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Impact of subculture cycles and inoculum sizes on suspension cultures of Vitis vinifera
CHINESE JOURNAL OF BIOTECHNOLOGY Volume 22, Issue 6, November 2006 Online English edition of the Chinese language journal Cite this article as: Chin J Biotech, 2006, 22(6), 984−989.
RESEARCH PAPER
Impact of Subculture Cycles and Inoculum Sizes on Suspension Cultures of Vitis vinifera QU Jun-Ge1,2, ZHANG Wei1,*, HU Quan-Li1, JIN Mei-Fang1 1
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
2
Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
Abstract: The commercial application of plant cell cultures is often hindered because of the instability of secondary metabolite biosynthesis, where the metabolite yield fluctuates and declines dramatically over the subculture cycles. This study proposed that such instability is due to the fluctuations of culture variables. To validate this hypothesis, the effects of the fluctuations of two culture variables (subculture cycle and inoculum size) on the biomass, anthocyanin biosynthesis, and intracellular carbon, nitrogen and phosphate during 10 continuous subculture cycles were investigated. The subculture cycle was fluctuated for 12 h over a 7-day cycle (6.5, 7, and 7.5 d), and the inoculum size was fluctuated by 20 % on the wet-weight basis of 2.00 g (1.60, 2.00, and 2.40 g). It was found that all the measured culture parameters fluctuated over the 10 subculture cycles. The fluctuation in terms of inoculum sizes had a greater effect on the stability of anthocyanin biosynthesis in suspension cultures of V. vinifera. Among all the subculture conditions investigated, 7-d subculture cycle and 1.60-g inoculum size were the best for maintaining relatively stable anthocyanin production. The anthocyanin yield showed a negative correlation with intracellular sucrose content or intracellular total phosphate content. Key Words:
Vitis vinifera; suspension culture; anthocyanin; subculture cycle; inoculum size
Plants represent one of the best chemical factories in nature, and over 100 000 compounds have been identified from plants, with approximately 4 000 new discoveries added every year[1]. Approximately 25 % of the drugs produced by pharmaceutical industries in USA are based on plant-derived chemicals[2]. Most of the bioactive chemicals are plant secondary metabolites. As a promising technology for the production of these bioactive chemicals, the products obtained using plant cell culture has many prominent advantages over those extracted from whole plants; for example, plant cell culture is not influenced by soil resource, season, climate, plant diseases, and insect pests. Moreover, the yields of plant secondary metabolites of interest can be selectively improved by regulation of their metabolic pathways[2,3]. Although plant cell culture is a promising technology for
the production of secondary metabolites, its commercial application has had only limited success[1,4]. The instability in terms of metabolite production in plant cell culture is one of the major problems in that hinders its commercialization[5–7], which is a universal phenomenon that has been observed in plant cell culture experiments conducted worldwide and therefore is a major impediment in this field[8,9]. Till now, the mechanism leading to this instability is ambiguous. A systematic investigation of anthocyanin accumulation in suspension cultures of Vitis vinifera as a model system has been initiated in the authors’ laboratory, and some studies in terms of the instability of biosynthesis of anthocyanins have been carried out[10]. During the long-term culture of plant cells, it is difficult to maintain the conditions, such as temperature, light irradiation,
Received: April 25, 2006; Accepted: June 13, 2006. * Corresponding author. Tel: +86-411-84379069; E-mail: [email protected] This work was supported by the grant from the National Natural Science Foundation of China (No. 20176058). Copyright © 2006, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.
QU Jun-Ge et al. / Chinese Journal of Biotechnology, 2006, 22(6): 984–989
subculture cycle, and inoculum size, constant. The tendency of these conditions to vary maybe one of the factors leading to the instability of secondary metabolites produced in plant cell cultures. In this article, the production of primary and secondary metabolites had been investigated in suspension cultures of Vitis vinifera in 10 successive subculture cycles, where subculture cycles and inoculum sizes varied, whereas the other conditions were maintained constant.
