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Article DOi: 10.5504/bbeq.2011.0129 A&eb

Biotechnol. & Biotechnol. eq. 25/2011/4, Suppl.

Biotechnol. & Biotechnol. eq. 2011, 25(4), Suppl., 34-38Keywords: sage; triterpenic acids; in vitro systems, somaclonal variation; polyploidy, flow cytometryAbbreviations:BAp: 6-benzylaminopurine;Kin: kinetin;nAA: 1-naphthylacetic acid;B5: Gamborg‘s B5 nutrient medium;DW: dry weight;oA: oleanolic acid;uA: ursolic acid

Introductioncytological abnormalities are a common occurrence in plant in vitro systems. This phenomenon is known as “somaclonal variation” and results from variability arising in cell cultures, regenerated plants and their progenies (12). the origin of somaclonal variation has been observed to be on the genetic level (appearance of heritable and irreversible changes, including: polyploidy, aneuploidy, insertions, deletions, translocations, mutations, etc.) and on the epigenetic level (appearance of heritable, but potentially reversible, changes in chromatin structure, DnA methylation, gene silencing or gene activation, etc.) (3, 7, 13). in general, genetic changes are attributed to endoreduplication, disturbances in plant cell cycle, deletions, activation of retrotransposons (5, 15). the most important factors inducing the appearance of such changes are considered to be the type and concentrations of phytohormones, media composition, callus age, genetic background of the used explants and cultivation conditions (5, 10, 15).

Salvia tomentosa Mill. (Lamiaceae) is widespread in the old World. it is a popular herb for tea preparation named “balsamic sage” in Europe (1) and used in traditional medicine for treatment of open cuts in Turkey under the name “tentürdiyot out” (2). essential oil and non-polar extracts possessed strong antimicrobial activity, whereas the polar extracts showed high antioxidant activity (6, 17). However, the chemical composition of plant extracts has not been investigated in details. Since S. tomentosa Mill. occurs in very limited populations on the territory of Bulgaria, its collection for commercial purposes has been prohibited (16). Development of in vitro systems of this protected species presents an attractive eco-friendly alternative for both studying and producing biologically active compounds without endangering their natural habitats.

Somaclonal variation in calli has been used as a powerful tool for random selection of highly productive cell lines via single cell cloning (14). on the other hand, the endopolyploidy caused by endoreduplication could significantly alter the secondary metabolism of plant cells (11, 19). the precise analysis of calli ploidy profiles are of great importance for selection of genetically stable lines. Flow cytometry is a fast, rapid and accurate method, which was successfully adapted for estimating nuclear DnA content in both plant organs and different plant in vitro systems (19).

In this study we present an efficient protocol for induction of callus cultures of Salvia tomentosa Mill., producing oleanolic and ursolic acids. the somaclonal variation in the obtained friable calli, based on changes in organization of their nuclear DNA was used for selection of a genetically stable high triterpenic acids producing callus line. To our knowledge, this is the first report for obtaining of triterpenes producing callus culture of S. tomentosa Mill.

PRODUCTION OF OLEANOLIC AND URSOLIC ACIDS BY CALLUS CULTURES OF SALVIA TOMENTOSA MILL.

Vasil Georgiev1, Andrey Marchev1, christiane haas2, Jost Weber2, Milena nikolova3, thomas Bley2, Atanas pavlov1, 4

1Bulgarian Academy of Sciences, the Stephan Angeloff institute of Microbiology, laboratory of Applied Biotechnologies, plovdiv, Bulgaria2technische universität Dresden, institute of Food technology and Bioprocess engineering, Dresden, Germany3Bulgarian Academy of Sciences, Institute for Biodiversity and Ecosystem Research, Sofia, Bulgaria4university of Food technologies, Department of organic chemistry and Microbiology, plovdiv, Bulgariacorrespondence to: Vasil Georgieve- mail: vasgeorgiev@gmail.com

ABSTRACTCallus cultures of Salvia tomentosa Mill. were induced and analyzed for their capacity to produce oleanolic and ursolic acids. The obtained callus lines showed high variability in their biosynthetic potentials. Flow cytometric investigations showed that this variability was due to polyploidization of plant cells in calli. Polyploidization of callus cell seems to be promoted by the growth regulators used for callus induction. One octaploid line (consisting of 8C, 16C and 32C cells) was selected as prospective producer of oleanolic (991.57 µg/g DW) and ursolic (641.85 µg/g DW) acids. To our knowledge, this is the first report for obtaining of triterpenes producing callus culture of S. tomentosa Mill.

35Who We ARe AnD WhAt We AchieVeD

Fig. 1. Responses of Salvia tomentosa Mill. explants cultivated on media with different concentrations of auxins and cytokinins.

Materials and MethodsPlant materialleaves from S. tomentosa Mill. Plants at the flowering stage were collected in June 2010 from the experimental field of the institute of Biodiversity and ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgaria.

