Protoplasts are plant cells without cell walls that can be isolated and cultured. The document discusses methods for isolating protoplasts from plant tissues using either mechanical or enzymatic methods. It also describes how protoplasts can be fused using electrofusion or polyethylene glycol to create somatic hybrids. The factors affecting protoplast culture and applications such as genetic engineering and crop improvement through somatic hybridization are summarized.
This document summarizes the process of isolating plant protoplast cells. It defines a protoplast as a plant cell without its cell wall. There are two main methods for isolating protoplasts - mechanical and enzymatic. The enzymatic method uses enzymes like pectinase and cellulase to gently dissolve the cell wall and is preferred. The steps of the enzymatic isolation process are described, including incubating tissue in the enzyme solution, filtering, washing, and testing viability. Finally, some applications of isolated protoplasts in research are mentioned like transformation and somatic hybridization.
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 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.
This document discusses callus and suspension cultures. Callus culture involves culturing explants on agar medium to form an unorganized cell mass called callus. Suspension cultures involve culturing tissues or cells in liquid medium, producing single cells and clumps. There are three types of suspension cultures: batch, continuous, and immobilized. Batch cultures use a limited nutrient supply until growth declines. Continuous cultures drain out used medium and add fresh medium to maintain a steady state. Immobilized cultures encapsulate plant cells in gels like agarose.
This document provides information on various methods of gene transfer in plants, including Agrobacterium-mediated gene transfer and direct gene transfer methods. Direct methods rely on delivering large amounts of DNA to plant cells through techniques like particle bombardment, electroporation, and microinjection. Agrobacterium-mediated gene transfer utilizes the bacterium Agrobacterium, which transfers genes into plant genomes. The document discusses several direct and Agrobacterium-mediated methods in detail and provides advantages and limitations of each approach.
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.
Its about how fruit ripening occurs and how we can manipulate ripening process by using biotechnology to delay ripening and to reduce postharvest losses
This document summarizes the process of isolating plant protoplast cells. It defines a protoplast as a plant cell without its cell wall. There are two main methods for isolating protoplasts - mechanical and enzymatic. The enzymatic method uses enzymes like pectinase and cellulase to gently dissolve the cell wall and is preferred. The steps of the enzymatic isolation process are described, including incubating tissue in the enzyme solution, filtering, washing, and testing viability. Finally, some applications of isolated protoplasts in research are mentioned like transformation and somatic hybridization.
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 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.
This document discusses callus and suspension cultures. Callus culture involves culturing explants on agar medium to form an unorganized cell mass called callus. Suspension cultures involve culturing tissues or cells in liquid medium, producing single cells and clumps. There are three types of suspension cultures: batch, continuous, and immobilized. Batch cultures use a limited nutrient supply until growth declines. Continuous cultures drain out used medium and add fresh medium to maintain a steady state. Immobilized cultures encapsulate plant cells in gels like agarose.
This document provides information on various methods of gene transfer in plants, including Agrobacterium-mediated gene transfer and direct gene transfer methods. Direct methods rely on delivering large amounts of DNA to plant cells through techniques like particle bombardment, electroporation, and microinjection. Agrobacterium-mediated gene transfer utilizes the bacterium Agrobacterium, which transfers genes into plant genomes. The document discusses several direct and Agrobacterium-mediated methods in detail and provides advantages and limitations of each approach.
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.
Its about how fruit ripening occurs and how we can manipulate ripening process by using biotechnology to delay ripening and to reduce postharvest losses
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.
Presented by- MD JAKIR HOSSAIN
Doctoral Research Scholar
Department of Agricultural Genetic Engineering ,
Faculty of Agricultural Sciences and Technologies,
Nigde Omer Halisdemir University, Turkey
E. Mail- mjakirbotru@gmail.com
1) Germplasm conservation involves preserving genetic material, such as seeds, cells, tissues, and body parts, through in-situ and ex-situ methods to maintain biodiversity and provide resources for breeding programs.
2) Cryopreservation at ultra-low temperatures in liquid nitrogen is an important ex-situ technique that can preserve germplasm long-term without subculturing. It involves preculturing plant materials, treating with cryoprotectants, and either slow-freezing or vitrification prior to storage in liquid nitrogen.
3) A case study demonstrates the successful cryopreservation of mint shoot tips using encapsulation-dehydration and PVS2-vitrification, with
This document provides an overview of various gene transformation techniques, including both vector-mediated and direct methods. It discusses natural transformation mechanisms like conjugation and transduction, as well as artificial vector-mediated techniques like Agrobacterium-mediated transformation. Direct methods like microinjection, electroporation, particle bombardment, and chemical methods using PEG or calcium phosphate are also covered. The applications, advantages, and limitations of different techniques are summarized. Overall, the document serves as an informative introduction to the key gene transfer methods used in plant biotechnology.
This document discusses 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.