1
Materials and methods
1.1 Cell lines and subculture conditions The original cell line in the experiments of this study was provided by Dr. Francois Cormier’s group (Quebec, Canada)[10], which was subcultured in the authors’ laboratory for four years. The maintenance medium was B5 medium[11], supplemented with 30 g/L sucrose, 250 mg/L casein hydrolysate, 0.1 mg/L α-naphthaleneacetic acid (NAA) and 0.2 mg/L kinetin (K). The pH was adjusted to 5.7–5.8 before autoclaving at 115 °C for 15 min. The suspension cells were subcultured on a weekly basis in 250-mL Erlenmeyer flasks enclosed with aluminum foil and containing 50 mL maintenance medium. The inoculum size was approximately 5.0 g wet cells prepared by filtering precultured 7-day-old suspension cells using a 50-µm mesh. The subcultures were maintained in dark on a reciprocating shaker at 100 r/min at (25±1) °C. 1.2 Impact of subculture cycles on suspension cultures of Vitis vinifera The approximately 7-day-old precultured suspension cells were filtered using 50-µm meshes, and 2.00 g wet cells were accurately weighed and inoculated in 100-mL Erlenmeyer flasks containing 20 mL maintenance medium. The subcultures were maintained in dark on a reciprocating shaker at 100 r/min at (25±1) °C and the subculture cycles were 6.5, 7, and 7.5 d. During each subculture, the suspension cells were inoculated in 6 flasks, of which half were used for resubculturing after a certain subculture time, whereas the other half were used for analysis. The 10 successive subcultures were examined. 1.3 Impact of inoculum sizes on suspension cultures of Vitis vinifera The approximately 7-day-old precultured suspension cells were filtered using 50-µm meshes, and 1.60 g, 2.00 g and 2.40 g wet cells were respectively weighed and inoculated in 100-mL Erlenmeyer flasks containing 20 mL maintenance medium. The subcultures were maintained in the dark on a reciprocating shaker at 100 r/min at (25±1) °C, and the subculture cycles were 7 d. During each subculture, the suspension cells were inoculated in 6 flasks, of which half were used for resubculturing after a certain subculture time, whereas the other half were used for analysis. The 10 successive subcultures were examined.
1.4 Biomass analysis When the suspension cells were cultured for a subculture cycle, they were harvested by vacuum filtration through filter paper, washed with MilliQ water and weighed to obtain the fresh cell weight (FCW, g/L). With some fresh cells being left for extraction of anthocyanins, the other cells were kept in an oven at 80 °C overnight to obtain the dry cell weight (DCW, g/L). 1.5 Extraction and analysis of anthocyanins To analyze anthocyanin content, 0.15 g fresh cells were sampled after vacuum filtration and were extracted with 50% acetic acid solution with a volume equivalent to 20 times the fresh cell weight for 1-h period at room temperature. The filtrate was filtrated through a 0.22-µm syringe filter. After filtering through a 0.22-µm filter, 1 mL filtrate was mixed with 3 mL McIIvaine’s buffer (14.7 g/L Na2HPO4·12H2O and 16.7 g/L anhydrous citric acid, pH 3.0). The absorbance of the resulting solution was measured at 535 nm with 50 % acetic acid: McIIvaine’s buffer (1:3) as the blank control. A color value (CV) of the pigment extract, which is a commercial indicator of anthocyanin content, was calculated by the following formula[12]: CV = 0.1× Absorbance × Dilution factor (CV/g − FCW) In the above-described procedure, the dilution factor was 80. 1.6 Intracellular sugar, protein, and phosphate content After the soluble sugars were extracted by alcohol, the sucrose and fructose contents were measured by the method of resorcin-hydrochloric acid[13] and the glucose content was measured by enzyme[13]. The soluble protein content was measured by Bradford method[14] after ultrasonic extraction[14]. The total phosphate was extracted by heating to a high temperature and measured by sodium molybdate[15]. 1.7 Data analysis All the data were analyzed by the software of Microsoft Excel 2000 or SPSS 13.0.
2
Results and analysis
2.1 Impact of different subculture cycles and inoculum sizes on biomass and anthocyanin accumulation in suspension cultures of Vitis vinifera As shown in Fig. 1 and Fig. 2, although the culture conditions were kept constant, both the biomass and anthocyanin accumulation in 10 successive subcultures in suspension cultures of Vitis vinifera, fluctuated for each subculture condition. The maximal fluctuation of biomass exceeded 50 % (Figs. 1A and 2A). The maximal fluctuation of anthocyanin content exceeded 100 % (Figs. 1B and 2B) and anthocyanin production exceeded 300 % (Figs. 1C and 2C). The results about the instability of the production of secondary metabolite in plant cell culture were similar to the previous reports[7].
QU Jun-Ge et al. / Chinese Journal of Biotechnology, 2006, 22(6): 984–989
As observed from the changing pattern of anthocyanin production in 10 successive subcultures, the instability showed cumulative effect. As shown in Fig. 1, for the subculture condition with respect to 6.5-d subculture cycle, the anthocyanin production decreased from 659 CV/L in the 8th subculture to 193 CV/L in the 10th subculture. For the
subculture condition with respect to 7-d subculture cycle, the anthocyanin production decreased from 996 CV/L in the 8th subculture to 279 CV/L in the 10th subculture. And for the subculture condition pertaining to the 7.5-d subculture cycle, the anthocyanin production increased from 315 CV/L in the 9th subculture to 974 CV/L in the 10th subculture.
Fig. 1 Cell growth and anthocyanin accumulation under different subculture cycles in successive subcultures of V. vinifera (A) Biomass (DCW); (B) Anthocyanin content; (C) Anthocyanin yield.
