Plant Cell Tiss Organ Cult (2011) 106:537–545 DOI 10.1007/s11240-011-9945-3
RESEARCH NOTE
Establishment of anthocyanin-producing cell suspension cultures of Cleome rosea Vahl ex DC. (Capparaceae) Claudia Simo˜es-Gurgel • Lı´via da Silva Cordeiro • Tatiana Carvalho de Castro • Ca´tia Henriques Callado Norma Albarello • Elisabeth Mansur
•
Received: 14 October 2010 / Accepted: 24 March 2011 / Published online: 17 April 2011 Ó Springer Science+Business Media B.V. 2011
Abstract The effects of different levels of Murashige and Skoog (MS) basal medium, 2,4-dichlorophenoxyacetic acid (2,4-D), and sucrose on anthocyanin production and biomass accumulation of cell suspension cultures of Cleome rosea were investigated. Cultures were established in liquid MS medium containing 30 g l-1 sucrose and supplemented with 0.90 lM 2,4-D. Proliferating cell suspension cultures achieved the highest growth capacity, a fourfold increase in biomass accumulation, following subculture at the exponential growth phase, 14–18 days of culture. Moreover, the presence of 2,4-D was essential for anthocyanin production and biomass accumulation. On the other hand, increasing levels of sucrose above 30 g l-1 resulted in a drastic reduction in biomass accumulation. Anthocyanin production was highest in cell suspension cultures grown on halfstrength MS medium (1/2 MS), 30 g l-1 sucrose, and 0.45 lM 2,4-D. These cell suspension cultures were mainly composed of small aggregates of spherical cells with similar morphology observed in anthocyanin-producing and non-producing cultures. Moreover, microscopic analysis of anthocyanin-producing cultures showed the presence of mixtures of non-pigmented, low-pigmented, and high-pigmented cells.
C. Simo˜es-Gurgel (&) L. d. S. Cordeiro T. C. de Castro N. Albarello E. Mansur Nu´cleo de Biotecnologia Vegetal, Universidade do Estado do Rio de Janeiro (UERJ), Rua Sa˜o Francisco Xavier, 524, PHLC, sala 509, Maracana˜, Rio de Janeiro, RJ CEP 20550-013, Brazil e-mail:
[email protected] C. H. Callado Laborato´rio de Anatomia Vegetal, Universidade do Estado do Rio de Janeiro (UERJ), Rio de Janeiro, Brazil
Keywords Cell aggregate Color value 2,4-dichlorophenoxyacetic acid Salt concentration Sucrose Abbreviations CV Color value 2,4-D 2,4-dichlorophenoxyacetic acid DW Dry weight FW Fresh weight
Protocols for cell suspension cultures have been established for many medicinal plant species, with specific interests in secondary metabolite production (Kolewe et al. 2008; Estrada-Zu´n˜iga et al. 2009; Boonsnongcheep et al. 2010; Bonfill et al. 2011), including those of plant pigments (Nawa et al. 1993; Filippini et al. 2003). Nevertheless, in vitro production of secondary metabolites remains difficult due to various biological and technical limitations resulting in low yields. Moreover, there is a negative correlation between cell growth and secondary metabolite production as has often been observed in plant cell cultures. In many cases, a two-stage culture regime is necessary to establish an efficient and productive protocol. The first stage involves inducting growth and maintaining a high cell density whereas the second stage involves the transfer of cells to another medium to induce the production of secondary metabolites (Wai-Leng and Lai-Keng 2004). Anthocyanins are pigments widely found in plant species providing scarlet to blue colors in flowers, fruits, leaves and storage organs. Interest in anthocyanins as food and beverage colorants has been increasing not only because they are less toxic than synthetic dyes but also because they exhibit significant antioxidant activity.
