Issn 2087-3948 | E-Issn 2087-3956|

| Nusantara Biosci | vol. 10 | no. 3 | pp. 137-202 | August 2018 |

| ISSN 2087-3948 | E-ISSN 2087-3956|

Rizaldo Arbet Rizaldo

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Sentulchicken of Balitnak

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NUSANTARA BIOSCIENCE ISSN: 2087-3948 Vol. 10, No. 3, pp. 137-141 E-ISSN: 2087-3956 August 2018 DOI: 10.13057/nusbiosci/n100301

The growth performances and the gut health parameters of Sentul chickens supplemented with various dosage of turmeric powder

INDRAWATI Y. ASMARA1, TUTI WIDJASTUTI1, IWAN SETIAWAN1, ABUN1, RUHYAT PARTASASMITA2,♥ 1Faculty of Animal Husbandry, Universitas Padjadjaran. Jl. Raya Bandung-Sumedang Km 21, Jatinangor, Sumedang 45363, West Java, Indonesia 2Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran. Jl. Raya Bandung-Sumedang Km 21, Jatinangor, Sumedang 45363, West Java, Indonesia. Tel. +62-22-7796412 ext. 104. Fax. +62-22-7794545, ♥email: [email protected]; [email protected]

Manuscript received: 22 May 2018. Revision accepted: 14 June 2018.

Abstract. Asmara IY, Widjastuti T, Setiawan I, Abun, Partasasmita P. 2018. The growth performances and the gut health parameters of Sentul chickens supplemented with various dosage of turmeric powder. Nusantara Bioscience 10: 137-141. The aim of this study was to investigate the effect of different levels of turmeric powder (0, 1, 2, 3 g/kg) on production performance and the number of Escherichia coli and Staphylococcus aureus bacteria in the intestine of Sentul chickens. The results showed that addition of 3 g/kg of turmeric powder to diet significantly reduced feed intake (FI) and improved feed conversion ratio (FCR) of Sentul Chickens aged 9-16 weeks. The birds fed with control diet had the highest number of S. aureus and E. coli, while the birds fed with the diets added with 2 and 3 g/kg turmeric powder had the lowest number of bacteria. The study reveals that increasing levels of turmeric powder in diets had positive effects on bird’s performance and the number of intestinal bacteria. It can be concluded that a level of 3 g/kg turmeric powder in diets gave the best result due to significantly improved FCR.

Keywords: Curcuma domestica, intestinal bacteria, performance, Sentul chickens, turmeric powder

INTRODUCTION prohibition of synthetic feed additives such as growth promoter antibiotics being used in chicken feeds. The Indonesia is one of country, which the number of natural feed additives derived from turmeric (Curcuma chicken domestication centers is largest in the world. The domestica Val) have been used as one of alternatives of diversity of chickens in Indonesia is high and differ from growth promoter antibiotics in poultry rations due to their other countries in Asia and in the world (Sulandari et al. safety for consumers. Turmeric is an essential component 2008). It was reported that more than 30 distinct groups of used in the Asian cuisines (Ganpati et al. 2011) as well as local chickens are located in Indonesia (Nataamijaya 2000; in Asian indigenous medicine as an antimicrobial agent Partasasmita et al. 2016). Some of them have specific (Lee et al. 2010; Moghadamtousi et al. 2014). plumage color and are regarded as an indigenous species i.e In animal farming, the use of turmeric and other herbs Sentul chickens (Diwyanto and Iskandar 1999), have positive effects on the feed intake, the secretion of Sentul Chicken is a local chicken from Ciamis region, digestive secretions and as anti-bacterial agents (Wenk West Java Province, Indonesia. Ciamis people also call 2003). Curcumin is identified as the main phytochemical in Sentul chicken as ‘Kulawu Chickens’, kulawu means grey turmeric, which is responsible for biological effects (Teow since the plumage colors of Sentul chickens are dominated et al. 2016). Tyagi et al. (2015) reported that curcumin, by grey (Universitas Padjadjaran 2012). Sentul chickens especially curcumin I is a significant component in had many advantages to be farmed because they are able to commercial curcumin. It is effective against Gram-positive adapt to harsh environment and remain productive in low bacteria such as Staphylococcus aureus and Gram-negative quality of diets (Widjastuti 1996). They produce 120-140 bacteria such as Escherichia coli. Both bacteria are eggs per year (Universitas Padjadjaran 2012). The weight commonly found in gastrointestinal of mammals and birds. of adult male chickens can reach 1300-3500 g, whereas the Some strains of E. coli may be pathogenic (Vidotto et al. weight of female adults is 800-2200 g (Sulandari et al. 1990) and cause of negative impact in commercial poultry 2007). Nowadays, Sentul chickens are raised for meat farms (Blanco et al. 1997), while S. aureus has ability to industry. They are sold when their weights are about 750 g produce entero-toxins which may cause food poisoning in and the age of 2.5 months (Asmara 2014). However, like human (Mamza et al. 2010). other local chickens such as Pelung and Kedu chickens, the It has been reported that turmeric influences growth population of Sentul chickens is declined and the status is performances of poultry. Turmeric may possess an in danger (Asmara 2014). antibacterial effect in chickens (Samarasinghe et al. 2003; Sentul chickens are potential to be developed as organic Lawhavinit et al. 2010; Akbarian et al. 2011; Ürüşan and poultry farms. One of characteristics of organic poultry Bölükbaş 2017), may depress abdominal fat (Samarasinghe farms is the use of natural feed additives and the et al. 2003), improve FCR in broilers (Ürüşan and 138 NUSANTARA BIOSCIENCE 10 (3): 137-141, August 2018

Bölükbaş 2017), increase health status in local ducks (Nova Data analyses and Yellita 2015) and increase egg production in local Analysis of variance was applied to the data using chickens (Widjastuti 2017). However, studies on turmeric statistical package program of SPSS version 19. powder in local chicken rations are rarely explored; thus, Significantly different means were separated by a Duncan’s investigations of beneficial effects of turmeric powder in multiple comparison test at 0.05 and 0.01 levels, local chickens are required. The purpose of this study was respectively. to test effects of turmeric powder supplemented in local chicken diets from 0 to 3 g/kg on the growth performances of chicken, i.e., feed intake (FI), weight gain (WG), feed RESULTS AND DISCUSSION conversion ratio (FCR) and intestinal bacteria of local chicken. Growth responses of local chickens on different dietary treatments were presented in Table 2. During week 1 until week 8 of study, dietary supplementation of 1, 2 or 3 g/kg MATERIALS AND METHODS had no significant effect on the growth parameters of birds, i.e., FI, WG, and FCR. the supplementation of turmeric in Experimental design the diets significantly (P<0.05) changed the value of FI and This study was designed in Completely Randomized FCR in week 9 until week 16, whereas the diets had no Design (CRD). A total of 100 day-old chicks (unsexed) of significant effect on WG. Grey Sentul Chickens was randomly distributed into 4 FCR values were significantly influenced by dietary groups. The groups consisted of control and 3 treatment turmeric (P<0.05) during week 9 to week 16 (Table 2). FI groups in which each group contained 5 sub-groups of five values of the groups with supplemented by 2 and 3 g/kg birds. In control group, the birds were fed with basal feed, turmeric powder were significantly lower than those in 0 while in treatment groups the birds were fed with the basal (control group) and 1 g/kg turmeric powder, respectively. diet with different levels of turmeric powder (1, 2, 3 g/kg, The lowest FCR was observed in the group with 3 g/kg respectively). The feed and water were provided ad- turmeric powder in diets, while the highest FCR was libitum. FI and WG were recorded weekly, while FCR was observed in the control group. It was found that the measured by dividing FI by WG. differences of FCR were significant between the groups The study was divided in two different phases and the (P<0.05). The best FCR occurred in the group with diet compositions for each phase was shown in Table 1. addition of 3 g/kg turmeric powder in diet (5.19). The local ingredients used to produce the diets and the The highest dry matter digestibility occurred in the energy metabolism and protein needs were formulated group supplemented with 3 g/kg turmeric powder; while based on Widjastuti (1996) diet formulation for Sentul the lowest one was found in the group with the addition of chicken. At the end of the study, five birds were randomly 2 g/kg turmeric powder (Table 3). It was observed that the selected from each treatment group and were slaughtered. highest crude protein digestibility occurred in the diets with A total of 20 carcasses were opened to collect samples of the addition of 3 g/kg turmeric powder. small intestine tracts of bird. All samples were proceeded at the same day for enumerating bacterial populations using Table 2. The effect of supplementation turmeric powder on methods reported by Safitri et al. (2008). performance of local chickens

Groups Table1. Feed ingredients and nutrient composition Variable Control 1 g/kg 2 g/kg 3 g/kg Age Aged 1-8 weeks Feed ingredients 1-8 weeks 9-16 weeks Feed intake (g) 1,802.60 1,871.93 1,757.93 1,586.27 Yellow corn (%) 56.00 58.00 Weight gain (g) 436.27 391.47 407.93 459.87 Soybean meal (%) 12.00 4.75 Feed conversion ratio 4.21 4.86 4.31 3.59 Aged 9-16 weeks Rice bran meal (%) 21.50 28.00 a a b b Fish meal (%) 9.25 8.00 Feed intake (g) 2,4012.39 23,698.12 22,250.04 21,429.36 Weight gain (g) 774.95 740.99 775.73 834.25 CaCo3 (%) 0.50 0.50 a a a b Bone meal (%) 0.75 0.75 Feed conversion ratio 6.23 6.41 5.74 5.19 Note: a, b, c, d: means within a row with no common superscript Nutrient composition: differ significantly (p<0.05) Metabolism energy (kcal/kg) 2,850 2,755 Crude protein (%) 17.00 15.08 Crude fat (%) 5.92 6.66 Crude fiber (%) 4.51 4.89 Table 3. Nutrient digestibility of diets Calcium (%) 1.16 1.05 Groups Phosphorus (%) 0.63 0.58 Variable Lysine (%) 1.21 0.97 Control 1 g/kg 2 g/kg 3 g/kg a b c d Methionine (%) 0.40 0.35 Dry matter (%) 59,86 55,75 53,86 66,05 a b ac c Methionine + lysine (%) 0.75 0.67 Crude protein (%) 75,67 72,79 75,78 76,80 Note: Proximate analysis conducted at Nutrition and Feed Note: a, b, c, d: means within a row with no common superscript Laboratory, Faculty of Animal Husbandry, Universitas differ significantly (p<0.05) Padjadjaran, Sumedang, Indonesia

ASMARA et al. – Growth and health of Sentul chickens supplemented with turmeric powder 139

Table 4. The number of Staphylococcus aureus and Escherichia contain 1.5-5% essential oils (Gupta et al. 2013). The coli in small intestine negative effect of turmeric to feed intake of chickens should be further investigated. This is because the results of Groups Variable studies on feed flavoring and taste research for poultry are Control 1 g/kg 2 g/kg 3 g/kg a b b b still debatable (Damron 2003). S. aureus (CFU/g) 1.49 1.17 1.11 1.07 Even though FI reduced significantly in present study, (x105) E. coli (CFU/g) 1.47a 1.15b 1.00c 0.96c WGs were similar in each treatment. In the present study, (x105) supplementation of turmeric powder to rations significantly a, b, c, d: means within a row with no common superscript differ improved the nutrient digestibility. As a result, it has significantly (p<0.05) increased feed digestion and nutrient utilization. These results may indicate some beneficial effects of turmeric powder in intestinal tract as it was found in broiler reported Table 4 shows the total number of bacteria observed in by Al-Sultan (2003), Samarasinghe et al. (2003), Durrani et small intestine of samples supplemented with different al. (2006), and Ürüşan & Bölükbaş (2017) as well as in concentration of turmeric powder. It shows that the number local chickens reported by Wang et al. (2015). These of bacteria decreased in line with the increase of turmeric previous studies stated similar argumentation that turmeric powder addition in the diet. The highest bacteria were may stimulate protein synthesis particularly increased the observed in the control group. The lowest number of S. secretion of digestive juices and improved the aureus was obtained in all groups supplemented with gastrointestinal condition. The improvement of bird different concentration of turmeric powder. While the performance by dietary supplementation of turmeric, may lowest number of E. coli was found in the group with the be due to mechanisms which were proposed by Platel and addition of 2 and 3 g/kg turmeric powder, respectively. Shrinivasan (1996; 2004). Platel and Shrinivasan (1996) reported that curcumin Discussion promote pancreatic digestive enzymes of rats such as The results of present study indicated that turmeric amylase, lipase, and proteases. Curcumin supplementation response depends on chicken age and usage dosage. The also stimulates the liver to secrete more bile enriched in turmeric powder has no effects on FI, WG and FCR for bile acids (Platel and Srinivasan 2004). Liver and pancreas local chickens aged 1 until 8 weeks, whereas it has are the main secretive organs in poultry digestive system significant effect on FI and FCR for chickens aged 9 to 16 (Dibner and Richards 2004). The avian liver provides weeks. From week 1 to week 8, Sentul chickens are exocrine secretions called bile to emulsify fats and raise the considered in early young period. Wenk (2003) stated that pH of the duodenal digest, while pancreas provides in the early age, metabolism and nutrient digestion of digestive enzymes and electrolytes, as well as endocrine animals are not yet functioned optimally while in the later secretions, consist of insulin and glucagon. The electrolytes age the digestion processes can be optimized and adapted resulted from pancreas is important in reducing the acidity to the available feedstuffs. At the time of hatching, of the chime. Higher pH in intestinal is required for the bacterial starts to colonize gastro-intestinal tracts of activity of the digestive enzymes produced by pancreas chickens. However, the bacterial colonialization seems to (Dibner and Richards 2004). Digestive enzymes produced be stable at older age (Rehman et al. 2007). The by pancreas break down different types of nutrient. composition of the bacterial community in intestinal is Amylase acts on starch, while lipase breakdown fats. influenced by many factors such as diet and age (Lu et al. Proteases are essential in protein digestion; however, they 2003). As a result, the response of turmeric addition in also have role in solubilization of fiber (Romero 2014). rations in the present study can be observed in age period The present study showed that the supplementation of of 9 to 16 weeks). turmeric improved FCR of local chickens at aged more At age of 9-16 weeks, the turmeric powder reduced FI than 8 weeks. In particular, supplementation of 3 g/kg significantly especially in the group with the addition of 2 turmeric powder to diet significantly produced optimum and 3 g/kg turmeric powder, respectively. These results FCR. This result was mainly due to the decreased trend of were found to be similar with the results on broiler FI was in line with the increase of turmeric powder in chickens reported by Ürüşan and Bölükbaş (2017) and local rations while BW was similar among treatments. FCR chickens reported by Wang et al. (2015). Ürüşan and improvements were found in some studies such as Al- Bölükbaş (2017) and Wang et al. (2015) stated that the Sultan (2003), Samarasinghe et al. (2003), Durrani et al. decrease of FI in their studies may be because of the effects (2006), Wang et al. (2015) and Ürüşan and Bölükbaş of turmeric aroma on the birds’ appetites. Wenk (2003) (2017). The low FCR in birds fed diets supplemented with reported that addition of seven different herbs or herb turmeric powder indicates the impact of phytogenic mixtures in basal diet result in a significant effect on feed products of turmeric that influence nutrient efficiency. intake due to anthraquinone derivatives. Aromatic taste and Optimization of pancreatic, as well as liver secretions due smell of turmeric are derived from its essential oils (Gupta to turmeric supplementation, may reduce time passage and et al. 2013). Aromatic oily liquids which are extracted by viscosity of diets. Acceleration of digestive process as a distillation from plant samples such as seeds and flowers result of increased availability of digestive enzymes and are known as essential oils (Adaszyńska-Skwirzyńska and bile acids causes the time reduction of feed transit. Nutrient Szczerbińska 2017). In general, dried turmeric rhizomes

140 NUSANTARA BIOSCIENCE 10 (3): 137-141, August 2018 absorption in the small intestine is more optimal in this to inhibit bacterial cell proliferation (Teow et al. 2016). situation (Platel and Shrinivasan 2004). However, a study reported different mechanism of The increased availability of digestive secretions may curcumin as anti-bacterial agent. Tyagi et al. (2015) also reduce viscosity of diets in gastrointestinal of chicken. reported that Curcumin I, a commercial curcumin, inhibits For example, increased proteases in intestinal are able to both S. aureus and E. coli. mainly by damaging the optimize disruption of proteins with starch and fiber in the bacterial membrane integrity. Fluorescent probes namely diets. Diet with high fiber increases viscosity of feed. The propidium iodide and calcein are used to examine the digestion rate would be expected to decrease, as intestinal bacterial membranes, while membrane leakage upon viscosity increases. In this situation, chicken would exposure to curcumin was also evaluated by fluorescence perceive a lower nutrient density diet (Bedford 1995). In and scanning electron microscopes. addition to previous mechanism, Rajput et al. (2013) stated The present study suggests that chicken with diets of that improved feed efficiency in chicken supplemented turmeric powder utilized feed more efficiently; therefore, with turmeric powder might be due to the larger villus area, resulting in a better feed conversion ratio. In addition, the which resulted in the improvement of nutrients absorption. turmeric powder may decrease the activities of some The villi are long folds of epithelial cells which is pathogenic bacteria for a better absorption of nutrients. In important for enzyme secretion and nutrient absorption particular, the level of 3 g/kg turmeric powder was found to (Dibner and Richards 2004). be beneficial to local chickens since the chickens improved The S. aureus and E. coli amount were less in the FCR due to positively affected gut bacteria. groups where the dosage of turmeric powder was increased in the ration. The dose level of 2 and 3 g/kg turmeric powder groups added in chicken diets resulted in the lowest ACKNOWLEDGMENTS number of bacteria. The finding is in agreement with the findings of Samarasinghe et al. (2003) and Lawhavinit et We would like to thank Universitas Padjadjaran, al. (2010). Samaringhe et al. (2003) reported that dietary Bandung, Indonesia through Academic Leadership Grant turmeric powder reduced E. coli bacteria in duodenum of (ALG) for funding this study. the broilers, while Lawhavinit et al. (2010) stated that turmeric could inhibit S. aureus of shrimp and chicken. 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Effect of therapeutic target of antibiotics. J Biol Chem 278 (45): 44424-44428. dietary supplementation of curcumin on growth performance, Wenk C. 2003. Herbs and botanicals as feed additives in monogastric intestinal morphology and nutrients utilization of broiler chicks. J animals. Asian-Aust J Anim Sci 16 (2): 282-289. Poult Sci 50: 44-52. Widjastuti T. 1996. Determination of Efficiency of Protein Use, Protein Rehman H, Vahjen W, Awad WA, Zentek J.2007. Indigenous bacteria and Energy Needs for Growth and Production of Sentul Chicken Eggs on bacterial metabolic products in the gastrointestinal tract of broiler Cage System and Litter System. [Dissertation]. Postgraduate chickens. Archives Anim Nutr 61 (5): 319-335. Program, Universitas Padjadjaran, Bandung. [Indonesian] Romero L. 2014. The role of feed enzymes in poultry gut health. Afma Matrix Oct 2014: 48-53.

NUSANTARA BIOSCIENCE ISSN: 2087-3948 Vol. 10, No. 3, pp. 142-145 E-ISSN: 2087-3956 August 2018 DOI: 10.13057/nusbiosci/n100302

Short Communication: Tetrazolium test for evaluating viability of Capsicum annum seeds

ADITYA KUSUMAWARDANA1,♥, BAMBANG PUJIASMANTO2, PARDONO2 1Program of Agronomy, Graduate Program, Universitas Sebelas Maret. Jl. Ir. Sutami 36A Surakarta 57126, Jawa Tengah, Indonesia. Tel./fax. +62-271- 632450, email: [email protected] 2Department of Agrotechnology, Faculty of Agriculture. Universitas Sebelas Maret. Jl. Ir. Sutami 36A Surakarta 57126, Central Java, Indonesia

Manuscript received: 14 Maret 2018. Revision accepted: 29 July 2018.

Abstract. Kusumawardana A, Pujiasmanto B, Pardono. 2018. Tetrazolium test for evaluating viability of Capsicum annum seeds. Nusantara Bioscience 10: 142-145. Seed quality is important in seeds production. This research was conducted to obtain information of topographical tetrazolium staining pattern of pepper (Capsicum annum) seeds. Tetrazolium test was conducted to determine the seed viability and plant growth vigor. Laboratory test for standard germination and field performances were performed on four lots (A, B, C, D) of pepper seeds. The viability categories pattern were determined by Root Mean Square (RMS), regression, and correlation analyses. Nine topographical patterns were recognized. The laboratory test results and field performances were compared with the topographical pattern. Combination of patterns 1,2 (embryonic axis and cotyledon completely stained) selected as viable category as it gave the least RMS value, the highest determination (R²) and correlation (r) coefficient with standard germination (RMS = 4, 06; R² = 0,761; r = 0,872). Combination of patterns 1,2 also gave the highest determination (R²) and correlation (r) coefficient with field stand (R² = 0,921; r = 0,959). The combination of patterns 1,2 is recommended for estimating plant growth performance in the field.

Keywords: Pepper seed, tetrazolium, topographical pattern, viability

INTRODUCTION MATERIALS AND METHODS

The quality of chili pepper seeds (Capsicum annum) Time and place must be known before they can be released to the market- The study was conducted from October 2017 to the sooner the quality identification, the better. The initial February 2018 at the laboratory and greenhouse of assay of chili pepper quality is the germination assay. Research Center for Development of Food Crop and However, through this assay, it takes at least 14 days to Horticulture Seed Quality Assessment (BB PPMB-TPH), know the viability of seeds. Thus, a convenient method is Ministry of Agriculture, Depok, West Java, Indonesia. necessary to quickly assess the quality of seeds and their germination. Experiments The majority of seed technologist defines seed viability Determination of seed quality as the capacity of a seed to germinate and form a normal Chili pepper seeds were obtained from several seed seedling. Therefore, the seed viability is tantamount to the companies. There were four lots of seeds analyzed in this germination capacity (Copeland and McDonald,1995). experiment, chili pepper variety Laskar (Lot A), variety Deminicis et al. (2014) suggest that tetrazolium test can be Sret (Lot B), variety Serambi (Lot C), and variety Madun chosen as an alternative to the conventional viability assay (Lot D). Lot B chili seeds have been stored for 12 months in Stizolobium aterrimum seeds. Tetrazolium test is a at 25°C. biochemical approach to assay seed viability based on the color change of plant life tissue upon treatment with Viability assay tetrazolium salts (Dina 2006). Upon staining with Seed viability assay was done by observing the tetrazolium salts, living cells will be dyed red whereas dead germination capacity of seeds and by tetrazolium-based cell will be colored white. The red color of the staining is method. For the germination assay, seeds were grown on a produced as the result of dehydrogenase enzyme activity filter paper in an electrical germination chamber at 20o- that releases H+ ions reacting with the tetrazolium salts and 30oC. Observation and recording of normal seedlings were forms stable, insoluble red formazan precipitate. The color done at day 7 and day 14 after seed placement (ISTA, intensity and the extent of the dyed area, also known 2016). For tetrazolium staining, seeds were put in a wet collectively as the topography, indicate the seeds viability paper at 20oC for 18 hours. Seeds were then wounded by (ISTA, 2016). The advantage of this staining method is that cutting the testa between the radicle and the cotyledon. it only requires 2-3 days to perform. Wounded seeds were immersed in 1% tetrazolium chloride Our study aimed to classify the staining topography of solution (in phosphate buffer) for 6 hours at 30oC, at dark tetrazolium assay on chili pepper seeds, as the basis to condition (ISTA, 2016). Seeds were cut in half, observed, determine the seed viability for future application in the field. and grouped based on their staining topographical pattern. KUSUMAWARDANA et al. – Tetrazolium test for evaluating viability of Capsicum annum seeds 143

The percentage of each topographical pattern group was Where, G is the percentage of germinated seedlings; P calculated. Each experiment used 50 seeds with 8 replicates. is the percentage of each topographical pattern group or a combination of multiple patterns; n is the number of seed Growth performance test lots (n = 4). G and P are obtained from the mean of values To assess the growth performance of chili peppers from eight replicates. varieties, seeds were grown uniformly in polybags in a controlled condition in a greenhouse. One seed was sown in each polybag at 2-3 cm depth. During the experiment, no RESULTS AND DISCUSSION refilling done on any dead plants. The observation of plant height was done at week 2 until week 6. This experiment Seed quality test results showed moisture value, weight aimed to verify the correspondence between the staining of 1000 grains and germination of consecutive lots A was topographical pattern with the plant viability standard. 7.5%, 4.34 g and 80%, lot B 9.4%, 3.41 g and 64%, C lots 7.4%, 4.66 g and 98% and lot D 5.5%, 4.49 g and 85% Data analyses (testing in October 2017). Tetrazolium assay can clearly All experiments used a completely randomized design indicate the structure and condition of a chili pepper. The with one factor, the seed lot. Data were analyzed using staining topography of the embryo upon 1% tetrazolium analysis of variance and Duncan Multiple Range Test at treatment can be observed under a microscope at 40x 5% significance level. Determination of topographical magnification. pattern of tetrazolium staining as the standard test for seed viability is based on the root mean square (RMS) value Viability assay by tetrazolium staining between the seedling viability test and tetrazolium staining Our tetrazolium staining experiment revealed at least 9 (Kuo et al. 1996; Pant et al. 1999). RMS was calculated topographical patterns in tested seeds. The patterns were using the following formula: sorted from the pattern that indicates the highest viability (pattern 1) to the lowest (pattern 9) (Figure 1).

Figure 1. Topographical pattern of tetrazolium

144 NUSANTARA BIOSCIENCE 10 (3): 142-145, August 2018

The percentage of each topographical pattern of Table 4 shows that lot C is the lot with the best growth tetrazolium staining is shown in Table 1. Pattern 1 and 2, performance, followed by lot D, A, and B, consecutively. collectively, are the predominant patterns observed on each Within 2 to 6 weeks after planting, lot C clearly produce seed tested: lot A 82%, lot B 69%, lot C 92%, and lot D the tallest plants 86%. Meanwhile, the percentage of pattern 3, 4, 5 Analysis of variance showed that the plant height of 4 occurrences was 5-6%; pattern 6 and 7 2-10%, pattern 8 seed lots from 2 to 6 WAP is significantly different (Table and 9 0-17%. 4). Lot C and D is not significantly different from each Our result indicates that there are 8 viable staining other, suggesting good vigor of the two plant groups (Table patterns (Table 2) with an RMS value less than 10. RMS 4). Analysis of regression and correlation between staining value greater than 10 shows that the representative pattern topology (patterns 1,2; 1,2,3; and 1,2,4) and plant height is considered as non-viable (not included in Table 2). was done. Correlation coefficient of each pattern Wibawa (2015) reported the RMS values obtained from the combination is shown in Table 5. Our result indicates a analysis of tetrazolium staining in viable papaya seeds are positive correlation between the tetrazolium staining below 10. pattern and the plant height. Pattern combination 1,2 shows Regression and correlation analyses between the the highest correlation among other combination (1,2,3 and tetrazolium staining test and germination test are shown in 1,2,4). The correlation coefficient of pattern 1,2 is 0.959. Table 3. Regression analysis can predict the dependent variable (Y) if the independent variable is known. The regression line equation describes the relationship between tetrazolium test (X axis) and the germination test (Y axis). The contribution of X variable to the Y value depends on the determination coefficient (R2) x 100%. The higher the R2, the higher the effect of the X variable to the Y variable. Table 3 shows that the determination coefficient for pattern 1,2 is 76,1% suggesting that the germination capacity is 76% determined by the pattern and 23.9% by other variables.

Plant growth performance assay Plant height is an indicator of plant growth performance in the field (Figure 2). The plant height of the four seed lots corresponds to their viability upon laboratory testing.

Tabel 1. Percentage of each topographical pattern of tetrazolium Figure 2. Plant growth in polybag staining in each seed lot

Topographical Lot A Lot B Lot C Lot D Table 3. Regression and correlation analyses of germination pattern (%) (%) (%) (%) assay and tetrazolium staining of three staining topography 1 42 36 54 46 combinations with least root mean square 2 40 33 38 40 3 3 1 3 2 Combination a b R² r 4 1 2 1 2 pattern 5 1 3 2 1 6 4 2 1 2 1, 2 -8.872 1.026 0.761 0.872 7 6 6 1 2 1, 2, 3 -13.63 1.105 0.755 0.869 8 3 9 0 3 1, 2, 4 -4.137 0.990 0.756 0.869 9 0 8 0 2 Note: a: interception, b: regression coefficient, R²: coefficient of determination, r: correlation coefficient

Table 2. Root mean square value of tetrazolium staining pattern Table 4. Height of plants grown in polybag of viable seeds Plant height (cm) Combination pattern Root mean square Seed lot 2 3 4 5 6 1, 2 4.06 WAP WAP WAP WAP WAP 1, 2, 3 4.44 Lot A 2.4b 4.0b 6.2b 10.7b 15.8b 1, 2, 4 4.79 Lot B 0.8c 1.7c 2.8c 4.1c 5.5c 1, 2, 5 4.82 Lot C 3.8a 6.3a 8.6a 13.7a 18.7a 1, 2, 3, 4 5.67 Lot D 3.7a 5.4a 8.1a 13.a 17.6a 1, 2, 3, 5 5.78 Note: Different letters indicate significant difference according to 1, 2, 4, 5 5.93 Duncan’s Multiple Range Test at 5% significance level; WAP: 1, 2, 3, 4, 5 7.17 week after planting.

KUSUMAWARDANA et al. – Tetrazolium test for evaluating viability of Capsicum annum seeds 145

Table 5. Analysis of regression and correlation between This poor growth performance of lot B corresponds to the tetrazolium staining assay and plant height result of the lot’s tetrazolium staining which indicates low seed viability. Combination a b R² r In conclusion, 1% tetrazolium staining assay revealed 9 pattern topographical patterns on chili pepper seeds. These patterns 1, 2 60.21 1.527 0.921 0.959 can be used as a reference to determine viable seeds from 1, 2, 3 60.59 1.657 0.927 0.963 1, 2, 4 62.44 1.476 0.919 0.958 non-viable ones. Staining patterns 1 and 2 show that the Note: a: interception, b: regression coefficient, R²: coefficient of whole cotyledon and embryonic axis are stained red evenly determination, r: correlation coefficient with or without dark red coloration at the tip of the radicle. Staining patterns 1 and 2 is highly correlated with seed’s germination capacity and plant height. Thus, staining Discussion patterns 1 and 2 can be utilized to estimate plant growth Combination of tetrazolium staining pattern to performance. Future studies using more seed lots from determine seed viability level uses correlation coefficient different varieties need to be done. (r). In this analysis, the tetrazolium staining pattern is the X-axis variable, and the germination capacity is the Y-axis variable. High correlation coefficient is demonstrated by REFERENCES pattern 1 and 2. The value of this correlation coefficient shows a value of 0.872 (close to 1). This result indicates a Basak O, Demir I, Mavi K, Matthews S. 2006. Controlled deterioration strong correlation between the tetrazolium staining result test for predicting seedling emergence and longevity of pepper (Capsicum annuum) seed lots. Seed Sci Technol 34:723-734. and the germination assay result. Correlation coefficient r Copeland OL, McDonald MB. 1995. Seed Science and Technology. =-1 suggests a perfect negative correlation, r=0 no Chapman & Hall, New York. correlation, and r=1 a perfect positive correlation (Mattjik Deminicis BB, Patricia DR, Bráulio PF, Henrique DV, Antônio DP, and Sumertajaya 2002). Guilherme SF. 2014. Tetrazolium test to evaluate Stizolobium aterrimum seeds quality. American Journal of Plant Sciences 5: 148- Seed vigor is indicated by the plant growth performance 152. in polybag. Our analysis showed that tetrazolium staining Dina. 2006. Uji Tetrazolium secara Kualitatif dan Kuantitatif sebagai and the seed vigor assay in the field are highly correlated Tolok Ukur Vigor Benih Kedelai (Glycine max L. Merr) serta with an r value of 0,0959 (Table 5). According to Hubungannya dengan Pertumbuhan Tanaman di Lapang. [Thesis]. Institut Pertanian Bogor, Bogor. [Indonesian] tetrazolium staining assay, seeds from lot C exhibit highly Murwantini E. 2013. Penggunaan Uji Konduktivitas sebagai Uji Vigor viable staining pattern (pattern 1,2) with a percentage of pada Benih Gandum (Triticum aesticum L). [Thesis]. Universitas 92% (Tabel 1). Meanwhile, the lot C plants grow tall with a Andalas, Padang. [Indonesian] height of up to 18,7 cm (Table 5). Viable seeds as ISTA [International Seed Testing Association]. 2016. International Rules for Seed Testing. International Seed Testing Association, Bassersdorf, determined by tetrazolium staining are highly likely to Switzerland. grow into plant with good growth performance in the field. Kuo WHJ, Yan AC, Leist N. 1996. Tetrazolium test for the seeds Salvia Kulik and Yaklich (1982) reported that tetrazolium staining splendens and Salvia farinacea. Seed Sci Technol 24: 17-21. can estimate the germination rate of soybean seeds in the Kulik, MM, Yaklich RW. 1982. Evaluation of vigor tests in soybean seeds: relationship of accelerated aging, cold, sand bench, and speed field. Pattern 1 and 2, characterized by the uniform red of germination tests to field performance. Crop Sci 22: 766-770. coloring in the embryonic axis and cotyledon, are sensitive Leist N. 2004. Seed vigour determination by means of the topographical enough to indicate seeds that will grow into normal and tetrazolium test. In: ISTA Seed Quality. International Seed Testing healthy seedlings. In line with our report in this current Association, Bassersdorf, Switzerland Mattjik AA, Sumertajaya IM. 2002. Perancangan Percobaan dengan study, previously, Leist (2004) stated that seeds with good Aplikasi SAS dan Minitab, Jilid I. IPB Press, Bogor. [Indonesian] vigor are stained bright red uniformly upon tetrazolium Pant NC, Purohit M, Lal RB. 1999. Tetrazolium test for the seeds of assay. Dendrocalanus strictus. Seed Sci Technol 27: 907-910. Poor plant vigor in the field can be caused by the Soetisna U. 2005. Studi anatomi benih sungkai (Peronema canescens Jack); perspektif viabilitas. Biodiversitas 6: 288-291. physiological deterioration of the seeds (Basak et al., Wibawa NF. 2015. Uji Tetrazolium Sebagai Tolok Ukur Viabilitas Dan 2006). This may be true for lot B seeds that had been in Vigor Benih Pepaya (Carica papaya L.). [Thesis]. Universitas long storage for about 12 months. Plants grown from lot B Sebelas Maret, Surakarta. [Indonesian] seeds exhibited the shortest stature among all group tested.

