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Selection of high rosmarinic acid producing Lavandula vera MM cell lines
Process Biochemistry 41 (2006) 2068–2071 www.elsevier.com/locate/procbio
Selection of high rosmarinic acid producing Lavandula vera MM cell lines M. Georgiev, A. Pavlov, M. Ilieva * Department of Microbial Biosynthesis and Biotechnologies, Laboratory in Plovdiv, Institute of Microbiology, Bulgarian Academy of Sciences, Plovdiv, Bulgaria Received 28 January 2006; received in revised form 3 May 2006; accepted 5 May 2006
Abstract Selection of stable Lavandula vera MM cell lines producing high amounts of rosmarinic acid (RA) was performed using m-F-D,L-phenylalanine and p-F-D,L-phenylalanine. As a result, two callus lines L. vera MF and L. vera PF were obtained, which produced 1.95 and 1.71 times more rosmarinic acid in comparison with the parent culture. After adaptation of the selected callus lines in liquid media the achieved yields of rosmarinic acid were 2808.4 mg L1 (in L. vera MF cell suspension) and 2594.6 mg L1 (in L. vera PF cell suspension). # 2006 Elsevier Ltd. All rights reserved. Keywords: Rosmarinic acid; Lavandula vera MM; Line selection; m-F-D,L-phenylalanine; p-F-D,L-phenylalanine
1. Introduction Optimization of secondary metabolite production in different plant in vitro systems is a difficult and complicated process. Frequently, the stage of the selection of highproducing cell lines is underestimated. However, the selection of high-producing cell lines using selective agents was found to be an effective strategy for the enhancement of the accumulation of aromatic compounds (phenolics and shikonin derivates) in cultured plant cells [1,2]. Rosmarinic acid (RA) is a secondary metabolite which possesses antiviral, antibacterial, anti-inflammatory, antiallergic activities [3–5] and prevents the proliferation of human cancer cells [6]. The interesting biological activities of RA and its low content in the intact plants impose the development of alternative ways for the production of RA. Plant cell and tissue cultures are considered prominent producers of RA [7] and, therefore, a number of strategies for yield improvement have been developed, such as nutrient medium optimization [8], elicitation [9], optimization of culture conditions [10], etc. However, there is no information available about the selection of high-producing rosmarinic acid cell lines of lavender.
* Corresponding author. Tel.: +359 32 642 430; fax: +359 28 700 109. E-mail addresses: [email protected], [email protected] (M. Ilieva). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.05.007
In this paper we report the data on the selection of Lavandula vera (hereafter simplified term L. vera MM) MM cell lines resistant to analogues of phenylalanine (m- and p-F-D,Lphenylalanine), their rosmarinic acid content, as well as on the adaptation of the obtained lines to the submerged cultivations. 2. Materials and methods 2.1. Plant cell culture and media L. vera MM callus culture was obtained from stems of the oil-bearing sort Lavender ‘‘Drujba’’ and maintained as it has been previously described [11]. It was cultivated on standard Linsmayer–Skoog (LS) media [12], supplemented with 30 g L1 sucrose, 0.2 mg L1 2,4-dichlorophenoxyacetic acid and 5.5 g L1 plant agar. For experiments on selection, the modified LS nutrient media (added with 60 g L1 sucrose and NO3/NH4+ ratio of 40) was used [8]. Callus culture was grown in a thermostat at 26 8C, in darkness; suspension culture was cultivated in Erlenmayer flasks with total volume 500 mL (working volume 100 mL) on a shaker (110 rad s1), at 26 8C in darkness. For inoculation, 20% (v/v) cell suspension cultivated under the above mentioned conditions for 7 days was used.