Chloroplasts can be genetically engineered through transformation because they have their own genetic systems and DNA located in structures called chloroplast nucleoids. Transformation involves delivering a chloroplast-specific expression vector containing the gene of interest, flanked by chloroplast DNA sequences to facilitate homologous recombination-mediated integration into the chloroplast genome. Successful methods for DNA delivery include biolistics and polyethylene glycol-mediated transformation, with biolistics being preferred. Transformed chloroplasts are called transplastomes and confer advantages like high-level expression of foreign genes without risks of transgene escape or gene silencing seen in nuclear transformation.
Somatic hybridization is a technique used to create hybrid plants by fusing isolated plant cells called protoplasts from two different plant species or varieties. This fusion occurs under in vitro conditions and can result in symmetric hybrids that contain chromosomes from both parents, or asymmetric hybrids that lose chromosomes from one parent. Cybrids are a type of hybrid where the nucleus comes from one species but the cytoplasm, including chloroplasts and mitochondria, comes from both parental species. Somatic hybridization and cybrid production allow for novel combinations of genes that can provide agricultural benefits like stress resistance but technical challenges remain in regenerating hybrid plants.
L21. techniques for selection, screening and characterization of transformantsRishabh Jain
This document discusses techniques for selecting and characterizing transformed cells containing recombinant DNA. Selectable marker genes allow transformed cells to survive in the presence of a selective agent, while reporter genes produce detectable gene products for identification. Common selectable markers include genes conferring resistance to neomycin or glyphosate. Common reporter genes encode enzymes like beta-galactosidase. Transformed clones can be identified through nucleic acid hybridization, PCR, or by detecting expressed protein products via techniques like western blotting.
1.What is plant tissue culture?
2.Production of virus free plants.
3.History.
4.Virus elimination by heat treatment.
5.Virus elimination by Meristem Tip culture.
6.Factor affecting virus eradication by Meristem Tip culture.
7.Chemotherapy.
8.Virus elimination through in vitro shoot-tip Grafting.
9.Virus Indexing.
10.Conclusion .
11.References .
Genetic engineering involves directly manipulating an organism's DNA using biotechnology. The DNA of interest is isolated from a source organism and inserted into a vector, which is then introduced into a host cell. Common vectors include plasmids, bacteriophages, cosmids, phagemids, and artificial chromosomes. Artificial chromosomes, such as Bacterial Artificial Chromosomes and Yeast Artificial Chromosomes, can carry large DNA fragments of up to 300,000 base pairs, making them useful for cloning and transforming large genes. However, constructing and maintaining artificial chromosomes can be challenging due to their size and potential for rearrangements.
This document discusses methods for producing haploid plants. It begins by defining haploid plants and their significance. It then describes the two main approaches for producing haploids - in vivo and in vitro. For in vivo, it outlines several techniques including androgenesis, gynogenesis, distant hybridization, and chemical/radiation treatments. For in vitro, it focuses on anther culture and microspore culture, providing details on the protocol for anther culture in tobacco including pre-treatment, culture conditions, and factors that influence success rates.
Androgenesis is the production of haploid plants through the culture of male gametophytes or microspores. There are two main methods - anther culture and isolated pollen/microspore culture. Anther culture involves excising anthers from flower buds and culturing them on nutrient media, while microspore culture isolates microspores from anthers. Several factors influence androgenesis success, including genotype, anther wall components, culture medium, growth regulators, and physical conditions. Androgenic haploids can develop directly from microspores or indirectly through a callus phase, following various developmental pathways. Androgenesis allows for the efficient production of haploid plants for breeding programs.
1. Callus culture involves growing undifferentiated plant cells and tissues on a nutrient medium under sterile conditions. This allows for the production of genetically identical clones without seeds or pollination.
2. A callus is an unorganized mass of cells formed from injured or cultured plant tissue. Successful callus culture requires selecting an explant, preparing sterile culture media, and regulating hormone levels to induce cell proliferation.
3. Callus cultures are maintained through periodic sub-culturing to replenish nutrients and prevent toxicity. The growth and characteristics of callus tissue can provide insights into plant cell metabolism, differentiation, and pathways for genetic engineering applications.
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.
Organellar genomes, such as those found in mitochondria and chloroplasts, can be manipulated. The mitochondria genome is maternally inherited and contains genes that code for proteins involved in respiration. The chloroplast genome is also maternally inherited and contains genes for photosynthesis-related proteins. Methods to transform these genomes include particle bombardment, PEG-mediated transformation, and Agrobacterium-mediated transformation. Manipulating organellar genomes has applications for crop improvement like developing cytoplasmic male sterility.
This document provides an overview of Agrobacterium-mediated gene transformation. It begins with an introduction to genetic transformation methods, including direct and indirect techniques. It then discusses Agrobacterium, including its classification, the history of using it for gene transformation, and features of its T-DNA and virulence genes. The document outlines the process of T-DNA transfer from Agrobacterium to plant cells. Finally, it describes some common methods for Agrobacterium-mediated gene transfer, such as infection through wounds, leaf disk, and co-cultivation techniques.
