma clonal variations:
• Somatic embryogenesis is an artificial
process in which a plant or embryo is
derived from a single somatic cell.
• Desired product can be obtained from plant
by somatic embryogenesis.
• Through somatic emryogenesis we get
genetically uniform cell.
• Bipolar in nature ( whole plant can grow)
• Disease free large scale production
• Synthatic seed production
• E.g; Somatic culture of tobacco plant to
extract nicotine.
Somatic embryogenesis:
• Morphogenesis (from the Greek
morphê shape and genesis creation,
literally "the generation of form") is the
biological process that causes a cell,
tissue or organism to develop its
shape.
• Organogenesis is the differenciation
of cells into organ system. In
monopolar growth either root or shoot
is grown at a time.
Morphogenesis:
9. METHODS OF PROPAGATION:
* According to a Survey
9
40-60%
Average Profit
Margin
$175,000
Average Annual
Revenue per Doctor
3%
Market Growth Per
Year
10. Cell suspension cultures are usually inoculated with the help of a callus fragment
put into a liquid medium. The minimal amount needed is about 2 to 3 g/l00 ml
medium.
10
PROTOPLAST CULTURE:
CELL SUSPENSION CULTURE :
1. Physical Methods
• Grinding with glass beads
2. Enzymatic Methods
• Cellulose
• Hemicellulose
• Pectinase
• The enzyme incubation can be carried out either successively in separate
enzyme solutions or in a mixture of these enzymes for a long time (5-8 h) at low
temperatures (8-25 °C), or alternatively for a short period ( < 2 h) at high
11. 11
GENERAL CULTURE TECHNIQUES :
1. Sterlization
• Sterlization of plant tissue by sodium hypochlorite, bezalkonium chloride, bromine
water.
• Sterlization of nutrient solution by autoclaving, tyndallization or sterile filtered.
• Sterlization of glassware and tools
• Air sterilization
2. Composition of media:
• Inorganic components
• Macronutrients
• Organic components
• Vitamins
• Growth regulators
12. 12
TISSUE CULTURE IS WIDELY USED IN:
PLANT TISSUE CULTURE:
• Plant tissue culture is a set of techniques for aseptic culture of cells, tissues, organs and
their components under desired physical and chemical conditions in vitro and controlled
conditions.
• It explore conditions that promote cell division and genetic reprogramming in vitro
conditions
• It is an important tool in basic and applied sciences as well as in commercial applications.
• Obtaining disease free plant.
• Rapid propagation of plants those that are difficult to propagate.
• Somatic hybridization.
• Genetics improvement of commercial plants e.g; transgenic plants
• Obtaining androgenic and gynogenic haploid plants for breeding programs.
13. 13
HISTORY:
• Gottlieb Haberlant justifiably is known as the father of plant tissue culture. He
predicted that eventually a complete and functional plant could be generated from
a single cell. (Totipotency)
• Other studies led to the culture of isolated root tips.
• The approach of using explants with meristematic cells produce the successful
and indefinite culture of tomato root tips.
• Only in 1985 was the sweetening agent from the flowers and leaves of the
verbena plant, Lippia dulcis, which exceeds saccharose l000-fold in its sweetening
power and which was used already by the Aztecs to sweeten their food, identified
as a colorless sesquiterpene oil, hernandulcin.
• The psychogenically active compounds from the "holy" fungi of the Indians,
psilocybin and psilocin from the leaves of the genus Psilocybe, are now being
utilized in modern medicine due to their structural similarity to the neurotransmitter
serotonin.
14. 14
CULTIVATION METHODS:
• Once protoplasts are separated from the isolation medium and transferred to a
suitable growth medium, adaptation and regeneration processes begin.
Adaptation is necessary because the growth conditions differ from those in the
tissue's previous environment.
Stabilization:
• The culture media for protoplasts are very similar to those for individual cells.
