Explant selection for spilanthes acmella (L) murr callus induction and optimization of the callus liquid culture

JOURNAL OF SCIENCE OF HNUE Natural Sci., 2010, Vol. 55, No. 6, pp. 128-133 EXPLANT SELECTION FOR Spilanthes acmella (L.) Murr. CALLUS INDUCTION AND OPTIMIZATION OF THE CALLUS LIQUID CULTURE Mai Sy Tuan, Mai Thi Hang, Nguyen Viet Thang and Dao Thi Hai Ly(∗) Hanoi National University of Education (∗)E-mail: hailybio@gmail.com Abstract. This paper focuses on researching of Spilanthes acmella (L.) Murr. callus culture. Different parts of the plant such as leaf, stem, flower and root have been cultured. Based on antimicrobial activity (5 - 20 mm on Staphylococcus and Vibrio) and radish root tip inhibition (82%), the callus derived from flowers is the best. Optimization of the callus liquid culture condition with 3 factors shaking rate, temperature and inoculum size as Box-Wilson model shows that the best condition is 115 rpm, 24◦C and 1.5g, respectively. Keywords: Spilanthes acmella (L.) Murr., callus liquid culture, optimiza- tion. 1. Introduction In Vietnam, because of tropical climate and complex terrain, there is a diver- sity valuable plant community including medicinal plants. Most medicinal plants have been used in traditional ways to treat diseases. They are collected in natural environments, some parts of trees to be used as components of traditional recipes. So it requires a lot of these raw materials. Harvesting a large amount of one plant stably is not easy. Therefore, applying biotechnology, especially plant tissue culture, is a potential approach to produce desirable bioactive compounds on an industrial scale from plant. Spilanthes acmella (L.) Murr. is a promising medicinal plant belonging to the family Asteraceae. It has been used as remedy for toothache, cough, flu, ra- bies diseases and tubeculosis. There were some reports about bioactive compounds from the plant such as N-isobutylamides (spilanthol, undeca-2E,7Z,9E-trienoic acid isobutylamide and undeca-2E-en-8,10-diynoic acid isobutylomide) [7]. Some medi- cal properties of the plant also were reported: Diuretic activity in rats [8]; Toxicity and electrophysiological effects on Periplaneta americana L. [5]. Micropropagation of the plant was done by Ang et al. [1] but no investigation of secondary metabolite production from the plant by tissue culture has been conducted so far. 128 Explant selection for Spilanthes acmella (L.) Murr. callus induction and optimization... In this paper, we report explant selection for callus formation and optimization of the callus liquid culture to obtain material for bioactive compound extracting. 2. Content 2.1. Material and method 2.1.1. Callus induction Spilanthes acmella (L.) Murr. was collected and planted in the herbal gar- den of Biology Faculty, Hanoi National University of Education. Different explants (root, stem, leaf, flower) of the plant were washed under running tap water for 30 minutes. They were then immersed in 70% alcohol for 30 seconds before sterilizing surface, using 0.1% HgCl2 (w/v) for 15 minutes. After rinsing three times with sterile distilled water, the explants were dried using sterile filter paper before in- oculating into basic MS culture medium [6] containing 0.5 mg/L BAP, 0.5 mg/L 2,4D, 3% sucrose (w/v). The pH of the medium was adjusted to 5.6 - 5.8, followed by addition of 0.8% agar before autoclaving at 121◦C, 1 at for 30 minutes. All the cultures were incubated at 25 ± 2◦C in the dark. The calli formed after 4 weeks of cultures being collected. 2.1.2. Bioactivity assay Calli derived from different explants were extracted by previously reported protocol [11]. Each extract was used for bioactivity assays to select which callus was the best. Bioactivity assays consists of antibacterial activity assay and radish root tip growth inhibition assay. Antibacterial activity was determined by using Bauer disc diffusion method [2]. The bacteria was obtained from the Culture Collection of Microbiology Department, Faculty of Biology, Hanoi National University of Educa- tion. Radish root tip inhibition assay were conducted as previous description using Raphanus salivus L. [11]. 