Organogenesis is the process by which plant organs like roots, shoots, flowers develop from explants. It is influenced by physical factors like light and temperature, and chemical factors like cytokinins and auxins. Organogenesis has advantages like being cheap, fast, and allowing for easy scaling up. Somatic hybridization involves fusing protoplasts from two plant species using techniques like PEG-mediated fusion. It allows for transferring genes between sexually incompatible plants but has challenges like low regeneration rates and hybrid viability. It differs from organogenesis in producing bipolar embryos without connections to parent tissue.
This document discusses embryo culture and embryo rescue techniques. It begins by defining an embryo and explaining that embryo culture involves growing plant embryos in artificial media to enhance survival. Embryo rescue involves culturing immature embryos to prevent abortion, especially for interspecific or intergeneric hybrids where endosperm development fails. The key steps of embryo culture include excising embryos, placing them in sterile media with suitable temperature, light and nutrients, and transferring viable plantlets to soil. Embryo culture has applications in shortening breeding cycles, overcoming dormancy, producing sterile seeds, and rescuing distant hybrids.
Protoplasts are plant cells that have had their cell walls removed, leaving the plasma membrane as the outermost layer. Protoplasts can be isolated from plant tissues using mechanical or enzymatic methods and cultured. During protoplast culture, the cells regenerate cell walls and can fuse with other protoplasts using techniques like PEG-induced fusion or electrofusion. Protoplast fusion allows for the transfer of genes between species and is used in crop improvement. Factors like plant species, age, and culture conditions affect successful protoplast isolation and culture.
Protoplasts are plant cells that have had their cell walls removed, leaving the plasma membrane as the outermost layer. Protoplasts can be isolated from plant tissues using mechanical or enzymatic methods and cultured. During protoplast culture, the cells regenerate cell walls and can fuse with other protoplasts using techniques like PEG-induced fusion or electrofusion. Protoplast fusion allows for the transfer of genes between species and is used in crop improvement. Factors like plant species, age, and culture conditions affect successful protoplast isolation and culture.
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.
Presented by- MD JAKIR HOSSAIN
Doctoral Research Scholar
Department of Agricultural Genetic Engineering ,
Faculty of Agricultural Sciences and Technologies,
Nigde Omer Halisdemir University, Turkey
E. Mail- mjakirbotru@gmail.com
1) Germplasm conservation involves preserving genetic material, such as seeds, cells, tissues, and body parts, through in-situ and ex-situ methods to maintain biodiversity and provide resources for breeding programs.
2) Cryopreservation at ultra-low temperatures in liquid nitrogen is an important ex-situ technique that can preserve germplasm long-term without subculturing. It involves preculturing plant materials, treating with cryoprotectants, and either slow-freezing or vitrification prior to storage in liquid nitrogen.
3) A case study demonstrates the successful cryopreservation of mint shoot tips using encapsulation-dehydration and PVS2-vitrification, with
This document provides an overview of various gene transformation techniques, including both vector-mediated and direct methods. It discusses natural transformation mechanisms like conjugation and transduction, as well as artificial vector-mediated techniques like Agrobacterium-mediated transformation. Direct methods like microinjection, electroporation, particle bombardment, and chemical methods using PEG or calcium phosphate are also covered. The applications, advantages, and limitations of different techniques are summarized. Overall, the document serves as an informative introduction to the key gene transfer methods used in plant biotechnology.
This document discusses 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.
Chloroplasts can be genetically engineered through transformation because they have their own genetic systems and DNA located in structures called chloroplast nucleoids. Transformation involves delivering a chloroplast-specific expression vector containing the gene of interest, flanked by chloroplast DNA sequences to facilitate homologous recombination-mediated integration into the chloroplast genome. Successful methods for DNA delivery include biolistics and polyethylene glycol-mediated transformation, with biolistics being preferred. Transformed chloroplasts are called transplastomes and confer advantages like high-level expression of foreign genes without risks of transgene escape or gene silencing seen in nuclear transformation.
Somatic hybridization is a technique used to create hybrid plants by fusing isolated plant cells called protoplasts from two different plant species or varieties. This fusion occurs under in vitro conditions and can result in symmetric hybrids that contain chromosomes from both parents, or asymmetric hybrids that lose chromosomes from one parent. Cybrids are a type of hybrid where the nucleus comes from one species but the cytoplasm, including chloroplasts and mitochondria, comes from both parental species. Somatic hybridization and cybrid production allow for novel combinations of genes that can provide agricultural benefits like stress resistance but technical challenges remain in regenerating hybrid plants.
L21. techniques for selection, screening and characterization of transformantsRishabh Jain
This document discusses techniques for selecting and characterizing transformed cells containing recombinant DNA. Selectable marker genes allow transformed cells to survive in the presence of a selective agent, while reporter genes produce detectable gene products for identification. Common selectable markers include genes conferring resistance to neomycin or glyphosate. Common reporter genes encode enzymes like beta-galactosidase. Transformed clones can be identified through nucleic acid hybridization, PCR, or by detecting expressed protein products via techniques like western blotting.
1.What is plant tissue culture?
2.Production of virus free plants.
3.History.
4.Virus elimination by heat treatment.
