African Journal of Biotechnology Volume 15 Number 46, 16 November 2016 ISSN 1684-5315
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African Journal of Biotechnology
Table of Content: Volume 15 Number 46 16 November, 2016
ARTICLES
Investigating the potential of Aucoumea klaineana Pierre sapwood and heartwood wastes to produce cellulosic ethanol 2587 Rodrigue Safou-Tchiama, Sébastien Ngwa Obame, Nicolas Brosse, Patrice Soulounganga and Timoléon Andzi Barhé
Comparison of seed priming techniques with regards to germination and growth of watermelon seedlings in laboratory condition 2596 Willams Ferreira Souza Barbosa, Fábio Steiner, Leandro Castro Matias de Oliveira and Paulo Henrique and Menezes das Chagas
Linkage disequilibrium and association mapping of drought tolerance in cotton (Gossypium hirsutum L.) germplasm population from diverse regions of Pakistan 2603 Abdelhafiz Adam Dahab, Muhammad Saeed, Nada Babiker Hamza, Bahaeldeen Babiker Mohamed and Tayyab Husnain
Inbreeding depression in crosses of coerulea clones of Walker’s Cattleya
(Cattleya walkeriana Gardner) 2613 Miléia Ricci Picolo, Ceci Castilho Custódio, Nelson Barbosa Machado-Neto
The protective role of honey against cytotoxicity of cadmium chloride in mice 2620 Asmaa Shahrour, Mohamed Zowail and Khaled Sharafeldin
Shelf life improvement of sorghum beer (pito) through the addition of Moringa oleifera and pasteurization 2627
Florence Adwoa Ayirezang, Courage Kosi Setsoafia Saba, Francis Kweku Amagloh and Hellie Gonu
Vol. 15(46), pp. 2587-2595, 16 November, 2016 DOI: 10.5897/AJB2016.15515 Article Number: 656B70761669 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2016 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Investigating the potential of Aucoumea klaineana Pierre sapwood and heartwood wastes to produce cellulosic ethanol
Rodrigue Safou-Tchiama1,2*, Sébastien Ngwa Obame1, Nicolas Brosse3, Patrice Soulounganga4 and Timoléon Andzi Barhé2
1Laboratoire de Recherche et de Valorisation du Matériau Bois (LaReVa Bois). Bât du Master Recherche en Sciences du Bois. Ecole Nationale des Eaux et Forêts. BP 3930, Libreville, Gabon. 2Laboratoire des Substances Naturelles et de Synthèse Organométalliques. Unité de Recherche en Chimie. Université des Sciences et Techniques de Masuku. BP. 941 Franceville, Gabon. 3Laboratoire d’Etudes et de Recherche sur le Matériau Bois, Faculté des Sciences et Techniques, Nancy-Université, Vandœuvre-lès-Nancy, France. 4Laboratoire Pluridisciplinaire des Sciences. Ecole Normale Supérieure. BP 17009, Libreville, Gabon.
Received 7 June, 2016; Accepted 24 October, 2016
This work investigates for the first time the different reactivity exhibited by sapwood and heartwood wastes under two conditions: They were pretreated with catalyzed ethanol organosolv in the presence of sulfuric acid and their pulps underwent enzymatic hydrolysis by Trichoderma reesei which causes them to release neutral sugars for the production of cellulosic ethanol. Aucoumea klaineana Pierre (Okoumé) wood wastes were used for the experiment. Organosolv pretreatment was performed to investigate how to harness the benefits of fermentable sugars and lignin, which were reacted at varying defined severity levels as follows: 160°C≤T≤210°C and 0%≤[H2SO4]≤2% (w/w). The highest ethanol organosolv lignin content was obtained at T=160°C and [H2SO4]=1% for sapwood (18.10%) and heartwood (19.46%) (w/w). Enzymatic hydrolysis of the pretreated wood samples displayed that sapwood and heartwood pulps released their highest sugars content under free acid pretreatment conditions. The maximum neutral sugar released by heartwood pulps pretreated at T=160°C and [H2SO4]=0% was 0.126 g/L while that from sapwood pretreated at T=185°C and [H2SO4]=0% was 0.125 g/L. It is noteworthy that, the neutral sugars from sapwood and heartwood do not have the same sensitivity to temperature increase. When pretreated at T=160°C and [H2SO4]=0%, sapwood pulps yielded the following neutral sugars: Xyl< AraGal < Man < Glu, while heartwood was: Ara < Gal < Xyl < Man < Glu. However, with increased temperature ( T=185°C and [H2SO4]=0%), sapwood yielded the following neutral sugars: Ara < Gal < Xyl < Man < Glu, while heartwood yielded the following: Xyl < AraGal < Man < Glu. Similar trend was observed in both sapwood and heartwood pulps when sulfuric acid concentration was increased.
Key words: Aucoumea klaineana Pierre, wood wastes, ethanol organosolv lignin, enzymatic hydrolysis, bioethanol. 2588 Afr. J. Biotechnol.
INTRODUCTION matter of plant, cellulose is potentially a huge renewable energy store and vast amounts of this material are Biofuels help to overcome the global warming concern routinely thrown away. Thus, ethanol cellulose from the because they use carbon from the atmosphere and give it wood waste timber industries is an opportunity for back to it, while fossil energies increase the carbon equatorial oil-producing developing countries of Congo content in the atmosphere. Biofuel was first mentioned in basin like Gabon to receive transfer of technologies. This 1903 and 1926, which corresponds to the beginning of will allow those countries to produce much ethanol at the automotive era (Payen, 1982). Nowadays, first cheaper rates to obtain fuels at a reduced cost. A generation bioethanol from sugarcane in Brazil or corn in prospective study has stated that ethanol (from sugar USA (Wheals et al., 1999) is blended with gasoline and cane, maize, sorghum and manioc fibers) or biodiesel used in all petrol engines without modifications (Miller, (from palm oil and Jatropha) demand in Gabon will be 12 2003; Steven and Verhe, 2004). and 46 millions of liters respectively, in the year 2020 Considering that the fossil fuels reservoirs will serve (Sielhorst et al., 2008). However, the first boards of trunk man’s needs for a very limited time period, scientific wastes from sawmill and the inner heartwoods from the search for alternatives has already been initiated. rotary cutting logs of Okoumé’s plywood industry which Amongst the renewable resources for emerging remain undervalorized can be suitable source for ethanol biotechnological strategies used to produce high energy- cellulose production. Previous studies have shown that less volume fuels, lignocellulose like cellulose which is ethanol can be obtained from wood specie like the most abundantly synthesized and stable Eucalyptus (Ferrari et al., 1992, Romani et al., 2012, carbohydrate of the biosphere has been identified. Yanez-S et al., 2014). However, the stable nature of the substrate and some Despite that hydrolysis of lignocelluloses by sulfuric fermentation difficulties of its monomeric products have acids is relatively old, it remains the most investigated been the major hurdles in using cellulose. But due to its process (Harris et al., 1945). Advantages and ubiquitous nature and abundant availability as renewable disadvantages of lignocellulose pretreatment process for resource, research on the utilization of cellulose for ethanol cellulose production with sulfuric acid have been obtaining bioethanol continues in diverse fields. extensively discussed by Taherzadeh and Karimi (2007). There are still many issues to be addressed in Nevertheless, Organosolv pretreatment released a considering the future of ethanol fuels. Many critics have cellulosic residue which can be used for ethanol charged that the energy balance of ethanol fuels is production, high neutral sugars yield in aqueous phase, flawed, arguing that the energy inputs exceed the energy better delignified fibers and relatively pure lignin for which content of the final product. However, a study released by interest grows in various industrials (Brosse et al., 2010; the United States Department of Agriculture in July of El Hage, 2010). 2002 refutes these claims and finds that ethanol has an However, the differences in the reactivity of sapwood output: input energy ratio of 1.34:1 (Shapouri et al., 2002). and heartwood pretreated with sulfuric acid and the Furthermore, another study found that the energy behavior of their partially delignified fibers through balance of gasoline is actually negative, giving ethanol a enzymatic hydrolysis have not been investigated. The 1.42:1 output energy ratio compared to gasoline aim of this study is to investigate the potential of sapwood (Alternative Fuels Data Center, 2003). On the other hand, and inner heartwood of Okoumé as biomass candidates converting the entire feedstock plant to useful end treated with ethanol organosolv lignin in an experimental products allows ethanol costs to decrease as design to find better conditions for lignin extraction. The manufacturing becomes more profitable. Besides being a neutral sugar contents obtained from enzymatic renewable fuel made from plants, with high octane at low hydrolysis of the partially delignified pulps shall be used cost, ethanol is a much cleaner fuel than petrol. Ethanol to discriminate between the two wood wastes. The blends dramatically reduce emissions of hydrocarbons, relative sensitivity of the cellulose-hemicellulose matrix (major sources of ground level ozone formation), cancer- sugars from sapwood and heartwood to temperature and causing benzene and butadiene, sulphur dioxide and sulfuric acid concentration increase will be investigated. particulate matter. However, the use of ethanol for fuel will almost certainly require cellulose technology. Likewise, MATERIALS AND METHODS Taherzadeh and Kairimi (2007) have recently indicated The raw material of Okoumé was collected in February 2016 at that lignocelluloses can be expected to be major SNBG (Société Nationale des Bois du Gabon). The air-dry material feedstocks for ethanol production in the future. Evans was ground and milled to a particle size of 0.25 mm (for Klason (2005) has explained that, being 50% of the total dry lignin) and 0.6 mm (for Ethanol Organosolv Lignin) using a Willey
*Corresponding author. E-mail: [email protected]. Tel: (+241)-02-45-77-65.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Safou-Tchiama et al. 2589
mill. It was stored at room temperature during the course of this Table 1. Experimental matrix of the face centered study. All chemical reagents used in this study were purchased composite design conditions used for the pretreatments of from Aldrich and VWR (France) and used as received. Trichoderma Okoumé’s heartwood and sapwood. reesei (product number C2730) was supplied by Sigma Aldrich. Other chemicals used include absolute ethanol, sulfuric acid, Number of Temperature [H2SO4] sodium hydroxide, potassium sodium tartrate, acetic acid glacial. reactions (°C) (%)
1 0 0 Extractives and ash content 2 -1 0 3 0 1 The extractive contents were obtained as follows: Triplicates of 1 g 4 1 1 of unextracted heartwood or sapwood sawdust were subjected to 5 0 0 soxhlet extraction with acetone of 70% (v/v) for 24 h to remove the extracts. The solid to liquid ratio was 2% (w/v). Then, the wood was 6 -1 1 oven-dried at 105°C for 24 h and cooled to ambient temperature in 7 0 0 a desiccator containing phosphorus pentoxide (P2O5). 8 1 0 The ash content of the wood samples was obtained at Tappi T 9 0 -1 211 cm-86 (2003) adapted as follows: 1 g of unextracted and dried heartwood or sapwood sawdust was weighted with an analytical 10 1 -1 and accurate balance of 0.1 mg. The samples were deposited in a 11 0 0 ceramic ashing crucible, and introduced in a muffle furnace 12 -1 -1 equipped with a thermostat set at 575±25°C equipped with optional ramping program. The wood biomass was ignited in a first step at Experimental conditions: 160°C≤T≤210°C such as -1=160°C; 250°C for 2 h, and then a second step was carried out at 500°C for 0=185°C and +1=210°C. 1%≤[H2SO4]≤2% (w/w) : -1=0% ; 24 h. At the termination of the pyrolysis, the ceramic ashing crucible 0=1% and 1=2%. The reaction time is 1hour. Ethanol was removed to the furnace directly into a desiccator containing (65%)/H2O=1v/2v and the solid to liquid ratio =1/7. The central point was R1; R5; R7 and R R11. P2O5 for cooling at room temperature until constant weight was obtained. The ash content was calculated as follows:
( ) MEOL is the weight of dried EOL and M0 is the moisten-free dry wood samples weight. For each residual heartwood or sapwood Mash is the weight of ash obtained after pyrolysis and M0 is the pulp obtained, the KL content was determined as described unextracted moisten-free dry wood samples weight. previously.
Klason lignin measurement Enzymatic hydrolysis of the pretreated wood samples
175 mg extractive-free and dried wood materials were hydrolyzed Enzymatic hydrolysis of the pulps obtained after EOL extraction with 72% (w/v) sulfuric acid and stirred for 1 h before autoclaving at was carried out using a published procedure with slight Tappi T 222 om-11 (2006). The autoclaved samples were filtered modifications (El Hage, 2010; Brosse et al., 2010). The sapwood with Buchner and the dried residue was weighed to give the Klason and heartwood pulps obtained after EOL treatment were grinded to lignin (KL) content; while the liquid fraction was keep at -5°C for the pass 0.5 mm, oven-dried at 103°C for 24 h and cooled at room neutral sugar content determination. temperature in a desiccator which contained P2O5. 2.0 g of the pretreated and dried pulps was poured into an Erlenmeyer flask with 98 mL of sodium acetate buffer (0.05 M; pH=4.8). Then, 0.38 mL of the enzymatic solution of 15 FPU per gram of substrate (2% Ethanol organosolv lignin extraction w/v) was added to the mixture and stirred. It was incubated at 50°C for 48 h in an air-bath shaking incubator at 150 rpm. About 0.2 mL 15 g of unextracted and dried material samples were treated with samples were drawn periodically, diluted in 1 mL distillated water, 65% (v/v) ethanol in the presence of sulfuric acid as catalyst and and placed in 1.5 mL Eppendorf safe-lock tube. In order to arrest different levels of temperature (Table 1), using a published protocol enzymatic activity, the samples in the Eppendorf tube were placed (Pan et al., 2006, 2007; El Hage, 2010). The solid to liquid ratio was in boiling water at 97°C for 5 min, cooled and kept in a refrigerator 1/7 (w/v). The process was optimized by an experiment design run ® at -5°C until further analysis. with the statistical JMP 11 Découvrir JMP. Cary, North Carolina: SAS Institute Inc.2013 software. The pre-treatments were carried out in a 1.0 L glasslined pressure Parr reactor equipped with a 4842 Monosaccharides and uronic acids reducing measurement temperature controller (Parr Instrument Company. Mole, IL) for 1 h
(manually controlled). The reactor was cooled until 30°C, and the An aliquot (2.5 mL) from the filtrates obtained after enzymatic pre-treated wood pulps were washed with warm (60°C) hydrolysis was freeze-dried and diluted to 10%, then the ethanol/water (2:1, 3x50.00mL). The washes were combined and 3 monosaccharide and uronic acids content in the filtrate were volumes of water were added to precipitate the Ethanol Organosolv quantified by the High Performance Anion Exchange Chromato- Lignin (EOL), which was collected by centrifugation (3000 rpm, 15 graphy with Pulsed Amperometric Detection (HPAEC-PAD) min and 10°C) and dried. The EOL content is calculated as follows: procedure. A Dionex ICS-3000 HPLC system was used to separate
and quantify neutral sugar and uronic acids. The system consisted ( ) of a SP gradient pump, an AS auto-sampler, an ED electrochemical
detector with gold working electrode, an Ag/AgCl reference electrode, 2590 Afr. J. Biotechnol.
Table 2. Dry density, ash, extractives and Klason lignin content of Okoumé’s sapwood and heartwood.
Samples Dry density (g/cm3)a Ash (%)a Extractives (%)a Klason lignin (%)a Sapwood 0.484±0.034b 0.232d 0.530±0.100e 29.43±1.00d Heartwood 0.429±0.046c 0.224d 1.912±0.090f 29.91±1.19d
Ash, extractives (n=3) and Klason lignin (n=3) content is based on dried wood samples. Density (n=6). Means ith the same letters are not statistically different at the 0.05 level of significance. a Mean±S.D
Table 3. Chemical composition of Okoumé as published by various authors.
Savard Lal et al. Brunck et al. Minkuè M’Eny Safou-Tchiama, 2005*; Medzegue Components (1959) (1977) (1990) (2000) Safou-Tchiama et al., 2007) (2007) Ash (%) 0.4 1.8 0.33 0.43 0.55 - Holocellulose (%) - 77.20 - 75.60 80.0±0.30 78.2±0.20 Hemicelluloses (%) - - - - 32.3±0.50* - Cellulose (%) - - - - 47.9±1.00* - Glucose (%) - - - - 55.70±3.70 - Xylose (%) - - - - 10.30±0.70 - Arabinose (%) - - - - 0.30±0.05 - Galactose (%) - - - - 0.30±0.04 - Mannose (%) - - - - 4.10±0.30 -
The ash content was based on percentage of dried wood sawdust. The sugar content was based on percentage of dried hollocellulose.
and Chromeleon software version 6.8 (Dionex Corp, USA). The previously observed for lodgepole pine (Campell et al., stationary phase used was a Carbopac PA20 (3 × 150 mm), Dionex 1990) and loblolly pine (Thompson et al., 2006). column with a guard column (3 × 50 mm, Dionex), while mobile Furthermore, the extractive content released by the phase was water, 250 mM NaOAc, and 1 M NaOH/20 mM NaOH. Monomers and uronic acids were separated using isocratic heartwood is close to those previously reported by Safou- conditions and linear gradient elution. All eluents were degassed Tchiama (2005) and Medzegue (2007) who obtained before use by flushing with helium for 20 min with 250 mM NaOH 2.1±0.1 and 2.0±0.4% respectively. No significant solution and re-equilibrated for 10 min in the starting conditions. difference was found (P>0.05) between the ash content Samples were injected through 25L full loop at a discharge rate of of sapwood and heartwood (Table 2) which is lower 0.4 mL/min and separations effected at 35°C column temperature. The pulse sequence for pulsed amperometric detection consisted of than those reported by previous works (Table 3). In potential +100 mV (0 to 200 ms), +100 mV integration (200 to 400 addition, no statistical difference (P>0.05) was found in ms), -2000 mV (410 to 420 ms) +600 mV (430 ms), and -100 mV the KL content of the selected samples. The lack of (440 to 500 ms). variability in the lignin content released by the sapwood and heartwood was observed for other wood species (Thompson et al., 2006). However, the KL content Statistical analysis obtained was in the same range with those published by
The data were analyzed by a one-way analysis of variance Brunck et al. (1990) and Minkuè ’Eny (2000) who found (ANOVA) followed by the Fischer’s LSD (last significant difference) 30 and 30.9% (g/dry weight), respectively. test at α=0.05 level of significance with Rr643.0.2 software. The hollocellulose deducted from the KL pointed out that the sapwood should be richer (70.04%) in cellulose- hemicelluloses complex than the heartwood (68.18%) as RESULTS AND DISCUSSION observed in lodgepole pine (Campbell et al., 1990). Nevertheless, the holocellulose content obtained from the Dry density, ash, extractives and Klason lignin inner heartwood is accordingly lower than that published content previously by Lal et al. (1977), Minkué M’Eny (2000), Medzegue, (2007); Safou-Tchiama, 2005 and Safou- The analysis of the biomass (Table 2) pointed out a Tchiama et al. 2007) who investigated the whole significant difference (P<0.05) between the sapwood and heartwood including mature wood assumed to be richer heartwood dry density. The former displayed the highest in fibers than the inner heartwood which is mainly dry density. However, the heartwood was more abundant composed of juvenile wood. The latter is poor in pulps in the extracts than the sapwood. Similar trend was (Campbell et al., 1990). The holocellulose content Safou-Tchiama et al. 2591
Table 4. Pretreatment conditions, ethanol organosolv lignin (EOL) and Klason lignin recovery from Okoumé’s sapwood (SW) and heartwood (HW).
