Mutagenic Action of Sodium Azide on Germination and Emergence in Landraces of Phaseolus vulgaris L.on the Jos Plateau Agro-Ecological Zone

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Phaseolus vulgaris (Common beans) landraces are one of the most cultivated legumes for human consumption on the montaneclimate of the Jos Plateau and is renowned for the production of reasonable quantities of the beans in Africa.Common beans is rich in fiber, micronutrients, minerals and has strong medicinal value. Despite its potential for improving nutrition and poverty reduction, it is highly under-utilized and research efforts to improve the genotypes of the plant found in this area for better productivity are few. A study was thus carried out to access the germination and emergence responses of three landraces of Phaseolus vulgaris (pinto, red kidney and navy) after treatment with sodium azide. Four doses of sodium azide were applied in concentrations of 0.1M, 0.04M, 0.03M and 0.02M for each landrace for both germination and emergence experiments. The results showed that at doses 0.02M and 0.03M, the pinto indicated significantly (p<0.05) higher mean germination which surpassed the control. All sodium azide doses in the red kidney and pinto had reduced mean germination. Sodium azide reduced the mean emergence of pinto, red kidney and navy at all the doses of sodium azide. The lethality of sodium azide was highest in navy and lowest in pinto. The decrease in germination and emergence was not dose related especially in red kidney and navy. The best response to the mutagen was observed in pinto with higher germination and the least percentage lethality. Sodium azide effectiveness and efficiency were strongest at the 0.1M dose of the mutagen.
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  • 1. IOSR Journal of Agriculture and Veterinary Science (IOSR-JAVS) e-ISSN: 2319-2380, p-ISSN: 2319-2372. Volume 10, Issue 2 Ver. I (February. 2017), PP 64-70 www.iosrjournals.org DOI: 10.9790/2380-1002016470 www.iosrjournals.org 64 | Page Mutagenic Action of Sodium Azide on Germination and Emergence in Landraces of Phaseolus vulgaris L.on the Jos Plateau Agro-Ecological Zone Sorishima Mlahaga Liamngee1* Joshua Jeremiah Ogah2 Kpadoo Terna Amagu3 Emmanuel Hala Kwon-Ndung4 Dennis Iorkor5 & Jeff Emmanuel Tervershima6 1 Cyto-genetics and Plant Breeding Research Unit, Department of Plant Science and Technology, University of Jos, Jos, Nigeria. 2 Department of Biological Sciences, Faculty of Science, Federal University, Kashere, Gombe State, Nigeria. 3 Department of Forestry Technology, College of Agriculture, Yandev, Benue State, Nigeria. 4 Department of Botany, Federal University, Lafia, Nasarawa State, Lafia. 5 Department of Plant Breeding and Seed Science, College of Agronomy, Federal University of Agriculture, Makurdi, Nigeria. 6 Department of Chemistry, Faculty of Science, Benue State University, Makurdi, Nigeria Abstract: Phaseolus vulgaris (Common beans) landraces are one of the most cultivated legumes for human consumption on the montaneclimate of the Jos Plateau and is renowned for the production of reasonable quantities of the beans in Africa.Common beans is rich in fiber, micronutrients, minerals and has strong medicinal value. Despite its potential for improving nutrition and poverty reduction, it is highly under-utilized and research efforts to improve the genotypes of the plant found in this area for better productivity are few. A study was thus carried out to access the germination and emergence responses of three landraces of Phaseolus vulgaris (pinto, red kidney and navy) after treatment with sodium azide. Four doses of sodium azide were applied in concentrations of 0.1M, 0.04M, 0.03M and 0.02M for each landrace for both germination and emergence experiments. The results showed that at doses 0.02M and 0.03M, the pinto indicated significantly (p<0.05) higher mean germination which surpassed the control. All sodium azide doses in the red kidney and pinto had reduced mean germination. Sodium azide reduced the mean emergence of pinto, red kidney and navy at all the doses of sodium azide. The lethality of sodium azide was highest in navy and lowest in pinto. The decrease in germination and emergence was not dose related especially in red kidney and navy. The best response to the mutagen was