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INTRODUCTION
Poiretia latifolia Vogel (Fabaceae), popularly known as "erva-de-touro", is traditionally used in the treatment of stomach and urinary problems, for flavoring traditional yerba mate and as an aphrodisiac (Müller, 1984; Amorim and Boff, 2009). Nanoemulsion, based on its essential oil, has potential as an anti-dermatophyte and anti-inflammatory agent (Porto et al., 2010; Pérez-Zamora et al., 2016; Paim et al., 2018). In addition, Poiretia latifolia could be an alternative income for farmers since its essential oil is rich in components that are important to the flavour and fragrance industry, such as carvone, dihydrocarvone, and limonene (Porto et al., 2010; Fernandes and Boff, 2017).
Poiretia latifolia naturally occurs on the South Plateau of Santa Catarina, Southern Brazil in the Ombrofila Mista Forest (Fernandes and Boff, 2017). The plant diversity of this region has the potential for numerous uses, for example, grazing, food, medicinal and cosmetic uses. However, this potential has been threatened by anthropogenic activities, such as cattle, fruit orchards, intensive cultivation of soybean and corn, and reforestation with Pinus sp. and Eucalyptus sp. (Pereira et al., 2006; Zank and Hanazaki, 2012). In order to strengthen plant diversity and enhance its potential, the most appropriate propagation technique for plants such as Poiretia latifolia is essential (Nogueira and Nogueira-Filho, 2011; Sarasan et al., 2011; Vodouhè et al., 2011).
Poiretia latifolia seeds have a germination rate above 80%; however, its production is severely damaged by insects, which destroy 54% of its flowers and 88% of its seeds (Maass et al., 2018). Vegetative propagation could overcome this problem and even facilitate the selection of the best plant characteristics (Rasmussen et al., 2015; Kaviani and Nagahdar, 2017; Wetzstein et al., 2018). However, vegetative propagation knowledge for P. latifolia is limited and unsuccessful (Teixeira et al. 2011; Amorim et al., 2012). The success of this technique relies on adventitious rooting, which is influenced by several factors, such as plant phenological stage, cutting type and levels of inhibitors and/or promoters of plant growth in the propagule (Guo et al., 2009; Zerche and Druege, 2009; Ludwig-Müller, 2011; Pacurar et al., 2014; Pigatto et al., 2018).
Exogenous plant growth regulators are widely used in vegetative propagation and can be synthetic or natural. However, when working with medicinal plants, natural alternatives are preferable. Yamashita et al. (2017) found that Cyperus rotundus extract (natural) and indol-3-butiric acid - IBA (synthetic) improved the rooting of Myrciaria cauliflora and Psidium guajava cuttings. High dilutions are another alternative that aid production, minimizing residues and reducing costs (Moreno, 2017). The homeopathic preparations Phosphorus 3CH and Kali muriaticum 12CH, selected based on the nutrients commonly used in plant fertilization, promoted increased root growth in eucalyptus seedlings. (Moraes et al., 2018). Cassol et al. (2017) also reported that the use of IBA and Arnica montana 12 CH stimulated the rooting of Cuphea gracilis.
The objective of this study was to evaluate the influence of phenological stages, segments of the branch and plant growth regulators on the vegetative propagation of P. latifolia using stem cutting.
MATERIALS AND METHODS
Collection and conduction locations
This study was conducted at the Lages Experimental Station of the EPAGRI (Agricultural Research and Rural Extension Agency of Santa Catarina State, Brazil). The vegetative material used in the experiments was collected from a natural population of Poiretia latifolia in the region of Vacas Gordas, district of Capão Alto, Santa Catarina (27°58'33.1" S and 50°30'26.5" W). Immediately after the sampling, the herbaceous shoots were immersed in water and transported in polystyrene boxes to avoid dehydration of the vegetal material.
Experiment design
In the first experiment, seven treatments with plant growth regulators were evaluated: immersion of the base of the cuttings for 20 s in four concentrations of indole-3-butyric acid (IBA) (1,000, 2,000, 3,000, and 4,000 mg L-1); aqueous extract of Cyperus rotundus, 10% concentration; high dilution of Calcarea phosphorica at 20CH (CH = hahnemannian centesimal dilution order); and pure water (control). Each treatment had three replicates of 20 cuttings in a completely randomized block design. The experiment was carried out in both phenological stages of P. latifolia: vegetative (14 December, 2016) and reproductive (18 January, 2017). In total, 840 cuttings were used in the experiment.
In the second experiment, the treatments consisted of the cutting type in terms of the segment of the P. latifolia branch, divided into the basal, median, and apical thirds. Each treatment had four replicates of 20 cuttings in a randomized block design, totaling 240 cuttings in the experiment. The cuttings were collected at the reproductive stage (22 February, 2017).