Temperature (°C) Sulfuric acid (%) EOL (%) Klason lignin (%) Reactions SW HW SW HW SW HW SW HW R1 185 185 1 1 16.77 15.43 73.44 81.42 R2 160 160 1 1 19.46 18.10 22.74 28.65 R3 185 185 2 2 17.24 15.02 73.75 76.72 R4 210 210 2 2 06.70 8.25 98.23 93.29 R5 185 185 1 1 11.15 13.98 91.37 83.60 R6 160 160 2 2 17.55 16.93 74.22 72.44 R7 185 185 1 1 11.46 14.75 90.43 83.87 R8 210 210 1 1 07.20 10.06 94.56 88.71 R9 185 185 0 0 09.83 4.02 22.26 23.80 R10 210 210 0 0 15.85 11.77 13.70 16.84 R11 185 185 1 1 14.44 12.36 97.57 90.71 R12 160 160 0 0 0.00 0.00 29.86 26.90
displayed by the sapwood and the inner heartwood of process severity. That result suggests a strong Okoumé is very close to that found for promising susceptibility of the sapwood and heartwood sugars to lignocellulose biomass like Miscanthus x Giganteus degradation, both at high temperature and acid which displayed sugar content of 70% (El Hage, 2010). concentration as observed for other lignocelluloses biomass (El Hage, 2010; Audu et al., 2012). This results in a pseudo-lignin formation which would increase Ethanol organosolv lignin strongly the KL content above the average value of untreated sapwood and heartwood as observed for other The EOL extracted from the wood samples is presented lignocellulosic materials (Hu et al., 2012; Li et al., 2007). in Table 4. Reactions R9 and R10 suggest that EOL could be obtained from the sapwood and heartwood of Okoumé by heating free acid at T=185°C and T=210°C, Enzymatic hydrolysis and sugar content respectively, and the highest acid free EOL content was yielded by sapwood heated at 210°C. Nevertheless, the The pretreated sapwood and heartwood pulps were KL released by R10 is very weak compared to the subjected to enzymatic hydrolysis and the total sugar untreated wood samples (Table 2). This suggests that a based on dried weigh per L (g/L) is presented in Figure 1. mass was lost when the sapwood and heartwood were Reactions R2 (T=160°C; [H2SO4]=1%), R6 (T=160°C; subjected to high temperatures. The polysaccharide [H2SO4]=2%) and R9 (T=185°C; [H2SO4]=0%) were the matrix of the sapwood was more sensitive to temperature most promising for the sapwood while the heartwood increase than the heartwood at [H2SO4]=0% (Table 4). exhibited its highest neutral sugars content under R2, Increasing acid concentration shows that both sapwood R10 (T=210°C; [H2SO4]=0%) and R12 (T=160°C; and heartwood released their highest EOL in R2 [H2SO4]=0%) pretreatment conditions. Those results have pretreatment (Table 4). Their respective content is very pointed out the variability between the sapwood and close to that obtained for a cellulose ethanol candidate heartwood of Okoumé under different temperature and like Pinus taeda which yielded 19% of EOL (Sannigrahi et acid concentrations. al., 2010). Nevertheless, the pretreatment conditions used for that wood specie were energy and acid consuming (T=170°C; [H2SO4]=1.1%). On the other hand, Effect of acid concentration on neutral sugar content both the wood wastes from Okoumé exhibited higher EOL than that extracted from Miscanthus (16%) (El The total sugar content released through enzymatic Hage, 2010). hydrolysis of the pulps is presented in Figure 1. The When both temperature (T>160°C) and sulfuric acid impact of acid concentration on enzymatic hydrolysis of concentration ([H2SO4]>1%) increased, the residual KL the pulps was studied in comparison with the acid free content of the sapwood and heartwood pulps increased reaction R12 (T=160°C; [H2SO4]=0%). It is obvious that as well (Table 4). Both the wood samples exhibited all the neutral sugars content released by the sapwood similar trend and their highest KL content was displayed are lower than those from heartwood. On the other hand, in R4 conditions which corresponds to the maximum the sugar yield order from the sapwood pretreated in R12 2592 Afr. J. Biotechnol.
140
120 sugars
100
total
/L) 80 3
10 60
neutral
(gx
40
Total yield 20 0 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R0 HW 94 26 13 13 13 14 13 13 123 27 126 95
SW 46 22 10 15 74 18 27 125 14 14 13 13
Figure 1. Total neutral total sugars yield obtained after enzymatic hydrolysis of pretreated heartwood (HW) and sapwood (SW) pulps at 160°C≤T≤210°C and 1%≤[H2SO4]≤2%. R0 (T=25°C; [H2SO4]=0%) is the reaction from which enzymatic hydrolysis was performed with unpretreated heartwood and sapwood.
Table 5. Monosaccharaides and uronic acids reducing content (mg/L) of the liquid fraction of Ethanol Organosolv Lignin pulps from Okoumé sapwood (SW) and heartwood (HW) enzymatically hydrolyzed according to the pretreatment conditions.
Rha Ara Gal Glu Xyl Man GalAc GluAc Reaction SW HW SW HW SW HW SW HW SW HW SW HW SW HW SW HW R2 0.00 0.00 1.90 1.33 0.44 0.34 39.28 85.97 2.94 0.61 0.91 5.37 0.26 0.00 0.12 0.22 R3 0.00 0.00 3.80 2.49 0.29 0.33 13.14 18.34 4.43 0.93 0.47 3.56 0.05 0.00 0.00 0.04 R4 0.00 0.00 0.90 0.37 0.30 0.40 7.62 8.12 1.19 0.16 0.52 4.13 0.07 0.00 0.12 0.05 R5 0.00 0.00 1.54 0.78 0.35 0.24 9.94 9.53 2.19 0.41 0.63 2.42 0.07 0.00 0.11 0.04 R6 0.00 0.00 0.93 0.38 0.32 0.39 66.93 8.24 0.40 0.15 4.42 4.24 0.00 0.00 0.16 0.05 R7 0.00 0.00 1.25 0.38 0.36 0.40 11.71 8.32 0.57 0.15 3.76 4.31 0.00 0.00 0.06 0.05 R8 0.00 0.00 1.80 0.37 0.49 0.39 17.66 8.08 0.68 0.15 5.89 4.13 0.00 0.00 0.08 0.05 R9 0.17 0.00 0.42 0.37 1.17 0.38 94.97 8.13 8.22 0.15 18.58 4.16 0.00 0.00 0.16 0.04 R10 0.00 0.00 0.38 0.12 0.41 0.46 8.32 99.97 0.15 4.82 4.32 13.99 0.00 0.00 0.05 0.07 R11 0.00 0.00 0.38 1.00 0.41 0.37 8.30 18.92 0.15 0.41 4.34 5.76 0.00 0.00 0.04 0.07 R12 0.00 0.22 0.37 0.73 0.37 0.93 8.15 96.63 0.15 7.63 4.18 17.68 0.00 0.00 0.04 0.36 R0 0.00 0.06 0.37 0.38 0.40 0.56 8.18 80.38 0.15 2.74 4.20 10.48 0.00 0.05 0.05 0.10
R0 (T=25°C; [H2SO4]=0%). Sulfuric acid concentration is based on (w/w). Rha, rhamnose; Ara, arabinose; Gal, galactose; Glu, glucose; Xyl, xylose; Man: mannose; GalAc, galacto-uronic acid; GluAc, glucuronic acid.
is as follows: Xyl< AraGal < Man < Glu while that from increase of the neutral sugar released by enzymatic the heartwood pulps is: Ara < Gal < Xyl < Man < Glu hydrolysis of the pulps in R2 (T=160°C; [H2SO4]=1%) and (Table 5). That result has pointed out that there is strong R6 (T=160°C; [H2SO4]=2%) compared to R12 (T=160°C; variability between the sapwood and heartwood pulps in [H2SO4]=0%). The neutral sugars yielded from R2 were: enzymatic hydrolysis. The enzymatic hydrolysis of the Gal < Man < Ara < Xyl < Glu (Table 5). The relative heartwood led to a release of a high mannose content, neutral sugars increase (Figure 2) shows clearly that confirming the abundance of mannose within Okoumé increasing acid concentration under the pretreatment cell wall fibers (Safou-Tchiama et al., 2007). conditions of R2 hydrolyzed strongly mannose of the Within the sapwood, Figure 1 shows that increasing sapwood pulps, while the enzymatic hydrolysis of all the acid concentration of the pretreatment leads to significant other neutral sugars was facilitated. The highest increase Safou-Tchiama et al. 2593
4900.00 (R2-R12)*100/R12 (R6-R12)*100/R12 3900.00 (R9-R12)*100/R12
(R10-R12)*100/R12 2900.00
1900.00 increase (%) increase
900.00 Relative neutral sugar yields yields sugar neutral Relative -100.00 SW HW SW HW SW HW SW HW SW HW
Ara Gal Glu Xyl Man
Figure 2. Relative neutral total sugar yield increase after enzymatic hydrolysis of sapwood (SW) and heartwood (HW) pulps from the promising pretreatments R2 (T=160°C, [H2SO4]=1%); R6 (T=160°C, [H2SO4]=2%), R9 (T=185°C; [H2SO4]=0%) and R10 (T=210°C; [H2SO4]=0%) in comparison with the free acid pretreated heartwood and sapwood pulps from reaction R12 (T=160°C; [H2SO4]=0%).
was observed for arabinose, glucose and xylose. noteworthy that xylose remained strongly sensitive to Furthermore, increasing acid concentration for +2% in R6 acid concentration at high temperature as previously leads to an increase in the neutral sugars yielded by the found in other biomass (Xiang et al., 2004). In addition, enzymatic hydrolysis of sapwood pulps (Figure 1). The glucose and mannose have pointed out a strong corresponding sugars were released as follows: Gal < sensitivity towards increased acid concentration within Xyl< Ara < Man < Glu (Table 5). Increasing acid the heartwood pretreated either in R2 (T=160°C; concentration under R6 conditions was favourable to the [H2SO4]=1%) or R6 (T=160°C; [H2SO4]=2%) as shown in enzymatic hydrolysis of glucose. Arabinose and xylose Figure2. Those results have pointed out that heartwood yields remained higher in R12 (Figure 2) whereas they exhibited an opposite response towards acid sulfuric were lower in R2 (Table 5). This suggests the inhibiting increase compared to sapwood. That should be effect of sulfuric acid concentration on enzymatic explained by the difference in chemical composition hydrolysis of arabinose and xylose within the sapwood between Aucoumea klaineana heartwood and sapwood. pulps pretreated in R6 (T=160°C; [H2SO4]=+2%). That The latter was found to be rich in terpens, furans or inhibiting effect was more marked for galactose (Figure phenol compounds bearing esterified 4-hydroxybenzoic 2). acid (unpublished data) described as inhibitors for Raising acid concentration either in R2 or R6 reduced enzymatic hydrolysis of wood pulps (Taherzadeh and the total sugar yielded from the heartwood fibers in R12 Karimi, 2007). (Figure 1). Nevertheless, in R2 reaction, the total sugar yielded from enzymatic hydrolysis of heartwood pulps is higher than that of sapwood (Figure 1); glucose and Effect of temperature on sugar content mannose remained the major neutral sugars within the heartwood pulps (Table 5). With the exception of The total neutral sugars yield (Table 5) shows that arabinose for which raising acid concentration according sapwood and heartwood pulps do not have the same to R2 increased its yield after enzymatic hydrolysis of the sensitivity towards temperature increase. Enzymatic heartwood pulps, the other neutral sugars content hydrolysis of the sapwood pulps pretreated under R9 decreased dramatically (Figure 2). That decrease was (T=185°C; [H2SO4]=0%) conditions released the highest observed for all the neutral sugars released by the total sugars content (Figure 1), and the following sugar enzymatic hydrolysis of the heartwood pulps from R6 yield order was obtained: Ara < Gal < Xyl < Man < Glu pretreatment; and the following sugar yields order was (Table 5). Raising temperature in R9 reaction increased obtained: Xyl < Ara < Gal < Man < Glu, while that from strongly the galactose, glucose, xylose and mannose R2 was: Gal < Xyl < Ara < Man < Glu (Table 5). It is content released from the enzymatic hydrolysis of 2594 Afr. J. Biotechnol.
sapwood pulps when compared to heartwood which the pretreatment process of the sapwood pulps leads to released one of the lowest neutral sugars yielded as an increase in the total neutral sugars content released follows: Xyl < AraGal < Man < Glu (Table 5). The by the enzymatic hydrolysis. Nevertheless, the heartwood results above have pointed out a strong sensitivity of all exhibited a strong sensitivity to temperature and sulfuric the heartwood neutral sugars to temperature increase. acid concentration increase; most of all the neutral They decreased dramatically under the pretreatment sugars tend to be hydrolyzed during the heartwood conditions of R12 (T=160°C; [H2SO4]=0%) (Figure 2). pretreatment process. But further investigations are Nevertheless, enzymatic hydrolysis of the wood pulps necessary for a better understanding of the molecular pretreated under R10 (T=210°C; [H2SO4]=0%) conditions composition and supramolecular organization of the two was favourable to heartwood. The latter exhibited one of EOL. The lignin-hemicellulose linkages as well as the highest total sugar content (Figure 1) and the same cellulose-hemicellulose interactions which control the sugars yield order like R12 pretreatment (Table 5). But access of chemicals and enzymatic hydrolysis shall be raising temperature from 160 to 210°C hydrolyzed performed. The yield of the fermentation process of the dramatically the arabinose, galactose, xylose and neutral sugars from the promising reactions will mannose of the heartwood pulps while enzymatic discriminate between the potential of sapwood and hydrolysis of glucose was favored (Figure 2); confirming heartwood of Okoumé to be used as source for cellulosic that pretreating the heartwood at T=185 or 210°C with ethanol production. [H2SO4]=0% hydrolyzed the neutral sugars in the same extent. Major neutral sugars like glucose and xylose were more sensitive to temperature hydrolysis when the Conflicts of Interests heartwood was pretreated at T=185°C. Finally, comparing the sensitivity of the wood samples The authors have not declared any conflict of interests. to temperature or sulfuric acid concentration increase has pointed out that sapwood and heartwood do not display the same behavior (Figure 2). The heartwood’s galactose, ACKNOWLEDGEMENT xylose and mannose yielded from the enzymatic hydrolysis decreased systematically when temperature This research was possible as a result of the RIFFEAC and sulfuric acid concentration increased in R12. The (Réseau des Institutions de Formation Forestière et same trend was observed not only for arabinose from R6, Environnementale en Afrique Centrale) Master of R9 and R10 pretreatment conditions; but also for the Science degree in Wood Sciences scholarship program. heartwood’s glucose from R2, R6 and R9 (Figure 2). The authors would like to thank ENEF Gabon (Ecole However in Figure 2, it is noteworthy that increasing Nationale des Eaux et Forêts) for his funding and all the temperature and acid concentration point out an inverse supports he brought to this research. The study was also trend for the sapwood. Arabinose, glucose and xylose supported by a fellowship granted by the ANBG (Agence yielded by enzymatic hydrolysis of the sapwood pulps Nationale des Bourses du Gabon). They thank the from R2, R6, R9 and R10 increased indeed, and the LERMAB (Laboratoire d’Etude et de Recherche sur le galactose yielded from R6, R9 and R10 increased in R12 Matériau Bois, Université de Loraine) for the scientific as well; whereas mannose content from the R6, R9 and and technical support. R10 pretreatment conditions increased compared to the heartwood. REFERENCES
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Vol. 15(46), pp. 2596-2602, 16 November, 2016 DOI: 10.5897/AJB2016.15279 Article Number: 6F97CFB61671 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2016 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Comparison of seed priming techniques with regards to germination and growth of watermelon seedlings in laboratory condition
Willams Ferreira Souza Barbosa, Fábio Steiner*, Leandro Castro Matias de Oliveira and Paulo Henrique and Menezes das Chagas
Mato Grosso do Sul State University - UEMS, Department of Agronomy, Rodovia MS 306, km 6.4, Cassilândia, Mato Grosso do Sul, Brazil.
Received 15 February, 2016; Accepted 11 November, 2016
Seeds of watermelon [Citrullus lanatus (Thunb.) Matsum and Nakai, cv. Crimson Sweet] were used to investigate the effects of different priming techniques on seed germination and early seedling growth. The seeds were soaked in solutions of 0.2% gibberellin (50 mg L–1 gibberellic acid), 0.2% cytokinin (90 –1 –1 –1 mg L kinetin), 0.2% potassium nitrate (2 g L KNO3), 0.2% calcium nitrate [2 g L Ca(NO3)2] or water (hydropriming) for 6 h at 25°C. After drying, five replicates of 25 seeds were distributed in plastic boxes with blotter paper and kept into a seed germinator at 26°C for 12 days. The different priming treatments significantly affect the measurements of the seed germination and growth of watermelon seedlings. The germination of watermelon seeds ranged from 0 to 100%, and was significantly greater when seeds were subjected to priming with GA3, KNO3, Ca(NO3)2 and water (control), and lower under cytokinin (CK) priming. The seed priming with 0.2% solution of CK resulted in 100% of abnormal seedlings, and therefore should not be used by watermelon growers. Seed priming with GA3, KNO3, Ca(NO3)2 and water (hydropriming) increased the shoot length, whereas GA3, Ca(NO3)2 and water priming improved the radicle length, as well as shoot dry matter watermelon seedlings. The KNO3 and water priming increased the root dry matter of watermelon seedlings. Seed priming with KNO3 and Ca(NO3)2 improved the germination rate and seedling vigor index. The results of this study show that seed priming with GA3, KNO3, Ca(NO3)2 and water (hydropriming) may be useful tools due to their positive effects on germination percentage and growth characteristics of watermelon seedlings.
Key words: Citrullus lanatus, cytokinin, gibberellin, hydropriming.
