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Plant Tissue Culture Application

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Plant Tissue Culture Application * * * * * * * * The only other source for genetic variation that I am aware of is polyploidy. As a breeder, you have to be aware of ... – PowerPoint PPT presentation

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Title: Plant Tissue Culture Application


1
Plant Tissue Culture Application
2
Development of superior cultivars
  • Germplasm storage
  • Somaclonal variation
  • Embryo rescue
  • Ovule and ovary cultures
  • Anther and pollen cultures
  • Callus and protoplast culture
  • Protoplasmic fusion
  • In vitro screening
  • Multiplication

3
Tissue Culture Applications
  • Micropropagation
  • Germplasm preservation
  • Somaclonal variation
  • Haploid dihaploid production
  • In vitro hybridization protoplast fusion

4
Micropropagation
5
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

6
Microcutting propagation
  • It involves the production of shoots from
    pre-existing meristems only.
  • Requires breaking apical dominance
  • This is a specialized form of organogenesis

7
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

8
COMPARISON OF CONVENTIONAL MICROPROPAGATION OF
VIRUS INDEXED REGISTERED RED RASPBERRIES
Conventional Micropropagation Duration 6
years 2 years Labor Dig replant every 2
years Subculture every 4 weeks unskilled
(Inexpensive) skilled (more expensive) Space
More, but less expensive (field) Less, but more
expensive (laboratory) Required to
prevent viral Screening, fumigation, spraying
None infection
9
Ways to eliminate viruses
  • Heat treatment.
  • Plants grow faster than viruses at high
    temperatures.
  • Meristemming.
  • Viruses are transported from cell to cell
    through plasmodesmata and through the vascular
    tissue. Apical meristem often free of viruses.
    Trade off between infection and survival.
  • Not all cells in the plant are infected.
  • Adventitious shoots formed from single cells can
    give virus-free shoots.

10
Elimination of viruses
Plant from the field
Pre-growth in the greenhouse
Active growth
Heat treatment 35oC / months
Adventitious Shoot formation
Virus-free Plants
Virus testing
Meristem culture
Micropropagation cycle
11
Indirect Somatic Embryogenesis
Explant ? Callus Embryogenic ? Maturation ?
Germination
  1. Callus induction
  2. Embryogenic callus development
  3. Maturation
  4. Germination

12
Induction
  • Auxins required for induction
  • Proembryogenic masses form
  • 2,4-D most used
  • NAA, dicamba also used

13
Development
  • Auxin must be removed for embryo development
  • Continued use of auxin inhibits embryogenesis
  • Stages are similar to those of zygotic
    embryogenesis
  • Globular
  • Heart
  • Torpedo
  • Cotyledonary
  • Germination (conversion)

14
Maturation
  • Require complete maturation with apical meristem,
    radicle, and cotyledons
  • Often obtain repetitive embryony
  • Storage protein production necessary
  • Often require ABA for complete maturation
  • ABA often required for normal embryo morphology
  • Fasciation
  • Precocious germination

15
Germination
  • May only obtain 3-5 germination
  • Sucrose (10), mannitol (4) may be required
  • Drying (desiccation)
  • ABA levels decrease
  • Woody plants
  • Final moisture content 10-40
  • Chilling
  • Decreases ABA levels
  • Woody plants

16
Plant germplasm preservation
  • In situ Conservation in normal habitat
  • rain forests, gardens, farms
  • Ex Situ
  • Field collection, Botanical gardens
  • Seed collections
  • In vitro collection Extension of
    micropropagation techniques
  • Normal growth (short term storage)
  • Slow growth (medium term storage)
  • Cryopreservation (long term storage
  • DNA Banks

17
In vitro Collection
  • Use
  • Recalcitrant seeds
  • Vegetatively propagated
  • Large seeds
  • Concern
  • Security
  • Availability
  • cost

18
Ways to achieve slow growth
  • Use of immature zygotic embryos
  • (not for vegetatively propagated species)
  • Addition of inhibitors or retardants
  • Manipulating storage temperature and light
  • Mineral oil overlay
  • Reduced oxygen tension
  • Defoliation of shoots

