Cryopreservation is a method for long-term preservation of plant genetic resources by storing them at ultra-low temperatures, typically in liquid nitrogen at -196°C. This stops biological activity and slows aging. The document discusses why preservation is important, various preservation methods, and the steps involved in cryopreservation including selection of plant material, addition of cryoprotectants, freezing, storage, thawing, and viability testing. Cryopreservation provides long-term storage of germplasm in a very small space and protects against loss from diseases, climate change, and other threats.
This document discusses the history and techniques of anther and pollen culture. It notes that anther and pollen culture were first developed in the 1950s and 1960s and can be used to produce haploid plants. The document outlines the procedures for anther and pollen culture, highlighting steps like collecting unopened flower buds, isolating microspores, and culturing on nutrient media. It also discusses factors that influence culture success like genotype, temperature, and physiological status of donor plants. The advantages of pollen culture over anther culture and various applications of anther and pollen culture are summarized.
This document discusses micropropagation, which is the rapid vegetative propagation of plants using modern tissue culture methods to produce genetically identical copies. It can be used to multiply genetically modified plants, overcome limitations of conventional breeding, and provide sufficient plantlets from stock plants that do not produce seeds or respond well to other propagation. The key methods are multiplication through meristematic tissue, adventitious shoots, somatic embryogenesis, and organogenesis. Micropropagation has commercial uses and significance in producing disease-free plants year-round, exchanging germplasm internationally, conserving genetics, and producing synthetic seeds. While expensive, it provides uniformity and allows maintaining germplasm stocks for years.
Acclimatization or acclimatisation (also called acclimation or acclimatation) is the process in which an individual organism adjusts to a change in its environment (such as a change in altitude, temperature, humidity, photoperiod, or pH), allowing it to maintain performance across a range of environmental conditions
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.
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."
This document discusses synthetic seeds, which are artificially encapsulated plant materials like somatic embryos, shoot buds, or cell aggregates that can be used for sowing like natural seeds. Synthetic seeds were originally only referred to somatic embryos for economic crop production, but now include other micropropagules. The first successful synthetic seed was produced in 1982 in carrot. There are two main types - desiccated synthetic seeds which are produced from desiccation tolerant species and hydrated synthetic seeds which encapsulate somatic embryos or shoots in hydrogels like sodium alginate. The encapsulation process involves a plant propagule, a gelling matrix that can include nutrients, and an artificial seed coat to develop the encapsulation system. Common encapsulation
This document discusses the production of synthetic seeds or artificial seeds through somatic embryogenesis and encapsulation. Synthetic seeds are somatic embryos encapsulated in a hydrogel to mimic seeds. They allow for direct delivery of tissue cultured plants, genetic uniformity, and large-scale production. The document outlines the procedure for synthetic seed production, including encapsulating somatic embryos, auxillary buds, or shoot tips in sodium alginate or other gels then germinating. Examples are provided for synthetic seed production in crops like papaya, banana, and carrot.
This document discusses the history and techniques of anther and pollen culture. It notes that anther and pollen culture were first developed in the 1950s and 1960s and can be used to produce haploid plants. The document outlines the procedures for anther and pollen culture, highlighting steps like collecting unopened flower buds, isolating microspores, and culturing on nutrient media. It also discusses factors that influence culture success like genotype, temperature, and physiological status of donor plants. The advantages of pollen culture over anther culture and various applications of anther and pollen culture are summarized.
This document discusses micropropagation, which is the rapid vegetative propagation of plants using modern tissue culture methods to produce genetically identical copies. It can be used to multiply genetically modified plants, overcome limitations of conventional breeding, and provide sufficient plantlets from stock plants that do not produce seeds or respond well to other propagation. The key methods are multiplication through meristematic tissue, adventitious shoots, somatic embryogenesis, and organogenesis. Micropropagation has commercial uses and significance in producing disease-free plants year-round, exchanging germplasm internationally, conserving genetics, and producing synthetic seeds. While expensive, it provides uniformity and allows maintaining germplasm stocks for years.
Acclimatization or acclimatisation (also called acclimation or acclimatation) is the process in which an individual organism adjusts to a change in its environment (such as a change in altitude, temperature, humidity, photoperiod, or pH), allowing it to maintain performance across a range of environmental conditions
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.
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."
This document discusses synthetic seeds, which are artificially encapsulated plant materials like somatic embryos, shoot buds, or cell aggregates that can be used for sowing like natural seeds. Synthetic seeds were originally only referred to somatic embryos for economic crop production, but now include other micropropagules. The first successful synthetic seed was produced in 1982 in carrot. There are two main types - desiccated synthetic seeds which are produced from desiccation tolerant species and hydrated synthetic seeds which encapsulate somatic embryos or shoots in hydrogels like sodium alginate. The encapsulation process involves a plant propagule, a gelling matrix that can include nutrients, and an artificial seed coat to develop the encapsulation system. Common encapsulation
This document discusses the production of synthetic seeds or artificial seeds through somatic embryogenesis and encapsulation. Synthetic seeds are somatic embryos encapsulated in a hydrogel to mimic seeds. They allow for direct delivery of tissue cultured plants, genetic uniformity, and large-scale production. The document outlines the procedure for synthetic seed production, including encapsulating somatic embryos, auxillary buds, or shoot tips in sodium alginate or other gels then germinating. Examples are provided for synthetic seed production in crops like papaya, banana, and carrot.
