This document provides instructions for five plant tissue culture experiments. Experiment 1 demonstrates the totipotency and nutritional requirements of shoot tip and root tip explants from aseptically germinated seedlings. When transferred to different media, the explants show varying growth responses correlated to the media contents. Experiment 2 examines the effects of hormone balance on explant growth and morphogenesis. Experiments 3-5 involve callus formation, suspension cultures, and anther culture techniques. The document provides detailed background information and step-by-step methods for each experiment.
Originally isolated from nature, but increasingly "improved" by genetic manipulation via mutagenesis and selection or recombinant DNA technology or protoplast fusion (fungi)
Environmental conditions for plant tissue culture laboratorySandhyaUpadhyay9
The document discusses the environmental conditions required for successful plant tissue culture. It outlines five key areas: 1) laboratory design and management, 2) environmental conditions, 3) sterilization techniques, 4) conditions in the inoculation area, and 5) conditions inside growth chambers. Specific requirements include maintaining temperature between 24-26°C, controlling air movement, using sterile culture vessels and media, and employing sterilization methods like autoclaving. The inoculation area must be inside a laminar flow hood with positive pressure and UV sterilization. Growth chambers need temperature control, lighting of 3000-8000 lux for 16 hours, and 40-60% humidity. Proper environmental protocols are necessary for contaminant-free culture and
This document provides information about plant tissue culture techniques at the Government Arts College in Coimbatore, India. It discusses the history and applications of plant tissue culture, characteristics of the techniques, important media components and their roles, effects of plant hormones, and basic procedures like media preparation and sterilization. The facility has equipment for sterile culture including laminar flow hoods, autoclaves, and culture racks. Plant tissue culture can be used for micropropagation, germplasm conservation, and production of transgenic plants.
This presentation provides an overview of plant tissue culture techniques. It discusses the basic principles of plant tissue culture, including totipotency and the three main stages of cell development. It also describes the basic steps, necessary equipment and facilities for setting up a tissue culture lab, including separate rooms for media preparation, aseptic transfer, culture, analysis, and acclimatization. Details are given on preparing different types of culture media and their components. The presentation covers various sterilization techniques for media, solutions, plant tissues, glassware and equipment. It concludes with references for further information.
This document discusses plant tissue culture and its various applications. It begins by defining tissue culture as the culture and maintenance of plant cells or organs in sterile conditions in vitro. Some key points made include: tissue culture produces clones with the same genotype; it is used commercially for plant propagation through micropropagation; and a reliable growth medium was developed in 1962 which helped tissue culture take off commercially. The document then discusses the various conditions needed for plant cells to multiply in vitro, such as appropriate explant tissue, a suitable nutrient medium, aseptic conditions, and growth regulators. It also describes processes like callus culture, organogenesis, somatic embryogenesis, and applications including seed culture, embryo culture, and organ culture.
Callus and meristem culture are tissue culture techniques used to grow plant cells or tissues under sterile conditions. Callus culture involves growing an unorganized mass of cells from isolated plant parts on nutrient media supplemented with hormones. Meristem culture uses the apical meristem to regenerate whole plants and is effective for eliminating viruses. Both techniques require aseptically preparing explant tissues, using suitable growth media and hormones, and incubating cultures under controlled conditions to initiate cell growth. The cultured tissues can be used to regenerate plants, preserve endangered species, and produce plants resistant to diseases.
This study establishs a basis for growing
plantlets without sucrose and investigating other
factors like carbon dioxide and light regime to
improve the in vitro growth performance.
This document provides an overview of plant tissue culture techniques. It defines plant tissue culture as growing plant cells, organs or tissues in a sterile environment with nutrient media. The key requirements for plant tissue culture are appropriate explant tissue, a suitable growth medium, aseptic conditions, growth regulators, and frequent sub-culturing. Plant tissue culture has advantages over working with intact plants like enabling large-scale growth and genetic modification. The document discusses regeneration methods like shoot regeneration and somatic embryogenesis that allow developing whole plants from cultured cells or tissues. It also covers the stages of micropropagation including multiplication, rooting, and acclimatization of plantlets.
Originally isolated from nature, but increasingly "improved" by genetic manipulation via mutagenesis and selection or recombinant DNA technology or protoplast fusion (fungi)
Environmental conditions for plant tissue culture laboratorySandhyaUpadhyay9
The document discusses the environmental conditions required for successful plant tissue culture. It outlines five key areas: 1) laboratory design and management, 2) environmental conditions, 3) sterilization techniques, 4) conditions in the inoculation area, and 5) conditions inside growth chambers. Specific requirements include maintaining temperature between 24-26°C, controlling air movement, using sterile culture vessels and media, and employing sterilization methods like autoclaving. The inoculation area must be inside a laminar flow hood with positive pressure and UV sterilization. Growth chambers need temperature control, lighting of 3000-8000 lux for 16 hours, and 40-60% humidity. Proper environmental protocols are necessary for contaminant-free culture and
This document provides information about plant tissue culture techniques at the Government Arts College in Coimbatore, India. It discusses the history and applications of plant tissue culture, characteristics of the techniques, important media components and their roles, effects of plant hormones, and basic procedures like media preparation and sterilization. The facility has equipment for sterile culture including laminar flow hoods, autoclaves, and culture racks. Plant tissue culture can be used for micropropagation, germplasm conservation, and production of transgenic plants.
This presentation provides an overview of plant tissue culture techniques. It discusses the basic principles of plant tissue culture, including totipotency and the three main stages of cell development. It also describes the basic steps, necessary equipment and facilities for setting up a tissue culture lab, including separate rooms for media preparation, aseptic transfer, culture, analysis, and acclimatization. Details are given on preparing different types of culture media and their components. The presentation covers various sterilization techniques for media, solutions, plant tissues, glassware and equipment. It concludes with references for further information.
This document discusses plant tissue culture and its various applications. It begins by defining tissue culture as the culture and maintenance of plant cells or organs in sterile conditions in vitro. Some key points made include: tissue culture produces clones with the same genotype; it is used commercially for plant propagation through micropropagation; and a reliable growth medium was developed in 1962 which helped tissue culture take off commercially. The document then discusses the various conditions needed for plant cells to multiply in vitro, such as appropriate explant tissue, a suitable nutrient medium, aseptic conditions, and growth regulators. It also describes processes like callus culture, organogenesis, somatic embryogenesis, and applications including seed culture, embryo culture, and organ culture.
Callus and meristem culture are tissue culture techniques used to grow plant cells or tissues under sterile conditions. Callus culture involves growing an unorganized mass of cells from isolated plant parts on nutrient media supplemented with hormones. Meristem culture uses the apical meristem to regenerate whole plants and is effective for eliminating viruses. Both techniques require aseptically preparing explant tissues, using suitable growth media and hormones, and incubating cultures under controlled conditions to initiate cell growth. The cultured tissues can be used to regenerate plants, preserve endangered species, and produce plants resistant to diseases.
This study establishs a basis for growing
plantlets without sucrose and investigating other
factors like carbon dioxide and light regime to
improve the in vitro growth performance.
This document provides an overview of plant tissue culture techniques. It defines plant tissue culture as growing plant cells, organs or tissues in a sterile environment with nutrient media. The key requirements for plant tissue culture are appropriate explant tissue, a suitable growth medium, aseptic conditions, growth regulators, and frequent sub-culturing. Plant tissue culture has advantages over working with intact plants like enabling large-scale growth and genetic modification. The document discusses regeneration methods like shoot regeneration and somatic embryogenesis that allow developing whole plants from cultured cells or tissues. It also covers the stages of micropropagation including multiplication, rooting, and acclimatization of plantlets.
The document discusses plant tissue culture. It defines plant tissue culture as the technique of growing plant cells, tissues, or organs in an artificial nutrient medium under sterile conditions. The key applications of plant tissue culture include commercial plant production, conservation of endangered species, plant breeding, production of valuable compounds, and crossing distantly related plant species. The document then provides details on preparing Murashige and Skoog medium, including composition, sterilization techniques, and procedures for inoculation of plant materials like seeds and rose buds.
The presentation covered the basic steps of plant tissue culture, including setting up a tissue culture lab with areas for media preparation, aseptic transfer, culture, analysis, and acclimatization. It discussed media components and preparation, sterilization techniques like filtration, radiation, and chemicals, and sterilizing plant tissues using chemicals like sodium hypochlorite. Proper cleaning of glassware and sterilization of equipment and materials are essential for maintaining aseptic conditions.
Plant tissue culture is a process where plant cells, tissues, or organs are cultured in an aseptic, nutrient-rich environment. This document discusses plant tissue culture, including its definition, history, requirements, processes, and applications. Specifically, it examines a study that analyzed phenolic content and antioxidant activity of extracts from rosemary (Rosmarinus officinalis) callus cultures, finding the highest yields using woody plant medium supplemented with specific hormones.
Plant tissue culture is a trending and a promising area due to its applications. hence knowledge of its basics and history would be helpful for the pioneers. ans this presentation also discusses about the structure and establishment of a commercial tissue culture laboratory.
Preparation of plant tissue culture media,types and SterilizationSubhas Picheli
The document discusses preparation of plant tissue culture media, types of media, and sterilization techniques. It covers:
- Types of media including Murashige and Skoog, White's Medium, and Gamborg Medium.
