Plant tissues and organs can be cryopreserved in liquid nitrogen at -196°C for long-term storage. This technique is useful for conserving germplasm of crops that do not produce seeds, like root and tuber crops. Cryopreservation involves culturing tissues in cryoprotectants like DMSO and sugars before freezing to increase freezing tolerance. Successful cryopreservation protocols have been developed for many plant cells, tissues, organs and other structures using techniques like slow cooling, rapid cooling, vitrification and encapsulation-dehydration. However, an optimal protocol applicable to all plant species has not been determined. The document provides detailed information on cryopreservation techniques and factors affecting successful recovery of cryopreserved plant materials
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.
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.
Cryopreservation is a process where biological materials like cells, tissues, and organs are preserved at very low temperatures, typically in liquid nitrogen at -196°C. This process stops all metabolic activities and allows long-term preservation. The key steps involve selection of suitable plant material, addition of cryoprotectants to prevent ice crystal formation, slow freezing or vitrification to solidify water in an amorphous glassy state without crystallization, storage in liquid nitrogen, and thawing for regeneration of plants. Cryopreservation has many applications in conservation of genetic resources, maintenance of disease-free stock, and long-term storage of cell cultures and germplasm in seed banks and gene banks.
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.
Introduction
Reason for cryopreservation
Selection of part of plant for cryopreservation
Technique of cryopreservation
Application
Limitation
Conclusion
In vitro pollination involves pollinating pistils or ovules that have been cultured in a nutrient medium such as Nitsch's medium. This technique can help overcome pre-fertilization barriers to hybridization between plant species. Key steps include sterilizing flower parts, collecting pollen, and applying pollen to excised pistils, ovaries, ovules, or stigmas depending on the method. Factors like culture medium, temperature, genotype, and physiological state of the explant can influence seed set. In vitro pollination has applications in plant breeding like overcoming self-incompatibility or cross-incompatibility barriers and producing haploid plants or hybrids.
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.
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.
Cryopreservation is a process where biological materials like cells, tissues, and organs are preserved at very low temperatures, typically in liquid nitrogen at -196°C. This process stops all metabolic activities and allows long-term preservation. The key steps involve selection of suitable plant material, addition of cryoprotectants to prevent ice crystal formation, slow freezing or vitrification to solidify water in an amorphous glassy state without crystallization, storage in liquid nitrogen, and thawing for regeneration of plants. Cryopreservation has many applications in conservation of genetic resources, maintenance of disease-free stock, and long-term storage of cell cultures and germplasm in seed banks and gene banks.
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.
Introduction
Reason for cryopreservation
Selection of part of plant for cryopreservation
Technique of cryopreservation
Application
Limitation
Conclusion
In vitro pollination involves pollinating pistils or ovules that have been cultured in a nutrient medium such as Nitsch's medium. This technique can help overcome pre-fertilization barriers to hybridization between plant species. Key steps include sterilizing flower parts, collecting pollen, and applying pollen to excised pistils, ovaries, ovules, or stigmas depending on the method. Factors like culture medium, temperature, genotype, and physiological state of the explant can influence seed set. In vitro pollination has applications in plant breeding like overcoming self-incompatibility or cross-incompatibility barriers and producing haploid plants or hybrids.
Callus is an unorganized mass of undifferentiated cells that can be cultured in vitro. It is produced when plant explants are cultured on medium containing auxin and cytokinin hormones under sterile conditions. Callus tissue lacks differentiation and is unable to perform photosynthesis. It can be maintained indefinitely and used for plant regeneration through processes like organogenesis and somatic embryogenesis. Successful callus culture requires aseptic preparation of explants, a nutrient medium with proper hormone balance, and controlled physical conditions for incubation.
This document discusses germplasm and its conservation. It begins by defining germplasm as a collection of genetic resources for an organism, such as a seed bank or gene bank, that contains the genetic information for a species. Germplasm conservation is important to preserve genetic diversity and provide plant breeders resources to develop new crop varieties. Methods of conservation include in situ conservation of plants in their natural habitat and ex situ conservation of seeds, tissues, cells or DNA stored outside the natural habitat. Cryopreservation in liquid nitrogen at -196°C is an effective long-term storage method that stops cellular metabolism. The document outlines the cryopreservation process and applications for conserving plant species and genetic variations.
The document provides an introduction to artificial seeds, including definitions and key concepts. It discusses the two main types of artificial seeds - desiccated and hydrated synthetic seeds. The production process involves establishing somatic embryogenesis, encapsulating somatic embryos or shoot buds, and planting the artificial seeds. Alginate is commonly used as the encapsulating material. Additives can be included to the matrix to serve as an artificial endosperm. The document outlines the potential uses and benefits of artificial seeds for propagation, germplasm preservation, and genetic engineering applications.
Genetic material of plants which is of value as a resource for present and future generations of people is referred to as plant genetic resources.
The whole library of different alleles of a species or sum total of genes in a species is known as gene pool, also called germplasm, genetic stock and genetic resources.
The term gene pool was coined by Dobzhansky in 1951.
The term germplasm was first used by Weismann in 1883.
Morphogenesis, organogenesis, embryogenesis & other techniquesHORTIPEDIA INDIA
The document describes the process of somatic embryogenesis. It involves 7 key steps:
1) Induction of embryogenesis from explant tissue on media supplemented with auxin
2) Development of somatic embryos through globular, heart, and torpedo stages of growth
3) Maturation of embryos with the formation of root and shoot meristems and cotyledons
4) Conversion of mature embryos to plantlets through germination on auxin-free media
Factors like explant type, growth regulators, and genotype influence the process. Somatic embryos differ from zygotic embryos in lacking a seed coat and having greater potential for propagation but weaker plantlets.
