Micropropagation by fjzhangxiaoquan



• Suggested reading: Vasil, ch. 3, Bhojwani, ch.
• Defn – asexual (vegetative) propagation of
  plants in vitro
• Criteria for commercial micropropagation
  – rapid multiplication vs. conventional
     • introducing new cultivars
     • ornamental foliage plants and some woodies
  – species that are otherwise difficult to propagate (e.g.,
              Micropropagation (contin)

• Criteria for commercial micropropagation
  – propagation of "pathogen-free" plants (e.g., potatoes,
    bananas, strawberries, raspberries, etc.)
  – rare or hard-to-find plants (i.e., allows a lab or nursery
    to offer a unique specialty)
• Key to commercial feasibility is balancing
  positives and negatives of micropropagation
             Micropropagation (contin)

• Positives and negatives of micropropagation
  – positives
     • rapid multiplication rates
     • low space requirement
  – negatives
     • labor costs
     • high overhead (equipment, facilities, supplies)
     • loss by contamination
     • danger of variation
             Micropropagation (contin)

• Stages of micropropagation
  – stage I – initiation or establishment of an aseptic
     • preferred explant type: shoot tip or ax. bud
     • medium: high salts for herbaceous spp, low salts
       for woodies
     • incubation: temp 24-26 C, photoperiod for veg.
       growth at 40-80 μmols/sec/m2 light intensity
              Micropropagation (contin)

• Stages of micropropagation
  – stage I problems
      • contamination
      • browning of the explant
      • dormant buds (woodies only)
  – stage II – multiplication – obtaining max. no. of
    propagules (tissue pieces or clumps used to generate
    future plantlets)
      • the goal: stimulate axillary shoot proliferation from
        meristems located in axils of primordial leaves
              Micropropagation (contin)

• stage II (contin)
   – cytokinin:auxin ratio adjusted to stimulate shoot
     production (usu. high cytokinin:auxin ratio)
   – can usu. use the same medium for stage I and II
   – problems
      • vitrification – a glassy appearance to leaves
      • acclimation to stage II – it takes some time to get
        to a rapid growth stage
      • generation of callus (potential for mutations)
              Micropropagation (contin)

• stage III – rooting (in vitro) or regeneration of
  roots on shoots from stage II cultures
   – cytokinin is usually removed; auxin may or may not
     be added to the medium
   – this stage is usu. skipped with many plants
   – for woodies, a pretreatment may be substituted
• stage IV – acclimatization – survival and
  establishment after transfer to soil (ex vitro)
              Micropropagation (contin)

• stage IV problems to overcome
  – infectious diseases in soil
  – desiccation – ways to overcome it
      • high humidity chamber
      • intermittent mist (alternating high/low humidity
      • mist under poly
        Production of pathogen-free plants

• Suggested reading: Bhojwani ch. 15
• Background
  – most vegetatively propagated plants are infected with
    1 or more systemic pathogens (usu. viruses)
  – yield increases from 30 to 300% can be achieved by
    replacement with virus-indexed plants
  – distribution of viruses is uneven in plant tissues
  – before "meristem culture" was widely adopted, heat
    therapy was used (but some viruses aren't susc.)
        Production of pathogen-free plants

• Basic procedure
  – shoot tip or meristem-tip dissected and plated on a
    MS-based medium
     • shoot tip must be less than 1 mm long
     • low conc. of auxin/cytokinins are used
     • the goal: to get growth of a shoot free of virus
  – culture 4-6 weeks or until shoot grows out
  – root shoot and transfer to greenhouse
         Production of pathogen-free plants

• Other conditions are sim. to micropropagation
• virus particles may be present in meristem
  tissue, but are often not present in the shoot that
  grows out
• Hypotheses to explain why meristem culture
   – viruses depend on vascular system to move efficiently
   – higher metabolic activity in the meristem perhaps
     interferes with virus replication
        Production of pathogen-free plants

• Hypotheses
  – "virus inactivating system" is most active in the
  – high endogenous auxin level in shoot apex may
    inhibit virus multiplication
• Methods of detecting viruses
  – goal: indexing or multiplication of plants verified free
    of a specific virus
  – visual virus symptoms
        Production of pathogen-free plants

• Methods of detecting viruses
  – sap transmission, by using an indicator plant
  – serological test (antigen-antibody reaction) such as
  – EM test – visually detecting virus particles in cells
• Maintenance of indexed stock
  – isolation from infected plants during multiplication
  – procedure
      • test and multiply in vitro
        Production of pathogen-free plants

• Maintenance of indexed stock
  – procedure
     • place stage IV plantlets in screened greenhouse
     • use 1st generation TC plants as a nuclear (stock)
       cutting block
     • propagules from cutting blocks are retested
     • production block – random samples tested
         Production of pathogen-free plants

• Productivity testing
   – check phenotypic integrity
   – check for abnormal growth
   – compare yield with conventionally propagated plants
      • vigor is usu. much better
      • potential for virus-indexed plants to be more susc.
        to other diseases
        Production of pathogen-free plants

• Other means of obtaining specific pathogen-free
  – chemicals that eliminate viruses
  – callus culture
  – virus-resistant transgenic plants
     • transformation and regeneration of a coat-protein
       gene results in plants that resist virus infection
     • papaya ringspot virus – cp gene of ringspot virus
       has been transferred into papaya transgenic

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