- Steps for preparing media including making stock solutions, adding ingredients, adjusting pH, dispensing, and autoclaving.
- Sources of contamination and methods for sterilizing glassware, equipment, media, explants, and the environment using techniques like autoclaving, dry heat, filtration, and surface sterilization.
To achieve the target of creating a new plant or a plant with desired characteristics, tissue culture is often coupled with recombinant DNA technology. The techniques of plant tissue culture have largely helped in the green revolution by improving the crop yield and quality.
The knowledge obtained from plant tissue cultures has contributed to our understanding of metabolism, growth, differentiation and morphogenesis of plant cells. Further, developments in tissue culture have helped to produce several pathogen-free plants, besides the synthesis of many biologically important compounds, including pharmaceuticals. Because of the wide range of applications, plant tissue culture attracts the attention of molecular biologists, plant breeders and industrialists.
Tissue culture is a process that clones plants through micropropagation. It involves culturing plant tissues in sterile conditions with specific nutrients and hormones. There are four main stages - initiation, multiplication, rooting, and acclimatization. The multiplication stage uses cytokinins to induce shoot growth from explants like leaves or stems. Rooting uses auxins to induce root formation from shoots. The process allows for mass production of genetically identical plants independent of seasons.
Plant tissue culture ⅱ isolation of protoplastbhoomishah45
Protoplast isolation involves removing the cell wall from plant cells to leave only the plasma membrane and intracellular components. The isolated protoplasts can be cultured and genetically manipulated. There are two main methods for isolating protoplasts - mechanical and enzymatic. The enzymatic method uses enzymes like cellulase and pectinase to break down the cell wall. Isolated protoplasts are purified through centrifugation and cultured in an osmotic medium to prevent bursting due to the lack of a cell wall. Protoplasts are useful for studies in cell fusion and genetic transformation.
Hello ever one i hope its useful for preparation of notes regarding plant tissue culture for Pharmacognosy .. B.pharm II yr IV sem.. plz give comments it may useful for me and i can rectify the things.
This lecture discusses tissue culture and the necessary equipment and environment. The basic equipment includes an incubator, hood, centrifuge, microscope, etc. The culture environment requires specific chemical factors in the media like essential elements, organic supplements, carbon sources, and plant growth regulators to control cell growth. The lecture outlines the objectives, key equipment, and factors that influence the cellular environment.
Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues, and organs under sterile conditions. The history of plant tissue culture began in the 1830s with theories of cell totipotency. Significant developments included the discovery of plant growth regulators in the 1920s-1940s and the development of plant cell differentiation and somatic embryogenesis in the 1950s-1960s. There are several types of plant tissue culture including shoot culture, callus culture, embryo culture, and meristem culture. Applications include germplasm conservation, large-scale production, disease eradication, genetic engineering, and more. The advantages are rapid propagation, disease-free plants, year-round growth, and conservation of endangered
This document provides an overview of plant tissue culture. It discusses the basics, including definitions and history. The key facilities, materials, and equipment needed are described. The composition and role of various media components such as macronutrients, micronutrients, carbon sources, and growth regulators are summarized. Different types of cultures and their applications are listed. The general steps involved in plant tissue culture are outlined.
Plant tissue culture involves growing plant cells, tissues, or organs in an artificial nutrient medium under sterile conditions. The document discusses the history, requirements, processes, and applications of plant tissue culture. It notes that plant tissue culture relies on the principles of totipotency, dedifferentiation, and cell competency. The key steps include collecting and sterilizing explants, producing callus tissue from explants, proliferating the culture, and subculturing callus. Applications include modifying plant chemistry, producing disease-free crops, improving crops through genetic engineering, and studying biosynthetic pathways.
Plant tissue culture is the process of growing plant cells, tissues or organs in an artificial nutrient medium under sterile conditions. It allows plants to be grown in vitro from small meristematic tissues. There are several types of plant tissue culture including callus culture, organ culture and cell suspension culture. The basic technique involves surface sterilization of explant tissue followed by establishment and subculture of the culture on nutrient media. Callus culture specifically produces an undifferentiated mass of plant cells called a callus from explants. Plant tissue culture has many applications including disease elimination, genetic studies, large scale propagation and crop improvement.
This document discusses plant tissue culture, including its history, essential facilities and equipment needed, composition of culture media, preparation of stock solutions and media, sterilization methods, measurement of growth, and types of culture. It notes that tissue culture developed from embryology techniques and involves growing small tissue explants aseptically on defined media. Essential facilities include an incubator, autoclave, microscope, and culture room, while media contains major and trace nutrients, vitamins, carbon source, and plant growth regulators.
This document provides information about plant tissue culture techniques. It discusses that plant tissue culture involves culturing small explant tissues in a sterile nutrient medium under controlled conditions. With the addition of hormones, new shoots and roots can be induced to grow from explants. Tissue culture is used for micropropagation to produce clones of plants. Single plant cells also have the ability to regenerate into whole plants given the right conditions. The document outlines the composition of solid and liquid culture media, different tissue culture techniques, types of culture, advantages, applications and nutritional requirements for plant tissue culture.
This document provides an introduction to plant tissue culture. It describes how plant parts can be grown in vitro in artificial nutrient media. The key aspects covered are the types of explants used, applications of plant tissue culture, essential operations, techniques like callus and suspension cultures, steps involved like preparation of media, sterilization, incubation, and acclimatization of plants. Common components of nutrient media and roles of plant hormones are also summarized.
Plant tissue culture provides several benefits for studying plant growth and development. It allows scientists to isolate plant parts and culture them in vitro, simplifying the study of controlling influences. Some key applications of plant tissue culture include clonal propagation of disease-free plants, studying plant cells' ability to regenerate whole plants from cultured cells, producing genetic variability through somaclonal variation, regenerating plants from pollen to create haploids, and rescuing hybrid embryos. Plant tissue culture also enables fundamental biological research, production of high-value biochemicals, and generation of transgenic plants.
This document discusses different types of inflorescences (flower arrangements) in plants. It begins by defining the technical term "inflorescence" as a cluster of flowers. There are two main types of inflorescences: racemose and cymose. Racemose inflorescences have an unlimited growth axis and acropetal flower arrangement, with examples given as raceme, spike, spadix, corymb, umbel and capitulum (head). Cymose inflorescences have a limited growth axis and basipetal flower arrangement, with examples of uniparous (monochasial), biparous and polychasial cymes. Specific plant examples are
The document discusses plant tissue culture. It defines plant tissue culture as the technique of growing plant cells, tissues, or organs in an artificial nutrient medium under sterile conditions. The key applications of plant tissue culture include commercial plant production, conservation of endangered species, plant breeding, production of valuable compounds, and crossing distantly related plant species. The document then provides details on preparing Murashige and Skoog medium, including composition, sterilization techniques, and procedures for inoculation of plant materials like seeds and rose buds.
The presentation covered the basic steps of plant tissue culture, including setting up a tissue culture lab with areas for media preparation, aseptic transfer, culture, analysis, and acclimatization. It discussed media components and preparation, sterilization techniques like filtration, radiation, and chemicals, and sterilizing plant tissues using chemicals like sodium hypochlorite. Proper cleaning of glassware and sterilization of equipment and materials are essential for maintaining aseptic conditions.
Plant tissue culture is a process where plant cells, tissues, or organs are cultured in an aseptic, nutrient-rich environment. This document discusses plant tissue culture, including its definition, history, requirements, processes, and applications. Specifically, it examines a study that analyzed phenolic content and antioxidant activity of extracts from rosemary (Rosmarinus officinalis) callus cultures, finding the highest yields using woody plant medium supplemented with specific hormones.
Plant tissue culture is a trending and a promising area due to its applications. hence knowledge of its basics and history would be helpful for the pioneers. ans this presentation also discusses about the structure and establishment of a commercial tissue culture laboratory.
Preparation of plant tissue culture media,types and SterilizationSubhas Picheli
The document discusses preparation of plant tissue culture media, types of media, and sterilization techniques. It covers:
- Types of media including Murashige and Skoog, White's Medium, and Gamborg Medium.
- Steps for preparing media including making stock solutions, adding ingredients, adjusting pH, dispensing, and autoclaving.
- Sources of contamination and methods for sterilizing glassware, equipment, media, explants, and the environment using techniques like autoclaving, dry heat, filtration, and surface sterilization.
To achieve the target of creating a new plant or a plant with desired characteristics, tissue culture is often coupled with recombinant DNA technology. The techniques of plant tissue culture have largely helped in the green revolution by improving the crop yield and quality.
The knowledge obtained from plant tissue cultures has contributed to our understanding of metabolism, growth, differentiation and morphogenesis of plant cells. Further, developments in tissue culture have helped to produce several pathogen-free plants, besides the synthesis of many biologically important compounds, including pharmaceuticals. Because of the wide range of applications, plant tissue culture attracts the attention of molecular biologists, plant breeders and industrialists.
Tissue culture is a process that clones plants through micropropagation. It involves culturing plant tissues in sterile conditions with specific nutrients and hormones. There are four main stages - initiation, multiplication, rooting, and acclimatization. The multiplication stage uses cytokinins to induce shoot growth from explants like leaves or stems. Rooting uses auxins to induce root formation from shoots. The process allows for mass production of genetically identical plants independent of seasons.
