Jumiati Asis, Nornasuha Yusoff and Nashriyah Mat

Transcription

1 J. Agrobiotech. Vol. 9(1S), 2018, p Universiti Sultan Zainal Abidin ISSN (Press) ISSN (Online) Asis et al. Allelopathic Assesments of Ficus deltoidea Varieties and Ficus microcarpa Allelopathic Assesments of Ficus deltoidea Varieties and Ficus microcarpa Jumiati Asis, Nornasuha Yusoff and Nashriyah Mat School of Agricultural Science and Biotechnology, Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin, Besut Campus, Besut, Terengganu Darul Iman, MALAYSIA. Corresponding author: Nashriyah Mat School of Agriculture Science and Biotechnology, Universiti Sultan Zainal Abidin, Besut, Terengganu, Malaysia Keywords: Allelopathic Ficus deltoidea Ficus microcarpa Sandwich method Methanolic bioassay method

2 223/ J. Agrobiotech. Vol. 9(1S), 2018, p ABSTRACT This study described allelopathic potential of two medicinal plant species; Ficus deltoidea varieties in comparison with Ficus microcarpa, by testing the leaf-litter through sandwich method and the leaf extracts through methanolic extraction method. These studies were carried out with three replications in Complete Randomized Design (CRD). The sandwich method was carried out by using 5 mg, 10 mg and 50 mg dry weight of the donor plants leaf. Among the eight donor plant tested, results showed that F. deltoidea var. kunstleri had the highest growth inhibition on lettuce radicle elongation (57.51%) as compared to control, followed by F. deltoidea var. trengganuensis (45.50%) and Ficus microcarpa (39.95%), respectively. Meanwhile, the methanolic extraction was done by using different concentrations of leaf extract (0.1 mg/ml, 1 mg/ml, 10 mg/ml and 100 mg/ml). Ficus deltoidea var. kunstleri and var. trengganuensis that have the highest inhibition on lettuce growth in Sandwich method, were further selected to be tested by using methanolic bioassay method. Ficus deltoidea var. kunstleri and F. deltoidea var. trengganuensis were observed to have high inhibition on lettuce radicle elongation at 66.63% and 64.95%, respectively, at concentration 100 mg/ml. Keywords: Allelopathic, Ficus deltoidea, Ficus microcarpa, Sandwich method, Methanolic bioassay method. ABSTRAK Kajian ini menerangkan potensi alelopati dua spesies tumbuhan perubatan, Ficus deltoidea yang dibandingkan dengan Ficus microcarpa, dengan menggunakan ujian daun gugur melalui kaedah sandwich dan ekstrak daun melalui kaedah pengeskstrakan metanol. Kajian ini dilakukan dengan tiga ulangan untuk setiap spesies penderma dengan menggunakan Reka Bentuk Rawak Lengkap (CRD). Kaedah sandwich dijalankan dengan menggunakan 5 mg, 10 mg dan 50 mg berat kering daun tumbuhan penderma. Di antara lapan jenis tumbuhan penderma yang diuji, keputusan menunjukkan bahawa F. deltoidea var. kunstleri mempunyai perencatan pertumbuhan tertinggi pada pemanjangan radikel salad (57.51%) berbanding dengan kawalan, diikuti oleh F. deltoidea var. trengganuensis (45.50%) dan Ficus microcarpa (39.95%). Sementara itu, pengekstrakan metanol dilakukan dengan menggunakan amaun ekstrak daun yang berbeza (0.1 mg/ml, 1 mg/ml, 10 mg/ml and 100 mg/ml. Berdasarkan kaedah Sandwich, F. deltoidea var. kunstleri dan var. Trengganuensis telah menunjukkan perencatan tertinggi pada pertumbuhan salad dan telah dipilih untuk diuji dengan kaedah bioasai metanol. Ficus deltoidea var. kunstleri dan F. deltoidea var. Trengganuensis telah menunjukkan perencatan pada kadar 66.63% dan 64.95% pada kepekatan 100 mg/ml. Kata kunci: Alelopati, Ficus deltooidea, Ficus microcarpa, Kaedah Sandwich, Bioasai ekstrak metanol INTRODUCTION Allelopathy was defined as inhibitory and/ or stimulatory effect whether directly or indirectly of one plant to another plant through the production of chemical compounds into the environment (Rice, 1984). Inhibitory effect of allelopathy which is the negative interaction occurred when one plant is inhibited but other plant is unaffected (Khanh et al., 2018). Allelopathy gives effects to the growth of plants due to the release of chemical compounds known as allelochemical mainly through root exudates and leachates during litter decomposition (Albuquerque et al., 2011). The phenomenon of allelopathy had becomes one of the potential alternative to minimize the dependency of herbicides in controlling weeds. The heavy use of herbicides leads to the evolution of weed resistance issue and environmental pollution problems (Soltys et al., 2013). There were several ways of allelopathic application in controlling weeds in the field which are mulching of crop residue, crop intercropping and become of the component in crop rotation. Although, the allelopathic activity in crop and weed species have been studied before, the exploitation of medicinal plants for their allelopathic activity become one of the interest recently. Fujii et al. (2003) suggested that plant which have medicinal properties, are also have strong allelopathic activity. Previous study conducted by Syed et al. (2014) had screened 19 medicinal plants of Pakistan using plant box and sandwich method. From sandwich method, the