INTRODUCTION
Watermelon [Citrullus lanatus (Thunb.) Matsum and (Cucurbitaceae). It is a native species from warm and dry Nakai] is an annual vine of the cucurbit family regions of Africa, and now widely cultivated throughout
*Corresponding author. E-mail: [email protected]. Tel: +55 67 3596 7622.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Barbosa et al. 2597
the world for its edible fruits (Filgueira, 2008). Among the Batista et al. (2015) studied the effect of different priming cucurbits, watermelon is the most popular in Brazil and in techniques on germination and growth of pepper and -1 the world. Optimum seed germination and seedling reported that GA3 at 200 mg L enhanced the germination emergence in watermelon occur at relatively high and seedling growth when compared to unprimed seeds. temperatures (25-28°C). Poor seed germination is a Iqbal et al. (2006) showed that application of CK at 100 or common phenomenon at sub-optimal temperatures 200 mg L-1 increased the germination rate and early (Demir and Mavi, 2004), which causes a great concern seedling growth of wheat when compared with for growers that growing watermelon seedlings in late hydropriming treatment. Cytokinins at 10 or 100 mg L-1 winter and early spring in the southern and south eastern significantly increased the germination rate of pigeon pea regions of the Brazil. These two regions were responsible seeds compared to unprimed seeds (Sneideris et al., for nearly 50% of Brazilian production of watermelon in 2015). However, seed priming with CK at 50 or 100 mg L- the 2014/2015 season (AGRIANUAL, 2015). Delayed and 1 inhibited the primary root development of maize reduced seedling emergence cause non-uniform stand seedlings compared to control. These and other establishment, which result in yield reductions (Singh et contradictory results seem to indicate an inherent al., 2001) and impairs the early watermelon markets in differential response among different species or the cool regions of Brazil. genotypes; therefore, justifying the need of conducting Many treatment techniques have been developed to more research in order to investigate the effects of seed improve the germination of watermelon seeds, especially priming with CK on germination and early growth of under improper conditions. There is no universal watermelon. technique for improving seed germination. Among the This research was carried out to investigate the effects methods used, pretreatment of seeds with plant growth of different priming techniques on seed germination and regulators and salts are considered the most appropriate initial growth of watermelon seedlings [C. lanatus and promising because of ease of application, scale of (Thunb.) Matsum and Nakai]. economies, and labor-saving attributes compared with methods in which the environment must be controlled for MATERIALS AND METHODS prolonged periods of time (Demir and Mavi, 2004; Nascimento, 2005; Ghassemi-Golezani and Esmaeilpour, Plant material and priming treatments 2008). Indeed, seed priming treatments using salts such The experiment was conducted in the Plant Propagation Laboratory as potassium nitrate (KNO3) have been effective in improving watermelon germination under improper of the Mato Grosso do Sul State University (UEMS), in Cassilândia, S, Longitude: 51°48'24'' W), during ''20'05؛MS, Brazil (Latitude: 19 conditions (Demir and Mavi, 2004; Nascimento, 2005). the month November 2015. Hydropriming treatment has also be successfully applied Seeds of watermelon [Citrullus lanatus (Thunb.) Matsum and to improve germination performance of watermelon Nakai, cv. Crimson Sweet] were surface sterilized with 1% (v/v) of (Huang et al., 2002) and cucumber seeds (Gurgel et al., sodium hypochlorite solution for 5 min and washed immediately 2009). However, few reports were documented on with distilled water many times. The sterilized seeds were then subjected to priming by direct immersion in solutions of 0.2% priming treatments using plant growth regulators (PGRs) -1 gibberellin (50 mg L gibberellic acid - GA3), 0.2% cytokinin (90 mg such as gibberellin and cytokinin in watermelon seeds. -1 -1 L kinetin - CK), 0.2% potassium nitrate (2 g L KNO3), and 0.2% -1 Cytokinin (CK) and gibberellin (GA3) are key hormones calcium nitrate [2 g L Ca(NO3)2] for 6 h at 25°C. A set of seeds controlling plant development. These plant hormones subjected to direct immersion in distilled water was taken as control. have an important role on several physiological and After priming period, seeds were put to dry in plastic boxes (11.0 × developmental processes, control of the cell cycle, apical 11.0 × 3.5 cm, type Gerbox) with germitest paper at room temperature (24-28°C) for 48 h (Eira and Marcos-Filho, 1990). dominance, including morphogenesis of shoots and roots, lateral root initiation, stem elongation, leaf and cotyledon expansion, and regulation of senescence (Al- Germination and growth conditions Al-Khassawneh et al., 2006; Taiz and Zeiger, 2010; Kerbauy, 2012). Seed priming with optimal Five replicates of 25 seeds were evenly distributed in plastic boxes with blotter paper, properly moistened with distilled water, in a concentrations of CK and GA3 has been shown to have volume equivalent to 2.5 times the mass of dry paper. The boxes beneficial effects on germination, growth and yield of a were then closed with lids to prevent evaporation and maintain the wide range of plant species (Jamil and Rha, 2007; relative humidity close to 100%. Germination was carried out in a Alonso-Ramirez et al., 2009; Nasri et al., 2012; Kandil et germination chamber under 12/12 h photoperiod (light/darkness), −2 −1 al., 2014). light fluence of 40 μmol m s photosynthetic photon flux density Gibberellin at 200 mg L–1enhanced the seed (PPFD), relative humidity of 90% (± 5%) and temperature of 26°C (± 2°C) for 12 days. Germinated seeds were recorded every 24 h germination and seedling growth of papaya (Lopes and for 12 days. Souza, 2008). Nasri et al. (2012) reported GA3 increased germination percentage of lettuce under salt stress Measurements of germination and seedling growth conditions. Albuquerque et al. (2009) reported GA3 increased the growth characteristic in sweet pepper. The germination (GC), germination rate index (GRI), early growth 2598 Afr. J. Biotechnol.
and vigor index of watermelon seedlings were measured. At 12 growth of the seedlings have been reported by other days were also measured the percentage of abnormal seedlings authors. Alonso-Ramirez et al. (2009) showed that GA3 and dead seeds. The Equations 1 and 2 and the parameters therein have strong stimulatory effect on seed germination, of were employed to express the process of seed germination: which their exogenous application was repeatedly found G (%) = SNG / SN0 × 100 (1) to promote germination of Arabidopsis seeds even under unfavorable stress conditions. Seed priming with GA3 Where G is germination percentage, SNG is the number of caused an increase in seed germination and seedling germinated seeds, and SN0 is the number of experimental seeds growth of sweet pepper (Albuquerque et al., 2009), with viability (25 seeds): lettuce (Nasri et al., 2012) and sugar beet (Jamil and GRI = Σ (ni / ti) (2) Rha, 2007; Kandil et al., 2014). Batista et al. (2015) showed that all the priming methods tested (that is, GA3, –1 Where, GRI is the germination rate index (seed day ), ni is the KNO3, Ca(NO3)2 and hydropriming) resulted in the number of germinated seeds on a given day, and ti is the time in improvement of germination rate of pepper seeds when days from the starting/sowing day (0) (Maguire, 1962). compared to unprimed seeds (control). Huang et al. The shoot and radicle length was measured in 15 normal seedlings randomly obtained after count of the total germination (2002) and Gurgel et al. (2009) reported that (12th day) using meter scale. The results were expressed in hydropriming treatment can be successfully applied on centimeter (cm). For the determination of dry matter of shoot and watermelon and cucumber seeds to improve germination roots, all normal seedlings obtained at the end of the germination performance, respectively. period were separated into shoot and roots, dried in a forced air The germination rate index ranged from 0 to 6.4 seed C, and then weighed. The day–1, and was significantly greater under KNO and؛circulation oven for three days at 60 results were expressed in mg seedling–1. 3 Vigor index of seedlings was calculated according to Zhang et al. Ca(NO3)2 priming, followed by GA3 priming and (2007) as shown in Equation 3: hydropriming (control), and lower under CK priming (Figure 1B). High values obtained for germination rate SVI = SL × Σ (ni / ti) (3) index indicate mean higher seed vigor of one treatment in relation to another. Marcos-Filho (2015) reported that the Where SVI is seedling vigor index, S is the shoot length in the L uniformity and speed of seedling emergence are twelfth day (cm), ni is the number of germinated seeds on a given day, and ti is the time in days from the starting/sowing day (0). important components of seed performance, thus directly affecting stand establishment. The seed priming with 0.2% solution of CK resulted in Statistical analyses 100% of abnormal seedlings (Figure 2), and was significantly greater than the other priming techniques The normality of data was previously tested by the Kolmogorov- Smirnov test and then data were submitted to analysis of variance used (Figure 1C). Although cytokinins are required for (ANOVA), and means of five priming treatments were compared by many growth and developmental processes in plants the Tukey test at the 0.05 level of confidence. For statistical such as cell division, morphogenesis of shoots and roots, analysis, the data expressed in percentage were previously apical dominance, chloroplast maturation, leaf and 0,5 transformed into arc sin (x/100) . The analyses were performed cotyledon expansion, and seed germination (Hirose et al., using Sisvar version 5.3 software for Windows (Statistical Analysis 2008; Taiz and Zeiger, 2010; Kerbauy, 2012), the Software, UFLA, Lavras, MG, BRA). exogenous application of supra-optimal cytokinin concentrations has remarkable effect on the inhibition of RESULTS AND DISCUSSION cell elongation process in both shoots and roots (Taiz and Zeiger, 2010). The inhibition of internode and root Effects of priming techniques on seed germination elongation induced by excess cytokinin is due to the production of ethylene triggered by the enzyme 1- The different priming treatment significantly affects the aminocyclopropane-1-carboxylic acid synthase (ACS) measurements of the germination process and rate of (Kerbauy, 2012). These results indicate that chances in watermelon seeds (Figure 1). The germination percentage endogenous CK concentration may negatively regulate values in the control treatment (Figure 1A) were higher elongation of shoots and roots, as seen in Figure 2. than the standard values (that is, 80%) for commercial Aragao et al. (2001) studied the effect of seed priming watermelon seeds in Brazil (BRASIL, 2012), indicating with CK on germination and growth of maize and that the seeds used in this study had high physiological reported that benzyl aminopurine (BAP) at 50 or 100 mg quality. L –1 inhibited the development of the primary root The germination percentage of watermelon seeds compared to control. However, seed priming with optimal ranged from 0 to 100%, and was significantly greater concentrations of CK has been shown to have beneficial when seeds were subjected to priming with GA3, KNO3, effects on germination and early growth of wheat (Iqbal et Ca(NO3)2 and water (control), and lower under CK al., 2006) and pigeon pea (Sneideris et al., 2015). priming (Figure 1A). The high efficiency of seed priming Another factor that may be related to inhibiting the with PGRs and salts in improving the germination and growth of watermelon seedlings is that the action of CK is Barbosa et al. 2599
a a a B 8 100 A ) a 1 a – a 7 80 6 b b (%) 5 60 4
40 3 Germination Germination 2 20 1
b c Germination rateindex (seedday 0 0 b 100 C D 100
80 80
60 60
40 40 Dead Dead seeds(%)
Abnormal Abnormal seedlings(%) 20 20 a a a b a a a ab ab 0 0 GA CK KNO3KNO3 Ca(NOCa(NO3)23)2 Control GA CK KNO3KNO3 Ca(NO3)2Ca(NO3)2 Control Treatments Treatments
Figure 1. Effect of different priming treatments on the germination percentage ; 12 days (A), germination rate index (B), abnormal seedlings (C), and dead seeds (D) of watermelon seeds [Citrullus lanatus (Thunb.) Matsum and Nakai, cv. Crimson Sweet]. Bars followed by the same lower case letters are not significantly different by Tukey test at the 0.05 level of confidence. Data refer to mean values (n = 4) standard error.
light-dependent. Changes in fluence rate of white light watermelon seedlings (Figure 3A). These results indicate were shown to have effect on the action of CK and that the seed priming with gibberellic acid, salts or water therefore one elongation of the stem and root. Under were adequate to promote the shoot growth of conditions of low light fluence, as in this study (40 μmol watermelon. Batista et al. (2015) also reported the −2 −1 m s PPFD), the cytokinin inhibits elongation of shoots efficiency of GA3, KNO3, Ca(NO3)2 and water priming to and roots (Kerbauy, 2012). enhance the shoot growth of per seedlings compared to The percentage dead seed varied from 0 to 5%, and unprimed seeds. was significantly greater under GA3 priming, and lower Radicle length of the watermelon seedlings was under CK and KNO3 priming (Figure 1D). The low favored under hydropriming (3.95 cm), followed by percentage of dead seeds is indicative of the high initial Ca(NO3)2 and GA3 priming, whereas the KNO3 priming viability of watermelon seeds used. had the lowest effect (2.62 cm) (Figure 3B). Shoot dry matter of watermelon seedlings was significantly higher under GA3, Ca(NO3)2 and water priming (Figure 3C), Effects of priming techniques on initial seedling whereas the higher dry matter of the roots was obtained growth with KNO3 and water priming (Figure 3D). Demir and Mavi (2004) and Nascimento (2005) reported that seed The growth of watermelon seedlings was significantly priming with KNO3 enhanced the seed germination and affected by different priming treatments (Figure 3). Seed growth of watermelon under improper conditions. The priming with GA3, KNO3, Ca(NO3)2 and water high efficiency of hydropriming treatments in improving (hydropriming) resulted in higher shoot length of the early seedling growth was also reported previously in 2600 Afr. J. Biotechnol.
10 mm
Figure 2. Abnormal seedlings proceeding from watermelon seeds of the cultivar Crimson Sweet subjected to priming by direct immersion in 0.2% cytokinin solution (90 mg L–1 of kinetin) at 12 days after sowing. The illustration shows seedlings with malformed roots, thickening of the hypocotyl, and without the formation of shoot.
watermelon (Huang et al., 2002), lentil (Ghassemi- seed priming with GA3, KNO3, Ca(NO3)2 and water Golezani et al., 2008) and cucumber (Gurgel et al., (hydropriming) can be successfully applied to improve the 2009). Seed priming with GA3 has been shown to have germination and initial growth of watermelon seedlings. beneficial effects on germination and growth of a wide Germination and seedling emergence stages are critical range of plant species (Jamil and Rha, 2007; Lopes and for crop production; rapid and uniform field emergence is Souza, 2008; Albuquerque et al., 2009; Alonso-Ramirez essential to achieve high yield and uniform plant stands, et al., 2009; Nasri et al., 2012; Kandil et al., 2014). resulted in early maturity and reduced disease attack The seedling vigor index ranged from 0 to 72.2, and (Singh et al., 2001; Subedi and Ma, 2005). was significantly greater under KNO3 and Ca(NO3)2 priming, followed by GA3 priming and hydropriming (control), and lower under CK priming (Figure 4). The Conclusions vigor tests allow identifying the seeds with higher or lower probability to show better performance in field conditions. Seed priming with GA3, KNO3, Ca(NO3)2 and water Vigorous seeds more efficiently mobilize reserves from (hydropriming) may can be successfully applied on storage tissues to the embryo axis and this capacity is watermelon seeds to improve germination performance reflected in higher seedling growth (Marcos-Filho, 2015). and growth characteristics of seedlings. Seed priming Therefore, vigor tests are important tools as an aid to with KNO3 and Ca(NO3)2 improved the germination rate germination test in research on physiological conditioning and seedling vigor index. The seed priming with 0.2% of seeds. solution of CK (90 mg L-1 kinetin) inhibited the In general, the results presented here indicate that germination and cell elongation process of the seedlings. Barbosa et al. 2601
A B 15 a 5 a a a 12 a 4 ab ab
(cm) b 9 3
6 2 Shoot length 3 1 Radicle length (cm)
b c 0 0
25 C D 5
)
)
1 1
a – – ab 20 ab 4 b a 15 3 ab bc c 10 2
5 1 Root dry Rootdry matter (mg seedling Shoot dry matter (mg seedling c d 0 0 GA CK KNO3KNO3 Ca(NOCa(NO3)23)2 Control GA CK KNO3KNO3 Ca(NO3)2Ca(NO3)2 Control Treatments Treatments
Figure 3. Effect of different priming treatments on shoot length (A), radicle length (B), shoot dry matter (C) and root dry matter (D) of watermelon seedlings [Citrullus lanatus (Thunb.) Matsum and Nakai, cv. Crimson Sweet]. Bars followed by the same lower case letters are not significantly different by Tukey test at the 0.05 level of confidence. Data refer to mean values (n = 4) standard error.
Conflict of interests a 80 a The authors have not declared any conflict of interests.
b 60 b REFERENCES
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Vol. 15(46), pp. 2603-2612, 16 November, 2016 DOI: 10.5897/AJB2015.15118 Article Number: 03BDE3A61673 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2016 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Linkage disequilibrium and association mapping of drought tolerance in cotton (Gossypium hirsutum L.) germplasm population from diverse regions of Pakistan
Abdelhafiz Adam Dahab1*, Muhammad Saeed2, Nada Babiker Hamza3, Bahaeldeen Babiker Mohamed1 and Tayyab Husnain4
1Environmental and Natural Resources Research Institute (ENRRI), National Centre for Research (NCR), Khartoum, Sudan. 2Department of Botany, Government College University, Faisalabad, Pakistan. 3Commission for Biotechnology and Genetic Engineering, National Centre for Research, Khartoum, Sudan. 4Centre of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore, Pakistan.
Received 22 November, 2015; Accepted 1 February, 2016
Drought stress is a major abiotic stress that limits crop production. Molecular association mapping techniques through linkage disequilibrium (LD) can be effectively used to tag genomic regions involved in drought stress tolerance. With the association mapping approach, 90 genotypes of cotton Gossypium hirsutum, from diverse regions of Pakistan were used. The morpho-physiological traits of all genotypes were evaluated in greenhouse under well-watered and drought stress conditions. Mean squares from analysis of variance for all morpho-physiological traits revealed highly significant variations (P≤0.05) between water levels and genotypes. Cotton varieties were screened for polymorphism with 180 simple sequence repeat (SSR) markers. Out of these 180 SSR markers, 95 were polymorphic. Genotyping of the selected 95 SSR primer pairs generated 57.5% polymorphism, and the number of polymorphic alleles per primer was 2.10. Population structure, linkage disequilibrium, and association mapping between pairs of SSR marker loci were studied. The significance of pairwise LD (P≤0.005) among all possible SSR loci was evaluated at significant threshold values (R2≥0.05); 7.1% of the SSR marker pairs showed significant pairwise LD in 90 accessions of G. hirsutum. Also we observed a significant (R2) LD between 13 pairs of SSR loci; each pair within the same chromosome in a range of 180 cM between NAU1230 and NAU3095 loci in chromosome (D5) and 1.612 cM between NAU462 and NAU3414 in chromosome A9. This indicates tight linkage between two alleles on the same chromosome. Markers, NAU3414, NAU2691, NAU1141 and NAU1190 were associated with more than single traits under drought treatments. Highest phenotypic variance explaining (R2) was ascribed to NAU3011 chromosome D13 significantly (p0.001) associated with root length under drought treatment.
Key words: Cotton Gossypium hirsutum, drought stress, association mapping, linkage disequilibrium (LD), simple sequence repeats (SSRs).
