CH 5. Micropropagation

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					CH 5.
Micropropagation
6-1. Introduction



   Rapid and mass production of healthy plants


   Micropropagation

   Clonal propagation

   In vitro propagation
                     culture
1. Meristem shoot tip ----------> virus-free plants

             organgenesis  ?
2. Explants ----------------> shoot ---> plants

            embryogenesis
                                      ?
3. Explants ---------------------> somatic embryos



                                      Artificial seeds
   6-2. Micropropagation through shoot tip culture


  Stage 1             Stage 2             Stage 3

*************        ******************   ****************
 plant          mass production -------> hardening
                                             root development

Explant                 cut ---> subculture
(leaf meristem)                          transplanting
                                            into soil
                        cut ---> subculture
Advantitious                               Acclimatization
shoots -------> cut ---> subculture
                                            Culture in field
 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
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
6-2. Micropropagation through shoot tip culture

       6-2-1. Stage 1: aseptic culture system




 Plantlets produced in the bract axil
6-2. Micropropagation through shoot tip culture

        6-2-2. Stage 2: rapid propagation


        * Repeatedly subculture
        * Propagation rate?
        * Efficiency? / Expense?

e.g.    One explant -----> 5 -----> 52 -----> 53 -----> 54 -----> 55
                      1st      2nd       3rd       4th      5th
                       ? <----- <----- <----- 15625 = 56 <-----
       shoot yield ?                                         6th
       period ?
       times ?
       activity ?   variation ?
e.g.

Weeks       No. shoots          Subculture within 24 wks              Total no. of
                                                                      shoots
---------   -----------------   -----------------------------------   -----------------

   2                   2                   2 12                       4096

   4                   4                   46                         4096

   6                   8                   84                         4096

   8                  14                  14 3                        2744

  12                  18                  18 2                        324

  24                  30                  30 1                        30
                                   -------------------                ----------------
                                       expense                        efficiency
    TIMECOURSE FOR PRODUCTION OF VIRUS INDEXED
    REGISTERED RED RASPBERRIES

Year          Conventinal        Micropropagation

1             Establish          Several
              screened nuclear   greenhouse stock
              block=30 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
 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:
6-2. Micropropagation through shoot tip culture

       6-2-1. Stage 1: aseptic culture system




 Dormant buds are cut from the stock plants and surfacesterilized. When
 placed on the appropriate growing medium, they begin the
 micropropagation process. This initial culture is referred to as "stage I"
6-2. Micropropagation through shoot tip culture

      6-2-2. Stage 2: rapid propagation
6-2. Micropropagation through shoot tip culture

      6-2-2. Stage 2: rapid propagation
6-2. Micropropagation through shoot tip culture

      6-2-2. Stage 2: rapid propagation
6-2. Micropropagation through shoot tip culture

      6-2-2. Stage 2: rapid propagation
6-2. Micropropagation through shoot tip culture

      6-2-2. Stage 2: rapid propagation
6-2. Micropropagation through shoot tip culture

      6-2-2. Stage 2: rapid propagation
6-2. Micropropagation through shoot tip culture

      6-2-2. Stage 2: rapid propagation
6-2. Micropropagation through shoot tip culture

           6-2-3. Stage 3: transplanting




At C&W, 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.
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.
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).
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.
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.
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
6-2. Micropropagation through shoot tip culture

      6-2-4. Commercial production - Scale up

      -- Quantity


      -- Quality

      Phenotype aberrations

      Impact on production scheduling
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
6-3. Artificial seeds



    Cultured cells
                            Single cell origin

           Somatic embryo

                 Encapsulation

                        Somatic seeds

                             Germination
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
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
6-3. Artificial seeds

      6-3-3. Germination


      -- Germination in vitro
      -- Germination in soil

 *    Synchrony

 *    Germination rate

 *    Develop into complete plants

 *    Negligible variation
6-4. Applications of micropropagation
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
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 ===> virus concentration

Why? -- high concentration of hormones in meristem
        -- competition between cell division and virus multiplication

       During culture of meristem ===> eliminate virus
             -- rapid growth of callus
             -- contact with the nutrient medium
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 ---> easy to culture
                            winter ---> easy to root
                            summer ---> virus-free
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
                   ===> less suffering from virus disease & better growth

      1969 Nyland & Goheen : review :
                  ===> 90 viruses & 30 diseases, curable by heat

     @ Heat pretreatment of plants ---> meristem culture
       e.g. Potato : heat 8 wks / 18 wks ---> 50% / 100% virus free

     @ Meristem culature ---> heat
       e.g. Chrysanthemum : heat 10 days / 30 days ----> 9% / 90% virus free
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:
6-4. Applications of micropropagation

       6-4-1. Virus-free plant production
* Detection of virus:
meristem culture ---> micropropagation ---> plants in soil ---> 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
        6-4. Applications of micropropagation

                                  plantlets differentiating from root tissue




shoot differentiation from leaf
tissue
6-4. Applications of micropropagation
6-4. Applications of micropropagation




                      The plant on the right is virus free.
6-4. Applications of micropropagation




                                        Agave




                  Aloe
6-5 Embryogenesis
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.
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.
 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.
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.
    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.
    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.
   Factors of embryogenic
          induction

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

				
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