Micropropagation and alkaloid production in Stemona sp

Stemona sp. has been used an antifungal, anti-parasitic, demulcent, and insecticidal herb. The objectives of the study were to select suitable explant and medium for micropropagation of Stemona sp. and to compare secondary metabolites found in natural tissue with those found in cultured tissue. The explants from terminal and axillary bud and bud adjacent to tuberous root were cultured on MS medium supplemented with 0, 1, 2 and 3 mg/L BA for 4 weeks and all transferred to 2 mg/L BA for another 8 weeks. The best growth was obtained from MS medium supplemented with 3 mg/L BA and followed by 2 mg/L BA which could induce the explants of bud adjacent to tuberous root to produce 71.25% callus and 70.09% multiple shoots. The calli were then cultured in liquid and on semi-solid MS medium supplemented with 0, 1, 2 and 3 mg/L NAA for 1 month to increase root formation. Semi-solid MS medium supplemented with 2 and 3 mg/L NAA stimulated 100% root induction from calli with an average of 12 and 14 roots/explant respectively. Different compounds from crude extracts of shoot, callus and root of Stemona sp. in dichloromethane were separated by thin layer chromatography. A mixture of toluene:ethyl acetate (80:20 v/v) was used as mobile phase and the separated substances were detected by UV light. The result indicated that natural root, callus and root from tissue culture had the same bands at Rf 0.02, 0.06, 0.14, 0.19, 0.42, 0.56, 0.69 and 0.85 and showed the semilar positive alkaloid bands at Rf 0.02. INTRODUCTION Stemona sp. is one of the most valuable oriental herbs. The root has been used as an antifungal, anti-parasitic, demulcent and insecticidal agent in countries such as China, Japan, and Thailand (Perry, 1980). Stemona sp. root has been reported to contain bibenzyl (Zhao et al., 1995), rotenoid (Roengsamran, 1973) and alkaloid (Lin et al., 1992). Sexual propagation by seeds of Stemona sp. is very poor and asexual propagation by planting tuberous root with attached bud cannot be done in cold season because of its dormancy requirements (Mathee, 2001). In this paper, we present for the first time a rapid in vitro clonal propagation system for Stemona sp. , which would be ideal for large-scale propagation and ex situ conservation and the secondary metabolites, found in natural plant tissues and cultured tissues were compared. MATERIALS AND METHODS Plant Materials Stemona sp. plants from Nakhon Ratchasima province, Thailand, were used as sources of explants. Terminal and axillary buds and buds adjacent to tuberous root explants were thoroughly washed with tap water and immersed in 0.2% carbendaxin for 5 min. The explants were then surface disinfected with 0.1% mercuric chloride solution for 3 min and three 5-min rinses in sterile distilled water. Subsequently, all explants were soaked in 0.01% citric acid for 5 min before transferring to the culture media. Media and Culture Condition The basal medium used for all studies was MS (Murashige and Skoog, 1962) with Proc. WOCMAP III, Vol 2: Conservation Cultivation & Sustainable Use of MAPs Eds.: A. Jatisatienr, T. Paratasilpin, S. Elliott, V. Anusarnsunthorn, D. Wedge, L.E. Craker and Z.E. Gardner Acta Hort. 676, ISHS 2005 68 3% sucrose, 0.2% gelrite (Sigma Chemical Company) and 0.01% myo-inositol. The pH of the media was adjusted to 5.8 prior to autoclaving for 15 min at 1.06 kg cm (121°C). Cultures were incubated at 25±2°C under 16 h daily illumination with white fluorescent light (approx. 45 μmol s). To obtain shoot and callus production in response to N-benzyladenine (BA), terminal and axillary buds and buds adjacent to tuberous root explants were cultured on MS supplemented with BA at the concentrations of 0, 1, 2, and 3 mg/L. To study root production in response to napthalene acetic acid (NAA), calli were grown on MS supplemented with 0, 1, 2, and 3 mg/L NAA without and with 0.2% gelrite. Ten replications were used for each treatment. Data were statistically analyzed using one way Analysis of Variance and the LSD values calculated at P=0.05 for comparing means of the treatments. Secondary Metabolites To compare secondary metabolites produced in natural tissues with those in the tissues from in vitro culture, different compounds from crude extracts of shoot, callus and root of Stemona sp. in dichloromethane were separated by thin layer chromatography (TLC). A mixture of toluene:ethyl acetate (80:20 v/v) was used as mobile phase. Separated substances were detected by UV light and sprayed with Dragendorff’s reagent for alkaloids screening test. RESULTS AND DISCUSSION Terminal and axillary buds and buds adjacent to tuberous root of Stemona sp. were cultured for 12 weeks with subculturing at every 4 weeks. At the end of week 4, the tissues were transferred to the same media with 2 mg/L BA thereafter. One week after culturing, only terminal buds and buds adjacent to tuberous root explants produced 1-2 shoots/explant, which was not different among all the media treatments. Only those explants on 2 mg/L BA were found to promote callus initiation at about 10 and 44%, respectively (Fig. 1 and 2). The calli were compact and green. Chonticha (2000) reported that S. tuberosa grown in the presence of 2,4-D and kinetin also promoted callus formation from leaf-base explants. Callus formation increased in all treatments after all the cultured tissues were transferred onto the medium with 2 mg/L BA for weeks 8-12. At week 12, the average highest percentages of 73.57% and 71.25% (Table 1) of callus induction were obtained from bud adjacent to tuberous root explants initially cultured on the medium with 2 (73.57%) and 3 (71.25%) mg/L BA. The terminal bud explant, produced low percentage of calli (47.06%) when the explants came from 1 mg/L BA medium. The axillary bud explant produced no regeneration. Calli from both explants produced 8-15 multiple shoots callus within 10-12 weeks. At week 12, the average highest percentage of 70.09% of multiple shoot induction was obtained from bud adjacent to tuberous root explants initially cultured on the medium with 3 mg/L BA and multiple shoots produced roots (Fig. 2c). Apparently cytokinin did not inhibit adventitious root formation and supplementary auxin was not required for root growth. These results support earlier findings in banana (Wong, 1986), gloxinia (Wuttisit and Kanchanapoom, 1998), and watermelon (Kanchanapoom et al., 1998). The difference in shoot production is probably due to the difference in levels of endogenous growth substances, presumably auxin, in these explants or to their insensitivity to the growth substances (Trewavas and Cleland, 1983). Axillary bud explants cultured on all concentrations of BA produced no shoot, root nor callus but only leaves or flowers. All calli from the bud adjacent to tuberous root explants from the medium with 2 mg/L BA for 4 weeks on MS medium with NAA (0, 1, 2, 3 mg/L) with and without 0.2% gelrite (100%) root formation. Media containing 2 or 3 mg/L NAA and 0.2% gelrite gave the highest root numbers (12-14 roots/explant) (Table 2 and Fig. 3), significantly higher than those induced by other auxin treatments. Our data showed that auxin in the form of NAA at 3 mg/L induced root formation in Stemona sp. Similar findings were also