Effect of Plant Growth Regulators on Micropropagation of Curcuma Aeruginosa Roxb

THAI JOURNAL OF BOTANY 2 (Special Issue): 135-142. 2010.

วารสารพฤกษศาสตรไทย 2 (ฉบับพิเศษ): 135-142. 2553.

Effect of plant growth regulators on micropropagation of Curcuma aeruginosa Roxb.

ORAWAN THEANPHONG1,*, THANAPAT SONGSAK1 & CHALERMPOL KIRDMANEE2

1 Faculty of Pharmacy, Rangsit University, Patumtani 12000, Thailand 2 National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Patumtani 12120, Thailand

ABSTRACT. Shoot explants of Curcuma aeruginosa Roxb. were cultured on Murashige and Skoog, 1962 (MS) agar medium supplemented with benzylaminopurine (BA) or Kinetin at the concentration of 1, 3, 5 and 7 mg/L, combination of BA (1, 3, 5 and 7 mg/L) and 0.5 mg/L naphthalene acetic acid (NAA) and combination of Kinetin (1, 3, 5 and 7 mg/L) and 0.5 mg/L NAA for 5 weeks. The results showed that MS agar medium supplemented with the combination of 1.0 mg/L BA and 0.5 mg/L NAA induced the highest number of new roots (10 roots/explant), new shoots (1.29 shoots/explant) and the highest length of shoot (3.56 cm).

KEYWORDS: Curcuma aeruginosa Roxb., benzylaminopurine, Kinetin, naphthalene acetic acid

INTRODUCTION plant are antiflatulent, antidysentery and antiulcer. Curcuma species belonging to the family Zingiberaceae have long been used In general, C. aeruginosa propagation as antifungal (Lee et al., 2007), antimicrobial rate is very low through underground (Bugno et al., 2007; Singh et al., 2002), rhizome. Thus, in vitro plant tissue culture antioxidant (Araujo et al., 2001; Mau et al., method is an interesting alternative to 2003; Cikrikci et al., 2008), anti-inﬂ ammatory accelerate the plant multiplication. In and analgesic agents (Mujumdar et al., 2000; addition, this method helps to maintain the Lee et al., 2007; Gupta et al., 2008). uniform and consistent production of true- to-type plants within a short period of time Curcuma aeruginosa Roxb. is 30-40 cm (Selvakkumar et al., 2007). in height. Its leaves are alternate, entire, red leaf sheath and red midrib. The rhizomes are In vitro multiplication of some Curcuma bullish violet. The ethnomedical uses of this species such as C. zedoaria, C. aromatica and

* Corresponding author: [email protected] 136 Orawan Theanphong et al.

C. longa has already been reported (Nayak, mg/L) of BA, Kinetin, either alone or in 2000; Islam et al., 2004; Miachir et al., combination with 0.5 mg/L NAA. The 2004). However, C. aeruginosa, an important concentrations of plant growth regulators species and medicinal plant, has not received were tabulated in Table 1. The pH of the the attention from tissue culture and no medium was adjusted to 5.7 with 2M KOH reports are available on in vitro multiplication before adding agar (10 g/L). Medium without of this important crop. Thus, the aim of this plant growth regulators were used as the work was to develop an efﬁ cient protocol control. All cultures were maintained at 25 for C. aeruginosa shoot multiplication ± 1°C with 12-hr photoperiod for 5 weeks. production. This technique would facilitate The effectiveness of the plant growth an alternative method for rapid large-scale regulators were evaluated from the numbers clonal propagation outdoor establishment of root and new shoot per explant and shoot and ex situ conservation of this plant. length.

