Study Site and Genetic Material

Research was performed in the experimental field “La María” of the Universidad Técnica Estatal de Quevedo (UTEQ), Ecuador, with certified turnip (Brassica napus L.) seeds obtained from the Company Agripac^® (Quevedo, Ecuador).

Homeopathic Treatments

Three homeopathic medicines were used, each one in two different centesimal Hahnemannian (6CH and 30CH) dynamizations of the trademark Similia^®, authorized by Health Ministry of Mexico for human use purchased in Farmacia Homeopática Nacional (FHN^®, CDMX, MX). During this research study, the following medicines were assessed: Silicea terra (SiT-6CH and SiT-30CH), Natrum muriaticum (NaM-6CH and NaM-30CH) and Phosphoricum acidum (PhA-6CH and PhA-30CH). The homeopathic treatments applied to turnip seeds were prepared in UTEQ by centesimal dilutions in aqueous base (7CH and 31CH) from each Similia^® medicine in alcoholic base (6CH and 30 CH) purchased in FHN^®. The previous procedure was performed in aseptic conditions by centesimal dilution (1:99) and dynamization (2 min) with vortex equipment, using distilled and deionized water as dilution/agitation vehicle. Thus, the procedures previously described (^{Mazón-Suástegui et al., 2017}; ^2018a, ^b; ^2019a, ^b; ^{Ortiz-Cornejo et al., 2017}) were applied, taking into account that described by Farmacopea Homeopática de los Estados Unidos Mexicanos (^{SSA, 2015}).

Experimental Design

Research was performed under a randomized complete experimental block design with factorial arrangement (2A × 3B + 1), considering the two homeopathic Centesimal Hahnemannian (7^th and 31^th) dynamizations as factor A (7CH and 31CH) and the three homeopathic medicines as factor B (SiT, NaM, PhA) with a total of six homeopathic treatments (3 × 2) and a control treatment (purified water [germination and emergence] and irrigation water with acceptable, CE = 0.22 ds m^-1 quality [plant development]) with three replicates. To sum up, the following homeopathic treatments were applied: T1: SiT-7CH; T2: NaM-7CH; T3: PhA-7CH; T4: SiT-31CH; T5: NaM-31CH; T6: PhA-31CH; and as Control treatment, purified water for germination and emergence, and irrigation water for plant development.

During the germination stage, sterile Petri boxes were used, placing filter paper as substrate base in the interior to preserve humidity, using 60 seeds per treatment and placing 20 seed per replicate. The seeds were submerged in the corresponding treatment for 20 min and subsequently placed in Petri boxes, which started the germination assays that were performed in darkness. The seeds were considered germinated when the radicle showed around 2 mm in length. During the emergence stage, an equal number of seeds were sown in flex-foam germinator boxes with 30% commercial substrate (NOVARBO^®, Finland) and 70% black soil, placing one seed in each one of the cavities at 1 cm in depth. The seeds were considered emerged when the seedling emerged passed through the substrate surface. For the vegetative development stage, plant transfer was performed once they showed an average height of 15 cm. For this purpose, seedlings were removed carefully from the seedbox, placing one seedling per furrow with a distance of 0.25 × 0.40 m. Once transplanted, they received daily irrigation. These treatments were applied in 21 plots of 4 m² with a population of 40 plants per plot, adding up to a total area of 225 m².

Variables Assessed

Germination percentage was determined for six days applying ^{Maguire’s (1962)} proposal.

where: n₁, n₂,…, n₆ = number of germinated seeds at time t₁,t₂,…, t₆ (in days).

At the end of the experiments, the plants were transferred to the laboratory where the root, stem and leaves were separated to assess their morphology. Length of stem (LT, for its abbreviation in Spanish) and root (LR, for its abbreviation in Spanish); fresh and dry biomass of the aerial part (stem and leaves) (BFPa, BSPa, for its abbreviation in Spanish) and root (BFR, BSR, for its abbreviation in Spanish). To determine fresh and dry biomass weight an analytical balance (Mettler Toledo^®, model AG204-USA) was used. To obtain dry biomass, the corresponding fresh biomass was placed in paper bags in separate samples, same which were introduced in a drying stove at a temperature of 70 °C until complete dehydration was obtained, approximately 72 h. To assess plant height during vegetative development, ten plants were taken randomly per each replicate, at 15, 30 and 45 days after sowing, time periods at which the number of leaves per plant were also recorded. Stem diameter was determined starting from 10 cm of the bulb in 10 plants taken at random and at 45 day after sowing. Weight was determined from 10 plants taken from the useful cultivation plot. For the leaf area, samples from the third leaf of each one of the 10 plants were taken from the whole net plot.