Plant tissue culture ⅱ isolation of protoplastbhoomishah45
Protoplast isolation involves removing the cell wall from plant cells to leave only the plasma membrane and intracellular components. The isolated protoplasts can be cultured and genetically manipulated. There are two main methods for isolating protoplasts - mechanical and enzymatic. The enzymatic method uses enzymes like cellulase and pectinase to break down the cell wall. Isolated protoplasts are purified through centrifugation and cultured in an osmotic medium to prevent bursting due to the lack of a cell wall. Protoplasts are useful for studies in cell fusion and genetic transformation.
Hello ever one i hope its useful for preparation of notes regarding plant tissue culture for Pharmacognosy .. B.pharm II yr IV sem.. plz give comments it may useful for me and i can rectify the things.
This lecture discusses tissue culture and the necessary equipment and environment. The basic equipment includes an incubator, hood, centrifuge, microscope, etc. The culture environment requires specific chemical factors in the media like essential elements, organic supplements, carbon sources, and plant growth regulators to control cell growth. The lecture outlines the objectives, key equipment, and factors that influence the cellular environment.
Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues, and organs under sterile conditions. The history of plant tissue culture began in the 1830s with theories of cell totipotency. Significant developments included the discovery of plant growth regulators in the 1920s-1940s and the development of plant cell differentiation and somatic embryogenesis in the 1950s-1960s. There are several types of plant tissue culture including shoot culture, callus culture, embryo culture, and meristem culture. Applications include germplasm conservation, large-scale production, disease eradication, genetic engineering, and more. The advantages are rapid propagation, disease-free plants, year-round growth, and conservation of endangered
This document provides an overview of plant tissue culture. It discusses the basics, including definitions and history. The key facilities, materials, and equipment needed are described. The composition and role of various media components such as macronutrients, micronutrients, carbon sources, and growth regulators are summarized. Different types of cultures and their applications are listed. The general steps involved in plant tissue culture are outlined.
Plant tissue culture involves growing plant cells, tissues, or organs in an artificial nutrient medium under sterile conditions. The document discusses the history, requirements, processes, and applications of plant tissue culture. It notes that plant tissue culture relies on the principles of totipotency, dedifferentiation, and cell competency. The key steps include collecting and sterilizing explants, producing callus tissue from explants, proliferating the culture, and subculturing callus. Applications include modifying plant chemistry, producing disease-free crops, improving crops through genetic engineering, and studying biosynthetic pathways.
Plant tissue culture is the process of growing plant cells, tissues or organs in an artificial nutrient medium under sterile conditions. It allows plants to be grown in vitro from small meristematic tissues. There are several types of plant tissue culture including callus culture, organ culture and cell suspension culture. The basic technique involves surface sterilization of explant tissue followed by establishment and subculture of the culture on nutrient media. Callus culture specifically produces an undifferentiated mass of plant cells called a callus from explants. Plant tissue culture has many applications including disease elimination, genetic studies, large scale propagation and crop improvement.
This document discusses plant tissue culture, including its history, essential facilities and equipment needed, composition of culture media, preparation of stock solutions and media, sterilization methods, measurement of growth, and types of culture. It notes that tissue culture developed from embryology techniques and involves growing small tissue explants aseptically on defined media. Essential facilities include an incubator, autoclave, microscope, and culture room, while media contains major and trace nutrients, vitamins, carbon source, and plant growth regulators.
This document provides information about plant tissue culture techniques. It discusses that plant tissue culture involves culturing small explant tissues in a sterile nutrient medium under controlled conditions. With the addition of hormones, new shoots and roots can be induced to grow from explants. Tissue culture is used for micropropagation to produce clones of plants. Single plant cells also have the ability to regenerate into whole plants given the right conditions. The document outlines the composition of solid and liquid culture media, different tissue culture techniques, types of culture, advantages, applications and nutritional requirements for plant tissue culture.
This document provides an introduction to plant tissue culture. It describes how plant parts can be grown in vitro in artificial nutrient media. The key aspects covered are the types of explants used, applications of plant tissue culture, essential operations, techniques like callus and suspension cultures, steps involved like preparation of media, sterilization, incubation, and acclimatization of plants. Common components of nutrient media and roles of plant hormones are also summarized.
Plant tissue culture provides several benefits for studying plant growth and development. It allows scientists to isolate plant parts and culture them in vitro, simplifying the study of controlling influences. Some key applications of plant tissue culture include clonal propagation of disease-free plants, studying plant cells' ability to regenerate whole plants from cultured cells, producing genetic variability through somaclonal variation, regenerating plants from pollen to create haploids, and rescuing hybrid embryos. Plant tissue culture also enables fundamental biological research, production of high-value biochemicals, and generation of transgenic plants.
This document discusses different types of inflorescences (flower arrangements) in plants. It begins by defining the technical term "inflorescence" as a cluster of flowers. There are two main types of inflorescences: racemose and cymose. Racemose inflorescences have an unlimited growth axis and acropetal flower arrangement, with examples given as raceme, spike, spadix, corymb, umbel and capitulum (head). Cymose inflorescences have a limited growth axis and basipetal flower arrangement, with examples of uniparous (monochasial), biparous and polychasial cymes. Specific plant examples are
PPT on Tissue Culture Class 10 CBSE Text Book NCERT.One Time Forever
This is a PPT Based on Class 10 Chapter How Do Organisms Reproduce, on a Small Topic of it That is Tissue Culture with easy and detailed explanation of each topic of tissue culture along with some pictures and some examples. Hopefully it Would Be Helpful To You. Thank You.
This document discusses pollination and was presented by the group Spartans. It defines pollination as the transfer of pollen from male to female reproductive organs in plants. The document outlines different types of pollination including self-pollination, cross-pollination, abiotic pollination via wind or water, and biotic pollination via insects, birds, bats, or humans. It discusses the importance and advantages and disadvantages of self-pollination and cross-pollination. The document also discusses factors that affect pollination, artificial means of pollination, problems with pollination, and solutions to improve pollination.
The document defines and provides examples of different types of inflorescences including:
- Spadix - A thick fleshy spike bearing clustered unisexual flowers found in arum plants.
- Raceme - An unbranched axis with pedicels of similar length.
- Umbel - Pedicels arise from the top of the peduncle.
- Head/Capitulum - Flowers grouped together without pedicels at the top of a peduncle like in daisies.
This document provides an overview of inflorescence structures in plants. It defines an inflorescence as a cluster of flowers on a stem and describes their general characteristics such as bracts, terminal flowers, phyllotaxis, and metatopy. It also explains the organization of simple inflorescences, distinguishing between indeterminate types like racemes, spikes, umbels and determinate types like cymes, corymbs, and heads. Specific examples are given for each type of inflorescence discussed.
Plant reproduction can occur through asexual and sexual methods. Asexual reproduction involves only one parent and produces genetically identical offspring through processes like fragmentation, budding, and spore formation. Sexual reproduction involves male and female gametes from two parents, producing offspring with new combinations of genes. In flowering plants, sexual reproduction takes place through flowers, which contain stamens that produce pollen and carpels containing ovules. Pollination leads to fertilization and seed formation, and seeds are then dispersed and germinate to produce new plants. Vegetative reproduction also allows for cloning of plants through propagation by cuttings, layering, and grafting of plant parts.
The document discusses two forms of plant reproduction: sexual reproduction which involves meiosis and fertilization, and asexual reproduction or vegetative propagation which involves mitosis. It provides details on sexual reproduction in angiosperms using flowers, conifers using cones, and ferns using spores. The document also lists different structures used in vegetative propagation like tubers, rhizomes, stolons, bulbs, and corms.
Chapter 16 Reproduction in Plants Lesson 2 - Pollinationj3di79
Pollination is the transfer of pollen grains from the anther to the stigma. It can occur through self-pollination within a flower or plant or cross-pollination between plants. Flowers have adapted traits for wind or insect pollination, with wind-pollinated flowers generally having small, dry pollen and insect flowers having nectar, scent and bright colors. Cross-pollination provides genetic diversity but requires two parents, while self-pollination only requires one but can lead to inbreeding over time. Plants use various mechanisms like separate male and female plants or maturation timing to encourage outcrossing.
This document discusses asexual reproduction in plants, which is the formation of new individuals from a single parent's cells and does not allow for genetic variation. It describes several methods of asexual reproduction including vegetative reproduction through bulbs, underground stems, runners, and leaves. It also discusses artificial propagation techniques like stem cuttings, grafting, layering, and plant tissue culture which can be used to reproduce plants without seeds.
This document provides an overview of plant reproduction, including pollination, fertilization, and seed dispersal. It defines pollination as the transfer of pollen from the anther to the stigma. The pollination process involves a bee or butterfly carrying pollen between flowers. Fertilization is the joining of pollen and ovule to form a seed. It defines key terms like stigma, style, ovary, and ovules. Seed dispersal methods help plants disperse their seeds through various means like wind, water, or animals to prevent overcrowding and establish new colonies.
The document discusses plant reproduction, including asexual and sexual reproduction. It covers the life cycles of non-flowering plants like mosses and ferns which have motile sperm that require moist environments. The document also discusses the life cycles of flowering plants, which have evolved relationships with insect pollinators and can live in more diverse environments as a result. Key terms defined include haploid, diploid, mitosis, and meiosis. The alternation of generations in plant life cycles is also explained.
