Protoplasts are naked plant cells without the cell wall, but they possess plasma membrane and all other cellular components. They represent the functional plant cells but for the lack of the barrier, cell wall. Protoplasts of different species can be fused to generate a hybrid and this process is referred to as somatic hybridization (or protoplast fusion). Cybridization is the phenomenon of fusion of a normal protoplast with an enucleated (without nucleus) protoplast that results in the formation of a cybrid or cytoplast (cytoplasmic hybrids).
This document discusses anther and pollen culture techniques. It provides a brief history of the development of these techniques from the 1950s onward. It then describes the process of anther culture, where anthers are cultured in nutrient medium to produce haploid callus or embryos. Pollen or microspore culture involves isolating pollen grains from anthers and culturing them. The goal is to produce haploid embryos or callus that can develop into haploid plantlets. Key factors that influence success include the genotype, microspore stage, culture medium, temperature, and physiological status of the donor plant. Anther culture has applications in mutation studies, plant breeding, and secondary metabolite production.
1) Germplasm conservation involves preserving genetic material, such as seeds, cells, tissues, and body parts, through in-situ and ex-situ methods to maintain biodiversity and provide resources for breeding programs.
2) Cryopreservation at ultra-low temperatures in liquid nitrogen is an important ex-situ technique that can preserve germplasm long-term without subculturing. It involves preculturing plant materials, treating with cryoprotectants, and either slow-freezing or vitrification prior to storage in liquid nitrogen.
3) A case study demonstrates the successful cryopreservation of mint shoot tips using encapsulation-dehydration and PVS2-vitrification, with
Ovule and seed culture techniques and their applications are summarized. Ovules and seeds can be isolated and cultured aseptically on nutrient media. This allows for applications like in vitro pollination and fertilization, hybridization of incompatible species, production of haploid callus, circumventing the need for host stimuli in parasitic plants, and inducing polyembryony. Seed culture is useful for propagating plants with reduced embryos, parasitic plants, orchids, and producing large numbers of uniform seedlings. However, in vitro culture still poses challenges like slow growth, low multiplication, poor rooting, and somaclonal variation.
Hairy root culture is a plant cell culture technique that uses the soil bacterium Agrobacterium rhizogenes to genetically transform plant cells. When plant tissues are infected by A. rhizogenes, its root-inducing plasmid integrates into the plant genome and causes unchecked root growth. These transformed roots, known as hairy roots, can be cultured in vitro and have several advantages over traditional plant cell cultures, including fast growth rates, genetic stability, and high production of secondary metabolites. Hairy root cultures are characterized by extensive branching, root hairs, absence of geotropism, and not requiring plant growth regulators. They have been used to produce valuable compounds and whole plants through regeneration.
This document describes the process of protoplast isolation, culture, and fusion from Ankita Singh and Vinars Dawane of the Government Holkar Science College in Indore. It provides an overview of protoplast isolation methods including mechanical, sequential enzymatic, and mixed enzymatic. Sources of protoplasts include leaves, callus cultures, and cell suspension cultures. The viability of isolated protoplasts can be tested through microscopy, tetrazolium reduction, fluorescein diacetate staining, and Evan's blue staining. Protoplasts are cultured through regeneration of cell walls, cell division, and development of callus/whole plants. Protoplast fusion can be spontaneous, mechanical, or
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.
Introduction
Reason for cryopreservation
Selection of part of plant for cryopreservation
Technique of cryopreservation
Application
Limitation
Conclusion
Cybrids are produced through the fusion of protoplasts from two different plant species, combining the cytoplasm of both but the nucleus of only one species. This technique allows for the transfer of cytoplasmic traits like male sterility between incompatible species. Protoplast isolation, fusion, selection, and regeneration of hybrid cells into whole plants are required to produce cybrids. Cybrids can be used to study cytoplasmic genes and transfer desirable agricultural traits, overcoming sexual incompatibility barriers in plant breeding.
This document discusses anther and pollen culture techniques. It provides a brief history of the development of these techniques from the 1950s onward. It then describes the process of anther culture, where anthers are cultured in nutrient medium to produce haploid callus or embryos. Pollen or microspore culture involves isolating pollen grains from anthers and culturing them. The goal is to produce haploid embryos or callus that can develop into haploid plantlets. Key factors that influence success include the genotype, microspore stage, culture medium, temperature, and physiological status of the donor plant. Anther culture has applications in mutation studies, plant breeding, and secondary metabolite production.
1) Germplasm conservation involves preserving genetic material, such as seeds, cells, tissues, and body parts, through in-situ and ex-situ methods to maintain biodiversity and provide resources for breeding programs.
2) Cryopreservation at ultra-low temperatures in liquid nitrogen is an important ex-situ technique that can preserve germplasm long-term without subculturing. It involves preculturing plant materials, treating with cryoprotectants, and either slow-freezing or vitrification prior to storage in liquid nitrogen.
3) A case study demonstrates the successful cryopreservation of mint shoot tips using encapsulation-dehydration and PVS2-vitrification, with
Ovule and seed culture techniques and their applications are summarized. Ovules and seeds can be isolated and cultured aseptically on nutrient media. This allows for applications like in vitro pollination and fertilization, hybridization of incompatible species, production of haploid callus, circumventing the need for host stimuli in parasitic plants, and inducing polyembryony. Seed culture is useful for propagating plants with reduced embryos, parasitic plants, orchids, and producing large numbers of uniform seedlings. However, in vitro culture still poses challenges like slow growth, low multiplication, poor rooting, and somaclonal variation.
Hairy root culture is a plant cell culture technique that uses the soil bacterium Agrobacterium rhizogenes to genetically transform plant cells. When plant tissues are infected by A. rhizogenes, its root-inducing plasmid integrates into the plant genome and causes unchecked root growth. These transformed roots, known as hairy roots, can be cultured in vitro and have several advantages over traditional plant cell cultures, including fast growth rates, genetic stability, and high production of secondary metabolites. Hairy root cultures are characterized by extensive branching, root hairs, absence of geotropism, and not requiring plant growth regulators. They have been used to produce valuable compounds and whole plants through regeneration.
