Rapid Micropropagation by Axillary Buds Cultures of Smilax china

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1 농업생명과학연구 44(6) pp Journal of Agriculture & Life Science 44(6) pp Rapid Micropropagation by Axillary Buds Cultures of Smilax china Hyun-Jin Song 1 Seon-Jeong Sim 1 Mi-Jin Jeong 1 Chang-Mi Heo 1 Hak-Gon Kim 1 Gwon-Yong Jeong 2 Su-Yeoung Heo 2 Yong-Weon Choi 2 Geun-Hye Park 2 Jae-Kyung Yang 2 Hyun-Shik Moon 1 Myung-Suk Choi 1* 1 Div. of Environ. Forest Sci., Gyeongsang National Uni, (Insti. of Agric. of Life Sci.), Jinju , Korea 2 Uiryeong-Gun Agricultural Technology Center, Uiryeong , Korea Received: OCT , Revised: DEC , Accepted: DEC ABSTRACT An efficient method for the rapid propagation of Smilax china from axillary buds was established. Plants with thick leafage were selected from Korea native S. china population. Axillary buds of S. china collected from selected plant and were cultured in various culture media (2MS, MS, 1/2MS, WPM, B5 and SH medium). Shoot was induced from axillary bud on MS basal medium after 4 weeks of culture. 1/2MS medium showed a higher growth rate than those of the others, while the lowest shoot growth was obtained in 2MS medium. Among the sucrose concentrations, 5% sucrose was the optimum level for shoots growth from axillay buds. Among cytokinins, 0.5 mg L -1 6-benzylaminopurine (BAP) treatment showed the best performance on shoot multiplication, yielding average shoot multiplication forming about 2.4. Rooting was induced directly near the base of the shoot on 1/2MS medium containing with three-auxins α-napthalene acetic acid (NAA), indole acetic acid (IAA) and β-indolebutyric acid (IBA) (0.5 and 1.0 mg L -1 ). The 1.0 mg L -1 IBA treatments induced earliest rooting with maximum of root number and root growth. These rooted plantlets were successfully transferred to pots for 4 weeks hardening process, and were transferred to soil with above 90% survival rate. Key words - Axillary buds, Shoot propagation, Multiple shoot, In vitro rooting I. INTRODUCTION Smilax china is a small perennial plant belonging to the Liliaceae frequently found in the warm temperate mountainous or hilly regions across eastern Asia (Bo et al., 2007). It has been well known that the rhizome of the genus Smilax has various pharmacological activities. It is commonly used traditional medicines used as anti-inflammatory (Shu et al., 2006), anti-cancer (Li et al., 2007), acute bacillary dysentery and chronic nephritis (Li et al., 2007), and antimicrobial activity (Song et al., 1998). Steroidal saponins are primary ingredients of S. china the main steroidal sapogen in being diosgenin. So S. china is likely a new resource of diosgenin to be used as a steroid intermediate in the pharmaceutical industry (Shu et al., 2004). The leaf is harvested in the summer and used roll material of Mangaeduck, traditional Korean rice cake. However, production of leaf of S. china are scarce because old aging of farmer and its low productivity. Propagation of S. china is very difficult because taking 2 or more years to germinate and low efficiency of rootstock sprouting. Therefore, this plant was grow slowly. So S. china must breed a plant with thick leafage. Micropropagation has been evaluated as an alternative to conventional vegetative propagation by several previous research groups (Tazawa & Sasahara, 2003; Mukherjee et al., 2010). Use of apices for the in vitro propagation of various vines species was documented earlier (Gray & Fischer, 1985). However, protocol for micropropagation of Smilax sp. has not been developed. There are only a few studies that have reported the in *Corresponding author: Myung-Suk Choi Tel: Fax: mschoi@gnu.ac.kr