This document discusses apomixis, which is the natural ability of some plant species to reproduce asexually through seeds. It describes the differences between sexual and apomictic reproduction in plants. It also outlines different types and methods of identifying apomixis, as well as the potential benefits it could provide, such as rapid hybrid variety development and economic hybrid seed production. The document then discusses current research on apomixis and related patents, and how introducing apomixis into crop plants could both accelerate hybrid variety development and benefit farmers globally.
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.
Flowering plants reproduce sexually through flowers. Flowers contain male stamen that produce pollen and female pistils. Fertilization occurs when pollen is transferred, or pollinated, to the stigma of the pistil. Pollination can be self-pollination within a flower or cross-pollination between flowers. After fertilization, the ovary develops into a fruit containing seeds. Seeds are dispersed by various methods and may germinate to form a seedling with root, shoot, and leaves developing from the radicle, hypocotyl, and epicotyl respectively.
Natural vegetative reproduction in plants occurs through underground stems, bulbs, corms, rhizomes, and leaves, allowing plants like ginger, onions, potatoes, heliconia, and bananas to produce offspring. Artificial vegetative reproduction is a man-made process using plant parts like stems, buds, and leaves to reproduce plants with desired traits. Common techniques include cuttings using stem cuttings, grafting to join parts from two plants, budding which grafts a small bud, and air layering which induces root growth on stem cuttings.
Apomixis is a type of asexual reproduction in which seeds are formed without fertilization. There are two main types - gametophytic apomixis, where an unreduced cell gives rise to an embryo sac, and sporophytic apomixis, where an unreduced cell develops directly into an embryo. Apomixis was first discovered in citrus seeds in 1719 and allows for the production of genetically identical offspring from a single parent. While apomixis has advantages for plant breeding like fixing desirable traits, it is also genetically complex and the level can be affected by environmental factors.
The document discusses different modes of reproduction in flowers, including self-pollination, cross-pollination, and apomixis. It describes staminate flowers that produce pollen for pollination and pistillate flowers that receive pollen for fertilization. Apomixis allows reproduction without fertilization and can occur in some flower types.
Vegetative propagation is a form of asexual reproduction used by plants to produce offspring without seeds, pollen, or flowers. The offspring are genetically identical to the parent plant. Vegetative propagation can occur naturally through modified plant structures like stems, roots, leaves, and buds, or can be induced artificially through methods like cuttings, grafting, and tissue culture. Vegetative propagation is advantageous for gardeners because it allows desirable plant traits to be reproduced rapidly and in large quantities.
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.
Essay on Plant Tissue Culture Contents:
the Definition of Plant Tissue Culture.
the History of Plant Tissue Culture.
the Basic Requirements of Plant Tissue Culture.
the General Techniques of Plant Tissue Culture.
the Basic Aspects of Plant Tissue Culture.
the Cellular Totipotency.
the Differentiation.
the Methods in Plant Tissue Culture.
the Applications of Plant Tissue Culture.
the Morphogenesis.
the Subculture or Secondary Cell Culture.
the Soma-Clonal Variation.
the Somatic Hybrids and Cybrids.
the Micro-Propagation.
the Artificial Seed.
the Cryopreservation.
Animal Tissue Culture
The foundation of animal cell and tissue culture was laid by Jolly (1903) when he showed that animal cells could not only survive but could divide in culture medium. The actual beginning of animal cell culture and tissue culture was made by Harrison (1907) and later by Carrel (1912) who used frog’s tissue in tissue culture. They successfully showed that animal cells can be grown indefinitely in culture medium just like microorganisms. Later tissues from warm blooded animals like chick and mammals were used as material for tissue culture purpose.
Plant tissue culture, also known as micropropagation, uses sterilized plant parts or seeds placed in sterile containers with nutrient-rich gel medium to propagate plants. The explants are prevented from infection by microorganisms during rooting or multiplying. Exact copies of donor plants can be created using this method, which is useful for cloning plants with desirable traits faster than traditional propagation. The process involves establishing an aseptic culture, multiplying propagules, preparing propagules for soil transfer, and establishing plants in soil. Tissue culture allows for rapid multiplication of plants from a single explant in a brief period.
Tissue culture is a technique where small pieces of plant or animal tissue are cultured in a sterile medium outside of the organism. It was first developed in 1885 and has since been used extensively in medicine, agriculture, and research. It allows for the rapid duplication of plant materials while eliminating diseases and maintaining genetic traits. However, it requires specialized facilities and equipment and reduces genetic diversity.
The document discusses the history and techniques of plant tissue culture. It begins with early experiments in the 1830s culturing plant cells outside of their natural environment. Significant developments include the discovery of growth hormones auxin and cytokinin in the 1950s, and the development of the Murashige and Skoog medium in 1962. The document outlines the basic requirements and procedures for plant tissue culture, including selecting an explant, preparing sterile media, inoculation, incubation, sub-culturing, and transferring plantlets. Applications include rapid clonal propagation, inducing mutations, producing disease-free plants, and conserving rare species.
cellular totipotency and callus cultureNeha Kakade
This ppt comprises a detailed information about cellular totipotency and callus culture in plant tissue culture . It has its applications, significance and procedure described in it . This explains about the property of totipotency. It describes stages of callus culture. It also descibes history of plant tissue culture
Plant bio 1 introduction to cell tissue cultureDr. Preeti Pal
Tissue culture is a method of growing plant cells, tissues or organs in vitro on artificial nutrient media under sterile conditions. Plant tissue culture involves exposing plant tissue to specific nutrients, hormones and light to produce many new cloned plants over a short period. The father of plant tissue culture is considered to be German botanist Gottlieb Haberlandt who conceived the concept of cell culture in 1902. A key aspect of plant tissue culture is initiation and maintenance of callus cultures, which are masses of unorganized proliferating cells grown on artificial media.
This document provides an overview of plant tissue culture methods and applications. It discusses the basic concepts of plant tissue culture, including plasticity and totipotency. The stages of micropropagation are outlined as initiation, multiplication, rooting, and transfer to soil. Micropropagation involves regeneration and multiplication of plants from explants like axillary buds and shoot tips. The document also discusses meristem culture, shoot tip culture, growth media, factors affecting tissue culture, applications like disease elimination and germplasm conservation, somaclonal variation, and cryopreservation.
Plant tissue culture broadly refers to the in vitro cultivation of plants, seeds and various parts of the plants (organs, embryos, tissues, single cells, protoplasm).
The cultivation process is invariably carried out in a nutrient culture medium under aseptic conditions.
Plant cells have certain advantages over animal cells in culture system.
Unlike animal cells, highly mature and differentiated plant cells retain the ability of totipotency i.e. the ability of change to meristematic state and differentiate into a whole plant
Plant tissue culture involves growing plant cells, tissues, organs, or whole plants in vitro on a nutrient medium under sterile conditions. It allows for mass propagation of plant materials, rapid plant breeding through selection of variants, and genetic manipulation. The key principles involve using plant hormones like auxin and cytokinin to induce cell differentiation and regeneration into whole plants. Advantages include rapid multiplication, disease elimination, genetic transformation, and conservation of endangered species.
Plant tissue culture is the process of growing plant cells, tissues or organs in sterile conditions on a nutrient medium. It has many applications like germplasm preservation of endangered plants, producing disease-free plants through micropropagation, and creating novel hybrids through protoplast fusion and somatic hybridization. However, issues remain like genetic instability of hybrids and lack of efficient selection methods. Overall, tissue culture is a valuable biotechnology tool with potential for crop improvement and conservation efforts.
Plant tissue culture is the process of growing plant cells, tissues or organs in sterile conditions on a nutrient medium. It has many applications like germplasm preservation of endangered plants, genetic improvement of crops, and production of secondary metabolites. The basic steps include selection of explant, initiation of culture on growth media, multiplication through cell division, and rooting and transfer to soil. Technologies such as micropropagation, somatic hybridization, and cryopreservation have been commercialized for mass propagation of crops. However, issues remain regarding genetic stability, selection of fused products, and regeneration efficiency.
Organ culture involves growing excised plant organs in vitro to allow differentiation while maintaining architecture and function. It provides an experimental system to study nutrient and growth factor requirements of organs and their interdependence. Callus culture involves growing isolated plant cells or tissues on artificial media to produce an unorganized proliferative cell mass called callus. Successful callus culture requires aseptically preparing explants, using appropriate media supplemented with auxins and/or cytokinins, and incubating under controlled conditions. Callus tissue has been important for fundamental and applied plant biology research, including vegetative propagation of important species.
1. The document discusses various techniques of in vitro plant tissue culture including organ culture, seed culture, meristem culture, embryo culture, ovary culture, anther culture, callus culture, cell suspension culture, and protoplast culture.
2. Key steps in in vitro culture include isolation of explant tissues, regeneration and callus formation, embryogenesis, and organogenesis.
3. Applications of these techniques include virus-free plant production, increasing seed germination efficiency, producing somatic embryos and haploid plants, enabling single cell studies, and facilitating somatic hybridization.
This document provides an introduction to animal cell culture. It discusses what cell and tissue culture are, how cell cultures are obtained through primary culture or purchasing established cell lines. It describes the characteristics of cultured cells, including their morphology and functional properties. Some of the key challenges in cell culture are avoiding contamination and providing an optimal environment for cell growth. The document also gives a brief overview of what cell culture is used for.
