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.
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
The document discusses two types of embryo culture: mature embryo culture and embryo rescue. Mature embryo culture involves isolating mature embryos from ripe seeds and culturing them in vitro. Embryo rescue involves culturing immature embryos to rescue them from unripe or hybrid seeds that fail to germinate. The document also discusses factors involved in embryo culture like the media, temperature, light, time of culture, and nutritional requirements that vary depending on the heterotrophic or autotrophic phase of embryo development. Various plant species that embryo culture has been employed for are also listed.
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
The document discusses haploid plant culture techniques. It describes anther/pollen culture and ovule culture as important methods to produce haploid plants. Anther/pollen culture involves culturing anthers or isolated pollen grains to develop into haploid embryos or callus. Ovule culture involves culturing isolated ovules. The document outlines factors that affect androgenesis (anther/pollen culture) and discusses using techniques like colchicine treatment or long-term callus culture to induce chromosome doubling and generate homozygous diploid plants from haploids.
This document discusses haploid plant production through anther culture. It begins by defining key terms like gametophyte, sporophyte, haploid and diploid plants. It then describes the two main methods of haploid production - anther culture and isolated microspore culture. For anther culture, it outlines the process of culturing immature anthers on nutrient media, including pretreatments, media composition and plant regeneration. Anther culture can result in direct or indirect embryogenesis and four pathways of pollen development are described. The document provides detailed steps of anther culture and potential issues like production of diploid plants.
OVARY CULTURE:-
"the in-vitro culturing of ovaries in an aseptic condition from the pollinated or un-pollinated flowers, in an appropriate nutrient medium and under optimal conditions." And
OVULE CULTURE:-
"Ovule culture is an experimental system by which ovules are aseptically isolated from the ovary and are grown aseptically on chemically defined nutrient medium under controlled conditions."
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
The document discusses two types of embryo culture: mature embryo culture and embryo rescue. Mature embryo culture involves isolating mature embryos from ripe seeds and culturing them in vitro. Embryo rescue involves culturing immature embryos to rescue them from unripe or hybrid seeds that fail to germinate. The document also discusses factors involved in embryo culture like the media, temperature, light, time of culture, and nutritional requirements that vary depending on the heterotrophic or autotrophic phase of embryo development. Various plant species that embryo culture has been employed for are also listed.
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
The document discusses haploid plant culture techniques. It describes anther/pollen culture and ovule culture as important methods to produce haploid plants. Anther/pollen culture involves culturing anthers or isolated pollen grains to develop into haploid embryos or callus. Ovule culture involves culturing isolated ovules. The document outlines factors that affect androgenesis (anther/pollen culture) and discusses using techniques like colchicine treatment or long-term callus culture to induce chromosome doubling and generate homozygous diploid plants from haploids.
This document discusses haploid plant production through anther culture. It begins by defining key terms like gametophyte, sporophyte, haploid and diploid plants. It then describes the two main methods of haploid production - anther culture and isolated microspore culture. For anther culture, it outlines the process of culturing immature anthers on nutrient media, including pretreatments, media composition and plant regeneration. Anther culture can result in direct or indirect embryogenesis and four pathways of pollen development are described. The document provides detailed steps of anther culture and potential issues like production of diploid plants.
OVARY CULTURE:-
"the in-vitro culturing of ovaries in an aseptic condition from the pollinated or un-pollinated flowers, in an appropriate nutrient medium and under optimal conditions." And
OVULE CULTURE:-
"Ovule culture is an experimental system by which ovules are aseptically isolated from the ovary and are grown aseptically on chemically defined nutrient medium under controlled conditions."
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.
Haploid Production - Techniques, Application & Problem ANUGYA JAISWAL
Haploid is applied to any plant originating from a sporophyte (2n) and containing (n) number of chromosomes.
Artificial production of haploids was attempted through distant hybridization, delayed pollination, application of irradiated pollen, hormone treatment and temperature shock.
The artificial production of haploids until 1964 was attempted through:
1. Distant hybridization
2. Delayed pollination
3. Application of irradiated pollen
4. Hormone treatments
5. Temperature shocks
The development of numerous pollen plantlets in anther cultures of Datura innoxia, first reported by two Indian scientists (Guha and Maheshwari, 1964, 1966), was a major breakthrough in haploid breeding of higher plants.
