Callus Induction and Influence of Culture Condition and Culture Medium on Growth of Thai Aromatic Rice, Khao Dawk Mali 105, Cell Culture

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1 World Applied Sciences Journal 5 (2): , 2008 ISSN IDOSI Publications, 2008 Callus Induction and Influence of Culture Condition and Culture Medium on Growth of Thai Aromatic Rice, Khao Dawk Mali 05, Cell Culture 2 3 Jaruwan Summart, Sanha Panichajakul, Preecha Prathepha and Pornthap Thanonkeo Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand 2 Department of Biotechnology, Faculty of Technology, Mahasarakham University, Mahasarakham 44000, Thailand 3 Department of Biotechnology, Fermentation Research Center for Value Added Agricultural Products, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand Abstract: The effect of different concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D) (, 2, 3, 4 and 5 mg l ) on callus induction of Thai aromatic rice seeds variety KDML05 cultivated on MS medium was investigated. The supplementation of medium with 2 mg l 2,4-D was found most efficient for callus induction, provided calli with quite good in texture and friable in nature. The influences of incubation temperatures (25±2 C and 30±2 C), light (dark and light), sucrose concentrations (2, 3 and 4% (w/v)) and culture media (MS, Gamborg-B5 (B5), Linsmair and Skoog (LS) and Chu medium (N6)) on growth of rice callus were investigated. Although sucrose at 4% and the N6 medium gave the highest growth of callus but the high quality of callus was obtained when it was grown on the MS medium containing 3% sucrose at 25 C under dark condition. Key words: Oryza sativa KDML05 Culture medium 2, 4-D INTRODUCTION 6.9 billion Baht in revenues. It is known not only for its aroma but also for its good cooking, i.e., soft, tender and Rice (Oryza sativa L.) has become an important fluffy when cooked and eating qualities [2]. model system with immediate convenient of applications KDML05 is a photo-sensitive variety which because of its economic and nutritional importance could be grown only one a year [3]. It is also known as around the world [, 2]. Rice has several advantages as a a salt- and drought-sensitive, giving low yield and poor model plant, e.g., it has a relatively small genome grain milling quality when it was grown under salinity soil (~430 Mb) which has been completely sequenced [3, 4] or dry condition. Therefore, attempts are being made in and it can be genetically modified by various order to develop new varieties that are superior to transformation methods [5-7]. Although it has a higher KDML05. gene density than other cereals, genetic and physical During the past few decades biotechnological maps of rice show significant structural and functional techniques such as somaclonal variation, in vitro similarities to other cereals [8-0]. selection, production of doubled haploid lines from anther Khao Dawk Mali 05 (KDML05), commonly known culture and genetic transformation are being employed in in food markets as Jasmine Rice or Thai Hom Mali Rice, is rice development for the creation of novel rice varieties one of the indica-type rice. KDML05 is the most popular [4]. However, the production of embryogenic calli and its aromatic rice variety grown in Thailand. The rice is very subsequent regeneration are the basic prerequisites for much in demand and flavor for authentic dishes of the potential use of these techniques. Successful Thai people as well as international market particularly in embryogenic calli induction is depened on many factors Asia, Africa, Europe and USA []. In 200, KDML05 such as plant genotype, explant type, culture medium, was exported to other countries all over the world for as plant growth regulator and culture environment [5]. In much as 7.5 million tons which generate approximately order to develop a reproducible and an efficient procedure Corresponding Author: Dr. Pornthap Thanonkeo, Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand 246