5.Virus elimination by Meristem Tip culture.
6.Factor affecting virus eradication by Meristem Tip culture.
7.Chemotherapy.
8.Virus elimination through in vitro shoot-tip Grafting.
9.Virus Indexing.
10.Conclusion .
11.References .
Genetic engineering involves directly manipulating an organism's DNA using biotechnology. The DNA of interest is isolated from a source organism and inserted into a vector, which is then introduced into a host cell. Common vectors include plasmids, bacteriophages, cosmids, phagemids, and artificial chromosomes. Artificial chromosomes, such as Bacterial Artificial Chromosomes and Yeast Artificial Chromosomes, can carry large DNA fragments of up to 300,000 base pairs, making them useful for cloning and transforming large genes. However, constructing and maintaining artificial chromosomes can be challenging due to their size and potential for rearrangements.
This document discusses methods for producing haploid plants. It begins by defining haploid plants and their significance. It then describes the two main approaches for producing haploids - in vivo and in vitro. For in vivo, it outlines several techniques including androgenesis, gynogenesis, distant hybridization, and chemical/radiation treatments. For in vitro, it focuses on anther culture and microspore culture, providing details on the protocol for anther culture in tobacco including pre-treatment, culture conditions, and factors that influence success rates.
Androgenesis is the production of haploid plants through the culture of male gametophytes or microspores. There are two main methods - anther culture and isolated pollen/microspore culture. Anther culture involves excising anthers from flower buds and culturing them on nutrient media, while microspore culture isolates microspores from anthers. Several factors influence androgenesis success, including genotype, anther wall components, culture medium, growth regulators, and physical conditions. Androgenic haploids can develop directly from microspores or indirectly through a callus phase, following various developmental pathways. Androgenesis allows for the efficient production of haploid plants for breeding programs.
1. Callus culture involves growing undifferentiated plant cells and tissues on a nutrient medium under sterile conditions. This allows for the production of genetically identical clones without seeds or pollination.
2. A callus is an unorganized mass of cells formed from injured or cultured plant tissue. Successful callus culture requires selecting an explant, preparing sterile culture media, and regulating hormone levels to induce cell proliferation.
3. Callus cultures are maintained through periodic sub-culturing to replenish nutrients and prevent toxicity. The growth and characteristics of callus tissue can provide insights into plant cell metabolism, differentiation, and pathways for genetic engineering applications.
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.
Organellar genomes, such as those found in mitochondria and chloroplasts, can be manipulated. The mitochondria genome is maternally inherited and contains genes that code for proteins involved in respiration. The chloroplast genome is also maternally inherited and contains genes for photosynthesis-related proteins. Methods to transform these genomes include particle bombardment, PEG-mediated transformation, and Agrobacterium-mediated transformation. Manipulating organellar genomes has applications for crop improvement like developing cytoplasmic male sterility.
This document provides an overview of Agrobacterium-mediated gene transformation. It begins with an introduction to genetic transformation methods, including direct and indirect techniques. It then discusses Agrobacterium, including its classification, the history of using it for gene transformation, and features of its T-DNA and virulence genes. The document outlines the process of T-DNA transfer from Agrobacterium to plant cells. Finally, it describes some common methods for Agrobacterium-mediated gene transfer, such as infection through wounds, leaf disk, and co-cultivation techniques.
Organogenesis is the process by which plant organs like roots, shoots, flowers develop from explants. It is influenced by physical factors like light and temperature, and chemical factors like cytokinins and auxins. Organogenesis has advantages like being cheap, fast, and allowing for easy scaling up. Somatic hybridization involves fusing protoplasts from two plant species using techniques like PEG-mediated fusion. It allows for transferring genes between sexually incompatible plants but has challenges like low regeneration rates and hybrid viability. It differs from organogenesis in producing bipolar embryos without connections to parent tissue.
This document discusses embryo culture and embryo rescue techniques. It begins by defining an embryo and explaining that embryo culture involves growing plant embryos in artificial media to enhance survival. Embryo rescue involves culturing immature embryos to prevent abortion, especially for interspecific or intergeneric hybrids where endosperm development fails. The key steps of embryo culture include excising embryos, placing them in sterile media with suitable temperature, light and nutrients, and transferring viable plantlets to soil. Embryo culture has applications in shortening breeding cycles, overcoming dormancy, producing sterile seeds, and rescuing distant hybrids.
Protoplasts are plant cells that have had their cell walls removed, leaving the plasma membrane as the outermost layer. Protoplasts can be isolated from plant tissues using mechanical or enzymatic methods and cultured. During protoplast culture, the cells regenerate cell walls and can fuse with other protoplasts using techniques like PEG-induced fusion or electrofusion. Protoplast fusion allows for the transfer of genes between species and is used in crop improvement. Factors like plant species, age, and culture conditions affect successful protoplast isolation and culture.