The technique of haploid production through anther culture ('anther - androgenesis') has been extended successfully to numerous plant species, including many economically important plants, such as cereals and vegetable, oil and tree crops.
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.
Somatic embryogenesis, in plant tissue culture 2KAUSHAL SAHU
Introduction
Types of somatic embryogenesis
Developmental stages
Factors affecting somatic embryogenesis
Importance
Conclusions
References
The process of regeneration of embryos from somatic cells, tissue or organs is regarded as somatic or asexual embryogenesis.
opposite of zygotic or sexual embryogenesis.
Embryo-like structures which can develop into whole plants in a way that is similar to zygotic embryos are formed from somatic cells.
Somatic embryogenesis is the process where embryos form from sporophytic cells in vitro rather than from a zygote. There are different types of embryos including zygotic, formed from fertilized eggs, and somatic embryos which form directly from other plant tissues and organs in culture. The correct developmental stage of the explant tissue is crucial for initiation of embryogenic callus formation in somatic embryogenesis, with young or juvenile explants producing more embryos than older explants.
The document discusses organogenesis, which is the development of adventitious organs or primordial from undifferentiated plant cell mass through differentiation. It describes the process, including dedifferentiation and redifferentiation stages. There are two types of organogenesis - direct organogenesis which does not involve callus formation, and indirect organogenesis which involves callus formation. Organogenesis is used in plant tissue culture to regenerate plants through shoot or root cultures and is influenced by factors like explant source and size, plant growth regulators, and culture conditions. It has commercial applications in micropropagation of plants.
1. Callus culture involves growing undifferentiated plant cells and tissues on a nutrient medium under sterile conditions. This allows for the production of genetically identical clones without seeds or pollination.
2. A callus is an unorganized mass of cells formed from injured or cultured plant tissue. Successful callus culture requires selecting an explant, preparing sterile culture media, and regulating hormone levels to induce cell proliferation.
3. Callus cultures are maintained through periodic sub-culturing to replenish nutrients and prevent toxicity. The growth and characteristics of callus tissue can provide insights into plant cell metabolism, differentiation, and pathways for genetic engineering applications.
This document discusses triploid production through endosperm culture and somatic embryogenesis. It defines endosperm culture as the in vitro development of isolated mature or immature endosperm tissue to obtain triploid plantlets. Two types of endosperm culture are described: mature and immature. The key steps and factors affecting endosperm culture are outlined. Somatic embryogenesis is defined as the development of embryos from somatic cells in vitro. The document compares somatic and zygotic embryos and describes the two routes of somatic embryogenesis: direct and indirect. The stages of somatic embryogenesis and factors influencing the process are summarized.
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 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.
Until two decades ago the genetic resources were getting depleted owing to the
It was imperative therefore that many of the elite, economically important and endangered species are preserved to make them available when needed.
The conventional methods of storage failed to prevent losses caused due to various reasons.
A new methodology had to be devised for long term preservation of material.
Aseptic techniques in plant tissue culturekumarkanika
Importance of practising Aseptic Techniques in plant tissue culture and what are these techniques what precautions should be taken when doing tissue culture
Establishment and maintenance of callus and suspension culture.pptxSujata Koundal
This document discusses callus and suspension cultures. Callus cultures involve growing loose aggregates of parenchyma cells on a solid nutrient medium. Suspension cultures grow tissues and cells in liquid medium with constant agitation. Batch cultures use a limited supply of nutrients until they are depleted, while continuous cultures maintain a steady state by draining out used medium and adding fresh medium. Both callus and suspension cultures need to be sub-cultured regularly to maintain healthy growth.
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 different types of plant organ culture, including root, shoot apical meristem, leaf, flower, and ovule cultures. Root culture involves culturing excised radical tips of aseptically germinated seeds. Shoot apical meristem culture involves culturing the shoot tip comprising the meristem and developing leaves. Flower culture involves culturing excised floral buds to produce full blooms. Ovule culture involves culturing isolated ovules to facilitate fertilization and embryo development. Organ cultures have various applications including studying organ development, production of secondary metabolites, and generating virus-free plants.
Cryopreservation is a process for long-term storage of biological material such as germplasm at ultra-low temperatures, typically using liquid nitrogen at -196°C. This preserves cells and tissues by stopping all biological activity. The document discusses the various steps involved, including selection of plant material, addition of cryoprotectants, controlled freezing and thawing processes, and techniques for determining viability after storage and thawing. Cryopreservation is important for long-term conservation of plant genetic resources.