Embryo rescue, Somaclonal Variation, CryopreservationAbhinava J V
This document discusses various techniques in plant biotechnology including embryo rescue, somaclonal variation, and cryopreservation. Embryo rescue involves culturing immature or weak embryos on artificial nutrient media to allow their development. Somaclonal variation refers to genetic and phenotypic changes that can occur in plants regenerated from tissue culture. Cryopreservation aims to preserve plant cells and tissues in a frozen state at ultra-low temperatures like liquid nitrogen. The key steps involve adding cryoprotectants, freezing, storage, thawing, and regeneration of plants. These techniques have various applications for breeding programs and conservation of plant genetic resources.
Micropropagation, also known as tissue culture, is a method of rapidly multiplying plant materials using aseptic laboratory techniques to produce many clonal progeny. Key aspects of micropropagation include taking explants from stock plants and culturing them on nutrient media, proliferating shoots in a multiplication stage, and rooting the shoots to produce clonal plantlets. This allows for mass production of genetically identical plant materials year-round while eliminating diseases.
The document discusses symmetric and asymmetric somatic hybrids and cybrids in plant tissue culture. It defines symmetric hybrids as retaining chromosomes from both parents and asymmetric hybrids as retaining chromosomes from only one parent. Recent examples of cybrid production are provided, including transferring cytoplasmic male sterility from Satsuma mandarin to seedy citrus cultivars and introducing transformed tobacco chloroplasts into petunia. Cybridization allows for combining plant species that cannot reproduce sexually by fusing protoplasts such that the nucleus of one species is combined with the cytoplasm of another.
Micropropagation is the process of rapidly multiplying stock plant materials using modern tissue culture methods. It involves 5 main stages: selection of stock plants, initiation and establishment of culture, multiplication of shoots, rooting of shoots, and establishment of plantlets. The two main approaches are multiplication through axillary buds/apical shoots and adventitious shoots. Organogenesis and somatic embryogenesis are also used, where organs or embryos are formed directly or indirectly from explants. Micropropagation is useful for cloning noble plants and providing sufficient plantlets from stock plants that do not produce many seeds or vegetatively propagate well.
Somaclonal Variation in Plant tissue culture - Variation in somaclones (somatic cells of plants)
Somaclonal variation # Basis of somaclonal variation # General feature of Somaclonal variations # Types and causes of somaclonal variation # Isolation procedure of somaclones via without in-vitro method and with in-vitro method with their limitations and advantages # Detection of isolated somaclonal variation # Application (with examples respectively related to crop improvement) # Advantages and disadvantages of somaclonal variations.
Also watch, Gametoclonal variation slides to understand, how to changes occur in gametoclones of plants.
https://www.slideshare.net/SharmasClasses/gametoclonal-variation
In-Vitro Pollination and Fertilization
The document discusses in-vitro pollination and fertilization techniques. It begins with a brief history, noting its development in 1902 and use to produce hybrids between incompatible species. It then describes barriers to pollination and fertilization that can be overcome through in-vitro methods. Several techniques are outlined, including ovule, ovary, and stigma cultures. Requirements for successful in-vitro fertilization include viable gametes and proper culture conditions. The document concludes by discussing applications in plant breeding like overcoming self-incompatibility and producing stress-tolerant hybrids.
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.
Cryopreservation in plant culture and techniques - introduction, definition , steps of cells cryopreservation, methods of cryopreservation, Techniques of cryopreservation , application, advantage and disadvantage, diagram of cryopreservation.
Cryopreservation involves storing biological material at ultra-low temperatures, usually in liquid nitrogen. This allows long-term preservation by stopping almost all metabolic activity in cells. Materials are frozen using slow freezing, rapid freezing, or stepwise freezing methods. They are then stored long-term at temperatures near -196°C. When needed, samples are thawed quickly in a warm water bath before use or analysis. Cryopreservation has many applications for preserving cells, tissues, blood, embryos and more.
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
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.
This document provides information on in vitro germplasm conservation. It discusses that germplasm conservation aims to preserve the genetic diversity of plants. There are several methods of in vitro conservation including cryopreservation, cold storage, and low pressure/low oxygen storage. Cryopreservation involves freezing plant cells and tissues at ultra-low temperatures like in liquid nitrogen to bring their metabolism to zero. It allows for long term conservation of large amounts of genetic material in a small space. Cold storage conserves germplasm at low non-freezing temperatures to slow growth. Low pressure and low oxygen storage reduce atmospheric pressure and oxygen concentration to inhibit plant tissue growth.
Conservation and preservation of germplasmIñnøcènt ÅñDi
The document discusses germplasm conservation, including both ex situ and in situ methods. Ex situ conservation involves maintaining genetic resources outside their natural habitat, such as in seed banks, field gene banks, DNA banks, botanical gardens, and through in vitro and cryopreservation methods. In situ conservation preserves species in their natural environments through biosphere reserves, national parks, wildlife sanctuaries, and on-farm conservation. Cryopreservation is described as a method to bring plant cells and tissues to a zero metabolism state through freezing at very low temperatures in liquid nitrogen.
The document discusses the objectives, purpose, principles, and stages of orthodox seed storage. The key points are:
1) The objectives of seed storage are to maintain initial seed quality like germination and vigor throughout storage by providing suitable storage conditions like low moisture and temperature.
2) The purpose of seed storage is to preserve the high germination and vigor of seeds from harvest until planting to ensure adequate plant stands and healthy, vigorous plants.
3) Principles of seed storage include maintaining cool, dry conditions as orthodox seeds can be dried to low moisture levels which increases their longevity, with lower moisture and temperatures extending seed life.