2 40 Journal of Agriculture & Life Science 44(6) vitro production of dioscin in adventitious root cultures of S. china (Kwon & An, 2008), only a multiple bud formation by anther cultures of S. oldhami (Tazawa & Sasahara, 2003). Therefore, it is important to develop an efficient micropropagation technique for S. china to rapidly disseminate superior trees. This study establishes a producible and rapid method for in vitro micropropagation of S. china through axillary buds. II. Materials and Methods 2.1. Plant material S. china selected in Jinju (Mt. Gajwa), Korea and axillary buds were collected. These are washed with Tween % (v/v) ethanol for 1 min. Surface disinfested with NaClO 3% (v/v) for 10 min, and rinsed five times with sterile distilled water. Plants were then placed in Petri dishes ( mm) containing 50 ml of regeneration medium. The medium consisted of 1/2MS (Murashige & Skoog, 1962), 30 g L -1 sucrose, and 3.8 g L -1 gerlite (Duchefa, Haarlem, Netherlands), and ph was adjusted to 5.7 before autoclaving at 121 C for 15 min. Each Petri dish ( mm) contained 4 explants, and all plates were incubated in dark at 25 ± 1 C. For each experiment described below, The axillary buds were replicated three times. After 4 weeks of initiation culture, adventitious shoots were transferred onto shoot elongation medium: half-strength MS (1/2MS) medium Determination of optimal culture conditions For determination of culture media on plant growth, internode of stem were cultured in several basal culture media; 2MS, MS, 1/2MS, WPM (Llyod & McCown, 1980), B5 (Gamberg et al., 1968), and SH (Schenk & Hildebrandt, 1972) medium with 3% (w/v) sucrose as carbon source. Plants were cut into 1.5 cm internode, and subcultured in various culture media (2MS, MS, 1/2MS, WPM, B5 and SH medium). Medium adjusted to ph 5.7 before the autoclaving at 121 C, 15 min. Each culture bottle (500m 3 ) contained one explant, and all culture bottles were incubated in dark at 25 ± 1 C. All cultures were grown under the conditions described above for a period of 8 weeks. A segment of stems was cultured in several sucrose containing media. The basal medium for shoot initiation consisted of 1/2MS. This medium was supplemented with various amounts of sucrose (1, 3, 5, 7 and 9%) as a carbohydrate source and shoot regeneration were also tested. Observations were made after 8 weeks and number of explants producing shoots were recorded. The experiments were repeated five times Determination of optimal BAP concentration on shoot multiplication Axillary buds of S. china and shoots were cultured containing 50 ml culture medium. The axillary buds from the stem node of S. china were decapitated to avoid apical dominance. For adventitious shoot induction, explants were cultured on 1/2MS culture media supplemented with 0, 0.1, 0.5, 1.0 and 2.0 mg L -1 BAP. All the cultures were carried out at 25 ± 1 C in chamber maintaining 16h light/8h dark photoperiod, 40 µmol/m 2 s. After 8 weeks of culture, the number of explants with adventitious shoots was counted. The growth of the stem and length from the top of the root to the end of the stem representing the height of the S. china were measured. The experiment was repeated three times Determination of optimal auxins on in vitro rooting and acclimation Rooting was induced directly near the base of the shoot on 1/2MS medium with NAA, IBA and IAA. Media consisting of various concentrations of threeauxins, NAA (0.5 and 1.0 mg L -1 ), IBA (0.5 and 1.0 mg L -1 ), and IAA (0.5 and 1.0 mg L -1 ) was treated. The multiplied shoots after elongation were cut and inoculated into petri dishes containing 50 ml solid media. The root numbers and length of root were scored after 8 weeks of cultures and the experiment was repeated three times.

3 Song et al : Rapid Micropropagation by Axillary Buds Cultures of Smilax china Statistical analysis All above experiments were conducted in five replications. The data generated were subjected to statistical analysis by using SAS for Window Version 6.12 (SAS Institute Inc., 2001). Duncan s multiple-range test (DMRT) was applied to evaluate differences among the treatments. III. RESULTS and DISCUSSION 1984). Here sucrose was investigated as carbon sources at concentration of 1, 3, 5. 7 and 9% (w/v) (Fig. 3). Feeding of sucrose was increased on shoot growth at all treatments. After 8 weeks among the three concentrations, 5% sucrose was the optimum level for shoots growth from axillary buds. Similar results were obtained in micropropagation of Quercus variabillis, when 3% sucrose was used as carbon source on shoot multiplication (Romano et al., 1995) Effects of culture medium on shoot growth Shoots were induced from axillary bud on 1/2MS basal medium after 1 weeks of culture (Fig. 1A). Shoots were further grown on 1/2MS basal medium (Fig. 1B). Various culture media such as 2MS, MS, 1/2MS, WPM, B5 and SH medium were used to optimize the shoot growth of S. china (Fig. 2). After 8 weeks of in vitro culture, MS medium showed a higher growth rate than those of the others, while the lowest shoot growth was obtained in 2MS medium. The optimal culture medium on shoot growth of S. china is either half-strength MS salts or low nutrient containing culture medium. 1/2MS medium was more effective compared to MS, WPM and B5 media may be explained by the fact that MS full strength medium contains very high doses of nitrogen (approximately 2- to 4-fold higher) than that of B5 and WPM. And also, there are differences in micronutrient compositions among MS, B5 and WPM media which might cause differences in shoot proliferation rate of different species (Lu, 2005). This result was in agreement with that reported in grape (Mukherjee et al., 2010) in which BAP was most effective among cytokinins in inducing bud break and shoot proliferation in the explants Effect of sucrose concentration on shoot growth Normally for the culture of cell, tissue or organ, it is necessary to incorporate a carbon energy source into the medium. Sucrose has almost invariably been found to be the best carbohydrate, although glucose is generally found to support growth well (George & Sherrington Fig. 1. In vitro propagation from axillary bud of S. china. (a) plant regeneration from axillary bud on MS medium supplemented without plant growth regulators (b) shoot elongation of S. china on 1/2MS medium supplemented without plant growth regulators (c) shoot multiplication of S. china on MS medium supplemented with 0.5 mg L -1 of BAP (d) in vitro rooting on 1/2MS medium supplemented with 1.0 mg L -1 of IBA (e) shoot elongation of S. china on 1/2MS medium supplemented without plant growth regulators and (f) acclimation of in vitro propagated plant at growth chamber.