Additional to the biometric analyses, an economic analysis was performed based on the productivity observed of the turnip plants that received the homeopathic experimental treatments and the control treatment (only water) without homeopathic medicine. This information made it possible to determine which of the homeopathic treatments had the capacity to offer a greater profitability to the producer. The cultivation yield was assessed starting from the weight of all the plants coming from the useful area an expressing their values in kilogram per hectare. From the information related to the benefit-cost relationship, the benefit of each one of the treatments in terms of percentage was determined: if the resulting value was greater than 1, it was considered acceptable or profitable; if the value was equal to 1, it was considered without financial benefit or loss; if the result was less than 1, it was considered as not profitable, so the project would be rejected.

Statistical Analysis

The corresponding database to all the morphometric variables was subjected first to normality and homoscedasticity tests, and subsequently to an analysis of variance to determine differences among treatments. Tukey’s test was also applied at 95% to establish statistical differences among the interaction (medicine dynamizations: 7CH and 31CH versus medicine type: SiT, NaM, PhA). The statistical software InfoStat ver. 2017 (FCA-UNC, Córdoba, AR) was used.

Initial Stage (Germination and Emergence)

For the germination percentage, significant differences (P < 0.05) were found (Figure 1). The greatest percentage (100%) was recorded with SiT-7CH, PhA-7CH, and NaM-7CH when compared with the 31^st centesimal Hahnemannian (31CH) dilution of these same medicines and the control group (83.5%). On the contrary, no significant differences were found as to the emergence percentage of the plants treated with SiT, PhA, and NaM, in the 7^th or 31^st centesimal Hahnemannian (7CH or 31CH) dynamizations. With this respect, ^{Betti et al. (1997)} showed the beneficial influence of Arsenicum album in wheat seed germination; ^{Meneses and Gonzales (2003)} showed that this same homeopathic medicine empowered coffee seed germination. ^{Panda et al. (2013)} concluded that the homeopathic medicines Arsenicum album and Baryta carbonica stimulated pea (Pisum sativum L.) seed germination, and that these treatments also improved plant growth and its photosynthetic activity. Apparently, the response to homeopathic treatments observed in different species, including turnip, could have been related to seed inhibition in the homeopathic medicines. This result indicated that the means of medicine application was adequate, and the treatments could have favored mobilizing the nutritional reserves in seeds, such as proteins, lipids, or carbohydrates (^{Meneses y González, 2003}). When seed germination starts and cells are sufficiently hydrated, the protein synthesis activation is produced, and thus the formation of hydrolytic enzymes that promote mobilization of the reserve substances (^{Barcelo et al., 1984}). These results corroborated that seed inhibition time is one of the factors that could be determining the favorable response of the medicines; however, further research should be performed taking into account other factors to elucidate the best mode of action of homeopathic medicine.

Figure 1: Percentage of germination of turnip seeds (Brassica napus L.), treated with three homeopathic medicines (SiT, PhA and NaM) in two different dynamizations (7CH and 31 CH). Values with similar letters do not show significant statistical differences for P < 0.05, according to Duncan’s multiple comparison procedure. 

For the morphometric variables, significant differences (P < 0.05) were observed during germination (Table 1). In stem growth, the plants treated with PhA-7CH and NaM-31CH reached the greatest average length (3.40 cm), compared with the rest of the treatments (7CH and 31CH), and the control (2.63 cm) group. As to radicle growth, no significant differences were found between the homeopathic treatments and the control group; however, the greatest values were recorded in the plants treated with SiT‑7CH (5.63 cm). In the emergence stage, no statistical differences were recorded among treatments; however, the greatest average stem length (8.36 cm) corresponded also to the plants provided with SiT-7CH. For plant fresh weight, those provided with the same treatment (SiT‑7CH) showed the greatest average value (0.025 g) when compared with the rest of the treatments and the control group which showed the least average value (0.015 g). With respect to dry weight, no statistical differences were recorded; nonetheless, the plants provided with the 7^th dynamization or power (7CH) of the three medicines (SiT, PhA, and NaM) showed the greatest average (Table 1) compared to the other groups (31CH and control).

Some references are available on the importance of Silicea terra in plant growth. For example, ^{Pulido et al. (2014)} reported better development in cabbage plants treated with SiT-30CH. In UTEQ different research has been performed in vegetables that show a positive effect of SiT by increasing growth in size and weight of cucumber, tomato, and pepper plants (unpublished data). According to ^{Tichavsky (2007)}, Silicea terra has a special importance in agrohomeopathy because of the wide spectrum of beneficial effects that SiT promotes in plants, and rules assimilation processes; influences in the epidermis and in the most diverse vegetal tissues determinant; dominates nutrition in general and helps to control important cell disturbances, for example, demineralization.