4) Seed storage involves different stages from physiological maturity on the plant
Cryopreservation allows for the long-term storage of biological materials like plant germplasm by storing them at ultra-low temperatures, typically in liquid nitrogen at -196°C. This stops all metabolic activities and allows preservation. The key steps are selection of plant material, addition of cryoprotectants to prevent freezing damage, controlled freezing typically via slow or stepwise freezing, long-term storage in liquid nitrogen, and thawing for viability testing and regeneration of plants. Cryopreservation is important for preserving genetic resources and makes them available for future use in plant breeding.
This document summarizes procedures for cryopreserving and reconstituting preserved cell lines. It discusses that cryopreservation allows indefinite storage of biological material at -196°C. Common cryoprotectants like DMSO and glycerol are added to cell suspensions to protect cells from ice crystal formation during freezing and thawing. The document provides protocols for freezing suspension and adherent cell cultures slowly at 1°C/minute then storing in liquid nitrogen. It also outlines two methods for rapidly thawing cells involving either direct plating or centrifugation to remove cryoprotectants before culturing.
Callus is an unorganized mass of undifferentiated cells that can be cultured in vitro. It is produced when plant explants are cultured on medium containing auxin and cytokinin hormones under sterile conditions. Callus tissue lacks differentiation and is unable to perform photosynthesis. It can be maintained indefinitely and used for plant regeneration through processes like organogenesis and somatic embryogenesis. Successful callus culture requires aseptic preparation of explants, a nutrient medium with proper hormone balance, and controlled physical conditions for incubation.
This document discusses germplasm and its conservation. It begins by defining germplasm as a collection of genetic resources for an organism, such as a seed bank or gene bank, that contains the genetic information for a species. Germplasm conservation is important to preserve genetic diversity and provide plant breeders resources to develop new crop varieties. Methods of conservation include in situ conservation of plants in their natural habitat and ex situ conservation of seeds, tissues, cells or DNA stored outside the natural habitat. Cryopreservation in liquid nitrogen at -196°C is an effective long-term storage method that stops cellular metabolism. The document outlines the cryopreservation process and applications for conserving plant species and genetic variations.
The document provides an introduction to artificial seeds, including definitions and key concepts. It discusses the two main types of artificial seeds - desiccated and hydrated synthetic seeds. The production process involves establishing somatic embryogenesis, encapsulating somatic embryos or shoot buds, and planting the artificial seeds. Alginate is commonly used as the encapsulating material. Additives can be included to the matrix to serve as an artificial endosperm. The document outlines the potential uses and benefits of artificial seeds for propagation, germplasm preservation, and genetic engineering applications.
Genetic material of plants which is of value as a resource for present and future generations of people is referred to as plant genetic resources.
The whole library of different alleles of a species or sum total of genes in a species is known as gene pool, also called germplasm, genetic stock and genetic resources.
The term gene pool was coined by Dobzhansky in 1951.
The term germplasm was first used by Weismann in 1883.
Morphogenesis, organogenesis, embryogenesis & other techniquesHORTIPEDIA INDIA
The document describes the process of somatic embryogenesis. It involves 7 key steps:
1) Induction of embryogenesis from explant tissue on media supplemented with auxin
2) Development of somatic embryos through globular, heart, and torpedo stages of growth
3) Maturation of embryos with the formation of root and shoot meristems and cotyledons
4) Conversion of mature embryos to plantlets through germination on auxin-free media
Factors like explant type, growth regulators, and genotype influence the process. Somatic embryos differ from zygotic embryos in lacking a seed coat and having greater potential for propagation but weaker plantlets.
Embryo rescue, Somaclonal Variation, CryopreservationAbhinava J V
This document discusses various techniques in plant biotechnology including embryo rescue, somaclonal variation, and cryopreservation. Embryo rescue involves culturing immature or weak embryos on artificial nutrient media to allow their development. Somaclonal variation refers to genetic and phenotypic changes that can occur in plants regenerated from tissue culture. Cryopreservation aims to preserve plant cells and tissues in a frozen state at ultra-low temperatures like liquid nitrogen. The key steps involve adding cryoprotectants, freezing, storage, thawing, and regeneration of plants. These techniques have various applications for breeding programs and conservation of plant genetic resources.
Micropropagation, also known as tissue culture, is a method of rapidly multiplying plant materials using aseptic laboratory techniques to produce many clonal progeny. Key aspects of micropropagation include taking explants from stock plants and culturing them on nutrient media, proliferating shoots in a multiplication stage, and rooting the shoots to produce clonal plantlets. This allows for mass production of genetically identical plant materials year-round while eliminating diseases.
The document discusses symmetric and asymmetric somatic hybrids and cybrids in plant tissue culture. It defines symmetric hybrids as retaining chromosomes from both parents and asymmetric hybrids as retaining chromosomes from only one parent. Recent examples of cybrid production are provided, including transferring cytoplasmic male sterility from Satsuma mandarin to seedy citrus cultivars and introducing transformed tobacco chloroplasts into petunia. Cybridization allows for combining plant species that cannot reproduce sexually by fusing protoplasts such that the nucleus of one species is combined with the cytoplasm of another.
Micropropagation is the process of rapidly multiplying stock plant materials using modern tissue culture methods. It involves 5 main stages: selection of stock plants, initiation and establishment of culture, multiplication of shoots, rooting of shoots, and establishment of plantlets. The two main approaches are multiplication through axillary buds/apical shoots and adventitious shoots. Organogenesis and somatic embryogenesis are also used, where organs or embryos are formed directly or indirectly from explants. Micropropagation is useful for cloning noble plants and providing sufficient plantlets from stock plants that do not produce many seeds or vegetatively propagate well.
