Characterization and Propagation of Some Medicinal Plants in the Central-South Region of Chile

G Model INDCRO-5529; No. of Pages 9 ARTICLE IN PRESS

Industrial Crops and Products xxx (2010) xxx–xxx

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Characterization and propagation of some medicinal plants in the central-south region of Chile

Susana Fischer a,∗, Marisol Berti a,f, Rosemarie Wilckens a, Marcelo Baeza b, Edgar Pastene c, Luis Inostroza d, Claudia Tramón e, W. Gonzalez a a Dep. Producción Vegetal, Facultad de Agronomía, Universidad de Concepción, Casilla 537, Chillán, Chile b Facultad de Ciencias Biológicas y Oceanográﬁca, Universidad de Concepción, Chile c Facultad de Farmacia, Universidad de Concepción, Chile d Instituto de Investigaciones Agropecuarias Quilamapu, Chillán, Chile e Facultad de Ingeniería Agrícola, Universidad de Concepción, Chile f Dep. of Plant Sciences, North Dakota State University, Fargo, ND, United States article info abstract

Article history: The increase of land use for crop cultivation and forest in South Central Chile, and the increasing wild- Received 10 October 2010 crafting of medicinal plants has resulted in a significant reduction of the plant population density of many Accepted 12 October 2010 native and endemic medicinal plants. Their cultivation and domestication is very limited, and there are Available online xxx no regulations or legislation for wildcrafting in Chile. The objectives of this study were to collect genetic material from five native medicinal plants (Adesmia emarginata, Buddleja globosa, Fabiana imbricata, Linum Keywords: chamissonis, and Sophora macrocarpa), characterize the environmental conditions in which these grow Habitat in the Bio-Bio Region, Chile, and to determine the content of specific bioactive molecules. A maximum Germination of 10 accessions of each species were collected in 2003, 2004, and 2005 in a longitudinal and transversal Seed ◦ ◦ ◦ Flavonoids transect of the Bio-Bio Region (36 00 –38 30 S; 71 W). Data of altitude, light conditions, soil chemical Rutin and physical characteristics and associated flora of the site collection were recorded. Seed germination requirements and the content of specific bioactive molecules (flavonoids or rutin) were also determined. In general, all these species are adapted to grow in poor soils with different pH values, P and K levels, low organic matter and N content. Flavonoids were determined in both A. emarginata (0.6–1%) and B. globosa (9.7–13.9%) leaves, while rutin concentration was determined in F. imbricata leaves and stems (1.3–5.3%). Results showed great variations for the content of active principles with medicinal activity, which indi- cates a corresponding variation in the quality of raw materials for the pharmaceutical industry. Due to the fact that information on the domestication, propagation, cultivation and agronomic practices can ensure good pharmaceutical quality, this study provides a basis for further research on Chilean medicinal plants.

1. Introduction Vogel, 2000; Conama, 2003). In addition, approximately 13% of the vascular flora has a potential use. The use of medicinal plants has increased in the last few years. Adesmia emarginata is used in traditional medicine mainly The current concern on better health has resulted in a higher because of its analgesic and diuretic properties. It is a perennial demand of medicinal plants worldwide and has also encouraged plant that usually stays under snow at least 5 months a year, the interest in the search for new active compounds in the flora depending on weather conditions. of tropical, sub-tropical and temperate flora, such as in Chile. As a Buddleja globosa is a perennial shrub or small tree, reaching 4 m long and narrow country, with different types of soil and climate, high, with a soft thin trunk and a ramified branch structure. The Chile has a highly endemic flora. In fact, 85% of Chilean vascular infusion of these leaves is mainly used because of it diuretic, anti- flora is native and approximately 44% is endemic (Cubillos, 1994; inflammatory and wound-healing properties. It is used as a remedy for hepatic affections and dysentery, and as a local antiseptic for intestinal ulceration (Montes, s/f; Munoz˜ et al., 2001). An ointment for healing cracks in the nipples of breastfeeding mothers is sold ∗ Corresponding author. Tel.: +56 42 208871; fax: +56 42 275009. in local shops in Chile. It can also be used for dyeing natural fibers E-mail address: sfi[email protected] (F. Susana). (Navas, 1979) and as an ornamental plant (Montenegro, 2000).

Please cite this article in press as: Susana, F., et al., Characterization and propagation of some medicinal plants in the central-south region of Chile. Ind. Crops Prod. (2010), doi:10.1016/j.indcrop.2010.10.012 G Model INDCRO-5529; No. of Pages 9 ARTICLE IN PRESS 2 F. Susana et al. / Industrial Crops and Products xxx (2010) xxx–xxx

