INITIATION AND MAINTENANCE OF CALLUS - PowerPoint Presentation

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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

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

.

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

.