This document discusses techniques for in vitro clonal propagation of fruit crops. It covers the basics of micropropagation, which involves four stages: establishment, shoot multiplication, root formation, and acclimatization. Various tissue culture techniques are described that can be used for clonal propagation, including meristem culture, shoot tip micrografting, anther culture, embryo culture, ovary/ovule culture, callus culture, cell suspension culture, and protoplast culture. Requirements for facilities, media preparation, and procedures for each stage of micropropagation are provided. The document aims to inform the reader about the various in vitro techniques that can be used for commercial clonal propagation of fruit crops.
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 in vitro pollination and artificial seeds. It begins by introducing in vitro pollination as a technique to produce hybrid embryos between plant species that cannot naturally cross-breed. It then describes different in vitro pollination techniques like stigmatic, ovarian, and ovular pollination. The document also covers the applications of in vitro pollination in plant breeding by overcoming self-incompatibility and cross-incompatibility. Next, it defines artificial seeds as embryos enclosed in a medium within a artificial seed coat, combining advantages of cloning and seed propagation. Finally, it outlines the procedure for synthetic seed production and advantages over somatic embryos for propagation.
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.
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.
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.
Techniques of in vitro clonal propagation for fruit cropsPawan Nagar
This document discusses techniques for in vitro clonal propagation of fruit crops. It describes the four stages of micropropagation: establishment, shoot multiplication, root formation, and acclimatization. The establishment stage involves disinfesting explants and initiating shoot development. In the multiplication stage, shoots are subcultured to expand into clusters. Root formation prepares plantlets for transplanting. Finally, acclimatization conditions plantlets for greenhouse and field conditions. The document also outlines types of tissue culture systems and developmental patterns for plantlet formation.
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 in vitro pollination and artificial seeds. It begins by introducing in vitro pollination as a technique to produce hybrid embryos between plant species that cannot naturally cross-breed. It then describes different in vitro pollination techniques like stigmatic, ovarian, and ovular pollination. The document also covers the applications of in vitro pollination in plant breeding by overcoming self-incompatibility and cross-incompatibility. Next, it defines artificial seeds as embryos enclosed in a medium within a artificial seed coat, combining advantages of cloning and seed propagation. Finally, it outlines the procedure for synthetic seed production and advantages over somatic embryos for propagation.
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.
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.
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.
Techniques of in vitro clonal propagation for fruit cropsPawan Nagar
This document discusses techniques for in vitro clonal propagation of fruit crops. It describes the four stages of micropropagation: establishment, shoot multiplication, root formation, and acclimatization. The establishment stage involves disinfesting explants and initiating shoot development. In the multiplication stage, shoots are subcultured to expand into clusters. Root formation prepares plantlets for transplanting. Finally, acclimatization conditions plantlets for greenhouse and field conditions. The document also outlines types of tissue culture systems and developmental patterns for plantlet formation.
- Ovule culture involves aseptically isolating ovules from ovaries and growing them on defined nutrient media under controlled conditions. This allows for studying embryo development from zygote to mature embryo.
- Ovaries and unfertilized ovules can be sources for haploid or doubled haploid production. An ovule contains a megaspore or egg cell that can be fertilized to form a zygote and eventually a mature embryo.
- The protocol involves collecting unfertilized or fertilized ovules, surface sterilizing the ovaries, isolating ovules using a spatula, and incubating them on solid or liquid media in light or dark conditions.
Conservation and preservation of germplasmIñnøcènt ÅñDi
The document discusses germplasm conservation, including both ex situ and in situ methods. Ex situ conservation involves maintaining genetic resources outside their natural habitat, such as in seed banks, field gene banks, DNA banks, botanical gardens, and through in vitro and cryopreservation methods. In situ conservation preserves species in their natural environments through biosphere reserves, national parks, wildlife sanctuaries, and on-farm conservation. Cryopreservation is described as a method to bring plant cells and tissues to a zero metabolism state through freezing at very low temperatures in liquid nitrogen.
Clonal Propagation: Introduction, Techniques, Factors, Applications and Disadvantages
Multiplication of Apical or Axillary bud, Shoot tip or meristem culture
Production of Disease free plants by Micropropagation techniques: their Advantages and Disadvantages
The document discusses various applications of tissue culture techniques including producing virus-free plants through heat treatment, meristemming, and using single cells to regenerate shoots; micropropagation to rapidly produce clones; somaclonal variation to induce genetic mutations; and synthetic seeds to efficiently transport and germinate plant materials. Micropropagation is described as being faster but more expensive than conventional propagation methods, and somaclonal variation can generate heritable or non-heritable variations for crop improvement.
Production of synthetic seed involves encapsulating somatic embryos, shoot buds, or cell aggregates using tissue culture techniques. This allows for the large-scale, low-cost propagation of plants while maintaining genetic uniformity. Synthetic seeds can be stored longer than traditional seeds and planted directly in fields without the need for transplanting. While synthetic seeds have advantages over traditional micropropagation methods, their production and germination rates can still be limited for some plant species.
Micropropagation and commercial exploitation in horticulture cropsDheeraj Sharma
Micro-propagation – principles and concepts, commercial exploitation in horticultural crops. Techniques - in vitro clonal propagation, direct organogenesis, embryogenesis, micrografting, meristem culture. Hardening, packing and transport of micro-propagules.
This document discusses plant tissue culture techniques, including the types of explants used and sterilization protocols. It describes that meristem culture uses meristematic tissues like shoot apical meristems. Seed and ovule culture uses fertilized ovules from plants with small or minute seeds. Callus culture produces an unorganized cell mass. Haploid culture generates haploid cells from tissues like pollen and anthers. Different sterilization chemicals like sodium hypochlorite and calcium hypochlorite are used depending on the explant type. Protocols vary in chemical concentration and duration to sterilize tissues without loss of viability. Cells in culture go through growth phases including exponential growth, before requiring sub-cult
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
INVITRO CULTURE: TECHNIQUES, APPLICATIOSNS & ACHIEVEMENTS.
INVITRO TECHNIQUES AND BIOTECHNOLOGY USE IN AGRICULTURE AND CROP IMPROVEMENT. APPLICATIONS OF VARIOUS BIOTECHNOLOGICAL TECHNIQUES AND METHODS. TISSUE CULTURE, MICROPROPAGATION, EMBRYO CULTURE, ANTHER CULTURE, POLLEN CULTURE, ENDOSPERM CULTURE, OVULE CULTURE, OVARY CULTURE, ETC.
Meristem, embryo, and protoplast culture are three methods of micro-propagation. Meristem culture uses small stem tips placed in media to produce disease-free plants. Embryo culture excises embryos and places them in media to overcome issues like embryo abortion. Protoplast culture isolates plant cells using enzymes and regenerates plants from cultured protoplasts. The main advantages are producing clones and disease-free plants, while disadvantages include high costs and not all plants being amenable to tissue culture.
This document discusses routine nursery practices in horticulture. It covers sanitation, drainage, training, and pruning techniques used in nurseries. It also discusses potting, repotting, and mulching of nursery plants. The use of plant growth regulators like auxins, gibberellins, and cytokinins to promote rooting and vegetative propagation is explained. Methods of applying growth regulators include soaking, quick dipping, and using lanoline paste. Proper nursery techniques help produce quality planting materials for transplanting.
This document discusses distant hybridization and various techniques used to produce haploid plants. Distant hybridization refers to crosses between individuals of different plant species or genera. Such crosses can result in fully fertile, partially fertile, or fully sterile offspring depending on chromosomal homology. Androgenesis and gynogenesis are techniques used to induce haploid plants from male and female gametes, respectively. Androgenesis involves culturing immature anthers or isolated microspores while gynogenesis involves culturing unpollinated flower parts. Wide hybridization is also used to induce maternal haploids. Factors like genotype, developmental stage, and culture conditions influence haploid induction and regeneration.
Tissue culture is the process of growing plant cells, tissues or organs in an artificial, sterile environment. It involves removing plant cells and placing them in a nutrient solution supplemented with hormones, vitamins and minerals. Key requirements include using appropriate explant tissue, a suitable growth medium, aseptic conditions, and growth regulators. The document outlines various tissue culture techniques such as micropropagation, callus culture, somatic embryogenesis, organogenesis, and anther/pollen/ovule/ovary culture. Factors affecting successful tissue culture include the plant genotype, explant source, nutrient composition of the growth medium, and control of environmental factors like light, temperature and sterility.