2 World Appl. Sci. J., 5 (2): , 2008 for callus induction of KDML05, the influences of 2,4-D the best cultural conditions for callogenesis, the following concentration, incubation temperature, light, sucrose parameters were tested; incubation temparature (25±2 C concentration and culture media were evaluated in this or 30±2 C), light (dark and light conditions), sucrose study. concentration (2, 3 and 4% w/v) and culture medium (MS B5, LS and N6 medium). After cultivation, calli MATERIALS AND METHODS samples were collected at certain time intervals and growth was determined. Plant Material: The seeds of Oryza sativa L. cv. KDML05 used in this study were kindly provided by The Growth Measurement: Growth of calli was determined by International Training Center for Agricultural measuring the increase in cell dry weight (DW). The calli Development, Khon Kaen, Thailand. were placed on a petri dish and dried at 50 C in an oven to a constant weight. Results were expressed as DW (g). Sterilization of Rice Seeds: Seeds of Oryza sativa L. cv. KDML05 were carefully dehusked manually. The seeds Experimental Design and Statistical Analysis: The were washed with detergent and rinsed thoroughly with experiments were conducted using Completely tap water. They were surface sterilized in 95% ethanol for Randomized Design (CRD) with three replications. 3 min, 5% (v/v) sodium hypochlorite (Clorox ) solution Analysis of variance (ANOVA) was performed using containing Tween 20 as wetting agent for 5 min and 30% SPSS computer package and the results are means ± Clorox solution containing Tween 20 for 5 min. After standard deviation (SD). surface sterilization, the seeds were rinsed three times with sterile double distilled water and were used for RESULTS AND DISCUSSION induction of callus. Effect of 2,4-D Concentration on Callus Induction of Establishment of Callus Culture: Induction of KDML05 KDML05 Rice: The KDML05 rice seeds cultivated on rice callus was performed by placing sterilized rice seeds MS medium without 2,4-D supplementation produced on MS medium (0.8% w/v agar) containing 3% (w/v) only seedling without formation of callus. When the sucrose and supplemented with 2 mg l 2,4-D. The seeds seeds were cultivated on MS medium supplemented with were incubated at 25±2 C with a 6/8 h light/dark cycle 2,4-D, however, the formation of callus was observed, 2 (light intensity 20 µmole m s, cool white fluorescent suggesting that 2,4-D play a crucial role in callus TLD Philips 8W/33). Calli ( g) formed from scutellar induction of rice as described by Chen et al. [6], Maeda region were transferred onto a new fresh media at every [7] and Bajaj [8]. Callus was initiated from the basal 4 weeks intervals. Subculturing was performed within (mesocotyl or coleoptile) region of the germinated seeds 4 cycles in order to select for a fast-growing callus lines. after one week of cultivation. The proliferation of callus The frequency of callus induction was calculated was continued until the fourth week of culture, then the according to the following formula: frequency of callus induction and size of callus were determined. The results are summarized in Table. The Callus induction frequency (%) = supplementation with 3 mg l 2,4-D gave the maximum No. of seeds produced calli callus induction frequency (00%), followed by the 00 No. of seeds cultured supplementation with 2 mg l (96.67%) and mg l 2,4-D (95.56%), respectively. Morphology of the generated calli Effect of 2,4-D Concentration on Callus Induction of KDML05 Rice: Rice seeds were placed on MS medium Table : Effect of different concentrations of 2,4-D on callus induction and callus size supplemented with various concentrations of 2,4-D 2,4-D Frequency (0,, 2, 3, 4, and 5 mg l ). The seeds were incubated at concentration of callus Size of 25±2 C with a 6/8 h light/dark cycle for callus induction. (mg l ) induction (%) callus (mm)* Four weeks after culture, the frequency of callus induction (%) and size of callus [(width + length)/2] were recorded. Effects of Temperature, Light, Sucrose Concentration and Culture Medium on Callus Growth: To find out for ± ± ± ± ± ± ± ± ± ± ± ±3.0 *mean±sd 247

3 World Appl. Sci. J., 5 (2): , 2008 was varied depended on 2,4-D concentration. High condition promoted higher growth than that cultivated concentration of 2,4-D (>2.0 mg l ) resulted in yellowish, under dark condition [25]. The differences between our small or shorted and compact apperance. However, results with those previously reported are lightly due to calli generated on MS medium supplemented with 2 mg l the difference in rice genotype. 