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Therefore, these pigments have potential applications in nutraceutical developments, and much attention has been focused on their multifaceted pharmacological potential, which includes specific biological activities, such as antiinflammatory (Wang and Mazza 2002), antitumor (Bagchi et al. 2004) and cardioprotective (Brindle and Timberlake 1996). In recent years, there has been a large number of publications reporting anthocyanins production by cell suspension cultures (Zhang et al. 2004; Gould et al. 2008). Cleome rosea is a Brazilian herbaceous annual species frequently found in coastal sandy plains, which are ecosystems intensely affected by human impact (Zamith and Scarano 2006). This species has been studied using plant cell culture strategies (Simo˜es et al. 2004, 2009a), in relation to its medicinal potential (Simo˜es et al. 2006). More recently, an anthocyanin-producing callus culture line has also been obtained (Simo˜es et al. 2009b). Considering that cell suspension cultures generally comprise more homogeneous cell populations and grow more rapidly than callus cultures (Saito and Mizukami 2002), the present study aimed to establish a single-stage culture regime for anthocyanin-producing fast growing cell suspension lines of C. rosea. Stem segments (0.5 cm) from in vitro-grown plants (Simo˜es et al. 2004) were incubated on solidified Murashige and Skoog (MS) (1962) medium containing 30 g l-1 sucrose and supplemented with 0.90 lM 2,4dichlorophenoxyacetic acid (2,4-D). Calli were subcultured using the same medium composition by transferring samples of approximately 4 g at 20-day intervals. Reddish-pink regions were observed on callus surface after 6–7 months in culture. These regions were isolated mechanically and subcultured, allowing the establishment of pigmented and non-pigmented callus lines. The pigments were identified as anthocyanins by spectrophotometric analysis (Simo˜es et al. 2009b). Cell suspension cultures were initiated by transferring 3 g of friable calluses from both callus lines to liquid MS medium with the same composition used for callus induction. Media were adjusted to pH 5.8 prior to autoclaving (121°C, 104 kPa) for 15 min and afterwards dispensed into 125-ml erlenmeyer flasks (25 ml of culture medium per flask) closed with double-aluminum caps. Suspension cultures were incubated on a gyratory shaker (100g) under a 16-h photoperiod provided by cool-white fluorescent tubes (80 lmol m-2 s-1), at 24 ± 2°C. Five flasks were used per treatment and the assays were repeated 3 times. To determine the growth kinetics, the cells were harvested by filtering through a nylon mesh (45 lm) using a filter unit (NalgeneTM Cat. No. 320–2533) connected to a vacuum pump (GASTÒ Manufacturing). The cell biomass accumulation was estimated by measurement of fresh (FW) and dry (DW) weights. Dry mass was determined after
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drying the cells at 45°C to constant weight. Samples were collected every 4 days during a 50-day period and each point of the growth curve represents the mean of three independent determinations (flasks). To investigate the effects of factors that influence anthocyanin production and biomass accumulation, cell suspension cultures were initiated from pigmented callus incubated on full-strength MS, half-strength MS (1/2 MS), or 1/4-strength MS (1/4 MS) medium supplemented with 0, 0.45, or 0.90 lM 2,4-D, and 30, 50, or 70 g l-1 sucrose. Five flasks were used per treatment, and a total of 20 treatment combinations were used. Cultures were maintained in a growth chamber under the same environmental conditions as described above. These cell suspension cultures were evaluated during the exponential growth phase in each of six passages (subcultures). Biomass accumulation, fresh weight (FW) and dry weight (DW), and anthocyanin content (CV index) were determined in each passage. All assays were repeated 3 times at each time point. Anthocyanin quantification was based on the color value index (CV), which is an indicator of total anthocyanins (Yoshinaga et al. 1999; Konczac-Islam et al. 2000), with small modifications. Briefly, samples of cell suspensions (100 mg) were extracted for 24 h at 4°C using 1% (v/v) HCl-methanol. The extracts were centrifuged (1,000g for 10 min) and the absorbance was measured at 525 nm using a UV–Vis spectrophotometer (Shimadzu UV–160). The CV index was calculated using the following equation: CV = 0.1 9 OD 525 9 40/1 g FW, where 0.1 9 OD 525 is 10% of the absorbance measured at 525 nm, 40 is the level of dilution (100 mg of cell cultures extracted in 4 ml of HCl-methanol) and FW is the fresh cell weight. The CV index was chosen to quantify the total anthocyanin content instead of the extinction coefficient of one particular type of anthocyanin, because the extracts from cell cultures are mixtures of various anthocyanins, which can vary under different culture conditions (Zhang et al. 2002). To analyze cell morphology, samples of pigmented and non-pigmented cell suspension cultures at exponential and stationary phases were analyzed with an image capture system Image-Pro Plus for Windows, using a videocamera Optronics attached to an Olympus BX40 microscope. The experiments were organized in a completely randomized design and were repeated three times. The data were analyzed using one-way analysis of variance (ANOVA) and the differences among means were tested by the Tukey test at 5% level of significance. The analyses were carried out with the software MSTAT-C (v.2.1; Michigan State University, MI, USA). Cell suspensions were characterized by a 6-day lag phase, followed by an exponential phase lasting until day 18 of culture, with the most significant growth occurring between days 14 and 18. During this period, the cultures