Production of synthetic seed involves encapsulating somatic embryos, shoot buds, or cell aggregates using tissue culture techniques. This allows for the large-scale, low-cost propagation of plants while maintaining genetic uniformity. Synthetic seeds can be stored longer than traditional seeds and planted directly in fields without the need for transplanting. While synthetic seeds have advantages over traditional micropropagation methods, their production and germination rates can still be limited for some plant species.
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.
Invitro culture of unpollinated ovaries and ovules represents an alternative for the production of haploid plant
First successful report on the induction of gynogenic haploid was in barley by San Noeum in 1976
Haploid plants are obtained from ovary and ovule culture of rice, wheat, maize, sunflower, tobacco, poplar, mulberry etc
Whites or MS or N6 inorganic salt medium supplement with growth substances are used
This document summarizes the culture of in-vitro pollination and fertilization. It describes the different types of in-vitro pollination including ovular, ovarian, placental and stigmatic pollination. The methods of in-vitro pollination and fertilization are outlined involving sterilization procedures and suitable media and explants. Applications include using in-vitro fertilization to overcome self-incompatibility in some plants or enable intergeneric crosses. The techniques used involve isolating pollen and egg cells, inducing fusion through electrofusion, and culturing the fertilized eggs on nutrient media to develop into plants. Considerations for successful in-vitro fertilization include the physiological state of
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.
The document discusses tissue culture techniques and their applications in plant breeding. It provides a historical background of tissue culture dating back to the early 20th century. The major steps involved in tissue culture are explained, including selection of explant tissue, sterilization, establishment in culture medium, multiplication through callus formation, root formation, and hardening of plantlets. Key techniques like protoplast culture, haploid culture, and micropropagation are also summarized along with their achievements in generating somatic hybrids, true breeding lines, and large-scale clonal propagation respectively.
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.
Synthetic seeds are encapsulated somatic embryos or shoot buds that can be used for planting like traditional seeds. They allow for clonal propagation of plants that are difficult to reproduce through traditional seeds, including some fruit crops. The production of synthetic seeds involves inducing somatic embryogenesis in callus cultures, maturing the embryos, and encapsulating them in a protective gel before planting. This allows genetic material to be stored and dispersed while avoiding issues with seed-borne diseases, low seed viability, and difficulties reproducing species that lack traditional seeds.
This document discusses meristem culture and shoot tip culture techniques. It describes the three stages of meristem culture: establishment, multiplication, and root regeneration. Shoot tips less than 1 mm are excised and cultured on medium supplemented with hormones like cytokinins and auxins to promote growth. Meristem culture allows for virus elimination, micropropagation, genetic resource preservation, and facilitates international plant exchange. It is an effective method for producing disease-free plants.
This document discusses micropropagation, which is the rapid multiplication of plant materials using tissue culture methods. It involves taking explants like shoot tips or buds and culturing them on growth media to produce many new plantlets. The process involves initiation, multiplication, rooting, and acclimatization stages. Approaches include multiplication from axillary buds/shoots or adventitious shoots. Applications are high rate propagation of disease-free plants, seed production in some crops, and cost effectiveness. Automation using bioreactors and robots can increase production scale but reduces flexibility.
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.
Meristem tip culture for the production of the virus free plantsArjun Rayamajhi
This presentation gives general idea on the meristem tip culture for the production of the virus free plants. The principles, methods and procedures of the meristem tip culture included. General idea on different in vitro culture techniques for virus elimination meristem tip culture viz. thermotherapy, cryotherapy,chemotherapy and electrotherapy are provided.
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).
Embryo culture involves growing plant embryos artificially in order to enhance survival rates. It is commonly used to rescue weak or immature embryos that may not otherwise survive to become viable plants. The process involves excising embryos from seeds or ovaries and placing them onto sterile nutrient-rich media under suitable temperature, light, and humidity conditions. Embryo culture has various applications in plant breeding, including shortening breeding cycles, overcoming seed dormancy, producing hybrids, and conserving plant germplasm. It is an important technique in modern plant breeding and development of new crop varieties.
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.
Cybrids are produced through the fusion of protoplasts from two different plant species, combining the cytoplasm of both but the nucleus of only one species. This technique allows for the transfer of cytoplasmic traits like male sterility between incompatible species. Protoplast isolation, fusion, selection, and regeneration of hybrid cells into whole plants are required to produce cybrids. Cybrids can be used to study cytoplasmic genes and transfer desirable agricultural traits, overcoming sexual incompatibility barriers in plant breeding.
The document discusses explants used for clonal propagation in biotechnology of horticultural crops. It defines an explant as a tissue taken from a mother plant and cultured under aseptic conditions on a defined medium. The choice of explant depends on the type of culture to be initiated, its purpose, and the plant species. Common explant sources include shoots, leaves, stems, and roots. Explants must be surface sterilized to remove contaminants and allow contamination-free culture initiation. Sub-culturing is needed when cells reach the stationary state of growth.
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.
Cryopreservation is a process where tissues, cells, or organs are preserved at very low temperatures, typically -80°C or -196°C, to bring metabolism and cell division to a halt. It involves selecting plant material, adding cryoprotectants, freezing the material, storing it in liquid nitrogen, thawing it, washing away cryoprotectants, and attempting to regenerate plants from the preserved material. Cryopreservation allows for indefinite storage of genetic resources in a minimal amount of space and with minimal labor required, helping to conserve endangered species, disease-free plants, and rare germplasm.