Initiation of in vitro culturesYoung leaves were sterilized by treatment with 70% ethanol for 20 sec. followed by treatment with 6 % (w/v) calcium hypochlorite for 6 min. and transferred on solid B5 nutrient media supplemented with 30 g/L sucrose (Duchefa,

the netherlands), 5.5 g/l ‘‘plant agar’’ (Duchefa, the Netherlands) and prepared in 132 variants with permutations of the auxins 2,4-dichlorophenoxyacetic acid (2,4-D; Sigma, Germany) or 1-naphthaleneacetic acid (nAA; Duchefa, the netherlands) and the cytokinins 6-benzylamynopurine (BAp; from Duchefa, the netherlands) or Kinetin (Kin; Duchefa, the netherlands) concentrations at levels of 0.0, 0.2, 0.5, 1.0, 2.0 and 4.0 mg/L. The variants with higher cytokinins concentrations were cultivated under illumination (16 h light/8 h dark), whereas the other variants were cultivated in darkness, both at 26 °C. When the calli appeared, they were separated from the plant explants and maintained at 26 °c, in darkness

36 Biotechnol. & Biotechnol. eq. 25/2011/4, Suppl.

with subcultivation periods of 21 days. All formed calli were evaluated on the basis of both their abilities for independent growth after separation from plant explants and the contents of oleanolic and ursolic acids. The fastest growing, friable calli were selected for the following analyses of ploidy profiles.

Extraction of ursolic and oleanolic acidslyophilized biomasses (0.1 – 0.2 g) from investigated plant leaves or 21-day-old callus cultures were extracted (in triplicate) with acetone (1:10 w/v) at 45 °C under sonification. The acetone fractions were filtrated by filter paper and evaporated to dryness (at 60 °C). The residues were dissolved in 500 µL methanol (Sigma, Germany), filtrated by 22 µm filter and analyzed by HPLC.

HPLC analysesThe triterpenes were analyzed by HPLC system consisting of Waters 1525 Binary pump (Waters, Milford, MA, uSA), Waters 2487 Dual λ Absorbance Detector (Waters, Milford, MA, USA), controlled by Breeze 3.30 software. Supelco Discovery HS C18 column (5 μm, 25 cm × 4.6 mm) operated at 26 °C was used for separation. The mobile phase consisted of 0.01 M Kh2po4 (pH 2.80) : methanol (12 : 88, v/v) with gradient of flow rate as follows: 0 min – 18 min (0.8 mL/min); 18 min-19 min (decrease to 0.6 ml/min); 19 min – 30 min (0.6 ml/min); 30 min – 31 min (rising back to 0.8 ml/min) and 31 min – 40 min (0.8 mL/min). Eluting compounds were detected by monitoring the eluate at 210 nm.

Flow cytometric investigationsCell nuclei were extracted from fresh leaves or callus cultures (20 – 100 mg) by using the Marie extraction buffer (50 mM glucose, 15 mM nacl (VWR, leuben, the netherlands), 5mM na2eDtA (Sigma, Steinheim, Germany), 50 mM sodium citrate (Roth, Karlsruhe, Germany), 0.5 % (v/v) Tween 20 (Merck, Darmstadt, Germany), 50 mM hepeS (Sigma) and 0.5 % (v/v) β-mercaptoethanol (Euroline, Pero, Italy) (9). The buffer’s pH was adjusted to 7.2 prior to analysis, the prepared buffer was stored at -20 °C. Analytical procedure included cutting plant material with a sharp blade in Marie buffer, filtration of extracted nuclei through a 30 µm mesh filter (CellTrics, Partec GmbH, Germany), staining with 60 µL propidium iodide (50 µg/ml pi; Sigma) and measurement by a flow cytometer after 30 s.

The flow cytometric analyses were performed by CyFlow SL Blue (20 mW solid state laser at 488 nm) flow cytometer (Partec GmbH, Münster, Germany) operated with FloMax (Partec GmbH) software. DNA histograms were recorded on a semilogarithmic scale. to reduce the level of debris, the nuclei were gated in SSC vs. red fluorescence channel dot plots. Cycle values were calculated as described previously (19).

Results and Discussionthe best calli formation of S. tomentosa Mill. leaves explants was achieved when combinations of NAA and BAP were used and cultivation was performed in darkness. The variants

including 2,4-D and KIN or BAP were not appropriate for callus induction and promoted explant necrosis in both cultivations under illumination and in darkness. in general, formation of in vitro culture occurred with 71 out of the 132 tested combinations of phytohormones. S. tomentosa Mill. cells responded to the effect of external phytohormones in the classical way. When explants were cultivated in media containing higher concentrations of cytokinines, compact morphogenic calli were formed, whereas when the media contained higher concentrations of auxins, root formations appeared (Fig. 1). the best variants featuring the formation of fast growing friable calli were the media containing near to equal concentrations of auxin and cytokinine (0.5 mg/l nAA and 0.5 mg/l BAp) (Fig. 1). Distributions of obtained calli concerning their content of oleanolic and ursolic acids are presented in Fig. 2. Only 6% of the obtained calli accumulated more than 1000 µg/g DW oleanolic acid, and 5% produced oleanolic acid in the range of 0-100 µg/g DW (Fig. 2a). on the other hand, most of the calli (41%) accumulated higher concentrations of ursolic acid (in the range of 501–1000 µg/g DW) and a very small number (6%) produced it in the lower range between 0 and 100 µg/g DW (Fig. 2b). these very important results allow us to choose several perspective callus cultures with the aim to select a high ursolic acid producing cell line. in comparison, during the screening of daidzein producing callus line, 217 calli derived from five different Psoralea species were analyzed and only 7.2% of them accumulated the desired isoflavone in higher concentrations (4). These data show that we were able to induce a rather larger number of calli from S. tomentosa Mill. that produce the metabolite of interest.

