This document provides information about plant tissue culture techniques at the Government Arts College in Coimbatore, India. It discusses the history and applications of plant tissue culture, characteristics of the techniques, important media components and their roles, effects of plant hormones, and basic procedures like media preparation and sterilization. The facility has equipment for sterile culture including laminar flow hoods, autoclaves, and culture racks. Plant tissue culture can be used for micropropagation, germplasm conservation, and production of transgenic plants.
This document discusses plant tissue culture techniques, including the types of explants used and sterilization protocols. It describes that meristem culture uses meristematic tissues like shoot apical meristems. Seed and ovule culture uses fertilized ovules from plants with small or minute seeds. Callus culture produces an unorganized cell mass. Haploid culture generates haploid cells from tissues like pollen and anthers. Different sterilization chemicals like sodium hypochlorite and calcium hypochlorite are used depending on the explant type. Protocols vary in chemical concentration and duration to sterilize tissues without loss of viability. Cells in culture go through growth phases including exponential growth, before requiring sub-cult
This document summarizes the process of plant genetic transformation using Agrobacterium tumefaciens. It describes how A. tumefaciens transfers T-DNA from its Ti plasmid into plant cells, integrating the T-DNA into the plant genome and expressing genes that cause crown gall disease. The document also outlines the key steps in the process, from gene transfer to the plant cell through regeneration of a transformed whole plant and methods to detect successful transformation events. Common genes inserted into transgenic crops are also listed, including genes for herbicide and insect resistance.
Plant tissue culture is the process of growing plant cells, tissues or organs in an artificial nutrient medium under sterile conditions. It allows plants to be grown in vitro from small meristematic tissues. There are several types of plant tissue culture including callus culture, organ culture and cell suspension culture. The basic technique involves surface sterilization of explant tissue followed by establishment and subculture of the culture on nutrient media. Callus culture specifically produces an undifferentiated mass of plant cells called a callus from explants. Plant tissue culture has many applications including disease elimination, genetic studies, large scale propagation and crop improvement.
This document discusses embryo culture and embryo rescue techniques. It begins by defining an embryo and explaining that embryo culture involves growing plant embryos in artificial media to enhance survival. Embryo rescue involves culturing immature embryos to prevent abortion, especially for interspecific or intergeneric hybrids where endosperm development fails. The key steps of embryo culture include excising embryos, placing them in sterile media with suitable temperature, light and nutrients, and transferring viable plantlets to soil. Embryo culture has applications in shortening breeding cycles, overcoming dormancy, producing sterile seeds, and rescuing distant hybrids.
Herbicide tolerance plants are developed to control weeds that compete with crops for resources and can reduce crop yields by 20% annually. Weeds are typically controlled mechanically or through herbicides. Glyphosate is a commonly used broad-spectrum herbicide that acts by inhibiting the shikimate pathway enzyme EPSP synthase, preventing aromatic amino acid production. Transgenic plants are made tolerant to glyphosate by introducing the aroA gene to allow continued shikimate pathway function. Phosphinothricin resistance is conferred by the bar gene, which allows detoxification of the glutamine synthase inhibitor phosphinothricin. Bromoxynil tolerance results from the bxn gene introducing nitril
Transformation is the process of altering an organism's genetic makeup by inserting new genes. Common transformation methods include Agrobacterium-mediated transformation, particle bombardment, protoplast transformation using polyethylene glycol or electroporation, and fibre-mediated DNA delivery. Agrobacterium transformation involves the bacteria transferring T-DNA from its Ti plasmid into the plant genome, while direct methods introduce naked DNA into plant cells using physical methods like particle bombardment or chemical treatments that make cell membranes permeable. Transformation allows improving crop traits like yield and stress resistance.
This document discusses plant tissue culture techniques, including the types of explants used and sterilization protocols. It describes that meristem culture uses meristematic tissues like shoot apical meristems. Seed and ovule culture uses fertilized ovules from plants with small or minute seeds. Callus culture produces an unorganized cell mass. Haploid culture generates haploid cells from tissues like pollen and anthers. Different sterilization chemicals like sodium hypochlorite and calcium hypochlorite are used depending on the explant type. Protocols vary in chemical concentration and duration to sterilize tissues without loss of viability. Cells in culture go through growth phases including exponential growth, before requiring sub-cult
This document summarizes the process of plant genetic transformation using Agrobacterium tumefaciens. It describes how A. tumefaciens transfers T-DNA from its Ti plasmid into plant cells, integrating the T-DNA into the plant genome and expressing genes that cause crown gall disease. The document also outlines the key steps in the process, from gene transfer to the plant cell through regeneration of a transformed whole plant and methods to detect successful transformation events. Common genes inserted into transgenic crops are also listed, including genes for herbicide and insect resistance.
Plant tissue culture is the process of growing plant cells, tissues or organs in an artificial nutrient medium under sterile conditions. It allows plants to be grown in vitro from small meristematic tissues. There are several types of plant tissue culture including callus culture, organ culture and cell suspension culture. The basic technique involves surface sterilization of explant tissue followed by establishment and subculture of the culture on nutrient media. Callus culture specifically produces an undifferentiated mass of plant cells called a callus from explants. Plant tissue culture has many applications including disease elimination, genetic studies, large scale propagation and crop improvement.
This document discusses embryo culture and embryo rescue techniques. It begins by defining an embryo and explaining that embryo culture involves growing plant embryos in artificial media to enhance survival. Embryo rescue involves culturing immature embryos to prevent abortion, especially for interspecific or intergeneric hybrids where endosperm development fails. The key steps of embryo culture include excising embryos, placing them in sterile media with suitable temperature, light and nutrients, and transferring viable plantlets to soil. Embryo culture has applications in shortening breeding cycles, overcoming dormancy, producing sterile seeds, and rescuing distant hybrids.
Herbicide tolerance plants are developed to control weeds that compete with crops for resources and can reduce crop yields by 20% annually. Weeds are typically controlled mechanically or through herbicides. Glyphosate is a commonly used broad-spectrum herbicide that acts by inhibiting the shikimate pathway enzyme EPSP synthase, preventing aromatic amino acid production. Transgenic plants are made tolerant to glyphosate by introducing the aroA gene to allow continued shikimate pathway function. Phosphinothricin resistance is conferred by the bar gene, which allows detoxification of the glutamine synthase inhibitor phosphinothricin. Bromoxynil tolerance results from the bxn gene introducing nitril
Transformation is the process of altering an organism's genetic makeup by inserting new genes. Common transformation methods include Agrobacterium-mediated transformation, particle bombardment, protoplast transformation using polyethylene glycol or electroporation, and fibre-mediated DNA delivery. Agrobacterium transformation involves the bacteria transferring T-DNA from its Ti plasmid into the plant genome, while direct methods introduce naked DNA into plant cells using physical methods like particle bombardment or chemical treatments that make cell membranes permeable. Transformation allows improving crop traits like yield and stress resistance.