The document discusses plant tissue culture and provides definitions, a brief history, and types of cultures including seed culture, callus culture, and anther culture. It describes the basic requirements for a plant tissue culture laboratory including equipment, facilities for washing/sterilization, media preparation, culture rooms, and data collection areas. The key steps in plant tissue culture procedures are outlined including explant preparation, surface sterilization, callus production, proliferation, subculture, and suspension culture. Applications in pharmacognosy and edible vaccines are mentioned. Nutritional requirements and functions of elements in culture media are also summarized.
Plant Tissue Culture..“Micropropagation Studies On Bambusa tulda”Manzoor Wani
I hereby declare that a dissertation work entitled ―Micropropagation studies on Bambusa tulda plant through nodal explant” Submitted to university in fulfillment for the award of degree in Bachelors Of Science (forestry) is carried out by me at State Research Institute Jabalpur Madhya Pradesh.
2. 152 Plant Tissue Culture
Contents
Introduction....................................................................................................................152
Terminology...................................................................................................................152
Laboratory Requirements for Tissue Culture ................................................................153
Demonstration of "in vitro" Morphogenesis and Totipotency of Seedling Explants ....154
Effects of Hormone Balance on Explant Growth and Morphogenesis..........................160
Callus Formation and Multiplication.............................................................................164
Establishment of Suspension Cultures...........................................................................167
Anther Culture ...............................................................................................................167
Acknowledgements........................................................................................................168
Literature Cited ..............................................................................................................169
Appendices A to E .........................................................................................................170
Introduction
Plant tissue culture techniques are essential to many types of academic inquiry, as well as to
many applied aspects of plant science. In the past, plant tissue culture techniques have been used in
academic investigations of totipotency and the roles of hormones in cytodifferentiation and
organogenesis. Currently, tissue-cultured plants that have been genetically engineered provide
insight into plant molecular biology and gene regulation. Plant tissue culture techniques are also
central to innovative areas of applied plant science, including plant biotechnology and agriculture.
For example, select plants can be cloned and cultured as suspended cells from which plant products
can be harvested. In addition, the management of genetically engineered cells to form transgenic
whole plants requires tissue culture procedures; tissue culture methods are also required in the
formation of somatic haploid embryos from which homozygous plants can be generated. Thus,
tissue culture techniques have been, and still are, prominent in academic and applied plant science.
The techniques demonstrated in these exercises range from simple ones that can easily be
performed by beginning students to those done by botany or physiology students. Experiment 1 and
2 employ plant material derived from aseptic seed germinations, while Experiments 3, 4, and 5 use
portions of large intact plants. Experiment 1 demonstrates "in vitro" morphogenesis and totipotency
and has been used successfully by beginning classes containing both biology majors and non-majors
(expected results are presented in Appendix A). The remaining experiments are designed for use by
more advanced students.
For further reading see Bottino (1981), Butcher and Ingram (1976), Dodds and Roberts (1985),
Street (1973), Sunderland and Roberts (1977), and Wetherell (1982).
Terminology
Aseptic Free from microorganisms
Callus Undifferentiated, swollen cell mass forming under the influence of elevated
plant hormone levels.
Etiolation Yellow and stretched plant; parts elongate until light is intercepted.
Explant Part of an organism used in "in vitro" culture.
3. Plant Tissue Culture 153
IAA Indoleacetic acid; a plant hormone increasing cell elongation and, under
certain circumstances, implicated in stimulating cell division and root
formation. IAA moves in a polar manner in plants forming an IAA
gradient in tissues. Orientation of plant organs, then, influence callus
formation and morphogenesis.
"in vitro" "In glass"; as in tissue culture methods
Morphogenesis Change in shape
Polarity Orientation in gravitational field.
Primordia The earliest detectable stage of an organ, such as a leaf, root or root branch.
Root hairs Epidermal cell extensions of young root that increase absorptive surface
area.
Totipotency The establishment of missing plant organs or parts; formation of a whole
plant from a few cells or small portion of a plant.
Wound response Formation of callus in wounded area.
Laboratory Requirements for Tissue Culture
General Organization
Localize each portion of the tissue culture procedure in a specified place in the laboratory. An
assembly-line arrangement of work areas (such as, media preparation, glassware washing,
sterilization, microscopy, and aseptic transfers) facilitates all operations and enhances cleanliness.
Media (tissue culture and nutrient agar) are available from Carolina Biological Supply Co.,
Burlington, NC. Laminar flow hoods are available from several suppliers.
Glassware
Use glassware that has only been used for tissue culture and not other experiments. Toxic metal
ions absorbed on glassware can be especially troublesome. Wash glassware with laboratory
detergent, then rinse several times with tap water and, finally, rinse with purified water.
High-purity Water
Use only high-purity water in tissue culture procedures. Double glass distilled water or
deionized water from an ion-exchanger are acceptable. Water should not be stored, but used
immediately. Regular maintenance and monitoring of water purification equipment are necessary.
Purified water for tissue culture can also be purchased.
4. 154 Plant Tissue Culture
Plant Material
Plants used in tissue culture need to be healthy and actively growing. Stressed plants,
particularly water-stressed plants, usually do not grow as tissue cultures. Insect and disease-free
greenhouse plants are rendered aseptic more readily, so contamination rate is lower when these
plants are used in tissue culture procedures. Seeds that can be easily surface sterilized usually
produce contamination-free plants that can be grown under clean greenhouse conditions for later
experimental use.
Aseptic Technique
The essence of aseptic technique is the exclusion of invading microorganisms during
experimental procedures. If sterile tissues are available, then the exclusion of microorganisms is
accomplished by using sterile instruments and culture media concurrently with standard
bacteriological transfer procedures to avoid extraneous contamination.
Media and apparatus are rendered sterile by autoclaving at 15 lbs/inch2 (121°C) for 15 minutes.
The use of disposable sterile plasticware reduces the need for some autoclaving. Alternative
sterilization techniques such as filter sterilization must be employed for heat-labile substances like
cytokinins.
Aseptic transfers can be made on the laboratory bench top by using standard bacteriological
techniques (i.e., flaming instruments prior to use and flaming the opening of receiving vessels prior
to transfer). Aseptic transfers are more easily performed in a transfer chamber such as a laminar
flow hood, which is also preferably equipped with a bunsen burner (Bottino, 1981).
If experimental tissues are not aseptic, then surface sterilization procedures specific to the
tissues are employed. Common sterilants are ethyl alcohol and/or chlorox with an added surfactant.
Concentration of sterilants and exposure time are determined empirically.
Experiment 1:
Demonstration of "in vitro" Morphogenesis and Totipotency of Seedling Explants
A simple exercise demonstrating plant totipotency as well as the nutritional requirements of
different plant organs employs shoot tip and root tip explants cut from aseptically germinated
seedlings. Each type of explant (excised part of the intact organism) is transferred to three simple
tissue culture media.
Background
During seed formation, the developing embryo and associated tissues tend to exclude pathogens
and foreign materials that may be in the parent plant. Contents of the seed, then, are essentially
aseptic and the resultant seedlings can be maintained in the aseptic condition if the outer surface of
the seed (seed coat) is sterilized with sodium hypochlorite (or other surface sterilant) prior to
germinating the seeds in a sterile petri dish.
Methods: Week 1
The manipulations that are required for the germination of aseptic seedlings are outlined below
and illustrated in Figure 9.1.
5. Plant Tissue Culture 155
Figure 9.1. The manipulations required for the germination of aseptic seedlings (Experiment 1,
Methods: Week 1).
6. 156 Plant Tissue Culture
1. Outside the laminar flow hood: Place several (5 to 10) tomato or lettuce seeds in a small petri
dish. Fill the petri dish with a 7% chlorox solution to which a drop of wetting agent has been
added. The soapy chlorox solution is usually a good surface sterilant. Swirl the seeds
intermittently during the 10- or 15-minute chlorox treatment.
2. Preparation for aseptic transfers: Begin by washing your hands and forearms with soap,
followed by swabbing with 70% ethyl alcohol (EtOH). Sterilize the laminar flow hood by
wiping the inside (top, sides, and bottom) with EtOH. Turn on the hood; 10–15 minute
operation of the hood before use insures aseptic conditions within the work area of the hood.
Continue to swirl the seeds intermittently during the chlorox treatment. Prior to actual aseptic
transfers inside the chamber, swab hands and forearms with EtOH again; also wipe the external
surface of the petri dish before placing it inside the hood. The hood should contain the
following: a large jar which can be used as a "sink," flasks of sterile water, forceps in a beaker
of ethanol, sterile filter paper (5–7 cm diameter filter paper can be sterilized in glass petri
dishes), and sterile petri dishes which can be used as the seed germination dishes.
3. Inside the laminar flow hood: Decant chlorox and replace with sterile H2O. Rinse this way
twice. Each rinse should rest 10 minutes. Prepare the sterile germinating petri dish by
retrieving a forceps from the 70% EtOH beaker. Using the sterile forceps remove three (3)
rounds of sterile filter paper from a sterile container and place them in the germinating dish
(sterile plastic petri dish). Finally, add 5–10 ml of sterile H2O to the seeds; decant seeds and
water into the sterile germinating dish and incubate at 25°C until the next laboratory. (Both
tomato and light-insensitive lettuce seeds germinate in the light. Since shoots become green but
roots remain white under these conditions, seedling morphology is recognized more easily when
light-germinated.)
