INTRODUCTION 1 A callus consists of an amorphous mass of loosely arranged thin walled parenchyma cells arising from the proliferating cells of the cultured explants 2 Frequently as a ID: 913345
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Slide1
INITIATION AND MAINTENANCE OF CALLUS
Slide2INTRODUCTION
1. A
callus
consists of an
amorphous mass of loosely arranged thin walled parenchyma cells
arising from the proliferating cells of the cultured explants.
2. Frequently
, as a
result of wounding
, a callus is formed at the cut end of a stem or root.
3. The
term
“callus”
should not be confused with
“
callose
”,
another botanical term. The latter refers to a polysaccharide associated primarily with sieve elements.
4. The
stimuli involved
in the initiation of wound callus are the
endogenous hormones
auxin
and
cytokinin
.
Slide35. In
addition to
mechanical injury
, callus may be produced in plant tissues following an invasion by certain microorganisms or by insect feeding.
6. Using
tissue culture techniques
, callus formation can be induced in numerous plant tissues and organs that do not usually develop callus in response to an injury.
7. Plant
material typically cultured includes vascular cambia, storage parenchyma,
pericycle
of roots, cotyledons, leaf
mesophyll
, and
provascular
tissue. In fact,
all
multicellular
plants are potential sources of explants
for callus initiation.
Slide4FIRST SUCCESSFUL CULTURE
1. In
1939
the first successful prolonged cultures of experimentally induced callus were achieved almost simultaneously at the research laboratories of
Gautheret
in Paris,
Nobecourt
in Grenoble, and
White
in Princeton.
2. These
cultures were originally derived from
explants of cambial tissue of carrot and tobacco
.
3. The
term
“tissue culture”,
as applied to such cultures, is a
misnomer
: A cultured tissue does not maintain its unique characteristics as a plant tissue, but reverts to a
disorganized
callus.
4. The
most important characteristics of callus, from a
functional viewpoint
, is that it has the potential to develop
normal roots, shoots, and
embryoids
that
can form plants
and, in addition, can be used to initiate a
suspension culture
.
Slide5ESTABLISHMENT OF A CALLUS
1
. Establishment
of a callus
from an
explant
can be divided roughly into
3 developmental stages
:
A. Induction
B. Cell
division
C. Differentiation
2. During
the initial induction phase
metabolism is stimulated prior to mitotic activity
. The length of this phase depends on the physiological status of the explants cells as well as the cultural conditions.
3. Afterward
, there is a phase of
active cell division
as the explants cells revert to a
meristematic
state
.
4. The
third phase involves the appearance of
cellular differentiation
and the expression of certain metabolic pathways that lead to the formation of secondary products.
Slide6GROWTH CHARACTERISTICS OF A CALLUS
1. Growth
of a callus involve a complex relationship
among
A.
The
plant material
used to
initiate
the
callus
B.
The
compostion
of the medium
C.
The
environmental
conditions during the incubation period
2. Some
callus growths are heavily
lignified and hard
in texture, whereas others break easily into small fragments.
3. The
fragile growths that crumble readily are termed
“friable cultures”.
Slide7PIGMENTATION
1. Callus
may appear
yellowish, white, green, or pigmented
with
anthocyanin
.
2. Pigmentation
may be uniform
throughout the callus or some regions may
remain un pigmented
.
3.
Anthocyanin
synthesizing and –
nonsynthesizing
cell lines
have been isolated
from carrot cultures
and a stable pigment producing strain of cultured
Euphorbia
sp. cells
was isolated after 24
clonal
selections and subcultures.
Slide8ANATOMY OF CALLUS CULTURES
1. A
homogenous callus
consisting
entirely of parenchyma cells is rarely found.
2.
Cytodifferentiation
(The morphological development of undifferentiated cells into more
specialised
ones) occurs in the form of
tracheary
elements, sieve elements,
suberized
cells,
secretory
cells, and
trichomes
.
3. Small
nests of dividing cells form vascular nodules
(
meristemoids
“A small, triangular
stomatal
precursor cell that
functions temporarily as
a stem cell in
a
meristem
”)
that may become centers for the formation of
shoot apices, root primordial, or incipient embryos.
Slide94.
Vascular
nodules
typically consist of discrete zones of
xylem and phloem separated by a cambium.
5. The
orientation of the xylem and phloem
with respect to the
cambial zone
is influenced by the
nature of the original tissue
.
6. The
location of the nodules
within the callus
can be modified
by
altering the composition of the medium.
7. Vascular
differentiation may also take the form of somewhat randomly arranged strands of
tracheary
elements.
Slide10HORMONAL REQUIREMENTS
1. The
hormonal requirements
for the initiation of callus depend on the
origin of the explants
tissue.
2.
Juice
vesicle from lemon fruits
, and explants containing cambial cells, exhibit callus growth
without the addition of any exogenous growth regulators.
3.
Most
excised tissues
, however, require the addition of one or more
growth regulators
in order to initiate callus formation.
4. Explants
can be classified according to their exogenous requirements, in the following manner:
A.
Auxin
B.
Cytokinin
C.
Auxin
and
Cytokinin
D. Complex
natural extracts.
Slide11SUBCULTURING OF THE CALLUS
1. After
the callus has been grown for a while in association with the original tissue, it becomes
necessary to subculture
the callus to
a
fresh
medium
.
2. Growth
on the same medium for an extended period will lead to a
depletion of essential
nutrients and to a gradual
desiccation
of the gelling agent.
3. Metabolites
secreted by the growing callus may
accumulate to toxic levels
in the medium.
4. The
transferred
fragment of callus
must be of a
sufficient size
to ensure renewed growth on the fresh medium.
5. If
the transferred
inoculum
is too small
, it may exhibit a very slow rate of growth or none at all.
6. Street
(1969) recommended that the
inoculum
be
5-10mm in diameter and weigh 20-100mg.
Slide127. Successive
subcultures are usually performed every
four to six weeks
with cultures maintained on an agar medium at
25
o
C
or above
.
8.
Passage
time
, however, is somewhat variable and
depends on the rate of growth of the callus.
9.
A
friable callus
can be subdivided with a thin spatula or scalpel, transferred directly to the surface of a sterile Petri dish, and sliced into fragments with a scalpel.
10.
Only
healthy tissue
should be transferred, and
brown or necrotic tissue must be discarded.
11. Interest
has been shown in developing alternative methods for long term maintenance of tissue cultures, for example,
freeze preservation
.
Slide13WHAT
ARE SOME OF THE BEST PLANT MATERIALS FOR THE INITIATION OF A CALLUS CULTURE?
1.
Young
healthy tissue
that are
rich in nutrients
, and possibly
endogenous hormones
, are the best choices for the induction of cell division; for example,
storage organs and cotyledons of seeds.
2. These
include tissues from
potato tuber
(
Solanum
tuberosum
),
storage roots of turnip
(
Brassica
rapa
),
sweet potato
(
Ipomea
batatas
),
and carrot
(
Daucus
carota
).
3. Callus
is easily started from the
cotyledons of
soyabean
(
Glycine
max
).
4.
Stem
pith parenchyma
from
lettuce
(
Latuca
sativa
) and
tobacco
(
Nicotiana
tabacum
) readily divides in the
presence of
auxin
and
cytokinin
.
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