1) The document describes a seminar presentation on protoplast isolation and somatic hybridization.
2) Key points include the history of protoplast isolation, properties of protoplasts, protocols for isolation and fusion, applications including generating novel hybrids, and limitations.
3) Examples are provided to illustrate identification and characterization of somatic hybrids using morphological, cytological, biochemical and molecular techniques.
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.
Morphogenesis, organogenesis, embryogenesis & other techniquesHORTIPEDIA INDIA
The document describes the process of somatic embryogenesis. It involves 7 key steps:
1) Induction of embryogenesis from explant tissue on media supplemented with auxin
2) Development of somatic embryos through globular, heart, and torpedo stages of growth
3) Maturation of embryos with the formation of root and shoot meristems and cotyledons
4) Conversion of mature embryos to plantlets through germination on auxin-free media
Factors like explant type, growth regulators, and genotype influence the process. Somatic embryos differ from zygotic embryos in lacking a seed coat and having greater potential for propagation but weaker plantlets.
The isolation, culture and fusion of protoplasts is a fascinating field in plant research. Protoplast isolation and their cultures provide millions of single cells (comparable to microbial cells) for a variety of studies.
1. Acclimatization is the process by which plants adapt to changes in their environment over multiple generations through natural selection.
2. It requires genetic variability in introduced plant materials and occurs more readily in cross-pollinated species and annual crops.
3. Examples of acclimatization include humans developing more red blood cells at high altitudes and plants surviving freezing temperatures if the temperature drops gradually over time rather than suddenly.
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.
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 methods for producing haploid plants through gynogenesis, or haploid production from female gametes. It describes two main methods - ovary culture and ovule culture - for inducing gynogenesis in vitro. Key steps include excising ovaries from plants and culturing them on nutrient media supplemented with hormones to induce parthenogenesis. Successful gynogenesis has been achieved in several plant families, with the frequency of responsive ovules typically being low, around 1-5%. Producing haploid plants provides benefits for genetic studies.
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.
Morphogenesis, organogenesis, embryogenesis & other techniquesHORTIPEDIA INDIA
The document describes the process of somatic embryogenesis. It involves 7 key steps:
1) Induction of embryogenesis from explant tissue on media supplemented with auxin
2) Development of somatic embryos through globular, heart, and torpedo stages of growth
3) Maturation of embryos with the formation of root and shoot meristems and cotyledons
4) Conversion of mature embryos to plantlets through germination on auxin-free media
Factors like explant type, growth regulators, and genotype influence the process. Somatic embryos differ from zygotic embryos in lacking a seed coat and having greater potential for propagation but weaker plantlets.
The isolation, culture and fusion of protoplasts is a fascinating field in plant research. Protoplast isolation and their cultures provide millions of single cells (comparable to microbial cells) for a variety of studies.
1. Acclimatization is the process by which plants adapt to changes in their environment over multiple generations through natural selection.
2. It requires genetic variability in introduced plant materials and occurs more readily in cross-pollinated species and annual crops.
3. Examples of acclimatization include humans developing more red blood cells at high altitudes and plants surviving freezing temperatures if the temperature drops gradually over time rather than suddenly.
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.
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 methods for producing haploid plants through gynogenesis, or haploid production from female gametes. It describes two main methods - ovary culture and ovule culture - for inducing gynogenesis in vitro. Key steps include excising ovaries from plants and culturing them on nutrient media supplemented with hormones to induce parthenogenesis. Successful gynogenesis has been achieved in several plant families, with the frequency of responsive ovules typically being low, around 1-5%. Producing haploid plants provides benefits for genetic studies.
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.
Embryo culture is a laboratory method for producing plant lets from a fertilized or unfertilized embryo in invitro condition. there are several advantages are associated with the embryo culture like production of haploid plants, making distant crosses successful, sometimes aborted embryos can be rescued from a unsuccessful hybridization.
This document discusses haploid plant production through anther culture. It begins by defining key terms like gametophyte, sporophyte, haploid and diploid plants. It then describes the two main methods of haploid production - anther culture and isolated microspore culture. For anther culture, it outlines the process of culturing immature anthers on nutrient media, including pretreatments, media composition and plant regeneration. Anther culture can result in direct or indirect embryogenesis and four pathways of pollen development are described. The document provides detailed steps of anther culture and potential issues like production of diploid plants.
This document discusses media used for plant tissue culture. It provides details on the components and purpose of various standard media including Murashige and Skoog medium, Gamborg's B5 medium, Nitsch medium, and others. It also discusses the composition and use of specialized media for banana, orchid, and woody plant tissue culture. The key components of media include minerals, vitamins, carbon sources, and plant growth regulators to provide nutrients and regulate growth for cultured plant cells and tissues.
The document discusses cytoplasmic and cytoplasmic-genetic male sterility in crop plants. It begins with an introduction to the topic and definitions of male sterility, including genetic, cytoplasmic, and cytoplasmic-genetic male sterility. It then discusses the genetics and molecular basis of cytoplasmic male sterility, including the role of aberrant mitochondrial genes. Examples are provided of specific CMS-causing genes and restoration mechanisms in different crops like rice, maize, and others. The presentation concludes with future perspectives on commercializing additional cytoplasmic sources and further understanding the genetic and molecular basis of CMS.