2.2. Experimental settings m-F-D,L-phenylalanine (MFP) and p-F-D,L-phenylalanine (PFP) (Sigma– Aldrich, USA) were dissolved in distilled water and sterilized by filtration (Stericup, GV Durapore Membrane, 0.2 mm). Selection was carried out using the method described by Gonzales and Widholm [13], which includes mixing of medium (containing the relevant amounts of selection agent and agar) with equal amounts of 7-day old suspension culture. After 42-days, the grown colonies were transferred to standard LS solid media and cultivated for another
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21 days. Their growth was checked by the scale 0 (without growth) to 5 (excellent growth). The rosmarinic acid content of the selected lines was checked as well.
Table 2 Rosmarinic acid content in m-F-D,L-phenylalanine selected Lavandula vera MM callus lines
2.3. Analysis
MFP concentration (mM)
2.3.1. Growth of the L. vera lines Growth of the culture lines was monitored by measurement of dry weight (60 8C to constant weight) [14]. 2.3.2. Rosmarinic acid extraction and determination Frozen cell biomasses were extracted with 50% ethanol at 70 8C for 1 h (3 20 min). Ethanolic extracts were combined and evaporated under reduced pressure (vacuum evaporator, Laborota 4002, Heidolph). The samples were dissolved in 70% ethanol and were stored for 24 h at 10 8C. The obtained precipitate was separated and the supernatant was used for analysis of rosmarinic acid. Quantitative determination of rosmarinic acid was performed spectrophotometrically (spectrophotometer UV/VIS Shimadzu 1240) at 327 nm [15]. As a standard, pure rosmarinic acid (Extrasynthese, Genay, France) was used. The data presented are the averages from two independent experiments, each repeated twice (S.D.). All determinations were performed in three replicates.
Line
Rosmarinic acid (mg g1 dry weight)
Percentage from control
–
Control
5.08 0.11
100
0.2 mM MFP
MF11 MF12 MF13 MF14 MF15
4.56 0.19 5.17 0.18 4.47 0.06 5.60 0.11 4.60 0.09
90 102 88 110 91
0.4 mM MFP
MF21 MF22 MF23 MF24 MF25
4.82 0.17 5.51 0.13 5.17 0.13 5.73 0.08 6.12 0.16
95 109 102 113 121
0.5 mM MFP
MF31 MF32 MF33 MF34
6.12 0.19 6.69 0.21 5.95 0.12 5.47 0.08
121 132 117 108
0.6 mM MFP
MF41 MF42 MF43
8.94 0.19 7.11 0.15 6.40 0.15
176 140 126
0.8 mM MFP
MF51 MF52 MF53
9.90 0.17 8.21 0.26 8.81 0.11
195 162 173
3. Results and discussion 3.1. Selection of L. vera MM callus lines with high and stable rosmarinic acid production The advantage of the amino acid analogues as selection agents is that they kill the majority of the wild type cells, while the desired variants (high phenolics producing cell lines) survive the treatment [16]. This phenomenon is attributed to the fact that only cells with increased phenylalanine ammonialyase activity (PAL) (key enzyme in phenolics biosynthesis pathway) can be detoxified by them and survive. As far as PAL is one of the key enzymes included in the biosynthetic pathway of RA, it can be expected that the cell lines with enhanced activity of PAL will produce higher amounts of RA [7]. As selective agents, m- and p-F-D,L-phenylalanine were used in concentration from 0.2 – 0.1 mM and 0.2 – 0.8 mM, respectively. After a 42-day cultivation, the colonies were
Table 1 Influence of selective agent’s concentrations on growth of Lavandula vera MM cell culture Selection agent
Isolated colonies
Growth
Without selection agent (control) 0.2 mM MFP 0.4 mM MFP 0.5 mM MFP 0.6 mM MFP 0.8 mM MFP 1.0 mM MFP 0.2 mM PFP 0.4 mM PFP 0.5 mM PFP 0.6 mM PFP 0.8 mM PFP
8 5 5 4 3 3 – 4 4 3 3 –
+++++ ++++ ++++ +++ ++ + – ++++ +++ ++ + –
+++++ excellent (5); ++++ very good (4); +++ good (3); ++ middle (2); + weak (1); – without growth (0).