19
Cryopreservation
Storage of living tissues at ultra-low
temperatures (-196C)
  • Conservation of plant germplasm
  • Vegetatively propagated species (root and tubers,
    ornamental, fruit trees)
  • Recalcitrant seed species (Howea, coconut,
    coffee)
  • Conservation of tissue with specific
    characteristics
  • Medicinal and alcohol producing cell lines
  • Genetically transformed tissues
  • Transformation/Mutagenesis competent tissues
    (ECSs)
  • Eradication of viruses (Banana, Plum)
  • Conservation of plant pathogens (fungi, nematodes)

20
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21
Cryopreservation Steps
  • Selection
  • Excision of plant tissues or organs
  • Culture of source material
  • Select healthy cultures
  • Apply cryo-protectants
  • Pre-growth treatments
  • Cooling/freezing
  • Storage
  • Warming thawing
  • Recovery growth
  • Viability testing
  • Post-thawing

22
Cryopreservation Requirements
  • Preculturing
  • Usually a rapid growth rate to create cells with
    small vacuoles and low water content
  • Cryoprotection
  • Cryoprotectant (Glycerol, DMSO/dimetil
    sulfoksida, PEG) to protect against ice damage
    and alter the form of ice crystals
  • Freezing
  • The most critical phase one of two methods
  • Slow freezing allows for cytoplasmic dehydration
  • Quick freezing results in fast intercellular
    freezing with little dehydration

23
Cryopreservation Requirements
  • Storage
  • Usually in liquid nitrogen (-196oC) to avoid
    changes in ice crystals that occur above -100oC
  • Thawing
  • Usually rapid thawing to avoid damage from ice
    crystal growth
  • Recovery
  • Thawed cells must be washed of cryo-protectants
    and nursed back to normal growth
  • Avoid callus production to maintain genetic
    stability

24
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25
Somaclonal Variation
  • Variation found in somatic cells dividing
    mitotically in culture
  • A general phenomenon of all plant regeneration
    systems that involve a callus phase
  • Some mechanisms
  • Karyotipic alteration
  • Sequence variation
  • Variation in DNA Methylation
  • Two general types of Somaclonal Variation
  • Heritable, genetic changes (alter the DNA)
  • Stable, but non-heritable changes (alter gene
    expression, epigenetic)

26
Epigenetic
the study of gene regulation that does not
involve making changes to the SEQUENCE of the
DNA, but rather to the actual BASES within the
nucleotides and to the HISTONES
  • The three main mechanisms for regulation are
  • CpG island methylation (meCGmeCGmeCGmeCGmeCGm
    eCGmeCGmeCG)
  • acetylation and methylation of histone H3
  • the production of antisense RNA

27
Somaclonal Breeding Procedures
  • Use plant cultures as starting material
  • Idea is to target single cells in multi-cellular
    culture
  • Usually suspension culture, but callus culture
    can work (want as much contact with selective
    agent as possible)
  • Optional apply physical or chemical mutagen
  • Apply selection pressure to culture
  • Target very high kill rate, you want very few
    cells to survive, so long as selection is
    effective
  • Regenerate whole plants from surviving cells

28
Requirements for Somaclonal Breeding
  • Effective screening procedure
  • Most mutations are deleterious
  • With fruit fly, the ratio is 8001 deleterious
    to beneficial
  • Most mutations are recessive
  • Must screen M2 or later generations
  • Consider using heterozygous plants?
  • But some say you should use homozygous plants to
    be sure effect is mutation and not natural
    variation
  • Haploid plants seem a reasonable alternative if
    possible
  • Very large populations are required to identify
    desired mutation
  • Can you afford to identify marginal traits with
    replicates statistics? Estimate 10,000 plants
    for single gene mutant
  • Clear Objective
  • Cant expect to just plant things out and see
    what happens relates to having an effective
    screen
  • This may be why so many early experiments failed

29
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30
Embryo Culture Uses
  • Rescuing interspecific and intergeneric hybrids
  • wide hybrids often suffer from early spontaneous
    abortion
  • cause is embryo-endosperm failure
  • Gossypium, Brassica, Linum, Lilium
  • Production of monoploids
  • useful for obtaining "haploids" of barley, wheat,
    other cereals
  • the barley system uses Hordeum bulbosum as a
    pollen parent