Plant tissue culture,its methods, advantages,disadvantages and applications.Komal Jalan
Plant tissue culture is the most widely used technique for growing very large number of plant using a very small part of the main plant(explant). Tissue culturing is very common for many popular and demanding crops.Few of them discussed here are Potato,Papaya,Pinepple,Banana,Gerbera,Sunflower,Orchids
Production of synthetic seed involves encapsulating somatic embryos, shoot buds, or cell aggregates using tissue culture techniques. This allows for the large-scale, low-cost propagation of plants while maintaining genetic uniformity. Synthetic seeds can be stored longer than traditional seeds and planted directly in fields without the need for transplanting. While synthetic seeds have advantages over traditional micropropagation methods, their production and germination rates can still be limited for some plant species.
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.
Invitro culture of unpollinated ovaries and ovules represents an alternative for the production of haploid plant
First successful report on the induction of gynogenic haploid was in barley by San Noeum in 1976
Haploid plants are obtained from ovary and ovule culture of rice, wheat, maize, sunflower, tobacco, poplar, mulberry etc
Whites or MS or N6 inorganic salt medium supplement with growth substances are used
This document summarizes the culture of in-vitro pollination and fertilization. It describes the different types of in-vitro pollination including ovular, ovarian, placental and stigmatic pollination. The methods of in-vitro pollination and fertilization are outlined involving sterilization procedures and suitable media and explants. Applications include using in-vitro fertilization to overcome self-incompatibility in some plants or enable intergeneric crosses. The techniques used involve isolating pollen and egg cells, inducing fusion through electrofusion, and culturing the fertilized eggs on nutrient media to develop into plants. Considerations for successful in-vitro fertilization include the physiological state of
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.
The document discusses tissue culture techniques and their applications in plant breeding. It provides a historical background of tissue culture dating back to the early 20th century. The major steps involved in tissue culture are explained, including selection of explant tissue, sterilization, establishment in culture medium, multiplication through callus formation, root formation, and hardening of plantlets. Key techniques like protoplast culture, haploid culture, and micropropagation are also summarized along with their achievements in generating somatic hybrids, true breeding lines, and large-scale clonal propagation respectively.
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.
Synthetic seeds are encapsulated somatic embryos or shoot buds that can be used for planting like traditional seeds. They allow for clonal propagation of plants that are difficult to reproduce through traditional seeds, including some fruit crops. The production of synthetic seeds involves inducing somatic embryogenesis in callus cultures, maturing the embryos, and encapsulating them in a protective gel before planting. This allows genetic material to be stored and dispersed while avoiding issues with seed-borne diseases, low seed viability, and difficulties reproducing species that lack traditional seeds.
This document discusses meristem culture and shoot tip culture techniques. It describes the three stages of meristem culture: establishment, multiplication, and root regeneration. Shoot tips less than 1 mm are excised and cultured on medium supplemented with hormones like cytokinins and auxins to promote growth. Meristem culture allows for virus elimination, micropropagation, genetic resource preservation, and facilitates international plant exchange. It is an effective method for producing disease-free plants.
This document discusses micropropagation, which is the rapid multiplication of plant materials using tissue culture methods. It involves taking explants like shoot tips or buds and culturing them on growth media to produce many new plantlets. The process involves initiation, multiplication, rooting, and acclimatization stages. Approaches include multiplication from axillary buds/shoots or adventitious shoots. Applications are high rate propagation of disease-free plants, seed production in some crops, and cost effectiveness. Automation using bioreactors and robots can increase production scale but reduces flexibility.
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.
Meristem tip culture for the production of the virus free plantsArjun Rayamajhi
This presentation gives general idea on the meristem tip culture for the production of the virus free plants. The principles, methods and procedures of the meristem tip culture included. General idea on different in vitro culture techniques for virus elimination meristem tip culture viz. thermotherapy, cryotherapy,chemotherapy and electrotherapy are provided.
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).
Embryo culture involves growing plant embryos artificially in order to enhance survival rates. It is commonly used to rescue weak or immature embryos that may not otherwise survive to become viable plants. The process involves excising embryos from seeds or ovaries and placing them onto sterile nutrient-rich media under suitable temperature, light, and humidity conditions. Embryo culture has various applications in plant breeding, including shortening breeding cycles, overcoming seed dormancy, producing hybrids, and conserving plant germplasm. It is an important technique in modern plant breeding and development of new crop varieties.
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.
Cybrids are produced through the fusion of protoplasts from two different plant species, combining the cytoplasm of both but the nucleus of only one species. This technique allows for the transfer of cytoplasmic traits like male sterility between incompatible species. Protoplast isolation, fusion, selection, and regeneration of hybrid cells into whole plants are required to produce cybrids. Cybrids can be used to study cytoplasmic genes and transfer desirable agricultural traits, overcoming sexual incompatibility barriers in plant breeding.
The document discusses explants used for clonal propagation in biotechnology of horticultural crops. It defines an explant as a tissue taken from a mother plant and cultured under aseptic conditions on a defined medium. The choice of explant depends on the type of culture to be initiated, its purpose, and the plant species. Common explant sources include shoots, leaves, stems, and roots. Explants must be surface sterilized to remove contaminants and allow contamination-free culture initiation. Sub-culturing is needed when cells reach the stationary state of growth.
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.