WHAT IS ARTIFICIAL SEED..?
Artificial seed can be defined as artificial encapsulation of somatic embryos, shoot bud or aggregates of cell of any tissues which has the ability to form a plant in in-vitro or ex-vivo condition.
Artificial seed have also been often referred to as synthetic seed.
HISTORY
Artificial seeds were first introduced in 1970’s as a novel analogue to the plant seeds.
The production of artificial seeds is useful for plants which do not produce viable seeds. It represents a method to propagate these plants.
Artificial seeds are small sized and these provides further advantages in storage, handling and shipping.
The term, “EMBLING” is used for the plants originated from synthetic seed.
• The use of synthetic varieties for commercial cultivation was first suggested in Maize (Hays & Garber, 1919).
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).
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.
Haploid Production - Techniques, Application & Problem ANUGYA JAISWAL
Haploid is applied to any plant originating from a sporophyte (2n) and containing (n) number of chromosomes.
Artificial production of haploids was attempted through distant hybridization, delayed pollination, application of irradiated pollen, hormone treatment and temperature shock.
The artificial production of haploids until 1964 was attempted through:
1. Distant hybridization
2. Delayed pollination
3. Application of irradiated pollen
4. Hormone treatments
5. Temperature shocks
The development of numerous pollen plantlets in anther cultures of Datura innoxia, first reported by two Indian scientists (Guha and Maheshwari, 1964, 1966), was a major breakthrough in haploid breeding of higher plants.
The technique of haploid production through anther culture ('anther - androgenesis') has been extended successfully to numerous plant species, including many economically important plants, such as cereals and vegetable, oil and tree crops.
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.
Somatic embryogenesis, in plant tissue culture 2KAUSHAL SAHU
Introduction
Types of somatic embryogenesis
Developmental stages
Factors affecting somatic embryogenesis
Importance
Conclusions
References
The process of regeneration of embryos from somatic cells, tissue or organs is regarded as somatic or asexual embryogenesis.
opposite of zygotic or sexual embryogenesis.
Embryo-like structures which can develop into whole plants in a way that is similar to zygotic embryos are formed from somatic cells.
Somatic embryogenesis is the process where embryos form from sporophytic cells in vitro rather than from a zygote. There are different types of embryos including zygotic, formed from fertilized eggs, and somatic embryos which form directly from other plant tissues and organs in culture. The correct developmental stage of the explant tissue is crucial for initiation of embryogenic callus formation in somatic embryogenesis, with young or juvenile explants producing more embryos than older explants.
The document discusses organogenesis, which is the development of adventitious organs or primordial from undifferentiated plant cell mass through differentiation. It describes the process, including dedifferentiation and redifferentiation stages. There are two types of organogenesis - direct organogenesis which does not involve callus formation, and indirect organogenesis which involves callus formation. Organogenesis is used in plant tissue culture to regenerate plants through shoot or root cultures and is influenced by factors like explant source and size, plant growth regulators, and culture conditions. It has commercial applications in micropropagation of plants.
1. Callus culture involves growing undifferentiated plant cells and tissues on a nutrient medium under sterile conditions. This allows for the production of genetically identical clones without seeds or pollination.
2. A callus is an unorganized mass of cells formed from injured or cultured plant tissue. Successful callus culture requires selecting an explant, preparing sterile culture media, and regulating hormone levels to induce cell proliferation.
3. Callus cultures are maintained through periodic sub-culturing to replenish nutrients and prevent toxicity. The growth and characteristics of callus tissue can provide insights into plant cell metabolism, differentiation, and pathways for genetic engineering applications.
This document discusses triploid production through endosperm culture and somatic embryogenesis. It defines endosperm culture as the in vitro development of isolated mature or immature endosperm tissue to obtain triploid plantlets. Two types of endosperm culture are described: mature and immature. The key steps and factors affecting endosperm culture are outlined. Somatic embryogenesis is defined as the development of embryos from somatic cells in vitro. The document compares somatic and zygotic embryos and describes the two routes of somatic embryogenesis: direct and indirect. The stages of somatic embryogenesis and factors influencing the process are summarized.
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 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.
Until two decades ago the genetic resources were getting depleted owing to the
It was imperative therefore that many of the elite, economically important and endangered species are preserved to make them available when needed.
