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CH 5. Micropropagation

CH 5. Micropropagation. 6-1. Introduction. Rapid and mass production of healthy plants   Micropropagation   Clonal propagation   In vitro propagation  . culture. virus-free plants. 1. Meristem shoot tip ----------> ? 2. Explants ----------------> shoot ---> plants

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CH 5. Micropropagation

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  1. CH 5. Micropropagation

  2. 6-1. Introduction Rapid and mass production of healthy plants   Micropropagation   Clonal propagation   In vitro propagation  

  3. culture virus-free plants 1. Meristem shoot tip ----------> ? 2. Explants ----------------> shoot ---> plants embryogenesis 3. Explants ---------------------> ? organgenesis somatic embryos Artificial seeds

  4. 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

  5. 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. 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

  7. 6-2. Micropropagation through shoot tip culture 6-2-1. Stage 1: aseptic culture system Plantlets produced in the bract axil

  8. 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 <-----   6th shoot yield ? period ? times ? variation ? activity ?

  9. 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  

  10. TIMECOURSE FOR PRODUCTION OF VIRUS INDEXED REGISTERED RED RASPBERRIES • Year Conventinal Micropropagation • 1 Establish Several • screened nuclear greenhousestock • block=30 plants plantsusedto • establishinvitro • 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

  11. 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:

  12. 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"

  13. 6-2. Micropropagation through shoot tip culture 6-2-2. Stage 2: rapid propagation

  14. 6-2. Micropropagation through shoot tip culture 6-2-2. Stage 2: rapid propagation

  15. 6-2. Micropropagation through shoot tip culture 6-2-2. Stage 2: rapid propagation

  16. 6-2. Micropropagation through shoot tip culture 6-2-2. Stage 2: rapid propagation

  17. 6-2. Micropropagation through shoot tip culture 6-2-2. Stage 2: rapid propagation

  18. 6-2. Micropropagation through shoot tip culture 6-2-2. Stage 2: rapid propagation

  19. 6-2. Micropropagation through shoot tip culture 6-2-2. Stage 2: rapid propagation

  20. 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. The microcuttings, as they are called, are trimmed up and soaked in a rooting hormone (auxin) solution...

  21. 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.

  22. 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).

  23. 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 hardeningoff.

  24. 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.

  25. 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

  26. 6-2. Micropropagation through shoot tip culture 6-2-4. Commercial production - Scale up -- Quantity -- Quality Phenotype aberrations Impact on production scheduling

  27. 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

  28. 6-3. Artificial seeds Cultured cells Single cell origin Somatic embryo Encapsulation Somatic seeds Germination

  29. 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

  30. 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

  31. 6-3. Artificial seeds 6-3-3. Germination -- Germination in vitro -- Germination in soil * Synchrony * Germination rate * Develop into complete plants * Negligible variation

  32. 6-4. Applications of micropropagation Virus-free plant production Germplasm preservation

  33. 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  

  34. 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

  35. 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  

  36. 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

  37. 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:  

  38. 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  

  39. plantlets differentiating from root tissue shoot differentiation from leaf tissue 6-4. Applications of micropropagation

  40. 6-4. Applications of micropropagation

  41. 6-4. Applications of micropropagation The plant on the right is virus free.

  42. 6-4. Applications of micropropagation Agave Aloe

  43. 6-5 Embryogenesis

  44. 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.

  45. 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.

  46. 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.

  47. 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.

  48. 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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