In Vitro Propagation of Mallotus repandus (Willd.) Muell. Arg.

S. Kaewsuwan, N. Soonthornchareonnon and S. Prathanturarug Faculty of Pharmacy Mahidol University Bangkok Thailand

Keywords: medicinal plant, anti-inflammatory drug

Abstract An in vitro propagation protocol was developed for Mallotus repandus (Willd.) Muell. Arg. (Euphorbiaceae), a potential medicinal plant for anti- inflammatory drug development. Nodal segments and shoot tips from 2 month-old appearing shoots were aseptically cultured on solid Murashige and Skoog (MS) basal medium supplemented with three cytokinins alone and in combination with 1- naphthylacetic acid (NAA) to induce shoot formation for 8 weeks. Of the three cytokinins tested (6-benzylaminopurine (BA), isopentenylaminopurine (2iP), and kinetin (Kin)), BA induced shoot development most efficiently. The best shoot regeneration rate (3.36 shoots/response explant) was obtained when the explants were cultured on MS medium supplemented with 4.44 µM BA. Addition of 0.54 µM NAA to the media suppressed shoot induction rate. The regenerated shoots were excised and then cultured on MS medium containing 10.74-53.71 µM NAA for rooting. The best root induction (73.08%) was revealed using MS medium supplemented with 32.23 µM NAA. Regenerated plants were successfully transferred to soil and grown under greenhouse conditions. The stem of the regenerants will be chemically analysed to compare with their mother plants.

INTRODUCTION Mallotus repandus (Willd.) Muell. Arg., a Euphorbiaceous climber, is found in Thailand, India, Ceylon, Philippines, Malay Islands, China, Australia, and New Caledonia (Hooker, 1885; Ridley, 1924). In Thailand, it has been traditionally used in a herbal recipe for the relief of muscle pain (Chuakul et al., 2000). An aqueous extract of the plant showed anti-inflammatory activity (Lin et al., 1992), while an alcoholic extract exhibited both anti-nociceptive and anti-inflammatory activities. Bergenin was proved to be a pharmacologically active compound for the anti-inflammatory activity (Chiewcherngka and Chaiyatham, 1999). Thus, Mallotus repandus shows potential for development as an analgesic or anti-inflammatory drug. But its wild crafted source is collected from natural forest areas where trees are being rapidly depleted. Plant tissue culture is an alternative method of commercial propagation and also genetic conservation of many medicinal plants, such as Aconitum carmichaeli Debx. (Hatano et al., 1988), Croton sublyratus Kurz. (Shibata et al., 1996), and Maytenus ilicifolia Mart. (Pereira, 1995). In this paper, an effective method for the in vitro propagation of Mallotus repandus was established.

MATERIALS AND METHODS

Plant Materials Seeds of Mallotus repandus were collected from Chacheongsao province in Thailand. Subsequently, they were germinated and maintained in soil under greenhouse conditions.

Preparation of Explants Explants were obtained from two month old shoots which subsequently had the leaves removed. Shoots were sterilized using 1.0% sodium hypochlorite solution containing a few drops of Tween 80 for 20 minutes, rinsed 3 times in sterile distilled

Proc. WOCMAP III, Vol 2: Conservation Cultivation & Sustainable Use of MAPs 95 Eds.: A. Jatisatienr, T. Paratasilpin, S. Elliott, V. Anusarnsunthorn, D. Wedge, L.E. Craker and Z.E. Gardner Acta Hort. 676, ISHS 2005 water before placement on shoot induction medium.

Shoot Induction Single-node and shoot tip explants were placed vertically on the shoot induction medium and cultured at (25-27°C) and under 8 hr dark-16 hr light for 8 weeks. Plant growth regulators, such as 6-benzylaminopurine (BA), isopentenylaminopurine (2iP), and kinetin (Kin) were tested to find out the optimum concentrations for shoot induction of the plant. A total number of 20-23 replications for each treatment with 1 explant were inoculated; therefore 20-23 explants per treatment were tested.

Rooting of Regenerated Shoots After 8 weeks of cultures on the shoot induction medium, regenerated shoots were excised and transferred to medium containing 1-naphthylacetic acid (NAA) for 1 week, then transferred to half-strength MS medium for 4 weeks. A total number of 20-25 replications for each treatment with 1 shoot were inoculated; therefore 20-25 shoots per treatment were tested.

Acclimatization Regenerated plantlets were transferred to the sand and peat (1:1) potting medium and maintained in a misting system. Survived plants were transferred to pot culture and maintained under greenhouse conditions.

