Callogenesis and Organogenesis from Inflorescence Segments of Curcuma Alismatifolia and Curcuma Hybrid Laddawan

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1 2013 American Transactions on Engineering & Applied Sciences. American Transactions on Engineering & Applied Sciences Anchalee Jala a* Callogenesis and Organogenesis from Inflorescence Segments of Curcuma Alismatifolia and Curcuma Hybrid Laddawan a Department of Biotechnology, Faculty of Science and Technology, Thammasat University, THAILAND A R T I C L E I N F O Article history: Received February 21, 2013 Received in revised form April 22, 2013 Accepted May 03, 2013 Available online May 09, 2011 Keywords: Curcuma sp.; Callogenesis; Shoot organogenesis; 2,4-Dichlorophenoxy acetic acid. A B S T R A C T Callogenesis of Curcuma alismatifolia and Curcuma hybrid Laddawan were proliferated from young inflorescences when cultured them on Murashige and Skoog (MS) medium supplemented with various concentration (8, 10, 12, and 14 mg/l) 2,4-dichlorophenoxyacetic acid (2,4-D) at dark period for 4 weeks to form somatic embryo. The highest percentage growth rate is 90 percent for somatic embryo proliferated on MS medium supplemented with 14 mg/l 2,4-D. When induced these somatic embryo to form new shoot organogenesis by culturing them on MS medium supplemented with various types ( glucose, sucrose and maltose ) of sugar, the highest percentage of new shoots were proliferated from Curcuma alismatifolia and Curcuma hybrid Laddawan with 0.25 g/l maltose which were and 46.67, respectively Am. Trans. Eng. Appl. Sci. 1. Introduction The genus Curcuma (Zingiberaceae) comprises more than 80 species of rhizomatous perennial *Corresponding author (A. Jala). Tel/Fax: Ext address: anchaleejala@yahoo.com American Transactions on Engineering & Applied 213 Sciences. Volume 2 No. 3 ISSN eissn Online Available at

2 herbs and is widespread in tropical Asia, extending to Africa and Australia (Purseglove et al. 1981). Within the genus Curcuma, Curcuma alismatifolia and Curcuma hybrid Laddawan is a highly valued ornamental, which is indigenous to Burma and Thailand (Apavatjrut et al. 1996; Schaffer et al. 2011). It is considered as a novel ornamental plant in the floricultural market and grows well after having been introduced to Guangzhou, China. It is usually used in tropical and subtropical landscape design as a potted, foliage, and flower plant. It has a long flowering period (April to November) and vase cut flowers can last 20 days. For these reasons it has seen an increasing demand in the world ornamental market. However, seeds obtained are limited and can t germinate during cold season due to their dormancy, as traditional multiplication involves propagation by rhizome. Since it is difficult to keep pace with the demands created by market exploitation, it is thus essential to establish an efficient propagation and regeneration system, preferably in vitro, to ensure the clonal propagation of desired genotypes. In this study, anthers were used as explants to induce somatic embryo, which was then used to form shoots organogesis. This study reports on an efficient callus and plant regeneration for Curcuma alismatifolia and Curcuma hybrid Laddawan. 2. Materiasls and Method Young inflorescence of Curcuma alismatifolia and Curcuma hybrid Laddawan were used as explants and cleaned surface with liquid detergent and washed with tap water for 2 min., immersed in 70% alcohol for 5 min. and sterilized with 20% sodium hypochlorite for 20 min. then with 5% sodium hypochlorite for 10 min and immersed in sterile distilled water 3 times for 2 min each. Petal and Sepal and the end of inflorescence were removed from the sterilized inflorescence. Sterilized inflorescence about cm long sections were inoculated on Murashige and Skoog (MS.1962) supplemented with 2mg/l BA. Explants were incubated on agarified MS medium. All medium contained 30 g m/l sucrose and ph was adjusted to 5.8 with 1.0 N HCl or 1.0 N NaOH before adding 2.5 gram Phytagel (Sigma, USA) and autoclaving at 121 C for 20 min and incubated at culture room at 27 ± 2 C exposed to 60 µmol m-2 s-1 fluorescent light (TLD 36W/ lm Philips Thailand) in a 16-hour photoperiod. All cultures were subcultured every two weeks, for three times to multiply callus. 214 Anchalee Jala

