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ORGANOGENESIS
 Plant regeneration by tissue culture techniques can be
achieved by either zygotic embryo culture, somatic
embryogenesis, or organogenesis.
 Organogenesis is employed in micropropagation from bud
and shoot material and in organ production from callus
and suspension cultures.
 Roots, shoots and flowers are the organs that may be
initiated from tissue cultures.
 Embryos are not classified as organs because these
structures have an independent existence, that is, embryos
do not have vascular connections with the parent plant
body.
FACTORS AFFECTING ORGANOGENESIS
 Organogenesis involves the interplay of a host of
factors:
1. Donor plant growth
2. Source of the explants
3. Culture medium
4. Supplements of growth regulators
5. Environmental conditions
EFFECT OF CHEMICALS
 The first major breakthrough came with the discovery
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that in vitro organogenesis in tobacco cultures could be
chemically regulated.
The addition of auxin to the medium served to initiate
root formation, where as shoot initiation was inhibited.
The inhibition of shoot formation can be partially
reversed by increasing the concentration of both sucrose
and inorganic phosphate.
Later it was found that adenine sulfate was active in
promoting shoot initiation, and this chemical reversed
the inhibitory effect of auxin.
The studies of Skoog’s group led to the hypothesis that
organogenesis is regulated by a balance between
cytokinin and auxin.
A relatively high auxin:cytokinin ratio induced root
formation in tobacco callus, where as a low ratio of the
same compounds favored shoot production.
EPIDERMAL AND SUBEPIDERMAL EXPLANTS
 The most precise regulation of organ formation has been
achieved with epidermal and subepidermal explants
consisting of a few cell layers in thickness.
 The formation of floral buds, vegetative buds and roots
have been demonstrated in thin cell –layer explants of
several species by regulating the auxin:cytokinin ratio,
carbohydrate supply, and environmental conditions.
 Primary explants consisting of three to six layers of
epidermal and subjacent collenchymas removed from the
region of the leaf midvein of Begonia rex produced shoots
or roots from the epidermal cells.
 Recent studies have shown that cell wall oligosaccharides
influence organogenesis in thin cell layer explants of
tobacco.
 The presence of plant growth regulators, pH and the ionic
environment are thought to be involved in the activation
of certain hydrolytic cell wall enzymes that, in turn,
release biologically active oligosaccharides.
OTHER GROWTH REGULATORS
 Gibberellins tend to suppress both root and shoot
initiation in cultures.
 Endogenous ethylene may be a factor in shoot initiation.
 One report indicates that ethylene blocks the early stages
of organogenesis, but enhances the further development
of primordia.
 Endogenous ethylene was identified as a factor in bud
induction arising from cultured tobacco cotyledons and
Lilium bulb.
ENVIRONMENTAL FACTORS
 Cultured explants are typically incubated in the dark for the
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initiation and subsequent development of callus, although low
–level illumination may be beneficial.
A light requirement has been reported for adventitious bud
formation in hairy roots of horse radish (Armoracia
lapathifolia).
The roots had been inoculated with Agrobacterium rhizogenes.
The hairy roots produced buds on a hormone – free medium in
the presence of red light, but not far-red light.
Thus phytochromes appeared to be involved in this
phenomenon.
Excised roots from non transformed plants did not exhibit this
response.
DICOT AND MONOCOT
 In comparison to dicots, monocot cultures are more difficult
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to regenerate.
For plantlet regeneration in many dicot callus cultures, the
callus is removed from the maintenance medium and sub
cultured on a shoot-induction medium.
Medium has a cytokinin : auxin ratio in the range of 10:1 to
100:1,in many cases by supplementing the medium with
cytokinin as the sole growth regulator.
Exogenous cytokinin may be unnecessary for the initiation
of shoots in monocot cultures.
The omission of auxin from the maintenance medium may
be sufficient to induce shoot formation, and two successive
transfers on auxin-free media have been recommended.
 Root initiation frequently occurs spontaneously after the
culture has initiated buds, and shoot development
undoubtedly alters the endogenous hormones within the
culture.
 Regenerated shoots are transferred to a root-inducing
medium. Auxin alone or in combination with a low level of
cytokinin will enhace root primordial formation.
 There is some evidence that phenolic compounds may act
with auxin to promote rooting.
 For example the combination of phloroglucinol with
indolebutyric acid was more effecting rooting than auxin
alone.