MATERIALS AND METHODS TABLE 1. Different concentration of plant Preparation of plant material growth regulators in the MS agar medium

Rhizomes of C. aeruginosa were obtained Plant growth regulators Cytokinin Auxin in June 2008 from Ratchaburi province, Medium Thailand. The rhizomes were cleaned with Kinetin NAA BA (mg/L) detergent before washing with tap water. (mg/L) (mg/L) They were surface sterilized with 0.2 % w/v M 1(control) - - - mercuric chloride solution and a few drops M 2 1 - - of Tween-20 for 8 min and thoroughly M 3 3 - - washed ﬁ ve times with sterile distilled water. M 4 5 - - Under aseptic conditions, the outer leaves M 5 7 - - were removed from the shoot tips. The M 6 - 1 - explants (1-1.5 cm long) consisting of a shoot M 7 - 3 - tip with a small portion of the rhizome were M 8 - 5 - used in this study. M 9 - 7 - M 10 1 - 0.5 In vitro multiplication M 11 3 - 0.5 The shoot explants were inoculated onto M 12 5 - 0.5 Murashige and Skoog, 1962 (MS) agar M 13 7 - 0.5 medium supplemented with 30 g/L sucrose M 14 - 1 0.5 for 2 weeks and then transferred to MS agar M 15 - 3 0.5 medium supplemented with 30 g/L sucrose M 16 - 5 0.5 and different concentrations (1, 3, 5 and 7 M 17 - 7 0.5 Eݦ ect oݦ plant growth regulators on micropropagation oݦ Curcuma aeruginosa 137

Statistical analysis cell differentiation in the root meristem. Cytokinins have been demonstrated to The experiment was completely determine root meristem size by controlling randomized design (CRD) with three cell differentiation rate at the vascular tissue replications. The numbers of root, new shoot transition zone (Dello et al., 2007; Perilli et per explant and shoot length were collected al., 2010). The balance between the rate of from 30 explants per treatment. The data cell division in the division zone and the rate were presented in the form of mean ± S.D. of cell differentiation at the transition zone The ANOVA was performed for analysis of determines root meristem size and the overall the data obtained for each experiment and rate of root growth (Dello et al., 2007; Perilli the means were tested by Duncan’s Multiple et al., 2010). Range Test (p < 0.05). The supplement of cytokinin and auxin

RESULTS AND DISCUSSION at low concentration into MS agar medium increase the number of new root and shoot The effects of various plant growth per explant and shoot length produced. It may regulators on the production of new roots, result from the synergistic effects of the new shoots per explant and the shoot length cytokinin and auxin at low concentration on are showed in Table 2. promoting the shoot proliferated and growth of cells. Coenen & Lomax (1997) reported Supplementation of the combination of that cytokinin and auxin act together 1 mg/L BA and 0.5 mg/L NAA (M 10) was to control various physiological and the most effective for initiating the highest development responses. Similar results were new shoots (1.29 ± 0.09 per explant) and new also observed by Hosoki & Sagawa (1997); roots (10 ± 0.11 per explant). In addition, this Shirin et al. (2000); Rahman et al. (2004) medium gave the highest average length of and Loc et al. (2005). shoot 3.56 ± 0.16 cm. Hormones free MS agar medium (M 1) was used as a control, This is the ﬁ rst report showed the effect which not induce new shoot. Figs. 1 & 2 of plant growth regulators: BA, Kinetin showed that the medium was supplemented and NAA, on micropropagation of with higher concentrations of BA (M 3-5, M C. aeruginosa shoot tip. 11-13) and Kinetin (M 7-9, M 15-17), a number of shoot multiplication and elongation CONCLUSION were found to be lower. This indicated that cytokinin increased the number of proliferated The micropropagation of C. aeruginosa shoots but retarded shoot elongation was established using MS agar media (Prathanturarug et al., 2004). Kyozuka supplemented with cytokinin and auxin at (2007) reported that the activity of cytokinins low concentration (1 mg/L BA and 0.5 mg/L was essential to maintain undifferentiated NAA). This condition gave the highest new cells in shoot apical meristem and to promote shoot, new root and the highest average 138 Orawan Theanphong et al.

FIGURE 1. Effect of plant growth regulators on shoot length (M1-M17 represents the different concentration of plant growth regulators in the MS agar medium) Eݦ ect oݦ plant growth regulators on micropropagation oݦ Curcuma aeruginosa 139

FIGURE 2. Effect of plant growth regulators on root initiation (M1-M17 represents the different concentration of plant growth regulators in the MS agar medium) 140 Orawan Theanphong et al.