3 224/ Asis et al. leachates of Withania somnifera and Sarcococca saligna showed strong inhibitory results which are more than 50% inhibition. Suwitchayanon et al. (2017) had screened 14 medicinal plants from Northern Thailand for their allelopathic potential activities using Sandwich method. It was found that Cymbopogon nardus completely inhibiting the bioassay species. In this study, Ficus deltoidea and Ficus microcarpa were chosen due to their medicinal properties. About 850 species of woody trees, shrubs, vines, epiphytes and hemiepipyhtes in the family Moraceae belong to the Ficus genus. Ficus deltoidea is commonly known to possess a wide range of medicinal value for human health. Its leaves and powdered roots cure wounds, rheumatism, sores, antidiabetic, after-birth tonic and also to treat leucorrhoe. Ficus deltoidea is commonly known as mistletoe fig or Mas cotek by the Malays in Peninsular Malaysia. It was a native to the Malayan Archipelago and found in Thailand, Indonesia and Malaysia. The F. deltoidea is found in Peninsular Malaysia usually as an epiphyte in lowland and mountain as well as terrestrial shrub (Turner, 1995). According to Turner (1995), there were seven varieties of Ficus deltoidea, namely var. angustifolia, var. trengganuensis, var. motleyana, var. intermedia, var. bilobata, var. kunstleri and var. deltoidea. In this present study, seven varieties of F. deltoidea were used together with Ficus microcarpa, which was most locally found. The objectives of this study were to investigate the allelopathic potential of leaves from seven varieties of Ficus deltoidea and the inhibitory effects of methanolic leaf extract from selected varieties of Ficus deltoidea in comparison with Ficus microcarpa (Indian laurel tree). MATERIALS AND METHODS Identification and Sample Collection The fresh leaves of Ficus deltoidea varieties of angustifolia, deltoidea, bilobata, kunstleri, trengganuensis, intermedia and motleyana were collected from the Ficus deltoidea Germplasm at Universiti Sultan Zainal Abidin (UniSZA), Besut Campus. Each of the variety was measured as three replicate plants. Each replicate was represented with their own accession number. The Ficus deltoidea varieties specimens used in this study which already existed in the Herbarium of Universiti Sultan Zainal Abidin were as shown in Table 1. For Ficus microcarpa, selected samples were collected from Kg. Tembila, Kg. Tempinis, and Kg. Mengkuang, Besut, Terengganu and prepared prior to deposition at the Herbarium (Table 2). Table 1 Accession, voucher number and location of Ficus deltoidea varieties as plant materials. Variety FD Accession Herbarium Voucher Location Number (UniSZA) Number (UniSZA) angustifolia FD Germplasm angustifolia FD Germplasm angustifolia FD Germplasm bilobata FD Germplasm bilobata FD Germplasm deltoidea FD Germplasm deltoidea FD Germplasm deltoidea FD Germplasm intermedia FD Germplasm intermedia FD 325b 306 Germplasm intermedia FD 325c 306 Germplasm kustleri FD Germplasm kustleri FD Germplasm kustleri FD Germplasm motleyana FD 234a 336 Germplasm motleyana FD 234b 337 Germplasm motleyana FD 234c 338 Germplasm trengganuensis FD Germplasm trengganuensis FD Germplasm

4 225/ J. Agrobiotech. Vol. 9(1S), 2018, p The bioassay test species used was the seeds of Lettuce (Lactuca sativa), that was obtained from market. All of these F. deltoidea varieties and F. microcarpa were the donor plants, whilst the seeds of Lettuce were the receptor (test) plants for this experiment. Herbarium Specimens The specimens were properly, pressed, dried and mounted on standard size herbarium sheets (Abbasi et al., 2013). Wooden frame, corrugated cardboard and folded newspaper were used for the pressing procedure. Specimen was mounted by separating it out. Lastly, the specimen voucher number was given to each sample and deposited at the Herbarium of Universiti Sultan Zainal Abidin (Table 2), that includes the existing specimens already deposited earlier (Table 1), as the recorded total specimens used