The conventional methods of storage failed to prevent losses caused due to various reasons.
A new methodology had to be devised for long term preservation of material.
Aseptic techniques in plant tissue culturekumarkanika
Importance of practising Aseptic Techniques in plant tissue culture and what are these techniques what precautions should be taken when doing tissue culture
Establishment and maintenance of callus and suspension culture.pptxSujata Koundal
This document discusses callus and suspension cultures. Callus cultures involve growing loose aggregates of parenchyma cells on a solid nutrient medium. Suspension cultures grow tissues and cells in liquid medium with constant agitation. Batch cultures use a limited supply of nutrients until they are depleted, while continuous cultures maintain a steady state by draining out used medium and adding fresh medium. Both callus and suspension cultures need to be sub-cultured regularly to maintain healthy growth.
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 different types of plant organ culture, including root, shoot apical meristem, leaf, flower, and ovule cultures. Root culture involves culturing excised radical tips of aseptically germinated seeds. Shoot apical meristem culture involves culturing the shoot tip comprising the meristem and developing leaves. Flower culture involves culturing excised floral buds to produce full blooms. Ovule culture involves culturing isolated ovules to facilitate fertilization and embryo development. Organ cultures have various applications including studying organ development, production of secondary metabolites, and generating virus-free plants.
Cryopreservation is a process for long-term storage of biological material such as germplasm at ultra-low temperatures, typically using liquid nitrogen at -196°C. This preserves cells and tissues by stopping all biological activity. The document discusses the various steps involved, including selection of plant material, addition of cryoprotectants, controlled freezing and thawing processes, and techniques for determining viability after storage and thawing. Cryopreservation is important for long-term conservation of plant genetic resources.
WHAT IS ARTIFICIAL SEED..?
Artificial seed can be defined as artificial encapsulation of somatic embryos, shoot bud or aggregates of cell of any tissues which has the ability to form a plant in in-vitro or ex-vivo condition.
Artificial seed have also been often referred to as synthetic seed.
HISTORY
Artificial seeds were first introduced in 1970’s as a novel analogue to the plant seeds.
The production of artificial seeds is useful for plants which do not produce viable seeds. It represents a method to propagate these plants.
Artificial seeds are small sized and these provides further advantages in storage, handling and shipping.
The term, “EMBLING” is used for the plants originated from synthetic seed.
• The use of synthetic varieties for commercial cultivation was first suggested in Maize (Hays & Garber, 1919).
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 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.
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 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.
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.
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
The document discusses methods for protoplast culture, including liquid culture, agar culture, droplet culture, and co-culture. It also describes techniques for testing protoplast viability, such as FDA staining, Evan's blue staining, and tetrazolium reduction tests. Protoplasts can be regenerated into whole plants and are useful for studies on genetic engineering, ultrastructure, membrane properties, and isolating mutants.
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
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.
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
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 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 the process of protoplast isolation, culture, and fusion. It discusses how protoplasts are isolated from plant tissues through enzymatic digestion of cell walls. The viability of isolated protoplasts is then tested before they are cultured on nutrient media and induced to regenerate new cell walls. Protoplast fusion allows the creation of somatic hybrids between plant species for crop improvement applications. The overall technique provides tools for genetic engineering and plant biotechnology research.
1) Anther and pollen culture techniques involve culturing microspores excised from anthers or pollen grains to produce haploid plants. This allows for the efficient production of fully homozygous lines.
2) Factors like genotype, culture medium, and pretreatments influence anther culture success. Haploids must be doubled to be fertile and useful.
3) Somatic hybridization fuses protoplasts from different plant species using techniques like PEG or electrofusion. This can combine traits not otherwise possible. Selection and regeneration are required to produce hybrid plants.
PROTOPAST ISOATION, PROTOPAST FUSION AND SOMATIC HYBRIDISATION.pptxMrChuha
Protoplasts are plant cells that have had their cell walls removed, leaving the plasma membrane as the outer layer. They can be isolated from various plant tissues like leaves and cell suspension cultures through enzymatic digestion of the cell walls using enzymes like cellulases, pectinases, and hemicellulases. There are two main methods for isolating protoplasts - sequential and mixed enzymatic methods. In the sequential method, pectinase treatment is followed by cellulase treatment, while the mixed method uses a combination of enzymes simultaneously. After enzymatic treatment, protoplasts are collected, washed, and cultured on appropriate media to induce cell division and regeneration of plants.
