Analele ştiinţifice ale Universităţii Al. I. Cuza Iaşi Tomul LVI, fasc. 1, s. II a. Biologie vegetală, 2010 STUDIES REGARDING IN VITRO REGENERATION OF ARNICA MONTANA L. FROM NATURAL POPULATIONS - BISTRITA VALLEY (EASTERN CARPATHIANS) CAMELIA PAULA STEFANACHE *, DOINA DANILA *, R. NECULA *, ELVIRA GILLE * Abstract. Arnica montana, is a rare and vulnerable species, traditionally used for medicinal purposes. Our approach aims at the possibility of using in vitro multiplication as a complementary component for the initiation and development of the species conservation strategy in the mountain valley of Bistrita in the Eastern Carpathians. For in vitro regeneration, the biological material used to initiate tissue cultures, comes from Arnica montana seedlings, resulting from germination of seeds collected from natural populations. On Murashige and Skoog (1962) solidified multiplication medium, supplemented with different amounts of BAP* and NAA**, we obtained a total of 2-3 neoplantlets/explant. Bud multiplication was succeeded by rooting phase, the neoplantlets being transferred for ex vitro acclimatization, with a survival rate of 75-80% after invigorate plants are transplanted in situ. Preliminary phytochemical analysis carried out by TLC and HPLC-UV in plants from natural populations of A. montana showed the presence of compounds of the polyphenolic and flavonoidic types. * Benzil-amoni-purine; ** Naphthalene acetic acid Keywords: Arnica montana, in vitro multiplication, conservation, HPLC Introduction Arnica montana is a traditional herb in Europe and North America being used for many centuries. The popularity A. montana oil has led to excessive collection, the species becoming increasingly rare. Habitat fragmentation, agricultural intensification and excessive collection of A. montana resulted in disappearance of many populations, species being introduced in Annex D of the EU Council Regulation No. 338/97 and Annex V (b) Habitats Directive [13] (92/43/EC - Directive for Conservation of Natural Habitats and Wild Fauna and Flora). A. montana is included in Red List of higher plants in the flora of Romania with the status of rare and vulnerable species [1]; Romania is still among the main countries supplying raw material. In this context, in 2004, began the initiation of the Conservation of medicinal plants in Eastern Europe: Arnica montana in Romania project, which aimed to increase motivation for the conservation of the habitats rich in biodiversity by demonstrating how it can be used for sustainable production of medicinal and aromatic plants in the benefit of gatherers and land owners [8]. In this purpose has been developed a special cultivation technology [3], but many companies prefer the raw material coming from natural populations because of high costs associated with cultivation. Considering this facts, it is likely many population to disappear. Regeneration from soil seed banks could be an important buffer against genetic impoverishment, although not for species with a transient seed bank. Genetic variation and * National Institute of R&D for Biological Sciences / Stejarul Research Centre, Piatra Neamt, Romania 33
population size could be increased by artificially adding seeds or plants from other populations [7]. In vitro multiplication studies for A. montana species were performed both at international - Conchou et a.l (1992), C. L. Lê (1998) and national level - Butiuc-Keul and Deliu (2001) [2, 5, 6]. The final aim of the study is the reintroduction of the species in the montane grasslands where the species has been fully disappeared, and the introduction of new crops (genes) in small, endangered populations (to increase the heterozys), especially because the micropropagation techniques provides the advantage of a better control of the genetic material, and a relative large number of crops in a short period of time. Material and methods 1. Culture media and conditions The basal medium consists of MS mineral salts and vitamins plus 2.5% sucrose, solidified with 0,80 0,85% agar. The medium ph was adjusted to 5,5 with NaOH before autoclaving (120 o C for 15 min). To initiate caulogenesis we used variants of MS medium supplemented with BAP (benzylaminopurine) and NAA (naphthaleneacetic acid): BN (1 mg/l BAP and 0,3 mg/l NAA), B 10 (1 mg/l BAP) and B 20 (2 mg/l BAP) medium. For root inducing we used MS basal medium, MR medium (supplemented with 1 mg/l NAA) and MSN medium (MS basal medium supplemented with 0,5 mg/l NAA). Cultures were maintained in the experimental culture room in a temperature of 22 + 1 C [10]. 