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Induction and Maintenance of Callus from Leaf Explants of Mirabilis Jalapa L

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Induction and Maintenance of Callus from Leaf Explants of Mirabilis Jalapa L ® Medicinal and Aromatic Plant Science and Biotechnology ©2009 Global Science Books Induction and Maintenance of Callus from Leaf Explants of Mirabilis jalapa L. Anna Pick Kiong Ling1* • Kuok-Yid Tang1 • Jualang Azlan Gansau2 • Sobri Hussein3 1 Department of Bioscience, Faculty of Engineering and Science, Tunku Abdul Rahman University, 53300 Setapak, Kuala Lumpur, Malaysia 2 School of Science and Technology, Universiti Malaysia Sabah, 88999 Kota Kinabalu, Sabah, Malaysia 3 Agrotechnology and Bioscience Division, Malaysian Nuclear Agency, Bangi, 43000 Kajang, Selangor, Malaysia Corresponding author : * [email protected] ABSTRACT Mirabilis jalapa L., commonly known as ‘four o’clock plant’ produces a strong, sweet smelling fragrance after the flowers open at late afternoon. It is a well known ornamental plant as the flowers of different colours can be found simultaneously on the same plant or an individual flower can be splashed with different colours. The colour-changing phenomenon is one of the unique characteristics of M. jalapa as it can display flowers with different colour when it matures. Apart from its ornamental value, it has also earned its place in herbal medicine practices around the world. Its array of biological activities continues to support its use worldwide for control of viruses, fungi and yeast. In this study, callus culture was initiated from the leaf explants of M. jalapa. The suitable strength of MS (Murashige and Skoog) nutrient media was determined and the effects of different types of auxins [4-amino-3,5,6-trichloro picolinic acid (picloram), 2,4- dichlorophenoxyacetic acid (2,4-D), -naphthaleneacetic acid (NAA)] and cytokinin [6-benzyl amino purine (BAP)] at 0.0 2.5, 5.0, 7.5, 10.0, and 20.0 M were investigated in order to determine the suitable callus induction and maintenance media. The establishment of callus culture was greatly influenced by the strength of MS medium, type as well as the concentration of plant growth regulators (PGRs) used. The best callus induction response was obtained on half strength (½) MS media supplemented with 20.0 )M picloram which produced healthy and friable callus. Meanwhile, ½ MS supplemented with NAA or BAP as well as PGR-free medium did not produce any callus; rather, explants became necrotic after 3 to 4 weeks of culture. Calli were successfully maintained in ½ MS supplemented with 10.0 M picloram. Callus in maintenance medium showed a sigmoid growth pattern and reached a maximum growth rate between weeks 1 and 3. _____________________________________________________________________________________________________________ Keywords: browning effects, callus cultures, growth curve, medicinal plant Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; BAP, 6-benzylamino purine; MS, Murashige and Skoog medium; NAA, - naphthaleneacetic acid; picloram, 4-amino-3,5,6-trichloro picolinic acid; PGR, plant growth regulator INTRODUCTION also valuable in speeding up conventional breeding and pro- pagation, reducing space and labour requirement and Mirabilis jalapa L. also known as the “four o’clock plant”, achieving manipulative goals that cannot be carried out via belongs to the Nyctaginaceae family. It is a tropical herb in vivo conditions. For instance, with the advances in plant commonly cultivated in North America, a perennial in the biotechnology, the extract of M. jalapa cultured cells and its south, warm west and an annual in the north. Long before precipitate fraction with 90% saturated ammonium sulfate the Spanish Conquest of Mexico, it was cultivated by the was found to show an anti-plant viral activity comparable to Aztecs for its medicinal properties and for its showy frag- that of the roots and leaves of the original plant (Ikeda et al. rant flowers (Le Duc 1986). In the traditional popular medi- 1987). In view of these facts, the present study was conduc- cine of the South American continent, particularly in Brazil, ted to determine and examine the suitable strength of MS the roots, shoots, leaves, fruits, and seeds of M. jalapa L. media, and the effects of different auxins and cytokinin at are employed for different affections (Lorenzi 1991; various concentrations on callus induction from leaf ex- Lorenzi and Souza 1995). For instance, traditionally healers plants. In addition, the best maintenance medium and the in Brazil use a paste of the root applied as a poultice to treat growth curve of leaf-derived callus of M. jalapa L. was also scabies and muscular swellings; the juice of the leaves is determined. used in the treatment of diarrhoea, indigestion and fevers (Diggs et al. 1999). MATERIALS AND METHODS Conventionally, M. jalapa reproduces via viable seeds but the low percentage of seed viability limits its natural Plant materials propagation since the hawk-moth population are the only species