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.
Discovery of An Apparent Red, High-Velocity Type Ia Supernova at 𝐳 = 2.9 wi...Sérgio Sacani
We present the JWST discovery of SN 2023adsy, a transient object located in a host galaxy JADES-GS
+
53.13485
−
27.82088
with a host spectroscopic redshift of
2.903
±
0.007
. The transient was identified in deep James Webb Space Telescope (JWST)/NIRCam imaging from the JWST Advanced Deep Extragalactic Survey (JADES) program. Photometric and spectroscopic followup with NIRCam and NIRSpec, respectively, confirm the redshift and yield UV-NIR light-curve, NIR color, and spectroscopic information all consistent with a Type Ia classification. Despite its classification as a likely SN Ia, SN 2023adsy is both fairly red (
�
(
�
−
�
)
∼
0.9
) despite a host galaxy with low-extinction and has a high Ca II velocity (
19
,
000
±
2
,
000
km/s) compared to the general population of SNe Ia. While these characteristics are consistent with some Ca-rich SNe Ia, particularly SN 2016hnk, SN 2023adsy is intrinsically brighter than the low-
�
Ca-rich population. Although such an object is too red for any low-
�
cosmological sample, we apply a fiducial standardization approach to SN 2023adsy and find that the SN 2023adsy luminosity distance measurement is in excellent agreement (
≲
1
�
) with
Λ
CDM. Therefore unlike low-
�
Ca-rich SNe Ia, SN 2023adsy is standardizable and gives no indication that SN Ia standardized luminosities change significantly with redshift. A larger sample of distant SNe Ia is required to determine if SN Ia population characteristics at high-
�
truly diverge from their low-
�
counterparts, and to confirm that standardized luminosities nevertheless remain constant with redshift.
Signatures of wave erosion in Titan’s coastsSérgio Sacani
The shorelines of Titan’s hydrocarbon seas trace flooded erosional landforms such as river valleys; however, it isunclear whether coastal erosion has subsequently altered these shorelines. Spacecraft observations and theo-retical models suggest that wind may cause waves to form on Titan’s seas, potentially driving coastal erosion,but the observational evidence of waves is indirect, and the processes affecting shoreline evolution on Titanremain unknown. No widely accepted framework exists for using shoreline morphology to quantitatively dis-cern coastal erosion mechanisms, even on Earth, where the dominant mechanisms are known. We combinelandscape evolution models with measurements of shoreline shape on Earth to characterize how differentcoastal erosion mechanisms affect shoreline morphology. Applying this framework to Titan, we find that theshorelines of Titan’s seas are most consistent with flooded landscapes that subsequently have been eroded bywaves, rather than a uniform erosional process or no coastal erosion, particularly if wave growth saturates atfetch lengths of tens of kilometers.
Candidate young stellar objects in the S-cluster: Kinematic analysis of a sub...Sérgio Sacani
Context. The observation of several L-band emission sources in the S cluster has led to a rich discussion of their nature. However, a definitive answer to the classification of the dusty objects requires an explanation for the detection of compact Doppler-shifted Brγ emission. The ionized hydrogen in combination with the observation of mid-infrared L-band continuum emission suggests that most of these sources are embedded in a dusty envelope. These embedded sources are part of the S-cluster, and their relationship to the S-stars is still under debate. To date, the question of the origin of these two populations has been vague, although all explanations favor migration processes for the individual cluster members. Aims. This work revisits the S-cluster and its dusty members orbiting the supermassive black hole SgrA* on bound Keplerian orbits from a kinematic perspective. The aim is to explore the Keplerian parameters for patterns that might imply a nonrandom distribution of the sample. Additionally, various analytical aspects are considered to address the nature of the dusty sources. Methods. Based on the photometric analysis, we estimated the individual H−K and K−L colors for the source sample and compared the results to known cluster members. The classification revealed a noticeable contrast between the S-stars and the dusty sources. To fit the flux-density distribution, we utilized the radiative transfer code HYPERION and implemented a young stellar object Class I model. We obtained the position angle from the Keplerian fit results; additionally, we analyzed the distribution of the inclinations and the longitudes of the ascending node. Results. The colors of the dusty sources suggest a stellar nature consistent with the spectral energy distribution in the near and midinfrared domains. Furthermore, the evaporation timescales of dusty and gaseous clumps in the vicinity of SgrA* are much shorter ( 2yr) than the epochs covered by the observations (≈15yr). In addition to the strong evidence for the stellar classification of the D-sources, we also find a clear disk-like pattern following the arrangements of S-stars proposed in the literature. Furthermore, we find a global intrinsic inclination for all dusty sources of 60 ± 20◦, implying a common formation process. Conclusions. The pattern of the dusty sources manifested in the distribution of the position angles, inclinations, and longitudes of the ascending node strongly suggests two different scenarios: the main-sequence stars and the dusty stellar S-cluster sources share a common formation history or migrated with a similar formation channel in the vicinity of SgrA*. Alternatively, the gravitational influence of SgrA* in combination with a massive perturber, such as a putative intermediate mass black hole in the IRS 13 cluster, forces the dusty objects and S-stars to follow a particular orbital arrangement. Key words. stars: black holes– stars: formation– Galaxy: center– galaxies: star formation
PPT on Direct Seeded Rice presented at the three-day 'Training and Validation Workshop on Modules of Climate Smart Agriculture (CSA) Technologies in South Asia' workshop on April 22, 2024.
