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Lecture 2: Applications of Tissue Culture to Plant Improvement

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Title: Lecture 2: Applications of Tissue Culture to Plant Improvement Author: Steve R. King Last modified by: ibm Created Date: 3/13/2003 5:16:30 AM – PowerPoint PPT presentation

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Title: Lecture 2: Applications of Tissue Culture to Plant Improvement


1
AGRICULTURAL BIOTECHNOLOGY
2
Stages of Biotechnology Development
  • Ancient biotechnology
  • early history as related to food and shelter
    Includes domestication
  • Classical biotechnology
  • built on ancient biotechnology Fermentation
    promoted food production, and medicine
  • Modern biotechnology
  • manipulates genetic information in organism
    Genetic engineering

3
Stages of Biotechnology Development
  • Traditional/old biotechnology
  • The conventional techniques that have been used
    to produce beer, wine, cheese, many other food
  • New/modern biotechnology
  • All methods of genetic modification by
    recombinant DNA and cell fusion techniques,
    together with the modern development of
    traditional biotechnological process

4
Areas of Biotechnology
  • Organismic biotechnology
  • uses intact organisms Does not alter genetic
    material
  • Molecular biotechnology
  • alters genetic makeup to achieve specific goals
  • Transgenic organism- an organism with
    artificially altered genetic material

5
Biotechnology A collection of technologies
6
The Applications of Biotechnology
  • Medical Biotechnology
  • Diagnostics
  • Therapeutics
  • Vaccines
  • Agricultural Biotechnology
  • Plant agriculture
  • Animal agriculture
  • Food processing
  • Environmental Biotechnology
  • Cleaning through bioremediation
  • Preventing environmental problems
  • Monitoring the environment

7
Plant agriculture
  • Crop production and protection
  • Genetically engineered (transgenic) crops
  • Using biological methods to protect crops
  • Exploiting cooperative relationships in nature
  • Nutritional value of crops
  • Improving food quality and safety
  • Healthier cooking oils by decreasing the conc. Of
    saturated fatty acids in vegetable oils
  • Functional foods foods containing significant
    levels of biologically active components that
    impart health benefits

Plant Biotechnology
8
PLANT BIOTECHNOLOGY
  • Manipulating plants for the benefit of mankind
  • A process to produce a genetically modified plant
    by removing genetic information from an organism,
    manipulating it in the laboratory and then
    transferring it into a plant to change certain of
    its characteristics

Technology
  • tissue culture
  • plant transformation

9
Plant Tissue Culture
the culture of plant seeds, organs, tissues,
cells, or protoplasts on nutrient media under
sterile conditions.
10
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11
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

12
Control of in vitro culture
Cytokinin
Leaf strip
Adventitious Shoot
Root
Callus
Auxin
13
Factors Affecting Plant Tissue Culture
  • Growth Media
  • Minerals, Growth factors, Carbon source, Hormones
  • Environmental Factors
  • Light, Temperature, Photoperiod, Sterility, Media
  • Explant Source
  • Usually, the younger, less differentiated the
    explant, the better for tissue culture
  • Different species show differences in amenability
    to tissue culture
  • In many cases, different genotypes within a
    species will have variable responses to tissue
    culture response to somatic embryogenesis has
    been transferred between melon cultivars through
    sexual hybridization

14
Three 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
  • Dedifferentiation
  • Capacity of mature cells to return to
    meristematic condition and development of a new
    growing point, follow by redifferentiation which
    is the ability to reorganize into new organ
  • Competency
  • the endogenous potential of a given cells or
    tissue to develop in a particular way

15
Types of In Vitro Culture
  • Culture of intact plants (seed and seedling
    culture)
  • Embryo culture (immature embryo culture)
  • Organ culture
  • 1. shoot tip culture
  • 2. root culture
  • 3. leaf culture
  • 4. anther culture
  • Callus culture
  • Cell suspension culture
  • Protoplast culture

16
Tissue Culture Applications
  • Micropropagation
  • dihaploid production
  • Protoplast fusion
  • Genetic engineering

17
Micropropagation
  • Embryogenesis
  • Direct embryogenesis
  • Indirect embryogenesis
  • Organogenesis
  • Organogenesis via callus formation
  • Direct adventitious organ formation
  • Microcutting
  • Meristem and shoot tip culture
  • Bud culture

