Mutagenic effects of hydroxylamine and hydroquinone on Some Agronomic and yield characters of soybean
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https://www.eduzhai.net/ International Journal of M odern Botany 2013, 3(2): 20-24 DOI: 10.5923/j.ijmb.20130302.02 Mutagenic Effects of Hydroxylamine and Hydroquinone on some Agronomic and Yield Characters of Soyabean (Glycine max (L.) Merr.) J. K. Mensah*, G. O. Okooboh, I. P. Osagie Department of Botany, Faculty of Natural Sciences, Ambrose Alli University, Ekpoma, Nigeria Abstract Dry and healthy seeds of soyabean (Glycine max (L.) Merr.) of about 10% moisture content were exposed to varying concentrations of 0-0.300% of Hydro xylamine and Hydroquinone for 24hours and their effects on germination, survival, chlorophyll content, agronomic and yield characters reported. Soyabean responded differentially to the chemicals for the parameters studied. The useful traits observed in the present work for both chemicals include increase in plant height/number of branches, number of flowers per plant, increase in the number of pods per plant. The two chemicals also increased the chlorophyll content and induced early maturity. Ho wever, these useful traits identified during the present study need to be tested further on a wider scale in later generations in order to isolate specific mutants with improved characters. Keywords Mutagenic Effects, Hydro xylamine, Hydroquinone, Yield Characters 1. Introduction The mutagenic property of mutagens was first demonstrated in 1927, when Hermann Muller discovered that X-rays can cause genetic mutation in fruit flies, producing phenotypic mutants as well as observable changes to the chromosomes. Similar work by Lewis Stadler also showed the mutational effect of X-rays on barley in 1928 and ultraviolet (UV) radiat ion on maize in 1936. Mutation breeding has been widely used for the imp rovement of plant characters in various crops. It is a powerful and effective tool in the hands of plant breeders especially for autogamous crops having narrow genetic base. The prime strategy in mutation breeding has been to upgrade the well adapted plant varieties by altering one or two major agronomic material traits which limit their productivity or enhance their quality. Mutation induction offers significant increase in crop production and the possibility of inducing desired attributes that either cannot be found in nature o r have been lost during evaluation. Treatment with mutagens alters genes or break chro mosomes. Induced mutations have been used to improve a wide variety of crops with different reproductive systems and at different ploidy levels. Ch emical mut agens were not d emo nstrated to cause mutation until the 1940s, when Charlotte Auerbach and J.M. Robson found that mustard gas can cause mutations in fruit * Corresponding author: email@example.com (J. K. Mensah) Published online at https://www.eduzhai.net Copyright © 2013 Scientific & Academic Publishing. All Rights Reserved flies. A wide range of chemicals have also been identified and tested for their mutagenicity in bio logical systems. These include nitrous acid, the alky lating agents, hydroxylamine, hydroquinone, sodium azide and the antibiotics–streptomycin, chloramphenicol and mito mycinc. Hydro xylamine has been used to induce pollen sterility and high yield ing variants in crop p lant[7-8]. Hydro xy lamine has previously been used to increase the seed yield and other agronomic characters leading to the production of kiyanka, a commercial soviet variety of wheat. Previous studies, have reported that hydroquinone positively affects the maturity time and pod yield per plant. Many chemical mutagens have been employed for obtaining useful mutants in various crop species. However the various workers emphasize that artificial induction of mutation by colchicines (Col), ethyl methane sulphonate (EMS), hydroquinone (HQ), hydro xylamine(HA) and sodium azide (SA) provides tool to overcome the limitat ions of variability in p lants especially mutations that induce specific imp rovement without disturbing their preferred attributes[9, 11, 12]. Soyabeans are one of the “biotech food” crops that have been genetically mod ified, and genetically mod ified soyabeans are being used in an increasing number of products. Soyabeans possesses good quality protein which is comparable to other protein foods and is suitable for all ages, infants to the elderly. Soyabean protein products also contain a high concentration of isoflavones, up to lg/kg. The