CH 5. Micropropagation

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CH 5. Micropropagation
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6-1. Introduction
    Rapid and mass production of healthy plants
    Micropropagation   Clonal propagation
  In vitro propagation  
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culture
virus-free plants
1. Meristem shoot tip ----------gt   
? 2. Explants ----------------gt
shoot ---gt plants embryogenesis
3. Explants ---------------------gt ?  
organgenesis
somatic embryos
Artificial seeds
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6-2. Micropropagation through shoot tip culture
  Stage 1 Stage 2 Stage 3
 
 plant mass production
-------gt hardening root development
   Explant cut ---gt subculture  (leaf
meristem) transplanting   into
soil   cut ---gt subculture
 Advantitious Acclimatization  shoots
-------gt cut ---gt subculture  
Culture in field  
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6-2. Micropropagation through shoot tip culture
Stage 1 establishment of the aseptic system
  Stage 2 multiplication of propagula by
repeated subculture   Stage 3 transfer the
plantlets into soil    
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6-2. Micropropagation through shoot tip
culture 6-2-1. Stage 1 aseptic culture system
Source of explant   Apical shoot apex
  Axillary bud   Undeveloped flower bud  
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6-2. Micropropagation through shoot tip
culture 6-2-1. Stage 1 aseptic culture system
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6-2. Micropropagation through shoot tip
culture 6-2-2. Stage 2 rapid propagation
  Repeatedly subculture   Propagation rate?
  Efficiency? / Expense?     e.g. One
explant -----gt 5 -----gt 52 -----gt 53
-----gt 54 -----gt 55   1st 2nd
3rd 4th 5th   ? lt----- lt-----
lt----- 15625 56 lt-----   6th

shoot yield ?
period ?
times ?
variation ?
activity ?
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e.g.
Weeks No. shoots Subculture within 24
wks Total no. of shoots   ---------
----------------- -------------------------------
---- -----------------  2 2 2
12 4096   4 4 4 6 4096  
6 8 8 4 4096   8 14
14 3 2744   12 18 18 2 324
  24 30 30 1 30  
------------------- ----------------
expense efficiency     
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TIMECOURSE FOR PRODUCTION OF VIRUS INDEXED
REGISTERED RED RASPBERRIES
Year Conventinal Micropropagation 1 Establis
h Several screened nuclear greenhouse
stock block30 plants plants
used to establish in vitro stages I,
II 5 stage II transfers (5 fold
multiplication/ transfer) 3125
plants greenhouse rooting and field
planting 2 Transplant 300 Harvest
31,000 suckers
suckers (foundation I) 4 Transplant
3000 suckers (foundation II) 6 Harvest
30,000
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COMPARISON OF CONVENTIONAL MICROPROPAGATION OF
VIRUS INDEXED REGISTERED RED RASPBERRIES Conv
entional 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
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6-2. Micropropagation through shoot tip
culture 6-2-1. Stage 1 aseptic culture system
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6-2. Micropropagation through shoot tip
culture 6-2-2. Stage 2 rapid propagation
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6-2. Micropropagation through shoot tip
culture 6-2-2. Stage 2 rapid propagation
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6-2. Micropropagation through shoot tip
culture 6-2-2. Stage 2 rapid propagation
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6-2. Micropropagation through shoot tip
culture 6-2-2. Stage 2 rapid propagation
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6-2. Micropropagation through shoot tip
culture 6-2-2. Stage 2 rapid propagation
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6-2. Micropropagation through shoot tip
culture 6-2-2. Stage 2 rapid propagation
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6-2. Micropropagation through shoot tip
culture 6-2-2. Stage 2 rapid propagation
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6-2. Micropropagation through shoot tip
culture 6-2-3. Stage 3 transplanting
At CW, as with many commercial labs, rooting in
culture (in vitro) is skipped in favor of rooting
out of culture (ex vitro). This tray of unrooted
microshoots has now been taken out of the sterile
environment. The microcuttings, as they are
called, are trimmed up and soaked in a rooting
hormone (auxin) solution...
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6-2. Micropropagation through shoot tip
culture 6-2-3. Stage 3 transplanting
...then planted in flats much like young bedding
plants. At this point they still have no roots.
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6-2. Micropropagation through shoot tip
culture 6-2-3. Stage 3 transplanting
Flats of unrooted microcuttings are transferred
to a fog chamber within a greenhouse. Directly
out of culture, the plantlets need high humidity
levels and reduced light (similar to the in vitro
environment).
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6-2. Micropropagation through shoot tip
culture 6-2-3. Stage 3 transplanting
Over the next 10-14 days, fogging is diminished
and the tent opened up, until the plants do not
need extra protection (ie. the plantlets on the
right). This process is known as acclimatization
or hardening off.
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6-2. Micropropagation through shoot tip
culture 6-2-3. Stage 3 transplanting
The microcuttings have rooted, transplanted into
larger cells, and increased greatly in size in
only a few weeks.
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6-2. Micropropagation through shoot tip
culture 6-2-3. Stage 3 transplanting
After all that work, the plants are ready for the
field. They are moved to a shade house at least
one week before planting to finish the
hardening-off process
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6-2. Micropropagation through shoot tip
culture 6-2-4. Commercial production - Scale up
-- Quantity   -- Quality   Phenotype
aberrations Impact on production scheduling
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6-2. Micropropagation through shoot tip
culture 6-2-4. Commercial production - Scale up
Some problems     Asynchronous
development   Vitrification
(hyperhydration, glassiness )   Physiological
disorder of shoot cultures   Water-soaked
appearance   still can grow, and multiply
  but can not be rooted   Chronic
contamination   Rooting  
Variation  
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6-3. Artificial seeds
Cultured cells   Single cell origin
    Somatic embryo   Encapsulation
  Somatic seeds   Germi
nation  
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6-3. Artificial seeds 6-3-1. Cell culture
Fast growing cells High frequencing for
embryogenesis Long-term potential for
embryogenesis Less somatic variation
Synchrony
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7-3. Artificial seeds 7-3-2. Encapsulation
Sodium alginate Embryoid in culture
medium ø 4mm 50 mM CaCl2
15-30 min Capsules ø 4.5 - 5.0 mm 0.8 -
1.2 g/cap Calcium
alginate Embryoid Nutrient
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6-3. Artificial seeds 6-3-3. Germination
-- Germination in vitro --
Germination in soil   Synchrony  
Germination rate   Develop into complete
plants   Negligible variation  
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6-4. Applications of micropropagation
Virus-free plant production
Germplasm preservation
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6-4. Applications of micropropagation 6-4-1.
Virus-free plant production
Original problem     Vegetatively
Transmitted   propagated plants Virus
disease to new plants Yield / Quality
   
