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Maqui (Aristotelia chilensis): Morpho-phenological characterization to design high-yielding cultivation techniques
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Maqui (Aristotelia chilensis): Morpho-phenological characterization to design high-yielding cultivation techniques b ˜ Hermine Vogel a,∗ , Patricio Penailillo , Ursula Doll c , Geo Contreras a , a Giordano Catenacci , Benita González a a b c
Facultad de Ciencias Agrarias, Universidad de Talca, Casilla 747, Talca, Chile Instituto de Ciencias Biológicas, Universidad de Talca, Casilla 747, Talca, Chile Facultad de Ciencias Forestales, Universidad de Talca, Casilla 747, Talca, Chile
a r t i c l e
i n f o
Article history: Received 4 July 2014 Accepted 23 September 2014 Available online xxx Keywords: Fruit set Floral meristem Phenology Cross pollination
a b s t r a c t Maqui (Aristotelia chilensis) is a species whose berries contain one of the highest levels of antioxidant currently known. At present the rising demand for fruit is being supplied exclusively by wild crafted raw material. Ongoing domestication includes studies of fruit production, in both wild populations and cultivated progenies from different provenances, as a basis for proposals of high-yielding cultivation techniques. Morphological characterization at the end of the first summer indicated significant differences between provenances for plant height (mean values from 99 to 133 cm), petiole length (1.8–2.5 cm), width of the leaf blade (3.6–5.1 cm), ratio leaf length/width (1.9–2.2) and leaf area (16–30 cm2 ). Plant width (69–91 cm), internode length (2.7–3.3 cm) and leaf blade length (7.8–9.9 cm) did not differ among clones of different provenances. Fruit set in wild populations ranged from 54% in a mountainous area to 61% in a coastal population. Some hermaphrodite plants even reached 69%. Covered floral branches set very few fruits, less than half the weight of uncovered branches (38 and 97 mg, respectively), while the pulp–seed relation was similar for both treatments, about 2:1. Histological studies revealed that transition from vegetative to floral meristem occurs in spring during fruit development on the branches that are formed as an elongation to the flowering and fruit bearing shoot. The establishment of phenological stages permits the visualization of the variation in sprouting, blooming, fruit ripening between different clones. The present results reveal a large variability for vegetative and fruit producing characteristic that would permit a successful selection of high yielding plants. At the same time, fundamental knowledge about plant architecture and phenology supports studies of cultivation techniques, such as optimizing density, harvest or pruning. © 2014 Elsevier GmbH. All rights reserved.
Abbreviations: m a.s.l., meters above sea level; DAF, days after bloom; FAA, formaldehyde–acetic acid–alcohol solution. ∗ Corresponding author. Tel.: +56 71 2200214; fax: +56 712200212. ˜ E-mail addresses: [email protected], [email protected] (H. Vogel), [email protected] (P. Penailillo), [email protected] (U. Doll), giordano [email protected] (G. Contreras), [email protected] (G. Catenacci), [email protected] (B. González). http://dx.doi.org/10.1016/j.jarmap.2014.09.001 2214-7861/© 2014 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Vogel, H., et al., Maqui (Aristotelia chilensis): Morpho-phenological characterization to design high-yielding cultivation techniques. J. Appl. Res. Med. Aromat. Plants (2014), http://dx.doi.org/10.1016/j.jarmap.2014.09.001
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1. Introduction Aristotelia chilensis (Molina) Stuntz, Elaeocarpaceae, is a Chilean medicinal plant commonly called “maqui” or “quëlón”. It is a sacred species of the indigenous Mapuche people, being a symbol of benevolent and peaceful intent (Mösbach, 1992). Leaves are traditionally used for wound healing and fruits to treat diarrhea and dysentery. The edible berries are consumed fresh or prepared as liquor. Its dark purple color has been used also to color red wine (Gupta, 1995; Hoffmann et al., 1992; Montes and ˜ et al., 1981). The species adapts Wilkomirsky, 1984; Munoz to high radiation during the summer in Central Chile by synthetizing polyphenols and specifically anthocyanins that protect the seeds in the small berries from damage caused by UV light. In the last years extraordinary antioxidant properties of the maqui fruits have been reported, some of the first being Miranda-Rottmann et al. (2002), followed by Araya et al. (2006), Avello et al. (2008) and Olate (2008). These results triggered recent studies and development of food, nutraceutical, cosmetical and pharmaceutical products (Avello et al., 2009; Céspedes et al., 2008, 2009, 2010; Gironés-Vilaplana et al., 2012; Rubilar et al., 2011; Schreckinger et al., 2010), all of them based until now on wild crafted maqui berries with increasing exploitation. Generally wild collection of maqui berries implies cutting the fruit bearing branches. But little is known about the reproductive structures or the moment of floral induction and development of the species. The question is whether cutting the branches for harvest affects the fruit production in the following season, making this practice unsustainable. For future cultivation it is also important to develop pruning techniques to maintain the plants stature while at the same time optimizing fruit production. The present work tries to provide answers to fundamental questions that may contribute to design crop management techniques of A. chilensis, such as pruning or density of male pollinators (that will not produce fruits but are necessary for a high yielding crop). A. chilensis is a small, up to 4 m high, tree with several flexible, thin stems, described by Rodríguez (2005) as evergreen, by Damascos and Prado (2001) as a wintergreen species, as some of the leaves remain for only one
winter. Its geographic distribution is reported in Chile from latitudes 30◦ to 43◦ S from sea level up to 2500 m elevation in the mountainous areas. It has been introduced to the Robinson Crusoe Island located in the Pacific Ocean, where it has become an invasive plant (López et al., 2013; Matthei, 1995). Maqui is known as a dioecious species (Hoffmann et al., 1992; Riveros et al., 1996) producing either male or female flowers grouped in axillar corymbs of 2–4 flowers in spring, between October and November (Rodríguez, 2005). The pale yellow flowers of female individuals have a trilocular ovary and a short style with stamen reduced to staminodes, whereas male flowers show 10–15 stamens with a rudimentary pistil (Verdi, 2004). The black, round berry is small, 4–6 mm, with four to eight angular seeds that ripen in summer, December and January (Rodríguez, 2005). Separation of male and female organs in different individuals implies cross mating and high genetic variability even within wild populations. The black maqui berries attract birds that contribute to the dispersion of the species (Mösbach, 1992; Vogel et al., 2008). Seeds eaten by birds have a higher germination rate than non-eaten ones (Valdivia and Simonetti, 2007). Even though maqui is reported to grow in moist areas, such as brooks, at the feet of hills or edges of woods (Rodríguez, 2005), it is quite resistant to dry periods. 2. Materials and methods The present studies were performed in clones of different provenances between latitudes 34◦ S and 41◦ S (Fig. 1, Table 1). Selected plants from these wild populations were propagated vegetatively by rooting cuttings. The clones obtained were established in spring of 2009 in the Experimental Station of Universidad de Talca (Panguilemo, 35◦ 22 S/71◦ 35 W; 120 m above sea level (m a.s.l.) characterized by a Mediterranean climate with the main precipitation during the winter months, from April to September (Fig. 2)). 2.1. Morphological characterization of vegetative traits in cultivated accessions For morphological characterization individuals cultivated in the Experimental Station Panguilemo coming from
Table 1 Morphological characterization of eight different provenances cultivated in Experimental Station Panguilemo. Provenance Latitude (S/W) Altitude (m) Number of plants Plant characteristics Height (cm) Width (cm) Internode length (cm) Leaf characteristics Petiole length (cm) Leaf blade length (cm) Leaf blade width (cm) Ratio length/width Leaf area (cm2 )
San Fernando ◦
◦
Romeral ◦
◦
San Clemente ◦
◦
Mulchén ◦
◦
Curacautín ◦
◦
Villarrica ◦
◦
Entrelagos ◦
◦
Puerto Montt
34 /70 530 7
34 /70 495 6
35 /71 275 4
37 /72 329 4
38 /71 607 4
39 /71 190 4
40 /72 165 7
41◦ /72◦ 92 7
109 ab 91 3.3
114 ab 81 2.9
99 b 69 3.0
119 ab 75 2.9
127 ab 74 3.1
111 ab 86 2.8
133 a 91 2.7
125 ab 86 3.2
2.1 ab 9.9 5.1 a 1.9 ab 30 a
2.1 ab 9.5 5.1 a 1.9 a 29 ab
1.8 b 8.1 4.2 ab 2.0 ab 21 ab
2.1 ab 9.5 4.9 ab 1.9 ab 28 ab
2.5 a 9.5 4.7 ab 2.0 ab 26 ab
2.1 ab 7.8 3.6 b 2.2 b 16 b
2.3 ab 9.1 4.3 ab 2.1 ab 23 ab
2.0 ab 9.2 4.3 ab 2.1 ab 23 ab
Different letters in the same line denote significant differences, p ≤ 0.05.
