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Effect of Plant Growth Regulators on Micropropagation of Curcuma Aeruginosa Roxb

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Effect of Plant Growth Regulators on Micropropagation of Curcuma Aeruginosa Roxb THAI JOURNAL OF BOTANY 2 (Special Issue): 135-142. 2010. วารสารพฤกษศาสตรไทย 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 regulato rs 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).
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