that act as its pollinator (Martinez and Búrquez The seeds of M. jalapa were obtained from Sabah, Malaysia. The 1996). Hence, alternative techniques like plant tissue cul- black coloured mature seeds were germinated on the soil with the ture were established to propagate a large number of in composition of nitrogen, phosphate, potassium at the ratio of 1: 3: vitro plants. A rapid and efficient in vitro regeneration sys- 2. The seeds germinated easily (with 96% of germination percen- tem has been developed from nodal segments (Zaccai et al. tage) within 3 to 7 days. The leaves were collected from one- 2007) and cotyledons (Xu et al. 2005) of M. jalapa. By month old healthy seedlings. using plant tissue culture technique, genetically similar plantlets can be produced in relatively short time. Besides, by maintaining genetic stability, tissue cultured plants are Received: 31 October, 2008. Accepted: 1 February, 2009. Original Research Paper Medicinal and Aromatic Plant Science and Biotechnology 3 (1), 42-47 ©2009 Global Science Books Surface sterilization In order to determine the best maintenance medium, callus was also transferred to ½ MS supplied with 0.0, 10.0, 20.0 and 30.0 One-month old leaves were collected and washed under running μM picloram. tap water for 30 min then initially surface sterilized in 0.2% (w/v) fungicide (Ancom Thiram, USA) for 30 min followed by a dip in Growth curve measurement 70% (v/v) ethanol for 1 min. Leaves were then immersed for 25 min in 15% (v/v) Clorox? containing 2 to 3 drops of Tween-20 Subsequently, the best treatment or maintenance medium (½ MS + (Amresco, USA) with gentle and continuous shaking. The leaves 10.0 μM picloram) was subjected to growth curve analysis with were then rinsed three times with sterile distilled water for 5, 10, the initial fresh weight of 0.01 g. The growth of leaf-derived callus and 15 min, respectively. was evaluated by measuring the fresh weight at one-week interval continuously for four weeks. The experiments were conducted Culture media with three replicates and repeated twice. Standard MS (Murashige and Skoog 1962) solid media was used Statistics analysis as the basal medium. To screen the suitable strength of MS media on callus induction of M. jalapa, full- and half-strength (½) MS The data collected in this study were subjected to the statistical media supplemented with various plant growth regulators (PGRs) analysis. One way ANOVA and Tukey’s Honestly Significant Dif- at different concentrations were prepared. Meanwhile, medium ference (Tukey’s HSD) test (p<0.05) were used to determine the without any PGR was used as the control. For the preparation of differences in mean number of all tested parameters. Statistical MS media, macronutrient, micronutrient, vitamin, FeNaEDTA analysis was performed using SPSS software (version 11.5) (SPSS were added and fortified with 3% (w/v) sucrose; PGRs were added Inc. USA). before autoclaving. The pH of the medium was adjusted to 5.7 ± 0.1 using 0.1 M NaOH or 0.1 M HCl and solidified with 0.8% RESULTS AND DISCUSSION (w/v) agar. The media were then autoclaved at 121°C for 15 min. Approximately 25 mL of the sterile medium were distributed into Effects of medium strength on callus induction each 100 mm × 15 mm sterile disposable Petri dish (Labchem, Malaysia). Different strengths of MS media produced different effects on callus induction (Table 1). The results were evaluated in Callus initiation terms of the day of callus induction, the intensity of callus growth as well as the morphology of the callus formed. It The surface-sterilized leaves were cut into approximately 5 mm2 was observed that all the full- and ½ MS medium sup- explants and cultured on either full- or ½ MS solid media sup- plemented with 2, 4-D (2.5, 5.0, 7.5, 10.0, and 20.0 μM) plemented with different PGRs – picloram (Duchefa, Netherlands), successfully induced the callus from the leaf explants of M. 2,4-D (Sigma, U.S.A), NAA (Duchefa, Netherlands), and BAP jalapa within 4 weeks of culture. In terms of days of callus (Duchefa, Netherlands) at 0.0, 2.5, 5.0, 7.5, 10.0, and 20.0 μM. A formation, explants cultured in ½ MS medium supplemen- total of 5 explants were used for each treatment and all the expe- ted with 7.5 μM 2, 4-D required the shortest period which riments were performed with three replicates and repeated twice. was 7 days of culture for callus formation. This treatment produced profuse growth of friable and whitish callus (Fig. Culture conditions 1A). On the other hand, full MS medium supplemented with 7.5 μM 2,4-D induced the callus after 10 days of cul- All the cultures were incubated and maintained at 26 ± 2°C under ture and the unhealthy yellowish watery callus (Fig. 1B) a light intensity of approximately 2000 Lux provided by cool were observed in this treatment. white fluorescent lamps under a 16-h photoperiod. Analysis of the From the results, it indicated that the callus induction percentage of callus induction, days of callus formation, morpho- from the leaf explants of M.
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