Study Site

During the development of this research study, the f irst stage (germination) was performed in the Microbiology and Molecular Biology Laboratory of the Universidad Técnica Estatal de Quevedo (UTEQ), located at the university campus “Manuel Haz Álvarez” on km 1.5 Quevedo - Santo Domingo. The second stage (emergence and vegetative development) was performed in the Experimental Field “La María”, property of UTEQ on the road to Mocache, km 7.5 to Quevedo-El Empalme, Cantón Quevedo, Province Los Ríos - Ecuador at 72 m.

Genetic Material and Homeopathic Mdicines

Certif ied seeds of tomato (Solanum lycopersicum L.) variety Floradade from the Company Agripac^® in Ecuador were used in this study. For its development, the homeopathic centesimal dynamizations (6CH and 12CH) of the homeopathic medicines Similia^® (CDMX, MX), authorized for humans and with registration in the Health Ministry in Mexico (^{SSA, 2015}) were used. The homeopathic treatments (7CH and 13CH) were prepared in UTEQ by centesimal dilution (1:99) in distilled and deionized water, followed by dynamization with vortex equipment (two min), starting from the respective (6CH and 12CH) officinal medicines in alcoholic dynamizations. The previous process was performed applying the basic homeopathic procedures (^{Mazón-Suástegui et al., 2017}; ^{Ortiz-Cornejo et al., 2017}).

Design and Experimental Development

The treatments were established under a randomized complete experimental block design with factorial arrangement (2A × 4B + 1), considering the dilutions as factor A (7CH and 13CH) and the homeopathic medicines as factor B (Silicea terra (SiT), Natrum muriaticum (NaM), Zincum phosphoricum (ZiP) and Phosphoricum acidum (PhA) with a total of nine treatments, including a control group (distilled water) (T1:SiT-7CH; T2:NaM-7CH; T3:ZiP-7CH; T4:PhA-7CH; T5:SiT-13CH; T6:NaM-13CH; T7:ZiP-13CH; T8:PhA-13CH; T9:Control) and three replicates. For germination, 30 seeds per replicate were placed on filter paper in Petri glass boxes. Previously, the seeds were subjected to an inhibition process in each one of the treatments for 20 minutes. Germinated seed counts were performed daily to assess germination rate and percentage. The experiment lasted for 10 days. The substrate was kept humid during the germination experiment. At the end of the experiment, 10 seedling were taken per replicate (30 for treatments) to assess the morphometric variable.

For the emergence stage, 30 seeds per replicate were placed in flex-foam germinator trays with the following characteristics: alveolus design 4 × 4 cm (50 alveoli per tray); external measurements of the tray 35 × 67 cm; alveolus length 8 cm, disinfected for 24 h with 5% chlorine, subsequently washed with water and filled with substrate mixture. Previous to sowing, the seeds were placed in each one of homeopathic treatments for 20 minutes. Irrigation was continuous to satisfy plant needs, applied in two stages, one in the morning and the second one in the afternoon to maintain humidity. Likewise, the application of the medicines was performed by spraying in the same stages in 15‑30 min lapses. Emerged seedlings were counted daily to assess emergence rate and percentage. The experiment lasted 15 days. At the end, 10 seedlings per replicate (30 for treatments) were taken to assess morphometric variables. Germination rate and percentage were determined according to that proposed by ^{Maguire (1962)}. Finally, for the vegetative development stage, 405 plants (45 per treatment; 15 per replicate) were used. Transplant was performed at 15 days (previous to acclimation) when plants showed an average stem height of 10 cm in plastic pots of approximately 1 kg with a mixture of substrate based on blonde peat with compost in a ratio of 40 and 60%, respectively. To guarantee transplant success, only one plant was placed in each pot, and once transplanted, irrigation with a water pot started, with the frequency described in the emergence stage. The treatments were applied at 15 days after transplant, each 72 h, directly on leaves using 200 mL atomizers. Each homeopathic medicine was used in the dose of 100 mL with 33 agitations for a uniform application on the plants. The experiment lasted 10 weeks until floral blooms appeared. At the end of this period 10 plants were taken per replicate to assess the morphometric variables.

Variables Assessed

At the end of the experiments, the plants were transferred to the laboratory where root, stem and leaves were separated to assess morphology, measuring stem (LT) and root (LR) length, fresh and dry biomass of the aerial part (stem and leaves) (BFPa, BSPa) and root (BFR), (BSR). To determine fresh and dry biomass, an analytical balance was used. For dry biomass determination, in all the stages tissues were placed in paper bags and introduced in a drying stove at a temperature of 70 °C until complete dehydration was obtained (approximately 72 h). Additionally, during the vegetative development fresh and dry leaf biomass (BFH) (BSH), stem diameter (DT), leaf number (nH), branch number (nR), and floral blooms (nB) were assessed (The acronyms of the variables are in Spanish).