Plant Cell Tiss Organ Cult (2011) 106:537–545
achieved a four-fold increase on biomass accumulation. The stationary phase was followed by a gradual reduction in cell density (Fig. 1). Biomass accumulation and pigment production were evaluated during six subcultures performed after 16 ± 2 days of culture. The anthocyanin synthesis by cell suspensions of C. rosea was more intense during the exponential phase. In many cell cultures, the secondary metabolite production is enhanced by nutrient depletion and usually begins when the cultures move from the exponential into the stationary phase. However, visual evaluation of cultures described in this study indicated that the anthocyanin content declined during the stationary phase, suggesting that the substrate supply is an important factor in the biosynthesis of these pigments in in vitro cultures. Similar results were observed by Nakamura et al. (1998), who suggested that the production of other secondary metabolites during the stationary phase could inhibit the anthocyanin biosynthesis. Production of other plant pigments in cell suspension cultures, such as betalains and carotenoids, is also growthassociated (Bourgaud et al. 2001). The cell suspension cultures initiated with non-pigmented calluses (Fig. 2a) achieved a fourfold increase on biomass accumulation at each subculture. On the other hand, cultures initiated with pigmented calluses, albeit achieving a high biomass accumulation, presented a significant reduction in the capability of anthocyanin production, with the maximum pigment concentration being observed during the second subculture. These cultures initially presented a pale pink color (Fig. 2c), but pigment production was halted after the fourth subculture (Table 2, M10). Reduction, loss or changes in anthocyanin production in cell suspensions have been reported by several authors (Vogelien et al. 1990; Hirasuma et al. 1991; Callebaut et al. 1997; Zhang et al. 2002; Qu et al. 2005). The manipulation of the culture environment can be effective in increasing secondary metabolites production, once many pathways are easily altered by external factors such as nutrient levels, stress factors and growth regulators
Fig. 1 Percentage of growth of cell suspension cultures of Cleome rosea obtained in liquid MS medium supplemented with 0.9 lM 2,4D, during 50 days in culture. FW fresh weight, DW dry weight
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(Rao and Ravishankar 2002; Bernabe´-Antonio et al. 2010; Nagella and Murthy 2011). Hence, MS strength, 2,4-D and sucrose concentrations were tested in order to establish cultures with high and stable anthocyanin production. To evaluate the influence of MS strength, cell cultures were initiated in the presence of 30 g l-1 sucrose and in the absence of 2,4-D. Cultures initiated in full MS medium achieved low values of CV, with a complete interruption of pigment production after the third subculture (Table 1, M1). On the other hand, the use of MS1/2 resulted in high anthocyanin production, but only during the first three subcultures (Table 1, M4). Nutritional restriction is an efficient strategy to induce anthocyanin synthesis in in vitro cultures based in the fact that many nutrient deficiencies, especially in nitrogen, phosphorus and sulfur, are accompanied by anthocyanin accumulation. Pigment production under these conditions is a strategy used by plants to avoid the over-accumulation of carbohydrates in tissues, in order to prevent physiological disorders (Barker and Pilbeam 2007). Although the use of MS1/2 showed to be beneficial to anthocyanin production, cell suspensions established in MS1/4 showed low pigment production and only up to the first subculture (Table 1, M6). Moreover, cultures initiated in media without hormonal supplementation presented an expressive reduction in biomass accumulation over time in culture. Anthocyanin-producing callus lines of C. rosea also achieved the highest values of CV when cultivated on MS medium with reduction on total salt concentration. However, unlike cell suspensions, calluses cultivated on MS1/4 achieved high production of anthocyanins, although with a significant reduction in biomass accumulation (Simo˜es et al. 2009b). In this work, the presence of 2,4-D proved to be critical for biomass accumulation on cell suspensions. Cultures established in full MS medium supplemented with this growth regulator maintained a high and constant cell density over the six subcultures (Table 2, M7 and M10). Nevertheless, although the supplementation with 2,4-D led to the production of pigments throughout the subcultures, especially at the concentration of 0.45 lM, the maximum values achieved were around 6 CV/gFW. Cultures established in MS1/4, even in the presence of 2,4-D, achieved low values of CV and a gradual reduction of biomass accumulation along the subcultures (Table 3, M17 and M19). Although 2,4-D has been shown to inhibit the production of secondary metabolites in many plant species, the role of this growth regulator in anthocyanin production is contradictory. Its presence was crucial in many cultures (Sakamoto et al. 1993; Meyer and van Staden 1995; Konczac-Islam et al. 2000), while in others its elimination or replacement by other growth regulator resulted in enhanced pigment production (Narayan and Venkataraman 2000; Narayan et al. 2005).