Fig. 2. Distributions of Salvia tomentosa Mill. callus lines concerning their content of oleanolic (a) and ursolic (b) acids.

As it was reported previously, the type and concentration of phytohormones could play an important role with respect to the accumulation of secondary metabolites in callus cultures (18). concentrations of biosynthesized oleanolic and ursolic

37Who We ARe AnD WhAt We AchieVeD

acids in calli are plotted as functions of auxin and cytokinin in the media in Fig. 3. In general, the lower auxin concentrations in combination with higher cytokinin concentrations stimulate the production of oleanolic acid (Fig. 3a), whereas the combination of lower equal concentrations of both auxins and cytokinines stimulated accumulation of ursolic acid (Fig. 3b).

Seven fast growing, friable callus lines which accumulated considerable amounts of oleanolic and/or ursolic acids were selected for the following analyses of their genetic stability. The data for their ploidy profiles compared to those of the plant leaves tissue is presented in Table 1. in contrast to the normal diploid plant tissue (2C = 2x = 16), from which they were induced, all callus lines were mixoploid (cycle values between 1.21 and 2.2) consisting of cells with tetraploid (4C = 4x = 32), octaploid (8c = 8x = 64) and hexadecaploid (16c = 16x = 128)

genomes (Table 1). The only exception was line (ST45), which is octaploid (8C = 8x = 64) and contains 16.29% hexadecaploid (16C = 16x =128) and 2.04% of 32C cells (32C = 32x = 256, cycle value 2.20). Endopolyploidy in callus cultures was due to activation of plant cell endocycle (8). endoreduplication process could be promoted by the type and concentrations of external phytohormones (10). in the case of callus line St45 probably the increased concentration of cytokinin BAp could be the reason for the increased ploidy levels. it should be noted that this callus line accumulated the highest amounts of both oleanolic and ursolic acids (Table 1). this is in agreement with our previous reports for increased production in Datura stramonium tetraploid hairy roots as compared to diploid ones (11). This confirms that polyplodization, either induced or spontaneous, is a promising strategy to increase yields of secondary metabolites of interest.

Fig. 3. Distributions of biosynthesized oleanolic (a) and ursolic (b) acids in Salvia tomentosa Mill. callus lines obtained on media with different concentrations of auxins and cytokinins.

TABLE 1Ploidy profiles and triterpene acids content of seven selected Salvia tomentosa Mill. callus lines

Lines Phytochormones, mg/L Triterpenes, µg/g DW Fractions of cells in the corresponding ploidy level (%) Cycle

valueNAA KIN BAP ОА UA 2C 4C 8C 16C 32CPlant leaves - - - 4462.13 8520.68 97.45 2.55 - - - 0.03ST8 0.2 0.2 - 106.34 0.00 - 17.13 73.43 9.45 - 1.92ST10 1.0 0.2 - 675.97 472.16 - 17.94 73.42 8.64 - 1.91ST38 0.2 - 0.2 0.00 287.88 - 62.95 32.3 4.75 - 1.42ST39 0.5 - 0.2 100.04 457.47 - 50.76 36.42 12.82 - 1.62ST41 2.0 - 0.2 25.20 257.57 - 79.76 18.52 1.73 - 1.22ST42 4.0 - 0.2 340.85 515.27 - 80.71 17.65 1.64 - 1.21ST45 0.5 - 0.5 991.57 641.85 - - 81.68 16.29 2.04 2.20

38 Biotechnol. & Biotechnol. eq. 25/2011/4, Suppl.

Conclusionscallus cultures of Salvia tomentosa Mill. were successfully induced on B5 medium. The best growth regulators for initiation of friable calli were NAA and BAP. Analyses of obtained callus lines showed high variability in their production of both oleanolic and ursolic acids. The flow cytometric investigations showed that this variability may be due to genetic changes, leading to polyploidy of plant cell nuclei. the observed reorganization of the plant cell genomes was probably due to activation of plant cells endocycle promoted by the growth regulators in the callus induction media. one octaploid line (consisting of 8c, 16c and 32c cells) produced the highest amounts of oleanolic (991.57 µg/g DW) and ursolic (641.85 µg/g DW) acids. It was selected as a perspective producer of these triterpenes.

AcknowledgementsThis work was supported by grant BG051PO001-3.3.04/32 financed by Operational Programme Human Resources Development (2007 – 2013) and co-financed by the European Social Fund of the european union.

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