An Efficient In Vitro Propagation of Zanthoxylum piperitum

An Efficient In Vitro Propagation of Zanthoxylum piperitum S.J. Hwang Department of Food and Biotechnology Dongshin University Naju 52-714 Korea Keywords: Zanthoxylum piperitum DC., shoot-tip culture, in vitro propagation Abstract A protocol is described for rapid multiplication of Zanthoxylum piperitum DC. (Rutaceae), an important aromatic and medicinal plant, through shoot-tip explant cultures. Murashige and Skoog (MS) medium supplemented with various concentrations of N-6-benzyladenine(BA), N-benzyl-9-(2-tetrahydropyrenyl) adenine (BPA) and thidiazuron (TDZ), either single or in combination with α- naphthaleneacetic acid (NAA), was used to determine the rate of shoot proliferation. N-6-benzyladenine (BA) used at.5 mg l -1 was the most effective in initiating shoot proliferation, producing an average of 23 microshoots per shoot-tip explant after 4 days of culture. Induction of rooting (98%) was achieved by transferring the shoots to the same basal medium containing 2 mg l -1 indole-3-butyric acid (IBA). The plantlets were transferred to soil after acclimatization and showed 63% survival. These results indicate that possible application for the mass production of plantlets is through in vitro culture system in Zanthoxylum piperitum DC. INTRODUCTION Zanthoxylum piperitum DC. (Rutaceae) distributed in Korea, Japan, and China is an aromatic plant used in traditional spice and as a medicinal plant to treat against cold, hypotension, neuralgia, paralysis and toothache (Go and Han, 1996). One of the possible biological activity detected in this species includes an anticancer effect. The GC-MS results of Zanthoxylum piperitum revealed the presence of over 1 volatile components. Major components were 1,8-cineol, limonene, geranyl acetate, myrcene in the fruit peel and citronellal, and citronellol in the leaves (Kim et al., 1989). Since the concentrations of volatiles and other compounds are different for each plant, the main goal of breeding is to select highly productive individuals and to propagate them vegetatively in order to maintain their valuable characteristics. Conventional propagation through seeds and stem and root cuttings is too slow to provide answers to meet the demand for this valuable plant. Propagation through seeds is an inadequate solution due to low viability, a low germination rate and delayed rooting of seedlings. Clonal propagation of plant germplasm through tissue culture for rapid production of medicinal plants is an important prerequisite for in vitro conservation (Lynch, 1999). Successful micropropagation of tree species is a relatively recent phenomenon (Mott, 1981; Thorpe, 199; Bajaj, 1997). Several woody species such as poplars, eucalyptus, and wild cherry are at present commercially propagated (Gavinlertvatana et al., 1987; Thorpe, 199; Bajaj, 1997), while for others such as loblolly pine, and Shorea, the protocols are being standardized for mass multiplication (Haissig et al., 1987). So far, no information is available on micropropagation of Z. piperitum. In this paper we describe the development of an in vitro propagation system for Z. piperitum. MATERIALS AND METHODS Plant Materials Mature seeds of Zanthoxylum piperitum were collected from trees growing in the forests at Kwang-yang, S. Korea. They were classified by the Department of Biology, Chonnam National University. 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 25 89