observed in pinto with higher germination and the least percentage lethality. Sodium azide effectiveness and efficiency were strongest at the 0.1M dose of the mutagen. Keywords:Emergence,Germination,Jos-Plateau,Landraces,Phaseolus vulgaris,Sodium azide I. Introduction Phaseolus vulgaris (Common beans) is one highly under-utilized plant species in Nigeria with most of the genotypes still existing as landraces. It has adapted to the montane Jos Plateau agro ecological zone which is a recognized bean production area in Africa [1]. It is also one of the most widely consumed legumes in the communities of the area. In Africa, cultivated genotypes are usually restricted to two or three per area[1], on the Jos plateau there are five stably cultivated genotypes. The most common include pinto, navy, red kidney and cranberry. Phaseolus vulgaris, is a typical legume. It belongs to the family Fabaceae. It is cosmopolitan in distribution [2]. In Nigeria, especially on the Jos Plateau, it is referred to as Mangu bean or Achishuru by the local communities.Common beans is a highly nutritive, relatively low-cost protein food. They are among the most sought after of foods for their high protein, minerals and medicinal value. They are also easy to cultivate, do not require much farm inputs as do other food crops and they in turn enrich the soil. Women are responsible for about 70% of its production. It is high in dietary fibre, micronutrients and a variety of phytochemicals [3]. Mature dry seeds are mostly consumed.Common beans is described as a temperate or subtropical crop. When they occur in the tropics, they are usually found in montane valleys. In other areas, lower down the plateau, very few common beans are grown. Temperatures soaring above 300 C can negatively affect flowering and completely prevent seed set above 350 C. In western more developed countries, the plant plays only a minor dietary role [3] unlike in the rural areas of the developing countries around the world where it is prized and serves as a traditional and staple food for local enclaves [4]. In many parts of the Jos Plateau, particular types of the beans are specially prepared and eaten during marriages and other festive periods. Phaseolus vulgaris has been recognized as a crop that would ensure food security in Sub-Sahara Africa [5] and is of great importance in
  • 2. Mutagenic Action of Sodium AzideonGermination and Emergence in Landraces of Phaseolus DOI: 10.9790/2380-1002016470 www.iosrjournals.org 65 | Page helping to alleviate the problem of malnutrition and diversification of diet in Nigeria and Sub-Sahara Africa as it contributes significantly to the amount of calories and other nutrients in the diet. However, the utilization and acceptance of Phaseolusbeans has always been hampered by many problems such as high amounts of synthesized phytic acid in the seeds and the Hard-To-Cook (HTC) phenomenon which have negatively affected its wider acceptability and utilization. Also, there are still not enough works done on the response of these local genotypes to induced mutation. Breeding efforts and improvement programmes on Phaseolus vulgaris in Nigeria have remained scarce even in areas of primordial and fundamental research. This study is therefore aimed at accessing the effect of sodium azide on the germination and emergence of three Phaseolus vulgaris landraces.The variability created by induced mutation in these genotypes is highly desired to improve its productivity. II. Materials And Methods 2.1 Location of Experimental Site This study was conducted at the Mista-Ali Fadama Experimental site located on the outskirts of Jos, the Plateau state capital. It is a small semi-rural settlement which has a rainy season that lasts from about April to October. The area has a mean annual temperature 26.810 c and a mean annual relative humidity of 82.29%, it is elevated above sea level by 1,220m and it is aligned along longitude 80 53’N and latitude 90 57’N [6]. 