transferred to standard LS solid media and after another 21 days their growth was checked (Table 1). Callus lines demonstrated different growth, but the tendency is inhibition of growth with increase of MFP and PFP concentration. However, when MPF was used, weaker inhibition of growth of L. vera MM cell culture was observed as compared with PFP. Isolated plant cell lines were analyzed for their rosmarinic acid content (Tables 2 and 3). It was established that in the variants with MFP concentrations between 0.2 and 0.4 mM, the Table 3 Rosmarinic acid content in p-F-D,L-phenylalanine selected Lavandula vera MM callus lines PFP concentration (mM)
Line
Rosmarinic acid (mg g1 dry weight)
Percentage from control
–
Control
5.08 0.11
100
0.2 mM PFP
PF11 PF12 PF13 PF14
8.16 0.18 7.95 0.22 8.68 0.16 6.56 0.19
161 156 171 129
0.4 mM PFP
PF21 PF22 PF23 PF24
7.16 0.25 5.47 0.13 5.43 0.15 7.82 0.26
141 108 107 154
0.5 mM PFP
PF31 PF32 PF33
5.25 0.18 3.65 0.05 4.43 0.09
103 72 87
0.6 mM PFP
PF41 PF42 PF43
3.82 0.11 3.08 0.07 2.47 0.03
75 61 49
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M. Georgiev et al. / Process Biochemistry 41 (2006) 2068–2071
Table 4 Rosmarinic acid content in L. vera MM (control), L. vera MF and L. vera PF after 5 and 10 passages
Control L. vera MF L. vera PF
RA (mg g1 dry weight)
RA after 5 passages (mg g1 dry weight)
Standard deviation (%)
RA after 10 passages (mg g1 dry weight)
Standard deviation (%)
5.08 9.90 8.68
4.91 0.14 10.07 0.20 8.38 0.10
3.3 1.7 3.5
4.95 0.10 9.55 0.13 8.25 0.11
2.6 3.5 5.0
amounts of biosynthesized RA were comparable with the control variant, while further enhancement of MFP concentration corresponded to the enhanced RA biosynthesis. Highest RA content (9.9 mg g1 dry weight or 1.95 times higher compared to the control) was shown by the line L. vera MM MF51. As a result of selection with PFP, 14 lines which showed different RA content were isolated (Table 3). From isolated lines, highest RA content (8.68 mg g1 dry weight or 1.71 times higher compared to the control) was shown by the line L. vera MM PF13. Similar enhancement of shikonin content (whose biosynthesis also passed through the phenylpropanoid pathway) was observed after selection of resistant to PFP Lithospermum erythrorhizon cell lines [1]. On the basis of the obtained results, two lines [L. vera MM MF51 (L. vera MF) and L. vera MM PF13 (L. vera PF)] were chosen for further experiments for their adaptation under submerged cultivation. The selection can be defined as successful when the obtained yields of desired metabolites are stable, as much as in some cases it has been established that selected lines lose their enhanced biosynthetic potential under subcultivation [17]. Therefore, RA content in the selected L. vera MF and L. vera PF was checked after 5 and 10 subcultivations and it was established that they showed a stable RA content (Table 4). 3.2. Submerged cultivation of the selected cell lines After 2 months of adaptation in LS liquid media (with period of subcultivation 7 days), selected lines were cultivated in LS modified liquid media [8]. It was established that the timecourses of growth of selected lines followed the profile of control variant (Fig. 1A): a short lag-phase and intensive growth during the exponential phase. The maximal growth was