Protoplasts are plant cells that have had their cell walls removed, leaving the plasma membrane as the outermost layer. Protoplasts can be isolated from plant tissues using mechanical or enzymatic methods and cultured. During protoplast culture, the cells regenerate cell walls and can fuse with other protoplasts using techniques like PEG-induced fusion or electrofusion. Protoplast fusion allows for the transfer of genes between species and is used in crop improvement. Factors like plant species, age, and culture conditions affect successful protoplast isolation and culture.
This document discusses protoplast fusion techniques in plant tissue culture. It begins by defining a protoplast as a naked plant cell without a cell wall. The key steps discussed are:
1) Isolating protoplasts from plant tissue using either mechanical or enzymatic methods. Enzymatic isolation using cellulase, pectinase and hemicellulase is preferred.
2) Fusing the protoplasts using techniques like electrofusion, PEG fusion or high pH/Ca2+ solutions.
3) Identifying and selecting hybrid cells using markers like pigmentation, chloroplast presence or nuclear staining.
4) Culturing the hybrid cells and regenerating hybrid plants
Protoplast is a naked cell (without cell wall) surrounded by a plasma membrane. It can regenerate cell wall, grow and divide.
Spheroplast cells have their cell wall only partially removed.
Is fragile but can be cultured and grow into a whole plant.
Cells can originate from any type of tissue (Mesophyll tissue - most suitable source ).
Can be applied in somatic hybridization.
Can be applied in biotechnology and microbiology.
Somatic hybridization is the development of hybrid plants through the fusion of somatic protoplasts of two different plant species/ varieties.
Somatic Hybridization was firstly introduced by Carlson in Nicotiana
glauca.
In 1960, E.C Cocking contributed to the enzymatic isolation and culture of protoplast.
Protoplast fusion involves isolating plant cells called protoplasts that have had their cell walls removed. This allows the fusion of protoplasts from different plant species using techniques like PEG or electrofusion. The fused protoplasts can regenerate into hybrid plants. Protoplast fusion is used for plant breeding to create hybrids of sexually incompatible species. It provides a way to combine genomes and study gene expression and inheritance. However, the process of isolating intact protoplasts can be challenging and yields may be low.
Protoplasm fusion involves removing the cell wall from plant or bacterial cells through enzymatic digestion to create protoplasts. Protoplasts can be fused through spontaneous fusion during isolation or induced fusion using various methods like mechanical manipulation, chemical fusogens, or electricity. Successful fusion results in a hybrid cell containing genetic material from both parent cells. Protoplast fusion allows for combining genes from sexually incompatible species and is useful for plant breeding and genetic engineering applications.
This document provides an overview of plant tissue culture techniques, including micropropagation and protoplast fusion. It discusses the various stages of micropropagation (initiation, multiplication, rooting, acclimatization). Explants can be surface sterilized and cultured on nutrient media. Protoplast fusion involves isolating protoplasts enzymatically, fusing them using techniques like PEG or electrofusion, and regenerating hybrid plants. The hybrids can be identified based on markers and ability to regenerate. Micropropagation allows mass production of clones while protoplast fusion enables hybridization of distantly related species.
The document discusses protoplast fusion, which involves removing the cell walls of plant cells to create naked protoplasts that can then be fused. It describes how to isolate protoplasts from plant tissues using either mechanical or enzymatic methods. The fusion of protoplasts from different species or varieties can create hybrid cells called heterokaryons or hybrids. Techniques to induce protoplast fusion include treatment with polyethylene glycol (PEG), calcium ions, electricity, or sodium nitrate. Successful somatic hybridization follows a procedure of isolating, fusing, regenerating cell walls, and selecting hybrid plant cells and colonies. Applications of protoplast fusion include combining genomes of sterile plants and
Somatic hybridization and Protoplast IsolationPABOLU TEJASREE
The document discusses protoplast isolation and somatic hybridization. It defines somatic hybridization as the development of hybrid plants through the fusion of somatic protoplasts from different plant species or varieties. The key steps in somatic hybridization are isolating protoplasts, fusing the protoplasts from desired species, identifying and selecting hybrid cells, culturing the hybrid cells, and regenerating hybrid plants. Protoplasts can be isolated using enzymatic or mechanical methods, and their viability and ability to form cell walls can be tested. Various factors like enzyme concentration, temperature, and pH affect protoplast isolation and viability. Protoplast fusion can occur spontaneously or be induced using methods like chemical treatment, electro
Protoplast fusion involves removing the cell walls of plant cells through enzymatic or mechanical means to create naked protoplasts. These protoplasts can then be fused using chemicals, electricity, or other methods. This allows the cytoplasms and sometimes nuclei of different plant cells to merge, creating hybrid cells. Successful fusion can generate hybrid plants through regeneration of cell walls and tissues. Protoplast fusion overcomes sexual incompatibility and is used to introduce traits like disease resistance between species. It remains a technically challenging process with limitations like genetic instability and uncertain expression of transferred traits.