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Fig. 2 Cell suspension culture lines from C. rosea. a Non-pigmented line established in MS medium supplemented with 0.9 lM 2,4-D. Bar 0.86 cm. b Cell aggregate from non-pigmented line. Bar 50 lm. c Low-pigmented line established in MS medium supplemented with
0.9 lM 2,4-D. Bar 0.86 cm. d Cell aggregate from low-pigmented line. Bar 50 lm. e High-pigmented line established in MS1/2 medium supplemented with 0.45 lM 2,4-D. Bar 0.80 cm. f Cell aggregate from high-pigmented line. Bar 50 lm
A continuous production of anthocyanin along the subcultures, together with a high biomass accumulation, was obtained in cell suspensions initiated in MS1/2 added with 30 g l-1 sucrose and supplemented with 2,4-D (Table 3, M13 and M15). These cultures showed an increase in anthocyanin production during the first three subcultures followed by a constant production, without statistical differences, until the sixth subculture. The highest values of CV were achieved by cultures initiated in medium supplemented with 0.45 lM 2,4-D (M13). These cultures were observed until the 25th subculture, showing the same levels of anthocyanin production and reaching an average of 20.32 ± 1.06 CV/g FW (Fig. 3). These were maintained for more than 2 years with high biomass accumulation and an intense purple color (Fig. 2e). The increase in sucrose levels above 30 g l-1 caused significant reduction on anthocyanin production and biomass accumulation, even in cultures initiated in MS1/2 with supplementation of 2,4-D (Table 1, M2, M3, M5; Table 2, M8, M9, M11, M12; and Table 3, M14, M16, M18, M20). These results contradict previous findings in callus cultures, where the highest anthocyanin content was
obtained on medium enriched with 70 g l-1 sucrose (Simo˜es et al. 2009b). This difference can be related to the fact that growth in liquid culture exposed the cells to an increased osmotic stress as compared to callus cultures. Sugar levels have been shown to affect the productivity of anthocyanins in cell suspensions in various ways according to the species. Cultures of Vitis vinifera were positively affected by the osmotic stress created by increased sucrose concentrations (Do and Cormier 1990), while concentrations up to 50 g l-1 reduced anthocyanin production in cell cultures of Aralia cordata (Sakamoto et al. 1993). An important aspect to be considered in suspension cultures is the cell morphology, which is strongly influenced by several parameters and often determines the profile of metabolite production. Cultures obtained from C. rosea were composed predominantly of spherical cells, forming small aggregates of 3–15 cells. A few isolated cells (spherical or elongated) were also found. It is well known that a degree of cell aggregation in cell suspension cultures influences secondary metabolite production (Zhao et al. 2003; Edahiro and Seki 2006). However, differences in cell aggregate size distribution patterns between non-
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13.12 ± 3.24 a
0
nd
5
6
nd
4.59 ± 0.31 d
4.61 ± 0.14 d
6.48 ± 0.18 c
12.74 ± 0.46 a 12.05 ± 0.46 b
FW (g)
nd
0.11 ± 0.01 d
0.12 ± 0.01 d
0.24 ± 0.01 c
0.33 ± 0.01 b 0.37 ± 0.03 a
DW (g)
nd
nd
nd
nd
0
10.21 ± 2.40 a 6.81 ± 1.30 b
nd
nd
nd
nd
4.43 ± 0.70 b
8.91 ± 0.91 a 4.92 ± 0.79 b
FW (g)
nd
nd nd
nd
5.75 ± 0.37 a
5.70 ± 1.62 a
FW (g)
nd
nd
nd
0.17 ± 0.01 b
0.37 ± 0.06 a 0.20 ± 0.03 b
DW (g)
nd
nd nd
nd
0.23 ± 0.01 a
0.20 ± 0.02 a
DW (g)
nd
nd
nd
0
2.14 ± 0.63 a 0
CV/gFW
M6
nd
nd nd
nd
0
0.89 ± 0.34 a
CV/gFW
M3
nd
nd
nd
3.38 ± 0.22 a
3.47 ± 0.27 a 3.27 ± 0.24 a
FW (g)
nd
nd nd
nd
5.12 ± 0.71 a
4.01 ± 0.83 a
FW (g)
nd
nd
nd
0.13 ± 0.01 a
0.14 ± 0.01 a 0.13 ± 0.01 a
DW (g)
nd
nd nd
nd
0.20 ± 0.02 a
0.18 ± 0.03 a
DW (g)
M1 MS ? 30 g l-1 sucrose; M2 MS ? 50 g l-1 sucrose; M3 MS ? 70 g l-1 sucrose; M4 MS1/2 ? 30 g l-1 sucrose; M5 MS1/2 ? 50 g l-1 sucrose; M6 MS1/4 ? 30 g l-1 sucrose
CV Color value, FW fresh weight, DW dry weight, nd not determined
Same letters in each column of each treatment are not significantly different by Tukey test at 5%
Data represent mean ± SD