This document discusses seeds, including that seeds are produced after fertilization within the mother plant and develop from the zygote and seed coat. It notes that angiosperms produce enclosed seeds within a fruit structure, while gymnosperms produce naked seeds that develop on cones and become covered by cone scales. The embryo is the fertilized ovule that will grow into a new plant, and the seed coat develops from maternal tissue to protect the embryo. Seed and seedling vigor tests conducted after one month can evaluate traits like shoot dry weight, root weight, height, and growth rates. Proper harvesting, handling, and storage are also important to minimize seed damage.
INTRODUCTION
WHAT IS ANDROGENESIS ?
HISTORY
TYPES OF ANDROGENESIS TECHNIQUES
ONTOGENY OF ANDROGENIC HAPLOIDS
GYNOGENESIS
FACTORS AFFECTING ANDROGENESIS
APPLICATIONS OF ANDROGENESIS
LIMITATIONS
REFERENCES
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.
Types of seed dormancy & Methods to overcome itAbarna Abi
This document discusses types of seed dormancy and methods to overcome dormancy. There are several types of dormancy including seed coat dormancy, dormancy due to rudimentary embryos, dormancy due to chemical inhibitors, and dormancy due to internal factors. Methods to overcome dormancy include mechanical scarification, soaking seeds in water, acid treatment, cold stratification, dry storage, and treatment with chemicals like gibberellic acid. Overcoming dormancy allows seeds to germinate when conditions are suitable.
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.
Plant tissue culture is a technique used to propagate plants asexually under controlled laboratory conditions. It involves culturing plant cells, tissues, or organs on artificial nutrient media. The key principles are totipotency, where plant cells can regenerate into whole plants, and plasticity, where plants can alter their growth to suit their environment. There are various types of plant tissue culture including embryo, seed, meristem, cell, protoplast, callus, pollen, and organ culture. Important applications include producing virus-free plants through meristem culture and generating novel hybrids through protoplast fusion.
Plant tissue culture is the process of maintaining or growing plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. It involves techniques like cell culture, organ culture or meristem culture to produce clones of a plant through micropropagation. The key steps are selection of explant tissue from a donor plant, sterilization, establishment of the explant on a culture medium, multiplication through cell division and shoot formation, rooting of shoots, and acclimatization of plantlets in soil. Micropropagation allows for rapid mass multiplication of plant materials while maintaining genetic uniformity.
- Ovule culture involves aseptically isolating ovules from ovaries and growing them on defined nutrient media under controlled conditions. This allows for studying embryo development from zygote to mature embryo.
- Ovaries and unfertilized ovules can be sources for haploid or doubled haploid production. An ovule contains a megaspore or egg cell that can be fertilized to form a zygote and eventually a mature embryo.
- The protocol involves collecting unfertilized or fertilized ovules, surface sterilizing the ovaries, isolating ovules using a spatula, and incubating them on solid or liquid media in light or dark conditions.
Conservation and preservation of germplasmIñnøcènt ÅñDi
The document discusses germplasm conservation, including both ex situ and in situ methods. Ex situ conservation involves maintaining genetic resources outside their natural habitat, such as in seed banks, field gene banks, DNA banks, botanical gardens, and through in vitro and cryopreservation methods. In situ conservation preserves species in their natural environments through biosphere reserves, national parks, wildlife sanctuaries, and on-farm conservation. Cryopreservation is described as a method to bring plant cells and tissues to a zero metabolism state through freezing at very low temperatures in liquid nitrogen.
Clonal Propagation: Introduction, Techniques, Factors, Applications and Disadvantages
Multiplication of Apical or Axillary bud, Shoot tip or meristem culture
Production of Disease free plants by Micropropagation techniques: their Advantages and Disadvantages
The document discusses various applications of tissue culture techniques including producing virus-free plants through heat treatment, meristemming, and using single cells to regenerate shoots; micropropagation to rapidly produce clones; somaclonal variation to induce genetic mutations; and synthetic seeds to efficiently transport and germinate plant materials. Micropropagation is described as being faster but more expensive than conventional propagation methods, and somaclonal variation can generate heritable or non-heritable variations for crop improvement.
Production of synthetic seed involves encapsulating somatic embryos, shoot buds, or cell aggregates using tissue culture techniques. This allows for the large-scale, low-cost propagation of plants while maintaining genetic uniformity. Synthetic seeds can be stored longer than traditional seeds and planted directly in fields without the need for transplanting. While synthetic seeds have advantages over traditional micropropagation methods, their production and germination rates can still be limited for some plant species.
Micropropagation and commercial exploitation in horticulture cropsDheeraj Sharma
Micro-propagation – principles and concepts, commercial exploitation in horticultural crops. Techniques - in vitro clonal propagation, direct organogenesis, embryogenesis, micrografting, meristem culture. Hardening, packing and transport of micro-propagules.
This document discusses plant tissue culture techniques, including the types of explants used and sterilization protocols. It describes that meristem culture uses meristematic tissues like shoot apical meristems. Seed and ovule culture uses fertilized ovules from plants with small or minute seeds. Callus culture produces an unorganized cell mass. Haploid culture generates haploid cells from tissues like pollen and anthers. Different sterilization chemicals like sodium hypochlorite and calcium hypochlorite are used depending on the explant type. Protocols vary in chemical concentration and duration to sterilize tissues without loss of viability. Cells in culture go through growth phases including exponential growth, before requiring sub-cult
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
INVITRO CULTURE: TECHNIQUES, APPLICATIOSNS & ACHIEVEMENTS.
INVITRO TECHNIQUES AND BIOTECHNOLOGY USE IN AGRICULTURE AND CROP IMPROVEMENT. APPLICATIONS OF VARIOUS BIOTECHNOLOGICAL TECHNIQUES AND METHODS. TISSUE CULTURE, MICROPROPAGATION, EMBRYO CULTURE, ANTHER CULTURE, POLLEN CULTURE, ENDOSPERM CULTURE, OVULE CULTURE, OVARY CULTURE, ETC.
Meristem, embryo, and protoplast culture are three methods of micro-propagation. Meristem culture uses small stem tips placed in media to produce disease-free plants. Embryo culture excises embryos and places them in media to overcome issues like embryo abortion. Protoplast culture isolates plant cells using enzymes and regenerates plants from cultured protoplasts. The main advantages are producing clones and disease-free plants, while disadvantages include high costs and not all plants being amenable to tissue culture.
This document discusses routine nursery practices in horticulture. It covers sanitation, drainage, training, and pruning techniques used in nurseries. It also discusses potting, repotting, and mulching of nursery plants. The use of plant growth regulators like auxins, gibberellins, and cytokinins to promote rooting and vegetative propagation is explained. Methods of applying growth regulators include soaking, quick dipping, and using lanoline paste. Proper nursery techniques help produce quality planting materials for transplanting.
This document discusses distant hybridization and various techniques used to produce haploid plants. Distant hybridization refers to crosses between individuals of different plant species or genera. Such crosses can result in fully fertile, partially fertile, or fully sterile offspring depending on chromosomal homology. Androgenesis and gynogenesis are techniques used to induce haploid plants from male and female gametes, respectively. Androgenesis involves culturing immature anthers or isolated microspores while gynogenesis involves culturing unpollinated flower parts. Wide hybridization is also used to induce maternal haploids. Factors like genotype, developmental stage, and culture conditions influence haploid induction and regeneration.
Tissue culture is the process of growing plant cells, tissues or organs in an artificial, sterile environment. It involves removing plant cells and placing them in a nutrient solution supplemented with hormones, vitamins and minerals. Key requirements include using appropriate explant tissue, a suitable growth medium, aseptic conditions, and growth regulators. The document outlines various tissue culture techniques such as micropropagation, callus culture, somatic embryogenesis, organogenesis, and anther/pollen/ovule/ovary culture. Factors affecting successful tissue culture include the plant genotype, explant source, nutrient composition of the growth medium, and control of environmental factors like light, temperature and sterility.
This document discusses seeds, including that seeds are produced after fertilization within the mother plant and develop from the zygote and seed coat. It notes that angiosperms produce enclosed seeds within a fruit structure, while gymnosperms produce naked seeds that develop on cones and become covered by cone scales. The embryo is the fertilized ovule that will grow into a new plant, and the seed coat develops from maternal tissue to protect the embryo. Seed and seedling vigor tests conducted after one month can evaluate traits like shoot dry weight, root weight, height, and growth rates. Proper harvesting, handling, and storage are also important to minimize seed damage.