The document discusses totipotency in plant cells. Totipotency refers to the ability of single plant cells to regenerate into a whole plant through cell differentiation and tissue culture techniques. The document outlines various tissue culture systems used to study totipotency, including callus culture, suspension culture, single cell culture, and protoplast culture. Factors that influence a cell's ability to express totipotency, such as the explant source and culture conditions, are also discussed.
Double haploids are produced by doubling the chromosomes of haploid cells. Haploid cells have half the number of chromosomes as the original organism due to meiosis. A doubled haploid would have the full chromosome number and be homozygous. There are two main methods to produce haploids - anther/pollen culture (androgenesis) and ovary/ovule culture (gynogenesis). The haploids can then be doubled using chemicals like colchicine to produce doubled haploids. Doubled haploids have benefits for plant breeding as they are fully homozygous in the first generation, allowing for faster breeding cycles.
Somaclonal and gametoclonal variation refer to genetic variations that arise in plants regenerated from cell and tissue cultures. There are two main types - somaclonal variation originating from somatic cells, and gametoclonal variation from gametic cells like pollen. Variations can be induced through long term culture, exposure to mutagens, or selection in media containing inhibitors or toxins. Somaclonal variants are isolated and screened using cytological, biochemical, and molecular markers to identify desirable heritable traits for commercial use in plant breeding programs.
INTRODUCTION
2. HISTORY
3. BASIC COMPONENT OF MEDIA
1. Inorganic nutrient
2. organic supplements
3. Carbon and energy source
4. Growth Regulators
5. Solidifying Agent
6. PH
4. TYPES OF MEDIA
5. MS MEDIA
6. IMPORTANCE
7. CONCLUSION
8. REFERANCE
A process where an embryo is derived from a single somatic cell or group of somatic cells. Somatic embryos (SEs) are formed from plant cells that are not normally involved in embryo formation.
Embryos formed by somatic embryogenesis are called Embryoids.
The process was discovered for the first time in Daucas carota L. (carrot) by Steward (1958), Reinert (1959).
Anther culture:- the in vitro culturing of anthers containing microspores or immature pollen grains on a nutrient medium for the purpose of generating haploid plantlets.
Culturing anthers for the purpose of obtaining Double Haploid is not easy with many field crop species, particularly with the cereals, cotton, and grain legumes.
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 .
☺INTRODUCTION
☺Bt COTTON
☺MAJOR PESTS OF COTTON
☺MODE OF ACTION OF Bt GENE
☺ADVANTAGES
☺DISADVANTAGES
☺CONCLUSION
☺REFERENCES
Genetically modified variety of cotton that produces an insecticide whose gene has been derived from a soil bacterium called Bacillus thuringiensis (Bt).
Three types of toxins.
A total of 229 cry toxins ( cry1Aa to Cry72Aa), cyt toxins ( cyt 11Aa to cyt3Aa) and 102 vip toxins( vip1Aa1 to vip4Aa1) have been discovered.
Viral infections in plants can be controlled through several strategies including using certified seed/plants, controlling weeds that harbor viruses, and insecticide use since most viruses are vector-borne. Transgenic virus resistance involves expressing viral genes including coat proteins, replicases, movement proteins, or antisense RNA to interfere with viral replication or movement. The papaya industry was saved through a transgenic papaya resistant to papaya ringspot virus. While virus resistance holds promise, risks like recombination or heterologous encapsidation must be monitored.
The document discusses organogenesis, which is the development of adventitious organs or primordial from undifferentiated plant cell mass through differentiation. It describes the process, including dedifferentiation and redifferentiation stages. There are two types of organogenesis - direct organogenesis which does not involve callus formation, and indirect organogenesis which involves callus formation. Organogenesis is used in plant tissue culture to regenerate plants through shoot or root cultures and is influenced by factors like explant source and size, plant growth regulators, and culture conditions. It has commercial applications in micropropagation of plants.
The document discusses somatic hybridization through the fusion of protoplasts from different plant species. It describes:
1. The process of somatic hybridization which involves isolating protoplasts from plant tissues, fusing the protoplasts from different species using chemical or electrical methods, selecting hybrid cells, culturing the hybrid cells and regenerating hybrid plants.
2. Methods for isolating viable protoplasts including enzymatic and mechanical methods. Enzymatic isolation uses cellulase, hemicellulase and pectinase enzymes.
3. Techniques for purifying isolated protoplasts such as filtration, centrifugation, flotation and density buffer methods to remove
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.
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
Clone is the progeny of a single plant, produced by asexual reproduction
Clonal selection is the selection of the most desirable members of a clone for continued vegetative propagation rather than for sexual reproduction.
The members of a clone keep up genetic constancy.
So by clonal selection and continued vegetative propagation, the desirable qualities of plants can be maintained for long.