achieved on day 10 of cultivation, however, the selected lines L. vera MF and L. vera PF accumulated 18% (28.3 g L1) and 12% (30.1 g L1) lower biomass, respectively, in comparison with the control variant. This is the main reason for the lower specific growth rate and the doubling time for L. vera MF and L. vera PF to be calculated (Table 5). The intensive biosynthesis of rosmarinic acid began on 6th day from the beginning of cultivation, as the maximum was reached on 11th (Fig. 1B). L. vera MF and L. vera PF biosynthesized 2804.4 mg L1 (about 159% higher RA in comparison with the control) and 2594.6 mg L1 (about 147% higher RA in comparison with the control), respectively. The amounts of biosynthesized RA, expressed as mg g1 dry weight, were 102.4 mg g1 dry weight (1.88 times higher compared with the control variant) in L. vera MF and 89.5 mg g1 dry weight (1.64 times higher compared with the control variant) in L. vera PF. Comparison between yields of biomass and rosmarinic acid with regard to the carbon source (sucrose) (Table 5) showed that the selection influenced the yield of rosmarinic acid to a higher extent than the yield of biomass. In our knowledge, the obtained yields of rosmarinic acid are of the highest reported up to now and they are good base for further scale-up of the process. Table 5 Specific growth rate (m), doubling time (td), growth yield (YB/S) and rosmarinic acid yield (YP/S) of cell suspensions L. vera MM (control), L. vera MF and L. vera PF Control 1
m (h ) td (h) YB/S (g g1 sucrose) YP/S (mg g1 sucrose)
0.69 10 100 0.63 36.37
L. vera MF 2
2
0.56 10 124 0.50 58.18
L. vera PF 0.51 102 136 0.55 53.78
Fig. 1. Time-courses of growth (A) and rosmarinic acid biosynthesis (B) during the cultivation of L. vera MF (&), L. vera PF (4) and L. vera MM (*) cell suspensions in modified LS liquid media. Error bars represent standard deviation.
M. Georgiev et al. / Process Biochemistry 41 (2006) 2068–2071
In conclusion, after selection with m- and p-F-D,Lphenylalanine, two lines (L. vera MF and L. vera PF) with stable growth and enhanced rosmarinic acid content were isolated. This demonstrated that selection with F-derivates of amino acids could be used as an effective strategy for obtaining of cell lines with enhanced biosynthesis of rosmarinic acid.
[7] [8]
[9]
Acknowledgement [10]
We thank Mrs. Sonya Kuzeva for her excellent technical assistance.
[11]
References [1] Bulgakov VP, Kozyrenko MM, Fedoreyev SA, Mischenko NP, Denisenko VA, Zvereva LV, et al. Shikonin production by p-fluorophenylalanine resistant cells of Lithospermum erythrorhizon. Fitoterapia 2001;72:394–401. [2] Quesnel AA, Ellis BE. Comparison of UV irradiation and p-fluorophenylalanine as selective agents for production of aromatic compounds in plant cell cultures. J Biotechnol 1989;10:27–38. [3] Makri O, Kintzios S. In vitro rosmarinic acid production: an update. In: Ramawat KG, editor. Biotechnology of medicinal plants. Vitalizer and therapeutic. USA/Plymouth, UK: Science Publishers/Inc. Enfield (NH); 2004. p. 18–31. [4] Osakabe N, Takano H, Sanbongi C, Yasuda A, Yanagisawa R, Inoue K, et al. Anti-inflammatory and anti-allergic effect of rosmarinic acid (RA); inhibition of seasonal allergic rhinoconjunctvitis (SAR) and its mechanism. BioFactors 2004;21:127–31. [5] Chun S-S, Vattem DA, Lin Y-T, Shetty K. Cranberry phenolics-mediated antioxidant enzyme response in oxidatively stressed porcine muscle. Process Biochem 2005;40:2225–38. [6] Yoshida M, Fuchigami M, Nagao T, Okabe H, Matsunaga K, Takata J, et al. Antiproliferative constituents from Umbelliferae plants. VII: active