RESULTS AND DISCUSSION

Shoot Induction Frequency of 2-month old explants responding in BA, 2iP, and Kin treatments were 20-75, 12-50, and 13-100%, respectively (Table 1-3). These variations may be due to the difference among initials plant materials. The maximum shoot induction rate was achieved with the application of 4.44 µM BA (5.10±1.07 shoots/explant) (Fig. 1A and 4A). Fig. 1B shows poor response in shoot multiplication when 2iP at 0.49-9.84 µM were added to the medium. The highest shoot induction rate was obtained at the concentration of 19.69 µM (2.25±0.40 shoots/explant). In the Kin experiment, the maximum shoot induction rate was obtained at the concentration of 18.59 µM (2.00±0.70 shoots/explant) (Fig. 1C). Addition of 0.54 µM NAA did not improved the shoot induction rate of BA (Fig. 1A) and 2iP (Fig. 1B). Controversial results were obtained when addition of 0.54 µM NAA to Kin (Fig. 1C). Fig. 2 shows the frequency of explant responding, shoot multiplication rate, and shoot length, when the explants were incubated on MS supplemented with BA, 2iP, and Kin at the selected concentration, alone or in combination with NAA. Among these three tested cytokinins, BA (4.44 µM), 2iP (19.69 µM), and Kin (18.59 µM), BA treatment gave higher percentage of explant responding than any other (Fig. 2A). Furthermore, BA induced the highest shoot induction rate for node and shoot tip explants (3.36±0.60 shoots explant) (Fig. 2B). It was suggested that BA (4.44 µM) was optimum concentration for shoot induction of the plant. NAA added to the media slightly induced shoot elongation. The shoot length of regenerated shoots, averaged across all cytokinin treatments or cytokinin and NAA treatments, was 0.47±0.08 and 0.63±0.12 cm per regenerated shoot, respectively (Fig. 2C).

Rooting of Regenerated Shoots Regenerated shoots were cultured on MS medium containing various concentrations of NAA (10.74-53.71 µM) for 1 week and transferred to half-strength MS medium for 4 weeks, the maximum frequency (73.08%) was obtained at the concentration of 32.23 µM NAA (Fig. 3A). However, the higher concentrations (42.97 and 53.71 µM)

96 produced slightly more number of roots per regenerated shoot (Fig. 3B).

Acclimatization The regenerated plants were transferred to the potting medium consisting of sand and peat (1:1) and maintained in a misting system with 50-70% survival after 4 weeks. Then the survived plants were transferred to pot filled with soil and maintained under greenhouse conditions (Fig. 4C). The regenerated plants were morphologically indistinguishable from one another.

CONCLUSIONS An effective method was developed for the in vitro propagation Mallotus repandus was shoot induction of single-node or shoot tip explants on MS medium supplemented with 4.44 µM BA for 8 weeks. Rooting of regenerated shoots on the media containing 32.23 µM NAA for 1 week, followed by half-strength media for 4 weeks were successfully transplanted to pot culture and grown in the green house.

ACKNOWLEDGEMENTS The authors sincerely thank the National Research Council of Thailand for financial support.

Literature Cited Chiewcherngka, P. and Chaiyatham, P. 1999. Pharmacological action of Mallotus repandus (Willd) Muell. Arg. A special project for the bachelor degree of science in pharmacy, Mahidol University. Chuakul, W., Saralamp, P. and Prathanturarug, S. 2000. Kokya Esan. Amarin Printing Group, Bangkok. Hatano, K., Kamura, K., Shoyama, Y. and Nishioka, I. 1988. Clonal Multiplication of Aconitum carmichaeli by tip tissue culture and alkaloid contents of clonally propagated plant. Planta Med. 54:152-155. Hooker, J.D. 1885. Flora of India. L. Reeve, London. Lin, C.C., Lin, J.M. and Chiu, H.F. 1992. Studies on folk medicine "Thang-kau-tin" from Taiwan (I) the anti-inflammatory and liver-protective effect. Am. J. Chin. Med. 20:37- 50. Pereira, A.M.S. 1995. Effect of phytoregulators and physiological characteristics of the explants on micropropagation of Maytenus ilicifolia. Plant Cell Tiss. Organ Cult. 42:295-297. Ridley, H.N. 1924. The Flora of the Malay Pennisula III. L. Reeve, London. Shibata, W., Murai, F., Akiyama, T., Siriphol, M., Matsunaga, E. and Moronto, H. 1996. Micropropagation of Croton sublyratus Kurz-a tropical tree of medicinal importance. Plant Cell reports. 16:147-152.