NUSANTARA BIOSCIENCE ISSN: 2087-3948 Vol. 10, No. 3, pp. 146-150 E-ISSN: 2087-3956 August 2018 DOI: 10.13057/nusbiosci/n100303

The effect of combination of indigenous phosphate solubilizing bacteria of Riau, Indonesia on the available phosphorus and phosphorus uptake of soybean

LUFITANUR ALFIAH1,♥, DELITA ZUL2, NELVIA NELVIA3 1Agrotechnology Department, Faculty of Agriculture, Universitas Pasir Pengaraian, Pasir Pengaraian, Rokan Hulu, Riau, Indonesia, 28557 ♥email: [email protected], Tel.: +62- 762-7392272 2Biology Department, Faculty of Mathematics and Natural Sciences, Universitas Riau, Pekanbaru, Indonesia, 28293 3Agrotechnology Department, Faculty of Agriculture, Universitas Riau, Pekanbaru, Riau, Indonesia, 28293

Manuscript received: 20 April 2016. Revision accepted: 1 August 2018.

Abstract. Alfiah L, Zul D, Nelvia N. 2018. The effect of combination of indigenous phosphate solubilizing bacteria of Riau, Indonesia on the available phosphorus and phosphorus uptake of soybean. Nusantara Bioscience 10: 146-150. Despite the abundant amount of phosphorus (P) in the soil, P uptake by the plants is very limited. In acidic soil, phosphorus (P) is bound to aluminum (Al) and iron (Fe), whereas in the alkaline soil, phosphorus (P) is bound to calcium (Ca). The improvement of efficiency and availability of P to plants can be made by utilizing a group of solubilizing phosphate microorganisms. Potency test to investigate the P solubility by Phosphate Solubilizing Bacteria (PSB) has been conducted by isolating the bacteria from peat soil at Biosphere Reserves of Giam Siak Kecil Bukit Batu, Riau, Indonesia. The semi-quantitative test revealed that the PSB were able to dissolve Ca3 (PO4)2, FePo4 and phosphate rock. However, the adaptation ability and potency of PSB from indigenous Riau peat soil inoculated into soybean (Glycine max L. Merr) plants in the mineral soil have not yet been investigated. The present study was carried out from March to June 2015 on the alluvial soil in Babussalam Village, Rambah Sub-district, Rokan Hulu District, Riau. The aim of this study was to determine the effect of PSB inoculation on bacterial population and phosphatase activity. The study also aimed to determine the available P and P uptake and their impact on soybean growth and production. The study employed a factorial experiment laid out in a completely randomized design (CRD) consisted of two factors, i.e., soil treatment and PSB. The first factor comprised of two levels, i.e., T0: non-sterilized soil, T1: sterilized soil. The second factor consisted of 4 levels, i.e., B0: without PSB inoculation, B1: inoculation using 2 isolates of PSB (BB_UB6 and BB_K9), B2: inoculation using 3 isolates of PSB (BB_UB6, BB_K9 and BB_K2), and B3: inoculation using 4 isolates of PSB (BB_UB6, BB_K9, BB_K2, and BB_HS13). The results showed that inoculation of starter 3 had the highest phosphatase activity rate of 12.43 μg p NP g-1 hour-1. The highest available P was produced by starter 2, while the P uptake on non-sterilized soil was higher than that on the sterilized soil at 2.63 mg plant-1. PSB inoculation and soil sterilization did not significantly affect the population of phosphate solubilizing bacteria.

Keywords: Available P, P uptake, phosphate solubilizing bacteria, soybean

INTRODUCTION substance such as IAA and gibberellins acid that can increase plant growth. Phosphate availability remains the main issue in the Potency test to examine the P solubility by PSB isolated cultivation of soybean (Glycine max L. Merr) plants. from peat soil at Biosphere Reserves of Giam Siak Kecil Available P in the soil is abundant, but the concentration of Bukit Batu, Riau, Indonesia has been conducted using a P that can be absorbed by plants is very low. The semi-quantitative approach resulted that the PSB were able concentration of available P in the soil is affected by the to dissolve Ca3 (PO4)2, FePO4 and phosphate rock. soil pH and soil type. In acidic soil, P is bound to However, a study focusing on the ability of adaptation and aluminum (Al) and iron (Fe), whereas in the alkaline soil, P the potency of PSB originated from indigenous Riau peat is bound to calcium (Ca) and magnesium (Mg). The soil and inoculated into soybean plants in the mineral soil presence of these bonds causes P to turn into chelate have not been investigated yet. Thus, this study aimed at compounds absorbed with soil colloids; therefore, P is not determining the effect of combined PSB isolates in available for plants. The provision of P fertilizer becomes improving available P and P uptake of soybean plants. less effective and efficient because a significant amount of P is bound through the absorption process. One possible effort to solve this problem is by using microorganisms MATERIALS AND METHODS from the phosphate solubilizing bacterial group (PSB) which can help release the P bond to make it available to This study was performed in two stages, namely the plants. The release of P bond is facilitated by the laboratory work and field work during March to June 2015. production of organic acids and phosphatase enzymes by Laboratory work was conducted in the Microbiology PSB. Additionally, PSB also produces growth promoting Laboratory of Biology Department, Faculty of Mathematics ALFIAH et al. – Effect of phosphate solubilizing bacteria on soybean 147 and Natural Sciences and Soil Laboratory of Faculty of polybag-1. After one week, the growing media was ready to Agriculture, University of Riau, Pekanbaru, Indonesia. be used. Field research was carried out on the alluvial soil in Babussalam Village, Rambah Sub-district, Rokan Hulu, Sterilization of seed surface, pre-germination, and District, Riau Province, Indonesia. inoculation The study employed a factorial experiment laid out in a The seeds were surfaced sterilized following the completely randomized design (CRD) consisted of two method of Hallmann et al. (1997). The soybean seeds were factors, where the first factor, the soil treatment included washed, and then immersed in 70% alcohol for one minute. two levels, i.e., T0: non-sterilized soil, T1: sterilized soil, The seeds were then immersed in NaOCl 2.5% for eight while the second factor, PSB inoculation, consisted of 4 minutes and rinsed with sterile water for three times. The levels, i.e., B0: without PSB inoculation, B1: inoculation sterilized seeds were then pre-germinated by placing the using 2 isolates of PSB (BB_UB6 and BB_K9), B2: seeds on the wet cotton until the seeds ruptured and inoculation using 3 isolates of PSB (BB_UB6, BB_K9 and germinated. Germinated seeds were then soaked with PSB BB_K2), and B3: inoculation using 4 isolates of PSB starter according to treatment for 2 hours, after which the (BB_UB6, BB_K9, BB_K2, and BB_HS13). seeds were ready to be planted.

Preparation of PSB starter Planting and maintenance Starter 1 (a mixture of 2 PSB isolates): Starter 1 was The soybean seeds were sown by making planting holes made from two PSB isolates (BB_UB6 and BB_K9) by on the prepared media in a three cm depth. Each hole was inoculating one ose of each bacterial isolate into 10 ml of then filled with 2 seeds of soybean. After the seeds grow, liquid Pikovskaya medium, which then incubated at room thinning was done and 1 plant polybag-1 was retained up to temperature for 24 hours with 150 rpm agitation. After the harvest. Polybag arranged with a distance of 40 cm x 40 incubation period, 10 ml of each PSB inoculum was cm. Fertilizer was added at a dose of 0.125 g urea polybag-1 inoculated into an Erlenmeyer containing 40 ml of liquid and 0.5 g KCl polybag-1 was done at 20-30 days after planting. Pikovskaya. The culture was then incubated for 24 hours at room temperature with 150 rpm agitation. After the Data analysis incubation period, the two inoculum were mixed so as to Observations were made to determine the bacterial produce inoculum 1 in 100 ml volume consisting of a population, phosphatase activity rate, soil available P, and mixture of 2 isolates with a ratio of 1: 1. P uptake. Obta8ned data were subjected to Analysis of Starter 2 (a mixture of 3 PSB isolates): Starter 2 was Variance (ANOVA) using SAS 9.1 program. The treatment made of three PSB isolates (BB_UB6, BB_K9, and means were separated using a Duncan Multiple Range Test BB_K2) by inoculating one ose of each bacterial isolate at  5% significance level. into 10 ml of liquid Pikovskaya medium, then was incubated at room temperature for 24 hours with 150 rpm agitation. After the incubation period, 5 ml of each PSB inoculum RESULTS AND DISCUSSION was inoculated into Erlenmeyer containing 45 ml of liquid Pikovskaya medium. The culture was then incubated for 24 PSB population tended to increase by the inoculations hours at room temperature with 150 rpm agitation. After using starter 1, 2, and 3 as compared to no inoculation the incubation period, the three inoculum were mixed so as treatment both in sterilized and non-sterilized soils. PSB to produce Starter 2 in 120 ml volume consisting of a population in non-sterilized soil tended to be two times mixture of 3 PSB isolates with a ratio of 1: 1: 1. higher than that in the sterilized soil. PSB population in Starter 3 (a mixture of 4 isolate of PSB): Starter 3 was PSB inoculation using starter 1 tended to be higher, increased made following the same procedures of starter 1 and starter by nine times of the control treatment (Figure 1). 2, except that at the end of the inoculum preparation process, is the four PSB inoculum were mixed so as to produce Starter 3 in 100 ml volume consisting of 4 PSB isolates with a ratio of 1: 1: 1: 1.

Preparation of growing media Topsoil was hoed for approximately 30 cm depth and then collected. The composite soil sample was then mashed using a shovel and sieved. The non-sterilized soil was put into the polybags as much as 5 kg polybag-1. The sterilized soil was prepared by employing the Tyndallization method, 0 by gradually evaporating soil at 100 C for 1 hour and repeating the same process three times within 24 hours. Starter 1 = Mixture of 2 PSB isolates (BB_UB6 and BB_K9) The non-sterilized and sterilized soils were then let sit for Starter 2 = Mixture of 3 PSB isolates(BB_UB6, BB_K9 and BB_K2) Starter 3 = Mixture of 4 PSB isolates(BB_UB6, BB_K9, BB_K2 and BB_HS13) about one day and then added with phosphate rocks at a dose of 0.75 g polybag-1 and sterilized manure of 25 g Figure 1. The PSB population on non-sterilized and sterilized soil 30 days after inoculation

148 NUSANTARA BIOSCIENCE 10 (3): 146-150, August 2018

Sterilized and non-sterilized soil media influenced the the colony proved that the bacteria dissolved P through the variation of PSB population in each treatment. On non- production of phosphatase enzymes. The area of the clear sterilized soil, inoculated PSB synergized positively with zone qualitatively shows the level of bacteria's ability to indigenous soil bacteria; therefore, the population of PSB dissolve P from the insoluble phosphate (Rachmiati 1995). on this type of soil was more abundant than that on the The highest level of phosphatase activity in this sterilized soil. A study by Cahyani (2009) revealed that soil experiment was produced by inoculation treatment using sterilization significantly influenced the population of soil Starter 3 (12.43 μgpNP g-1 hour-1). The result of the present bacteria. Soil microbes affect one another resulting in very study is higher than that reported by Suliasih and Rahmat dynamic conditions in the rhizosphere (Lines-Kelly 2005). (2007), where the activity of inoculated PSB phosphatase Bacteria within a community convey their presence to each only ranged from 1.818 to 1.947 μg pNP g-1 hour-1. On the other by producing, detecting, and responding to small other hand, is the present results of phosphatase activity are signal molecules called autoinducers, and the process of lower than that reported by Tamad et al. (2013) at 30 intercommunication is called quorum sensing (Miller and μgpNP g-1 hour-1. Fitriatin et al. (2009) reported that the Bassler 2001). Quorum sensing enables the starter bacteria highest phosphatase activity was obtained in the and indigenous bacteria to interact positively, thereby inoculation of combined solubilizing phosphate bacteria causes the increase of bacterial activity. This study also with a phosphate solubilizing function of 201.50 μg pNP g- indicated that PSB originating from peat soil was able to 1 hour-1. adapt to the new environment on mineral soils. The tendency of PSB population increase may affect The PSB population found in this study is higher than the increase of phosphatase enzyme activity. The that obtained by Fitriatin et al. (2009), where the correlation between PSB population and phosphatase population was only 1.70 x 106. On the other hand, the enzyme activity is presented in Figure 2. present study results are lower than the results obtained by The presented scatter plot shows that there was a Lestari et al. (2011) which ranged at 41-72 x 108 at the end positive correlation between the two variables, which of the incubation period. Bacteria have the ability to grow means the higher population of bacteria; the higher is and adapt according to their growing conditions and can enzyme phosphatase activity. This condition proves that the utilize the nutrient sources contained in the substrate on the increase in PSB population will also increase phosphatase growth medium (Lestari et al. 2011). enzyme activity. This finding also indicates the occurrence of quorum sensing where the behavior of bacteria depends Phosphatase enzyme activity on the population. Bacteria, in high population, convey Soil sterilization has a significant effect on phosphatase their abilities, including phosphate solubilizing bacteria enzyme activity in soil (Table 1). Phosphatase enzyme (PSB) (DeAngelis 2006). This notion is also in line with activity on non-sterilized soil increased 1.5 times higher than that on the sterilized soil. These results indicate that Table 1. Phosphatase enzyme activity on non-sterilized and non-sterilized soil has higher phosphatase enzyme activity sterilized soils with PSB inoculation as a result of indigenous soil bacteria allowing the positive interaction among bacteria. Phosphatase enzyme activity (µg pNP g-1 hour-1) Mean phosphatase enzyme activity on sterilized soil Soil type Mean was found lower than on the non-sterilized soil. The soil Without Starter Starter Starter PSB 1 2 3 heating might have caused this during the sterilization a a a a a process which affected the phosphatase enzyme in the soil. Non-sterilized 8.72 13.60 9.87 16.43 12.15 Sterilized 5.80a 9.91a 8.32a 8.43a 8.11b Nelson and Cox (2000) suggested that during enzymatic c ab bc a Mean 7.26 11.75 9.09 12.43 reactions, the rise in temperature increases the kinetic Note: The numbers in the same rows and columns which is energy of reacting molecules; thereby it accelerates followed the same lowercase letter are not significantly different collisions among molecules. Collisions will facilitate the at α 5% DMRT. formation of substrate enzymes so that the product formed will be much more. At an optimum temperature, the collision between the enzyme and the substrate is very effective, so that the formation of the enzyme-substrate is easier and more products are formed. However, at a too high temperature, the collision will accelerate the damage to the formation of active enzyme groups (enzyme denaturation) in interacting with the substrate and the enzyme catalytic activity will decrease (Kilara and Harwalkar, 1996). Santosa (2009) also mentioned that soil sterilization has a significant effect on PSB activities. Phosphatase enzyme activity was found higher in PSB- inoculated growing media than that in non- inoculated growing media. This indicates that the PSB-inoculated growing media were capable of producing phosphatase Figure 2. The correlation between PSB population and enzymes. The formation of a clear zone (holozone) around phosphatase activity.

ALFIAH et al. – Effect of phosphate solubilizing bacteria on soybean 149

Table 2. Available P (ppm) and P uptake (mg.plant-1) on was the result of higher bacterial diversity. However, the sterilized and non-sterilized soils 30 days after inoculation role of indigenous microbes in providing soil P is insufficient to increase soil P uptake. The high available P Treatment found in this study was not in line with the high absorption Soil type Without Starter Starter Starter Mean of P by the soybean plants, which is indicated by the low PSB 1 2 3 mean P absorption by soybean plants due to sterilization -1 Available P (ppm) that ranged from 0.51 to 2.63 mg plant . Saidi (2002) Non-sterilized 20.23a 24.88a 36.94a 36.30a 29.59a stated that soil P is closely related to P uptake; thus the Sterilized 8.41a 26.34a 29.25a 22.49a 21.62a higher is the soil P, the higher is the P uptake in plants. Mean 14.32b 25.61ab 33.10a 29.40a Jones et al. (1991) suggested that P uptake is categorized as low in the range of 1.6 to 2.6 mg plant-1. P uptake (mg plant-1) The low absorption of P may be caused by the nutrient a a a a a Non-sterilized 1.90 1.52 0.69 6.42 2.63 limiting factor in the soil. The low content of sodium and Sterilized 0.51a 0.67a 0.51a 0.36a 0.51b a a a a magnesium in the soil before the study caused the Mean 1.21 1.10 0.60 3.39 inhibition of P absorption by plants. Besides, Mg is a Note: The numbers in the same rows and columns which is followed the same lowercase letter are not significantly different crucial nutrient that plays a role in the photosynthesis at α 5% DMR test. process. Syafii (2008) stated that Mg has a vital role in chlorophyll synthesis. The low content of Mg element may decrease the level of chlorophyll and, hence the photosynthesis rates; thus the photosynthesis products are the notion of Tamad et al. (2013) which stated that the PSB not optimally produced. The P absorption by soybean population was positively correlated with phosphatase plants in this study was lower than that reported by Lestari activity. Phosphatase enzyme activity in the soil is closely et al. (2011) (58.3 mg plant-1). We concluded from the related to microbial activity because microbial biomass is present study findings that PBS inoculation and soil the primary source of enzymes in the soil. Suliasih et al. sterilization can increase soil phosphatase activity and (2010) reported that phosphatase enzyme activity on available P. The results also indicate that the two variables tomato-growing soil increased in line with the increasing have no significant effect on bacterial population and P population of solubilizing phosphate bacteria. uptake of soybeans.

Available P PSB inoculation increased the available P of the soil as ACKNOWLEDGEMENTS much as twice higher than that without inoculation (Table 2). Starter 2 treatment produced the highest available P by The authors appreciate and thank Nia Sufika Indriani 33.10 ppm. However, the major effect of soil sterilization for the significant assistance provided to the author during and the interaction of the two treatments did not this research. significantly increase the soil available P. Non-sterilized soils tended to contain higher available P compared to sterilized soil. REFERENCES A significant increase of available P indicates that PSB from Bukit Batu peat soil are able to adapt well to mineral Cahyani VR. 2009. Influence of soil sterilization methods on nutrient soil in Rokan Hulu District. The increase in soil available P status, microbiota population, potential of mycorrhizal infection and in this study is much higher than the increase in available P plant growth. Soil Science 6 (1): 43-52 De Angelis KM. 2006. Microbial Community Ecology and Bacterial from the adding of PSB in Sei Garo (Lestari et al. 2011) at Quorum Sensing as Control Points in Rhizosphere Nitrogen Cycling. 16.2 ppm. Bacterial isolates have different adaptability to [Dissertation]. The University of California. Berkeley, CA. their newly grown environment. Widyati (2007) stated that Fitriatin BN, Joy B, Subroto T. 2009. The Influence of Organic the ability of bacteria in dissolving P depends on the Phosphorous Substrate on Phosphatase Activity of Soil Microbes, Proceeding International Seminar of Chemistry, Universitas process of their own isolate metabolism. PSB activity in Padjadjaran, Jatinangor, Indonesia, 30-31 Oktober 2008. dissolving P is highly dependent on soil temperature, soil Hallmann J, Quadt-Hallmann A, Mahaffee WF, Kloepper JW. 1997. moisture, pH, food supply and environmental conditions Bacterial endophytes in agricultural crops. J Microbiol 43(10): 895- during their growth (Widawati and Suliasih 2006). 914. Jones JB, Wolf B, Mills HA. 1991. Plant Analysis Handbook.Micro- Table 2 shows that the main effect of soil sterilization Macro Publishing, Inc. Georgia. USA. increased P uptake in soybean plants. It may be concluded Kilara A, Harwalkar VR. 1996. Denaturation. In Food Proteins: Properties that soybean plants were able to absorb P better in non- and Characterization. VCH Publishers. New York. sterilized soil than in sterilized soil. Available P in non- Lestari, Wahyu TM, Linda, Martina A. 2011. The ability of seigaro's phosphate solubilizing bacteria in provision of dissolved phosphate sterilized soil was five times higher than that in sterilized and its absorption on soybean plants. Biospecies 4 (2): 1-5. soil. However, inoculated PSB isolate mixture and Lines-Kelly R. 2005. Defend The Rhizosphere and Root Against treatment interaction did not significantly increase the P Pathogenic Microorganism. Department of Primary Industries, State uptake by soybean. of New South Wales, Sydney. Miller MB, Bassler BL. 2001. Quorum sensing in bacteria. Ann Rev This condition indicates the influence of indigenous soil Microbiol 55: 99-165. microbes in providing P nutrient to the non-sterilized soil Nelson DL, Cox MM. 2000. Lehninger Principles of Biochemistry. 3rd ed. Worth Publisher, New York.

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Rachmiati Y. 1995. Phosphate solubilizing bacteria from plant rhizosphere Syafi S. 2008. Morphological and Physiological Response of Different and its ability in dissolving phosphate. Proceedings of the National Genotypes Seeds of Jatropha (Jatropha curcas L.) to Drought Stress. Congress VI HITI Volume 1. Land Use as a Spatial Planning Tool to [Thesis]. Bogor Agricultural University, Bogor. [Indonesia] Improve Human's Welfare, Soil Science Association of Indonesia. Tamad, Ma’as A, Radjagukguk B, Hanudin E, Widada J. 2013. Jakarta, 12-15 December 1995. Availability of phosphorus in andisol soil for maize (Zea mays L.) by Saidi D. 2002. Isolation and characterization of cellulolytic bacteria and phosphate solubilizing bacterial inoculum. J Agron. 41 (2): 112-117 phosphate solubilizer from andisol as biological fertilizer agent. Widawati S, Suliasih. 2006. Augmentation of potential Phosphate Habitat 8 (4): 201 - 211. Solubilizing Bacteria (PSB) for growth of spur caysim (Brasicaca Santosa E. 2009. The activity of Some Bacteria Solubilizing Phosphate ventis Oed.) in marginal land. Biodiversitas 7 (1): 10-14. Isolatesat Various Levels C-Organic in Ultisols Soil. Soil Research Widyati E. 2007. Utilization of sulfate-reducing bacteria for Institute. Bogor. bioremediation of coal mine former land. Biodiversitas 8 (4): 283- Suliasih R. 2007. Phosphatase activity and calcium phosphate dissolution 286. by some Phosphate Solubilizing Bacteria. Biodiversitas 8: 23-26.

NUSANTARA BIOSCIENCE ISSN: 2087-3948 Vol. 10, No. 3, pp. 151-158 E-ISSN: 2087-3956 August 2018 DOI: 10.13057/nusbiosci/n100304

Male gametophyte development steps in Pistacia vera L.

ELAHE SADEGHIRAD1, AHMAD MAJD2,, ALIREZA IRANBAKHSH1, AMANOLLAH JAVANSHAH3 1Department of Biology, Faculty of Sciences, Science and Research Branch, Islamic Azad University. Hesarak, Tehran, Iran 2Department of Biology, Faculty of Sciences, North Tehran Branch, Islamic Azad University, South Makran Street, Tehran, Iran. email: [email protected] 3Pistachio Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Kerman, Iran

Manuscript received: 7 April 2018. Revision accepted: 3 August 2018.

Abstract. Sadeghirad E, Majd A, Iranbakhsh A, Javanshah A. 2018. Male gametophyte development steps in Pistacia vera L. Nusantara Bioscience 10: 151-158. Salinity affects the growth and development of plant. It also affects the development steps of male gametophyte in pistachio plant. Pistachio (Pistachio vera) is a member of family Anacardiaceae and order Sapindales. To study the effects of salinity on those steps, an experiment was performed in two locations in Golshan Anar with commensurate circumstances, namely: a control area (A) which was well-irrigated with fresh water, and the other area (B) which was well-irrigated with salty water added with NaCl solution with EC values of 14 dS.m-1. The flowers sampling was done in two Golshan Anar regions on the springtime based on a completely randomized design with three replications. The development steps of male gametophyte in pistachio plant were observed using conventional cell histology techniques and light microscopy observations and were then contrasted with samples subjected to no salinity stress. The results represented that several steps of male gametophyte development are as follows: (1) the anther experiencing normal growth which is tetrasporangiate, (2) cytokinesis taking place simultaneously with meiosis in the microspore mother cell, the tetrahedral tetrads, (3) microspores being delivered after meiosis by microsporogenesis were more or less irregular in shape during the contraction period. Finally, the abnormal shape and structure of the number of cases reviewed in three replicates of pollens can be one of the significant factors influencing the decline in the product.

Keywords: Anther, Pistacia, salinity

INTRODUCTION concentration of soil solution and the other is the impact of toxic ions which hinder enzymatic activity in key processes Adversely, salinity influences the growth and (Zhang and Blumwald, 2001). development of plant and it is a major environmental stress Plants utilize various mechanisms to reduce the impacts limiting agricultural production (Saini 1997). Reproductive of these environmental stresses. One of these mechanisms outgrowth starting from meiosis in the spore mother cells is the imperfect establishment of eggs or pollen that leads for fertilization and untimely seed formation is highly to the displacement of the food founts from the sensitive to various stresses, such as drought, heat, cold, reproductive organs into metabolic reactions and leads to flooding, and nutrient insufficiencies (Salter and Goode, stress resistance. The aging of the reproductive organs can 1967; O’Toole and Moya, 1981; Saini and Aspinall, 1981, also be stimulated or predated by salinity stress. Asch and 1982; Westgate and Boyer 1986; Satake and Yoshida 1978; Wopereis (2001) stated that 45% of the rice harvest is Schoper et al. 1987; Morrison 1993; Hayase et al. 1969; lessened by salinity stress as an impact of the clusters Brooking 1976, Matsushima, 1962; Zavadskaya and sterility and the seed weight reduction in formed seeds. Skazkin 1960; Campbell and Leyshon, 1980; Sharma et al. Salinity becomes a major cause of defective grain and 1987; Azouaou and Souvré 1993; Lardon and Triboi- product and quality reduction in cotton (Davidonis, Blondel, 1994). These stresses lead to several structural and Johnson, and Landivar, 2000). functional deformities in reproductive organs, causing The studies of Namuco and O'Toole (1986) and failing of fertilization or premature abortion of seed or Westgate and Boyer (1986) indicated that during plant fruit. Thus, the breakage to fecundity from stress at this growth microspores have elevated sensitivity to salinity. To phase is notably critical for crops in which the economic the mind of Sun et al. (2004), on salinity stress conditions, outcome is the result of sexual prolificacy, as elevation in microsporocytes of Arabidopsis plants did not grow into water shortage possibly characterizes as the most mature pollens grains but these cells became vacuole and significant environmental element restricting global crop got old in two days. Moreover, the remaining pollen grains fecundity (Fischer and Turner, 1978; Boyer, 1982). Among stay in the fallen anthers. Prolonged periods of salinity these stresses, salinity is a main question since the past stress gave no effect on mature pollen grains. It verifies centuries. Around 20% of land under implantation and that the effect of salinity on the pollen grains relies on the nearly 50% of watered lands in the world are influenced by developmental phases of anthers. Male reproductive ion concentration (Zhu 2002). Elevated salinity has two perfection in plants is exceptionally susceptive to water spoiling impacts on plants. The first is the impact induced deficiency during meiosis in the microspore mother cells by the water deficiency as a product of escalating (Saini 1997). Pistachio (Pistachio vera) a member of the 152 NUSANTARA BIOSCIENCE 10 (3): 151-158, August 2018 family Anacardiaceae and order Sapindales (Al-Saghir, Chelli-Chaabouni et al. (2010), and Bastam et al. (2013). 2010). Morphologically, pistachio (P. vera) is recognized as the oldest species of this genus (Baninasab and Mobli, 2008). P. vera is a dioecious woody plant with imparipinnate MATERIALS AND METHODS leaves which fall in autumn (Al-Saghir, 2010). The male and female anthesis have 450-500 and 150- This comparative study was carried out on two locals 250 flowers, successively. A very long life is owned by this with identical conditions involving control garden (A) tree whose height can reach 7-10 meters (Asaja, 2006). The which was watered with fresh well water and other garden surface of the stigma receives pollen when the female (B) which was watered with salty water with EC (electric flowers are exposed, then pollination takes place that will conductivity) value of 14 dS.m-1 NaCL solution. Based on produce fruit and seeds. The female flower buds located on a completely randomized design with three replications, a the branches of a one-year-old tree begin to bloom in late sampling of flowers was taken place in two gardens of March and 100 to 300 flowers are pollinated in every Golshan Anar area in the springtime. Golshan is a village inflorescence during the first two weeks of April (Polito located around Anar in the suburb of Rafsanjan city, and Kallsen, 2005). Researchers have conducted many Kerman Province. The study area is 14 km southeast of different studies on this species including morphological Anar, at 30º48´N and 55º21´W, and the average height of and developmental studies on female flowers and 1408 m with a desert and hot-dry climate. The sampling of embryogenesis of the genus Pistacia L (Al-Saghir, 2010; soils was conducted on the surface up to a depth of 120 cm, Bachelier and Endress, 2007; Grundwag, 1976) and species it was to determine the soil texture, and, by this, the P.vera (Lin et al. 1984; Martinez-Palle and Herrero, 1998; percentage of each of the three particles i.e. sand, silt, and Shuraki and Sedgley, 1996; Shuraki and Sedgley, 1997), clay were analyzed in the soil laboratory of Rafsanjan's Endress and Bachelier (2007) have examined the genus Pistachio Research Center. Then, by the technique of soil Pistacia L. and reported that female flowers have 5-8 textural triangle, the percentage of sand was determined as excrescences similar to calyx, the female portion holds a shown in table 1 (Mohammadi, 2006). large round ovary with short vigor and trifurcation of To learn about the developmental phases of anthers, stigma (two lobes on each branch). Grundwag (1976) sampling from male inflorescence buds was carried out stated that this genus has one ovule per ovary, a downward- until the opening of anthers and pollination. Morphology of moving ovary, a monolayer, and many nuclei. The inflorescence and male flowers was examined using a embryonic sac of the genus (Grundwag, 1976) and species dissecting microscope. To learn about the anatomy and the (Lin et al. 1984) have Polygonum-type. Yet, there are development, the samples were soaked in FAA solution various studies on male flowers (Al-Saghir, 2010; Asaja, (90% ethanol 70 + 5% acetic acid+ 5% formaldehyde) for 2006; Azouaou and Souvré, 1993; Bachelier and Endress, 24-72 hours (Johansen, 1940), and then placed in the 2007; Shiyan et al. 2001; Li et al. 2011; Qiu et al. 2010). running water for 24 hours. The samples were dried in Various researches have been done on the morphological ethanol with series of increasing concentrations and then structure, but less attention has been provided to the soaked in alcohol 70%. For paraffin embedding, the founding of sapidity in pistachios. Other researchers carried samples were first dried using ethanol 70, and then the out morphological studies on pistachio pollen and declared ethanol in tissues was little by little substituted with toluene that their morphology was diversified among different (paraffin solvent) by soaking the samples in solutions with varieties (Afshari et al. 2008; Davarynejad et al. 1996; Li et increasing toluene level for 20 minutes. Next, molding was al. 2011; Qiu et al. 2010). carried out on samples by putting them in molten paraffin Pistachio beans own a special economic significance in for at least 7 hours. Sectioning was performed using a the family Anacardiaceae, genus Pistacia L. in accordance rotational microtome with a thickness of 0.8 micrometers. with Molecular Data Bank of Iranian Pistachio reported in The slides were deparaffinized by toluene and blemished 2008, pistachio is the first non-oil exported product in Iran with Hematoxylin-Eosin according to the protocol suggested (IPMD). Several techniques and studies have been carried by Meyer (Yeung 1984). To cling the samples, the sections out to establish the knowledge about this precious species were first dried in distilled water and increasing ethanol as well as to determine distinction among diverse varieties. series and until they became transparent in toluene. Finally, Yet, the morphological and developmental evaluation of the permanent slides were acquired using Entalen glue and flowers, particularly male flowers, are very finite. The coverslip. Microscopic examination of samples was essential characteristics of reproductive organs and performed under a light microscope (Olympus BH2 Japan) understanding the developmental phases of gametophytes and the suitable samples were photographed with a digital find a substantial significance in botanical sciences since camera (Canon IXUS 120 IS USA). they are the proper equipment for the recognition and categorization of plants. The hodiernal inquiry arranges to Table 1. Soil particles percentage and type of soil texture learn anatomical characteristics of male flower and Soil particles developmental phases of anthers in pistachios and the Area of Clay Silt Sand impact of salinity on these phases in natural circumstance Pistachio Type of soil texture with no entry of any natural and experimental factors. (%) (%) (%) Biological stress of Pistacia was observed by Seydi et al. A Sandy loam (light) 19.8 25.6 54.6 B Sandy loam (light) 19.8 17.6 62.6 (2015), Parsa and Karimian (1975), Ranjbar et al. (2002),