Cell suspension culture involves growing single plant cells or small cell aggregates in agitated liquid medium. It allows studying cellular events during growth and development without the limitations of callus culture. An ideal suspension culture consists of only uniformly growing single cells. It is established by transferring friable callus pieces to agitated medium, then filtering and subculturing the dispersed cells. Suspension culture offers insights into cell physiology and is useful for cloning, secondary metabolite production, and mutagenesis studies. While it addresses issues with callus culture, cell suspension cultures can have decreasing productivity over time and slow growth.
This document discusses callus and suspension cultures. Callus culture involves culturing explants on agar medium to form an unorganized cell mass called callus. Suspension cultures involve culturing tissues or cells in liquid medium, producing single cells and clumps. There are three types of suspension cultures: batch, continuous, and immobilized. Batch cultures use a limited nutrient supply until growth declines. Continuous cultures drain out used medium and add fresh medium to maintain a steady state. Immobilized cultures encapsulate plant cells in gels like agarose.
Molecular markers can be used to characterize plant genetic resources and assist in pre-breeding for climate change. Various marker techniques are described, including hybridization-based RFLP and PCR-based RAPD, ISSR, SSR, AFLP, EST, and SCoT. Molecular markers reflect heritable DNA differences and have advantages like being ubiquitous, stable, and not affecting phenotypes. Data from markers can be analyzed to construct genetic similarity matrices and dendrograms to study genetic diversity and relationships. Molecular markers have applications in fingerprinting, diversity studies, marker-assisted selection, genetic mapping, and gene tagging.
This document discusses meristem culture and shoot tip culture techniques. It describes the three stages of meristem culture: establishment, multiplication, and root regeneration. Shoot tips less than 1 mm are excised and cultured on medium supplemented with hormones like cytokinins and auxins to promote growth. Meristem culture allows for virus elimination, micropropagation, genetic resource preservation, and facilitates international plant exchange. It is an effective method for producing disease-free plants.
Shoot tip culture is a plant tissue culture technique used to produce virus-free plants by culturing the meristematic tissue at the tip of a plant shoot. This allows production of new plants that are genetically identical to the donor plant but free of viruses, as viruses are unable to move between cells in the meristem. The protocol involves surface sterilizing and culturing shoot tip explants less than 1mm on agar media, with stages of culture establishment, shoot proliferation, and root regeneration using cytokinins and auxins. Shoot tip culture has applications in micropropagation, storage of plant genetic resources, quarantining imported plant materials, and eliminating viruses from infected plants.
1. Plants have internal mechanisms for tolerating variations in external environments like water deficit, cold, and heat stress.
2. Engineering stress tolerance focuses on producing osmoprotectants like glycine betaine and sorbitol to reduce osmotic stress from water deficit. Glycine betaine is produced through pathways using enzymes like choline monooxygenase.
3. Salt tolerance has been engineered by transforming plants with genes for vacuolar antiport proteins like NHX1 to transport sodium ions out of cells.
This document provides an overview of various gene transformation techniques, including both vector-mediated and direct methods. It discusses natural transformation mechanisms like conjugation and transduction, as well as artificial vector-mediated techniques like Agrobacterium-mediated transformation. Direct methods like microinjection, electroporation, particle bombardment, and chemical methods using PEG or calcium phosphate are also covered. The applications, advantages, and limitations of different techniques are summarized. Overall, the document serves as an informative introduction to the key gene transfer methods used in plant biotechnology.
This document discusses cell suspension culture, which involves growing plant cells, tissues, or organs in liquid nutrient medium. There are two main types of cell suspension cultures: batch culture and continuous culture. Batch culture involves growing cells in a finite volume of agitated medium, while continuous culture replaces old medium continuously to maintain cells in steady growth. The document outlines different methods for each type of culture and their characteristics, advantages, and importance for studying plant cell physiology and biochemistry.
This document summarizes a seminar presentation on improving fruit quality through the use of elicitors and bio-molecules. The presentation outlines the seminar topics which include introduction to elicitors and bio-molecules, their mechanisms of action, and case studies on their use in pomegranate and citrus fruits. It also summarizes results from studies showing elicitors can increase phenolic content and bioactive compounds in fruits, thereby improving quality attributes such as color, firmness, and reducing decay during storage.
Tissue culture involves growing plant cells, tissues, or organs in an artificial nutrient medium under sterile conditions. This allows for cloning plants at a large scale. Key applications include producing virus-free plants, rapidly multiplying biomass, and generating transgenic plants through techniques like Agrobacterium transformation which transfers DNA into plant cells. Tissue culture is an essential part of plant biotechnology and micropropagation to regenerate whole plants from small explants for commercial propagation of new cultivars with desirable traits.
Somaclonal variation refers to genetic variations that can arise during plant tissue culture and regeneration. When plant cells or tissues are cultured in vitro, genetic and epigenetic changes can occur, resulting in phenotypically different regenerated plants (somaclones) compared to the original plant. Somaclonal variation is caused by factors like culture conditions, genotype, explant source, and selection method used. It can generate variations in chromosome structure, number, and gene mutations. Somaclonal variation has been used to develop novel variants with improved traits like disease resistance, abiotic stress tolerance, and altered plant morphology. However, extensive field testing is required to evaluate variants due to possible genetic instability and undesirable effects.
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.
The MTT assay and the MTS assay are colorimetric assays for measuring the activity of enzymes that reduce MTT or close dyes (XTT, MTS, WSTs) to formazan dyes, giving a purple color The main application allows to assess the viability (cell counting) and the proliferation of cells (cell culture assays)
It can also be used to determine cytotoxicity of potential medicinal agents and toxic materials, since those agents would stimulate or inhibit cell viability and growth
Somatic hybridization is a technique used to produce hybrid plants by fusing protoplasts (plant cells without cell walls) from two different plant species or varieties. There are several key steps:
1. Isolation of protoplasts from plant tissues using either mechanical or enzymatic methods. Enzymatic methods using cellulase and pectinase enzymes are more common.
2. Fusion of the protoplasts using chemical fusogens like polyethylene glycol (PEG) or physical methods like electrofusion. This results in hybrid cells called heterokaryons.
3. Selection and culture of the hybrid cells using techniques like antibiotic resistance or genetic markers.
4. Regeneration
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 provides an overview of plant tissue culture techniques, including micropropagation and protoplast fusion. It discusses the various stages of micropropagation (initiation, multiplication, rooting, acclimatization). Explants can be surface sterilized and cultured on nutrient media. Protoplast fusion involves isolating protoplasts enzymatically, fusing them using techniques like PEG or electrofusion, and regenerating hybrid plants. The hybrids can be identified based on markers and ability to regenerate. Micropropagation allows mass production of clones while protoplast fusion enables hybridization of distantly related species.
Plant tissue culture techniques allow for the culture and maintenance of plant cells or organs in sterile conditions. Tissue culture produces clones with identical genotypes. Commercial uses include plant propagation, known as micropropagation. Key requirements for plant tissue culture include appropriate explant tissue, a nutrient growth medium, sterile conditions, and growth regulators like auxins and cytokinins. Plant cells in culture can form callus tissue or regenerate into whole plants through organogenesis or somatic embryogenesis depending on the hormone ratios and explant source tissue used.