97 Tables

Table 1. Effect of BA concentration and 0.54 µM NAA on the frequency of explant responding.

Concentration (µM) % Explant responding ± S.E. BA BA + 0.54 µM NAA 0.44 75.00 ± 11.18 72.73 ± 14.08 2.22 53.85 ± 14.39 46.67 ± 13.33 4.44 55.56 ± 12.05 25.00 ± 13.06 8.88 43.75 ± 12.81 37.50 ± 12.50 17.76 23.08 ± 12.16 20.00 ± 10.69 PGR-free 50.00 ± 13.87

Table 2. Effect of 2iP concentration and 0.54 µM NAA on the frequency of explant responding.

Concentration (µM) % Explant responding ± S.E. 2iP 2iP + 0.54 µM NAA 0.49 50.00 ± 13.87 22.22 ± 14.70 2.46 30.77 ± 12.84 11.76 ± 8.05 4.92 22.22 ± 14.70 23.08 ± 12.16 9.84 33.33 ± 12.60 15.38 ± 10.42 19.96 28.57 ± 12.53 20.00 ± 13.33 PGR-free 25.00 ± 13.06

Table 3. Effect of Kin concentration and 0.54 µM NAA on the frequency of explant responding.

Concentration (µM) % Explant responding ± S.E. Kin Kin + 0.54 µM NAA 0.46 80.00 ± 13.37 54.55 ± 15.75 2.32 54.55 ± 15.75 37.50 ± 18.30 4.65 100.00 ± 0.00 22.22 ± 14.70 9.29 100.00 ± 0.00 37.50 ± 18.30 18.59 91.67 ± 8.33 12.50 ± 12.50 PGR-free 58.33 ± 14.86

98 Figures

7.00 A 6.00

5.00 4.00

3.00

2.00

Shoots / response explant 1.00 0.00 0.44 2.22 4.44 8.88 17.76 PGR-free

BA concentration (µ M)

BA BA + NAA PGR-free

3.00 3.00 B C 2.50 2.50

2.00 2.00

1.50 1.50

1.00 1.00 0.50 0.50 Shoots /response explant Shoots /response explant 0.00 0.00 0.49 2.46 4.92 9.84 19.69 PGR-free 0.46 2.32 4.65 9.29 18.59 PGR-free 2iP concentration (µM) Kin concentration (µM) 2iP 2iP + NAA PGR-free Kin Kin + NAA PGR-free

Fig. 1. Effect of BA, 2iP, and Kin alone or in combination with 0.54 µM NAA on shoot induction of Mallotus repandus node and shoot tip explants. Vertical lines indicate S.E.

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100.00 A

g 80.00

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% Explant respondin % Explant 20.00

0.00 BA 4.44 2iP 19.69 Kin 18.59 PGR-free Cytokinin concentration (µ M)

Cytokinin Cytokinin + NAA PGR-free

5.00 B 1.00 C

4.00 0.80

3.00 0.60

2.00 0.40

1.00 Shoot length (cm) 0.20 Shoots / response explant 0.00 0.00 BA 4.44 2iP 19.69 Kin 18.59 PGR-free BA 4.44 2iP 19.69 Kin 18.59 P GR-free Cytokinin concentration (µM) Cytokinin cencentration (µM)

Cytokinin Cytokinin + NAA PGR-free Cytokinin Cytokinin + NAA PGR-free

Fig. 2. Effect of cytokinins and 0.54 µM NAA on shoot induction and shoot elongation of Mallotus repandus node and shoot tip explants. Vertical lines indicate S.E.

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100.00 A 4.00 B 4.00 t 3.50 3.50 80.00 ) 3.00 3.00

2.50 2.50 60.00 2.00 2.00

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% Rooting 1.00 1.00 Length / root (cm 0.50 0.50 20.00 Roots / regenerated shoo Roots 0.00 0.00 PGR-free 10.74 21.48 32.23 42.97 53.71 0.00 PGR-free 10.74 21.48 32.23 42.97 53.71 NAA concentrati on (µM) NAA concentration (µM) Roots / regenerated shoot Length / root (cm)

Fig. 3. Effect of NAA on rooting frequency, root induction rate and root length of regenerated shoots of Mallotus repandus. Vertical lines indicate S.E.

A C

B

Fig. 4. In vitro propagation of Mallotus repandus. (A) Shoot induction after 8 weeks of culture on MS medium containing 4.44 µM BA, (B) A regenerant, (C) A regenerated plant grown under greenhouse conditions, 4 weeks after transplantation. Scale bars = 1 cm.

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