2. Plant material The explants were represented by plantlets obtained through seed germination in aseptic condition. The seeds were collected from A. montana natural populations in Dorna Arini area, during august 2009. 3. Thin Layer Chromatography The preliminary identification of the polyphenolic, flavonoidic, triterpenic and phytosterolic compounds was made by means of TLC. 4. High Performance Liquid Chromatography For the HPLC analysis, we used an Agilent 1200 series system, equipped with a diodearray UV detector (DAD) and autosampler. To separate the compounds, we used an analytic chromatography column with reverse phase of the Zorbax Eclipse XDB-C18 type (granulation 5 µm, 150 x 4.6 mm d.i.). The column temperature was maintained at 30 C. For the elution we applied a gradient with two solvents: acetonitryl (solvent A) and an acetic buffer solution (solvent B), prepared starting from a watery solution of sodium acetate (2mM) adjusted to a ph=3.5 with acetic acid of 99% purity. The initial conditions were 2%A and 98%B. The linear gradient programme was of 2-14-20-30-25% solvent A for 0-20-40-50-60 min, after which we switched back to the initial conditions. The discharge used was of 1.0 ml/min. The sample injecting (100 µl) was done by help of the autosampler. As standard substances we used chlorogenic, caffeic, ferulic, and rozmarinic acids, the flavonoids rutosid, apigenol, apigenol 7-O-glucoside, luteolin, luteolin 7-O-glucoside, cvercetol, hyperoside (furnished by the LG-Standards company). The stock solutions of the standards were diluted with methanol and analyzed in the same conditions to achieve the standard curves. To identify the peaks we compared both the values of the retention time (RT) and the UV absorbtion spectra of the compounds decelated in the analyzed samples, with those of the standards used. For the quantitative results calculation we used the peak areas in the standard curve method. 34
Results and discussions 1. The phytochemical characterization of the donor plants 1.1 Qualitative phytochemical analysis for triterpenes and phytosterols from dichloromethane extracts The phytochemical analysis, performed by TLC, showed that the highest number of fractions / 13 are found in samples 1 and 2, and the lowest number in sample 3 (tab. 2). A similar status can be observed in samples 4 and 5 (rhizome / roots), where, by size and the intensity of the spot, the compound with Rf = 0,72, and β-sitosterol prevail. It is found in certain amounts in most samples analyzed, except sample 3. In the roots / rhizomes (samples 4 and 5) are found some volatile compounds that have revealed in the chromatograms for the volatile oils, natural appearance as volatile compounds have common biogenetic pathway of terpene derivatives, some volatile fractions can belong to the monoterpenoids. Comparative analysis of rhizomes and roots harvested in autumn (sample 4-5), in the spring (sample 8) respectively, have a qualitatively identical biosynthetic spectrum, the differences being only quantitative. 1.2 Phytochemical analysis of the polyphenolic compounds in absolute methanolic extract TLC analysis showed a different spectrum for polyphenolic and flavonoid compounds - detected / identified. In leaf and basal rosette extracts the chlorogenic acid is well evidenced, Rf = 0,52 (blue fluorescent spot), compared with root extract where it is in the form of traces. In the absolute methanolic extracts of A. montana (plant material collected in 2010) were identified by HPLC technique, reverse phase with UV detection, following polyphenolic compounds: apigenol and derivatives of phenolic acids, characterized by UV spectrum compared with available standards. Data from literature indicates the presence of essential oil in rhizomes, roots and inflorescence. The volatile oil constituents include the thymol and its derivatives, and fatty acids (palmitic, linoleic, myristic, linolenic). Fatty acid composition of leaf essential oil was also evaluated [12]. Chiritoiu M., in 2009 obtained a quantity of volatile oil of 0,5-1,5% in roots and 0,04-3,8% in flowers [4]. Other compounds found in A. montana include the bitter compound arnicin, caffeic acid, carotenoids (α-and β-carotene, cryptoxanthin, luteolin), phytosterols, resin, tannins, lignans, and anthoxanthine [9, 11]. 