Statistical Analysis

Data related to morphometric variables were subjected to an analysis of variance (ANOVA) and Tukey’s test (P ˂ 0.05) to establish statistical differences among the interactions of the treatments and their dynamizations. Normality and homocedasticity tests were previously performed. The statistical software InfoStat Ver. 2017 was used.

Germination Stage

No significant differences were observed among the homeopathic treatments assessed on germination rate and percentage, with the exception of a percentage increase obtained with the treatment ZiP-7CH (58%) above the control group (52%) (Figure 1). In this sense, previous studies performed with seeds have shown the efficiency of other homeopathic medicines on germination. For example, when Arsenicum album 30CH was applied in coffee seeds, germination was boosted and the time required for this purpose decreased (^{Meneses et al., 2004}). Nonetheless, in this study, the main effect on germination was inhibition, above all with the application of NaM-7CH, which could have been associated to the dynamization applied or seed inhibition time. Silveira (2008²) determined that the germination percentage depended on seed inhibition time with the homeopathic treatment, observing that the highest germination percentage (94%) of Crotalaria juncea L. (Madrás hemp) occurred at two hours. Some homeopathic medicines have been reported to have inhibitory effects on germination of different species. For example, ^{Rivas et al. (1996)} assessed Lycopodium 201CH, Sulphur 201CH, and Natrum sulphur 202CH in wheat and tomato seed germination, finding that no increase in seed germination was achieved in both cases. However, they observed an increase in vigor and wheat seedling size (50%) with Sulphur 202CH and Cuprum 202CH, whereas no stimulating or inhibiting effects were found in germination, but an increase in seedling length was observed. In this study similar effects were observed; that is, no significant differences were found on germination rate and percentage, but a positive effect was observed in all the morphometric variables and significantly in seedling dry weight. The seedlings treated with Zincum phosphoricum in 7CH dynamization showed the greatest measurements in total length (5.5 ± 0.98 cm), fresh weight (120 ± 10 mg), and dry weight (5.8 ± 0.7 mg) of the aerial part, whereas those treated with Natrum muriaticum 13 CH showed the greatest values in BFPa (120 ± 10 mg) and BSPa (5.8 ± 0.7 mg). The Silicea terra 7CH treatment also boosted the variables BFPa (120 ± 10 mg) and LR (8.2 ± 2.6 cm) whereas those in the control group (without homeopathic medicine) showed the lowest average value in the variables BFPa (90 ± 10 mg), BSPa (2.7 ± 0.5 mg) and BSR (0.6 ± 0.4 mg) (Table 1). This same effect on the morphological characteristics of cultivations were observed with the homeopathic treatment Arnica montana 6CH that increased root biomass in watermelon (Citrullus lanatus) and melon (Cucumis melo L.) seedlings during germination (Brunetto, 2017³). Additionally, the effects of the homeopathic medicines Arsenicum álbum and Baryta carbonica were assessed on germination, growth and photosynthetic activity of Pisum sativum L., observing that both increased germination rate, particularly in 202CH dynamization. Likewise, positive effects on growth and photosynthetic activity were observed with the highest power, showing an increase in chlorophyll and carotenoids. The authors highlighted that the best results were obtained after diluting the medicine beyond Avogadro’s Number. On the contrary, Casas (2008⁴) mentioned that no significant differences were observed in the effect of Calcarea carbonica, Arsenicum album and Sulphur, all of them applied in a 200CH dynamization on Ferocactus histrix (Cactaceae) seed germination.

Figure 1: Germination rate and percentage of tomato seeds (Solanum lycopersicum L.) treated with four homeopathic medicines in two different dynamizations. 

Emergence Stage

With respect to the effects of the homeopathic treatments on emergence rate and percentage of tomato seedlings, no significant differences were observed. However, with the application of Silicea terra and Zincum phosphoricum in 7CH dynamizations, the greatest values were obtained in both variables (higher than 25%), and a rate close to three seeds per day with respect to the control group (23% and a rate of 2.5 seeds per day) and the other treatments. It is worth to mention that the plants treated with 7CH dynamizations showed greater percentages than those treated with the 13CH dynamization of the same medicine (Figure 2). Some authors have pointed out that the effect of homeopathic medicines, in a great measure, depends on the dynamizations used (^{Meneses, 2017}), which could be related to the stress condition in which the plants treated are found. Nonetheless, further research is needed to assess the specific effects and differences that the dynamizations or power of each medicine could have.