2.2 Cultural practices, Plot Layout and Experimental Design The experimental site was neatly cleared out and beds for the seeds were made. Using an experimental field size of 14.8m in length and 8.5m in width, the experimental field comprised of 3 three equal blocks. Spacing between the blocks was 0.4m. Each block had 15 equal plots measuring 2m x 0.98m numbering a total of 45 plots for the three blocks. All the treatments were applied on each of the three genotypes including the control which gave 15 treatment combinations in all. They were replicated in the three blocks and completely randomized within the blocks. The Phaseolus vulgaris landraces used were; Pinto (Black in colour), Red kidney (Red in colour), and Navy (White in colour). The treatments are as follows; Dose 1 (D1) = 0.1M Dose 2 (D2) = 0.02M Dose 3 (D3) = 0.03M Dose 4 (D4) = 0.04M Control (D0) = No treatment The seeds were planted 15 per plot, in 3 rows and 5 columns with 0.3m spacing between the column and rows. The seeds were planted to a depth of 2.5cm [7]. The experiment was thus laid out in a Randomized Complete Block Design (RCBD).The plants were sprayed with insecticides to kill insect pest on the 6th Week After Planting (WAP). Weeding was done using the African hand hoe on the 4th and 8th Weeks After Planting (WAP). The plants were watered thrice weekly till harvest. 2.3 Treatment of Phaseolus vulgaris Genotypes with Sodium Azide for Germination Experiment Dry, healthy Phaseolus vulgaris seeds of the pinto, red kidney and navy genotypes were pre-soaked in distilled water up to ten times their volume, according to their varieties for 4 hours. While they were soaking, the mutagen solutions were prepared for four doses (D1 = 0.1M, D2 = 0.02M, D3 = 0.03M, D4 = 0.04M, Control = No dose) for each of the 3 genotypes. After soaking, the seeds were air dried for 20 minutes then soaked in the mutagen solutions.According to their genotypes for 11 /2 hours. Petri-dishes were prepared by washing with distilled water and cotton wool was placed in each petri dish and was moderately soaked with distilled water. The petri-dishes were then labeled according to the landraces and their respective doses.After the treatment time was over (11 /2 hours) the seeds were washed under running water for 30 minutes. 10 seeds were then arranged in each of the prepared petri-dishes according to their varieties and doses. They were then stored away in an airy well lit lab area for observation. The seeds were watered with distilled water every two days. Readings were taken every day for 16 days by counting the number of germinated seeds in each treatment for each of the three landraces. Germination was measured by the emergence of the radicle form the split testa. Percentage survival was scored on day 16. 2.4 Treatment of Phaseolus vulgaris Genotypes with Sodium Azide for Field Experiment Phaseolus vulgaris seeds of the Pinto, Red kidney and Navy genotypes were pre-soaked in distilled water up to ten times their volume, according to their genotypes for 4 hours. While they were soaking, the mutagen solutions were prepared for four doses (D1 = 0.1M, D2 = 0.02M, D3 = 0.03M, D4 = 0.04M, Control = No dose ) for each of the 3 genotypes. After soaking, the seeds were air dried for 20 minutes then soaked in the mutagen solutions. According to their genotypes for 11 /2 hours. After the treatment time was over the seeds were
  • 3. Mutagenic Action of Sodium AzideonGermination and Emergence in Landraces of Phaseolus DOI: 10.9790/2380-1002016470 www.iosrjournals.org 66 | Page washed under running water for 30 minutes. The seeds were immediately taken to the field for planting. Percentage survival was scored on the week of harvest. Chlorophyll mutants were scored in the third week, 19 Days After Planting (DAP) and classified according to [8]. Mutation frequency was calculated as percentage of M1 plants per dose while mutagenic effectiveness and efficiency were calculated based on the formula suggested by [9]. III. Results Table 1: Analysis of Variance for Germination in three genotypes of P. vulgaris treated with Sodium Azide Source of variation Degree of freedom Sum of squares Mean squares F calc. F tab. Doses 4 128.5 32.12 30.5 2.69* Genotype 2 468.8 234.4 223.2 3.32* Dose x Genotype 8 182.1 22.76 21.67 2.27* Error Total 30 44 31.53 748.8 1.05 LSD=1.70 Table 2: Effect Of Sodium AzideOn Germination in the Pinto Genotype OfP.vulgaris Dose of Sodium Azide(M) Mean Germination Percentage Increase In Germination(%) Percentage Decrease In Germination(%) Percentage lethality(%) Percentage survival(%) 0.04M 8.0a --- 3.6 20.00 80.00 Control 8.3ab --- --- --- 83.33 0.1M 8.6ab 30.83 --- 13.33 86.66 0.02M 0.03M Mean 9.3b 10.0c 10.75 17.00 --- --- 6.66 0.00 12.49 93.33 100.00 71.10 LSD=1.70 *Means with same lettering are not significantly different from each other Table 3: Effect Of Sodium AzideOn Germination in the Red Kidney Genotype of P. vulgaris Dose of sodium azide Mean germination Percentage increase in germination(%) Percentage decrease in germination(%) Percentage lethality(%) Percentage survival(%) 0.1M 0.00a --- 100 100.00 0.00 0.03M 1.0ab --- 90 90.00 10.00 0.02M 2.0b --- 80 80.00 20.00 0.04M Control Mean 2.0b 10.0c --- --- 80 --- 80.00 --- 87.5 20.00 100.00 30.00 LSD=1.70 *Means with same lettering are not significantly different from each other Table 4: Effect of Sodium Azide on Germination in the Navy Genotype of P. vulgaris Dose of sodium azide Mean germination Percentage increase in germination(%) Percentage decrease in germination(%) Percentage lethality(%) Percentage survival(%) 0.1M 0.00a --- 100 100.00 0.00 0.03M 0.3a --- 93.47 96.66 3.33 0.04M 0.3a --- 93.47 96.66 3.33 0.02M Control Mean 1.3a 4.6b --- --- 71.73 --- 86.66 94.99 13.33 46.66 13.33 LSD=1.70 *Means with same lettering are not significantly different from each other Table 5: Interaction between Dose of Sodium Azide and Genotype in the Emergence of Phaseolus vulgaris Seeds After 19 Days Pinto Red kidney Navy Mean Control 25 30 14 23d 0.02M 28 6 4 16c 0.03M 30 3 1 11.3b 0.04M 24 6 1 10.3ab 0.1M 26 0 0 8.6a Mean 26c 9b 4a
  • 4. Mutagenic Action of Sodium AzideonGermination and Emergence in Landraces of Phaseolus DOI: 10.9790/2380-1002016470 www.iosrjournals.org 67 | Page 3.1 Germination tests of Three Landraces of Phaseolus vulgaris Treated with Sodium Azide The pinto germinated the most, also indicating the highest survival percentage among the three genotypes (71.10%) (Table 2). Sodium azide was least injurious on pinto as its percentage lethality was also the least among the three genotypes (12.49) (Table 2). Doses 0.02M and 0.03M significantly (p<0.05) increased beyond the control in their germination percentage, 0.03M having the highest mean germination (Table 2). Dose 0.1M had a 100% lethality percentage in the red kidney and navy as no single seed germinated (Table 3 and 4). In the red kidney, the control percentage survival was 100% (Table 3). This dropped to 46.66% in the navy (Table 4). The highest percentage survival apart from the control among the red kidney treatments was 20% (0.02 and 0.03M) (Table 3). The highest percentage survival in the Navy treatments was the least dose (0.02M) at 13.33% apart from the control (Table 4). Sodium azide treatment lead to a decrease in germination of all the doses in both red kidney and navy. Interaction between the dose of sodium azide and the genotype in the study was significant (P<0.05). Table 6: Analysis Of Variance for Emergence in three genotypes of P. vulgaris treated with Sodium Azide Source of variation Degree of freedom Sum of squares Mean squares F calc. F tab. Doses 4 237.2 59.3 10.4 2.69* Genotype 2 276.85 138.42 24.2 3.32* Dose x Genotype 8 63.15 4.15 0.79 2.27NS Error Total 30 44 171.08 763.32 5.70 LSD=3.99 Table 7: Effect of Sodium Azide on Emergence in the Pinto Genotype of P. vulgaris Dose of sodium azide Mean emergence Percentage increase in emergence (%) Percentage decrease in emergence (%) Percentage lethality(%) Percentage survival(%) 0.04M 4.33a - 65.63 71.11 28.88 0.1M 4.66a - 63.01 68.88 31.11 0.02M 6.33a - 49.76 57.77 48.88 0.03M 7.33a - 41.8 51.11 48.88 Control Mean 12.60b - - - 62.21 84.44 47.10 LSD=3.99 *Means with same lettering are not significantly different from each other Table 8: Effect of Sodium Azide on Emergence in the Red kidney Genotype of P. vulgaris Dose of sodium azide Mean emergence Percentage increase in emergence (%) Percentage decrease in emergence (%) Percentage lethality(%) Percentage survival(%) 0.1M 0.00a - 100 100.00 0.00 0.03M 1.00a - 88.45 86.66 6.66 0.04M 1.33a - 84.64 82.22 8.88 0.02M 2.33a - 73.09 84.44 15.55 Control Mean 8.66b - - - 88.31 55.55 17.32 LSD=3.99 *Means with same lettering are not significantly different from each other Table 9: Effect of Sodium Azide on Emergence in the Navy Genotype of P. vulgaris Dose of sodium azide Mean emergence Percentage increase in emergence (%) Percentage decrease in emergence (%) Percentage lethality(%) Percentage survival(%) 0.02M 0.66a - 80.18 95.55 4.44 0.04M 0.66a - 80.18 95.55 4.44 0.1M 0.66a - 80.18 95.55 4.44 0.03M 1.00a - 69.98 86.66 6.66 Control Mean 3.33a - - - 93.32 22.22 8.44 LSD=3.99 *Means with same lettering are not significantly different from each other Table 10: Mutagen Effectiveness and Efficiency per dose of Sodium Azide in Phaseolus vulgaris Dose of sodium azide (mM) Percentage lethality per dose (L) Mutation frequency (M) Mutagen effectiveness M x 100 C x t Mutagen efficiency M x 100 L 100 71.11 13 8.66 18.25 20 57.77 2 6.66 3.48 30 62.22 3 6.66 3.21 40 65.55 3 5.0 3.05
