Callus Tissue: Morphology, Internal Structure and Characteristics (With Diagram)

Let us make in-depth study of the morphology, internal structure and characteristics of the callus tissue.

Morphology:

Callus tissue proliferates as amorphous mass of cells having no regular shape. So, it is very difficult to describe its external morphology.

Ap­parently, all callus tissue derived from different plants looks alike i.e. hazardous mass of cells, but they can be distinguished on the basis of other characteristics such as texture, coloura­tion, hormone requirements etc. On that basis, even callus tissue initiated from explants (such as stem, leaf, root, petiole, flower etc.) of the same plant species may show considerable variations.

Internal Structure:

Internal structure of the callus tissue is re­vealed by light microscopy and electron micros­copy.

Light Microscopic Study:

Microtome section or squash preparation of the callus tissue shows that the cellular com­position of the callus tissue is extremely het­erogenous ranging from small cells with dense cytoplasm to large cells with vacuolated cytopla­sm. The shape of the cells within the callus tis­sue varies from spherical to markedly elongate (Fig 3.2).

Large elongated cells are generally non-dividing cells having a large central vacuole whereas the small actively dividing cells are with reduced vacuole size and dense cytoplasm. Elon­gated cells within the callus tissue may differenti­ate into lignified xylem tracheids or phloem-like cells.

Formation of xylem and phloem within the callus tissue is known as cytodifferentiatiori. It has also been observed that some groups of meristematic cells constituting the active loci de­velop some small nests, scattered throughout the callus tissue. These are called meristemoids. The meristemoid may differentiate into either shoot or root primordia or embryoids.

Electron Microscopic Study:

Electron microscope also reveals that the cells at their non-dividing state have a large cen­tral vacuole and thin peripheral cytoplasm. The number of organelles is minimal in the cyto­plasm. The synthesis of new cytoplasm and re­duction in size of the central vacuole takes place when the cells enter into the dividing state.

The­re is an increase of the endoplasmic reticulum, mitochondria, Golgi bodies and ribosome. The endoplasmic reticulum occurs as sheets running parallel with the cell wall. The ribosome occurs as polyribosome groups. The cell wall of the actively growing cells is thin. Sometimes cell walls develops irregular cellulosic thicken­ings which projects inside the cytoplasm.

Micro­tubules are associated with the walls of actively growing cells. Cells from the green coloured zone contain chloroplasts, but generally the internal system of the grana is poorly developed. More frequently the plastid acts as amyloplasts. Cul­tured cells contain membranous myelin-like bod­ies and membrane enclosed groups of vesicles which are known as multi-vesicular bodies.

Characteristics:

Texture:

Callus tissue can vary considerably in ap­pearance and texture. On the basis of texture, callus tissue can be divided into two categories such as soft callus and hard callus. Soft callus is friable in nature and is made of heterogenous mass of cells having minimal con­tact (Fig 3.3). On the other hand, hard cal­lus consists of giant cells, tracheid-like cells and closely packed cells i.e. compact in nature (Fig 3.3b). Hard callus may be nodular in form.

Colouration:

Generally callus tissue is creamish yellow or white in colour. Sometimes callus tissue may be pigmented. Pigmentation may be uniform or patchy. Callus tissue may be green in colour. Sometimes, white callus tissue grown under dark condition turns green after transferring in light condition. Green colour develops due to develop­ment of chloroplastid in the cells of callus tissue e.g. callus tissue from the cotyledons of the soy­a bean.

Callus may be yellow possibly due to syn­thesis of carotenoid pigments, e.g. callus tissue of Nigella sativa grown under dark condition. In some cauliflower cultures, callus tissue is purple in colour due to accumulation of anthocyanin in vacuoles or due to production of oxidized form of DoPA (3, 4 dihydroxy phenylalanine).

Brown colour frequently develops in the ex- plant and subsequently in the callus tissue. This is mainly due to excretion of phenolic substances. Plant tissues contain large number of phenolic compounds and also contain polyphenol oxidase remaining spatially separated from the phenols.

When the plant tissue is cut during explant pre­paration, the enzyme comes in contact with phe­nols which are then oxidized to quinones. These quinones then subsequently polymerize to form brown products. The formation of such quinones is responsible for the browning of the callus tis­sue. Excretion of phenols generally inhibits the growth of the callus tissue.