Fig. 2 Cell growth and anthocyanin accumulation under different inoculum sizes in successive subcultures of V. vinifera (A) Biomass (DCW); (B) Anthocyanin content; (C) Anthocyanin yield.
To compare the impact of different subculture conditions on anthocyanin accumulation of Vitis vinifera, the instability coefficient, δ, was used to express the extent of instability of anthocyanin accumulation in 10 successive subculture cycles. 9
( Xn − 1 − Xn )
n =1
Xn X
δ= ∑
(1)
As shown in Eq. (1), δ reflected not only the influence of differences among samples, but also the whole samples, and it was an integrated consideration on the instability of secondary metabolite production. Table 1 showed the instability coefficients of anthocyanin contents in 10 successive subcultures under different subculture conditions. When the
inoculum size was kept constant, the instability coefficients of different subculture cycles were nearly similar (δ was 0.65, 0.68 and 0.62, respectively). When the subculture cycle was kept constant, the instability coefficients of different inoculum sizes considerably varied. At the inoculum size of 2.00 g, δ was 0.68. When inoculum sizes were 1.60 g and 2.40 g, δ were 0.44 and 0.54, respectively. Among all the inoculum sizes, 1.60 g showed the best stability. This showed that when compared with subculture cycles, inoculum sizes had greater influences on instability. Among all the subculture conditions that were investigated, 7-d subculture cycle and 1.60-g inoculum size was the best one to maintain the relatively stable anthocyanin production.
Table 1 Instability coefficients of anthocyanin contents under different subculture conditions in 10 successive subcultures of V. vinifera Subculture Condition
(6.5d, 2.0g )
(7d, 2.0g )
(7.5d, 2.0g )
(7d, 1.6g )
(7d, 2.4g )
δ
0.65
0.68
0.62
0.44
0.54
Variation coefficient is the ratio of standard deviation to average. The higher the variation coefficient, the more instable
the samples[10]. Table 2 compared the variation coefficients of the anthocyanin contents with respect to different subculture
QU Jun-Ge et al. / Chinese Journal of Biotechnology, 2006, 22(6): 984–989
conditions. Under each subculture, 9 data of 3 different subculture cycles or 3 different inoculum sizes (3 replicates of each condition) were used to calculate the variation coefficient. The variation coefficient of each subculture cycle showed the instability of anthocyanin accumulation in different subculture cycles or different inoculum sizes. As shown in Table 2, the variation coefficients of different subculture cycles were higher than those of different inoculum sizes in 10 successive subculture cycles. The sum of variation coefficients of different subculture cycles in 10 successive subculture cycles
was 1.9, while that of different inoculum sizes was 1.4. Taking Table 1 into account, it could be seen that the above result was obtained when the inoculum sizes were all 2.00 g when different subculture cycles were investigated. And 2.00 g was the most instable inoculum size for anthocyanin accumulation. It was only because of the excessive instable factor that the variation coefficient of subculture cycles was higher than that of inoculum sizes. This also showed inoculum sizes had a greater influence on instability than subculture cycles in suspension cultures of Vitis vinifera.
Table 2 Variation coefficients of anthocyanin content under all the subculture cycles and inoculum sizes in 10 successive subcultures of V. vinifera No.
1
2
3
4
VC of SC
0.21
0.08
0.18
0.13
VC of IS
0.15
0.09
0.14
0.15
5
6
7
8
9
10
Sum
0.20
0.13
0.25
0.17
0.22
0.31
1.9
0.18
0.14
0.11
0.14
0.13
0.16
1.4
No.: subculture number VC of SC: variation coefficients of subculture cycle VC of IS: variation coefficients of inoculum size
2.2 Anthocyanin accumulation and intracellular sugar, protein and total phosphate content The relationship between anthocyanin accumulation and intracellular sugar, protein or phosphate content was illustrated with the culture conditions of 7-d subculture cycle and 2.0-g inoculum size. As shown in Fig. 3, A represented anthocyanin accumulation in 10 successive subcultures; B–F
represented sucrose, fructose, glucose, soluble protein, and total phosphate, respectively. With the fluctuation of anthocyanin biosynthesis, the intracellular sugar, protein and phosphate contents showed instability of varying degrees. But it was difficult to summarize the rules only by the trendlines, and therefore, correlation analysis of SPSS was applied here.
