44. In vitro screening of the effects of antimitotics on differentiating rice tissues

A number of antimitotic chemicals have been used for induction and selection of aneuploids and/or haploids in a variety of microbial systems, i.e., p-fluoro- phenylalanine (FPA) which is an amino acid analogue (Lhoas 1967; Day and Jones 1971; Verma and Sinha 1973), chloral hydrate which is an aliphatic alcohol (Singh and Sinha 1976; Sinha and Jha 1985) and griseofluvin which is an antifungal compound (Sinha and Jha 1985). But this line of studies are few in higher plants. Out of many antimitotics so far reported, FPA only has been studied as to its effect on higher plants. This antimetabolite has been found to change the level of ploidy in some angiosperms, namely, Nicotiana tabacum (Gupta and Carlson 1972), Allium cepa (Sinha and Bhojwani 1976), Oryza punctata (Niizeki 1977), Oryza alta (Niizeki and Fukui 1978, 1983), Festuca X Lolium (Nitzsche 1980) and Zea mays (Banks et al. 1982). However, most of these studies have been confined to cyto-morphological analysis, and the underlying mechanisms have not been explored. The changes in different metabolic processes affecting various macro-molecules have not been investigated. In this note, we report the effects of two other antimitotics in addition to FPA, chloral hydrate and ethionine, on the growth and metabolism of in vitro reared explants (root, shoot, endosperm and embryo) of rice (Oryza sativa L., Indica varieties, Sita and Rajendra Dhan 201). The levels of different macro-molecules were monitored in order to have an insight into the mode of action of these antibiotics.

The growth and regeneration of cultured root, shoot, endosperm and embryo explants were inhibited when allowed to differentiate on MS medium (sucrose 3% w/v; agar 8% w/v; pH 5.8) containing different concentrations of chloral hydrate (82.7, 165.4 and 827 mg/1), FPA (25, 50 and 100 mg/1) or ethionine (5, 10 and 15 mg/l; Sharma and Sinha 1988). Growth retardation was obvious from decreases in fresh and dry weights of the treated calli and also from the decreased elongation of roots and shoots differentiated from treated embryos. This suggests a decrease in the rate of cell division during their growth and morphogenesis. Chlorophyll was adversely affected and the differentiated plantlets became pale- yellow to whitish, and finally black in 21 days of incubation. The extent of growth inhibition depended on the chemical used and its concentration. Of the three chemicals, ethionine was most effective. The higher the concentration, the more pronounced the adverse effects leading to necrosis and curling of roots and shoots.

Morphological changes induced by these antimitotics were also manifested at the macro-molecular level. These chemicals brought about a marked reduction in the total activity of peroxidases. Such macro-molecular alterations result in retarded growth of treated embryos. Decreases in the amount of proteins and carbohydrates indicate an overall disturbance in metabolism. The decrease in peroxidase activity would reduce the rate of oxidation of IAA, increasing its indogeneous level. The anomalous and retarded growth of treated embryos could be attributed to induced hormonal imbalance. Curling of roots and shoots would take place on account of unequal rates of cell division along their axes. Changes in total DNA were also observed. This suggests that changes in ploidy have taken place in the treated tissues. We are now engaged in exploitation of the use of antimitotic chemicals in tissue culture of rice.

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