Principles and Techniques of Micropropagation

Cards (49)

  • tissue culture: the initial step of micropropagation where plant cells or organs are grown on a nutrient medium in a sterile environment
  • tissue culture steps:
    1. selection of an explant
    2. culturing on a nutrient medium
    3. forming a callus
    4. developing it into a specific organ or cell
  • Tissue culture is used for fusion of plant cells, increase in the number of sets of chromosomes, and production of pathogen-free plants
  • micropropagation: a technique of tissue culture that involves growing plantlets in sterile conditions
  • micropropagation steps:
    1. selection of a mother plant
    2. multiplication of plantlets in a culture
    3. pretransplant (preparing of plantlets for transfer from the culture to soil)
  • Micropropagation is used for the production of large quantities of identical plantlets
  • Tissue culture is the starting step of micropropagation where plant cells or organs are grown on a nutrient medium, whereas micropropagation is the production of thousands of identical plantlets
  • totipotency: a single cell has the genetic program to grow an entire plant
  • cell potency: a cells ability to differentiate into other cell types
  • Totipotent stem cells can differentiate into any cell
  • Pluripotent stem cells can differentiate many cell types but not all
  • Multipotent stem cells can differentiate into some cell types
  • Unipotent stem cells can differentiate into only one cell type
  • A high cytokinin to auxin ratio tends to promote shoot organogenesis
  • A low cytokinin to auxin ratio promotes rooting
  • More equal concentrations of auxin and cytokinin tend to promote both shoots and roots
  • structures formed in tissue culture:
    • plantlets
    • seedlings
    • callus
    • somatic embryos
  • micropropagation stages:
    • Stage 0: donor plant selection
    • Stage I: establishment
    • Stage II: shoot multiplication
    • Stage III: root formation
    • Stage IV: acclimitization
  • Things to pay attention to with donor plant selection:
    • genetic and epigenetic characteristics of the source plant
    • control of pathogens
    • physiological conditioning of the source plant to optimize its ability to establish in a culture
  • recalcitrant: species or plants non-responsive to tissue culture
  • important aspects of the establishment stage:
    • explant type
    • explant disinfestation
    • culture medium
    • stabilization
  • in vitro: production in a test tube or other similar vessel where culture conditions and medium are controlled for optimum growth during tissue culture
  • ex vitro: rooting of microcuttings occurs using greenhouse growing medium rather than rooting in vitro where cuttings are rooted on agar in a test tube
  • explant disinfestation: the process of removing microbial contaminants from the surface of explants
  • primary disinfectants for explant disinfestation: alcohol, 10-20% bleach, mercuric chloride, silver nitrate, hydrogen peroxide
  • culture media usually includes:
    • a semisolid support (agar, gelrite, etc)
    • a basal medium of inorganic nutrient elements both macronutrients and micronutrients
    • an energy source (sucrose)
    • hormones, vitamins, etc
  • most commonly used media:
    • Murashige & Skoog (MS): media for herbaceous plants
    • woody plant medium (WPM): media for woody plants
  • apical dominance: elongation of the main terminal shoot
  • Explant initially grows by apical dominance
  • Juvenile explants stabilize more easily
  • Multiple microshoots can be separated and subcultured to multiply the shoots exponentially or can be utilized to form roots
  • Shoot multiplication medium is usually supplemented with cytokinin
  • Cytokinin and auxin are used to support a basic level of growth
  • Shoot initiation is strongly supported by cytokinin concentration
  • Auxin is usually low or absent in Stage II
  • in vitro rooting: roots are formed in the culture medium in a test tube (reduced cytokinin and increased auxin)
  • ex vitro rooting: microcuttings are treated by dipping in auxin for some time and followed by inserting into filled plug trays
  • acclimatization: the process of gradually moving tissue culture grown plantlets to the outdoor environment
  • Maintaining high humidity around roots is a major requirement for acclimatization
  • During acclimatization, plants shift from heterotrophic to autotrophic