In Vitro Developmental Pathways

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In Vitro Developmental Pathways
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Explants

• Sterile pieces of a whole plant from which
  cultures are generally initiated
• Types of explant
• Generally all plant cells can be used as an
  explant, however young and rapidly growing
  tissue (or tissue at an early stage of
  development) are preferred.

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• Root tip
• Root cultures can be established from explants
  of the root tip of either primary or lateral
  roots.
• Shoot tip
• The shoot apical meristem from either axillary
  or adventitious buds can be cultured in vitro.
• Embryo
• Both immature and mature embryos can be used as
  explants to generate callus cultures or somatic
  embryos.
• Immature, embryo-derived callus is the most
  popular method of monocot plant regeneration.
• Haploid tissue
• Male gametophyte (Pollen in anthers) or female
  gametophyte (the ovule) can be used as an
  explant.
• Haploid tissue cultures can produce haploid or
  di-haploid plants due to doubling of chromosomes
  during the culture periods.

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Callus

• Definition It is an unspecialized and
  unorganized, growing and dividing mass of cells,
  produced when explants are cultured on the
  appropriate solid medium, with both an auxin and
  a cytokinin and correct conditions.
• During callus formation there is some degree of
  dedifferentiation both in morphology and
  metabolism, resulting in the lose the ability to
  photosynthesis.

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Callus

• Callus cultures may be compact or friable.
• Compact callus shows densely aggregated cells
• Friable callus shows loosely associated cells and
  the callus becomes soft and breaks apart easily.
• Habituation it is the lose of the requirement
  for auxin and/or cytokinin by the culture during
  long-term culture.

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Cell-suspension cultures

• When friable callus is placed into the
  appropriate liquid medium and agitated, single
  cells and/or small clumps of cells are released
  into the medium and continue to grow and divide,
  producing a cell-suspension culture.
• The inoculum used to initiate cell suspension
  culture should neither be too small to affect
  cells numbers nor too large too allow the build
  up of toxic products or stressed cells to lethal
  levels.
• Cell suspension culture techniques are very
  important for plant biotransformation and plant
  genetic engineering.

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Three stages of callus culture

• Induction
• Cells in explant dedifferentiate and begin to
  divide
• Proliferative Stage
• Rapid cell division
• Morphogenesis stage
• Differentiation and formation of organized
  structures specifically processes that lead to
  plant regeneration from somatic cells

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Induction
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Division
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Plant morphogenesis

• Organogenesis
• The formation of organs (such as leaves,
  shoots, roots) on a plant organ, usually of a
  different kind.
• 1. Enhancement of axillary bud proliferation/
• development
• 2. Adventitious shoot formation
• 3. Adventitious root formation
• Somatic embryogenesis
• Embryo initiation and development from somatic
  cells

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Organogenesis

• Shoot initiation and development with subsequent
  formation of adventitious roots
• Enhancement of axillary bud proliferation and
  development - stimulation of the shoot apical
  meristem in vitro that includes proliferation of
  lateral buds
• Adventitious shoot formation - dedifferentiation
  and/or differentiation and development of shoots
  from non-meristematic cells (one or more than
  one) either directly or indirectly
• Adventitious root formation - roots are initiated
  adventitiously at the base of the shoot apex and
  a vascular continuum is established to complete
  plant regeneration.

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Somatic Embryogenesis

• Dedifferentiation is typically minimal but a
  meristemoid-like tissue can be formed in the
  latter case
• Histogenesis of somatic embryogenesis is
  characterized by the formation of a bipolar
  structure, in contrast to adventitious
  organogenesis
• Single cell origin of somatic embryos makes
  chimerism infrequent adventitious shoots can
  arise from more than one cell

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Somatic Embryos

• Bipolar
• Not connected to explant or callus cells by
  vascular tissue
• In most woody plants, tissue must be juvenile or
  reproductive

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Cell Developmental Phases Leading to Morphogenesis