The following points highlight the chemical, physical and other factors affecting cytodifferentiation qualitatively and quantitatively.
(1) The chemical factors are: (a) Auxin (b) Cytokinin (c) Gibberellin (d) Effect of Sugars. (2) The physical factors are: (a) Light (b) Temperature (c) Pressure and (d) Water Stress. (3) The other factors are: (a) Morphactins (b) Methionine and (c) Irradiation.
(1) Chemical Factors:
Generally phytohormones and sugars influence greatly on Cytodifferentiation. It is evident that auxin, cytokinin and gibberellin are involved in the process of Cytodifferentiation.
Auxin at low concentration stimulates xylogenesis. There is an inverse relationship between the degree of xylem differentiation and auxin concentration. The influence of auxin in xylem differentiation has been demonstrated under in vitro by many workers.
Grafting a small vegetative bud on the upper surface of the callus tissue, it has been observed that, after few days, the differentiation of vascular tissue in callus tissue takes place below the bud (Fig 6.2(a)). This observation suggests that the stimulus of vascular tissue differentiation in callus tissue is provided by the vegetative bud.
Now, if the direct contact between the bud and the callus tissue is broken by placing a semi-permeable membrane at the site of the graft, still the differentiation of vascular tissue occurs. This experiment indicates that the nature of stimulus is of diffusible chemicals. Further, a piece of callus tissue is placed on a medium which do not favour the differentiation of vascular tissue.
The callus tissue is totally undifferentiated and is free from vascular elements. Now a V-shaped incision is made on the upper surface of the callus tissue and the V-shaped cavity is filled up with 1% agar block containing auxin and sucrose. The appearance of vascular tissue is again noticed in the callus issue (Fig 6.2(b)). But in the control set the callus tissue is without any vascular tissue. Therefore, the above experimental evidence clearly indicates that auxin plays an important role in Cytodifferentiation.
There are some evidences to put forward that cytokinin may also be involved in Cytodifferentiation. It has been demonstrated that kinetin added medium containing coconut water enhances tracheidal differentiation in the callus tissue of Nicotiana tabacum. The stimulatory effect of cytokinin in xylogenesis has also been observed in the cytoledonary callus tissue of soybean.
Nitsch and Nitsch (I960) on the other hand, have shown that storage parenchyma of Jerusalem artichoke tuber produced a natural cytokinin and cell division and Cytodifferentiation could be the outcome of a complex interaction between exogenous auxin and endogenous eyel of cytokinins.
Similarly, the exogenous cytokinins in combination with an auxin do markedly increase the quantity of tracheary elements has been shown by Bergmann. But in some cases, kinetin shows some inhibitory effect on xylogenesis e.g. Coleus stem callus, callus tissue of Helianthus and Linum. Therefore, the exact role of cytokinin is not clear and remains to be determined in several forms of tissue system.
The influence of gibberellin in cell division and xylem differentiation has been investigated under m vitro conditions in order to established some quantitative relationship. Gibberellin interacting with auxin is effective in cell enlargement and tracheidal differentiation but not when used alone.
For cultured pea root segments in auxin-cytokinin media, the percentage of xylem differentiation is rarely up to 20 in respect to total cell population, whereas auxin-cytokinin-gibberellin treatments increase the response giving a higher percentage of differentiated cell compared to other combinations. The hormonal interactions among the three and, in particular, of the role of gibberellin in presence of auxin or auxin-cytokinin in xylogenesis, needs further exploration.
d. Effect of Sugars:
The sugar, particularly sucrose, in the medium is very essential for Cytodifferentiation. Even the effect of auxin on vascular differentiation seems to be closely dependent on the presence of sugar in the medium. Sucrose as an energy source is very important in Cytodifferentiation. At lower levels of sucrose (1.5 to 2.5%) only xylem is formed and concentration above 4% favours a balance of xylem and phloem elements. Only disaccharides which contain an a-glucosyl radical at the non-reducing end induce vascularized nodules in callus as in Phaseolus.
As a carbon source, sugar serves a dual purpose- production and deposition of cellulose and concurrently of lignification of cell i.e. deposition of lignin in the lattices of the cellulosic micro fibrils in secondary walls during Cytodifferentiation of vessels and tracheids. Fructose, mannose, xylose, rhamnose, arabinose, galactose and mannitol (2%) have not shown any positive effect in vascular differentiation as compared to sucrose.
Xylem differentiation to some extent occurs with the incorporation of cellobiose, lactose-raffinose and glucose (2%). Maltose (2%) could partially replace sucrose in xylem and phloem nodule differentiation. Glucose used alone causes the development of scattered xylem elements. Soluble starch (4%) stimulates xylem formation.
It is interesting to note that only those carbohydrates which support significant cell division a so support tracheary element formation. With stem callus of Parihenocissus iricuspidata the tissue remains parenchymatous at lower concentration of sucrose (1%) in the medium, but with increase in its concentration (2.5%), the number of xylem arcs and the number of tracheary member in each are proportionally high and each arc is flanked by an internal cambium.
(2) Physical Factors:
There are very few reports of the effect of physical factors i.e. light, temperature etc. on vascular differentiation.
In general light has proved to be inhibitory in xylogenesis although in exceptional cases as in carrot, it can be a requirement but replaceable by cytokinin. The response to light varies depending on the nature and source of the tissue, being inhibitory in some and promotive in others.
The nature of vascular differentiation is influenced by temperature conditions-whereas high temperature (35°C) proves stimulatory to xylogenesis and formation of compact wood, as in Jerusalem artichoke (Hehanthus tuberosus), low temperature causes the development of undifferentiated new tissue.
Factors such as increased pressure have been shown to be stimulatory to xylem differentiation. Probably through induced ethylene production.
d. Water Stress:
Water stress is also a controlling factor in the initiation of experimentally induced xylogenesis in cultured explants of lactuca.
(3) Other Factors:
There are some other factors which play either positive or negative role for Cytodifferentiation.
Chlorflurenol inhibits xylogenesis in pith explants of lettuce possibly through inhibition of auxin transport. However, addition of cysteine to Chlorflurenol medium serves to nullify the inhibitory effect.
The culture medium supplemented with methionine (0.025 to 0.5 µM) enhances the induction of tracheid differentiation in majority of cases. It has been suggested that enhanced production of ethylene due to the use of methionine as a substrate, plays a role in xylem differentiation.
Tissues subjected to ionising radiations have shown enhanced xylem differentiation. However, studies on the effect of X-irradiation (4000r) on protein synthesis suggest irradiation might inhibit xylogenesis by disrupting the normal cellular biochemical pathway for protein synthesis as xylem differentiation is thought to be dependent on protein synthesis.