Evidence of Jet Activity from the Secondary Black Hole in the OJ 287 Binary S...Sérgio Sacani
Wereport the study of a huge optical intraday flare on 2021 November 12 at 2 a.m. UT in the blazar OJ287. In the binary black hole model, it is associated with an impact of the secondary black hole on the accretion disk of the primary. Our multifrequency observing campaign was set up to search for such a signature of the impact based on a prediction made 8 yr earlier. The first I-band results of the flare have already been reported by Kishore et al. (2024). Here we combine these data with our monitoring in the R-band. There is a big change in the R–I spectral index by 1.0 ±0.1 between the normal background and the flare, suggesting a new component of radiation. The polarization variation during the rise of the flare suggests the same. The limits on the source size place it most reasonably in the jet of the secondary BH. We then ask why we have not seen this phenomenon before. We show that OJ287 was never before observed with sufficient sensitivity on the night when the flare should have happened according to the binary model. We also study the probability that this flare is just an oversized example of intraday variability using the Krakow data set of intense monitoring between 2015 and 2023. We find that the occurrence of a flare of this size and rapidity is unlikely. In machine-readable Tables 1 and 2, we give the full orbit-linked historical light curve of OJ287 as well as the dense monitoring sample of Krakow.
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.
3. Modules
Introduction
Historical development
Protoplast isolation & Methods
Protoplast purification & viability test
Protoplast fusion & Techniques
Protoplast culture
Culture methods
Regeneration of protoplasts
Applications
Advantages & Disadvantages
Summary
4. What is protoplast?
The protoplasm of a living plant or bacterial cell
whose cell wall has been removed.
Protoplasts are plant cells that have been stripped
of their cell walls through the action of pectinases
and cellulases.
In
case
of
Plants
6. PROTOPLAST ISOLATION
Protoplasts are isolated by two techniques
The essential step of protoplast isolation is the proper use
of Osmoticum.
Mechanical Method
Enzymatic Method
7. Sources of Explant for Protoplast Isolation
Protoplasts can be isolated directly from the
different parts of whole plant which bears the soft
parenchymatous tissue (e.g., young fully expanded
soft leaves) or indirectly from the in vitro grown
plant tissue (e.g., callus tissue).
Before isolation of protoplast, the source material
if it is from in vivo grown plant then it should be
properly surface sterilized using the proper method
of sterilization. Then any of the methods either
mechanical or enzymatic can be used to isolate the
protoplast
8. Mechanical Method
Any soft parenchymatous tissue is kept in a plasmolyticum.
The plasmolyzed tissue is then finely chopped into pieces and the intact
cells (plasmolyzed) are released into the medium from the cut surface.
The suspension is then allowed for deplasmolysis and the released
protoplasts attain their original size.
10. Limitations Of Mechanical method
Yield of protoplasts and their viability is low.
It is restricted to certain tissues with vacuolated cells.
The method is laborious and tedious
11. Enzymatic Method
Young fully expanded soft leaves, or in vitro grown callus tissue or
cell suspension culture grown cells can be used as the source
material.
The tissues or cells are incubated in plasmolyticum for 1 hr.
before enzymatic treatment.
The intact tissue materials cut into smaller pieces to increase the
surface area of enzymatic activity.