SADEGHIRAD et al. – Male gametophyte development steps in Pistacia vera 153

RESULTS AND DISCUSSION period, the color of anthers is red and it later changes during development phases. At the pollination period, they Results are completely yellow or their pedicel is yellow, but the tip The study on morphology and anatomy of male flowers remains red. The color of anthers changes starting from the indicated that the formation of male inflorescences is in the ventral surface of inflorescence to the dorsal surface form of complex and lateral panicle on the branches before (Figure 1.B-C). the leaves appeared (Figure 1.A). During the flowering

B C D 2000 µm A 1500 µm 1500 µm 700 µm

F G I 700 µm E 700 µm 500 µm 500 µm

500 µm J

Figure 1.A-I. Morphological and anatomical structure of inflorescence and male pistachio flowers (P. vera): A. Emerging male inflorescence laterally before the leaves; B. Dorsal surface of the male inflorescence bridge along with red anthers; C. Surface of male inflorescence showing color changes of anthers to yellow; D-E. Male flower panicles with a short peduncle and large anthers with 2 sepal 1 bract; F. Flower with of dehiscent anther, G. Longitudinal section of inflorescence, the arrow refers to the secretory ducts in the side of vascular bundles; H-I. Cross-section of inflorescence, narrow arrow points flower with five anthers, thick arrow shows flower with four anthers and five anther arrowhead shows flower with six anthers and seven anthers, A = Anther, S = Sepal, B = Bract

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Four to six stamens with large and bulky anthers are From division and differentiation of one or more epidermal owned by male flowers, plus two or three sepals and 0-1 anthers cell (s), microsporangium wall and spore-forming bract; the sepals and the bracts are recognizable by the size. tissue are produced. By dividing high colorable cells and Filaments are short and huge. In length and depth close to dense cytoplasm, spore-forming tissue is detected (Figure the central vascular bundle of anthers, the dehiscence of 2.A). The distance of two dorsal sporangia is shorter than anthers occurs (Figure 1.D-F). The number of anthers per that of the two ventral sporangia; at this stage, flower was confirmed by cross sections of male microsporangia located in a theca are far away from each inflorescence and it resulted that the four-anther was other, and they are also split up from each other by septum dominant (Figure 1.H-I). The arrangement of flowers as in depth. As the spore-forming tissue divisions process panicle is confirmed by longitudinal section of occur, tangential divisions of some cells obtained from inflorescence. In addition, the secretory ducts in the epidermal cell division take place to form the anthers walls. vascular bundles are exposed (Figure 1.G). Pre-meiotic stage is finished by halting mitosis of spore- Three steps, namely, pre-meiotic, meiosis and post- forming tissue and transformation of spore-forming cells to meiotic involves in the development of anther and they are microspore mother cells that are large cells with bulk concluded from the examination of microscopic sections. nucleus and dense cytoplasm (Figure 2.B).

St PMC

200 µm A a B 200 µm b 200 µm 200 µm

50 µm 200 µm C 50 µm c 50 µm D d

En Me Ts

Tp Bs

E F 50 µm G H 50 µm 50 µm 50 µm

Figure 2. A-H. Stages of forming tetrad microspores inside of anthers in normally grown pistachio (P. vera) and in plants under NaCl treatment: A. Entire cross-section view of anther at the mitosis division of spore-forming tissue cells phase, in plants under salinity treatment on the right figure anthers were small, shriveled; B. Stopping of mitosis in spore-forming tissue cells and microspore mother cells, in plants under salinity treatment on the right figure anthers were small, shriveled and distancing from nutritional layer; E. Spacing between nutritional layer and microspore mother cells with high magnification; C. On the right figure entire view of anthers with microspores mother cells in meiosis; F. Anther wall of microspore mother cell meiosis time, which is composed of the epidermis, a mechanical layer, more than three middle layers and taptom; G. PMC in two and four-core stage, D&H. Cytoplasm division simultaneously in the microspore mother cells and production of tetrahedral tetrad surrounded by a thick layer of transparent callus, ST = spore-forming tissue, PMC = microspore mother cells, Ts = quad-core microspore mother cells (tetrad cell), Bs = a dual-core microspore mother cells (dyad cell), TP = nutritional layer

SADEGHIRAD et al. – Male gametophyte development steps in Pistacia vera 155

EP En

Tp EP En

200 µm A 200 µm B 500 µm C 20 µm D

200 µm E 50 µm F G 20 µm

Figure 3. A-D. Anatomical structure of anther cross-sections of normally grown pistachio (P. vera), and E-G. in plants under salinity treatment since the production of free microspore to flourish anther: A. Complete decomposition of callus wall surrounding the tetrad and production of free microspores entire. Cross-section view of anther in the mature pollen. Arrowhead points out to the break down of the walls of a cavity for anther dehiscence. Anthers wall is composed of an epidermis layer, a mechanical wall layer, three middle layers and a tapetum layer, and entire view of anther cross-section at free microspores stage and disappearance of nutritional layer; B. Microspores feeding nutrition layer, leading disappearance of nutritional layers with the middle layer cells; C. Entire view of dehiscent anther; D. Mature pollen; E. Entire cross-section view of anther in the mature pollen grains. Arrowhead points out to break down the walls of a cavity for anther dehiscence in plants under salinity treatment, F. Anther with mature pollen Arrowhead points out to break down the walls of a cavity for anther dehiscence in plants under salinity treatment, G. Mature pollen under salinity treatment; EP = epidermis, En = mechanical layers, ML = middle layer

To begin the Meiotic stage was by taking away of the microsporangium at this stage, microspore mother cells microspore mother cells from tapetum cells. At this stage, with two and four cores are available successively after central indentation of each anther theca is completely deep meiosis I and II (Figure 2.G). After meiosis, the cytoplasm and reaches to the central vascular bundle near to anther, so cleavage is performed by the setting up channels of the that the two microsporangia in the theca are completely microspore mother cell in which these cells are oriented as separated from each other and the septum is hidden (Figure tetrahedral type and are called tetrahedral tetrad. All tetrads 2.B). As the phase of meiosis I happen, pectocellulosic wall are of tetrahedral type and four cells are situated in a of microspore mother cells is hydrolyzed and replaced by common callus wall and also are divided by a callus new callus walls. These walls keep away microspore (Figure 2.H). Post-meiotic phase starts after the splintering mother cells from interacting with each other during of the callus wall and passing from tetrad phase to free meiosis. At the time of meiosis phase of microspore mother microspore phase. At the commencement of this process, cell, anther wall is composed of epidermis, a mechanical microspore is still in tetrad configuration. Microspores hold layer, more than three middle layers and nutritional layer a certain nucleus with marginal place and congested (Figure 2.F). In plants under salinity treatment, although cytoplasm after being exempted from tetrad at the florets appeared usual, but the growth of the anthers was beginning of discrepancy. Then, the callus lid vanishes unusual, and more anthers were small and whitered (Figure entirely and the anther wall possesses mechanical layer, 2.A-D right). During meiosis of these cells, Cytoplasm three middle layers and nutritional layer (Figure 3.A). At cleavage happens at the same time. Inside of each last,, middle and tapetum layers vanish with the

156 NUSANTARA BIOSCIENCE 10 (3): 151-158, August 2018 discrepancy of fully grown pollen grains; tapetum is this characteristic. The cytoplasmic classification was glandular (Figure 3.B). Then the impact of condensing of synchronous after meiosis of microspore mother cell, mechanical layer walls become clearly visible, except the conformable to that of the polygonum type; this finding epidermis wall that causes to the impression of a U-shaped and glandular tapetum layer are in line with results of Li et motif layer (Figure 3.D). Fully grown anther wall owns the al. (2011) on P. vera and results of Qiu et al. (2010) on P. mechanical layer with U-shaped motif along with the traces chinensis. In this research, the researchers examined that of tapetum layer and the middle layer. The separation of cytoplasmic partition after meiosis is carried out the two middle walls of each sac with the central part of synchronously by setting up channels from around anther causes to dehiscence in length and depth (Figure microspore mother cell toward the center of cell and 3.A-C, E). Anther was shriveled and pollen grains owned building up four microspores. These cells are oriented unusual form (Figure 3.E-G). tetrahedrally. In this study, all tetrads were tetrahedral. Li et al. (2011) discovered that tetrad of P. vera is isobilateral, Discussion so there are both types of tetrad in varieties of P. vera. Qiu Pistachio is a plant with high economic value and it is et al. (2010) informed that P. chinensis has both types of native to Iran, as plant in cultivation, it is widely planted in tetrahedral and isobilateral tetrads. In pre-meiotic phase, various locations. It is so significant to do research on the parts of microsporangia situated in one theca are secluded bioecological and ecological issues of the plant, including from each other via the septum. But in meiotic phase, the sorts of stress in Iran, such as the NaCl stress which is middle indentation of each theca of anther turns into totally one of the major issues in cities and in the locations for deep until it arrives in the central vascular bundle of pistachio cultivation. Biological stress of Pistacia was anthers, so that the two microsporangia in a theca are observed by Parsa and Karimian (1975), Ranjbar et al. totally secluded from each other. This stuff is also found in (2002), Chelli-Chaabouni et al. (2010), Bastam et al. few plants. In most cases, the septum totally secludes two (2013), and Seydi et al. (2015). Al-Saghir (2010) in the pollen sacs until the end of the maturation phase. At last, study of the genus Pistacia L. informed that the flowers are the two sacs are attached and let pollens out via shallow small, monoecious, with no petal and panicle type of dehiscence pore. The post-meiotic phase starts with inflorescences. This research also informed that the flowers disintegration of callus wall and passing tetrad phase to are monoecious and are born from female pedicel in microspore phase. At last, middle stratum and tapetum complex panicle and on separate pedicels, as well as hold stratum vanish with distinction of fully-grown pollens. 4-6 stamens, 2 or 3 sepals and the 0-1 bract. The sum of Fully-grown anther wall is equipped with the mechanical stamens in the genus Pistacia were 4-6 pcs which has been stratum with U-shaped motif along with the traces of the informed by Hormaza and Polito (1996), 3-5 pcs by Shiyan tapetum stratum and the middle stratum. Separation of the (2001), and 4-5 pcs in species P. chinensis by Qiu et al. two middle walls of each sac with the central part of anther (2010). Al-Saghir (2010) in the research of genus Pistacia leads to dehiscence in length and depth. The pollens are L. exerted bracteole term for non-bracteal excrescences detached across the deep cleavage locations. surrounding flowers. Male and female flowers of this genus Crops manage the establishment of pollen grains, eggs, hold 1-3 small bracts and 2-7 bracteoles. Moreover, and grains in reaction to environmental circumstance Bachelier and Endress (2007) familiarized non-bracteal change. In extreme environmental circumstance, excrescences as sepals and informed its numbers in male establishment of pollen grains will fail. In this research, the flowers of P. lenticus are identical with their stamens male gametophyte growth phase in pistachio plant was numbers, which was 4-6. These researchers informed that examined with standard methods and plant reaction to they feel doubt if these excrescences are sepals or bract, NaCl stress at this phase was observed. The findings they also informed that 5-10 sepals-like organs in P. yielded from this study is in a mutual accord with the result terbinthus and only two of them in P. mexicana. In of prior studies on the preventative effect of salinity stress addition, as the flowers of the genus Pistacia are pollinated on male gametophyte growth in plants (Namuco and by wind, it indicates the tendency of evolution route O'Toole 1986, Moss and Downey 1971, O'Toole and Moya towards being dioecious and losing perianth (petals 1981, Saini 1981, Sheoran 1996, Westgate and Boyer shortage and diminished sepals). In our review studies, 1986). Namuco and O'Toole (1986) revealed that only a few numbers of reports were discovered about microspores are susceptive to salinity stress during the growth phases of anther to deliver pollen in the genus development phase. In some plants, including beans, pistachios. there was individual study in the P. vera species canola, corn, and soybeans, stress circumstances lead to the done by Li et al. (2011). In this study, during meiosis, the mortality of plant cells in the mature gametophytes anther wall of microspore mother cell consists of the (Kokubun 2001, Moss and Downey 1971, Sage and epidermis, a mechanical layer with more than three middle Webster 1990, Young 2004). Consequently, the natural layers, and a glandular tapetum layer. The stability feature growth of gametophyte, embryos, and pollen in plants can of middle layers is important in this species. Because in be stopped or thwarted by environmental stresses, but the most crops, middle layers vanish at untimely phase and fiasco in the establishment phases relies on the phase where before the establishment of the tapetum layer (Sanders et stress is applied. Male reproductive growth in plants is very al. 1999), but in this species, middle layers vanish within susceptive to salinity stress and dehydration in PMC during tapetum layer at the pollen grains discrepancy, and this is meiosis. During this stage, the establishment of most of in agreement with Li et al. (2011) who have also notified microspores or pollen grains is impeded by the water

SADEGHIRAD et al. – Male gametophyte development steps in Pistacia vera 157 deficiency and it leads to male sterility. A direct impact on wrinkled, discolored, and small. The untimely cracking of reproductive tissues is seemingly not the cause of these the anther wall, the shrinkage of pollen grains, and the injuries but an indirect impact of water deficiency in establishment of pollen with unusual shapes and properties different organs like leaves is. The mechanism of this verify that the salinity stress lessens the yield of Canola reaction may involve a long distance molecular warning in through impacting the male gametophyte development organs that are under pressure and influences fertility in (Mahmoodzadeh and Bemani 2008). Learning separate reproductive tissues. A lot of studies notify the complicity environmental circumstances in the future and changes that of abscisic acid in this matter, but more clue is needed to take place during the growth phases of the pistachio and its verify the participation of this hormone in the induction of reproductive organs is nominated. Saini et al. (1984) and male sterility (Morgan 1980, McRae 1985). Lalonde et al. (1997) examined the anomalies of anthers The discontinuation of male gametophyte growth growth due to salinity stress and water deficit at the time of caused by stress circumstances is as a result of disruption meiosis of carbohydrates metabolism and distribution within the anther, and the stoppage of the sugar hydrolysis key enzyme, which is invertase. The glucose level can organize REFERENCES the gene expression of invertase. Salinity stress can cause the decrease in photosynthesis that will lead to the decrease Afshari H, Talaei A, Panahi B, Hokmabadi H. 2008. 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NUSANTARA BIOSCIENCE ISSN: 2087-3948 Vol. 10, No. 3, pp. 159-163 E-ISSN: 2087-3956 August 2018 DOI: 10.13057/nusbiosci/n100305

Antibacterial potency of simple fractions of ethyl acetate extract of Begonia baliensis

HARTUTININGSIH-M. SIREGAR1,♥, R.S. PURWANTORO1, PRAPTIWI2, A. AGUSTA2 1Center for Plant Conservation-Bogor Botanic Gardens, Indonesian Institute of Sciences. Jl. Ir. H. Juanda No. 13, Bogor 16122, West Java, Indonesia. Tel./fax.: +62-251-8322187. ♥email: [email protected] 2Laboratory of Natural Products, Research Center for Biology-Indonesian Institute of Sciences. Jl. Raya Jakarta-Bogor Km. 46, Cibinong 16911, West Java, Indonesia

Manuscript received: 28 June 2018. Revision accepted: 9 August 2018.

Siregar HM, Purwantoro RS, Praptiwi, Agusta A. 2018. Antibacterial potency of simple fractions of ethyl acetate extract of Begonia baliensis. Nusantara Bioscience 10: 159-163. Balinese people utilized Begonia baliensis Girm. (Begoniaceae) as traditional medicine to relieve cough. It was applied in a unique way by inserting the plant material into a bamboo column and then burning it in the fire. The liquid produced by the combustion process was used as a cough medicine. Based on this traditional knowledge, it is expected that B. Baliensis has antibacterial activity. B. baliensis plant collected from Bukit Sangyang, Penebel, Tabanan-Bali has been used in this study. The chemical compounds of ethyl acetate extracts were isolated/ separated by column chromatography. The obtained fractions were analyzed for antibacterial activity by disc diffusion assay against Escherichia coli, Bacillus subtilis, Staphylococcus epidermidis and Staphylococcus aureus. Chromatographic column yielded 14 simple fractions, whereas antibacterial test results showed 5 active fractions. Fraction 3 (F3) was active against S. epidermidis, fraction 5 (F5) against E. coli and S. epidermidis, while fractions 10, 11 and 12 were active only against Bacillus subtilis. Isolation and purification of the active components likely increased their potential as antibacterials.

Keywords: Antibacterial, Begonia baliensis, disc diffusion, simple fractions

INTRODUCTION medication expenses (Khunaifi 2010; Purwantoro et al. 2010). Herbs have been used as traditional medicine for In order to combat bacterial infections, antibiotics play generations by communities and are still in use. Bioactive an important role in eliminating infection-causing bacteria. compounds present in the plant can be detected with However, antibiotic itself can barely cope with bacterial pharmacological approach.The study of medicinal plants as infections due to poor selection of antibacterial, and the a source of pharmacologically active compounds has emergence of resistance issues. Therefore, alternative increased worldwide (Surasak 2009). According to Ali et al medicines from plant resources need to be found. (2011) development of resistance in human pathogens Many species that belong to Begoniaceae are against conventional antibiotic necessitates searching for commonly known as ornamental plants. However, some indigenous medicinal plants having antibacterial property, species have been used as vegetables and medicinal herbs. medicinal plants used actively in folklore, ayurvedic and Begonia glabra is used to heal a fresh wound, while B. traditional system of medicine in Pakistan. Escherichia fimbristipula reduces heat or fever, used as cough coli, Bacillus subtilis, Staphylococcus epidermidis and medicine, pain reliever and processed into fresh beverage Staphylococcus aureus are amongst strong pathogen of as bitter tea (Tebbit 2005). B. grandis is used as a herbal their genera. They frequently show resistance to various medicine to clean wounds, to reduce swelling and to treat a medicines leading to complication in determining suitable number of diseases. B. lailana B. baramensis, B. stenogyna anti-microbial selection for therapy. Treatment for and B. lazat are used as vegetables in Kuching (Kiew et al. infectious disease caused by antibiotic-resistant bacteria 2015). In West Java, Indonesia, B. multangula and B. requires research for new potential cures. Study on robusta are used as tamarind substitutes in cooking because antibacterial substance is needed to find new antibiotic to of their sour taste. inhibit and terminate growth of antibiotic-resistant bacteria. In Balinese society, Begonia baliensis with local name The infectious disease still ranked the highest for cause of Bacem Kebo has been used as a traditional medicine and illness and death in developing countries, including vegetable. Bacem kebo identified as B.baliensis is a new Indonesia. Infectious disease results in physical suffering Begonia species endemic to Bali (Girmansyah 2008). This and decrease in working performance and productivity, species grows with only a limited population size on the which in the end leads to proliferation of loss. For the slopes of Bukit Tapak, Candikuning, Tabanan, Bali. State, the level of infectious disease in community will lead (Satyanti et al 2013). Continuous harvest from its natural to decrease of national productivity in general, and on the habitat will cause declining wild population. Sustainable other hand, government also needs to spend more for 160 NUSANTARA BIOSCIENCE 10 (3): 159-163, August 2018 harvest management supported by intensive cultivation will incubated for 24 hours at 37°C. Observation of growth help to maintain natural population. inhibition by test bacteria was done after incubation was Raw young leaves and stems are traditionally eaten as completed. The antibacterial activity was determined by vegetables. This plant is also considered as a traditional measuring the diameter of clear zone of inhibition. medicine to treat cough (Siregar 2008). Stems and leaves are inserted in bamboo, then burnt on fire. The discharge is used as a cough medicine. RESULTS AND DISCUSSION Previous research has shown that Begonia muricata, B. multangula, B. baliensis have antibacterial activity against Morphological characters Escherichia coli and Staphylococcus aureus (Purwantoro et Begonia baliensis Girmansyah. Stem brownish green to al. 2011). Based on the previous results, this study is reddish brown, erect and cane-like, succulent, rhizomatous, carried out to determine the antibacterial activity of the hairy, herbaceous, 15-50 cm tall. Stipules pale green. simple fractions of B. baliensis extract. Leaves distant; 5-15 cm apart, petiole pale green to reddish brown, hairy, asymmetric, 14-21 x 11-18 cm, basal lobe rounded, apex acuminate; venation palmate-pinnate. MATERIALS AND METHODS Inflorescences axillary, few flowered, shorter than the leaves, protandrous, peduncle 2-8 cm long, green, glabrous; Material bract absent. Male flowers 4, with a pale reddish green Begonia baliensis plant was collected from the forest pedicel 1.3-2.5 cm long; tepals 4, white and red around the area of Shangyang Hill, Jatiluwih, Tabanan, Bali. middle, inner two similar but smaller, milky white, 11-14 x B.baliensis plant was cleaned and cut into small pieces, 8-9 mm, anthers pale yellow. Female flowers 7, with a pale then dried under the sun. The dried samples were ground green pedicel 4-5 mm long; ovary dark green with reddish into powder. brown at the larger wing, thick and fleshy, 7-9 x 5-8 mm, locules 3, placentas 2 per locule; tepals 5, milky white, Extraction outer two reddish white, broadly obovate, margin not B. baliensis powder (207.13 grams) was extracted by toothed. Fruit a berry, pendant on a stiff fleshy pedicel, 4-5 maceration method using three different solvents namely, mm long, green when ripe, fleshy (Figure 1). Distribution. n-hexane, ethyl acetate and methanol with different Bali. Habitat. Humid forest, along trails at 1300-1800m polarities. Maceration in each solvent was performed 3 (Girmansyah 2008) times, the filtrate was collected and concentrated with a The rendement of extract was presented in Table 1. The rotary evaporator. The extract was weighed to know the highest result was in methanol extract (13.04%), followed rendement extract. Rendement extract is the weight of the by n-hexane extract (1.67%) and ethyl acetate (4.57%). extract divided by the weight of the sample multiplied by 100%. Isolation of bioactive compounds from ethyl acetate Begonia baliensis extract Isolation of bioactive compounds Previous research showed that ethyl acetate extract has The chemical compounds of ethyl acetate extract of B. antibacterial activity against Bacillus subtilis and baliensis (1 g) were separated by column chromatography Pseudomonas aeruginosa (Siregar et al. 2014). In the using silica gel (300 g) as the stationary phase. The mobile present study, separation of chemical compounds of ethyl phase was a mixture of dichloromethane-methanol in a acetate extract of B. baliensis was done by column ratio of 30:1 to 1:1 and finally 100% methanol. The eluates chromatography. Figure 2. showed that ethyl acetate were monitored with TLC. Eluate with similar TLC extract of B. baliensis contained several chemical patterns combined into one as a simple fraction. The simple compounds indicated by several spots on TLC plate. fraction was then tested for its antibacterial activity. The chromatogram profile of simple fractions from ethyl acetate extract of B. baliensis (Figure 3) showed that Antibacterial test one fraction still contained several chemical compounds Simple fractions of ethyl acetate extract was tested for indicated by the existence of several 'spots' in the fraction. antibacterial activity against Escherichia coli, Bacillus subtilis, Staphylococcus epidermidis and Staphylococcus Antibacterial properties of simple fraction aureus. All isolates were cultured on Mueller Hinton Agar All simple fractions of ethyl acetate extract were in petri dish. Each simple fraction was tested for assayed for their antibacterial activity against E. coli, S. antibacterial activity using the disc diffusion method. epidermidis, and B. subtilis by the paper disc diffusion Sterile blank disc (6.0 mm in diameter) were impregnated method. The antibacterial test results are shown in Figure 4 with different concentrations (50 µg, 100 µg, and 200 µg) and Table 1. The results of the antibacterial test show that 5 of fraction. The impregnated disc was placed on Mueller fractions have antibacterial activity, i.e., fraction (F) 3 is Hinton Agar in petri dish that had been inoculated with the capable of inhibiting the growth of S. epidermidis, F5 is test bacteria. All antibacterial work was carried out active against E. coli and S. epidermidis, whereas F10, F11, aseptically in the laminar air flow. The petri dish was then and F12 are active against B. subtilis.

SIREGAR et al. – Antibacterial effect of Begonia baliensis 161

A C

Figure 1. Morphological features of Begonia baliensis Girm. A. Leaves, B. Flowers, C. Fruit

Sample : ethyl acetate extract of Begonia baliensis TLC : Merck GF254 Solvent : dichloromethane-methanol (10: 1) Stain reagent: 2% CeSO4 in 10% H2SO4 Figure 4. Inhibitory zone of simple fractions of ethyl acetate Begonia baliensis extract against Bacillus subtilis and Staphylococcus epidermidis by the paper disc diffusion. The clear Figure 2. TLC profile of ethyl acetate extract of Begonia zone indicated bacterial growth inhibition. baliensis

Table 1. Weight of Begonia baliensis leaf extract

Weight extract (g) Rendement (%) Extract B. baliensis n-hexane 3.46 1.67 Ethyl acetate 9.46 4.57 Methanol 27 13.04

Extraction of B. baliensis was carried out using different solvents with different polarity, i.e., hexane, ethyl acetate, and methanol. According to Sani et al. (2014), the solvent plays an important role in producing a high yield of Figure 3. Chromatogram of simple fractions of ethyl acetate extract. The chemical compounds will be extracted in a extract Begonia baliensis. Mobile phase: Dichloromethane: solvent with the same polarity. The result showed that the methanol (10:1). Stain reagent: Cerium sulfate

162 NUSANTARA BIOSCIENCE 10 (3): 159-163, August 2018 highest percentage of extract or rendement was methanol Agustini (2007), Gram-positive bacteria have a simpler cell extract. This is because almost all chemical compounds can wall so that it might be more sensitive to antibacterial be dissolved in methanol. According to Marcus and compounds. Gram-negative bacteria have more complex Glikberg (1986), methanol is a good solvent for polar cell walls with layer of lipoproteins on the outside of their organic substances. The highest percentage of methanol cell walls (Chan et al. 2005). Therefore Gram harmful extract indicated the relatively high content of polar bacteria become less sensitive to antibacterial compounds. chemical compounds (Yuhernita and Yuniarti 2011). The result (Table 2) showed the tendency of increasing The chemical compound in ethyl acetate extracts was inhibition of bacterial growth by increasing fraction separated by thin layer chromatography (TLC). According concentration. This is because the increase in concentration to Sashidaran et al. (2011), the TLC is a simple, of the extract increased the concentration of antibacterial inexpensive and quick method to determine the chemical compounds. According to Zuhud et al. (2001), the higher compounds in the extract. concentration of extract and the duration of contact resulted In the earlier research, it was found that the ethyl in the easier penetration of antibacterial compounds acetate extract of B. baliensis was able to inhibit the growth through the bacterial cell wall. of Bacillus subtilis and Pseudomonas aeruginosa. This In conclusion, Begonia baliensis contains many polar indicated that the ethyl acetate extract contains chemical compounds, in which the yield of methanol extract is the compounds having antibacterial activity. According to highest one (13.04%). However, the active extract is ethyl Suresh et al. (2016), the Indian medicinal plants tested, acetate extract. Five simple fractions of ethyl acetate Acalypha indica and A. lanata showed a significant extracts namely, F3, F5, F10, F11, and F12 showed the antibacterial activity. A. indica showed potential activity antibacterial activities, and only F5 was active against against Staphylococcus aureus and E. coli. A. lanata Escherichia coli. Fraction three (F3) was active against significantly exhibited antibacterial activity against E. coli, Staphylococcus epidermidis, F5 was active against Salmonella typhi, and Pseudomonas aeruginosa. Escherichia coli and Staphylococcus epidermidis, while The fractionation with chromatography column yielded F10, F11, and F12 are selectively active against Bacillus 14 simple fractions. Several fractions have antibacterial subtilis. Isolation and purification of the active compounds activity, i.e., F3, F5, F10, F11, and F12. The growth of E. might improve their potency as antibacterials. A simple coli was only inhibited by F5 at the concentration of 200 fraction of ethyl acetate of B. baliensis extract which has ug. The antibacterial assay showed that Gram-negative (E. antibacterial activity needs to be further analyzed for the coli) bacteria were less sensitive than gram-positive isolation and characterization of potentially antibacterial bacteria (B. subtilis, S. epidermidis and S. aureus) to simple chemical components. fractions of Begonia baliensis. According to Kusmiyati and

Table 2. Weight of simple fraction of ethyl acetate Begonia baliensis extract and its inhibitory zone

Inhibitory zone (mm) against

E.coli B.subtilis S.epidermidis S.aureus No. Weight 50 100 200 50 100 200 50 100 200 50 100 200 fraction (mg) µg µg µg µg µg µg µg µg µg µg µg µg

1 24.60 ------2 29.30 ------3 46.00 ------8 - - - 4 96.70 ------5 21.90 - - 7 - - - - 8 9 - - - 6 7.80 ------7 23.20 ------8 41.40 ------9 49.50 ------10 22.60 - - - - 8 8 ------11 98.70 - - - 7 7 8 ------12 23.00 - - - - 9 11 ------13 9.90 ------14 16.2 ------Acetone - - - - Methanol - - - -

SIREGAR et al. – Antibacterial effect of Begonia baliensis 163

ACKNOWLEDGEMENTS Kusmiyati, Agustini NWS. 2007. Activity test of antibacterial compounds from microalgae Porphyridium cruentum. Biodiversitas 8 (1): 48-53. [Indonesian] Authors acknowledge the Head of Center for Plant Purwantoro RS, Hartutiningsih-M. Siregar, Sudarmono, Praptiwi. 2010. Conservation-Bogor Botanic Gardens, Indonesian Institute Antibacterial test Lasianthus (Rubiaceae) as a medicinal plant and its of Sciences and DGHE, Indonesia. This work was propagation efforts. Buletin Kebun Raya 13 ( 2): 86-93. [Indonesian] conducted within the framework of Research Incentive Purwantoro RS, Siregar HM, Satyanti A, Wihermanto, Kusuma YWC, Sumanto. 2011. Characterization of active components in Begonia Project for Researcher and Engineer, Synergy Activity of baliensis extract and Lasianthus obscurus (Rubiaceae) as medicinal Research and Development of Science and Technology raw materials and conservation aspects. Proposal of Researcher and 2009-2010. Thanks to Prof. Dedy Darnaedi who helped Engineer Incentive Activities LIPI, Research and Technology, LIPI, correct the manuscript. Thanks also go to Dr. Sudarmono, Bogor.