However, in the former it is necessary to create conditions favoring the formation
of cell walls and ensuring the stability of naked cells. Thus, the addition of
polyethylene glycol (PEG) 1500 to the medium often accelerates the uniform
deposition of micro fibrils.
Wall Formation:
• Polysaccharides such as hemicellulose synthesized for cell wall formation are
initially largely excreted into the medium. Studies on the structure of newly formed
1. PROTOPLAST CULTURE:
15. 15
CULTIVATION METHODS:
Medium Components:
Particular attention is paid to the effect of ion concentrations on protoplast division.
Depending on the origin, the auxin/cytokinin ratio must also be specifically adapted.
Highly differentiated mother cells (e.g. leaf cells) usually require a low auxin/kinetin
ratio, while protoplast cultures obtained from actively growing cell cultures or
meristematic tissue require high auxin/cytokinin ratios.
Sensitivity:
Further, a particularly great sensitivity to certain components of the medium and
environmental conditions (light, shearing forces) is characteristic of protoplast
cultures. For example, freshly isolated protoplasts are usually highly light-sensitive,
at least during the first 4-7 days.
1. PROTOPLAST CULTURE
16. 16
CULTIVATION METHODS:
The preferred donor materials for individual cells are young leaves, calli and
protoplasts. Cultures of N. tabacum and Phaseolus vulgaris, filtered off by a
mesh of 0.1 to 0.3 mm, consist of 90% single cell.
The three basic techniques:
• nurse culture
• plating technique
• culture in a microchamber
2. CULTURES OFSINGLE CELLS:
18. 18
CULTIVATION METHODS:
Nurse Culture:
The isolated cells to be cultured are fixed on a piece of filter paper on the surface of
an actively growing nurse callus. The contact through the absorbent paper is
sufficient to maintain a supply of nutrients and unknown growth factors. Once the
developing microcalli have attained the size necessary for survival (200-400 µm),
they are individually cultivated on a fresh medium.
2. CULTURES OFSINGLE CELLS:
19. 19
CULTIVATION METHODS:
Plating and Feeder-Layer Technique; Culture in a Microchamber:
• Conditioned Medium:
During a nurse culture, unknown growth factors accumulated in a suspension culture
or on the supporting filter paper are transmitted by the medium from cell to cell. In
this way, the medium is enriched with these substances. Such a medium is thus
called conditioned if it contains such substances excreted by any living cells which
ensure survival and reproduction of cells cultivated at a suboptimal density. The
effectivity of such media depends in part on the age of the nurse culture. However,
nutrients depleted from the medium during preculturing must be replaced, otherwise
they may be inhibitory.
2. CULTURES OFSINGLE CELLS:
20. 20
CULTIVATION METHODS:
• Plating and Feeder-Layer Techniques:
Plating Technique.
In this technique, cultures with a cell density less than the critical density are mixed
with the medium containing agar (0.6%) at a temperature of 30-35 °C and poured
into petri dishes to a depth of 1 mm. The low agar density makes it possible to follow
the development of single cells through an inverse microscope.
The culture may be divided into individual cubes, called agar beads. Plating success
is determined by the ratio of the number of cells in the original suspension to the
number of colonies developing within the following 21 days.
The goal is to attain the highest possible plating efficiency (PE), the number of
colonies per plated single cells, at the least possible cell density. The lower boundary
has so far been around 5000/ml.
2. CULTURES OFSINGLE CELLS:
21. 21
CULTIVATION METHODS:
• Plating and Feeder-Layer Techniques:
Feeder-Layer Technique:
In order to add a nutrient supply and stimulate division, living cells or protoplasts
unable to divide are added to the agar medium as a feeder layer.
2. CULTURES OFSINGLE CELLS:
22. 22
CULTIVATION METHODS:
• Culture in a Microchamber:
In micro chambers, the problem of insufficient cell density is solved by reducing the
amount of medium. Starting from the minimum concentration that ensures plating
success, the amount was reduced to 0.6 µl, 0.25-0.5 µl and even to microdroplets of
10-25 nl Such mini-cultures of a single cell or protoplast may be cultivated by either
hanging them from a slide held in place by the medium's surface tension (hanging
drop culture), or on a slide or in mini-agar caverns (1 x 1 x 1.75 mm) open above.
