Effect of chemical mutagens on growth of Okra (Abelmoschus esculentus L. Moench)

The purpose of the present study was to evaluate the effects of mutagens i.e. Ethyl methanesulfonate (EMS) and Sodium azide (SA) on the morphological growth of Okra. Seeds of two Okra varieties Subzpari and Pahuja were first pre-soaked in distilled water and then treated with 0.0 (Control), 0.1, 0.2 and 0.3% Sodium azide (SA) and Ethyl methanesulfonate (EMS) separately. The results showed that the germination rate was significantly decreased with increased lethality (%) and mutation frequency (%) in M1 of both varieties with increasing concentrations of EMS and SA against control. The results showed that different concentrations of EMS and SA affected morphological attributes of both okra varieties as the increase of mutagen concentration showed variable results in M1 generation compared with control, but M2 generation showed useful improvement in various traits. Such as the M2 generation of Subs pari from 0.1% SA treatment showed a decrease in flowering time while an increase in the number of fruits per plant, fruit size, seeds yield (g) per plant and 100 seeds weight.

, mutations occur in the eukaryotic genome as 10 -8 base pair per generation. Mutations can be induced artificially by using physical and chemical mutagens. Induced mutations have been used as an important tool for the improvement of certain traits in the existing germplasm. After Muller [10] discovered the mutagenic effects of X-rays on the fly, induced mutations were used for plant breeding. Ethyl Methanesulfonate is a carcinogenic organic compound, a popular and powerful mutagen that produces random point mutations in the genomes through nucleotide substitutions [11,12]. Therefore, okra is of high nutritional value with medical applications and economic importance hence needs to improve the production, quality and quantity of fruit. Taking into account the importance of okra, the present study was done with the objectives of evaluating the germination and growth attributes of M1 and M2 generations okra through the treatment of sodium azide (SA) and ethyl methane sulfonate (EMS) under different concentrations.

Materials and methods
Seeds of two varieties of Okra (Pahuja and Subzpari) were obtained from registered seed companies, Hyderabad, Pakistan. The experiment was performed during the year 2015-16 at IBGE, University of Sindh, Jamshoro, Pakistan. Treatment of Okra seeds For chemical mutagenesis, 0.1% (0.5g/50ml), 0.2% (1.0g/50ml) and 0.3% (1.5g/50ml) solutions of ethyl methane sulfonate (ACROS) and sodium azide (ACROS) were prepared separately. For each treatment, 250dry, healthy and uniform sized seeds of both okra varieties; Pahuja and Subzpari were initially presoaked in 100 ml distilled water in the lab at room temperature for six hours to ensure complete hydration. After removing the excess water presoaked 60seeds of each Pahuja and Subzpari varieties were submerged in 50 ml solutions containing sodium azide and ethyl methane sulfonate separately with shaking (70rpm) in the dark for 60 minutes at room temperature [13]. After chemical treatment, seeds were washed with tap water and were sown in pots (5 seeds/pot) containing 10kg of sand and clay (1:1 ratio). For control, untreated seeds were soaked in distilled water for seven hours. The experiment was repeated twice and the total 120 seeds were used for each treatment. For M2 generation study, mature, dry and uniform seeds of each treatment and control obtained from M1 generation were grown in pots under the same soil conditions as in M1 generation. The experiment was performed two times, thus a total 120 seeds for each treatment in the M2generation study. The mutagenic effect of ethyl methanesulfonate (EMS) and sodium azide (SA) on M1and M2 generations of both okra varieties was assessed. Various growth attributes including germination (%), shoot and root lengths, stem diameter, number of branches, flowering time, number of fruits, fruit length, seeds per fruit and 100 seeds weight were observed. The germination of seeds was observed after every week for three weeks and the germination percentage was calculated after three weeks by the formula as follows: The shoot length of each plant was measured after 30 days of sowing and expressed in cm while root length was measured after harvesting of fruits and expressed in cm. The shoot diameter was measured on maturity time while number of branches was counted at maturity. The fruit size was measured at maturity, number of fruit per plant, number of seeds per fruit and 100 seeds weight was determined.
The experiment was performed according to the Randomized Complete Block Design (RCBD) manner. The collected data were expressed as the mean of triplicates ± standard deviation (SD) which was calculated.
S=sample standard deviation N=the number of observations =the observed values of a sample item ̅ =the mean value of the observations Results and discussion Germination of seeds is a key parameter used to evaluate the impact of the mutagen on plants. The inhibition of seed germination after the treatment of seeds with various mutagens is a very effective method to study the effects of mutagens on plants.
The germination of seeds was found to be the highest in control plants as compared to other treated plants during M1 and M2 generations which showed that mutagenic treatments inhibited the germination in both varieties. The results revealed that germination was significantly reduced as the dose of mutagen was increased. The lowest germination response was noted in M1 plants of Subs pri when seeds were treated with 0.3% of EMS and SA as 45% and 40% respectively compared to control which showed 90% germination (Table 1). Similarly,both varieties Subs pri and Puhja showed sensitivity to higher doses of mutagens as 38% and 40% lethality observed in 0.3% of SA and EMS respectively in Subs pri and 40% and 47% lethality in 0.3% SA and EMS treated M1 plants of Puhja. Similarly, the highest % of survived mutants was in 0.1% EMS and SA treated plants of Subspri and Puhja with 92% and 93% mutant frequency respectively (Table 1). On the other hand, seed germination response was good in all seeds obtained from M1 plants which showed 59% to 75% germination response in M2 plants compared with control 84-88% germination (Table 1). Similarly, M2 plants showed less lethality and mutant frequency than M1 plants. Seed germination is a significant trait for measuring the response of plants to mutagenic treatment [15]. As observed in the present study, Ettheret al. [16] also reported the promotion of biological parameters by low-dose SA and EMS. Contrary to our results, seed germination was reduced to all doses of sodium azide in Eruca sativa L [17] and in Helianthus annum [18]. Delays or inhibition of physiological activities including enzymatic activity, hormonal imbalance and mitotic activity essential for germination of seeds have explained the reduction in germination caused by mutagenesis [17,19]. The results revealed that there is a linear association between increased doses of mutagens (SA and EMS) and reduced germination of seeds. This was also reported in black gram [20], wheat [21] and Okra [22]. The shoot length in two okra varieties was observed to exhibit marginal differences in mutagens treated and control. The effect of EMS on shoot length of Subz pari was noted as a significant increase in shoot length (17 0±1.2). An increase in number of fruits was noted due to 0.1 and 0.2% levels of EMS and 0.1% SA. Similar results were also obtained in cowpea [29].
The promotion of biological parameters by EMS and low-dose gamma-rays was previously reported in Vicia faba L. and Cicer arietinum [16,30].
The results show that the effects of the two mutagenesis treatments are different and the varieties are diverse and passed down from generation to generation. According to results (