Methods: Week 2
Examine the contents of the aseptic germinating dish without opening the lid. If there is no
fungal or bacterial contamination around the seedlings, proceed; if contamination exists, request a
dish of aseptic seedlings from the instructor. The manipulation required for the transfer of seedling
explants to Mineral Salts (M) and Minimal Organic (O) growth media are outlined below and
illustrated in Figure 9.2.
1. Swab chamber, hands, and upper/lower surfaces of petri dish with 70% ethanol.
2. Place germinating dish in transfer chamber.
3. Remove scalpel or scissors from the ETOH beaker already in the hood. Slip instrument between
sheets of sterile toweling to remove ETOH (ethanol).
4. Lift one edge of lid and cut off no more than 10 mm of root tip. Excise two root tips. Lower
lid. Place scalpel back into ETOH beaker.
5. Place tubes with sterile media into the transfer chamber. (Media formulae are given in
Appendix B.) Use one tube of Minimal Organic Medium (O) and one of Mineral Salts Medium
(M). Loosen these caps.
7. Plant Tissue Culture 157
Figure 9.2 The manipulations required for the transfer of seedling explants to Mineral Salts (M)
and Minimal Organic (O) growth media (Experiment 1, Methods: Week 2).
8. 158 Plant Tissue Culture
6. Remove forceps or inoculating loop from ETOH. Slip between sterile toweling to remove
ETOH.
7. Remove excised root tip from germinating dish and transfer to the surface of the Minimal
Organic Medium (O). Transfer second root tip to the surface of the Mineral Salts Medium (M).
Caution: pick up root tip by the severed end; damage to the apical meristem disrupts mitosis!
Measure or estimate length of root tips. Record.
8. Using aseptic technique as above, prepare and transfer one shoot tip into each type of media.
Pick up the shoot tip by the severed end and insert it part way into the medium with an overall
vertical orientation of the cotyledons and shoot tip. Record size and shape of shoot tip.
9. Place the four tubes in a slant rack under lights.
10. Examine cultures each week. Record observations on the amount of growth and morphogenesis
of both root and shoot cultures.
Observations
As cultures progress it should be possible to correlate size/shape changes with the nutrient
content of the medium. A third medium, the B-deficient Medium, contains the same mineral
constituents as does the Mineral Salts Medium (M) and the same amount of sucrose as the Minimal
Organic Medium (O), but is devoid of B vitamins. Thus, this medium is referred to as B-deficient (-
B). Shoot tips and root tips have been transferred to this demonstration medium. Growth on this
medium can be evaluated and compared with growth on student experimental media M and O. The
Mineral Salts Medium (M) is the basal growth medium, supplying essential mineral nutrients for
autotrophic plant growth (Appendix C).
Predict the resultant growth in each circumstance, then monitor the growth and development of
root and shoot explants in each medium (M, O, and -B) and evaluate the following (record
observations in Table 9.1):
1. Effect of B-vitamins on:
a) shoot growth (increase in size) and morphology (change of shape), and
b) root growth and morphology.
2. Effect of organic medium containing sucrose on:
a) shoot growth and morphology, and
b) root growth and morphology.
Optional: media M, O, and -B are set up with root tip and shoot tip explants in darkness. This
set of samples can be observed along with those in the light to evaluate the effect of light as well as
media contents on the growth and development of plant organs. A row labeled "etiolation" would
be added to the bottom of Table 9.1.
9. Plant Tissue Culture 159
Experimental observations should include the following:
1. Parameters: temperature; light quality, duration, and intensity.
2. Drawings to scale.
3. Gross measurements (length, biomass accumulation, extent of morphogenesis, totipotency,
primordia, number of branches) after 1 week and 2 weeks.
4. Net changes (Table 9.1).
5. Does the irregular orientation of the shoot explant change the growth pattern? How can these
observations be explained?
Table 9.1. Use blank table to record results from Experiment 1.
Shoot Root
Media Mineral B-deficient Organic Mineral B-deficient Organic
Cap color White Red Blue White Red Blue
Change in
length
Change in
Biomass
Extent of
morphogenesis
Totipontecy
Primordia
Number of
Branches
Questions
1. Are all portions of the seedling totipotent?
2. Is autotrophic and heterotrophic growth of different plant organs apparent?
3. How could the experimental design be changed to more completely evaluate B-vitamin and
extra-sucrose effects?
4. If B vitamins seem important to root growth and development, how are vitamins probably
supplied to plant roots in intact plants?
10. 160 Plant Tissue Culture
Experiment 2:
Effects of Hormone Balance on Explant Growth and Morphogenesis
Background
Plant hormones, like animal hormones, are relatively small molecules that are effective at low
tissue concentrations. The two types of plant hormones used in this experiment are cytokinins and
auxins.
Cytokinins are derived from adenine and produce two immediate effects on undifferentiated
cells: the stimulation of DNA synthesis and increased cell division (Ting, 1982). Cytokinins also
produce a delayed response in undifferentiated tissue which is the formation of shoot primordia.
Both naturally occurring cytokinins, such as zeatin and synthetic analogs, such as kinetin,
demonstrate cytokinin effects (Figure 9.3). Although low tissue concentrations of cytokinins (e.g., 1
× 10-8 M zeatin) have noticeable effects, higher concentrations are found in actively dividing tissues
such as those of plant embryos and developing fruits.
Auxins are indole or indole-like compounds that stimulate cell expansion, particularly cell
elongation. Auxins also promote adventitious root development. Indoleacetic acid (IAA), a
naturally occurring auxin, and napthaleneacetic acid (NAA), a synthetic auxin, are depicted in
Figure 9.3. Only small amounts of auxin (1 × 10-6 M) are required to demonstrate an IAA response
and even smaller amounts of synthetic auxin (e.g., NAA) are required for a tissue response. The
likely reason for potency of synthetic auxins is their stability in plant tissue (i.e., the enzymes and
processes that degrade IAA do not "recognize" synthetic auxins). Synthetic auxins, then, are more
effective hormones that also last for an extended length of time. Furthermore, light influences the
physiological activity of IAA while synthetic auxins are not as light sensitive.
Plant hormones do not function in isolation within the plant body, but, instead, function in
relation to each other. Hormone balance is apparently more important than the absolute
concentration of any one hormone. Both cell division and cell expansion occur in actively dividing
tissue, therefore cytokinin and auxin balance plays a role in the overall growth of plant tissue. Since
hormone balance is presumably important to the overall effect on growth and morphological
changes, then the hormone differentials in each of the experimental media (A, B, and C) should
produce somewhat different effects on the growth and development of excised explants.
Source of Aseptic Explant Material
During seed development the embryos are formed with a placenta-like interface of intervening
tissues between parental vascular supply and the embryo proper. This circumstance depresses
passive migration of most foreign bodies and microorganisms into the developing embryo. If the
embryo which often develops aseptically is released from the seed by aseptic germination
procedures, then aseptic seedlings result. Any part of the aseptic seedling can be used as "in vitro"
experimental material. In this experiment three explant types will be used: hypocotyl (undeveloped
lower stem), epicotyl (shoot apex), and cotyledons.
11. Plant Tissue Culture 161
Figure 9.3. Structural formulae of plant growth and differentiation hormones used in Experiment 2.
Media Formulae
Media A, B, C, D, and E each contain the same complement of minerals, that is, salt base as in
Medium D (Appendix D). The effect of minerals alone on explant growth and development
constitute "basal growth rate" against which the effects of other media constituents can be
measured. Medium D, then, serves as the base-line control for endogenous growth. Medium E,
containing both essential minerals plus sucrose, constitutes the organic and inorganic control which
can be used as the base-line indicator of explant growth when both minerals and sucrose are
supplied. In addition to the substrate, sucrose, Medium E contains two organic growth factors,
inositol and thiamine, which promote sugar metabolism and general anabolic growth processes.
Medium E also contains additional phosphate thereby matching the phosphate concentrations of the
experimental media (A, B, C). The experimental media contain similar inorganic and organic
complements, but differ in hormone content.
Since cytokinins are derived from adenine, adenine sulfate has been added to each of the
experimental media (A, B, C). In addition, either kinetin or 2iP ([2-isopentenyl]-adenine), both of
which are synthetic cytokinins having immediate hormone activity, are supplemental cytokinins in
media A, B, and C. Of the three experimental media, Medium A contains the highest amount of
active cytokinin (30 mg/liter), while media B and C contain much lower amounts (2 mg/liter and 1
mg/liter, respectively).
Conversely, Medium A contains only a small amount of auxin (0.3 mg IAA/liter), while
Medium B contains a higher amount (2 mgIAA/liter). Medium C contains the lowest absolute
concentration of auxin (0.1 mg NAA/liter), but this synthetic auxin is more efficient in promoting
cell expansion and root formation than the naturally occurring auxin, IAA. Medium C, then, may
actually represent the formula with the highest physiological auxin activity.
Since cytokinin/auxin balance is reportedly important to the final overall effect on growth and
development, the results for each experimental media may be expected to differ. The balance
12. 162 Plant Tissue Culture
represented by Medium A is decidedly skewed towards a high cytokinin/low auxin ratio. Medium
B represents a more even distribution of cytokinin and auxin, while Medium C may have an
effectively higher auxin than cytokinin ratio because of the "in vivo" stability of NAA as well as its
effectiveness as an auxin.