Fabiana imbrincata is a shrub with numerous upright branches, S. macrocarpa seeds were scarified by soaking them into sulfu- attaining a height of 3 m. Leaves are small and sessile, emitting ric acid (98–99%, v/v) for 15 or 30 min. After scarification, seeds when crushed an aromatic and resinous odor. The infusion obtained were washed with running water for 16 h to remove the acid. For from the bark and stems of this shrub has gastroprotector, chola- mechanical scarification, seeds were placed in a cylinder, covered gogue, liver stimulant, sedative, antiseptic, soothing, and diuretic with sand paper and agitated for 10 min. Then, seeds were placed effects (Montes, s/f; Munoz˜ et al., 2001; Navas Bustamante, 2001). in plastic trays of 14 cm × 19 cm on folded filter paper humidified This infusion is also used to treat Fasciola hepatica infections in goats with distilled water. and sheep in Chilean folk medicine (Navas Bustamante, 2001). In L. chamissonis: seeds were washed with Benomil 10% (w/v) to Chile, wild plants are harvested for flower shops. In other coun- avoid pathogens. Seeds were then placed in Petri dishes with humid tries, this shrub is used as an ornamental plant in gardens (Sánchez filter paper. de Lorenzo-Cáceres, s/f) or in flowerpot (Grüneberg, 1993, 1994, Three germination chambers were used. For the experiment 1995). under dark conditions, Petri dishes were covered with aluminum Linum chamissonis whole plant or dry seeds, which contain both paper. Metal Petri dishes were used and the evaluation was car- oil and glucosides, are used in Chilean folk medicine as laxative and ried out in a dark chamber with red light in order to avoid the emollient, for indigestion, painful dyspepsia, cold, intestinal disor- stimulation of light in the germination process. Observations on ders, antispasmodic, and irritable bowel syndrome (Munoz˜ et al., the number of germinated seeds were made on a daily basis and 2001). Whole plant is used to diminish local inflammations (Munoz˜ records were kept. et al., 2001). Sophora macrocarpa is a perennial small tree or shrub that can 2.3. Evaluated parameters reach 3 m in height (Rodríguez et al., 1983). It contains flavonoids that promote pharmacological activities on capillary fragility and Germination percentage was calculated, as follows: anti-inflammatory activity on blood vessels. It also presents anti- = . / . × spasmodic and antipyretic effects (Backhouse and Delporte, 1977). GP (No ofgerminatedseeds) (No ofsownseeds) 100 (1) Because of their hard seed coat, S. macrocarpa seeds are not able to where GP is the germination percentage. germinate (Rodríguez et al., 1995). Vigor index was calculated using the formula described by The increase of land used for crop and forest cultivation in South Anfirud and Schneiter (1984): Central Chile, and the increasing wild crafting of medicinal plants has resulted in the decrease of plant population density of many A1 A2 An IV = + +···+ native and endemic medicinal plants. There are no regulations or X1 X2 Xn legislation for wildcrafting in Chile (Vogel et al., 2005). In order where A = number of seeds that germinated daily in each experi- to prevent the extinction of some endemic plants, actions need to 1 mental unit from the first day, when the first seed germinated, to be taken. On this respect, only a few attempts have been made to the day when the last seed germinated for each experimental unit; domesticate and cultivate them. X = correlative day since the first germinated seed was observed in This preliminary study aimed to characterize the environment, any experimental unit (day 1), until the day in which the last seed rescue, propagate, and characterize plants both in situ and ex situ germinated; N = day of the last germination. and to determine the content of specific bioactive molecules of Germination rate was determined according to the formula used some native medicinal species grown in the Bio-Bio Region, in by Quintero et al. (1999):TG=(N × T + N × T + Nn × Tn)/(No. total South Central Chile, in order to contribute to the maintenance of 1 1 2 2 of germinated seeds) where: N = germinated seed number on day regional genetic heritage. i i = 1,2, ..., nTi = time in days TG50: time (days) required to achieve 2. Materials and methods 50% of germination (Olivares et al., 1999).

2.1. Characterization of soil and environment 2.3.1. Determination of total flavonoids A sample of 400 mg of pulverized dry plant material (180 mesh) Accessions of each of the following species were collected was extracted in a water bath with 15 mL of methanol for 15 min between 2003 and 2005: A. emarginata (six accessions), B. globosa, (Nieva-Moreno et al., 2000). The extracts were further sonicated F. imbricata, L. chamissonis (10 accessions of each one) and S. macro- during 15 min, concentrated by rotary evaporation and brought to carpa (nine accessions). These were sampled along a longitudinal 50 mL methanol in a volumetric flask. A 500 ␮L aliquot of the sam- and transversal transect of the Bio-Bio Region (36◦00–38◦30S; 71◦00W). Altitude, light conditions, associated flora, and chemical and physical characteristics of the soil were recorded from the collection site.

2.2. Germination assay

A completely randomized experimental design with a factorial arrangement was used. Treatments consisted of two factors: light conditions (light or darkness), and temperature (5, 12, and 20 ◦C), with 4 replications of 30 seeds each for A. emarginata, 10 seeds for L. chamissonis, and 25 seeds each for S. macrocarpa. A. emarginata seeds were classified according to the color of the seed cover into 3 groups: dark brown, grayish brown and very pale brown seeds. Seeds separated by color were submitted to mechanical scarification with sand paper for 3 and 6 min, or water immersion at 30 ◦C and at 40 ◦C for 3, 7, and 10 min (30 seeds for each treatment). Then, seeds were placed in Petri dishes on filter paper with distilled water. Fig. 1. Adesmia emarginata plants with flowers and fruit.