This document provides an overview of plant tissue culture. It discusses key topics such as the history of tissue culture including Gottlieb Haberlandt's pioneering work. The widely used Murashige and Skoog medium is described. The document outlines different types of tissue culture like callus culture and meristem culture. Applications include plant conservation and propagation, breeding, and producing valuable compounds. The conclusion emphasizes performing tissue culture ethically.
Cell suspension culture involves growing single plant cells or small cell aggregates in agitated liquid medium. It allows studying cellular events during growth and development without the limitations of callus culture. An ideal suspension culture consists of only uniformly growing single cells. It is established by transferring friable callus pieces to agitated medium, then filtering and subculturing the dispersed cells. Suspension culture offers insights into cell physiology and is useful for cloning, secondary metabolite production, and mutagenesis studies. While it addresses issues with callus culture, cell suspension cultures can have decreasing productivity over time and slow growth.
This document discusses callus and suspension cultures. Callus culture involves culturing explants on agar medium to form an unorganized cell mass called callus. Suspension cultures involve culturing tissues or cells in liquid medium, producing single cells and clumps. There are three types of suspension cultures: batch, continuous, and immobilized. Batch cultures use a limited nutrient supply until growth declines. Continuous cultures drain out used medium and add fresh medium to maintain a steady state. Immobilized cultures encapsulate plant cells in gels like agarose.
Molecular markers can be used to characterize plant genetic resources and assist in pre-breeding for climate change. Various marker techniques are described, including hybridization-based RFLP and PCR-based RAPD, ISSR, SSR, AFLP, EST, and SCoT. Molecular markers reflect heritable DNA differences and have advantages like being ubiquitous, stable, and not affecting phenotypes. Data from markers can be analyzed to construct genetic similarity matrices and dendrograms to study genetic diversity and relationships. Molecular markers have applications in fingerprinting, diversity studies, marker-assisted selection, genetic mapping, and gene tagging.
This document discusses meristem culture and shoot tip culture techniques. It describes the three stages of meristem culture: establishment, multiplication, and root regeneration. Shoot tips less than 1 mm are excised and cultured on medium supplemented with hormones like cytokinins and auxins to promote growth. Meristem culture allows for virus elimination, micropropagation, genetic resource preservation, and facilitates international plant exchange. It is an effective method for producing disease-free plants.
Shoot tip culture is a plant tissue culture technique used to produce virus-free plants by culturing the meristematic tissue at the tip of a plant shoot. This allows production of new plants that are genetically identical to the donor plant but free of viruses, as viruses are unable to move between cells in the meristem. The protocol involves surface sterilizing and culturing shoot tip explants less than 1mm on agar media, with stages of culture establishment, shoot proliferation, and root regeneration using cytokinins and auxins. Shoot tip culture has applications in micropropagation, storage of plant genetic resources, quarantining imported plant materials, and eliminating viruses from infected plants.
1. Plants have internal mechanisms for tolerating variations in external environments like water deficit, cold, and heat stress.
2. Engineering stress tolerance focuses on producing osmoprotectants like glycine betaine and sorbitol to reduce osmotic stress from water deficit. Glycine betaine is produced through pathways using enzymes like choline monooxygenase.
3. Salt tolerance has been engineered by transforming plants with genes for vacuolar antiport proteins like NHX1 to transport sodium ions out of cells.
This document provides an overview of various gene transformation techniques, including both vector-mediated and direct methods. It discusses natural transformation mechanisms like conjugation and transduction, as well as artificial vector-mediated techniques like Agrobacterium-mediated transformation. Direct methods like microinjection, electroporation, particle bombardment, and chemical methods using PEG or calcium phosphate are also covered. The applications, advantages, and limitations of different techniques are summarized. Overall, the document serves as an informative introduction to the key gene transfer methods used in plant biotechnology.
This document discusses cell suspension culture, which involves growing plant cells, tissues, or organs in liquid nutrient medium. There are two main types of cell suspension cultures: batch culture and continuous culture. Batch culture involves growing cells in a finite volume of agitated medium, while continuous culture replaces old medium continuously to maintain cells in steady growth. The document outlines different methods for each type of culture and their characteristics, advantages, and importance for studying plant cell physiology and biochemistry.
This document summarizes a seminar presentation on improving fruit quality through the use of elicitors and bio-molecules. The presentation outlines the seminar topics which include introduction to elicitors and bio-molecules, their mechanisms of action, and case studies on their use in pomegranate and citrus fruits. It also summarizes results from studies showing elicitors can increase phenolic content and bioactive compounds in fruits, thereby improving quality attributes such as color, firmness, and reducing decay during storage.
Tissue culture involves growing plant cells, tissues, or organs in an artificial nutrient medium under sterile conditions. This allows for cloning plants at a large scale. Key applications include producing virus-free plants, rapidly multiplying biomass, and generating transgenic plants through techniques like Agrobacterium transformation which transfers DNA into plant cells. Tissue culture is an essential part of plant biotechnology and micropropagation to regenerate whole plants from small explants for commercial propagation of new cultivars with desirable traits.
Somaclonal variation refers to genetic variations that can arise during plant tissue culture and regeneration. When plant cells or tissues are cultured in vitro, genetic and epigenetic changes can occur, resulting in phenotypically different regenerated plants (somaclones) compared to the original plant. Somaclonal variation is caused by factors like culture conditions, genotype, explant source, and selection method used. It can generate variations in chromosome structure, number, and gene mutations. Somaclonal variation has been used to develop novel variants with improved traits like disease resistance, abiotic stress tolerance, and altered plant morphology. However, extensive field testing is required to evaluate variants due to possible genetic instability and undesirable effects.
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.
The MTT assay and the MTS assay are colorimetric assays for measuring the activity of enzymes that reduce MTT or close dyes (XTT, MTS, WSTs) to formazan dyes, giving a purple color The main application allows to assess the viability (cell counting) and the proliferation of cells (cell culture assays)
It can also be used to determine cytotoxicity of potential medicinal agents and toxic materials, since those agents would stimulate or inhibit cell viability and growth
Somatic hybridization is a technique used to produce hybrid plants by fusing protoplasts (plant cells without cell walls) from two different plant species or varieties. There are several key steps:
1. Isolation of protoplasts from plant tissues using either mechanical or enzymatic methods. Enzymatic methods using cellulase and pectinase enzymes are more common.
2. Fusion of the protoplasts using chemical fusogens like polyethylene glycol (PEG) or physical methods like electrofusion. This results in hybrid cells called heterokaryons.