2. Determination of germination capacity The germination capacity of the seeds harvested from the donor plants was tested in 3 variants: on filter paper, culture medium, and on soil. Highest germination degree was obtained for the seeds inoculated on filter paper moistened with distilled water supplemented with 0,02 mg/l kinetin and 2 mg/l gibberellic acid. The aseptic isolation of seeds germinated on filter paper and MS culture medium was done using mercuric chloride (0,1%), different action times. 3. Plant regeneration and multiplication Plant regeneration and root induction from the inoculated explants after 4 week of culture were 95% in all medium variants. The use of the media supplemented with 1 mg/l BAP (B 10 ), 2 mg/l BAP (B 20 ) respectively, has resulted in the best multiplication rates, achieving 3-4 plantlets per explant in 4 weeks. The media supplemented with 1 mg/l BAP and 0,3 mg/l NAA (BN), showed lower multiplication rates. Subcultures were performed every 4 weeks. 35
For the root induction and growth, an optimum morphogenetic response was achieved when we used MS basal medium supplemented with 0,5 mg/l NAA (MSN), in 4 weeks 95% of the plantlets formed a vigorous root system. 4. Acclimatization of in vitro-regenerated plants For ex vitro acclimatization, the neoplantlets with a vigorous root system were transferred to pots with sterile soil and covered with transparent glasses to ensure a vapor saturated atmosphere. After 4-5 weeks of acclimatization, the plantlets have been transferred in field, the survival rate being 75-80%. Conclusions 1. In accordance with the standards used (CSS), in dichloromethane extracts were identified: the betta-sitosterol, present in all samples analyzed; oleanolic acid in samples of leaf / basal rosettes; ursolic acid mainly in senescence leaf, in form of traces in roots. 2. The phytochemical analysis of the absolute methanolic extract for polyphenolic compounds revealed (CSS) that the majority of factions - 11 in number, belong to polyphenols, and flavonoids are absent, except in the leaf - derivatives of apigenin and luteolin 3. The comparative analysis (HPLC) of the absolute methanolic extract, showed (mg/100g fresh plant material) an amount higher in basal rosette and leaf samples for apigenol (3,33-10,83) and the total amount of phenolic acids and derivatives (157,33-244,40); the apigenol fall below detection limit in roots and rhizomes, in which were revealed phenolic acids and derivatives expressed in chlorogenic acid equivalents (40,96-110,23). 4. Regarding the phytochemical analysis, these are preliminary studies that will be further deepened through multiannual comparative analysis. 5. The plantlets obtained by germinating seeds in sterile conditions are an optimal explant source for micropropagation. 6. In vitro multiplication technique can be used as an alternative for reintroduction of the species in mountain meadows / introduction of new individuals in populations with low numbers of individuals; further studies should be performed. REFERENCES 1. BOŞCAIU N., COLDEA GHE., HOREANU C, 1994, Lista Roşie a plantelor vasculare dispãrute, periclitate, vulnerable şi rare din Flora României. Ocrotirea Naturii şi a Mediului Inconjurãtor. Bucureşti. Editura Academiei Române. 38 (1): 45-56. 2. BUTIUC-KEUL A.L., DELIU C, 2001, Clonal propagation of Arnica montana L., a medicinal plant, In Vitro Cell Dev. Biol. - Plant, 37, 581-585. 3. CASSELLS A.C., WALSH C, BELIN M., CAMBORNAC M., ROBIN J.R., LUBRANO C, 1999, Establishment of a plantation from micropropagated Arnica chamissonis a pharmaceutical substitute from the endangered A. montana, Plant Cell, Tissue and Organ Culture, 56, 139-144. 4. CHIRITOIU M., 2009, The remake of the Arnica montana L. population in Arges County, Analele Universitatii din Craiova, seria Agricultura - Montanologie - Cadastru, Vol. XXXIX, 85-88. 5. CONCHOU O., NICHTERLEIN K., VÖMEL A., 1992, Shoot Tip Culture of Arnica montana for Micropropagation, Planta Med, 58. 6. LÊ C.L., 1998, In vitro clonal multiplication of Arnica montana L., Acta Horticulturae, 457; 7. LUIJTEN H. SHEILA, KÉRY M., OOSTER MEIJER J.G.B., DEN NIJS HANS CM., 2002, Demographic consequences of inbreeding and outbreeding in Arnica montana: a field experiment, Journal of Ecology, 90, 593-603. 36