With respect to the emergence stage (Table 2), no significant differences were observed as to stem growth; however, it is worth to highlight that the plants treated with NaM-7CH showed greater average (8.64 cm) than the control group (7.33 cm). In the case of radicle length, the treatment NaM-7CH obtained significant differences (P < 0.05) compared to the control group (2.74 cm) and the rest of the treatments. ^{Mazón-Suástegui et al. (2018b)} demonstrated that the application of NaM-7CH and NaM-13CH) in basil promoted an increase in the morphometric variables assessed with the best results for NaM-7CH in the variety Napoletano. Likewise, Batirola (2007⁴) found that NaM-9CH increased primary root length in maize (Zea mays) plants; according to ^{Tichavsky (2007)}, this medicine regulated inhibition of protoplasma and cell nuclei in water, which allowed assimilating, regulating and conserving other salts. According to ^{Marques (2009)}, these positive results reached with NaM with respect to root growth, had to deal with a greater anchoring of the plant and greater nutrient absorption that optimized its growth.

Vegetative Development

During this stage, significant differences (P < 0.05) were obtained (T₃₀ y T₄₅) in the morphometric variables assessed only at 30 and 45 days. Table 3 shows plant height at 15, 30, and 45 days. At the end of the study (45 days), favorable effects were observed with all the homeopathic treatments. Particularly, the plants treated with NaM-7CH reached an average height of 43.2 cm, higher than the control treatment (water) without homeopathy (31.17 cm). No significant differences were recorded with respect to the number of leaves per plant; nonetheless, the highest values were observed with the treatment SiT-7CH with a final average of 17 leaves per plant (Table 3). In the same manner, the highest value for stem diameter was obtained with SiT‑7CH, with a measurement of 8.6 cm, higher than the control group (6.4 cm) without homeopathic medicine (Table 4).

For radicle length variability in the vegetative development stage, no significant differences were found; however, the highest averages were achieved with the application of a high centesimal dilution (NaM-31CH), which recorded an average of 17.82 cm, without differing statistically (P < 0.05) with the other treatments (13.62 to 16.62 cm), including the control group (Table 4). In agreement with our results, ^{Pulido et al. (2017)} reported greater root and dry radicle biomass of broccoli plants cultivated in greenhouse conditions when ultradiluted (30CH) dynamizations were applied beyond the limit established by the Avogadro Theory (1 × 10^-23) of the same medicine (SiT-30CH). Supporting the previous report, ^{Panda et al. (2013)} found that homeopathic medicines in high dynamizations (32 CH and 200 CH) of Arsenicum album and Baryta carbonica improved pea growth and its photosynthetic activity. These results agree with the beneficial effect observed when high dynamizations (31CH) were applied in this study since the turnip plants treated with NaM-31CH and SiT-31CH showed greater root growth, implying a greater capacity for nutrient intake. The serial dilution degree of NaM‑31CH and SiT-31CH was equivalent to the 62^nd decimal (1 × 10^‑62) dilution, which went beyond the Avogadro Theory limit previously mentioned (1 × 10^‑23) that assumed no molecules of the initial concentrate solution or “Mother Tincture” (MT) existed in the treatments 31CH. Nonetheless, several physical-chemical studies have demonstrated the unequivocal presence of MT nanoparticles in high centesimal dilutions, such as 30CH and 200CH (^{Chikramane et al., 2010}). Nevertheless, other response variables should be taken into account to better dilucidate the effect of the homeopathic treatments applied in B. napus L.

With respect to the weight variable, the results indicated significant differences (P < 0.05) in plant fresh and dry weight (PFP and PSP). Better PFP was obtained with SiT-7CH (1020 g) compared with the control (400 g) and with this same treatment, higher PSP value (49.63 g) was also obtained compared with the control group (32.19 g). Significant differences (P < 0.05) were observed with respect to dry root weight but not with fresh root weight. For dry weight all the treatments influenced root growth positively compared to the control group (water) without homeopathy (Table 4). In this respect, Almeida (2002⁵) recorded an increase in basil (Ocimum basilicum L.) fresh weight when SiT-30CH was applied, and ^{Meneses (2009)} concluded that SiT impacted favorable in plant growth. More information on the effect of homeopathic medicines are available in ^{Carneiro et al. (2011)} and ^{Meneses (2009)}.

Additional to the biometric analyses, a basic economical analysis was performed to compare among treatments, finding statistical differences (P < 0.05) among yields (Table 5), with an average of 34 250 kg ha^-1 for SiT-7CH compared with the control group (water) without homeopathic treatment (18 575 kg ha^-1). The greatest benefit/cost (B/C) relationship corresponded to the plants with SiT-7CH, with a B/C of 1.7 and profitability of 71.33%, compared with the other treatments and the control group, whose B/C relationship was 1.2, with a profitability of 23.24%. The biological effect of Silicea terra (SiT-7CH) agrees with the results obtained with respect to economic yields and profitability of turnip cultivation. CV = coefficient of variation (%). Different letters denote significant differences (P < 0.05) using Duncan’s statistical test.