TABLE 2. Effect of the plant growth regulators on the number of root and new shoot per explants and shoot length of C. aeruginosa

Medium shoot length No. of root / explants No. of new shoot / (cm ± S.D.) (mean ± S.D.) explants (mean ± S.D.) M 1 (control) 1.17 ± 0.18a 0.14 ± 0.38 a 0.00 a M 2 2.49 ± 0.79abcd 3.86 ± 0.35bcde 0.57 ± 0.13abc M 3 1.24 ± 0.87a 1.57 ± 0.99abc 0.71 ± 0.09bcd M 4 1.77 ± 0.85ab 0.86 ± 0.26ab 0.29 ± 0.16abc M 5 1.76 ± 0.79ab 1.29 ± 0.98abc 0.29 ± 0.09abc M 6 2.21 ± 0.45abcd 4.23 ± 0.72cde 0.43 ± 0.13abc M 7 3.33 ± 0.33cd 2.43 ± 0.90abcd 0.43 ± 0.13abc M 8 3.23 ± 0.64cd 2.00 ± 0.15abcd 0.29 ± 0.09abc M 9 2.74 ± 0.18bcd 1.86 ± 0.90abcd 0.29 ± 0.09abc M 10 3.56 ± 0.16d 10.00 ± 0.11 f 1.29 ± 0.09d M 11 2.46 ± 0.27abcd 5.71 ± 0.21 e 0.86 ± 0.19cd M 12 1.74 ± 0.77ab 3.71 ± 0.42bcde 0.71 ± 0.16bcd M 13 1.40 ± 0.97ab 0.71 ± 0.76ab 0.29 ± 0.09abc M 14 2.61 ± 0.11abcd 4.86 ± 0.18de 0.29 ± 0.09abc M 15 2.61 ± 0.48abcd 3.29 ± 0.76abcde 0.29 ± 0.16abc M 16 1.94 ± 0.83abc 2.86 ± 0.21abcde 0.29 ± 0.09abc M 17 1.64 ± 0.14ab 2.14 ± 0.90abcd 0.14 ± 0.08ab Mean in the column followed by the difference latter are signiﬁ cantly different at the 0.05 probability level according to Duncan’s Multiple Range Test (p < 0.05). length of shoot. The present experiment REFERENCES described a micropropagation protocol for Araujo, C.A.C. & Leon L.L. 2001. Biological the rapid propagation of C. aeruginosa. activities of Curcuma longa. Memórias do Instituto Oswaldo Cruz 96(5): 723–728. ACKNOWLEDGEMENT Bugno, A., Nicoletti, M.A., Almodovar, A.A.B., Pereira, T.C. & Auricchio, M.T. 2007. An- This work was supported by the program timicrobial activity of Curcuma zedoaria for Pharmaceutical senior project in Rangsit extract as assesses by linear regression University in 2008 and the authors are compared with commercial mouthrinses. grateful to Dr. Thaya Jenjittikul, Department Brazilian Journal of Microbiology 38: of Plant Science, Faculty of Science, Mahidol 440–445. University for her kindness, meaningful advice and valuable information. Eݦ ect oݦ plant growth regulators on micropropagation oݦ Curcuma aeruginosa 141