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Fig. 1. Geographical locations of provenances of A. chilensis cultivated in the Experimental Station of Universidad de Talca, Panguilemo.
different provenances (Fig. 1, Table 1) were studied at the end of the summer (January) of the first growing season. Internode length was determined on three branches per plant, leaf characteristics were taken as the mean of 25 expanded leaves per plant, and leaf area was estimated by the formula: (Ll − Lw /2)Lw /2 + /2(Lw /2)2 , where Ll is leaf blade length and Lw is leaf blade width. For statistical analysis means were separated by Tukey test (p ≤ 0.05).
plants established in 2009 in the Experimental Station Panguilemo. This involved one male and four females, two of them of the San Fernando provenance, one from Romeral and another from Puerto Montt. The profiles were covered with a black anti-mulch tissue and isolated with Styrofoam to prevent heating. Periodically the glass-wall was uncovered and photographed in order to count the number of white roots appearing.
2.2. Root development
2.3. Fruit set in wild populations
At the end of August of 2013 mini glass-wall profiles (45 cm × 35 cm) were placed in the soil, facing east, of five
In two wild populations of maqui located in Central Chile, one at the coast (Pelluhue 35◦ S/72◦ W, altitude
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monthly rainfall mean monthly minimum temperature
Rainfall [mm]
Temperature [°C]
mean monthly maximum temperature
Fig. 2. Precipitation and mean temperature in the Experimental Station Panguilemo, seasons 2012/13 and 2013/14.
Fig. 3. Vegetative stages of A. chilensis: (A) apical vegetative bud dormant; (B) swollen apical bud with yellowish tips; (C) initiation of sprouting, apical bud bust, green leaves appearing; (D) sprouting, the form of the apical bud is changing; (E) end of sprouting, leaves emerge from the apical bud.
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Fig. 4. Phenological stages of the reproductive cycle of A. chilensis: (A) axillary inflorescence bud dormant; (B) inflorescence buds emerging; (C) pre-anthesis with floral buds clearly separated; (D) anthesis, full blooming; (E) end of blooming, vestiges of flowers in male plants or not pollinated females; (F) beginning of fruit development, green fruits without vestiges of flowers; (G) marking: >50% green fruits, some reddish; (H) ripening: >50% fruits of dark color, no green fruits; (I) end of ripening: all fruits black purple.
200 m, with Mediterranean climate and maritime influence) and the other in the foothills of the Andes (El Picazo 35◦ S/71◦ W, 400 m a.s.l.; with higher radiation and marked seasonal and daily temperature differences), flower production and fruit set were evaluated in 20 female and 20 male individuals of each population during the 2012/13 season. In each plant, three representative branches were marked and the number of flowers and fruit set registered. In the coastal population three hermaphrodite plants were detected. Statistical analysis among populations was performed by Mann–Whitney (p ≤ 0.05) and among male, female and hermaphrodites by Kruskal–Wallis (p ≤ 0.05). 2.4. Cross and self-pollination As A. chilensis is not a perfect dioecious species, we also studied sporadic self-pollination. Four cultivated female
clones from Central Chile (34◦ S/70◦ W) were selected. In each plant six branches with abundant floral buds were marked, three of them covered completely with frost protection fleece before anthesis to prevent cross pollination, the other three uncovered as a control treatment. The number of fruits per plant, as well as fruit weight and number of seeds were registered. Statistical analysis among covered and uncovered branches was performed by Mann–Whitney nonparametric test (p ≤ 0.05). 2.5. Floral induction and development Floral induction and development was studied in three clones of different provenances (Romeral, Mulchén and Puerto Montt, see Fig. 1) all established in the Experimental Station of Universidad de Talca, during the third season. Samples of the young, vegetative long shoots which
Please cite this article in press as: Vogel, H., et al., Maqui (Aristotelia chilensis): Morpho-phenological characterization to design high-yielding cultivation techniques. J. Appl. Res. Med. Aromat. Plants (2014), http://dx.doi.org/10.1016/j.jarmap.2014.09.001
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Fig. 5. Fruit set in two wild populations located at the coast (Pelluhue) and in the foothills of the Andes (El Picazo) for male (♂), female (♀) and hermaphrodite (♀♂) individuals during the 2012/13 season (no. of flowers or fruits per flowering shoot system).
contained the axillary buds were taken weekly from the moment of full bloom (>80%), in one of the clones (Romeral) even one week before, from September 27 to January 10. The moment of sampling was established as days after full bloom (DAF) determined in two branches of each plant. The sprouts developed during the previous season were fixed
immediately with FAA and buds removed for histological studies. Histological sections of the buds (10–15 m) were made by a cryostat (LEICA CM 1510S) at −20 ◦ C. The sections were observed in an optical epifluorescence microscope (OPTIKA) at different magnifications (4×, 10×, 40× and 100×). The size of the meristem was measured
90 80
Fruit weight (mg)
70 60 50
Pulp 40
Seed
30 20 10 0
Uncovered
Covered
Fruits per flowering shoot system
250
200
150
100
50
0
Uncovered
Covered
Fig. 6. Fruit development on covered and uncovered branches in cultivated maqui.
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Fig. 7. Floral induction and development in three clones of A. chilensis from full bloom (0 DAF) until floral differentiation (DAF = days after bloom). Provenances A = Romeral, B = Mulchén, C = Puerto Montt. Stage 1: vegetative meristem, stage 2: inflorescence meristem, and stage 3: early differentiated inflorescence. Bar = 100 m.
by means of the software ImageJ (1.47i National Institutes of Health, USA). Meristems of lateral shoots (flowering shoot system) were classified into three stages: stage 1 being a narrow, flat vegetative meristem, stage 2 a domed inflorescence meristem, and stage 3 an early differentiated inflorescence. At the moment of sample collection the length of the young apical sprout was measured. At the same time the number of expanded leaves was registered. To correlate the phenological stages with the climatic
situation, accumulated day degrees above 10 ◦ C were registered from September 1 to March 31. 2.6. Phenological studies Phenological stages were observed in different clones coming from the wild populations San Fernando, Romeral, San Clemente, Mulchén, Curacautín, Villarrica, Entrelagos and Puerto Montt (Fig. 1), all established with two
Fig. 8. Pheno-morphological cycle of A. chilensis.
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San Fernando Romeral
PROVENANCES
San Clemente Mulchén Curacautín Villarrica Entrelagos Puerto Montt 0
100
200
Apical and Floral Sprouting Buds Anthesis Marking
300
400
500
600
Floral Buds Emerging Fruit Development End of Ripening
700
800
DAY DEGREES
Fig. 9. Phenological stages of different provenances in the Experimental Station Panguilemo during spring 2011.
individuals per clone at the Experimental Station of Universidad de Talca, Panguilemo, during August of 2009. The plantation was irrigated from the first week in October to the end of April by a drip irrigation system according to the weekly measured evapotranspiration. The phenophases were evaluated weekly from August 1 to December 28 during the seasons 2012 and 2013 in seven and four female clones, respectively (Fig. 10). One apical vegetative branch and three reproductive branches were studied in two individuals per clone. The same procedures were realized for male individuals. The number of open flowers was recorded in three flowering shoot systems, one in apical position, the others in the middle and lower zone of three randomly selected branches. A phenomorphological diagram (Fig. 8) representing qualitative abstractions of the annual cycle of female plant was constructed by observation of the different phenological phases in female plants of maqui that grew in Panguilemo during the 2012 season. The symbols used represent plant organs according to Montenegro et al. (1989). The different stages in maqui were established by Catenacci (2012). Fig. 3 shows the vegetative and Fig. 4 the generative development of the buds.
