Principle and Techniques of Plant Tissue Culture

• axillary shoot formation: formation of shoots from the explant where buds exist (meristematic tissue)
• adventitious shoot formation: use of explant source that does not contain an axillary bud like roots, stem internodes, leaves, and bulb scales (non-meristematic tissue)
• In vitro axillary shoot formation has the most direct application for plant propagation via micropropagation
• meristem culture: using the smallest part of the shoot tip as the explant, including the meristem dome and a few subtending leaf primordia
• Meristem culture is mostly used for virus elimination
• shoot culture: like miniaturized stems, divisions, or layers that grow from lateral and terminal meristems
• shoot culture is most often used in micropropagation systems
• different growth patterns for shoot cultures:

  • axillary branching
  • nodal cultures
  • stool shoots
  • proliferation of protocorm-like bodies
  • minitubers
  • micrografting
• apical dominance is inhibited by removing the shoot tip
• cytokinin is known to inhibit apical dominance
• Nodal cultures

  • plants with very strong apical dominance are repeatedly subcultured through their growing internodes
  • long shoots are cut into single nodes and planted vertically in the medium
  • axillary buds at each node elongate and grow in length
• stool shoots: a shoot with several nodes is cultured to form multiple shoots
• protocorms are structures formed when the orchid seeds germinate
• In tissue culture, growing points of orchids produce clusters of protocorm-like bodies. The PLB can be subcultured by dividing into clusters or cutting into sections. When they are transferred to a medium with lower levels of auxin and cytokinin, the single PLB will produce shoots and roots
• Lateral meristems from nodal segments of potato plants in culture can form miniature tubers. These tubers are storage protein and can be harvested and used to grow potato plants. They are virus free
• Micrografting is comparable to conventional grafting and budding of woody plants. It offers benefits like creation of disease-free plants, determining graft incompatibility, and propagating novel plants
• adventitious shoot formation

  • nonmeristematic tissue culture systems
  • shoots originating from non nodal explants
  • shoots can originate directly from explant or indirectly from callus
  • used to regenerate diploid plants and plants with different ploidy levels
• diploid plant regeneration systems:

  • leaf pieces
  • stem internodes
  • root pieces
  • thin-layer explants
  • homogenized tissue
  • fragmented shoot apices
  • cotyledons and hypocotyls
  • young needle fascicles
  • immature inflorescences on flower stems
  • bulb scales and other storage structures
• thin-layer explants: epidermal peels or thin traverse sections that are only a few cell layers thick
• homogenized tissue: tissue mechanically homogenized in a blender as initial explants
• anther and pollen culture: used for plant breeding to produce haploid plants; anthers and pollens have half the ploidy level of the original plant
• endosperm culture: endosperm is the seed's storage organ and is triploid; this technique is used to generate triploid plants
• In vitro culture can be used for:

  • aseptic seed culture
  • embryo culture and embryo rescue
  • ovule and ovary culture
• seeds are cultured on tissue culture medium for many purposes:

  • to give certain chemical treatments to seedlings for experiments
  • orchid seeds are very tiny and lack stored food reserves, which means they require external food reservoir to regenerate
  • generate microbe free plants for follow up experiments
• embryo rescue: the excision of the embryo of seeds and then culturing on a medium to generate shoot and root
• embryos generated during wide hybridization abort at early stages
• ovary: female plant part
• Unfertilized ovule culture is used for haploid production
• Unfertilized ovules can be excised and fertilized in vitro with pollen
• Fertilized ovule culture is a form of embryo culture but in the very early stages of embryo development
• callus: an undifferentiated mass or group of cells formed from dedifferentiation of cultured cells
• Callus cultures can be used for developing roots, shoots, and/or complete plantlets
• Cell suspension cultures can be developed by growing homogenized tissues in liquid cultures
• protoplasts: cells without cell wall
• Protoplasts are isolated from tissues by digesting the cell wall with cell wall degrading enzymes
• Protoplasts are very useful for introducing foreign DNA segments into cells as they are lacking cell wall and DNA enters easily into cells
• Protoplasts can be hybridized to each other and are used to form somatic hybrids or cyhybrids
• One of the less common but highly efficient use of tissue culture is generate somatic embryos and synthetic seeds
• somatic embryos: embryos generated from somatic cells and these somatic embryos like the original embryos can produce shoots and roots
• synthetic seed production: encapsulation of somatic embryos into some capsules or coatings to sell them as seeds