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Plant Tissue Culture:

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Plant Tissue Culture: History; Schwann and Schleiden (1838) - Totipotency theory Haberlandt (1902) - Concept of cell culture published in first report Experiments ... – PowerPoint PPT presentation

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Title: Plant Tissue Culture:


1
  • Plant Tissue Culture
  • History
  • Schwann and Schleiden (1838) - Totipotency theory
  • Haberlandt (1902) - Concept of cell culture
    published in first report Experiments on the
    culture of isolated plant cells
  • Skoog and Miller (1957) - Predicted hypothesis
    Regulation of initiation of shoot and root in
    cultured callus by auxin and cytokinin
  • Steward (1958) and Reinert (1959) - Somatic
    embryogenesis in carrot cultures
  • Murashige and Skoog (1962) - MS medium
  • Chilton (1983) - Production of transgenic tobacco
  • 1984 onwards - Commercial multiplication of
    crops, disease free crops, release of GM crops
    like tomato, soybean, corn, cotton, canola,
    potato etc.
  • What is plant tissue culture?
  • It is a collection of techniques used to
  • maintain or grow plant cells, tissues or organs
    under sterile conditions on a nutrient culture
    medium of known composition i.e. in vitro

2
  • Plant tissue culture is widely used to produce
    clones of a plant through a method known as
    microprogagation
  • Different techniques in plant tissue culture may
    offer certain advantages over traditional methods
    of propagation, including
  • production of exact copies of plants that produce
    particularly good flowers, fruits, or have other
    desirable traits
  • quick production of mature plants
  • production of multiples of plants in the absence
    of seeds or necessary pollinators to produce
    seeds
  • regeneration of whole plants from plant cells
    that have been genetically modified
  • The production of plants in sterile containers
    that allows them to be moved with greatly reduced
    chances of transmitting diseases, pests, and
    pathogens
  • The 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
  • Plant tissue culture relies on the fact that
  • many plant cells have the ability to regenerate a
    whole plant (totipotency)

3
  • Single cells, plant cells without cell walls
    (protoplasts), pieces of leaves, stems or roots
    can often be used to generate a new plant on
    culture media given the required nutrients
    and plant hormones
  • Fig 7.2 Alberts 5th Ed
  • Basis for Plant Tissue Culture
  • Two hormones affect plant differentiation
  • Auxin - stimulates root development
  • Cytokinin - stimulates shoot development
  • Generally, the ratio of these two hormones can
    determine plant development
  • auxin ?cytokinin - root development
  • cytokinin ?auxin - shoot development
  • auxin cytokinin - callus development
  • Figs downloaded
  • Factors Affecting Plant Tissue Culture
  • Growth Media - Minerals, Growth factors, Carbon
    source

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Control of in vitro culture
Cytokinin
Leaf strip
Adventitious Shoot
Root
Callus
Auxin
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  • Environmental Factors - Light, Temperature,
    Photoperiod
  • Explant Source - Usually, the younger, less
    differentiated the explant, the better for tissue
    culture like
  • Shoot tips
  • Axillary buds
  • Seeds
  • Hypocotyl from germinated seed
  • Leaves
  • Fundamental Abilities of Plants
  • Totipotency
  • The potential or inherent capacity of a plant
    cell to develop into an entire plant if suitably
    stimulated
  • It implies that all the information necessary for
    growth and reproduction of the organism is
    contained in the cell
  • Differentiation
  • The capacity of mature cells to return to
    meristematic condition and development of a new
    growing point, followed by redifferentiation
    which is the ability to reorganize into new
    organs

9
  • Competency
  • The endogenous potential of a given cell or
    tissue to develop in a particular way
  • Types of in vitro Culture
  • Culture of intact plants (Seed orchid culture)
  • Embryo culture (embryo rescue)
  • Organ culture
  • Shoot tip culture
  • Root culture
  • Leaf culture
  • Anther culture
  • Callus culture
  • Cell suspension and single cell culture
  • Protoplast culture
  • Fig
  • Breeding Applications of Tissue Culture
  • Micropropagation

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  • Germplasm preservation
  • Somaclonal variation
  • Embryo culture
  • Haploid and dihaploid production
  • In vitro hybridization - protoplast fusion
  • Plant genetic engineering
  • Micropropagation
  • Embryogenesis
  • Direct embryogenesis
  • Indirect embryogenesis
  • Organogenesis
  • Organogenesis via callus formation
  • Direct adventitious organ formation
  • Microcutting
  • Meristem and shoot tip culture
  • Bud culture

13
  • Somatic Embryogenesis
  • The process of initiation and development of
    embryos or embryo-like structures from somatic
    cells
  • The production of embryos from somatic or
    non-germ cells
  • Usually involves a callus intermediate stage
    which can result in variation among seedlings
  • Not a common micro-propagation technique but is
    currently being used to produce superior pine
    seedlings
  • Tissue culture maintains the genetic of the cell
    or tissue used as an explant
  • Tissue culture conditions can be modified to
    cause to somatic cells to reprogram into a
    bipolar structure
  • These bipolar structures behave like a true
    embryo - called somatic embryos
  • Organogenesis
  • The process of initiation and development of a
    structure that shows natural organ form and/or
    function
  • Figs