Somaclonal Variation in Plant tissue culture - Variation in somaclones (somatic cells of plants)
Somaclonal variation # Basis of somaclonal variation # General feature of Somaclonal variations # Types and causes of somaclonal variation # Isolation procedure of somaclones via without in-vitro method and with in-vitro method with their limitations and advantages # Detection of isolated somaclonal variation # Application (with examples respectively related to crop improvement) # Advantages and disadvantages of somaclonal variations.
Also watch, Gametoclonal variation slides to understand, how to changes occur in gametoclones of plants.
https://www.slideshare.net/SharmasClasses/gametoclonal-variation
In-Vitro Pollination and Fertilization
The document discusses in-vitro pollination and fertilization techniques. It begins with a brief history, noting its development in 1902 and use to produce hybrids between incompatible species. It then describes barriers to pollination and fertilization that can be overcome through in-vitro methods. Several techniques are outlined, including ovule, ovary, and stigma cultures. Requirements for successful in-vitro fertilization include viable gametes and proper culture conditions. The document concludes by discussing applications in plant breeding like overcoming self-incompatibility and producing stress-tolerant hybrids.
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.
Cryopreservation in plant culture and techniques - introduction, definition , steps of cells cryopreservation, methods of cryopreservation, Techniques of cryopreservation , application, advantage and disadvantage, diagram of cryopreservation.
Cryopreservation involves storing biological material at ultra-low temperatures, usually in liquid nitrogen. This allows long-term preservation by stopping almost all metabolic activity in cells. Materials are frozen using slow freezing, rapid freezing, or stepwise freezing methods. They are then stored long-term at temperatures near -196°C. When needed, samples are thawed quickly in a warm water bath before use or analysis. Cryopreservation has many applications for preserving cells, tissues, blood, embryos and more.
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
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.
This document provides information on in vitro germplasm conservation. It discusses that germplasm conservation aims to preserve the genetic diversity of plants. There are several methods of in vitro conservation including cryopreservation, cold storage, and low pressure/low oxygen storage. Cryopreservation involves freezing plant cells and tissues at ultra-low temperatures like in liquid nitrogen to bring their metabolism to zero. It allows for long term conservation of large amounts of genetic material in a small space. Cold storage conserves germplasm at low non-freezing temperatures to slow growth. Low pressure and low oxygen storage reduce atmospheric pressure and oxygen concentration to inhibit plant tissue growth.
Conservation and preservation of germplasmIñnøcènt ÅñDi
The document discusses germplasm conservation, including both ex situ and in situ methods. Ex situ conservation involves maintaining genetic resources outside their natural habitat, such as in seed banks, field gene banks, DNA banks, botanical gardens, and through in vitro and cryopreservation methods. In situ conservation preserves species in their natural environments through biosphere reserves, national parks, wildlife sanctuaries, and on-farm conservation. Cryopreservation is described as a method to bring plant cells and tissues to a zero metabolism state through freezing at very low temperatures in liquid nitrogen.
The document discusses the objectives, purpose, principles, and stages of orthodox seed storage. The key points are:
1) The objectives of seed storage are to maintain initial seed quality like germination and vigor throughout storage by providing suitable storage conditions like low moisture and temperature.
2) The purpose of seed storage is to preserve the high germination and vigor of seeds from harvest until planting to ensure adequate plant stands and healthy, vigorous plants.
3) Principles of seed storage include maintaining cool, dry conditions as orthodox seeds can be dried to low moisture levels which increases their longevity, with lower moisture and temperatures extending seed life.
4) Seed storage involves different stages from physiological maturity on the plant
Cryopreservation allows for the long-term storage of biological materials like plant germplasm by storing them at ultra-low temperatures, typically in liquid nitrogen at -196°C. This stops all metabolic activities and allows preservation. The key steps are selection of plant material, addition of cryoprotectants to prevent freezing damage, controlled freezing typically via slow or stepwise freezing, long-term storage in liquid nitrogen, and thawing for viability testing and regeneration of plants. Cryopreservation is important for preserving genetic resources and makes them available for future use in plant breeding.
This document summarizes procedures for cryopreserving and reconstituting preserved cell lines. It discusses that cryopreservation allows indefinite storage of biological material at -196°C. Common cryoprotectants like DMSO and glycerol are added to cell suspensions to protect cells from ice crystal formation during freezing and thawing. The document provides protocols for freezing suspension and adherent cell cultures slowly at 1°C/minute then storing in liquid nitrogen. It also outlines two methods for rapidly thawing cells involving either direct plating or centrifugation to remove cryoprotectants before culturing.
Cryopreservation is the technique of freezing cells and tissues at very low sub-zero temperatures to preserve them. This stops biological activity and keeps materials genetically stable. Cryopreservation relies on cryoprotectants, which protect cells from freezing damage by penetrating cells and replacing water. The main cryopreservation procedures are slow freezing and vitrification. Slow freezing uses gradual cooling and cryoprotectants allow water to leave cells, while vitrification rapidly freezes cells to glass transition. Cryopreservation has applications in fertility preservation, assisted reproduction research, and biodiversity conservation.
Cryopreservation is a method of preserving living cells and tissues by cooling them to very low sub-zero temperatures, usually using liquid nitrogen at -196°C. This stops all biological activity, preventing cell death. The cells can survive freezing and thawing if the process is carefully controlled to prevent ice crystal formation inside cells, which can damage membranes. Cryopreservation involves harvesting samples, adding cryoprotectants like glycerol to reduce freezing damage, slowly freezing samples, storing in liquid nitrogen, and slowly thawing them to revive cells. It allows long-term storage of biological materials like cells, tissues, embryos and organs at ultra-low temperatures.