Cryopreservation is a process where tissues, cells, or organs are preserved at very low temperatures, typically -80°C or -196°C, to bring metabolism and cell division to a halt. It involves selecting plant material, adding cryoprotectants, freezing the material, storing it in liquid nitrogen, thawing it, washing away cryoprotectants, and attempting to regenerate plants from the preserved material. Cryopreservation allows for indefinite storage of genetic resources in a minimal amount of space and with minimal labor required, helping to conserve endangered species, disease-free plants, and rare germplasm.
Plant tissue culture,its methods, advantages,disadvantages and applications.Komal Jalan
Plant tissue culture is the most widely used technique for growing very large number of plant using a very small part of the main plant(explant). Tissue culturing is very common for many popular and demanding crops.Few of them discussed here are Potato,Papaya,Pinepple,Banana,Gerbera,Sunflower,Orchids
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.
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.
Dr. Ehsan Dulloo discusses conservation strategies to respond to the global loss of plant genetic resources at the 29th International Horticulture Congress, including ex situ conservation, in situ conservation, cryopreservation, seed banks and the importance of crop wild relatives.
http://www.bioversityinternational.org/research-portfolio/conservation-of-crop-diversity/
This document discusses plant cell culture and preservation techniques. It provides information on:
- Growing plant cells, tissues, and organs in artificial media for research and production of products.
- Techniques for maintaining and preserving plant cells including minimal growth at reduced temperatures, cryopreservation at liquid nitrogen temperatures, and various cryopreservation protocols like slow cooling, vitrification, and encapsulation-dehydration.
- Assessing viability of cryopreserved plant cells through tests like FDA, TTC, and Evan's blue staining. Regeneration of plants from stored tissues or cells is the ultimate test of survival.
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.
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.
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.
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.
The document discusses ex situ conservation methods for germplasm, including seed banks, gene banks, tissue culture banks, cryopreservation, and botanical gardens. It focuses on seed banks, which preserve dried seeds at low temperatures; gene banks, which maintain collections of seeds, plants, and animals; tissue banks, which conserve buds and meristematic cells; cryobanks, which preserve seeds and embryos at very low temperatures; and field gene banks, which conserve genetic diversity outdoors. The document also provides details on the techniques, mechanisms, and applications of cryopreservation for long-term germplasm conservation.
Cryopreservation is a method for long-term conservation of plant genetic resources by storing plant materials like seeds, tissues, cells, pollen, etc. at ultra-low temperatures, usually in liquid nitrogen at -196°C. This preserves the viability and genetic integrity of the materials. There are several advantages like maintaining a large number of accessions in a small space and providing pathogen-free plant materials. Successful cryopreservation involves pretreatment with cryoprotectants, controlled freezing and thawing, then regeneration of plants from the stored materials. It allows preservation of plant genetic diversity for future use in breeding programs.
Cryopreservation is a process where tissues, cells, or organs are preserved at very low temperatures, typically -80°C or -196°C, to bring metabolism to zero and prevent damage. It involves selecting plant material, adding cryoprotectants like sucrose or glycols, freezing the material slowly or rapidly in liquid nitrogen, storing it in liquid nitrogen, thawing it, washing away cryoprotectants, and attempting to regenerate plants from the preserved material. Cryopreservation allows for indefinite storage of plant genetic resources and has major advantages like minimal space and labor requirements.
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 involves preserving biological material such as sperm, cells or tissues at sub-zero temperatures, usually using liquid nitrogen.
- There are several techniques used in cryopreservation including slow freezing, vitrification and step-wise freezing to prevent ice crystal formation and cell damage.
- Cryopreservation has many applications including sperm banks, fertility preservation, conservation of plant and animal species, and controversial techniques like cryonics where human bodies are preserved at very low temperatures.
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.
Cryopreservation is the process of preserving living cells and tissues by cooling them to very low sub-zero temperatures (typically -196°C using liquid nitrogen). The document discusses the history, materials, methods, and applications of cryopreservation for various plant materials including plant protoplasts, shoot tips, meristems, seeds, and the establishment of plant cell banks and pollen banks for long-term preservation. Some of the key achievements highlighted include the cryopreservation of plant cell lines, pollen and pollen embryos, excised meristems, and recalcitrant seeds. The main difficulties are damage to plant cells during freezing and thawing due to ice crystal formation.
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 discusses different methods of germplasm conservation including in situ and ex situ conservation. In situ conservation involves protecting genetic resources in their natural habitats through national parks, biosphere reserves, gene sanctuaries and sacred forests. Ex situ conservation involves maintaining genetic resources outside their natural habitats through seed banks, gene banks, tissue culture, cryopreservation and botanical gardens. The document provides details on various types of in situ and ex situ conservation methods.
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 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.
Introduction
Reason for cryopreservation
Selection of part of plant for cryopreservation
Technique of cryopreservation
Application
Limitation
Conclusion
Cryopreservation is the process of preserving living cells and tissues by cooling them to very low sub-zero temperatures (typically -196°C using liquid nitrogen). The key steps involve pre-treatment of plant materials with cryoprotectants and dehydration, slow or rapid freezing, storage in liquid nitrogen, thawing, and regeneration of plants. Cryopreservation allows for long-term storage of plant genetic resources and endangered species. While it has enabled conservation of many plant species, some recalcitrant plants remain difficult to cryopreserve. Recent developments include vitrification and encapsulation-dehydration techniques.
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.