10.87 ± 0.60 b
0
3
4
10.24 ± 2.14 b 16.77 ± 2.30 a
CV/gFW
CV/gFW
1 2
M5
M4
Passage/ subculture
nd
nd nd
nd
nd
0.17 ± 0.03 b nd
6
4.73 ± 1.16 b nd
0.46 ± 0.17 b
0.23 ± 0.02 b
1.89 ± 0.34 a 0
0 nd
6.99 ± 2.64 b
0.36 ± 0.04 a 0.31 ± 0.07 a
3
8.62 ± 1.33 a
4 5
3.93 ± 0.80 a
1.36 ± 0.57 b
1
CV/gFW
DW (g)
CV/gFW
FW (g)
M2
M1
2
Passage/ subculture
Culture medium
Table 1 Anthocyanin content (CV) and biomass accumulation (FW and DW) in cell suspension cultures of Cleome rosea maintained in MS medium containing different salt and sucrose concentrations
Plant Cell Tiss Organ Cult (2011) 106:537–545 541
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123
0
0
5
6
12.65 ± 2.60a
14.40 ± 2.80a
13.11 ± 3.26a
14.58 ± 1.77a
14.12 ± 2.71a
15.45 ± 2.62a
FW (g)
0.32 ± 0.08a
0.37 ± 0.02a
0.31 ± 0.06a
0.34 ± 0.04a
0.33 ± 0.01a
0.39 ± 0.10a
DW (g)
0.32 ± 0.06b
nd
nd
nd
0
0
4.53 ± 0.46a
nd
nd
nd
nd
3.38 ± 0.32b
6.32 ± 0.94a
6.48 ± 0.18a
FW (g)
nd
nd
nd
5.50 ± 0.47a
6.51 ± 0.83a
6.33 ± 0.77a
FW (g)
nd
nd
nd
0.13 ± 0.01a
0.24 ± 0.10ab
0.24 ± 0.01a
DW (g)
nd
nd
nd
0.21 ± 0.02a
0.25 ± 0.03a
0.17 ± 0.02b
DW (g)
nd
nd
nd
0
0
3.19 ± 1.26a
CV/gFW
M12
nd
nd
nd
0
0
2.16 ± 0.76a
CV/gFW
M9
nd
nd
nd
4.32 ± 1.46a
4.02 ± 0.54a
4.14 ± 1.97a
FW (g)
nd
nd
nd
4.76 ± 0.60a
4.99 ± 1.26a
5.08 ± 0.23a
FW (g)
nd
nd
nd
0.15 ± 0.02a
0.20 ± 0.02a
0.20 ± 0.11a
DW (g)
nd
nd
nd
0.15 ± 0.03a
0.16 ± 0.05a
0.19 ± 0.04a
DW (g)
M7 MS ? 0.45 lM 2,4-D ? 30 g l-1 sucrose; M8 MS ? 0.45 lM 2,4-D ? 50 g l-1 sucrose; M9 MS ? 0.45 lM 2,4-D ? 70 g l-1 sucrose; M10 MS ? 0.90 lM 2,4-D ? 30 g l-1 sucrose; M11 MS ? 0.90 lM 2,4-D ? 50 g l-1 sucrose; M12 MS ? 0.90 lM 2,4-D ? 70 g l-1 sucrose
CV Color value, FW fresh weight, DW dry weight, nd not determined
Same letters in each column of each treatment are not significantly different by Tukey test at 5%
Data represent mean ± SD
3.56 ± 0.39c
2.40 ± 0.14d
3
6.32 ± 0.43a
4
5.19 ± 0.24b
2
CV/gFW
CV/gFW
1
M11
M10
Passage/ subculture
13.06 ± 2.24a
nd nd
3.01 ± 0.22b
0.38 ± 0.02b 0.35 ± 0.05b
6
13.61 ± 1.49a 12.21 ± 0.97a
4.72 ± 1.15ab
0
0
2.00 ± 0.26a
4.51 ± 0.48b
0.35 ± 0.02b
0.34 ± 0.04b
0.49 ± 0.03a
4
14.86 ± 1.21a
14.64 ± 1.83a
15.47 ± 0.85a
5
4.05 ± 0.62b
6.32 ± 0.43a
2
3
6.08 ± 1.16a
CV/gFW
DW (g)
CV/gFW
FW (g)
M8
M7
1
Passage/ subculture
Culture medium
Table 2 Anthocyanin content (CV) and biomass accumulation (FW and DW) in cell suspension cultures of C. rosea maintained in MS medium containing different sucrose and 2,4-D concentrations
542 Plant Cell Tiss Organ Cult (2011) 106:537–545
FW (g)
DW (g)
FW (g)
DW (g)
1.56 ± 0.62bc 5.43 ± 0.68bc 0.30 ± 0.03ab nd
1.22 ± 0.72bc 5.04 ± 0.76bc 0.30 ± 0.02ab nd
5
6
0
5.79 ± 0.24bc 0.31 ± 0.01ab nd
0.25 ± 0.01b
DW (g)
CV/gFW
M19
nd nd
nd
nd nd
nd
5.77 ± 1.25b
0.29 ± 0.04a 0.29 ± 0.03a
0.28 ± 0.03a
nd
FW (g)
nd
nd
DW (g)
nd
nd
nd
nd
0
3.56 ± 0.61a 0.08 ± 0.01a
nd
nd
nd
nd
nd
nd
3.26 ± 0.24a 0.10 ± 0.02a
M13 MS1/2 ? 0.45 lM 2,4-D ? 30 g l-1 sucrose; M14 MS1/2 ? 0.45 lM 2,4-D ? 50 g l-1 sucrose; M15 MS1/2 ? 0.90 lM 2,4-D ? 30 g l-1 sucrose; M16 MS1/2 ? 0.90 lM 2,4-D ? 50 g l-1 sucrose; M17 MS1/4 ? 0.45 lM 2,4-D ? 30 g l-1 sucrose; M18 MS1/4 ? 0.45 lM 2,4-D ? 50 g l-1 sucrose; M19 MS1/4 ? 0.90 lM 2,4-D ? 30 g l-1 sucrose; M20 MS1/4 ? 0.90 lM 2,4D ? 50 g l-1 sucrose
CV Color value, FW fresh weight, DW dry weight, nd not determined
1.19 ± 0.40c
5.01 ± 0.78b
3.30 ± 0.75b 5.99 ± 0.97b 1.25 ± 0.69c
4.84 ± 0.79a 0.18 ± 0.02a 5.23 ± 0.55a 0.19 ± 0.01a
3.33 ± 1.30a 3.41 ± 0.24a 0.10 ± 0.02a
CV/gFW
0.12 ± 0.04b 0
0.22 ± 0.04a
DW (g)
3.67 ± 0.20a 0.12 ± 0.01a 3.20 ± 0.92b 6.89 ± 0.30ab 0.29 ± 0.03a
4.95 ± 0.95b
9.14 ± 2.56a
FW (g)
M20
17.80 ± 1.24a 11.47 ± 1.27a 0.32 ± 0.03a nd
3.89 ± 0.31a 0.16 ± 0.05a 1.36 ± 0.57c
Same letters in each column of each treatment are not significantly different by Tukey test at 5%
Data represent mean ± SD
2.11 ± 0.69b
4
7.13 ± 0.36b
FW (g)
nd
4.00 ± 0.54b 0.15 ± 0.04b 18.07 ± 0.72a 11.60 ± 0.44a 0.32 ± 0.01a nd nd
4.12 ± 0.89a 3.97 ± 0.34a 0.13 ± 0.02a 7.10 ± 1.44a 0
4.41 ± 1.66b
0.15 ± 0.07b
3
10.45 ± 1.82a 0.58 ± 0.30a
4.81 ± 0.70c
9.08 ± 0.62a
2.20 ± 1.21b
1
CV/gFW
2
DW (g)
FW (g)
M18