Methods: Week 1 (Aseptic Seed Germination)
Materials:
cucumber seeds (tomato may also be used; sterilization procedure, Experiment 1)
95% ethanol
sterile jars, sterile water
25% chlorox, freshly prepared
sterile forceps or spatula
Procedure:
1. Sterilize seeds (cucumber) for 1 minute with 95% EtOH.
2. Rinse in sterile water
3. Sterilize in 25% bleach for 5 minutes and rinse three times with sterile water.
4. Transfer 10 seeds with a sterile forceps or spatula to a nutrient agar plate.
5. Incubate for 1 week (20–23°C).
Methods: Week 2
Seedling explants (approximately 1 cm in length) of aseptically germinated cucumber (or
tomato) can be cut from the seedlings as shown in Figure 9.4. Aseptic techniques including the use
of the laminar flow hood are necessary to evaluate growth experiments in which nutrient rich media
are used. An explant (hypocotyl, epicotyl, or cotyledon) should be placed on each of the following
experimental media:
A) Murashige Shoot Multiplication, Medium A,
B) Murashige Shoot Multiplication, Medium B, and
C) Murashige Shoot Multiplication, Medium C,
which contain different concentrations of growth hormones (Appendix D). For comparison, one
explant of each type should be placed on each control media:
D) Murashige and Skoog Salt Base, Medium D, and
E) Murashige Minimal Organic Medium + Sucrose with NaH2PO4⋅H20, Medium E.
Seal the petri dishes with parafilm to prevent desiccation and incubate at low light intensity until
next week. Record incubation conditions.
13. Plant Tissue Culture 163
Figure 9.4. The transfer of explants to differential growth media using aseptic techniques
(Experiment 2, Methods: Week 2).
Discussion Questions
What effect does a hormone balance that is applied pharmacologically "in vitro" have on
seedling explant growth and morphogenesis? Which media formulations produce callus? To what
extent? On which explants? Were these results predictable? What media produce anomalous
14. 164 Plant Tissue Culture
growth rather than callus? How is this explained? Are adventitious roots formed on all explants in
Medium C? Which explants are readily totipotent on control media (D, E)? Is a wound response
associated with observed totipotency? Does the hormone balance of experimental media (A, B, C)
influence the extent of totipotency? Are the organs that form during organogenesis complete and
apparently functional? Do microscopic observations of cell size, dimension, and orientation
facilitate the interpretation of hormone effects? Do these explants grown in Medium A (high
cytokinin, low auxin) show rapid cell division without cell expansion? Are shoot primordia forming
on the explants in Medium A? Are callus cells disorganized and oversized?
Additional Studies
How does explant orientation in the medium influence results? If callused explants are placed
on Medium A, will shoot primordia develop? Is it possible to generate an entire plant from a
callused explant? Are serial transfers and the multiplication of callus possible? On which media?
Is it possible to clone whole plants from multiplied callus? Under what conditions?
Experiment 3:
Callus Formation and Multiplication
Callus is defined as an unorganized tissue mass growing on solid substrate. Callus forms
naturally on plants in response to wounding, infestations, or at graft unions (Bottino, 1981). Since
extensive callus formation can be induced by elevated hormone levels, tissue culture media
designed to produce callus contain pharmacological additions of cytokinins and auxins.
Callus formation is central to many investigative and applied tissue culture procedures. Callus
can be multiplied and later used to clone numerous whole plants. Additionally, various genetic
engineering protocols employ callus initiation procedures after DNA has been inserted into cells;
transgenic plants are then regenerated from transformed callus. In other protocols callus is
generated for use in biotechnological procedures such as the formation of suspension cultures from
which valuable plant products can be harvested.
Callus Formation
Explants from several parts of large intact plants can be used to form callus. The most
successful explants are often young tissues of one or a few cell types. Pith cells of young stem are
usually a good source of explant material. Initially, callus cells proliferate without differentiating,
but eventually differentiation occurs within the tissue mass. Actively dividing cells are those
uppermost and peripheral in the callus. The extent of overall differentiation usually depends on the
hormone balance of the support medium and the physiological state of the tissue.
15. Plant Tissue Culture 165
Callus Multiplication
Actively growing callus can be initiated on culture media with an even physiological balance of
cytokinin and auxin (Tobacco Callus Initiation Medium; Appendix E). After callus biomass
increases two to four times (after 2–4 weeks of growth), callus can be divided and placed on fresh
Tobacco Callus Initiation Medium for callus multiplication. Multiplication procedures can be
repeated several times (up to eight sequential transfers) before gross chromosome instability (or
contamination) occurs.
Differentiation and Plant Regeneration
Multiplied callus can be stimulated to form shoots by increasing the cytokinin concentration and
decreasing auxin content of culture media (Tobacco Shoot Development Medium; Appendix E).
Shoot masses can be cut apart and transferred to rooting medium. Once rooted, regenerated plants
can be acclimatized to natural rather than "in vitro" growth conditions. Regenerated plants are
especially valuable if the parent plant was itself unique or if the plants were genetically engineered.
If, for example, multiplied callus was first used to form suspension cultures on which genetic
engineering or cell selection was accomplished, resultant regenerated plants via tissue culture could
possess special traits or capabilities.
Materials and Methods
Callus Formation (Bottino, 1981)
1. Obtain a 5-cm section of tobacco stem.
2. Cut off all leaves.
3. Immerse it in a beaker of 95% ethanol for 15 seconds.
4. In the laminar flow hood, expose the pith by cutting away epidermis, cortex, and vascular
tissue with a sterile scalpel (Figure 9.5).
5. Slice the exposed length of pith into a sterile petri dish.
6. Cover the dish to keep pith sterile.
7. Aseptically slice 5-mm cross-sections of pith.
8. Transfer one cross-section to each plate of Tobacco Callus Initiation Medium.
9. Cover the dishes, seal with parafilm, and place in an incubator at 22–25°C.
Callus Multiplication
1. Obtain a plate of tobacco callus.
2. Aseptically divide the callus into smaller pieces.
3. Transfer divided callus pieces to fresh Tobacco Callus Initiation Medium.
16. 166 Plant Tissue Culture
Figure 9.5. By using pith explant and plating procedures, callus can be generated from pith cells
placed on Tobacco Callus Initiation Medium (Bottino, 1981).
17. Plant Tissue Culture 167
Experiment 4:
Establishment of Suspension Cultures
Background
Suspension cultures are suspensions of individual plant cells and small cell clusters (microcalli)
grown in liquid media. Suspension cultures are established by transferring small pieces of callus to
liquid medium which is subsequently placed on a gyratory shaker. Within a few days individual
plant cells and microcalli should be detached from the original inoculum and growing in the
constantly agitated medium. Suspension cultures grow best if the larger pieces of callus are
removed after the culture has been initiated (Bottino, 1981).
Methods
1. Break up tobacco callus into small pieces and transfer to liquid medium (Tobacco Callus
Initiation Medium, without the agar). Use 25–50 ml medium in 250-ml flask for adequate aeration
for the swirling culture.
2. A thick suspension of callus should continue to proliferate within the next week on a gyratory
shaker.
3. Remove large pieces of callus from suspension culture by passing through a sterile sieve. Large
pieces of callus are usually detrimental to the maintenance of a suspension culture.
4. Suspension cultures can be multiplied by diluting 5 ml of the original strained suspension
culture to 50 ml total volume (1:10 dilution, vol:vol) of fresh medium. The growth rate and cell
generation time can be determined.
Usefulness of Suspension Cultures
Suspension cultures are used in a number of biotechnological procedures such as inoculum for
plant bioreactors, which resemble biofermentors. Valuable plant products can be extracted from
bioreactors during the growth of plant cells as cell suspensions. This type of biotechnology is still in
the initial stages of development and industrial scale-up. Suspension cultures treated with enzymes
that degrade cell walls produce protoplast cultures which can be used in DNA transformation
experiments as well as in protoplast fusion (somatic hybridization) experiments.
Experiment 5: Anther Culture
Background
The purpose of anther and pollen culture is the production of haploid plants by the induction of
embryogenesis from repeated divisions of microspores or immature pollen grains (Dodds and
Roberts, 1985). The chromosome complement of these haploids can be doubled by colchicine
treatment or other techniques to yield fertile homozygous diploids. The resultant diploids can be
used in plant breeding to improve crop plants (Sunderland and Cocking, 1978).
18. 168 Plant Tissue Culture
The haploid nature of embryoids should be determined by standard chromosome staining
procedures (acetocarmine or Feulgen reaction) prior to colchicine treatment. Similarly, the effect of
colchicine on chromosome doubling needs to be monitored by chromosome staining.
Materials
tobacco buds
sharp pointed forceps, surgical scissors
95% ethanol
petri dishes of culture medium (1/2 strength MS, 2% sucrose 0.8% agar, glutamine [800
mg/liter], serine [100 mg/liter])
Methods
1. Obtain two buds at the appropriate stage. This occurs in tobacco when the sepals and the petals
in the bud are the same length.