INTRODUCTION
Global climate change poses serious problems for deficit for agricultural production, development of drought sustained crop production. Due to the continuous water tolerant crop to meet the food and fibre demand has 2604 Afr. J. Biotechnol.
become a necessity (Saeed et al., 2012). Increasing 2012), bean (Shi et al., 2011), sugar beet (Würschum et aridity of semi-arid regions and limited water resources al., 2011), cotton fiber quality traits (Abdurakhmonov et have led to a crucial necessity for improving crop drought al., 2009) and cotton salinity stress tolerance (Saeed et resistance (Passioura, 2007). There is extensive al., 2014). Cotton Gossypium spp. is widely used for research on genetic and breeding programs of cotton and natural fiber in the textile industry. The worldwide it has a long history of improvement through conventional commercial effect of cotton production is ~$500 billion per breeding and selection with frequent long-term yield annum with consumption of ~115-million bales or ~27- achievements. The identification of drought-related QTLs million metric tons (MT) of fibre (Chen et al., 2007). plays an essential role in crop improvement through Cotton is one of the main warm-season cultivars, grown marker assisted selection. DNA marker studies have laid during summer in arid and semiarid regions where water basis for revealing the molecular basis for the traits is limited (Singh et al., 2007). The adaptive traits of plants related to drought tolerance (YongSheng et al., 2009). to unfavourable environmental conditions include Among the variety of genetic markers, SSR markers have numbers of physiological, morphological and biochemical shown high potential to detect polymorphism (Dongre et features of whole plant (Saeed et al., 2012). Genes al., 2011; Dahab et al., 2013) and have been used involved in molecular mechanisms can be tagged with extensively for cotton genome mapping and marker the help of molecular mapping approaches. In our assisted selection (He et al., 2007). Researchers have present study, we assessed extent of LD in the G. mapped QTLs for morphological traits (Liang et al., hirsutum germplasm from diverse regions of Pakistan. 2014), physiological traits (Saeed et al., 2011), earliness The aims of this study were to assess the population (Li et al., 2013), yield (LiFang et al., 2010) and fibre traits structure, linkage disequilibrium (LD), and association of (Islam et al., 2014). Abdelraheem et al. (2015) assessed molecular markers with drought stress tolerance of cotton a backcross inbred line (BILs) population derived from a (G. hirsutum L.) in a collection of 90 elite cotton cross between Pima cotton, ‘Pima S-7’ (Gossypium germplasm accessions. barbadense) and Upland cotton ‘Sure- Grow 747’ (Gossypium hirsutum) for their drought tolerance morphological traits using PEG induced osmotic stress. A MATERIALS AND METHODS total of six QTLs were detected for plant height (PH), fresh shoot and root weight (RW), explaining 10.9 to Plant 19.2% of the phenotypic variation (PV). Rodriguez-Uribe et al. (2014), studying drought tolerance of cotton, stated The plant material consisted of 90 genotypes of cotton G. hirsutum that, of a total of 110 drought-responsive genes identified (Table 1) collected from diverse regions of Pakistan. The cotton varieties were grown in green house during March-April 2010. Four through a microarray analysis, there was 79% seeds of each genotype were sown after soaking overnight at field expression. Saeed et al. (2011) evaluated F2, F2:3 and capacity of 2-3 cm3 depth in 8 polythene bags of 25×5 cm2 , filled F3:4 populations derived from an intraspecific cross with ~ 1.15 kg of compost soil (peat, sand, soil, 1:1:1). They were between two G. hirsutum lines for morphological, arranged in a randomized complete block design with three physiological and yield traits. Seven QTLs were detected replications and two treatments. One set consists of 4 bags kept as for osmotic potential, osmotic adjustment, plant height, a control (W1) and the other as water stressed (W2). After germination, only one plant/bag was kept for data recording. and seed cotton yield. Association mapping, based on Standard pH (6.5), temperature (25 ± 2°C), humidity (50%) and light linkage disequilibrium (LD), is a new methodology which requirements (13 h photoperiod) for cotton growth were maintained examines thousands of polymorphisms for assessing throughout the total duration of experiment. Seedlings grown under QTL effect. It is more effective compared to linkage both stress and non-stress conditions were irrigated and fertilized till analysis since it does not require generation of the development of the first true leaf, and thereafter, seedlings grown under control condition (W1) were watered daily to keep the segregating populations/large numbers of progeny. soil at field capacity. Water stress condition was developed by Association mapping has three advantages: increased withholding water supply to the seedlings grown under water stress mapping resolution, reduced research time, and greater condition (W2), and the effect of water stress was monitored visually allele numbers. It is a powerful technique used to identify and with soil moisture meter (HH2 Theta Probe Type, Delta-T genomic regions linked to specific variants of a device, Cambridge, England). At initial wilting stage (observed phenotypic trait (Saeed et al., 2014). Genome-wide visually), when soil had 14 to 16% soil moisture content, the stressed plants were watered to relieve the sign of wilting but not association in plants has wide range of use and there are enough to reach field capacity. The experiment was continued for many reports of association studies on many crops such 45 days from the date of emergence till the 3rd main stem leaf was as barley (Gutiérrez et al., 2011; Cockram et al., 2010), fully expanded. The plants grown under normal water supply and rice (Shao et al., 2011), bread wheat (Yu et al., 2012), stressed conditions were measured for morphological and maize (Poland et al., 2011), triticale (Niedziela et al., physiological parameters.
*Corresponding author. E-mail: [email protected].
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Dahab et al. 2605
Table 1. List of cultivars used in the study.
S/N Cultivar name S/N Cultivar name S/N Cultivar name 1 CIM-496 31 MNH-806 61 XU-ZHOU-142 2 CIM-482 32 MNH-802 62 XING TAI-68-71 3 CIM-473 33 MNH-770 63 XIAO-VEMIAN 4 CIM-448 34 MNH-720 64 VH-148 5 CIM-446 35 MNH-638 65 UA-73 6 CIM-4/99 36 MNH-636 66 UA-7-25/46 7 CIM-1100 37 MNH-6070 67 UA-31-102 8 CIM-109 38 MNH-554 68 U-4(5143) 9 CHINA 39 MNH-552 69 U-4 10 BT-3701 40 MNH-147 70 U-276 11 BT-2009 41 MG-66 71 TH-35/99 12 BT-1573 42 M-944-0243 72 Stoneville-825 13 BS-1 43 M-944-00-0030 73 Stoneville-213 14 BH-118 44 L-S-S 74 Stonevilla 15 B-557 45 LA-208 75 STAMP-82 16 AYT-85094 46 IS-7-F1 76 SLS-1 17 AYT-85094 47 IRMA-1480 77 SLH-284 18 Australia-407721 48 GR-156- 78 S-14 19 4 F 49 FH-901 79 S-12 20 362-F 50 FH-900 80 S-11 21 299F 51 FH-682 81 Rehmani 22 268-F 52 FH-125 82 N-KARSHOMG 23 268 F 53 FH-113 83 NIBGE-4 24 1027 ALF 54 FH-1000 84 NIBGE-4 25 1021(KIVI) 55 841/52 85 NIAB--78 26 65090 56 CRSM-38 86 MS-93 27 6040 57 CRSM-38 87 MS-40 28 3996 58 CRS-2009 88 MNH-814 29 2616 59 CIM-534 89 MNH-812 30 814 60 CIM-506 90 MNH-807
Phenotyping of plant materials method described (Zhang et al., 2000). The DNA samples were stored at -20°C until further use. The DNA quality was evaluated On 26th April, 2010 green-house experiment was completed and with 1% agarose gel electrophoresis prepared using 0.5X TAE the following parameters were measured. First, plant length (PH) buffer, and ethidium bromide (10 ng/100 ml) was added to the gel and plant fresh weight (PFW) were recorded after the plants were to stain the DNA bands. The samples were electrophoresed for separated into shoot and root parts, and data were recorded for approximately 30 min after which the products were viewed using shoot length (SL), root length (RL), fresh shoot weight (FSW) and an ultraviolet trans- illuminator and photographed using the fresh root weight (FRW). The respective shoots and roots of all Syngene Gel Documentation System. The DNA concentration was plants were then oven-dried at 70°C till a constant dry weight was estimated by the absorbance at 260 nm. The working DNA samples reached. The dry weight of shoot and root of respective plants were (containing 50 ng μL-1) were stored at 4°C for genotyping. SSR recorded and summed up to get the dry plant weight (DPW). The primer pairs used were from different sources: NAU from Nanjing root shoot ratio (RSR) was calculated using the formula: RSR= Agricultural University, Nanjing, China (Han et al., 2006); BNL DRW/DSW. Relative water contents (RWC) were calculated using primers from Research Genetics Co. (Huntsville, AL, USA, the following formula: RWC = [(Fresh weight - Dry weight) / (Turgid http://www.resgen.com); JESPR from sequences of Reddy et al. weight – Dry weight)] × 100. (2001); TM from Dr. John Yu, USDA-ARS, Crops Germplasm Research Unit, TE, USA; CIR from Nguyen et al. (2004) (Table 2). Details about these markers can be found at SSR genotyping www.cottonmarker.org. All 90 accessions were genotyped using a 180 core set of SSR marker primers. These chromosome-specific For extraction of the genomic DNA from each accession group, 4 to primer pairs were selected using the results of different laboratories 5 young fully expanded leaves from each plant were collected and and published papers (Siu et al., 2000; Han et al., 2006; Shen et stored at −80°C. The genomic DNA was isolated from the frozen al., 2005; Abdurakhmonov et al., 2007). They were based on leaf tissues using the cetyltrimethyl ammonium bromide (CTAB) information related to important QTLs and chromosome distribution. 2606 Afr. J. Biotechnol.
Table 2. List of SSR markers used in the study.
S/N Primers chr.0 Position S/N Primers chr.0 Position 1 BNL1053 - - 49 NAU2691-275 D3(ch.17) 81.063 2 BNL1421 A1(ch.1) 163.5 50 NAU2697-165 D13(ch.18) 87.399 3 BNL1606-180 D3(ch.17) 50.727 51 NAU2714-450 D6(ch.25) 112.068 4 BNL1672-110 A9(ch.9) 60.015 52 NAU2974_195 D7(ch.16) 101.7 5 BNL226 D2(ch.14) 120 53 NAU2980-270 D13(ch.18) - 6 BNL2569-170 A6(ch.6) 111.001 54 NAU2995-195 A7(ch.7) 70.732 7 BNL3089-125 A4(ch.4) 3.399 55 NAU3011-330 D13(ch.18) 98.587 8 BNL3280-195 D10(ch.20) 107.214 56 NAU3053-190 D7(ch.16) 30.704 9 BNL3359-210 A6(ch.6) 134.565 57 NAU3084-235 D12(ch.26) 130.319 10 BNL3590-180 A2(ch.2) 61.01 58 NAU3092-245 D5(ch.19) 9.968 11 BNL409-100 A13(Chr.13) 127.092 59 NAU3095-225 D5(ch.19) 180.972 12 BNL448 D4(ch.22) 22.7 60 NAU3100-195 D9(Chr.23) 25.886 13 BNL946-335 D10(ch.20) 61.17 61 NAU3206-500 A6(ch.6) 36.353 14 JESPR153-130 A13(ch.13) 62.641 62 NAU3207-275 A8(ch.8) 70.619 15 JESPR220-200 D4(ch.22) 102.872 63 NAU3209-265 D2(ch.14) 104.477 16 JESPR274-105 A9(ch.9) 42.802 64 NAU3254-1600 A1(ch.1) 102.873 17 JESPR291-175 D8(ch.24) 152.036 65 NAU3306-2400 D6(ch.25) 39.547 18 JESPR92 D12(ch.26) 132.1 66 NAU3385-500 A1(ch.1) 74.005 19 NAU1070-165 D2(ch.14) 13.512 67 NAU3414-225 A9(ch.9) 119.156 20 NAU1102 D5(ch.19) 0 68 NAU3424-210 D7(ch.16) - 21 NAU1103 D11(ch.21) 7.5 69 NAU3519-215 A12(ch.12) 146.714 22 NAU1125 D8(ch.24) 47.7 70 NAU3529-295 A5(ch.5) 117.035 23 NAU1141-200 A13(ch.13) 31.193 71 NAU3606 - - 24 NAU1167-200 A3(ch.3) 105.325 72 NAU3608-220 D7(ch.16) 121.531 25 NAU1190_400 A3(ch.3) 62.9 73 NAU3654-350 A7(ch.7) 23.067 26 NAU1200 A5(ch.5) 16.6 74 NAU3695_280 A11(ch.11) 125.6 27 NAU1218-150 A6(ch.6) 57.568 75 NAU3703_660 A11(ch.11) 67.3 28 NAU1230 D5(ch.19) 0.2 76 NAU3860_250 A12(ch.12) 85 29 NAU1233 A10(ch.10) 40.2 77 NAU3901_280 D1(ch.15) 117.3 30 NAU1254-305 A8(ch.8) 21.557 78 NAU3903_300 D2(ch.14) 110.1 31 NAU1266 - - 79 NAU3916-320 D10(ch.20) 30.947 32 NAU1350-305 D8(ch.24) 107.342 80 NAU3961-210 D12(ch.26) 48.569 33 NAU1366-700 D11(ch.21) 142.709 81 NAU4073-160 A1(ch.1) 53.3 34 NAU1369-410 A8(ch.8) 43.809 82 NAU418 D9(ch.23) 99.6 35 NAU2016-250 A11(ch.11) 18.128 83 NAU437-290 A2(ch.2) 52.833 36 NAU2169-700 D8(ch.24) 77.052 84 NAU4516 D13
37 NAU2190-360 D2(ch.14) 24.1 85 NAU453-180 D10(ch.20) - 38 NAU2265-380 A2(ch.2) 5.902 86 NAU4565 - - 39 NAU2306-380 D8(ch.24) 50.968 87 NAU458-190 D1(ch.15) 12.422 40 NAU2317-220 A10(ch.10) 2.554 88 NAU462-650 A9(ch.9) 117.544 41 NAU2336-215 D2(ch.14) 54.308 89 NAU483-250 A3(ch.3) 27.986 42 NAU2355b - - 90 NAU4956-240 D7(ch.16) 50.973 43 NAU2439-150 D8(ch.24) 70.516 91 NAU5091-160 D11(ch.21) 66.492 44 NAU2443-150 D13(ch.18) 61.754 92 NAU5189-280 D9(ch.23) 140.07 45 NAU2477-200 A4(ch.4) 82.172 93 NAU6106 - - 46 NAU2503-250 D5(ch.19) 175.169 94 NAU6191 - - 47 NAU2527 A10(ch.10) 31.7 95 TMP20-205 A11(ch.11) 72.758 48 NAU2540_160 D10(ch.20) 25.4
base pairs (bp) for gels. DNA bands of SSRs were developed with silver staining, and recorded. Dahab et al. 2607
Table 3. Mean squares of the ANOVA of morpho-physiological traits.
Genotypes Water Levels × Trait/Source of variation Block Water Levels (WL) Error Coeff Var. % (G) Genotypes Shoot Length (cm) 445.7589 21182.3** 12.91 67.8** 18.7* 14.1 Root Length (cm) 56.1126 3463.6** 18 44.47** 17.73n.s. 22.2 Fresh Shoot Weight (g) 13.8 2409.6** 1.46 3.93** 2.25** 29.7 Fresh Root Weight (g) 0.29 34.0** 0.04 0.07** 0.048n.s. 39.4 Dry shoot weight (g) 2.1758 60.04** 0.089 0.25** 0.14** 34 Dry root weight (g) 0.0136 0.0072** 0.0044 0.0101** 0.0064.n.s. 51 Relative Water Contents % 151 14307** 29.11 96.25** 71.13** 12
*= P≤0.05; ** = P ≤0.01; *** = P ≤ 0.001
The PCR amplifications were performed in a 10 μl reaction mix STRUCTURE2.2 into TASSEL software package (Bradbury et al., containing 1 μl 10× PCR buffer, 0.2 μl dNTPs (5 mM each), 0.1 μl 2007). To assess significant marker-trait associations P-marker 25 mM MgCl2, 0.1 μl Taq DNA polymerase, and 1 μl (50 ng) 0.05 was used. genomic DNA. The microsatellites were amplified using the standard polymerase chain reaction (PCR) procedures described by Zhang et al. (2000). Two millilitre of PCR products was RESULTS separated vertically on denaturing 16% polyacrylamide gels with 5× TBE buffer at 180 V for 45 min and stained with silver (Bassam et Phenotypic variation al., 1991). A 50 bp DNA ladder was used to estimate allele sizes in The cotton varieties under study revealed a wide range of Silver staining and development of bands phenotypic variation in morpho-physiological traits under both control and drought treatments (Table 3). Mean After a specific migration of the band on the gel, the gels were placed in fixing solution (40% ethanol and 10% glacial acetic acid) squares from analysis of variance for all morpho- for 20 min. They were washed three times with distilled water and physiological traits revealed highly significant variation stained with silver staining solution (0.2% AgNO3) for 20 min. After (P≤0.05) with respect to water levels and genotypes. staining, the gels were again washed three times with distilled water However, the interaction between water levels and for 20 s, and the developing solution (3% NaOH and 0.05% genotypes was significant for shoot length, fresh shoot formaldehyde) was applied for 3 to 5 min. weight and dry shoot weight (Table 1). Mean value of all 90 genotypes indicated significant reduction in all Inference of population structure for association mapping seedling traits. The considerable amount of genotypic variance apparent in all traits shows that variance under The STRUCTURE software is a DOS, Windows, UNIX (Solaris) and Linux based database that performs a model-based clustering water stress conditions is genetically determined and method for gathering the occurrence of population structure, finding selection of varieties\lines for drought tolerance on the diverse genetic populations, allocating individuals to populations, basis of seedling traits is possible. Correlation and classifying migrants and admixed individuals. Complementary coefficients between means in the stressed and non- studies on genotypic data for evaluating the population structure stressed conditions were positive and highly significant (P before continuing with LD analysis were performed by a model- ≤ 0.01) for most of the traits (Table 4). The frequency based approach; they were implemented in the software package STRUCTURE (Pritchard et al., 2000) to identify subgroups in cotton distribution of morpho-physiological traits under control cultivars under study. Admixture model under independent allele (W1) and water stressed (W2) treatments indicated frequencies using the burn-in time of 50,000, and a number of considerable genotypic variance (Figure 1). MCMC repeats at 100,000 were used (Pritchard et al., 2000), with the K ranging from 2 to 15. SSR genotyping, population structure, pairwise linkage disequilibrium and LD decay Extent of linkage disequilibrium and marker-trait association analysis The selected SSR primer pairs generated a total of 241 SSR alleles, of which 147 (60%) were polymorphic, The genome-wide LD between pairs of SSR marker loci was studied using the software package TASSEL ver. 2.1. Linkage resulting in 57.5% polymorphism. The average number of disequilibrium was estimated by a weighted average of squared polymorphic alleles per primer was 2.10. To determine allele-frequency correlations (R2) between SSR loci. The the population structure and number of subgroups in significance of pairwise LD (P-values≤0.005) among all possible cotton cultivars under study, a model-based approach, SSR loci was evaluated using TASSEL. The LD values between all implemented in the software package STRUCTURE pairs of SSR loci were plotted as LD plots using TASSEL to estimate the general view of genome-wide LD patterns and to (Pritchard et al., 2000), the distribution of log probability evaluate LD structures. The marker-trait associations were of data, LnP(D), did not show a clear peak against any calculated by GLM association test incorporating Q matrices from value of K, but by the use of parameter ΔK, rate of 2608 Afr. J. Biotechnol.
Table 4. Correlation coefficients of morpho-physiological traits under well-watered (W) and drought-stress (W2) treatment.