Fig. 3 Accumulation of primary and secondary metabolites in successive subcultures of V. vinifera
As shown in Table 3, anthocyanin production and intracellular sucrose, fructose, glucose, protein, and phosphate showed correlation of varying degrees. Among all these primary metabolites, sucrose and phosphate showed
remarkable correlation. For example, under the condition of 6.5-d subculture cycle and 2.0-g inoculum size as well as 7.5-d subculture cycle and 2.0-g inoculum size, anthocyanin production and phosphate content showed negative correlation
QU Jun-Ge et al. / Chinese Journal of Biotechnology, 2006, 22(6): 984–989
at the level of 0.05. Under the condition of 7-d subculture cycle and 2.0-g inoculum size as well as 7-d subculture cycle and 2.40-g inoculum size, anthocyanin production and sucrose content showed negative correlation at the level of 0.05. And
under the condition of 7-d subculture cycle and 1.6-g inoculum size, anthocyanin production and sucrose content as well as phosphate content showed negative correlations at the level of 0.05.
Table 3 Correlation between anthocyanin yield and sucrose, fructose, glucose, protein, and phosphate content in successive subcultures of V. vinifera Sucrose (6.5 d, 2.0 g) (7 d, 2.0 g) (7.5 d, 2.0 g ) (7 d, 1.6 g ) (7 d, 2.4 g )
Fructose
Glucose
Protein
Phosphate
Pearson Correlation
−0.097
0.172
−0.021
−0.484
−0.643*
Sig. (2-tailed)
0.789
0.634
0.955
0.156
0.045 −0.510
*
Pearson Correlation
−0.690
−0.336
0.263
0.217
Sig. (2-tailed)
0.027
0.343
0.463
0.547
0.132
Pearson Correlation
−0.203
−0.482
−0.554
−0.339
− 0.646*
Sig. (2-tailed)
0.574
0.159
0.097
0.337
0.044
*
−0.212
0.191
−0.261
−0.652*
Sig. (2-tailed)
0.012
0.557
0.598
0.466
0.041
Pearson Correlation
−0.722*
−0.416
−0.202
−0.122
−0.597
Sig. (2-tailed)
0.018
0.231
0.577
0.736
0.068
Pearson Correlation
−0.751
* P=0.05.
3
Conclusions
The instability of secondary metabolites accumulation in plant cell culture is one of the bottlenecks for the commercialization of secondary metabolites production using plant cell culture. In the previous study by the authors, it was found that the anthocyanin accumulation in different anthocyanin-producing cell lines of Vitis vinifera exhibited fluctuation in 33 successive suspension subcultures[10]. For example, the highest anthocyanin content of A cell line in 33 subcultures was 4.89 CV/g-FCW, whereas the lowest was 0.28 CV/g-FCW. The former was more than 17 times of the later. Vogelien and Hirasuma also reported the instabilities of anthocyanin accumulation in suspension cultures of Vitis vinifera and Daucus carota[16,17]. The results of all these findings were in accordance with the results of the studies by the authors. The biomass, anthocyanin accumulation, and contents of intracellular C, N and P were instable in 10 successive suspension cultures of Vitis vinifera for different subculture cycle and inoculum size conditions (Figs. 1–3). It was difficult to maintain the subculture conditions, such as temperature, light irradiation, subculture cycle, and inoculum size, constant. It can be concluded from this research that both subculture cycle and inoculum size affect the instability of suspension cultures of Vitis vinifera. Inoculum sizes had greater influence on instability than subculture cycles (Table 1). Among all the subculture conditions investigated, 7-d subculture cycle and 1.60-g inoculum size was the best to maintain relatively stable anthocyanin production. For the given plant cell lines, the conditions can be optimized to maintain the high and stable production of secondary metabolites by screening temperature,
light irradiation, subculture cycle, inoculum size, inoculum age, shear force, and so on. This is the key to the commercialization of plant cell culture. By the correlation analysis of anthocyanin production and intracellular parameters in suspension cultures of Vitis vinifera, it is found that anthocyanin yield showed a negative correlation with intracellular sucrose content or intracellular total phosphate content (Table 3). The previous reports indicated that the phosphate content in the medium affected the activity of PAL[18,19]. The sucrose content in medium affected anthocyanin content by regulating osmotic pressure[20,21]. The extracellular nutrients will influence the intracellular contents in plant cell culture. In the subsequent study, the authors will investigate the correlation of sucrose and phosphate contents in medium with intracellular sucrose and phosphate contents. Based on the above research, corresponding techniques will be designed to control intracellular sucrose and phosphate contents by controlling their extracellular contents. The high and stable anthocyanin accumulation will be realized in suspension cultures of Vitis vinifera. At present, the further research is underway. REFERENCES [1]
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related cultured cells of carrot. Plant Cell, Tissue and Organ
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[10] Qu JG, Zhang W, Yu XJ, et al. Instability of anthocyanin accumulation in Vitis vinifera L. var Gamay Fréaux suspension cultures. Biotechnology and Bioprocess Engineering, 2005, 10: 155–161. [11] Gamborg OL, Miller RA, Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research, 1968, 50: 151–156. [12] Zhang W, Curtin C, Kikuchi M, et al. Integration of jasmonic acid and light irradiation for enhancement of anthocyanin biosynthesis in Vitis vinifera suspension cultures. Plant Science,
of enzyme activities and product accumulation. Plant Cell, Tissue and Organ Culture, 1983, 2: 333–340. [20] Do CB, Cormier F. Accumulation of anthocyanins enhanced by a high osmotic potential in grape (Vitis vinifera L.) cell suspensions. Plant Cell Reports, 1990, 9: 143–146. [21] Do CB, Cormier F. Accumulation of peonidin 3-glucoside enhanced by a high osmotic stress in grape (Vitis vinifera L.) cell suspension. Plant Cell, Tissue and Organ Culture, 1991, 24: 49–54.