3. Results and discussion 3.1. Morphological plant and leaf characteristics in clones of different provenances The study of vegetative characteristics gives an overview of the variability of some morphological traits (Table 1). Plants coming from southern Chile (Entrelagos) are growing taller than those from San Clemente. Plant width, an indicator needed for distance between plants, is highly variable and, during the first year, reached maximum mean values up to 91 cm without differing among provenances.
Leaf area was almost twice as large in plants coming from the most northern site, San Fernando, than those from Villarrica. The characteristics which show significant differences among provenances may indicate their genetic determination as all accessions were cultivated under the same environmental conditions. Mean values for length and width of the leaves were higher than reported in the literature. Gupta (1995) and Rodríguez (2005) described the size of maqui leaves as 3–8 cm long and 1.5–3.5 cm wide, Hoffmann et al. (1992) reported 4–9 cm for leaf length and Damascos and Prado (2001) averages of 7.1 and 7.3 cm for new and old leaves, respectively. These differ from present results with individual values ranging from 6.8 to 11.7 cm for the length and 3.1 to 6.2 cm for the width, which could reflect the effect of management techniques, as our cultivated clones were subjected to cultivation practices such as irrigation, whereas the description in the literature is based on wild plants. The lack of differences between provenances in some of the morphological characteristics indicates a high natural variability even within the same natural population, and was to be expected because of dioecism. Similar results have been reported previously in other wild crafted medicinal plants native to Chile, such as boldo (Peumus boldus) (Vogel et al., 2011a), Buddleja globosa (Vogel et al., 2011b) or bailahuen (Haplopappus sp.) (González et al., 2012).
3.2. Root development Root growth showed a marked reduction at the beginning of flowering and during fruit growth, coincident with the drying of the soil. This indicates a critical moment where restriction of water in the soil may reduce the production of fruits.
Please cite this article in press as: Vogel, H., et al., Maqui (Aristotelia chilensis): Morpho-phenological characterization to design high-yielding cultivation techniques. J. Appl. Res. Med. Aromat. Plants (2014), http://dx.doi.org/10.1016/j.jarmap.2014.09.001
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Fig. 10. Phenology of male and female clones of different provenances during 2012 and 2013 in the Experimental Station of Universidad de Talca (Panguilemo).
3.3. Fruit set in wild populations Fruit set in the wild populations studied is illustrated in Fig. 5 reaching 69% in the hermaphrodite plants and 61% in the females, with no significant difference between the two groups. Male individuals showed no fruit set, except one single berry at the coastal population (Pelluhue). On the other hand fruit set at the coastal population was significantly higher (68%) than in the Andes (54%). It is interesting that male plants had significantly more flowers per flowering shoot system (20.6 ± 10.5) than females (12.9 ± 6.7) and hermaphrodites (16.8 ± 3.4), considering both wild populations. These mean values are all much higher than the 2–4 flowers per corymb reported by Rodríguez (2005) indicating a high variability in the number of flowers per flowering shoot system. The same study showed that flowering and fruiting of the Andean population (El Picazo) occurs about one month later than at the coastal population (Pelluhue).
3.4. Cross pollination In dioecious species cross pollination is obligatory for fruit development. Nevertheless in our maqui gene bank,
as well as in some wild populations, plants with some hermaphrodite flowers have been detected, even male individuals with some single fruits can be observed occasionally. That’s why a study of cross and self-pollination was established in four female clones from central Chile. Fig. 6 shows clearly that the main pollination system is out-crossing. Only a few fruits developed on covered branches. This result was to be expected as A. chilensis is commonly known as a dioecious species. Moreover, fruit weight is significantly higher in berries harvested on the uncovered control branches (97 mg composed by 64% pulp and 36% seeds) compared with the covered ones (38 mg; 69% pulp and 31% seeds) which means that fruits obtained by self-pollination even have lower quality.
3.5. Transition from vegetative to floral meristem in cultivated provenances Full bloom (0 DAF) was observed in the Romeral provenance on October 4 and in the clones from Mulchén and Puerto Montt on October 18. At the moment of full bloom all axial buds showed flat meristems indicating a vegetative stage of the bud (Fig. 7). The moment of transition
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differed according to the provenance: the most northern, from Mediterranean climate zones, Romeral and Mulchén, initiated floral development (stage 2) on November 9 (35 and 28 DAF) and differentiation (stage 3) 70 DAF, whereas the most southern clones from Puerto Montt, coming from temperate climate zones, developed stage 2 (pre-floral) one week later and did not reach stage 3 until 70 DAF. The present study indicates that transition of the vegetative to floral meristem occurs in spring, during fruit development. At the moment of fruit harvest, the lateral buds exhibit morphological evidence of reproductive structures. So, if wild collectors cut the branches of A. chilensis to simplify harvest berries of maqui, they put at risk the production of the next year. 3.6. Architecture and phenology Maqui is a wintergreen bush according to Damascos and Prado (2001). Its structure is modular, each module formed by long shoots (Fig. 8). The long shoots (dolichoblasts) are sprouts with an apical vegetative bud and opposite leaves. The axillary buds develop a flowering shoot system that is protected by bud scales during dormancy. These buds will activate in spring, sprouting, stretching and developing flowering shoot systems (in sensu Classen-Bockhoff and ˜ 2013) of limited growth that carry the flowers Bull-Herenu, in corymbs, and die after fruit development and wilting. Concomitant with the bloom, the long shoots of the season are formed from the vegetative buds at the end of the branch developed the year before. Their growth is indeterminate. When the apical bud is frozen, died back or removed, one to four apical buds will continue shooting up. When breaking the monopodial growth, sympodial system is initiated. Fig. 9 illustrates the phenological stages of different provenances in function of the day degrees during spring 2011, whereas Fig. 10 shows the weekly development of male and female individuals cultivated at the Experimental Station of Universidad de Talca, Panguilemo, during the growing seasons 2012 and 2013. The female plants are of different provenances, while the males all coming from the Romeral population. Clones from different provenances may differ in initiating sprouting: in 2011 clones coming from Curacautín only started flowering when all others had finished blooming and were developing fruits. This provenance was the only one that did not ripen until end of December. As Curacautín is from the highest location of the studied populations from the South (607 m a.s.l.) plants from this provenance may be adapted to the colder climate conditions and may require more low temperature accumulation to activate bud development. These findings may be beneficial for selection for late ripening commercial clones that would permit the extension of harvest. Differentiation in the phenological stages between female clones was also observed in 2012 with first signs of activation from mid-August for clones from latitude 34◦ S, whereas the southern provenances (latitudes 40◦ S and 37◦ S) remained dormant until the third week of September (Fig. 10). The flowering of the females was covered from end of September until mid-October by the male clones 19,