20.47 ± 4.42a 12.07 ± 0.97a 0.41 ± 0.08a nd
6
Passage/ M17 subculture CV/gFW
21.43 ± 2.89a 11.80 ± 1.01a 0.35 ± 0.02a 0
5
3.66 ± 0.49b 0.12 ± 0.02b 18.40 ± 1.39a 12.11 ± 0.94a 0.34 ± 0.03a 0
22.69 ± 5.60a 13.33 ± 0.36a 0.38 ± 0.06a 2.79 ± 1.02b 4.97 ± 0.48b 0.21 ± 0.02b 19.53 ± 4.69a 11.49 ± 0.87a 0.38 ± 0.04a 0
28.71 ± 2.20a 11.79 ± 1.22a 0.34 ± 0.02a 0
3
4
12.29 ± 0.71a 0.35 ± 0.02a 4.65 ± 1.02a 5.39 ± 2.92a 0.20 ± 0.07a
CV/gFW
7.93 ± 1.21b
DW (g)
11.09 ± 0.67b 12.50 ± 0.89a 0.35 ± 0.01a 3.97 ± 0.15b 5.30 ± 2.13b 0.18 ± 0.05b 10.92 ± 0.55b 12.40 ± 0.78a 0.34 ± 0.01a 3.50 ± 0.73a 6.09 ± 0.37a 0.21 ± 0.01a
FW (g)
1
CV/gFW
M16
2
CV/gFW
11.92 ± 0.52a 0.36 ± 0.12a 6.14 ± 0.67a 8.91 ± 0.92a 0.37 ± 0.06a 7.29 ± 0.44b
DW (g)
M15
FW (g)
M14
Passage/ M13 subculture CV/gFW
Culture medium
Table 3 Anthocyanin content (CV) and biomass accumulation (FW and DW) in cell suspension cultures of C. rosea maintained in MS medium containing different salt and sucrose concentrations, and supplemented with 2,4-D
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Plant Cell Tiss Organ Cult (2011) 106:537–545
vitro conditions. The system described here represents a suitable and reliable approach for pigment production in cell suspension cultures of C. rosea, associated with high biomass accumulation in a single-stage culture regime. Moreover, the anthocyanin production in cell suspensions was more efficient when compared to the callus cultures reported previously (Simo˜es et al. 2009b), since high levels of productivity were achieved with less culture manipulation.
Fig. 3 Anthocyanin production in cell suspension cultures of C. rosea maintained during 25 subcultures in MS1/2 medium supplemented with 0.45 lM 2,4-D. Data are means ± SD, and those with an asterisk are not significantly different by Tukey test at 5%
pigmented (Fig. 2b) and pigmented cultures of C. rosea were not observed (Fig. 2d, f). Moreover, no significant differences on cell morphology were observed during both exponential and stationary phases. The predominance of aggregates formed by a small number of cells is a positive factor with regard to the production of secondary metabolites, especially anthocyanic pigments. Some authors have observed a reduction in anthocyanin production in enlarged cell aggregates (Madhusudhan and Ravishankar 1996; Pe`pin et al. 1999). Because anthocyanin biosynthesis is also strongly influenced by light intensity, which affects the activity of key enzymes involved in their biosynthetic pathway, such as phenylalanine ammonia-lyase and chalcone synthase (Zhang et al. 2002), it is possible that increased aggregate size can cause a lack of light at the core cells. In addition, other factors such as oxygen supply, differential exposure to the microenvironmental factors and local concentration gradients might alter the cell growth and secondary metabolite production in large cell aggregates (Zhong et al. 1993; Meyer et al. 2002). The suspension cultures of C. rosea presented nonpigmented, low-pigmented and high-pigmented cells, suggesting that these cultures are heterogeneous systems, containing a range of cell populations, with variable anthocyanin biosynthetic capabilities. Moreover, the main difference between cultures presenting a low (Fig. 2d) and a high (Fig. 2f) anthocyanin production was the amount of pigment in the producing cells and not the number of these cells. This differentiated capability of cells to accumulate anthocyanin, indicating an extensive variation in the pigment biosynthesis among cell populations, may involve modifications of the control mechanisms determining intracellular accumulation more than changes in the genetic information of the cells (Filippini et al. 2003). Much effort has been expended in developing alternative methods to improve anthocyanin biosynthesis under in
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Acknowledgments The authors are grateful to Maria Francisca Santoro Assunc¸a˜o for the valuable technical assistance. This work was supported by Fundac¸a˜o Carlos Chagas Filho de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq).