14
Somatic embryogenesis from Pro-embryonic masses
(PEMs)
Auxin leads to high Putrescine
PEM
Single cells sloughed off the surface
Development and cycling of Pro-embryonic masses
Putrescine to Spermidine
Remove Auxin Polyamine Inter-convesions
Eg. Carrot, Monocots, some conifers
Spermidine to Spermine
15
Secondary embryo formation - Mostly in dicots
Abundant Secondary Embryos
Charcoal ABA
Cytokinin
-Cytokinin
Early embryo
16
  • The ability of non-meristematic plant tissues to
    form various organs de novo
  • The production of roots, shoots or leaves
  • These organs may arise out of pre-existing
    meristems or out of differentiated cells
  • This, like embryogenesis, may involve a callus
    intermediate but often occurs without callus
  • Fig
  • Somatic embryogenesis and Organogenesis Both of
    these technologies can be used as methods of
    micro-propagation
  • Not always desirable because they may not always
    result in populations of identical plants
  • The most beneficial use of somatic embryogenesis
    and organogenesis is in the production of whole
    plants from a single cell or a few cells
  • Tissue Culture Applications
  • Micropropagation
  • Germplasm preservation
  • Somaclonal variation

17
Tissue culture of Lilium speciosum (source
Chang et al., 2000)
18
Regeneration in Callus Cultures
Different stages of embryo formation
4- Stages of embryos
Callus Regeneration
19
  • Synthetic/Artificial seeds
  • Embryo culture
  • Haploid and dihaploid production
  • In vitro hybridization protoplast fusion
  • Industrial products from cell cultures
  • Plant genetic engineering
  • Micropropagation
  • The art and science of plant multiplication in
    vitro
  • Usually derived from meristems or vegetative
    buds) without a callus stage
  • Tends to reduce or eliminate somaclonal
    variation, resulting in true clones
  • Can be derived from other explant or callus but
    these are often problematic
  • Steps of Micropropagation are
  • Stage 0 i.e. selection and preparation of the
    mother plant
  • sterilization of the plant tissue takes place

20
  • Stage I  i.e. Initiation of culture - explant
    placed into growth media
  • Stage II i.e. Multiplication - explant
    transferred to shoot media shoots can be
    constantly divided
  • Stage III i.e. Rooting - explant transferred to
    root media
  • Stage IV i.e. Transfer to soil - explant returned
    to soil hardened off
  • Fig
  • Features of Micropropagation
  • Clonal reproduction - Way of maintaining
    heterozygozity
  • Multiplication Stage can be recycled many times
    to produce an unlimited number of clones -
    Routinely used commercially for many ornamental
    species, some vegetatively propagated crops
  • Easy to manipulate production cycles -Not limited
    by field seasons/environmental influences
  • Disease-free plants can be produced - Has been
    used to eliminate viruses from donor plants
  • Fig
  • Germplasm Preservation
  • Two methods

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In Vitro Clonal Propagation of Plants
23
  • Slow growth techniques
  • ? Temp., ? Light, media supplements (osmotic
    inhibitors, growth retardants), tissue
    dehydration
  • Medium-term storage (1 to 4 years)
  • Cryo-preservation
  • Ultra low temperatures - liquid nitrogen (-196oC)
    in presence of Cryoprotective agents like
    Glycerol, DMSO, PEG
  • Stops cell division and metabolic processes
  • Very long-term (indefinite)
  • Somaclonal Variation
  • A general phenomenon of all plant regeneration
    systems that involve a callus phase
  • Two general types of Somaclonal Variation
  • i. Heritable, genetic changes (alter the DNA)
  • ii. Stable, but non-heritable changes (alter
    gene expression, epigenetic)
  • Done in cell suspension culture

24
  • Then apply physical or chemical mutagen and
    selection pressure to culture
  • Regeneration of whole plants from surviving cells
  • Fig
  • It Important alternative of creation of
    additional genetic variability in crops where
    tissue culture and regeneration system has been
    established
  • Somaclonal mutants can be enriched during in
    vitro culture include
  • - Resistance to disease and herbicides
  • Tolerance to environmental stress and chemical
    stress
  • Increased seedling vigor in lettuce
  • Joint less pedicels in tomato
  • Increase production of secondary metabolites
  • New varieties have been developed through
    somaclonal variation in tomato, sugar cane,
    celery, Brassica, sorghum
  • Synthetic / Artificial Seeds
  • Synthetic or artificial seeds are somatic
    embryos engineered for use in the commercial
    propagation of plants

