Melastoma malabathricum, belongs to the Melastomaceae family, is an important medicinal plant widely distributed from Madagascar to Australia, that is used in traditional remedies for the treatment of variousailments. Besides its medicinal properties, it has been identified as a potential source of anthocyanin production.The present study was carried out to investigate the effect of sucrose and methyl jasmonate and feeding time oncell biomass yield and anthocyanin production in cell suspension culture of M. malabathricum. Addition of differentconcentrations of sucrose into the cell culture of M. malabathricum influenced cell biomass and pigment accumulation. The addition of methyl jasmonate was found to have no effect on cell biomass but the presence of higher amount (12.5-50mg/L) had caused a reduction in anthocyanin production and accumulation. MS medium supplemented with 30g/L sucrose and 3.5 mg/L of MeJA added on cero day and 3rd day produced high fresh cell mass at the end of nine days of culture but did not support the production of anthocyanins. However, cells cultured in the medium supplemented with 45g/L sucrose without MeJA showed the highest pigment content (0.69±0.22Cv/g-FCM). The cells cultured in MS medium supplemented with 30 g/L sucrose with 3.5mg/L MeJA added on the 3rd and 6th day of culture, showed the lowest pigment content (0.37-0.40Cv/g-FCM). This study indicated that MeJA was not necessary but sucrose was needed for the enhancement of cell growth and anthocyanin production in M. malabathricum cell cultures. Rev. Biol. Trop. 59 (2): 597-606. Epub 2011 June 01.
Resumen Melastoma malabathricum pertenece a la familia de las melastomáceas, es una planta medicinal importante ampliamente distribuida desde Madagascar hasta Australia, que se utiliza en remedios tradicionales para el tratamiento de diversas dolencias. Además de sus propiedades medicinales, se ha identificado como una fuente potencial de producción de antocianinas. En esta investigación se estudió el efecto de la sucrosa, el metil jasmonato y el tiempo de ingestión en la producción de biomasa de las células y la producción de antocianinas, en el cultivo de células en suspensión de M. malabathricum. La adición de diferentes concentraciones de sucrosa al cultivo de células de M. malabathricum influencia la biomasa de las células y la acumulación de pigmento. La adición de metil jasmonato no tuvo ningún efecto sobre la biomasa celular, pero la presencia de una cantidad más alta (12.5-50mg/L) causó una reducción en la producción y acumulación de antocianinas. El medio MS complementado con sucrosa 30g/L y 3.5mg/L de MeJA en el día cero y el tercer día produjo una gran masa de células frescas al final de los nueve días de cultivo pero no se pudo mantener la producción de antocianinas. Sin embargo, las células cultivadas en el medio complementado con 45g/L de sucrosa sin MeJA mostró el mayor contenido de pigmento (0.69±0.22cv/g-fcm). Las células cultivadas en el medio MS complementado con 30 g/L de sucrosa y con 3.5 mg/l MeJA en el tercer y sexto día de cultivo, mostró el menor contenido de pigmentos (0.37-0.40cv/g-fcm). Este estudio indicó que MeJA no era necesario pero la sucrosa sí se necesitaba para mejorar el crecimiento celular y la producción de antocianinas en cultivos de células de M. malabathricum.
Palabras clave: Melastoma malabathricum, suspensión de cultivo celular, sucrosa, metil jasmonato, antocianinas. ]]>
Melastoma malabathricum L. (Melastomataceae) is one of the important medicinal plants widely distributed throughout Malaysia. Different plant parts of M. malabathricum are used in traditional remedies for the treatment of various ailments. The crude extract has been used in the treatment of diarrhea, postpartum treatment, hemorrhoids, to ]]>
et al. 2000, Lohezic-Le et al. 2002, Cheng et al. 2006, Susanti et al. ]]>
2006,Zakaria et al. 2006).
Generally, most of the plant species only produce small quantity of secondary metamolites. Cell suspension culture technique has been used as the alternative for the production of secondary metabolites including plant pigments. Elicitation has been shown to be the most efficient strategy that direct to the enhancement in anthocyanin production in plant cell cultures (Zhang & Furusaki 1999). The ]]>
et al. 1998, Fang et al. 1999). Methyl jasmonate (MJ) has successfully used as an elicitor in other plant species for enhancing the production of secondary metabolites in the cell cultures (Aoyagi et al. 2001, Kim
et al. 2004, Thanh et al. 2005). Similarly, the manipulation in the components of the culture medium (e.g. carbon source, nitrogen and phosphate) also found to be effective for the production of secondary metabolites (Trejo-Tapia et al. 2001). Mori & Sakurai (1994) reported the enhancement of anthocyanin accumulation and cell growth in strawberry cell suspension cultures with the supplement of glucose, sucrose and fructose in the culture medium.