3 2.1 Somatic Embryo Induction Proliferated callus was transferred to MS medium supplemented with various concentration (2, 4, 6, 8, 10, 12 and 14 mg/l ) 2,4-D, 3% sucrose and ph was adjusted to 5.8 with 1.0 N HCl or 1.0 N NaOH before adding 2.5 gram Phytagel (Sigma, USA) and autoclaving at 121 C for 20 min and incubated at culture room at 27±2 C in dark period for four weeks to induce somatic embryogenesis. Percentage number of somatic embryos is recorded. 2.2 Shoot Organogenesis Callus from Curcuma alismatifolia and Curcuma hybrid Laddawan which cultured on MS medium supplemented with 14 mg/l 2,4-D and 0.25 gram per liter of different types of sugar (glucose, sucrose, maltose ) and ph was adjusted to 5.8 with 1.0 N HCl or 1.0 N NaOH, before adding 2.5 gram Phytagel (Sigma, USA) and autoclaving at 121 C for 20 min and incubated at culture room at 27 ± 2 C exposed to 60 µmol m-2 s-1 fluorescent light (TLD 36W/ lm Philips Thailand) in a 16-hour photoperiod. All cultures were subcultured every three weeks for four times. Shoot proliferation was investigated after culturing for 45 days. 3. Data Analysis Somatic embryo experiment was used Randomized Complete Block Design with 8 treatments three replicates and each replication was 10 explants. Shoot Induction was used Randomized Complete Block Design with three treatments three replicates and each replication was 10 explants. Means were analyzed by Analysis of Variance and significant differences between means were compared by the Duncan s New Multiple Range Test (DMRT) using SPSS v For all comparisons, statistical significance was considered at p< Result Somatic embryos from Curcuma alismatifolia and Curcuma hybrid Laddawan were proliferated after cultured on MS medium supplemented with different concentration of 2,4-D in dark period for 4 weeks. It was found that number of somatic embryos proliferated on MS medium supplemented with 8, 10, 12 and 14 mg/l 2,4 D. When compared average percentage *Corresponding author (A. Jala). Tel/Fax: Ext address: anchaleejala@yahoo.com American Transactions on Engineering & Applied Sciences. Volume 2 No. 3 ISSN eissn Online Available at 215

4 Table 1 Effect of 2,4-D which induced somatic embryo from young inflorescence of Curcuma alismatifolia and Curcuma hybrid Laddawan after cultured for 4 weeks. 2,4-D (mg/l) Percentage of somatic embryo proliferated from young inflorescence of Curcuma (%) C. alismatifolia** C. hybrid Laddawan.** e 0.00 d e 0.00 d e 0.00 d e 0.00 d d c c bc b b a a ** - highly Significant difference p 0.01 abc - in the same row was not significant difference when compared their mean with DMRT at p 0.01 Figure 1 Young inflorescence Curcuma alismatifolia and Curcuma hybrid Laddawan cultured on MS Medium supplemented with 12 and 14 mg/l 2,4-D after cultured for 4 weeks 1a.Compact callus ( C. alismatifolia) proliferated on MS medium supplemented with 12 mg/l 2,4-D 1b.Friable callus(c. alismatifolia) proliferated on MS medium supplemented with 14 mg/l 2,4-D 1c.Compact callus(c. hybrid Laddawan ) proliferated on MS medium supplemented with 12 mg/l 2,4-D 1d. Friable callus (C. hybrid Laddawan) proliferated on MS medium supplemented with 14 mg/l 2,4-D. number of somatic embryos in statistic. They were highly significant difference (p<0.01) and in MS medium supplemented with 14 mg/l 2,4-D gave the highest average percentage number of somatic embryos in both Curcuma alismatifolia and Curcuma hybrid Laddawan which are 90 as shown in Table 1. When compared type of callus in 12 and 14 mg/l 2,4-D in each Curcuma alismatifolia and Curcuma hybrid Laddawan, they showed that characteristic of callus which proliferated in 12 mg/l 2,4-D their cells are smaller and more compact than from 14 mg/l 2,4-D as shown in Figure Anchalee Jala