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6-4. Applications of micropropagation 6-4-1.
Virus-free plant production
Unevenly distribution of virus in plants  
  Holme, 1948 Kassanis, 1957   Close to
meristem shoot apical / root gt virus
concentration     Why? -- high concentration
of hormones in meristem   -- competition
between cell division and virus multiplication
    During culture of meristem gt eliminate
virus   -- rapid growth of callus   --
contact with the nutrient medium
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6-4. Applications of micropropagation 6-4-1.
Virus-free plant production
Meristem culture for producing virus-free
plants     1. Explant size Large
/ Small ?     2. Bud location Apical
/ Axillary ?     3. Season e.g.
Carnation early spring / late autumn ---gt easy
to culture   winter ---gt easy to
root   summer ---gt virus-free  
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6-4. Applications of micropropagation 6-4-1.
Virus-free plant production
Meristem culture for producing virus-free
plants   4. Heat treatment 1890 Kobus
sugarcane 50 - 52 oC water 30 min. 1936
Kunkel peach 50 oC water 10 min. / 35 - 38
oC air 2-4 weeks gt less suffering from
virus disease better growth 1969 Nyland
Goheen review gt 90 viruses 30
diseases, curable by heat _at_ Heat
pretreatment of plants ---gt meristem culture
e.g. Potato heat 8 wks / 18 wks ---gt 50 /
100 virus free _at_ Meristem culature ---gt
heat e.g. Chrysanthemum heat 10 days / 30
days ----gt 9 / 90 virus free  
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6-4. Applications of micropropagation 6-4-1.
Virus-free plant production

• Meristem culture for producing virus-free plants
   
• 5. Antiserum treatment inactivate
  virus  
• 6. Medium  

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6-4. Applications of micropropagation 6-4-1.
Virus-free plant production
Detection of virus   meristem
culture ---gt micropropagation ---gt plants in soil
---gt detection of virus
  Virus-free plants     e.g. Asparagus Goose
berry Rhubarb   Banana Horse
radish Strawberry   Cassava Pea Sugarcane
  Cauliflower Potato Sweet potato
  Garlic Rasp berry Yams  
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6-4. Applications of micropropagation
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6-4. Applications of micropropagation
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6-4. Applications of micropropagation
The plant on the right is virus free.
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6-4. Applications of micropropagation
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6-5 Embryogenesis
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• In somatic embryogenesis the embryos regenerate
  from somatic cells, tissue or organs either de
  nove directly from the tissues, which is the
  opposite of zygotic or sexual embryogenesis.

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Various terms for non-zygotic embryos

• Adventious embryos somatic embryos arising
  directly from other organs or embryos.
• Parthenogenetic embryos formed by the
  unfertilized egg.
• Androgenetic embryos formed by the male
  gametophyte.

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Somatic embryogenesis differs from organogenesis

• Bipolar structure with a closed radicular end
  rather than a monopolar structure.
• The embryo arises from a single cell and has no
  vascular connection with the mother tissue.

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• The initiation and development of embryos from
  somatic tissues in plant tissue culture was first
  recognized by Steward et al. (1958) and Reinert
  (1958, 1959) in culture of Daucus carota.

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Two routes to somatic embryogenesis
(Sharp et al., 1980)

• Direct embryogenesis
• The embryos initiate directly from explant in the
  absence of callus formation.
• Indirect embryogenesis
• Callus from explant takes place from which
  embryos are developed.

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Types of embryogenic cells

• Pre-embryogenic determined cells, PEDCs
• The cells are committed to embryonic development
  and need only to be released. Such cells are
  found in embryonic tissue.
• Induced embryogenic determined cells, IEDCs
• In majority of cases embryogenesis is through
  indirect method. Specific growth regulator
  concentrations and/or cultural conditions are
  required for initiation of callus and then
  redetermination of these cells into the
  embryogenic pattern of development.

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Factors of embryogenic induction

• Floral or reproductive tissue
• Auxin (2,4-D)
• Reduced nitrogen in the medium.

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Somatic embryogenesis as a means of propagation
is seldom used

• High probability of mutations
• The method is usually rather difficult.
• Losing regenerative capacity become greater with
  repeated subculture
• Induction of embryogenesis is very difficult with
  many plant species.
• A deep dormancy often occurs with somatic
  embryogenesis.