This document describes the process of protoplast isolation, culture, and fusion from Ankita Singh and Vinars Dawane of the Government Holkar Science College in Indore. It provides an overview of protoplast isolation methods including mechanical, sequential enzymatic, and mixed enzymatic. Sources of protoplasts include leaves, callus cultures, and cell suspension cultures. The viability of isolated protoplasts can be tested through microscopy, tetrazolium reduction, fluorescein diacetate staining, and Evan's blue staining. Protoplasts are cultured through regeneration of cell walls, cell division, and development of callus/whole plants. Protoplast fusion can be spontaneous, mechanical, or
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.
Introduction
Reason for cryopreservation
Selection of part of plant for cryopreservation
Technique of cryopreservation
Application
Limitation
Conclusion
Cybrids are produced through the fusion of protoplasts from two different plant species, combining the cytoplasm of both but the nucleus of only one species. This technique allows for the transfer of cytoplasmic traits like male sterility between incompatible species. Protoplast isolation, fusion, selection, and regeneration of hybrid cells into whole plants are required to produce cybrids. Cybrids can be used to study cytoplasmic genes and transfer desirable agricultural traits, overcoming sexual incompatibility barriers in plant breeding.
Introduction
History
Importance of protoplast
Sources of protoplast
Isolation of protoplast
Viability test
Fusion of protoplast
Selection of hybrid cells
Genetic consequences
Symmetric & asymmetric Hybrids
Cybrids
Advantages
Disadvantages
Conclusion
References
Somatic embryogenesis is the artificial process where an embryo develops from a somatic cell rather than a gamete. It does not involve fertilization or the formation of endosperm or a seed coat. The first observations of somatic embryogenesis were in carrot cells in 1958. Now it has been achieved in over 300 plant species. It is a valuable technique for rapid large scale propagation of plants, including for commercial and conservation purposes. The process involves four key steps - induction, maintenance, development and regeneration.
Callus is an unorganized mass of undifferentiated cells that can be cultured from plant explants. It is produced when explants are cultured on medium containing auxin and cytokinin hormones under sterile conditions. Callus cells lose their specialized structures and ability to photosynthesize. Callus cultures can be used to regenerate whole plants and produce secondary metabolites, providing significance to research. Proper sterilization, hormone balance, and handling are important for successful callus formation and growth.
plant tissue culture / paper raft nurse techniqueAlvin karthi
This document describes the paper raft nurse technique for single cell culture. The key steps are:
1) Isolated single cells are placed onto sterile filter paper squares that have been placed on actively growing callus tissue a few days prior.
2) The filter paper acts as a "raft" to provide nutrients and moisture to the single cells from the underlying callus tissue.
3) The single cells are then incubated and allowed to divide and form colonies on the filter paper raft before being transferred to fresh medium to generate single cell clones.
This document provides an overview of plant tissue culture techniques. It discusses that plant tissue culture involves growing plant cells, tissues or organs in a sterile environment with nutrient media. The techniques rely on two principles - totipotency, the ability of plant cells to regenerate into a whole plant, and plasticity, the ability of plants to alter their growth in response to their environment. Explants from various plant tissues are sterilized and placed on culture media, which are composed of inorganic salts, organic nutrients and plant hormones. The culture media, explant source, and plant species can affect regeneration efficiency. Applications of plant tissue culture include commercial plant production, conservation of rare species, screening for desirable traits like herbicide resistance, and
The isolation, culture and fusion of protoplasts is a fascinating field in plant research. Protoplast isolation and their cultures provide millions of single cells (comparable to microbial cells) for a variety of studies.
Synthetic seeds are encapsulated somatic embryos or shoot buds that can be used for planting like traditional seeds. They allow for clonal propagation of plants that are difficult to reproduce through traditional seeds, including some fruit crops. The production of synthetic seeds involves inducing somatic embryogenesis in callus cultures, maturing the embryos, and encapsulating them in a protective gel before planting. This allows genetic material to be stored and dispersed while avoiding issues with seed-borne diseases, low seed viability, and difficulties reproducing species that lack traditional seeds.
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
Suspension Culture and Single Cell Cultures, Culturing methods, maintenance a...Ananya Sinha
Suspension Culture and Single Cell Cultures, Culturing methods, maintenance and application
Generally, suspension culture is a one stop technology to produce secondary metabolites on a large scale in-vitro, irrespective of the climatic condition or nutrient availability (as required in field plants).
In this presentation, we will see the importance of suspension culture, culturing methods and it's application (mostly with respect to plants) and also focus on what exactly is a single cell culture.
1) Shoot tip culture involves culturing the terminal portion of a shoot tip, comprising the meristem and developing leaves and stem tissue.
2) It is used to produce virus-free plants by removing viruses that cannot move between cells, for micropropagation, and to store plant genetic resources.
3) The protocol involves surface sterilizing and culturing small shoot tip explants through stages of culture establishment, shoot proliferation, and root regeneration. Factors like explant size and physiological condition affect the process.
This document discusses somaclonal variation, which refers to genetic variation that arises during tissue culture or plant regeneration from cell cultures. It provides definitions and history of the term as coined by Larkin and Scowcroft in 1981. The document outlines the various causes and types of somaclonal variation including physiological, genetic, and biochemical causes. It also describes methods for generating somaclonal variation both with and without in vitro selection. Finally, it discusses applications for detecting and isolating somaclonal variants, particularly for developing disease resistance in various crop species.
Organogenesis, in plant tissue cultureKAUSHAL SAHU
Introduction
Definition
Types of organogenesis
Organogenesis through callus formation (indirect organogenesis)
Growth regulators for indirect organogenesis
Organogenesis through adventitious organ (direct organogenesis)
Growth regulators for direct organogenesis
Factor affecting the soot bud differentiation
Organogenic differentiation
Application of organogenesis
Conclusion
References
The document discusses plant protoplast isolation, purification, and culturing. Some key points:
- Protoplasts are plant cells that have had their cell walls removed, leaving just the plasma membrane. They allow for plant cell fusion and regeneration.
- Protoplasts are typically isolated from plant tissues like leaves using enzymatic digestion with cellulase and pectinase. This yields more protoplasts than mechanical methods.
- Isolated protoplasts are purified by centrifugation and washing to remove cell debris. They are then cultured in liquid or solid nutrient media and tested for viability before regeneration.
This document discusses methods for producing haploid plants. It begins by defining haploid plants and their significance. It then describes the two main approaches for producing haploids - in vivo and in vitro. For in vivo, it outlines several techniques including androgenesis, gynogenesis, distant hybridization, and chemical/radiation treatments. For in vitro, it focuses on anther culture and microspore culture, providing details on the protocol for anther culture in tobacco including pre-treatment, culture conditions, and factors that influence success rates.
