In this article, we will discuss about the meaning of cytodifferentiation, primary steps in cytodifferentiation, protocol for the study of cytodifferentiation and conclusion.
What is the Meaning of Cytodifferentiation?
In plant tissue culture, during growth and maturation of the callus tissue or free cells in suspension culture, few dedifferentiated cells undergo cytoquiescence and cytosenescence and these twin phenomena are mainly associated with differentiation of vascular tissue, particularly tracheary elements.
The whole developmental process is termed as Cytodifferentiation.
Primary Steps in Cytodifferentiation:
The fate of an individual cell in culture is variable and hence unpredictable. Amongst a group of cells within the callus tissue or free cells in cell suspension culture, a few cells become morpho-genetically competent for Cytodifferentiation which cannot be identified at the early stage in advance. Cytodifferentiation occurs either spontaneously or under the stimulus of specific nutritional or hormonal factors. So, it is not conditioned by a single regular event.
Preliminary steps in Cytodifferentiation are reflected by a series of histological and biochemical changes in the cell. On the basis of numerous observations and publications, the proposed fate of the cultured cells during Cytodifferentiation can be divided into three steps. The callus tissue at the time of its initiation and further growth shows a mixed population of small, more rounded oval and few elongated cells with dense cytoplasm.
With an increase in subculture number, few cells become more elongate with a thick wall and the calli are friable. By further increasing the subculture number, callus tissue shows maximum xylogenesis with tracheary elements having a continuous spiral deposition of secondary wall materials (Fig 6.1 A-C). Actually, the initiation of xylogenesis takes place from mitotically blocked and elongated cells.
In ultra-structural studies of Cytodifferentiation, it is evident that a chain of intracellular degradative changes are associated with Cytodifferentiation. Auto-destruction of cellular organelles such as chloroplast, endoplasmic reticulum, dictyosomes, ribosomes and mitochondria leading to loss of entire protoplasmic mass are the main of the degradative changes.
The separation of bounding membrane of organelles are the first step in cytoquiescence leading to cytosenescence. These auto-phagic activity within the cell in turn is closely linked with certain hydrolytic enzymes. Acid phosphatase, a hydrolytic enzyme, is commonly present in the cell and has been detected in association with the cell wall, distycome, plastids and lysosomal systems.
It is evident that the synthesis of acid phosphatase is indicative of autolysis of the protoplast during cytoquiescence and cytosenescence of the cell. Therefore, the transformation of living cells into the dead, empty tracheid during cellular differentiation and the biosynthesis of the acid phosphatase enzyme are functionally related to the autolysis of the cell contents and lignin biosynthesis for spiral deposition of secondary wall materials of the developing tracheary elements.
Protocol for the Study of Cytodifferentiation:
Actually there is no specific methodology for the study of Cytodifferentiation. During the maintenance of callus culture, or when the callus tissue is transferred in another medium for plant regeneration, differentiation of xylem elements may be frequently observed in the squashed preparation of the callus tissue grown in particular medium. Such medium with some modification can be used as medium for Cytodifferentiation of a particular plant material.
Differentiation of xylem element in the callus tissue of Cowpea (Vigna unguiculata) is described below:
1. The hypocotyl portion of aseptically grown seedling of Vigna unguiculata can be used as initial explant.
2. The explant is cultured in Murashige and Skoog’s medium supplemented with 2, 4-D (2-4 mg/L) and kinetin (0.5 mg/L) at 25°C with 16 hrs. light.
3. Cultures are maintained in a serial subcultures with 28 days passage duration.
4. Time to time, callus tissue are harvested and macerated in 4% aqueous solution of NaOH at 50°C. This treatment clears and softens the tissue.
5. NaOH solution is carefully replaced by 0.04% aqueous solution of safranin.
6. After 30 minutes, the dye solution is replaced by IN HCL at 50°C.
7. After one hour, HCL is removed and glycerol is added. The acid destains the parenchyma cells but the lignified xylem retains the red dye.
8. Finally, a slide is made and observed under microscope.
There are still some lacuna in our understanding of the cellular and biochemical aspects of Cytodifferentiation. Tissue culture techniques offer not only an excellent opportunity to study the factors that elicit the Cytodifferentiation but also allow investigation of factors controlling the differentiation of tracheary elements.