This document provides information on cryopreservation and reconstitution of preserved cell lines. It discusses that cryopreservation involves storing live material at ultra-low temperatures to suspend biological processes indefinitely. This allows long-term storage of cells without deterioration. The document then describes techniques for cryopreserving cell lines, benefits of freezing cells, types of cryoprotectants used, and mechanisms of cryoprotectant action. It provides protocols for freezing and thawing both suspension and adherent cell cultures, emphasizing the importance of controlled cooling and rapid thawing. The document concludes by outlining suggested procedures for thawing cryopreserved cells directly or after centrifugation to remove cryoprotectants.
This document summarizes procedures for cryopreserving and reconstituting preserved cell lines. It describes cryopreservation as storing live material at ultra-low temperatures to suspend biological processes. Cryopreservation allows indefinite storage of cells without deterioration. The document outlines techniques for cryopreserving cell lines using cryoprotectants like DMSO to prevent ice crystal formation during freezing and thawing. It provides protocols for freezing and thawing suspension and adherent cell cultures, emphasizing the importance of controlled cooling and rapid thawing to minimize cell damage.
Cryopreservation is the process of preserving living cells and tissues by cooling them to very low sub-zero temperatures. This summary discusses the history, methods, applications and case studies of cryopreservation:
1. Cryopreservation has been used since the mid-20th century to conserve genetic resources like plant seeds, cells, and tissues through freezing and storage in liquid nitrogen.
2. Key methods include addition of cryoprotectants, slow freezing techniques, vitrification, desiccation, and storage in liquid nitrogen.
3. Cryopreservation is used in seed banks, gene banks, and for research applications like breeding disease-resistant crops and conserving endangered plant species.
4. Case
Cryopreservation is the process of preserving biological materials such as cells, tissues, organs, embryos, and sperm at very low temperatures. It allows for long-term storage of biological samples by suspending their metabolic activities. Samples are typically stored in liquid nitrogen at -196°C. Cryopreservation aims to cool samples without the formation of ice crystals that can damage cells. Cryoprotectants are used to protect cells from freezing damage. Common cryopreservation methods include storage at -196°C, above -196°C, freeze drying, and vitrification. Cryopreservation finds applications in fertility treatments and preservation of genetic materials.
Cryopreservation Prepared by Md. Ali HaidarAli Haidar
I am Md. Ali Haidar student at faculty of Agriculture, EXIM Bank Agricultural University Bangladesh. I am a future Agriculturist. I published my Presentation for helping other student.
Cryopreservation involves the viable freezing of biological material at ultra-low temperatures (-150 to -196°C) in liquid nitrogen for long-term storage. It represents a safe and cost-effective method for conserving germplasm. The key principles are removing water from tissues using cryoprotectants to prevent ice crystal formation during freezing and storage. Common tissues preserved include seeds, embryos, and cell cultures. Liquid nitrogen is widely used as the storage medium due to its inert and inexpensive properties. Conventional and newer methods like vitrification and encapsulation-dehydration aim to protect cells from freezing damage. Cryopreservation has many applications for genetic conservation of endangered species and disease-free stocks.
Cryopreservation is a technique used to preserve biological materials such as cells, tissues, organs and embryos at ultra-low temperatures using liquid nitrogen. The first successful cryopreservation was of chicken sperm in the 1950s. Cryopreservation is important for conserving genetic resources as it allows long-term storage of plant and animal species. The key steps involve collecting and preparing the biological material, adding cryoprotectants to prevent ice crystal formation, freezing the material at controlled rates, storing it in liquid nitrogen, and thawing it for use. Cryopreservation has many applications including conserving endangered species and cells, blood banking, stem cell storage, and assisted reproduction technologies.
Cryopreservation is a process that preserves biological material such as cells, tissues, organs, and embryos at very low temperatures. It allows for long-term storage. Key aspects covered in the document include:
- A brief history of cryopreservation including early pioneers and discoveries.
- Cryoprotectants like glycerol and DMSO are used to prevent ice crystal formation and reduce cell damage during freezing and thawing.
- Different cryopreservation techniques exist like slow freezing, rapid freezing, and stepwise freezing which control ice formation.
- Cryopreserved materials can be stored long-term in liquid nitrogen at -196°C or other cryogenic temperatures where biological activity is effectively stopped
Germplasm refers to the genetic material of an organism. This document outlines methods for conserving plant germplasm, specifically cryopreservation which involves freezing plant tissues in liquid nitrogen. The key steps in cryopreservation include selecting suitable plant material, pre-freezing treatments using techniques like preculture or desiccation, freezing the material, storing it in liquid nitrogen, thawing it, and assessing viability. Cryopreservation allows for long-term storage of plant genetic resources and clonal propagation of plant varieties.
Low temperatures are used to preserve food by slowing microbial growth and chemical reactions. There are several methods of cold storage including common storage below 15°C, chilling storage just above freezing, and freezing storage which prevents microbial growth entirely. Freezing involves either quick freezing under -18°C within 30 minutes to form small ice crystals, or slow freezing over longer periods to form larger crystals. During freezing, ice crystals form which can damage cells, while chemical and enzymatic reactions are slowed. Frozen storage further slows these processes but can cause quality changes over long periods.
The document discusses various methods for preserving microorganisms. Short term methods include periodic transfer to fresh medium, storage in saline suspension, and refrigeration. Long term methods involve storage under mineral oil, lyophilization (freeze drying), cryopreservation in liquid nitrogen, and storage in sterile soil or silica gel. Lyophilization works by freezing and then reducing moisture content through sublimation and desorption. It allows storage at room temperature for many years but can damage some microbes. Cryopreservation in liquid nitrogen at -196°C also enables long term storage of over 10-30 years without genetic change.