in this study. Table 2 Herbarium specimens of Ficus deltoidea varities and Ficus microcarpa that were deposited at the Herbarium of Universiti Sultan Zainal Abidin. Plant Species Voucher Latitude Longitude (Accession Number) Number Ficus microcarpa Ficus microcarpa Ficus microcarpa Ficus deltoidea var. bilobata (FD296) Ficus deltoidea var. trengganuensis (FD090) Preparation of Plant Materials The experimental samples of each of the seven varieties of Ficus deltoidea and Ficus microcarpa (tested donor species) were freshly collected, separately placed in a paper bag and oven-dried at 60 C for approximately 24 hours. The potential allelopathic activities of all varieties were determined under laboratory condition by using sandwich and methanolic extract bioassay methods. Agar (0.7% w/v) (Nacalai tesque agar) was used as a growth medium in the sandwich method. Sandwich Method The Sandwich bioassay method was used to evaluate the potential allelopathic effect of the leaf-litter leachate in the laboratory (Fujii et al., 2003). Through this method, seven varieties of F. deltoidea along with F. microcarpa were collected and investigated for their potential allelopathic activities. To carry out this method, six multi-well plastic dishes were used. Six-welled multi-dish plastic plates were filled with different amount with leaf litter of tested donor species (i.e; 5 mg, 10 mg and 50 mg). Five ml of agar was added in two successions to form layers above and below the plant samples. Five pre-germinated lettuce seeds were sowed on the solidified agar. The multi-well dishes were labeled, covered with plastic tape and incubated at 22 C for three days. The radicle and hypocotyl lengths of lettuce were measured and expressed as a percentage of the control. The treatments were replicated three times and data was presented as the mean of tree replicates. Agar with no plant material was set as the control. Methanolic Extract Bioassay Method The methanolic extract bioassay was used to evaluate the inhibitory activities of potential allelopathic plants using extract from methanol. In this method, tested donor species which showed the highest inhibitory percentage from previous experiment (which is sandwich method) were selected for bioassay using methanolic extract. Therefore, the leaves of Ficus deltoidea var. kunstleri and var. trengganuensis were further selected and collected from the Germplasm of UniSZA, Besut Campus, Terengganu. The plant materials were oven-dried at 60 C for 24 hours. Then, the oven dried plant materials were soaked in 80% methanol at 4 C for 72 hours prior to be dried using the rotary evaporator (modified from Takemura et al., 2013). The methanolic leaf extract was used for bioassays after filtration. Filter paper was placed in a glass Petri dish. Test solution was added to the filter paper in the petri dish and evaporated overnight at room temperature. After that, the distilled water of 1 ml was added and ten pre-

5 226/ Asis et al. germinated lettuce were placed on the filter paper, and incubated for 52 hour at 20 C in the dark. The control treatment did not contain any plant sample in the petri dish. The concentration of test solution was ranged from 0 mg/ml to 100 mg/ml fresh weight. The radicle and hypocotyl lengths were determined after three days. Data Analysis All experiments were conducted by using the Completely Randomized Design (CRD) with three replicates. The experimental data was subjected to the analysis of variance (ANOVA) which is a one-way ANOVA, and means was compared using the Duncan Multiple Range Test (DMRT) at the 5% level of significance. The statistical analysis was done by using the SPSS software version 21. RESULTS AND DISCUSSION Allelopathic evaluation of the leaf litter leachate of Ficus deltoidea varieties and Ficus microcarpa using Sandwich Method Table 3 and Table 4 indicated the growth inhibition percentage of tested Ficus deltoidea varieties and Ficus microcarpa on the radicle and hypocotyl length of lettuce seeds respectively. The percentage inhibitions on the radicle and hypocotyl lengths of lettuce seedling were concentration and varieties dependent. The growth of radicle and hypocotyl were significantly decreased (p<0.05) as the amount of tested leaf litter increased (Table 3 and Table 4). From Table 3, the lettuce radicle inhibition was observed to be significant by more than 50% respectively as compared to the control when grown with 50 mg of leaf-litter leachate of all species except from both var. motleyana and var. deltoidea. However, the