4 42 Journal of Agriculture & Life Science 44(6) well, and healthy roots induced from shoot base after 8 weeks. Fig. 4 shows the influence of BAP levels on the production of number of shoot bud per explant and shoot elongation of S. china. When shoots were cultured on 1/2MS basal medium without BAP, there was no shoot multiplication, but they elongated. Multiple shoot bud formation appeared near the base of the explant. 0.5 mg L -1 of BAP treatment was adjudged as the optimal for shoot multiplication forming about 2.4 multiple shoots. Fig. 2. Effect of culture media on shoot growth of S. china. Initial shoot length was about 1.0 cm long. Each value represented the means ± SE Fig. 3. Effect of sucrose concentration on shoot growth of S. china. Shoots were cultured on 1/2MS basal medium with various concentration of sucrose. Each value represented the means ± SE 3.3. Determination of optimal BAP concentration on shoot multiplication Four week after inoculation, the axillary buds began to sprout from the nodes of the explants and continued to multiply (Fig. 1C). Multiple shoots were induced on basal shoots. Multiplied shoots were cut on basal shoots, and then transferred to dish. On fresh medium shoots developed further. The shoots elongated very Fig. 4. Effect of BAP on shoot number and growth of S. china. Shoots were cultured on 1/2MS medium with various concentration of BAP. Each value represented the means ± SE The concentrations of exogenous cytokinin appear to be the main factor affecting shoot multiplication. The BAP is the most commonly preferred cytokinin (Vuylsteke, 1989). Our experimental results indicated that BAP concentration significantly influenced shoot multiplication and elongation. Low concentrations of BAP increased the shoot proliferation rate, but high concentrations decreased shoot multiplication. Increasing concentrations of BAP increased multiple microshoots of grape, but high concentrations of BAP depressed shoot elongation (Sajid et al., 2001). Further, BAP appeared

5 Song et al : Rapid Micropropagation by Axillary Buds Cultures of Smilax china 43 to be more effective than other growth regulators for production of more shoot buds per explants (Rao & Purohit, 2006). Similarly, for shoot primordial growth of S. china in liquid culture medium, BAP was more effective on shoot primordial growth than zeatin or TDZ (Ishii, 2002). Our results are in agreement with the above observations, demonstrating that BAP is suitable for stimulation of shoot multiplication. The studies of this kind may be used to develop strategies for large-scale propagation of elite S. china In vitro rooting and acclimation The shoots with about 1.5 cm long were excised and placed on the rooting medium. In the experiments on root induction, different concentrations of NAA, IBA and IAA influenced the time of root formation, shoot growth (Fig. 5). The culture of 1.0 mg L -1 IBA was better than 0.5 mg L -1 NAA, 1.0 mg L -1 NAA, 0.5 mg L -1 IBA, 1.0 mg L -1 IAA, and 0.5 mg L -1 IAA in terms of the frequency of rooting. Roots were induced directly at the base of the shoot at all treatments. 1.0 mg L -1 of IBA, proved to be the best rooting medium. The growth of root increased pararell with root number and reached 50% after 8 weeks (Fig. 5). Among treatments, root growth was also high on 1.0 mg L -1 IBA treatment. Even the shoot growth was optimal on this medium. Therefore, we performed in culture medium without plant growth regulator for elongation of shoot after rooting (Fig. 1E). These rooted plantlets were successfully transferred to pots for 4 weeks hardening process (Fig. 1F). Later these plants were transferred to soil with above 90% survival rate. Generally, it is well known that vines plants are difficult to undergo in vitro propagation. Especially, in vitro propagation of vines is even more difficult. In the present protocol we have established a rapid in vitro propagation system of plantlets for S. china. This rapid propagation rate could only be achieved using micropropagation rather than the conventional in vivo propagation method. The regenerated plants, grown in net house, did not exhibit any detectable variations in morphology in comparison with their donor plant. Fig. 5. Effect of auxins on root number and growth of S. china. Shoots were cultured on 1/2MS medium with various concentration of auxins. Each value represented the means ± SE Ⅳ. Acknowledgements This work was supported by the Region Agriculture Specialization Program grant funded by Rural Development Administration, Korea. Literature cited Bo, S., G. Hongzhu, C. Yajun, Y. Min, H. Jian, and G. Dean Steroidal saponins from Smilax china and their anti-inflammatory activities. Phytochem. 68: Gamberg, O. L., R. A. Miller, and K. Ojimaki Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Res. 50: George, F. and P. D. Sherrington Plant propagation by tissue culture. In Handbook and Directory of Commercial laboratories. pp. 709.

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