Annisa Satyanti, Wihermanto, YWC Kusuma, WH Ardi Sasidharan S, Chen Y, Saravanan D, Sundram KM and Yoga Latha L. and Sumanto who were willing to share their knowledge 2011. Extraction, isolation, and characterization of bioactive and Andi Saptaji Kamal for his help in Natural Product compounds from plant extracts. Afr J Tradit Complement Altern Med Laboratory, Research Center for Biology, Indonesian 8 (1): 1-10. Satyanti A, Siregar HM. 2012. Microclimate preference and habitat of Institute of Sciences (LIPI), Bogor, Indonesia. Begonia in Bedugul Bali. Biotropia 19 (2): 80-91. Siregar HM, Purwantoro RS, Sudarmono, Fijriyanto IA, Satyanti A,Agusta A. 2014. Bio pharmacy potential of Begoniaceae (Begonia muricata Blume, B. multangu Blume, B. baliensis Girmansyah) REFERENCES through in vitro antibacterial test and antifungal test. Proceedings The 2nd International Symposium on Temulawak. The 40th Meeting of Ali NH, Shaheen F, Shahana UK. 2011. Antibacterial activity in spices National Working Group on Indonesian Medicinal Plant. Institute of and local medicinal plants against clinical isolates of Karachi, Research and Community Services-Bogor Agricultural University, Pakistan. Pharmaceut Biol 49 (8): 833-839. Indonesia: 190-195. Chan ECS, Pelczar JrMJ, Krieg NR. 2005. Microbiology. 5th ed. Tata Siregar HM. 2008.Knowing and caring Begonia. PT Agromedia Library. McGraw-Hill Education Pvt. Ltd., New Delhi. Jakarta. [Indonesian] Girmansyah D. 2008. A taxonomic study of Bali and Lombok Begonia Surasak L, Sanan S, Supayang PV. 2009. Medicinal plants with (Begoniaceae). Reinwardtia 12 (5): 419-434. significant activity against important pathogenic bacteria. Jeeva S, Marimuthu J, Antonisamy. 2012. Anti-bacterial and Pharmaceutical Biology 47 (8): 683-689. phytochemical studies on methanolic extracts of Begonia floccifera Suresh M, Mohammad SAA, Pradipta KR, Panneerselvam A, Thajuddin Bedd. Flower. Asian Pac J Trop Biomed 1 (1): S151-S154 N. 2016. Screening and antibacterial efficacy of selected Indian Khunaifi M. 2010. Test of antibacterial activity of binahong leaf extract medicinal plants. Asian Pac J Trop Biomed 6 (3): 185-191. (Anredera cordifolia (Ten) Steenis against Staphylococcus aureus Tebbitt MC. 2005. Begonias. Cultivation, Identification, and Natural bacteria and Pseudomonas aeruginosa. [Thesis]. Department of History. Timber Press, USA. Biology, Faculty of Science and Technology, State Islamic University Yuhernita, Juniarti. 2011. Analysis of secondary metabolite compounds Maulana Malik Ibrahim, Malang. [Indonesian] from leaf methanol extract surian potential as an antioxidant. Makara Kiew R, Sang J, Repin R, Ahmad JA. 2015. A Guide to Begonias of Sains 15 (1): 48-52. Borneo. Natural History Publications (Borneo). Sdn. Bhd., Kota Zuhud EAM, Rahayu WP, Wijaya CH, Sari PP. 2001. Antimicrobial Kinabalu, Sabah, Malaysia. activity of kedawung extract (Parkia roxburghii G. Don) to pathogenic bacteria. Jurnal Teknologi & Industri Pangan 12 (1): 6.

NUSANTARA BIOSCIENCE ISSN: 2087-3948 Vol. 10, No. 3, pp. 164-169 E-ISSN: 2087-3956 August 2018 DOI: 10.13057/nusbiosci/n100306

Total phenolic content and antioxidant activity of ginger extract and SNEDDS with eel fish bone oil (Anguilla spp.)

IMAS IFRIAN WIJAYANTI1,♥, AGUNG BUDIHARJO1,2,♥♥, ARTINI PANGASTUTI1,2, FEA PRIHAPSARA3,♥♥♥, ANIF NUR ARTANTI3 1Bioscience Graduate Program, Universitas Sebelas Maret. Jl. Ir. Sutami 36A Surakarta 57 126, Central Java, Indonesia. Tel./fax.: +62-856- 786686168/+61-857-43210550, ♥email: [email protected] 2Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sebelas Maret. Jl. Ir. Sutami 36A Surakarta 57 126, Central Java, Indonesia. ♥♥email : [email protected] 3Department of Pharmacy, Faculty of Mathematics and Natural Sciences, Universitas Sebelas Maret. Jl. Ir. Sutami 36A Surakarta 57 126, Central Java, Indonesia. ♥♥♥ email: [email protected]

Manuscript received: 17 June 2018. Revision accepted: 14 August 2018.

Abstract. Wijayanti II, Budiharjo A, Pangastuti A, Prihapsara F, Artanti AN. 2018. Total phenolic content and antioxidant activity of ginger extract and SNEDDS with eel fish bone oil (Anguilla spp.). Nusantara Bioscience 10: 164-169. Aims of this study were to measure phenolic content of ginger (Zingiber officinale) and to observe the comparison of antioxidant activity between pure ginger extract and ginger extract in SNEDDS (Self Nanoemulsifying Drug Delivery System) with bone of eel fish oil (Anguilla spp.) as a conductor. Analysis was done based on IC50 value using DPPH (2,2 diphenyl-1-picrylhydrazyl) method and activity of Superoxide Dismutase (SOD) in vivo using Adrenochrome Assay. Determination of total phenolic content of ginger extract was measured by UV/Vis Spectrophotometric using Folin-Ciocalteau reagents then the absorbances were measured at wavelength 756 nm. Calculation of IC50 DPPH of the ginger extract and SNEDDS were measured using spectrophotometer UV/Vis at 517 nm wavelength. Measurements of SOD was based on the ability of SOD inhibit spontaneous auto reduction of epinephrine using Adrenochrome Assay at 12 male Wistar rats that divided into 4 treatment groups with 3 replicates for each group. Total phenolic content of ginger extract from 3 replications were 4,8519 ± 0,037 µg/mL equivalent with gallic acid standard solutions (GAE) or 485,1847 ± 3,7045 mgGAE/100 g dry weight. Antioxidant scavenging activity by DPPH assay of ginger was higher than SNEDDS. IC50 of ginger was 385.4 mg/mL, and IC50 of SNEDDS was 428.4 mg/mL. The result of descriptive analysis of Kruskal Wallis of SOD activity assay indicated significant differences between treatment groups (P = 0.001 (P <0.05)). SOD activity test performed in vivo showed that ginger extract made into SNEDDS system with eel fish oil used as the conductor has a significant influence compared with ginger extract group.

Keywords: Anguilla, DPPH, eel fish, phenolic content, SOD, SNEDDS, Zingiber officinale

INTRODUCTION on hypertensive test animals showed that the use of ginger at certain doses can lower blood pressure. Ginger causes Oxidative stress is a condition where the formation of vasodilation in mice and rabbits, and inhibits calcium Reactive Oxygen Species (ROS) and long-lasting channel-blocking activity as verapamil (Nicoll and Henein antioxidant defense systems are imbalanced (Juranek and 2009). Bezek 2005; Ahmad 2013). One of the results of the The compounds in ginger have been proven to be one reaction between free radicals and epithelial cell of the antioxidant agents that inhibit ROS, which become phospholipid membranes is malondialdehyde (MDA). one of the causes of various diseases. However, many MDA formation can be prevented by antioxidant chemical compounds have low solubility in water despite compounds, Superoxide Dismutase (SOD), catalase (CAT), high intestinal permeability, so that compounds are and Glutathione Peroxidase (GPx). However, the first body classified into class II drugs according to reserves in overcoming oxidative stress are SOD Biopharmaceutical Classifications System (BCS). The (Rajkumar et al. 2008). So that SOD is very important in current lipid-based drug delivery system has been widely the body to scavenge free radicals. developed as it demonstrates the ability to increase the The use of ginger in certain doses is known to have bioavailability of drug compounds. One type of lipid-based high antioxidant activity, which has been widely tested to drug delivery is SNEDDS (Self-Nano Emulsifying Drug counteract various types of free radicals and protect cell Delivery System), which is a mixture of oils, surfactants, membrane lipids from oxidation activity. Several studies co-surfactants, and drugs that can form emulsions in water using rat test animals, showed that ginger can decrease solvents with nanometer-sized droplets when it is dissolved lipid peroxidation activity as well as improve the in liquid medium (Patel et al. 2010; Christophersen 2014). performance of antioxidant enzymes and serum In this study, the SNEDDS system was prepared by glutathione. Other studies found an antioxidant effect of mixing ginger extract with eel bone oil together with ginger is equivalent to ascorbic acid. Research conducted surfactant and co-surfactant to reduce surface tension so WIJAYANTI et al. – Phenolic content and antioxidant activity of ginger 165 that the two substances can be completely mixed. Ginger MATERIALS AND METHODS extract made into SNEDDS is expected to optimize the performance of the compound in ginger and form a Extraction of ginger and SNEDDS preparation nanometer-sized emulsion to enhance bioavailability. Extraction of ginger was prepared by dissolved 100 g of The delivery used in SNEDDS is eel bone oil. ginger powder into 500 mL of ethanol 96% for 3 days, then Utilization of fish bone is expected to reduce the effect of the extract solution was filtered for evaporation using a fish waste obtained from the existing waste disposal in eel rotary evaporator to obtain a thickened ginger extract. Then fish ponds in Surakarta. Eel fish (Anguilla bicolor) is a the preparation of ginger SNEDDS with eel fish bone oil very popular fish consumption in many countries especially was prepared by mixed 1000 mg of ginger extract into 0.56 Japan, China, Germany, and France. It contains vitamin A, g of eel fish bone oil, 3.89 g of tween 80 as surfactant and B1, B2, B6, C, D, albumin protein, DHA 0.56 g of PEG 400 as cosurfactant. It was then mixed in (Docosahexaenoic acid) and EPA (Eicosapentaenoic acid) vortex for 2 minutes, sonication for 15 minutes and water that is better known as omega-3, as well as some other bath for 10 minutes at 45oC. minerals (Rovara et al. 2010). Eel fish is one of the cultivated fish that has high Measurement of total phenolic content economic value. Sidat has a high nutrient content. Eel Measurement of total phenolic content by colorimetric energy content reached 270 kcal/100 g, vitamin A content assay based on procedure of Chun et al. (2003) and Malik of sidat reached 4700 IU-/100 g seven times more than et al. (2015) with few modifications using gallic acid chicken eggs, 45 times that of cow's milk. Vitamin B1 of (GAE) as a standard. (i) Preparation of Na2CO3 7%. As eel equivalent to 25 times of vitamin B1 content of cow's much as 3.5 g Na2CO3 was diluted into aquabidest until 50 milk. While vitamin B2 eel is equal to 5 times of vitamin mL. (ii) Preparation of gallic acid. As much as 1000 ppm of B2 content of cow's milk. Compared with salmon, eel gallic acid was made by diluted 10 mg of gallic acid into contains DHA (Docosahexaenoic acid, for child growth) of methanol p.a until 10 mL. From this stock solution, as 1,337 mg/100 g while salmon is only 820 mg/100 g or much as 2,5 mL was taken into methanol until 25 mL to mackerel 748 mg/100 g. Sidat contains EPA make 100 ppm of gallic acid solution. From this solution (Eicosapentaenoic Acid) of 742 mg/100 g while salmon is series of amount taken solution i.e 1, 2, 3, 4, 5 mL was only 492 mg/100 g or mackerel 409 mg/100 g (Baedah added into methanol p.a until 10 mL for each so that their 2010). concentrations were 10, 20, 30, 40 and 50 ppm of gallic Cardiovascular protection as effects of fish oil and acid solutions. (iii) Measurement of gallic acids polyunsaturated fatty acids (PUFA) can make lower blood absorbances. Each concentration of gallic acid was added pressure and prevent the development of hypertension. into 0.4 mL Folin-Ciocalteau reagent, which were then Effects of Cardiovascular Protective by fish oil due to 2 mixed and incubated for 4-8 minutes. After 4-8 minutes, as fatty acids contained in fish oil include-Eicosapentaenoic much as 4 mL of Na2CO3 7% was added into each solution, acid (EPA) and Docosahexaenoic acid (DHA). Dietary followed by incubation for 90 minutes. Determinations of supplements contained in both these fatty acids may gallic acid solutions absorbances were using UV/Vis provide cardiovascular protection effects and lower blood spectro-photometric at wavelength 756 nm. The calibration pressure. DHA is known to be effective in lowering blood curve was made from absorbances measuring of gallic acid pressure and cardiovascular disease risk factors (Frenoux et standard solutions. al. 2001). Preparation of ginger solution extract was made by Antioxidant status is a factor associated with several adding 10 mg of extract into 10 mL of methanol p.a. diseases such as cardiovascular pathology. The Determination of total phenolic content of ginger extract concentration of oxidized LDL plasma is strongly was measured using UV/Vis Spectrophotometric by adding associated with cardiovascular disease. This LDL is 1 mL of ginger extract into 0,4 mL Folin-Ciocalteau destroyed by macrophage and contributes to the formation reagent then they were mixed and incubated for 4-8 of cell formation, accumulation in the vessel wall and the minutes. After 4-8 minutes, 4 mL of Na2CO3 7% was development of atherosclerosis. PUFAs such as GLA, added into the mixture and incubated for 90 minutes. The EPA, and DHA inhibit lipid hypersensitivity and absorbances were measured at wavelength 756 nm. peroxidation that promote atherogenic particles that neutralize the effects of blood pressure, platelet metabolism Free radical scavenging activity determination and lipid profiles (Fernoux et al. 2001; Inoue et al. 2015). DPPH was used for the determination of free radical The presence of eel bone oil as a conductor in SNEDDS scavenging activities based on Kwon and Kim (2003). As ginger extract is expected to optimize the performance of much as 1 mL of extract solutions and ginger SNEDDS phenolic compounds of ginger in its utilization as an solutions for different concentration (1000; 500; 250; 125; antioxidant agent. Thereafter comparing the antioxidant 62,5; and 31,25 µg/mL ) in test tube was added into1 mL of activity of pure ginger extract with ginger extract in 0,3 mM DPPH in methanol. The mixtures were mixed and SNEDDS with bone of eel fish oil as a conductor. then incubated in a dark chamber for 30 minutes. The Antioxidant activity was measured by DPPH (2,2 diphenyl- absorbances were measured at 517 nm. The percentage of 1-picrylhydrazyl) assay and superoxide dismutase enzyme scavenging activity was calculated using formula: activity with Adrenochrome Assay. % scavenging activity = x 100%

166 NUSANTARA BIOSCIENCE 10 (3): 164-169, August 2018

Where: Where: A : Absorbance of control A : Absorbance of control B : Absorbance of sample B : Absorbance of sample

Determination of superoxide dismutase activity Then the SOD activity was measured using formula: In vivo preparation of the samples was done as follows: A total of 12 male Wistar rats were divided into 4 treatment SOD activity = x Fp groups with 3 replicates for each group. The groups were divided into a control group in which this group received no special treatment, the dex group was the group receiving 30 μg/kg B dexamethasone injection daily for 18 days. RESULTS AND DISCUSSION Group 3 was given oral extract 200 mg/kg for 18 days, which were then injected with dexamethasone on days 5- Phenolic content of ginger (Zingiber officinale) extract 18. Group 4 received SNEDDS orally at 300 μg/kg for 18 Measurement of Total Phenolic Content was done using days, which were then injected with dexamethasone on gallic acid to create standard curve based on Ahmad et al. days 5-18. The detailed schedule of in vivo treatment is (2015). The absorbance of five gallic acid concentrations presented in Table 1. was measured at wavelength of 765 nm. The results were SOD activity was measured using Adrenochrome Assay then used to form regression curve and get the regression method as an easy method which has a high sensitivity to equation to measure the ginger phenolic content. The measure SOD activity. Measurements based on the ability standard curve of gallic acid is presented in Figure 1. of SOD inhibit spontaneous auto reduction of epinephrine. Ginger extract solution was measured at wavelength 756 Epinephrine solution in acidic state will be stable, but it nm. The result was presented in Table 2. will be spontaneously oxidized with the increase of pH. Auto-oxidation occurs most rapidly accompanied by the Table 1. Schedule and treatment groupings in vivo formation of adrenochrome with linear velocity at pH 10.2 o Group Days- and temperature 30 C. 1st 2nd 3rd 4th 5th until 18th Determination of superoxide dismutase activity was Control (1) Without treatment measured based on Fatimah et al. (2010). As much as 1 mL Group 2 Dexamethasone of blood 50% of each replication were incubated at Group 3 Ginger extract Dex + ginger extract 200 temperature of 37oC for 15 minutes, then they were mg/kg body weight centrifuged with acceleration 3000 rpm for 5 minutes. Group 4 SNEDDS (300 mg) Dex + SNEDDS 300 mg Afterward, the pellet was washed off by NaCl 0,9% Note: dexamethasone on the group 3 and 4 is given as much as 30 solutions and centrifuged with acceleration 3000 rpm for 5 μg/kg body weight injection daily for 14 days. SNEEDS and minutes. This part was done 3 times. As much as 500 µL of ginger extract were given using intragastric tube supernatant was added into 800 µL chloroform-ethanol

(3:5), they were subsequently mixed for 1 minutes and centrifuged for 10 minutes. Supernatant was kept at the refrigerator for Blanko, As much as 2800 µL buffer carbonate was added into 100 µL aquadest and 100 µL epinephrine. This mixing was then mixture and measured its absorbance at wavelength of 480 nm and at temperature 30oC. For sample tube, as much as 5 µL of sample was added into 2800 µL buffer carbonate, 100 µL aquadest and Absorbances 100 µL epinephrine, then the absorbance was measured at wavelength 480 nm at temperature 30oC. The percentage of SOD activity was calculated using formula: Concentration

% scavenging activity = x 100% Figure 1. Regression curve of absorbance of gallic acid on concentration

Table 2. Result of total phenolic content of ginger solution

Gallic acid equivalent standard Total Phenolic content Concentration Absorbance (µgGAE/mL) (mgGAE/100 g dry weight) 1 mg/mL 0.65 4.888889 488.889 0.648 4.814815 481.480 0.649 4.851852 485.185 Mean 4.851852 485.1847 SD 0.037037 3.7045

WIJAYANTI et al. – Phenolic content and antioxidant activity of ginger 167

Based on the regression curve, it showed that regression Table 3. Percentage of DPPH scavenging activity of SNEDDS equation of gallic acid at concentration 10, 20, 30, 40, and and ginger 50 ppm was y = 0.027x + 0.518, which the standard solution of phenolic compound was linear between Concentration Activity of Activity of absorbances and concentrations with coefficient of (µg/mL) SNEDDS ginger correlation (r) was 0.9205. Result of measurement of 31.25 37% 37% phenolic content of ginger (Zingiber officinale Rosc) 62.5 37% 45% showed that it has phenolic content as much as 4.8519 ± 125 44% 65% 0.037 µg/mL equivalent with gallic acid standard solutions 250 62% 83% (GAE) or 485.1847 ± 3.7045 mgGAE/100 g dry weight. 500 79% 94% 1000 90% 94% DPPH scavenging activity DPPH scavenging activity of ginger and SNEDDS was measured based on the formula to obtain the percentage of DPPH scavenging activity that was presented in Table 3. DPPH scavenging activity of ginger and SNEDDS was

made into a regression curve to obtain the regression

equation used to measure IC50 between ginger and

%) ( SNEDDS. The graph of the regression curve between ginger and SNEDDS can be seen in Figure 2. Based on the regression graphic above, it showed that that antioxidant Scavenging activity by DPPH assay of ginger was higher than SNEDDS. IC50 was measured based

on the regression equation of each ginger, and SNEDDS cavenging activity cavenging result of IC50 of ginger was 385.4 mg/mL, and IC50 of SNEDDS was 428.4 mg/mL. IC50 values denoted the concentration of the sample which is required to scavenge S DPPH 50% DPPH free radicals. Higher IC50 values denoted lower antioxidant activities. It means that SNEDDS antioxidant activities based on DPPH assay was lower than pure ginger extract. Concentration

Superoxide dismutase activity Figure 2. Regression curve of ginger and SNEDDS activity on SOD activity was measured with adrenochrome assay. DPPH It showed that the activity of SOF of SNEDDS was higher than other groups. The activity of SOD is presented in Figure 3. Control group was the group of rats without any special treatment, then the dexamethasone group was the group of rats which got dexamethasone injection subcutaneously 30

µg/kg body weight for 18 days. Ginger group got ginger extract orally for 18 days (50 mg/kg body weight) and dex

injection for 14 days. Then the SNEDDS group got

unit/mL) ( SNEDDSS orally for 18 days (50 mg/kg body weight) and dex injection for 14 days.

The graph above showed that the normal control group activity had a SOD activity of ±0.646 units/mL. The

dexamethasone group had the lowest SOD activity of SOD

±0.065 units/mL. In the ginger group, mice treated with dexamethasone and treated with oral ginger extract

increased the activity of SOD to ± 0.326 units/mL. In the Ginger treatment group with dexamethasone and SNEDDS SNEDDS

treatment, the activity of SOD increased to ±1.21 units/mL. Normal control Normal

The result of descriptive analysis of Kruskal Wallis Dexamethasone expressed that Asymp sig value P = 0.001 (P <0.05) Group indicated that there were significant differences between treatment groups. SOD activity test performed in vivo Figure 3. Activity of superoxide dismutase on dexamethasone- showed that ginger extract made into SNEDDS system induced rat with eel fish oil as the conductor has a significant influence compared with ginger extract group.

168 NUSANTARA BIOSCIENCE 10 (3): 164-169, August 2018

Discussion standard solution of gallic acid. Compounds contained in Aims of the study were to measure the total phenolic ginger have high antioxidant activity. In the antioxidant content of ginger (Zingiber officinale) extracted using 96% activity test using DPPH showed IC50 extract of pure ethanol solvent. The ethanol solvent is chosen because of ginger was higher than SNEDDS but in vivo test using the ability of ethanol to bind secondary metabolite adrenochrome assay to measure superoxide dismutase compounds in the sample (Harborne 1998). The result of enzyme activity, SNEDDS activity was higher than other the measurement of phenolic content showed a high yield treatment groups. of 4.85 mg/mL equivalent with standard solution of gallic acid. The compounds in ginger have been proven to prevent the causes of various diseases (Nicoll and Henein ACKNOWLEDGMENTS 2009; Akinyemi et al. 2014). Ginger extract in SNEDDS with eel fish oil conductor This study was supported by the Directorate General for can form nanoparticle-sized emulsion droplets in the Strengthening Research and Development, Community gastrointestinal tract so that in the same dose of extract, the Service, Ministry of Research and Higher Education of the ability of cells to absorb drug compounds is greater to Republic of Indonesia. provide optimum efficiency. The ability of SNEDDS drug delivery systems is expected to optimize the absorption of drugs in the gastrointestinal tract to optimize the effects of REFERENCES compounds in small doses. The antioxidant activity of ginger extract and SNEDDS Ahmad A, Singhal U, Hossain MB, Islam N, Rizvi I. 2013. The role of the was measured using DPPH acceleration showed that IC50 endogenous antioxidant enzymes and malondialdehyde in essential ginger was lower than SNEDDS. It showed that in vitro hypertension. J Clin Diagn Res 7 (6): 987-990. 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NUSANTARA BIOSCIENCE ISSN: 2087-3948 Vol. 10, No. 3, pp. 170-177 E-ISSN: 2087-3956 August 2018 DOI: 10.13057/nusbiosci/n100307

The potential of agroforestry as an adaptation strategy to mitigate the impacts of climate change: A case study of Kiine Community, Kenya

MUNENE ANNE NYARUAI, JOHN K. MUSINGI, BONIFACE N. WAMBUA Department of Geography and Environmental Studies, University of Nairobi. Nairobi, Kenya. email: [email protected], [email protected]

Manuscript received: 5 April 2018. Revision accepted: 16 August 2018.

Abstract. Nyaruai MA, Musingi JK, Wambua BN. 2018. The potential of agroforestry as an adaptation strategy to mitigate the impacts of climate change: A case study of Kiine Community, Kenya. Nusantara Bioscience 10: 170-177. This study has a purpose of evaluating the agroforestry potent as a conformation policy to the effects of climate change in the location of the study. One hundred farmers were used as study sample in collecting data with stratified sampling technique. To achieve data from individual farmers, both arranged and disarranged questionnaires were used. The study utilized questionnaires and observation timetable to collect data from individual farmers associated with the study objectives. The study found out that more preferable practices in agroforestry were planting the trees and shrubs as windbreakers, riparian forest buffers, silvopasture, and boundary planting while the less preferable practices were forest farming, alley cropping, and woodlots. It also found that the coaching to identify both indigenous and exotic agroforestry tree species is needed. In particular, 94% and 90% of the respondents got a feeling that the coaching on agroforestry practices and incorporation of exotic species is needed very much. The reason is that the feeling felt by respondents could give contribution to shortening the prolonged production time of trees on farm. On the contrary, 90% of the respondents are confident that agroforestry can increase catchment yield in rivers and streams, ameliorate the micro-climate, increase wood production as well as increase livestock health and products. The result showed that agroforestry has a direct link in increasing subsistence of people in the study area. Food (fruits), fodder, fuelwood, medicinal substances, gums, tannins, essential oils, fibers and waxes are the examples of agroforestry products sold by the surrounding farmers. The money will be used to provide second-tier facilities such as paying the tuition for their children or even getting healthcare facilities. The result shows that agroforestry is a method in agricultural production which can decrease the effects of human activities and climate change on the local environment. Agroforestry can increase the endurance of agricultural outturn to contemporary climate variance as well as prolonged climate change by means of the utilization of trees for intensification, diversification and supporting of farming systems.

Keywords: Conformation policy, agroforestry, climate change, Kenya

INTRODUCTION destitution and/or to eliminate famine. Diverse researches have shown the relation of poverty to the environment such The World Summit on Social Development (UN 1995) as those conducted by Agarwal (1997), Cleaver (1997), and describes poverty as a "condition characterized by severe UNDP (2000). deprivation of basic human needs, including food, drinking Substantially, in the 'poor' population, the surrounding water, sanitation facilities, health, shelter, education, and in that locality is often more downgraded. Such downgrade information. It depends not only on income but also on is caused by varied activities involving populace accretion access to services". To worsen the circumstance is the as well as technological improvement. Such activities poor’s lineal conviction on the surrounding and its services provide a significant difference between the needs and their for their subsistence. fulfillment in services such as energy, food, housing, Poverty brings to release of environmental goods at the transport, water, sewerage facilities, etc. Unquestionably, prices which are frequently higher than equipment prices. the consequence has been the unwanted alterations in land The consequence is a degraded surrounding which cannot utilization such as deforestation, substandard farming fulfill the requirements of the present population and also techniques, decadence of air and water grade, instable will threaten the availability of mankind’s requirements in generation and rubbish management, and rapid elevation of the future. Heretofore, extensive destitution and poverty-stricken city. The following are some of the environmental degradation keeps on to be examined in accurate effects of environmental downgrade, (i) spite of attempts performed by governments, non- Widespread effects to climate change particularly on the governmental organizations or even civil societies. countries in developing phase (DFID, EC, UNDP and Frequently contrived by deficient protection, World Bank 2002). (ii) Elevation in the establishment and malnourishment, elevating numbers of infant mortality, the expansion of vector-borne illness including malaria deficiency of earnings as well as unhealthy condition of (WRI 1998). (iii) Appearance of acute respiratory life, this situation keeps on burdening developmental infections in women who frequently use fuelwood in attempts plotted at various objectives involving to decrease cooking (Ezzati and Kammen 2001). (iv) Crucial NYARUAI et al. – The potential of agroforestry to mitigate climate change 171 decrement in subsistence alternatives as most of the favors MATERIALS AND METHODS provided by the environment will be remarkably decreased (Brocklesby and Hinshelwood 2001). It is undeniable to Scope of the research express that the above circumstances will be worsened by This study was performed in Kiine, a sub-location in climate change considering the average global temperature Kirinyaga county, Kenya (Figure 1). In accordance with the has risen by 0.3-0.6C over the past Century (IPCC 1990). 2009 Kenya Population and Housing Census report (KPHC The risen temperature is contributed to greenhouse gases 2009), the population of the county is "528,054 and has an which are mainly resulted from human activities such as annual growth rate of 1.5% and projected to be 595,379 in fossil fuel combusting, deforestation and substandard 2017, as compared to 552,359 recorded in 2012". The agricultural practices. Annually, about 13 million Ha of density of population of this County was 488 people per forest has been turned into land for agricultural activities km2 in 2012 and hoped to be elevated higher to 524 people (FAO 2006). The surface temperature increases to about per km2 in 2017. The Kenya Integrated Household Budget 30°C due to the availability of greenhouse which traps heat Survey (KIHBS 2005/2006) has it that 67% of land parcels released from the earth's surface (Pearce 2003) and in the county have title deeds, while 23% of farmers farm Pierrehumbert (2004)). Tremendously brought about by on land owned by the National Irrigation Board. pollution, the effects of climate change varied in various Furthermore, the County has a destitution rate of 36% countries, regions, and continents (IPCC 2007). compared with national average of 46%. ICRAF (2013) clarifies agroforestry as an agricultural The Kirinyaga County Transition Implementation Plan method that unifies plants, bushes, and animals on a farm (KCTIP 2014) records that Kirinyaga County pervades a resulting in various advantages. These advantages are region of 1,478.1 square kilometers and is categorized into multiple such as the providing of food for animals and three ecological zones; the lowland areas that embrace timber for fuelwood, the soil enrichment as well as the 1,158-2,000 m asl., the midland areas laying 2,000-3,400 m medicinal products (Sanches 2000; Kwesiga et al. 2003). asl. and the highland embracing areas of 3,400 metres to Various types of Agroforestry methods are found 5,380 metres above sea level. The lowland area is worldwide, such as forest farming, riparian buffers, alley described by gentle rolling plains that cover most of Mwea cropping, silvopasture, as well as forest. According to constituency. The midland area comprises of Ndia, Agroforestry Research Trust (2010), various researches Gichugu and Kirinyaga Central constituencies. The have delivered the advantage of integrated farming highland area pervades the upper areas of Ndia, Gichugu methods in comparison to monoculture method since they and Central constituencies and the whole of the mountain intensify the variety in localities of food production and the area. Kiine belongs to Ndia constituency in Kirinyaga subsistence mainly through the selling of farming products. County. Despite its location in the midlands, this area was Agroforestry is a promising agricultural system in selected, since it has experienced the negative effects of resolving many challenges of our time. The same thing climate change combined with the reality that about 87% of occurs in Kirinyaga County. Based on Kirinyaga County its people are hired in the agricultural sector and puts up First County Integrated Development Plan for 2013-2017, 72% of household earnings. Farmers in the study area the main causes of environmental downgrade in the County supplied the required data used for examining the purposes are deforestation, substandard solid waste processing, river of this research. bank cultivation, and pollution from industries and farmers. Furthermore, this report reveals that these causes have Research design brought to immense climate variability and change The research utilized a descriptive survey design to influencing the agricultural and health fields due to, among collect data for analysis. Kombo and Tromp (2006) other things, uncertain rainfall schemes, recession of the clarified that a descriptive survey as a method which glaciers on Mt Kenya which could be assumed as water primary purpose is to gather and analyze data to develop tower for this county. particular details. This method was more applicable, since The country keeps on experiencing the elevating bad it was efficient in data collection especially in wide area of consequences of climate change and their effects. It gets research. Moreover, the design was used because the worsened by the nowadays environmental circumstances method does no compromise on the population under study (undervalued soils, substandard agricultural methods) as (Kothari 2004). well as destitution. Hence, this study tries to clearly specify the potent of agroforestry as an adaptive action plans to the Target population negative impact of climate change in the research area. The research concentrated on a sample size of 100 The purposes of this study were (i) to examine the most farmers in Kiine. The sample size was excellent and a appropriate agroforestry practices giving maximum reasonable representation of the farmer populace. Also, the advantages with regard to conformation to effects of sample size was excellent because of the time span and the climate change in the research area, (ii) to recognize main financial plans obtainable for the research. The primary agroforestry expenses and advantages in conformation to center of attraction is on Karima region, which has around climate change, (iii) to decide whether agroforestry can 450 farmers. remarkably assist the enhancements of subsistence of the community in the research area.