23 and 27F1, whereas the late females could be pollinized until end of October by clones 10F1 and 22F1. In 2013 blooming started about one week later but showed the same behavior as in the season before: northern provenances were blooming at the beginning of October, whereas the Entrelagos provenance started the last week of October until the first of November. 4. Conclusions Maqui berry production has, and is, up to now all being provided by wild crafting where the fruit bearing branches are cut to retrieve the fruits. Our studies of floral induction show that the generative buds are developing on these long shoots, and are growing as an elongation to the flowering/fruit bearing shoot system during fruit development. Cutting these branches therefore involves removing the next season’s potential harvest. That’s why in wild maqui plants not more than half of the branches should be cut and, as a long-term solution, a non-destructive harvest system, of only removing fruit, should be developed. The pollination studies confirm our hypothesis of strong cross-fertilization, as self-pollination implies important qualitative and quantitative losses in fruit production. For the different clones, suitable male pollinators are required to assure good cross-fertilization during the different blooming periods considering the existence of early, normal or late flowering clones, respectively. Our studies of morphological traits and phenology indicate the presence of wide variability with possibilities to select the most suitable clones, for instance types with high flowering shoot system production and at the same time inhibited vigor of vegetative growth that would permit extraordinary fruit production in a high density crop. Acknowledgements The present research was financially supported by CONICYT Chile, Project FONDEF D10I1252 in collabora˜ tion with Fundación Chile and the companies AgroQueni, Ana María, Bayas del Sur, Domingo Echegaray, Hortifrut, ˜ for helping in the Surfrut. Thanks to Kester Bull-Herenu interpretation of lateral shoots and bibliographic support, to Pamela González for providing the graphic of the phenolmorphological cycle, and to Luis Letelier for designing the map. We also like to thank Peter Caligari for English revision. References Araya H, Clavijo C, Herrera C, 2006. Capacidad antioxidante de frutas y verduras cultivadas en Chile. Archivos Latinoamericanos de Nutrición 56 (4), 361–364. ˜ Avello M, Valladares R, Ordonez J, 2008. Capacidad antioxidante de Aristotelia chilensis (Molina) Stuntz. Revista Cubana de Plantas Medicinales 13 (4), 1–7. Avello M, Valdivia R, Sanzana R, Mondaca MA, Mennickent S, Aeschlimann V, Bittner M, Becerra J, 2009. Extractos antioxidantes y antimicrobianos de Aristotelia chilensis y Ugni molinae y sus aplicaciones como preservantes en productos cosméticos. Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas 8 (6), 479–486. Catenacci G, (Thesis Facultad de Ciencias Agrarias) 2012. Determinación del crecimiento, fenología, producción y características de bayas en clones de Maqui, Aristotelia chilensis (Molina) Stuntz, según distintas
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G Model JARMAP-16; No. of Pages 11
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procedencias e inclinación de ramillas. Universidad de Talca, Talca, Chile. Céspedes CL, El-Hafidi M, Pavón N, Alarcón J, 2008. Antioxidant and cardioprotective activities of phenolic extracts from fruits of Chilean blackberry Aristotelia chilensis (Elaeocarpaceae), Maqui. Food Chemistry 107, 820–829. Céspedes CL, Alarcón J, Valdez-Morales M, Paredes-López O, 2009. Antioxidant activity of an unusual 3-hydroxyindole derivative isolated from fruits of Aristotelia chilensis (Molina) Stuntz. Zeitschrift für Naturforschung, Section C, Biosciences 64 (9–10), 759–762. Céspedes CL, Alarcón J, Avila J, Nieto A, 2010. Antiinflammatory activity of Aristotelia chilensis (Molina) Stuntz (Elaeocarpaceae). Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas 9 (2), 127–135. ˜ K, 2013. Towards an ontogenetic underClassen-Bockhoff R, Bull-Herenu standing of inflorescence diversity. Annals of Botany 112, 1523–1542. Damascos MA, Prado CH, 2001. Leaf phenology and its associated traits in the wintergreen species Aristotelia chilensis (Molina) Stuntz (Elaeocarpaceae). Revista Chilena de Historia Natural 74, 805–815. Gironés-Vilaplana A, Mena P, García-Viguera C, Moreno DA, 2012. A nobel beverage rich in antioxidant phenolics: Maqui Berry (Aristotelia chilensis) and lemon juice. LWT – Food Science and Technology 47, 279–286. González B, Vogel H, Razmilic I, San Martín J, Doll U, 2012. Biomass, resin and essential oil content and their variability in natural populations of the Chilean crude drug Bailahuén (Haplopappus spp.). BOLETIN Latinoamericano y del Caribe de Plantas Medicinales y Aromaticas 11 (1), 66–73. Gupta MP (Ed.), 1995. 270 Plantas Medicinales Iberoamericanas. CYTEDSECAB, Santafé de Bogotá, Colombia, pp. 261–263. Hoffmann A, Farga C, Lastra J, Veghazi E, 1992. Plantas medicinales de uso común en Chile. Ediciones Fundación Claudio Gay, Santiago, Chile, pp. 130–133. ˜ López P, Baeza CM, Penailillo P, Stuessy TF, Greimler J, Ruiz E, Novoa P, Gatica A, 2013. Introduced plant species in the biosphere reserve Alejandro Selkirk Island, Robinson Crusoe Archipelago, Chile, and their threat to the native and endemic vegetation. Memoirs New York Botanical Garden 108, 211–231. Matthei O, 1995. Manual de las malezas que crecen en Chile. Alfabeta Impresores, Santiago, Chile, pp. 219–220. Miranda-Rottmann S, Aspillaga AA, Pérez DD, Vásquez L, Martínez ALF, Leighton F, 2002. Juice and phenolic fractions of the berry Aristotelia chilensis inhibit LDL oxidation in vitro and protect human endothelial cells against oxidative stress. Journal of Agricultural and Food Chemistry 50, 7542–7547.
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Montenegro G, Avila G, Aljaro ME, Osorio R, Gómez M, 1989. Chile. In: Orshan G (Ed.), Plant Phenomorphological Studies in Mediterraneantype Ecosystems. Kluwer Academic Publishers, The Netherlands, pp. 347–387. Montes M, Wilkomirsky T, 1984. Medicina tradicional chilena. Editorial Universidad de Concepción, Chile. Mösbach EW, 1992. Botánica indígena de Chile. Editorial Andrés Bello, Santiago, Chile. ˜ M, Barrera E, Meza I, 1981. El uso medicinal y alimenticio de planMunoz tas nativas y naturalizadas en Chile. Publicación Ocasional del Museo Nacional de Historia Na 33, 3–91. Olate V, (Thesis Facultad de Ciencias de la Salud) 2008. Estudio químico y actividad antioxidante de los antocianos presentes en los frutos de maqui. Universidad de Talca, Chile. ˜ AM, Arroyo MK, 1996. Sistemas de reproducción en Riveros M, Humana especies del bosque valdiviano (40◦ Latitud Sur). Phyton 58, 167–176. Rodríguez R, 2005. Elaeocarpaceae Juss. ex DC. In: Marticorena C, Rodríguez R (Eds.), Flora de Chile 2 (3), 15–17. Rubilar M, Jara C, Poo Y, Acevedo F, Gutiérrez C, Sineiro J, Shene C, 2011. Extracts of Maqui (Aristotelia chilensis) and Murta (Ugni molinae Turcz.): sources of antioxidant compounds and ␣-Glucosidase/␣Amylase inhibitors. Journal of Agricultural and Food Chemistry 59, 1630–1637. Schreckinger ME, Wang J, Yousef G, Lila MA, González de Mejía E, 2010. Antioxidant capacity and in vitro inhibition of adipogenesis and inflammation by phenolic extracts of Vaccinium floribundum and Aristotelia chilensis. Journal of Agricultural and Food Chemistry 58, 8966–8976. Valdivia CE, Simonetti JA, 2007. Decreased frugivory and seed germination rate do not reduce seedling recruitment rates of Aristotelia chilensis in a fragmented forest. Biodiversity and Conservation 16, 1593–1602. Verdi V, (Thesis Facultad de Ciencias Agrarias) 2004. Evaluación del período de receptividad del estigma en maqui (Aristotelia chilensis (Molina) Stuntz) y murta (Ugni molinae Turcz.). Universidad Austral de Chile, Valdivia, Chile. Vogel H, Razmilic I, San Martín J, Doll U, González B, 2008. Plantas Medicinales Chilenas, Segunda Edición. Editorial Universidad de Talca, Talca, Chile. Vogel H, González B, Razmilic I, 2011a. Boldo (Peumus boldus) cultivated under different light conditions, soil humidity and plantation density. Industrial Crops and Products 34, 1310–1312. Vogel H, Jeldres P, Razmilic I, Doll U, 2011b. Morphological characters, yields and active principles in wild and cultivated accessions of the Chilean medicinal plant Buddleja globosa Hope. Industrial Crops and Products 34, 1322–1326.