31
Bulbosum Method
Hordeum bulbosum Wild relative 2n 2X 14
Hordeum vulgare Barley 2n 2X 14
X
?
Embryo Rescue
Haploid Barley 2n X 7 H. Bulbosum chromosomes
eliminated
  • This was once more efficient than microspore
    culture in creating haploid barley
  • Now, with an improved culture media (sucrose
    replaced by maltose), microspore culture is much
    more efficient (2000 plants per 100 anthers)

32
Bulbosum technique
  • H. vulgare is the seed parent
  • zygote develops into an embryo with elimination
    of HB chromosomes
  • eventually, only HV chromosomes are left
  • embryo is "rescued to avoid abortion
  • Excision of the immature embryo
  • Hand pollination of freshly opened flowers
  • Surface sterilization EtOH on enclosing
    structures
  • Dissection dissecting under microscope
    necessary
  • Plating on solid medium slanted media are often
    used to avoid condensation

33
Culture Medium
  • Mineral salts K, Ca, N most important
  • Carbohydrate and osmotic pressure
  • Amino acids
  • Plant growth regulators

34
Culture Medium
  • Carbohydrate and osmotic pressure
  • 2 sucrose works well for mature embryos
  • 8-12 for immature embryos
  • transfer to progressively lower levels as embryo
    grows
  • alternative to high sucrose auxin cyt PGRs
  • amino acids
  • reduced N is often helpful
  • up to 10 amino acids can be added to replace N
    salts, incl. glutamine, alanine, arginine,
    aspartic acid, etc.
  • requires filter-sterilizing a portion of the
    medium

35
Culture Medium
  • natural plant extracts
  • coconut milk (liquid endosperm of coconut)
  • enhanced growth attributed to undefined hormonal
    factors and/or organic compounds
  • others extracts of dates, bananas, milk, tomato
    juice
  • PGRs
  • globular embryos require low conc. of auxin and
    cytokinin
  • heart-stage and later usually none required
  • GA and ABA regulate "precocious germination
  • GA promotes, ABA suppresses

36
Wide crossing of wheat and rye requires embryo
rescue and chemical treatment to double the
number of chromosomes.
Triticale
37
Haploid Plant Production
  • Embryo rescue of interspecific crosses
  • Creation of alloploids
  • 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)

38
Specific Examples of DH uses
  • Evaluate fixed progeny from an F1
  • Can evaluate for recessive quantitative traits
  • Requires very large dihaploid population, since
    no prior selection
  • May be effective if you can screen some
    qualitative traits early
  • For creating permanent F2 family for molecular
    marker development
  • For fixing inbred lines (novel use?)
  • Create a few dihaploid plants from a new inbred
    prior to going to Foundation Seed (allows you to
    uncover unseen off-types)
  • For eliminating inbreeding depression
    (theoretical)
  • If you can select against deleterious genes in
    culture, and screen very large populations, you
    may be able to eliminate or reduce inbreeding
    depression
  • e.g. inbreeding depression has been reduced to
    manageable level in maize through about 50 years
    of breeding this may reduce that time to a few
    years for a crop like onion or alfalfa

39
Somatic Hybridization
Development of hybrid plants through the fusion
of somatic protoplasts of two different plant
species/varieties
40
Somatic hybridization technique
1. isolation of protoplast

2. Fusion of the protoplasts of desired
species/varieties

3. Identification and Selection of somatic hybrid
cells

4. Culture of the hybrid cells

5. Regeneration of hybrid plants
41
Isolation of Protoplast (Separartion of
protoplasts from plant tissue)
2. Enzymatic Method
1. Mechanical Method
42
Mechanical Method
Plant Tissue
Cells Plasmolysis
Microscope Observation of cells
Release of protoplasm
Cutting cell wall with knife
Collection of protoplasm
43
Mechanical Method
  • Used for vacuolated cells like onion bulb scale,
    radish and beet root tissues
  • Low yield of protoplast
  • Laborious and tedious process
  • Low protoplast viability