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Kirinyaga

Nairobi

Figure 1. Location of Kiine Community ( ) in the Kirinyaga County, Kenya

Sampling procedure and sample size Questionnaires Sample size The written questionary was characterized by both overt The research concentrated on a sample size of 100 and closed-ended inquiries and was separated into five farmers in Kiine. The sample size was excellent because of sections. The questionnaires were brought to the the time span and the financial plans obtainable for the respondents and were collected one week later after replies research. had been given. The technique was preferable because it permitted the respondents sufficient time to answer the Selection of study sample questionnaires. The utilization of questionnaires was This research utilized stratified sampling technique to beneficial for the study as it is easier to manage as well as pick out farmers to be sampled. Stratified sampling to analyze the data supplied by the respondents. technique principally categorizes a certain population based on described characteristics which are then utilized to Interviews acquire samples (Cochran 1963). This technique confirms An interview was performed on illiterate respondents or equitable deputation of sub-groups in the chosen samples. on respondents who gave incomplete response to the questionnaires due to the lack of time. Hence, the research Methods of data collection assistant would meet those respondents and would renew To simplify details collection in answering to the the questionnaires based on the feedback provided. This research inquiries, the research utilized three types of technique was highly beneficial for respondents who had equipment namely questionnaires, interviews and inability in writing or reading for one reason or another. observation. Before doing data collection from the respondents, the researcher acquired a research approval Observation from the chief and other regional authorities. This solemn Further examination schedules were applied to approval gave assistance to the researcher in socializing accomplish response resulted from the questionnaires, with the farmers in an effort to obtain replies that will give based on the feedback. For this technique, the natural assistance in achieving the research objectives. The settings of the study area were examined by the research researcher then came to see the settlements in which the assistants and responses against the set items captured in research would be performed to introduce herself as well as the interview schedule sheet were provided by them. to create affinity with the respondents.

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Instrument validity years, 50% were between the ages of 41-50 years, while the Mugenda and Mugenda (2003) elucidate that validity as remaining 22.22% were above 50 years old. This indicates the righteousness of outcomes. Validity was assured by the that the larger part of the farmers (50%) are between the consultation with research experts who are supervisors to age of 41-50. make sure that the instruments of data collection can It is significant that the middle age group in the context perform their measurement correctly. of the respondents (41-50) developed the bulk of agroforestry practitioners who would become the main Instrument reliability actor in setting up competence in agroforestry practices and The ability of a research instrument to present fixed whom would remarkably affect its uptake. The age range results upon several trials is called reliability. The 41-50 years posed the larger part of respondents (50%), and reliability of the questionnaires was verified through test- has remarkable level of education, a factor that would retest method, namely, the same questionnaires to fill were escalate the favorable outcome of new agroforestry given to the same respondents two times in an in-between inventions to be introduced in the area. period of two weeks. The instruments were applied on a few selected subjects. Level of education of farmers The respondents were questioned to inform their level Data analysis of education. 11.11% were illiterate, 11.11% had owned The collected data were examined with a method of primary level of education, 50% had achieved secondary descriptive statistics. The analysis process demanded an level of education, 27.78 had reached tertiary level of appraisal of the data captured in the questionnaires and education. Table 3 shows the level of education observation timetables and detecting any inaccuracies and finally coding the responses in a manner that would assist The number of years that farmers have spent in Kiine area in further analysis. The analysis conveyed the findings in The majority (38.89%) of interviewed farmers had lived terms of percentages and frequencies of occurrence for 10-19 years in the village while a few (5.55%) had lived utilizing the Statistical Package of Social Sciences (SPSS). for 50 and 59 years in the village (Table 4).

The climate of Kiine area RESULTS AND DISCUSSION The respondents were questioned to inform the climate of their area. 5.56% said that climate is dry and hot, Response rate 22.22% said that the climate is cool and wet, 44.44% said The sample population consisted of 100 farmers in that the climate is cool and dry, while the remaining Kiine, Kirinyaga County. 10 out of 100 farmers gave back 27.78% said that the climate has been wet and hot. This unfilled questionnaires, thus response rate was 90 %. This represents that the climate of Kiine area in Kirinyaga high number of participants increases the findings as these County is cool and dry as the majority (44.41%) stated the would be discerned as more than sufficient representative climate as cool and dry (Figure 2). 60% of the population of the population under study. consider that climate is not the same as the climate lately, while the remaining 40% think that climate is always the Demographic profile of the respondents same as it is today (Table 5). This item looked for the farmers' gender, age, level of education and the length of time they have been practicing farming. This was significant since, on a study about a particular population, certain facts must be understood first. Table 1. Distribution of farmers by gender The demographic profile of the respondents will show the way they fulfill the research appliance. The outcomes are Gender Frequency Percentage as described below. Male 60 66.7 Female 30 33.3 Farmers gender Total 90 100 The farmers were questioned to inform their gender and the outcomes are assumed up in Table 1. As described in Table 1, most farmers (66.7%) were male and some (33.3%) were female. This could be accredited to land Table 2. Ages of the respondents tenancy and demesne issues which in many African regions belong to men. Age group No. of farmers Percentage below 20 0 0 Age of the farmers 21-30 10 11.11 The respondents were questioned to inform their age. 31-40 15 16.67 Table 2 shows the age of the farmers who were the samples 41-50 45 50 in the study area. As shown, none of the farmers were over 50 20 22.22 under the age of 20 years, 11.11% were between 21-30 Total 90 100 years old, and 16.67% were between the ages of 31-40

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This shows that the climate has undergone changes over Table 3. Level of education of farmers the years due to human activities having a negative impact on the environment. Regarding the impacts of climate Level of education No. of farmers Percentage change in their region, 65% stated that the impact was very Illiterate 10 11.11 serious, 25% said the impact was serious while the Primary 10 11.11 remaining 10% informed that climate change was not secondary 45 50.00 serious. This finding implies that the impact of climate Tertiary 25 27.78 change in the Kiine region is a serious problem (Figure 3). Total 90 100 The majority (80%) had a feeling that the local microclimate can be made better. The remaining 20% had the feeling that it cannot be fixed. Those who feel that it Table 4. Number of years the respondents have been living in can be enhanced proposed some methods such as Kiine area, Kenya environmental conservation, afforestation, the use of cheaper energy in agroforestry, and the punishment for No. of years No. of farmers Percentage environmental polluters. 0 to 9 3 16.67 10 to 19 7 38.89 Investigation of agroforestry practices which provide 20 to 29 5 27.78 maximum benefits in terms of adaptation of climate 30 to 39 2 11.11 change impacts in the study area 40 to 49 1 5.55 To test the preferred agroforestry technology, a five- 50 to 59 0 0 Total 18 100 point Likert scale is used and added by the range from strongly agree, agree, unsure, disagree and strongly disagree, explaining that 1 is the most preferred while 5 is Table 5. Climatic change in Kiine area, Kenya the least preferred. They were then questioned to evaluate various agroforestry technologies in their village. Table 6 No. of Response Percentage presents how farmers evaluate several agroforestry farmers practices. It shows that most farmers like the placement of Climate has not always been as it is 54 60 trees and shrubs that are very high as windbreaks, and the The climate has changed 36 40 planting of riparian forest buffers as trees, shrubs or grasses and tree combinations with livestock. This is very different from Silvopasture which is a combination of plants with foliage and livestock on the same land, the planting short- rotation wood (woodlots) in humid areas and the Alley planting-rows of trees with a very broad space that creates alleyway of plants on the side of the hill that receives the smallest ranking. Table 6 shows clearly that the most preferred agroforestry practices are the planting of trees and shrubs as windbreaks, planting buffer for forests reparation, silvo grazing, boundary planting, home gardening and a combination of trees with food crops. On the contrary, the most unpopular agroforestry practices are forest agriculture, alley planting and planting of woodlots in humid areas. Figure 2. Climate of Kiine area, Kenya Major agroforestry costs and benefits in adaptation to climate change Agroforestry costs The respondents were inquired to evaluate agroforestry expenses that they could associate to on a five-point scale. The expense in this occurrence points to find that was needed to allow the respondents adopting agroforestry practices and should not be apprehended in monetary equivalent. First, the respondents were inquired if the coaching of residents on agroforestry should be intensified. From those being inquired, 44.4% strongly agreed that such coaching should be intensified, 33.33% agreed, 5.56 were not sure whether such coaching should be intensified.

Figure 3. Impact of climate change in Kiine area, Kenya

NYARUAI et al. – The potential of agroforestry to mitigate climate change 175

Table 6. Agroforestry practices

Category Category 1 2 3 4 5 Total Planting trees and shrubs as windbreakers No. of farmers 35 25 15 5 10 90 % of farmers 38.89 27.8 16.67 5.56 11.11 100 Planting of riparian forest buffers No. of farmers 30 30 10 10 10 90 % of farmers 33.33 33.3 11.11 11.11 11.11 100 Silvopasture No. of farmers 40 20 15 10 5 90 % of farmers 44.44 22.2 16.67 11.11 5.56 100 Forest farming No. of farmers 15 10 10 30 25 18 % of farmers 16.67 11.1 11.11 33.33 27.78 100 Alley cropping No. of farmers 4 4 1 4 5 18 % of farmers 22.22 22.2 5.56 22.22 27.78 100 Boundary planting No. of farmers 40 25 5 15 5 90 % of farmers 44.44 27.8 5.56 16.67 5.56 100 Home garden No. of farmers 7 6 1 2 2 18 % of farmers 38.89 33.3 5.56 11.11 11.11 100 Planting of woodlots in moist region No. of farmers 10 10 5 30 35 90 % of farmers 11.11 11.1 5.56 33.33 38.89 100 Combination of trees with food crops No. of farmers 30 35 5 15 5 90 % of farmers 33.33 38.9 5.56 16.67 5.56 100 Note: 1. Strongly agree, 2. Agree, 3. Don’t know, 4. Disagree, 5. Strongly disagree

Table 7 Agroforestry costs

Agroforestry costs Category 1 2 3 4 5 Total Coaching on agroforestry be enhanced No. of farmers 40 30 5 10 5 90 % of farmers 44.44 33.33 5.56 11.11 5.56 100 Knowledge regarding agroforestry needed No. of farmers 35 40 0 5 10 90 % of farmers 38.89 44.44 0.00 5.56 11.11 100 Indigenous and exotic species needed No. of farmers 45 35 0 5 5 90 % of farmers 50.00 38.89 0.00 5.56 5.56 100 Resistance to agroforestry practices evident No. of farmers 35 30 15 5 5 90 % of farmers 38.89 33.33 16.67 5.56 5.56 100 Residents adapted to the current conditions No. of farmers 10 10 5 30 35 90 % of farmers 11.11 11.11 5.56 33.33 38.89 100 Note: 1. Strongly agree, 2. Agree, 3. Don’t know, 4. Disagree, 5. Strongly disagree

or not, 11.11% disagree while the remaining 5.56% products has been brought up by Young (1987) as; “It is strongly disagree that coaching of residents on agroforestry widely argued that the lengthy production period and the should be intensified. These encourage to an understanding incidence of most of the costs at the time of establishment, that farmers in the study area are not well-informed on create financial problems for farmers in adopting practices agroforestry farming implementations. The government involving tree growing”. and other stakeholders should thus create mechanisms to Other findings about agroforestry expenses are set appropriate coaching in this area. Even more, there was described circumstantially in Table 7. The findings of this finding that when the respondents were inquired if the study reveal that there is discrepancy to agroforestry profound knowledge regarding agroforestry was required, a practices. This is supported by evidence at the time the majority (44.4%) stated that such knowledge is required, respondents were questioned to asses the issue of while only a few (11.11%) stated that such knowledge is discrepancy to agroforestry practices (Table 8). needed. Certainly, to invest in enhancing the capability of farmers is imperative. The respondents were also questioned if they needed both indigenous and exotic Table 8. Discrepancy to agroforestry species of trees and crops. A majority (50%) strongly Strongly Strongly agreed that they needed both indigenous and exotic trees Response Agree Neutral Disagree agree disagree and plants species while a minority (5.56%) strongly No. of 45 20 5 10 10 disagree with this program. One of the interesting people arguments about species needs and usage of agroforestry Percentage 50 22.23 5.55 11.11 11.11

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A majority (50%) strongly agree that there was materials that are recycled and used as natural fertilizers; evidence of discrepancy to agroforestry praxes, while the helping to regulate the water cycle; and providing shade". minority (11.11%) strongly disagree that such discrepancy On the request to express their feelings on the in agroforestry praxes existed. This discrepancy can be agroforestry and increased yield in food, craft, and emerged due to many factors such as capital, land tenure medicinal crops, a majority 50% agree, 33.33% strongly and tree ownership, social economic stratification, agree, 11.11% disagree while the remaining 5.56% strongly technology and the length of time for trees to be ready for disagree. This is a sign that agroforestry raises the harvest. On the question if the inhabitants had done an production of food, craft, and medicinal crops. adaptation to the current conditions, 11.11% strongly agree, 11.11% agree, 5.56% were unsure, 33.33% disagree while Agroforestry contribution on subsistence raise the remaining 5.56% strongly disagree. This is a sign that The respondent was handed with a 5 point Likert scale the deforestation and environmental downgrade negatively which is used to appraise agroforestry contribution on the affect inhabitants of Kiine and comprehensive efforts are rise of their subsistence. The result is showed in Table 10. needed to recover the neighborhood to its original The research gave the following conclusions: on the conditions. statement that agroforestry is needed to enhance the market network between producers and consumers, 44.44% Advantages of Agroforestry strongly agree, 27.77% agree, 5.56% were unsure, 11.11% To discover the advantages of Agroforestry, the disagree and the remaining 11.11% strongly disagree. On respondents were handed out with various positive effects the question if the processing, handling, and marketing of of Agroforestry and requested to appraise them. Table 9 products yielded by traditional agroforestry practices shows the responses of the farmers on advantages of should be enhanced, 38.33% strongly agree, 33.33% agree, agroforestry. 11.11% were unsure, 5.56% disagree and the remaining Table 9 shows that 50% of sample strongly agree that 11.11% strongly disagree (Table 10). Table 10 also agroforestry can make the climate of the area better. This presents the result on the request to appraise the issue of finding is reinforced by researches carried out by escalated catchments areas for rivers, streams, wells due to Torquebiau, (1994) which resumed that “Agroforestry can agroforestry, the majority (55.56%) strongly agree. improve the resilience of agricultural production to current As described in Table 10, on the matter if nutrition and climate variability as well as long-term climate change health of households should be enhanced through fruits through the use of trees for intensification, diversification based Agroforestry practices, a majority of the respondents and buffering of farming systems. For example, trees (27.77%) strongly agree. On the contrary, 11.11% of them improve soil quality and fertility by contributing to water strongly disagree with this approach. On the occasion that retention and by reducing water stress during low rainfall the respondents were inquired if improving, processing, years. Trees can also reduce the impacts of weather handling, and marketing of products from agroforestry will extremes such as droughts or torrential rain”. boost adoption of Agroforestry practices, 33% strongly Most of interviewee (33.33 %) strongly agree that agree, 38.88% agree, 11% were unsure, 5.56% disagree agroforestry raises the production of wood for fuel, while the remaining 11%% strongly disagree. On the construction, craft, (Table 9). This is reinforced by Raintree question if putting in new germplasm for Agroforestry with (1991), who expresses that “agroforestry is an approach to a focus on trees with high economic benefit, 50% strongly agricultural production that can reduce the impacts of agree, 33%% agree, 11% disagree and the remaining human activities and global climate change on the local 5.56% strongly disagree. A majority of the respondents environment. Agroforestry systems integrate commercial (39%) strongly agree that adaptive capability of drylands crop production into the natural forest environment, farming to climate changes should be increased. harnessing trees for a variety of benefits: improving soil Conversely, about 6% of the respondents strongly disagree structure, drainage, and nutrient levels; preserving with that thought. biodiversity; increasing forage, firewood and other organic

Table 9. Advantages of agroforestry

Advantages of agroforestry Category 1 2 3 4 5 Total Increased catchment for rivers, streams, wells No. of farmers 50 20 5 10 5 90 % of farmers 55.56 22.22 5.56 11.11 5.56 100 Improved climate No. of farmers 45 25 5 5 10 90 % of farmers 50.00 27.78 5.56 5.56 11.11 100 Increased wood No. of farmers 30 30 5 15 10 90 % of farmers 33.33 33.33 5.56 16.67 11.11 100 Increased food output, craft and medicinal crops No. of farmers 30 45 0 10 5 18 % of farmers 33.33 50.00 0.00 11.11 5.56 100 Improved livestock health and livestock products No. of farmers 35 35 10 10 10 90 % of farmers 38.89 38.89 11.11 5.56 5.56 100

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Table 10. Methods of enhancing agroforestry

Category 1 2 3 4 5 Total Enhancing the producers-consumers market network, value addition, and Freq. 8 5 1 2 2 18 processing of agroforestry products % 44 28 6 11 11 100 Improving nutrition and health for households through integration of fruit tree Freq. 7 6 2 1 2 18 species % 39 33 11 6 11 100 Improving the production, processing, handling, and marketing of agroforestry Freq. 5 5 3 3 2 18 products % 28 28 17 17 11 100 Enhancing forest farming Freq. 6 7 2 1 2 18 % 33 39 11 6 11 100 Introducing a new agroforestry germplasm Freq. 9 6 0 2 1 18 % 50 33 0 11 6 100 Enhancing adaptive capacity of dryland farming to climate change Freq. 7 7 1 1 2 18 % 39 39 6 6 11 100

The findings of this research result the following Ezzati M, Kammen DM. 2001. Quantifying the effects of exposure to conclusion. Agroforestry practices can remarkably buttress indoor air pollution from biomass combustion on acute respiratory infections in developing countries. Environ Health Perspect 109 (5): the residents in dealing with undesirable impacts of climate 481-488. change at the present while at the same time increasing IPCC. 1990. The Science of Climate Change. Key Findings of IPCC tenacity against future impacts. The result of study done by Working Group 1. Intergovernmental Panel on Climate Change, other scholars support this conclusion, for example, Oram Geneva, Switzerland. IPCC. 2007. Climate Change: Impacts, Vulnerabilities and Adaptation in (1993) who stated that agroforestry practices make the Developing Countries. Intergovernmental Panel on Climate Change. farmers and their families able to diversify their farm Intergovernmental Panel on Climate Change, Geneva, Switzerland. practices and set up network to guarantee livelihood ICRAF [World Agroforestry Centre]. 2013. Strategy 2013-2022: sustainability, like the availability of products to be traded Transforming lives and landscapes with trees. World Agroforestry Centre, Nairobi. in the market; Torquebiau (1994) who found that in KCTIP. 2014. Kirinyaga County Transition Implementation Plan. Office Sumatra, for example, some people grow plants as a fount of the Governor, Kirinyaga County, Kutus, Kenya of food, diversified it with rubber plants in their fallow KIHBS. 2005/2006. Kenya Integrated Household Budget Survey 2005- fields; while in Borneo, some people cultivate rattan in rice 2006. Kenya National Bureau of Statistics (KNBS), Nairobi, Kenya Kombo DK, Tromp DL. 2006. Proposal and Thesis Writing. An fields during the last rice season and the rattan which is a Introduction. Paulines Publications, Nairobi. very aggressive vine will use any neighboring trees as Kothari CR. 2004. Research Methodology: Methods and Techniques. 2nd supports. He added further that rattan is a very profitable ed. New Age International Ltd., New Delhi. crop and can be harvested after 8-10 years. KPHC. 2009. 2009 Kenya Population and Housing Census Reports. Kenya National Bureau of Statistics (KNBS), Nairobi, Kenya Kwesiga F, Akinnifesi FK, Mafongoya PL, McDermott MH, Agumya A. 2003. Agroforestry research and development in southern Africa REFERENCES during the 1990s: Review and challenges ahead. Agrofor Syst 59: 173-186. Mugenda A, Mugenda O. 2003. Research Methods; Quantitative and Agarwal B. 1997. Gender, environment, and rural poverty interlinks Qualitative Approaches. African Centre for Technology Studies, regional variations and temporal shifts in rural India, 1971-1991. Nairobi. World Dev 25 (1): 23-52. Oram P. 1993. Global Perspective on Population, Resources and Agroforestry Research Trust. 2010. Heartland Institute 2009 International Agricultural. Proceedings of the 7 Australian Agronomy Conference, Conference on Climate Change, March 8-10, 2009, New York. Adelaide, 1993. Australian Society of Agronomy, Carlton, Australia. Brocklesby MA, Hinshelwood E. 2001. Poverty and the environment: Pearce F. 2003. Land of the midnight sums. New Scientist 177: 2379. What the poor say: An assessment of poverty-environment linkages in Pierrehumbert RT. 2004. Warming the World. Nature 432: 677. participatory poverty assessments. Centre for Development Studies, Raintree JB. 1991. Socio-economic attributes of trees and tree planting University of Wales, Swansea, UK, practices. Community Forestry Note 9. FAO, Rome. CIDP. 2013. Kirinyaga County First County Integrated Development Plan Sanchez P.A. 2000. Linking climate change research with food security 2013-2017. Office of the Governor, Kirinyaga County, Kutus, Kenya and poverty reduction in the tropics. Agric Ecosyst Environ 82: 371- Cleaver, K. 1997) Rural development strategies for poverty reduction and 383. environmental protection in Sub-Saharan Africa. World Bank, Torquebiau E. 1994. Agroforestry Research for Integrated Land Use: An Washington D.C. Introduction to the Concept of Agroforestry. ICRAF, Nairobi, Kenya. Cochran WG. 1963. Sampling Techniques. 2nd ed. John Wiley and Sons, UN. 1995. World Summit for Social Development, Copenhagen, Inc., New York. Denmark, 6-2 March 1995. DFID, EC, UNDP, World Bank. 2002. Linking Poverty Reduction and UNDP. 2000. Human Development Report 2000: Human Rights and Environmental Management Policy Challenges and Opportunities. Human Development. United Nations Development Programme & Department for International Development, United Kingdom (DFID), Oxford University Press. New York. Directorate General for Development, European Commission (EC), Young A. 1987. The Potential of Agroforestry as a Practical Means of United Nations Development Programme (UNDP), The World Bank, Sustaining Soil Fertility. ICRAF, Nairobi, Kenya. July 2002.

NUSANTARA BIOSCIENCE ISSN: 2087-3948 Vol. 10, No. 3, pp. 178-182 E-ISSN: 2087-3956 August 2018 DOI: 10.13057/nusbiosci/n100308

Influence of NAA and coconut water with variation of soaking duration on the growth of yellow bamboo branch cutting

TIA SETIAWATI, AGINTA PUTRI REHULINA KELIAT, RULY BUDIONO, RUHYAT PARTASASMITA, JOHAN ISKANDAR Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran. Jl. Sukarno-Hatta Km 21, Jatinangor, Sumedang, 45363, West Java, Indonesia. email: [email protected];  [email protected]

Manuscript received: 10 July 2018. Revision accepted: 22 August 2018.

Abstract. Setiawati T, Keliat APR, Budiono R, Partasasmita R, Iskandar J. 2018. Influence of NAA and coconut water with variation of soaking duration on the growth of yellow bamboo branch cutting. Nusantara Bioscience 10: 178-182. Yellow bamboo (Bambusa vulgaris Schrad Ex. var. Striata) is one kind of bamboo used for industrial and household raw materials. Yellow bamboo propagation with branch cuttings has relatively low percentage of growth. To increase the growing success of yellow bamboo branch cuttings can be used growth regulators substance, such as NAA and coconut water. This study aimed to obtain the best combination of NAA and coconut water with the soaking duration in the growth of yellow bamboo branch cuttings. The study was conducted using Completely Randomized Design (CRD) of 6 × 3 factorial arrangement with five replications. The first factor was the combination of NAA and coconut water (CW), which consisted of six levels, namely 100% CW, 20% NAA + 80% CW, 40% NAA + 60% CW, 60% NAA + 40% CW, 80% NAA + 20% CW and 100% NAA. The second factor was the soaking duration which consists of three levels, namely 12 h, 24 h, and 36 h. Parameters observed included shoot emerging time, shoot number, shoot length, leaf area, root number and root length. The data obtained were analyzed using the Analysis of Variance (ANOVA) and Duncan's Multiple Range Test (DMRT) α=5%. The results showed that the combination of 80% NAA + 20% CW gave the best yellow bamboo branch cuttings growth with an average shoot length of 1.44 cm, leaf area 41.29 cm2, root number 42.87 and root length 23.70 cm. The interaction of 80% NAA + 20% CW with soaking duration of 36 h resulted in average the fastest shoot emerging time of 2.02 days after planting.

Keywords: Branch cutting, coconut water, yellow bamboo, NAA

INTRODUCTION ideal for bamboo species with thick-walled branches such as Bambusa spp. or Dendrocalumus spp. (Banik 1995; Bamboo is a perennial wood grass that grows very fast Islam et al. 2011). Adventive root formations are important and produces very high biomass. Bamboo can absorb in vegetative propagation through cuttings which often carbon faster than other similar tree species that have rapid result in significant economic losses (Pandey et al. 2011; growth. Bamboo has many utilities, among others as a Ibironke 2016), so it takes rooting hormones to increase source of local economy, structural raw materials, animal root growth. As for the statement of Tiwari and Das (2010) feed and fiber source for papermaking, home industry and that to improve sprouting, rooting and the survival of stem household purposes (Banik 1995; Devi 2013). cuttings, plant growth regulators can be used. In most bamboo-producing Asian countries, there is a Hormones that aid in adventitious root growth is auxin, shortage of supply and in recent years there is a widening such as an Indolebutyric acid (IBA) and Naphthalene gap between supply and demand (Devi 2013). Over- Acetic acid (NAA), both are the most widely used exploitation occurs with the increased harvesting of commercial synthetic auxin for promoting root formation bamboo from the wild or rural environments (Banik 1995; on stem cuttings (Muller and Leyser 2011: Khudhur and Devi 2013). The demand for bamboo increases steadily due Omer 2015). But synthetic rooting hormone is still to its versatile usage (Goyal et al. 2010). Yellow bamboo is relatively expensive so it takes alternative substances that one of the bamboo species of 19 species or groups of are cheaper but effective in supporting the growth of root species agreed by The International Network for Bamboo cuttings. Natural substances that have the ability to and Rattan (INBAR) which cooperate with the stimulate rooting of cuttings, as a source of hormones such International Plant Genetic Resources Institute, as research as auxin (IAA/indole-3-acetic acid), cytokinin, and material which is worthy to be focused and wider use gibberellin can be found in coconut water (Yong 2009; (Banik 1995). Karunarathna and Harris 2016). Yong et al. (2013) state In general, bamboo propagation is done vegetatively that coconut water contains cytokinin which helps through rhizomes, stem and branches cuttings. The benefits stimulate root and shoot growth. The cytokinin found in of bamboo propagation through branch cuttings are the coconut water support cell division, and along with other high availability and ease of handling (Banik 1995; chemical components promotes plant growth (Muhammad Kaushal et al. 2011; Bhol and Parida 2015). This method is et al. 2015). In addition, the duration of soaking cuttings in SETIAWATI et al. – Effect of NAA and coconut water on the growth of yellow bamboo 179 active substance should be considered to obtain optimal Emergence time of shoot and shoot number growth on cuttings. Ibironke (2016) reported that root Based on the result of DMRT, the effect of NAA+ CW initiation on Boungainvillea cuttings can be increased when combination and soaking duration on emergence time and soaked with indole -3-butyric acid or coconut water for 5- number of shoot on branch cutting. Table 2 showed that in 10 minutes. The coconut water treatment on cuttings have the combination of 80% NAA + 20% CW with soaking the significant effects in increase shoot length, the girth of duration of 36 hours generated the fastest emerging time of shoots, number of leaves, dry and fresh weight of root and bud was 2.02 days after planting and the highest shoot root length. number was 2.05. In this treatment, it was suspected that NAA and plant growth regulator (PGR) contained in coconut water can be effectively translocated to the plant MATERIALS AND METHODS part that became the point of bud initiation. It was seen that in all combinations with longer soaking duration, the The materials used in this research were coconut water average time of shoot appears was faster and the number of (Cocos nucifera), yellow bamboo branch as cuttings shoot was also higher. material,. Research used an experimental method in In general, the time of shoot emergence was faster and greenhouse with Completely Randomized Design (CRD) more shoots produced with increasing the duration of two factors. The first factor was six concentration soaking. Soaking for 36 h allowed the increase of hormone combination of NAA and coconut water (CW) i.e. 0% solution absorbed by the cuttings, otherwise on 12 hours, NAA + 100% CW, 20% NAA + 80% CW, 40% NAA + PGR absorbed was not able to spur the growth of cuttings 60% CW, 60% NAA + 40% CW, 80% NAA + 20% CW to the maximum. It seems the soaking duration is related to and 100% NAA + 0% CW. The second factor was three the amount of nutrients and PGR absorbed by the cuttings. treatment of soaking duration, i.e., 12, 24 and 36 h. Each Soaking will determine the length of contact time of the treatment was repeated five times. material to the solution so that it will affect the number of Variations of NAA concentrations used were 20%, solutions absorbed by the plant which will ultimately affect 40%, 60%, and 80%, taken from a stock solution of NAA the effectiveness of PGR (Islam et al. 2011; Razvi et al. 500 ppm, then mixed with coconut water according to a 2011). Therefore the duration of soaking should be predetermined combination. Each mixed solution (NAA + adjusted to the concentration of solution used. coconut water) was prepared as much as 1000 mL. The combination of NAA 20% + 80% CW was done by mixing 200 mL of NAA 500 ppm with 800 mL CW, for other Table 1. The ANOVA results of NAA + coconut water combinations were done in the same way. The branch was combinations and soaking duration effect on growth traits of cut off the end so that the length of the cuttings was ± 75 yellow bamboo branch cuttings. cm with 4 segments. The base of the bamboo branch was Combination Soaking Interaction soaked in each treatment solution with different soaking Parameters of NAA + duration of C×T periods i.e. 12, 24, and 36 h. Branch cuttings were grown CW (C) (T) on mixed media of soil, sand, and manure (2:1:1) in Emergence time of shoot * * * polybag with vertical position. Leaf area * ns ns Observations were made at 60 days after planting on Length of root * ns ns buds emergence time, number and length of the shoot, leaf Length of shoot * ns ns area, and number and length of the root. The data were Number of root * ns ns statistically analyzed using SPSS 16.0. Analysis of Number of shoot ns ns ns variance (ANOVA) was performed to determine significant Note: * significant; ns = non significant differences. Mean separation was carried out by the

Duncan’s Multiple Range Test (DMRT) α=5%. Table 2. The average emergence time of shoot and shoot number of branch cutting on the various combination of NAA + CW and RESULTS AND DISCUSSION soaking duration

Emergence time of The effect of NAA + coconut water combinations and Combination of Number of shoot shoot (hour) soaking duration on the growth of branch cuttings. Table 1 NAA + CW showed that the combination of NAA + CW, the duration 12 h 24 h 36 h 12 h 24 h 36 h 100% CW 6.80 d 3.00 b 2.65 ab 0.53 1.05 1.50 of soaking and its interaction had no significant effect on 20% NAA + 80% CW 7.35 de 4.90 bc 2.45 ab 0.85 1.20 1.10 the number of shoots, but had significant effect on the 40% NAA + 60% CW 6.20 d 3.30 b 2.30 ab 0.70 0.75 1.35 emergence time of shoot. The shoot length and leaf area 60% NAA + 40% CW 4.90 bc 4.95 bc 2.30 ab 0.75 1.20 1.40 were significantly affected by the combination of NAA + 80% NAA + 20% CW 6.70 d 4.75 bc 2.02 a 1.00 1.95 2.05 CW. In root growth, the combination of NAA + CW had 100% NAA 6.20 d 4.80 bc 2.70 ab 0.70 1.10 1.55 significant effect on number and length of root. There was Note: the mean followed by the same letters on the same column no interaction effect on root growth. showed no significant difference according to DMRT 5%.