2. CULTURES OFSINGLE CELLS:
24. 24
CULTIVATION METHODS:
From the point of view of an application-oriented industry, experiments performed on
the scale of petri dishes and Erlenmeyer flasks cannot be economically utilized. Even
in laboratory fermenters with a capacity of 5-501, questions of scaling-up to industrial
scale are usually unanswerable. Therefore, industrial production conditions are
usually simulated in so-called pilot plants at a scale up to 1000 l. The results thus
obtained can be applied to larger reactors, provided the geometric and dynamic
parameters remain constant in the scaling factor. However, this is possible without
further difficulties only if a single parameter is rate-determining. This factor must be
kept constant during scaling-up.
Determining Factors:
The aspects that must be considered in culturing large volumes may be classified as
process and culture conditions. The extent of scaling-up is determined by factors of
physical, chemical, biochemical and biological nature.
3. MASS CULTIVATION METHOD:
27. 27
GROWTH OF CELL AND TISSUE CULTURE:
Measurement methods:
Changes in growth may be measured by fresh and dry weight, cell mass, cell
number, mitotic index or indirectly by the conductivity of the medium.
Fresh Weight: The method of fresh-weight determination neglects the various water
contents of the material. Therefore, the values of callus cultures, frequently
determined as total weight of callus, medium layer and petri dish, experience large
variations due to evaporation via the medium's surface. More exact values are
obtained by determining the weight after complete separation from the culture
medium. This is possible when the material is cultured on separating layers of
cellulose or nylon.
Dry Weight: Quantification by means of dry weight excludes error due to varying
endogenous water contents. This requires repeated drying, usally at 60°C, to the
point of constant weight. Up to fresh weights of 500 mg, a linear relationship between
fresh and dry weight is assumed.
GROWTH PROCESS:
28. 28
GROWTH OF CELL AND TISSUE CULTURE:
Cell Mass:
This may be determined by densification by centrifugation (ca. 2000 g, 5 min) of a
particular percentage of the volume (ca. 4-7 ml) in graduated, conical centrifuge
tubes. In order to avoid errors due to water absorption by the cells, the so-called
packed cell volume (PCV) must be recorded immediately following the separation
process.
Cell Number:
To determine the number of cells per unit volume, existing cell clumps or aggregates
must be separated into isolated cells - not only in callus cultures but also in most
suspension cultures. This is commonly done using chrome-trioxide alone or in
combination with hypochlorous acid. Possible alternatives are EDT A and pectinase.
GROWTH PROCESS:
29. 29
GROWTH OF CELL AND TISSUE CULTURE:
Conductivity:
The inverse relationship between the conductivity and fresh or dry weight of the
medium allows the determination of growth without taking samples (which would
affect the sterility of the culture). In fully synthetic media, conductivity is determined
almost exclusively by salt concentrations. As long as the pH of the medium remains
above 3 (CH + < 10-3 mol/I), the concentration of hydrogen ions does not affect
conductivity.
Cellulose Concentration:
Calcofluor-white ST (0.1 % aqueous) allows monitoring of changes in the
concentrations of cell wall polymers from fJ-glycosidic bound glucose molecules such
as cellulose or callose. The textile brightener specifically binds to fJ-l,4-glucans and
intensely fluoresces following stimulation with shortwave blue light. In this way, even
traces of these compounds may be identified
GROWTH PROCESS:
31. SECONDARY METABOLITES:
31
Secondary metabolites, also called specialised metabolites,
toxins, secondary products, or natural products, are organic compounds
produced by bacteria, fungi, or plants which are not directly involved in the
normal growth, development, or reproduction of the organism.