3. Selection and culture of the hybrid cells using techniques like antibiotic resistance or genetic markers.
4. Regeneration
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 provides an overview of plant tissue culture techniques, including micropropagation and protoplast fusion. It discusses the various stages of micropropagation (initiation, multiplication, rooting, acclimatization). Explants can be surface sterilized and cultured on nutrient media. Protoplast fusion involves isolating protoplasts enzymatically, fusing them using techniques like PEG or electrofusion, and regenerating hybrid plants. The hybrids can be identified based on markers and ability to regenerate. Micropropagation allows mass production of clones while protoplast fusion enables hybridization of distantly related species.
Plant tissue culture techniques allow for the culture and maintenance of plant cells or organs in sterile conditions. Tissue culture produces clones with identical genotypes. Commercial uses include plant propagation, known as micropropagation. Key requirements for plant tissue culture include appropriate explant tissue, a nutrient growth medium, sterile conditions, and growth regulators like auxins and cytokinins. Plant cells in culture can form callus tissue or regenerate into whole plants through organogenesis or somatic embryogenesis depending on the hormone ratios and explant source tissue used.
This document provides an overview of plant tissue culture. It discusses key topics such as the history of tissue culture including Gottlieb Haberlandt's pioneering work. The widely used Murashige and Skoog medium is described. The document outlines different types of tissue culture like callus culture and meristem culture. Applications include plant conservation and propagation, breeding, and producing valuable compounds. The conclusion emphasizes performing tissue culture ethically.
tissue culture, a method of biological research in which fragments of tissue from an animal or plant are transferred to an artificial environment in which they can continue to survive and function. The cultured tissue may consist of a single cell, a population of cells, or a whole or part of an organ.
Applications include:
micropropagation using meristem and shoot culture to produce large numbers of identical individuals.
screening programmes of cells, rather than plants for advantageous characters.
large-scale growth of plant cells in liquid culture as a source of secondary products.
Tissue culture involves the use of small pieces of plant tissue (explants) which are cultured in a nutrient medium under sterile conditions. Tissue culture is in vitro maintenance and propagation of isolated cells tissues or organs in an appropriate artificial environment.
This document provides an overview of plant tissue culture techniques. It discusses the history and milestones of tissue culture, including the development of the Murashige and Skoog medium. The document describes the various types of tissue culture, choice of explant, regeneration pathways, and applications. It also discusses hairy root culture and the recognition of tissue culture facilities. The techniques of sterilization, culture growth, and plant regeneration are outlined. Advantages and disadvantages of tissue culture are presented.
The document provides details on the history and development of plant tissue culture techniques. Some key points:
- Plant tissue culture was pioneered in the early 20th century with efforts to culture plant cells in vitro. The field has since developed techniques to culture various plant tissues, organs, and cells.
- Important media like MS and B5 were developed to provide nutrients for cultured plant cells and tissues. These media contain macronutrients, micronutrients, vitamins, carbon sources, and growth regulators.
- Growth regulators like auxins and cytokinins are critical components added to media to induce cell division and organogenesis. Different growth regulator combinations induce formation of callus, roots, or shoots.
- Techn
This document provides an overview of plant tissue culture. It defines plant tissue culture as the in vitro culture of plant cells, tissues, or organs on an artificial nutrient medium under sterile conditions. The key requirements for plant tissue culture are discussed, including necessary equipment, media preparation, sterilization techniques, and growth regulators. Plant cells have three fundamental abilities - totipotency, dedifferentiation, and competency - that allow regeneration of whole plants in culture. A brief history of developments in plant tissue culture is also presented.
This document provides an overview of plant tissue culture. It discusses the basics, including definitions and history. The key facilities, materials, and equipment needed are described. The composition and role of various media components such as macronutrients, micronutrients, carbon sources, and growth regulators are summarized. Different types of cultures and their applications are listed. The general steps involved in plant tissue culture are outlined.
1. The document reports on developing a micropropagation protocol for Bacopa monnieri (Brahmi).
2. Various tissue culture techniques were tested, including callus culture and meristem culture, to optimize shoot initiation and proliferation using different concentrations of BAP and TDZ.
3. The results showed that MS medium supplemented with 2% BAP produced the highest number and length of shoots, making it the most effective treatment for shoot multiplication.
Plant biotechnology also known as green biotechnology is the use of biotechnology in plant or crop production. There are several techniques used such as ell culturing. Organ culture, explant culture, cell suspension culture are some culture types. This is a very useful technology in which have several applications like synthetic seed production, somaclonal variation, cybridization, hybridization.
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.
This document discusses plant tissue culture, including its history, essential facilities and equipment needed, composition of culture media, preparation of stock solutions and media, sterilization methods, measurement of growth, and types of culture. It notes that tissue culture developed from embryology techniques and involves growing small tissue explants aseptically on defined media. Essential facilities include an incubator, autoclave, microscope, and culture room, while media contains major and trace nutrients, vitamins, carbon source, and plant growth regulators.
This document provides an overview of bacterial spores, including their definition, structure, important spore-forming bacteria, sporulation process, properties, resistance, germination, and uses. Key points include:
- Bacterial spores are dormant, highly resistant forms of bacteria that form in response to starvation or stress.
- Important spore-forming genera include Bacillus and Clostridium, which include pathogenic species.
- Spores have a protective multilayer structure and properties like low water content that make them highly resistant to heat, chemicals, radiation, and desiccation.
- Spores can germinate into active vegetative cells in response to certain nutrients after a triggering process called activation.
Plant tissue culture media contain essential nutritional components to support in vitro plant growth. These include macro and micro nutrients, iron, vitamins, carbon sources, organic nitrogen, and a gelling agent. The Murashige and Skoog (MS) medium is commonly used and contains specific concentrations of ammonium nitrate, potassium nitrate, calcium chloride, magnesium sulfate, potassium phosphate, micronutrients, iron-EDTA, vitamins, sucrose, and agar. Plant growth regulators like auxins, cytokinins, and gibberellins are also added to influence cell growth and differentiation. The media is prepared under sterile conditions and its pH is adjusted to 5-5.8 for successful tissue culture.
General steps in biotechnology: and Various sterilization techniques followed in a tissue culture lab space, such as autoclaving, filtering, flame sterilization, chemical sterilization, UV radiation etc.
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,
Cell culture is the process of growing cells in a controlled environment outside of a living organism. Some key developments in cell culture include the use of antibiotics to prevent contamination, the use of trypsin to detach and subculture cells, and the use of defined culture media. Cell culture is commonly used for research in areas like cancer, virology, genetics, and toxicology testing. The basic requirements for cell culture include a substrate, nutrients provided in culture media, controlled environmental factors like temperature and CO2 levels, and sterile conditions maintained through aseptic technique.
Cell culture is the process of growing cells in a controlled environment outside of a living organism. Some key developments in cell culture include the use of antibiotics to prevent contamination, the use of trypsin to detach and subculture cells, and the use of defined culture media. Cell culture is commonly used for research in areas like basic cell biology, toxicity testing, cancer research, virology, genetic engineering, and gene therapy. There are different types of cell cultures, including primary cultures derived directly from tissue and continuous cell lines that can be passaged indefinitely. Maintaining proper cell culture requires a controlled environment, appropriate culture media and supplements, and aseptic technique to prevent contamination.