The enzymes can be used either sequentially in two step method
or in a single step by mixed enzymatic method.
13. Enzymes
The enzymes used are of three main categories:
Cellulase
Hemicellulase
Pectinase
The concentration of enzymes used and the time period of
incubation varies greatly depending on the tissue type
14. Advantages of enzymatic method
Used for variety of tissue and organs such as fruits, roots,
petioles and leaves
Osmotic shrinkage is minimum
Cells remain intact and not injured
Protoplast readily obtained
15. Purification of protoplasts
For purification, the protoplasts suspended in osmoticum
are centrifuged using sucrose (20%) solution.
The viable protoplasts float on the top surface of sucrose
solution forming a band.
These protoplasts are then collected, re-suspended in
osmoticum and washed several times.
counting the number with the help of hemocytometer
16. Protoplast Viability Test
1. Fluorescein
diacetate (FDA)
dissolved in
acetone is used
at a conc. of
0.01% and intact
viable protoplasts
only fluoresce
when observed
under UV.
2. Phenosafranine
is also used at a
conc. of 0.01 %,
which is specific
for dead
protoplast that
shows red in
color.
17. Protoplast fusion
Somatic fusion, also called
protoplast fusion, is a type
of genetic modification in
plants by which two distinct
species of plants are fused
together to form a new
hybrid plant with the
characteristics of both, a
somatic hybrid.
19. ELECTROFUSION
Mild electro stimulation
Two glass capillary
microelectrodes
An electric field of low strength
Leads to pearl chain
arrangement of protoplast
Application of high intensity
electric impulse for some
microseconds
Breakdown of membrane and
subsequent fusion
20. peg
This chemical has been
identified as a possible fusogen
Has a molecular weight of
about 1500-6000
Usually PEG solution of about
28-50% is used
This polymer binds to the lipid
membrane of the cell and thus
induces fusion
Fusion takes place for 45 min in
incubation
22. Protoplast culture
Isolated protoplast can be cultured in an appropriate medium to
reform cell wall and generate callus
Protoplasts are cultured either in
Agar medium
Liquid medium
23. 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.
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.
24. LIQUID CULTURE
Liquid culture is the preferred method for protoplast cultivation for
the following reasons:
It is easy to dilute and transfer.
Density of the cells can be manipulated as desired
For some plant species, the cells cannot divide in agar medium,
therefore liquid medium is the only choice.
Osmotic pressure of liquid medium can be altered as desired.
25. Protoplast culture
Protoplast cultured in suitable nutrient media first generate a new
cell wall
The formation of a complete cell with a wall is followed by an
increase in size, number of cell organelles and incubation of the
cell division
The first cell division may occur within 2 to 7 days of culture
Resulting in small clumps of cells, also known as micro colony
with in 1 to 3 weeks
From such clumps, there are 2 ways to generate a complete plant
Through organogenesis
Osmatic embryo converted into whole plant through germination
26. Culture Methods
Micro drop culture
Co-culture of protoplasts
Feeder layer technique
The culture techniques of
protoplasts are almost the
same that are used for cell
culture with suitable
modifications.
27. 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.
28. 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.
29. 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.
30. Regeneration of Protoplasts
Protoplast regeneration which may also be regarded as protoplast
development occurs in two stages:
1. 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.
31. Regeneration of Protoplasts
2. 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.
32. Applications
STUDY OF OSMOTIC BEHAVIOUR
STUDY OF IAA action
Study of cell wall formation
Organelle isolation
Study of morphogenesis
Virus uptake and replication
Gene transfer
Induction of Mutation and Genetic Variability
Implantation of Chloroplast
Transplantation of Nuclei
Transplantation of Chromosome
Somatic Hybridization
33. Study of Osmotic Behavior
Influence of different
environmental factors on
the osmotic behavior can
be studied using plant
protoplasts.
34. Study of IAA Action
When growth promoters like IAA are
applied to plants, they act directly
on plasma membrane of the cell and
increase the permeability of the
membrane to water resulting in cell
elongation. This can be established
by the use of protoplast in vitro.
When IAA is applied to the
plasmolyticum containing
protoplasts they expand rapidly and
finally burst due to too much
vacuolation. Further, it can be
verified by using anti-auxins that
suppress this bursting, indicating
that the site of action of IAA is the
plasma-lemma of the plant cell.