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Maqui (Aristotelia chilensis): Morpho-phenological characterization to design high-yielding cultivation techniques b ˜ Hermine Vogel a,∗ , Patricio Penailillo , Ursula Doll c , Geo Contreras a , a Giordano Catenacci , Benita González a a b c
Facultad de Ciencias Agrarias, Universidad de Talca, Casilla 747, Talca, Chile Instituto de Ciencias Biológicas, Universidad de Talca, Casilla 747, Talca, Chile Facultad de Ciencias Forestales, Universidad de Talca, Casilla 747, Talca, Chile
a r t i c l e
i n f o
Article history: Received 4 July 2014 Accepted 23 September 2014 Available online xxx Keywords: Fruit set Floral meristem Phenology Cross pollination
a b s t r a c t Maqui (Aristotelia chilensis) is a species whose berries contain one of the highest levels of antioxidant currently known. At present the rising demand for fruit is being supplied exclusively by wild crafted raw material. Ongoing domestication includes studies of fruit production, in both wild populations and cultivated progenies from different provenances, as a basis for proposals of high-yielding cultivation techniques. Morphological characterization at the end of the first summer indicated significant differences between provenances for plant height (mean values from 99 to 133 cm), petiole length (1.8–2.5 cm), width of the leaf blade (3.6–5.1 cm), ratio leaf length/width (1.9–2.2) and leaf area (16–30 cm2 ). Plant width (69–91 cm), internode length (2.7–3.3 cm) and leaf blade length (7.8–9.9 cm) did not differ among clones of different provenances. Fruit set in wild populations ranged from 54% in a mountainous area to 61% in a coastal population. Some hermaphrodite plants even reached 69%. Covered floral branches set very few fruits, less than half the weight of uncovered branches (38 and 97 mg, respectively), while the pulp–seed relation was similar for both treatments, about 2:1. Histological studies revealed that transition from vegetative to floral meristem occurs in spring during fruit development on the branches that are formed as an elongation to the flowering and fruit bearing shoot. The establishment of phenological stages permits the visualization of the variation in sprouting, blooming, fruit ripening between different clones. The present results reveal a large variability for vegetative and fruit producing characteristic that would permit a successful selection of high yielding plants. At the same time, fundamental knowledge about plant architecture and phenology supports studies of cultivation techniques, such as optimizing density, harvest or pruning. © 2014 Elsevier GmbH. All rights reserved.
Abbreviations: m a.s.l., meters above sea level; DAF, days after bloom; FAA, formaldehyde–acetic acid–alcohol solution. ∗ Corresponding author. Tel.: +56 71 2200214; fax: +56 712200212. ˜ E-mail addresses: [email protected], [email protected] (H. Vogel), [email protected] (P. Penailillo), [email protected] (U. Doll), giordano [email protected] (G. Contreras), [email protected] (G. Catenacci), [email protected] (B. González). http://dx.doi.org/10.1016/j.jarmap.2014.09.001 2214-7861/© 2014 Elsevier GmbH. All rights reserved.
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1. Introduction Aristotelia chilensis (Molina) Stuntz, Elaeocarpaceae, is a Chilean medicinal plant commonly called “maqui” or “quëlón”. It is a sacred species of the indigenous Mapuche people, being a symbol of benevolent and peaceful intent (Mösbach, 1992). Leaves are traditionally used for wound healing and fruits to treat diarrhea and dysentery. The edible berries are consumed fresh or prepared as liquor. Its dark purple color has been used also to color red wine (Gupta, 1995; Hoffmann et al., 1992; Montes and ˜ et al., 1981). The species adapts Wilkomirsky, 1984; Munoz to high radiation during the summer in Central Chile by synthetizing polyphenols and specifically anthocyanins that protect the seeds in the small berries from damage caused by UV light. In the last years extraordinary antioxidant properties of the maqui fruits have been reported, some of the first being Miranda-Rottmann et al. (2002), followed by Araya et al. (2006), Avello et al. (2008) and Olate (2008). These results triggered recent studies and development of food, nutraceutical, cosmetical and pharmaceutical products (Avello et al., 2009; Céspedes et al., 2008, 2009, 2010; Gironés-Vilaplana et al., 2012; Rubilar et al., 2011; Schreckinger et al., 2010), all of them based until now on wild crafted maqui berries with increasing exploitation. Generally wild collection of maqui berries implies cutting the fruit bearing branches. But little is known about the reproductive structures or the moment of floral induction and development of the species. The question is whether cutting the branches for harvest affects the fruit production in the following season, making this practice unsustainable. For future cultivation it is also important to develop pruning techniques to maintain the plants stature while at the same time optimizing fruit production. The present work tries to provide answers to fundamental questions that may contribute to design crop management techniques of A. chilensis, such as pruning or density of male pollinators (that will not produce fruits but are necessary for a high yielding crop). A. chilensis is a small, up to 4 m high, tree with several flexible, thin stems, described by Rodríguez (2005) as evergreen, by Damascos and Prado (2001) as a wintergreen species, as some of the leaves remain for only one
winter. Its geographic distribution is reported in Chile from latitudes 30◦ to 43◦ S from sea level up to 2500 m elevation in the mountainous areas. It has been introduced to the Robinson Crusoe Island located in the Pacific Ocean, where it has become an invasive plant (López et al., 2013; Matthei, 1995). Maqui is known as a dioecious species (Hoffmann et al., 1992; Riveros et al., 1996) producing either male or female flowers grouped in axillar corymbs of 2–4 flowers in spring, between October and November (Rodríguez, 2005). The pale yellow flowers of female individuals have a trilocular ovary and a short style with stamen reduced to staminodes, whereas male flowers show 10–15 stamens with a rudimentary pistil (Verdi, 2004). The black, round berry is small, 4–6 mm, with four to eight angular seeds that ripen in summer, December and January (Rodríguez, 2005). Separation of male and female organs in different individuals implies cross mating and high genetic variability even within wild populations. The black maqui berries attract birds that contribute to the dispersion of the species (Mösbach, 1992; Vogel et al., 2008). Seeds eaten by birds have a higher germination rate than non-eaten ones (Valdivia and Simonetti, 2007). Even though maqui is reported to grow in moist areas, such as brooks, at the feet of hills or edges of woods (Rodríguez, 2005), it is quite resistant to dry periods. 2. Materials and methods The present studies were performed in clones of different provenances between latitudes 34◦ S and 41◦ S (Fig. 1, Table 1). Selected plants from these wild populations were propagated vegetatively by rooting cuttings. The clones obtained were established in spring of 2009 in the Experimental Station of Universidad de Talca (Panguilemo, 35◦ 22 S/71◦ 35 W; 120 m above sea level (m a.s.l.) characterized by a Mediterranean climate with the main precipitation during the winter months, from April to September (Fig. 2)). 2.1. Morphological characterization of vegetative traits in cultivated accessions For morphological characterization individuals cultivated in the Experimental Station Panguilemo coming from
Table 1 Morphological characterization of eight different provenances cultivated in Experimental Station Panguilemo. Provenance Latitude (S/W) Altitude (m) Number of plants Plant characteristics Height (cm) Width (cm) Internode length (cm) Leaf characteristics Petiole length (cm) Leaf blade length (cm) Leaf blade width (cm) Ratio length/width Leaf area (cm2 )
San Fernando ◦
◦
Romeral ◦
◦
San Clemente ◦
◦
Mulchén ◦
◦
Curacautín ◦
◦
Villarrica ◦
◦
Entrelagos ◦
◦
Puerto Montt
34 /70 530 7
34 /70 495 6
35 /71 275 4
37 /72 329 4
38 /71 607 4
39 /71 190 4
40 /72 165 7
41◦ /72◦ 92 7
109 ab 91 3.3
114 ab 81 2.9
99 b 69 3.0
119 ab 75 2.9
127 ab 74 3.1
111 ab 86 2.8
133 a 91 2.7
125 ab 86 3.2
2.1 ab 9.9 5.1 a 1.9 ab 30 a
2.1 ab 9.5 5.1 a 1.9 a 29 ab
1.8 b 8.1 4.2 ab 2.0 ab 21 ab
2.1 ab 9.5 4.9 ab 1.9 ab 28 ab
2.5 a 9.5 4.7 ab 2.0 ab 26 ab
2.1 ab 7.8 3.6 b 2.2 b 16 b
2.3 ab 9.1 4.3 ab 2.1 ab 23 ab
2.0 ab 9.2 4.3 ab 2.1 ab 23 ab
Different letters in the same line denote significant differences, p ≤ 0.05.