Obtaining and applying plant growth regulators
The IBA was dissolved in 1N NaOH and adjusted to the concentrations with distilled water. The aqueous extract of Cyperus rotundus was obtained by blending 100 g of fresh plant bulbs in 1,000 mL of pure water in a blender for 10 min. The high dilution of Calcarea phosphorica was obtained from a pharmacy, and the last two levels of dynamization were made with pure water, following the methodology described in the Brazilian Homeopathic Pharmacopoeia (2011). The plant growth regulators were applied with immersion for 20 s with 1/3 of the base of the cuttings in the solution. The high dilution of Calcarea phosphorica at 20CH was applied to the aerial part of the cuttings with spraying twice a week at a volume of 50 mL per replicate until the end of the experiment. During this period, the other treatments received pure water.
Cutting preparation and planting
The herbaceous P. latifolia shoots were segmented into cuttings with two lateral buds. In the first experiment, all branch segments were used and placed randomly inside the blocks, producing cuttings with varied lengths and diameters. In the second experiment, the diameter and length of the cuttings varied according to the characteristics of the segment evaluated: basal, median, or apical.
The preparation of the cuttings for all experiments and treatments followed the protocol described by Bettoni et al. (2014b). At the base of the cuttings, a transverse cut was made, with 20% of the total length of the cutting remaining below the lateral bud. At the top end of the cutting, an angle cut was made, with 30% of the total length of the cutting remaining above the lateral bud. Afterwards, at the base of the cuttings, two longitudinal lesions were made, with approximately 30% of the total length of the cuttings. A leaflet was maintained on each cutting.
After preparation, the cuttings were transplanted to Styrofoam honeycomb trays with 60 cells with sand and vermiculite substrate (1:1, v/v). The trays were arranged in plastic boxes (60 x 40 x 20 cm) and covered with a transparent plastic film to maintain moisture saturation. They were watered with spraying twice per day and kept in a greenhouse at 25°C. No control for pests and diseases was carried out.
Statistical analysis and assessment
Survival rate, rooting rate, shoot sprouting rate, number of roots and shoots, and length of the four larger roots and the two larger shoots were assessed 35 d after planting the cuttings. In order to analyze survival, rooting, and sprouting, generalized linear models were used with the binomial model or the binomial model with a dispersion parameter (quasibinomial), both considering the logit link function. The means were compared with confidence intervals (95%) for Tukey contrasts of the linear predictors of the fitted model. All analyses were performed using the R environment v 3.2.1 (The R Foundation, 2015).
RESULTS AND DISCUSSION
The joint analysis of the experiment with plant growth regulators in the vegetative and reproductive phases of P. latifolia plants showed the simple effect of the treatments (Tab. 1), meaning that the plant growth regulators performed similarly in the two phenological stages: vegetative and reproductive.
Table 1 Descriptive levels (p-values) of the joint analysis of propagation with Poiretia latifolia cuttings and plant growth regulators in the vegetative and reproductive stages, Lages, SC, 2018.
| Factors | SURV. | R | R/C | LR | S | S/C | LS |
|---|---|---|---|---|---|---|---|
| PE | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | 0.0430 | 0.3806 |
| PE/Block | 0.0371 | 0.0230 | 0.1738 | 0.0085 | 0.0352 | 0.7835 | 0.1011 |
| GR | 0.0417 | 0.0293 | <0.0001 | <0.0001 | 0.0369 | 0.0438 | 0.0069 |
| GR*PE | 0.0724 | 0.0699 | 0.6407 | 0.5178 | 0.1897 | 0.9826 | 0.2279 |
PE = phenological stage, GR = growth regulators, SURV. = survival rate of cuttings, R = rooting rate, R/C = roots per cutting, LR = mean length of the four largest roots, S = sprouting rate, S/C = shoots per cuttings, LS = mean length of the two largest shoots.
The phenological stage of the P. latifolia plants influenced the cutting viability through the rate of surviving, rooting, and sprouted cuttings; the vegetative stage was the lowest (Tab. 2). The plant growth regulators did not increase the cuttings viability. Cuttings from the medial and basal branch sections performed better than those from the apical section. The sprouting rate was still better for the basal section than for the median section (Tab. 2).