35. Study of Cell Wall Formation
The early deposition of
cellulosic micro-fibril and
their orientation at the
protoplast surface can be
followed using both light and
electron microscope and
has also provided much
basic information
concerning cell wall biology.
36. Organelle Isolation
Protoplasts are very convenient material for the isolation of
chloroplasts, mitochondria, nuclei and even chromosomes. It has
been demonstrated that chloroplasts particularly isolated from
cereal protoplast have higher capacity for CO2 fixation than those
obtained by mechanical grinding.
37. Study of Morphogenesis
Isolated protoplast provides an ideal single cell system. Under
suitable condition, protoplast regenerates its own wall and
become the walled cells. Cell division followed by plant
regeneration may occur from such unique single cell system either
through organogenesis or embryogenesis.
38. Virus Uptake and Replication
But after the innovation of protoplast isolation and its culture, this problem is
almost solved. Protoplast can directly be inoculated with pathogenic virus in
the medium. The process of uptake of virus particle, their replication inside
the protoplasts and their mode of action at the molecular and cellular level
are made possible by the aid of protoplasts.
The plant virus interrelationships in the past were not clearly known due to
lack of suitable experimental systems that can easily infect the cells.
39. Isolation of Bacteroides from Root Nodule Protoplast:
Viable Bacteroides from root nodules of legumes has been
isolated by first preparing nodule protoplast and then rupturing
them either mechanically or by lowering suddenly the concen-
tration of the plasmolyticum in the surrounding medium. This
method ensures the freedom of the preparation of bacteria from
the infection thread.
40. Induction of Mutation and Genetic Variability:
It has been repeatedly observed that plant cell in culture show a
wide range of genetic diversity. This phenomena can be exploited
by plant breeders and geneticists for inducing variability in
protoplast culture. The recessive characters can be detected in
the regenerated plants derived from haploid protoplasts.
Therefore, haploid protoplast would make an ideal system for
studying the effect of irradiation and for the induction of mutation
by plating them in media supplemented with various chemical
mutagens.
From this method, mutant line can be selected.
41. Implantation of Chloroplast
Plant protoplasts have ability to
uptake the isolated chloroplasts
by the process of endocytosis.
Several reports have described
uptake of chloroplasts.
Chloroplasts isolated from
Vaucheria dichotoma were
implanted into carrot cell
culture protoplasts. The
chloroplasts may enter the
cytoplasm enclosed in
membrane-bound vesicles,
although the enclosing
membrane in some cases is
absent
42. Transplantation of Nuclei:
Isolated nuclei can be introduced into the protoplasts. Both intra
and inter-specific nuclear transplantation have been observed in
Petunia hybrida, Nicotiana tabacum and Zea mays. Retention,
normal function or degradation of the incorporated nuclei is not
known. But it is really opening up new avenues for the study of
nuclear- cytoplasmic interaction if fertile plants with foreign
nuclei could be regenerated from such protoplasts.
43. Transplantation of Chromosome
The uptake of isolated metaphase chromosomes has proven
successful in plant protoplast. This procedure provides a valuable
method for genetic information transfer and gene analysis
44. Somatic Hybridization:
Fusion of protoplast that facilitates the mixing of 2 whole
genomes and could be exploited in crosses at
Intergenic,intekingdom and interspecific level
Somatic hybridization is used to produce hybrids from sexually
incompatible species
This method could also be used to produce selection procedures
46. limitations
Intergenic process between widely related plants which are not
compatible sexually are not possible
In certain wide crosses , elimination of chromosomes from hybrid
cell is another limitation of somatic hybridization
In protoplast fusion experiments ,the percentage of fusion
between two different parental protoplast is very low
For hybrid identification, selection and isolation at the culture
level ,there is no standardized method which is applicable for all
materials
47. Advantages
It facilitates the mixing of two genomes and can be used in
crosses at interspecific intraspecific or even intergenic
level
To create new strains with desired properties and for strain
improvement
Mixing two genomes open the door to gene transfer and a
study of gene expression , stability of several traits and cell
genetic changes
48. summary
In a nutshell, protoplast culture has found its many
uses and many applications in fields such as
genetic engineering and crop breeding. genetic
transformation by introduction of transgene DNA
somatic hybridization by protoplast fusion of
species or subspecies resistant to traditional cross
breeding and isolation of sub cellular organelles
are the examples of how the development of
protoplast system has led to an increase in the
versatility of plants.