2,4-D exhibited good in texture (i.e., creamish, big size) and friable in nature as compared to other 2,4-D Effect of Sucrose Concentration on Callus Growth: concentrations tested (Fig. ). The results of this study Sucrose is generally used as the major source of carbon are in good agreement with those of Pandey et al. [9], and energy in culture media of rice [29-3]. In this study, Thadavong et al. [20] and Abeyaratne et al. [2] who the different concentrations of sucrose on cell growth of reported that 2,4-D at 2.0 mg l was better for callus KDML05 were tested and the results are shown in Fig. 4. induction from mature rice seeds. However, the responds The maximum growth was obtained when cells were of rice seeds to lower and higher 2,4-D concentration for cultured on MS medium containing 4% sucrose. However, callus induction were also reported [22, 23]. Raina [24] the morphology of cells on this medium was relatively dry reported that 2,4-D is the most suitable auxin for callus and compact in nature as compared with those observed induction of rice in tissue culture, however the optimum on the medium containing 2 and 3% sucrose (soft, concentration of 2,4-D varied depending on the explant creamish and friable). This cell type character may be source and genotype. caused by the influence of sucrose on the humidity of in vitro culture conditions [32]. Effect of Incubation Temperature on Callus Growth: No significant different in growth of calli (as cell dry weight) Effect of Culture Medium on Callus Growth: The was observed when they were cultured at 25±2 C or influence of different culture media (MS, LS, B5 and N6) 30±2 C (Fig. 2), but the callus morphology was remarkably on cell growth of KDML05 was investigated and the different. Calli grown at 25±2 C were relatively dry, results are shown in Fig. 5. The maximum average cell yellowish in color and compact in appearance, whereas mass was observed when cells were cultured on N6, calli grown at 30±2 C were soft, creamish in color and followed by LS, B5 and MS medium, respectively. These friable in nature (data not shown). These results results are similar to those of Niroula et al. [23] who suggested that temperature had an affect on KDML05 demonstrated that N6 medium was most efficient for callus cell morphology, like those observed in other rice induction, cell proliferation and plant regeneration from cultivars such as Nipponbare and Kitaake [25] or Hassawi seeds of various rice genotypes. Abdullah et al. [33] [26, 27]. reported that MS medium did not support growth of Morinda elliptica cell culture even though it supports the Effect of Light Condition on Callus Growth: The effect of production of anthraquinones. However, the MS medium light condition on growth of the KDML05 cell culture promotes both cell growth and jaceosidin production in was illustrated in Fig. 3. Calli grown under dark Saussurea medusa [34]. It is evident from these findings condition had higher cell mass than that under light that plant genotype and composition of the culture condition (6/8 h light/dark cycle). With respect to the medium influenced growth of plant cell culture. callus morphology, cells grown under light condition were Calli cultivated on MS and N6 medium were relatively relatively friable as compared to that grown under dark soft, creamish and friable in nature, whereas calli grown condition (data not shown). on LS and B5 were mainly compact. In addition, calli Light is a very important physical factor for callus cultivated on B5 also produced root which is not induction, cell growth and production of plant secondary flavorable for cells growth. Although N6 medium gave the metabolites. However, the degree of responsibility to light highest cell mass but the viability of cell was shorter than is depended on cell type and plant species. In glutinous that observed on MS medium. Further study in order to rice cultivar TDK, seeds cultured under light condition find out for the best culture medium for growth of gave higher average callus formation frequency (92.03%) KDML05 cells such as modification of the culture and larger average size of callus (3.88 mm) than those medium may be needed. Ogawa [35] and Rachmawati and cultured under dark condition (87.02% and 3.44 mm, Anzai [36] reported that C-modified medium is applicable respectively) [20]. Similar results were also observed in for callus induction and plant regeneration to a wider the indica rice cultivar RD6 [28]. In Japonica rice cultivars range of cultivars including Indica, Japonica and Javanica Nipponbare and Kitaake, cultivation of cells under light subspecies. 248