addition of soy protein in d iet or replacing animal protein in the diet with soy lowers blood cholesterol and hence the risk International Journal of M odern Botany 2013, 3(2): 20-24 21 of coronary diseases and cancer. Earlier works[14-16] reported an important link between soy consumption and a reduced risk of certain types of cancer. Asian women, who typically eat a soy based diet, have a much lower incidence of breast cancer than western women. Significant changes in the menstrual cycle of women who were fed on soy diet have also been reported. The objective of this study is to enhance the genetic variability in soyabean (Glycine max) using hydroxylamine and hydroquinone to determine the optimal conditions for the induction of this genetic variability, in order to shorten the generation cycle of the crop and to increase yield through genetic modificat ion. 2. Materials and Method Healthy and uniform seeds of soyabean were selected fro m a lot obtained fro m Ekpo ma Main Market, Ekpoma, Esan West Local Govern ment Area, Edo State, Nigeria. Sets of 30 seeds were exposed to concentrations of 0 (Control), 0.019 %, 0.038 %, 0.075 %, 0.150 %, 0.300 % of Hydro xylamine and Hydroquinone in Petri d ishes for 24 hours with intermittent shaking. The treated seeds were washed with distilled water to remove excess chemical and toxic products and sown directly into potting bags up to the point of harvest. During the course of studies, the following parameters were carefully measured. 2.1. Germination and Survi val Studies The study of germination was carried out fro m the second to forth day. The total nu mber of emerged seeds in the different concentrations of the two chemicals (hydroxy lamine and hydroquinone) was expressed as a mean of the control. Seeds showing the emergence of cotyledon fro m the soil surface were recorded as having emerged. Plants that survived the 21st day were recorded as having survival and the results of the treated plants was expressed as a percentage of the control 2.2. Agronomic Studies The parameters studied include: Plant Height, Nu mber of days to flower Nu mber of Leaves, Number of flowers per Plant, Nu mber of pods per Plant 2.3. Biochemical Study Determinati on of Chlorophyll Content of Leaves Chlorophyll determination took p lace at the onset of flower bud init iation. Fro m each experiment, Leaves from five rando mly selected plants were collected fresh very early in the morn ing (7.00 am) for each treatment, stored in ice packs in the field and transported to the laboratory for analysis. Chlorophyll determination was carried out following the method of Lichtenthaler and Buschmann. Two leaf d iscs of 3mm rad ius obtained from the third youngest leaf of each plant fro m each of the treat ments were ground, extracted with 5ml o f 80% w/w acetone and 20% water and centrifuged at 2,500r.p.m for three minutes. The clear supernatant was collected and the absorbance was determined at 645n m and 663n m on a Bosch and lamb spectronic 20 spectrophotometer using 80% w/w acetone and 20% water as reference b lank. Calcul ati on of total chl orophyll in two leaf punches For each plant, the chlorophyll was extracted fro m two leaf punches in 5 ml of solvent. The total Ch l a and b concentrations in the 2 leaf punch extract was calculated based on Lichtenthaler and Buschmann . Chl a = 12.25 A665 - 2.79 A645 = μg/ ml in e xtract Ch l a =[12.25 A665 - 2.79 A645] x 5 = total μg in 2 leaf punches Chl b = 21.5 A645 - 5.1 A665 = μg/ml in extract Ch l b =[21.5 A645 - 5.1 A665] x 5 = total μg in 2 lea f punches For each leaf punch, radius = 3 mm = 0.003 m and s urface area = π r2 = 3.14 x 0.0032 = 0.0000282 m2 Co mbined surface area of 2 leaf punches: 0.0000565 m2 Therefore, the total amount of Chl a+b (in μg) obtained fro m 2 leaf punches was multip lied by 1/0.0000565 = 17699.1 to obtain the concentration of Chl a+b in μg m-2. The value obtained was then converted to mg m-2. The average concentration for each treatment was then recorded as the total chlorophyll. 3. Result and Discussion The effects of hydroxy lamine and hydroquinone on the parameters studied are presented below. 