Cikrikci, S., Mozioglu, E. & Yilmaz, H. 2008. Loc, N.H., Due, D.T., Kwon, T.H. & Yang, M.S. Biological activity of curcuminoids isolated 2005. Micropropagation of zedoary from Curcuma longa. Records of Natural (Curcuma zedoaria Roscoe) – a valuable Products 2(1): 19–24. medicinal plant. Plant Cell Tissue and Coenen, C. & Lomax, T.L. 1997. Auxin-cytokinin Organ Culture 81: 119–122. interactions in higher plants: old problem Mau, J., Lai, E.Y.C., Wang, N., Chen, C., Chang, and new tools. Trends in Plant Science C. & Chyau, C. 2003. Composition 2(9): 351–356. and antioxidant activity of the essential Dello, I.R., Nakamura, K., Moubayidin L., Perilli, oil from Curcuma zedoria. Journal of S., Taniguchi, M., Morita, M.T., Aoyama, Ethnopharmacology 82: 581–591. T., Costantino, P. & Sabatini, S. 2007. Miachir, J.I., Romani, V.L.M., Amaral, A.F.C., Cytokinin determine Arabidopsis root- Meool, M.O., Crocomo, O.J. & Melo, M. meristem size by controlling cell 2004. Micropropagation and callogenesis of differentiation. Current Biology 17: 678– Curcuma zedoaria Roscoe. Scientia Agricola 682. (Piracicaba, Brazil) 61: 427–432. Gupta, S.K., Agarwal, R., Srivastava, S., Mujumdar, A.M., Naik, D.G., Dandge, C.N. & Agarwal, P., Agrawal, S.S., Saxena, R. & Puntambekar, H.M. 2000. Antiinﬂ ammatory Galpalli, N. 2008. The anti-inﬂ ammatory activity of Curcuma amada Roxb. in albino effects of Curcuma longa and Berberis rats. Indian Journal of Pharmacology 32: aristata in endotoxin-induced uveitis in 375–377. rabbits. Investigative Ophthalmology and Murashige, T. & Skoog, F. 1962. A revised medium Visual Science 49: 4036–4040. for rapid growth and bioassays with tobacco Hosoki, T. & Sagawa, Y. 1977. Clonal propagation tissue cultures. Physiologia Plantarum of ginger (Zingiber officinale Rosc.). 15: 473–497. Horticulture Science 12: 451–452. Nayak, S. 2000. In vitro multiplication and Islam, M.A., Kloppstech, K. & Jacobsen, H.J. microrhizome induction in Curcuma 2004. Efficient procedure for In vitro aromatica Salisb. Plant Growth Regulation microrhizome induction in Curcuma longa 32:41–47. L. (Zingiberaceae) – a medicinal plant of Perilli, S., Moubayidin, L. & Sabatini, S. 2010. Tropical Asia. Plant Tissue Culture 14: The molecular basis of cytokinin function. 123–134. Current Opinion in Plant Biology 13: Kyozuka, J. 2007. Control of shoot and root 21–26. meristem function by cytokinin. Current Prathanturarug, S., Angsumalee, D., Pongsiri, N., Opinion in Plant Biology 10: 442–446. Suwacharangoon, S. & Jenjittikul, T. 2004. Lee, Y., Weng, C. & Mau, J. 2007. Antioxidant In vitro propagation of Zingiber petiolatum properties of ethanolic and hot water extracts (Holttum) I. Theilade, A rare zingiberaceous from the rhizome of Curcuma aromatica. plant from Thailand. In Vitro Cellular & Journal of Food Biochemistry 31: 757– Development Biology-Plant 40: 317–320. 771. 142 Orawan Theanphong et al.

Rahman. M.M., Amin, M.M., Jahan, H.S. & Shirin, F., Kumar, S. & Mishra, Y. 2000. In vitro Ahmed, R. 2004. In vitro regeneration of plantlet production system for Kaemferia plantlets of Curcuma longa Linn. a valuable galanga, a rare Indian medicinal herb. Plant spice plant in Bangladesh. Asian Journal Cell Tissue and Organ Culture 63: 193– of Plant Science 3: 306–309. 197. Selvakkumar, C., Balakrishnan, A. & Lakshmi, Singh, R., Chandra, R., Bose, M. & Luthra, P.M. B.S. 2007. Rapid in vitro microprapagation 2002. Antimicrobial activity of Curcuma of Alpinia ofﬁ cinarum Hance, an important longa rhizome extract on pathogenic medicinal plant, through rhizome bud bacteria. Current Science 83(6): 737–740. explants. Asian Journal of Plant Science 6: 1251–1255.