This document discusses cell suspension culture, which involves growing plant cells in liquid nutrient medium. It describes two types of cell suspension cultures: batch culture and continuous culture. Batch culture involves growing cells in a fixed volume of medium until growth ceases due to limiting nutrients or oxygen. Continuous culture continuously supplies fresh medium while maintaining a constant culture volume, and can be open or closed. The document outlines the importance and advantages of cell suspension culture for studying cell physiology and biochemistry.
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.
The document discusses protoplast culture, beginning with definitions and historical development. Protoplasts are isolated through either mechanical or enzymatic methods from plant tissues. Isolated protoplasts can be purified, cultured, and regenerated through cell division to form callus or whole plants. Protoplast culture has various applications, including studying cell walls, organelle isolation, and somatic hybridization. While limitations exist, protoplast culture provides advantages for genetic engineering and crop improvement.
Introduction
History
Importance of protoplast
Sources of protoplast
Isolation of protoplast
Viability test
Fusion of protoplast
Selection of hybrid cells
Genetic consequences
Symmetric & asymmetric Hybrids
Cybrids
Advantages
Disadvantages
Conclusion
References
Somatic embryogenesis is the artificial process where an embryo develops from a somatic cell rather than a gamete. It does not involve fertilization or the formation of endosperm or a seed coat. The first observations of somatic embryogenesis were in carrot cells in 1958. Now it has been achieved in over 300 plant species. It is a valuable technique for rapid large scale propagation of plants, including for commercial and conservation purposes. The process involves four key steps - induction, maintenance, development and regeneration.
Callus is an unorganized mass of undifferentiated cells that can be cultured from plant explants. It is produced when explants are cultured on medium containing auxin and cytokinin hormones under sterile conditions. Callus cells lose their specialized structures and ability to photosynthesize. Callus cultures can be used to regenerate whole plants and produce secondary metabolites, providing significance to research. Proper sterilization, hormone balance, and handling are important for successful callus formation and growth.
plant tissue culture / paper raft nurse techniqueAlvin karthi
This document describes the paper raft nurse technique for single cell culture. The key steps are:
1) Isolated single cells are placed onto sterile filter paper squares that have been placed on actively growing callus tissue a few days prior.
2) The filter paper acts as a "raft" to provide nutrients and moisture to the single cells from the underlying callus tissue.
3) The single cells are then incubated and allowed to divide and form colonies on the filter paper raft before being transferred to fresh medium to generate single cell clones.
This document provides an overview of plant tissue culture techniques. It discusses that plant tissue culture involves growing plant cells, tissues or organs in a sterile environment with nutrient media. The techniques rely on two principles - totipotency, the ability of plant cells to regenerate into a whole plant, and plasticity, the ability of plants to alter their growth in response to their environment. Explants from various plant tissues are sterilized and placed on culture media, which are composed of inorganic salts, organic nutrients and plant hormones. The culture media, explant source, and plant species can affect regeneration efficiency. Applications of plant tissue culture include commercial plant production, conservation of rare species, screening for desirable traits like herbicide resistance, and
The isolation, culture and fusion of protoplasts is a fascinating field in plant research. Protoplast isolation and their cultures provide millions of single cells (comparable to microbial cells) for a variety of studies.
Synthetic seeds are encapsulated somatic embryos or shoot buds that can be used for planting like traditional seeds. They allow for clonal propagation of plants that are difficult to reproduce through traditional seeds, including some fruit crops. The production of synthetic seeds involves inducing somatic embryogenesis in callus cultures, maturing the embryos, and encapsulating them in a protective gel before planting. This allows genetic material to be stored and dispersed while avoiding issues with seed-borne diseases, low seed viability, and difficulties reproducing species that lack traditional seeds.
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
Suspension Culture and Single Cell Cultures, Culturing methods, maintenance a...Ananya Sinha
Suspension Culture and Single Cell Cultures, Culturing methods, maintenance and application
Generally, suspension culture is a one stop technology to produce secondary metabolites on a large scale in-vitro, irrespective of the climatic condition or nutrient availability (as required in field plants).
In this presentation, we will see the importance of suspension culture, culturing methods and it's application (mostly with respect to plants) and also focus on what exactly is a single cell culture.
1) Shoot tip culture involves culturing the terminal portion of a shoot tip, comprising the meristem and developing leaves and stem tissue.
2) It is used to produce virus-free plants by removing viruses that cannot move between cells, for micropropagation, and to store plant genetic resources.
3) The protocol involves surface sterilizing and culturing small shoot tip explants through stages of culture establishment, shoot proliferation, and root regeneration. Factors like explant size and physiological condition affect the process.
This document discusses somaclonal variation, which refers to genetic variation that arises during tissue culture or plant regeneration from cell cultures. It provides definitions and history of the term as coined by Larkin and Scowcroft in 1981. The document outlines the various causes and types of somaclonal variation including physiological, genetic, and biochemical causes. It also describes methods for generating somaclonal variation both with and without in vitro selection. Finally, it discusses applications for detecting and isolating somaclonal variants, particularly for developing disease resistance in various crop species.
Organogenesis, in plant tissue cultureKAUSHAL SAHU
Introduction
Definition
Types of organogenesis
Organogenesis through callus formation (indirect organogenesis)
Growth regulators for indirect organogenesis
Organogenesis through adventitious organ (direct organogenesis)
Growth regulators for direct organogenesis
Factor affecting the soot bud differentiation
Organogenic differentiation
Application of organogenesis
Conclusion
References
The document discusses plant protoplast isolation, purification, and culturing. Some key points:
- Protoplasts are plant cells that have had their cell walls removed, leaving just the plasma membrane. They allow for plant cell fusion and regeneration.
- Protoplasts are typically isolated from plant tissues like leaves using enzymatic digestion with cellulase and pectinase. This yields more protoplasts than mechanical methods.
- Isolated protoplasts are purified by centrifugation and washing to remove cell debris. They are then cultured in liquid or solid nutrient media and tested for viability before regeneration.
This document discusses methods for producing haploid plants. It begins by defining haploid plants and their significance. It then describes the two main approaches for producing haploids - in vivo and in vitro. For in vivo, it outlines several techniques including androgenesis, gynogenesis, distant hybridization, and chemical/radiation treatments. For in vitro, it focuses on anther culture and microspore culture, providing details on the protocol for anther culture in tobacco including pre-treatment, culture conditions, and factors that influence success rates.