2. Holding the bud by the pedicel between the thumb and first finger, dip the entire bud in 95%
ethanol for 15 seconds
3. Remove bud and allow excess alcohol to drip off.
4. With a pair of sterile forceps, remove the outer layer of tissue, the sepals.
5. Next, remove the inner layer of tissue, the petals, exposing the anthers.
6. Open the petri dish containing the medium for the induction of haploids. Remove each anther
from the bud and drop it onto the medium. Do not damage the anther or include any filament
tissue.
7. Repeat for another bud.
8. When finished, seal the plates and place in incubator (25°C).
9. In 2–3 weeks examine for somatic embryo initiation. Embryoid-forming cells are characterized
by dense cytoplasmic contents, large starch grains and a relatively large nucleus. Embryoids
appear opaque among translucent cells. Embryoids also exhibit high dehydrogenase activity
and can be detected by tetrazolium staining (Dodds and Roberts, 1985).
Acknowledgements
Paul Bottino (Botany Department, University of Maryland, College Park) provided the
sterilization procedure for Experiment 2 and Methods for Experiments 4 and 5.
19. Plant Tissue Culture 169
Literature Cited
Bottino, P. J. 1981. Methods in plant tissue culture. Kemtec Educational Corp., Kensington,
Maryland, 72 pages.
Butcher, D. N., and D. S. Ingram. 1976. Plant tissue culture. Arnold, London, 67 pages.
Dodds, J. H., and L. W. Roberts. 1985. Experiments in plant tissue culture. Second edition.
Cambridge University Press, New York, 232 pages.
Street, H. E. 1973. Plant tissue and cell culture. Blackwell Scientific Publications, Oxford, 320
pages.
Sunderland, N., and E. C. Cocking. 1978. Plant tissue culture in China—major change ahead?
Nature, 274:643-44.
Sunderland, N., and M. Roberts. 1977. New approach to pollen culture. Nature, 270:236-238.
Ting, I. P. 1982. Plant physiology. Addison-Wesley, Reading, Massachusetts, 642 pages.
Wetherell, D. F. 1982. Introduction to "in vitro" propagation. Avery Publishing Group Inc.,
Wayne, New Jersey, 16 pages.
20. 170 Plant Tissue Culture
APPENDIX A
Expected Results of Experiment 1
21. Plant Tissue Culture 171
APPENDIX B
Media Formulae Used in the
Culture of Aseptic Seedling Explants (Root and Shoot Tips)
Mineral Medium = M = white cap Murashige Minimal Organic Medium
Murashige and Skoog Salt Base (MS) with Agar and Sucrose = O = blue cap
with Agar
Components mg/liter Components mg/liter
NH4NO3 1, 1,900.000 Components
MS Salt Base 4,303.530
KNO3 650.000 100.000
CaCl2 (Anhydrous) 333.000 i-Inositol
Thiamine HCl 0.400
MgSO4 (Anhydrous) 181.000
KH2PO4 170.000
FeHaEDTA 36.700 Sub Total 4,403.930
H2BO3 6.200 Agar 10,000.000
MnSO4⋅H2O 16.900 Sucrose 30,000.000
ZnSO4⋅7H2O 8.600
Total
Kl 0.830 44,403.930
Total
Na2MoO4⋅2H2O 0.250
CuSO4⋅5H2O 0.025
Reference: Huang, L. C., and T. Murashige.
CoCl2⋅6H2O 0.025 1976. TCA Manual, 3(1):539-548.
Sub Total 4,303.530
Agar (non-nutritional gel) 10,000.000 B-deficient = -B = red cap
Total 14,303.530 Demonstration Medium
Reference: Murashige, T., and F. Skoog. Components mg/liter
1962. Physiologia Plantarium, 15:473-497. MS Salt Base 4,303.530
i-Inositol 100.000
Sucrose 30,000.000
Agar 10,000.000
Total 44,403.530
See Appendix C, the general nutrient requirements for plant growth.
Both MS and Minimal Organic (O) media are available as preweighed packets from Carolina Biological
Supply Co., Burlington, NC. The mineral medium is Murashige and Skoog Salt Base (MS) to which agar has
been added. The organic medium is Murashige Minimal Organic Medium with added agar and sucrose; the
salts in this medium are the same as those in MS. The organic medium, then, contains a full inorganic
complement plus three organic constituents—sucrose (as a metabolic energy source) and two growth factors,
inositol and thiamine. The B1-deficient medium is the same as the Minimal Organic Medium (O) except
thiamine is absent. This B-deficient medium is made by using preweighed MS and adding the other
components.
Media preparation: Add preweighed salts to nearly one liter of purified water. Mix until dissolved. Add any
other ingredients one at a time. Mix each until dissolved. Add agar and the remainder of the water. Heat
until boiling begins. Dispense 10-ml aliquots into 20-mm diameter culture tubes. Add cap and autoclave.
The pH of tissue culture media should be between 5.0 and 6.5. (These media formulae are printed with the
cooperation of Carolina Biological Supply Co.).
22. Plant Tissue Culture 172
APPENDIX C
Nutrient Requirements of Plants for Autotrophic Growth
Relative Proportion
Nutrients Assimilation Form Required Function
Nitrogen NO1 160 Structural component of amino
acids, nucleic acids, hormones,
coenzymes, proteins, etc.
Phosphorus PO3 40 Structural components of nucleic
acids, phospholipids, ATP,
coenzymes, etc.
Potassium K+ 125 Activator of various enzymes;
regulation of stomatal opening and
closing
Sulfur SO2 75 Structural component of amino
acids, vitamins, enzymes, etc.
Magnesium Mg++ 50 Cofactor for many enzyme reactions
of carbohydrate metabolism;
structural component of chlorophyll
Calcium Ca++ 50 Enzyme activator, required for
mitosis, forms Ca pectates of
cell wall
Iron Fe++/Fe+++ 2 Structural component of hemes
(cytochromes, etc.) and electron
acceptors in light reaction
Others and Traces Mn, Bo, Cl, Na, I, Cu, and Mo
Trace Elements enhance activity of various enzymes
Carbon CO2 or HCO Gets fixed into carbohydrates
Hydrogen as H2O Solvates constituents of tissues
Oxygen and is split by photolysis-light
reaction, photosynthesis
* Number of pounds necessary to produce 100 bushels of corn.
23. Plant Tissue Culture 173
APPENDIX D
Media Formulae Used in the
Culture of Aseptic Seedling Explants "in vitro"
Murashige Shoot Multiplication Murashige and Skoog Salt Base (MS)
Medium A Medium D = Control, inorganic
(plus added sucrose) Components mg/liter
Components mg/liter 1,650.000
NH4NO3
MS Salt Base 4,303.530 KNO3 1,900.000
NaH2PO4⋅H2O 170.000 CaCl2 (Anhydrous) 333.000
Adenine Sulfate 80.000 MgSO4 (Anhydrous) 181.000
2iP 30.000 KH2PO4 170.000
IAA 0.300 FeHaEDTA 36.700
i-Inositol 100.000 H2BO3 6.200
Thiamine HCl 0.400 MnSO4⋅H2O 16.900
Sucrose 20,000.000 ZnSO4⋅7H2O 8.600
Kl 0.830
Murashige Shoot Multiplication Na2MoO4⋅2H2O 0.250
Medium B CuSO4⋅5H2O 0.025
(plus added sucrose) CoCl2⋅6H2O 0.025
Components mg/liter Total 4,303.530
MS Salt Base 4,303.530
NaH2PO4⋅H2O 170.000 Murashige Minimal Organic Medium
Adenine Sulfate 80.000 with NaH2PO4⋅H2O and Sucrose
IAA 2.000 Medium E
i-Inositol 100.000 (= Control, organic and inorganic)
Kinetin 2.000 mg/liter
Components
Thiamine HCl 0.400
Sucrose 20,000.000 MS Salt Base 4,303.530
NaH2PO4⋅H2O 170.000
Murashige Shoot Multiplication i-Inositol 100.000
Medium C Thiamine HCl 0.400
(plus added sucrose) Sucrose 20,000.000
Components mg/liter
MS Salt Base 4,303.530
NaH2PO4⋅H2O 85.000
Adenine Sulfate 40.000
i-Inositol 100.000
Kinetin 1.000
NAA 0.100
Thiamine HCl 0.400
Sucrose 20,000.000
All media are available as preweighed packets from Carolina Biological Supply Co., Burlington, NC, and other suppliers.
Specific additions to preweighed packets, as indicated above, suppliment Media A, B, C, and E. One percent (1%) agar was
used to solidify each medium.
24. 174 Plant Tissue Culture
APPENDIX E
Formulae for Tobacco Callus Initiation Medium and
Tobacco Shoot Development Medium
Tobacco Callus Initiation Medium
Components mg/liter
MS Salt Base 4,303.530
i-Inositol 100.000
Nicotinic Acid 0.500
Pyridoxine HCI 0.500
Thiamine HCI 0.400
Glycine 2.000
Casein Hydrolysate 1,000.000
IAA 2.000
Kinetin 0.200
Tobacco Shoot Development Medium
Components mg/liter
MS Salt Base 4,303.530
i-Inositol 100.000
Nicotinic Acid 0.500
Pyridoxine HCI 0.500
Thiamine HCI 0.400
Glycine 2.000
Casein Hydrolysate 1,000.000
IAA 0.030
Kinetin 1.000
Premade media in sterile culture jars are
available from Carolina Biological Supply Co., Burlington, NC, and other suppliers.