INTRODUCTION
WHAT IS ANDROGENESIS ?
HISTORY
TYPES OF ANDROGENESIS TECHNIQUES
ONTOGENY OF ANDROGENIC HAPLOIDS
GYNOGENESIS
FACTORS AFFECTING ANDROGENESIS
APPLICATIONS OF ANDROGENESIS
LIMITATIONS
REFERENCES
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.
Types of seed dormancy & Methods to overcome itAbarna Abi
This document discusses types of seed dormancy and methods to overcome dormancy. There are several types of dormancy including seed coat dormancy, dormancy due to rudimentary embryos, dormancy due to chemical inhibitors, and dormancy due to internal factors. Methods to overcome dormancy include mechanical scarification, soaking seeds in water, acid treatment, cold stratification, dry storage, and treatment with chemicals like gibberellic acid. Overcoming dormancy allows seeds to germinate when conditions are suitable.
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.
Plant tissue culture is a technique used to propagate plants asexually under controlled laboratory conditions. It involves culturing plant cells, tissues, or organs on artificial nutrient media. The key principles are totipotency, where plant cells can regenerate into whole plants, and plasticity, where plants can alter their growth to suit their environment. There are various types of plant tissue culture including embryo, seed, meristem, cell, protoplast, callus, pollen, and organ culture. Important applications include producing virus-free plants through meristem culture and generating novel hybrids through protoplast fusion.
Plant tissue culture is the process of maintaining or growing plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. It involves techniques like cell culture, organ culture or meristem culture to produce clones of a plant through micropropagation. The key steps are selection of explant tissue from a donor plant, sterilization, establishment of the explant on a culture medium, multiplication through cell division and shoot formation, rooting of shoots, and acclimatization of plantlets in soil. Micropropagation allows for rapid mass multiplication of plant materials while maintaining genetic uniformity.
This document provides an overview of plant tissue culture. It defines tissue culture as the in vitro cultivation of plant cells or tissues under aseptic conditions on a nutrient medium. The history and key figures in the development of plant tissue culture are discussed. Details are provided on nutrient requirements, preparation and sterilization of culture media, basic laboratory requirements, establishment of cultures from explants, and types of growth and cultures. The advantages and applications of plant tissue culture are also summarized.
The document discusses micropropagation, which is a method of vegetative propagation used to rapidly produce multiple genetically identical copies of plants through tissue culture techniques. It describes the five main stages of micropropagation as preparatory, initiation of culture, multiplication, rooting of shoots, and transplantation. The multiplication stage involves approaches like callus formation, adventitious bud formation, and enhanced axillary branching to produce many new shoots from an explant. Micropropagation offers advantages like producing large numbers of disease-free clones from a single plant in a relatively short time and small space.
This document contains protocols for various plant tissue culture techniques. It discusses the introduction to plant tissue culture, sterilization techniques used, and then outlines 8 specific protocols: 1) tissue culture media preparation, 2) explant preparation and surface sterilization, 3) embryo culture, 4) culture of anther for haploid production, 5) meristem culture, 6) meristem tip culture for virus-free plants, 7) induction of somatic embryogenesis, and 8) protoplast isolation, culture, and regeneration. The goal of these protocols is to describe the principles and procedures of different plant tissue culture methods.
Tissue culture is a technique where cells, tissues or whole plants are grown in a sterile nutrient culture medium under controlled conditions. It allows for rapid vegetative propagation of plants. Key steps include sterilizing equipment and explants, preparing nutrient medium, subculturing to promote growth, and rooting and hardening plantlets. Tissue culture has many applications like mass multiplication of crops and plants, eliminating diseases, and genetic modification. It is used commercially for propagating crops but contamination and rooting difficulties can be issues.
Rejuvenation techniques like pruning, manuring, thinning shoots, and controlling pests and diseases can help restore productivity and vitality to old orchards. Top working methods such as cleft grafting and wedge grafting involve grafting desirable scions or shoots onto the branches or trunks of existing trees to convert them to more profitable varieties. Bridge grafting can repair tree injuries by grafting scions between the damaged section and healthy crown to restore nutrient transport.
This document provides a list of 21 architectural works and the architects or theorists associated with them. It includes seminal works that advanced modern and postmodern architecture in the 20th century, addressing topics like ornamentation, functionalism, complexity, diagramming, and relationships between buildings and landscapes. Each entry lists a short description or key concept related to the architectural work.
Development of cancer therapeutics is often carried out in 2D cultures prior to testing on animal model. In comparison to 2D cultures, discuss the potential of using 3D in vitro models for drug efficiency testing.
Mitosis and meiosis are both cell division processes in eukaryotes. Mitosis produces two identical daughter cells through chromosome duplication and separation, while meiosis reduces the chromosome number through two cell divisions. Meiosis results in four haploid daughter cells through homologous chromosome separation in meiosis I and sister chromatid separation in meiosis II. Both processes involve the duplication of chromosomes followed by their orderly separation through different stages including prophase, metaphase, anaphase and telophase.
The document discusses different types of cell cultures, including primary cultures derived from animal tissue, continuous cultures comprised of cell lines, and normal diploid cells with a finite lifespan. It describes common cell lines used in research, including HeLa cells from cervical carcinoma and CHO cells from hamster ovary. The key components of maintaining cell cultures are discussed, such as temperature, atmosphere, sterile conditions, culture medium, and growth equipment. Tasks demonstrate observing cell morphology, quantifying cell density using a hemocytometer, and counting chromosomes in CHO cells, which are shown to have an aneuploid number due to long-term cultivation.
This document discusses various propagation techniques for small cardamom. It describes seed propagation methods including seed collection, treatment, sowing, and nursery practices. Vegetative propagation through division of rhizomes is also covered. The document outlines micropropagation stages from selection of explants to rooting and acclimatization. Micropropagation provides a means to rapidly multiply planting material through tissue culture. Various growth media and plant growth regulators are used to induce shoot formation, elongation, and rooting.
This document discusses in vitro plant breeding techniques. It describes how plant cells, tissues, and organs can be cultured under controlled conditions in glass or plastic vessels with defined growth media. Plant cells have three key abilities - totipotency, dedifferentiation, and competency - that allow regeneration of whole plants in tissue culture. The ratio of auxin and cytokinin plant hormones can determine whether roots or shoots develop. Somatic embryogenesis is described as the formation of embryo-like structures from somatic cells that can develop into whole plants similarly to zygotic embryos.
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.
Dr. Rehab Al Mousa. Plant Tissue CultureRehab Moussa
Plant tissue culture is a technique for growing plant cells, tissues or organs in vitro on artificial nutrient media under sterile conditions. It allows for the clonal propagation of plants as well as applications in plant breeding such as haploid production, somatic hybridization and genetic modification. Some challenges include contamination, hyperhydricity, phenolic exudation, shoot tip necrosis and somaclonal variation. Tissue culture has many uses in micropropagation, plant breeding, germplasm preservation, plant physiology and production of secondary metabolites.
Micropropagation is the process of rapidly multiplying plant materials in vitro. It involves taking plant cells, tissues or organs as explants and culturing them on nutrient media to stimulate growth. Common micropropagation techniques include axillary shoot proliferation from pre-existing meristems using cytokinin treatment, shoot organogenesis from leaf or stem explants, and somatic embryogenesis from callus or suspension cells. Micropropagation has advantages like rapid multiplication, disease elimination, and production of propagules with desirable traits, but also limitations like high equipment and expertise costs. It has applications for quickly increasing stocks of new varieties and producing disease-free plants.
Commercial exploitation of micro its technologyPawan Nagar
Micropropagation is a tissue culture technique where whole plants are regenerated from small plant tissues or cells grown in a sterile nutrient culture medium. It allows for rapid multiplication of elite plant varieties in a short period of time compared to traditional propagation methods. The document outlines the various steps involved in micropropagation including selection of explant material, surface sterilization, initiation and establishment of aseptic culture, multiplication of shoots, rooting of plantlets, and acclimatization of plantlets in the greenhouse. Micropropagation has many advantages for commercial horticulture including production of disease-free plants, continuous propagation year-round, export of pathogen-free plants, and long-term germplasm storage.
