propagation by indirect regeneration · 2,4-d together with cytokinins is used primarily for callus...
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PROPAGATION BY INDIRECT REGENERATION
Organs are said to be regenerated indirectly when they are formed on previously unorganised callus, or in cell cultures.
Callus culture Callus is a coherent and amorphous tissue,
formed when plant cells multiply in a disorganised way.
It is often induced in or upon parts of an
intact plant by wounding, by the presence of
insects or microorganisms, or as a result of
stress
Induction of callus growth
An auxin is generally required for the induction of callus from explants.
Applied auxins seem to be capable of fundamentally altering the genetically programmed physiology of whole plant tissues, which had previously determined their differentiated state.
Cells, which respond to auxin, revert to a dedifferentiated state and begin to divide.
Which type of auxins induce dedifferentiate state in plant cells?
The auxin most frequently employed to initiate callus cultures is 2,4 D.
However, since cultures maintained on 2,4-D may become genetically variable, some investigators prefer NAA or IAA, or a transfer of callus to a medium containing one of these alternative compounds once it has been initiated by 2,4-D.
Examples of use of the auxins
2,4-D together with cytokinins is used primarily for callus induction and the formation and maintenance of suspension cultures, being replaced by NAA and IBA when morphogenesis is required.
NAA is favoured auxins for shoot culture.
2,4,5 trichlorophenoxyacetic acid (2,4,5-T) is used only rarely in tissue cultures, and then almost exclusively for the induction of callus and indirect embryogenesis in monocotyledons such as Avena, Oryza, and Panicum.
Explants from dicotyledons and gymnosperms
In most herbaceous broad leafed plants, it is possible to initiate
morphogenically competent callus cultures from explants derived
from many different tissues.
Leaf, stem or root segments, pieces of storage tissue (e.g. tubers),
seed embryos, shoot tips and seedling tissues have been used at
various times.
In many tree species, including gymnosperms, is frequently used
tissues near the vascular bundles or the cambium of stem or root
sections.
Explants from monocotyledons In most cereals, for example, callus
growth can only be obtained from
organs such as:
– zygotic embryos,
– germinating seeds,
– seed endosperm or the seedling
mesocotyl,
– and very young leaves or leaf
sheaths,
– immature inflorescences
but so far never from mature leaf
tissue.
Classification of callus
Colour
Green
White
Other
Degree of compaction
Friable
Compact
Morphogenetic potential:
Translucent, watery callus is seldom morphogenic,
whereas nodular callus frequently is
Degree of compaction
Auxins promote cell dispersion in suspension cultures while cytokinins tend to cause cell aggregation.
The relatively high levels of auxin added to liquid media to obtain dispersion will prevent morphogenesis, but might induce embryogenesis if the cells are still competent.
Soft callus was obtained from
leaves. Explants were collected
form 45 days old plants growing in
vivo condition.
S.lycopersicum
M82 ac. LA0722
Explants on growth medium
based on MS mineral formulation,
Sucrose 30 g/l, 2,4D (0,442
mg/l) and BAP (0,225 mg/l).
0 I II III
Callus stages
Sel 6
S.pimpinellifolium
Callus induction on some species of Solanum spp.
Explants on growth medium
based on MS mineral formulation,
Sucrose 30 g/l, 2,4D (0,442
mg/l) and BAP (0,225 mg/l).
Fasi di sviluppo del callo
Stages and age of callus: the
stage III is 60 days old
0 I
II III
Callus induction from fruit of Solanum lycopersicon
Stage II: propagation of callus
•Once a morphogenic callus has been
isolated, propagation is carried out either by
callus subdivision, or by the preparation of
cell suspensions.
•The success of each technique depends on
the subcultured tissues or cells continuing to
regenerate shoots.
Callus subdivision:Callus is cut into smaller pieces which
increase in size when subcultured in a
liquid or an agar-solidified medium.
The organogenic capacity of callus is
easily lost on repeated subculture.
Use of high growth regulator levels
can encourage the proliferation of
non-regenerative callus which will
displace tissues having the
competence to form new shoots
(e.g. in Pelargonium; Holdgate,
1977).
Genetic stability in indirect organogenesis
In some crop plants, the genetic differences between plants derived from callus and suspension cultures are considerable, and are sufficient to have attracted the interest of plant breeders as a new source of selectable variability.
Subsequent exposure to high levels of growth substances such as 2,4-D should also be avoided as far as possible.
Genetic stability of plants from highly competent callus cultures may be assisted by the continual presence of
superficial meristems.