References Bagchi D, Sen CK, Bagchi M, Atalay M (2004) Anti-angiogenic, antioxidant and anti-carcinogenic properties of a novel anthocyanin-rich berry extract formula. Biochemistry 69:75–78 Barker AV, Pilbeam DJ (2007) Handbook of plant nutrition. CRC Press, Boca Raton Bernabe´-Antonio A, Estrada-Zu´n˜iga ME, Buendı´a-Gonza´lez L, ´ vila VM, Cruz-Sosa F (2010) Reyes-Chilpa R, Cha´vez-A Production of anti-HIV-1 calanolides in a callus culture of Calophyllum brasiliense (Cambes). Plant Cell Tiss Org Cult 103:33–40 Bonfill M, Mangas S, Moyano E, Cusido RM, Palazo´n J (2011) Production of centellosides and phytosterols in cell suspension cultures of Centella asiatica. Plant Cell Tiss Org Cult 104:61–67 Boonsnongcheep P, Korsangruang S, Soonthornchareonnon N, Chintapakorn Y, Saralamp P, Prathanturarug S (2010) Growth and isoflavonoid accumulation of Pueraria candollei var. candollei and P. candollei var. mirifica cell suspension cultures. Plant Cell Tiss Org Cult 101:119–126 Bourgaud F, Gravot A, Milesi S, Gontier E (2001) Production of plant secondary metabolites: a historical perspective. Plant Sci 161:839–851 Brindle P, Timberlake CF (1996) Anthocyanins as natural food colors: selected aspects. Food Chem 58:103–109 Callebaut A, Terahara N, de Haan M, Decleire M (1997) Stability of anthocyanin composition in Ajuga reptans callus and cell suspension cultures. Plant Cell Tiss Org Cult 50:195–201 Do CB, Cormier F (1990) Accumulation of anthocyanins enhanced by a high osmotic potential in grape (Vitis vinifera L.) cellsuspensions. Plant Cell Rep 9:143–146 Edahiro JI, Seki M (2006) Phenylpropanoid metabolite supports cell aggregate formation in strawberry cell suspension culture. J Biosci Bioeng 102:8–13 Estrada-Zu´n˜iga ME, Cruz-Sosa F, Rodrı´guez-Monroy M, VerdeCalvo JR, Vernon-Carter EJ (2009) Phenylpropanoid production in callus and cell suspension cultures of Buddleja cordata Kunth. Plant Cell Tiss Org Cult 97:39–47 Filippini R, Caniato R, Piovan A, Cappelletti EM (2003) Production of anthocyanins by Catharanthus roseus. Fitoterapia 74:62–67 Gould K, Davies K, Winefield C (2008) Anthocyanins: biosynthesis, function and applications. Springer, New York Hirasuma TJ, Shuler ML, Lackney VK, Spanswick RM (1991) Enhance anthocyanin production in grape cell culture. Plant Sci 78:107–120
Plant Cell Tiss Organ Cult (2011) 106:537–545 Kolewe ME, Gaurav V, Roberts SC (2008) Pharmaceutically active natural product synthesis and supply via plant cell culture technology. Mol Pharm 5:243–256 Konczac-Islam I, Yoshinaga M, Nakatami M, Terahara N, Yamakawa O (2000) Establishment and characteristic of an anthocyaninproducing cell line from sweet potato storage root. Plant Cell Rep 19:472–477 Madhusudhan R, Ravishankar GA (1996) Gradient of anthocyanin in cell aggregates of Daucus carota in suspension cultures. Biotechnol Lett 18:1253–1256 Meyer HJ, van Staden J (1995) The in vitro production of an anthocyanin from callus culture of Oxalis linearis. Plant Cell Tiss Org Cult 40:55–58 Meyer JE, Pe´pin MF, Smith MAL (2002) Anthocyanin production from Vaccinium pahalae: limitations of the physical microenvironment. J Biotechnol 93:45–57 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497 Nagella P, Murthy HN (2011) Effects of macroelements and nitrogen source on biomass accumulation and withanolide-A production from cell suspension cultures of Withania somnifera (L.) Dunal. Plant Cell Tiss Org Cult 104:119–124 Nakamura M, Seki M, Furusaki S (1998) Enhanced anthocyanin methylation by growth limitation in strawberry suspension culture. Enzyme Microb Technol 22:404–408 Narayan MS, Venkataraman LV (2000) Characterisation of anthocyanins derived from carrot (Daucus carota) cell culture. Food Chem 70:361–363 Narayan MS, Thimmaraju R, Bhagyalakshmi B (2005) Interplay