They were labelled secondary compounds in contradistinction to primary
compounds due to:
1. an apparently limited taxonomic distribution
2. Synthesis occurring only under certain conditions
3. An apparent lack of function
4. No apparent necessity for life.
32. 32
SOME PLANT SECONDARY METABOLITES AND THEIR
APPLICATION:
Metaobolites Application Species
Ajmalicine Circulation Catharanthus roseus
Atropine Anti-cholinergic Atropa belladonna
Hyoscyamine
Hyoscine
Theophylline
Anti-cholinergic
Anti-cholinergic
Hyoscyamus spp.
Datura spp.
Camellia sinensis
Diosgenin Contraceptive Dioscorea spp.
Quinine Anti-Malarial Cinchona spp
Eugenol
Local anesthetic
Syzygium aromaticum
Morphine Analgesics Papaver somniferum
34. 34
STORAGE OF SECONDARY METABOLITES:
Accumulation:
Turnover: Depending on the object, secondary compounds are either secreted
into the surrounding medium or stored intracellularly. There they experience
turnover processes with characteristic half-lives. Their degradation was first
proven in cell suspension cultures. Degradation and synthesis often occur
simultaneously. The extent of their accumulation is mainly determined by three
cell capacities: synthetic capacity, storage capacity and the capacity to
metabolize the compounds in transport and detoxification processes.
Individual plant organs differ in their significance in this process.
35. 35
PHARMACEUTICALLY ACTIVE NATURAL PRODUCT
SYNTHESIS VIA PLANT CELL CULTURE TECHNOLOGY:
Most secondary metabolites are often present in extremely low
amounts in the plant, often less than 1% of the total carbon.
This paucity can make natural harvestation impractical for bulk
production, especially in the case of slow growing species. The
significant engineering challenge is then to find a means by which
to produce the desired natural products in a way that is both
sustainable and financially feasible. Production of these products
in a microbial or fungal host by transferring the biosynthetic
pathway is possible.
36. PRODUCTION OPTIONS FOR NATURAL
PRODUCTS:
36
Chemical synthesis of natural products is possible and commercially feasible,
particularly for those with relatively simple chemical structures such as
aspirin (derived from the natural product salicylic acid) and ephedrine. In many
cases, however, the metabolite has a complex structure, which can include
multiple rings and chiral centers, so that a synthetic production process
becomes prohibitively costly. Many natural products used in cancer treatment,
including compounds such as paclitaxel, vinblastine, and camptothecin, fall into
this latter class, so an alternative method of supply is necessary.
37. 37
PRODUCTION OPTION FOR NATURAL PRODUCTS :
• Depending on the nature of the plant, extraction directly from harvested
plant tissue may be an option. Especially if a plant can be cultivated en
masse, this can be attractive on a commercial basis.
• The anticancer drugs vincristine and vinblastine, among other medicinally
valuable metabolites such as ajmalicine and serpentine, are found in the
Madagascar periwinkle Catharanthus roseus.
• Even though these important alkaloids, particularly vincristine and
vinblastine, naturally occur at very low levels in C. roseus less than 3 g per
metric tons the fast growing nature of the periwinkle makes field cultivation
most practical at the present time.
• However, the relative inefficiency and high cost of whole plant extraction
implies that an improved method of supply would be useful for these
valuable anticancer agents.
38. 38
PRODUCTION OPTION FOR NATURAL PRODUCTS :
• When natural supply is limited due to a combination of low yields and slow
growth rates, in vitro cultures provide an attractive alternative. Most plant
species can be cultured in vitro in either an undifferentiated or differentiated
state.
• As many secondary metabolites are produced by specialized cells, organ
cultures such as shoots or roots can exhibit similar metabolite profile
patterns compared to the native plant, whereas undifferentiated cultures
often accumulate secondary metabolites to a lesser extent, and sometimes
not at all.
