Lab 7

• tissue culture: the process of propagating plants from small tissues in a sterile environment
• tissues: shoot tip, leaves, buds, callus, roots, single cell
• culture vessels: test tubes, petri dishes, jars
• medium: water and agar + a source of sugar, macro and micronutrients, hormones, vitamins, antibiotics, etc
• agar: a polysaccharide mixture that causes the liquid medium to turn into a gel
• Murashige & Skoog: media for herbaceous plants
• Woody Plant Medium: media for woody plants
• stages of tissue culture process:

  • stage 0: donor plant selection
  • stage I: establishment
  • stage II: shoot multiplication
  • stage III: rooting
  • stage IV: acclimatization
• establishment: sterile explant is sealed in a container
• shoot multiplication: shoot production is induced by increasing concentration of cytokinin in the culture media
• rooting: placed in rooting media containing higher concentrations of auxin to cytokinin
• acclimatization: newly rooted plantlets are introduced to the growing environment: heavily shaded, cool, moist, and highly humid
• factors affecting success of cutting propagation:

  • time of year
  • tissue maturity
  • exogenous treatments
  • wounding
  • environmental factors
• cutting propagation: a portion of stem, root, or leaf is cut from the parent or stock plant and induced to form roots and shoots by chemical, mechanical, and/or environmental manipulation
• Cutting propagation is expensive
• types of cutting propagation:

  • stem cuttings
  • leaf-bud cuttings
  • leaf cuttings
  • root cuttings
• stem cuttings: segments of shoots containing lateral or terminal buds are obtained, and under the proper conditions adventitious roots will develop, and produce independent plant
• important factors in satisfactory rooting of stem cuttings:

  • type of wood
  • stage of growth
  • time of year
• types of stem cuttings:

  • hardwood
  • semi-hardwood
  • softwood
  • herbaceous
• herbaceous stem cuttings: non-woody, herbaceous plants; 3-5 inch stem is cut from parent plant
• softwood stem cuttings: soft, succulent, new growth of woody plants; May, June, July
• semi-hardwood stem cuttings: partially mature wood of the current season's growth, just after a flush of new growth; mid-July to early fall
• hardwood stem cuttings: taken from dormant, mature stems in late fall, winter, or early spring; fully dormant with no sign of active growth
• root cuttings

  • proximal ending should always be up
  • may also be planted horizontally
  • adventitious shoots form at proximal end
• leaf cuttings

  • adventitious buds, shoots, and roots form at base
  • original leaf does not become part of the new plant
• Auxin triggers root initiation
• root initiation stages:

  1. root initiation
  2. auxin active: auxin is required for root formation
  3. auxin inactive: auxin is not required for roots
  4. root elongation (auxin not required)
• micropropagation: the process of propagating plants from small tissues in a sterile environment
• You can use shoot tips, leaves, buds, callus, roots, and single cells in tissue culture
• Tissues in tissue culture are placed on a medium typically composed of water and agar with some source of sugar
• Murashige & Skoog is a media used for herbaceous plants
• Woody Plant Medium is a medium used for woody crops
• Tissue culture steps:

  1. establishment
  2. multiplication
  3. rooting
  4. acclimatization
• establishment: requires the propagator to ensure the sterility of the plant material that will enter the closed tissue culture system
• laminar flow hood: hood provides positive pressure toward the propagator, thus decreasing the likelihood of tissue contamination by microbes in the atmosphere or being passed on by the propagator
• The laminar flow hood must be cleaned thoroughly prior to the attempt at propagation
• Gloves and masks must be worn at all times and each time the propagator's hands are brought out of the hood, they should reapply 70% ETOH
• All water, containers, media, tools, paper towels, etc should be autoclaved prior to the propagation attempt
• Plant materials should be disinfected thoroughly
• Containers with media should never be opened unless inside the hood