inhibition percentages of the radicle of lettuce when grown with 5 mg and 10 mg of leaf-letter leachate as shown, were significantly inhibited by less than 50% respectively as compared to control except in var. kunstleri that was grown in 10 mg leaf-litter leachate. Based on the average inhibition percentage in Table 3, the highest lettuce radicle inhibition percentage compared to control was observed from Ficus deltoidea var. kunstleri (57.51%), followed by var. trengganuensis (45.50%) and Ficus microcarpa species (39.95%). Only one species which is Ficus deltoidea var. kunstleri showed the lettuce inhibition percentage more than 50%. Based on the results in Table 3, it is indicated that F. deltoidea var. kunstleri is the strongest lettuce radicle inhibitor among tested donor species using Sandwich method. According to Zakiah and Harun (2015), F. deltoidea var. kunstleri has several phytochemical constituents such as tannins, phlobatannins, saponins, flavonoids, steroids, terpenoids, alkaloids, anthraquinones and polyphenols which act as the plant secondary metabolites. Ficus deltoidea var. kunstleri has larger leaf area with a network of vein and this variety also is taller compared to other variety of F. deltoidea tested in this study. According to Singh et al. (2003), the allelochemicals in the higher plant has the ability to inhibit the growth of other neighboring plants. Based on Table 4, when the lettuce seedlings were grown with 5 mg dry leaf-litter leachate of Ficus deltoidea varieties and Ficus microcarpa, the highest percentage inhibition of hypocotyl was observed from F. deltoidea var. kunstleri (4.21%), followed by F. deltoidea var. deltoidea (1.31%) and the four other F. deltoidea varieties and F. microcarpa showed significant stimulation of hypocotyl length. When the lettuce were grown with 10 mg dry leaf litter, only F. deltoidea var. deltoidea (3.32%) showed significant inhibitory effect of lettuce hypocotyl elongation whilst, stimulation in hypocotyl length of lettuce was observed when grown with 10 mg of other tested donor species (Table 4). In contrast, the lettuce hypocotyl length was significantly inhibited when grown with 50 mg of F. deltoidea var. deltoidea, F. deltoidea var. motleyana, F. deltoidea var. bilobata and F. deltoidea var. intermedia. However, based on percentage of mean inhibition of hypocotyl length in Table 4, only two species showed inhibitory effect on the hypocotyl length which were F. deltoidea var. deltoidea and F. deltoidea var. motleyana. Based on the results obtained in the Sandwich method, it can be summarized that the percentage inhibition on the radicle length of tested donor plants were higher compared to the hypocotyl length. Besides, some varieties of Ficus deltoidea exhibited stimulatory effect on the hypocotyl length (Table 3 and Table 4). This finding was similar with the outcomes that had been reported by Fujii et al. (2003), who screened the allelopathic potential of 239 medicinal plant species in Japan. From the results of this screening, the highest inhibition of

6 227/ J. Agrobiotech. Vol. 9(1S), 2018, p lettuce radicle growth length was by more than 50% respectively. However, more tendencies were to inhibit radicle growth as compared to hypocotyl growth (Fujii et al., 2003). Table 3 Growth inhibition percentage of leaf litter leachate from Ficus deltoidea varieties and Ficus microcarpa on lettuce radicle length Name of tested donor Amount of leaf from tested donor 5 mg 10 mg 50 mg Percentage of mean inhibition of radicle length (%) var. kunstleri c d 66.24d e var. trengganuensis c c e F. microcarpa b c bc var. angustifolia b c bc var. deltoidea a b b var. bilobata 0.00 b a 53.43b cd var. intermedia a a cd var. motleyana a 1.25 a a Note: Means within the columns followed by the same alphabet were not significantly different (p>0.05) according to DMRT. Plants were ranked in order of their inhibitory activity. Table 4 Growth inhibition percentage of leaf litter leachate from Ficus deltoidea varieties and Ficus microcarpa on lettuce hypocotyl length Name of tested donor Amount of leaf from tested donor 5 mg 10 mg 50 mg Percentage of mean inhibition of hypocotyl length (%) var.deltoidea 1.31 ab 3.32 bc cd var. motleyana a abc 7.60 bc var. kunstleri c c abc F. microcarpa ab abc a var. bilobata -5.68b c bc 2.99d e var. angustifolia a a ab var. intermedia ab a e var. trengganuensis abc ab -1.63d e Note: Means within the rows followed by same alphabet were not significantly different (p>0.05) according to DMRT. Plants were ranked in