3.1. Germination and Survi val Studies The germination studies showed that there were reductions or remarkable decrease in germination with increasing concentration of hydroxy lamine and hydroquinone. Table 1. Effectsof hydroxylamine and hydroquinone on mean germination and survival Tre atments Con centra tion) Hydroxylamine Hydroquinone Hydroxylamine Hydroquinone GERMINATIO N (%) Control 0.019 0.038 0.075 30.0 29.5 27.5 23.5 30.0 29.0 25.0 22.5 SURVI VAL 9.3 7.3 6.7 6.0 9.3 6.7 5.3 4.3 0.150 22.5 21.5 5.3 3.5 0.300 18.5 19.0 4.0 3.0 It is known in mutagenic e xperiments that the germination is inversely proportional to the dosage. The higher the mutagenic dosage, the lower the germination[19-20, 9]. In the present investigation, similar t rends were observed. Survival was based on the number of surviving plants on the 21st day after planting (DAP) and the results followed similar trends as that of the germination as shown in Table 1. It is apparent that the control plants had the highest mean survival when compared with other concentrations of the mutagens. 22 J. K. M ensah et al.: M utagenic Effects of Hydroxylamine and Hydroquinone on some Agronomic and Yield Characters of Soyabean (Glycine max (L.) M err.) Generally, the survived plants decreased with increasing concentrations of the chemical mutagens under investigation. Previous reports[21, 22] have shown that survival trends are similar to that of germination but only a litt le lower as a result of the lethal effects of the physiological damage due to the mutagens. 3.2. Plant Height Table 2. Effect s of Hydroxylamine and Hydroquinone on mean plant height of soyabean Tre atment Mean plant height ( cm ) Hy droxyl amine Control 0.019 % 0.038 % 0.075 % 0.150 % 0.300 % Hydroquinone Control 0.019 % 0.038 % 0.075 % 0.150 % Week 1 18.9±1.41 20.1±1.37 18.6±5.47 29.7±0.9 21.5±3.01 19.3±0.87 22.1±1.74 23.1±4.13 19.5±1.80 24.4±4.00 17.1±1.50 Week 2 23.0±0.17 24.5±1.58 22.2±6.44 25.2±0.87 21.7±3.73 18.9±0.53 26.9±1.80 27.1±4.60 24.0±2.45 26.5±0.60 27.6±3.05 Week 3 28.9±1.14 30.2±2.79 28.5±5.43 30.3±1.48 27.0±4.64 23.7±0.27 30.8±3.42 33.8±6.37 29.3±2.98 31.4±2.37 25.9±6.95 Week 4 35.6±1.01 36.9±2.79 35.2±5.41 36.3±1.10 23.7±4.68 30.5±0.26 35.9±3.41 40.7±6.20 31.7±3.20 32.7±2.45 21.2±6.9 0.300 % 15.7±0.64 27.4±2.05 29.1±3.96 30.3±3.94 It has been observed from the present investigation that there were reductions with increasing concentrations of both chemicals (hydroxy lamine and hydroquinone) except 0.019% and 0.075% of both chemicals wh ich showed increase in height when co mpared to the controls of both chemicals. The reduced plant height was associated with loss of vigour and delayed emergence of the first floral leaf. Similar observations have been recorded in sunflower. In the present investigation, plants treated with hydroquinone induced more stunted growth compared to plants treated with hydro xylamine. The lowest concentration of Hydroxy lamine appears to slightly stimulate plant heights while all other concentrations of both chemicals appear to lower the plant height as shown in Table 2. 3.3. Number of Leaves / Plant The effect o f hydro xylamine and hydroquinone on number of leaves is presented in Table 3. Increasing concentrations of the chemical mutagens (hydroxylamine and hydroquinone) led to gradual reduction in the number of leaves. Control treatments of both chemicals recorded the highest number of leaves. However, the chemical mutagens caused a slight increase in 0.3% in Week 5. 3.4. Number of Days to Fl ower The effects of hydro xylamine and hydroquinone on mean number of days to flower is presented in Table 4 Increasing concentration of both chemical mutagens led to gradual reductions in the number of days to flower in 0.075 % by stimu lating early init iation of flo wer bud. However, h igher concentration of both chemical mutagens caused a delay in the number of days to flower in 0.150 % and 0.300 %. In the present study, early flowering variants have been recorded in both chemical mutagens under lower concentrations (0.019 % and 0.038 %). Early maturity mutants have previously been isolated under mutagenic treatment of some legu me p lants[24-25]. 3.5. Number of Fl owers Per Plant In the present investigation, 0.019% of both chemical mutagens recorded the highest number of flo wers per plant when compared to the control as shown in table 4. Increase concentrations of the chemical mutagens led to decrease in the number of flo wer per plant. Table 3. Effect s of Hydroxylamine and Hydroquinone on mean number of leaves of soyabean Treatment Hydroxylamine Control 0.019 % 0.038 % 0.075 % 0.150 % 0.300 % Hydroquinone Control 0.019 % 0.038 % 0.075 % 0.150 % 0.300 % Week 1 4.0±0.2 4.0±0.05 5.4±0.29 3.1±0.47 2.1±0.1 2.4±1.23 4.2±0.59 3.8±0.29 2.5±0.71 3.2±0.14 3.0±1.0 2.6±0.43 Week 2 7.8±0.24 7.7±0.47 6.9±1.35 7.9±0.1 8. ±0.1 7.4±0.53 7.9±0.1 8.0±0.13 7.7±0.47 8.0±0.0 75±0.5 7.5±0.71 