M. malabathricum has also been identified as a potential source for anthocyanin production (Janna et al. 2006). Anthocyanins are well known alternatives to synthetic food colours/ dyes and beneficial to health with potential physiological benefits (Mazza & Brouillard 1990, Downham & Collins 2000). However, until now there is no report ]]>
M. malabathricum. Therefore, the present study was undertaken with an aim to investigate the effect of sucrose and methyl jasmonate acted as elicitors of cell biomass yield and anthocyanin production in cell suspension culture of M. malabathricum.
Material and Methods
Establishment of cell suspension cultures of M. malabathricum: The cell suspension culture of M. malabathricum was prepared by inoculating 0.75g (fresh weight) of nine-day old cells into liquid MS (Murashige ]]>
et al. 2008). Sterile stainless steel sieve with 850μm pore size was used to filter the initial established cell cultures to obtain homogenous cell suspension culture. Individual cells or small cell aggregates that passed through the sieve (with diameter <850μm) were subsequently filtered through Whatman no. 1 filter paper using an air suction pump (Aspirator 3A-S-Eyela, Raikakikai Co. Ltd, Tokyo) to separate the cells from the medium. The cell cultures were placed on an orbital shaker (new Brunswick ]]>
oC under continuous illumination with cool white fluorescent tubes at a light intensity of 6.3-11.9mmol photons m-2sec-1. After nine days, the cells were harvested and used as plant materials for subsequent studies.
Elicitation with sucrose and methyl jasmonate: A cell biomass of 0.5g ]]>
The same cell mass of 0.5g obtained through similar method was utilized ]]>
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The cells were harvested at the end of nine days. Cell aggregates and the liquid medium were separated as previously mentioned and the fresh cell mass was determined after the cells were harvested. The pigment content and pigment production was determined from the harvested cells as stated below.
Effect of sucrose ]]>
Cell mass of 0.5g was inoculated into two different culture media: (i) liquid MS medium supplemented with 0.25mg/L BA+0.5mg/L nAA+30g/L sucrose (the normal sucrose content used in MS medium) and (ii) liquid MS medium supplemented with 0.25mg/L BA+0.5mg/L nAA+45g/L sucrose (best sucrose concentration that stimulated the highest pigment production). MeJA concentration of 3.5mg/L was added into the medium at day zero, day three and day six of culture. The addition of MeJA solution at the beginning of cell culture (0 day) was used as control. The cultures were maintained under continuous light with intensity of 6.3-11.9mmol ]]>
-2sec-1 and the cells were harvested at the end of nine days of culture. The cell mass, pigment content and production were then determined.
Quantification of pigment content and pigment production: The total anthocyanins, represented by pigment content and pigment production of each culture, was quantified based on its colour value (Cv)/g-DCM ]]>
oC for 24 hours. Optical absorbance (or optical density, OD) of the supernatant of each sample was determined at 535nm wavelength using the Pharmacia novaspec II Rapid spectrophotometer against the blank which consisted of the solvent. Pigment content, Cv/g-DCM, was adapted from Mori et ]]>
(1993) as follow:
Pigment content = 0.1 x OD535 x dilution factor (Cv/g-DCM)
Pigment production, ]]>
Data collection and analysis: Seven to eight replicates were used for each of the treatment study and each individual study was carried out using complete randomized design (CRD). The fresh cell biomass, the pigment content and pigment production was determined for each sample ]]>
Results
Effect of Sucrose: Different added concentration of sucrose influenced ]]>
M. malabathricum. However, control (without sucrose) caused cell death. Medium supplemented with 15g/L sucrose induced the highest fresh cell weight, (1.20±0.11g) which was significantly different from the cells cultured in medium supplemented with 45g/L sucrose. On the contrary, significantly higher dried cell weight was obtained from the cells cultured in the medium supplemented with 45g/L sucrose as compared to the cells cultured in the medium supplemented with 15g/L (Fig. 1A). ]]>
The cells cultured in medium supplemented with 15g/L sucrose exhibited the lowest pigment content (0.29CV/g-FCM) and pigment production (0.35CV/flask). The cells cultured in medium supplemented with high sucrose concentration (90g/L) produced very high pigment content of 1.62±0.44CV/g-FCM. However, its cell biomass yield was very low ]]>
M. malabathricum could be used for enhancement of ]]>
Fig. 1B).
Elicitation with Methyl Jasmonate: The results showed that addition of MeJA at concentrations between 0 and 50mg/L at day zero did not influence the cell biomass at the end of nine days of culture (Fig. 2A). The control culture and the cells cultured in MS medium ]]>
Fig. 2B). Additional result obtained indicated that a concentration of 3.5mg/L MeJA was found to be the best concentration that could induce the highest pigment production ]]>
Fig. 3).