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triterpenes and rosmarinic acid from Centella asiatica. Biol Pharm Bull 2005;28:173–5. Petersen M, Simmonds MSJ. Rosmarinic acid. Phytochemistry 2003;62:121–5. Pavlov A, Panchev I, Ilieva M. Nutrient medium optimization for rosmarinic acid production by Lavandula vera MM cell suspension. Biotechnol Prog 2000;16:668–70. Szabo E, Thelen A, Petersen M. Fungal elicitor preparations and methyljasmonate enhance rosmarinic acid accumulation in suspension cultures of Coleus blumei. Plant Cell Rep 1999;18:485–9. Pavlov A, Georgiev M, Ilieva M. Optimisation of rosmarinic acid production by Lavandula vera MM plant cell suspension in a laboratory bioreactor. Biotechnol Prog 2005;21:394–6. Ilieva-Stoilova M, Pavlov A, Kovacheva-Apostolova E. Further research into Lavandula species: cell cultures of L. vera and rosmarinic acid production. In: Lis-Balchin M, editor. Lavender: the genus Lavandula. London/ New York: Taylor/Francis; 2002. p. 214–26. Linsmayer EM, Skoog F. Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 1965;18:100–27. Gonzales RA, Widholm JM. Selection of plant cells for desirable characteristics: inhibitor resistance. In: Dixon RA, editor. Plant cell culture—a practical approach. Oxford: IRL Press Limited; 1985. p. 67–78. Dixon RA. Isolation and maintenance of callus and cell suspension culture. In: Dixon RA, editor. Plant cell culture—a practical approach. Oxford: IRL Press Limited; 1985. p. 1–20. Lopez-Arnaldos T, Lopez-Serrano M, Barcelo RA, Calderon AA, Zapata JM. Spectrophotometric determination of rosmarinic acid in plant cell cultures by complexation with Fe2+ ions. Fresen J Anal Chem 1995; 351:311–4. Berlin J. Screening and selection for variant cell lines with increased levels of secondary metabolites. In: Charlwood BV, Rhodes MJC, editors. Secondary products from plant tissue culture. Oxford: Clarendon Press; 1990. p. 119–38. Deus-Neumann B, Zenk MH. Instability of alkaloid production in Catharanthus roseus cell suspension cultures. Planta Med 1984;50: 427–31.
Selection of high rosmarinic acid producing Lavandula vera MM cell lines M. Georgiev, A. Pavlov, M. Ilieva * Department of Microbial Biosynthesis and Biotechnologies, Laboratory in Plovdiv, Institute of Microbiology, Bulgarian Academy of Sciences, Plovdiv, Bulgaria Received 28 January 2006; received in revised form 3 May 2006; accepted 5 May 2006
Abstract Selection of stable Lavandula vera MM cell lines producing high amounts of rosmarinic acid (RA) was performed using m-F-D,L-phenylalanine and p-F-D,L-phenylalanine. As a result, two callus lines L. vera MF and L. vera PF were obtained, which produced 1.95 and 1.71 times more rosmarinic acid in comparison with the parent culture. After adaptation of the selected callus lines in liquid media the achieved yields of rosmarinic acid were 2808.4 mg L1 (in L. vera MF cell suspension) and 2594.6 mg L1 (in L. vera PF cell suspension). # 2006 Elsevier Ltd. All rights reserved. Keywords: Rosmarinic acid; Lavandula vera MM; Line selection; m-F-D,L-phenylalanine; p-F-D,L-phenylalanine
1. Introduction Optimization of secondary metabolite production in different plant in vitro systems is a difficult and complicated process. Frequently, the stage of the selection of highproducing cell lines is underestimated. However, the selection of high-producing cell lines using selective agents was found to be an effective strategy for the enhancement of the accumulation of aromatic compounds (phenolics and shikonin derivates) in cultured plant cells [1,2]. Rosmarinic acid (RA) is a secondary metabolite which possesses antiviral, antibacterial, anti-inflammatory, antiallergic activities [3–5] and prevents the proliferation of human cancer cells [6]. The interesting biological activities of RA and its low content in the intact plants impose the development of alternative ways for the production of RA. Plant cell and tissue cultures are considered prominent producers of RA [7] and, therefore, a number of strategies for yield improvement have been developed, such as nutrient medium optimization [8], elicitation [9], optimization of culture conditions [10], etc. However, there is no information available about the selection of high-producing rosmarinic acid cell lines of lavender.