The ability of an explant to regenerate into a whole plant under in vitro asceptic conditions by providing a proper artificial nutrient medium is called as Plant Tissue Culture.
The debris of the ‘last major merger’ is dynamically youngSérgio Sacani
The Milky Way’s (MW) inner stellar halo contains an [Fe/H]-rich component with highly eccentric orbits, often referred to as the
‘last major merger.’ Hypotheses for the origin of this component include Gaia-Sausage/Enceladus (GSE), where the progenitor
collided with the MW proto-disc 8–11 Gyr ago, and the Virgo Radial Merger (VRM), where the progenitor collided with the
MW disc within the last 3 Gyr. These two scenarios make different predictions about observable structure in local phase space,
because the morphology of debris depends on how long it has had to phase mix. The recently identified phase-space folds in Gaia
DR3 have positive caustic velocities, making them fundamentally different than the phase-mixed chevrons found in simulations
at late times. Roughly 20 per cent of the stars in the prograde local stellar halo are associated with the observed caustics. Based
on a simple phase-mixing model, the observed number of caustics are consistent with a merger that occurred 1–2 Gyr ago.
We also compare the observed phase-space distribution to FIRE-2 Latte simulations of GSE-like mergers, using a quantitative
measurement of phase mixing (2D causticality). The observed local phase-space distribution best matches the simulated data
1–2 Gyr after collision, and certainly not later than 3 Gyr. This is further evidence that the progenitor of the ‘last major merger’
did not collide with the MW proto-disc at early times, as is thought for the GSE, but instead collided with the MW disc within
the last few Gyr, consistent with the body of work surrounding the VRM.
Unlocking the mysteries of reproduction: Exploring fecundity and gonadosomati...AbdullaAlAsif1
The pygmy halfbeak Dermogenys colletei, is known for its viviparous nature, this presents an intriguing case of relatively low fecundity, raising questions about potential compensatory reproductive strategies employed by this species. Our study delves into the examination of fecundity and the Gonadosomatic Index (GSI) in the Pygmy Halfbeak, D. colletei (Meisner, 2001), an intriguing viviparous fish indigenous to Sarawak, Borneo. We hypothesize that the Pygmy halfbeak, D. colletei, may exhibit unique reproductive adaptations to offset its low fecundity, thus enhancing its survival and fitness. To address this, we conducted a comprehensive study utilizing 28 mature female specimens of D. colletei, carefully measuring fecundity and GSI to shed light on the reproductive adaptations of this species. Our findings reveal that D. colletei indeed exhibits low fecundity, with a mean of 16.76 ± 2.01, and a mean GSI of 12.83 ± 1.27, providing crucial insights into the reproductive mechanisms at play in this species. These results underscore the existence of unique reproductive strategies in D. colletei, enabling its adaptation and persistence in Borneo's diverse aquatic ecosystems, and call for further ecological research to elucidate these mechanisms. This study lends to a better understanding of viviparous fish in Borneo and contributes to the broader field of aquatic ecology, enhancing our knowledge of species adaptations to unique ecological challenges.
Phenomics assisted breeding in crop improvementIshaGoswami9
As the population is increasing and will reach about 9 billion upto 2050. Also due to climate change, it is difficult to meet the food requirement of such a large population. Facing the challenges presented by resource shortages, climate
change, and increasing global population, crop yield and quality need to be improved in a sustainable way over the coming decades. Genetic improvement by breeding is the best way to increase crop productivity. With the rapid progression of functional
genomics, an increasing number of crop genomes have been sequenced and dozens of genes influencing key agronomic traits have been identified. However, current genome sequence information has not been adequately exploited for understanding
the complex characteristics of multiple gene, owing to a lack of crop phenotypic data. Efficient, automatic, and accurate technologies and platforms that can capture phenotypic data that can
be linked to genomics information for crop improvement at all growth stages have become as important as genotyping. Thus,
high-throughput phenotyping has become the major bottleneck restricting crop breeding. Plant phenomics has been defined as the high-throughput, accurate acquisition and analysis of multi-dimensional phenotypes
during crop growing stages at the organism level, including the cell, tissue, organ, individual plant, plot, and field levels. With the rapid development of novel sensors, imaging technology,
and analysis methods, numerous infrastructure platforms have been developed for phenotyping.
The technology uses reclaimed CO₂ as the dyeing medium in a closed loop process. When pressurized, CO₂ becomes supercritical (SC-CO₂). In this state CO₂ has a very high solvent power, allowing the dye to dissolve easily.
EWOCS-I: The catalog of X-ray sources in Westerlund 1 from the Extended Weste...Sérgio Sacani
Context. With a mass exceeding several 104 M⊙ and a rich and dense population of massive stars, supermassive young star clusters
represent the most massive star-forming environment that is dominated by the feedback from massive stars and gravitational interactions
among stars.
Aims. In this paper we present the Extended Westerlund 1 and 2 Open Clusters Survey (EWOCS) project, which aims to investigate
the influence of the starburst environment on the formation of stars and planets, and on the evolution of both low and high mass stars.
The primary targets of this project are Westerlund 1 and 2, the closest supermassive star clusters to the Sun.
Methods. The project is based primarily on recent observations conducted with the Chandra and JWST observatories. Specifically,
the Chandra survey of Westerlund 1 consists of 36 new ACIS-I observations, nearly co-pointed, for a total exposure time of 1 Msec.
Additionally, we included 8 archival Chandra/ACIS-S observations. This paper presents the resulting catalog of X-ray sources within
and around Westerlund 1. Sources were detected by combining various existing methods, and photon extraction and source validation
were carried out using the ACIS-Extract software.
Results. The EWOCS X-ray catalog comprises 5963 validated sources out of the 9420 initially provided to ACIS-Extract, reaching a
photon flux threshold of approximately 2 × 10−8 photons cm−2
s
−1
. The X-ray sources exhibit a highly concentrated spatial distribution,
with 1075 sources located within the central 1 arcmin. We have successfully detected X-ray emissions from 126 out of the 166 known
massive stars of the cluster, and we have collected over 71 000 photons from the magnetar CXO J164710.20-455217.
Current Ms word generated power point presentation covers major details about the micronuclei test. It's significance and assays to conduct it. It is used to detect the micronuclei formation inside the cells of nearly every multicellular organism. It's formation takes place during chromosomal sepration at metaphase.
The ability to recreate computational results with minimal effort and actionable metrics provides a solid foundation for scientific research and software development. When people can replicate an analysis at the touch of a button using open-source software, open data, and methods to assess and compare proposals, it significantly eases verification of results, engagement with a diverse range of contributors, and progress. However, we have yet to fully achieve this; there are still many sociotechnical frictions.
Inspired by David Donoho's vision, this talk aims to revisit the three crucial pillars of frictionless reproducibility (data sharing, code sharing, and competitive challenges) with the perspective of deep software variability.