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Fig. 1. Geographical locations of provenances of A. chilensis cultivated in the Experimental Station of Universidad de Talca, Panguilemo.
different provenances (Fig. 1, Table 1) were studied at the end of the summer (January) of the first growing season. Internode length was determined on three branches per plant, leaf characteristics were taken as the mean of 25 expanded leaves per plant, and leaf area was estimated by the formula: (Ll − Lw /2)Lw /2 + /2(Lw /2)2 , where Ll is leaf blade length and Lw is leaf blade width. For statistical analysis means were separated by Tukey test (p ≤ 0.05).
plants established in 2009 in the Experimental Station Panguilemo. This involved one male and four females, two of them of the San Fernando provenance, one from Romeral and another from Puerto Montt. The profiles were covered with a black anti-mulch tissue and isolated with Styrofoam to prevent heating. Periodically the glass-wall was uncovered and photographed in order to count the number of white roots appearing.
2.2. Root development
2.3. Fruit set in wild populations
At the end of August of 2013 mini glass-wall profiles (45 cm × 35 cm) were placed in the soil, facing east, of five
In two wild populations of maqui located in Central Chile, one at the coast (Pelluhue 35◦ S/72◦ W, altitude
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monthly rainfall mean monthly minimum temperature
Rainfall [mm]
Temperature [°C]
mean monthly maximum temperature
Fig. 2. Precipitation and mean temperature in the Experimental Station Panguilemo, seasons 2012/13 and 2013/14.
Fig. 3. Vegetative stages of A. chilensis: (A) apical vegetative bud dormant; (B) swollen apical bud with yellowish tips; (C) initiation of sprouting, apical bud bust, green leaves appearing; (D) sprouting, the form of the apical bud is changing; (E) end of sprouting, leaves emerge from the apical bud.
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Fig. 4. Phenological stages of the reproductive cycle of A. chilensis: (A) axillary inflorescence bud dormant; (B) inflorescence buds emerging; (C) pre-anthesis with floral buds clearly separated; (D) anthesis, full blooming; (E) end of blooming, vestiges of flowers in male plants or not pollinated females; (F) beginning of fruit development, green fruits without vestiges of flowers; (G) marking: >50% green fruits, some reddish; (H) ripening: >50% fruits of dark color, no green fruits; (I) end of ripening: all fruits black purple.
200 m, with Mediterranean climate and maritime influence) and the other in the foothills of the Andes (El Picazo 35◦ S/71◦ W, 400 m a.s.l.; with higher radiation and marked seasonal and daily temperature differences), flower production and fruit set were evaluated in 20 female and 20 male individuals of each population during the 2012/13 season. In each plant, three representative branches were marked and the number of flowers and fruit set registered. In the coastal population three hermaphrodite plants were detected. Statistical analysis among populations was performed by Mann–Whitney (p ≤ 0.05) and among male, female and hermaphrodites by Kruskal–Wallis (p ≤ 0.05). 2.4. Cross and self-pollination As A. chilensis is not a perfect dioecious species, we also studied sporadic self-pollination. Four cultivated female
clones from Central Chile (34◦ S/70◦ W) were selected. In each plant six branches with abundant floral buds were marked, three of them covered completely with frost protection fleece before anthesis to prevent cross pollination, the other three uncovered as a control treatment. The number of fruits per plant, as well as fruit weight and number of seeds were registered. Statistical analysis among covered and uncovered branches was performed by Mann–Whitney nonparametric test (p ≤ 0.05). 2.5. Floral induction and development Floral induction and development was studied in three clones of different provenances (Romeral, Mulchén and Puerto Montt, see Fig. 1) all established in the Experimental Station of Universidad de Talca, during the third season. Samples of the young, vegetative long shoots which
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Fig. 5. Fruit set in two wild populations located at the coast (Pelluhue) and in the foothills of the Andes (El Picazo) for male (♂), female (♀) and hermaphrodite (♀♂) individuals during the 2012/13 season (no. of flowers or fruits per flowering shoot system).
contained the axillary buds were taken weekly from the moment of full bloom (>80%), in one of the clones (Romeral) even one week before, from September 27 to January 10. The moment of sampling was established as days after full bloom (DAF) determined in two branches of each plant. The sprouts developed during the previous season were fixed
immediately with FAA and buds removed for histological studies. Histological sections of the buds (10–15 m) were made by a cryostat (LEICA CM 1510S) at −20 ◦ C. The sections were observed in an optical epifluorescence microscope (OPTIKA) at different magnifications (4×, 10×, 40× and 100×). The size of the meristem was measured
90 80
Fruit weight (mg)
70 60 50
Pulp 40
Seed
30 20 10 0
Uncovered
Covered
Fruits per flowering shoot system
250
200
150
100
50
0
Uncovered
Covered
Fig. 6. Fruit development on covered and uncovered branches in cultivated maqui.
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Fig. 7. Floral induction and development in three clones of A. chilensis from full bloom (0 DAF) until floral differentiation (DAF = days after bloom). Provenances A = Romeral, B = Mulchén, C = Puerto Montt. Stage 1: vegetative meristem, stage 2: inflorescence meristem, and stage 3: early differentiated inflorescence. Bar = 100 m.
by means of the software ImageJ (1.47i National Institutes of Health, USA). Meristems of lateral shoots (flowering shoot system) were classified into three stages: stage 1 being a narrow, flat vegetative meristem, stage 2 a domed inflorescence meristem, and stage 3 an early differentiated inflorescence. At the moment of sample collection the length of the young apical sprout was measured. At the same time the number of expanded leaves was registered. To correlate the phenological stages with the climatic
situation, accumulated day degrees above 10 ◦ C were registered from September 1 to March 31. 2.6. Phenological studies Phenological stages were observed in different clones coming from the wild populations San Fernando, Romeral, San Clemente, Mulchén, Curacautín, Villarrica, Entrelagos and Puerto Montt (Fig. 1), all established with two
Fig. 8. Pheno-morphological cycle of A. chilensis.
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San Fernando Romeral
PROVENANCES
San Clemente Mulchén Curacautín Villarrica Entrelagos Puerto Montt 0
100
200
Apical and Floral Sprouting Buds Anthesis Marking
300
400
500
600
Floral Buds Emerging Fruit Development End of Ripening
700
800
DAY DEGREES
Fig. 9. Phenological stages of different provenances in the Experimental Station Panguilemo during spring 2011.
individuals per clone at the Experimental Station of Universidad de Talca, Panguilemo, during August of 2009. The plantation was irrigated from the first week in October to the end of April by a drip irrigation system according to the weekly measured evapotranspiration. The phenophases were evaluated weekly from August 1 to December 28 during the seasons 2012 and 2013 in seven and four female clones, respectively (Fig. 10). One apical vegetative branch and three reproductive branches were studied in two individuals per clone. The same procedures were realized for male individuals. The number of open flowers was recorded in three flowering shoot systems, one in apical position, the others in the middle and lower zone of three randomly selected branches. A phenomorphological diagram (Fig. 8) representing qualitative abstractions of the annual cycle of female plant was constructed by observation of the different phenological phases in female plants of maqui that grew in Panguilemo during the 2012 season. The symbols used represent plant organs according to Montenegro et al. (1989). The different stages in maqui were established by Catenacci (2012). Fig. 3 shows the vegetative and Fig. 4 the generative development of the buds.