18
Somatic Embryogenesis
  • The production of embryos from somatic or
    non-germ cells.
  • Usually involves a callus intermediate stage
    which can result in variation among seedlings

19
Peanut somatic embryogenesis
20
Organogenesis
  • 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.

21
Microcutting
  • This is a specialized form of organogenesis
  • It involves the production of shoots from
    pre-existing meristems only.
  • Requires breaking apical dominance
  • Microcuttings can be one of three types
  • Nodal
  • Shoot cultures
  • Clump division

22
Steps of Micropropagation
  • Stage 0 Selection preparation of the mother
    plant
  • sterilization of the plant tissue takes place
  • Stage I  - Initiation of culture
  • explant placed into growth media
  • Stage II - Multiplication
  • explant transferred to shoot media shoots can be
    constantly divided
  • Stage III - Rooting
  • explant transferred to root media
  • Stage IV - Transfer to soil
  • explant returned to soil hardened off

23
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24
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25
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

26
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
  • These techniques have been further developed for
    the production of plants from embryos developed
    by non-sexual methods (haploid production
    discussed later)

27
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28
Haploid Plant Production
  • Embryo rescue of interspecific crosses
  • Creation of alloploids (e.g. triticale)
  • Bulbosum method
  • Anther culture/Microspore culture
  • Culturing of Anthers or Pollen grains
    (microspores)
  • Derive a mature plant from a single microspore
  • Ovule culture
  • Culturing of unfertilized ovules (macrospores)
  • Sometimes trick ovule into thinking it has been
    fertilized

29
Anther/Microspore Culture
30
Anther/Microspore Culture Factors
  • Genotype
  • As with all tissue culture techniques
  • Growth of mother plant
  • Usually requires optimum growing conditions
  • Correct stage of pollen development
  • Need to be able to switch pollen development from
    gametogenesis to embryogenesis
  • Pretreatment of anthers
  • Cold or heat have both been effective
  • Culture media
  • Additives, Agar vs. Floating

31
Ovule Culture for Haploid Production
  • Essentially the same as embryo culture
  • Difference is an unfertilized ovule instead of a
    fertilized embryo
  • Effective for crops that do not yet have an
    efficient microspore culture system
  • e.g. melon, onion
  • In the case of melon, you have to trick the
    fruit into developing by using irradiated pollen,
    then x-ray the immature seed to find developed
    ovules

32
What do you do with the haploid?
  • Weak, sterile plant
  • Usually want to double the chromosomes, creating
    a dihaploid plant with normal growth 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 dihaploids
  • Can be confirmed using flow cytometry

33
Protoplast
  • Created by degrading the cell wall using enzymes
  • Very fragile, cant pipette

34
Protoplast fusion
  • Protoplasts are made from two species that you
    want to cross
  • The membranes are made to fuse
  • osmotic shock, electrical current, virus
  • Regenerate the hybrid fusion product
  • Contain genome from both organisms
  • Very, very difficult

35
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36
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37
Uses for Protoplast Fusion
  • Combine two complete genomes
  • Another way to create allopolyploids
  • Partial genome transfer
  • Exchange single or few traits between species
  • May or may not require ionizing radiation
  • Genetic engineering
  • Micro-injection, electroporation, Agrobacterium
  • Transfer of organelles
  • Unique to protoplast fusion
  • The transfer of mitochondria and/or chloroplasts
    between species

38
Callus
  • A mass proliferation of an unorganised mass of
    cells
  • Requirement for support ensures that scale-up is
    limited

39
Cell suspension culture
  • When callus pieces are agitated in a liquid
    medium, they tend to break up.
  • Suspensions are much easier to bulk up than
    callus since there is no manual transfer or solid
    support.

40
Introduction of callus into suspension
  • Friable callus goes easily into suspension.
  • 2,4-D
  • Low cytokinin
  • semi-solid medium
  • enzymic digestion with pectinase
  • blending
  • Removal of large cell aggregates by sieving.
  • Plating of single cells and small cell aggregates
    - only viable cells will grow and can be
    re-introduced into suspension.

41
Introduction into suspension
Sieve out lumps 1 2
Initial high density

Subculture and sieving
Pick off growing high producers
Plate out
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