This document discusses cell suspension culture, which involves growing plant cells in liquid nutrient medium. It describes two types of cell suspension cultures: batch culture and continuous culture. Batch culture involves growing cells in a fixed volume of medium until growth ceases due to limiting nutrients or oxygen. Continuous culture continuously supplies fresh medium while maintaining a constant culture volume, and can be open or closed. The document outlines the importance and advantages of cell suspension culture for studying cell physiology and biochemistry.
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.
The document discusses protoplast culture, beginning with definitions and historical development. Protoplasts are isolated through either mechanical or enzymatic methods from plant tissues. Isolated protoplasts can be purified, cultured, and regenerated through cell division to form callus or whole plants. Protoplast culture has various applications, including studying cell walls, organelle isolation, and somatic hybridization. While limitations exist, protoplast culture provides advantages for genetic engineering and crop improvement.
This document summarizes methods for isolating and culturing plant protoplasts. Protoplasts are plant cells that have had their cell walls removed. The document describes two methods for isolating protoplasts - mechanical and enzymatic. It also discusses purifying the protoplasts, assessing their viability, culturing them in liquid media to regenerate cell walls, and the potential applications of protoplast culture and regeneration including plant breeding techniques.
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
This document discusses protoplast fusion techniques in plant tissue culture. It begins by defining a protoplast as a naked plant cell without a cell wall. The key steps discussed are:
1) Isolating protoplasts from plant tissue using either mechanical or enzymatic methods. Enzymatic isolation using cellulase, pectinase and hemicellulase is preferred.
2) Fusing the protoplasts using techniques like electrofusion, PEG fusion or high pH/Ca2+ solutions.
3) Identifying and selecting hybrid cells using markers like pigmentation, chloroplast presence or nuclear staining.
4) Culturing the hybrid cells and regenerating hybrid plants
Protoplast culture refers to the process in which whole plants are developed from the culture of cells without cell wall. This techniques widely used in plant breeding and crop improvement.
Isolation of protoplast in plant tissue culture.sadiakarim8
The document discusses the isolation of protoplasts from plant cells. There are two main methods for isolating protoplasts - mechanical and enzymatic. The enzymatic method uses enzymes to digest the cell wall and is more widely used as it works for a variety of plant tissues and causes less damage to the cells. Key steps in the enzymatic method include incubating plant tissue in enzyme solutions, filtering to separate protoplasts from debris, and centrifugation to purify the protoplasts. Isolated protoplasts can be used for cell fusion between unrelated plant species and for genetic modification of plants. Factors like plant species, age of donor tissue, and pre-treatment of tissue affect the viability
The document discusses somatic hybridization through the fusion of protoplasts from different plant species. It describes:
1. The process of somatic hybridization which involves isolating protoplasts from plant tissues, fusing the protoplasts from different species using chemical or electrical methods, selecting hybrid cells, culturing the hybrid cells and regenerating hybrid plants.
2. Methods for isolating viable protoplasts including enzymatic and mechanical methods. Enzymatic isolation uses cellulase, hemicellulase and pectinase enzymes.
3. Techniques for purifying isolated protoplasts such as filtration, centrifugation, flotation and density buffer methods to remove
Protoplast fusion involves isolating plant cells called protoplasts that have had their cell walls removed. This allows the fusion of protoplasts from different plant species using techniques like PEG or electrofusion. The fused protoplasts can regenerate into hybrid plants. Protoplast fusion is used for plant breeding to create hybrids of sexually incompatible species. It provides a way to combine genomes and study gene expression and inheritance. However, the process of isolating intact protoplasts can be challenging and yields may be low.
Plant tissue culture ⅱ isolation of protoplastbhoomishah45
Protoplast isolation involves removing the cell wall from plant cells to leave only the plasma membrane and intracellular components. The isolated protoplasts can be cultured and genetically manipulated. There are two main methods for isolating protoplasts - mechanical and enzymatic. The enzymatic method uses enzymes like cellulase and pectinase to break down the cell wall. Isolated protoplasts are purified through centrifugation and cultured in an osmotic medium to prevent bursting due to the lack of a cell wall. Protoplasts are useful for studies in cell fusion and genetic transformation.
This document summarizes a presentation on protoplast isolation, culture, and fusion. It discusses how protoplasts are isolated from plant tissues using enzymatic digestion of cell walls. Isolated protoplasts can be cultured and induced to regenerate new cell walls and undergo cell division. Protoplasts from different plant species or varieties can be fused using techniques like PEG or electrofusion to create somatic hybrid plants. Selection methods are used to identify fused hybrid protoplasts from unfused parental protoplasts based on differences in growth characteristics, antibiotic resistance, or fluorescent labeling. The techniques described have applications in plant genetic engineering and crop improvement.
Protoplast is a naked cell (without cell wall) surrounded by a plasma membrane. It can regenerate cell wall, grow and divide.
Spheroplast cells have their cell wall only partially removed.
Is fragile but can be cultured and grow into a whole plant.
Cells can originate from any type of tissue (Mesophyll tissue - most suitable source ).
Can be applied in somatic hybridization.
Can be applied in biotechnology and microbiology.
Somatic hybridization is the development of hybrid plants through the fusion of somatic protoplasts of two different plant species/ varieties.
Somatic Hybridization was firstly introduced by Carlson in Nicotiana
glauca.
In 1960, E.C Cocking contributed to the enzymatic isolation and culture of protoplast.
This document summarizes a seminar on protoplast culture. It defines protoplasts as plant cells without cell walls that are capable of regenerating cell walls and dividing. The document outlines the general procedure for protoplast culture, including isolating protoplasts enzymatically from plant tissues, culturing them in nutrient media, testing viability, and regenerating plants from protoplast-derived callus tissue. The importance of protoplast culture is mentioned as a tool for crop improvement through somatic hybridization and genetic engineering.
The document discusses protoplasts, which are plant cells that have had their cell walls removed, leaving the cell membrane and organelles. It describes methods for isolating protoplasts from plant tissues using either mechanical or enzymatic methods. The enzymatic method uses enzymes like pectinase and cellulase to break down the cell wall. Protoplasts have various applications including isolating cell organelles and studying cell structures. The document also discusses immobilizing enzymes by binding them to inert matrices, which has benefits like reusability and stability. Methods of immobilization include adsorption, covalent binding, and entrapment in gels.