3. Results and discussion 3.1. Morphological plant and leaf characteristics in clones of different provenances The study of vegetative characteristics gives an overview of the variability of some morphological traits (Table 1). Plants coming from southern Chile (Entrelagos) are growing taller than those from San Clemente. Plant width, an indicator needed for distance between plants, is highly variable and, during the first year, reached maximum mean values up to 91 cm without differing among provenances.
Leaf area was almost twice as large in plants coming from the most northern site, San Fernando, than those from Villarrica. The characteristics which show significant differences among provenances may indicate their genetic determination as all accessions were cultivated under the same environmental conditions. Mean values for length and width of the leaves were higher than reported in the literature. Gupta (1995) and Rodríguez (2005) described the size of maqui leaves as 3–8 cm long and 1.5–3.5 cm wide, Hoffmann et al. (1992) reported 4–9 cm for leaf length and Damascos and Prado (2001) averages of 7.1 and 7.3 cm for new and old leaves, respectively. These differ from present results with individual values ranging from 6.8 to 11.7 cm for the length and 3.1 to 6.2 cm for the width, which could reflect the effect of management techniques, as our cultivated clones were subjected to cultivation practices such as irrigation, whereas the description in the literature is based on wild plants. The lack of differences between provenances in some of the morphological characteristics indicates a high natural variability even within the same natural population, and was to be expected because of dioecism. Similar results have been reported previously in other wild crafted medicinal plants native to Chile, such as boldo (Peumus boldus) (Vogel et al., 2011a), Buddleja globosa (Vogel et al., 2011b) or bailahuen (Haplopappus sp.) (González et al., 2012).
3.2. Root development Root growth showed a marked reduction at the beginning of flowering and during fruit growth, coincident with the drying of the soil. This indicates a critical moment where restriction of water in the soil may reduce the production of fruits.
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Fig. 10. Phenology of male and female clones of different provenances during 2012 and 2013 in the Experimental Station of Universidad de Talca (Panguilemo).
3.3. Fruit set in wild populations Fruit set in the wild populations studied is illustrated in Fig. 5 reaching 69% in the hermaphrodite plants and 61% in the females, with no significant difference between the two groups. Male individuals showed no fruit set, except one single berry at the coastal population (Pelluhue). On the other hand fruit set at the coastal population was significantly higher (68%) than in the Andes (54%). It is interesting that male plants had significantly more flowers per flowering shoot system (20.6 ± 10.5) than females (12.9 ± 6.7) and hermaphrodites (16.8 ± 3.4), considering both wild populations. These mean values are all much higher than the 2–4 flowers per corymb reported by Rodríguez (2005) indicating a high variability in the number of flowers per flowering shoot system. The same study showed that flowering and fruiting of the Andean population (El Picazo) occurs about one month later than at the coastal population (Pelluhue).
3.4. Cross pollination In dioecious species cross pollination is obligatory for fruit development. Nevertheless in our maqui gene bank,
as well as in some wild populations, plants with some hermaphrodite flowers have been detected, even male individuals with some single fruits can be observed occasionally. That’s why a study of cross and self-pollination was established in four female clones from central Chile. Fig. 6 shows clearly that the main pollination system is out-crossing. Only a few fruits developed on covered branches. This result was to be expected as A. chilensis is commonly known as a dioecious species. Moreover, fruit weight is significantly higher in berries harvested on the uncovered control branches (97 mg composed by 64% pulp and 36% seeds) compared with the covered ones (38 mg; 69% pulp and 31% seeds) which means that fruits obtained by self-pollination even have lower quality.
3.5. Transition from vegetative to floral meristem in cultivated provenances Full bloom (0 DAF) was observed in the Romeral provenance on October 4 and in the clones from Mulchén and Puerto Montt on October 18. At the moment of full bloom all axial buds showed flat meristems indicating a vegetative stage of the bud (Fig. 7). The moment of transition
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differed according to the provenance: the most northern, from Mediterranean climate zones, Romeral and Mulchén, initiated floral development (stage 2) on November 9 (35 and 28 DAF) and differentiation (stage 3) 70 DAF, whereas the most southern clones from Puerto Montt, coming from temperate climate zones, developed stage 2 (pre-floral) one week later and did not reach stage 3 until 70 DAF. The present study indicates that transition of the vegetative to floral meristem occurs in spring, during fruit development. At the moment of fruit harvest, the lateral buds exhibit morphological evidence of reproductive structures. So, if wild collectors cut the branches of A. chilensis to simplify harvest berries of maqui, they put at risk the production of the next year. 3.6. Architecture and phenology Maqui is a wintergreen bush according to Damascos and Prado (2001). Its structure is modular, each module formed by long shoots (Fig. 8). The long shoots (dolichoblasts) are sprouts with an apical vegetative bud and opposite leaves. The axillary buds develop a flowering shoot system that is protected by bud scales during dormancy. These buds will activate in spring, sprouting, stretching and developing flowering shoot systems (in sensu Classen-Bockhoff and ˜ 2013) of limited growth that carry the flowers Bull-Herenu, in corymbs, and die after fruit development and wilting. Concomitant with the bloom, the long shoots of the season are formed from the vegetative buds at the end of the branch developed the year before. Their growth is indeterminate. When the apical bud is frozen, died back or removed, one to four apical buds will continue shooting up. When breaking the monopodial growth, sympodial system is initiated. Fig. 9 illustrates the phenological stages of different provenances in function of the day degrees during spring 2011, whereas Fig. 10 shows the weekly development of male and female individuals cultivated at the Experimental Station of Universidad de Talca, Panguilemo, during the growing seasons 2012 and 2013. The female plants are of different provenances, while the males all coming from the Romeral population. Clones from different provenances may differ in initiating sprouting: in 2011 clones coming from Curacautín only started flowering when all others had finished blooming and were developing fruits. This provenance was the only one that did not ripen until end of December. As Curacautín is from the highest location of the studied populations from the South (607 m a.s.l.) plants from this provenance may be adapted to the colder climate conditions and may require more low temperature accumulation to activate bud development. These findings may be beneficial for selection for late ripening commercial clones that would permit the extension of harvest. Differentiation in the phenological stages between female clones was also observed in 2012 with first signs of activation from mid-August for clones from latitude 34◦ S, whereas the southern provenances (latitudes 40◦ S and 37◦ S) remained dormant until the third week of September (Fig. 10). The flowering of the females was covered from end of September until mid-October by the male clones 19,