Effect of sucrose concentration and day of elicitation of methyl jasmonate: The results clearly showed that culture medium supplemented with 30g/L sucrose and 3.5mg/L of MeJA, added on different day did not ]]>
Fig. 4A). Cell proliferation medium containing 30g/L or 45g/L sucrose with or without the addition of 3.5mg/L MeJA did not affect pigment production. However, cells cultured in the medium supplemented with 45g/L sucrose ]]>
Fig. 4B).
Discussion
Effect of Sucrose: The high sucrose content reduced water content in ]]>
M. malabathricum. Similar trend was reported by Sato et al. (1996) that supplementing the culture medium of strawberry cell suspension culture with >0.09M sucrose resulted in a decrease in cell growth and attributed this to the inhibition of nutrient uptake due to an increase in the osmotic ]]>
Perilla frutescens cell cultures, they reported that growth rates increased with increase in initial sucrose concentration (15, 30, 45 and 60g/L) in the medium for both cultures with initial inocula of 15 and 50 g-wet cells/L. Our result thus confirmed that higher concentration of sucrose ]]>
The cells culture supplemented with 15g/L sucrose exhibited the lowest pigment content and pigment production. However, the culture supplemented with 90g/L sucrose concentration produced high pigment content. On the other hand, this concentration (90g/L sucrose) reduces the cell biomass and pigment production. It is possible that the high ]]>
M. malabathricum cell cultures.
The cell culture medium supplemented with 45, 60 and 75g/L sucrose show high pigment production. Therefore, we recommend that sucrose content in the culture medium should not be exceeded 75g/L. Zhong & Yoshida ]]>
Perilla frutescens, the medium that produced higher cell densities had higher sucrose levels as compared to the medium that produced higher pigment accumulation. This observation was the reverse for cell suspension culture of M. malabathricum, whereby the medium that enhanced pigment production had higher sucrose levels compared to the medium that produced the highest biomass production. A relatively higher concentration of sucrose was also reported to be favorable for ]]>
Anchusa officinalis cell culture (De-Eknambul & Ellis 1985), the ajmalicine, serpentine, and tryptamine in Catharanthus roseus cell cultures (Merillon et al. 1984), the carotenoid production by carrot cells (Yun et al. 1990), the betacyanin accumulation in suspension cultures of Phytolacca americana as well as anthocyanin production by grape cells (Do & Cormier ]]>
Camptotheca acuminate cells (Pasqua et al. 2005). Product formation could be attributed to a certain level of osmotic stress caused by higher initial sucrose concentration (Zhong & Yoshida 1995). The positive effects of osmotic potential, provoked by high sucrose concentrations in medium, on the accumulation of anthocyanins was demonstrated in cell cultures of Vitis vinifera, where an increase in osmotic potential of the medium from -0.5 to 0.9MPa resulted in a ]]>
Elicitation with methyl jasmonate: Addition of different concentrations of MeJA showed different trends in pigment content and pigment production. Lower concentrations of MeJA (2-5mg/L) showed significantly higher pigment content and pigment production as compared to the higher concentrations of MeJA (12.5-50mg/L). Our results were in consistent ]]>
et al. (1999) who reported that addition of MeJA influenced the anthocyanin accumulation depending on its concentrations in Vaccinium pahalae. Similarly, addition of 200μM MeJA considerably increased the ginsenoside content in Panax ginseng and the increasing concentration (above 200μM) declined the accumulation of ginsenosides (Thanh et al. 2005). It was reported that the increased pigment accumulation caused by MeJA in Alkanna ]]>
was not due to increased biomass growth but to stimulation of the cells to its synthesis (Urbanek et al. 1996). For M. malabathricum cell culture, since the addition of MeJA did not increase the cell biomass but pigment production, it did suggest that MeJA stimulated the synthesis of anthocyanin production via induction of particular enzymes that catalyzes the synthesis of anthocyanin. As reported by Mizukami et al. ]]>
(1993), Jasmonic acid and its derivatives were involved in a part of the signal transduction pathway that induced particular enzymes catalyzing biochemical reactions for the synthesis of secondary metabolites.
Effect of Sucrose concentration and day of elicitation of Methyl Jasmonate: Culture medium supplemented with 30g/L, 45g/L ]]>
M. malabathricum. But the presence of sucrose was needed for both cell biomass and production of pigments. Our earlier result did show that when MeJA was added into the culture medium it did affect the production of pigment, ]]>
et al. (2002) who found that addition of jasmonic acid at different feeding time influenced the anthocyanin accumulation in cell suspension cultures of Vitis vinifera.
]]>
Acknowledgments
We thank Universiti Sains Malaysia forresearch funding and facilities.
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