* Corresponding author. Tel.: +359 32 642 430; fax: +359 28 700 109. E-mail addresses: [email protected], [email protected] (M. Ilieva). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.05.007
In this paper we report the data on the selection of Lavandula vera (hereafter simplified term L. vera MM) MM cell lines resistant to analogues of phenylalanine (m- and p-F-D,Lphenylalanine), their rosmarinic acid content, as well as on the adaptation of the obtained lines to the submerged cultivations. 2. Materials and methods 2.1. Plant cell culture and media L. vera MM callus culture was obtained from stems of the oil-bearing sort Lavender ‘‘Drujba’’ and maintained as it has been previously described [11]. It was cultivated on standard Linsmayer–Skoog (LS) media [12], supplemented with 30 g L1 sucrose, 0.2 mg L1 2,4-dichlorophenoxyacetic acid and 5.5 g L1 plant agar. For experiments on selection, the modified LS nutrient media (added with 60 g L1 sucrose and NO3/NH4+ ratio of 40) was used [8]. Callus culture was grown in a thermostat at 26 8C, in darkness; suspension culture was cultivated in Erlenmayer flasks with total volume 500 mL (working volume 100 mL) on a shaker (110 rad s1), at 26 8C in darkness. For inoculation, 20% (v/v) cell suspension cultivated under the above mentioned conditions for 7 days was used.
2.2. Experimental settings m-F-D,L-phenylalanine (MFP) and p-F-D,L-phenylalanine (PFP) (Sigma– Aldrich, USA) were dissolved in distilled water and sterilized by filtration (Stericup, GV Durapore Membrane, 0.2 mm). Selection was carried out using the method described by Gonzales and Widholm [13], which includes mixing of medium (containing the relevant amounts of selection agent and agar) with equal amounts of 7-day old suspension culture. After 42-days, the grown colonies were transferred to standard LS solid media and cultivated for another
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21 days. Their growth was checked by the scale 0 (without growth) to 5 (excellent growth). The rosmarinic acid content of the selected lines was checked as well.
Table 2 Rosmarinic acid content in m-F-D,L-phenylalanine selected Lavandula vera MM callus lines
2.3. Analysis
MFP concentration (mM)
2.3.1. Growth of the L. vera lines Growth of the culture lines was monitored by measurement of dry weight (60 8C to constant weight) [14]. 2.3.2. Rosmarinic acid extraction and determination Frozen cell biomasses were extracted with 50% ethanol at 70 8C for 1 h (3 20 min). Ethanolic extracts were combined and evaporated under reduced pressure (vacuum evaporator, Laborota 4002, Heidolph). The samples were dissolved in 70% ethanol and were stored for 24 h at 10 8C. The obtained precipitate was separated and the supernatant was used for analysis of rosmarinic acid. Quantitative determination of rosmarinic acid was performed spectrophotometrically (spectrophotometer UV/VIS Shimadzu 1240) at 327 nm [15]. As a standard, pure rosmarinic acid (Extrasynthese, Genay, France) was used. The data presented are the averages from two independent experiments, each repeated twice (S.D.). All determinations were performed in three replicates.