5 The callus originated from the young inflorescence surface and not from inner tissue in Curcuma alismatifolia (Figure 1a) and Curcuma hybrid Laddawan (Figure 1c). The callus which proliferated from MS medium supplemented with 14mg/l 2,4-D are friable, their cells are bigger and uninucleated which ready to form somatic embryo in both Curcuma alismatifolia (Figure 1.b) and Curcuma hybrid Laddawan (Figure 1d). 4.1 Shoot Induction from Somatic Embryo The callus originated from the young inflorescence surface and not from inner tissue in Curcuma alismatifolia and Curcuma hybrid Laddawan and formed somatic embryo.when culture these somatic embryo on MS medium supplemented with 0.25g/l different types of sugar (glucose sucrose and maltose) for 12 weeks. It was found that somatic embryo responded to different types of sugar. They proliferated to young shoot in MS medium supplemented with different type of sugar. When compared the result in statistic, induction shoot from MS medium supplemented with 0.25g/l maltose was highly significant difference (p 0.01) from glucose and sucrose as shown in Table 2. The highest average percentage of shoot induction in Curcuma alismatifolia and Curcuma hybrid Laddawan were and 46.67, respectively. Table 2 Shoot Induction of Curcuma alismatifolia and Curcuma hybrid Laddawan from somatic embryo which cultured on MS medium supplemented with different types of sugar after cultured for 12 weeks. Types of sugar Percentage of shoot induction (%) (0.25 g/l) C. alismatifolia ** C. hybrid Laddawan ** No sugar 0.00 c 0.00 c glucose b c sucrose b b maltose a a ** - highly Significant difference p 0.01 abc - in the same row was not significant difference when compared their mean with DMRT at p 0.01 *Corresponding author (A. Jala). Tel/Fax: Ext address: anchaleejala@yahoo.com American Transactions on Engineering & Applied Sciences. Volume 2 No. 3 ISSN eissn Online Available at 217

6 Figure 2 Shoot induction from somatic embryo on MS medium supplemented with 0.25g/l maltose on Curcuma alismatifolia and Curcuma hybrid Laddawan after culturing 12 weeks: (a) Somatic embryo proliferation in C. alismatifolia (b) Differentiation of Somatic embryo in C. alismatifolia (c) Shoot proliferation in C. alismatifolia (d) Somatic embryo proliferation. In C. hybrid Laddawan (e) Differentiation of Somatic embryo in C. hybrid Laddawan (f) Shoot proliferation in C. hybrid Laddawan 5. Discussion Young inflorescence of Curcuma alismatifolia and Curcuma hybrid Laddawan cultured on MS medium supplemented with different concentration of 2,4-D in dark period for 4 weeks showed different responses. Callus proliferated on MS medium supplemented with 8, 10, 12 and 14 mg/l 2,4-Dand MS medium supplemented with 14mg/l 2,4-D gave the highest average percentage of friable callus which were 90 in both curcuma. As worked of Yaping et al. (2013) showed that 2,4-D in the induction medium could induce a high percentage of callus (56.2 %). This callus was compact and friable. In C. alismatifolia, regeneration has been reported from young inflorescences (Wannakrairoj, 1997). Also, Toppoonyanont et al. ( 2005) had reported that C. alismatifolia inflorescences were used as explants and, when inoculated on MS basal medium containing 44.0M BA and 0.57M NAA for 1 month they developed and reverted to vegetative shoots directly from flower organs (or floral buds) and not via callus. Mohanty et al. (2008) reported that plant regeneration from callus cultures of C. aromatica was possible with 2,4-D (9.1M) and 2.3M KT. Callus could be induced from the base of adventitious shoots of C. kwangsiensis on MS medium containing 1.4M TDZ, 4.4M BA and 2.3M 2,4-D (Zhang et al. 2011). Plant regeneration from the culture of immature of C. longa inflorescences was possible by direct shoot development on MS basal medium supplemented with BA (22.0 or 44.0M) in combination with 0.9M 2,4-D or 0.54M NAA and 4.5 or 9.0M TDZ in combination with 0.57M 218 Anchalee Jala