RESEARCH PAPER
Impact of Subculture Cycles and Inoculum Sizes on Suspension Cultures of Vitis vinifera QU Jun-Ge1,2, ZHANG Wei1,*, HU Quan-Li1, JIN Mei-Fang1 1
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
2
Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
Abstract: The commercial application of plant cell cultures is often hindered because of the instability of secondary metabolite biosynthesis, where the metabolite yield fluctuates and declines dramatically over the subculture cycles. This study proposed that such instability is due to the fluctuations of culture variables. To validate this hypothesis, the effects of the fluctuations of two culture variables (subculture cycle and inoculum size) on the biomass, anthocyanin biosynthesis, and intracellular carbon, nitrogen and phosphate during 10 continuous subculture cycles were investigated. The subculture cycle was fluctuated for 12 h over a 7-day cycle (6.5, 7, and 7.5 d), and the inoculum size was fluctuated by 20 % on the wet-weight basis of 2.00 g (1.60, 2.00, and 2.40 g). It was found that all the measured culture parameters fluctuated over the 10 subculture cycles. The fluctuation in terms of inoculum sizes had a greater effect on the stability of anthocyanin biosynthesis in suspension cultures of V. vinifera. Among all the subculture conditions investigated, 7-d subculture cycle and 1.60-g inoculum size were the best for maintaining relatively stable anthocyanin production. The anthocyanin yield showed a negative correlation with intracellular sucrose content or intracellular total phosphate content. Key Words:
Vitis vinifera; suspension culture; anthocyanin; subculture cycle; inoculum size
Plants represent one of the best chemical factories in nature, and over 100 000 compounds have been identified from plants, with approximately 4 000 new discoveries added every year[1]. Approximately 25 % of the drugs produced by pharmaceutical industries in USA are based on plant-derived chemicals[2]. Most of the bioactive chemicals are plant secondary metabolites. As a promising technology for the production of these bioactive chemicals, the products obtained using plant cell culture has many prominent advantages over those extracted from whole plants; for example, plant cell culture is not influenced by soil resource, season, climate, plant diseases, and insect pests. Moreover, the yields of plant secondary metabolites of interest can be selectively improved by regulation of their metabolic pathways[2,3]. Although plant cell culture is a promising technology for
the production of secondary metabolites, its commercial application has had only limited success[1,4]. The instability in terms of metabolite production in plant cell culture is one of the major problems in that hinders its commercialization[5–7], which is a universal phenomenon that has been observed in plant cell culture experiments conducted worldwide and therefore is a major impediment in this field[8,9]. Till now, the mechanism leading to this instability is ambiguous. A systematic investigation of anthocyanin accumulation in suspension cultures of Vitis vinifera as a model system has been initiated in the authors’ laboratory, and some studies in terms of the instability of biosynthesis of anthocyanins have been carried out[10]. During the long-term culture of plant cells, it is difficult to maintain the conditions, such as temperature, light irradiation,
Received: April 25, 2006; Accepted: June 13, 2006. * Corresponding author. Tel: +86-411-84379069; E-mail: [email protected] This work was supported by the grant from the National Natural Science Foundation of China (No. 20176058). Copyright © 2006, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.
QU Jun-Ge et al. / Chinese Journal of Biotechnology, 2006, 22(6): 984–989
subculture cycle, and inoculum size, constant. The tendency of these conditions to vary maybe one of the factors leading to the instability of secondary metabolites produced in plant cell cultures. In this article, the production of primary and secondary metabolites had been investigated in suspension cultures of Vitis vinifera in 10 successive subculture cycles, where subculture cycles and inoculum sizes varied, whereas the other conditions were maintained constant.