8. MICHLER B., 2007, Conservation of Eastern European Medicinal plants Arnica montana in Romania, Case study Garda de Sus, Management Plan, 12-20. 9. SCHMIDT TJ, STAUSBERG S, RAISON JV, BERNER M, WILLUHN G., 2006, Lignans from Arnica species. Nat Prod Res.; 20(5): 443-453. 10. STEFANACHE C., DANILA D., GILLE E., TOMESCU C., 2010, In Vitro multiplication used in preserving the Arnica montana L. species in the Romanian Bistrita Mountains, Planta Med., 76, 1380. 11. VANHAELEN M., 1973, Identification of carotenoids in Arnica montana. Planta Med.; 23(4):308-311. 12. WILLUHN G., 1972, Fatty acids of the essential oil from leaves of Arnica montana and Arnica longifolia. Z Naturforsch B.; 27(6):728. 13. [ *** ] Directive for Conservation of Natural Habitats and Wild Fauna and Flora 92/43/EC, Annex V (b) - Plant and animal species of Community interest whose taking in the wild and exploitation are likely to be subject to management measures. Thanks The authors thank Prof. Dr. Nicolae Stefan Univ. Al. I. Cuza, Iasi, for his help in identifying A. montana populations. Acknowledgements The work is sustained in the PNCDI2 (51055/2007) program financed by the Romanian Government - National R&D Agency. EXPLANATION OF PLATES PLATE I Table 1; Fig. 1. Estimation of some bioproduction parameters in populations of Arnica montana from Dorna-Arini - Bistrita Valley (O - Ortoaia Valley (S1-S2), A - Arini Valley (S1- S3)). Table 2. Samples of A. montana phytochemical analyzed. PLATE II Fig. 2a. Donor plants. Fig. 2b. Plantlets resulted after seed germination in aseptic conditions. Fig. 2c. Tissue culture initiation. Fig. 2d.; Fig. 2e. Multiplication and growth of in vitro Arnica montana regenerated shoots. Fig. 2f. The rooting phase. Fig. 2g.; Fig. 2h. Acclimatization and field transfer. PLATE III Fig. 3a.; Fig. 3b. Thin layer chromatography for triterpenes and phytosterols of the analyzed samples (Standards: β-sito - betta-sitosterol; Stg. - Stigmasterol; A.O. - Oleanolic acid; A.U. - Ursolic acid); Fig. 3c. Thin layer chromatography for volatile oil fractions of the analyzed samples. 37
PLATE IV Fig. 4a; Fig. 4b. Thin layer chromatography for polyphenolcarboxilyc compounds of the analyzed samples (Standards: Caf Caffeic acid; Cl Chlorogenic acid; Fe Ferulic acid; OC o-coumaric acid). Fig. 4c.; Fig. 4d. Thin layer chromatography for flavonoidic compounds of the analyzed samples (Standards: Cv Quercetin; R Rutosid; L Luteolin; Ap - Apigenol). PLATE V Fig. 5a. HPLC for A. montana (sample 1- bazal rosette). Fig. 5b. HPLC for A. montana (sample 7- leafs, average sample). Fig. 5c. HPLC for A. montana (sample 8 - Rhyzomes + roots, plants harvested in the spring). 38
DOINA DANILA, CAMELIA STEFANACHE PLATE I Table 1. Nr. plants/ plot Calathids 4m 2 20m 2 100 inflorescence (g d.w.) I Arini Valley Slope1 54.04 193.14 10.87 Slope2 34.21 245.72 12.75 Slope3 61.32 170.54 11.48 Mean 49.86 203.13 11.7 II Ortoaia Valley Slope1 5.34 22.81 11.15 Slope2 2.15 11.22 8.31 Mean 3.75 17.02 9.73 Fig. 1 Table 2. Sample no. Samples 1 Basal rosette (from seeds - year I) 2 Basal rosette (from rhyzomes - year II) 3 Senescence leafs (from rhyzomes > year II) 4 Rhyzomes - plants harvested in the autumn 5 Roots - plants harvested autumn 6 Basal rosette (from rhyzomes > year II) 7 Leafs - average sample 8 Rhyzomes + roots - plants harvested in the spring 39
CAMELIA STEFANACHE, DOINA DANILA PLATE II Fig. 2a Fig. 2b Fig. 2c Fig. 2d Fig. 2e Fig. 2f Fig. 2g Fig. 2h 40
ELVIRA GILLE PLATE III Fig. 3a Fig. 3b Fig. 3c Fig. 4a Fig. 4b Fig. 4c Fig. 4d 41
RADU NECULA PLATE IV mau DAD1 A, Sig=320,4 Ref=500,80 (ANTIOX\2010-10-01_12-52-40 2010-OCT-01\ANTIOX_001105.D) 26.504 17.5 15 12.5 10 27.598 7.5 25.262 30.407 46.001 5 2.5 12.320 35.387 45.025 46.912 47.678 48.674 54.166 0 Fig. 5a. 10 20 30 40 50 60 min mau DAD1 A, Sig=320,4 Ref=500,80 (ANTIOX\2010-10-01_12-52-40 2010-OCT-01\ANTIOX_001111.D) 26.475 25 20 15 27.716 10 5 11.102 35.365 45.003 46.912 47.673 48.662 25.211 12.294 45.990 30.262 54.127 0 Fig. 5b. 10 20 30 40 50 60 min mau DAD1 A, Sig=320,4 Ref=500,80 (ANTIOX\2010-10-01_12-52-40 2010-OCT-01\ANTIOX_001112.D) 14 12 26.489 27.746 10 8 6 16.039 4 2 11.114 12.305 14.873 25.228 30.062 45.996 46.934 47.675 48.667 0 Fig. 5c. 10 20 30 40 50 60 min 42