This document provides an introduction to somatic hybridization, which is the fusion of somatic protoplasts from two different plant species or varieties to create hybrid plants. It discusses the history and development of the technique, including early work isolating protoplasts. The key steps in somatic hybridization are described: isolating protoplasts using either mechanical or enzymatic methods, fusing the protoplasts spontaneously or through induction methods, identifying and selecting hybrid cells, culturing the hybrid cells, and regenerating hybrid plants. Advantages include creating novel hybrids and transferring desirable traits between species, while limitations include low regeneration rates and viability of fused products.
Protoplasts are plant cells that have had their cell walls removed, allowing for genetic manipulation and fusion. The document details methods for isolating protoplasts from plant tissues using either mechanical or enzymatic methods. Once isolated, protoplast viability and density can be determined before attempting to culture the protoplasts and induce cell wall regeneration. Applications include somatic hybridization through protoplast fusion to transfer genes between species.
The document discusses plant protoplast isolation, purification, and culturing. Some key points:
- Protoplasts are plant cells that have had their cell walls removed, leaving just the plasma membrane. They allow for plant cell fusion and regeneration.
- Protoplasts are typically isolated from plant tissues like leaves using enzymatic digestion with cellulase and pectinase. This yields more protoplasts than mechanical methods.
- Isolated protoplasts are purified by centrifugation and washing to remove cell debris. They are then cultured in liquid or solid nutrient media and tested for viability before regeneration.
This document summarizes the process of isolating plant protoplast cells. It defines a protoplast as a plant cell without its cell wall. There are two main methods for isolating protoplasts - mechanical and enzymatic. The enzymatic method uses enzymes like pectinase and cellulase to gently dissolve the cell wall and is preferred. The steps of the enzymatic isolation process are described, including incubating tissue in the enzyme solution, filtering, washing, and testing viability. Finally, some applications of isolated protoplasts in research are mentioned like transformation and somatic hybridization.
This document summarizes a seminar on protoplast culture. It defines protoplasts as plant cells without cell walls that are capable of regenerating cell walls and dividing. The document outlines the general procedure for protoplast culture, including isolating protoplasts enzymatically from plant tissues, culturing them in nutrient media, testing viability, and regenerating plants from protoplast-derived callus tissue. The importance of protoplast culture is mentioned as a tool for crop improvement through somatic hybridization and genetic engineering.
Protoplasts are naked plant cells without the cell wall, but they possess plasma membrane and all other cellular components. They represent the functional plant cells but for the lack of the barrier, cell wall. Protoplasts of different species can be fused to generate a hybrid and this process is referred to as somatic hybridization (or protoplast fusion). Cybridization is the phenomenon of fusion of a normal protoplast with an enucleated (without nucleus) protoplast that results in the formation of a cybrid or cytoplast (cytoplasmic hybrids).
Protoplasts are naked plant cells that have had their cell walls removed through enzymatic treatment. This document outlines methods for isolating and culturing protoplasts from plant tissues like leaves. Protoplasts are isolated using cell wall degrading enzymes like cellulase and pectinase, and stabilized in an osmotic solution like mannitol. Various culture techniques exist, including agar embedding and hanging drop cultures. Protoplasts can regenerate new cell walls and divide, or be fused with other protoplasts through techniques like PEG fusion to create somatic hybrids for applications like transferring disease resistance between plant species.
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3. Introduction - The Protoplast
● Is a naked cell surrounded by a plasma membrane. It can
regenerate cell wall, grow and divide.
● Is fragile but can be cultured and grow into a whole plant.
● Cells can originate from any type of tissue.
● Is important for crop improvement.
● Via somatic hybridisation, new hybrids are produced
● Is used by biotechnologists, microbiologists, pathologists,...
● Somatic Hybridization was firstly introduced by Carlson in
Nicotiana 'glauea'
● In 1960, E.C Cocking contributed to the enzymatic isolation
and culture of protoplast
4. Protoplast isolation
● Refers to the separation of protoplast from plant tissue
● Important to isolate viable and uninjured protoplast as gently
and as quickly as possible
● Involves two methods:
○ Mechanical
○ Enzymatic
5. Mechanical method
● Tissue is immersed in 1.0
M sucrose until
protoplasm shrunk away
from their enclosing cell
wall (Plasmolysis)
● Plasmolysed tissue is cut
with a sharp knife at such
a thickness that only cell
walls are cut
Plasmolysed cell
6. Mechanical method
● Undamaged protoplast in
strips are released by
osmotic swelling when
placed in a low
concentration of sucrose
solution
● Problem encountered:
some cells release uncut
complete protoplast
while the rest produces
broken dead protoplasts.