7 IAA (Salvi et al. 2000). Moreover, they located at the same positions and were arranged spirally within the bracteole, similarly to ex vitro shoots (Udomdee et al. 2003). All of these reports showed that callus were induced from different explants by using of 2,4-D single or NAA in combination with BA, KT or TDZ. The callus doubled in size in less than two weeks when subcultured in a fresh MS medium supplemented with 1 mg/l 2,4-D. In this treatment, the concentration was maintained at 1 mg/l due to its response in producing favorable rapid results. In plant tissue culture, sucrose is the most commonly used carbohydrate source because of the wide spread of this disaccharide as a transporter molecule, and its high solubility in water. Many in vitro studies have proven that sucrose supports near optimum rates of growth and also plays multiple roles in the provision of carbon and energy that promotes cell growth and division (Balachandran et al. 1990). Viu et al. (2009) reported that Callus was suspended in a liquid medium to enhance proliferation. For initiation, only friable callus was used. Prior to any new treatment, 1 g of the friable callus was proliferated in a liquid medium with same concentration as callus induction medium (MS medium supplemented with 1mg l-1 2,4-D and 30g /l sucrose). The highest average percentage of shoot proliferated from somatic embryo of Curcuma alismatifolia and Curcuma hybrid Laddawan, when cultured on MS medium supplemented with 0.25g per liter of maltose which are and 46.67, respectively. But Wannakrairoj(1997) and Jala, (2012) reported that plant regeneration from the culture of immature of C. longa inflorescences was possible by direct shoot development on MS basal medium supplemented with BA (22.0 or 440 M) in combination with 0.9 M 2,4-D in combination with 30g/l sucrose. And the same as Zhang et al (2011) report that callus could be induced from the base of adventitious shoots of C. kwangsiensis on MS medium containing 1.4M TDZ, 4.4 M BA and 2.3M 2,4-D. Normally they used sucrose for carbon source. Sucrose is the most commonly used carbohydrate in plant tissue or cell culture (Vu et al., 1993). Many factors affecting the growth of excised plant organs, tissues and cells in vitro have been studied extensively over the last 30 years, including plant growth regulators, light, temperature, medium ph, humidity, gas exchange and the presence of harmful microorganisms (Leifert et al., 1995). On the other hand, plant carbohydrate *Corresponding author (A. Jala). Tel/Fax: Ext address: anchaleejala@yahoo.com American Transactions on Engineering & Applied Sciences. Volume 2 No. 3 ISSN eissn Online Available at 219

8 requirements in vitro have rarely been studied in detail and still are poorly understood (Leifert et al., 1995; Swedlund & Locy, 1993). It has also been well documented that certain plant tissues may contain and/or utilize different carbohydrates at the same time. It is then not surprising that carbon sources other than sucrose might be effective in promoting in vitro tissue specific growth responses in a given species (Swedlund & Locy, 1993). Furthermore, several reports have pointed out these carbon sources as inducers of cell differentiation in some plant species (Lemos & Blake, 1996; Karhu, 1997; Tamil et al. 2012). Galactose, glycerol and sorbitol are examples of alternative carbon sources that have been tested in vitro for different purposes in a wide range of plant species (Swedlund et al.1993). 6. Conclusion Young inflorescence of Curcuma alismatifolia and Curcuma hybrid Laddawan could proliferated to somatic embryo when cultured on MS medium supplemented with 8, 10, 12, and 14 mg/l 2,4-D in dark condition for 4 weeks and got the highest average percentage at 14mg/l 2,4-D which are 90 percent. Somatic embryo proliferated to shoot organogenesis from friable callus that cultured on MS medium supplemented with 0.25g/l maltose. The highest average percentage of shoot proliferated in Curcuma alismatifolia and Curcuma hybrid Laddawan were and 46.67, respectively. 7. References Apavatjrut P, T. Sirisawad, P. Sirirugsa, P. Voraurai,C. Suwanthada. (1996) Studies on chromosome number of seventeen Thai Curcuma species. In: Proceedings of 2nd national conference on flower and ornamental plant. 2: Balachandran, S.M., S.R. Bhat, and K.P.S. Chandel In vitro clonal multiplication of turmeric (Curcuma spp.) and ginger (Zingiber officinale Rosc). Plant Cell Reports. 8: Jala, A. (2011) Effects of NAA BA and Sucrose On Shoot Induction and Rapid Micropropagation by Trimming Shoot Of Curcuma Longa L. American Transactions on Engineering & Applied Sciences, 3: (2) : Karhu, S.T. (1997) Sugar use in relation to shoot induction by sorbitol and cytokinin in apple. Journal of the American Society for Horticultural Science. 122: Leifert, C., K.P. Murphy, P.J. Lumsden. (1995) Mineral and carbohydrate nutrition of plant 220 Anchalee Jala

