Vegetative propagation and proposal for sustainable management of Valeriana carnosa Sm., a traditional medicinal plant from Patagonia

Journal of Applied Research on Medicinal and Aromatic Plants 14 (2019) 100218

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Vegetative propagation and proposal for sustainable management of Valeriana carnosa Sm., a traditional medicinal plant from Patagonia

T

Nicolás Nagahamaa,b,c, , María M. Manifestod, Renée H. Fortunatoc,d,e ⁎

a

Estación Experimental Agroforestal Esquel (INTA), Esquel, Chubut, Argentina Facultad de Ciencias Naturales y Ciencias de la Salud, Universidad Nacional de la Patagonia San Juan Bosco, Esquel, Chubut, Argentina c Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina d Instituto de Recursos Biológicos, CIRN- INTA, Hurlingham, Buenos Aires, Argentina e Facultad de Agronomía y Ciencias Agroalimentarias, Universidad de Morón, Buenos Aires, Argentina b

ARTICLE INFO

ABSTRACT

Keywords: Agamic multiplication Indole-3-butyric acid In situ cultivation Sustainability α-Naphthaleneacetic acid

Valeriana carnosa is an important medicinal herb in the Patagonian popular medicine. The underground parts of this native species are used for the preparation of various formulations in the traditional health system for treatment of hepatic, circulatory, urinary and digestive disorders as well as having analgesic, anti-inflammatory and anti-depressive properties. Although V. carnosa is one of the most used medicinal plants in Patagonia, currently the raw material (roots and rhizomes) is obtained from natural populations. The aim of this study was to generate novel tools for stimulating in situ cultivation of V. carnosa to improve the sustainable management of this phytomedicinal resource and the quality control of its commercial plant material. The agamic multiplication of V. carnosa was tested, applying different concentrations of indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA) and a commercial rooting mix product (containing NAA 3000 ppm) in leafless and leafy cuttings. The application of NAA was more effective than IBA to stimulate roots development in stem cuttings. The results indicate that leafy cuttings treated with NAA (1000 ppm and 3000 ppm) give 40–60 propagules from each adult mother plant, ensuring significantly (p < 0.05) higher rooting percentages. To determine the effect of the phytohormones application on the root system development, five morphological root variables were analyzed by non-parametric analysis of variance (Kruskal–Wallis) at P-value (0.05) significance levels and differences were observed depending on the treatment used. In this study we propose: 1- an efficient and inexpensive protocol for vegetative multiplication, and 2- provide information for in situ cultivation of V. carnosa in Patagonia.

1. Introduction The World Health Organization (WHO) has estimated that more than 80% of the world’s population in developing countries depends primarily on herbal medicine for basic healthcare needs (Vines, 2004). In the world, the demand and the commercial trade of a wide variety of those wild species are increasing, and being over-exploited, their introduction into cultivation systems being recommended by different agencies (Lambert et al., 1997; World Health Organization (WHO, 1993). The cultivation of medicinal plants is the only means of meeting ever-increasing demand and supplying the growing industry (Schippmann et al., 2002; Jeelani et al., 2017). Given the importance of valerians as medicinal plant, propagation studies of native species of Valeriana have been developed in several countries (de Andrade Salles et al., 2002; Enciso-Rodríguez, 1997; Mathur and Ahuja, 1991). The roots and rhizomes of many valerians contain various compounds used ⁎

as sedatives and relaxants, to reduce nervousness and agitation associated with stress (Nahrstedt, 1984). The Eurasian species, V. officinalis L., is used worldwide for treating anxiety and mild sleep disorders (Murphy et al., 2010; Patočka and Jakl, 2010). In some countries sources of secondary metabolites have been sought in their native species as alternatives to V. officinalis. Some examples are V. edulis Nutt. ex Torr. & A. Gray subsp. procera (Kunth) F. G. Mey. in Mexico (Oliva et al., 2004), V. glechomifolia F. G. Mey. in Brazil (de Andrade Salles et al., 2002), V. jatamansi Jones in India (Kaur et al., 1999) and V. prionophylla Standl. in Guatemala (Holzmann et al., 2011). Fifty species of Valeriana grow in Argentina and twenty of them in Patagonia region (Borsini et al., 1999; Kutschker, 2011; Nagahama et al., 2016). V. carnosa Sm. (Valerianoideae, Caprifoliaceae) or “Ñanculahuén” is an evergreen shrubby herb of up to 60 cm in height, ramified, with woody rhizomatous stems and thickened main roots. This is one of the most used plants by Patagonian regional ethnic groups and