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  • These techniques have been further developed for
    the production of plants from embryos developed
    by non-sexual methods (haploid production
    discussed later)
  • Very useful technology where
  • True seeds are not used or readily available for
    multiplication (e.g. potato)
  • hybrid plants (e.g. hybrid rice)
  • Vegetatively propagated plants are more prone to
    infections (e.g. day lily, garlic, potato,
    sugarcane, sweet potato, grape and mango).
  • Synthetic seeds are also useful for
    multiplication of
  • Genetically engineered plants (transgenic plants)
  • Somatic and cytoplasmic hybrids (obtained through
    protoplast fusion techniques)
  • Sterile and unstable genotypes
  • preservation of desirable elite genotypes
  • Embryo Culture
  • Embryo culture developed from the need to rescue
    embryos (embryo rescue) from wide crosses where
    fertilization occurred, but embryo development
    did not occur

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Somatic embryos encapsulated in gel
29
  • These techniques have been further developed for
    the production of plants from embryos developed
    by non-sexual methods (haploid production
    discussed later)
  • It is use to
  • rescue F1 hybrid from a wide cross
  • overcome seed dormancy, usually with addition of
    hormone to media (GA)
  • overcome immaturity in seed
  • speed generations in a breeding program
  • rescue a cross or self (valuable genotype) from
    dead or dying plant
  • Fig
  • Haploid Plant Production
  • Anther culture/Microspore culture - culturing of
    anthers or pollen grains (microspores) - derive a
    mature plant from a single microspore
  • Fig
  • Ovule culture - culturing of unfertilized ovules
    (macrospores)
  • Effective for crops that do not yet have an
    efficient microspore culture system. For example
    Melon, onion

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Anther/Microspore Culture
32
Anther Culture
33
  • What do you do with the haploid?
  • Weak, sterile plant
  • Usually want to double the chromosomes, creating
    a di-haploid plant with normal growth and
    fertility
  • Chromosomes can be doubled by
  • Colchicine treatment
  • Spontaneous doubling
  • Tends to occur in all haploids at varying levels
  • Many systems rely on it, using visual observation
    to detect spontaneous di-haploids
  • Can be confirmed using flow cytometry
  • Protoplast Fusion
  • Figs
  • It is used to
  • Combine two complete genomes - another way to
    create allopolyploids

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, Electrofusion, Ca ions
36
  • Partial genome transfer - exchange single or few
    traits between species which may or may not
    require ionizing radiation
  • Genetic engineering - micro-injection,
    electroporation, Agrobacterium
  • Transfer of organelles - unique to protoplast
    fusion i.e. the transfer of mitochondria and/or
    chloroplasts between species
  • Fig
  • Examples are
  • - Protoplast fusion between male sterile
    cabbage and normal cabbage was done, and cybrids
    were selected that contained the radish
    mitochondria and the cabbage chloroplast
  • - Current procedure is to irradiate the
    cytoplasmic donor to eliminate nuclear DNA
    routinely used in the industry to re-create male
    sterile brassica crops
  • Industrial Products
  • Secondary metabolites produced by plants -
    alkaloids, terpenoids, steroids, anthocyanins,
    anthraquinones, polyphenols
  • Often restricted production - specific species,
    tissue or organ
  • Plant Genetic Engineering

37
Possible Result of Fusion of Two Genetically
Different Protoplasts
chloroplast
mitochondria
Fusion
nucleus
heterokaryon
cybrid
hybrid
cybrid
hybrid
38
  • Over All Applications
  • Plant tissue culture is used widely in the plant
    sciences, forestry, and in horticulture.
    Applications include
  • The commercial production of plants used as
    potting, landscape, and florist subjects, which
    uses meristem and shoot culture to produce large
    numbers of identical individuals
  • To conserve rare or endangered plant species
  • A plant breeder may use tissue culture to screen
    cells rather than plants for advantageous
    characters, e.g. herbiside resistance/tolerance.
  • Large-scale growth of plant cells in liquid
    culture in bioreactors for production of valuable
    compounds, like plant derived secondary
    metabolites  and recombinant proteins used
    as biopharmaceuticals
  • To cross distantly related species by protoplasm
    fusion and regeneration of the novel hybrid
  • To rapidly study the molecular basis for
    physiological, biochemical, and reproductive
    mechanisms in plants, for example in vitro
    selection for stress tolerant plants, and in
    vitro flowering studies.
  • To cross-pollinate distantly related species and
    then tissue culture the resulting embryo which
    would otherwise normally die (Embryo Rescue)?

39
  • For chromosome doubling and induction
    of polypoidy. For example doubled
    haploids, tetraploids, and other forms
    of polyopoloids. This is usually achieved by
    application of antimiotic agents such
    as colchicine or oryzalin
  • As a tissue for transformation, followed by
    either short-term testing of genetic constructs
    or regeneration of transgenic plants
  • Certain techniques such as meristem tip culture
    can be used to produce clean plant material from
    virused stock, such as potatoes and many species
    of soft fruit
  • Production of identical sterile hybrid species
    can be obtained
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