The document discusses protoplast fusion, which involves removing the cell walls of plant cells to create naked protoplasts that can then be fused. It describes how to isolate protoplasts from plant tissues using either mechanical or enzymatic methods. The fusion of protoplasts from different species or varieties can create hybrid cells called heterokaryons or hybrids. Techniques to induce protoplast fusion include treatment with polyethylene glycol (PEG), calcium ions, electricity, or sodium nitrate. Successful somatic hybridization follows a procedure of isolating, fusing, regenerating cell walls, and selecting hybrid plant cells and colonies. Applications of protoplast fusion include combining genomes of sterile plants and
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.
Somatic hybridization and Protoplast IsolationPABOLU TEJASREE
The document discusses protoplast isolation and somatic hybridization. It defines somatic hybridization as the development of hybrid plants through the fusion of somatic protoplasts from different plant species or varieties. The key steps in somatic hybridization are isolating protoplasts, fusing the protoplasts from desired species, identifying and selecting hybrid cells, culturing the hybrid cells, and regenerating hybrid plants. Protoplasts can be isolated using enzymatic or mechanical methods, and their viability and ability to form cell walls can be tested. Various factors like enzyme concentration, temperature, and pH affect protoplast isolation and viability. Protoplast fusion can occur spontaneously or be induced using methods like chemical treatment, electro
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.
The problems attract worldwide attention K/a Global Environmental Problems.
The top three environmental problems are: (1) Greenhouse Effect and Global Warming (2) Depletion of Ozone and (3) Acid Rain.
Aim1: To study the method of genome identification through ENSEMBL browser.
Aim2: To study the method of genome identification through VISTA.
Aim3: To study the method of genome identification through UCSC Genome Browser.
Aim4: To study the method of genome and amino acid sequences through UCSC Genome Browser.
Intracellular Components
We will now begin our discussion of intracellular organelles. As we have mentioned, only eukaryotic cells have intracellular sub-divisions, so our discussion will exclude prokaryotic cells. We will also focus on animal cells, since plant cells have a number of further specialized structures. In this section we will discuss the importance of the cell nucleus, mitochondria, peroxisomes, endoplasmic reticulum, golgi apparatus, and lysosome.
Types of Receptors
Receptors are protein molecules in the target cell or on its surface that bind ligands. There are two types of receptors: internal receptors and cell-surface receptors.
Microbial biomass conversion processes take advantage of the ability of microorganisms to consume and digest biomass and release hydrogen. Depending on the pathway, this research could result in commercial-scale systems in the mid- to long-term timeframe that could be suitable for distributed, semi-central, or central hydrogen production scales, depending on the feedstock used.
The cells derived from root apical and shoot-apical meristems and cambium differentiate and mature to perform specific functions. This act leading to maturation is termed as differentiation. During differentiation, cells undergo few to major structural changes both in their cell walls and protoplasm. The living differentiated cells, that by now have lost the capacity to divide can regain the capacity of division under certain conditions. This phenomenon is termed as dedifferentiation. For example, formation of meristems – interfascicular cambium and cork cambium from fully differentiated parenchyma cells. While doing so, such meristems / tissues are able to divide and produce cells that once again lose the capacity to divide but mature to perform specific functions, i.e., get redifferentiated.
Meat and milk from farmed animals including livestock (cattle, goat and buffalo) and poultry are sources of high quality protein and essential amino acids, minerals, fats and fatty acids, readily available vitamins, small quantities of carbohydrates and other bioactive components.1 The Food and Agriculture Organization (FAO) 2008 estimate shows that meat consumption has grown with increase in population. The average global per capita meat consumption is 42.1 kg/year with 82.9 kg/year in developed and 31.1 kg/year in developing countries in a recommended daily animal-sourced protein per capita of 50 kg per year2. Milk on the other hand is consumed in various forms: liquid, cheese, powder, and cream at a global per capita consumption of 108 kg per person per year which is way below the FAO recommended daily consumption of 200 kg.
This document provides instructions and safety guidelines for working in a laboratory. Key points include:
- Punctuality, cleanliness, organization and responsibility are important for lab work. Proper protective equipment like lab coats and gloves must be worn.
- Chemicals, glassware and equipment should be handled carefully and stored properly. Eating, drinking and loose items are prohibited on lab benches.
- Accidents and injuries should be reported immediately. Basic first aid procedures are outlined for issues like chemical exposure, burns and cuts.
- Safety precautions are required for hazards like fires, electricity, compressed gases, toxins and carcinogens. Proper ventilation, labeling and disposal are important.
Polyclonal antibodies are produced by injecting an antigen into an animal to elicit an immune response. They recognize and bind to multiple epitopes on an antigen. Monoclonal antibodies are produced through cell fusion and screening to identify antibodies that bind to a single epitope. Polyclonal antibodies are useful for detection applications due to binding multiple epitopes, while monoclonal antibodies are useful for research and treatment due to their high specificity for a single epitope. Both have advantages and disadvantages depending on the application.
There are many characteristics of biological data. All these characteristics make the management of biological information a particularly challenging problem. Here mainly we will focus on characteristics of biological information and multidisciplinary field called bioinformatics. Bioinformatics, now a days has emerged with graduate degree programs in several universities.
Hormones, Proteins, etc. present in blood in minute concentration can be assayed by the recent advanced technique of “Enzyme Immuno Assay” without involving any disadvantage. The basic reaction is the interaction between an antibody and an antigen.
Meat and milk from farmed animals including livestock (cattle, goat and buffalo) and poultry are sources of high quality protein and essential amino acids, minerals, fats and fatty acids, readily available vitamins, small quantities of carbohydrates and other bioactive components.1 The Food and Agriculture Organization (FAO) 2008 estimate shows that meat consumption has grown with increase in population. The average global per capita meat consumption is 42.1 kg/year with 82.9 kg/year in developed and 31.1 kg/year in developing countries in a recommended daily animal-sourced protein per capita of 50 kg per year2. Milk on the other hand is consumed in various forms: liquid, cheese, powder, and cream at a global per capita consumption of 108 kg per person per year which is way below the FAO recommended daily consumption of 200 kg.