Line
Rosmarinic acid (mg g1 dry weight)
Percentage from control
–
Control
5.08 0.11
100
0.2 mM MFP
MF11 MF12 MF13 MF14 MF15
4.56 0.19 5.17 0.18 4.47 0.06 5.60 0.11 4.60 0.09
90 102 88 110 91
0.4 mM MFP
MF21 MF22 MF23 MF24 MF25
4.82 0.17 5.51 0.13 5.17 0.13 5.73 0.08 6.12 0.16
95 109 102 113 121
0.5 mM MFP
MF31 MF32 MF33 MF34
6.12 0.19 6.69 0.21 5.95 0.12 5.47 0.08
121 132 117 108
0.6 mM MFP
MF41 MF42 MF43
8.94 0.19 7.11 0.15 6.40 0.15
176 140 126
0.8 mM MFP
MF51 MF52 MF53
9.90 0.17 8.21 0.26 8.81 0.11
195 162 173
3. Results and discussion 3.1. Selection of L. vera MM callus lines with high and stable rosmarinic acid production The advantage of the amino acid analogues as selection agents is that they kill the majority of the wild type cells, while the desired variants (high phenolics producing cell lines) survive the treatment [16]. This phenomenon is attributed to the fact that only cells with increased phenylalanine ammonialyase activity (PAL) (key enzyme in phenolics biosynthesis pathway) can be detoxified by them and survive. As far as PAL is one of the key enzymes included in the biosynthetic pathway of RA, it can be expected that the cell lines with enhanced activity of PAL will produce higher amounts of RA [7]. As selective agents, m- and p-F-D,L-phenylalanine were used in concentration from 0.2 – 0.1 mM and 0.2 – 0.8 mM, respectively. After a 42-day cultivation, the colonies were
Table 1 Influence of selective agent’s concentrations on growth of Lavandula vera MM cell culture Selection agent
Isolated colonies
Growth
Without selection agent (control) 0.2 mM MFP 0.4 mM MFP 0.5 mM MFP 0.6 mM MFP 0.8 mM MFP 1.0 mM MFP 0.2 mM PFP 0.4 mM PFP 0.5 mM PFP 0.6 mM PFP 0.8 mM PFP
8 5 5 4 3 3 – 4 4 3 3 –
+++++ ++++ ++++ +++ ++ + – ++++ +++ ++ + –
+++++ excellent (5); ++++ very good (4); +++ good (3); ++ middle (2); + weak (1); – without growth (0).
transferred to standard LS solid media and after another 21 days their growth was checked (Table 1). Callus lines demonstrated different growth, but the tendency is inhibition of growth with increase of MFP and PFP concentration. However, when MPF was used, weaker inhibition of growth of L. vera MM cell culture was observed as compared with PFP. Isolated plant cell lines were analyzed for their rosmarinic acid content (Tables 2 and 3). It was established that in the variants with MFP concentrations between 0.2 and 0.4 mM, the Table 3 Rosmarinic acid content in p-F-D,L-phenylalanine selected Lavandula vera MM callus lines PFP concentration (mM)
Line
Rosmarinic acid (mg g1 dry weight)
Percentage from control
–
Control
5.08 0.11
100
0.2 mM PFP
PF11 PF12 PF13 PF14
8.16 0.18 7.95 0.22 8.68 0.16 6.56 0.19
161 156 171 129
0.4 mM PFP
PF21 PF22 PF23 PF24
7.16 0.25 5.47 0.13 5.43 0.15 7.82 0.26
141 108 107 154
0.5 mM PFP
PF31 PF32 PF33
5.25 0.18 3.65 0.05 4.43 0.09
103 72 87
0.6 mM PFP
PF41 PF42 PF43
3.82 0.11 3.08 0.07 2.47 0.03
75 61 49
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M. Georgiev et al. / Process Biochemistry 41 (2006) 2068–2071
Table 4 Rosmarinic acid content in L. vera MM (control), L. vera MF and L. vera PF after 5 and 10 passages
Control L. vera MF L. vera PF
RA (mg g1 dry weight)
RA after 5 passages (mg g1 dry weight)
Standard deviation (%)
RA after 10 passages (mg g1 dry weight)
Standard deviation (%)
5.08 9.90 8.68
4.91 0.14 10.07 0.20 8.38 0.10
3.3 1.7 3.5
4.95 0.10 9.55 0.13 8.25 0.11
2.6 3.5 5.0
