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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:
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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.
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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.
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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.
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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.
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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.
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(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.
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(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.