Seed Sterilization, Germination and Production of Aseptic Seedling Following a 5 min treatment with 7% ethanol, seeds were surface-sterilized with an aqueous solution of 4% (v/v) sodium hypochlorite plus 1-2 drops of Tween 2 for 1 min and then rinsed five times for 1 min in sterile distilled water. After sterilization, seeds were cultured on MS (Murashige and Skoog, 1962) basal medium supplemented with 2% (w/v) sucrose and.3% (w/v) Phytagel (Phytagel, Sigma Co., USA). The ph was adjusted to 5.7 before autoclaving at 121 C and 15 kpa for 2 min. The cultures were incubated at 26 C with a 16-h photoperiod under cool white fluorescent light (4 μmol m -2 s -1 ). These culture conditions were used in all the experiments unless otherwise stated. Shoot Multiplication After 3 months in culture, 1-1.5 cm shoots were excised from seedling cultures and transferred into a medium for shoot induction. The basal media tested were MS, B5 (Gamborg et al., 1968) and WPM (Lloyd and McCown, 1981) containing 3 g l -1 sucrose and 3 g l -1 Phytagel and supplemented with BA, BPA or thidiazuron at concentrations of.5, 1., or 2. mg l -1 in combination with or.5 mg l -1 NAA. To optimize the level of sucrose, the shoot multiplication medium was supplemented with different concentration (1-5%). Explants were oriented vertically in the culture media and 3 replicates each in time. The percentage of explants producing shoots and the average number of shoots produced per explant were recorded after 6 weeks. Rooting and Acclimatization To induce rooting, shoots obtained with just state what the plant growth regulator concentrations were excised and cultured in hormone-free MS basal medium or MS medium plus IBA or NAA at concentrations of.5, 1., 2., or 3. mg l -1, respectively. Elongated shoots 2-2.5 cm long were excised to test the effects of various auxin treatments on root induction. After 6 weeks of culture, the rooting percentage, the number of roots and the maximum length of roots were evaluated. Subsequently, plantlets were removed from cultures, washed in running tap water and transferred to plastic pots containing autoclaved vermiculite. The pots were kept covered with plastic bags for 2 weeks and finally moved to the greenhouse. RESULTS AND DISCUSSION Within 2 weeks, 2 percent seeds of Z. piperitum DC. germinated on MS basal medium containing 3% sucrose. The propagation of shoots was regulated by the culture conditions. The important factors reported were the types of the plant growth regulator added to the culture medium, the concentration of carbon source, the strength of macronutrients and the age of explants. Among the cytokinins used, BA was highly suitable compared to other cytokinins. The addition of NAA to cytokinins generally did not promote shoot multiplication (Table 1). Kinetin was not effective in inducing shoot bud development (data not shown). The rate of multiplication was maximal (23 shoots per shoot) on medium supplemented with.5 mg l -1 BA (Fig. 1). Shoot length, however, was not affected at any BA concentration. On MS, B5, or WPM, new shoots developed within 2-3 days of inoculation. Among the three basal media, MS proved to be the best, where 98% of explants formed new shoots with an average of 23.4 ± 1.5 shoots per explant (Table 2) and an average shoot length of 1-1.5 cm (data not shown). Therefore, for the subsequent experiments MS medium was used. Most media used in plant tissue culture contain sucrose as energy source and sucrose concentration was found to influence plant regeneration (Chen and Chang, 22). In the absence of sucrose, shoot development was inhibited. The 3% of sucrose proved to be the most effective of concentration tested for in vitro cultivation of Z. piperitum (Table 3). Proliferated shoots were subcultured for every 3-4 weeks, and new shoots were harvested periodically. The production of shoots was further promoted by repeated 9

subculturing of original explants on fresh multiplication medium supplemented with.5 mg l -1 BA after each harvest of the newly formed shoots. It was clearly indicated that induction of in vitro shoot multiplication in Z. piperitum can be accomplished by using shoot tips isolated from aseptic seedling. Rooting and transplantation of plantlets to the field is the most important but different task in micropropagation (Murashige, 1974). For a micropropagation system to be useful in a breeding program, a high frequency of successful rooting and establishment in the soil are necessary. Different plant species might vary in their requirement of auxin type for adventitious root formation. As shown in Table 4, IBA was more suitable for root induction than NAA (Fig. 2). Root length showed only very minor differences amongst the various auxin concentrations (data not shown). According to Nickell (1982), the slow movement and slow degradation of IBA facilitates its localization near the site of application and thus its better function in inducing roots. When cultured on MS medium supplemented with 2 mg l -1 IBA alone, each shoot developed an average of six roots (Table 4). The effectiveness of IBA in rooting has been reported for medicinal plants like Hemidesmus indicus (Sreekumar et al., 2), Decalepis hamiltonii (Giridhar et al., 23), Melia azedarach (Thakur et al., 1998), and Taxus mairei (Chang et al., 21). The addition of NAA alone or IBA with NAA facilitated callus formation. Rooted planlets, when transferred to soil, showed about 73% survival. The protocol described here for the micropropagation of Zanthoxylum piperitum through microshoot multiplication facilitates the rapid propagation of this valuable medicinal plant. It will also be of great use in conservation and genetic transformation studies aimed at plant improvement. Literature Cited Bajaj, Y.S.P. 1997. Biotechnology in agriculture and forestry. Vol. 39. Berlin, Heidelberg, NY: Springer Verlag. p.3-16. Chang, S.H., Ho, C.K., Chen, Z.Z. and Tsay, J.Y. 21. Micropropagation of Taxus mairei from mature trees. Plant Cell Reports 2:496-52. Chen, J.T. and Chang, W.C. 22. Effects of tissue culture conditions and explant characteristics on direct somatic embryogenesis in Oncidium Gower Ramsey. Plant Cell, Tissue and Organ Culture 69:41-44. Gamborg, O.L., Miller, R.N. and Ojima, K. 1968. Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research 5:151-158. Gavinlertvatana, P., Matheson, A.C. and Sim, A.P. 1987. Feasibilty study on tissue culture of multipurpose forest tree species. Bankok: Winrock Intl. p.1-78. Giridhar, P., Ramu, D.V., Reddy, B.O., Rajasekaran, T. and Ravishankar, G.A. 23. Influence of phenylacetic acid on clonal propagation of Decalepis hamiltoni Wight & Arn: An endangered shrub. In Vitro Cellular and Developemental Biology-Plant 39:463-467. Go, Y.S. and Han, H.J. 1996. Chemical constituents of Korean chopi and sancho. Korean J. Food Sci. and Technol. 28:19-27. Haissig, P.E., Nelson, N.D. and Kidd, G.H. 1987. Trends in the use of tissue culture in forest improvement. BioTechniques 5:52-57. Kim, J.H., Lee, K.S. and Kim, K.R. 1989. Flavor components of the fruit peel and leaf oil from Zanthoxylum piperitum DC. Korean J. Food Sci. and Technol. 21:562-568. Lloyd, G.B. and McCown, B.H. 1981. Commercially feasible micropropagation of mountain laurel (Kalmia latifolia) by use of shoot tip culture. Comb. Proc. Intl. Plant Propagators Soc. 3:421-427. Lynch, P.T. 1999. Tissue culture techniques in in vitro plant conservation. p.41-62. In: E.E. Benson (ed.), Plant conservation biotechnology, London, Taylor & Francis. Mott, R.L. 1981. Trees. p.217-254. In: B.V. Conger (ed.), Cloning agricultural plants via in vitro techniques, Florida CRL Press. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497. 91