amounts of biosynthesized RA were comparable with the control variant, while further enhancement of MFP concentration corresponded to the enhanced RA biosynthesis. Highest RA content (9.9 mg g1 dry weight or 1.95 times higher compared to the control) was shown by the line L. vera MM MF51. As a result of selection with PFP, 14 lines which showed different RA content were isolated (Table 3). From isolated lines, highest RA content (8.68 mg g1 dry weight or 1.71 times higher compared to the control) was shown by the line L. vera MM PF13. Similar enhancement of shikonin content (whose biosynthesis also passed through the phenylpropanoid pathway) was observed after selection of resistant to PFP Lithospermum erythrorhizon cell lines [1]. On the basis of the obtained results, two lines [L. vera MM MF51 (L. vera MF) and L. vera MM PF13 (L. vera PF)] were chosen for further experiments for their adaptation under submerged cultivation. The selection can be defined as successful when the obtained yields of desired metabolites are stable, as much as in some cases it has been established that selected lines lose their enhanced biosynthetic potential under subcultivation [17]. Therefore, RA content in the selected L. vera MF and L. vera PF was checked after 5 and 10 subcultivations and it was established that they showed a stable RA content (Table 4). 3.2. Submerged cultivation of the selected cell lines After 2 months of adaptation in LS liquid media (with period of subcultivation 7 days), selected lines were cultivated in LS modified liquid media [8]. It was established that the timecourses of growth of selected lines followed the profile of control variant (Fig. 1A): a short lag-phase and intensive growth during the exponential phase. The maximal growth was
achieved on day 10 of cultivation, however, the selected lines L. vera MF and L. vera PF accumulated 18% (28.3 g L1) and 12% (30.1 g L1) lower biomass, respectively, in comparison with the control variant. This is the main reason for the lower specific growth rate and the doubling time for L. vera MF and L. vera PF to be calculated (Table 5). The intensive biosynthesis of rosmarinic acid began on 6th day from the beginning of cultivation, as the maximum was reached on 11th (Fig. 1B). L. vera MF and L. vera PF biosynthesized 2804.4 mg L1 (about 159% higher RA in comparison with the control) and 2594.6 mg L1 (about 147% higher RA in comparison with the control), respectively. The amounts of biosynthesized RA, expressed as mg g1 dry weight, were 102.4 mg g1 dry weight (1.88 times higher compared with the control variant) in L. vera MF and 89.5 mg g1 dry weight (1.64 times higher compared with the control variant) in L. vera PF. Comparison between yields of biomass and rosmarinic acid with regard to the carbon source (sucrose) (Table 5) showed that the selection influenced the yield of rosmarinic acid to a higher extent than the yield of biomass. In our knowledge, the obtained yields of rosmarinic acid are of the highest reported up to now and they are good base for further scale-up of the process. Table 5 Specific growth rate (m), doubling time (td), growth yield (YB/S) and rosmarinic acid yield (YP/S) of cell suspensions L. vera MM (control), L. vera MF and L. vera PF Control 1
m (h ) td (h) YB/S (g g1 sucrose) YP/S (mg g1 sucrose)
0.69 10 100 0.63 36.37
L. vera MF 2
2
0.56 10 124 0.50 58.18
L. vera PF 0.51 102 136 0.55 53.78
Fig. 1. Time-courses of growth (A) and rosmarinic acid biosynthesis (B) during the cultivation of L. vera MF (&), L. vera PF (4) and L. vera MM (*) cell suspensions in modified LS liquid media. Error bars represent standard deviation.
M. Georgiev et al. / Process Biochemistry 41 (2006) 2068–2071
In conclusion, after selection with m- and p-F-D,Lphenylalanine, two lines (L. vera MF and L. vera PF) with stable growth and enhanced rosmarinic acid content were isolated. This demonstrated that selection with F-derivates of amino acids could be used as an effective strategy for obtaining of cell lines with enhanced biosynthesis of rosmarinic acid.
[7] [8]
[9]
Acknowledgement [10]
We thank Mrs. Sonya Kuzeva for her excellent technical assistance.
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