This document discusses various types of plant tissue culture techniques. It defines explants as sterile pieces of plant material used to initiate cultures. Young, rapidly growing tissue is preferred for explants. Common types of in vitro culture discussed include callus culture, cell suspension culture, organ culture, and protoplast culture. Aseptic technique and sterilization methods like heating, filtration, and chemicals are used to prevent contamination during culture.
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 types of tissue culture can be grouped by the structures formed in culture.
Plantlets
Seedlings
Callus
Somatic EmbryogenesisPlantlet formationThis is the most common form of micropropagation. Uses a portion of the stem with one to several nodes
1. Axillary shoot formation Meristem culture Shoot culture
2. Adventitious shoot formation Diploid plant regenerationPseudocorms
Pseudocorms are the structures initiated after seed germination in orchids Haploid and triploid regeneration
This document discusses plant tissue culture techniques. It begins by explaining how plant tissue culture has become popular for horticultural and industrial applications as well as studying plant growth and development. It then describes the history and development of plant tissue culture, including its earliest uses and current commercial applications like micropropagation. The rest of the document details the various techniques involved in plant tissue culture, including quantitative and qualitative improvement approaches, micropropagation stages and methods, and troubleshooting browning issues.
Commercial Exploitation of Micro-propagation in fruit crops & its TechniquesPawan Nagar
Micropropagation is a tissue culture technique where plantlets are regenerated from small plant parts like shoot tips, nodes, and meristems. It allows for the rapid multiplication of plant materials in a relatively short period of time compared to traditional propagation methods. The process involves sterilizing and culturing explants on nutrient media, multiplying shoots through subculture, rooting the shoots, and acclimatizing the plantlets. Micropropagation has various advantages like producing disease-free plants, conserving germplasm, and facilitating the export of plants. It has been commercialized for many horticultural crops in India like banana, citrus, grapes, guava, papaya, and strawberry through research institutes.
Plant tissue culture is a technique of growing plant cells, tissues, organs, seeds, or other plant parts in a sterile
environment on a nutrient medium.
Tissue culture had its origins at the beginning of the 20th century with the work of Gottlieb Haberlandt
(plants).
WHY PLANT TISSUE CULTURES ARE DONE ??
The production of clones of plants that produce particularly good flowers, fruits, or have other desirable traits.
To quickly produce mature plants.
The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds.
The regeneration of whole plants from plant cells that have been genetically modified.
The production of plants in sterile containers reduces disease transmission
Allows production of plants from seeds that otherwise have very low chances of germinating and growing, i.e.: orchids and Nepenthes.
To clean particular plants of viral and other infections and to quickly multiply these plants as 'cleaned stock' for horticulture and agriculture.
***For PTC, the laboratory must have the following facilities:
Washing facility for glassware and ovens for drying glassware.
Medium preparation room with autoclave, electronic balance and pH meter.
Transfer area sterile room with laminar air-flow bench and a positive pressure ventilation unit called High Efficiency Particulate Air (HEPA) filter to maintain aseptic condition.
Culture facility: Growing the explant inoculated into culture tubes at 22-28° C with illumination of light 2400 lux, with a photoperiod of 8-16 hours and a relative humidity of about 60%.
*****Based on the explants some other plant tissue culture types are:
1. Organ culture
2. Meristem culture
3. Protoplast culture
4. Cell culture.
Micropropagation is an advanced vegetative propagation technology for producing a large number of transplants in a limited time and space.
STAGES
Stage 0 — Mother Plant Selection:
Stage I — Establishment of Aseptic Culture:
Stage II — Multiplication of shoots:
Stage III — In Vitro Rooting:
Stage IV — Transplantation or Hardening:
APPLICAIONS
Commercial exploitation of micropropagation& its tech in fruit cropsPawan Nagar
Micropropagation is a process where a whole plant can be regenerated from small plant tissues or cells in a suitable culture medium. It allows for rapid multiplication of plants in a relatively short time and small space. The process involves 5 main steps - selection of explant, initiation and establishment of aseptic culture, multiplication of shoots, rooting, and acclimatization of plantlets. Some key advantages of micropropagation are that it enables fast commercial propagation of new varieties, provides a continuous supply of young pathogen-free plants, and allows for storage and exchange of germplasm. Micropropagation has been developed for many horticultural crops in India like banana, citrus, grapes, and strawberries.
The document discusses micropropagation, which is a type of tissue culture used to rapidly produce many uniform plants from small plant parts like meristems. It involves four stages: establishing an aseptic culture, multiplying propagules in nutrient media, pre-transplant rooting, and transplanting to soil. Micropropagation has advantages like producing many clean, genetically identical plants quickly and independently of seasons. It requires specialized facilities and expertise. Potential problems include contamination, tissue browning, abnormal growth, and shock from in vitro to in vivo conditions. Micropropagation has applications for rapidly increasing stocks of superior varieties and producing disease-free plants.
This document discusses micropropagation, which is the rapid multiplication of plant materials using tissue culture methods. It involves taking explants like shoot tips or buds and culturing them on growth media to produce many new plantlets. The process involves initiation, multiplication, rooting, and acclimatization stages. Approaches include multiplication from axillary buds/shoots or adventitious shoots. Applications are high rate propagation of disease-free plants, seed production in some crops, and cost effectiveness. Automation using bioreactors and robots can increase production scale but reduces flexibility.
Micropropagation is a method of vegetative plant propagation that uses small plant parts or cells to produce many new plants in a short period of time. The process involves selecting disease-free mother plants, establishing aseptic cultures, multiplying shoots in culture media, rooting the shoots in vitro, and hardening the plantlets for transplantation. Micropropagation takes advantage of plant cells' natural ability to regenerate entire new plants from single cells through a process called totipotency. This allows for large-scale production of identical disease-free plants, creation of new plant varieties, and conservation of endangered species.
This document discusses micropropagation as a method of clonally propagating plants. It begins by explaining traditional clonal propagation methods and their limitations. It then describes the benefits of micropropagation, which allows for rapid multiplication of plants using small explant tissues in sterile conditions. The document outlines the five main stages of micropropagation: preparation, initiation of cultures, multiplication, rooting, and transplantation. It provides details on each stage, focusing on choices of explants, factors influencing successful culture initiation, and methods of multiplication like regeneration from callus or direct shoot formation. Micropropagation offers advantages like high multiplication rates, disease elimination, and cryopreservation of plant materials.
Plant tissue culture involves growing plant cells, tissues or organs under sterile conditions on a nutrient medium. It has a history dating back to the 1930s and was pioneered by scientists like Haberland and White. Techniques include using explants from plants, sterilizing them, and culturing them on nutrient media containing salts, sugars and growth regulators. This allows for dedifferentiation of cells and regeneration of whole plants. Micropropagation specifically refers to rapidly multiplying shoots in culture. It has five stages - selection of stock plants, initiation of culture, shoot multiplication, rooting, and hardening. It has advantages like producing many pathogen-free plants quickly from small explants.
Micropropagation is a method of rapidly multiplying plant materials through tissue culture techniques. It involves four main stages: selection and sterilization of explant material, multiplication of propagules in culture, regeneration of plantlets, and acclimatization of plantlets to soil conditions. The method allows for large-scale production of disease-free plants in a controlled environment and offers advantages such as year-round production and transport of materials across borders. However, it also has limitations such as high costs, need for specialized facilities and skilled personnel, and lack of confirmed genetic stability in some methods.
Micropropagation is the process of rapidly multiplying plant materials in an aseptic laboratory environment. It involves culturing small pieces of plant tissue on nutrient media containing hormones and sugars. The ratio of auxin and cytokinin hormones determines whether shoots or roots develop. Micropropagation has several advantages over traditional propagation methods, including producing many identical clones of plants that are disease-free and genetically uniform. The process involves initiation, multiplication, rooting, and transfer to soil stages. Common micropropagation techniques are meristem culture, callus culture, and embryogenesis.
Micropropagation is a tissue culture technique used for the rapid asexual propagation of plants. It involves culturing small pieces of plant tissues or organs on growth media in controlled conditions. The process includes initiation of cultures from explants, multiplication through shoot proliferation or somatic embryogenesis, rooting of shoots, and transplantation of plantlets to soil. Micropropagation allows for large-scale production of genetically identical clones in a short period of time. It has applications in producing disease-free plants, conserving rare species, and commercializing new plant varieties.
