Other Plant Tissue Topics

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Other Plant Tissue Topics
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Somaclonal Variation

• Somaclonal variation is a general phenomenon of
  all plant regeneration systems that involve a
  callus phase
• There are two general types
• Heritable, genetic changes (alter the DNA)
• Stable, but non-heritable changes (alter gene
  expression, epigenetic)
• With or without mutagen

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Somaclonal/Mutation Breeding

• Advantages
• Screen very high populations (cell based)
• Can apply selection to single cells
• Disadvantages
• Many mutations are non-heritable
• Requires dominant mutation (or double recessive
  mutation) most mutations are recessive
• Can avoid this constraint by not applying
  selection pressure in culture, but you lose the
  advantage of high through-put screening have to
  grow out all regenerated plants, produce seed,
  and evaluate the M2

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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
• 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

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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?
• 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

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Introduction into suspension
Sieve out lumps 1 2
Initial high density

Subculture and sieving
Plate out
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Synchronization

• Cold treatment 4oC
• Starvation deprivation of an essential growth
  compound, e.g. N ?accumulation in G1
• Use of DNA synthesis inhibitors thymidine,
  5-fluorodeoxyuridine, hydroxyurea
• Colchicine method arresting the cells in
  metaphase stage, measured in terms of mitotic
  index ( cells in the mitotic phase)

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Selection

• Select at the level of the intact plant
• Select in culture
• single cell is selection unit
• possible to plate up to 1,000,000 cells on a
  Petri-dish.
• progressive selection over a number of phases

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Selection Strategies

• Positive
• Negative
• Visual
• Analytical Screening

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Positive selection

• Add into medium a toxic compound e.g. hydroxy
  proline, kanamycin
• Only those cells able to grow in the presence of
  the selective agent give colonies
• Plate out and pick off growing colonies.
• Possible to select one colony from millions of
  plated cells in a days work.
• Need a strong selection pressure - get escapes

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Negative selection

• Add in an agent that kills dividing cells e.g.
  chlorate / BUdR.
• Plate out leave for a suitable time, wash out
  agent then put on growth medium.
• All cells growing on selective agent will die
  leaving only non-growing cells to now grow.
• Useful for selecting auxotrophs.

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Visual selection

• Only useful for colored or fluorescent compounds
  e.g. shikonin, berberine, some alkaloids
• Plate out at about 50,000 cells per plate
• Pick off colored / fluorescent-expressing
  compounds (cell compounds?)
• Possible to screen about 1,000,000 cells in a
  days work.

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Analytical Screening

• Cut each piece of callus in half
• One half subcultured
• Other half extracted and amount of compound
  determined analytically (HPLC/ GCMS/ ELISA)

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Targets for Somaclonal Variation

• Herbicide resistance and tolerance
• Specific amino acid accumulators
• Screen for specific amino acid production
• e.g.Lysine in cereals
• Abiotic stress tolerance
• Add or subject cultures to selection agente.g.
  salt, temperature stress
• Disease resistance
• Add toxin or culture filtrate to growth media

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T-DNA Tagging

• T-DNA is inherited as a single dominant gene
  locus
• Insertion of the T-DNA can result in a mutation
• Large T-DNA tag populations have been generated
  a primary goal is to disrupt the function
  (mutate) every gene, it is estimated that300,000
  random insertions will saturate the genome with
  mutations
• Tag insertion of the T-DNA tags the region
  in the genome because the T-DNA sequence is known
  and it can be located in the genome

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Embryo Culture
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Embryo Culture Uses

• Rescue F1 hybrids from wide crosses
• Overcome seed dormancy, usually with addition of
  hormone (GA) to medium
• To overcome immaturity in seeds
• To speed generations in a breeding program
• To rescue a cross or self (valuable genotype)

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Haploid Plant Production

• Embryo rescue of interspecific crosses
• 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)

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Androgenesis

• History
• 1964, 1966 Datura innoxia (Guha and Maheshwari)
• 1967 Nicotiana tabacum (Nitsch)
• Critical factor - change in developmental
  pattern from mature pollen to embryogenesis

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Factors influencing androgenesis

• Genotype of donor plants
• Anther wall factors
• Culture medium and culture density
• Stage of microspore or pollen development
• Effect of temperature and/or light
• Physiological status of donor plant

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Tetrad
Pollen mother cell
Pollen forming
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Bulbosum Method
Hordeum bulbosum Wild relative 2n 2X 14
Hordeum vulgare Barley 2n 2X 14
X
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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)

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Ovule Culture

• Haploids can be induced from ovules
• The number of ovules is less and thus is used
  less than anther culture
• May be by organogenesis or embryogenesis
• Used in plant families that do not respond to
  androgenesis
• Liliaceae
• Compositae

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Haploids

• Typically 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

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Protoplasts

• Created by degrading the cell wall using enzymes
• Very fragile

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• Protoplasts can be induced to fuse with one
  another
• Electrofusion A high frequency AC field is
  applied between 2 electrodes immersed in the
  suspension of protoplasts- this induces charges
  on the protoplasts and causes them to arrange
  themselves in lines between the electrodes. They
  are then subject to a high voltage discharge
  which causes their membranes to fuse where they
  are in contact.
• Polyethylene glycol (PEG) causes agglutination
  of many types of small particles, including
  protoplasts which fuse when centrifuged in its
  presence
• Addition of calcium ions at high pH values

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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

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chloroplast
mitochondria
Fusion
nucleus
heterokaryon
cybrid
hybrid
cybrid
hybrid
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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

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Germplasm Preservation

• Extension of micropropagation techniques
• Two methods
• 1. Slow growth techniques
• ?Temp., ?Light, media supplements (osmotic
  inhibitors, growth retardants), tissue
  dehydration, etc
• Medium-term storage (1 to 4 years)
• 2. Cryopreservation
• Ultra low temperatures. Stops cell division
  metabolic processes
• Very long-term (indefinite?)

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Why not seeds?

• Some crops do not produce viable seeds
• Some seeds remain viable for a limited duration
  only and are recalcitrant to storage
• Seeds of certain species deteriorate rapidly due
  to seed borne pathogens
• Some seeds are very heterozygous not suitable for
  maintaining true to type genotypes
• Effective approach to circumvent the above
  problems may be application of cryopreservation
  technology

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Cryogenic explants

• Undifferentiated plant cells
• Embryonic suspension
• Callus
• Pollen
• Seeds
• Somatic embryos
• Shoot apices

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Cryopreservation

• Requirements
• PreculturingUsually a rapid growth rate to
  create cells with small vacuoles and low water
  content
• CryoprotectionGlycerol, DMSO, PEG, etc, to
  protect against ice damage and alter the form of
  ice crystals
• FreezingThe most critical phase one of two
  methods
• Slow freezing allows for cytoplasmic dehydration
• Quick freezing results in fast intercellular
  freezing with little dehydration

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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

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Synthetic Seeds
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