The document discusses protoplast fusion, which involves removing the cell walls of plant cells to create naked protoplasts that can then be fused. It describes how to isolate protoplasts from plant tissues using either mechanical or enzymatic methods. The fusion of protoplasts from different species or varieties can create hybrid cells called heterokaryons or hybrids. Techniques to induce protoplast fusion include treatment with polyethylene glycol (PEG), calcium ions, electricity, or sodium nitrate. Successful somatic hybridization follows a procedure of isolating, fusing, regenerating cell walls, and selecting hybrid plant cells and colonies. Applications of protoplast fusion include combining genomes of sterile plants and
Protoplast fusion technology involves removing the cell walls from cells, leaving only the cell membrane. This allows the fusion of the cell membranes and cytoplasm of two different cell types, transferring genes. There are three main methods of inducing protoplast fusion - mechanical fusion using microtools, chemical fusion using substances like polyethylene glycol, and electrofusion using electric fields. Enzymes like cellulase and pectinase are used to break down plant cell walls, while lysozyme degrades bacterial cell walls. Protoplast fusion has applications in strain improvement for industrial purposes by creating cells with desired properties.
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.
Embryo culture is a laboratory method for producing plant lets from a fertilized or unfertilized embryo in invitro condition. there are several advantages are associated with the embryo culture like production of haploid plants, making distant crosses successful, sometimes aborted embryos can be rescued from a unsuccessful hybridization.
This document discusses haploid plant production through anther culture. It begins by defining key terms like gametophyte, sporophyte, haploid and diploid plants. It then describes the two main methods of haploid production - anther culture and isolated microspore culture. For anther culture, it outlines the process of culturing immature anthers on nutrient media, including pretreatments, media composition and plant regeneration. Anther culture can result in direct or indirect embryogenesis and four pathways of pollen development are described. The document provides detailed steps of anther culture and potential issues like production of diploid plants.
This document discusses media used for plant tissue culture. It provides details on the components and purpose of various standard media including Murashige and Skoog medium, Gamborg's B5 medium, Nitsch medium, and others. It also discusses the composition and use of specialized media for banana, orchid, and woody plant tissue culture. The key components of media include minerals, vitamins, carbon sources, and plant growth regulators to provide nutrients and regulate growth for cultured plant cells and tissues.
The document discusses cytoplasmic and cytoplasmic-genetic male sterility in crop plants. It begins with an introduction to the topic and definitions of male sterility, including genetic, cytoplasmic, and cytoplasmic-genetic male sterility. It then discusses the genetics and molecular basis of cytoplasmic male sterility, including the role of aberrant mitochondrial genes. Examples are provided of specific CMS-causing genes and restoration mechanisms in different crops like rice, maize, and others. The presentation concludes with future perspectives on commercializing additional cytoplasmic sources and further understanding the genetic and molecular basis of CMS.
The document discusses totipotency in plant cells. Totipotency refers to the ability of single plant cells to regenerate into a whole plant through cell differentiation and tissue culture techniques. The document outlines various tissue culture systems used to study totipotency, including callus culture, suspension culture, single cell culture, and protoplast culture. Factors that influence a cell's ability to express totipotency, such as the explant source and culture conditions, are also discussed.
Double haploids are produced by doubling the chromosomes of haploid cells. Haploid cells have half the number of chromosomes as the original organism due to meiosis. A doubled haploid would have the full chromosome number and be homozygous. There are two main methods to produce haploids - anther/pollen culture (androgenesis) and ovary/ovule culture (gynogenesis). The haploids can then be doubled using chemicals like colchicine to produce doubled haploids. Doubled haploids have benefits for plant breeding as they are fully homozygous in the first generation, allowing for faster breeding cycles.
Somaclonal and gametoclonal variation refer to genetic variations that arise in plants regenerated from cell and tissue cultures. There are two main types - somaclonal variation originating from somatic cells, and gametoclonal variation from gametic cells like pollen. Variations can be induced through long term culture, exposure to mutagens, or selection in media containing inhibitors or toxins. Somaclonal variants are isolated and screened using cytological, biochemical, and molecular markers to identify desirable heritable traits for commercial use in plant breeding programs.
INTRODUCTION
2. HISTORY
3. BASIC COMPONENT OF MEDIA
1. Inorganic nutrient
2. organic supplements
3. Carbon and energy source
4. Growth Regulators
5. Solidifying Agent
6. PH
4. TYPES OF MEDIA
5. MS MEDIA
6. IMPORTANCE
7. CONCLUSION
8. REFERANCE
A process where an embryo is derived from a single somatic cell or group of somatic cells. Somatic embryos (SEs) are formed from plant cells that are not normally involved in embryo formation.
Embryos formed by somatic embryogenesis are called Embryoids.
The process was discovered for the first time in Daucas carota L. (carrot) by Steward (1958), Reinert (1959).
Anther culture:- the in vitro culturing of anthers containing microspores or immature pollen grains on a nutrient medium for the purpose of generating haploid plantlets.
Culturing anthers for the purpose of obtaining Double Haploid is not easy with many field crop species, particularly with the cereals, cotton, and grain legumes.
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 .
☺INTRODUCTION
☺Bt COTTON
☺MAJOR PESTS OF COTTON
☺MODE OF ACTION OF Bt GENE
☺ADVANTAGES
☺DISADVANTAGES
☺CONCLUSION
☺REFERENCES
Genetically modified variety of cotton that produces an insecticide whose gene has been derived from a soil bacterium called Bacillus thuringiensis (Bt).