This document discusses tissue culture techniques for banana micropropagation. It begins by providing background on the importance of banana as a crop in India and challenges with conventional propagation methods. It then describes the various stages of banana micropropagation in tissue culture, including explant preparation, initiation, multiplication, rooting, and hardening. Key details are provided on media composition and conditions for each stage. The overall process takes approximately 10 months to produce hardened plantlets ready for field planting. Tissue culture techniques allow for large-scale production of disease-free, uniform banana planting material.
Micropropagation is the rapid vegetative propagation of plants under controlled conditions of light, temperature, and nutrients. It involves multiplying plant parts through tissue culture in multiple stages: initiation and establishment of explants, shoot multiplication, root development, and acclimatization. The technique allows for mass production of disease-free plants from small amounts of tissue year-round and international exchange without disease risk. It provides an alternative for species resistant to conventional propagation and helps conserve endangered species.
Definition of hairy root culture ,multiple shoot culture ,Production of hairy root and multiple shoot , advantages an disadvantages of hairy root and multiple shoot culture, Sterilization and sterilizing agents wit concentration and exposure time
Mass multiplication procedure for tissue culture and PTC requirementDr. Deepak Sharma
This presentation include basic Micropropagation protocol: Application and advantages of mass multiplication. Beside this the requirement of tissue culture are there (Nutrient, gelling agent, energy source, vitamins and PGRs) are also included.
This document provides information about a seminar on micropropagation techniques in fruit crops. It discusses the need for micropropagation due to issues like seasonal limitations and virus transmission. The advantages of micropropagation include producing true-to-type plants, overcoming seasonal constraints, and allowing large-scale multiplication. The document outlines the stages of micropropagation including establishment, proliferation, rooting, and acclimatization. It also describes different approaches like axillary budding and somatic embryogenesis. Several case studies demonstrate the use of micropropagation in plants like date palm, mango, and lemon.
Micropropagation is the process of cloning plants through tissue culture techniques. It involves five main stages: selection of source plants, establishment of cultures through sterilization and growth in medium, proliferation through transfer to multiplication medium, rooting and hardening of shoots, and transfer of plantlets to soil. Various micropropagation methods are used, including meristem culture, somatic embryogenesis, and organogenesis. While it allows for mass production of disease-free clones, micropropagation requires specialized facilities and training and can result in genetic variability or contamination issues.
Meristem and shoot tip culture in horticultural cropsHORTIPEDIA INDIA
This document outlines the process of meristem and shoot tip culture in horticultural crops. It discusses (1) the establishment of explants in culture media, (2) the multiplication of propagules through axillary shoot proliferation using cytokinins, and (3) the regeneration of adventitious roots using auxins to complete the tissue culture process. Meristem and shoot tip culture is an effective method for cloning plant material and producing disease-free plants for agriculture and industry.
ISO/IEC 27001, ISO/IEC 42001, and GDPR: Best Practices for Implementation and...PECB
Denis is a dynamic and results-driven Chief Information Officer (CIO) with a distinguished career spanning information systems analysis and technical project management. With a proven track record of spearheading the design and delivery of cutting-edge Information Management solutions, he has consistently elevated business operations, streamlined reporting functions, and maximized process efficiency.
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Date: May 29, 2024
Tags: Information Security, ISO/IEC 27001, ISO/IEC 42001, Artificial Intelligence, GDPR
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A Strategic Approach: GenAI in EducationPeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
it describes the bony anatomy including the femoral head , acetabulum, labrum . also discusses the capsule , ligaments . muscle that act on the hip joint and the range of motion are outlined. factors affecting hip joint stability and weight transmission through the joint are summarized.
Introduction to AI for Nonprofits with Tapp NetworkTechSoup
Dive into the world of AI! Experts Jon Hill and Tareq Monaur will guide you through AI's role in enhancing nonprofit websites and basic marketing strategies, making it easy to understand and apply.
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আমাদের সবার জন্য খুব খুব গুরুত্বপূর্ণ একটি বই ..বিসিএস, ব্যাংক, ইউনিভার্সিটি ভর্তি ও যে কোন প্রতিযোগিতা মূলক পরীক্ষার জন্য এর খুব ইম্পরট্যান্ট একটি বিষয় ...তাছাড়া বাংলাদেশের সাম্প্রতিক যে কোন ডাটা বা তথ্য এই বইতে পাবেন ...
তাই একজন নাগরিক হিসাবে এই তথ্য গুলো আপনার জানা প্রয়োজন ...।
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Exploiting Artificial Intelligence for Empowering Researchers and Faculty, In...Dr. Vinod Kumar Kanvaria
Exploiting Artificial Intelligence for Empowering Researchers and Faculty,
International FDP on Fundamentals of Research in Social Sciences
at Integral University, Lucknow, 06.06.2024
By Dr. Vinod Kumar Kanvaria
A review of the growth of the Israel Genealogy Research Association Database Collection for the last 12 months. Our collection is now passed the 3 million mark and still growing. See which archives have contributed the most. See the different types of records we have, and which years have had records added. You can also see what we have for the future.
How to Build a Module in Odoo 17 Using the Scaffold MethodCeline George
Odoo provides an option for creating a module by using a single line command. By using this command the user can make a whole structure of a module. It is very easy for a beginner to make a module. There is no need to make each file manually. This slide will show how to create a module using the scaffold method.
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How to Manage Your Lost Opportunities in Odoo 17 CRMCeline George
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1. Pradeep Kumar Vishwakarma
M.Sc. (Horti.) Fruit science,
IInd Semester
REG. NO: 04-2691-2015
Techniques of In vitro clonal
propagation for fruit crops
2. In vitro:
The culture of plant cells or organs in culture vessels (like
test tubes) under controlled environment and nutritive growth medium.
In vitro clonal propagation (Micropropagation):
Micropropagation is the production of new plants under the ultra-
controlled environment within the culture vessel (in vitro). Commercial
micro-propagation began in the United States in 1965 with orchid
production.
Uses for micropropagation:
Mass propagation of specific clones
Production of pathogen-free plant
Clonal propagation of parental stock (inbred lines) for hybrid seed
production
Year-round nursery production
Germplasm preservation.
Techniques of In vitro clonal propagation for fruitcrops
3. Commercial Tissue culture propagation is particularly
useful in the following situations:
Where the propagation rate is slow by conventional means
New cultivars with high market demand
Cultivars with high market value
Plants which is difficult to propagate clonally
Conservation of endangered species.
4. Disadvantages of Tissue culture propagation:
High labor costs
A requirement for expensive and sophisticated facilities, trained
personnel, and specialized techniques
A high-volume, more or less continuous distribution system, or
adequate storage facilities to stockpile products, is required
Pathogen contamination or insect infestation can cause high
losses in a short time
Variability and production of off-type individuals can be a risk
in the products emerging from micropropagation
Careful rouging field testing of new products, and continuing
research and development are essential to decrease this risk
Economics and marketing are key to the success of commercial
operations
5. General laboratoryfacilities andprocedures:
Facilitiesand Equipment:
Facilities for micropropagation may be placed into three
categories as determined by their scope, size, sophistication, and
cost. These categories are:
Research laboratories where precise work requires highly
sophisticated equipment
Large commercial propagation facilities where several
million plants can be mass-produced annually and
limited facilities for small research laboratories, individual
nurseries, or hobbyists where a relatively small volume of
material is handled.
Regardless of size, the facility should include separate
preparation) transfer, and growing areas. In addition, there may
be a need for service areas, office, and cold storage.
6. MediaPreparation:
Success of in vitro technology depends on the nutritional
media. It is essential to understand the nutritional requirement for
optimal growth of a particular tissue. Medium ingredients can be
grouped into the following categories:
Inorganic salts
Organic compounds
Gelling agents
There are basically four stages to the micropropagation process.
Stage I. Establishment
Stage II. Shoot multiplication
Stage III. Root formation
Stage IV. Acclimatization
Micropropagation procedures:
7. Stage I-Establishment:
The function of this stage is to disinfest the explant, establish it in
culture, and then stabilize the explants for multiple shoot development.
Preparation for Establishing Cultures:
Handling Stock Plants:
A principal consideration for handling stock plants is reducing the
potential for contamination by fungi, viruses and other pathogens.