Traits PDW PFW PH RDW RFW RFW RSR SDW SFW
W1 0.89958** PFW W2 0.6296**
W1 0.7083** 0.7527** PH W2 0.4677** 0.5698**
W1 0.7779** 0.7261** 0.5532** RDW W2 0.3963** 0.1202NS 0.1075NS
W1 0.6880** 0.6861** 0.4361** 0.7831** RFW W2 0.2841* 0.5756** 0.2715* 0.1734NS
W1 0.4815** 0.5279** 0.7717** 0.4702** 0.3889** RL W2 0.1851NS 0.1836NS 0.7498** 0.0888NS 0.1043NS
W1 -0.3833** -0.3175* -0.3001* 0.2084NS 0.0163NS -0.0793NS RSR W2 -0.1579NS -0.2843* -0.2024NS 0.8251** -0.0110NS -0.0482NS
W1 0.9963** 0.8927** 0.7066** 0.7221** 0.6512** 0.4689** 0.4522** SDW W2 0.9692** 0.6499** 0.4753** 0.1597NS 0.2585** 0.1740NS -0.390**
W1 0.8862** 0.9954** 0.7596** 0.6854** 0.6135** 0.5216** -0.3470* 0.8837** SFW W2 0.6367** 0.9897** 0.5733** 0.1001NS 0.4530* 0.1818NS -0.3085* 0.6636**
W1 0.6569** 0.6852** 0.8517** 0.4349** 0.3295* 0.3241* -0.381** 0.6648** 0.7007** SL W2 0.5268** 0.6762** 0.8041** 0.0795NS 0.3097** 0.2097NS -0.2562* 0.5481** 0.6825**
PH, Plant length; PFW, Plant fresh weight ;SL, shoot length; RL, Root length; SFW, shoot fresh weight; shoot dry weight; SDW, RDW, root dry weight; RFW, Root fresh weight; RSR, root shoot ratio; RWC, relative water contents.
change in the log probability of the data, graph peaked maize (10%) (Remington et al., 2001) and sorghum against a value of K = 5. This confirmed 5 subpopulations (8.7%) (Hamblin et al., 2004). A high recombination rate in the germplasm at significant threshold values in allopolyploid cottons was reported (Brubaker et al., (R2≥0.05). 7.1% of the SSR marker pairs showed 1999) and it might be one of the factors explaining the significant pairwise LD in 90 accessions of G. hirsutum in observed low level of pairwise LD in cotton, along with our study. Plots for pairwise LD between SSR markers mutation, selection, and genetic drift that occurred in the demonstrated significant LD blocks in the genome-wide domestication of G. hirsutum germplasm. LD analysis. We observed a significant (R2) LD between
13 pairs of SSR loci within the same chromosome in Marker-trait association for morpho-physiological range of 180 cM between NAU1230 and NAU3095 loci in traits chromosome D5 and 1.612 cM and between NAU462 and NAU3414 in chromosome A9. Triangle plots for A total of 21 marker loci identified by GLM analysis were pairwise LD between SSR markers demonstrated significantly associated (P≤0.001) with phenotyped traits significant LD blocks in the genome-wide LD analysis. under both control and drought treatment (Table 6). Out Genome-wide LD decay was assessed by plotting r2 LD of these 21 markers, NAU3414, NAU2691, NAU1141 and values as a function of genetic distance in cM. Two long NAU1190 were associated with more than one morpho- stretches of LD blocks were observed on chromosomes physiological trait under drought treatments (Table 4). A3 and D9, extending to a distance of 180 and 77 cM Phenotypic variance explained (R2) value ranging from respectively (Table 5). Genome-wide LD at r2 > 0.1 9.91 to 19%. Highest phenotypic variance explaining (R2) rapidly decayed within ~1.61 to 11 cM, indicating a strong was ascribed to NAU3011 chromosome D13 significantly potential for association mapping (Saeed et al., 2014). (P≤0.001) associated with root length under drought The percentage of SSR loci pairs in LD observed in 90 G. treatment. This locus appeared to be a major locus as it hirsutum (7.1%) was comparable with reports in cotton is associated with highest phenotypic variance. NAU3414 (11 and 12%) (Abdurakhmonov and Addukarimov, 2008) located on chromosome A9 was associated with Dahab et al. 2609
Figure 1. Frequency distribution of morpho-physiological traits under control (W1) and water stressed (W2) treatments. 2610 Afr. J. Biotechnol.
Table 5. The pairwise genome linkage disequilibrium (LD) between pairs of SSR markers in same chromosomes and their distances.
Locus Name1 Locus Name2 R2 DPrime pDiseq Chr cM NAU3095 NAU1230 0.181971 0.827381 0.000386 D5 180.772 NAU2691 BNL1606 0.107744 0.444444 0.047173 D3 30.336 NAU3011 NAU2697 0.098475 0.373626 0.02466 D13 11.188 NAU2336 NAU2190 0.083965 0.576803 0.014481 D2 30.208 NAU462 NAU3414 0.083056 0.288194 0.020138 A9 1.612 NAU4105 NAU2697 0.080887 0.355656 0.013277 D13 29.299 NAU483 NAU1167 0.076643 0.835045 0.003996 A3 77.339 NAU1230 NAU1102 0.065643 0.367246 0.036619 D5 - NAU462 BNL1672 0.064808 0.280702 0.036295 A9 57.529 NAU3703 NAU3695 0.0625 0.4375 0.022808 A11 58.3 NAU3254 NAU2083 0.061728 0.333333 0.03903 A1 75.02 NAU3011 NAU2980 0.060445 0.340909 0.078648 D13 - NAU2336 NAU1070 0.048902 0.511364 0.102041 D2 40.796
Table 6. Marker-trait associations assessed by GLM analysis with their phenotypic variance explained (R2) values (P ≤ 0.001).
Traits Marker Chr. W1 W2 Relative value NAU3414 A9 0.113 - - BNL1606 D3 - - 0.118 SL BNL1421 A1 - - 0.1232 NAU2691 D3 0.0793 - - NAU2697 D13 0.1021 - - RL NAU3011 D13 - 0.1904 - PL NAU5091 D11 - 0.1529 - NAU1190 A3 0.1357 - - FSW NAU3414 A9 - 0.1791 - NAU3903 D2 0.0996 - - JESPR220 D4 0.1687 - - NAU6191 A6 - 0.102 - FRW NAU1141 A13 0.0942 - - NAU3100 D9 - - 0.1133 NAU1190 A3 0.1263 - - FPW NAU2974 D7 - - 0.1096 NAU1190 A3 0.1226 - - NAU1070 D2 - - 0.1219 DSW NAU3414 A9 - 0.1129 - NAU2974 D7 - - 0.0991 DRW NAU2265 A2 0.1068 - - NAU1167 A3 0.1193 - - RSR NAU1369 A8 0.1193 - - NAU1141 A13 0.1096 - - RWC NAU2016 A11 - 0.1068 -
maximum number of traits (shoot length, shoot fresh DISCUSSION weight and dry shoot weight whose value ranged from 11.3, 17.9 and 11.29% respectively). Six markers were Currently, association mapping methods has been used exclusively associated with drought treatment (W2). in diverse plant species such as bread wheat (Yu et al., Dahab et al. 2611
2012; Phumichai et al., 2012), barley (Cockram et al., be a major locus as it is associated with highest 2008), triticale (Niedziela et al., 2012) and bean (Shi et phenotypic variance. NAU3414 located on chromosome al., 2011). For molecular studies, there should be a A9 was associated with maximum number of traits (shoot reasonable degree of variability present among the length, shoot fresh weight and dry shoot weight whose organism of interest; only then the molecular approaches value ranged from 11.3, 17.9 and 11.29% respectively). can identify the genetic case underlying this variability. As Six markers were exclusively associated with drought there was a significant variability shown in our treatment (W2). This study also proved that association experimental material under greenhouse conditions, thus mapping approach has strong potential to assess our molecular study findings are of future significance. In significant marker-trait associations, save much time and our cotton germplasm, the number of polymorphic alleles cost compared to linkage mapping approach. detected per primer pair ranged from one to eight, with 2.10 alleles per primer pair. The SSR markers revealed a considerable amount of variation in the sampled genome, Conflict of interests even though the overall polymorphism detected for these cotton cultivars was relatively low. The narrow genetic The authors have not declared any conflict of interests. base of cotton has been mentioned in many studies using such molecular markers as SSRs (Bertini et al., 2006; Abbreviations Zhang et al., 2011; Kalivas et al., 2011), within Upland cultivars, which generally reveal a low level of genetic QTL, Quantitative trait loci; SSR, simple sequence variety. There was little variation in the estimation of the repeat; TASSEL, trait analysis by association, evolution molecular diversity among the upland cultivars (G. and linkage; GLM, general linear model; PL, plant length, hirsutum). However, Abdurakhmonov et al. (2007) PFW, plant fresh weight, SL, shoot length, RL, root reported that the genetic distance for the Upland cultivars length, SFW, shoot fresh weight, SDW, shoot dry weight, was in 0.01 to 0.28 range. The simple sequence repeats RDW, root dry weight, RFW, root fresh weight, RSR, root (SSR) allelic diversity found in our population for shoot ratio; RWC, relative water contents; LD, linkage association analysis is approximately the same as the disequilibrium. total diversity presented in more extended studies. The same mean number of alleles per locus as in our study was found in a collection of 106 accessions, with 2.13 REFFERENCES SSR alleles (Guo et al., 2006). In this study at significant threshold values (R2≥0.05), 7.1% of the SSR marker Abdelraheem A, Hughs SE, Jones DC, Zhang JF (2015). Genetic analysis and quantitative trait locus mapping of PEG-induced osmotic pairs showed significant pairwise LD in a total of 90 stress in cotton. Plant Breed. 134:111-120 accessions of G. hirsutum. This is comparable with Abdurakhmonov IY, Abdukarimov A (2008). Application of association previous reports on cotton: 11 to 12% of SSR loci pairs in mapping to understanding the genetic diversity of plant germplasm the exotic G. hirsutum accessions (Abdurakhmonov and resources. Int. J. Plant Genom. 2008:18. Abdurakhmonov IY, Saha S, Jenkins JN, Buriev ZT, Shermatov SE, Abdukarimov, 2008), 4% SSR markers in G. hirsutum Scheffler BE, Pepper AE, John ZY, Kohel RJ, Abdukarimov A (2009). variety accessions (Abdurakhmonov et al., 2009) and 3% Linkage disequilibrium based association mapping of fiber quality cotton germplasm from China and USA (Saeed et al., traits in G. hirsutum L. variety germplasm. Genetica 136:401-417. 2014). In our study, cotton germplasms used are from Abdurakhmonov IY, Kohel R, Saha S, Pepper A, Yu J, Buriev Z, Abdullaev A, Shermatov S, Jenkins J, Scheffler B (2007). Pakistan; whereas in previous reports, the cotton Genomewide linkage disequilibrium revealed by microsatellite germplasm used were of African, Australian, and Latin markers and association study of fiber quality traits in cotton, American, Mexican, Uzbek, China and USA ecotypes. Proceedings of the 15th Plant and Animal Genome Conference. 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Vol. 15(46), pp. 2613-2619, 16 November, 2016 DOI: 10.5897/AJB2016.15586 Article Number: A6B8C3A61675 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2016 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Inbreeding depression in crosses of coerulea clones of Walker’s Cattleya (Cattleya walkeriana Gardner)
Miléia Ricci Picolo, Ceci Castilho Custódio, Nelson Barbosa Machado-Neto*
Department of Agronomy, Universidade do Oeste Paulista (UNOESTE), Presidente Prudente, São Paulo, Brazil.
Received 25 July, 2016; Accepted 21 September, 2016
Orchids are among the most beautiful flowers and endangered due to habitat destruction and over- collection. Cattleya walkeriana is one of the most beautiful flowers joining the small sized plant with medium large and heavily scented flowers. It is widely known and appreciated by its beautiful clones and it has much to offer to breeders because their plants have besides other attributes as small habit and big flowers, many colour variations, form and precocity, becoming flower only four years in ex vitro culture. However, in some of the original places it is becoming a red listed species. Notwithstanding, very little is known about the genetics of these flowers and the variability in the species that is widespread in the Brazilian territory. The aim of this work was to estimate the variability among cultivated materials using the F statistics and to verify if there was inbreeding in plant crosses with similar characteristics, employing as a tool the RAPD simple methodology. The results obtained showed that RAPD was good enough to estimate the variability in C. walkeriana. The selected primers were able to define colour group, especially the coerulea. Inbreeding will occur in crosses of clones with the same colour.
Key words: Orchidaceae, deoxyribonucleic acid (DNA), random amplified polymorphic DNA (RAPD), variability, domestication.
INTRODUCTION
Currently, molecular markers have been used in plant 2009; Oliveira et al., 2010; Xue et a l., 2010; Machado breeding for several objectives, which makes possible Neto and Vieira, 2011; Sharma et al., 2011; Manners et more detailed and consistent analysis of their genetics. al., 2013). The establishment of deoxyribonucleic acid (DNA) mole- The variability estimated by these markers can be cular patterns serves as a parameter for identification of used, as any other markers, to estimate the population clones and varieties and as a tool for a better genetics (F statistics) (Wright, 1978). However, these taxonomical classification and variability determination in tools (F statistics) are barely used to follow populations in orchids (Chung et al., 2006; Minoo et al., 2006, 2008; plant breeding (Sallam et al., 2015). Marker Assisted Parab et al., 2008; Niknejad et al., 2009; Verma et al., Selection (MAS) has been a useful tool for plant breeders,
*Corresponding author. E-mail: [email protected]. Tel: (+55 18) 32292000.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 2614 Afr. J. Biotechnol.
but has had limited success in improving complex traits to offer to breeders because their plants have small habit, (Heffner et al., 2010). large flowers, many colour variations (type: (pink), alba, According Minoo et al. (2008), random amplified poly- semi-alba and coerulea, Figure 1), form and precocity morphic DNA (RAPD) technique has some advantages (Menezes, 2011), becoming flower only four years in ex over other techniques as the easy, rapid achievement of vitro culture. However, in some of the original places, it is polymorphic markers, low cost, absence of hybridization, becoming a red listed species (Brasil, 2008). detection of polymorphism in highly repetitive genome, Very few is known about the genetics of Cattleya and high level of polymorphism compared to other species; Cattleya intermedia (Machado-Neto and Vieira, molecular markers. However, either RAPD or any other 2011) using RAPD; Catleya elongata (Cruz et al., 2011) single primer amplification method (SPAR) (Gupta et al., with isozymes and ISSR; Cattleya coccinea (Novello et 1994), present a few disadvantages, such as ambiguity in al., 2013); with ISSR in Cattleya bicolor, Cattleya labiata the interpretation of the bands co-migrating fragments of and Cattleya schofieldiana (Fajardo et al., 2014) with equal size or close and dominant character in most of the SSR and one in C. labiata using RAPD and ISSR markers obtained, what cannot be a penalty for this (Pinheiro et al., 2012). And lesser is known about the (Simmons, 2007). In the orchids study, this method can inheritance of some characters especially because these be used to indicate genetic similarity between these plants are perennials and the time between one plants, their hybrids and wild ancestors, to offspring generation and the following is almost 5 years. prediction of a cross, based on the information from the Plant breeding and evolution are related for two genotype of early and efficiently, as well as helping in the reasons; the first is that plant breeding might be defined species classification, or just to measure the raw as evolution guided by man and the second is that both variability (Costa et al., 2006; Minoo et al., 2008; Niknejad processes have their basis in, and a major effect on, et al., 2009; Oliveira et al., 2010; Xue et al., 2010; biodiversity (Ceccarelli, 2009). Machado-Neto and Vieira, 2011; Manners et al., 2013). In populations submitted to constant selection, where The analysis at molecular level is an advantage for just the superior individuals were promoted for studies on perennial plants, enabling the evaluation of reproduction, the alleles controlling characters of interest genetic similarity between genotypes (Ambiel et al., 2008, had their frequency increased, leading to diversity loss in 2010; Machado-Neto and Vieira, 2011) and to collect crop plants. In orchids, flower shape and colours have information on the level of genetic diversity of wild orchids been improved by breeding (Machado Neto and Vieira, to enable a better conservation of the species (Manners 2011). While in wild specimens of C. walkeriana, another et al., 2013). colour than the type are often not found; it is common to In the world flora, orchids are the second largest family, find alba, coerulea and different colours in bred C. with almost 736 genera and over 26,000 species (Chase walkeriana with exceptionally well-shaped flowers. This et al., 2015), ranging from 7 to 10% of the flowering plant species has much to offer for breeders, but it counts just species (Cowan et al., 2006) distributed in five with 109 direct hybrid offspring and it is not much used as subfamilies: Apostasioideae, Cypripedioideae, Epiden- parent; for example two related species, Cattleya droideae, Orchidoideae, and Vanilloideae (Cameron, loddigesii and C. intermedia, counts with 230 and 217 2006). Cattleya together with Cymbidium, Oncidium, primary hybrids respectively (RHS, 2016).. Phalaenopsis and Dendrobium are important commercial This study aimed at the measurement of the variability ornamental species due to its large spectrum of colours and inbreeding in a population submitted to selection and and relatively high cross ability with other genera. Orchid directional crosses of coerulea clones of C. walkeriana, commercialization, both pot plants and cut flowers, is by means of F statistics (Wright, 1978; Sallam et al., highly significant worldwide, about US$504 million (De 2015), and to evaluate the ability of RAPD markers in and Medhi, 2015) and is increasing year after year. grouping phenotypes of these plants and measure the The number of described species in Cattleya is still a fixation of the coerulea characteristic in the species and matter of debate, ranging from 49 to 114 species among clones. To our knowledge, this work is the first of (excluding Guarianthe and Cattleyella and including this kind with this species. Sophronitis (Van den Berg, 2014) and Brazil has 98 endemics (Forzza et al., 2013). MATERIALS AND METHODS Cattleya walkeriana belongs to the unifoliate group of the genus and within which it can be considered a small Plant plant. It has a stout rhizome, with three internode pseudobulbs, close to each other. It has long roots, thick In this work, young and adult plants of C. walkeriana, coerulea (blue) colour, type (pink), semi-alba and alba (white flowers), were and often branched (Menezes, 2011). It is widely known used. Plants have diverse origin, except the offspring. They are and appreciated by its beautiful clones. It is found in listed in Table 1. different regions of Brazil, growing over rocks or trees in the states of Goiás, São Paulo, Mato Grosso do Sul, and DNA extraction Minas Gerais, nearby lakes, rivers or swamps and it could be easily cultivated (Menezes, 2011). It has much DNA extraction and amplifications were done as in Machado-Neto Picolo et al. 2615
Figure 1. Colour forms of Cattleya walkeriana Gardner.
Table 1. List of Cattleya walkeriana plants, their colour and origin.
Plant Colour Origin
Patrícia (parental P1) Coerulea Prata - Minas Gerais
Blue City (parental P2) and ABC (parental for Backcross - BC1) Coerulea Offsprings of selfed DICK (ESALQ) clone from Itajubá, MG.