In shotgun sequencing the genome is broken randomly into short fragments (1 to 2 kbp long) suitable for sequencing. The fragments are ligated into a suitable vector and then partially sequenced. Around 400–500 bp of sequence can be generated from each fragment in a single sequencing run. In some cases, both ends of a fragment are sequenced. Computerized searching for overlaps between individual sequences then assembles the complete sequence.
Sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology cannot read whole genomes in one go, but rather reads small pieces of between 20 and 30,000 bases, depending on the technology used. Typically the short fragments, called reads, result from shotgun sequencing genomic DNA, or gene transcript (ESTs).
The problem of sequence assembly can be compared to taking many copies of a book, passing each of them through a shredder with a different cutter, and piecing the text of the book back together just by looking at the shredded pieces. Besides the obvious difficulty of this task, there are some extra practical issues: the original may have many repeated paragraphs, and some shreds may be modified during shredding to have typos. Excerpts from another book may also be added in, and some shreds may be completely unrecognizable.
Vaccine (L. vacca = cow) is a preparation/suspension or extract of dead/attenuated (weakened) germs of a disease which on inoculation (injection) into a healthy person provides temporary/permanent active/passive immunity by inducing antibodies formation.
Thus antibody provoking agents are called vaccines.
This document discusses the treatment of municipal waste and industrial effluents through various biological processes. It describes the four main treatment processes: 1) preliminary treatment to remove solids, 2) primary treatment using sedimentation to remove settleable organic materials, 3) secondary or biological treatment using microorganisms to break down dissolved and suspended organic matter, and 4) tertiary or advanced treatment for additional removal of nutrients or contaminants. Key biological treatment methods discussed include activated sludge processes, trickling filters, and rotating biological contactors.
The genetic variations found in the in vitro cultured cells are collectively referred to as somaclonal variations.
The plants derived from such cells are referred to somaclones. Some authors use the terms calliclones and proto-clones to represent cultures obtained from callus and protoplasts respectively.
The growth of plant cells in vitro is an asexual process involving only mitotic division of cells. Thus, culturing of cells is the method to clone a particular genotype. It is therefore expected that plants arising from a given tissue culture should be the exact copies of the parental plant.
The occurrence of phenotypic variants among the regenerated plants (from tissue cultures) has been known for several years. These variations were earlier dismissed as tissue culture artefacts. The term somaclonal variations was first used by Larkin and Scowcraft (1981) for variations arising due to culture of cells, i.e., variability generated by a tissue culture. This term is now universally accepted.
As described elsewhere the explant used in tissue culture may come from any part of the plant organs or cells. These include leaves, roots, protoplasts, microspores and embryos. Somaclonal variations are reported in all types of plant tissue cultures.
In recent years, the term gametoclonal variations is used for the variations observed in the regenerated plants from gametic cells (e.g., anther cultures). For the plants obtained from protoplast cultures, proto-clonal variations is used.
Solid waste management is a polite term for garbage management. As long as humans have been living in settled communities, solid waste, or garbage, has been an issue, and modern societies generate far more solid waste than early humans ever did.
The chemical compounds produced by plants are collectively referred to as phytochemicals. Biotechnologists have special interest in plant tissue culture for the large scale production of commercially important compounds. These include pharmaceuticals, flavours, fragrances, cosmetics, food additives, feed stocks and antimicrobials.
Most of these products are secondary metabolites— chemical compounds that do not participate in metabolism of plants. Thus, secondary metabolites are not directly needed by plants as they do not perform any physiological function (as is the case with primary metabolites such as amino acids, nucleic acids etc.). Although the native plants are capable of producing the secondary metabolites of commercial interest, tissue culture systems are preferred.
This presentation was provided by Steph Pollock of The American Psychological Association’s Journals Program, and Damita Snow, of The American Society of Civil Engineers (ASCE), for the initial session of NISO's 2024 Training Series "DEIA in the Scholarly Landscape." Session One: 'Setting Expectations: a DEIA Primer,' was held June 6, 2024.
How to Setup Warehouse & Location in Odoo 17 InventoryCeline George
In this slide, we'll explore how to set up warehouses and locations in Odoo 17 Inventory. This will help us manage our stock effectively, track inventory levels, and streamline warehouse operations.
How to Make a Field Mandatory in Odoo 17Celine George
In Odoo, making a field required can be done through both Python code and XML views. When you set the required attribute to True in Python code, it makes the field required across all views where it's used. Conversely, when you set the required attribute in XML views, it makes the field required only in the context of that particular view.
LAND USE LAND COVER AND NDVI OF MIRZAPUR DISTRICT, UPRAHUL
This Dissertation explores the particular circumstances of Mirzapur, a region located in the
core of India. Mirzapur, with its varied terrains and abundant biodiversity, offers an optimal
environment for investigating the changes in vegetation cover dynamics. Our study utilizes
advanced technologies such as GIS (Geographic Information Systems) and Remote sensing to
analyze the transformations that have taken place over the course of a decade.
The complex relationship between human activities and the environment has been the focus
of extensive research and worry. As the global community grapples with swift urbanization,
population expansion, and economic progress, the effects on natural ecosystems are becoming
more evident. A crucial element of this impact is the alteration of vegetation cover, which plays a
significant role in maintaining the ecological equilibrium of our planet.Land serves as the foundation for all human activities and provides the necessary materials for
these activities. As the most crucial natural resource, its utilization by humans results in different
'Land uses,' which are determined by both human activities and the physical characteristics of the
land.
The utilization of land is impacted by human needs and environmental factors. In countries
like India, rapid population growth and the emphasis on extensive resource exploitation can lead
to significant land degradation, adversely affecting the region's land cover.
Therefore, human intervention has significantly influenced land use patterns over many
centuries, evolving its structure over time and space. In the present era, these changes have
accelerated due to factors such as agriculture and urbanization. Information regarding land use and
cover is essential for various planning and management tasks related to the Earth's surface,
providing crucial environmental data for scientific, resource management, policy purposes, and
diverse human activities.
Accurate understanding of land use and cover is imperative for the development planning
of any area. Consequently, a wide range of professionals, including earth system scientists, land
and water managers, and urban planners, are interested in obtaining data on land use and cover
changes, conversion trends, and other related patterns. The spatial dimensions of land use and
cover support policymakers and scientists in making well-informed decisions, as alterations in
these patterns indicate shifts in economic and social conditions. Monitoring such changes with the
help of Advanced technologies like Remote Sensing and Geographic Information Systems is
crucial for coordinated efforts across different administrative levels. Advanced technologies like
Remote Sensing and Geographic Information Systems
9
Changes in vegetation cover refer to variations in the distribution, composition, and overall
structure of plant communities across different temporal and spatial scales. These changes can
occur natural.