Three types of toxins.
A total of 229 cry toxins ( cry1Aa to Cry72Aa), cyt toxins ( cyt 11Aa to cyt3Aa) and 102 vip toxins( vip1Aa1 to vip4Aa1) have been discovered.
Viral infections in plants can be controlled through several strategies including using certified seed/plants, controlling weeds that harbor viruses, and insecticide use since most viruses are vector-borne. Transgenic virus resistance involves expressing viral genes including coat proteins, replicases, movement proteins, or antisense RNA to interfere with viral replication or movement. The papaya industry was saved through a transgenic papaya resistant to papaya ringspot virus. While virus resistance holds promise, risks like recombination or heterologous encapsidation must be monitored.
The document discusses organogenesis, which is the development of adventitious organs or primordial from undifferentiated plant cell mass through differentiation. It describes the process, including dedifferentiation and redifferentiation stages. There are two types of organogenesis - direct organogenesis which does not involve callus formation, and indirect organogenesis which involves callus formation. Organogenesis is used in plant tissue culture to regenerate plants through shoot or root cultures and is influenced by factors like explant source and size, plant growth regulators, and culture conditions. It has commercial applications in micropropagation of plants.
The document discusses somatic hybridization through the fusion of protoplasts from different plant species. It describes:
1. The process of somatic hybridization which involves isolating protoplasts from plant tissues, fusing the protoplasts from different species using chemical or electrical methods, selecting hybrid cells, culturing the hybrid cells and regenerating hybrid plants.
2. Methods for isolating viable protoplasts including enzymatic and mechanical methods. Enzymatic isolation uses cellulase, hemicellulase and pectinase enzymes.
3. Techniques for purifying isolated protoplasts such as filtration, centrifugation, flotation and density buffer methods to remove
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.
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
Clone is the progeny of a single plant, produced by asexual reproduction
Clonal selection is the selection of the most desirable members of a clone for continued vegetative propagation rather than for sexual reproduction.
The members of a clone keep up genetic constancy.
So by clonal selection and continued vegetative propagation, the desirable qualities of plants can be maintained for long.
The document discusses protoplast fusion, which involves removing the cell walls of plant cells to create naked protoplasts that can then be fused. It describes how to isolate protoplasts from plant tissues using either mechanical or enzymatic methods. The fusion of protoplasts from different species or varieties can create hybrid cells called heterokaryons or hybrids. Techniques to induce protoplast fusion include treatment with polyethylene glycol (PEG), calcium ions, electricity, or sodium nitrate. Successful somatic hybridization follows a procedure of isolating, fusing, regenerating cell walls, and selecting hybrid plant cells and colonies. Applications of protoplast fusion include combining genomes of sterile plants and
Protoplast fusion technology involves removing the cell walls from cells, leaving only the cell membrane. This allows the fusion of the cell membranes and cytoplasm of two different cell types, transferring genes. There are three main methods of inducing protoplast fusion - mechanical fusion using microtools, chemical fusion using substances like polyethylene glycol, and electrofusion using electric fields. Enzymes like cellulase and pectinase are used to break down plant cell walls, while lysozyme degrades bacterial cell walls. Protoplast fusion has applications in strain improvement for industrial purposes by creating cells with desired properties.
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.
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.
Somatic hybridization is a technique used to produce hybrid plants by fusing protoplasts (plant cells without cell walls) from two different plant species or varieties. There are several key steps:
1. Isolation of protoplasts from plant tissues using either mechanical or enzymatic methods. Enzymatic methods using cellulase and pectinase enzymes are more common.
2. Fusion of the protoplasts using chemical fusogens like polyethylene glycol (PEG) or physical methods like electrofusion. This results in hybrid cells called heterokaryons.
3. Selection and culture of the hybrid cells using techniques like antibiotic resistance or genetic markers.
4. Regeneration
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.
This document discusses protoplast isolation and fusion. It defines a protoplast as a plant, bacterial, or fungal cell that has had its cell wall removed, leaving the plasma membrane intact. The document outlines the history of protoplast isolation, from the first isolation in 1892 to the use of commercial enzyme mixtures in the 1960s. It also describes how protoplasts can be fused using polyethylene glycol or other fusogens to create somatic hybrids for plant breeding purposes.
This document describes the process of protoplast isolation, culture, and fusion from Ankita Singh and Vinars Dawane of the Government Holkar Science College in Indore. It provides an overview of protoplast isolation methods including mechanical, sequential enzymatic, and mixed enzymatic. Sources of protoplasts include leaves, callus cultures, and cell suspension cultures. The viability of isolated protoplasts can be tested through microscopy, tetrazolium reduction, fluorescein diacetate staining, and Evan's blue staining. Protoplasts are cultured through regeneration of cell walls, cell division, and development of callus/whole plants. Protoplast fusion can be spontaneous, mechanical, or
Somatic ybridization and its applicationPawan Nagar
This document discusses somatic hybridization, which involves fusing plant protoplasts from two different species or varieties to create a hybrid plant. It describes the process of somatic hybridization, including isolating protoplasts, fusing them using spontaneous or induced methods, selecting hybrid cells, and regenerating plants from hybrid callus tissue. The advantages are producing novel hybrids and transferring genes between incompatible species. The limitations include low regeneration rates and viability of fused cells. Somatic hybridization has applications in crop improvement by introducing traits like disease resistance from wild relatives.