44
Enzymatic Method
Leaf sterlization, removal of epidermis
Plasmolysed cells
Plasmolysed cells
Pectinase cellulase
Pectinase
Protoplasm released
Release of isolated cells
Protoplasm released
cellulase
Isolated Protoplasm
45
Enzymatic Method
  • Used for variety of tissues and organs including
    leaves, petioles, fruits, roots, coleoptiles,
    hypocotyls, stem, shoot apices, embryo
    microspores
  • Mesophyll tissue - most suitable source
  • High yield of protoplast
  • Easy to perform
  • More protoplast viability

46
Protoplast Fusion (Fusion of protoplasts of two
different genomes)
1. Spontaneous Fusion
2. Induced Fusion
Intraspecific
Intergeneric
Electrofusion
Mechanical Fusion
Chemofusion
47
Uses for Protoplast Fusion
  • Combine two complete genomes
  • Another way to create allopolyploids
  • In vitro fertilization
  • 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

48
Spontaneous Fusion
  • Protoplast fuse spontaneously during isolation
    process mainly due to physical contact
  • Intraspecific produce homokaryones
  • Intergeneric have no importance

49
Induced Fusion
Chemofusion- fusion induced by chemicals
  • Types of fusogens
  • PEG
  • NaNo3
  • Ca 2 ions
  • Polyvinyl alcohol

50
Induced Fusion
  • Mechanical Fusion- Physical fusion of protoplasts
    under microscope by using micromanipulator and
    perfusion micropipette
  • Electrofusion- Fusion induced by electrical
    stimulation
  • Fusion of protoplasts is induced by the
    application of high strength electric field
    (100kv m-1) for few microsecond

51
Possible Result of Fusion of Two Genetically
Different Protoplasts
chloroplast
mitochondria
Fusion
nucleus
heterokaryon
cybrid
hybrid
cybrid
hybrid
52
Identifying Desired Fusions
  • Complementation selection
  • Can be done if each parent has a different
    selectable marker (e.g. antibiotic or herbicide
    resistance), then the fusion product should have
    both markers
  • Fluorescence-activated cell sorters
  • First label cells with different fluorescent
    markers fusion product should have both markers
  • Mechanical isolation
  • Tedious, but often works when you start with
    different cell types
  • Mass culture
  • Basically, no selection just regenerate
    everything and then screen for desired traits

53
Advantages of somatic hybridization
  • Production of novel interspecific and intergenic
    hybrid
  • Pomato (Hybrid of potato and tomato)
  • Production of fertile diploids and polypoids from
    sexually sterile haploids, triploids and
    aneuploids
  • Transfer gene for disease resistance, abiotic
    stress resistance, herbicide resistance and many
    other quality characters
  • Production of heterozygous lines in the single
    species which cannot be propagated by vegetative
    means
  • Studies on the fate of plasma genes
  • Production of unique hybrids of nucleus and
    cytoplasm

54
Problem and Limitation of Somatic Hybridization
  1. Application of protoplast technology requires
    efficient plant regeneration system.
  2. The lack of an efficient selection method for
    fused product is sometimes a major problem.
  3. The end-product after somatic hybridization is
    often unbalanced.
  4. Development of chimaeric calluses in place of
    hybrids.
  5. Somatic hybridization of two diploids leads to
    the formation of an amphiploids which is
    generally unfavorable.
  6. Regeneration products after somatic hybridization
    are often variable.
  7. It is never certain that a particular
    characteristic will be expressed.
  8. Genetic stability.
  9. Sexual reproduction of somatic hybrids.
  10. Inter generic recombination.

55
TYPICAL SUSPENSION PROTOPLAST LEAF PROTOPLAST
PEG-INDUCED FUSION


56
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57

NEW SOMATIC HYBRID PLANT

58
True in vitro fertilization
A procedure that involves retrieval of eggs and
sperm from the male and female and placing them
together in a laboratory dish to facilitate
fertilization
  • Using single egg and sperm cells and fusing them
    electrically
  • Fusion products were cultured individually in
    'Millicell' inserts in a layer of feeder cells
  • The resulting embryo was cultured to produce a
    fertile plant

59
In Vitro Fertilization
60
Requirements for plant genetic transformation
  • Trait that is encoded by a single gene
  • A means of driving expression of the gene in
    plant cells (Promoters and terminators)
  • Means of putting the gene into a cell (Vector)
  • A means of selecting for transformants
  • Means of getting a whole plant back from the
    single transformed cell (Regeneration)
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