Table 2 Viability of Poiretia latifolia cuttings collected in different phenological stages, treated with plant growth regulators and from different sections of the branch. Lages, SC, 2018.
| Treatments | Survival (%) | Rooting (%) | Sprouting (%) |
|---|---|---|---|
| Phenological stages of plant collection1 | |||
| Vegetative | 53.81±3.16 b | 55.71±3.01 b | 49.52±3.25 b |
| Reproductive | 81.90±2.22 a | 82.62±2.09 a | 70.00±2.50 a |
| Plant growth regulators1 | |||
| Control | 68.33±5.43 ab | 68.33±5.11 ab | 60.83±3.52 ab |
| 1,000 mg L-1 IBA | 57.50±9.20 b | 58.33±8.63 b | 47.67±8.82 b |
| 2,000 mg L-1 IBA | 66.67±8.72 ab | 70.00±8.66 ab | 63.33±8.23 ab |
| 3,000 mg L-1 IBA | 64.17±7.46 ab | 67.50±6.42 ab | 60.83±8.41 ab |
| 4,000 mg L-1 IBA | 72.50±6.68 ab | 74.17±7.00 ab | 60.00±4.83 ab |
| C. rotundus | 66.67±10.46 ab | 66.67±9.80 b | 64.17±10.36 ab |
| Calc. phos. 20CH | 79.17±5.54 a | 79.17±5.39 a | 72.50±4.23 a |
| Sections of the branch2 | |||
| Apical | 2.50±1.44 b | 2.50±1.44 b | 5.00±0.00 c |
| Median | 16.25±1.25 a | 32.50±2.50 a | 17.50±1.44 a |
| Basal | 12.50±2.50 a | 46.25±2.39 a | 12.50±2.50 b |
Control = distilled water, C. rotundus = aqueous extract of Cyperus rotundus, Calc. phos. 20CH = High dilution of Calcarea phosphorica at 20CH (CH = Hahnemannian centesimal dilution order). Different letters in the columns present a statistical difference between treatments, Tukey's contrast (P≤0.05). Means ± standard error; 1 Trials analyzed together; 2 Independent trial.
The vegetative stage, contrary to that found for viability (Tab. 2), increased the number of roots, average length of the four larger roots, number of shoots, and average length of the two bigger shoots (Tab. 3).
Table 3 Quality of viable Poiretia latifolia cuttings collected in different phenological stages, treated with plant growth regulators and from different sections of the branch. Lages, SC, 2018.
| Treatments | R/C (n°) | LR (cm) | S/C (n°) | LS (cm) |
|---|---|---|---|---|
| Phenological stages of plant collection1 | ||||
| Vegetative | 21.46±0.82 a | 18.39±0.65 a | 1.35±0.06 a | 8.18±0.46 a |
| Reproductive | 16.77±0.63 b | 15.67±0.49 b | 1.19±0.04 b | 7.78±0.32 b |
| Plant growth regulators1 | ||||
| Control | 16.97±1.35 bc | 15.35±1.22 b | 1.25±0.10 ab | 7.59±0.43 b |
| 1,000 mg L-1 IBA | 19.58±1.24 abc | 17.46±1.14 ab | 1.27±0.11 ab | 8.04±0.71 ab |
| 2,000 mg L-1 IBA | 20.90±1.26 ab | 17.57±0.41 b | 1.31±0.08 ab | 8.27±0.79 ab |
| 3,000 mg L-1 IBA | 22.47±1.54 a | 17.66±0.94 ab | 1.07±0.09 b | 6.81±0.67 b |
| 4,000 mg L-1 IBA | 21.63±1.42 a | 20.46±1.07 a | 1.10±0.06 ab | 6.79±0.52 b |
| C. rotundus | 15.83±1.81 c | 14.16±1.37 b | 1.40±0.07 ab | 8.13±0.68 b |
| Calc. phos. 20CH | 16.42±1.14 c | 16.58±0.61 b | 1.52±0.11 a | 10.26±0.67 a |
| Sections of the branch2 | ||||
| Apical | 4.00±2.00 a | 11.90±9.10 a | 1.00±0.00 a | 3.00±2.00 a |
| Median | 7.58±0.64 a | 13.12±1.76 a | 1.17±0.10 a | 5.04±0.83 a |
| Basal | 7.37±1.40 a | 9.63±1.07 a | 1.12±0.12 a | 3.12±1.16 a |
Control = distilled water, C. rotundus = aqueous extract of Cyperus rotundus, Calc. phos. 20CH = high dilution of Calcarea phosphorica at 20CH (CH = Hahnemannian centesimal dilution order), R/C = roots per cutting, LR = mean length of the four larger roots, S/C = shoots per cuttings, LS = mean length of the two larger shoots. Different letters in the columns present a statistical difference between treatments, Tukey's contrast (P≤0.05). Means ± standard error; 1 Trials analyzed together; 2 Independent trial.
The IBA concentrations 3,000 and 4,000 mg L-1 had higher root numbers than the treatments without synthetic auxin applications. IBA at 4,000 mg L-1 was superior to 2,000 mg L-1 but did not differ from 3,000 mg L-1 for the length of the four larger roots (Tab. 3). Increases in the IBA concentration increased the number of roots per cutting and the average length of the four larger roots, which had an inverse relationship with the shoot number and the length of the two larger shoots (Tab. 3).