Pathogens are not automatically eliminated by in vitro technique unless
this goal is built into the system. Selected “virus-free” or pathogen-indexed
stock plants should be used.
Choice of Explant
The kinds of explant and where and how they are collected varies
with the purpose of the culture, the species, and often the cultivar.
Disinfestation:
Disinfestation is the process of removing contaminants from the
surface of the explant. A typical procedure would be to cut the plant
material into small pieces and wash them in running tap water. For
materials that are difficult to disinfest, a quick dip in alcohol may be
helpful. Wrapping the shoots with small squares of sterile gauze will hold
them intact during the treatment.
8. Pretreatment of Explants:
Exudation of phenolic and other substances from the cut explant
surface can inhibit initial shoot development. Using liquid media in the
initial stage or frequent transfers on agar medium for several days may leach
out the toxic materials. Likewise, using antioxidants, such as ascorbic acid
and citric acid, in the preliminary washing may be useful.
Initial Shoot Development:
Depending on the explant, shoots will initiate from:
(a) The stimulation of axillary shoots
(b) The initiation of adventitious shoots on excised shoots, leaves, bulb,
scales, flower stems, cotyledons, and other organs
(c) The initiation from callus on the cut surfaces. Which medium is selected
varies with the species, cultivar, and kind of explant to be used
Some woody plant species can take up to a year to complete Stage I, this
phenomenon is called stabilization. A culture is stabilized when explants
produce a consistent number of “normal” shoots after subculturing.
“Abnormal” shoots usually are arrested in development and fail to elongate.
10. Stage II-Shoot Multiplication:
In the multiplication stage each explant has expanded into a
cluster of microshoots arising from the leaf axils or base of the
explant. The separate microshoots are transplanted into a new culture
medium, which is a process called subculturing. During the
multiplication stage, cultures are subcultured every 4 to 8 weeks.
The kind of medium used depends on the species, cultivar,
and type of culture. The basal medium is usually the same as in Stage
I, but often the cytokinin and mineral supplement level is increased.
Adjustments may need to be made after some experimentation.
11. Stage III- Root Formation:
Shoots developed during the multiplication stage do not usually
have roots. There are some exceptions (like African violet and poplar)
that spontaneously root on the multiplication medium, but for most other
species, single shoots (micro-cuttings) must be moved to a medium or
suitable environment to induce roots. Therefore, the purpose of Stage III
is to prepare plantlets for transplanting from the tissue culture
environment of the test tube to a free-living existence in the greenhouse
and on to their ultimate location. Therefore, Stage III may not only
involve rooting, but also conditioning the plantlet to increase its potential
for acclimation and survival during transplanting, for example, by
increasing agar and or sucrose concentration. Light intensity is
sometimes increased during Stage III. Some plants respond to an
“elongation” phase between Stages II and III, achieved by placing the
microshoot into an agar medium for 2 to 4 weeks without cytokinins (or
at very low levels) and, in some cases gibberellic acid, which reduces the
influence of cytokinin. The microshoots are then rooted in a cytokinin
and GA-free medium.
13. Stage IV- Acclimatization to Greenhouse Conditions:
Once plantlets are well rooted, they must be acclimatized to the
normal greenhouse environment. In vitro rooted plants are removed from
the culture vessel and the agar is washed away completely to remove a
potential source of contamination. Plantlets are transplanted into a
standard pasteurized in a shaded, high humidity tent or under mist or fog.
Several days may be required for new functional roots to form.
Plantlets should be gradually exposed to a lower relative humidity and
higher light irradiance. Any dormancy or resting condition that develops
may need to be overcome as part of the establishment process. In
summary, micropropagation involves the vegetative propagation of
plants through (1) establishment, (2) multiplication, (3) rooting, and (4)
acclimatization (transplanting) of clones in vitro starting with small
explants and ending with a rooted plant established in a container or in
the ground.
15. Types of tissue culture systems
The procedures used for tissue culture that are associated
with propagation. These include the formation of:
1. Plantlets
2. Seedlings
3. Callus
4. Somatic embryos
Of these techniques, the process of plantlet formation via
micropropagation has the most direct application for plant
propagation and will be emphasized in this chapter.
16. Micropropagation of Plantlets from Tissue Culture:
Cultural systems described under plantlet formation are based
on the maintenance and multiplication of microshoots in culture to
produce rooted micro cuttings. A distinction is made between
microshoots originating from axillary buds and those arising
adventitiously directly from tissue without axillary buds (i.e., leaf or
petiole) or indirectly from callus developed from the original explant.
Developmental Stages in Micropropagation:
Success of micropropagation is largely due to separating
different developmental aspects of culture into stages, each of which is
manipulated by media modification and environmental control. Four
distinct stages are recognized for most plants.
Stage I: Establishment
Stage II: Shoot multiplication
Stage III: Root formation
Stage IV: Acclimatization
17. There are basically two developmental patterns for plantlet
formation in tissue culture, described by the way shoots originate from the
initial explant in Stages I and II. Once shoots have formed, the rooting and
acclimatization stages are the same for the various developmental patterns.
Meristem culture:
A procedure to eliminate diseases from plants, using a very small piece
of tissue from the shoot tip as the initial explant
Meristem culture utilizes the smallest part of the shoot tip as the
explant, including the meristem dome and a few subtending leaf
primordia
The number of additional structures depends on the length of the
excised stem. The primary reason for this procedure is to produce a
plantlet that is free of systemic viruses, virus-like organisms, and
superficial fungi and bacteria
The meristem is usually free of disease organisms; therefore, the
smaller the explant, the more effective the elimination of pathogens
Invitro techniques of clonal propagation(Tissue culture):
18.
19. Shoot-tipmicrografting in vitro
Grafting very small meristem tips comparable to those
described in the preceding section can be used as an alternative
method to produce virus-free materials for various woody plants, such
as Citrus and Prunus. For example, this procedure is important in
citrus not only because it is successful, but also because explants can
be used from ontogenetically mature trees, which avoids the juvenile
phenotype of nucellar seedlings also used in virus cleanup in citrus.
The technique for micrografting can be illustrated with citrus.
Citrus embryos are excised from rootstock seeds, surface
disinfested, and planted in standard inorganic salt medium with 1
per cent agar
Embryos germinate in the dark in 2 weeks
Seedlings are then removed and decapitated to a 1 to 1.5 cm
length; cotyledons and lateral buds are excised with a mounted
razor blade
20. A 0.14 to 0.18 mm tip with 3 leaf primordia is used as the scion
and this gives reasonable success, and the shoot tip eliminates
viruses
An inverted T-bud cut is made in the seedling root stock, cutting
1 mm down the stem, followed by a horizontal cut on the
bottom
The excised shoot tip is placed inside the flap next to the
cambium
Grafted plants are placed in a liquid medium
A filter paper bridge with a center hole supports the stem
Cultures are kept in the light for 3 to 5 weeks to heal. When two
expanded leaves appear on the scion, the grafted plant is
transplanted
A similar procedure has been used for apple and plum
Rootstocks are either seedling plants or rooted stems
Shoot-tip scions taken from cultured plants reduce
contamination problems
22. Anther culture:
Anther culture is a procedure for obtaining haploid plants
from normally diploid plants
A flower bud from Nicotiana tabacum is excised just as the
petals are emerging from the bud
The immature anther is removed aseptically and planted on
agar with a standard nutrient medium without hormones
Pollen should be at the uninucleate, microspore stage of
development
Somatic embryos develop from callus derived from the
haploid microspores
Haploid (1n) embryos germinate to form plantlets
It is used for plant breeding to produce haploid plants
24. EmbryoCulture and EmbryoRescue:
Embryo culture is a procedure involving tissue culture of
immature embryos that require controlled conditions to
complete development
It is most commonly used by plant breeders for embryo rescue
of genetic crosses that would not form seed on the plant
Embryo culture involves the excision of an embryo from a
seed and germinating it in aseptic culture
As early as 1904, the first attempts were made to grow
immature embryos in nutrient solution
These immature crucifer embryos germinated, but growth was
weak. Hannig called this “precocious germination.” A principal
use of embryo culture is to “rescue” embryos that would have
aborted within the seed before the fruit was mature
Much interspecific and intergeneric hybridization are initially
successful but the embryo aborts during development.