order of their inhibitory activity. Allelopathic effect of methanolic leaf extract from selected varieties of Ficus deltoidea Based on Sandwich method that had been done (Table3 and Table 4), it was found that F. deltoidea var. kunstleri and var. trengganuensis showed the highest inhibitory effect on the radicle length of lettuce. Both species were further tested using bioassay method. Tables 5 and 6 indicate that the inhibition of radicle and hypocotyl lengths of lettuce seedlings was concentration dependent. Results show significant inhibition towards the radicle and hypocotyl length of lettuce as the concentration of methanolic leaf extract F. deltoidea var. kunstleri and var. trengganuensis increased (Table 5 and Table 6). Table 5 shows that the radicle and hypocotyl length of lettuce were stimulated when tested with 0.1 and 1.0 mg/ml F. deltoidea var. kunstleri methanolic leaf extract. However, at concentration 100 mg/ml of F. deltoidea var. kunstleri, the maximum inhibition of both radicle (66.63%) and hypocotyl (4.82%) growth was shown Rank Rank

7 228/ Asis et al. respectively. This finding was also similarly recorded by Gilani et al. (2010) in which inhibitory activities increased as concentration increased. Besides, this finding was also consistent with Fujii et al. (2003), that the medicinal plant could inhibit the growth of lettuce by more than 50% respectively. The leaf extract of F. deltoidea var. trengganuensis showed the maximum inhibition of both radicle (64.95%) and hypocotyl (31.60%) growth, respectively at concentration 100 mg/ml (Table 6). From Table 6, it shows that the radicle length of lettuce was stimulated at concentration 0.1 and 1.0 mg/ml F. deltoidea var. trengganuensis methanolic leaf extract. In comparison with methanolic leaf extract F. deltoidea var. kunstleri and var. trengganuensis, F. deltoidea var. kunstleri shower higher inhibitory effect on the radicle length of lettuce at concentration 100 mg/ml but F. deltoidea var. trengganuensis showed higher inhibitory effect on the hypocotyl length at concentration 100 mg/ml compared to F. deltoidea var. kunstleri (Table 5 and Table 6). However, both varieties exhibited more than 50% inhibitory effect on the radicle length of lettuce compared to the hypocotyl length. Previous study found that the F. deltoidea var. kunstleri had phytochemical constituents such as alkaloids, anthraquinones, tannins, phlobatannins, saponins, flavonoids, steroids, terpenoids and polyphenols (Zakiah & Harun, 2015). This phytochemical contains with respect to the role of this plant as traditional medicine. According to the previous study by Olofsdotter et al. (2002), phenolic compounds are considered to be responsible for inhibition of competing plants. This study showed that different varieties of Ficus deltoidea possibly contain different types of phenolic compounds. Table 5 Percentage of inhibitory effects from methanolic leaf extracts of Ficus deltoidea var. kunstleri on the radicle and hypocotyl length of lettuce Concentration (mg/ml) Inhibition of radicle (%) Inhibition of hypocotyl (%) a a a ab b ab c 4.82 b Note: Means within the columns followed by same alphabet were not significantly different (p>0.05) according to DMRT. Table 6 Percentage of inhibitory effects from methanolic leaf extracts of Ficus deltoidea var. trengganuensis on the radicle and hypocotyl length of lettuce Concentration (mg/ml) Inhibition of radicle (%) Inhibition of hypocotyl (%) a ab a b b 3.88 b c 31.6 c Note: Means within the columns followed by same alphabet were not significantly different (p>0.05) according to DMRT. CONCLUSION The screening of allelopathic potential of leaf-litter leachate through Sandwich and methanolic extraction methods has proven the presence of allelochemicals in the tested two medicinal plant species, from Besut District of Terengganu, Malaysia. The allelochemicals caused unfavorable condition on the growth of radicle and hypocotyl of the lettuce seedlings, as the receptor species. Through Sandwich method, the leaf-litter leachate of F. deltoidea var. kunstleri and var. trengganuensis showed the highest allelopathic activity compared to Ficus microcarpa. In the methanolic extraction method, the var. kunstleri have higher allelopathic activity compared to F. deltoidea var. trengganuensis Further research regarding identification and isolation of these allelochemicals could be carried out by using this information. This would contribute towards effective development of natural botanical herbicide by using medicinal plants.