180 NUSANTARA BIOSCIENCE 10 (3): 178-182, August 2018

In this study, the lowest coconut water concentration, In Table 3, 100% NAA treatment lead in the growth i.e., 20% was able to induce shoots faster with the highest and development restriction of shoot and leaf expansion. number. According to Davies (1990) plant hormones are Similar results were found in a study by Rahdari et al. organic substances which influence physiological processes (2014) that on treated stem cutting of Cordyline terminalis at low concentrations. The formation of shoots with with increased NAA concentrations from 1000 to 2000 and coconut water treatment in this study may be due to the 3000 ppm resulted in a decrease the number of branches/ presence of cytokinin that has major role in cell division, leaf that could indicate that auxins in high concentrations shoot formation, and activity of shoot meristem (Yong et could inhibit shoot and branch growth. According to Raj et al. 2009). The presence of cytokinin as reported by Davies al. (2013) and Sarmiento et al. (2016), a high auxin (1990) may induce bud formation and the release of lateral concentration stimulates the biosynthesis of ethylene, buds from apical dominance. At 20% coconut water which further triggers the production of abscisic acid concentration, the need for cytokinin to induce buds, not (ABA). The ABA together with ethylene, accelerates leaf only from exogenous cytokinin, but also endogenous senescence and ultimately death of leaf. In this condition, cytokinin that is naturally produced by cuttings. Both will leaf death allows leaf abortion which further leads to interact to produce physiological responses such as shoot decreased leaf area. Likewise, the addition of coconut formation. Wroblewska (2015) stated that the growth and water with higher concentrations up to 100% inhibit the development of plant are influenced by the interaction and growth of leaf area due to the concentration of PGR such as balance between exogenous and endogenous hormones. cytokinin, auxins, and gibberellins contained in it will be in In addition, the application of NAA in this study supra-optimal concentration (Table 3) thus exceeding the contributes to shoot formation. According to Tiwari and required PGR concentration of the plant tissue. Das (2010), the use of hormones or auxins plays an important role in affecting growth/sprouting and survival of Number and length of roots stem cuttings. The formation of shoots/leaves can also be The effect of NAA+ CW combination on number and associated with rooting system. Nasri et al. (2015) length of roots. Table 4 showed that in the combination suggested that increased production of new leaves in treatment of 80% NAA + 20% CW produced the highest cuttings may be associated with an increase in the number root number of 42.87 with the root length of 16.55 cm. In and length of roots in auxin (IBA)-treated cuttings allowing this combination, the increase in roots number was cuttings to absorb more water and nutrients from rooting followed by the decrease in root length. The highest root media, which in turn will contribute to better growth and length of 23.70 cm was found in 100% NAA but had the new leaves formation. In all treatments of CW-combined lowest root number of 7.67 cm. This phenomenon is NAA showed higher root number than treatment without similar to that reported by Rahdari et al. (2014) that an NAA. increase in the number of roots in Cordilyn graft treated with auxin (IBA and NAA) is accompanied by a decrease Length of shoot and leaf area of branch cutting in long growth. Based on the result of DMRT, the effect of NAA+ CW combination on length of shoot and leaf area of branch Table 3. The average length of shoot and leaf area of branch cutting. Table 3 indicated that the highest average shoot cutting on the various combination of NAA+CW length of 1.44 cm was found in the combination of 80% Length of shoot Leaf area NAA + 20% CW, significantly different from the other Combination of NAA + CW (cm) (cm2) combinations. In this combination, the effect of cytokinin 100% CW 0.80 a 6.72 a hormone contained in the coconut water allowed initiate 20% NAA + 80% CW 0.74 a 11.93 a the formation of buds more quickly (Table 2), so the shoot 40% NAA + 60% CW 0.53 a 11.94 a growth will also be faster and allowed longer buds to be 60% NAA + 40% CW 0.78 a 23.93 b produced. This condition can not be separated also from the 80% NAA + 20% CW 1.44 b 41.29 c contribution of auxin in elongation of cells and cytokinin in 100% NAA 0.53 a 4.77 a cell division so that buds become longer. Note: the mean followed by the same letters on the same column In the combination of 80% NAA + 20% CW has also showed no significant difference according to DMRT 5%. generated the highest average leaf area of 41.29 cm2 that was significantly different from other combinations. In this treatment, endogenous auxin (NAA) and cytokinin Table 4. The average roots number and roots length of branch cutting on the various combination of NAA+CW hormones in coconut water were sufficient for branch cuttings to promote cell division and differentiation in Number of Length of Combination of NAA + CW forming new shoots, so that no more exogenous hormones roots roots (cm) were required in high concentrations. The leaf area is the 100% CW 4.47 a 2.99 a result of cell enlargement induced by the presence of 20% NAA + 80% CW 13.27 bc 3.80 ab cytokinin and related to the root growth. The total leaf area 40% NAA + 60% CW 15.80 c 6.59 ab is adjusted to compensate for the extent of root growth, as 60% NAA + 40% CW 24.67 d 8.65 b the amount of cytokinin reaching the shoot will reflect the 80% NAA + 20% CW 42.87 e 16.55 c extent of the root system (Davies 1990). 100% NAA 7.67 ab 23.70 d Note: the value followed by the same letters on the same colomn show no significant difference according to DMRT 5%.

SETIAWATI et al. – Effect of NAA and coconut water on the growth of yellow bamboo 181

In this study, all combinations of NAA + CW increased Regional Forest Tree Improvement Project (FORTIP) and Bangladesh the number of roots (Table 5). NAA synergize with Forest Research Institute (BFRI). Bhol N, Parida S. 2015. Influence of growth regulators on propagation of cytokinin contained in coconut water induce of the root culm- and branch cuttings of Bambusa vulgaris. J Tree Sci. 34 (1): growth. The role of cytokinin in spurring root growth, 64-68. especially by stimulating the differentiation of root Davies PJ. 1990. The plant hormones: their nature, occurrence and meristem cells (Muller and Leyser 2011), whereas the role functions. In. Davies PJ. (ed.). Plant Hormones and their Role in Plant Growth and Development. Kluwer, Boston. of auxin has been reported as the central mediator of organ Devi YR. 2013. Bamboo forest resources of India and its role in food development by promoting cell division, cell extension, security-a review. Agri Reviews 34 (3): 236-241. DOI- and cell differentiation (Ditengou 2006). Auxin has proven 10.5958/j.0976-0741.34.3.009. to accelerate and improve the rooting percentage of the Goyal AK, Middha SK, Usha T, Chatterjee S, Bothra AK, Nagaveni MB, Sen A. 2010. Bamboo-infoline: a database for north Bengal bamboos. stem cuttings (Kasim and Rayya 2009) while cytokinin Bioinformation 5 (4): 184-185. according to Jonas et al. (2012) is phytohormone that Hartmann HT, Kester DE, Davies FT, Geneve RL. 1997. Plant stimulates water uptake, promote cell division and organ Propagation: Principles and Practices. Prentice Hall International, development and auxin has the role in root initiation. Nasri London, UK. Ibironke OA. 2016. Effects of rooting hormones on the propagation of et al. (2015) revealed that exogenous auxin applications bougainvillea from cuttings. Int J Res Agri Forest 3 (1): 57-62 may have an indirect influence by increasing the Islam MA, Bhuiyan MK, Hossain MM, Hossain MA. 2011. Clonal translocation rates and sugar movement to stem cuttings propagation of Bambusa vulgaris by leafy branch cuttings. J Forest and promote root growth. Res 22 (3): 387-392. Jonáš M, Salaš P, Baltazár T. 2012. Effect of exogenously application Table 4 also showed that the increase in NAA selected phytohormonal substances on the physiological and concentrations up to 100% lead to the decrease in roots morphological indicators of Philadelphus x hybrid in containers. Acta number. It was due to the auxin required had exceeded the Universitatis Agriculturae Et Silviculturae Mendelianae Brunensis 12 optimum concentration to support the root growth. (8): 109-118 Karunarathna B, Harris KD. 2016. Effect of coconut water on the cutting Karunarathna and Haris (2016) reported that auxin induces establishment of Ixora (Ixora cocciniea L.). Int J Adv Res Rev 1 (11): the adventive rooting but at the high concentrations of 27-33. auxin often causes damage. Hartmann et al. (1997) Kasim NE, Rayya A. 2009. Effect of different collection times and some mentioned that the higher concentration of auxins inhibits treatments on rooting and chemical in terminal constituents of bitter almond hardwood cutting. J Agri Biol Sci 5 (2): 116-122. induction and elongation of roots and stimulates plant cells Kaushal R, Gulabrao YA, Tewari SK, Chaturvedi S, Chaturvedi OP. to produce ethylene. Davies (1990) also reported that root 2011. Rooting behaviour and survival of bamboo species propagated growth restriction caused by high concentration of auxin is through branch cuttings. Indian J Soil Conserv 39 (2): 171-175. mediated by auxin-produced ethylene. It is supported by Khudhur SA, Omer TJ. 2015. Effect of NAA and IAA on stem cuttings of Dalbergia. J Biol Life Sci 6 (2): 208-220. the statement of Muller and Leyser (2011) that auxin Muhammad K, Gul Z, Jamal Z, Ahmed M, Asif ur RK, Khan ZU. 2015. applications depend on the type and concentration, auxin Effect of coconut water from different fruit maturity stages, as natural can stimulate rooting, hamper the growth of shoot or substitute for synthetic PGR in vitro potato micropropagation. Intl J produce herbicidal effects. Biosci 6 (2): 84-92. Muller D, Leyser O. 2011. Auxin, cytokinin and the control of shoot The combination of 80% NAA + 20% CW was the best branching. Ann Bot 107: 1203-1212. treatment in increasing the growth of yellow bamboo Nasri F, Fadakar A, Saba MK, Yousefi B. 2015. Study of indole Butyric branching cuttings significantly on the variables of shoot Acid (IBA) effects on cutting rooting improving some of wild emergence time, shoot length, leaf area, root number, and genotypes of Damask roses (Rosa damascena Mill.). J Agri Sci 60 (3): 263-275. root length. The soaking duration did not significantly Pandey A, Tamta S, Giri D. 2011. Role of auxin on adventitious root affect all variables observed, except in the shoot emergence formation and subsequent growth of cutting raised plantlets of Ginkgo time which showed the fastest time with soaking for 36 h. biloba L. Int J Biodvers Conserv 3 (4): 142-146 The effect of interaction between the combination of 80% Rahdari P, Khosroabadi M, Delfani K. 2014. Effect of different concentration of plant hormones (IBA and NAA) on rooting and NAA + 20% CW with soaking duration of 36 h occurred growth factors in root and stem cuttings of Cordyline terminalis. J on shoot emergence time. Med Bioeng 3 (3): 190-194 Raj XJ, Ballabh B, Murugan MP, Dhar P, Tayade AB, Warhgat AR, Chaurasia OP, Srivastava RB. 2013. Eeffect of auxins on adventitious rooting from hardwood cuttings of Hippophae rhamanoides under ACKNOWLEDGEMENTS Ladakh, Himalayas. Indian Forester, 139 (3): 228-231. Razvi S, Nautiyal S, Bakshi M, Bhat JA, Pala NA. 2011. Influence of This research was financially supported by ALG season and phytohormones on rooting behavior of green bamboo by cuttings. Int J Conserv Sci 2 (3): 199-206. (Academic Leadership Grant) of Prof. Johan Iskandar. We Sarmiento AIP, de Souza PVD, Schwarz SF. 2016. Collection season and take this opportunity to express our special appreciation to auxin treatment in the propagation by cuttings of mandarin hybrids. Prof. Tri Hanggono Achmad, a rector of the Padjadjaran Pesq Agropec Trop Goiânia 46 (2): 215-221. University who has supported the ALG program and Tiwari RKS, Das K. 2010. Effect of stem cuttings and hormonal pre- treatment on propagation of Embelia tsjeriam and Caesalpinia encouraged conduct of the research. bonduc, two important medicinal plant species. J Med Plants Res 4 (15): 1577-1583. Wróblewska K. 2015. Benzyladenine effect on rooting and axillary shoot REFERENCES outgrowth of Gaura lindheimeri Engelm. A. 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Yong WHJ, Liya G, Yan Fei N, Swee NT. 2013. The composition of plant USA 3Natural Sciences & Science Education, Nanyang growth regulators in coconut water. Parsons Laboratory, Department Technological University, Nanyang Walk, Singapore. of Civil & Environmental Engineering, MIT, Cambridge, MA 02139,

NUSANTARA BIOSCIENCE ISSN: 2087-3948 Vol. 10, No. 3, pp. 183-192 E-ISSN: 2087-3956 August 2018 DOI: 10.13057/nusbiosci/n100309

Effect of sucrose and plant growth regulators on callogenesis and preliminary secondary metabolic of different explant Myrmecodia tuberosa

YANTI PUSPITA SARI1,♥, EKO KUSUMAWATI1, CHAIRUL SALEH2, WAWAN KUSTIAWAN3, SUKARTINGSIH3 1Department of Biology, Faculty of Mathematics and Natural Sciences, Mulawarman University. Jl. Barong Tongkok No. 4, Kampus Gn. Kelua, Samarinda, 75119, Kalimantan Timur, Indonesia. Tel.: +62-541 747974, Fax: +62-541-747974, ♥email: [email protected] 2Department of Chemistry, Faculty of Mathematics and Natural Sciences, Mulawarman University. Jl. Barong Tongkok No. 4, Kampus Gn. Kelua, Samarinda, 75119, Kalimantan Timur, Indonesia 3Faculty of Forestry, Mulawarman University. Samarinda 75119, East Kalimantan, Indonesia

Manuscript received: 24 July 2018. Revision accepted: 24 August 2018.

Abstract. Sari YP, Kusumawati E, Saleh C, Kustiawan W, Sukartingsih. 2018. Effect of sucrose on callogenesis and preliminary secondary metabolic of different explant (Myrmecodia tuberosa). Nusantara Bioscience 10: 183-192. Myrmecodia tuberosa Jack is a medicinal plant that contains bioactive compounds, such as flavonoids, tannins, tocopherols, phenols, and an abundance of minerals, that are useful as antioxidants. With the constant increases in popularity of the medicinal plant, the M. tuberosa is threatened by extinction if over-exploitation continues. Thus, the effort to conserve this plant is vital. Tissue culture is an alternative method to conserve and produce active compounds that are similar to those of the native ant nest plant with callus. The addition of certain compounds such as sucrose can affect the secondary metabolite content through in vitro plant or callus. The aim of this research was to find the explant sources (cotyledon, stem, tuber, and root), determine the best growth regulator to produce the callus, and evaluate the optimum sucrose concentration to enhance secondary metabolite production of the callus. The results showed that callus was obtained from all explant sources and all growth regulators. The best callus that was marked by a friable green and yellowish green callus was provided by cotyledon with the growth regulator of 2 mg⋅L-1 of 2.4-dichlorophenoxyacetic acid (2.4-D) and 2 mg⋅L-1 of kinetin. Calli treated with 30 g of sucrose resulted in the best secondary metabolites, containing alkaloids, phenols, flavonoids, saponins, and steroids.

Keywords: 2.4-D, 2.4-dichlorophenoxyacetic acid, callus, Myrmecodia tuberosa, kinetin, secondary metabolite, sucrose

INTRODUCTION provide raw material to produce a medicinal compound to achieve great benefits in the pharmaceutical aspect (Radha The ant nest plant (Myrmecodia tuberosa) is a herbal et al. 2011) and to select genotypes that have considerable medicine that contains bioactive compounds, such as amounts of bioactive compound (Cantelmo et al. 2013). glycosides, vitamins, minerals, flavonoids, tocopherols, Generally, organ culture, cell suspension, and callus culture polyphenols, and tannins (Engida et al. 2013; Sanjaya et have been used to study secondary metabolite synthesis al. 2014; Sudiono et al. 2015), which are useful as using the tissue culture method (George and Sherrington antioxidants and anticancer compounds. Several 1984; Smetanska 2008). Massive development through researchers have shown that the ant nest plant inhibits the tissue culture indicates that it does not affect the active growth of various types of cancer cells, such as those in compound content from the plants. ovarian cancer (Hasanuddin et al. 2015), oral tongue To assess biomass accumulation and synthesis of squamous cell carcinoma (OTSCC) (Supriatno 2014), lung secondary compounds in cultures, various strategies have and colon cancer (Manoi 2008; Subroto and Saputro been developed. Biomass accumulation and metabolite 2008), HeLa and MCM-B2 cell cancer (Soeksmanto et al. biosynthesis are two-stage events. The first stage is to 2010). Another report showed that the ant nest plant has control the parameters, including regulation the growth anti-microbial bioactivity and antioxidants and multiplication of both cultured cells/organs and (Prachayasittikul et al. 2008). biomass accumulation, as well as parameters that related The popularity of the ant nest plant as a pharmaceutical to biosynthesis of metabolites which are controlled in the plant is undoubtedly, causing many people to explore next step. Further, to select high-producing cells or organ directly from nature. The over exploration of the clones; optimize medium parameters such as suitable Myrmecodia plant without cultivation might decrease the medium, salt, sugar, nitrogen, phosphate, and plant population. Thus, there is an urgent need to restore the growth regulator levels; and physical factors such as natural population of this plant and use an alternative temperature, illumination, light quality, medium pH, pharmaceutical source without taking it directly from agitation, aeration, and environmental gas (e.g., oxygen, nature. The tissue culture method is an alternative to carbon dioxide, and ethylene) can be controlled in the 184 NUSANTARA BIOSCIENCE 10 (3): 183-192, August 2018 early stage of the culture process. Meanwhile, in the Based on the information above, the purpose of this second stage of the culture process, elicitation, research was to determine the optimum combination of replenishment of nutrient and precursor feeding, growth regulators (2.4-D: 0.5, 1, 1.5, and 2 mg⋅L-1 and permeabilization, and immobilization strategies that assist kinetin 2 mg⋅L-1) and explant source (cotyledon, stem, with the accumulation of metabolites can be applied. In tuber, and root) on callus production in the ant nest plant addition, in line with stage-specific strategies, to produce using an in vitro method. The research was also aimed to huge number of biomass in accordance with an increase evaluate the optimum sucrose concentration to enhance in the accumulation of secondary compounds can be also secondary metabolite production from the callus. possible (Murthy et al. 2014). The successful tissue culture depends on factors such as the medium, growth regulator, explant source, and MATERIALS AND METHODS environment. In addition, the successful tissue culture has been also affected by the types and concentration of the Plant materials growth regulator added in the medium (Shi 2014). Auxin, Seeds of the ant nest plant (Myrmecodia tuberosa L. cytokinin, and gibberellin are growth regulators usually family Rubiaceae) were provided from the ripe fruit, which used in tissue culture (Davies 2010; Coggins and Lovatt were collected from Antutan village, Tanjung Palas 2014; Erland et al. 2017). One of the auxins, 2.4- regency, Bulungan district, North Borneo. The seeds dichlorophenoxyacetic acid (2.4-D) which is a synthetic collection has been granted and permitted by the head of growth regulator, is beneficial to induce callus formation local authority (reference 025/RT-004/ATT/XI/2017) (Welsh and Mogea 1991; Phua et al. 2016). Nevertheless, proper interaction between auxin and cytokinin should be Sample preparation considered to enhance callus growth. Seeds were sterilized using 70% alcohol for 1 minute, Past researchers have stated that the highest weight of followed by 30%, 20%, and 10% natrium hypochlorite -1 callus was obtained from 1.00 mg⋅L of BA in combination (v/v) for 10 minutes each and washed three times for 5 -1 with 0.99 mg⋅L of 2.4-D in Solanum khasianum (Tambe minutes in sterile conditions. For germination, seeds were -1 2013) and 3.0 mg⋅L of 2.4-D in Gardenia latifolia placed in MS medium and agar (7.5 g L-1) with 30 g L-1 (Reddy and Saritha 2012). Meanwhile, Sari and Kusuma sucrose (Murashige and Skoog 1962). (2015) revealed that an ant nest plant callus was formed in solid and liquid Murashige and Skoog (MS) medium with Seed germination -1 -1 2.4-D (2 mg⋅L ) and kinetin (2 mg⋅L ). In addition, a high Sterile seeds of the ant nest plant were grown in glass quality of callus can be produced using specific nutrients bottles, containing MS0 (basal medium without plant and manipulate nutrient components in the culture medium growth regulators), incubated in a culture room with a light (e.g., carbon source, nitrogen, and phosphate) to induce cell intensity of ±1000-2000 Lux with a temperature of 20ºC- growth for effectiveness of secondary metabolite 25ºC. After one month, the seedling plant was used as an production (Tapia et al. 2001). explant source, which was grown in a callus formation Sucrose has a positive effect on the production of medium. The cotyledon, hypocotyl, tuber, and root were secondary metabolites both in cell and organ cultures used as explant sources. (Paiva and Janick 1982; Fowler 1983). The effect of −1 sucrose concentration (i.e. 20, 30, 40 and 60 g L ) was Callus formation investigated in suspension cultures of Panax Cotyledon from a month-old plant was prepared to use notoginseng for production of ginseng saponin (secondary as an explant source. The explant was grown in MS with a metabolite) and polysaccharide (primary metabolite). In growth regulator combination of 2.4-D (0.5, 1, 1.5, and 2 addition, a sugar feeding strategy was formulated to mg⋅L-1) and kinetin (2 mg⋅L-1), incubated in a tissue culture enhance the saponin accumulation by P. notoginseng cells room with a light intensity of ±1000-2000 lux with a (Zhang et al. 1996). Sucrose concentration (5, 7, and 9% temperature of 20ºC-25ºC for 10 weeks. All growth w/v) showed increased accumulation of phenols, regulation concentration was selected as described in flavonoids, chlorogenic acid, and total hypericin in previous experiment (Sari and Kusuma 2015). All adventitious root cultures of Hypericum perforatum L treatment was done in triplicates. (Cui et al. 2010). Sucrose levels below 6-9% did not significantly alter the specific alkaloid content, but biomass Sucrose treatment production by Solanum aviculare hairy roots was The best callus from induction was used in sucrose maximum and about 60% higher than in 3% sucrose treatment. The 0.5 g callus was weighed and placed in the medium when the initial sucrose concentration was 4-6% MS medium that was added with different concentrations (Yu et al. 1996). The suitable medium for both biomass of sucrose (30, 60, 90, and 120 g). After 8 weeks, Brahmi (Bacopa monnieri) (6.31 ± 0.12 g fresh weight secondary metabolite content was determined from each and 250 ± 5.00 mg dry weight) and bacoside A −1 callus. accumulation (13.09 mg g dry wt) found in MS medium supplemented with 2% sucrose and pH set at 4.5 (Naik et al. 2010).

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Secondary metabolite extraction intensity. For the phytochemical test, the presence or The callus was extracted using ethanol as a solvent for absence of phytochemicals such as alkaloids, flavonoids, 48 h. The extraction process continued until the extraction phenols, saponins, triterpenoids, and steroids in the extract solution become clear (± 48 h) followed by filtration using was indicated as positive (presence) or negative (absence). Whatman paper (Whatman 2; Sigma-Aldrich, Germany). Weight of callus which were obtained after various After filtration, a rotary evaporator was used to evaporate concentration of sucrose treatment were analysed by using the remaining solvent. The extracted ethanol was then ANOVA (SPSS 22, Inc., USA), followed by Tukey’s post collected to be used as a sample extract in the hoc test to evaluate any significant differences at p<0.05. phytochemical test to determine the phytochemical contents in the ant nest plant extract. RESULTS AND DISCUSSION Phytochemical test To detect the presence of possible phytochemicals in Seed germination the sample extract, some phytochemicals, such as alkaloids, The seeds of the ant nest plant that were grown in MS0 flavonoids, phenols, saponins, and steroids/triterpenoids, started to grow on day 3, reaching full growth as a plant at were tested, following the methods of Pal et al. (2012). For day 30 (Figure 1). the alkaloid test (Mayer's test), 1 mL of Wagner's reagent Seed germination is a mechanism related to the was added to the extracted sample. The formation of a alteration of both morphological and physiological aspects, white precipitate indicates a positive test. In the flavonoid which is caused by embryo activation. The process of test (Shinoda's test), 5 mL of extract and 1 mL of embryo expansion and elongation is a process that is concentrated hydrochloric acid and magnesium ribbon initiated by absorbing water by the seed before were mixed and shaken. The appearance of a pink-red color germination. The germination process is terminated when indicates the presence of flavonoids. For the phenolic test, the radicle has completely grown out to cover the seed the extracted sample was mixed with a few drops of 1% layers (Hermann et al. 2007). solution of FeCl3. A blue-green or black coloration Seed germination of the ant nest plant using tissue indicates the presence of phenol. In the saponin test (foam culture was affected by two types of factors: internal and test), the extracted sample was added to 10 mL of distilled external factors. The internal factors include the endosperm water, cooled, air dried, and shaken vigorously for 10 s. (food stores) and the level of seed maturity. In agreement The appearance of stable foam (1-3 height) indicates the with the findings by Miransari and Smith (2009), for the presence of saponin. In the steroid/triterpenoid test seed to germinate, the availability of starches, proteins, (Liebermann-Burchard test), CH3COOH glacial and lipids, and nutrients must be completed in the seed embryo absolute H2SO4 were added to the sample extract with a through the activity of specific enzymes and pathways. ratio of 20:1. The appearance of a blue or green color Müller et al. (2013) added that the activity of the enzyme indicates the presence of steroids, and the appearance of a pectin methylesterases is also influenced in seed red or brown color indicates the presence of triterpenoids. germination. Further, the homogalacturonans of the wall, a methyl esterified, are facilitated by the enzyme that affects Data analysis the cell-wall porosity and elasticity, causing cell growth The results were presented as descriptive data. Callus and water uptake. In addition, the expansion of the cell wall induction was observed at the first growth of the callus of the radicle and the tissues occurs during the process of along with the percentage of callus growth, callus seed germination. morphology (texture and color), and callus growth

A B

Figure 1. Seed germination of the ant nest plant (Myrmecodia tuberosa) in MS0 medium. A. 3 days, B. 30 days. Bar = 1 cm

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Besides internal factors, seed germination is also the anion NO3 or the cation NH4. According to Malmgren affected by external factors, such as plant growth (1996), the ammoniated form of nitrogen is more beneficial regulators, light, and medium composition. The successful than the nitrate form, which is proved by the fastest growth seed germination of the ant nest plant in a brief time is of the Dactylorhiza species seedlings at 50-100 mg⋅L−1 influenced by the MS medium composition, which contains NH4NO3. Similar results were found in past research, several amino acids that might boost seed germination. which revealed that an efficient concentration of organic Waes and Debergh (1986) reported that the addition of and inorganic nitrogen sources can be used to promote the amino acids, such as serine, glutamic acid, peptone, growth of explants (Chen and Chang 2002). The 30-day hydrolysate casein, and yeast extract, in the medium induce old ant nest plant was used as an explant source to be seed germination. In comparison with other salt formation, grown in the initial medium treatment. MS inorganic salts have high contents of nitrate, potassium, and ammonium. The MS medium also contains high macro Callus induction and microelements and inorganic salts that are sufficient to Callus induction of cotyledon, stem, tuber, and root support the optimum growth of the plant. In addition, the explants of the ant nest plant was affected by the concentration and quality of nitrogen in the MS medium combination of growth regulators 2.4-D and kinetin (Table contribute to the prolific growth obtained with plant- 1). derived seed types that are incubated in this medium. Thus, the nitrogen supplied in the medium is in inorganic form as

Table 1. Effects of 2.4-D and kinetin composition on the callus induction of the ant nest plant (Myrmecodia tuberosa Jack) from the cotyledon, stem, tuber, and root explants at week 10

Growth regulator Percentage of Callus Callus morphology Callus Explant 2.4-D Kinetin callus formation formation time Color Texture intensity mg⋅L-1 mg⋅L-1 (%) (week) Cotyledon 0.5 0.0 100 2 Yellow Friable ++ 1.0 0.0 100 2 Yellow Friable ++ 1.5 0.0 100 2 Yellowish green Friable ++ 2.0 0.0 100 2 yellowish green Friable ++ 0.5 2.0 100 2 Yellowish green Friable +++ 1.0 2.0 100 2 Yellowish green Friable +++ 1.5 2.0 100 2 Yellowish green Friable +++ 2.0 2.0 100 2 Green-yellowish green Friable +++

Stem 0.5 0.0 100 2 Yellowish green Friable ++ 1.0 0.0 100 2 Yellow Friable ++ 1.5 0.0 100 2 Yellowish green Friable ++ 2.0 0.0 100 2 Yellow Friable ++ 0.5 2.0 100 2 Yellow Friable +++ 1.0 2.0 100 2 Yellowish green Friable +++ 1.5 2.0 100 2 Yellowish green Friable +++ 2.0 2.0 100 2 Yellowish green Friable +++

Tuber 0.5 0.0 100 2 Yellowish green Friable ++ 1.0 0.0 100 2 Yellowish green Friable ++ 1.5 0.0 100 2 Yellowish green Friable ++ 2.0 0.0 100 2 Yellow Friable ++ 0.5 2.0 100 2 Yellowish green Friable +++ 1.0 2.0 100 2 Yellow Friable +++ 1.5 2.0 100 2 Yellow Friable +++ 2.0 2.0 100 2 Yellowish green Friable +++

Root 0.5 0.0 100 2 Yellowish green Friable + 1.0 0.0 100 2 Yellowish green Friable + 1.5 0.0 100 2 Yellowish green Friable ++ 2.0 0.0 100 2 Yellowish green Friable ++ 0.5 2.0 100 2 Yellowish green Friable +++ 1.0 2.0 100 2 Yellowish green Friable +++ 1.5 2.0 100 2 Yellowish green Friable +++ 2.0 2.0 100 2 Yellowish green Friable +++ Note: + = low, ++ = intermediate, +++ = high. Three explants were used for each PGRs combination