26. Ovary and Ovule Culture:
Ovary and ovule culture include the aseptic culture of the excised
ovary, ovule (fertilized or unfertilized), and placenta attached
within the ovule
Although this technique was first utilized to investigate problems
of fruit and seed development, adaptations of the procedure have
uses in propagation, particularly in genetic improvement
Unfertilized ovules have been excised, grown in culture, supplied
with pollen, and subsequently fertilized in vitro
Such procedures are most successful with plants that have multiple
ovules
Pollen can be placed directly on the placenta inside the ovule,
where pollen tubes can develop and grow immediately into the
ovules without passing down the style
Cultured ovules are useful for rescuing embryos that abort at a very
young stage if not separated from the plant
This technique is simpler than culturing isolated embryos.
28. Callus Culture:
Callus results from cell division in non-differentiated parenchyma
cells
Eventually a callus culture does form stratified cell layers with outer
meristematic layers and inner cells that can form vascular tissue.
Callus is produced on explants in vitro as a response to wounding
and growth substances, either within the tissue or supplied in the
medium
Explants from almost any plant structure or part seeds, stems, roots,
leaves, storage organs, or fruits can be excised, disinfested, and
induced to form callus
Continued subculture at three to four week intervals of small cell
clusters taken from these callus masses can maintain the callus
culture for long periods
30. Cell Suspensions culture:
A suspension culture is started by placing a piece of friable callus or
homogenized tissue in liquid medium so that the cells disassociate from each
other
Batch cultures, cells are grown in a flask placed on a shaking device that allows
air and liquid to mix. Rotating devices that result in continuous bathing of the
tissue are available
Another device, called a chemostat or turbidostat, continuously cycles the media
through the cell culture essentially in the same manner as in the culture of
microorganisms
In a third method, cells on a filter paper layer are placed on a shallow liquid
medium in a Petri dish with no agitation
Growth of cells follows a typical pattern based on changes in rates of cell
division
Cells first divide slowly (lag phase), then more rapidly (exponential), increasing
to a steady state (linear), followed by a declining rate (deceleration) until a
stationary state is reached
When cells are transferred to a new liquid medium, the process will be repeated.
Under proper environmental conditions with media control, the process can go
on indefinitely.
32. Protoplast Culture:
Protoplasts are the living parts of plant cells, containing the
nucleus, cytoplasm, vacuole and various cellular structures
surrounded by a semipermeable membrane (plasmalemma)
but with the cell wall removed protoplasts can be obtained
from cells in suspension or derived directly from mesophyll
leaf cells
The major advance that permitted protoplast cultures to be
made was the discovery that plant cell walls could be
removed by enzymes that digest pectin and allow the
protoplast surrounded by its cellular membrane to survive.
33.
34.
35.
36. 1.Banana:
Method: Shoot-tipcultures:
Banana is commercially propagated by shoot tip culture in vitro.
StageI: Initiationof shoot cultures:
Shoot cultures of banana start conventionally from any plant part
that contains a shoot meristem, i.e. the parental pseudo-stem, small
suckers, peepers and lateral buds. The apex of the inflorescence and
axillary flower buds are also suitable explants for tissue culture initiation.
Overall, it is important to select explant material from preferably mature
individuals whose response to environmental factors is known, and
whose quality traits governed by genotypic and environmental effects
have been identified. For rapid in vitro multiplication of banana, shoot
tips from young suckers of 40100 cm height are most commonly used as
explants.
37. Media and its combination:
The explant is placed directly on a multiplication inducing culture
medium. For banana micropropagation, MS based media are widely
adopted. Generally, they are supplemented with sucrose as a carbon source
at a concentration of 3040 g/l. Banana tissue cultures often suffer from
excessive blackening caused by oxidation of poly phenolic compounds
released from wounded tissues. These undesirable exudates form a barrier
round the tissue, preventing nutrient uptake and hindering growth.
Therefore, during the first 46 weeks, fresh shoot tips are transferred to new
medium every 12 weeks.
Stage II: Multiplication of shoot tip cultures:
The formation of multiple shoots and buds is promoted by
supplementing the medium with relatively high concentrations of
cytokinins. In banana, BA is the preferred cytokinin and is usually added in
a concentration of 0.120 mg/l. For the multiplication of propagules, we use
the same medium as for the initiation of shoot cultures (p5 medium
containing 2.25 mg/l BA and 0.175 mg/l IAA).
38. Stage III: Regenerationof plants:
Individual shoot or shoot clumps are transferred to a nutrient
medium which does not promote further shoot proliferation but
stimulates root formation. The cytokinin in the regeneration medium is
greatly reduced or even completely omitted. Within 2 weeks, shoot tips
develop into un-rooted shoots. To initiate rhizo-genesis IAA, NAA
(naphthalene acetic acid) or IBA (indole3butyric acid) are commonly
included in the medium at between 0.1 and 2 mg/l. We use the same
auxin concentration as in the proliferation medium (0.175 mg/l IAA), but
a tenfold lower BA concentration (0.225 mg/l). For some genotypes
(Musa spp. ABB and BB group) that produce compact proliferating
masses of buds, activated charcoal (0.10.25%) is added to the
regeneration/rooting medium to enhance shoot elongation and rooting.
Hardening:
After rooting, plants are hardened in vitro for 24 extra weeks on
the regeneration/rooting medium prior to transplantation to soil.
39. Invitro propagationof banana:
Flow chart:
Selection of superior clone
↓
Culture initiation in the production lab
↓
2nd/3rd sub-cultures
↓
Virus-free certified material
↓
Multiplication of shoots up to a maximum of 8 passages
↓
Rooting (in vitro)
↓
Hardening inside the greenhouse, polyhouse & shade area Nursery
↓
Dispatch
40. Tissue culture technique is providing a rapid system for production
of large number of genetically uniform and disease free plantlets for
agriculture and forestry. Although there have been previous reports on date
palm micropropagation through the organogenesis and somatic
embryogenesis the protocols needs to be improved. The objective of the
work is to develop reliable method of organogenesis for date palm.
Method:
Plant Material:
The propagation material is used the offshoots were collected. The
selected offshoots were 4-5-year-old, each weighting approximately 6-8 kg.
Cleaning of explants:
To remove the attached soil and other debris, the offshoots were
washed with the tap water and the outer large leaves and fibers were
carefully removed with the sharp knife until the shoot tip zone was exposed.
Shoot tips were then trimmed to approximately 6-7 cm in length and 4-6 cm
in width.
2. Date palm
41. Disinfectionand antioxidanttreatment:
The excised shoot tips were washed three-four times with
double distilled water. Thereafter, the cleaned shoot tips were subjected
to two steps of disinfection: a) the washed shoot tips were dipped for 20
minutes in a fungicide (Benlate, 5 g l-1) solution; b) the shoot tips were
dipped in 33% commercial clorox solution for 25-30 minutes. The
explants were then rinsed three times with autoclaved distilled water in
a laminar flow hood. The disinfected explants were then soaked in an
antioxidant solution to minimize oxidation of phenolic compounds
(responsible for the browning of tissues), and to protect them from
desiccation.
Explant preparation:
After the proper disinfection and antioxidant treatments, the
shoot tips were cut into 1-1.5 cm pieces under the laminar flow hood.
42. Callusinduction:
Two – four shoot tip pieces were placed on MS medium (Murashige
and Skoog, 1962) solidified with 3% agar-agar and supplemented various
auxins types and concentrations, viz.: 0.0-54.28 μM 2,4-
dichlorophenoxyacetic acid (2,4-D), 0.0-46.96 μM 2,4-5-
trichlorophenoxyacetic acids (2,4,5-T), 0.0-64.31 μM chlorophenoxy-acetic
acid (CPA). 0.3 g l-1 of activated charcoal was added to the medium in
order to remove the phenolic compounds.
Regenerationand shoot multiplication:
Callus (80-90 mg) was cultured on MS liquid and solid medium
supplemented with beznzylaminopurine (BAP) at concentrations 0.0-8.96
μM and kinetin (KN) at concentrations 0.0-9.28 μM. Data were recorded on
percentage of calli producing shoots (%), shoot number per a callus and
shoot length.
Rooting:
After 14 weeks in the regenerating medium, shoots were separated and
placed on MS liquid and solid medium supplemented with various
concentrations of IBA (0.0-29.52 μM), IAA (0.0-34.24 μM) and NAA (0.0-
32.22 μM).