A presentation covering the process of protoplast culture including protoplast isolation, protoplast fusion, culture of protoplast, its application, factors affecting protoplast culture and the future of protoplasts.
This document summarizes a seminar on doubled haploids (DH). It defines a DH as an individual with a doubled set of chromosomes from a haploid cell. It discusses the history of DH development, including early work in the 1920s. It also covers methods for producing haploids, identifying haploids, doubling chromosomes, and applications of DHs in plant breeding like QTL mapping, backcrossing, hybrid sorting, and cultivar development. DHs allow fixing of traits in one or two generations, faster development of pure lines and cultivars compared to conventional methods.
This presentation discusses the history and process of plant domestication. It begins with an overview of the origins and timeline of agriculture, noting that domestication of major crops like rice, wheat and maize was completed by 4000 BC. The presentation then covers centers of domestication, key domestication traits, genes controlling traits, and modern techniques like genome sequencing, GWAS, and NGS that are helping to further understand domestication.
The document discusses using an aerobic submerged attached growth bioreactor for intermediate wastewater treatment. It presents results from experiments on organic matter and ammonia removal efficiency under different solid and salinity conditions. The bioreactor showed potential for secondary treatment, with remediation rates dependent on bacterial growth intensity. Higher salinity retarded bacterial growth and remediation. Small tire chips performed best as the packing material due to their spherical shape allowing better bacterial attachment than rectangular materials.
This document provides information on several plants commonly used in integrated pest management including their plant type, hardiness zone, size, sunlight and moisture requirements, bloom time and color, uses in landscaping, and other key details. It includes profiles of dogwood, hosta, knockout rose, foxglove, forsythia, English ivy, goldenrod, and redbud.
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.
This document discusses the isolation of protoplasts from plant cells and the factors that influence the yield and viability of protoplasts during the isolation process. Some key steps in the isolation process include enzymatic degradation of the plant cell wall using commercial enzymes to produce naked viable cells. The yield and viability of protoplasts can be affected by the plant species, plant tissue, isolation conditions, and culture medium after isolation. Viability is assessed using methods like observation of cytoplasmic streaming, oxygen uptake, photosynthetic activity, and staining.
This document discusses the design and construction of bioreactors. It explains that bioreactors provide optimal conditions for growing microorganisms by maintaining sterility and mixing. The key components of bioreactors include the vessel, agitator, sparger, temperature, pH and foam probes, cooling jacket, heating coil, and controls for dissolved oxygen and pressure. Proper monitoring and control of factors like temperature, pH, oxygen levels, and shear forces are necessary to support microbial growth and product formation.
This document discusses sunflower botany and production. It notes that sunflower is an important oilseed crop domesticated in the US. It describes sunflower's taxonomy, morphology, flowering biology, and methods for selfing and crossing varieties. The document outlines the development of hybrid varieties in India, including popular hybrids and their parent lines. It provides details on sunflower research stations, commercially grown varieties and hybrids, and the economic importance of sunflower oil.
Protoplast fusion involves removing the cell walls of plant cells through enzymatic or mechanical means to create naked protoplasts. These protoplasts can then be fused using chemicals, electricity, or other methods. This allows the cytoplasms and sometimes nuclei of different plant cells to merge, creating hybrid cells. Successful fusion can generate hybrid plants through regeneration of cell walls and tissues. Protoplast fusion overcomes sexual incompatibility and is used to introduce traits like disease resistance between species. It remains a technically challenging process with limitations like genetic instability and uncertain expression of transferred traits.
Somatic hybridization and Protoplast IsolationPABOLU TEJASREE
The document discusses protoplast isolation and somatic hybridization. It defines somatic hybridization as the development of hybrid plants through the fusion of somatic protoplasts from different plant species or varieties. The key steps in somatic hybridization are isolating protoplasts, fusing the protoplasts from desired species, identifying and selecting hybrid cells, culturing the hybrid cells, and regenerating hybrid plants. Protoplasts can be isolated using enzymatic or mechanical methods, and their viability and ability to form cell walls can be tested. Various factors like enzyme concentration, temperature, and pH affect protoplast isolation and viability. Protoplast fusion can occur spontaneously or be induced using methods like chemical treatment, electro
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Protoplasts are naked plant cells without the cell wall, but they possess plasma membrane and all other cellular components. They represent the functional plant cells but for the lack of the barrier, cell wall. Protoplasts of different species can be fused to generate a hybrid and this process is referred to as somatic hybridization (or protoplast fusion). Cybridization is the phenomenon of fusion of a normal protoplast with an enucleated (without nucleus) protoplast that results in the formation of a cybrid or cytoplast (cytoplasmic hybrids).
This document 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.