Corresponding author at: Estación Experimental Agroforestal Esquel (INTA), Esquel, Chubut, Argentina. E-mail address: [email protected] (N. Nagahama).

https://doi.org/10.1016/j.jarmap.2019.100218 Received 17 April 2019; Received in revised form 6 August 2019; Accepted 7 August 2019 Available online 07 September 2019 2214-7861/ © 2019 Elsevier GmbH. All rights reserved.

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the rural creole. It is considered a sacred plant and the breadth of its reputed effects has led to its being known as “the plant that cures the seven illnesses” (Molares and Ladio, 2009; Molares et al., 2018). The underground parts of V. carnosa have been cited as a remedy for hepatic, respiratory, circulatory, urinary and digestive disorders as well as having analgesic, anti-inflammatory, anti-depressive and woundhealing properties (Estomba et al., 2006; Molares and Ladio, 2009). Studies suggest that secondary metabolites in V. carnosa are mostly valepotriates, flavonoids, phenolic acids, essential oils, among others (Guajardo et al., 2018; Bach, 2014; Kutschker et al., 2010). Currently, the V. carnosa raw material (roots and rhizomes) sold in health food stores and by herbalists is collected from wild populations and its gathering and commercialization has increased rapidly over the last decades (Cuassolo, 2009). Ñanculahuén is propagated by rhizomes and seeds and, as it has been described for other Valeriana species, the sexual reproduction is not an attractive multiplication practice, since the seeds remain dormant for a long time, have low viability and germinate slowly (Kaur et al., 1999). On that basis different methodologies of agamic multiplication have been proposed for valerian species with phytomedicinals uses, e.g. in vitro micropropagation protocols in V. officinalis, V. jatamansi and V. glechomifolia (Kaur et al., 1999; Mathur and Ahuja, 1991; de Andrade Salles et al., 2002). In order to optimize and accelerate the vegetative propagation process, auxins or different commercial products (most containing synthetic auxin) have been developed, which are plant growth regulators that are applied to stem and leaf cuttings to form adventitious roots (Blythe et al., 2007). In the present study, we report an efficient methodology for the multiplication of V. carnosa, evaluating basal (leafless) and apical (leafy; with 3–5 leaves) stem cuttings treated with two pure rooting hormones (IBA and NAA) at different concentrations (250, 500 and 1000 ppm), hormone mix NAA:IBA (500:500 ppm) and with a commercial rooting mix product Nafusaku® PR (NAA at 3000 ppm with diluents and adjuvants). The development of a multiplication method is the first step in any domestication effort. Keeping this fact in view this study was carried out for developing the protocol for vegetative propagation of this important medicinal plant species so that methodology can be efficiently disseminated to the farmers for adopting commercial cultivation of V. carnosa on their farmland in Patagonia.

