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Nitrate ions are very important ions in plants because they are essential for the synthesis of chlorophylls, cytochromes, nitrogen bases (purines and pyrimidines), proteins, anthocyanins, nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in plants for healthy growth. Despite this, nitrate ions cannot be used by the plants directly. It is first converted into ammonia before being incorporated into organic compounds by a process of nitrate assimilation. It is a reverse process of nitrification. Nitrate assimilation process is completed in two steps. In the first step, the nitrate ion is reduced to nitrite ion by an enzyme called nitrate reductase. This enzyme is a flavoprotein and contains molybdenum. It takes place in the cytoplasm of plants. In the second step, nitrite ions are then reduced to ammonia by an enzyme which is called nitrite reductase. Therefore, nitrate reductase is one of the most important enzymes in the assimilation of exogenous nitrate, the predominant form of nitrogen available to green plants growing in soil. Activity of this enzyme in plants gives a good estimate of the nitrogen status of the plant and is very often correlated with growth and yield. (Srivastava, 1980). The term ‘antimutagen’ was used originally to describe those agents which reduce the frequency or rate of spontaneous or induced mutation independent of the mechanism involved (Novick and Szilard, 1952). Studies on antimutagenic factors (about 200 compounds) were initially carried out in the 1950s in the field of microbial genetics (Clarke and Shankel, 1975). In the present study, antimutagenicity of vitamin-E on the nitrate reductase activity have been determined against in calli raised from diethyl sulphate treated seeds of Trigonella foenum-graecum L.

The testing of antimutagen (vitamin-E) in in vitro plant culture using enzymatic activity (Nitrate reductase activity) as parameters was investigated for the first time. Nevertheless, testing of mutagens and antimutagens in T. foenum-graecum L. have been investigated by few researchers (Rawat and Mahna 2001, Rawat and Bhati, 2013, Rawat, 2019 and Rawat, 2021). According to Stavric (1994) antimutagenic compounds can act at cellular level by enhancing the activities of enzymes involved in detoxification of mutagens, inhibiting the activities of enzymes involved in formation of mutagens metabolites, trapping of electrophiles, scavenging reactive oxygen species, inhibiting metabolic activation and protecting nucleophilic sites of DNA. Sharma et al., (2016) reported that in vitro antimutagenic effect on aqueous extract/fraction of Parkinsonia aculeata L. (Fabaceae) against 4-Nitro-o-phenylenediamine, sodium azide (direct acting mutagens) and 2-Aminofluorene (indirect acting mutagen) in TA98 and TA100 strains of Salmonella typhimurium by employing Ames assay and DNA nicking assay. Leaves of P. aculeata L. exhibited significant DNA protecting property against FR and antimutagenic activity against promutagen and direct acting mutagens in TA98 and TA100 strains of S. typhimurium in a dose dependent manner.

Table-1. Nitrate reductase activity of calli raised from seeds treated in DES alone treatments and combinations (post-treatments) with vitamin-E: -

 

 Tabulated ‘r’ for 3 d.f. at p=0.05 is 0.878       

Tabulated ‘r’ for 2 d.f. at p=0.05 is 0.950

Reported values are mean ±SE of 3 replicates

* Non-Significant 

r= Karl Pearson’s coefficient of correlation.   

Dry pure line viable seeds of T. foenum‐ graecum L. were surface sterilized with 0.1% (w/v) mercuric chloride (HgCl2) solution for 3 min. The seeds were thoroughly washed with sterilized distilled water so as to remove the traces of mercuric chloride and were pre-soaked in distilled water for 4 h at 25±10C. The experiments were designed to have the following three sets‐ 1. Control or Untreated‐ In this set, some of the pre-soaked seeds were kept in distilled water for 8 h at 25±10C. 2. Treated with diethyl sulphate (DES; pH 7.41) alone‐ In this set, some of seeds from control (untreated) were treated with freshly prepared three concentrations of DES (0.25%, 0.50% and 0.75%), prepared in water, for a period of 8 h at 25±10C. 3. Post‐treated with vitamin-E‐ In this set, some of the DES treated seeds (with 0.50% concentration level of DES) were post‐treated with freshly prepared two alcoholic concentrations of vitamin-E (0.25% and 0.50%) separately, for a period of 8 h at 25±10C. For each set, 30 seeds were used and were replicated thrice. Seeds of all the experimental sets were transferred to the flask containing 30 ml of MS plant tissue culture media (Murashige and Skoog, 1962) for raising the calli and were allowed to grow for four weeks under in vitro conditions. After four weeks, brown and dark tissues were removed from the explants and calli were sub-cultured on freshly prepared MS media and analysis the antimutagenic effects of vitamin-E on nitrate reductase activity in calli raised from DES mutagenized seeds of T. foenum-graecum L. Nitrate reductase activity was estimated by using the method of Scholl et al., 1974.

The values presented in the Table-1, revealed that DES treatment series (0.25%=0.315; 0.50%=0.170; 0.75%=0.156) produced a negative effect in all concentration levels as compared to the control (0.308). With the except in 0.25% DES treated series where a positive effect was recorded. In present study, one concentration level of DES, i.e., 0.50% alone was used for the treatment of T. foenum-graecum L. seeds which resulted in 0.170±0.29 (h/250mg/fw) nitrate reductase activity as compared to 0.308±0.35 (h/250mg/fw) in the untreated used as a control. Marked elevation in nitrate reductase activity were observed in combination with vitamin-E post-treated series, but the improvement was not gradual. Post-treatment of DES treated T. foenum-graecum L. seeds with two concentration levels of vitamin-E (0.25% and 0.50%) showed promotary effects on the nitrate reductase activity which it was recorded 0.175±0.36 (h/250mg/fw) and 0.172±0.38 (h/250mg/fw) for the two concentration levels of vitamin-E, respectively. Maximum activity in the nitrate reductase activity (0.175±0.36 (h/250mg/fw) was observed in 0.50% DES+0.25% vitamin-E post-treated series and minimum activity in the nitrate reductase activity (0.172±0.38 (h/250mg/fw) was observed in 0.50% DES+0.50% vitamin-E post-treated series as compared to 0.308±0.35 (h/250mg/fw) in the untreated used as a control.

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