Our observation is that multiple layers — hardware, operating systems, third-party libraries, software versions, input data, compile-time options, and parameters — are subject to variability that exacerbates frictions but is also essential for achieving robust, generalizable results and fostering innovation. I will first review the literature, providing evidence of how the complex variability interactions across these layers affect qualitative and quantitative software properties, thereby complicating the reproduction and replication of scientific studies in various fields.
I will then present some software engineering and AI techniques that can support the strategic exploration of variability spaces. These include the use of abstractions and models (e.g., feature models), sampling strategies (e.g., uniform, random), cost-effective measurements (e.g., incremental build of software configurations), and dimensionality reduction methods (e.g., transfer learning, feature selection, software debloating).
I will finally argue that deep variability is both the problem and solution of frictionless reproducibility, calling the software science community to develop new methods and tools to manage variability and foster reproducibility in software systems.
Exposé invité Journées Nationales du GDR GPL 2024
Immersive Learning That Works: Research Grounding and Paths ForwardLeonel Morgado
We will metaverse into the essence of immersive learning, into its three dimensions and conceptual models. This approach encompasses elements from teaching methodologies to social involvement, through organizational concerns and technologies. Challenging the perception of learning as knowledge transfer, we introduce a 'Uses, Practices & Strategies' model operationalized by the 'Immersive Learning Brain' and ‘Immersion Cube’ frameworks. This approach offers a comprehensive guide through the intricacies of immersive educational experiences and spotlighting research frontiers, along the immersion dimensions of system, narrative, and agency. Our discourse extends to stakeholders beyond the academic sphere, addressing the interests of technologists, instructional designers, and policymakers. We span various contexts, from formal education to organizational transformation to the new horizon of an AI-pervasive society. This keynote aims to unite the iLRN community in a collaborative journey towards a future where immersive learning research and practice coalesce, paving the way for innovative educational research and practice landscapes.
ESPP presentation to EU Waste Water Network, 4th June 2024 “EU policies driving nutrient removal and recycling
and the revised UWWTD (Urban Waste Water Treatment Directive)”
2. Plant Tissue Culture
PG and Research Department of Botany
Government Arts College (Autonomous)
Coimbatore 641 018
Dr. K. KALIMUTHU, M.Sc., M.Phil., Ph.D.
Assistant Professor
3. •Plant Tissue Culture refers to the technique of growing
plant cells, tissues, organs, seeds or other plant parts in
a sterile environment on a nutrient medium.
•It is mainly based on the totipotency of the cell. That is
every living cell, of a multicellular organism is capable of
independent development, when provided with suitable
conditions (white 1963). Morgan (1901) coined the term
totipotency.
4. Differentiation (De-)
The physiological and
morphological changes that occur in
a cell, tissue, or organ during
development.
Organogenesis
The development of tissues and/or organs from
individual cells not from pre-existing meristems.
5. Characteristic of Plant Tissue Culture Techniques
Environmental condition optimized
nutrition,
light,
temperature.
Ability to give rise to callus,
embryos,
adventitious roots and shoots.
Ability to grow as single cells
(protoplasts, microspores,
suspension cultures.
6. HISTORY
German botanist Gottlieb Haberlandt (1902),
regarded as the father of plant tissue culture, first
developed the concept of in vitro cell culture. He was
the first to culture isolated and fully differentiated plant
cells in a nutrient medium.
Gautheret, White and Nobecourt largely contributed to the
developments made in plant tissue culture (1934-1940).
Steward and Reinert (1959) first discovered somatic embryo production
in vitro. Maheswari and Guha (1964) from India were the first to develop
anther culture and pollen culture for the production of haploid plants.
7. Applications of Plant Tissue Cultures
•Micropropagation
•Somatic embryogenesis and artificial seeds production.
•Recovery of pathogen free stocks.
•Germplasm conservation and transfer.
•Embryo rescue and culture.
•Haploid and triploid production.
•Secondary metabolite production.
•Protoplast isolation, culture and fusion.
•Vector mediated gene transfer.
8. Advantages
In terms of product development and
enhancement
•It is the only method of rapid, high volume and less space
occupying plant multiplication system (e.g.) Foliage
ornamentals.
•Plantlets produced are uniform, true to type or with
improved phenotype (e.g.) Syngonium
•Plantlets are disease free (e.g.) Banana, Chrysanthemum
•Only way through which genetically engineered products
of the future can be brought into market place.
9. In terms of product format and marketability
•A variety or choice is offered as rooted, clumps or
hardened plants specifically for exports.
•Movement of plantlets easy due to relatively less
cumbersome quarantine.
•No seasonal production in artificial environment 24
hours a day can be contemplated.
•New plant species can be introduced quickly and in
large quantities to the market.
•In general, a balance between the plants for both local
and export markets and reasonable pricing can pave the
way for a sustained, viable Plant Tissue Culture
Industry.
21. SOME OF THE IMPORTANT BASAL MEDIA USED IN
PLANT TISSUE CULTURE
1. Murashige and Skoog medium (MS)
2. Linsmaer and Skoog medium (LS)
3. Gamborg’s medium (B5)
4. Woody plant medium (WPM)
5. Vacin and Went medium (VW)
6. Knudson ‘C’
7. White’s medium
22.
23. S.No Item Chemical Formula 1 lit 5 lit 10 lit 25 lit 50 lit
Dissolve
in
1 Major
Ammonium
nitrate
1.65g 8.25g 16.5g 41.25g
25lit/5
00
ml
25lit
500ml
20ml/litPotassium
nitrate
KNO3 1.9g 9.5g 19g 47.5g
Potasssium di
hydrogen
orthophosp
hate
KH2PO4 0.17g 0.85g 1.7g 4.25g
Calcium
chloride
CaCl2 2H2O 0.44g 2.2g 4.4g 11g
Magnesium
sulphate
MgSO4.7H2O 0.37g 1.85g 3.7g 9.25g
2 Minor
Manganese
sulphate
MnSO4.4H2O 16.8m
g
84mg 168m
g
420mg 840mg 50 lit
100
ml
2ml/lit
Zink sulphate ZnSO4.6H2O 8.6 mg 43 mg 86 mg 125 mg 430 mg
Boric acid H3BO3 6.2 mg 31 mg 62 mg 155 mg 310 mg
MS MEDIUM - STOCK COMPOSITION
26. ONE LITRE MEDIUM PREPARATION
• Add stocks as follows
900 ml of distilled water in 1L flask
Major stock - 20 ml
Add CaCl2. 2H2O - 440 mg
Minor stock - 2 ml
Micro stock - 2 ml
Vitamins stock - 2 ml
Amino acid - 2 ml
KI stock - 2 ml
Iron EDTA stock - 10 ml
Myo- inositol - 100 mg
Add Sucrose - 30 g
• Adjust pH to 5.8 to 6 with 0.5N HCL or 0.2N NaOH and make up to
1000 ml.
27. • After add required amount of growth regulators.
• Keep the medium over a micro wave/ heater and dissolve the Agar
with constant stirring.