NAA:IBA 500:500 ppm (NAA/IBA) and the commercial rooting product Nafusaku® PR by S. Ando & Cía. S.A. (NAF; α-naphthaleneacetic acid at 3000 ppm, 0.3 g of NAA and 100 g of diluents and adjuvants). The concentrations of rooting hormones were prepared with the addition of inert talc, and the control cuttings were inoculated only with inert talc. 2.3. Propagation conditions The propagation study was conducted at a Field Research Station (EEA-INTA Esquel), during July-October, 2015. The station is located at -43.12 latitude and -71.55 longitude, 531 m a.s.l., about 35 km southwest of the natural population from which the stem cuttings were obtained. Propagation assay was placed in a polyhouse for 60 days (JulyAugust), with two micro-sprinkler irrigations per day (one minute each). Later, all trays were placed outside under nethouse/shade for 90 days (September-November; Fig. 1C), because, in previous tests, prolonged exposures at temperatures higher than 40 °C caused wilting and death of propagules. Under nethouse/shade, propagated plants were watered by micro-sprinkler irrigation for two minutes four times per day. Average temperatures inside the greenhouse and outside under nethouse/shade were recorded (Table 1). 2.4. Survival, rooting rate and root system development The survival and rooting averages were calculated for each replicate and these values were used for statistical analyses. To compare root development between different treatments, the numbers of primary, secondary and tertiary roots (N°R1, N°R2 and N°R3, respectively), length of primary roots (R1-L) and diameter of primary roots (R1-D) were measured. Only the primary roots that had secondary roots were counted and simple adventitious rootlets were not considered as N°R1. For the variables R1-L and R1-D, the larger developed primary roots were measured. A digital caliper (Electronic IP65, 797B Series, Starrett®) was used for the measurements. For the analysis of roots system development, we excluded the treatment IBA250 because the rooting rate was 11.7%, and only seven cuttings rooted with this treatment. To determine differences in rooting parameters among treatments, a work data set including 7 treatments (CONT, NAA/IBA, NAA250, NAA500, NAA1000, IBA500 and IBA1000) and 5 variables (N°R1, N°R2, N°R3, R1-L and R1-D). Not rooted and dead individuals were considered as missing data. Because individuals that did not develop third order roots were assigned values of zero, a transformation of the original variables was performed using the square root algorithm of (n + 1).

2. Material and methods 2.1. Plant material The propagation material was collected from healthy mother V. carnosa plants in June 2015, in the northwest Chubut province, Argentina (−42.89 latitude and −71.31 longitude), at 808 m a.s.l. (Fig. 1A-B). To obtain the cuttings, we made a cut in the base of each mother plant, leaving the tuberous root in the ground. We obtained 40–60 cuttings from each adult plant (0.6–1 m diameter), using sterile pruning scissors and stored these immediately in damp cloth to prevent drying until the treatment with rooting hormone powder and subsequent planting. While preparing the cuttings a diagonal cut at 5 mm below a bud on the proximal end was made.

2.5. Statistical analyses

2.2. Experimental design

3.1. Survival and rooting rate

One thousand and eighty stem cuttings, 6–10 cm long, were selected from 23 mother plants, 540 of which were leafless and 540 leafy (with 3–5 leaves). Cuttings (60 for each treatment) were randomly planted (6 replicates of 10 plants each) under sixteen treatments in black polystyrene trays (size 27 × 32 cm) with 30 compartments (200 ml each well), which were filled with volcanic sand/peat (3:1). Cuttings were inoculated with powder hormones, α-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) at 250, 500 and 1000 ppm; hormone mix

Propagated stem cuttings of V. carnosa were evaluated after 152 days (Fig. 1D-E). All propagules obtained from leafless stem cuttings wilted and died between 30 and 60 days. In these propagules, rapid leaf regrowth was observed between the first 10 and 30 days, but they did not survive. In contrast, propagules obtained from leafy stem cuttings (with 3–5 leaves) showed different survival and rooting rates (Fig. 2; Table 2). The percentages of propagules survival and rooting were different among treatments (p < 0.05; Table 2). Without application of

The Shapiro-Wilks statistic (Shapiro and Wilks, 1965) was used for testing normality, which rejected the hypothesis of normality (p < 0.0001). Therefore, the differences among treatments was tested using multiple comparisons after a Kruskal–Wallis test (Conover, 1999) at p ≤ 0.05 level. All data were analyzed using the Infostat v. 2010 program (Di Rienzo et al., 2010). 3. Results

2

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Fig. 1. Valeriana carnosa. A, site of mother plants population. B, adult plant shows underground organs. C, general view of propagation assay under nethouse/shade after 90 days. D–E, leafy stems propagated using NAA1000. D, root system developed in propagules obtained after 152 days. E, roots detail.

rooting hormones (CONT) propagules survival was 100% and significantly different to NAA250, IBA250 and NAF treatments (70 ± 7.75, 76.67 ± 4.94 and 88.33 ± 4.77, respectively). Rooting rate was significantly (p < 0.05) higher for NAF and NAA1000 treatments (Table 2).