Please cite this article in press as: Susana, F., et al., Characterization and propagation of some medicinal plants in the central-south region of Chile. Ind. Crops Prod. (2010), doi:10.1016/j.indcrop.2010.10.012 G Model INDCRO-5529; No. of Pages 9 ARTICLE IN PRESS F. Susana et al. / Industrial Crops and Products xxx (2010) xxx–xxx 3

Table 1 Physical and chemicals properties of soil where accessions of Adesmia emarginata, Buddleja globosa, Fabiana imbricata, Linum chamissonis and Sophora macrocarpa were collected.

Species Accession Sand (%) Silt (%) Clay (%) pH O.M. (%) N-NO3 P Olsen K interc. Al extractable (mg kg−1) (mg kg−1) (cmol kg−1) (mg kg−1)

Adesmia emarginata P 101 57.9 29.7 12.4 5.55 1.16 6 4.2 0.39 390 P 102 92.7 4.2 3.1 5.73 3.66 3.3 5.9 0.17 303 P 103 93.8 3.1 3.1 6.06 1.8 3.8 2.5 0.05 188 P 104 93.7 3.1 3.2 5.84 2.57 4.8 3.4 0.03 504 P 105 95.6 3 1.4 6.08 0.02 0.5 0.9 0.01 34 P 106 92 5 3 5.92 3.8 2 1.7 0.04 365

Buddleja globosa MA 101 92.4 5.1 2.5 6.46 0.71 5 4.5 0.1 13 MA 102 58.7 29.4 11.9 6.55 4.37 5.5 9.6 0.4 340 MA 103 64.5 24.8 7.7 6.7 3.85 11.8 15.1 0.48 192 MA 104 73.3 22.6 4.1 5.77 20.09 29 3.2 0.32 850 MA 105 38.9 43.6 17.5 4.79 1.09 1.5 4.6 0.67 1850 MA106 42.7 40.3 17 4.66 8.62 10.5 6 0.23 295 MA107 72.2 21.4 6.4 5.73 3.06 9.5 0.5 0.59 185 MA108 54.3 35.9 9.8 6.24 13 5 2.5 0.39 328 MA110 47.9 37.7 14.4 5.06 3.35 4 2.9 0.99 –

Fabiana imbricata PI 101 56 40.5 3.5 5.81 11.06 8.5 1.6 0.1 688 PI 102 71.9 24.9 3.2 5.96 8.73 4.8 21.8 0.4 300 PI 103 94 3 3 5.61 2.15 2.3 1.9 0.03 291 PI 104 74.6 23.6 1.8 5.88 1.89 1.5 1.5 0.08 478 PI 105 90 8 1.4 6.01 0.35 0.8 2.3 0.08 97 PI 106 52.6 39.5 7.9 6.15 5.32 5.8 3.4 0.3 401 PI 107 94.2 4.1 1.7 6.52 0.34 0.3 1.4 0.03 75 PI 108 38.8 41 20.2 6.12 4.14 2.5 2.8 0.29 490 PI 109 72.3 23.9 3.8 5.86 14.84 6.2 1 0.19 1600 PI 110 63.6 25.7 10.7 5.37 2.63 1 8.1 0.15 349

Linum chamisonis NA101˜ 34.0 35.1 30.9 6.0 2.9 2.3 1.7 0.27 156 NA102˜ 43.7 35.4 20.9 5.8 2.2 4.3 0.1 0.11 111 NA103˜ 38.9 24.6 36.5 6.0 2.2 2.0 0.3 0.26 106 NA104˜ 51.2 30.0 18.8 5.7 4.4 3.3 2.1 0.47 80 NA105˜ 56.2 28.0 15.8 5.7 3.9 4.0 1.7 0.24 70 NA106˜ 51.6 24.2 24.2 5.9 3.4 3.3 2.8 0.28 58 NA107˜ 43.3 22.2 34.5 6.2 7.8 3.3 4.0 0.37 120 NA108˜ 40.1 21.2 38.7 6.0 1.9 2.8 2.6 0.68 147 NA109˜ 43.7 26.1 30.2 6.1 0.7 2.5 0.1 0.1 82 NA110˜ 14.7 29.7 55.6 5.7 4.7 5.3 1.5 0.64 226

Sophora macrocarpa SO 101 47.9 40.3 11.8 5.74 10.42 11 5 0.11 988 SO 102 – – – 6.11 11.57 15.5 2.2 0.45 1620 SO 103 70.6 20.4 9.0 5.57 4.89 192.5 5.8 0.44 246 SO 104 35.7 26.9 37.4 4.81 5.67 157.5 2.3 0.56 600 SO 105 80.6 14.7 4.7 5.33 3.08 25.5 1.1 0.08 325 SO 106 86.0 12.6 1.4 5.61 4.42 2.5 10.2 0.16 – SO 107 76.0 23.1 0.9 5.63 10.79 2.8 5.7 0.13 – SO 108 20.7 31.5 47.8 5.19 4.62 10.5 2.3 0.7 – SO 109 14.8 36.5 48.7 5.21 5.41 16.3 5.7 2 –