Habituation of Callus Tissue:

Generally, callus tissue needs growth hor­mones in the nutrient medium in order to grow as long as they are maintained through serial sub­cultures. But it has been observed that the callus tissue in some plant species may become habit­uated after prolonged culture.

This means that the callus tissue is able to grow on a standard maintenance medium or basal medium which is devoid of growth hormones. This property of the callus tissue is known as habituation and the callus tissue is known as habituated callus tissue. The actual cause of habituation is not fully known.

But it has been suggested that the cells in habituated callus tissue appear to have developed the capacity to synthesis ade­quate amount of auxins and/or cytokinins which probably accounts for their independence of exogenously supplied hormones. Habituated callus tissue cannot be distinguish from the normal cal­lus tissue except in their hormone requirement.

The plant tumour tissue can be isolated from the plant and cultured aseptically. In cul­ture, the tumour tissue is capable of growing on simple basal medium (i.e. hormone-free)-like the habituated callus. These tissues differ from the habituated callus in their mode of origin. In case of wound tumour, the virus remains and multi­ple within the cells and may disappear after pro­longed periods in culture.

Crown gall tumour tissues are made free from bacteria artificially for culture. Otherwise the microorganisms soon overgrow the cells in culture. Alternatively, secondary tumours can be cultured directly. Presence of bacterial DNA in the genome of the crown gall tumour cells pos­sibly makes them hormone-independent.

Chromosomal Variation in Callus Tissue:

Chromosomal variation may occur geneti­cally or epigenetically in the cells of the callus tissue.

(a) Genetical basis of Chromosomal Variation:

Callus tissue is obtained from root, shoot, leaves and other organs. These organs are made of numerous cells which remain in dif­ferent states of differentiation. Normally, in vivo meristematic diploid cells undergo selective division for the growth of an or­gan. On the other hand, endoreduplication is of frequent occurrence in the differtiated tissues of higher plants and the endoreduplicated cells remain in mitotically blocked condition.

The degree of endoreduplication depends upon the degree of cellu­lar differentiation. Therefore, the genomic constituent is heterogeneous in original ex- plant. Callus tissue may get such genomic heterogeneity possibly due to non-selective induction of asynchronous division of both diploid and endoreduplicated cells.

So, the pre-existing genomic heterogeneity of explant may be a source of chromosomal variation in the callus tissue. Variation of chromosome number ranges from aneuploidy to different level of polyploidy, such as tetraploid, hexaploid and so on. Occur­rence of both diploid and different level of polyploid cells in the same callus tissue is known as mixaploid cell population.

(b) Epigenetic basis of Chromosomal Variation:

It has also been observed that at the early stage of callus growth, the percentage of diploid cell is generally higher than the per­centage of polyploid cells. The number of polyploid cells may increase or decrease through serial subcultures. Again, highly meristematic cells are expected to be all diploid. But the callus tissue derived from meristem also shows the variation in chro­mosome number.

Crepis is a plant in which cellular differentiation occurs without endoreduplication. Callus tissue obtained from the explant of such plant shows that all cells are not diploid. Thus, it appears that variation in chromosome number is not always derived from original explant.

But it may come from the interaction of both genome and cytoplasm. Their interaction may bring about mitotic disturbance. In culture of pea, J. G. Torrey showed that kinetin encouraged the development of polyploid cells in culture. There is another common observation that strong auxin-like 2,4-D induces the polyploidy in callus cul­ture.

It is also found that prolonged subculture may lead to the establishment of the one kary­otype and others are gradually eliminated. In most of the cases polyploid cells are found. Therefore, whatever may be the cause of chro­mosomal instability; in fact, it is more or less a common cytological feature in most of the cul­ture.

Sometimes, structural changes of chromo- some-like deletion, translocation etc. may oc­cur in culture. Gould (1982) used C-banding technique to show that three years old culture of Brachycoma dischromosomatica (2n = 4) was pseudodiploid. There are also many other ex­amples where structural change of chromosome occurs. An ideal callus culture is characterised by the possession of numerical or structural stabil­ity in long term culture. But it is very rare. Cells of the callus tissue may be haploid if it is derived from microspore culture.