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All treatment combinations of growth regulators kinetin resulted in the largest callus of Cephaelis resulted in calli with a percentage of 100%. A callus is a ipecacuanha and Melaleuca alternifolia (Kiong et al. collective form of disorganised cell masses. The formation 2007). Stella and Braga (2002) used a combination of of callus via growth and accumulation of cells has a auxin (picloram) and cytokinin (kinetin) in the callus of relationship with wounding. Further, a single differentiated Rudgea jasminoides. The best callus was found through the cell and other totipotent cells could produce a callus that addition of 2 mg⋅L-1 of 2.4-D and 2 mg⋅L-1 of kinetin in all can regenerate the whole plant body (Nagata and Takebe explants that were used. In addition to the larger callus size, 1971). The callus is formed at 2 weeks of age, marked by the resulting callus had a more intensive green color with explant swelling and callus formation in the edge, slice, or friable callus texture. The large callus size might be due to lesion of the explant. The callus is fully formed at the leaf the corresponding concentration of growth regulators, surface after a month. This is related to the uptake of causing rapid cell division in the ant nest explants. The nutrition in the medium by the explant. The absorption of callus can be provided with two plant hormones, auxin, and nutrients is even better with direct contact between the cytokinin in a balanced condition. Pande et al. (2015) also medium and explant. got highest callus induction frequency on MS media This result was in line with previous research supplemented with 1.5 mg L−1 2, 4-Dichlorophenoxy- performed by Morini et al. (2000), which revealed that the acetic acid, and 1.5 mg L−1 of Benzyl amino purine. callus formation of quince leaves was found only in the abaxial surface part. The appearance of the callus on the Callus color injured part might be because of the excitement of the The results of the observation for 10 weeks after tissue on the explant to cover the wound. Explant response planting found that the combination of growth regulator on the treatment medium is started with explant swelling or 2.4-D and kinetin on the MS medium induced the elongation. The size of the explant becomes bigger from formation of a callus on various explants from the ant nest the beginning, and the callus begins to form on part of the plants (Figure 2). The color of the calli at all treatments injured explant. The combination of auxin (2.4-D) and ranged from yellow and yellowish green to green. The cytokinin (kinetin) as growth regulators on the MS medium resulting color variations might be due to the diverse types can induce callus formation, both in single and of growth regulators, the difference in growth regulator combination treatments. The callus began to appear on the concentration, and the type of explant. Compared to only edge of the explants and on the wounded parts and auxin, a combination of auxin and cytokinin resulted in a continued to grow until the end of the observation at 10 callus color that was more green, caused by cytokinin, weeks after planting. In addition, Stobbe et al. (2002) which tends to promote chlorophyll formation (Edwin F revealed that the wound-induced calli regenerate new George et al. 2008). According to Afshari et al. (2011), organs or new tissues, suggesting that they are highly various callus color conditions could be caused by the pluripotent. pigmentation, the influence of light, and the plant parts In 2.4-D without kinetin, callus growth response in all used as the source explant. explants (cotyledon, stem, tuber, and root) with a small Based on the callus color (Figure 2), the addition of callus needed extended time to grow bigger. This suggests 2 mg⋅L-1 of 2.4-D and 2 mg⋅L-1 of kinetin resulted in that the ant nest plant callus cannot grow optimally with a assorted colors from pale green and yellowish green to single auxin administration without the addition of a green. The green color of the callus of the ant nest plant growth regulator from the cytokine group. According to was because of the chlorophyll content. The chlorophyll Kala et al. (2014), the growth regulators cannot induce a was formed in the callus due to the addition of the growth callus from C. parviflorum leaf explants. However, the regulator 2.4-D and kinetin. Kinetin from the cytokinin combinations of growth regulators resulted in optimum group is involved in the chlorophyll formation in the callus callus production. Callus formation is strongly influenced along with the existence of light. This finding was in line by the type and concentration of the growth regulator. with previous research by Leupin et al. (2000), which Zulfiqar et al. (2009) revealed that the growth and reported that the color change in callus to green was morphogenesis of plants in vitro are controlled by the because of chlorophyll formation. balance and interaction of the growth regulator that was Meanwhile, the yellow to yellowish green callus (Table absorbed from the media. Auxins play a role in stimulating 1) was found because of the kinetin concentrations added the growth of explant cells; thus, auxin tends to form a to the media, which had a lower concentration than 2.4-D. callus that begins from cell division in the meristematic Kinetin as a cytokinin stimulated chlorophyll formation, area. At the beginning of the growth response, auxin whereas auxin can be an inhibitor. According to George triggered the elongation of cells through loosening the and Sherrington (1984), the decrease in the formation of cellulosic cell wall. This elongation of the cell was due to chlorophyll with 2.4-D was found in the culture of peas and the response of 2.4-D, but the cell cannot divide rapidly potatoes. In addition, the yellow color callus was also because there was no addition of kinetin. The combination caused by the chlorophyll degradation process, lack of of the growth regulators, 2.4-D, and kinetin, in all explant kinetin, and low kinetin concentration. In addition, kinetin sources (cotyledons, stems, tubers, and roots) resulted in a plays a role in the formation of chlorophyll, causing the larger callus size compared to only 2.4-D. In line with past green color to appear. research (Rout et al. 2000), the combination of 2.4-D and

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A B

C D

Figure 2. Optimum calli at a concentration of 2 mg⋅L-1 of 2.4-D and 2 mg⋅L-1 of kinetin at 10 weeks after planting in explant. A. Cotyledon, B. Stem, C. Tuber, D. Root. Bar = 1 cm

Callus texture In line with Chakraborty et al. (2013), the MS medium with Table 1 shows friable callus was found in calli with all 2.4-D (0.5 mg⋅L-1) and kinetin (0.2 mg⋅L−1) resulted in the treatments. The friable callus was formed through the best explant Withania somnifera (L.) for callusing with growth of cells at a small size and loose cell interaction that soft, friable, and greenish white features. The addition of was affected by the occurrence of auxin. Previous growth regulators in the medium caused the callus cells to researchers reported that 2.4-D stimulated cell elongation be active in cell division and cell enlargement, raised the by increasing of plasticity of the cell wall to become loose, osmotic pressure, and increased protein synthesis. causing water to easily flow to the inner cell by osmosis, The current visual results indicated that the appearance causing the cell to become elongated (Robbiani et al. of the callus with only the 2.4-D and with the 2.4-D and 2010). Thus, friable calli contain much water because the kinetin combination produced a friable callus and formed wall cell has not reached lignification yet, and the group of nodules (globular). This indicated that the callus can be cells can be easily separated from the others. The callus further grown into shoots or plantlets. Purnamaningsih texture from the explant can be distinguished as friable and (2016) stated that the callus structure usually describes non-friable. The non-friable callus has compact and tight regeneration potency to form buds and roots. The friable cells that are difficult to separate. In contrast, a friable callus has a higher ability to form buds than the compact callus from an explant has loose cell interaction that is callus. In this case, the growth regulator increased callus easily detached using tweezers. regeneration. In addition, the growth of the callus was also The addition of growth regulator with a combination of greatly affected by the nutrient balance. 2.4-D and kinetin on various explants formed a friable texture in the callus (Figure 2). The friable callus was also Sucrose treatment obtained in a study conducted by Sari and Kusuma (2015), The weight gain of the callus was significantly affected which reported that the friable callus was also obtained on by different concentrations of sucrose. The average weight ant nest cotyledons planted in solid and liquid MS media. gain of the callus is shown in Table 2.

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Table 2. Callus morphology and average of weights of the callus produced secondary metabolites is probably due to a of the ant nest plant (Myrmecodia tuberosa) treated with different competition between primary metabolism and secondary concentrations of sucrose for 8 weeks metabolism for the same substance. The M. tuberosa plant is a medicinal plant for which the callus formation in the Sucrose Callus morphology Mean weight of tissue culture also has the same secondary metabolite (g) Color Texture callus (g) content with the original plant in nature. 30 Green Friable 2.12 ± 0.40d 60 Green-yellowish Friable 1.60 ± 0.18c The current results found that the callus color ranged green from green to yellowish green. The callus color indicates 90 Yellowish green Friable 1.15 ± 0.28b the presence of chlorophyll in the tissues. The green callus 120 Yellowish green Friable 0.68 ± 0.69a was present in MS-treated medium with a sucrose Note: Different letters (a, b, c) indicate significantly different concentration of 30 and 60 g L-1. The greener color callus means for different treatments at p < 0.05 using Tukey’s test indicates more chlorophyll content, which can support the growth of the callus. The yellowish green callus was also found in the sucrose treatment with different concentrations: 90 and 120 g⋅L-1. The resulting color Callus morphology variation was caused by the difference in sucrose Callogenesis is the initial response, characterized by the concentration in each treatment. This finding is in formation of the callus, which starts from the edge of the agreement with that of George and Sherrington (1984) in explant (wounded part) at the top and bottom that has direct which sucrose in the tissue culture media can inhibit contact with the medium. The callus is formed faster on the chlorophyll synthesis with varying levels of inhibition, part that has direct contact with the media. This is probably depending on the tissue and plant species. The related to the process of nutrient uptake in the medium by accumulation of sucrose in cells can also inhibit the process the explant. The appearance of the callus on the wounded of photosynthesis because of the accumulation of sucrose part might be caused by the excitement of the tissue on the in the cell, causing the demand of sugar in the cell to be explant to cover the wound. George and Sherrington (1984) fulfilled. As a result, the cells inhibit photosynthesis and stated that the cell division that leads to the callus the formation of chlorophyll. formation occurs from the injuries and both the natural and Growth in tissue culture can be characterized by the artificial hormone supply from the outside into the explant. increase of the wet weight of the callus. Physiologically, Light is an external factor that influences callus the weight of the wet callus consists of water and formation. The color change that exists in the callus was carbohydrates that have a relationship with sucrose in the because of pigment, nutrients, and environmental factors, culture medium as a carbon source and osmotic regulator, such as light (Evans et al. 2003). George and Sherrington which is critical for embryoid and callus formation (Last (1984) stated that white light could induce callus formation and Brettell 1990). Sucrose can be rapidly hydrolyzed to and organogenesis in the plant tissue. A callus that has form glucose and fructose, increasing the osmolality of the yellowish green and green color was formed with the medium. In this study, the sucrose-treated medium entered addition of kinetin. The green color was because of the plant cell through diffusion and osmosis processes. chlorophyll, resulting from the 2.4 D interaction with Shahnewaz and Bari (2004) revealed that the effect of kinetin, mainly because kinetin (cytokinin) has a function sucrose concentration influences the callus induction in the formation of chlorophyll in the callus and due to frequency that might be due to its contribution to the environmental factors, such as exposure to light. Leupin et osmotic potential of the medium instead of its utilization as al. (2000) claimed that the color change in the callus from a carbon source. white to green was due to chlorophyll formation. In cell metabolism, glucose and fructose enter into the glycolysis and Krebs cycle to form ATP to be used for Callus growth callus growth. Perry et al. (1987) revealed the change in The largest mean (2.12 g) of callus weight of the ant -1 solute content (e.g. carbon source) could cause various nest plant was found in sucrose-treated medium at 30 g L change in turgor and osmotic turgor. In tissue culture, (Table 2). However, the sucrose-treated medium leads to -1 turgor pressure might cause elongation and magnification reduced callus weight. In the treatment of 60 g L , sucrose of callus cells. Furthermore, turgor pressure might be decreased the weight of the callus to 1.60 g. This was due different in each cell, and the cell growth response to the to the increasing concentration of medium that inhibited the addition of carbohydrates also varies for each species. absorption of water and minerals. These inhibitory effects -1 were found in the 90 and 120 g L sucrose-treated medium, Phytochemical test resulting in a mean weight of callus of 1.15 and 0.68 g. The phytochemical test results of the callus of the ant Based on the current results, it can be estimated that the -1 nest plant with different concentrations of sucrose are sucrose-treated medium above 30 g L can inhibit the shown in Table 3. growth of the M. tuberosa callus. Lindsey and Yeoman (1983) stated that the inhibition of growth in cultures that

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A B

C D

Figure 3. Callus of the ant nest plant (Myrmecodia tuberosa) with different concentrations of sucrose. A. 30 g⋅L-1, B. 60 g⋅L-1, C. 90 g⋅L-1, D. 120 g⋅L-1 for 8 weeks. Bar = 1 cm

Table 3. Phytochemical screening test of the ant nest plant callus with different sucrose concentrations

Sucrose (g) Flavonoid Phenol Alkaloid Saponin Steroid Triterpenoid 30 ++ ++ ++ ++ ++ - 60 + + + + + - 90 + + + + + - 120 + + + + + -

The phytochemical test on the ant nest plant calli body defense against pest attacks. The compounds are treated with different sucrose concentrations found that utilized by the plants as a self-defense system, while for metabolite content was positive from each treatment, which humans, they are used as an active ingredient in medicine. contained flavonoids, phenols, alkaloids, and steroids, According to Subroto and Saputro (2008), ant nest plants whereas a negative result was found for triterpenoid. The are rich in tocopherol antioxidants (vitamin E) and some best sucrose treatment was obtained with the addition of important minerals (calcium) for the body. 30 g, resulting in a more concentrated test color than the Koes et al. (2005) stated that flavonoids can be others. There was no change in metabolite content between synthesized in all parts of the plant that have a pivotal role the ant nest plant callus from the tissue culture and from in providing color, fragrance, and taste to the fruits, nature. Sari et al. (2017) stated that all parts of the ant nest flowers, and seeds, which makes them attractants for plant (tuber, stem, and leaves) from nature contained insects, birds, or mammals and aids in pollen or seed phenols, flavonoids, saponins, and alkaloids but not transmission. In addition, Blount et al. (1992) stated that triterpenoid. According to Fowler (1983), the tissue culture flavonoids that are very important in plant resistance method can be used to produce chemical compounds that against pathogenic bacteria and fungi also have are usually derived from the native plant as well as new antipathogenic properties that can be non-specific and compounds synthesis, which is not produced by the plant. result, in part, from their antioxidative properties. The secondary metabolites synthesized from the explant Moreover, Beckman (2000) added that flavonoids affect with the tissue culture depend on the culture condition. The the tightening of the plant structures and tissues by proper culture produces secondary metabolites that are stimulating auxin (IAA) activity, which promotes the similar to those of the native plant. Based on the utilization differentiation of tissues and promotes of callus and tylose aspect, ant nest plants have various chemical compounds. formation and the closure of the vascular system to protect The chemical compounds from this plant might have a role against pathogen infection. in the activity of pathogenic resistance, allelopathy, and

SARI et al. – Callogenesis and secondary metabolite of Myrmecodia tuberosa 191

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NUSANTARA BIOSCIENCE ISSN: 2087-3948 Vol. 10, No. 3, pp. 193-202 E-ISSN: 2087-3956 August 2018 DOI: 10.13057/nusbiosci/n100310

Incorporation of dietary palm date pits in all-male Nile tilapia (Oreochromis niloticus) diets

NEDAL MOHAMMED SIDDIG SWAR, ASAAD HASSAN WIDAA MOHAMED College of Graduate Studies, Sudan University of Science and Technology. Khartoum, Sudan. email: [email protected], [email protected]

Manuscript received: 4 July 2018. Revision accepted: 31 August 2018.

Abstract. Swar NMS, Mohamed AHW. 2018. Incorporation of dietary palm date seeds in all-male Nile tilapia (Oreochromis niloticus) diets. Nusantara Bioscience 10: 193-202. The study utilizes a 45-days randomized factorial design 3×2, three levels (25, 50 and 75%) of palm date seeds, three levels (non, with and without) of 3% bakery yeast (Saccharomyces cerevisiae) and three replicates. The observation was carried out in twenty-one plastic aquaria. All-male Nile tilapia (Oreochromis niloticus) fingerlings were put in each aquarium. Each aquarium was provided with well-aerated and triggered dechlorinated tap water with an average weight of 1.9±1.11 g/fish (10 fish/aquaria) and a total length of 5.18±0.69 cm/fish. Fish were given food three times/day (10 days, at 8.30,11.30, 3.30 A.M) at a rate of 12, 8 and 4% of body weight, to examine the impact of partial substitution of animal protein (fish meal) in the diet on growth achievement, carcass contexture, feed utilization, condition factor (k) and feed expense. Seven experimental diets were prepared; control diet (T0, CP 36.84) concluding 45% (fish meal) as animal protein and 0% (palm date seed meal) plant protein. Tested diets (T1, T2 and T3) concluding 25, 50, 75% with 3% bakery yeast (CP 35.71, 35.53, 34.74) and 25, 50, 75% without yeast (CP36.01, 36.27 and 35.57) respectively ; so, they substitute about 75, 50, and 25 of fish meal diet respectively. The outcomes showed that, tilapia were given food on T1, T2 and T4 diets (25, 50, and 25%) substitution with and without yeast respectively registered the greater growth achievement, feed and protein utilization than other experimental diets such as control diet, also they indicate the highest condition factor (K) grades which state that the fish are in decent health. Tilapia were given food by diet T3 (25% fish meal) T5, T6 (50 and 25% fish meal) with and without yeast respectively possessed deficient growth and diverged remarkably (p>0.05) from the other diets. Diet contexture remarkably had an impact on carcass contexture. These data prompted that Palm date seed with and without yeast S. cerevisiae can, to a certain extent, substitute fish meal (animal protein) in a diet for all-male Nile tilapia fingerlings at level-up to 50 and 25% with and without yeast respectively, without any unfavorable result on accretion achievement. Furthermore, fish diet, partially substituted with 3% yeast, resulted from superior accretion achievement than other diets within the present experimental circumstance. This study showed that there is an economic efficiency of confounding palm date seed (plant protein) as partial substitution of fish meal (animal protein) with and without yeast in all-male Nile tilapia, that it could decrease the expense of feeds.

Keywords: Date palm, diet, Nile tilapia, Oreochromis niloticus

INTRODUCTION poultry (El Hag et al. 1993; Tag El-Din and Nour 1993; Elgasim et al. 1995; El Hag and El Khanjari 2000; Yagoub Feed fabrication is the primary problem faced by and Elemam 2011; Sulaiman 2014), furthermore, for rats aquaculture implementation. Today, dietary gives (Vandepopuliere et al. 1995; Hussein et al. 1998; Ali et al. contribution of about 60-85% of the element of operating 1999), and for fish such as mullet, carp and tilapia species expense in NRC fish farm (1993), hence the potential (Yousif et al. 1996a; Belal and Al-Owafeir 2004). Wu utilization of low-cost uncommon autochthonous (2003) stated that approximately, 50-70% of the accretion feedstuffs, such as herbs protein and their extracts has been of tilapia in ponds is caused by natural food mesh, while expanding, There were many issued researches that have the rest derives from either supplementary, completely ensured that the supplementation of herbs or their extracts commercial or therapeutic feed, depending on the system (e.g aquatic plant, cottonseed, groundnut, sesame cake, etc) of aquaculture. the habit of fish in feeding can alter when in the feed holds a advantageous consequence in boosting there is an alteration in the environment. Yosif (2004) accretion parameters and prevention from infections in claimed that we can artificially alter the habits in the aquaculture (Temesgen 2004; Shalaby 2004; Sasmal et al. feeding of the fishes and take benefit of them if we can 2005; Johnson and Banerji 2007; Ebrahim et al. 2007; Abd comprehend the biological, physiological characteristics of el Hakim et al. 2008; Tartiel et al. 2008; Khan et al. 2013; accretion and digestion of fish, the patterns of feeding and Labib et al. 2015). The great value of Nitrogen Free Extract the nutritional requirement of the fishes. (NFE) in date pits has drawn the awareness of many The main methods of food combinations are to consider researchers to examine its probable usage in animals meal, the local circumstances, to use the locally obtainable with propitious outcomes. The inclusion of date pits into materials, to perform coalesced usage, and to do the best in dietary remarkably increased the accretion of and feed thriving food founts. The selection of crude stuff for diet utilization on sheep, calves and Sudanese desert lambs and should be done to expand and make use of economic and 194 NUSANTARA BIOSCIENCE 10 (3): 193-202, August 2018 sustainable crude stuff to keep comparative formula. Crude factory). All diets were formulated with commonly used stuff granularity must be fine enough to make sure ingredients such as fish meal, groundnut cake, wheat bran, convenient digestibility and absorption. Appropriate bread flour, starch, yeast, mineral mix and vegetable oil, manufacturing provision must be elected and plausible and they were presented in Table 4. The experiments of manufacturing techniques must be utilized to ensure the this research were performed in the Freshwater Fisheries similarity and the quality of the diet. The granularity of Laboratory and Hatchery, Sudan University of Science and crushed stuff of feed should be fine (100<40 mesh, 90<60 Technology, Khartoum. It was conducted for one month mesh) (Wu 2003). and a half, from 19 August to 2 October 2015. Manufactured diet for fish must be in the shape of The date seeds were washed and immersed in tap water pellet, granule, crumbles or sticky paste. Fish' habitat is in for three days and got dried. Some were ground in the water so the diet requires proper steadiness in water after home blender, while the others were ground by being applied that it could stand for at least 20 to 30 professional coffee grinder in the central Khartoum market. minutes without becoming disorganized, dissolute and lost. Chemical Analysis of date seeds were shown in Table 1. Wu (2003) stated that the manufactured diet should not The additional additive yeast was added in the make pollution in the environment (Zero-pollution experimental diets with a rate of 3 gram (by substituting 2g deliverance is needed). Many types of research were of starch) for diets T1, T2, T3 respectively, in dried form. performed worldwide to examine palm date seeds as The chemical analysis of the yeast was shown in Table 2. replacement for fish food in Nile tilapia (Oreochromis The chemical composition of fish meal was shown in Table niloticus) diet, but none of them was done in Sudan. For 3. that reason, this research was performed in the Fisheries Laboratory and hatchery of the Sudan University Science Method of preparation and Technology to appraise the accretion achievement, Dry stuffs were shown in Table 4 and their percentages carcass contexture, feed utilization, condition factor (k) and in the mixture were shown in Table 5. After being ground, feed expense of all-male Nile tilapia fingerlings, the feed first of all, they were sifted into very fine particles, then with various levels of date seeds, as partial substitution of they were weighed by using electrical balance (Model: fish protein, such as 25, 50, and 75%; 75, 50, and 25% with 2003, Max: 200 g, cl: 0.001 g, AC: 220W/50HZ, S/N: 11 g, and without bakery yeast (Saccharomyces cerevisiae) (with SF: 400). Based on the prearranged mixing, all dried stuff the aim of making the date seeds fiber easily absorbed by was mixed well for homogeneity and the vegetable oil, tilapia fingerlings) respectively, and also to maximize the vitamins and mineral were supplemented to that mixture. profits from the sectional stuffs which are considered as Tap water was boiled and additional starch (10 g/100 g) by-products and may caused socio-economic and were supplemented to make a gelatinized binder. The water environmental difficulties in order to lower the expense of was added more (depend on the ingredient) to have a feeds. preferential paste with about 10% moisture content. Pellets The purposes of this research were (i) to inquire the were produced by manual meat grinder with 0.6 mm- impact of the coalescence of date palm seeds levels (25, 50 diameter and later were dried in the lab for 24 h and and 75%) with and without yeast respectively in all-male subsequently broken into crumbled form and each diet was Nile tilapia feed in the accretion achievement, carcass packed in a plastic bag and kept under the ambient room contexture, feed utilization, condition factor (K) and feed temperature until the experiment started. The samples of expense. If it results well it can motivate scientists and each diet were taken for the chemical analysis and the researchers to do innovative studies; to look up for new results were shown in Table 4. fonts to lessen the feed expense; to enhance a new taste for cultured species; to accomplish the problems brought about by by-products; to improve fish dietary and fish farming RESULTS AND DISCUSSION profit in Sudan and thus it will improve food stability, (ii) to inquire the impact of coalescence of date palm seeds Experimental fish levels (25, 50 and 75%) with and without yeast respectively All-Male fingerlings of Nile tilapia were purchased in all-male Nile tilapia dietary with the consideration on from Aljwariss private farm (south Jebel Awliaa dam) with total length and water quality parameters during the around 2 months old in average and average weight of 0.5- experiments. 2.0 g (these fish were kept in a pool with high density and fed for only to keep them alive and not to gain weight until they were sold). They were about 500 fingerlings and kept MATERIALS AND METHODS in oxygenated plastic bag. They were acclimatized for half an hour in the surface of the plastic containers filled by tap Experimental diets water and then those fish were kept in plastic containers The stuff for the diets was gathered from the local (aquarium) in fisheries laboratory and fed on a basal diet market (Koko market). Seven investigational diets were for 18 days (from 1-18 August) for the purpose of formalized with different levels of fresh wet date seeds adaptation and once the adaptation period was finished, (MeshriK Wad Lagai) 0, 25, 50, and 75% for diets T0, T1, some initial samples were taken to the laboratory of the T2, T3, T4, T5 and T6 respectively in which the date seeds institution research center of animal resources (Soba) for the came from Abu Alama date packaging factory (a local chemical analysis, and the result was presented in Table 6.

SWAR & MOHAMED – Dietary palm date pits in male nile tilapia diets 195

Experimental design Table 3. Chemical composition (% DM) of fish meal The fish (10/Aquaria) were disparted randomly to 21 of 3 36 L plastic containers (36×34×31 cm ) (aquarium). The Type Moisture DM CP% EE% CF% Ash NFE containers were filled with precipitated tap water (in the Fish meal 7.85 92.15 61.29 8.15 0.6 9.5 12.61 hatchery). Fish were bulk weighed, and their initial weights Note: DM: Dry Matter. CP: Crude Protein. EE: Either extract. CF: and total length (from the tip of the mouth till the end of Crude Fiber. NFE: Nitrogen-free extract the caudal fin) were recorded for random sample. Using Digital scale (electronic kitchen, auto zero, power 1.5v*2.AA battery, 5000g*1g/177oz*0.10z) and ruler respectively, the results were as follows, the average weight was1.9±1.11g, average length was 4.9± 0.77cm. Table 5. Nutritional Percentage: (2×3×3₊3 (control) Factorial =21

Table 1. Chemical composition of date seeds (% DM) Plant protein Date seeds%+Yeast (g) Total N-Ex-D

Type Moisture DM CP% EE% CF% Ash NFE D-P with (group A) 25+ 3g 50+3g 75+3g 3 3 Date seeds 1.5 98.5 6.27 2.9 3.5 2 83.83 D-P without (group B) 25 50 75 3 3 Gross 398.609 Control (non) 0 0 0 1 1 energy/kcal/100g Animal protein% 75 50 25 - - Digestible 225.11 Total - - - 7 7 energy/kcal/100g Note: D-P-with-Y : Date seeds with yeast. D-P-without-Y: Date Note: Nitrogen-free extract (NFE) = 100- (moisture + crude seeds without yeast. N-Ex-D : Number of experiments diet protein + crude fat + crude fiber + ash). Gross energy (kcal/100g), based on 5.7 kcal/g protein, 9.5 kcal/g lipid, and 4.0 kcal/g carbohydrate. Digestible energy (kcal/100g), based on 5.0 kcal/g protein,9.0 kcal/g lipid, 2.0 kcal/g carbohydrate. DM: Dry Matter. Table 6. Carcass composition (% DM) of all-male Nile tilapia CP: Crude Protein. EE: Either extract. CF: Crude Fiber

Table 2. Chemical composition (% DM) of yeast Type DM Moisture CP EE ASH NFE

Type Name Moisture DM CP EE CF Ash NFE Caracas 78 22 17.35 6.125 1.325 53.2 G/E (kcal/100 g) 369.8825 S. cerevisiae Bakery- 9.2 90.8 51.5 6.3 1.8 6.8 24.4 Note: DM: Dry Matter, CP: Crude Protein, EE: Either extract, CF: yeast Crude Fiber, NFE: Nitrogen-free extract Note: DM: Dry Matter, CP: Crude Protein, EE: Either extract, CF: Crude Fiber, NFE: Nitrogen-free extract

Table 4. The stuff and chemical composition of the experimental diets/100g

Treatments Ingredient Control With (group A) Without (group B) 0% 25% 50% 75% 25% 50% 75% Fish-meal (60 cp) 45 33.75 22.5 11.25 33.75 22.5 11.25 Date-seeds (6.27 cp) 0 11.25 22.5 33.75 11.25 22.5 33.75 Groundnut cake (34.5 CP) 21 21 21 21 21 21 21 Wheat bran (15.7 CP) 10 10 10 10 10 10 10 Bread flour 10 10 10 10 10 10 10 Starch 5 2 2 2 5 5 5 Yeast 0 3 3 3 0 0 0 Mineral mix 4 4 4 4 4 4 4 Vegetable oil 5 5 5 5 5 5 5

Chemical composition (% DM) Moisture 6.5 8.5 6.0 12.5 7.5 6.0 6.50 DM 93.5 91.5 94.0 87.5 92.5 94.0 93.5 CP 36.84 35.71 35.53 34.74 36.01 36.27 35.57 EE 3.85 3.55 3.45 3.40 3.95 3.70 3.60 CF 3.03 2.97 2.96 2.90 2.80 2.90 2.92 Ash 15.0 13.5 8.50 12.50 9.50 7.50 9.50 NFE 34.79 35.78 43.57 33.97 40.24 43.64 41.92 G/Ekcal/100g 385.70 380.34 409.53 366.18 403.74 416.40 404.60 D/Ekcal/100g 288.42 282.025 295.805 272.23 296.08 301.90 294.10 P Energy 54.44 53.51 49.45 54.08 50.84 49.65 50.11 ERE 1.883 2.71 1.61 2.17 3.81 4.86 2.16 DM: Dry Matter, CP: Crude Protein, EE: Ether Extract, NFE: Nitrogen free Extract’s\E: Gross Energy, D/E: Digestible Energy. Protein Efficiency Ratio (PER) = wet weight gain (g)/Amount of protein given (g). Protein productive value (PPV%) = (P2-PI) ×100/Protein intake (g) where: P2: Protein content in fish carcass at the end; P1: Protein content at the start. Protein-energy = (energy in protein/gross energy) X 100. Energy retention efficiency ERE (EU%) = [Final body energy (Kcal)-Initial body energy (Kcal) ÷ Dietary energy consumption (Kcal)] X 100

196 NUSANTARA BIOSCIENCE 10 (3): 193-202, August 2018

The fish in each aquarium were fed on the experimental P2 = Protein content in fish carcass at the end; diets according to the feeding control, three times/day (at PI = Protein content at the start. 8.30-11.30-3.30 o’clock). On first 20 days the fish were fed with a ratio of 12% of body weight, and then it was Energy retention efficiency reduced to 8% in third 10 days and into 4% in the last 10 ERE (EU%) = (E2-EI) ×100/Energy intake (kcal) days. The daily nourishing quota was adjusted every 10 days and also the bulk fish weighing was done every 10 Where: days with a sampling method. At the end of the diet ET = Energy in fish carcass (kcal) at the end experiment, each fish were weighed individually with an EI = Energy in fish carcass (kcal) at the start electrical balance. The total length was measured on some Energy retention efficiency (%) = [final body energy sample randomly, and some fish were taken as a sample (Kcal)-initial body energy (Kcal)/dietary energy from each treatment and kept in the freezer for the consumption (Kcal)] X 100 chemical analysis. All the dirt found inside plastic containers were siphoned twice daily before feeding time to Statistical analysis prevent accumulation of fecal matter and waste (since Feedstuff, experimental feeds and carcasses were accessories and personal hygienic were taken good care of, examined for proximate composition following the no disease appeared during the experiment), also the guidance of AOAC (2003). Statistical analyses of data containers were brushed and cleaned weekly based on their were done by SPSS (version 15) 2007 (general model cleanliness conditions. Approximately 10% (2-3 litter at multivariate). To evaluate specific differences between one time) of fresh water was added to restore the water treatment means at (p>0.05), Duncan’s multiple range tests volume and also the containers were drained at each were applied. To describe the data in figures, the excel sampling and refilled again with completely new water. On sheet was used. the day of sampling, no feed was provided for fish. At this time, all plastic containers were equipped with aerations nets using an air blower. Water quality parameters such as RESULTS AND DISCUSSION oxygen, temperature, total ammonia, nitrites and nitrates, and pH were measured by using a dissolved oxygen meter Growth performance (Model: DO-5509), manual thermometer, kits, Eco Tester Results in Table 7 indicated that the overall mean ± SD PH1 (Range 0.0-14.0, power 4*1.5”VA76” micro alkaline of the final weight, (WG%), (WG g/fish), (DWG g/fish), batteries) respectively (water sample was stored in freezer (RWG) and (SGR) of all-male Nile tilapia fed on diets with till the end of the trial). Each sampling was measured. partially substituted by palm date seeds with and without yeast escalated remarkably (p>0.05) with escalating of date Growth performance parameters palm seeds percentages (with yeast) in T1 and T2, and also Weight gain (WG%) = W2/W1x100 escalated remarkably (p>0.05) with increasing of date palm Average daily gain (ADG) (g/fish day-1) = total gain/ seeds substitution in T4 (without yeast). However, the duration period (days). greatest values of final weight (36.15±5.22), WG% Average daily feed intake (ADF/IN) = Total feed (193.60±25.26), WG (g/fish (17.48±4.2), WG (g) intake/duration period. 4-Specific growth rate (SGR)% (0.39±0.11), RWG% (93.60±25.26) and SGR% (1.45± day-1) = 100 × (Ln W2-Ln WI)/n. 0.30) were achieved by fish fed on T2, and the greatest values of final body weight (g) (34.76± 6.52), PWG% Where: (181.42±22.76), PWG (g) (15.76± 4.86), DWG (g) (0.35± Ln: Natural logarithms, n: is the duration period 0.13), RWG% (81.42± 22.76) and SGR (1.31 ± 0.29) were achieved by fish fed on T4. Relative weight gain (RWG%) = Weight gain/initial weight ×100 The increment weight Survival Rate (%) = 100× (Final number of fish/initial Results in Table 8 indicated that the result of mean ± number of fish) 7-condition factor (K)= 100×W/L3 SD of increased weight of All-Male Nile tilapia fed on the experimental diet control, T1, T2%, T3%, and T4%,T5%, Where: T6%/days increase in control (18.76±1.28 to 39.03± 1.79), W = Weight of the fish in gram in diets contain palm date seeds with T1 (16.67± 1.28 to L = Total length of fish in centimeter. 33.17±2.5), T2 (18.67± 1.74 to 36.15± 5.2) and T4 (19.00± 1.67 to 34.76± 6.52) without yeast, but this result decrease Feeds efficiency in T3 (19.67± 0.97 to 23.41± 13.42), T5 (22.33± 0.48 to Feed conversion ratio (FCR) = dry matter intake 27.92± 13.24) and T6 (21.33± 1.74 to 18.02± 4.66). (g)/total gain (%) Protein efficiency ratio (PER) = total gain (g)/protein Particular accretion rate (%) intake (g) Results in Table 9 indicated that the mean ± SD of Protein productive value (PPV%) = (P2-PI) ×100/ particular accretion rate (%) of all-male Nile tilapia fed on protein intake (g) the experimental diet control, T1, T2%, T3%, and T4%,