F1 (first generation, 7 plants) Type (Patrícia x Blue City)
F2 (second generation, F1xF1, 11 plants) Unknown - BC1 (first backcross generation - 23 plants) Unknown (F1 x ABC) Rancho Sereno x Patrícia (4 plants) (RSP) Coerulea - Alba (CA) white unknown Puanani (Pu) Semi alba - Unnamed clone (SA) Semi alba (semi alba “Goiaba” x albescens “Denise Cavasini” – from Guará-SP) Twins (CWT) Light pink -
Prata and Itajubá are nearly 600 km apart.
and Vieira (2011). To identify markers, 120 primers from Operon 40 cycles of 1 min at 94°C, 1 min at 37°C for primer annealing and (Alameda, USA) were initially tested in four plants, one 90 s at 72°C for chain elongation in a PTC-100 Thermocycler. An representative of each generation (Parental, F1, F2, BC1) and 33 extra step of 5 min at 72°C for final elongation was included. were primarily chosen (A1, A2, A5, A10, A11, A14, A18, A19, A20, Amplification products were separated by electrophoresis in 1.5% C1, C2, C4, C5, C6, C7, C8, C11, C12, C14, C16, D1, D2, D13, G2, agarose gel. Gels were stained with ethidium bromide and G3, G5, G6, G7, G8, G11, G12, G14, G16) as they were visualized using Electrophoresis Analysis System (Biosystems). polymorphic. PCR was carried out in a reaction volume of 25 μl Each amplification reaction was repeated at least twice and only containing Tris buffer (20 mM Tris-HCl, pH 8.4), 50 mM KCl, 1.5 clearly distinct and reproducible bands were scored. Weak or low mM MgCl2, 0.4 μM of primer, 0.2 μM of each dNTP, 1U Taq intensity bands were not considered to avoid ambiguous polymerase and two concentrations of template DNA 25 and 50 ng. interpretations. The analysis of the bands was performed with the RAPD amplifications were performed in a thermo cycler under the Quantum program - Capt (Vilber -Lourmat) to determine the electro- following conditions: 94°C for 3 min for initial denaturation and then phoretic pattern. The primers selected for the final analysis were 2616 Afr. J. Biotechnol.
Table 2. Nucleotide sequences of the primers, number of bands, polymorphism and size of amplified fragments in Cattleya walkeriana.
Primers Nucleotide sequence (5’ 3’) Polymorphic bands Fragment size (pb) A2 TGC CGA GCT G 10 280 - 1380 A5 AGG GGT CTT G 9 300 - 1190 A10 GTC ATC GCA G 10 250 - 1370 G5 CTG AGA CCG A 10 280 - 1380 G11 TGC CCG TCG T 8 400 - 1460 G13 CTC TCC GCC A 7 350 - 1060 Total - 54 -
A2, A5, A10, G5, G11 and G13, because they successfully “Rancho Sereno” and “Patricia” were in the same branch, amplified a total of 54 fragments. Polymerase chain reaction (PCR) because both plants are coerulea. All coerulea plants was performed as described earlier. showed up together in a larger branch. In another clade, Bands were used to construct a similarity matrix based on the Jaccard coefficient, coding 1 as presence and 0 as absence. The F1 plants, although the same phenotype, were separated grouping analysis was done using the Unweighted Pair-Group by branches indicating genetic proximity, but no similarity Method Using an Arithmetic Average (UPGMA) algorithm. This (group II – Figure 2). The CWT plant showed next to analysis was performed with the software NTSYS 2.1 (Rohlf, 2004). semi-alba (group III – Figure 2), these last sharing the Molecular variance analysis (AMOVA) was calculated by total same clade, and the plant CA in other branch bellow, but decomposition of its components among and between accessions using the square distances with the Arlequin software (Excoffier et close to the semi-alba (group IV – Figure 2). This figure al., 2015). shows the clustering of plants that have flourished The fixation index or F statistics of Wright (FST) was generated by (relatives, control and F1) and plants that have not the Arlequin software (v. 3.5). The inbreeding coefficient (FIS) was flowered (F2 and BC1 generations). Some F2 plants were calculated by the formula: close to F1 ones which indicates a probable phenotypic similarity. The proximity of these plants with those of (1 FIT ) already known phenotype may indicate that their flower F 1 IS will have the same colour. (1 FST ) As shown in Table 3, for the population studied, there and the general fixation index (FIT) was calculated by the formula: was a high variability, indicated by the overall FST (0.017), considering Wright (1978) in which FST > 0.25 was Ho considered low variability. However, even using coerulea
FIT 1 ( ) of different origins an increase in the FST values was He showed (0.337 for the parents, 0.539 for F and 0.567 for 1 where Ho and He were the observed and waited heterozygosity F2, 0.465 for all the coerulea, and 0.492 for BC1) respectively, obtained in Arlequin software. explained by the fact that plants with similar characteristics were crossed and there was a decreasing in the variability meaning a strong differentiation between RESULTS AND DISCUSSION those plants and the population (FST 0.017). The selection of plants with the same colour for the initial Table 2 shows the nucleotide sequences used. The cross led to endogamy showed in this study by the cross fragments generated ranged from 150 to 2500 bp, lying between Patricia x Blue City (Table 1), but it could within the boundaries according to Xue et al. (2010), in happen in nature, as a pollinator would choose flower which the RAPD technique has good reproducibility. with the same colour, by chance, creating an inbreed After primers selection, the construction of a population. dendrogram was made with all RAPD markers selected. In Cattleya intermedia (Machado-Neto and Vieira, 2011) According to the band analysis, it was possible to and Cattleya elongata (Cruz et al., 2011) FST values were estimate the ability of these markers to group plants by low indicating a high gene flux among plants (0.016 and their colour, that efficiency is demonstrated by the 0.100 respectivelly), but for Fajardo et al. (2014) these dendrogram (Figure 2), which exhibit a group of coerulea values are much higher (from 0.177 in Cattleya labiata to plants, where P1 and RSP1 are in the same branch, but in 0.322 in Cattleya granulosa) indicating loss of variability a different location of P2 and RSP (P). That both P1 and in the last case. P2 plants could be regarded as coerulea but to different The FIS values shown (Table 3) are also very subclades, P1 for group IA and P2 for group IB. informative, as the values are closer to -1, in the overall It was expected that all plants from a cross between sample, meaning that there is more heterozygosity in Picolo et al. 2617
Figure 2. Grouping of plants of Cattleya walkeriana progeny F1, F2, BC1 and other colour forms.
Table 3. Wright’s measure of population differentiation (FST) and inbreeding (FIS), observed (Ho) and estimated Heterosigosity (He) using RAPD markers for Cattleya walkeriana offsprings.
Source of variation FST FIS HO (±SD) HE (±SD) Parents 0.377** -0.403** 0.496 (0.273) 0.353 (0.140)
F1 0.539** -0.022 0.456 (0.197) 0.446 (0.126)
F2 0.567** -0.320** 0.454 (0.198) 0.344 (0.145) Coerulea 0.465** -0.243** 0.496 (0.213) 0.399 (0.135)
BC1 0.492** -0.447** 0.450 (0.211) 0.311 (0.149) Overall 0.017 -0.734** 0.463 (0.211) 0.267 (0.153)
**P<0.01.
this. On the other hand, there was more homozygosis as FIS, there were indications that general population has a the values approaches to zero, exemplified by -0.022 in good gene flux and driven crosses led to gene diversity the F1 population. Intermediate values as the parents (- loss. 0.403), F2 (-0.320), coerulea (-0.243) and BC1 (0.447) Li and Ge (2006) using RAPD markers found low were more heterozygous than F1. These kind of data are genetic diversity within populations and high among the very useful for perennials (Guries and Ledig, 1981). The studied populations of Changnienia amoena (an orchid values found in this work for Ho and He were not species). These results were due to small population statistically different for the populations. In both, FST and size, the local extinction because of habitat destruction 2618 Afr. J. Biotechnol.
and restricted gene flow. Chase MW, Cameron KM, Freudenstein JV, Pridgeon AM, Salazar G, In Platanthera leucophaea, another species of orchid, van den Berg C, Schuiteman A. (2015). An updated classification of Orchidaceae. Bot. J. Lin. Soc 177(2):151-174. rare and endangered species, the values of FST for RAPD Chung SY, Choi SH, Kim MJ, Yoon KE, Lee GP, Lee JS, Ryu KH and isoenzymes (0.889 and 0.754, respectively) showed (2006). Genetic relationship and differentiation of Paphiopedilum and a large amount of inbreeding consistent with each other Phragmipedium based on RAPD analysis. Sci. Hortic. 109(2):153- (Holsinger et al., 2002). Moreover, in a study by Pressoir 109. Costa FR, Pereira TNS, Vitória AP, Campos KP, Rodrigues R, Silva and Berthaud (2004), the allelic fixation index in corn DH, Pereira MG (2006). Genetic diversity among Capsicum landraces showed little variation between populations accessions using RAPD markers. Crop Breed. App. Biotechnol. (FST 0.003 to 0.011). 6(1):18-23. Ambiel et al. (2008, 2010) estimated in Brachiaria Cowan RS; Chase MW; Kress W; Savolainen V (2006). 300,000 species to identify: problems, progress, and prospects in DNA brizantha, an apomitic species, lower values of FST (0.216 barcoding of land plants. Taxon 55:611-616. and 0.276) indicate a high gene flow. According to Wright Cruz DT, Selbach-Schnadelbach A, Lambert SM, Ribeiro PL, Borba EL (1978) populations with low levels of selection showed (2011). Genetic and morphological variability in Cattleya elongata lower values of F . Sallam et al. (2015) used the F Barb. Rodr.(Orchidaceae), endemic to the campo rupestre vegetation ST in northeastern Brazil. Plant Syst. Evol. 294(1-2):87-98. statistics to follow selection during a barley breeding De LC, Medhi RP (2015). Orchid – a diversified component of farming process, so this could be a very useful tool to follow systems for profitability and livelihood security of small and marginal improvement during breeding program, especially in farmers. J. Glob. Biosci. 4(2):1393-1406. perennials as orchids. Excoffier L, Laval G, Schneider S (2015). Arlequin ver 3.5 An Integrated Software Package for Population Genetics Data Analysis (software) Wild and cultivated populations differ statistically in Bern: University of Berne Computational and Molecular Population various characters likely to be human selected, although Genetics Lab (CMPG), 2015. Available at: some cultivated plants are morphologically
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Vol. 15(46), pp. 2620-2626, 16 November, 2016 DOI: 10.5897/AJB2016.15426 Article Number: C6E387861701 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2016 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
The protective role of honey against cytotoxicity of cadmium chloride in mice
Asmaa Shahrour, Mohamed Zowail and Khaled Sharafeldin*
Department of Zoology, Faculty of Science, Benha University, Benha, Egypt.
Received 21 April, 2016; Accepted 16 August, 2016
The present study aimed to investigate the honey (HY) protective role in opposition to cadmium (Cd) induced chromosomal aberrations of bone marrow and sperm abnormalities. Forty five (45) adult male albino mice were caged into six groups. Mice were injected, i.p, 300 mg HY/kg and/or 0.67 mg CdCl2/kg b.w for 96 h, separately and alternated. The alternated trials were continued for consecutive eight days. Results show that mice injected with cadmium had significant increase in the frequency of aberrant chromosomes as fragment, centric fusion, gap, stickiness and aneuploidy and in sperm abnormality. The administration of HY improved the frequency of the chromosomal aberrations and sperm abnormality induced by Cd.
Key words: Cadmium, Honey, sperm, chromosome aberrations.
INTRODUCTION
The toxic adverse of cadmium (Cd) is known as also causes severe soft tissues and bone damages environmental and industrial pollutant. Its physical and (Cucu et al., 2011; Ercal et al., 2001; Ersan et al., 2008). chemical properties constitute the industrial individuality Cadmium is known to enhance the production of reactive for applications (Krichah et al., 2003). As a heavy metal, oxygen species (ROS) (Liu et al., 2001). The toxic effect Cd causes severe injuries (Suzuki et al., 1989). People of Cd is controlled by the oxidation of cellular organelles often develop health disorders starting from vomiting, by generating ROS reactions which lead to lipid stomach pain and diarrhea to bone fracture, lung and peroxidation, apoptosis, damage of DNA and altered reproductive failure, particularly those who live in vicinity gene expression (Stohs et al., 1999; Wu et al., 2002; of factories that release cadmium or work in metal Thévenod, 2003). Therefore, to relieve Cd adverse effect refinery industry (Nordberg, 2009). Amara et al. (2011) the antioxidants induction is considered as an important and Singh et al. (2007) mentioned that the damaged therapeutic approach (Renugadevi and Prabu, 2010; central nervous system and DNA or cancer progression Sinha et al., 2009). appeared as consequences of Cd exposure. Cadmium At least 181 substances are included in the composition
*Corresponding author. E-mail: [email protected].
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License
Shahrour et al. 2621
of honey (Wang and Li, 2011). This composition varies minced together in isotonic medium then filtered to exclude large primarily depending on the floral source rather than fragments. The cells' smears were prepared and stained with 5% seasonal and environmental factors. Glucose (31%) and Eosin Y. Light microscope (100x) green filter was used to examine smears. Thousand sperms were assessed for each animal to fructose (38%) represents the main contributors of honey investigate the morphology of sperm abnormality according to the solution. Minor constituents of honey have antioxidant criteria of Wyrobek and Bruce (1975). Any overlay or contact properties as phenolic acids, enzymes, ascorbic acid, sperms or heads without tails were ignored. carotenoid-like substances and flavonoids (Andrade et al., 1997; Ferreres et al., 1992; Wang and Li, 2011; Statistical analysis Cherchi et al., 1994). The present study aimed to investigate the protective role of honey (HY) opposing the Mean ± SE was expressed to all values where five animals were chromosomal and sperm abnormalities induced by evaluated, n=5, in each group. Statistical analysis of cytogenetic cadmium. was performed on SPSS software (version 18) using one-way analysis of variance (ANOVA) test. Significance was considered when P values less than 0.05. MATERIALS AND METHODS
Cadmium was used as cadmium chloride (CdCl2) (Oxford RESULTS Laboratory, Mumbai, India). The concentrations used was 0.67 mg/ kg or 1/10 LD50 according to Bench et al. (1999). Honey (HY) clover Various chromosomal aberrations were observed in the flower was purchased from Isis Company, Cairo, Egypt. 300 mg HY/kg b. w. was used as an optimal dose according to El-Raby bone marrow cells of albino mice injected with cadmium. (2010). These were of structural and numerical type, were identified and quantitated relative to non-treated control (Figure 1A). Animals Structural aberration included chromatid deletions (Figure 1B); fragments (Figure 1C); centric fusion (Figure In this study, 30 adult Swiss albino mice (MUS musculus) were used, varying from 20 to 25 g in weight and aged three month old. 1D); centromeric attenuation (Figure 1E); end to end These mice were obtained from the National Research Center (Figure 1F); rings; gap (Figure 1G) (Figure 1H); (N.R.C.), Dukki, Cairo, Egypt, were caged individually under polyploidy (Figure 1I); stickiness (Figure 1J). A chromatid standard conditions of light, temperature, humidity and fed with was considered to have a gap when it had an unstained standard pellet diet and water ad libitum. area shorter than its diameter or equal to it (Figure 1G). End to end association was scored when two chromatids
Experiment of different chromosomes appeared attached this could result from reciprocal translocation or stickiness (Figure The experiment was categorized in six groups of five animals each. 1F). The stickiness is considered as assort of chromo- Group I was the control group, group II: mice were i.p injected with somal agglutination of unknown nature which resulted in 0.5 ml saline solution daily for 96 h, as positive control, group III: a pycnotic or sticky appearance of chromosome (Figure mice were i.p injected, as a single dose, with 0.67 mg CdCl2/kg b.w. dissolved in 0.5 ml saline solution, for 96 h, group IV: mice were 1J). injected i.p, with 300 mg honey (HY)/kg b.w. dissolved in 0.5 ml As shown in Table 1, the mean number nuclei with saline solution daily for 96 h, group V (HYCd): mice were injected chromosomal aberration in mice treated with cadmium i.p with HY daily for 96 h, then single dose of CdCl2 for 96 h (as a appear high significant of chromosomal aberrations in all protective trial), and group VI (CdHY): mice were i.p injected, single animals compared with control. The more types of dose, CdCl2 for 96 h then followed by four consecutive honey aberration appeared with chromosomal fragments, doses, for 96 h (as a treatment trial). centric fusion, gaps, stickiness and aneuploidy. Also the mitotic activity shows decrease in the treated groups with Cytogenetical study cadmium chloride when compared to control. Honey administration has the same results as control of Colchicine was injected intraperitoneally 2 to 3 h before sacrificing. chromosomes abnormalities and the rate of cell division. Bone-marrow was extracted from femur bone and metaphases Honey as a protective or treatment dose lead to decrease according to the method of Yosida and Amano (1965). The preparations of mitotic chromosome were made according to Ford of chromosome aberration with increase in cell division and Hamerton (1956). Giemsa stain (7%) in phosphate buffer (pH that shows Cd HY and HY Cd groups could protect the 6.8) was used for slides. Hundred spreads metaphases per animal body from cytotoxicity arising from cadmium. were investigated for chromosomal aberration analysis. Figure 2 shows normal sperm (a), hummer sperm (b), without hook (c) and banana shape (d). Honey recorded
Sperm head morphology assay increased in number normal sperm head (Figure 3). Honey as protective or treatment group (Cd HY or HY Cauda epididymides were excised and both epididymides were Cd) could protect the body from cytotoxicity arising from
2622 Afr. J. Biotechnol.
A B C Normal chromosome Deletion Fragment
D E F Centric fusion Centric attunation End to end
G H I Gap Ring Polyploidy
J Stickiness
Figure 1. Metaphase spread from mouse bone marrow cells showing: (a) normal chromosomes spread, (b) deletion, (c) fragment, (d) centric fusion, (e) centromeric attenuation, (f) centric attenuation, (g) end to end, (h) ring, (i) polyploidy, (j) stickiness. All aberrations are indicated by arrows.
Shahrour et al. 2623
Table 1. Average of chromosomal aberration observed in bone marrow cells of male mice treated with Cd and HY.
Group Fragment Centric fusion Gap Stickiness Trisomy Total % Controla 3.0±0.5b,d 8.2±0.7d 0.0±0.0d 0.0±0.0d,e 0.2±0.8d 12.4±2dd 1.24 +Controlb 5.8±0.7a,c,e,f 8.6±1.0c,d 0.0±0.0d 0.0±0.0d,e 0.2±0.6d 14.6±2.3 1.46 HYc 4.8±0.6b 5.4±0.7b,d 0.2±0.2 0.0±0.0d,e 0.2±0.7d 10.6±2.2 1.1 Cdd 6.8±0.4a 11.8±1.5a,b,c,e,f 0.4±0.2a,b,e,f 1.0±0.3a,b,c 1.4±0.3a,b,c 21.4±2.7a,b,c,e,f 2.1 CdHYe 5.0±1.0b 8.0±1.1d 0.0±0.0d 0.8±0.4a,b,c 0.8±0.3 7.4±2.8d 0.74 HYCdf 5.0±0.7b 9.0±1.2d 0.0±0.0d 0.6±0.2 0.6±0.3 15.2±2.4d 1.52
Average was expressed as mean±standard error. The significances were indicated as follow: asignificant with control, bsignificant with +control, csignificant with HY, dsignificant with Cd, esignificant with Cd Hy, fsignificant with HY Cd. Significant means P<0.05.