This document provides an overview of cryopreservation, which involves preserving biological material such as cells, tissues, organs, and embryos at ultra-low temperatures, typically in liquid nitrogen. It discusses the history, principles, mechanisms, and applications of cryopreservation. Key aspects covered include the use of cryoprotectants to prevent freezing damage to cells, various freezing and thawing methods, long-term storage in liquid nitrogen, and viability testing after thawing to regenerate plants or animals from preserved material. Cryopreservation has important applications in biobanking, conservation of endangered species, and preservation of disease-free agricultural crops.
Cryopreservation is the process of preserving living cells and tissues by cooling them to very low sub-zero temperatures. This stops all biological and chemical processes, halting the living material in a state of suspended animation. There are several key steps in cryopreservation including preculturing materials, adding cryoprotectants, slow or stepwise freezing, storage in liquid nitrogen at -196°C, rapid thawing, and then reculturing. Common cryopreservation methods include slow freezing, vitrification, encapsulation-dehydration, and cryopreservation has many applications for preserving genetic resources like semen, embryos, oocytes, and more.
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.
This presentation discusses cryopreservation of gametes. Cryopreservation is a process that uses very low temperatures, typically with liquid nitrogen at -196°C, to preserve living cells and tissues. Cryoprotective agents are used to protect cells from freezing damage. Techniques discussed include slow freezing, rapid freezing and vitrification. Applications include sperm banking, embryo freezing and ovarian tissue cryopreservation. Both benefits and limitations of cryopreservation are mentioned such as the ability to preserve biological materials long-term but also the risk of cell damage from ice formation or toxic effects of cryoprotectants.
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Speaker: Diego Blas (IFAE/ICREA)
Title: Gravitational wave detection with orbital motion of Moon and artificial
Abstract:
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1. Cryopreservation of PlantTissues and Organs
Plant tissues and organs can be frozen and stored in
liquid nitrogen (LN) at -196°C for long-term storage of
germplasm. This would be of great value in the
conservation of germplasm of those crops, which
normally do not produce seeds, e.g., root and tuber
crops produce recalcitrant seeds, or where it may not
be desirable to store seeds.
2. The preservation of cells, tissues and organs in liquid
nitrogen is called cryopreservation, and the science
pertaining to this activity is known as cryobiology. Many
studies have been carried out on cryopreservation of
plant cells and organs, and the approach appears to have
considerable promise in germplasm conservation.
3. At -196°C, since at this temperature, all metabolic
processes and growth are suppressed, and the occurrence
of genetic, karyotypic, morphological and biochemical
changes are also prevented. Cryopreservation has proved
to be the most reliable method for long-term
preservation of cell cultures.
4. Calli, cell suspensions, protoplasts, pollen shoot-tips and
embryos have all been successfully preserved.
Drawback:
A serious drawback of the technique is that a general
protocol applicable to all species and explants is not
available. In addition, survival tends to decline with
storage period in most of the cases, most likely due to
injuries sustained by cells during the freezing step.
5. Choice of Material:
Material chosen for cryopreservation should be, as far as possible, in
meristematic state. Cell cultures are generally preserved in lag or early
exponential phase of growth. Cells in the early lag/stationary phase
may be susceptible to cryoinjury because of their arrest in the G1
phase.
Cultures in the exponential phase contain cells in different stages of
mitosis and cytokinesis; such cells suffer from injury during freezing
and thawing. In comparison, cells in the late lag phase are
predominantly in G2 and ready to embark upon cell division. In some
species, it may be important to use highly embryogenic cell cultures
since nonembryogenic or poorly embryogenic cultures show poor or no
re-growth after thawing.
6. Preculture
Culture of cells/tissues/organs in the presence of amino acids like proline,
sugars like sucrose and mannitol or at low temperature prior to freezing
initiates in them important physiological changes, which increase their
freezing tolerance. These changes relate to membrane fluidity to facilitate
dehydration and accumulation of substances like proline, which protect
the cells from toxicity of high solute concentrations.
Plantlets may be hardened by growing them at a near freezing
temperature, e.g., for one week at 22°C for 8 hr and -1°C for 16 hr (each
day). Cold-hardened tissues show increased water efflux, which permits a
rapid dehydration of the cells; this prevents the formation of large
intracellular ice crystals during freezing.
7. Cold-hardening pretreatments have been quite useful in
cryopreservation of temperate plant species. Sucrose (30-
240 gl-1) is the most commonly used sugar for the
pretreatment of cultures. But mannitol can also be used. A
3-4 day- preculture on 2-4% proline either alone or in
combination with some other cryoprotectant like DMSO
markedly improves the survival of suspension cultures.
8. Sugars act as osmotically effective agents, although
they do not penetrate inside the cells. Dehydration of
cells/tissues occurs in the presence of sugars during
the preculture, which prevents lethal ice crystal
formation during freezing. Proline may act by
reducing the level of latent injury to the cells or it may
actively participate in recovery metabolism.
9. Cryoprotection
The two major sources of cryoinjury are mechanical
damage due to ice crystals and toxic solution effects
due to the excessive concentration of intracellular
solutes resulting from ice formation. Water loss during
freezing may reduce the cell volume below a critical
level necessary for survival. In addition, dislocation of
structural water, which protects the integrity of cellular
components like membranes may also be involved in
cryoinjury. In fact, a critical amount of liquid water is
essential for membrane integrity and for cells to survive
freezing.
