Transcript Cell-suspension cultures

In Vitro Developmental
Pathways
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.
a) Root tip:
Root cultures can be established from explants of the root tip of
either primary or lateral roots.
b) Shoot tip:
The shoot apical meristem from either axillary or adventitious buds
can be cultured in vitro.
c) 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.
d) 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.
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.
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.
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 cellsuspension 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.
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
Induction
Division
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
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 nonmeristematic 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.
Somatic Embryogenesis
 Dedifferentiation is typically minimal but a meristemoidlike 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
Somatic Embryos
• Bipolar
• Not connected to explant or callus cells by
vascular tissue
• In most woody plants, tissue must be
juvenile or reproductive
Cell Developmental Phases
Leading to Morphogenesis