Therefore this multiplication protocol represents a low-cost technique and it could be implemented by small farmers in Patagonia. We obtained a great quantity of V. carnosa plants by macropropagation using phytohormones, thus enabling inexpensive, fast and effective plant production for their introduction to cultivation. In addition, the propagation technique is sustainable because mother plants from which the propagules were obtained regrew a few weeks after the cuts were made and all survived, and thus the multiplication methodology using wild population plants is not extractive. The mother plants from which the plant material (cuttings) were obtained developed many new shoots 30 days after pruning and in the following summer (January – March, 2016), we observed that all the mother plants survived, developed a lot of foliage, flourished and fructified. Sand/peat (3:1) substrate was selected for the present experiments because previous assays showed that other low cost substrata (pine sawdust, heavy forest soil and humus) were not as adequate due lower drainage. When experimens were carried out in pots, we observed that intense irrigation and excessive moisture of the substrate in the summer months caused the progressive rotting of roots, but this did not happen in the cold months (rainy season). It was observed that, for the efficient propagation of V. carnosa, it is necessary to have a good drainage substratum, as well as some active leaves retained on the cuttings. Similar results using leafy cuttings were observed for macropropagation

3.2. Root system development The Kruskal–Wallis test for the rooting parameters evaluated showed significant differences among treatments for N°R1, N°R2, N°R3 and R1-D (p < 0.05; Table 3). The length of primary root (R1-L) showed no significant differences among treatments (p = 0.18). Stem cuttings treated with NAF developed a significantly higher number of primary roots (N°R1; 8.75 ± 0.28), followed by NAA1000 (5.44 ± 0.33). For N°R2, the highest number of roots was observed for the treatments NAF, NAA250, NAA500, NAA1000, IBA500 and IBA1000 (Table 3). The variables N°R3 and R1-D were significantly lower for the NAF treatment (Table 3). 4. Discussion This study was carried out under local environmental conditions (using only a greenhouse) and without using expensive reagents. Table 1 Temperature data of each month and experimental condition. Experimental conditions

Month

Mean temperature (°C)

Absolute minimun temperature (°C)

Absolute maximun temperature (°C)

Greenhouse

July August September October November

7.9 9.6 7.2 9.6 13.7

−5.2 −2.7 −4.5 −4.7 −2.3

33.3 36.7 21.4 25.3 31.4

Nethouse/shade

3

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dry season (low water availability, mainly in surface layers) in Patagonia. The results reveal that the exogenous application of auxins, mainly NAA (NAA1000 and NAF), can stimulate root development in V. carnosa. Due to the wide distribution of V. carnosa and the chemical/genetic variability observed among different populations (Guajardo et al., 2018; Bach et al., 2014; Kutschker et al., 2010), this methodology may be very useful for maintaining clonal orchards of genotypes/chemotypes of interest for further genetic breeding studies. An important consideration for the cultivation of V. carnosa is that there are populations of this species that are geographically isolated or located in particular environments. Some authors have suggested the existence of local adaptations of plant populations at phenotypic and genotypic levels for different species (Becker et al., 2006; Reckinger et al., 2010). Local adaptation in populations from geographically separate locations may also involve a reduction of the performance or fitness of individuals, when subjected to ecologically different environmental conditions (Schmitt and Gamble, 1990). Reciprocal transplantation experiments revealed a clear relationship between plant performance and the distance from their original location (Montalvo and Ellstrand, 2001). The introduction of nonlocal individuals may even cause outbreeding depression, which breaks down coadapted gene complexes when local and nonlocal individuals interbreed (Keller et al., 2000). Adaptations to specific habitat conditions get lost and the fitness of the crossbred offspring decreases (Vander Mijnsbrugge et al., 2010). Considering the allogamous reproduction system of V. carnosa, the propagation protocol herein described can facilitate multiplication of natural populations from local materials, avoiding potential outbreeding depression processes. The use of V. carnosa has high cultural and symbolic value for the Mapuche and Tehuelche ethnic groups and the intensive cultivation (monoculture) of this species is not an accepted practice by the aboriginal communities (because the plants would lose their "healing powers"). An alternative to intensive cultivation is in situ cultivation, as a tool for repopulating the natural populations from where V. carnosa is traditionally harvested. The cultivation system proposed here for V. carnosa consists in the propagation of selected plants and the revegetation of natural populations.