(a) ( b)
(d0 (c)
Figure 2. (a) Normal sperm, (b) hummer sperm, (c) without hook, (d) Banana shape.
2624 Afr. J. Biotechnol.
50 45 40 35 hummer 30 banana 25 amorphous 20 without hook 15 10 5 0
Control- Control+ Hy Cd CdHy HYCd
Figure 3. Incidence of total abnormally shaped sperms per thousand after injection of HY and Cd.
cadmium. The same values of normal sperm were and declined mitotic index. Abnormal sperm population recorded (Figure 3). was observed in the present study. Due to the effect of Cd-induced ROS on specific gene loci of germ cell chromosomes, it may dysfunction the maintenance of DISCUSSION normal sperm structure. Most of the germ cells have been destroyed in Cd-treated mice due to either Currently, chromosomal fragment, centric fusion, gap, membranous or macromolecular damage incurred by stickiness and aneuploidy were produced by Cd injection. formation of ROS leading to declined sperm count and Other study revealed that Cd induced chromosomal ultimately testicular weight loss (Acharya et al., 2003; break, centric fusion, terminal association and C-mitosis Oldereid et al., 1994); as well as the decreased sperm in Oreochromis mossambica (Chandra and Khuda- count and alterations in motility have been associated Bukhszh, 2004). Cd burden has been correlated with with cigarette smoking (Kulikauskas et al., 1985). chromosomal aberrations (IARC 1993). Singh et al. Honey administration has the same results as control of (2007) showed that low dose of Cd (1 mg/kg/day) for 30 chromosomes abnormalities and the rate of cell division. days resulted in chromosomal aneuploidy, breaks, gaps Honey as protective or treatment dose lead to decrease and centromeric fusion. However, dose of 25 mg in chromosome aberration with increase in cell division Cd/kg/day for 20 days and 200 mg/kg/day for five days that shows Cd HY and HY Cd groups could protect the resulted in severe damage of chromosome. Singh and body from cytotoxicity arising from cadmium. Honey Sankhla (2010) have assured that Cd increased the could be used to prevent and eliminate mutation caused number of chromosomal aberrations and declined the by aflatoxins (Sharmanov et al., 1986). This was in mitotic index. On the other hand, the Cd exposure has accordance with reducing the chromosomal aberrations been shown to stimulate free radical production, resulting induced by aflatoxins (Sharmanov et al., 1986). The in various pathological conditions in humans and animals chromosomal aberrations induced by mycotoxins could (El-Demerdash et al., 2004; Shaikh et al., 1999). Acute or be minimized by honey administration (Ezz El-Arab et al., chronic exposure of Cd has been associated with 2006). Using 300 mg HY/kg against the brodifacoum increased lipid peroxidation in testes, erythrocytes, and pesticide, mitomycin-c anticancer drug and zinc other soft tissues (Manca et al., 1991; Sarkar et al., phosphide pesticides lead to chromosome aberration, 1997). Free-radicals can originate from exposure to micronucleus formation and sperm head abnormalities cadmium, resulting in elevated chromosomal aberrations (El-Raby, 2006, 2007, 2010). The association of honey
Shahrour et al. 2625
with cyclophosphamide (CPM) ameliorates the evaluated rats. reduced glutathione (GSH), malondialdehyde (MDA) and chromosomal aberrations. Zoheir et al. (2015) also stated that honey inhibited the cytotoxic and genotoxic risks Conclusion associated with the treatment by CPM in mice. Royal jelly has shown a protective effect in opposition to From the current study, we can conclude that both the chromosomal abnormalities in bone marrow and treatment and protection with honey ameliorated the histological alterations in kidney produced by valproic adverse effects of Cd on chromosome and sperm. The acid in male mice (Galaly et al., 2014). The treatment cover honey as monofloral honey was the type of honey with flavonoids significantly reduced the chromosome collected by most beekeepers and consumed by abnormalities and delayed tumorigenesis in adult mice Egyptians. exposed to fetal irradiation (Uma Devi and Satyamitra, 2004). Honey sugar displayed complex behavior toward the enhanced or inhibited mutagenic activity in a model Conflict of Interests by cooked food systems (Skog, 1993). Royal jelly (RJ) had shown highly efficient antioxidant where it scavenges The authors have not declared any conflict of interests. the produced free radicals (Cemek et al., 2010; El- Nekeety et al., 2007; Türkmen, 2009). El-Monem (2011) REFERENCES also revealed that RJ caused a significant recovery in antioxidant status. However RJ reduced glutathione Acharya UR, Acharya S, Mishra M (2003). Lead acetate induced (GSH) and inhibited malondialdehyde production which cytotoxicity in male germinal cells of Swiss mice. Ind. Health ameliorated DNA damage and genotoxicity induced by 41(3):291-4. Amara S, Douki T, Garrel C, Favier A, Rhouma KB, Sakly M, malathion in rat cells. Inoue et al. (2003) and Narita et al. Abdelmelek H (2011). 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Effect of cellular defenses, and enhances the lipid peroxidation process in different doses of Malaysian honey on reproductive parameters in primary cultures of rat Leydig cells. Reprod.Toxicol. 16(4):379-384. adult male rats. Andrologia 44(s1):182-186. Wyrobek A, Bruce W (1975). Chemical induction of sperm abnormalities Narita Y, Nomura J, Ohta S, Inoh Y, Suzuki KM, Araki Y, Okada S, in mice. Proceed. Natl. Acad. Sci. 72(11):4425-4429. Matsumoto I, Isohama Y, Abe K, Miyata T (2006). Royal jelly Yosida TH, Amano K (1965). Autosomal polymorphism in laboratory stimulates bone formation: physiologic and nutrigenomic studies with bred and wild Norway rats. Rattus norvegicus, found in Misima. mice and cell lines. Biosci. Biotechnol. Biochem. 70(10):2508-2514. Chromosoma 16(6):658-667. Nordberg GF (2009). Historical perspectives on cadmium toxicology. Zoheir BA, Creaser RA, Lehmann B (2015). Re-Os geochronology of Toxicol. Appl. 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Vol. 15(46), pp. 2627-2636, 16 November, 2016 DOI: 10.5897/AJB2016.15581 Article Number: D7BC1BA61703 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2016 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Shelf life improvement of sorghum beer (pito) through the addition of Moringa oleifera and pasteurization
Florence Adwoa Ayirezang1*, Courage Kosi Setsoafia Saba1, Francis Kweku Amagloh2 and Hellie Gonu1
1Department of Biotechnology, Faculty of Agriculture, University for Development Studies, Tamale, Ghana. 2Food Processing Technology Unit, Faculty of Agriculture, University for Development Studies, Tamale, Ghana.
Received 22 July, 2016; Accepted 7 September, 2016
Pito is a traditional alcoholic beverage that is mostly brewed in the three northern regions of Ghana. Although widely consumed and used in many festivities, poor storability limits its economic potential as an income-generating venture for most women. This study was carried out to improve the shelf-life of pito through the addition of Moringa oleifera leaf extract and pasteurization (75 to 80°C). Microbial enumeration, physico-chemical parameters (pH, extract (%) and alcohol) and consumer preference scores were used as quality indices of each pito treatment carried out. There was a general decline in coliform and fungi growth and in the physico-chemical (pH, extract (%) and alcohol) properties during the 56 days of storage. Microbial load, extract (%), alcohol content and pH were significantly different (P<0.05) among treatments. Pasteurized moringa pito had the least microbial load. The treated pito samples had higher values in pH, extract (%) and alcohol content than the untreated pito during storage. There was high consumer acceptability of pasteurized pito from the 0 day to the 28 days of storage, with a mean score of 4.27 ± 0.75 to 3.61 ± 1.36. However, the moringa treated pito (pasteurized moringa pito and moringa pito) was less preferred (with a mean score of 2.86 ± 1.19 to 1.87 ± 0.92) from the 0 day to the 28 days of storage. The untreated pito was also acceptable for a period of seven days. Based on the findings of this research, it can be concluded that pasteurization and/or the addition of M. oleifera leaf extract can improve the shelf-life of pito for four weeks, but addition of moringa extract in pito reduced consumer preference for it. Further research using other antimicrobial plants is recommended as consumers did not like pito with the M. oleifera leaf extract.
Key words: Moringa, pasteurization, pito, shelf-life, storage.
INTRODUCTION
The brewing and drinking of traditional beverages are Traditional brewed beverages are characterized by good intrinsic part of the culture of the African people. mineral composition such as calcium, magnesium,
*Corresponding author. E-mail: [email protected]. Tel: +233(0) 543336674.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 2628 Afr. J. Biotechnol.
sodium, zinc, potassium and iron, which are necessary Information on the use of M. oleifera in the brewing of for regulating and building the living cells (Kolawole et al., sorghum beer has not received any attention. Therefore, 2007; Duodu et al., 2012) and also contain probiotic using pasteurization and a natural preservative (M. properties (Moslehi-Jenabian et al., 2010). One of such oleifera), which is easily accessible in Ghana and in the traditionally brewed beverage in Ghana is pito (sorghum tropics (Fahey, 2005; Quarcoo, 2008) in pito beer). Pito is brewed and consumed by people of the comparatively can be less expensive. West African sub regions (Demuyakor and Ohta, 1993). In light of the highlighted aforementioned problem Pito brewing is usually processed at households by associated with the pito industry, ways are needed to women and linked with the people of northern (Upper supply it to the citizens in a more quality and presentable West, Upper East and Northern Region) Ghana. It is an manner. Hence the relevance of this study is to improve income generating business which serves as a source of the shelf-life of pito by the addition of M. oleifera leaves employment in areas in which they are produced. under producers’ condition, combined with pasteurization However, pito production is limited by short shelf-life; as a at 75 to 80°C for 15 min. result, the product needs to be consumed within a day
(Demuyakor, 1994). MATERIALS AND METHODS The causes of short shelf-life of pito have been intensively studied. Microbial contamination and activity Sample collection from poor pito brewing practices is mainly the cause of Moringa oleifera processing spoilage (Novellie, 1962; Ekundayo, 1969; Faparusi et al., 1973; Dirar, 1978; Tisekwa, 1989). These microbes Fresh young green moringa (M. oleifera) leaves were collected from include diverse array of bacteria and wild yeasts with the Nyankpala community into a clean polyethylene bag. The lactic acid bacteria being the dominant spoilers leaves were washed thoroughly under tap running water, and dried at a room temperature (29 ± 2°C) and relative humidity of 46.5 to (Sakamoto and Konings, 2003). Spoilage of pito is 61.7% (Ghana Meteorological Agency, 2014) for 72 h. The dried attributed to undesirable changes in sensory charac- leaves were milled into fine powder using Philips blender teristics in terms of texture, smell, taste or appearance HR2000/16. The moringa powder was collected into a clean airtight (Doyle, 2001) which lead to discarding the whole product. bowl, and then stored in a refrigerator at 5°C.
Microbial spoilage of alcoholic beverages is of critical importance hence, different methods have been adapted Obtaining Moringa oleifera extracts to reduce spoilage. Among these methods are thermal treatment (pasteurization), filtration and chemical Dried moringa powder of 50 g was added to 500 ml distilled water in 1 litre conical flask, stoppered and kept for 1 week in a treatment (addition of artificial preservatives) (Ellis et al., refrigerator (5°C) with periodical manual shaking. The extract was 2005; Onaghise and Izuagbe, 1989; Osseyi et al., 2011). filtered using a clean, sterilized muslin cloth, and then boiled for 30 Instances when pasteurized at 75°C for 30 min and min with continuous stirring. addition of sorbic acid concentration of 5% improved the shelf-life of pito for a period of four weeks (Onaghise and Sourcing of pito Izuagbe, 1989). Some of these methods can be sophisticated and expensive for the small scale operator Dagarti pito samples were obtained from a commercial brewer in to adopt. Ellis et al. (2005) also improved the shelf-life of Nyankpala Township, in the Tolon District of Northern Region. Dagarti pito was prepared as shown in the flow chart. filtered pito for eight weeks by pasteurization (60 to 70°C for 15 min) and adding sodium metabisulphite. Increasing the shelf life of pito via chemical means such as sodium Treatments metabisulphite can have adverse effects such as In this study, four treatments were applied thus: untreated pito respiratory tract irritation and anaphylactic symptoms (control), pasteurised pito, moringa pito (moringa leaf extract + pito) which is life threatening (Pavord et al., 1991; Vally et al., and pasteurized moringa pito. Nine (9) litres of each treatment was 2009). dispensed from kegs into sterilised 300 ml glass bottles and Although synthetic antimicrobial and antioxidant agents crowned immediately using a manual hand crowner, then packaged into its respective box and labelled. Moringa pito treatments had M. are approved in many countries, its usage has created oleifera extracts and pito in the ratio 1:3 (v/v). The ratio of moringa environmental and health concerns, which has called for to pito was achieved through preliminary study of moringa pito, natural, safe and effective preservatives by consumers which showed that 25% moringa extract composition of the and producers (Ortega-Ramirez et al., 2014; Regnier et beverage was preferred by consumers. All pasteurised treatment al., 2012). Ortega-Ramirez et al. (2014) proposed that samples were carried at 75 to 80°C for 15 min using a water bath. medicinal plants traditionally used to treat health Pasteurised samples were allowed to cool before storage. The samples were stored for two months at an ambient temperature. disorders and prevent diseases can serve as a source of Analysis were carried during 0, 7, 14, 28 and 56 days of storage. bioactive compounds for food additives. This is because these medicinal plants are rich in antimicrobial phyto- Microbial analysis of samples chemicals. M. oleifera leaf, in that regard is also found to possess antimicrobial properties (Eilert et al., 1981). Microbial analysis was carried out for each treatment thus untreated Ayirezang et al. 2629
pito, pasteurised pito, moringa pito, and pasteurized moringa pito There was a general decline of coliform growth during during 0, 7, 14, 28 and 56 days of storage. MacConkey agar and storage. Pasteurized pito, moringa pito and pasteurized potato dextrose agar were used to determine coliforms and fungi moringa pito had similar (P > 0.05) coliform count. The (moulds and yeast), respectively. MacConkey agar and potato dextrose agar media were prepared using the manufacturer’s treated pito (pasteurized, moringa and pasteurized protocol from Sigma-Aldrich. The pour plate method as described moringa pito) showed a relative reduction of coliform by Hoben and Somasegaran (1982) was used in the microbial growth as compared to the untreated pito 0 day of count. storage. The reduction of pasteurized moringa pito was two times that of the pasteurized pito and four times that
Analysis of physico-chemical properties of the moringa pito. The untreated pito recorded the highest coliform growth than the treated pito samples. Four replicates of each treatment (untreated pito, pasteurized pito, As in the coliform growth, there was also a general moringa pito and pasteurized moringa pito) were sampled and decline of fungi growth during storage (Figure 3). The examined for pH, sugar and alcohol levels at 0, 7, 14, 28 and 56 study reveals the presence of fungi in each treatment. days of storage. The pH was determined using basic 20 pH meter Mycological count of samples of untreated pito, and the hand held refractometer was used to measure extract % in pasteurized pito, moringa pito and pasteurized moringa mass saccharose. The alcohol content was carried out using the 5 5 5 specific gravity method described by Mathapati et al. (2010). pito ranged between 4.6 × 10 to 37× 10 , 2.1 × 10 to 0.9 5 5 5 5 5 × 10 , 21 × 10 to 1.4 × 10 and 0.7 × 10 to 0.2 × 10 , respectively within 56 days of storage as shown in Figure Sensory evaluation of bottled pito 5. Pasteurized pito and pasteurized moringa pito had the low fungi load compared to untreated pito and moringa Sensory evaluation of each treatment (untreated pito, pasteurised pito, moringa pito, and pasteurized moringa pito) was assessed by pito prior to storage and after storage. There was 150 subjects (taste panel). The panel consists of students and significance difference (P< 0.05) in fungi population lecturers of UDS Nyankpala Campus, as well as community among treatments. Generally, pasteurized moringa pito members from Nyankpala, selected based on their familiarity with had relatively low fungi load. pito. Consumer preferences of the samples listed were compared at 0, 7, 14, 28 and 56 days of storage. Sensory ballot sheet was provided for each subject and the sensory scale adapted was the 5- point hedonic scale, just-about right scale and the forced-choice Physico-chemical properties (yes/no) scale (Stone and Sidel, 2004). Samples were coded and presented to the assessors to indicate their preferences. Samples pH levels were served in clean, transparent plastic containers with tight lids. The pH value of pito treatments during storage is
Statistical analysis presented in Figure 1. Untreated pito, pasteurized pito, morringa pito and pasteurized pito had pH between 3.2 to Data obtained from the paired preference test were analysed using 3.4, 3.2 to 3.5, 3.5 to 3.2 and 3.2 to 3.6, respectively the Microsoft Excel Programme. All other data were subjected to during the entire period of storage (Figure 4). pH values analysis of variance (ANOVA) for variation of means of treatments were decreased during storage. The pasteurized pito and with their respective period of storage, using the Genstat Discovery pasteurized moringa pito showed significantly (P < 0.05) edition 4. Multiple mean comparisons were also carried out with the Minitab. Statistical significance was set at P ˂ 0.05. higher pH values than the untreated pito from the 0 day to the 56 days of storage and with moringa pito from 0 day to the 28 days of storage. The level of pH reduction RESULTS in the untreated pito, moringa pito and pasteurized pito was 0.2, 0.1 and 0.04 times that of pasteurized moringa Microbial quality of samples during storage pito, respectively.
Microbial quality of pito beverage in this study reveals the presence of enterobacteriaceae (coliforms) when Levels of extract in % mass saccharose samples were analysed on MacConkey. Coliform count of samples of untreated pito, pasteurized pito, moringa pito Levels of extract (%) in the various treatment of pito and pasteurized moringa pito were between 1.5 × 105 to during storage are shown in Figure 5. There was a 40 ×105, 2.6 × 105 to 3.1 ×105, 1.3 ×105 to 7.1 ×105 and general decline in the levels of extract (%) in each 0.8 ×105 to 1.5 ×105, respectively during 56 days of treatment. Levels of extract (%) in untreated pito, storage (Figure 5). Coliform growth/population was pasteurized pito, moringa pito, and pasteurized moringa significanlty different (P < 0.05) among treatment during pito during 56 days of storage ranged from 5.00 to 7.00, storage. Coliform growth and population reduced in each 6.00 to 7.00, 4.20 to 6.52 and 5.00 to 6.28%, pito treatment during the period of storage (Figure 2). respectively. Pasteurized moringa pito were significantly Varied levels of coliforms were, however, observed with (P < 0.05) higher in the extract (%) than in the untreated respect to treatments as well as the duration of storage. pito from the 7 days to the 56 days of storage, and for the 2630 Afr. J. Biotechnol.