−1 ple was mixed with 1.5 mL ethanol 95% (v/v), 100 ␮L of 10% AlCl3 solved in 50 mL methanol (final concentration 0.05 mg mL ). −1 −1 in 95% (v/v), 100 ␮Lof1MCH3COONa and 2.8 mL dd H2O. To deter- Samples (5 ␮L ) and standard (1–5 ␮L = 50–250 ng ␮L ) were mine the absorbance of samples, 10% of AlCl3 was substituted by the placed in plates with a 4 mm band of Linomat IV (CAMAG; Mut- same amount of dd H2O in blank. Absorption measurements were tenz, Switzerland). Plates were developed in a CAMAG HPTLC made with AlCl3 and without AlCl3 at 415 nm (Jasco V530, Japan). twin-trough chamber and analyzed at 350 nm using a TLC Scan- Results were compared to the blank consisting of 500 ␮LddH2O, ner CAMAG III for rutin detection (Razmilic et al., 1994; Pastene et 1.5 mL ethanol 95% (v/v), 100 ␮L of 10% AlCl3 in 95% (v/v), 100 ␮Lof al., 1997, 2001). 1MCH3COONa and 2.8 mL dd H2O. The total flavonoid content was determined using a standard curve with quercetin (Darmstadt, Ger- 2.4. Data analysis many) (10–100 mg L−1). The mean of two samples were used and expressed as total flavonoids (%) (Nieva-Moreno et al., 2000). In order to determine differences among treatments, results of each trial were analyzed by analysis of variance (ANOVA) 2.3.2. Determination of rutin with the SAS (Statistical Analysis System) program and the Tukey A sample of 100 mg of pulverized plant (180 mesh) was test (P ≤ 0.05). The germination data were transformed using extracted three times with 15 mL methanol reflux for 15 min. The G = (PG + 0.5)1/2 (Little and Hills, 1976). extracts were mixed and brought to 50 mL methanol in a volumetric flask. Rutin content was determined by planar chromatrography 3. Results (HPTLC) (silica gel 60 F254, 10 cm × 10 cm HPTLC plates from Merck; mobile phase: ethyl acetate:formic acid:water: 9+1+1, 3.1. A. emarginata Clos (Fabaceae) Paramela (Fig. 1) v/v/v). Plates were previously washed with KH2PO4 0.1 M/50% methanol (v/v) and activated before analysis at 100 ◦C for 60 min. Habitat: Paramela grows in the Andes mountains above 1500 Standard: 2.50 mg rutin (quercetin-3-O-rhamnoglucoside) was dis- m.a.s.l., stony, well drained soils with a good soil aeration, on slopes

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Table 2 Location, altitude, environmental, and ecological characteristics of ﬁve native medicinal plants and their accessions in the Bio-Bio Region, Chile.

Species/accession Latitude, S Longitude, W Altitude Surrounding environment and soil Disruption Main species present number (m.a.s.l.) description