2.1.3. Submerge culture and optimization of the culture process The best callus obtained above was used for the initiation of suspension cul- tures. The callus was inoculated into 250 mL erlenmeyer flask containing 80 mL of callus induction medium without agar. The cultures were grown in a shaking incubator in darkness. Optimization of culture condition (three factors temperature, inoculum size and shaking rate) by classical method involves changing one independent variables while maintaning all others at a fixed level was conducted. Based on these re- sults (not reported here but optimum temperature was 27◦C, inoculum size was 3 g, shaking rate was 110 rpm), we use Box-Wilson experimental design to develop a statistical model for callus production [3]. Shaking rate (X1, rpm), tempera- ture (X2), inoculum size (X3, g) are chosen as independent factors in the exper- imental design. Yield of callus (g) is the dependent output variable. For conve- 129 Mai Sy Tuan, Mai Thi Hang, Nguyen Viet Thang and Dao Thi Hai Ly nience the independent variables in the model are utilized in their coded forms. The variables Xi are coded as xi according to the equation: xi = (Xi - X 0 i )/4Xi, where xi is the coded value of the variable Xi, X 0 i is the value of Xi at the center point of the investigated area, and 4Xi is the step size. In this study, we have: x1 = (X1 - 110)/5; x2 = (X2 - 27)/3; x3 = (X3 - 3)/1.5. The coded values for the independent variables and the corresponding real value are given in Table 1. A Box-Wilson experimental plan, with six star points (α = 1.682) [4] and with six replicates at the centre point with a total number of 20 experiments is employed (Table 2). Table 1. Real and coded values of independent variables in the experimental plan Variables Center point Step size Real values Coded values X1 110 5 105 110 115 -1 0 +1 X2 27 3 24 27 30 -1 0 +1 X3 3 1.5 1.5 3 4.5 -1 0 +1 Table 2. Experimental design and experimental yield of callus Exp Coded values Real values Yield of callus (g) No. x1 x2 x3 X1 X2 X3 1 -1 -1 -1 105 24 1.5 24.22 ± 0.32 2 -1 -1 +1 105 24 4.5 22.48 ± 0.64 3 -1 +1 -1 105 30 1.5 24.87 ± 0.57 4 -1 +1 +1 105 30 4.5 23.68 ± 0.41 5 +1 -1 -1 115 24 1.5 27.41 ± 0.51 6 +1 -1 +1 115 24 4.5 26.02 ± 0.44 7 +1 +1 -1 115 30 1.5 24.16 ± 0.92 8 +1 +1 +1 115 30 4.5 20.47 ± 0.88 9 - α 0 0 101.6 27 3 15.65 ± 0.47 10 + α 0 0 118.4 27 3 11.5 ± 0.53 11 0 - α 0 110 22 3 18.81 ± 0.94 12 0 + α 0 110 32 3 4.86 ± 0.71 13 0 0 - α 110 27 5.5 14.69 ± 0.72 14 0 0 + α 110 27 0.5 18.58 ± 0.68 15 0 0 0 110 27 3 24.01 ± 1.09 16 0 0 0 110 27 3 24.89 ± 0.79 17 0 0 0 110 27 3 24.43 ± 1.02 18 0 0 0 110 27 3 24.8 ± 0.97 19 0 0 0 110 27 3 24.04 ± 0.64 20 0 0 0 110 27 3 24.71 ± 1 130 Explant selection for Spilanthes acmella (L.) Murr. callus induction and optimization... 2.2. Results and discussion 2.2.1. Explant selection via bioactivity assays Crude extracts of the callus derived from stem, leaf, root and flower of Spi- lanthes acmella (L.) Murr. being tested for antibacteria and inhibition of radish root tip. Obtained results in Table 3 show that the callus extracts could inhibit many bacteria belonging to Vibrio and Staphylococcus genera with various degree of sensitivity. The callus induced from flower is the most strongly active. Table 3. Antibacterial activity of the callus extract Bacteria Diameter of inhibition zone (D - d, mm) Root Stem Leaf Flower S. aureus 0 0 0 5 Vibrio parahaemolyticus 10 18 16 20 Vibrio furnissii 15 12 15 16 S. aureus 30 sd 2 3 1 4 S. aureus 29 sd 0 0 0 2 S. epidomidis 7 6 9 11 S. aureus 7 yb 5 0 7 6 The second bioactive test was inhibition of radish root tip. Figure 1 indicates that the