Originally isolated from nature, but increasingly "improved" by genetic manipulation via mutagenesis and selection or recombinant DNA technology or protoplast fusion (fungi)
SDSS1335+0728: The awakening of a ∼ 106M⊙ black hole⋆Sérgio Sacani
Context. The early-type galaxy SDSS J133519.91+072807.4 (hereafter SDSS1335+0728), which had exhibited no prior optical variations during the preceding two decades, began showing significant nuclear variability in the Zwicky Transient Facility (ZTF) alert stream from December 2019 (as ZTF19acnskyy). This variability behaviour, coupled with the host-galaxy properties, suggests that SDSS1335+0728 hosts a ∼ 106M⊙ black hole (BH) that is currently in the process of ‘turning on’. Aims. We present a multi-wavelength photometric analysis and spectroscopic follow-up performed with the aim of better understanding the origin of the nuclear variations detected in SDSS1335+0728. Methods. We used archival photometry (from WISE, 2MASS, SDSS, GALEX, eROSITA) and spectroscopic data (from SDSS and LAMOST) to study the state of SDSS1335+0728 prior to December 2019, and new observations from Swift, SOAR/Goodman, VLT/X-shooter, and Keck/LRIS taken after its turn-on to characterise its current state. We analysed the variability of SDSS1335+0728 in the X-ray/UV/optical/mid-infrared range, modelled its spectral energy distribution prior to and after December 2019, and studied the evolution of its UV/optical spectra. Results. From our multi-wavelength photometric analysis, we find that: (a) since 2021, the UV flux (from Swift/UVOT observations) is four times brighter than the flux reported by GALEX in 2004; (b) since June 2022, the mid-infrared flux has risen more than two times, and the W1−W2 WISE colour has become redder; and (c) since February 2024, the source has begun showing X-ray emission. From our spectroscopic follow-up, we see that (i) the narrow emission line ratios are now consistent with a more energetic ionising continuum; (ii) broad emission lines are not detected; and (iii) the [OIII] line increased its flux ∼ 3.6 years after the first ZTF alert, which implies a relatively compact narrow-line-emitting region. Conclusions. We conclude that the variations observed in SDSS1335+0728 could be either explained by a ∼ 106M⊙ AGN that is just turning on or by an exotic tidal disruption event (TDE). If the former is true, SDSS1335+0728 is one of the strongest cases of an AGNobserved in the process of activating. If the latter were found to be the case, it would correspond to the longest and faintest TDE ever observed (or another class of still unknown nuclear transient). Future observations of SDSS1335+0728 are crucial to further understand its behaviour. Key words. galaxies: active– accretion, accretion discs– galaxies: individual: SDSS J133519.91+072807.4
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.
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.
Microbial interaction
Microorganisms interacts with each other and can be physically associated with another organisms in a variety of ways.
One organism can be located on the surface of another organism as an ectobiont or located within another organism as endobiont.
Microbial interaction may be positive such as mutualism, proto-cooperation, commensalism or may be negative such as parasitism, predation or competition
Types of microbial interaction
Positive interaction: mutualism, proto-cooperation, commensalism
Negative interaction: Ammensalism (antagonism), parasitism, predation, competition
I. Mutualism:
It is defined as the relationship in which each organism in interaction gets benefits from association. It is an obligatory relationship in which mutualist and host are metabolically dependent on each other.
Mutualistic relationship is very specific where one member of association cannot be replaced by another species.
Mutualism require close physical contact between interacting organisms.
Relationship of mutualism allows organisms to exist in habitat that could not occupied by either species alone.
Mutualistic relationship between organisms allows them to act as a single organism.
Examples of mutualism:
i. Lichens:
Lichens are excellent example of mutualism.
They are the association of specific fungi and certain genus of algae. In lichen, fungal partner is called mycobiont and algal partner is called
II. Syntrophism:
It is an association in which the growth of one organism either depends on or improved by the substrate provided by another organism.
In syntrophism both organism in association gets benefits.
Compound A
Utilized by population 1
Compound B
Utilized by population 2
Compound C
utilized by both Population 1+2
Products
In this theoretical example of syntrophism, population 1 is able to utilize and metabolize compound A, forming compound B but cannot metabolize beyond compound B without co-operation of population 2. Population 2is unable to utilize compound A but it can metabolize compound B forming compound C. Then both population 1 and 2 are able to carry out metabolic reaction which leads to formation of end product that neither population could produce alone.
Examples of syntrophism:
i. Methanogenic ecosystem in sludge digester
Methane produced by methanogenic bacteria depends upon interspecies hydrogen transfer by other fermentative bacteria.
Anaerobic fermentative bacteria generate CO2 and H2 utilizing carbohydrates which is then utilized by methanogenic bacteria (Methanobacter) to produce methane.
ii. Lactobacillus arobinosus and Enterococcus faecalis:
In the minimal media, Lactobacillus arobinosus and Enterococcus faecalis are able to grow together but not alone.
The synergistic relationship between E. faecalis and L. arobinosus occurs in which E. faecalis require folic acid
CLASS 12th CHEMISTRY SOLID STATE ppt (Animated)eitps1506
Description:
Dive into the fascinating realm of solid-state physics with our meticulously crafted online PowerPoint presentation. This immersive educational resource offers a comprehensive exploration of the fundamental concepts, theories, and applications within the realm of solid-state physics.