4 World Appl. Sci. J., 5 (2): , 2008 [a] [b] [c] [d] [e] Fig. : Morphological phenotypes of KDML05 calli oc 30 oc Fig. 2: Effect of incubation temperature on growth of callus Days darkness light Days Fig. 3: Effect of light or dark condition on growth of callus Fig. 4: Effect of sucrose concentration on growth of callus Fig. 5: Effect of different culture media on growth of callus 249

5 World Appl. Sci. J., 5 (2): , 2008 In summary, the quality and frequency of callus 7. Zhang, W. and R. Wu, 988. Efficient Regeneration of induction and growth of KDML05 cell culture ultimately depend on concentration of plant growth regulator, culture medium and culture environment. Therefore, selection of efficient culture medium and culture conditions like MS medium supplemented with 2 mg l 2,4-D or MS medium containing 4% sucrose and cultivation at 25±2 C under dark condition would offer great promise for the induction of higher quality of callus or the promotion of cell growth, respectively, for various means of biotechnology approach for improving this world s staple food crop either to increase yield or to improve nutritional quality. Transgenic Plants from Rice Protoplasts and ACKNOWLEDGMENTS This work was supported by the Agro-industry Consortium of Thailand, for a Ph.D. grant to J. Summart. We also thank Dr. Sudarat Thanonkeo for technical supports. REFERENCES. Harlan, J.R., 995. The Living Fields: Our Agricultural Heritage. Cambridge University Press, New York, pp: Khush, G.S., 997. Origin, Dispersal, Cultivation and Variation of Rice. Plant Mol. Biol., 35: Goff, S.A., D. Ricke, T.H. Lan, G. Presting, R. Wang, M. Dunn, J. Glazebrook, A. Sessions, P. Oeller, H. Varma et al., A Draft Sequence of the Rice Genome (Oryza sativa L. ssp japonica). Science, 296: Yu, J., S. Hu, J. Wang, G.K. Wong, S. Li, B. Liu, Y. Deng, L. Dai, Y. Zhou, X. Zhang and others, A Draft Sequence of the Rice Genome (Oryza sativa L. ssp. indica). Science, 296: Christou, P., T.L. Ford and M. Kofron, 99. Production of Transgenic Rice (Oryza sativa L.) Plants from Agronomically Important Indica and Japonica Varieties via Electric Discharge Particle Acceleration of Exogenous DNA into Immature Zygotic Embryos. Biotechnology, 9: Enriquez-Obregon, G.A., D.L. Prieto-Samsonov, G.A. de la Riva, M. Perez, G. Selman-Housein and R.I. Vazquez-Padron, 999. Agrobacterium-mediated Japonica Rice Transformation: A Procedure Assisted by an Antinecrotic Treatment. Plant Cell Tissue and Organ Culture, 59: Correctly Regulated Expression of the Foreign Gene in the Plants. Theor Appl. Gene.t, 76: Ahn, S., J.A. Anderson, M.E. Sorrells and S.D. Tanksley, 993. Homeologous Relationships of Rice, Wheat, and Maize Chromosomes. Mol. Gen. Genet., 24: Goff, S.A., 999. Rice as a Model for Cereal Genomics. Curr Opin Plant Biol, 2: Kurata, N., G. Moore, Y. Nagamura, T. Foote, M. Yano, Y. Minobe and M. Gale, 994. Conservation of Genome Structure between Rice and Wheat. Biotechnology, 2: Wongpornchai, S., K. Dumri, S. Jongkaewwattana and B. Siri, Effects of Drying Methods and Storage Time on the Aroma and Milling Quality of Rice (Oryza sativa L.) cv. Khao Dawk Mali 05. Food Chem., 87: Lanceras, J.C., Z.L. Huang, O. Naivikul, A. Vanavichit, V. Ruanjaichon and S. Tragoonrung, Mapping of Genes for Cooking and Eating Qualities in Thai Jasmine Rice (KDML05). DNA Res., 7: Pongtongkam, P., S. Peyachoknagul, P. Sripichit, A. Thongpan, K. Klakkhaeng, S. Ketsagul and K. Lertsirirungson, Effects of L-lysine on Callus Formation, Plant Regeneration and Flowering of Thai Rice c.v. KDML05. Kasetsart J. (Nat. Sci.), 38: Ram, H.H. and H.G. Singh, 998. Crop Breeding and Genetics. Kalyani Pulishers, New Delhi, pp: Khanna, H.K. and S.K. Raina, 998. Genotype x Culture Medium Interaction Effects on Regeneration Response of Three Indica Rice Cultivars. Plant Cell Tissue and Organ Culture, 52: Chen, Y., L.T. Liang, J. Zhu, R.F. Wang, S.Y. Li, W.Z. Tian and S.W. Zheng, 974. Studies on Induction Conditions and Genetic Expression of Pollen Plants in Rice. Sci. Sin. : Maeda, E., 980. Organogenesis and Cell Culture in Rice Plants under Sterile Condition (part I). Japan Agril. Res. Quarterly, 4: Bajaj, Y.P.S., 99. Biotechnology in Rice Improvement. In Biotechnology in Agriculture and Forestry 4. Eds. Y.P.S. Bajaj. Rice Spriger-Verlag, pp: Pandey, S.K., B. Ramesh and P.K. Gupta, 994. Study on Effect on Genotype and Culture Medium on Callus Formation and Plant Regeneration in Rice (Oryza sativa L.). Indian J. Genet, 54:

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