• Dispense the medium in culture bottles.
• Cover tightly with non-absorbent cotton wool/ autoclavable caps
and cling wrap the bottles.
• Store the medium in a sterile environment.
28. A SELECTED LIST OF ELEMENTS AND THEIR FUNCTIONS IN PLANTS
ELEMENT FUNCTION (S)
Nitrogen
Essential component of proteins, nucleic acids and some coenzymes.
(Required in most abundant quantity)
Calcium Synthesis of cell wall, membrane function, cell signaling.
Magnesium Component of chlorophyll, cofactor for some enzymes.
Potassium Major inorganic cation, regulates osmotic potential.
Phosphorus
Component of nucleic acids and various intermediates in respiration and
photosynthesis, involved in energy transfer.
Sulfur
Component of certain amino acids (methionine, cysteine and cystine, and
some cofactors).
Manganese Cofactor for certain enzymes.
Iron Component of cytochromes, involved in electron transfer.
Chlorine Participates in photosynthesis.
Copper Involved in electron transfer reactions, cofactor for some enzymes.
Cobalt Component of vitamin B12.
Molybdenum
Component of certain enzymes (e.g., nitrate reductase), cofactor for some
enzymes.
Zinc Required for chlorophyll biosynthesis, cofactor for certain enzymes.
30. PLANT HORMONES IN PLANT TISSUE CULTURE
Auxins
Main effects in tissue culture
systems
Modulators of metabolism, action or
transport
indole-3-acetic acid (IAA)
indole-3-butyric acid (IBA)
1-naphthaleneacetic acid (NAA)
phenyl acetic acid (PAA)
2,4-dichlorophenoxyacetic acid
(2,4-D)
2,4,5-trichlorophenoxyacetic acid
(2,4,5-T)
picloram
dicamba
p-chlorophenoxyacetic acid (CPA)
1.Adventitious root formation
(at high conc.).
2. Adventitious shoot formation
(at low conc.).
3. Induction of somatic embryos
(in part. 2,4-D).
4. Cell division.
5. Callus formation and growth.
6. Inhibition of outgrowth of
axillary buds
7. Inhibition of root growth.
1. 2,3,4-Triiodobenzoic acid (TIBA) and 1-
N-naphthylphthalamic acid (NPA) inhibit
polar auxin transport.
2. p-Chlorophenoxyisobutyric acid (PCIB)
inhibits auxin action as a genuine antiauxin
by binding to the auxin receptor.
3.Phenolic compounds (e.g. ferulic acid or
phloroglucinol) inhibit auxin oxidation.
4. riboflavin strongly promotes
photooxidation of IBA and IAA.
Cytokinins
Main effects in tissue culture
systems
Modulators of metabolism, action or
transport
zeatin (Z)
zeatinriboside (ZR)
isopentenyladenine (iP)
isopenenyladenosine (iPA)
6-bezylaminopurine (BAP)
kinetin
thidiazuron (TDZ)
N-(2-chloro-4-pyridyl)-N’-
phenylurea (CPPU)
1.Adventitious shoot formation
(at high conc.).
2. Adventitious root formation.
3. Cell division.
4. Callus formation and growth.
5. Stimulation of outgrowth of
axillary buds.
6. Inhibition of shoot elongation.
7. Inhibition of leaf senescence.
Compounds have been described that inhibit
cytokinin synthesis (lovastatin), degradation
and action. The various effects are,
however, not yet well studied or ambiguous.
31. Gibberellins
Main effects in tissue
culture systems
Modulators of metabolism,
action or transport
gibberellic acid (GA3)
gibberllin 1 (GA1)
gibberellin 4 (GA4)
gibberellin 7 (GA 7)
1.Shoot elongation.
2. Release from dormancy in
seeds, somatic embryos, apical
buds and bulbs.
3. Inhibition of adventitious root
formation.
4. Synthesis-inhibitors promote
root formation.
5. Synthesis-inhibitors promote
tuber, corm and bulb formation.
6. Synthesis-inhibitors facilitate
acclimatization.
Paclobutrazol and ancymidol inhibit
gibberellin sysnthesis and thereby result in
short shoots.
Ethylene Main effects in tissue
culture systems
Modulators of metabolism,
action or transport
1.Senescence of leaves.
2. Ripening of fruits.
3. Promotion or inhibition of
adventitious regeneration
(depending on the time of
application or on the genotype?).
1.1-aminocyclopropane-1-carboxylic acid
(ACC) is a precursor of ethylene and is
metabolized by plant tissues to ethylene.
2. Aminoethoxyvinylglycine (AVG)
inhibits ethylene synthesis. Co2+, ?-
aminooxy-acetic acid and ?-
aminoisobutyric acid also inhibit ethylene
synthesis but have a lower efficiency.
3. Silver inhibits ethylene action. Silver is
applied preferably as silverthiosulphate
(STS).
32. Abscisic acid
Main effects in tissue
culture systems
Modulators of metabolism,
action or transport
1.Maturation of somatic
embryos.
2. Facilitation of
acclimatization.
3. Bulb and tuber formation.
4. Promotion of the
development of dormancy.
Fluridone inhibits ABA synthesis. As it
acts by inhibiting an early step in
carotenoid synthesis, plants are unable to
synthesize chlorophyll. However,
fluridone does not seem to be toxic.
Paclobutrazol also inhibits ABA
synthesis.
Polyamines
Main effects in tissue
culture systems
Modulators of metabolism,
action or transport
putrescine
spermidine
spermine
1. Promotion of adventitious
root formation.
2. Promotion of shoot
formation.
3. Promotion of somatic
embryogenesis.
1. DL-a-difluoromethylarginine
(DFMA) and a –
difluoromethylornithine (DFMO)
block the synthesis of putrescine.
2. Methylglyoxal-bis-guanylhydrazone
(MGBG) and cyclohexylamine (CHA)
block the synthesis of spermidine and
spermine.
3. Amino-guanidine (AG) blocks the
degradation of putrescine.
33. Support Systems
Agar (from seaweed)
Agarose
Phytagel
Mixtures (Phytagar)
Mechanical (bridges, rafts)
Sand
34. Natural substances in tissue culture media
Coconut water
Yeast extract
Malt extract
Potato extract
Banana homogenate
35. Charcoal
• Activated charcoal is used as a detoxifying agent.
Detoxifies wastes from plant tissues, impurities
– Impurities and absorption quality vary
– Concentration normally used is 0.3 % or lower
• Charcoal for tissue culture
– acid washed and neutralized
– never reuse
36. MEDIA STERILIZATION
Media is sterilized at 121ºC (1.12kg/cm2) for 20 minutes
Filter sterilization
Compound such as certain amino acid, vitamins and hormones are
usually destroyed during autoclave (eg) calcium pantothenate, IAA (40%
loss), IBA (20% loss), zeatin, kinetin and thiamine HCl (high above [pH
5.5).