From crystalline structures to semiconductor devices, this presentation delves into the intricate principles governing the behavior of solids, providing clear explanations and illustrative examples to enhance understanding. Whether you're a student delving into the subject for the first time or a seasoned researcher seeking to deepen your knowledge, our presentation offers valuable insights and in-depth analyses to cater to various levels of expertise.
Key topics covered include:
Crystal Structures: Unravel the mysteries of crystalline arrangements and their significance in determining material properties.
Band Theory: Explore the electronic band structure of solids and understand how it influences their conductive properties.
Semiconductor Physics: Delve into the behavior of semiconductors, including doping, carrier transport, and device applications.
Magnetic Properties: Investigate the magnetic behavior of solids, including ferromagnetism, antiferromagnetism, and ferrimagnetism.
Optical Properties: Examine the interaction of light with solids, including absorption, reflection, and transmission phenomena.
With visually engaging slides, informative content, and interactive elements, our online PowerPoint presentation serves as a valuable resource for students, educators, and enthusiasts alike, facilitating a deeper understanding of the captivating world of solid-state physics. Explore the intricacies of solid-state materials and unlock the secrets behind their remarkable properties with our comprehensive presentation.
When I was asked to give a companion lecture in support of ‘The Philosophy of Science’ (https://shorturl.at/4pUXz) I decided not to walk through the detail of the many methodologies in order of use. Instead, I chose to employ a long standing, and ongoing, scientific development as an exemplar. And so, I chose the ever evolving story of Thermodynamics as a scientific investigation at its best.
Conducted over a period of >200 years, Thermodynamics R&D, and application, benefitted from the highest levels of professionalism, collaboration, and technical thoroughness. New layers of application, methodology, and practice were made possible by the progressive advance of technology. In turn, this has seen measurement and modelling accuracy continually improved at a micro and macro level.
Perhaps most importantly, Thermodynamics rapidly became a primary tool in the advance of applied science/engineering/technology, spanning micro-tech, to aerospace and cosmology. I can think of no better a story to illustrate the breadth of scientific methodologies and applications at their best.
Authoring a personal GPT for your research and practice: How we created the Q...Leonel Morgado
Thematic analysis in qualitative research is a time-consuming and systematic task, typically done using teams. Team members must ground their activities on common understandings of the major concepts underlying the thematic analysis, and define criteria for its development. However, conceptual misunderstandings, equivocations, and lack of adherence to criteria are challenges to the quality and speed of this process. Given the distributed and uncertain nature of this process, we wondered if the tasks in thematic analysis could be supported by readily available artificial intelligence chatbots. Our early efforts point to potential benefits: not just saving time in the coding process but better adherence to criteria and grounding, by increasing triangulation between humans and artificial intelligence. This tutorial will provide a description and demonstration of the process we followed, as two academic researchers, to develop a custom ChatGPT to assist with qualitative coding in the thematic data analysis process of immersive learning accounts in a survey of the academic literature: QUAL-E Immersive Learning Thematic Analysis Helper. In the hands-on time, participants will try out QUAL-E and develop their ideas for their own qualitative coding ChatGPT. Participants that have the paid ChatGPT Plus subscription can create a draft of their assistants. The organizers will provide course materials and slide deck that participants will be able to utilize to continue development of their custom GPT. The paid subscription to ChatGPT Plus is not required to participate in this workshop, just for trying out personal GPTs during it.
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2
Why preservation is
important ?
Until two decades ago the genetic resources were getting depleted owing to the
continous depredation by man.
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.
Usually, s
convenien
germplasm
Because m
through se
small spac
transporte
3. Germplasm
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3
A germplasm is a collection of genetic resources
for an organism, it is a hereditary material
trasmitted to off spring through germ cell.
For plants, the germplasm may be stored as a seed
collection (even a large seed bank).
For trees, in a nursery.
4. HISTORY
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Theoreticians of cryopreservation was James Lovelock
(born 1919).
Osmotic stress
Salt concentration
Christopher Polge, carried out cryopreservation of first
fowl sperm.
The concept of physical basis of heredity expressed by
the 19th-century Biologist August Weismann in 1883.
According to his theory, germplasm, which is
independent from all other cells of the body, is the
essential element of germ cells and is the hereditary
material that is passed from generation to generation.
5. 1. ATTACK BY PEST AND
PATHOGENS
2. CLIMATE DISORDER
3. NATURAL DISASTERS
4. POLITICAL AND ECONOMIC
CAUSES
CRYOPRESERVATION-deya 25-Jul-16
5
The conventional methods of germplasm
preservation are prone to possible
catastrophic losses because of:
6. 1 . IN-SITU PRESERVATION:
PRESERVATION OF THE GERMPLASM
IN THEIR NATURAL ENVIRONMENT
BY ESTABLISHING BIOSPHERES,
NATIONAL PARKS ETC.
2. EX-SITU PRESERVATION:
IN THE FORM OF SEED OR IN VITRO
CULTURES.
CRYOPRESERVATION-deya 25-Jul-16
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The conservation of germplasm can
be done by two methods.
7. IN-SITU conservation
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In situ conservation means “on site” conservation.
It is a coservation of genetic resources in a natural
population of plants, such as forests genetic resoures in
natural population of tree species.
It is the process of procecting an endangered plant in its
natural habitat
It is applied to conservation of
agriculture biodiversity in
agro ecosystem by farmers.
Especially these using
unconventional forming practice.