The growth regulators did not influence the number of shoots per cutting. However, Calcarea phosphorica 20CH increased the length of the two larger shoots of the cuttings but did not differ from the IBA concentrations 1,000 and 2,000 mg L-1 (Tab. 3). The different branch sections did not differ in the quality of the viable P. latifolia cuttings (Tab. 3).
During the plant collection, the branches were more lignified in the reproductive stage than in the vegetative stage. Cuttings from older plants that are more lignified, besides having more nutrient reserves because of the tissue consistency, are more resilient to environmental stresses, such as temperature and humidity (Villa et al., 2017). This may explain the better cutting viability for the reproductive stage and the medial and basal branch segments of P. latifolia plants (Tab. 2).
However, the vegetative stage had better cutting quality than the reproductive stage, the opposite of that found for cutting viability. According to Maia et al. (2008), more lignified cuttings may impede rooting, observing better rooting in less lignified cuttings of the apical segment of Hyptis suaveolens branches. Even though lower nutritional reserves and less resistance to stresses impede cutting viability, cuttings from the vegetative stage may have a better hormonal balance, improving rooting quality in surviving cuttings (Ludwig-Müller, 2011; Pacurar et al., 2014).
The IBA effects on root quality in this study were also observed by Bettoni et al. (2014a) and Amorim et al. (2012), who observed that, although IBA did not change the cutting viability of grapevine rootstocks and Poiretia latifolia cuttings, it improved the quality of the surviving cuttings. Amorim et al. (2012) suggested that studies on higher IBA concentrations and other rooting inducers. However, the application of IBA or increases its doses does not necessarily promote the quality of cuttings. Benzylaminopurine (BAP) concentrations from 0.2 to 3.0 mg L-1 did not enhance Poiretia latifolia vegetative propagation, and although there was not a statistical difference, the treatment with 2,000 mg L-1 of AIB promoted a better rooting rate than the treatment with 4,000 mg L-1 (Teixeira et al., 2011).
The high dilution of Calcarea phosphorica at 20CH promoted shoot growth and did not interfere with root quality (Tab. 3). Calcarea phosphorica, when applied at a young age, strengthens plants, improving nutrient absorption and resilience when suffering a hydric stress (Moreno, 2017). However, choosing a high dilution that provides the desired effect is somewhat complex and depends on several factors, such as the preparation method and the potency, application, concentration and nature of the preparation (Hanif and Dawar, 2015; Giesel et al., 2017). Giesel et al. (2017) reported that both the potency and the dynamization method influence the effect of a high dilution on the foraging of leaf-cutting ants (Acromyrmex laticeps Emery). According to Broetto et al. (2011), the quality of seedlings is associated with the development of both the aerial part and roots. Thus, more studies are needed to achieve results similar to that of the study by Pulido et al. (2014), who was able to stimulate both aerial growth and cabbage roots. The homeopathic preparations Phosphorus 3CH and Kali muriaticum 12CH, selected based on the nutrients commonly used in plant fertilization, promoted increased root growth in eucalyptus seedlings (Moraes et al., 2018). Cassol et al. (2017) also reported that the use of IBA and Arnica montana 12 CH, usually used to help an organism cope with traumatic events such the cutting process, stimulated the rooting of Cuphea gracilis.
The results obtained from the P. latifolia cuttings treated with C. rotundus extracts were similar to those of Yamashita et al. (2017), who tested five concentrations of the extract (0, 25, 50, 75, and 100%) and did not observe a difference in the number of roots in Myrciaria cauliflora and Psidium guajava. The authors suggested that the auxin concentration in the aqueous extract of C. rotundus is not sufficient to increase the number of roots. Dias et al. (2012) also warned that, depending on the cutting immersion time, the extract may have toxicity effects.
Even though there was no increase in the rooting rate with the plant growth regulator treatments, the application of 4,000 mg L-1 of IBA provided better development of the root system in the P. latifolia cuttings. The high dilution of Calcarea phosphorica at 20CH provided better development of the aerial part of the P. latifolia cuttings. The methodology for the cutting collection and preparation, as well as the conduction period of the rooting, provided a higher rooting rate (79.17%) than in previous studies (around 10 and 40%) (Amorim et al., 2012; Teixeira et al., 2011).
CONCLUSION
Calcarea phosphorica achieved a rooting rate of 79.17%. The high dilution improved the aerial part of the P. latifolia cuttings, while the AIB enhanced the rooting quality. More studies on these two plant growth regulators, both individually and combined, should be carried out to improve the vegetative propagation of this species. More lignified Poiretia latifolia cuttings are more viable because of the phenological stage and the position of the branch segment. The vegetative propagation used in this study is a feasible option for the domestication, preservation, and cultivation of Poiretia latifolia.