Somatic hybridization involves the fusion of isolated plant protoplasts to generate hybrid cells and plants combining the genetic material of both parental species. It allows for the production of novel interspecific and intergeneric hybrids not achievable through sexual hybridization. The key steps are isolating protoplasts using enzymatic methods, fusing the protoplasts using techniques like PEG or electrofusion, selecting hybrid cells, culturing the hybrid cells, and regenerating hybrid plants. Somatic hybridization has various applications in plant breeding and crop improvement.
This document describes the process of protoplast isolation, culture, and fusion. It states that protoplasts are plant cells without cell walls that are bound only by the plasma membrane. It outlines two methods for isolating protoplasts - mechanical and enzymatic. The enzymatic method uses cell wall degrading enzymes to isolate protoplasts from plant tissues over 16-18 hours. The isolated protoplasts can then be cultured, causing them to regenerate cell walls and undergo cell division. Protoplast fusion techniques like PEG or electrofusion can fuse protoplasts from different plant species to form somatic hybrids, allowing for plant modification studies.
This document summarizes the process of protoplast isolation, culture, and fusion. It discusses how protoplasts are isolated from plant tissues through enzymatic digestion of cell walls. The viability of isolated protoplasts is then tested before they are cultured on nutrient media and induced to regenerate new cell walls. Protoplast fusion allows the creation of somatic hybrids between plant species for crop improvement applications. The overall technique provides tools for genetic engineering and plant biotechnology research.
This document summarizes methods for isolating and culturing plant protoplasts. Protoplasts are plant cells that have had their cell walls removed. The document describes two methods for isolating protoplasts - mechanical and enzymatic. It also discusses purifying the protoplasts, assessing their viability, culturing them in liquid media to regenerate cell walls, and the potential applications of protoplast culture and regeneration including plant breeding techniques.
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This document discusses protoplast isolation, culture, and fusion. It defines protoplasts as plant, fungal, or bacterial cells without cell walls. Protoplasts can be isolated from plant tissue through either mechanical or enzymatic methods, with enzymatic isolation being more common. The document outlines steps for protoplast isolation, purification, and assessing viability. It also discusses protoplast culture techniques and methods for inducing protoplast fusion, including electrofusion and PEG treatment. Applications of protoplast techniques in plant research include genetic engineering and crop breeding.
This document discusses the morphology, structure, and identification of bacteria. It begins by describing the various shapes bacteria can take, such as rod-shaped, spherical, and spiral. It then details the internal structures of bacterial cells, including the cytoplasm, ribosomes, nucleoid, plasma membrane, cell wall, and flagella. Gram staining is introduced as an important method for initially classifying bacteria as either Gram-positive or Gram-negative based on differences in cell wall structure. The document provides diagrams and explanations of key bacterial cellular components.
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 analytical biochemistry techniques for isolating and sequencing proteins. It covers cell disruption methods like sonication, centrifugation types including differential and density gradient, and spectrophotometry. Cell disruption is needed to extract proteins from cells. Centrifugation separates particles based on size, shape and density. Differential centrifugation separates with increasing g-force while density gradient centrifugation uses a medium with increasing density. Spectrophotometry analyzes light absorption of substances using Lambert's and Beer's laws.
Protoplast technology and plant cell immobilizationPradnya Wadekar
This document discusses plant protoplast isolation and fusion. It describes how protoplasts can be isolated from plant tissues enzymatically or mechanically by removing the cell wall. The stages of protoplast fusion are outlined, including isolation, fusion using chemicals or electricity, selection of hybrid cells, culture and regeneration. Identification methods for hybrids and cybrids include chloroplast presence, nuclear staining and cytoplasmic markers.
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.
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6. 5/28/2012
HISTORY
Klercker isolated protoplast in 1892 by cutting plasmolaised
cells of tobacco leaf.
1960 Cocking used cellulase to isolate protoplas from fungi
1960 Power and Cocking isolated protoplast by single step
method.
1968 Takebe et al. used pectinase and cellulase to isolate
protoplast from tobacco by two step method.
1972 Power and Cocking fused the protoplast of 2 Nicotiana
sp and regenerated somatic hybrid. 6
7. 5/28/2012
PROPERTIES OF PROTOPLAST
Lack cell wall and have plasma membrane
Semi permeable and selectively permeable.