8. EX-SITU conservation
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Ex-site conservation means “off-site” conservation.
It is the process of protecting an endangered species
of plants out side of its natural habitat.
For example by removing part of the population
from a threatened habitat and placing it in a new
location
9. Ex-situ has following disadvantages
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Some plants do not produce fertile seeds.
Loss of seed viability
Seed destruction by pests, etc.
Poor germination rate.
This is only useful for seed propagating plants.
It’s a costly process.
10. Advantages
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Small areas can store large amount of material.
The germplasm preserved can be maintained in an
environment free from pathogens.
It can be protected against the natural hazard.
From the germplasm stock large number of plants
can be obtained whenever needed
11. Ex situ conservation can be carried out by using
several methods
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11
Seed gene bank
In vitro storage
DNA storage
Pollen storage
Field bank
Botanical garden
12. 12
In vitro method of germplasm
convervation◊ in vitro method
employing shoots
,meristems and
embryos are ideally
suited for the
conservation of
germplasm
◊ the plant with
recalcitrant seed
and genetically
engineered can also
be preserved by this
in vitro approach
25-Jul-16CRYOPRESERVATION-deya
13. various methods of in vitro
conservation
1. Cryopreservation - generally involves storage in liquid nitrogen.
2. Cold storage - it involves storage in low and non freezing temperature.
3. Low pressure – it involves partially reducing the atmospheric pressure of
surrounding.
4. Low oxygen storage - it involves reducing the oxygen level but
maintaining the pressure.
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14. It literally means preservation in “frozen state.”
The principle - to bring plant cells or tissue to a
zero metabolism and non dividing state by reducing
the temperature in the presence of cryoprotectant.
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Cryopreservation
Cryo is Greek word. (krayos –
frost)
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Cryopreservation is a non-lethal storage of biological
material at ultra-low temperature. At the
temperature of liquid nitrogen (-196 degree) almost
all metabolic activities of cells are ceased and the
sample can then be preserved in such state for
extended peroids.
However, only few biological materials can be frozen
to (-196 degree) without affecting the cell viability.
16. It can be done
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Over solid carbon dioxide (at -79 degree)
Low temperature deep freezer (at -80 degree )
In vapor phase nitrogen (at -150 degree)
In liquid nitrogen (at -196 degree)
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Why Liquid nitrogen ?
Chemically inert
Relatively low cost
Non toxic
Non flammable
Readily available
Liquid nitrogen is most widely used material for
cryopresevation.
Dry ice can also be used.
18. Mechanism of cryopreservation
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The technique of freeze preservation is based on the
transfer of water present in the cells from a liquid to
solid state.
Due to the presence of salts and organic molecules
in the cells, the cell water requires much more lower
temperature to freeze (even up to -68°C) compared
to the freezing point of pure water (around 0°C) .
When stored at low temperature , the metabolic
processes and biological deteriorations in the
cells/tissues almost come to standstill.
23. SELECTION OF PLANT MATERIAL
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Morphological and physiological conditions of
plant material influence the ability of explants to
survive during cryopreservation.
Different types of tissues can be used for
cryopreservation such as:
Ovules
Anther/pollen
Embryos
Endosperm
Protoplast, etc.
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o Callus derived from tropical plant is more
resistant to freezing damage.
o A rapidly growing stage of callus shortly after 1 or 2
weeks of subculture is best for cryopreservation.
o Old cells at the top of callus and blackened area
should be avoided.
o cultured cells are not ideal for freezing. Instead,
organized structures such as shoots apices,
embryos or young plantlets are preferred.
o Water content of cell or tissue used for
cryopreservation should be low freezable water,
tissues can withstand extremely low temperatures
26. PREGROWTH
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Pregrowth treatment protect the plant tissues
against exposure to liquid nitrogen.
Pregrowth involves the application of additives
known to enhance plant stress tolerance.
E.g.
abscisic acid
praline
trehalose
(OTHER EXAMPLES??)
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Partial tissue dehydration can be achieved by
the application of osmotically active compounds.
The addition of low concentration of DMSO (1-5%)
during pre-growth often improves shoot tip
recovery,
E.g.
C. roseus cells are precultured in medium
containing 1M sorbitol before freezing. (Chen et al.,
1984)
Digitalis cells were precultured on 6% Mannitol
medium for 3 days before freezing. (Seitz et al.,
1983)
Nicotiana sylvestris with 6% sorbitol for 2-5 days
before freezing. (Maddox et al., 1983)
28. ADDITION OF A CRYOPROTECTANT
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A cryoprotectant is a substance that is used to
protect biological tissue from freezing & thawing
damage (damage due to ice formation).
They acts like antifreeze
They lower freezing temperature
Increase viscosity and
Prevents damage to the cells.
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There are two potential sources of cell
damage during cryopreservation.
1. Formation of large ice crystals inside the
cell.
2. Intracellular concentration of solutes
increase to toxic levels before or during
freezing as a result of dehydration.
31. VITRIFICATION
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The term “vitrification” refers to any process
resulting in “glass formation”, the transformation
from a liquid to a solid in the absence of
crystallization.
According to this definition, cells that are properly
slow frozen become “vitrified”.
A process where ice formation cannot take place
because the aqueous solution is too concentrated to
permit ice crystal nucleation. Instead, water
solidifies into an amorphous ‘glassy’ state.