39. 39
PRODUCTION OPTION FOR NATURAL PRODUCTS :
• The anticancer compound camptothecin, produced by the ornamental tree
Camptotheca acuminata as well as Nothapodytes fetida and Ophiorrhiza
pumila among other species, has been shown to accumulate in
undifferentiated cultures in very low or even undetectable amounts
compared to root cultures in which production levels were comparable to the
intact plant.
• Similarly, no artemisinin, a potent antimalarial drug, was found in cell
suspension cultures of Artemisia annua, while trace amounts were detected
in shoot cultures.
40. 40
PRODUCTION OPTION FOR NATURAL PRODUCTS :
• Root cultures can be transformed into hairy roots using the soil dwelling
bacteria Agrobacterium rhizogenes, resulting in cultures which are
genetically stable, capable of unlimited growth without additional hormones,
and have an increased capacity for secondary metabolite accumulation.
• Undifferentiated suspension cultures, which can be more easily scaled to
levels suitable for commercial production. There are currently 14 plant cell
culture processes which have been commercialized for production of
secondary metabolites (including products used in applications other than
pharmaceuticals such as food and cosmetics).
42. PLANT SUSPENSION CELL CULTURE TECHNOLOGY:
42
• Production of metabolites via plant cell suspension culture is renewable, environmentally
friendly, and from a processing standpoint, amenable to strict control, an advantage in
regards to meeting Food and Drug Administration manufacturing standards. Technology
developed for other cell culture and fermentation systems (e.g., mammalian and yeast) can
be readily adapted for large scale applications with plant cells, easing difficulties associated
with scale-up.
• A notable example of the success of plant cell culture systems, due in large part to
innovative research and the application of novel technologies, is paclitaxel synthesis and
supply. Paclitaxel, produced by Taxus spp., is an important anticancer agent used as a first
line treatment for several types of cancer, including breast, ovarian, and non small cell lung
cancer, and has also shown efficacy against AIDS-related Kaposi sacoma. Production of
paclitaxel via cell culture technology has been studied since the 1980s as an alternative
supply source to harvest of the slow growing yew tree, since a single dose of 300 mg
requires the sacrifice of a 100 year old tree
44. 44
PLANT SUSPENSION CELL CULTURE TECHNOLOGY :
• The primary challenges impeding regular commercial application of plant
cell culture technology are low and variable yields of metabolite
accumulation.
• Some metabolites do not accumulate in appreciable quantities in
undifferentiated cells. In these cases, manipulation of genes within the
biosynthetic pathway is needed to utilize plant cell cultures for bulk
production.
45. 45
HAIRY ROOT CULTURES ASASOURCE OF SECONDARY
METABOLITES
• The hairy root system based on inoculation with Agrobacterium
rhizogenes has become popular in the two last decades as a method of
producing secondary metabolites synthesized in plant roots.
• The hairy root phenotype is characterized by fast hormone-independent
growth, lack of geotropism, lateral branching, and genetic stability. The
secondary metabolites produced by hairy roots arising from the infection of
plant material by A. rhizogenes are the same as those usually synthesized
in intact parent roots, with similar or higher yields.
• This feature, together with genetic stability and generally rapid growth in
simple media lacking phytohormones, makes them especially suitable for
biochemical studies not easily undertaken with root cultures of an intact
plant.
46. 46
HAIRY ROOT CULTURES ASASOURCE OF SECONDARY
METABOLITES
• During the infection process, A. rhizogenes transfers a part of the DNA
(transferred DNA, T-DNA) located in the root-inducing plasmid Ri to plant
cells, and the genes contained in this segment are expressed in the same
way as the endogenous genes of the plant cells.
47. 47
METABOLIC ENGINEERING AND DIRECTED BIOSYNTHESIS
• The engineering of biosynthetic pathways within a plant cell
to enhance accumulation of a constitutively produced metabolite
is an appealing strategy in which exciting progress has been
made in the past decade.
• A variety of tools have been employed to both identify unknown
genes and characterize secondary metabolite pathway
regulation, including precursor feeding, gene over
expression, application of metabolic inhibitors, and mutant
selection.