Such thermolabile compounds are sterilized by ultrafilteration
through size or Millipore filtration unit with 0.2um or .45 um pore size
filters. Media prepared for polypropylene containers/liquid media are filter
sterilized using this technique (or) alternatively by using UV or electrostatic
filters
STORAGE
Autoclaved medium is stored at room temperature to cool in sterile
environment.
37. Functions of medium
Provide water
Provide mineral nutritional needs
Provide vitamins
Provide growth regulators
Access to atmosphere for gas exchange
Removal of plant metabolite waste
38. TYPES OF EXPLANTS
• Seeds (eg.) Orchids, Cacti, Pterocarpus, Jatropha etc.
• Tender explants like meristems, leaf bits, shoot tips buds of
herbaceous plants. (eg.) Chrysanthemum, Strawberry,
Crossandra, Asparagus etc.
• Moderately hard explants like shoot tips, nodal sections of
shrubs etc. (eg.) Rose, Sugarcane, Dieffenbachia, Bamboo,
Phillodendron.
• Hardwood explants like nodal / shoot tip sections of trees
like Teak, Eucalyptus Pterocarpus, Jatropha etc.
• Explants from plants with compressed internodes length
(or) Gerbera, Timonium, Anthurium etc.
• Suckers, Corms, tubers, rhizome and bulb explant (eg,)
Banana, Caladium, Potato, Ginger, Turmeric, Calla lily,
Gladioli etc.
39. EXPLANT STERILIZATION
S.
NO.
CHEMICAL CONCENTRATION EXPOSU
RE TIME
(min)
Surface Sterilant
1.
Ethyl alcohol (enables removal of waxy epidermal coating and better
surface sterilant contact.)
70 0.5-5
2. Sodium hypochlorite (bleaches the explant and needs correct conc./time) 0.5-5 5-30
3. Mercuric chloride (Carcinogenic and needs careful handling) 0.1-2 1-15
4. Commercial bleach (bleaches the explant and needs correct conc./time) 10-20 5-30
Detergent
5. Teepol (for large, hardy explants) 1-2 2-10
6.
Tween 20 (mild, safe wetting agent – reducing surface tension, cleans dust / dirt
and facilitates better surface contact.)
One or few
drops
5-15
Fungicide
7. Bavistin (carbendazin) (for large explants like corms, suckers etc) 0.1-0.2 5-15
Antibiotics
(rarely employed as it promotes contamination on withdrawal (or) with a lag phase
in latter stages)
8. Rifampicin (for explant treatment) 5-10 ml /100 ml 5-30
9. Streptomycin (for explant treatment) 25–50 mg/100 ml 5-30
10. Ampicillin (for explant treatment) 25–50 mg/100 ml 5-30
40. STAGES OF MICROPROPAGATION
• Stage O – Mother Plant selection and preparation
• Stage I – Initiation
• Stage II – Multiplication
• Stage III – Shooting
• Stage IV – Rooting
• Stage V – Hardening
41. Micropropagation
Direct Indirect
Node and Shoot tip Leaf, Petiole, Stem and root
Surface sterilization Surface sterilization
Inoculation
( MS + Growth regulators
Observation and
subculturing
42. (Node, Shoot tip, internode,
leaf, petiole and root)
(Node, Shoot tip, internode,
leaf, petiole and root)
in vivo in vitro
Explant
Inoculation
(MS + Growth regulators)
Subculturing & Observation
Surface
sterilization
Steps Involved in Micropropagation
45. SEED CULTURE
Seed culture is the type of tissue culture that is primarily used for
plants.
Advantages
1. The production of exact copies of plants that produce
particularly good flowers, fruits or have other desirable
traits.
2. The production of plants in sterile conditions with greatly
reduced chances of transmitting diseases, pests and
pathogens.
3. The production of plants from seeds that otherwise have low
chances of germinating and growing.
4. Mass propagate plants for commercial use.
5. Disease-free plants
49. EMBRYO CULTURE
Mature embryos are isolated from ripe seeds and cultured in
artificial medium.
Advantage
Recovery of distant hybrids.
Recovery of haploid plants from interspecific crosses
Propagation of orchids
Shortening the breeding cycle
Overcoming dormancy
Ovule and ovary can also be cultured
58. GF
A B
Ceropegia pusilla - Habit observation
A- Ebbanad hills. B and C- Plants with tubers. D and E -Plants with flowers.
F -Plants with follicles G -Seed dispersal. H - Plant with flower. I - Seed.
I
SEED
ED
C
84. SOMATIC EMBRYO CULTURE
The developmental pathway of numerous well-organized,
embryoids resembling to zygotic embryo from the embryogenetic
potential somatic plant cells of the callus, tissue or cell suspension
culture is known as somatic embryogenesis.
History
J. Reinert (1958-59): Reported his first observation of in vitro
somatic embryogenesis in Daucus carota (Carrot).
N. S. Rangaswami (1961): Studied in detail the somatic
embryogenesis in Citrus sp.
Advantages
1. Production of artificial seed
2. Production of adventitious embryo
3. Mutagenic studies
4. Free of viral and other pathogenic infection
89. OVARY / OVULE CULTURE
Ovule culture is an elegant experimental system by which are aseptically isolated from the ovary
and are grown aseptically on chemically defined nutrient medium under controlled conditions.
Advantages
1. Produce haploid plants and embryology study
2. Study of genetic recombination in higher plants
3. Mutation study
4. Heritability studies are simplified (recessive mutation are easily
identified.
90. ANTHERS / MICROSPORE CULTURE
Anthers culture
Culturing of anther obtained from unopened flower bud in the
nutrient medium under aseptic condition.
Callus tissue or embryoids that give rise to haploid plantlets
either through organogenesis or embryogenesis.
Microspore culture
Pollen or microspore culture is in vitro technique by which the
pollen grains preferably at uninucleated stage, are squeezed
out aseptically from the intact anther and then culture on
nutrient medium.
The micropores develop into haploid embryoids or callus tissue
that give rise to haploid plantlets by embryogenesis or
organogenesis.
91. History of anther culture
1964, 1966 Datura innoxia (Guha and maheshwari) the Indian Scientist
1967 (Bourign and Nitsch): 1st haploid plants from isolated
anthers Nicotiana.
Advantage of anther culture
1. Simple
2. Less time consuming
3. Responsive
4. Utility of anther and pollen
culture for basic research
5. Cytogenetic studies
6. Genetic recombination in
higher plants
7. Controlling pollen
embryogenesis of higher plant
92. SHOOT TIP / MERISTEM CULTURE
Shoot tip culture may be described as the culture of
terminal portion of a shoot comprising the meristem together
with primordial and developing leaves and adjacent stem tissue.
Advantages
Virus elimination
Storage genetic recourses
Use in pant breeding
Quarantine
100. Cell suspension culture
When callus pieces are agitated in a liquid medium, they tend to
break up.
Suspensions are much easier to bulk up than callus since there is
no manual transfer or solid support.
101.
102. HAIRY ROOT CULTURE Normal roots from leaf segments
Hairy roots from callus culture
Typical hairy root development