43. Mediaand itscombination:
Modified Murashige and Skoog (MS) medium (Table 2) with 3%
sucrose supplemented with 2,4-D (100 mgl-1), NAA (3 mgl-1), 2iP (3 mgl-
1) and kinetin (3 mgl-1) is being successfully used for date palm tissue
culture. pH of the medium should be adjusted to 5.6 and solidified with 8.0
gl-1 of agar. Twenty five ml of medium is dispensed into 40 mm culture
tubes and sterilized for 15 minutes at 121o C. Activated charcoal (1.5 gl-1)
is added to the medium to avoid browning.
Acclimatizationand hardening:
A sterilized medium comprising peat moss, vermiculite and coarse
sand in the ratio 1:1:1 is ideal for soil transfer. It is recommended to
irrigate immediately with 50% Hoagland solution or 10% MS solution to
avoid dehydration of plants. Containers and potting media must be
adequately cleaned and sterilized. Moisture content of the medium must be
regulated to minimum, and relative humidity of the growth tub must be
maintained high. Acclimatization of date palm is lengthy processes, which
require several subculture steps and careful attention. Otherwise, casualty
rate will be very high. Fifty to ninety percent loss has been reported in date
palm culture during acclimatization.
44. Pomegranate is an economically important fruit crop of the
tropical and subtropical regions of the world that is cultivated for its
delicious fruits. Pomegranate is conventionally propagated by hard wood
and soft wood cuttings. But, this traditional propagation method does not
ensure disease-free and healthy plants. In addition, this method is a very
time-consuming and labor-intensive process. Hence, there is need to
develop an efficient in vitro technique for the propagation of this fruit
trees.
Method- Shoot tip culture:
Plant material and explants preparation:
Shoot tip and nodal segments (0.5 - 1.0 cm long) were excised
from two-year-old plants grown in a greenhouse. Following removing of
the leaves, explants were washed with running tap water for 30 minutes,
then were disinfected for 10 min in a 0.1% (w/v) calcium hypochlorite
solution with 2-3 drops of Tween 20 and rinsed three times in autoclaved
distilled water. The sterilized explants (three explants/jar) were vertically
cultured in induction medium.
3. Pomegranate
45. Culture mediaand culture conditions
Two different media; MS and WPM were used. Sucrose was
added at 30 g/L and myoinstol at 0.1 g/L. The pH of the prepared media
was then adjusted to 5.6 to 5.8 with 0.1 N NaOH, before adding 0.6%
agar.
In vitrorooting:
Shoot tips with 2-4 cm long developed in vitro were excised and
cultured in half-strength WPM medium containing 50 mg/L myo-
inositol, 15 g/L sucrose and 3 g/L agar. The medium was further
supplemented with 0, 2.5, 4.9 and 9.8 μM IBA or 0, 2.7, 5.4 and 10.8 μM
NAA. After 8 - 10 days, the rooted shoots were transferred to an auxin-
free half-strength medium for further elongation of the roots. Each auxin
treatment consisted of seven replicate jars with one shoot in each. After 4
weeks in culture, number and length of roots per rooted shoot were
evaluated.
46. Acclimatization and Hardeningof regenerated plantlets:
Well rooted explants were removed from the culture
medium. The roots were washed gently with tap water to remove
agar and then transferred to small plastic pots containing autoclaved
cocopeat-perlite mixture. The pots were covered with polyethylene
bags to maintain high humidity and kept at 25 ± 1°C in artificial
light (50 μmol/m2/s) provided by white fluorescent tubes for 3 to 4
weeks. To harden the plants, polyethylene bags opened gradually,
from a few minutes a day until normal conditions. Plants were then
transferred to larger pots (18 cm diameter) containing garden soil
(soil: compost, 1: 1); kept under shade for another 2 weeks and then
transferred to direct sunlight condition. The survival rate was
examined 40 days after transfer.
47. A retrospective overview of work done on the in vitro shoot tip grafting
in citrus in the world was taken up. In vitro shoot tip grafting is the most
reliable method to recover free citrus saplings from infected parental source.
The technique of in vitro shoot tip grafting is a suitable method for the
elimination of graft transmissible diseases in citrus. Preliminary studies showed
that certain pathogens are difficult to be eliminate from mother plant like citrus
exocortis and stubborn, which might be eliminated by a process of shoot tip
grafting in-vitro. Plant obtained by micro-grafting do not have the same
problems as nucellar plants such as reversion to the juvenile state, excessive
thorniness, vigorous and upright habit of growth, slowness to fruiting, alternate
bearing in early years and physical differences in fruit characteristics.
Method- In vitroshoot tip grafting in citrus species:
Procedure of in vitro shoot tip grafting: Shoo tip grafting in vitro
consists of grafting, under aseptic conditions with a small shoot tip of 0.1 to 0.2
mm, onto a young etiolated seedling rootstock. The technique has the following
steps: preparation of rootstock, preparation of scion, procedure of grafting,
growing of grafted plants in vitro, and transfer to soil.
4. Citrus
48. Preparation of rootstock forin vitroshoot tip grafting:
Chand L. (2013) conducted an experiment on shoot tip grafting
(STG). They reported that 30-50 per cent frequency of successful grafts was
obtained by using two week old dark grown rootstock seedlings and 0.14 to
0.18 mm long shoot tips as scions. The shoot tip was inserted into an
inverted-T cut made at the top of the decapitated rootstock epicotyls. Most
scion cultivars gave satisfactory grafts on Troyer citrange whereas lemon,
lime and citron yielded successful grafts only on Rough lemon. The age of
the rootstock also has an important influence on grafting success. The
highest rate of successful grafts using Troyer citrange as rootstock was
obtained with two week old seedlings, most shoot tips grafted on younger
seedlings (one week old) were covered with precocious callus formation by
the rootstock, whereas most shoot tips grafting on rootstock.
The scion taken from in vitro cultured buds produced healthy plants
than scions from whole tree. The frequency of successful grafts increased
with the size of the shoot tips but the per cent of virus free plants declined,
although this depended on the pathogen. In citrus, the shoot terminals
excised from mature bearing trees, actively growing shoot flushes from field
or greenhouse.
49. Special problems encountered by in vitro culture:
Hyperhydricity:
Hyperhydricity (originally called vitrification) is characterized by a
translucent, water-soaked, succulent appearance that can result in cultures that
deteriorate and fail to proliferate. Physiologically, expression involves excess
water uptake : (“waterlogging”) and inhibition of lignin and cellulose
synthesis. Hyperhydricity appears to be a consequence of the difference
between the water potential in the medium and developing shoots, as well as
a low nitrate to ammonium ratio. Hyperhydricity can also be caused by
hormonal imbalances, especially ethylene.
Internal Pathogens:
Even though micropropagation is considered to be a pathogen-free
system, complete absence of pathogens should not be taken for granted. The
original source material should be indexed for specific viruses even if the
original selection is from shoot-tip cultures or micrografts.
For example, in the initial enthusiasm for orchid micropropagation, the
importance of initial meristem treatment was not appreciated and shoot-tip
culture was assumed to control viruses. As a result, many of the commercial
sources of orchid cultivars were soon found to be infected, causing
considerable economic loss.
50. Excessive Exudation:
One of the most frequently encountered problems with
establishing explants (especially woody perennials) in culture is the
production of exudates by the explants. The exudates are usually
considered to be various phenolic compounds that oxidize to form a
brown material in the medium that tends to be inhibitory to
development. Treatments to minimize this problem include treating the
explant with an antioxidant (citric or ascorbic acid), including an
adsorbent material in the medium (polyvinylpyrrolidone or activated
charcoal), and frequent transfers to a new medium.
Shoot-Tip Necrosis:
Actively growing shoot tips sometimes develop “tip die- back”
usually caused by calcium deficiency that can be corrected by refining
the basic nutrient medium to include more calcium
51. Tissue Proliferation:
Tissue proliferation (TP) is the formation of gall-like
growths on the stem of micropropagated plants (146). It may
occur while plants are still in culture or may not be seen until
after plants have been moved to the greenhouse or nursery.
Habituation:
Habituation is the autotrophic growth in cultures that
had previously required auxin or cytokinin for growth. For
example, shoot cultures may become habituated to cytokinin
and continue to proliferate even after the culture has been
transferred to a medium without any growth regulator.