Adesmia emarginata P 101 36◦5406.6 71◦2406.6 1896 South side hill exposure;, high Fragmented- eroded Adesmia emarginata radiation, SED, gravelly, 75% slope P 102 36◦5221.8 71◦2805.8 1538 Flat highland within ancient lava ﬂow Fragmented Adesmia emarginata and South side exposure hill, SED, gravely, 2% slope P 103 1950 Highland meadow, SED, very gravelly, Cultivated Nothofagus anctartica 40% slope P 104 1500 West side exposure hill, SED, Very Adesmia emarginata gravelly P 105 37◦2644.3 71◦1951.8 1568 North side creek side, SED, very – Adesmia emarginata gravelly, 45% slope P 106 37◦0039.6 71◦3626.7 1537 West side mountain lagoon, sand and – Nothofagus anctartica rocky area, SED, very gravely, 20% slope Buddleja globosa MA 101 36◦5853,9 71◦3033.3 774 River side and dried mountain lagoon, – Nothofagus dombeyi, N. SED, very gravelly, 15% slope obliqua MA 102 30◦3606 71◦1650 840 South side hill exposure, moist by a Cultivated, Fragmented Lomatia hirsuta spring, MWD, gravelly, 90% slope MA 103 37◦5915.8 31◦225 538 West side of creek, SED, very gravelly, Cultivated-Eroded 30% slope MA 104 37◦0052.9 71◦3558.5 1393 Rocky hillside by tree line, SED, Very Cultivated Nothofagus dombeyi gravelly, 20% slope MA 105 37◦1113.1 78◦3414.9 146 Hillside by Paciﬁc ocean, MWD, Fragmented Chusquea quila gravelly, 10% slope MA 106 38◦0384 78◦1530.7 578 Roadside by planted Pinus radiata, Cultivated, Fragmented Pinus radiata, Gevuina MWD, gravelly, 10% slope avellana, Dasiphyllum diacanthoides MA 107 37◦0135.9 71◦3211.2 1324 Small rocky creek, SED, very gravely, – Fuchsia magallanica, 35% slope Crateagus oxyacantha MA 108 37◦0506.1 71◦3706.1 1082 River side, SED, very gravelly, 5% slope – Chusquea quila, Adesmia emarginata, Rosa moschata MA 109 36◦3716.4 72◦3450.8 87 Granitic soil, MWD, nongravelly, 0% Eroded Peumus boldus, Prunus slope domestica MA 110 36◦3642.8 72◦5257.6 327 Road side, SED, nongravelly, 50% slope Forestation Fuchsia magallanica, Luma apiculata, Aristotelia chilensis, Adiantum chilensis Fabiana imbrincata PI 101 36◦5853.3 71◦3028.5 787 North hillside exposure., MWD, Lomatia hirsuta gravelly, 30% slope PI 102 Hillside by road side, SED, gravelly, 20% Cultivated Nothofagus alpina slope PI 103 1700 North exposure hillside, red volcanic rock, SED, very gravely, 80% slope PI 104 700 Highland Chillan river, SED, 10% slope PI 105 37◦2328 71◦2727.6 924 Close by creek, SE, very gravelly, 30% Cultivated Nothofagus dombeyi slope PI 106 37◦4157.5 71◦1926.7 820 North Exposure Queuco river, MWD, Eroded Escallonia rubra gravelly, 60% slope PI 107 36◦54 02.3 71◦3207.4 1045 Road side, SED, gravelly, 15% slope Fabiana imbricata PI 108 36◦4139.3 71◦4350.1 550 North exposure hillside eroded, SED, Cultivated Pinus radiata nongravelly, 70% slope PI 109 37◦0041.2 71◦3602.9 1440 South exposure hillside, SED, very Nothofagus anctartica gravelly, 20% slope PI 110 37◦0141.4 71◦3159.8 1308 orilla camino fondo valle, SED, very Fabiana imbricata gravelly, 5% slope Linum chamisonis NA˜ 101 36◦4133 72◦1523.2 88 Dry coastland mountain range, SED, Cultivated Linum ussitatisimun, gravelly, 0% slope Leontodon saxalis, Avena fatua, Trifolium auricum NA˜ 102 37◦1326.9 72◦2314.3 86 Roadside, SED, nongravelly, 45% slope Cultivated Capsella bursa pastori, Avena fatua, Rosa moschata, Bromus sp. NA˜ 103 36◦2122.2 72◦2136.4 98 North exposure roadside, SED, Cultivated Linum chamisonis, Avena nongravelly, 95% slope fatua, Linum sp. NA˜ 104 36◦1141.2 72◦3246.7 56 Drain border, SED, nongravelly, 70% Fragmented Linum widatisum, Vulpia slope sp., Schinus polygamus NA˜ 105 36◦1242.9 72◦3107.3 55 Road side, SED, gravelly, 70% slope Cultivated Avena fatua, Brisa minor, Hipericum perforatum, Plantago lanceolata, Cortaderia rudiuscula NA˜ 106 36◦2822.4 72◦2303.3 114 Road side degraded soil, SED, Fragmented Avena fatua, Brisa mayor, nongravelly, 10% slope linum ussitatisimum, Bromo sp., Plantago lanceolata

Please cite this article in press as: Susana, F., et al., Characterization and propagation of some medicinal plants in the central-south region of Chile. Ind. Crops Prod. (2010), doi:10.1016/j.indcrop.2010.10.012 G Model INDCRO-5529; No. of Pages 9 ARTICLE IN PRESS F. Susana et al. / Industrial Crops and Products xxx (2010) xxx–xxx 5

Table 2 (Continued)

Species/accession Latitude, S Longitude, W Altitude Surrounding environment and soil Disruption Main species present number (m.a.s.l.) description

NA˜ 107 36◦3723.4 72◦3900 302 Road side, SED, nongravelly, 90% slope Cultivated Brassica sp., Hipericum perforatum, Rumex acetosella NA˜ 108 36◦3634.8 72◦3807.9 326 Road side by vineyards, SED, Cultivated Brisa mayor, Leontodon nongravelly, 90% slope taraxacoides, Avena fatua, Linum ussitatisinum, Hipoueris glabra NA˜ 109 36◦3711.3 72◦2051.5 120 Road side, SED, gravelly, 90% slope – Brisa mayor, Brisa minor NA˜ 110 36◦4736.0 73◦1024.9 65 Coastal terrace, SED, nongravelly, 5% Fire slope Sophora macrocarpa SO 101 36◦3546.2 71◦2923.7 503 – Deforestation Sophora macroparpa, Rosa moschata – –– – –– – –– SO 105 36◦2330.7 72◦2540 135 heavy soil, close by a creek, MWD, no Rosa moschata, Crateagus nongravelly, 0% slope sp., Maytenus boaria SO 106 36◦4353.7 71◦4932.1 NR River side, SED, gravelly, 70% slope Camping area Peumus boldus, Rubus ulmifolius, Teline monspesulana L, Chusquea quila SO 107 36◦4746.7 71◦4010.5 593 Hil lside, SED, gravelly, 90% slope Forestation Nothofagus obliqua, Otholobium glandulosus, Lithraea caustica SO 108 37◦1605,3 73◦2528.7 102 River side, grazing, SED, nongravelly, NR Luma apiculata, Rubus 5% slope ulmifolius,, Peumus bolus SO 109 37◦1142,7 73◦3329.5 15 Road side, SED, nongravelly, 70% slope, forestación Peumus boldus, Aristotelia well drained, no stony, pendiente70% chilensis Pinus radiata, Gevuina avellana