23 and 27F1, whereas the late females could be pollinized until end of October by clones 10F1 and 22F1. In 2013 blooming started about one week later but showed the same behavior as in the season before: northern provenances were blooming at the beginning of October, whereas the Entrelagos provenance started the last week of October until the first of November. 4. Conclusions Maqui berry production has, and is, up to now all being provided by wild crafting where the fruit bearing branches are cut to retrieve the fruits. Our studies of floral induction show that the generative buds are developing on these long shoots, and are growing as an elongation to the flowering/fruit bearing shoot system during fruit development. Cutting these branches therefore involves removing the next season’s potential harvest. That’s why in wild maqui plants not more than half of the branches should be cut and, as a long-term solution, a non-destructive harvest system, of only removing fruit, should be developed. The pollination studies confirm our hypothesis of strong cross-fertilization, as self-pollination implies important qualitative and quantitative losses in fruit production. For the different clones, suitable male pollinators are required to assure good cross-fertilization during the different blooming periods considering the existence of early, normal or late flowering clones, respectively. Our studies of morphological traits and phenology indicate the presence of wide variability with possibilities to select the most suitable clones, for instance types with high flowering shoot system production and at the same time inhibited vigor of vegetative growth that would permit extraordinary fruit production in a high density crop. Acknowledgements The present research was financially supported by CONICYT Chile, Project FONDEF D10I1252 in collabora˜ tion with Fundación Chile and the companies AgroQueni, Ana María, Bayas del Sur, Domingo Echegaray, Hortifrut, ˜ for helping in the Surfrut. Thanks to Kester Bull-Herenu interpretation of lateral shoots and bibliographic support, to Pamela González for providing the graphic of the phenolmorphological cycle, and to Luis Letelier for designing the map. We also like to thank Peter Caligari for English revision. References Araya H, Clavijo C, Herrera C, 2006. Capacidad antioxidante de frutas y verduras cultivadas en Chile. Archivos Latinoamericanos de Nutrición 56 (4), 361–364. ˜ Avello M, Valladares R, Ordonez J, 2008. Capacidad antioxidante de Aristotelia chilensis (Molina) Stuntz. Revista Cubana de Plantas Medicinales 13 (4), 1–7. Avello M, Valdivia R, Sanzana R, Mondaca MA, Mennickent S, Aeschlimann V, Bittner M, Becerra J, 2009. Extractos antioxidantes y antimicrobianos de Aristotelia chilensis y Ugni molinae y sus aplicaciones como preservantes en productos cosméticos. Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas 8 (6), 479–486. Catenacci G, (Thesis Facultad de Ciencias Agrarias) 2012. Determinación del crecimiento, fenología, producción y características de bayas en clones de Maqui, Aristotelia chilensis (Molina) Stuntz, según distintas
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G Model JARMAP-16; No. of Pages 11
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procedencias e inclinación de ramillas. Universidad de Talca, Talca, Chile. Céspedes CL, El-Hafidi M, Pavón N, Alarcón J, 2008. Antioxidant and cardioprotective activities of phenolic extracts from fruits of Chilean blackberry Aristotelia chilensis (Elaeocarpaceae), Maqui. Food Chemistry 107, 820–829. Céspedes CL, Alarcón J, Valdez-Morales M, Paredes-López O, 2009. Antioxidant activity of an unusual 3-hydroxyindole derivative isolated from fruits of Aristotelia chilensis (Molina) Stuntz. Zeitschrift für Naturforschung, Section C, Biosciences 64 (9–10), 759–762. Céspedes CL, Alarcón J, Avila J, Nieto A, 2010. Antiinflammatory activity of Aristotelia chilensis (Molina) Stuntz (Elaeocarpaceae). Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas 9 (2), 127–135. ˜ K, 2013. Towards an ontogenetic underClassen-Bockhoff R, Bull-Herenu standing of inflorescence diversity. Annals of Botany 112, 1523–1542. Damascos MA, Prado CH, 2001. Leaf phenology and its associated traits in the wintergreen species Aristotelia chilensis (Molina) Stuntz (Elaeocarpaceae). Revista Chilena de Historia Natural 74, 805–815. Gironés-Vilaplana A, Mena P, García-Viguera C, Moreno DA, 2012. A nobel beverage rich in antioxidant phenolics: Maqui Berry (Aristotelia chilensis) and lemon juice. LWT – Food Science and Technology 47, 279–286. González B, Vogel H, Razmilic I, San Martín J, Doll U, 2012. Biomass, resin and essential oil content and their variability in natural populations of the Chilean crude drug Bailahuén (Haplopappus spp.). BOLETIN Latinoamericano y del Caribe de Plantas Medicinales y Aromaticas 11 (1), 66–73. Gupta MP (Ed.), 1995. 270 Plantas Medicinales Iberoamericanas. CYTEDSECAB, Santafé de Bogotá, Colombia, pp. 261–263. Hoffmann A, Farga C, Lastra J, Veghazi E, 1992. Plantas medicinales de uso común en Chile. Ediciones Fundación Claudio Gay, Santiago, Chile, pp. 130–133. ˜ López P, Baeza CM, Penailillo P, Stuessy TF, Greimler J, Ruiz E, Novoa P, Gatica A, 2013. Introduced plant species in the biosphere reserve Alejandro Selkirk Island, Robinson Crusoe Archipelago, Chile, and their threat to the native and endemic vegetation. Memoirs New York Botanical Garden 108, 211–231. Matthei O, 1995. Manual de las malezas que crecen en Chile. Alfabeta Impresores, Santiago, Chile, pp. 219–220. Miranda-Rottmann S, Aspillaga AA, Pérez DD, Vásquez L, Martínez ALF, Leighton F, 2002. Juice and phenolic fractions of the berry Aristotelia chilensis inhibit LDL oxidation in vitro and protect human endothelial cells against oxidative stress. Journal of Agricultural and Food Chemistry 50, 7542–7547.
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Montenegro G, Avila G, Aljaro ME, Osorio R, Gómez M, 1989. Chile. In: Orshan G (Ed.), Plant Phenomorphological Studies in Mediterraneantype Ecosystems. Kluwer Academic Publishers, The Netherlands, pp. 347–387. Montes M, Wilkomirsky T, 1984. Medicina tradicional chilena. Editorial Universidad de Concepción, Chile. Mösbach EW, 1992. Botánica indígena de Chile. Editorial Andrés Bello, Santiago, Chile. ˜ M, Barrera E, Meza I, 1981. El uso medicinal y alimenticio de planMunoz tas nativas y naturalizadas en Chile. Publicación Ocasional del Museo Nacional de Historia Na 33, 3–91. Olate V, (Thesis Facultad de Ciencias de la Salud) 2008. Estudio químico y actividad antioxidante de los antocianos presentes en los frutos de maqui. Universidad de Talca, Chile. ˜ AM, Arroyo MK, 1996. Sistemas de reproducción en Riveros M, Humana especies del bosque valdiviano (40◦ Latitud Sur). Phyton 58, 167–176. Rodríguez R, 2005. Elaeocarpaceae Juss. ex DC. In: Marticorena C, Rodríguez R (Eds.), Flora de Chile 2 (3), 15–17. Rubilar M, Jara C, Poo Y, Acevedo F, Gutiérrez C, Sineiro J, Shene C, 2011. Extracts of Maqui (Aristotelia chilensis) and Murta (Ugni molinae Turcz.): sources of antioxidant compounds and ␣-Glucosidase/␣Amylase inhibitors. Journal of Agricultural and Food Chemistry 59, 1630–1637. Schreckinger ME, Wang J, Yousef G, Lila MA, González de Mejía E, 2010. Antioxidant capacity and in vitro inhibition of adipogenesis and inflammation by phenolic extracts of Vaccinium floribundum and Aristotelia chilensis. Journal of Agricultural and Food Chemistry 58, 8966–8976. Valdivia CE, Simonetti JA, 2007. Decreased frugivory and seed germination rate do not reduce seedling recruitment rates of Aristotelia chilensis in a fragmented forest. Biodiversity and Conservation 16, 1593–1602. Verdi V, (Thesis Facultad de Ciencias Agrarias) 2004. Evaluación del período de receptividad del estigma en maqui (Aristotelia chilensis (Molina) Stuntz) y murta (Ugni molinae Turcz.). Universidad Austral de Chile, Valdivia, Chile. Vogel H, Razmilic I, San Martín J, Doll U, González B, 2008. Plantas Medicinales Chilenas, Segunda Edición. Editorial Universidad de Talca, Talca, Chile. Vogel H, González B, Razmilic I, 2011a. Boldo (Peumus boldus) cultivated under different light conditions, soil humidity and plantation density. Industrial Crops and Products 34, 1310–1312. Vogel H, Jeldres P, Razmilic I, Doll U, 2011b. Morphological characters, yields and active principles in wild and cultivated accessions of the Chilean medicinal plant Buddleja globosa Hope. Industrial Crops and Products 34, 1322–1326.
Please cite this article in press as: Vogel, H., et al., Maqui (Aristotelia chilensis): Morpho-phenological characterization to design high-yielding cultivation techniques. J. Appl. Res. Med. Aromat. Plants (2014), http://dx.doi.org/10.1016/j.jarmap.2014.09.001