10. Culturing of cells/tissues in the presence of some high
molecular weight substances like dimethyl- sulphoxide
(DMSO), sugar alcohols like glycerol, sorbitol and amino
acids like proline for a period of time, prepares them to
tolerate cryoinjury. In majority of freeze preservation
protocols a mixture of DMSO, glycerol and sucrose is
used. The general order of cryoprotection seems to be
proline > DMSO + glycerol = DMSO + proline > DMSO.
11. Cryoprotective compounds differ remarkably in their
structure, and possibly in their mode of action. Some
cryoprotectants like DMSO penetrate the plant cell
quickly; others like glycerol are rather slow to enter cells,
while still others like sorbitol may not enter the cell at all.
Theoretically, a cryoprotectant may protect living cells
against freezing injury at the following three locations:
extracellular, cell membrane and intracellular sites. It may
also help to stabilize the intra- and inter- molecular
arrangements of cellular components and prevent the
removal of water molecules associated with cell
membranes during freezing.
12. DMSO (5-10%) is widely used either alone or in combination
with 5-20% glycerol dissolved in water or sugar solution; it is
also used in combination with sucrose, sorbitol or other
sugar alcohols. A combination of DMSO with other
cryoprotectants may be beneficial because they may act in
complementary manne.
For example, compounds like sorbitol do not enter the cells;
these would reduce cellular water content and, thereby,
decrease the rate of initial ice crystal formation. DMSO
itself enters the cells, and it reduces cellular dehydration
during freezing. Thus both the initial ice crystal formation
and subsequent dehydration during freezing are reduced
and, as a result, the survival may be increased.
13. Importance of DMSO
DMSO is the most commonly used cryoprotectant; it is
added gradually over a period of 30-60 min and the
temperature of the cultures in maintained at around 0°C.
Often, cells and tissues are suitably pre-cultured/pretreated
before freezing; this makes them hardy and improves their
survival. The frozen cells and tissues are stored in a liquid
nitrogen refrigerator; the temperature must not rise above
– 130°C otherwise ice crystals may be formed.
14. Different tissues have different sensitivities for cooling rates.
Ideally, cooling should be such that damaging ice crystals are
not formed.
In general, there are three strategies for freezing:
(1) Slow cooling,
(2) Rapid cooling, and
(3) Freezing, following dehydration.
Freezing:
15. Slow Cooling:
The material is cooled at the rate of 0.5-4°C min-1 up to -
40° to-100°C, and held at this temperature for 20-45 min
before it is plunged into liquid nitrogen. Initial slow
cooling reduces the amount of intracellular water, since
ice is first formed outside the cell and unfrozen
protoplasm loses water due to vapour deficit between
super-cooled protoplasm and extracellular ice. This
approach is particularly suitable for cell suspensions.
16. Rapid Cooling:
Some tissues cannot survives intermediate or slow
cooling rates and require rapid cooling of 200-l, 000°C
min-1. For example, shoot apices of strawberry and
Solatium goniocalyx did not survive cooling rates below
1,000°C min-4. In rapid cooling, the critical temperature
zone of ice crystal formation is passed so rapidly that ice
crystals of lethal size are not formed.
17. Freezing following Dehydration:
Excised single node segments (5 mm) from 6 to 8 week-old
plantlets of Asparagus officinalis were pre-cultured for 2 days
on 0.7 M sucrose, and then dehydrated with silica gel before
immersing them in LN. Explants dehydrated to 20% moisture
showed more than 60% survival as compared to around 17%
in case of non-dehydrated explants. Similarly, dehydrated
(18-30% moisture) embryogenic cell masses of sweet potato
showed 1.5 to 2- fold increases in survival on rapid freezing by
direct immersion in LN.
18. Extensions of this approach are the protocols for
vitrification and dehydration following encapsulation,
where tissues are dehydrated to threshold water levels.
In vitrification, the material is dehydrated using highly
concentrated aqueous solutions of cryoprotective
agents, whereas encapsulated cells/tissue may be
dehydrated by preculture on high sucrose concentration
or by exposure in a laminar air low. The dehydrated
materials are directly transferred into LN.
19. Thawing is the bringing of cryopreserved
materials back to the normal state in such a way
that damaging ice crystal formation does not
take place. Generally, the frozen material is
plunged into a waterbath at 37-40°C for 1-2
minutes; this gives the initial warming rate of
1,450°C min-1, and of 120°C min-1 between -50°C
and -10°C.
Thawing:
20. Viability of cells and tissues is usually determined by re-
growth, but chemical tests like staining with fluorescein
diacetate (FDA) or 2, 3, 5-triphenyltetrazolium chloride
(TTC) may be used to assess cell viability immediately
after thawing. Phenosafranine has also been used for
checking cell viability.
21. The post-thaw treatments should be such as to provide the
best possible conditions for recovery of cells from cryoinjury
and for resuming growth. The cryoprotectants may be
removed either by washing with the culture medium or by
plating the thawed cultures on to a filter paper kept on the
medium. Washing of the cells is considered deleterious in
many cases, and should preferably be avoided.
Reculture:
22. The permeability of plasma membrane increases due to
freezing and essential cell constituents may leak out of
the cells into the cryoprotectant solution. Washing may,
as a result, reduce the viability of cultures. Washing of cell
suspensions and embryo-genic callus cultures of
Gossypium hirsutum prevented their re-growth. But
when unwashed cells were placed on a filter paper, they
exhibited re-growth within two weeks of plating.
Similarly, washing of Saccharum cells or their re-culture
directly on the culture medium without washing was
deleterious.
However, healthy tissue was formed when the cells were
first plated on a filter paper discs and after 5 hr the discs
along with the cells were transferred to a fresh medium.