Fig. 2. Propagules rooted under different treatments after 152 days. A–B, NAF; C–D, NAA1000; E, IBA1000; F, NAA500; G, IBA500; H, NAA250; I, IBA250; J, NAA/IBA; K, CONT (not rooted live propagule); L, CONT (rooted propagule). Table 2 Kruskal-Wallis analysis. Survival and rooting percentages of V. carnosa leafy stem cuttings under different treatments after 152 days. Mean values (n = 6 replicates) ± standard error; different letters in the same column differ significantly by the Pair Comparison test. (p < 0.05). Treatment

n

mean percentage survival ± SE

mean percentage rooting ± SE

CONT NAA/IBA NAF NAA250 NAA500 NAA1000 IBA250 IBA500 IBA1000

6 6 6 6 6 6 6 6 6

100 ± 0.00 d 93.33 ± 2.11 bcd 88.33 ± 4.77 abc 70.00 ± 7.75 a 91.67 ± 3.07 abcd 96.67 ± 2.11 cd 76.67 ± 4.94 ab 95 ± 2.24 cd 96.67 ± 2.11 cd

50 ± 48.33 88.33 46.67 45 ± 81.67 11.67 31.67 38.33

8.16 b ± 6.01 ± 4.77 ± 5.58 2.24 b ± 8.33 ± 3.07 ± 3.07 ± 4.77

b c b c a ab b

5. Conclusions

in other medicinal plants (Abidin and Metali, 2015; Kipkemoi et al., 2013). Vegetative propagation by leafy stem cuttings in V. carnosa may solve not only the problems of long-term seed dormancy, low seed viability, poor germination and slow seedling growth, but may also reduce the time taken by the plant to reach a larger root system. Deep and well-developed roots are very important for plants at the time of transplantation to the field, due to the adverse climatic conditions in

Based on the results obtained, the application of NAA (NAA 1000 and NAF) showed to be more effective than IBA to stimulate roots development in V. carnosa, using leafy cuttings. The advantages of in situ cultivation of V. carnosa are the acceptance of the methodology by traditional users and the low maintenance costs. Since V. carnosa has an allogamous reproduction system and to avoid potential outbreeding depression processes, we suggest that the plant material to propagate should be collected from surrounding

Table 3 Root development variables with different hormones treatments for propagation in V. carnosa. Different letters mean significant differences among treatments (P < 0.05). Asterisks indicate the excluded treatment in the analysis. N°R1, number of primary roots; N°R2, number of secondary roots; N°R3, number of tertiary roots; R1-L, square root of (length of primary roots + 1); R1-D, square root of (diameter of primary roots + 1). Treatment

n

N°R1

CONT NAA/IBA NAF NAA250 NAA500 NAA1000 IBA250 IBA500 IBA1000

30 29 53 28 28 46 * 19 23

3.59 3.46 8.75 2.83 4.14 5.44 * 3.25 2.70

N°R2 ± ± ± ± ± ±

0.29 0.36 0.28 0.26 0.34 0.33

ab ab d a b c

± 0.38 ab ± 0.21 a

2.70 3.09 4.39 4.21 4.73 5.12 * 4.48 4.61

N°R3 ± ± ± ± ± ±

0.17 0.33 0.18 0.29 0.29 0.28

a a b b bc c

1.81 1.91 1.25 1.58 1.92 2.01 * 1.77 1.73

± 0.51 bc ± 0.32 bc

4

R1-L ± ± ± ± ± ±

0.09 0.19 0.07 0.15 0.15 0.11

bc bc a b bc c

± 0.15 bc ± 0.18 bc

9.84 9.36 9.76 9.08 9.56 9.63 * 9.11 9.78

R1-D ± ± ± ± ± ±

0.49 0.48 0.12 0.41 0.25 0.26

a a a a a a

± 0.70 a ± 0.41 a

1.57 1.58 1.34 1.44 1.49 1.52 * 1.55 1.48

± ± ± ± ± ±

0.03 0.03 0.01 0.03 0.02 0.02

c c a b bc c

± 0.10 bc ± 0.03 bc

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natural populations in which propagules will be transplanted. Finally, small-scale production of V. carnosa through in situ cultivation can reduce the extent to which wild populations are harvested and is an alternative to supply the regional markets demand.

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