  • 5. Mutagenic Action of Sodium AzideonGermination and Emergence in Landraces of Phaseolus DOI: 10.9790/2380-1002016470 www.iosrjournals.org 68 | Page 3.2Emergence of Three Landraces of Phaseolus vulgaris Treated with Sodium Azide The sodium azide doses reduced the emergence of all the pinto treatments used in the study. The highest survival percentage apart from the control was 0.03M and 0.02M (48.88%) (Table 7). The highest lethality was 71.11% at dose 0.04M with the least mean emergence of 4.33 seedlings. The control almost doubled the highest survival percentage (48.88% in 0.03M, 0.02M) at 84.44% (Table 7). Some seeds germinated but died before they completely emerged. Sodium azide dose 0.1M was completely lethal in the red kidney genotype as there were no emerged seedlings (Table 8). No dose performed better than the control. The best performing Navy dose were 0.02M, 0.04M and 0.1M at 4.44% survival rate. The best performing dose in the Navy treatment was 0.03M at 6.66% survival percentage. The increase in lethality was not dose related. IV. Discussion 4.1 Germination tests of Three Landraces of Phaseolus vulgaris Treated with Sodium Azide Germination is the re-activation of metabolic activity in a seed which results in the growth of the embryo by the emergence of first the radicle, then the plumule from the split testa. This process divided into three distinct stages which are imbibition, cell elongation and cell division [10] each one leading to the next. These processes are associated with the formation and utilization of enzymes which mediate crucial biochemical transformations such as the conversion of stored proteins, carbohydrates or oils depending on the kind of seed in the endosperm to simple ready-to-use substances used to fuel primary growth. The inhibition of respiration [11] disruption of enzyme formation and utilization are common abilities of sodium azide. This could explain the 100 percent lethality in the 0.1M dose of red kidney and navy (Table 3 and 4). At this dose sodium azide is able to inhibit energy supply and utilization, disrupt enzyme activity such as those of amylase, proteases and lipase responsible for mediating the conversions of starch to maltose, proteins to amino acids and lipids to fatty acids thus preventing essential food processes that can lead to the death of the embryo. Gibberellic acid is known to induce germination [12]. And since the presence of water and gibberellic acid initiates the formation of amylase, sodium azide might have disruptive ability when present with an enzyme or the ions which act as the enzyme cofactor thus bringing the germination process to a complete halt. These activities may still occur on a reduced scale in lower doses of sodium azide. The mean number of germinated seeds as observed in the experiment did not show a dose dependent decrease in the trend of germination with an increased mutagenic concentration of sodium azide especially in red kidney and navy. This observation disagreed with the work of [13], [14],[15]. The percentage germination showed that pinto had the most germination followed by the red kidney then Navy showing the least germination and obviously been affected the most by the doses of sodium azide.Pinto had two doses (0.02M and 0.03M) which increased significantly (P<0.05) in the number of germinated seeds. This result was also observed by [16] when they recorded an increase in the number of germinated seeds in tomato when treated with sodium azide. The explanations for an increase in germination rates after treatment with sodium azide at doses may lie in the fact that lower doses at certain optimal points in the cell cycle can affect cell metabolic activities differently from normal sodium azide action which has well known lethal effects. [17]
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