Spherical in shape
Sensitive to osmotic presser
Phagocytises and phenocytises
Fusion property
Regenerate the cell wall (totipotent) 7
8. 5/28/2012
SOMATIC HYBRIDIZATION TECHNIQUE
1. Isolation & purification of protoplast
2. Fusion of the protoplasts of desired species
3. Identification and Selection of hybrid protoplasts
4. Culture of the hybrid protoplasts
5. Regeneration & characterization of hybrid plants
8
11. 5/28/2012
1. MECHANICAL METHOD
Plant Tissue Cells Plasmolysis
Microscope Observation of cells
Cutting cell with knife Release of protoplasm
Collection of protoplasm
11
12. 5/28/2012
CONTED.,
Used for vacuolated cells like onion bulb
scale, radish and beet root tissues
Low yield of protoplast
Laborious and tedious process
Low protoplast viability
12
13. 5/28/2012
2. ENZYMATIC METHOD
Leaf sterlization, removal of
epidermis
Plasmolysed Plasmolysed
cells cells
Pectinase
Pectinase +cellulase
Release of Protoplasm
isolated cells released
Protoplasm released
cellaulase
Isolated
Protoplasm 13
14. 5/28/2012
CONTED.,
Used for variety of tissues and organs
Mesophyll tissue - most suitable source
High yield of protoplast
Easy to perform
More protoplast viability
14
19. 5/28/2012
SPONTANEOUS FUSION
Protoplast fuse spontaneously during isolation process
mainly due to physical contact
• Intraspecific produce homokaryones
• Interspecific and Intergeneric have no importance
19
21. 5/28/2012
Concentration of Replications A Replications B Replications C Mean
PEG Fusion Fusion Fusion Fusion
and Time of frequency (%) frequency (%) frequency (%) frequency
incubation (%)
PEG20% & 15 min 2.0 0.8 4.0 2.26
PEG 20% & 20 min 1.4 1.2 3.9 2.5
PEG 25% & 15 min 2.9 1.5 5.04 3.14
PEG 25% & 20 min 8.06 6.56 9.1 7.1
PEG 30% & 15 min 6.9 6.76 9.6 7.75
PEG 30% & 20 min 8.13 5.86 13.2 9.06
Kilima et al., 2009
21
30. 5/28/2012
CULTURE OF THE HYBRID CELLS
Hybrid cells are cultured on suitable medium provided with
the appropriate culture conditions.
culture in liquid medium
30
42. 5/28/2012
Production of unique nuclear cytoplasmic combinations
Production of heterozygous lines in vegetatively propagated
crops Eg:- Sugar cane, potato, other tubers root crops
Protoplast of sexually sterile plants
(haploid, triploid, anueploid) can be fused to produce fertile
diploids and polyploidy
Photosynthetic efficiency of plants can be enhanced through
the transfer of efficient foreign chloroplast into the plants
having less photosynthetic systems.
42
44. 5/28/2012
CONTI….
For cpDNA For nuclear DNA
Suthern bloting Peroxidase isozyme pattern44
45. 5/28/2012
Production of novel intergeneric hybrids
Fig. 1 (a) M. incana; (b) A somatic hybrid plant (pot cultivation); (c) B. oleracea; (d) Leaves
of (from left to right) M. incana (M), hybrids (H1 and H3), nonfusion plant
(R1), and B. oleracea(K)
Xiaoguang et al., 2008
45
46. 5/28/2012
Characterisation of somatic hybrids with molecular
markers
Brassica oleracea=K and Matthiola incana=M
RAPD
RAPD
Xiaoguang et al., 2008
46
SRAP
47. 5/28/2012
1. T1 +T2 +S1
2. T1 +T2 +S2
Fusion combinations
3. T1 +T2 +S3
4. T1 +T2 +S4
Hybrid lines
cell line No. 6 in combination 2.
cell line No. 5 in combination 2.
cell line No. 3 in combination 1.
cell line No. 1 in combination 1. Xiang et.al., 2004
47
49. 5/28/2012
TRANSFER OF DESIRABLE GENES
Transfer of PLRV resistance gene from
Solanum verrucosum to potato
(S. tuberosum L.)
Carrasco et al.,2000
49
50. clone A405nm Response
S. tuberosum (ver R3064) 0.5675 0.03 S
S. verrucosum(Ver 1340) 0.154 0.005 R
VR1 0.142 0.001 R
5/28/2012
VR2 0.149 0.002 R
VR3 0.133 0.002 R
VR4 0.146 0.005 R
VR5 0.136 0.001 R
VR6 0.153 0.001 R
VR7 0.287 0.003 S
VR8 0.309 0.002 S
VR9 0.146 0.001 R
VR10 0.130 0.001 R
VR11 0.143 0.002 R
VR12 0.138 0.001 R
VR14 0.376 0.005 S
VR16 0.140 0.001 R
VR17 0.136 0.001 R
Kennebec 0.689 0.011 S
50
Uninoculated Cntrol 0.115 0.001 ………
Carrasco et al.,2000
55. 5/28/2012
Atrazin resistance from S. nigrum to
tomato(VF36)
Fusion combinations
1. S. nigrum + tomato(VF36)
2. UV irradiated S. nigrum + tomato
3. UV irradiated S. nigrum + iodoacetate
treated tomato
4. Back fusion of tomato with somatic
hybrid
Jain et al.(1988)
55
61. 5/28/2012
Hybrids can be produced in vegetatively
propagated crops
In completely sterile plants
Ex : Monoploids
Breeding duration can be reduced by 6
years in transfer of cytoplasm
Hybrids can be produced in juvenile
phase of perennial crops
61
62. 5/28/2012
Poor regeneration(somatic incompatibility)
Non-viability of fused products
Production of unfavorable hybrids
Lack of an efficient method for selection of
hybrids
No confirmation of expression of
particular trait in somatic hybrids
Sexual reproduction of somatic hybrids. 62
63. 5/28/2012
Species Useful traits transferred Reference
Brassica
Qian et al.
