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.