• Additionally, elicitation, in relation to improving bulk yields in cell
culture, can also be used as a powerful tool to investigate
pathway regulation based on gene expression.
48. 48
METABOLIC ENGINEERING TOOLS:
• A metabolic engineering approach involves the manipulation of
targets within a cell. Techniques are therefore needed both for
the identification of these targets (i.e., genes, proteins,
metabolites) as well as for their exploitation.
• As many secondary pathways are still partially undefined,
elucidating pathway genes and their control elements is an
active research area.
• The subsequent identification of rate influencing steps within
a biosynthetic pathway can then be useful in providing targets for
a rational engineering strategy.
49. 49
METABOLIC ENGINEERING TOOLS:
• Plant cell cultures, including both suspension cultures and hairy
root cultures, have proven to be an extremely useful platform for
metabolic studies, as a fast growing and renewable source of
material.
• Whole plants can also be valuable, particularly as models to
study complex spatial and temporal control mechanisms
associated with environmental stimuli and morphogenesis from
a global metabolic perspective.
50. 50
METABOLIC ENGINEERING TOOLS:
• Several approaches have been used to identify the enzymes and their
corresponding genes which catalyze biosynthetic pathway steps.
• For the paclitaxel pathway, a successful approach utilized by the Croteau
laboratory incorporated feeding cell free Taxus extracts with precursors to
isolate and identify intermediate metabolites and enzymes.
• This approach led to the identification of taxadiene synthase, which
catalyzes the first committed step of the taxane pathway.
• Genes were subsequently identified from a cDNA library using PCR
amplification based on degenerate primers designed to recognize
conserved regions from homologous enzymes in other plants whose DNA
sequences were known.
51. 51
METABOLIC ENGINEERING TOOLS:
• Differential display methods (via reverse transcription and
PCR) comparing mRNA transcripts between elicited and
unelicited cells supplemented with a homology-based search of
a cDNA library from elicited cells, as well as random
sequencing of the same induced library, have also proven to be
extremely effective in gene discovery (for a comprehensive
review of molecular genetics in Taxus.
52. 52
TRADITIONAL STRATEGIES TO IMPROVE CELL CULTURE
YIELDS:
Optimization of cultural conditions:
• Number of chemical and physical factors like media components,
phytohormones, pH, temperature, aeration, agitation, light affecting
production of secondary metabolites affect culture productivity.
• Several products were found to be accumulating in cultured cells at a
higher level than those in native plants through optimization of cultural
conditions. Manipulation of physical aspects and nutritional elements
in a culture is perhaps the most fundamental approach for optimization of
culture productivity.
• For example, ginsenosides by Panax ginseng and Dixon, rosmarinic acid
by Coleus bluemei, shikonin by Lithospermum, ubiquinone-10 by Nicotiana
tabacum, berberin by Coptis japonica were accumulated in much higher
levels in cultured cells than in the intact plants.
53. 53
TRADITIONAL STRATEGIES TO IMPROVE CELL CULTURE
YIELDS:
Precursor feeding:
Exogenous supply of a biosynthetic precursor to culture medium may also
increase the yield of the desired product. This approach is useful when the
precursors are inexpensive. The concept is based on the idea that any
compound, which is an intermediate, in or at the beginning of a secondary
metabolite biosynthetic route, stands a good chance of increasing the yield of
the final product. Attempts to induce or increase the production of plant
secondary metabolites, by supplying precursor or intermediate compounds,
have been effective in many cases.
54. 54
TRADITIONAL STRATEGIES TO IMPROVE CELL CULTURE
YIELDS:
Precursor feeding:
For example,
• amino acids have been added to cell suspension culture media for
production of tropane alkaloids, indole alkaloids etc.
• Addition of phenylalanine to Salvia officinalis cell suspension cultures
stimulated the production of rosmarinic acid.
• Addition of the same precursor resulted stimulation of taxol production in
Taxus cultures.
