Download Initiation and maintenance of callus

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 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.
5. 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.
FIRST 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.
ESTABLISHMENT 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.
GROWTH 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”.
PIGMENTATION
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.
ANATOMY 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.
4. 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.
HORMONAL 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.
SUBCULTURING 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.
7. Successive subcultures are usually performed every
four to six weeks with cultures maintained on an agar
medium at 25 oC 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.
WHAT 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.