This slide is special for master students (MIBS & MIFB) in UUM. Also useful for readers who are interested in the topic of contemporary Islamic banking.
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This presentation includes basic of PCOS their pathology and treatment and also Ayurveda correlation of PCOS and Ayurvedic line of treatment mentioned in classics.
1. Protoplsat Culture
Dr. Naveen Gaurav
Associate Professor and Head
Department of Biotechnology
Shri Guru Ram Rai University
Dehradun
2. Protoplsat Culture
Protoplasts are naked plant cells without the cell wall, but they possess plasma membrane
and all other cellular components. They represent the functional plant cells but for the lack
of the barrier, cell wall. Protoplasts of different species can be fused to generate a hybrid
and this process is referred to as somatic hybridization (or protoplast fusion). Cybridization
is the phenomenon of fusion of a normal protoplast with an enucleated (without nucleus)
protoplast that results in the formation of a cybrid or cytoplast (cytoplasmic hybrids).
Historical developments:
The term protoplast was introduced in 1880 by Hanstein. The first isolation of protoplasts
was achieved by Klercker (1892) employing a mechanical method. A real beginning in
protoplast research was made in 1960 by Cocking who used an enzymatic method for the
removal of cell wall.
Rakabe and his associates (1971) were successful to achieve the regeneration of whole
tobacco plant from protoplasts. Rapid progress occurred after 1980 in protoplast fusion to
improve plant genetic material, and the development of transgenic plants.
Importance of Protoplasts and Their Cultures:
The isolation, culture and fusion of protoplasts is a fascinating field in plant research.
Protoplast isolation and their cultures provide millions of single cells (comparable to
microbial cells) for a variety of studies.
Protoplasts have a wide range of applications; some of them are listed below:
3. 1. The protoplast in culture can be regenerated into a whole plant.
2. Hybrids can be developed from protoplast fusion.
3. It is easy to perform single cell cloning with protoplasts.
4. Genetic transformations can be achieved through genetic engineering of protoplast DNA.
5. Protoplasts are excellent materials for ultra-structural studies.
6. Isolation of cell organelles and chromosomes is easy from protoplasts.
7. Protoplasts are useful for membrane studies (transport and uptake processes).
8. Isolation of mutants from protoplast cultures is easy.
Isolation of Protoplasts:
Protoplasts are isolated by two techniques
1.. Mechanical method
2. Enzymatic method
Mechanical Method:Protoplas isolation by mechanical method is a crude and tedious
procedure. This results in the isolation of a very small number of protoplasts.
The technique involves the following stages (Fig. 44.1):1. A small piece of epidermis from a
plant is selected.
2. The cells are subjected to plasmolysis. This causes protoplasts to shrink away from the cell
walls.
4. 3. The tissue is dissected to release the protoplasts.
Mechanical method for protoplast isolation is no more in use because of the following
limitations:
i. Yield of protoplasts and their viability is low.
ii. It is restricted to certain tissues with vacuolated cells.
ii. The method is laborious and tedious.
However, some workers prefer mechanical methods if the cell wall degrading enzymes (of
enzymatic method) cause deleterious effects to protoplasts.
Enzymatic Method:
Enzymatic method is a very widely used technique for the isolation of protoplasts. The advantages
of enzymatic method include good yield of viable cells, and minimal or no damage to the
protoplasts.
Sources of protoplasts:
Protoplasts can be isolated from a wide variety of tissues and organs that include leaves, roots,
shoot apices, fruits, embryos and microspores. Among these, the mesophyll tissue of fully
expanded leaves of young plants or new shoots are most frequently used. In addition, callus and
suspension cultures also serve as good sources for protoplast isolation.
Enzymes for protoplast isolation:The enzymes that can digest the cell walls are required for
protoplast isolation. Chemically, the plant cell wall is mainly composed of cellulose, hemicellulose
and pectin which can be respectively degraded by the enzymes cellulose, hemicellulose and
pectinase. The different enzymes for protoplast isolation and the corresponding sources are given
in Table 44.1.
In fact, the various enzymes for protoplast isolation are commercially available. The enzymes are
usually used at a pH 4.5 to 6.0, temperature 25-30°C with a wide variation in incubation period
that may range from half an hour to 20 hours.
5. The enzymatic isolation of protoplasts can be carried out by two approaches:
1. Two step or sequential method:
The tissue is first treated with pectinase (macerozyme) to separate cells by degrading middle
lamella. These free cells are then exposed to cellulose to release protoplasts. Pectinase breaks up
the cell aggregates into individual cells while cellulose removes the cell wall proper.
2. One step or simultaneous method:
This is the preferred method for protoplast isolation. It involves the simultaneous use of both the
enzymes — macerozyme and cellulose.
Isolation of protoplasts from leaves:
Leaves are most commonly used, for protoplast isolation, since it is possible to isolate uniform
cells in large numbers.
The procedure broadly involves the following steps (Fig. 44.2):
1. Sterilization of leaves.
2. Removal of epidermal cell layer.
3. Treatment with enzymes.
4. Isolation of protoplasts.
Besides leaves, callus cultures and cell suspension cultures can also be used for the isolation of
protoplasts. For this purpose, young and actively growing cells are preferred.
Purification of protoplasts:
The enzyme digested plant cells, besides protoplasts contain undigested cells, broken protoplasts
and undigested tissues. The cell clumps and undigested tissues can be removed by filtration. This is
followed by centrifugation and washings of the protoplasts. After centrifugation, the protoplasts
are recovered above Percoll.
6.
7. There are several methods to assess the protoplast viability:
1. Fluorescein diacetate (FDA) staining method—The dye accumulates inside viable
protoplasts which can be detected by fluorescence microscopy.
2. Phenosafranine stain is selectively taken up by dead protoplasts (turn red) while the
viable cells remain unstained.
3. Exclusion of Evans blue dye by intact membranes.
4. Measurement of cell wall formation—Calcofluor white (CFW) stain binds to the newly
formed cell walls which emit fluorescence.