1
Materials and methods
1.1 Cell lines and subculture conditions The original cell line in the experiments of this study was provided by Dr. Francois Cormier’s group (Quebec, Canada)[10], which was subcultured in the authors’ laboratory for four years. The maintenance medium was B5 medium[11], supplemented with 30 g/L sucrose, 250 mg/L casein hydrolysate, 0.1 mg/L α-naphthaleneacetic acid (NAA) and 0.2 mg/L kinetin (K). The pH was adjusted to 5.7–5.8 before autoclaving at 115 °C for 15 min. The suspension cells were subcultured on a weekly basis in 250-mL Erlenmeyer flasks enclosed with aluminum foil and containing 50 mL maintenance medium. The inoculum size was approximately 5.0 g wet cells prepared by filtering precultured 7-day-old suspension cells using a 50-µm mesh. The subcultures were maintained in dark on a reciprocating shaker at 100 r/min at (25±1) °C. 1.2 Impact of subculture cycles on suspension cultures of Vitis vinifera The approximately 7-day-old precultured suspension cells were filtered using 50-µm meshes, and 2.00 g wet cells were accurately weighed and inoculated in 100-mL Erlenmeyer flasks containing 20 mL maintenance medium. The subcultures were maintained in dark on a reciprocating shaker at 100 r/min at (25±1) °C and the subculture cycles were 6.5, 7, and 7.5 d. During each subculture, the suspension cells were inoculated in 6 flasks, of which half were used for resubculturing after a certain subculture time, whereas the other half were used for analysis. The 10 successive subcultures were examined. 1.3 Impact of inoculum sizes on suspension cultures of Vitis vinifera The approximately 7-day-old precultured suspension cells were filtered using 50-µm meshes, and 1.60 g, 2.00 g and 2.40 g wet cells were respectively weighed and inoculated in 100-mL Erlenmeyer flasks containing 20 mL maintenance medium. The subcultures were maintained in the dark on a reciprocating shaker at 100 r/min at (25±1) °C, and the subculture cycles were 7 d. During each subculture, the suspension cells were inoculated in 6 flasks, of which half were used for resubculturing after a certain subculture time, whereas the other half were used for analysis. The 10 successive subcultures were examined.
1.4 Biomass analysis When the suspension cells were cultured for a subculture cycle, they were harvested by vacuum filtration through filter paper, washed with MilliQ water and weighed to obtain the fresh cell weight (FCW, g/L). With some fresh cells being left for extraction of anthocyanins, the other cells were kept in an oven at 80 °C overnight to obtain the dry cell weight (DCW, g/L). 1.5 Extraction and analysis of anthocyanins To analyze anthocyanin content, 0.15 g fresh cells were sampled after vacuum filtration and were extracted with 50% acetic acid solution with a volume equivalent to 20 times the fresh cell weight for 1-h period at room temperature. The filtrate was filtrated through a 0.22-µm syringe filter. After filtering through a 0.22-µm filter, 1 mL filtrate was mixed with 3 mL McIIvaine’s buffer (14.7 g/L Na2HPO4·12H2O and 16.7 g/L anhydrous citric acid, pH 3.0). The absorbance of the resulting solution was measured at 535 nm with 50 % acetic acid: McIIvaine’s buffer (1:3) as the blank control. A color value (CV) of the pigment extract, which is a commercial indicator of anthocyanin content, was calculated by the following formula[12]: CV = 0.1× Absorbance × Dilution factor (CV/g − FCW) In the above-described procedure, the dilution factor was 80. 1.6 Intracellular sugar, protein, and phosphate content After the soluble sugars were extracted by alcohol, the sucrose and fructose contents were measured by the method of resorcin-hydrochloric acid[13] and the glucose content was measured by enzyme[13]. The soluble protein content was measured by Bradford method[14] after ultrasonic extraction[14]. The total phosphate was extracted by heating to a high temperature and measured by sodium molybdate[15]. 1.7 Data analysis All the data were analyzed by the software of Microsoft Excel 2000 or SPSS 13.0.
2
Results and analysis
2.1 Impact of different subculture cycles and inoculum sizes on biomass and anthocyanin accumulation in suspension cultures of Vitis vinifera As shown in Fig. 1 and Fig. 2, although the culture conditions were kept constant, both the biomass and anthocyanin accumulation in 10 successive subcultures in suspension cultures of Vitis vinifera, fluctuated for each subculture condition. The maximal fluctuation of biomass exceeded 50 % (Figs. 1A and 2A). The maximal fluctuation of anthocyanin content exceeded 100 % (Figs. 1B and 2B) and anthocyanin production exceeded 300 % (Figs. 1C and 2C). The results about the instability of the production of secondary metabolite in plant cell culture were similar to the previous reports[7].
QU Jun-Ge et al. / Chinese Journal of Biotechnology, 2006, 22(6): 984–989
As observed from the changing pattern of anthocyanin production in 10 successive subcultures, the instability showed cumulative effect. As shown in Fig. 1, for the subculture condition with respect to 6.5-d subculture cycle, the anthocyanin production decreased from 659 CV/L in the 8th subculture to 193 CV/L in the 10th subculture. For the
subculture condition with respect to 7-d subculture cycle, the anthocyanin production decreased from 996 CV/L in the 8th subculture to 279 CV/L in the 10th subculture. And for the subculture condition pertaining to the 7.5-d subculture cycle, the anthocyanin production increased from 315 CV/L in the 9th subculture to 974 CV/L in the 10th subculture.