• Feeding ferulic acid to cultures of Vanilla planifolia resulted in increase
in vanillin accumulation.
• Furthermore, addition of leucine, led to enhancement of volatile
monoterpenes in cultures of Perilla frutiscens, where as addition of geraniol
to rose cell cultures led to accumulation of nerol and citronellol
55. 55
TRADITIONAL STRATEGIES TO IMPROVE CELL CULTURE
YIELDS:
Elicitation:
Plants produce secondary metabolites in nature as a defense
mechanism against attack by pathogens. Elicitors are signals
triggering the formation of secondary metabolites.
Perhaps the most notable strategy for improving metabolite yields
is elicitation. An elicitor can be defined as any compound that
induces an upregulation of genes. Some elicitors target secondary
metabolic genes, which are often associated with defense
responses to perceived environmental changes.
56. 56
TRADITIONAL STRATEGIES TO IMPROVE CELL CULTURE
YIELDS:
Elicitation:
• Elicitors include natural hormones, nutrients, and many fungi-derived
compounds. In particular, jasmonic acid and its methyl ester methyl
jasmonate (MJ), are naturally occurring hormones involved in the
regulation of defense genes as part of a signal transduction system.
• Applied exogenously, they have been shown to induce secondary metabolic
activity and promote accumulation of desired metabolites in numerous plant
systems, including Taxus spp. and C. roseus.
• Different elicitors may act on different segments of the biosynthetic pathway.
For instance, MJ elicitation compared to salicylic acid elicitation in
Taxus spp. cultures resulted in different relative increases of
metabolic intermediates, suggesting that each elicitor preferentially directs
flux toward, and possibly away from, different intermediate taxanes.
57. 57
TRADITIONAL STRATEGIES TO IMPROVE CELL CULTURE
YIELDS:
Elicitation:
While many of the specific targets of elicitors have yet to be conclusively
identified, elicitation can be an extremely useful tool in conjunction with gene
expression profiling for identifying rate-influencing steps in secondary
biosynthetic pathways.
58. 58
TRADITIONAL STRATEGIES TO IMPROVE CELL CULTURE
YIELDS:
Immobilization of plant cell cultures:
• It has long been considered for increasing metabolite accumulation, as the
potential of higher cell densities, continuous removal of products/inhibitors, and
protection for shear-sensitive plant cells provide a number of advantages.
• Immobilization can be simply achieved using a gel matrix such as alginate;
however this becomes costly at a larger scale, especially when the product of
interest is not secreted and must be released using sonication or treatments with
an organic solvent.
59. 59
TRADITIONAL STRATEGIES TO IMPROVE CELL CULTURE
YIELDS:
Immobilization of plant cell cultures:
• Recently, immobilization of T. baccata cells in calcium-alginate beads was shown
to produce one of the highest reported levels of paclitaxel accumulation among
academic laboratories (43 mg/L).
• Immobilization also has the potential to simplify product extraction and
purification, as immobilized cultures of Linum usitatissimum excrete the
pharmaceutically active metabolite dehydrodiconiferyl alcohol 4--D-glucoside
(DCG) to a greater extent than suspension cultures.
60. Stafford, A., Morris, P., & Fowler, M. (1986). Plant cell biotechnology: a perspective.
Enzyme and microbial technology, 8(10), 578-587.
Endress, R., & Endress, R. (1994). Plant cell biotechnology: Springer.
Kolewe, M. E., Gaurav, V., & Roberts, S. C. (2008). Pharmaceutically active natural
product synthesis and supply via plant cell culture technology. Molecular pharmaceutics,
5(2), 243-256.
Aboujaoude, E., Salame, W., & Naim, L. (2015). Telemental health: a status update.
World psychiatry, 14(2), 223-230.
Mulabagal, V., & Tsay, H.-S. (2004). Plant cell cultures-an alternative and efficient
source for the production of biologically important secondary metabolites. Int J Appl Sci
Eng, 2(1), 29-48.
60
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