5. Oxygen uptake by protoplasts can be measured by oxygen electrode.
6. Photosynthetic activity of protoplasts.
7. The ability of protoplasts to undergo continuous mitotic divisions (this is a direct
measure).
8. Culture of Protoplasts:
The very first step in protoplast culture is the development of a cell wall around the
membrane of the protoplast. This is followed by the cell divisions that give rise to a small
colony. With suitable manipulations of nutritional and physiological conditions, the cell
colonies may be grown continuously as cultures or regenerated to whole plants. Protoplasts
are cultured either in semisolid agar or liquid medium. Sometimes, protoplasts are first
allowed to develop cell wall in liquid medium, and then transferred to agar medium.
Agar culture:
Agarose is the most frequently used agar to solidify the culture media. The concentration of
the agar should be such that it forms a soft agar gel when mixed with the protoplast
suspension. The plating of protoplasts is carried out by Bergmann’s cell plating technique .In
agar cultures, the protoplasts remain in a fixed position, divide and form cell clones. The
advantage with agar culture is that clumping of protoplasts is avoided.
Liquid culture:
Liquid culture is the preferred method for protoplast cultivation for the following reasons:
1. It is easy to dilute and transfer.
2. Density of the cells can be manipulated as desired.
3. For some plant species, the cells cannot divide in agar medium, therefore liquid medium is
the only choice.
4. Osmotic pressure of liquid medium can be altered as desired.
Culture Media:
The culture media with regard to nutritional components and osmoticum are briefly
described.
9. Nutritional components:
In general, the nutritional requirements of protoplasts are similar to those of cultured plant cells
(callus and suspension cultures). Mostly, MS and B5 media with suitable modifications are used.
Some of the special features of protoplast culture media are listed below:
1. The medium should be devoid of ammonium, and the quantities of iron and zinc should be less.
2. The concentration of calcium should be 2-4-times higher than used for cell cultures. This is
needed for membrane stability.
3. High auxin/kinetin ratio is suitable to induce cell divisions while high kinetin/auxin ratio is
required for regeneration.
4. Glucose is the preferred carbon source by protoplasts although a combination of sugars (glucose
and sucrose) can be used.
5. The vitamins used for protoplast cultures are the same as used in standard tissue culture media.
Osmoticum and osmotic pressure:
Osmoticum broadly refers to the reagents/ chemicals that are added to increase the osmotic
pressure of a liquid.
The isolation and culture of protoplasts require osmotic protection until they develop a strong cell
wall. In fact, if the freshly isolated protoplasts are directly added to the normal culture medium,
they will burst.
Thus, addition of an osmoticum is essential for both isolation and culture media of protoplast to
prevent their rupture. The osmotica are of two types — non-ionic and ionic.
Non-ionic osmotica:
The non-ionic substances most commonly used are soluble carbohydrates such as mannitol,
sorbitol, glucose, fructose, galactose and sucrose. Mannitol, being metabolically inert, is most
frequently used.
10. Ionic osmotica:
Potassium chloride, calcium chloride and magnesium phosphate are the ionic substances in use to
maintain osmotic pressure. When the protoplasts are transferred to a culture medium, the use of
metabolically active osmotic stabilizers (e.g., glucose, sucrose) along with metabolically inert osmotic
stabilizers (mannitol) is advantageous. As the growth of protoplasts and cell wall regeneration occurs, the
metabolically active compounds are utilized, and this results in the reduced osmotic pressure so that
proper osmolarity is maintained.
Culture Methods:
The culture techniques of protoplasts are almost the same that are used for cell culture with suitable
modifications. Some important aspects are briefly given.
Feeder layer technique:For culture of protoplasts at low density feeder layer technique is preferred. This
method is also important for selection of specific mutant or hybrid cells on plates. The technique consists
of exposing protoplast cell suspensions to X-rays (to inhibit cell division with good metabolic activity) and
then plating them on agar plates.
Co-culture of protoplasts:Protoplasts of two different plant species (one slow growing and another fast
growing) can be co- cultured. This type of culture is advantageous since the growing species provide the
growth factors and other chemicals which help in the generation of cell wall and cell division. The co-
culture method is generally used if the two types of protoplasts are morphologically distinct.
Micro drop culture:Specially designed dishes namely cuprak dishes with outer and inner chambers are
used for micro drop culture. The inner chamber carries several wells wherein the individual protoplasts
in droplets of nutrient medium can be added. The outer chamber is filled with water to maintain
humidity. This method allows the culture of fewer protoplasts for droplet of the medium.
Regeneration of Protoplasts:
Protoplast regeneration which may also be regarded as protoplast development occurs in two stages:
1. Formation of cell wall.
2. Development of callus/whole plant.
11. Formation of cell wall:
The process of cell wall formation in cultured protoplasts starts within a few hours after isolation that
may take two to several days under suitable conditions. As the cell wall development occurs, the
protoplasts lose their characteristic spherical shape. The newly developed cell wall by protoplasts can be
identified by using calcofluor white fluorescent stain.
The freshly formed cell wall is composed of loosely bound micro fibrils which get organized to form a
typical cell wall. This process of cell wall development requires continuous supply of nutrients,
particularly a readily metabolised carbon source (e.g. sucrose).
Cell wall development is found to be improper in the presence of ionic osmotic stabilizers in the medium.
The protoplasts with proper cell wall development undergo normal cell division. On the other hand,
protoplasts with poorly regenerated cell wall show budding and fail to undergo normal mitosis.
Development of Callus/whole Plant:
As the cell wall formation around protoplasts is complete, the cells increase in size, and the first division
generally occurs within 2-7 days. Subsequent divisions result in small colonies, and by the end of third
week, visible colonies (macroscopic colonies) are formed. These colonies are then transferred to an
osmotic-free (mannitol or sorbitol-free) medium for further development to form callus.
With induction and appropriate manipulations, the callus can undergo organogenic or embryo genic
differentiation to finally form the whole plant. A general view of the protoplast isolation, culture and
regeneration is represented in Fig. 44.2.
Plant regeneration can be done from the callus obtained either from protoplasts or from the culture of
plant organs. There are however, certain differences in these two calluses. The callus derived from plant
organs carries preformed buds or organized structures, while the callus from protoplast culture does not
have such structures.
The first success of regeneration of plants from protoplast cultures of Nicotiana tabacum was achieved
by Takebe et al (in 1971). Since then, several species of plants have been regenerated by using
protoplasts (Table 44.2).