B. napus (+) B. rapa Increased biomass and yield
(2003)
B. napus (+) Crambe Increased erucic acid content in Wang et al.
abyssinica seeds* (2003)
B. napus (+) Orychophragmus Improved fatty acid composition Hu et
violaceus in seeds* al(2002b)
Enhanced resistance to
Hu et al.
B. napus (+) Sinapsis arvensis Blackleg (Leptosphaeria
(2002)
maculans)*
B. oleracea (+) Moricandia Introduction of the C3–C4 Ishikawa et al.
arvensis intermediate trait† (2003)
Raphanus sativus (+) Diplotaxis Introduction of the C3–C4 Bang et al. 63
†
64. Solanum 5/28/2012
S. melongena (+) S. Resistance to bacterial wilt Collonnier et al.
aethiopicum (Ralstonia solanacearum)* (2001)
S. melongena (+) S. Resistance to bacterial and fungal Collonnier et al.
sisymbrifolium wilts* (2003)
S. tuberosum (+) S. Gavrilenko et al.
Resistance to potato virus Y*
etuberosum (2003)
Resistance to potato blight Szczerbakowa
S. tuberosum (+) S. nigrum
(Phytophthora infestans)† et al. (2003)
S. tuberosum (+) S. Resistance to bacterial wilt (R. Fock et al.
stenotomum solanacearum)* (2001)
Citrus
C. amblycarpa (+) Citroncirus Improved rootstock for Mexican Medina-Urrutia
webberri C35 lime† et al. (2004)
C. limonia (+) C. sunki cv. Tolerance to citrus blight, tristeza Costa et al.
Tanaka virus, and Phytophthora† (2003)
Production of mixoploid plants
C. reticulata cv. Blanco (+) C. Liu and Deng
tolerant to citrus exocortis virus
paradisi (2002)
(CEV)†
C. reticulata cv. Blanco (+) C. Tolerance to citrus blight, tristeza Costa et al. 64
65. 5/28/2012
C. reticulata cv. Blanco (+) Resistance to CEV†
Guo et al. (2002)
Poncirus trifoliata
C. sinensis cv. Rohde Red (+) Tolerance to citrus blight, tristeza Costa et al.
C. volkameriana virus, and Phytophthora† (2003)
C. sinensis cv. Ruby Blood (+) Tolerance to citrus blight, tristeza Costa et al.
C. volkameriana virus, and Phytophthora† (2003)
C. sinensis (+) Fortunella Cheng et al.
Increased plant vigour*
crassifolia (2003)
Tolerance to citrus blight, tristeza Costa et al.
C. sinensis (+) F. obovata
virus, and Phytophthora† (2003)
C. sinensis (+) Clausena
Production of triploid plants* Fu et al. (2003)
lansium
C. unshiu cv. Guoqing No. 1 (+)
Generation of seedless cybrids† Guo et al. (2004)
C. grandis cv. Buntan Pink
C. unshiu cv. Guoqing No. 1 (+)
Generation of seedless cybrids† Guo et al. (2004)
C. reticulata cv. Blanco
C. unshiu cv. Guoqing No. 1 (+)
Generation of seedless cybrids† Guo et al. (2004)
C. reticulata×C. sinensis
Michael et al., 200565
66. 5/28/2012
Reference
Species Application
Gene recognition mechanisms Leister and Katagiri
Arabidopsis thaliana
involved in plant pathogenicity (2000)
Elucidation of plant signal
A. thaliana/Zea mays Sheen (2001)
transduction mechanisms
Electrophysiological studies of
Brassica chinensis Fan et al. (2003)
outward K+ channels
Yasukawa et al.
Bryopsis plumosa Electrochemical assays of metabolic
(2002), Zhou et al.
(marine green alga) flux; enzyme (peroxidase) activity
(2003)
Cucurbita pepo Viral pathogenicity Choi et al. (2003)
Synthetic peptide import through the
Helianthus annuus Cormeau et al. (2002)
plasma membrane
Hibiscus cannabinus Viral replication processes Liang et al. (2002)
Comparison of stress mechanisms in 66
Hordeum vulgare Zhou et al. (2000)
67. Nicotiana Shanka
5/28/2012 et al.
Viral recombination and replication
benthamiana (2004)
Nicotiana Genetic basis of developmental regulation and Chesnokov et al.
plumbaginifolia specificity (2002)
Nicotiana Regulation of osmotic water transport across cell
Ding et al. (2004)
tabacum membranes
Oryza
sativa/Pisum
sativum/Sorghu Ishikawa et al.
Membrane permeability and tolerance to Al3+
m (2001)
bicolor/Triticum
vulgare/Z. mays
Phaseolus Electrophysiological studies of inward-rectifying Etherton et al.
vulgaris K+ channels (2004)
Raphanus Immunocytochemical evaluation of aquaporin
Suga et al. (2003)
sativus accumulation
Fluorometric analysis of photosynthetic electron
Vicia faba Goh et al. (2002)
transport
Intracellular responses to drought and salinity
Vigna radiata Kim et al. (2004)
stress
Vermeer et al.
V. unguiculata Studies on plasma membrane organisation
Shapka et al., 200467
(2004)