Low [sucrose soln]
7. Enzymatic method
● Refers to the use of enzymes to dissolve the cell wall for
releasing protoplasts
● Advantages:
○ Used for variety of tissues and organs such as fruits, roots,
petioles, leaves…
○ Osmotic shrinkage is minimum
○ Cells remain intact and not injured
○ Protoplast readily obtained
● Involves two methods:
○ Direct method (One step only)
○ Sequential method (Two step method)
8. 1. Direct method
● Incubation of leaf segments overnight in enzyme solution
● Mixture is filtered and centrifuged
● Protoplast forms pellet
● Then washed with sorbitol and re-centrifuged
● Clean protoplasts float
● They are pipetted out
9. 2. Sequential method
● Two enzyme mixtures(mixture A and mixture B) are used one
after the other
● Leaf segments with mixture A (Macerozyme in manifold at
pH 5.8) are vacuumed infiltrated for 5 mins, transferred to a
water bath at 25°C and subjected to slow shaking
● The enzyme mixture is then replaced by fresh ‘enzyme
mixture A’ and leaf segments are incubated for another hour
10. 2. Sequential method
● The mixture is filtered using nylon mesh and centrifuged for 1
min
● Washed 3 times with 13% mannitol
● Cells are then incubated with ‘enzyme mixture B’ (Cellulase
in mannitol solution at pH 5.4) for above 90 mins at 30°C
● The mixture is centrifuged for 1 min so that protoplast form a
pellet and clean 3 times with sorbitol
11. Purification of protoplast
● Protoplasts are purified by removing:
○ Undigested material (debris)
○ Bursts protoplasts
○ Enzymes
● Debris are removed by filtering the preparation through a
nylon mesh
● Enzymes are removed by centrifugation whereby the
protoplasts settle to the bottom of the tube and the
supernatant removed with the help of a pipette
● Intact protoplasts are separated from broken protoplasts
through centrifugation and removed by a pipette as they are
collected at the top of tube
12. Protoplast Culture
● Isolated protoplast can be cultured in an appropriate medium
to reform cell wall and generate callus
● Optimal culture conditions:
1. Optimal density to the culture.
2. Optimal auxin to cytokinin ratio, glucose and sucrose.
3. Maintain osmoprotectant in the medium
4. Temperature: 20-28°C
pH: 5.5-5.9
0.25% Casein hydrolysate
BAP and NAA
13. Culture of protoplasts
● Protoplasts cultured in suitable nutrient media first generate a
new cell wall
● The formation of a complete cell with a wall is followed by an
increase in size, number of cell organelles, and induction of
cell division
● The first cell division may occur within 2 to 7 days of culture
● Resulting in small clumps of cell, also known as micro
colony, within 1 to 3 weeks
14. Culture of protoplasts (continued)
● From such clumps, there are two routes to generate a
complete plant (depending on the species)
1. Plants are regenerated through organogenesis from callus
masses
2. The micro calli can be made to develop into somatic
embryos, which are then converted into whole plant through
germination
15. Importance of Protoplast Culture
● Study of Osmotic behavior
● Study of IAA action
● Study of Plasmalemma
● Study of Cell wall formation
● Organelle isolation
● Study of Morphogenesis
● Virus uptake and replication
● Study of photosynthesis
● Gene Transfer
17. Factors affecting protoplast culture
1. Plant species and varieties
● Small genetic difference leads to varying protoplast
responses to culture conditions
2. Plant age and organ
● Age of donor plant and its developmental stage
○ Stages are germinating embryos, plantlets, leaves…
3. Pre-culture conditions
● Protoplast culture are highly influenced by climatic factors
and different culture of seedlings yields protoplast having
different responses when cultured
18. Factors affecting protoplast culture
4. Pre-treatment to the tissue,before isolating protoplasts
● Cold treatment,plasmolysis and hormone increases the
chance of recovery of viable protoplasts and their plating
efficiency
5. Density
● Crucial as it influences plating efficiency and surviving of
protoplasts. At higher density, protoplasts compete with one
another while at lower density losses of metabolites from
protoplasts is more.
20. Electrofusion
● Mild electrical stimulation is being used to fuse protoplasts.
● Two glass capillary microelectrode are placed in contact with the
protoplasts.
● An electric field of low strength give rise to dielectrophoretic pole
generation within the protoplast suspension.
● This leads to pearl chain arrangement of protoplasts.
● No of protoplasts within a pearl chain depends upon the population
density of the protoplast and the distance between the electrodes.
● Subsequent application of high intensity electric impulse for some
microseconds results in the electric breakdown of membrane and
subsequent fusion.
● There are two types of current that are used here:
○ A.C current (fq=0.5-5.0 MHz and field strength=50-300V/cm)
○ D.C current (t=5-50 us and field strength=500-1000V/cm)
● Apply A.C-->Pearl chain-->D.C-->Pores form-->Fusion-->A.C
22. PEG (Polyethylene glycol) Fusion
● This chemical has been identified as a possible/potent fusogen.
● It has a high molecular weight about 1500-6000.
● Usually a PEG solution of about 28-50% is used for protoplast
fusion.
● This polymer binds to the lipid membrane of cells and thus induces
fusion
● Fusion takes place for 45 min in
incubation at r.t.p
● Fusion takes place slowly during
elusion of PEG with liquid culture
medium.
24. Application of Protoplast
Protoplasts can be used:
● In the production of Cybrid
● For Somatic Hybridization to overcome sexually
incompatible species
● Ingesting “Foreign” material into cytoplasm
● For DNA transformation
● Used to study wall synthesis and decomposition
● Studied as Single Cell System
25. - Production of Cybrid
Cybrid contain nuclear and cytoplasmic genome of one parent
and only the cytoplasmic genome of the second.