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Dehydration reduces the amount of water
Depresses
its freezing
temperature and
Promotes
vitrification
If cells are sufficiently dehydrated they may be able to
withstand immersion in liquid hydrogen.
Ice formationIncreases the
osmotic
pressure
34. ENCAPSULATION AND DEHYDRATION
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This involves the encapsulation of tissues in
calcium alginate beads.
Which are pre-grown in liquid culture media
containing high concentration of sucrose.
After these treatments the tissues are able to
withstand exposure to liquid nitrogen without
application of chemical cryoprotectants.
Cryoprotectants used in cryopreservation
35. FREEZING: RAPID FREEZING
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The plant material is placed in vials and plunged into
liquid nitrogen and decrease of -300 to -10000c or more
occurs.
The quicker the freezing is done , the smaller the
intracellular ice crystals are.
Dry ice can also be used in a similar manner.
This method is technically simple and easy to handle.
Rapid freezing has been employed for cryopreservation
of shoot tips of potato , strawberry , brassica species
36. SLOW FREEZING
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Tissue is slowly frozen with decrease in temperature from -0.1
to 10°c/min.
Slow cooling permits the flow of water from the cells to the
outside , thereby promoting extracellular ice formation
instead of lethal intracellular freezing.
This method has been successfully employed for
cryopreservation of meristems of peas , potato , cassava ,
strawberry etc. In a normal ice making process, the surface of
the cube freezes up much faster than the interior.
Which “cramps” the interior, clouding it.
By using very hot (and pure) water inside an insulated
environment, you are assuring yourself a very slow freezing
that allows the interior to cool down at a rate far closer to that
of the exterior, and that lack of “cramping” is what produces
such clear ice.
37. STEPWISE FREEZING
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In this method slow freezing down to -20 to 40c.
A stop for a period of approximately 30 min and
then additional rapid freezing to -196c is done by
plunging in liquid nitrogen.
Slow freezing permits protective dehydration of the
cells and rapid freezing prevents the growing of big
ice crystals.
The Stepwise freezing gives excellent results in
strawberry and with suspension cultures.
38. STORAGE
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Storage of frozen material at correct temperature is as
important as freezing.
The frozen cells/tissues are kept for storage at temperature
ranging from -70 to -196°c.
Temperature should be sufficiently low for long term
storage of cells to stop all the metabolic activities and
prevent biochemical injury.
Long term storage is best done at -196°c. …
39. THAWING
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It is done by putting ampoule containing the sample in a
warm water bath (35 to 40°c).
Frozen tips of the sample in tubes or ampoules are
plunged into the warm water with a vigorous swirling
action just to the point of ice disappearance.
It is important for the survival of the tissue that the tubes
should not be left in the warm water bath after ice melts .
just a point of thawing quickly transfer the tubes to a
water bath maintained at room temperature and
continue the swirling action for 15 sec to cool the warm
walls of the tube.
Tissue which has been frozen by
encapsulation/dehydration is frequently thawed at
ambient temperature.
40. DETERMINATION OF
SURVIVAL/VIABILITY
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Regrowth of the plants from stored tissues or cells is
the only test of survival of plant materials.
Various viability tests include Fluorescien diacetate
(FDA) staining , growth measurement by cell
number , dry and fresh weight.
Important staining methods are:
Triphenyl Tetrazolium Chloride (TTC)
Evan’s blue staining.
41. 25-Jul-16CRYOPRESERVATION-deya
Cell survival is measured by
amount of red formazan
product formed due to
reduction of TTC assay which
is measured
spectrometrically.
Only the viable cells which
contain the enzyme
mitochondrial dehydrogenase
which reduces TTC to red
formazan will be stained and
dead cells will not take up the
dye.
41
One drop of 0.1% solution of
Evan’s blue is added to cell
suspension on a microscope
slide and observed under light
microscope.
Only non viable cells (dead
cells) stain with Evan’s blue.
% of viable cells = Number of
fluorescent cells × 1oo total no
of cells(viable + non-viable).
Individual cell viability
assayed with Evan's blue dye
and fluorescein diacetate.
Staining methods
TRIPHENYL TETRAZOLIUM
CHLORIDE (TTC) ASSAY
EVAN’S BLUE STAINING
42. SEED BANK
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A seed bank stores seeds as a source for planting in case
seed reserves elsewhere are destroyed.
It is a type of gene bank.
The seeds stored may be food crops, or those of rare
species to protect biodiversity.
The reasons for storing seeds may be varied. Seeds are
dried to a moisture content of less than 5%.
The seeds are then stored in freezers at -18°C or below.
Because seed (DNA) degrades with time, the seeds need
to be periodically replanted and fresh seeds collected for
another round of long-term storage.
43. GENE BANK
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Gene banks are a type of biorepository which
preserve genetic material.
In plants, this could be by freezing cuts from the plant, or
stocking the seeds.
In animals, this is the freezing of sperm and eggs
in zoological freezers until further need.. In an effort to
conserve agricultural biodiversity, gene banks are used to
store and conserve the plant genetic resources of major
crop plants and their crop wild relatives.
There are many gene banks all over the world, with the
Svalbard Global Seed Vault being probably the most
famous one.
44. Application
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It is ideal method for long term conservation of material.
Disease free plants can be conserved and propagated.
Recalcitrant seeds can be maintained for long time.
Endangered species can be maintained.
Pollens can be maintained to increase longitivity.
Rare germplasm and other genetic manipulations can be stored.