Finally, after 24 hr the cells were scraped off the filter
paper and transferred on to a fresh culture medium.
23. Many other authors have reported the beneficial effects of a
gradual removal of cryoprotectants by use of the filter paper
disc culture technique, following their placement under low
light intensity or in total darkness for two to three weeks.
24. At a sufficiently low temperature, highly concentrated
aqueous solutions of cryoprotective agents become so viscous
that they solidify into a metastable glass state, without ice
crystal formation at practical cooling rates; this phenomenon
is called vitrification.
It eliminates the need for controlled slow freezing as cells and
meristems are first dehydrated by a treatment with a plant
vitrification solution (PVS) of suitable concentration and then
cryopreserved by direct transfer into LN. It has been used for
cryopreservation of cell cultures, shoot-tips and protoplasts.
Vitrification:
25. Vitrification solutions themselves may cause toxicity, which
depends mainly on their osmotic potential. Therefore,
formulation of a PVS of suitable osmotic potential, manipulation
of duration and temperature during exposure of cells to PVS,
and subsequent dilution procedures would minimize toxicity.
Freezing does not appear to cause cellular injury in addition to
that produced due to dehydration with PVS. The PVS toxicity to
Wasabia japonica apical meristems was markedly reduced by
use of cyroprotectants.
26. Shoot apices cryoprotected with 2 M glycerol + 0.4 M sucrose
prior to their dehydration with PVS2 showed shoot formation
in 80-90% of the explants after their immersion in LN. In
contrast, only 12.5% of the uncryoprotected shoot apices
dehydrated with PVS2 and frozen by immersion in LN showed
shoot formation.
27. Vitrification has been used to the greatest effect for
cryopreservation of germplasm of such plant species that
are recalcitrant to the traditional cryopreservation
methods based on controlled freezing, e.g., tropical crop
plants, recalcitrant seed producing forest trees and
tropical fruit crops, and certain clonally propagated
tropical fruit crops.
28. In contrast, successful temperate crop plant
cryopreservation methods are based on cold hardening
pretreatments. In addition, optimized embryo rescue
techniques used in combination with cryopreservation
and efficient re-culture procedures are promising in
conservation of recalcitrant germplasm.
Cryopreservation of plant tissues and cell cultures has
advanced to a level where it can be used as a routine tool
to conserve germplasm derived from wild relatives,
ancient and modern cultivars, and biotechnologically
derived genotypes of at least some plants species.
29. Encapsulation Dehydration:
In another approach, explants are first encapsulated in a
suitable matrix like alginate and then subjected to
dehydration. Generally, tolerance to dehydration is induced
by pre-culturing the encapsulated meristems in a medium
enriched with sucrose (about 0.7-1 M) or a mixture of sucrose
and glycerol.
30. The beads may be dehydrated under laminar air how to
critical water level, which varies among different species,
placed in a cryotube and plunged directly into LN. Survival
of encapsulated cells of Catharanthus roseus subjected to
rapid freezing increased many-fold when they were
dehydrated to 25% moisture prior to freezing. Apical
meristems, zygotic embryos and cell cultures of several
species have been cryopreserved by this method.
31. This procedure eliminates the need of cryoprotection that is
necessary when vitrification solutions are used and may
improve survival. Shoot primordia of horseradish (Armoracia
rusticana) were encapsulated in 2% alginate;, the beads were
pre-cultured on MS agar medium having 0.5 M sucrose or 1 M
glycerol for 1 day, and then dehydrated by silica gel/partial
vacuum to 70% of their weight after pre-culture.
32. The beads were then kept in cryotubes and plunged into LN.
More than 90% of shoot primordia formed normal shoots when
thawed and re-cultured. But vitrification of encapsulated shoot
primordia with PVS2 before plunging them into LN gave only
69% survival. However, in some species, dehydration of
encapsulated tissues itself may reduce survival.
Somatic embryos of Camellia japonica did not survive freezing
in either nonencapsulated or encapsulated states and subjected
to various cryoprotective treatments. However, a proportion of
zygotic embryos, with or without desiccation, survived rapid
freezing. Thus somatic embryos of C. japonica appear to be in a
physiological state not suitable for cryopreservation.
33. Shoot-tips isolated from preconditioned shoot cultures of
Holostemma annulare (H. ada-kodien), an endangered
medicinal plant of the Indian subcontinent, were encapsulated,
dehydrated and precultured in the dark on 0.5-0.75 M sucrose
and 3-5% DMSO before being cryopreserved.
Pre-culture in sucrose alone for 2-3 days resulted in 46-56%
survival and shoot regeneration in 42-52% of the shoot-tips that
survived cryopreservation. A 3-day pre-culture with 3 and 5%
DMSO enhanced the survival rates to 72-76% and shoot
regeneration rates to 67-68%, but the entire enhancement
came from cells producing callus.
34. Materials subjected to cryopreservation may show some special
requirements during re-culture. For example, shoot-tips from
freeze-preserved seedlings of tomato required GA3for
developing into shoots (they formed callus in the absence of
GA3), while normal shoot-tips of tomato do not require GA3.
Similarly, survival of carrot plantlets was greatly improved by
activated charcoal. It is, therefore, necessary to determine the
optimum conditions for re-culture of different plant species,
particularly when commonly used regimes fail.
35. Cell cultures, shoot-tips, somatic/zygotic embryos and even
plantlets of a number of species have been successfully frozen
and stored for variable periods (from a few minutes to several
months). In general, meristematic cells survive better during
freeze preservation than do mature differentiated cells. The
techniques for freeze preservation of shoot-tips are being refined
for germplasm storage over very long periods.