SED, somewhat excessively drained; MWD, moderately well drained. of 20–75% inclination and exposed to high solar radiation. Soil is are likely to be found at harvest time. Mature seeds (dark brown) acid with pH values between 5.5 and 6.1, low soil organic matter have a hard, water-resistant seed coat. Results showed that dark- (0.02–3.8%) and low soil N, P, and K content (Tables 1 and 2 ). Pop- brown seeds reached the highest germination (58%). However, seed ulations were collected in places where there is snow cover during germination did not increase either with the mechanical scarifi- the winter. After snow melts, plants regenerate from subterranean cation or water immersion treatments compared to the control. organs (roots) or seeds. Due to human interference (extraction Mechanical scarification had no influence on germination between and grazing), only six accessions could be collected. No popula- treatments. Dark brown seeds showed the highest vigor index (0.8) tions were found at high altitudes in the Bio-Bío watershed. Plant and the best germination rate (30 days) when compared to grayish height: 15–35 cm. Distribution: mainly irregularly distributed or in brown and very pale brown seeds (Table 3). homogeneous pure groups. Multiplication: seeds mature at differ- Total flavonoid content (Table 4) varied between 0.6 and 1.0%. ent times. Dark-brown, grayish-brown and very pale-brown seeds No relation was found between the flavonoid content and the altitude of the collection site.

Table 3 Seed germination rate in Adesmia emarginata of three different colors, subjected to 3.2. B. globosa Hoppe (Buddlejaceae) Matico, pañil, palguin mechanical scarification. (Fig. 2) Treatment Germination (%) Vigor Germination index rate Habitat: Matico thrives in both Andes Mountains and Chilean − (seed d 1) Coastal Range, between 90 and 1400 m.a.s.l., on light-textured soil, Color seed high or low pH values, organic matter content of 0.7–20%, low N, Dark brown 58 a 0.80 a 30 a variable P and K content. It also grows in humid environments, Grayish brown 39 b 0.50 b 35 b showing moderate to good growth in shady, half-shady to sunny Very pale 30 b 0.38 b 35 b locations (Tables 1 and 2). Plant height: 1–5.5 m. Distribution: in Scarification Control 46 ab 0.58 ab 34 ab homogeneous groups. Multiplication: seeds are very small. Germi- ◦ ◦ Mechanical, 3 min sand 52 a 0.80 a 32 ab nation fluctuated between 84.6% at 5 C and 70% at 20 C under light paper conditions. Mechanical, 7 min sand 54 a 0.82 a 28 a Flavonoid content fluctuated from 9.7% in leaves of plants from paper Water inmersion 30 ◦C/3 min 30 b 0.40 b 32 ab the Andes mountains (accessions MA-101; MA-102; MA-103; MA- Water inmersion 30 ◦C/7 min 42 ab 0.58 ab 34 ab 104) to 13.9% in plants from the Chilean Coastal Range (accessions Water inmersion 43 ab 0.55 ab 36 ab MA-105; MA-106), except for the MA-107 accession sampled in the ◦ 30 C/10 min Andes mountains (Table 4). Water inmersion 40 ◦C/3 min 44 ab 0.59 ab 34 ab Water inmersion 40 ◦C/7 min 38 ab 0.41 b 37 b Water inmersion 22 ab 0.35 b 32 ab 3.3. F. imbricata R. et P. (Solanaceae) Pichi, pichi-romero (Fig. 3) 40 ◦C/10 min

Means followed by different letters in the same column are signiﬁcantly different Habitat: This species grows in the foothills of the Andes moun- (P < 0.05). tains, between 700 and 1700 m.a.s.l., on light-textured soil, low pH,

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Fig. 4. Linum chamissonis ﬂowers.