Murashige, T. 1974. Plant propagation through tissue cultures. Annu. Rev. Plant Physiol. 25:135-166. Nickell, G.L. 1982. Kirk-othmer: Encyclopedia of chemical technology. Vol. 18. Wiley, NY. p.32-45. Sreekumar, S., Seeni, S. and Pushpangadan, P. 2. Micropropagation of Hemidesmus indicus for cultivation and production of 2-hydroxy 4-methoxy benzalhyde. Plant Cell, Tissue and Organ Culture 62:211-218. Thakur, R., Rao, P.S. and Bapat, V.A. 1998. In vitro plant regeneration in Melia azedarach. Plant Cell Reports 18:127-131. Thorpe, T.A. 199. The current status of plant tissue culture. p.1-33. In: S.S. Bhojwani (ed.), Plant tissue culture: applications and limitations, Amsterdam, Elsevier Science. Tables Table 1. Effect of PGRs on in vitro shoot proliferation of Z. piperitum seedling shoot axises. PGRs (mg l -1 ) No. of microshoot/ explant Explant initiating shoot (%) NAA. BA.5 1. 2. 23.4 ± 1.5 17.3 ± 1.9 6.3 ±.7 98.2 87.6 31.2 NAA.5 BA.5 1. 2. 2.1 ±.7 8.3 ±.5 23.4 28.2 NAA. BPA.5 1. 2. 14.5 ± 1.5 13.7 ± 1.7 61. 58.4 NAA.5 BPA.5 1. 2. 11.7 ± 1.6 43.3 NAA. TDZ.5 1. 2. 7.2 ±.7 5. ±.6 38.4 28.3 NAA.5 TDZ.5 1. 2. 12.2 ± 1.3 43.7 *Data (mean ± SE) scored after 6 weeks for 2-25 explants per treatment. 92

Table 2. Effect of basal medium on in vitro shoot proliferation of Z. piperitum seedling shoot axises. Media No. of microshoot/explant Explant initiating shoot (%) MS 23.4 ± 1.5 98.2 WPM 15.7 ± 2.1 78.3 B5 3.4 ± 1.3 42.3 *Data (mean ± SE) scored after 6 weeks for 2-25 explants per treatment. Table 3. Effect of sucrose concentration on in vitro shoot proliferation of Z. piperitum seedling shoot axises. Sucrose (%) No. of microshoot/explant Explant initiating shoot (%) 1 14.3 ±.3 48.2 3 23.4 ± 1.5 98.2 5 11.3 ± 1.3 42.3 *Data (mean ± SE) scored after 6 weeks for 2-25 explants per treatment. Table 4. Effect of auxins on rooting of in vitro formed shoots of Z. piperitum. Auxins (mg l -1 ) No. of root/shoot explant Rooting (%) Control 1.2 ±.3 23.4 IBA 1. 2. 3. 2.9 ±.4 6.2 ±.3 1.2 ±.6 87.1 98.7 55.4 NAA 1. 2. 3..7 ±.4 21.8 *Data (mean ± SE) scored after 6 weeks for 2-25 explants per treatment. 93

Figures Fig. 1. Shoot multiplication in MS medium supplemented with.5 mg l -1 BA. Fig. 2. Rooted shoot in MS medium supplemented with 2 mg l -1 IBA. 94