of growth regulators during solid-state and liquid-state batch cultivation of anthocyanin producing cell line of Daucus carota. Process Biochem 40:351–358 Nawa Y, Asano S, Motoori S, Ohtani T (1993) Production of anthocyanins, carotenoids and proanthocyanidins by culture cells of rabbiteye blueberry (Vaccinium ashei Reade). Biosci Biotech Biochem 57:770–774 Pe`pin MF, Smith MAL, Reid JF (1999) Application of imaging tools to plant cell culture: relationship between plant cell aggregation and flavonoid production. In Vitro Cell Dev Biol Plant 35:290–295 Qu J, Zhang W, Yu X, Jin M (2005) Instability of anthocyanin accumulation in Vitis vinifera L. var. Gamay Fre´aux suspension cultures. Biotechnol Bioprocess Eng 10:155–161 Rao SR, Ravishankar GA (2002) Plant cell cultures: chemical factories of secondary metabolites. Biotechnol Adv 20:101–153 Saito K, Mizukami H (2002) Plant cell cultures as producers of secondary metabolites. In: Oksman-Caldentey K-M, Barz WH (eds) Plant biotechnology and trangenic plants. Marcel Dekker, New York
545 Sakamoto K, Iida K, Sawamura K, Hajiro K, Asada Y, Yoshikawa T, Furuya T (1993) Effects of nutrients on anthocyanin production in cultured cells of Aralia cordata. Phytochemistry 33:357–360 Simo˜es C, Santos AS, Albarello N, Figueiredo SFL (2004) Shoot organogenesis and plantlet regeneration from stem explants of Cleome rosea Vahl (Capparaceae). J Plant Biotech 6:199–204 Simo˜es C, Mattos JCP, Sabino KCC, Caldeira-de-Arau´jo A, Coelho MGP, Albarello N, Figueiredo SFL (2006) Medicinal potential from in vivo and acclimatized plants of Cleome rosea Vahl ex DC. (Capparaceae). Fitoterapia 77:94–99 Simo˜es C, Albarello N, Callado CH, Castro TC, Mansur E (2009a) New approaches for shoot production and in vitro root cultures of Cleome rosea Vahl. Plant Cell Tiss Org Cult 98:79–86 Simo˜es C, Bizarri CHB, Castro TC, Coutada LCM, Silva AJR, Albarello N, Mansur E (2009b) Anthocyanin production in callus cultures of Cleome rosea: modulation by culture conditions and characterization of pigments by means of HPLC-DAD/ ESIMS. Plant Physiol Biochem 47:895–903 Vogelien DL, Hrazdina G, Reevers S, Dougall DK (1990) Phenotypic differences in anthocyanin accumulation among clonally related cultured cells of carrot. Plant Cell Tiss Org Cult 22:213–222 Wai-Leng L, Lai-Keng C (2004) Establishment of Orthosiphon stamineus cell suspension culture for cell growth. Plant Cell Tiss Org Cult 78:101–106 Wang J, Mazza G (2002) Inhibitory effects of anthocyanins and other phenolic compounds on nitric oxide production in LPS/IFN-cactive RAW 2647 macrophages. J Agric Food Chem 50:850–857 Yoshinaga M, Yamakawa O, Nakatani M (1999) Genotypic diversity of anthocyanin content and composition in purple-fleshed sweet potato (Ipomoea batatas (L.) Lam. Breeding Sci 49:43–47 Zamith LR, Scarano FR (2006) Restoration of a restinga sandy coastal plain in Brazil: survival and growth of planted woody species. Restoration Ecol 14:87–94 Zhang W, Curtin C, Kikuchi M, Franco C (2002) Integration of jasmonic acid and light irradiation for enhancement of anthocyanin biosynthesis in Vitis vinifera suspension cultures. Plant Sci 162:459–468 Zhang W, Franco C, Curtin C, Conn S (2004) To stretch the bondary of secondary metabolite production in plant cell-based bioprocessing: anthocyanin as a case study. J Biomed Biotechnol 5:264–271 Zhao D, Huang Y, Jin Z, Qu W, Lu D (2003) Effect of aggregate size in cell cultures of Saussurea medusa on cell growth and jaceosidin production. Plant Cell Rep 21:1129–1133 Zhong JJ, Yoshida M, Fujiyama K, Seki T, Yoshida T (1993) Enhancement of anthocyanin production by Perilla frutescens cells in a stirred bioreactor with internal light irradiation. J Ferment Bioeng 75:299–303
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