3.4. Linum chamisonis Schiede (syn. L. obtusifolium Phil., L. Fig. 2. Buddleja globosa leaves and inﬂorescence. ramossissimum Gay, L. aquilinum Mol. subsp. macrei) (Linaceae) (Nanco)˜ (Fig. 4)

Habitat: Nanco˜ grows only on the oriental slope of the Chilean Coastal Range, between 55 and 350 m.a.s.l., usually in dry areas with generally with low organic matter and low N, P, and K content. It high slope. Soils in which it grows are marginal, heavy-textured, usually grows on slopes of 5–80% inclination, along roads and hill- low fertility and highly eroded soils, with low content of organic sides, on stony soils that sometimes have deﬁcient drainage. Plant matter, N, P, and K (Tables 1 and 2). Plant height: 20 cm. Distribution: height: 30–150 cm. Distribution: homogeneous or irregular groups. irregularly distributed or in homogenous groups. Multiplication: Multiplication: germination of very tiny seeds was very low (3%) at the highest germination percentage (20–87%, depending on the ◦ 20 ◦C/24 h light. Seeds did not germinate at lower temperatures or accession) was obtained 12 C in light or dark conditions. After a in darkness. few minutes in contact with water, the mucilage covered the seed No relation was found between the altitude of the collection site and the rutin content (Table 4). Table 4 Total ﬂavonoid content in six accessions of Adesmia emarginata, eight accessions of Buddleja globosa and rutin content in 10 accessions of Fabiana imbricata from the Bio-Bio Region, Chile.

Specie Accession number Bioactive molecule Total ﬂavonoids (%)

Adesmia emarginata P101 1 P102 0.9 P103 0.7 P104 0.6 P105 0.6 P106 0.8

Buddleja globosa MA 101 11.3 MA 102 10.5 MA 103 11.8 MA 104 9.7 MA 105 13.5 MA 106 13.6 MA 107 13.9 MA 108 12.3

Specie Accession number Bioactive molecule Total rutin (%)

Fabiana imbricata PI101 5.2 PI102 3.6 PI103 4.5 PI104 5.0 PI105 3.7 PI106 3.3 PI107 5.3 PI108 5.0 PI109 1.3 Fig. 3. Fabiana imbricata ﬂowers and leafs. PI110 5.3

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Table 5 Table seed germination, vigor index, and germination rate in Linum chamisonis treated with different light condition.

Accesion Treatment Germination (%) Vigor index Germination rate (seed d−1)

12 ◦C20◦C12◦C20◦C12◦C20◦C

Na˜ 109 With light 30 Aa 5 Ba 0.242 Aa 0.077 Ba 22 – Darkness 37.5 Aa 7.5 Ba 0.358 Aa 0.057 ba 12 –

Na˜ 110 With light 52.5 Aa 60 Ba 0.609 Ba 2.176 Aa 13 17 Darkness 75 Aa 60 Ba 0.888 ba 1.814 Aa 15 13

Means followed by different capitol letters in the same row are signiﬁcantly different (P < 0.05). Means followed by different small case letters in the same column are signiﬁcantly different (P < 0.05)

Table 6 Seed germination, vigor index, and germination rate in Sophora macrocarpa treated with different scariﬁcation treatment-temperature combinations.

Scariﬁcation treatment Germination (%) Vigor index Germination rate (seed d−1)

5 ◦C12◦C20◦C5◦C12◦C20◦C5◦C12◦C20◦C

Control 5 bA 5 cA 0 bA 0.61 0.81 0.0 1.88 3.13 0.00 Sulfuric acid 15 min 44 aB 64 aB 2 bC 2.17 2.75 0.05 11.50 12.53 0.50 Sulfuric acid 30 min 64 aB 89 aA 13 aC 3.14 3.45 1.72 9.87 12.66 3.15 Sand paper 55 aA 53 bA 5 baB 2.83 2.68 1.02 11.13 12.26 2.13

Means followed by different capital letters in the same row are significantly different (P < 0.05). Means followed by different small case letters in the same column are significantly different (P < 0.05). coat. Seed germination started after 8 days, reaching the highest from a andesitic–basaltic sand of volcanic (Stolpe, 2006; Smaling germination around the 15th day. Germination rate increased at et al., 1996). 20 ◦C. Vigor index values depended on temperature and accession Both mechanical scarification and water immersion showed (Table 5). that seed color had influence on germination. This result suggests that probably seed dormancy of A. emarginata is probably asso- 3.5. S. macrocarpa J.e Sm. Fabaceae (mayú) (Fig. 5) ciated with different levels of seed coat permeability (Fischer et al., 2009), or with an embryo dormancy. Seeds of other Adesmia Habitat: Mayu grows in the foothills of Andes mountains, val- spp. also show low water permeability of the seed coat (Scheffer- ley, and the Chilean Coastal Range, between 15 and 590 m.a.s.l., Basso and Vendrusculo, 1997; Tedesco et al., 2001). It is possible on humid soil either heavy or light-textured, with low or medium that these seeds require a period of time after ripening to pH and low content of organic matter, N, P, and K, along riversides accelerate the germination process and also exposure to mois- or roads. Plant height: 1–2 m. Multiplication: the highest germina- ture and low temperature (stratification) to increase germination tion percentage (89%) was observed when seeds were scarified in (Pérez and Martínez-Laborde, 1994; Geneve, 2003). This behav- concentrated sulfuric acid for 30 min and germinated at 12 ◦C. Seed ior might probably allow a more persistent and long-lived seed germination started after16 days and germination continued until bank. 30 days (Table 6). 4.2. B. globosa 4. Discussion B. globosa grows in soils with different chemical and physi- 4.1. A. emarginata cal properties: sandy, loamy sand and loam soils (Vidal, 2007). Therefore, the accessions collected are exposed to a wide range These species grow in acid soil with low values of nutrient lev- of nutrients levels and organic matter. els, explained by the high proportion of sand (Psamments) derived The low temperature required by B. globosa seeds to germinate in a few days could be associated to the environment in which this species grows. Since the seed is small in size, it is recommended to sow them in winter in plug trays under low temperature and transfer them to a greenhouse after a few days. High flavonoid concentration in leaves of plants collected on the Chilean Coastal Range differs from the results reported by Havaux and Kloppstech (2001), who demonstrated an increase of flavonoid pigments when the plants are submitted to high light incidence under chilling temperatures in Arabidopsis thaliana mutants. This condition is likely to occur in spring and summer in the Andes mountains more than in the Chilean Coastal Range. It seems that plants from the Andes Mountains grow in shaded areas. Chaves et al. (1997) determined that in these conditions the amount of flavonoids in Cistus ladanifer was lower than in plants of open areas, what could happen on the Chilean Coastal Range. In the case of B. globosa, the biosynthesis of flavonoids is controlled by a broad temperature range and light if the temperature is low, as suggested the Fig. 5. Sophora macrocarpa leaves and flowers. results reported by Bilger et al. (2007).

