Principles and Techniques of Micropropagation

• 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