Fig. 1 Cell growth and anthocyanin accumulation under different subculture cycles in successive subcultures of V. vinifera (A) Biomass (DCW); (B) Anthocyanin content; (C) Anthocyanin yield.
Fig. 2 Cell growth and anthocyanin accumulation under different inoculum sizes in successive subcultures of V. vinifera (A) Biomass (DCW); (B) Anthocyanin content; (C) Anthocyanin yield.
To compare the impact of different subculture conditions on anthocyanin accumulation of Vitis vinifera, the instability coefficient, δ, was used to express the extent of instability of anthocyanin accumulation in 10 successive subculture cycles. 9
( Xn − 1 − Xn )
n =1
Xn X
δ= ∑
(1)
As shown in Eq. (1), δ reflected not only the influence of differences among samples, but also the whole samples, and it was an integrated consideration on the instability of secondary metabolite production. Table 1 showed the instability coefficients of anthocyanin contents in 10 successive subcultures under different subculture conditions. When the
inoculum size was kept constant, the instability coefficients of different subculture cycles were nearly similar (δ was 0.65, 0.68 and 0.62, respectively). When the subculture cycle was kept constant, the instability coefficients of different inoculum sizes considerably varied. At the inoculum size of 2.00 g, δ was 0.68. When inoculum sizes were 1.60 g and 2.40 g, δ were 0.44 and 0.54, respectively. Among all the inoculum sizes, 1.60 g showed the best stability. This showed that when compared with subculture cycles, inoculum sizes had greater influences on instability. Among all the subculture conditions that were investigated, 7-d subculture cycle and 1.60-g inoculum size was the best one to maintain the relatively stable anthocyanin production.
Table 1 Instability coefficients of anthocyanin contents under different subculture conditions in 10 successive subcultures of V. vinifera Subculture Condition
(6.5d, 2.0g )
(7d, 2.0g )
(7.5d, 2.0g )
(7d, 1.6g )
(7d, 2.4g )
δ
0.65
0.68
0.62
0.44
0.54
Variation coefficient is the ratio of standard deviation to average. The higher the variation coefficient, the more instable
the samples[10]. Table 2 compared the variation coefficients of the anthocyanin contents with respect to different subculture
QU Jun-Ge et al. / Chinese Journal of Biotechnology, 2006, 22(6): 984–989
conditions. Under each subculture, 9 data of 3 different subculture cycles or 3 different inoculum sizes (3 replicates of each condition) were used to calculate the variation coefficient. The variation coefficient of each subculture cycle showed the instability of anthocyanin accumulation in different subculture cycles or different inoculum sizes. As shown in Table 2, the variation coefficients of different subculture cycles were higher than those of different inoculum sizes in 10 successive subculture cycles. The sum of variation coefficients of different subculture cycles in 10 successive subculture cycles
was 1.9, while that of different inoculum sizes was 1.4. Taking Table 1 into account, it could be seen that the above result was obtained when the inoculum sizes were all 2.00 g when different subculture cycles were investigated. And 2.00 g was the most instable inoculum size for anthocyanin accumulation. It was only because of the excessive instable factor that the variation coefficient of subculture cycles was higher than that of inoculum sizes. This also showed inoculum sizes had a greater influence on instability than subculture cycles in suspension cultures of Vitis vinifera.
Table 2 Variation coefficients of anthocyanin content under all the subculture cycles and inoculum sizes in 10 successive subcultures of V. vinifera No.
1
2
3
4
VC of SC
0.21
0.08
0.18
0.13
VC of IS
0.15
0.09
0.14
0.15
5
6
7
8
9
10
Sum
0.20
0.13
0.25
0.17
0.22
0.31
1.9
0.18
0.14
0.11
0.14
0.13
0.16
1.4
No.: subculture number VC of SC: variation coefficients of subculture cycle VC of IS: variation coefficients of inoculum size
2.2 Anthocyanin accumulation and intracellular sugar, protein and total phosphate content The relationship between anthocyanin accumulation and intracellular sugar, protein or phosphate content was illustrated with the culture conditions of 7-d subculture cycle and 2.0-g inoculum size. As shown in Fig. 3, A represented anthocyanin accumulation in 10 successive subcultures; B–F
represented sucrose, fructose, glucose, soluble protein, and total phosphate, respectively. With the fluctuation of anthocyanin biosynthesis, the intracellular sugar, protein and phosphate contents showed instability of varying degrees. But it was difficult to summarize the rules only by the trendlines, and therefore, correlation analysis of SPSS was applied here.