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4.3. F. imbricata Acknowledgements

This species grows under similar soil characteristics as B. glo- Funding for this research was provided by Chile’s Agriculture bosa, but differing in the altitude where it grows. Most of natural and Livestock Service (SAG), Ministry of Agriculture, Chile. Project populations of Pichi are adapted to grow in the foothills or in the name: Rescue, characterization and propagation of some native valley. medicinal plants in the Eighth Región, SAG C3-83-08-34. Seeds seem to require higher temperatures for germination. Authors acknowledge the valuable collaboration of technicians Results were similar to those obtained by Thomson and Witt and students on plot planting, management, data collection, and (1987) for seeds of cutleaf groundcherry (Physalis angulata), smooth analysis. groundcherry (Physalis virginiana) and eastern black nightshade (Solanum ptycanthum), where light or darkness completely inhibited germination at temperatures of 5 and 10 ◦C but increased References at 20 and 10/20 ◦C, respectively. In general, the seeds of species Anfirud, M.N., Schneiter, A.A., 1984. Relationship of sunflower germination and vigor of Solanaceae are sensitive to photoperiod, especially when the tests to field performance. Crop Sci. 24 (2), 341–344. ◦ temperature varies between 20 and 30 C. Germination of With- Atwater, B.R., Vivrette, N.J., 1987. Natural protective blocks in the germination of ania somnifera seeds was found to be both temperature and seeds. Acta Hortic. 202, 57–67. Backhouse, E.N., Delporte, C., 1977. Estudio de flavonoides en Sophora macrocarpa light-dependent. Pre-chilling treatments reduced seed germina- Sm. Tesis Químico Farmacéutico. Universidad de Chile, Fac. Cienc. Farmac, San- ◦ tion, while exposure to constant temperatures between 25 C and tiago, Chile. 45 ◦C in dark conditions completely inhibited germination (Kambizi Bilger, W., Rolland, M., Nybakken, L., 2007. UV screening in higher plants induced by low temperature in the absence of UV-B radiation. Photochem. Photobiol. Sci. 6, et al., 2006). Light intensity and time of exposure, both impor- 190–195. tant factors for germination, were not considered in this study Conama, 2003. Estrategia nacional de biodiversidad. Comisión Nacional del Medio due to the fact that a photoperiod of 24 h at low irradiance inten- Ambiente, Santiago de Chile. ␮ −2 −1 Cubillos, A., 1994. Recursos fitogenéticos de la biodiversidad chilena: Una proposi- sity (20 mol m s ) was used. Pearson et al. (2002) determined ción de priorización para su preservación. Simiente 64 (4), 229–235. a relation between germination and seed size. In small-seeded Chaves, N., Escudero, J.C., Gutierrez-Merino, C., 1997. Role of ecological variables in species (seed mass<2mg),germination increased at an irradiance the seasonal variation of flavonoid content of Cistus ladanifer exudate. J. Chem. of 22.3 ␮mol m−2 s−1 when compared to germination in complete Ecol. 23 (3), 579–603. D’Antuono, L.F., Rossin, F., 1995. Experimental estimation of linseed (Linum usitatis- darkness. As seeds of Fabiana imbricate are small, it might be nec- simum L.) crop parameters. Ind. Crops Prod. 3, 261–271. essary to study if seed germination percentage increases when Fischer, S., Ramirez, C., Wilckens, R., Finot, V.L., 2009. Características morfo- soaking seeds in gibberellic acid, like Solanum vespertilio seeds anatómicas y germinación de semillas de paramela (Adesmia emarginata Clos, Fabaceae). Agro-Ciencia. Rev. Chil. 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