26. - Somatic Hybridization
Fusion of protoplast that facilitates the mixing of 2 whole
genomes and could be exploited in crosses at:
intergeneric, interkingdom and interspecific levels
Somatic hybridization is used to produce hybrids from sexually
incompatible species.
This method could also be used to study selection procedures.
28. Limitations of Somatic Hybridization
1. Intergeneric crosses between widely related plants which
are not compatible sexually are not possible.
2. In certain wide crosses, elimination of chromosomes from
hybrid cell is another limitation of somatic hybridisation.
3. In protoplast fusion experiments, the percentage of fusion
between two different parental protoplast is very low.
4. For hybrid identification, selection and isolation at the
culture level, there is no standardized method which is
applicable for all material.
29. Ingestion of foreign material
Protoplast being wall-less show high pinocytic activity and can
ingest biological active foreign bodies such as
DNA, plasmids, bacteria , viruses etc.
➢ results into modified cells.
Advantageous to plant breeder in getting more efficient crop
varieties in near future.
30. Advantages of Protoplast fusion
1. It facilitates the mixing of two genomes and can be used in
crosses at interspecific, intergeneric or even intraspecific
levels
2. To create new strains with desired properties and for strain
improvement
3. Mixing two genomes opens the door to gene transfer and a
study of gene expression, stability of several traits and cell
genetic changes
31. Disadvantages of Protoplast Fusion
During the mechanical method of isolation of protoplasts:
1. It yields a very small amount of protoplasts after a rather
tedious procedure
2. It is not suitable for isolating protoplasts from meristematic
and less vacuolated cells
● During and subsequent to digestion of the cell wall, the
protoplast becomes very sensitive to osmotic stress. Thus,
cell wall and protoplast storage must be done in an isotonic
solution to prevent rupture of the plasma membrane
33. Protoplast - Up to now
● Transfer of useful genes from one species to another.
● Technique used in strain improvement for bringing genetic
recombinations.
● Technique used in microbial engineering; new strains
produced for industrial purposes.
● Protoplast fusion used by:
○ Molecular biologists
○ Biotechnologists
○ Tissue Culturists
○ Industries
34. Alcohol Production
● Creation of new strain of Saccharomyces from
S.cerevisiae and T.reesei
○ Alcohol production- up to 80% from cellulose
material
○ Less dependent on Fossil Fuels
● Strains from S.uvarum
○ Can produce low carbohydrate beer of acceptable
flavour
○ Production of Better beer
35. Crop Production
● Palm Oil production (Elaeis guineensis, tenera oil palm)
○ Largest source of edible oil before soybean oil in the
world, contributes 31.8% of the world’s production of oils
and fats
○ Takes 8 years to fully grow and produce fruits.
○ Protoplast culture helps to grow the trees faster to satisfy
the increasing demands. Plantlets obtained within 2
years!
● Citrus
○ Subtropical fruit crop in the world
○ C. sinensis & C. reticulata = New Seedless citrus fruit
(Cybrids) Faster than c.breeding
36. Crop Production (cntd)
● Date fruit production (Phoenix dactylifera L.)
○ Deglet nour and Takerboucht (2 varieties)
○ Deglet nour-excellent fruit quality
○ Takerboucht-resistant against “bayoud” disease
○ Combination to give new strain- Protoplast fusion
● Potato production
○ Solanum tuberosum & S.brevidens (incompatible)
○ Protoplast fusion to introduce resistance to potato leaf roll
virus.
○ Hybrids showed resistance and were female fertile.
37. Conclusion
In a nutshell, protoplast culture has found its many uses and
applications in fields such as genetic engineering and crop
breeding. Genetic transformation by introduction of trans-gene
DNA, somatic hybridization by protoplast fusion of species or
subspecies resistant to traditional cross-breeding, and isolation
of sub-cellular organelles, are all examples of how the
development of protoplast systems has led to an increase in the
versatility of plants.
38. References
● http://en.wikipedia.org/wiki/Protoplast - 8 Nov 2013
● http://en.wikipedia.org/wiki/Somatic_fusion - 10 Nov 2013
● www.sciencedirect.com - 12 Nov
● http://books.google.mu/books?
id=BEH1czIoVAgC&pg=PA62&lpg=PA62&dq=protoplast+fusion+co
st&source=bl&ots=omqLqZrWt1&sig=yv3-
3Z7MJTITM5M8cp0fFBf9GuY&hl=en&sa=X&ei=QxmCUsDEC4eCr
AfnyoG4AQ&ved=0CE8Q6AEwBA#v=onepage&q&f=false - 12 Nov
2013
● http://agriinfo.in/default.aspx?page=topic&superid=3&topicid=1948
- 12 Nov 2013
● http://www.slideshare.net/naghmashehzadi/presentation-of-plant-
protoplasts?from_search=4 -12 nov 2013
● http://agriinfo.in/default.aspx?page=topic&superid=3&topicid=1946
12 Nov 2013