Milled Sorghum Malt
Water Yolga (Anogeisus leo carpus) Sedimentation of mix
Sediment Supernatant
Boil (1 hour)
Keep Over Night
Sediment Supernatant
Boil (5 hours)
Cool
Crude Yeast from Previous Brew
Ferment (14 hours)
Figure 1. A flow chart of the brewing process of Dagarti pito (Demuyakor, 1994).
80 Untreated pito P = 0.001 70 Pasteurized pito 60 Moringa pito
50 Pasteurized moringa pito
40 a
30 cfu/ml ) 5
2010 b b ( b 10 b b b b b b b 0 b b b b b b b b 0 day 7 days 14 daysb 28 days 56 days
-10 Viable number of colonies of coliforms coliforms of colonies of number Viable -20 Days of storage
Figure 2. Viable number of coliforms when conventional and treated pito were stored for two months. Values are means and standard deviations of colony forming unit per millilitres; values that do not share the same letter are significantly different (P < 0.05). All treatments were stored at an ambient room temperature. Ayirezang et al. 2631
70
P = 0.001 Untreated pito 60 Pasteurized pito Moringa pito
) cfu/ml ) cfu/ml 50
5 Pasteurized moringa pito
10
( 40 a 30 ab
20 abc bcd bcd 10 bcd bcd cd cd cd cd cd d cd d d Viable number of fungi of number Viable d 0 d d d 0 day 7 days 14 days 28 days 56 days -10 Days of storage
Figure 3. Viable number of fungi for conventional and modified pito stored for two months. Values are means and standard deviations of fungi forming units per millilitres; values that do not share the same letter are significantly different (P< 0.05). All treatments were stored at an ambient room temperature.
3.7 P = 0.001
3.6 ab ab Untreated pito 3.5 bc bc bc bcd cde Pasteurized pito 3.4 cd def def efg 3.3 fgh ghi Moringa pito ghi hij 3.2 ij ij ij j Pasteurized 3.1 j moringa pito
pH levels levels pH forsamples pito 3.0 2.9 0 day 7 days 14 days 28 days 56 days Days of storage
Figure 4. Levels of pH during 56 days. Values are the means ± standard deviation of quadruple determinations. Values that do not share the same letter are significantly different (P< 0.05). All treatments were stored at an ambient room temperature.
moringa pito from the 14 days to the 56 days of storage. difference of 19, 11 and 8%, in that order with respect to The extract (%) in all the treatments was reducing with pasteurized moringa pito during storage. From 0 day to increasing duration of storage. Untreated pito, moringa 14 days of storage, there was no significant (P > 0.05) pito and pasteurized pito, recorded a percentage reduction of extract (%) of the pasteurized pito and 2632 Afr. J. Biotechnol.
7.50 a a a p = 0.001 7.00
a 6.50 b b bc b cd cd Untreated 6.00 cd cd d pito 5.50 e Pasteurized 5.00 e ef pito h gef 4.50 Moringa pito
4.00 fg Levels Levels extract (%) of pito of 3.50 Pasteurized moringa pito 3.00 0 day 7 days 14 days 28 days 56 days Days of storage
Figure 5. Extract (%) during 56 days of storage. Values are the means ± standard deviation of quadruple determinations. Values that do not share the same letter are significantly different (P< 0.05). All treatments were stored at an ambient room temperature.
4.50 P = 0.001 Untreated pito
Pasteurized pito 4.00 a a a b Moringa pito Pasteurized moringa pito 3.50 c c c c 3.00 d e e e e 2.50 e e
f f
Levels % ofABV ofpito 2.00 f f g 1.50 0 day 7 days 14 days 28 days 56 days
Days of storage
Figure 6. Percentage of alcohol by volume (% ABV) during 56 days of storage of untreated pito and treated pito. Values are the means ± standard deviation of quadruple determinations. Values that do not share the same letter are significantly different (P < 0.05). All treatments were stored at an ambient room temperature.
pasteurized moringa pito. Alcohol levels were between 1.96 and 3.93, 2.62 and 3.93, 2.10 and 3.44 and 2.62 and 3.28% in untreated pito, pasteurized pito, moringa pito and pasteurized Alcohol levels moringa pito, respectively. Pasteurized pito, moringa pito and pasteurized moringa pito had higher alcohol content The alcohol content decreased during storage (Figure 6). than the untreated pito from 7 days to 56 days of storage. Ayirezang et al. 2633
140 120 100 80 Not sour 60 40 Just-about- 20 right 0
Very sour
7day 7day 7day 7day
0 day 0 day 0 day 0 day 0
14days 28days 56days 14days 28days 56days 14days 28days 56days 14days 28days 56days Number of respondants of Number Untreated pito Pasteurized Moringa pito Pasteurized pito moringa pito Treatments
Figure 7. The degree of sourness of pito samples. Market acceptability of pito samples.
160 140
120 100 80 60 40 20 yes 0
No
7day 7day 7day 7day
0 day 0 day 0 day 0 day 0
14 days 14 days 28 days 56 days 14 days 28 days 56 days 14 days 28 days 56 days 14 days 28 days 56 Number of respondants of Number Untreated pito Pasteurized pito Moringa pito Pasteurized moringa pito Treatments
Figure 8. Preference of pito samples in the Ghanaian market.
There was significant difference (P < 0.05) in alcohol showed that untreated pito samples were very sour after value in the samples investigated. The percentage 0 day of storage; followed by moringa pito, then the difference of the untreated pito during the 56 days of pasteurized pito samples (pasteurized pito and storage with respect to pasteurized moringa pito was pasteurized moringa pito) which became very sour after 33%; and moringa pito, pasteurized pito, recorded 24 and 14 and 28 days of storage, respectively. The preference 20%, respectively. Statistically, no significant (P > 0.05) of pito samples in the Ghanaian market (Figure 8) reduction in the alcohol levels was observed when the showed that respondents favoured pito samples without treated pito samples: pasteurized pito, moringa pito and moringa extract in the Ghanaian market than those with pasteurized moringa pito, were stored for seven days. the moringa extract. The findings of this study (Table 1) clearly showed significant difference (P < 0.05) in the overall degree of Sensory evaluation of bottled pito liking. Pito samples without moringa extract were highly
Consumer preference for the degree of sourness of acceptable by the assessors than those with moringa pito samples extract. The untreated pito was disliked extremely by assessors after a week of storage. Pasteurized pito was The degree of sourness of pito samples (Figure 7) extremely liked throughout a storage period of 4 weeks. 2634 Afr. J. Biotechnol.
Table 1. Presentation of the acceptability of treatments during storage.
Pito sample Parameter Storage (days) Fpr < 0.05 Untreated pito Pasteurized pito Moringa Pito Pasteurized moringa pito 0 4.41±0.70a 4.27±0.75ab 2.86±1.19efg 2.83±1.27efg 0.001 Overall 7 3.87±0.99bc 4.29±0.69ab 2.60±1.22g 2.78±1.24fg degree 14 3.26±1.23de 4.23±0.73ab 2.69±1.08fg 2.65±1.11fg of 28 3.30±1.38de 3.61±1.36cd 2.55±1.37g 2.65±1.31fg liking 56 2.51±1.51g 3.08±1.36ef 1.87±0.92h 1.91±1.09h
Values are means and standard deviations of triplicate determinations; values within interactions of individual sensory attributes that do not share the same letter are significantly different (P< 0.05). A 5-hedonic scale was used (1= dislike extremely/least acceptable; 3= neutral; 5= like extremely/ highly acceptable).
DISCUSSION in the case of coliforms. Generally, the addition of moringa extract and/or the heat treatment might have Moringa pito exhibited antimicrobial activity which is a been the major contributing factor, influencing the overall characteristic of moringa extract hence contributed to the reduction of microbial growth in moringa pito, pasteurized reduction of coliforms in pito at zero day compared to the pito and pasteurized moringa pito. high coliform load in untreated pito. The heat shock from In order to improve the shelf life of sorghum beer (pito), pasteurized pito and pasteurized moringa pito reduced the addition of M. oleifera to and pasteurization of coliform load in samples prior to storage. The lowest sorghum beer were employed in comparison with reduction of microbial load in pasteurized moringa pito traditionally brewed sorghum beer (pito). The pH values can be attributed to the cocktail of treatment hence can for all pito samples (untreated pito, pasteurized pito, enhanced microbial stability in pito during 56 days of moringa pito, and pasteurized moringa pito) were within storage. However the reduction of coliform load in the stipulated pH range for sorghum beer of 3 to 4 as untreated pito during storage would be as a result of indicated in previous study of increasing shelf of pito and spontaneous microbial activity; this include accumulation microbial assessment of pito during storage (Fadahunsi of lactic acid and acetic acids during storage produced by et al., 2013; Ellis et al., 2005; Kalawole et al., 2007). bacteria, which are detrimental to some sensitive bacteria However, each treatment lacked stability with respect to (Ekundayo, 1969; Fadahunsi et al., 2013) as well as the pH (3.2 to 3.5) value, which can be attributed to activities lowering of pH values in pito during the storage which of microbes with the pito samples during the period of some coliforms cannot survive (Ray and Bhunia, 2013) storage. Microbial activities were much more in untreated hence explaining the drastic reduction of coliform pito which led to a lower pH value compared to moringa population in untreated pito samples. Similar inference pito, pasteurized pito and pasteurized moringa pito which could be made for the reduction of coliform in various had higher pH values. The lower pH can also be treatments applied to the sorghum beer (pito). attributed to organic acid produced by some Unlike coliform, the mould population did not reduce microorganisms (bacteria, moulds and yeast) that were very significantly in moringa pito. This implied that, isolated in the pito treated samples and also suggested antimicrobial activity of moringa varied with the type of by Fadahunsi et al (2013). The pH values recorded did microbe which confirms the findings made by some not corroborate with Ellis et al. (2005) and Osseyi et al. researchers, on the inhibitory effect of M. oleifera leaf (2011) reports in which sorghum beer pasteurized at 60 extract on some selected fungal strains (Bukar et al., to 70°C had a stabilized pH value of 3.4 and 3.45 for 8 2010; Devendra et al., 2011) but a combination of weeks and 6 months, respectively during storage. The pasteurization and moringa extract treatment can reduce high pH in moringa pito, pasteurized pito and pasteurized mould population to abitarily acceptable levels. This moringa pito signifies low microbial activity in the treated corroborates with previous reports that pasteurization is pito samples. Comparatively, pH variation in moringa capable of inactivating microbial activity in traditionally pito, pasteurized pito, and pasteurized moringa was due brewed sorghum beers (Ellis et al., 2005; Osseyi et al., to the difference in each treatment carried out. Moringa 2011). The significant reduction of the number of fungi pito suggested that Moringa oleifera inclusion only could growth in the untreated pito during storage might have minize microbial activity to some extent when compared resulted from the exhaustion of nutrients in the products, to untreated pito. Both pasteurized pito, and pasteurized thus reducing the overall food availability for the moringa pito were better off in reducing microbial activity microorganisms as reported by other researchers hence having a higher pH value than moringa pito and (Fadahunsi et al., 2013). Also, the growth of fungi might untreated pito. This implies that pasteurized pito, and have been impeded by unfavourable conditions as stated pasteurized moringa pito would be less acidic compared Ayirezang et al. 2635
to moringa pito and untreated pito, since it has been produced more acids contributing to the sourness of pito reported that the souring in sorghum beer is owned to the during storage hence the low pH recorded for each presence of lactic acid bacteria or acetic acid bacteria sample during storage. The reduction of alcohol in all (Ekundayo, 1969; Demuyakor, 1994; Steinkraus, 2004; samples during storage would be linked to alcohol Lyumugabe et al., 2012). degrading microbes. Similar inference could be made for Levels of extract (% mass saccharose) in sorghum untreated pito. The significant reduction of alcohol in the beer (pito) brewing were determined by fermentation untreated pito led to early spoilage. This observation period and yeast cells activities in pito brewing. The indicated that for the efficient conversion of sugar to decline of the extract (%) was expected because sugar alcohol involves other factors (Demuyakor and Ohta, was converted into alcohol. The extract (%) showed a 1993; Lin et al., 2012). The stability of alcohol level in decline in all the treatments. This implied that untreated pito might have resulted from inadequate fermentation was still on-going, hence the presence of carbon source for the microorganisms present and also some microbes and yeast cell as revealed in the the low pH of the medium might not have been microbiology of the pito sampled. The decline of the appropriate for some microbial activity. This study extract (%) which showed a reduction in sugar content in suggests that moringa and/or pasteurization treatments pito during 56 days storage corroborates with earlier were capable of maintaining the alcohol content in the report of Demuyakor and Ohta, (1993), thus the sugar pito during storage for seven days of storage. content in pito reduced during storage since it served as The undesirable sensory characteristics of sorghum carbon source for energy by the microorganisms present. pito are as a result of microbial presence and activity The low extract (%) levels in moringa pito and (metabolic processes) that leads to the sorghum beer pasteurized moringa prior to storage might be due to the spoilage. The presence of coliforms and moulds in the 25% reduction in the volume of pito to allow for the pito samples may have resulted from unhygienic addition of the moringa extract. Despite Moringa oleifera practices during and after the brewing process of the pito, leaves known to contain carbohydrate (Mustapha and causing the relatively high increase of coliform and mould Babura, 2009), conversion rate into fermentable sugars growth at the initial stage of storage in the untreated pito. may be low because of Saccharomyces cerevisiae The high coliform and fungi count in the fresh pito might inability to convert starch/complex carbohydrate to simple have led to the present coliform in the pito samples after sugars and later to alcohol as well as the large proportion pasteurization and/or the addition of moringa extract. of carbohydrate in malted sorghum prior to fermentation The intensity of the sourness is a reflection of the (Lyumugabe et al., 2010). In additon, M. oleifera extract product acidity. The right degree of sourness is might have influenced S. cerevisiae activity hence considered as part of the general characteristics of a translating into the low percentages of sugar in the pito at good pito (Demuyakor, 1994); if it becomes very sour it is the initial stage of storage when treated with moringa an indication of deterioration. Also, the untreated pito was extract (moringa and pasteurized moringa pito). Untreated very sour after seven days of storage indicating the pito encountered high microbial activity hence resulting in impact of the decrease of pH level during storage. The the rapid decline of the amount of sugar for 14 days of panelists reported that the sourness was just about right storage. Pasteurized pito had minimum about of microbial for pasteurized pito, moringa pito and pasteurized load hence resulting in sugar stability for 14 days of moringa pito, until after 28 days of storage. storage. This period could be described as lag The overall degree of liking and potential of products periods/phase as sugar usage was almost negligible. The being accepted on the Ghanaian market mirrored the decline in extract (%) in pasteurised pito from day 14 to choices made by panels in each of the sensory attributes 28 might be due to microbial build up hence sugar earlier discussed. On the whole, pasteurized pito was utilization increased. mostly liked up to 28 days of storage. The untreated pito It was expected that, the amount of the sugar utilized was further not preferred after seven days of storage. should be equivalent to alcohol produced but this was not The reason may be attributed to off-flavours which alter observed. Stability of alcohol level observed for a week the quality of the beer causing it to deteriorate (Harayama (seven days) in pasteurized pito, pasteurized moringa et al., 1991; Rodrigues et al., 2011). Also the pito treated pito and moringa pito could be attributed to treatment with moringa extract which was less liked throughout the shock on microbes and change in the physico-chemical storage period shows that consumers are not familiar properties of the medium (pito), and hence sugar with the product, and this confirms report by Barcellos et utilization as a carbon source by microbes and yeast cell al. (2009) that, some consumers find it very difficult to was not efficient for the first seven days. Unlike untreated change. pito, it was characterized by alcohol reduction from day 1 to 14. Pasteurized pito, pasteurized moringa pito and moringa pito had a decrease in alcohol level after seven Conclusion days. This implied that alcohol reducing microbes were associated with each sample. The reduction of alcohol The addition of moringa extract and/or pasteurization had 2636 Afr. J. Biotechnol.
reduced the fungi and coliform growth in the treated pito Faparusi SI, Olofinboba MO, Ekwundayo JA (1973). The microbiology samples than the untreated pito. The physical of burukutu beer. Z. Allg. Mikrobiol. 13:563-568. Harayama K, Hayase F, Kato H (1991). Evaluation by multivariate composition, that is, pH, sugar and alcohol levels were analysis of off-flavor in headspace volatiles formed during storage of significantly influenced by pasteurization and the addition beer (food and nutrition). Agric. Biol. Chem. 55(2):393-398. of Moringa oleifera leaf extract: The untreated pito had Hoben H, Somasegaran P (1982). Comparison of the pour, spread, and lower pH than the treated pito samples during storage. drop plate methods for enumeration of Rhizobium spp. in inoculants made from presterilized peat. Appl. Environ. Microbiol. 44(5):1246- Comparatively causing the untreated pito to be very sour 1247. than the treated pito samples. Also the pasteurized pito, Kolawole O, Kayode R, Akinduyo B (2007). Proximate and microbial moringa pito and pasteurized moringa pito had higher analyses of burukutu and pito produced in Ilorin, Nigeria. Afr. J. sugar and alcohol content than the untreated pito during Biotechnol. 6(5):587-590. Lin Y, Zhang WLC, Sakakibara K, Tanaka S, Kong H (2012). Factors storage. Although pito with the moringa did improve the affecting ethanol fermentation using Saccharomyces cerevisiae shelf life, organoleptically, it was less liked by the BY4742. Biomass Bioenergy. 47:395-401. assessors. The sensory results showed that pasteurized Lyumugabe F, Gros J, Nzungize J, Bajyana E, Thonart P (2012). pito was more preferred by the consumers. Pasteurized Characteristics of African traditional beers brewed with sorghum malt: a review. Biotechnol. Agron. Soc. Environ. 16(4):510-526. pito was liked for the four weeks of storage. Based on the Lyumugabe F, Kamaliza G, Bajyana E, Thonart P (2010). findings of this study, the research concludes that the Microbiological and physico-chemical characteristics of Rwandesese shelf life of pito can be improved through pasteurization traditional beer. Afr. J. Biotechnol. 9(27):4241-4246. and/or the addition of Moringa oleifera leaf extract for 28 Mathapati PR, Ghasghase NV, Kulkarni MK (2010). Study of Saccharomyces cerevisiae 3282 for the production of tomato wine. days, however pito samples that contained the moringa Int. J. Adv. Chem. Sci. Appl. 1(1):5-15. extract was less favoured by consumers. Moslehi-Jenabian S, Pedersen LL, Jespersen L (2010). Beneficial effects of probiotic and food borne yeasts on human health. Nutrients 2(4):449-473. Mustapha Y, Babura S (2009). Determination of carbohydrate and β- Conflict of Interests carotene content of some vegetables consumed in Kano metropolis, Nigeria. Bayero J. Pure Appl. Sci. 2(1):119-121. 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