9 cell and tissue cultures. Critical Reviews in Plant Science.14: Lemos, E.E.P., J. Blake. (1996) Micropropagation of juvenile and mature Annona muricata L. Journal of Horticultural Science.71: Mohanty S, M.K. Panda, E. Subudhi, S. Nayak. (2008) Plant regeneration from callus culture of Curcuma aromatica and in vitro detection of somaclonal variation through cytophotometric analysis. Biol Plant 52: Murashige, T. and F. Skoog. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum. 15: Purseglove JW, E.G. Brown, C.L. Green, S.R. Robbins. (1981) Spices. Longman, London, New York. Salvi N.D., L. Geoge, S. Eapen. (2000) Direct regeneration of shoots from immature inflorescence cultures of turmeric. Plant Cell Tissue Org Cult. 62: Schaffer M, P.M. Schaffer, J. Zidan, G.B. Sela. (2011) Curcuma as a functional food in the control of cancer and inflammation. Curr Opin Clinical Nutrition Metabolic Care. 4: Swedlund B and R.D. Locy. (1993) Sorbitol as the primary carbon source for the growth of embryogenic callus of maize. Plant Physiol. 103: Tamil C.M. Sundram, M. Suffian, M. Annuar and Norzulaani Khali.( 2012) Optimization of culture condition for callus induction from shoot buds for establishment of rapid growing cell suspension cultures of Mango ginger (Curcuma mangga) Australian Journal of Crop Science. 6(7): Toppoonyanont N, Chongsang S, Chujan S, Somsueb S, Nuamjaroen P. (2005) Micropropagation scheme of Curcuma alismatifolia Gagnep. Acta Hort (ISHS) 673: Udomdee W, S. Fukai, L. Petpradap, J.A. Teixeira da Silva. (2003) Curcuma: studies on tissue culture, pollen germination and viability, histology and flow cytometry. Prop Ornamental Plants. 3: Viu A.F.M., M.A.O. Viu, A.R. Tavares, F. Vianello, G.P.P. Lima. (2009) Endogenous and exogenous polyamines in the organogenesis in Curcuma longa L. Sci Hortic. 121: Vu, J.C.V.; R.P. Niedz; G. Yelenosky. ( 1993) Glycerol stimulation of chlorophyll synthesis, embryogenesis and carboxylation and sucrose metabolism enzymes in nucellar callus of "Hamlin" sweet orange. Plant Cell, Tissue and Organ Culture. 33: Wannakrairoj S (1997) Clonal micropropagation of patumma (Curcuma alismatifolia gagnep). *Corresponding author (A. Jala). Tel/Fax: Ext address: anchaleejala@yahoo.com American Transactions on Engineering & Applied Sciences. Volume 2 No. 3 ISSN eissn Online Available at 221

10 Witthayasan Kasetsart. 31: Yaping Kou; Guohua Ma, A. Jaime, Teixeira da Silva, Nian Liu. (2013). Callus induction and shoot organogenesis from anther cultures of Curcuma attenuata Wall. Plant Cell, Tissue and Organ Culture (PCTOC).112 : 1-7. Zhang S.J, A.W. Liu N, G.H. Sheng, Ma, G.J. Wu. (2011) In vitro plant regeneration from organogenic callus of Curcuma kwangsiensis Lindl. (Zingiberaceae). Plant Growth Regul. 64: Dr.Anchalee JALA is an Associate Professor in Department of Biotechnology, Faculty of Science and Technology, Thammasat University, Rangsit Campus, Pathumtani, THAILAND. Her teaching is in the areas of botany and plant tissue culture. She is also very active in plant tissue culture research. Peer Review: This article has been internationally peer-reviewed and accepted for publication according to the guidelines given at the journal s website. 222 Anchalee Jala