Where: T5%, T6%/days escalate with no coalesced palm date seeds

SWAR & MOHAMED – Dietary palm date pits in male nile tilapia diets 197 in the diet, with and without yeast in the control showed that the nutritive value of date seed and their (2.78±19.41 to 3.61±8.10), T1 (2.80±4.35 to 3.49±15.07) , hormone-accretion content are more appropriate for fish T2 (2.79±13.71 to 3.16±75.52) with yeast and T4 nourishment. (2.67±13.57 to 3.14±49.41), T5 (2.67±8.59 to 3.31±19.02) and T6 (2.74±4.92 to3.33±51.10) without yeast, but this Feed efficiency result was lessened in T3 (2.93±18.08 to 2.90±108.75), but Results in Table 11 showed that the mean ± SD of meal overall there is no remarkable discrepancy (p>0.05). This intake, feed efficiency, food conversion ratio, protein means that all-male Nile tilapia grow normally when efficiency ratio, protein productive value, condition factor various levels of palm date seeds coalesced in the diets. (K%) and survival rate. The smaller remarkable rate of meal intake (the death fish did not lower the mortality rate, The weight of group/individual (g) so it was excluded) was 84.27± 7.9 and 81.10± 10.8 which Result in Table 10 showed that the mean ± SD of final was obtained by fish fed on diet T1 (which gave low FCR) weight \ individual (g/fish) of All-Male Nile tilapia groups and T6 (which gave high FCR). The mean rates of PER control (Non) (4.17±1.61), group A (3.82±1.57) and group increased in T0, T1, T5 and T6 significantly (p>0.05) as B (4.03±1.76) are not remarkably dissimilar (p>0.05) and the increase of palm date seed substitution. the mean weight/fish for 45 days was 4.00 g. These result

Table 7. Growth performance of all-male Nile tilapia fingerlings fed on the experimental diets (g/fish)

Treatments Items Non With Without Sig T0 (0.0%) T1 (25%) T2 (50%) T3 (75%) T4 (25%) T5 (50%) T6 (75%) Initial weight 19.00±1.11 0.23 Final weight** 39.03± 1.79 33.17±2.5 36.15± 5.22 23.41± 13.42 34.76± 6.52 27.92± 13.24 18.02± 4.66 0.00 WG (%) 209.45±6.98a 199.12±6.82b 193.60±25.26b 122.30±85.92e 181.42±22.76c 149.70 ±68.78d 113. 88± 22.72e 0.01 WG (g/fish) 20.37±0.69a 16.51±1.43b 17.48± 4.20b 3.74± 14.37e 15.76± 4.86c 5.59± 12.91d 3.32± 4.09e 0.00 DWG (g) 0.45± 0.02a 0.37± 0.04b 0.39± 0.11b 0.08±0.38d 0.35± 0.13b 0. 25± 0.34c 0.07± 0.11d 0.22 RWG (%) 109.45± 7.07a 99.12± 6.82b 93.60± 25.26b 22.30± 85.92d 81.42± 22.76c 24.24± 68.78d 15.92±22.72e 0.01 SGR 1.64± 0.07a 1.53± 0.08a 1.45± 0.30b 0.24± 2.46d 1.31 ± 0.29b 0.87 ± 1.62c 0.28± 0.57d 0.05 Note: Data are represented as mean of three samples replicates ± standard error. Means in the same row with the same letter are not remarkably different (P>0.05)

Table 8. The increment weight of all-male Nile tilapia (g/fish) as affected with date pit incorporation in diets/days

Weight of fish (g) at different times Diets 0 time 11 days 21 days 31 days 41days Increment (g) To (0.0%) 18.67±1.28d 24.00±2.21c 32.00±1.45b 39.00±3.02a 39.03±1.79a 20.36±1.95 T1 (25%) 16.67±1.28d 24.00±0.84c 28.00±6.32b 32.67±4.61a 33.17±2.5a 16.50±3.11 T2 (50%) 18.67±1.74d 24.33±0.48c 28.67±1.28b 33.67±3.95a 36.15±5.22a 17.48±2.53 T3 (75%) 19.67±0.97c 28.33±1.74a 24.67±7.12b 22.00±12.88b 23.41±13.42 3.74±7.20 T4 (25%) 19.00±1.67c 26.67±5.04b 29.00±5.49b 32,00±6.03a 34.67±6.52a 15.67±4.10 T5 (50%) 22.33±0.48c 31.00±5.09a 28.33±7.21b 25.67±9.98c 27.92±13.24b 5.59±6.86 T6 (75%) 21.33±1.74b 24.00±2.90a 21.33±5.44b 22.33±5.44a 18.02±4.66c 3.31±3.20 Note: Data are represented as mean of three samples replicates ± standard error. Means in the same row with the same letter are not significantly different (P>0.05)

Table 11. Growth and food utilization efficiency of all-male Nile tilapia fingerlings fed on the experimental diets

Treatment Item Non With Without Sig T0 (0.0%) T1 (25%) T2 (50%) T3 (75%) T4 (25%) T5 (50%) T6 (75%) Feed intake 92.40± 5.72a 84.27± 7.9d 88.00± 4.1b 89.47± 7.1b 90.80± 14.7a 96.76± 15.7a 81.10± 10.8d 0.10 FEED/EFF 0.22±0.02a 0.20±0.2a 0.20±0.0 5a 0.035±0.19d 0.17±0.34b 0.11 ±0.17c 0.04±0.60d 0. 02 FCR 0.44± 0.04a 0.42± 0.06a 0.46± 0.06a 1.48± 1.64d 0.49± 0.04a 0.65 ± 0.60c 0.71± 0.29c 0.18 P.E Ratio 1.67a 1.43a 1.48a 0.35d 1.31b 0.46c 0.28d - PPV 39.24a 38.60b 37.29c 37.13d 37.63c 38.59b 38.52b - K (%) 14.68a 12.95a 13.16a 9.87d 11.66b 11.38c 8.86e - Survival rate (%) 93.33a 90.00a 86.67b 66.67c 86.67b 63.33c 50.0d - Note: Data are represented as mean of three samples replicates ± standard error; Means in the same row with the same letter are not significantly different (P>0.05)

198 NUSANTARA BIOSCIENCE 10 (3): 193-202, August 2018

Table 9. Particular accretion rate of all-male Nile tilapia (g/fish) fed on the diet containing 50% date seeds substitution, as affected with date seed coalescence in diets/days lower body dry matter was yielded. On the fish fed on the diet containing 25% and 50% date seeds substitution with Weight of fish (g) at different times Diets and without yeast, the highest significant (p>0.05) in 0-11 days 11-21 days 21-31 days 31-41 days protein body content (31.15 and 31.35) respectively was To (0.0%) 2.78±19.41 3.33±9.36 3.22±7.96 3.61±8.10 produced. The lowest significant (p>0.05) fat (6.40 And T1 (25%) 2.80±4.35 3.29±9.91 3.19±19.02 3.49±15.07 T2 (50%) 2.79±13.71 3.20±31.93 2.89±61.13 3.16±75.52 6.55) was presented in fish fed on the diet with 75% T3 (75%) 2.93±18.08 3.00±41.32 2.50±101.40 2.90±108.75 substitution with and without yeast, and the lowest gross T4 (25%) 2.67±13.57 3.06±42.32 2.91±39.10 3.14±49.41 energy body contents (376.47 and 377.825) were found in T5 (50%) 2.67±8.59 3.27±17.53 3.94±19.61 3.31±19.02 fish fed on diets with 50% and 75% substitution with yeast. T6 (75%) 2.74±4.92 3.27±4.06 3.18±14.47 3.33±51.10 The highest number of ash body contents in the dry matter Sig 0.29 0.64 0.57 0.79 (2.40 and 2.20) were found in fish fed on diet containing Note: Data are represented as mean of three samples replicates ± 25% and 75% date seeds substitution with yeast. standard error. Means in the same row with the same letter are not significantly different (P>0.05) The water quality

The mean ± SD values of water quality parameters

Table 10. The weight of all-male Nile tilapia group/individual (g) assessed in the nurturing plastic containers (aquarium) are summed up in Table 14 The mean water temperature was o Control Group A (with Group B 27.55 C±0.82). This temperature has been informed as of Treatment (Non) yeast) (without east) the optimum range for tilapia growth and yield (Meske 4.17±1.61a 3.82±1.57a 4.03±1.76a 1985). The mean dissolved oxygen was 5.81ppm± 2.21. Sig 0.57 Siddiqui et al. (1989) claimed that tilapia demands a low Note: Data are represented as mean of three samples replicates ± oxygen and can stay alive at low oxygen values. Riche and standard error. Means in the same row with the same letter are not Garling (2003) informed that dissolved oxygen values significantly different (P>0.05) should be kept above 5.0 ppm for optimum accretion. The pH of water influences a lot of water quality parameters and the rates of many biological and chemical processes. However, the diets T1, T2 and T4 substitution with and Thus, pH is determined as significant parameters to be without yeast yielded a higher rate of FCR respectively inspected and managed in the aquaculture system (Losordo (0.42± 0.06, 0.46 ± 0.06 and 0.49±0.04) and the higher rate et al. 1998). of PER (1.43, 1.48 and 1.31) in T1, T2 and T4 palm date In this research, the mean value of pH was 7.64 ±0.36. seeds; T1 and T5 substitution yielded higher rate of PPV Pompa and Masser (1999) informed that tilapia can stay for the palm date seed (with and without yeast) respectively alive at pH ranging from 5 to 10 but they perform optimum (38.65 and 38.59). The greatest rate (p>0.05)of K was at pH 6 to 9. Ammonia and Nitrite are a problem in (14.68, 13.16, 12.95 and 11.66) in T0, T2, T1 and T4 aquaculture systems and should be observed continually. respectively. The greatest survival value was in T0, T1, T2 Ammonia establishment is shortly had to do with and T4. nourishing and relies on the grade of meal, nourishing rate, fish size and temperature (Riche and Garling, 2003). In this Feeding rate (regime%) research, ammonia (NH3) was 0.33±0.43 (optimum rate Results in Table 12 showed the mean ±SD of feeding was 0.2 ppm), nitrites (NO2) was 0.24±1.24 (optimum rate administration% for hormone-treated all-male Nile tilapia was 0.3 mg/l) and nitrate (NO3) was 0.99±1.34 (optimum fed on the experimental diets. The decrease in feeding rate was 200-300 ppm). The concentration in the nurturing intake caused the decrease of feed rate in the sample, and containers was highly significant (p>0.05) but was not the initial highest feed intake was in T5 (26.80±0.58), T6 within the safe range for tilapia culture. Accretion (25.60±2.09) and T3 (23.60±1.16). Although they indicated achievement parameters for tilapia fingerlings after the a lower percentage at the final feeding administration, nourishing experiments are presented in Table 7. It is namely 10.00±4.40, 9.60±1.46 and 8.80±5.15, respectively. obvious that there is a notable impact on experimental diets The differences occurred here were due to electricity on accretion achievement of the experimental fish. problem which led to the loss of their appetite and, in the end, to the mortality of them. The mortality rate was higher Length during experiment than that in the rest diets T1 (20.00±1.53), T2 Results in Table 15 exhibit that mean ± SD of the initial (22.40±2.09), T4 (22.80±2.01) and control (22.40±1.53). length (cm) and final length (cm) of all-male Nile tilapia fed on control, 25%, 50%, 75%, 25%, 50% and 75% diets Body composition showed no significant differences (P>0.05). The mean± SD of the chemical contexture of the entire fish body as influenced by partial substitution at the end of The economic evaluation the experimental period is described in Table 13. In all The outcome in Table 16 showed that the costs of groups involving date seeds diets, fish body dry matter and experimental diets based on the diet substances in the local protein content became better than that in control diet market during the experiment, the diet substances, price which has high protein and lower dry matter. On the fish (Sudanese Currency/kg), handlings, food conversion ratio

SWAR & MOHAMED – Dietary palm date pits in male nile tilapia diets 199 and the total price (Sudanese currency/kg). The lower accretion achievement and nutrient utilization. This is in significance price (p>0.05) was shown (3.38, 3.70 and agreement with so many works. Mabrouk et al. (2011) 3.95) in the 25%, 50% date seeds substitution with yeast resumed that wet cull date may be expense-effective when and 25% without yeast respectively. The highest price was partially substitutes yellow corn in tilapia meals as energy 11.91 and 5.72 in 75% date seeds substitution (with and fount, and enhances fish performances when they were without yeast) respectively; in comparison to the control, added with 0.03% digestion. Khadr (2006) informed that i.e., 3.54. There is no remarkable difference (p>0.05). This tilapia fed by diet bearing 75% date seeds revealed almost- outcome showed that the incorporation of date seeds 25%, economical outcomes in comparison with control. This 50% and 25% as the partial substitution of fish meal yield result was 25%, 50% and 25% as mentioned above. better economical and efficiency outcomes as well as better

Table 12. Feeding rate (regime%) intake by all-male Nile tilapia/sample

Feed intake (%) at different sample Diets 0 (12%) 1 (12%) 2 (8%) 3 (4%) Control (0%) 22.40±1.53 28.80±2.66 25.60±1.16 15.60±1.21 T1 (25%) 20.00±1.53 28.80±1.00 22.40±5.05 13.07±1.84 T2 (50%) 22.40±2.09 29.20±0.58 22.93±1.02 13.47±1.58 T3 (75%) 23.60±1.16 34.00±2.09 23.07±2.68 8.80±5.15 T4 (25%) 22.80±2.01 32.00±6.05 23.20±4.39 12.80±2.41 T5 (50%) 26.80±0.58 37.20±6.11 22.67±5.77 10.00±4.40 T6 (75%) 25.60±2.09 28.80±3.34 17.07±4.35 9.60±1.46 Note: Data are represented as mean of three samples replicates ± standard error. Means in the same row with the same letter are not significantly different (P>0.05)

Table 13. The end carcass composition (% DM) of all-male Nile tilapia fed experimental diet

Diets D.M Moisture C.P E.E ASH NFE G.E/kcal/100g T0 (0%) 73.5 26.5 31.81 6.85 2.15 32.695 377.144 T1 (25%) 75.0 25.0 31.15 6.70 2.40 34.75 380.205 T2 (50%) 74.0 26.0 30.60 6.70 2.10 34.6 376.47 T3 (75%) 75.0 25.0 30.25 6.40 2.20 36.15 377.825 T4 (25%) 76.0 24.0 30.90 6.75 2.15 36.25 385.255 T5 (50%) 77.0 23.0 31.35 6.75 2.10 36.8 390.02 T6 (75%) 74.5 25.5 31.05 6.55 2.05 34.85 378.61

Table 14. Show the water quality parameter during the experiment

T0 T1 T2 T3 T4 T5 T6 Treatment Sig 0% 25% 50% 75% 25% 50% 75% Temp. 27.58±0.9 27.75±1.0 27.42±0.7 27.58±0.9 27.5 ±1.0 27.50±0.8 27.50±0.5 0.98 O2 5.41±2.3 5.80±2.1 5.80±2.1 5.60±2.5 5.74±2.5 5.90±2.2 6.40±1.9 0.96 pH 7.60±0.5 7.74±0.4 7.65±0.3 7.64±0.3 7.50±0.4 7.60±0.3 7.73±0.4 0.63 * NH3 0.79±0.9 0.38±0.5 0.15±0.2 0.13±0.1 0.44±0.8 0.27±0.4 0.13±0.2 0.03 NO2 0.00±0.0 0.00±0.0 0.86±1.9 0.42±1.4 0.00±0.0 0.00±0.0 0.42±1.4 0.32 ** NO3 0.08±0.3 1.83±2.4 2.50±2.6 2.08±2.6 0.00±0.0 0.42±1.4 0.03±0.1 0.00 Note: Data are represented as mean of three samples replicates ± standard error. Means in the same row with the same letter are not significantly different (P>0.05)

Table 15. Length of all-male Nile tilapia during the experiment

Item T0 0% T1 25% T2 250% T3 75% T4 25% T5 50% T6 75% Sig IL (cm) 5.03±0.6a 4.23±0.4a 5.60±0.8a 5.20±0.6a 5.50±1.6a 5.47±0.2a 5.20±1. 2a 0.59 FL (cm) 6.43±0.3a 6.35±0.2a 6.50±0.2a 6.19±0.1a 6.68±0.3a 6.26±1.3a 5.88±0.2a 0.98 Note: Data are represented as mean of three samples replicates ± standard error. Means in the same row with the same letter are not significantly different (P>0.05)

200 NUSANTARA BIOSCIENCE 10 (3): 193-202, August 2018

Table 16. The economic evaluation of the experimental yeast respectively; without decreasing the accretion rates of diets/Sudanese currency all-male Nile tilapia, but further enhancing dietary date fiber to 300 g/kg and this resulted in notable persecution in Price Total price Item Treatments FCR all parameters (Belal et al. (2015)). SDG/kg SDG This outcome is in agreement with Omoregie and Fish meal 4.000 T0 (0%) 0.44 3.54 Ogbemudie (1993) reporting that the substitution of 15% Date-seeds 2.000 T1 (25%) 0.42 3.38 palm kernel/25% fish meal (28.56% dietary raw protein Groundnut cake 5.000 T2 (50%) 0.46 3.70 and 13.49% dietary crude fiber) gives the best accretion Wheat bran 4.000 T3 (75%) 1.48 11.91 and food utilization inversely to 30% palm kernel 10% fish Wheat bran 6.000 T4 (25%) 0.49 3.95 meals (27.86% dietary raw protein and 18.99% dietary raw Starch 5.000 T5 (50%) 0.65 5.23 fiber); this might be caused by the high raw fiber, fish age, Mineral mix 35.000 T6 (75%) 0.71 5.72 Vegetable oil 18.000 size, environment and low raw protein level. Similar Yeas 1.500 outcome was gained in Saudi Arabia by Al Amoudi et al. Total 80.500 (2001) who tested local Palm Kernel Meal (LPKM) as a Note: *The price of date seeds according to the price of rejected partial or a total substitution of fish meal on tilapia dates (Oreochromis spillurm) diets. They discovered that the substitution from 0 to 32.5% of palm kernel increased the mass of Nile tilapia, while 100% substitution gave lower weight. This research utilized four iso-nitrogenous Discussion isocaloric diets containing 0, 100, 200 and 300 g/kg date This research showed the potential of palm date seeds fiber as the substitution of wheat bran. They were given to to be added in commercial all-male Nile tilapia feeds as Nile tilapia fingerlings (0.65 g). Gaber et al. (2014) stated fish meal, as well as to be an important component for feed that the average final weight (g/fish), SGR (%/day), feed production in Sudan. There is little knowledge in the scientific literature regarding the utilization of date seeds in conversion ratio, PPV and PER were significantly (p≤0.05) the diets of Nile tilapia, particularly in the level of research influenced by the level of fresh date and level of and in the commercially profitable levels. Most tilapia uses digestragrom and the best diet was achieved by the diet starch efficiently from 22 to 46% dietary starch and 22% is with level 30% fresh date and 0.03 of digesragrom; it is considered the most advantageous level for juvenile tilapia presented in Table 9 and 11. Yosif (2012) also informed (Wang et al. 2005). There is significant advantage in the that the wheat flour was substituted by Dehydrated relation of the partial substitution of the fish feed currently Entromorpha, Prosopis cineraria pods and date seeds at 0, used in diets. A valuable sum of research has been carried 10, 20 and 30%. Diets of date seeds achieved the best out on the substitution of fish meal with plant meal as the survival rate (p>0.05). The carcass protein levels were alike protein found in diets of Nile tilapia (Temesgen 2004; in all treatments. Different from all the above result was Shalaby 2004; Sasmal et al. 2005; Johnson and Banerji mentioned by Mabrouk et al. (2011) which stated that by 2007; Ebrahim et al. 2007; Abd el Hakim et al. 2008; utilizing two forms of cull date and date seeds as energy Tartiel et al. 2008; Khan et al. 2013; Labib et al. 2015). fount, added with feed additives (phytogenics), to be Gohl (1975) informed that Local Palm Kernel Meal partially substituted (13.5%) by yellow corn (YC), iso- (LPKM) used in Indonesia and Malaysia contains 20 to nitrogenous (30.43% crude protein), isocaloric (436.43 kcal 25% of raw protein. In this study, however, the analysis of GE/100 g)) in feeding Nile tilapia fingerlings, the specific palm date seeds has represented that they bear only 6.27% accretion rate of fish and feed utilization can be increased. of crude protein, but they remarkably influence in the However, the substitution with either dry cull date or date experimental diets (T1, T2, T3, T4, T5 and T6 Table 4) and seed reduced tilapia achievements; meanwhile, date seeds in the accretion achievements of all-male Nile tilapia at the resulted from the worst values which are in contrary to the time they are incorporated in various levels. This result of this study as seen in table 4.1 and 4.5. It may be recommends that the availability of accretion hormone; due to the high Fiber content as seen in Table 1; which is steroid compounds, estrogen, progesterone, and estriol regularly indigestible to most cichlids especially because which has been notable since the 1950s (Barreveld 1993) they do not have the needed enzymes for fiber digestion would supply fishes with the significant sum of accretion and the movement of native food from the stomach is low rates. Assem et al. (2014) stated that the use of five and fast. Then, higher meal frequency led to better isoenergetic-isonitrogenous diets bearing 0, 0.5, 1.0, 1.5 carbohydrate usage for hybrid tilapia (Tung and Shiau and 2.0 g/100 g of fungi Trichoderma reesei reduced date 1991). Image period also affects the accretion of tilapia and seeds (FDDP) of Nile tilapia diets as substitution of α- El-Sayed, and Kawanna (2004) boosted the accretion of cellulose; the Oestradiol increases linearly with FDDP tilapia by practicing longer image period. Osman et al. content in diets indicating that an FDDP may have (2001) stated that the use of acid-treated date seeds as phytoestrogenic roll. carbohydrate fount for Nile tilapia fingerlings, i.e., with the The present research obviously testified that as much as substitution by 50% (15% of the total diet) and by 100% 25%, 50% (33.17±2.5 and 36.15± 5.22) and 25% (34.76± (30% of the total diet) for treated and untreated date seeds, 6.52), (p>0.05) of the fish meal protein, as seen in Table 7, respectively. Accretion achievement, feed conversion ratio, could be substituted by palm date seeds with and without and protein productive value was significantly (p>0.05)

SWAR & MOHAMED – Dietary palm date pits in male nile tilapia diets 201 higher in fish fed by 50% treated date seeds. Khadr (2006) (5.81 ppm± 2.21). Siddiqui et al. (1989) informed that claimed that the utilization of date seed meal (DSM) tilapia has a low oxygen need and can stay alive at small instead of yellow corn as an unusual energy fount in Male oxygen levels. Riche and Garling (2003) informed that the of Nile tilapia diets at level 0, 25, and 75% respectively levels of dissolved oxygen should be kept above 5.0 ppm resulted well. The diet holding 75% date seed meal showed for best accretion. Water pH has an effect on many water best accretion achievement. quality parameters and the values of many biological and Table 11 showed that the mean ±SD of meal intake and chemical procedures. In the present research, the average nourishing administration (% values) tend to give higher value of pH was (7.64±0.36) in which it was the optimum result in fish fed on diets control and on T5 (50% date rate of Nile tilapia culture. Pompa and Masser (1999) seeds without yeast). Fish food intake might be affected by informed that tilapia can remain alive at pH of 5 to 10 but many environmental factors such as water temperature, they stay their best at pH of 6 to 9. Ammonia and nitrite food concentration, stocking density, fish size and fish make a problem in the aquaculture system and must be behavior (Houlihan et al. 2001). Khadr (2006), stated that watched frequently. Ammonia establishment is directly cumulative feed consumption was influenced by feeding connected to feeding and is conditional on the grade of the date seed meal. The diet comprising 75% of date seed meal meal, the number of feeding, fish size, and temperature showed better accretion achievement as unusual energy (Riche and Garling 2003). In the present study, ammonia found, than the current result and it may be caused by the (NH3) was 0.33±0.43, nitrites (NO2) was 0.24±1.24 and various purpose of the inclusions. The least value of food nitrate (NO3) was 0.99±1.34, so they were not within the conversion rate (FCR) was in T3 and T6 as shown in Table safe range for tilapia culture, as the concentrations in the 4.5. Al Amoudi et al. (2001) informed that the highest nurturing containers are highly important (p>0.05). For this value of FCR was recorded on fish fed on control, i.e.1.73 reasons, some treatments showed poor accretion and on diet with 25% with yeast, namely 0.42. achievement, and reduced the survival rate as shown in The condition factor (K) was utilized as a barometer of Table 11. Accretion achievement parameters for tilapia health in fishing biology research, such as accretion and fingerlings after the feeding experiments are shown in nourishing frequency. this factor gives information on the Table 4.1; It is obvious that there is a remarkable impact of variety of physiological status of fish which can be utilized experimental diets on accretion achievement of the in the comparison of populations fed on certain diets. experimental fish. The economic evaluation on Table 16 Ighwela et al. (2011) informed that the use of diet with indicates that the smaller remarkable fare (p>0.05) was various maltose levels (0.0, 20%,25%, 30% and 35%) (3.38, 3.70 and 3.95) represented in the T1 (25%), T2 given to fingerlings of Nile tilapia resulted from higher (50%) date seeds substitution with yeast and T4 (25%) values of k in all treatment (1.64, 1.77, 1.72 and 1.79) than without yeast respectively. These results are consistent with the present result from other diets. Table 11 showed that so many previous researches. Mabrouk et al. (2011) the highest mean ±SD (p≤0.05) of factor (k) was in T0 resumed that call date may be expense-effective at the time it partially substitutes yellow corn in tilapia dietary as (control), T2 and T4, but all treatment results in good energy fount, and it also enhances fish achievements at the health. On the contrary, digestgarom addition raised fish time it increases 0.03% digestion. Khadr (2006) informed achievements either fed on diets of fresh cull date or that tilapia fed on the diet containing 75% wet cull date control diets (YC). Mabrouk et al. (2011) stated that this showed more economical outcomes than on control. result in accordance with the previous result of the study In conclusion, the substantial discrepancies in the done by Mabrouk with the yeast additives. Carcass analysis results noted in advance for the most advantageous dietary notably presented (p>0.05) smaller raw protein level in fish protein needs for optimum accretion might be caused by given by treated date seeds than the groups given by dissimilarities in fish genetics, size , age, stocking density, untreated date seeds. Osman et al. (2001) presented the raw stuff, protein quality, hygiene and environmental result of the initial carcass analysis was 17.35. Table 6 circumstances or other unidentified factors, which conceal showed that there is a notable discrepancy (p> 0.05). The the standardization of the parameters (Ahmad et al. 2004). greatest number of ash (p>0.05) was gained from 50% date We presumed that the addition of 25%, 50% and 25% date seeds replacement with yeast as shown in Table 13. It was seeds (Phoenix dactylifera) substitution with and without dissimilar from Yousif (2012) who reported that the ash yeast respectively is a great herbal protein fount, which level was higher (p>0.05) in the group fed on control diet could partially substitute fish nourishment in all-male Nile than on other. Dry matter and ash level in acid treated date tilapia (Oreochromis niloticus) for its positive impact in seeds were not influenced, as claimed by Osman et al. accretion achievement, feed utilization and economically (2001). The dampness level was inversely associated to fat decreasing the feed expense; at the same time, it can be content, irrespective of treatments, Yosif (2012), the concluded from the present study in general that bakery- greatest level of dampness was gained from 50% date seed yeast could perform as an excellent feed additive in all- substitution (without yeast), while the highest value of fat male Nile tilapia dietary. was gained from 50% date seed substitution (with yeast). Table 14 shows the result of the measurement in the nurturing plastic containers (aquarium) during the experiment describing the mean values of water quality REFERENCES parameters. The mean value of water temperature was Assem A, Khalifa M, ElShalia M. 2014. Physiological and microbiological (27.55C ± 0.82). The mean value of dissolved oxygen was indices as indicators of evaluating dietary fungi degraded date pits as

202 NUSANTARA BIOSCIENCE 10 (3): 193-202, August 2018

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| Nusantara Biosci | vol. 10 | no. 3 | pp. 137-202 | August 2018| | ISSN 2087-3948 | E-ISSN 2087-3956|

ISEA Journal of Bio l o g i c a l S c i e n c e s

The growth performances and the gut health parameters of Sentul chickens supplemented 137-141 with various dosage of turmeric powder INDRAWATI Y. ASMARA, TUTI WIDJASTUTI, IWAN SETIAWAN, ABUN, RUHYAT PARTASASMITA Short Communication: Tetrazolium test for evaluating viability of Capsicum annum seeds 142-145 ADITYA KUSUMAWARDANA, BAMBANG PUJIASMANTO, PARDONO The effect of combination of indigenous phosphate solubilizing bacteria of Riau, Indonesia on 146-150 the available phosphorus and phosphorus uptake of soybean LUFITANUR ALFIAH, DELITA ZUL, NELVIA NELVIA Male gametophyte development steps in Pistacia vera L. 151-158 ELAHE SADEGHIRAD, AHMAD MAJD, ALIREZA IRANBAKHSH, AMANOLLAH JAVANSHAH Antibacterial potency of simple fractions of ethyl acetate extract of Begonia baliensis 159-163 HARTUTININGSIH-M. SIREGAR, R.S. PURWANTORO, PRAPTIWI, A. AGUSTA Total phenolic content and antioxidant activity of ginger extract and SNEDDS with eel fish 164-169 bone oil (Anguilla spp.) IMAS IFRIAN WIJAYANTI, AGUNG BUDIHARJO, ARTINI PANGASTUTI, FEA PRIHAPSARA, ANIF NUR ARTANTI The potential of agroforestry as an adaptation strategy to mitigate the impacts of climate 170-177 change: A case study of Kiine Community, Kenya MUNENE ANNE NYARUAI, JOHN K. MUSINGI, BONIFACE N. WAMBUA Influence of NAA and coconut water with variation of soaking duration on the growth of 178-182 yellow bamboo branch cutting TIA SETIAWATI, AGINTA PUTRI REHULINA KELIAT, RULY BUDIONO, RUHYAT PARTASASMITA, JOHAN ISKANDAR Effect of sucrose and plant growth regulators on callogenesis and preliminary secondary 183-192 metabolic of different explant Myrmecodia tuberosa YANTI PUSPITA SARI, EKO KUSUMAWATI, CHAIRUL SALEH, WAWAN KUSTIAWAN, SUKARTINGSIH Incorporation of dietary palm date pits in all-male nile tilapia (Oreochromis niloticus ) diets 193-202 NEDAL MOHAMMED SIDDIG SWAR, ASAAD HASSAN WIDAA MOHAMED

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