8 229/ J. Agrobiotech. Vol. 9(1S), 2018, p ACKNOWLEDGEMENT The authors wish to express their gratitude to the laboratory staff at School of Agriculture Science and Biotechnology, Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin for their invaluable assistance throughout the study. REFERENCES Albuquerque, M. B., Santos, R. C., Lima, L. M., Filho, P. A. M., Nogueira, R. J. M. C., Câmara, C. A. G. & Ramos, A. R. (2011). Allelopathy, an alternative tool to improve cropping systems: A review. Agronomy for Sustainable Development 31(2): Abbasi, A., Khan, M., Shah, M. H., Shah, M., Pervez, A. & Ahmad, M Ethnobotanical appraisal and cultural values of medicinally important wild edible vegetables of Lesser Himalayas-Pakistan. Journal of Ethnobiology and Ethnomedicine 9(1): 66. doi: / Fujii, Y., Parvez, S. S., Parvez, M., Ohmae, Y. & Iida, O. (2003). Screening of 239 medicinal plant species for allelopathic activity using the sandwich method. Weed Biology and Management 3(4): Gilani, S. A., Fujii, Y., Shinwari, Z. K., Adnan, M., Kikuchi, A. & Watanabe, K. N. (2010). Phytotoxic studies of medicinal plant species of Pakistan. Pakistan Journal of Botany 42(2): Khanh T. D., Anh L. H., Nghia L. T., Trung K. H., Hien P. B., Trung D. M. and Xuan T. D. (2018). Allelopathic responses of rice seedlings under some different stresses. Plants. 7(40). doi: /plants Olofsdotter, M., Jensen, L. B. & Courtois, B. (2002). Improving crop competitive ability using allelopathy an example from rice. Plant Breeding 121(1): 1-9. Rice, E. L. (1984). Allelopathy 2nd ed. New York Academic Press. USA. 422 pp. Singh, H. P., Batish, D. R. & Kohli, R. K. (2003). Allelopathic interactions and allelochemicals: New possibilities for sustainable weed management. Critical Reviews in Plant Sciences 22(3-4): Soltys, D.; Krasuska, U.; Bogatek, R.; Gniazdowska, A. (2013). Allelochemicals as bioherbicides Present and perspectives. In Herbicides-Current Research and Case Studies in Use; Intech: Rijeka, Croatia, p Suwitchayanon, P., Kunasakdul, K. & Kato-Noguchi, H. (2017). Screening the Allelopathic Activity of 14 Medicinal plants from Northern Thailand. Environment Control Biology 55(3): Syed, S. A. M. I., Ahmed, Z. I., Razzaq, A. & Akmal, M. (2014). Root exudates and leaf leachates of 19 medicinal plants of Pakistan exhibit allelopathic potential. Pakistan Journal of Botany 46(5): Turner, I. M. (1995). A catalogue of the vascular plants of Malaya. National Park Board. Singapore Botanic Garden. The Gardens Bulletin Singapore 47: Takemura, T., Sakuno, E., Kamo, T., Hiradate, S. & Fujii, Y. (2013). Screening of the growth-inhibitory effects of 168 plant species against lettuce seedlings. American Journal of Plant Sciences 4(05): Zakiah, M. & Harun, H Pytochemical constituents in leaves and callus of Ficus deltoidea Jack var. kunstleri (King) Corner. Journal of Agrobiotechnology 12(5):