Fig. 3 Accumulation of primary and secondary metabolites in successive subcultures of V. vinifera
As shown in Table 3, anthocyanin production and intracellular sucrose, fructose, glucose, protein, and phosphate showed correlation of varying degrees. Among all these primary metabolites, sucrose and phosphate showed
remarkable correlation. For example, under the condition of 6.5-d subculture cycle and 2.0-g inoculum size as well as 7.5-d subculture cycle and 2.0-g inoculum size, anthocyanin production and phosphate content showed negative correlation
QU Jun-Ge et al. / Chinese Journal of Biotechnology, 2006, 22(6): 984–989
at the level of 0.05. Under the condition of 7-d subculture cycle and 2.0-g inoculum size as well as 7-d subculture cycle and 2.40-g inoculum size, anthocyanin production and sucrose content showed negative correlation at the level of 0.05. And
under the condition of 7-d subculture cycle and 1.6-g inoculum size, anthocyanin production and sucrose content as well as phosphate content showed negative correlations at the level of 0.05.
Table 3 Correlation between anthocyanin yield and sucrose, fructose, glucose, protein, and phosphate content in successive subcultures of V. vinifera Sucrose (6.5 d, 2.0 g) (7 d, 2.0 g) (7.5 d, 2.0 g ) (7 d, 1.6 g ) (7 d, 2.4 g )
Fructose
Glucose
Protein
Phosphate
Pearson Correlation
−0.097
0.172
−0.021
−0.484
−0.643*
Sig. (2-tailed)
0.789
0.634
0.955
0.156
0.045 −0.510
*
Pearson Correlation
−0.690
−0.336
0.263
0.217
Sig. (2-tailed)
0.027
0.343
0.463
0.547
0.132
Pearson Correlation
−0.203
−0.482
−0.554
−0.339
− 0.646*
Sig. (2-tailed)
0.574
0.159
0.097
0.337
0.044
*
−0.212
0.191
−0.261
−0.652*
Sig. (2-tailed)
0.012
0.557
0.598
0.466
0.041
Pearson Correlation
−0.722*
−0.416
−0.202
−0.122
−0.597
Sig. (2-tailed)
0.018
0.231
0.577
0.736
0.068
Pearson Correlation
−0.751
* P=0.05.
3
Conclusions
The instability of secondary metabolites accumulation in plant cell culture is one of the bottlenecks for the commercialization of secondary metabolites production using plant cell culture. In the previous study by the authors, it was found that the anthocyanin accumulation in different anthocyanin-producing cell lines of Vitis vinifera exhibited fluctuation in 33 successive suspension subcultures[10]. For example, the highest anthocyanin content of A cell line in 33 subcultures was 4.89 CV/g-FCW, whereas the lowest was 0.28 CV/g-FCW. The former was more than 17 times of the later. Vogelien and Hirasuma also reported the instabilities of anthocyanin accumulation in suspension cultures of Vitis vinifera and Daucus carota[16,17]. The results of all these findings were in accordance with the results of the studies by the authors. The biomass, anthocyanin accumulation, and contents of intracellular C, N and P were instable in 10 successive suspension cultures of Vitis vinifera for different subculture cycle and inoculum size conditions (Figs. 1–3). It was difficult to maintain the subculture conditions, such as temperature, light irradiation, subculture cycle, and inoculum size, constant. It can be concluded from this research that both subculture cycle and inoculum size affect the instability of suspension cultures of Vitis vinifera. Inoculum sizes had greater influence on instability than subculture cycles (Table 1). Among all the subculture conditions investigated, 7-d subculture cycle and 1.60-g inoculum size was the best to maintain relatively stable anthocyanin production. For the given plant cell lines, the conditions can be optimized to maintain the high and stable production of secondary metabolites by screening temperature,
light irradiation, subculture cycle, inoculum size, inoculum age, shear force, and so on. This is the key to the commercialization of plant cell culture. By the correlation analysis of anthocyanin production and intracellular parameters in suspension cultures of Vitis vinifera, it is found that anthocyanin yield showed a negative correlation with intracellular sucrose content or intracellular total phosphate content (Table 3). The previous reports indicated that the phosphate content in the medium affected the activity of PAL[18,19]. The sucrose content in medium affected anthocyanin content by regulating osmotic pressure[20,21]. The extracellular nutrients will influence the intracellular contents in plant cell culture. In the subsequent study, the authors will investigate the correlation of sucrose and phosphate contents in medium with intracellular sucrose and phosphate contents. Based on the above research, corresponding techniques will be designed to control intracellular sucrose and phosphate contents by controlling their extracellular contents. The high and stable anthocyanin accumulation will be realized in suspension cultures of Vitis vinifera. At present, the further research is underway. REFERENCES [1]
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