Mean number of leaves Week 3 10.4±0.29 10.5±0.41 9.3±1.31 10.2±0.70 9.9±0.73 10.1±0.65 Week 4 15.0±0.99 14±0.16 13.8±0.64 13.6±1.11 13.3±1,45 14.6±1.77 10.7±0.42 10.5±0.08 10.4±0.49 10.5±0.75 10.4±0.65 10.5±0.71 13.4±0.38 13±0.62 14.2±1.26 14±0.21 15±1.0 13.9±0.9 Week 5 17.8±0.53 17.6±0.22 15.7±0.54 17.8±1.30 18.5±1.60 19.8±4.40 16.7±0.27 17.1±0.93 17.8±1.00 17.7±1.28 20±3.0 21.2±4.55 International Journal of M odern Botany 2013, 3(2): 20-24 23 Table 4. Effect of hydroxylamine and hydroquinone on some agronomic characters of soyabean Treatment (%) Hydroxylamine Control 0.019 0.038 0.075 0.150 0.300 Hydroquinone Control 0.019 0.038 0.075 0.150 0.300 Number of daysto flower 37 35 35 40 40 44 37 36 38 39 42 48 Number of flowers per plant 6.81±0.59 7.1±2.0 7.5±0,97 11.9±1,89 8.9±0.19 7.1±0.18 6.71±0.31 6.9±2.08 8.8±1.15 7.6±1.09 7.7±0.15 7.0±0.89 Number of pods per plant 6.4 6.6 7.0 9.0 6.4 5.0 6.2 6.8 7.2 6.4 6.0 6.0 Table 5. Effects of Hydroxylamine and Hydroquinone on the total chlorophyll content of soyabean.( mg/g-1 fresh wt) Treatment Hydroxylamine Hydroquinone Co n centrat ion Control 0.019 % 0.038 % 0.075 % 0.150 0.300 % Control 0.019 % 0.038 % 0.075 % 0.150 % 0.300 % Chlorophyll a 0.56±0.35 1.24±0.60 0.63±0.51 0.52 ± 0.45 0.45±0.38 0.41±0.13 0.56±0.35 1.00±0.58 0.40±0.22 0.47±0.37 0.17±0.08 0.11±0.02 Chlorophyll b 2.74±0.04 2.97±1.00 0.79±0.16 0.85±0.22 1.07±0.37 1.52±0.21 2.74±0.04 1.54±0.33 1.02±0.14 0.73±0.33 1.48±0.56 0.54±0.32 Total (a+b) 3.30±0.12 4.51±0.67 1.41±0.49 1.37±0.61 1.52±0.60 1.94±0.12 3.30±0.12 2.54±0.34 1.43±0.29 1.20±0.38 1.62±0.63 0.65±0.33 3.6. Number of Pods Per Pl ant The effects of hydro xylamine and hydroquinone on mean number of pods per plant is presented in Tab le 4. Lower concentrations stimulated pod production whereas high concentrations reduced number of pods per plant compared to the control plants. The most effective concentration for hydroxylamine was 0.075 %. However for hydroquinone, the effective concentration wh ich stimulates pod production was 0.038 %. The stimulatory effects of the chemicals were more pronounced in hydro xylamine than hydroquinone treated plants. in the present work were early flo wering under 0.019% and 0.038 % in both chemicals; both chemicals increased plant height under lower concentrations (0.019% and 0.075%), increase in the number of flo wers per plant under 0.019% of both chemicals, increase in the nu mber o f pods per plant under 0.075% and 0.038% of hydro xylamine and hydroquinone respectively. Further studies would assist to have better understanding of inheritance pattern of these agronomic characters in soyabean so as to improve its genetic quality for the local farmers and hence improve on the present level of productivity of the crop. 3.7. Chl orophyll Content The effects of hydroxylamine and hydroquinone on chlorophyll content are presented in Table 5. The general trend of both chemicals on chlorophyll content showed slight decreases with increasing concentration. However, 0.019% treated plants of hydroxyamine contain more chlorophyll content than the control plants. There was a decrease in the germination and survival of the plants as the concentrations increased. On the basis of the observation made on the effect of hydro xylamine and hydroquinone on soyabean (Glycine max (L.) Merr.), on the agronomic and yield parameters, it can be concluded that the two chemicals (hydro xy lamine and hydroquinone) induced variability in the crop under study. The useful traits observed 4. Conclusions There was a decrease in the germination and survival of the plants as the concentrations of the two chemicals increased. On the basis of the observations made on the effects of hydroxy lamine and hydroquinone on soyabean (Glycine max (L.) Merr.), on the agronomic and yield parameters, it can be concluded that the two chemicals (hydroxy lamine and hydroquinone) induced variability in the crop under study. The useful traits observed in the present work were early flo wering under 0.019 % and 0.038 % in both chemicals; both chemicals increased plant height under lower concentrations (0.019 % and 0.075 %), increase in the number of flowers per plant under 0.019 % of 24 J. K. M ensah et al.: M utagenic Effects of Hydroxylamine and Hydroquinone on some Agronomic and Yield Characters of Soyabean (Glycine max (L.) M err.) both chemicals, increase in the nu mber o f pods per plant under 0.075% and 0.038% of hydro xylamine and hydroquinone respectively. 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