callus extracts all affected on development of radish root tip. The flower, leaf, stem, root callus extracts reduce the development by 82%, 72%, 66% and 46% respectively. Figure 1. Inhibition of callus extracts derived from different explants on radish root tip Through these observations, the callus from flowers was the best and was chosen for further studies. There have been some reports about activity of Spilanthes acmella (L.) Murr. flower [8, 9] and in other studies, our data demonstrates that flower extract possesses higher activity against microorganism than other parts of the plant. This agrees with researchers that the more active part of intact plant, the more potential to culture it is for getting bioactive metabolites [10]. 131 Mai Sy Tuan, Mai Thi Hang, Nguyen Viet Thang and Dao Thi Hai Ly 2.2.2. Optimization of Callus liquid culture Experimental results at each point based on the experimental design are shown in Table 2. Each data point shown in this Table is the average of 10 flasks. The Box-Wilson experimental design are a general series of experiments that have been developed to efficiently serve as a basis for deriving the mathematical model of a physical process. The model of regression fitted is Y = b0 ∑ i bixi + ∑ i≤j ∑ bijxixj where Y is the predicted response, subscripts i, j vary from number 1 to the number of variables, bo is the intercept term, bi are linear coefficients, bij are quadratic coefficients. This is a square regression model in terms of coded values. Parameters of this equation are evaluated from experimental results of specific experiments designed to determine their value (Table 2) with MINITAB 15. The resultant functional relationship in terms of coded values for predicting yield of callus values is Y = 24.13− 0.30x1 − 2.23x2 − 0.11x3 − 1.58x21 − 2.19x22 − 0.50x23 −1.33x1x2 − 0.27x1x3 − 0.22x2x3 The coefficients of this equation show that factor x2 is the most effective on the culture process. In Figure 2, the twelfth experiment was conducted at highest degree (32◦C), so the callus weight only increased 4.86 g. The factor x1 affected to the yield of callus more than the factor x3 did. It meant that temperature should be controlled accurately during the culture, then shaking rate. Inoculum size was the least significant. The first and the second experiment or the third and the forth experiment were inoculum size difference, but the results were very little unequalness. Figure 2. Yield of Callus cultured in different experiment conditions Based on the result shown in Figure 2, the best condition to obtain callus is in experiment No. 5, the temperature is 24◦C, shaking rate is 115 rpm, and inoculum size is 1.5 g. In comparison with callus gained from the experiment conducting at each optimum factor (27◦C, 110 rpm, 3 g), the best condition is higher. It indicates 132 Explant selection for Spilanthes acmella (L.) Murr. callus induction and optimization... that there is interaction between factors. Combination of all optimum factors may not be the best condition. 3. Conclusion Among 4 kinds of explant, flower is the best to callus culture because of high activity against bacteria and strong inhibition activity against radish root tip. Optimum condition to liquid culture of the callus in erlenmeyer flask is shaking rate at 115 rpm, temperature at 24◦C and inoculum size at 1.5 g. REFERENCES [1] Ang B. H. and Chan L. K, 2003. Micropropagation of Spilanthes acmella L., a bio-insecticide plant, through proliferation of multiple shoots. J. Appl. Hort., 5(2), pp. 65-68. [2] Bauer A. W., Kirby W. M., Sherris J. C., Turck M., 1966. Antibiotic suscepti- bility testing by a standardized single disk method. American Journal of Clinical Pathology, 45, pp. 493-496. [3] Box G. E. P., Wilson K. B., 1951. On the experimental attainment of optimum conditions. J. Roy. Stat. Soc B13, pp.1-45. 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