Figure 2: Emergence rate and percentage of tomato seedlings (Solanum lycopersicum L.) treated with four homeopathic medicines in two different dynamizations. 

On the other hand, the treatments showed significant differences with respect to their impact on the morphometric variables assessed (Table 2). The treatment Silicea terra 7CH favored greater seedling growth (6.6 cm) than that of the control group (4.6 cm) whereas the seedlings treated with Phosporicum acidum 13CH showed fresh and dry weight of the aerial part (105 and 12.5 mg) and that of root (18 and 6.1 mg). With respect to silica or raw matter from which the homeopathic medicine Silicea terra is produced, it has been considered one of the main mineral components of rocks, which has been proven necessary for optimum plant development (^{Tichavsky, 2007}). In its homeopathic presentation (SiT), silica is recommended for treating stunted plants with slow or interrupted growth, with delay in production or stressed by mildew-type fungi and similar (^{Meneses, 2017}). These effects of Silicea terra (SiT) have been evident in improving cabbage head production (^{Pulido et al., 2014}).

Vegetative Development Stage

During this stage, the best response of all the homeopathic treatments assessed was observed on tomato plants since the majority of the morphometric variables assessed showed significant differences. Those that received the treatment Phosphoricum acidum 7CH showed the greatest values in stem length (94 cm); stem diameter (5.94 cm) with fresh stem and root (17 and 10.4 g weight, respectively); dry stem and root (1.83 and 1 g weight, respectively); fresh and dry leaf (30 and 2 g weight, respectively); leaf# (131 leaves), bud# (6 flower buds) and branch# (12 branches), compared with the control group, which showed the lowest values in all the response variables studied. It was even notorious to confirm that in the group of plants without homeopathic treatment (control), no floral buds were observed. The application of the treatment Silicea terra 13CH improved DT (6.06 mm) and BST (1.90 g) significantly and in general, a positive effect of the homeopathic treatments could be observed in the rest of the morphometric variables assessed (Tables 3 and 4). With respect to the relationship with the medicine Phosphoricum acidum (PhA), the favorable effects of this treatment on the plant could be explained from the phosphoric acid composition and existing knowledge on its beneficial effects in agriculture. Phosphoric acid is used as fertilizer for plants because it provides phosphorus, which is a very important element for the photosynthetic process and synthesis of organic compounds besides taking part in cellular respiration, storing energy, and forming flowers. Based on the previous information and deriving from the analysis of the results obtained in this research study, the use of this homeopathic medicine can be related to the positive impact on plant vigor, making some of its metabolic processes more efficient. To this respect, Castro (2002⁵), stated that Phosphorus improves the general characteristics in wheat plants and Duarte (2007⁶) found a larger stem diameter in the white eucalyptus Eucalyptus globulus (Myrtaceae), with the application of this medicine. Similarly, ^{Lovatt and Mikkelsen (2006)} mentioned that phosphite (phosphoric acid) was absorbed faster by the plant tissues, which caused a greater growth, leaf development, and fruit size besides favoring the concentration of anthocyanins and promoting a greater photosynthetic activity. In this stage, the medicines Natrum muriaticum (produced from seasalt), Zincum phosphoricum (zinc phosphate), and Silicea terra (silica) also improved the health status of the plants treated, increasing stem diameter and fresh and dry stem biomass (Tables 3 and 4). These homeopathic medicines have already been assessed in other cultivation; for example, Natrum muriaticum 7CH and 13CH improved growth in basil Ocimum basilicum L. plants under salinity stress conditions (^{Mazón-Suástegui et al., 2018}). Similar results were obtained by ^{Pulido et al. (2014)} with Silicea terra 30CH since they observed better development in cabbage plants. Additionally, silica, as a non-essential nutrient, performs a reinforcing role in plant cellular wall, which could provide greater plant resistance to pests and diseases and greater tolerance to drought, improving in general the quality of cultivations treated with SiT. With respect to Zincum phosphoricum, no reports were found in vegetables, except for ^{Alvarado-Mendoza et al. (2017)} who reported that this homeopathic medicine applied in 7CH dynamization inhibited growth significantly of the fungus Fusarium oxysporum f. sp. lycopersici, a very harmful pathogenic agent that attacks tomato plants.

Finally, it is important to note that the dynamization degree (dilution/succussion) and application frequency of the homeopathic medicines have an impact on treatment efficiency. In agriculture, the most common is the use of low power, from 3 to 12 CH, taking into account the limit of Avogrado Number (equivalent to one dilution (1 × 10^-23) where the solvent does not contain any chemical molecule of the original solute in the initial concentrated solution or mother tincture (^{Tichavsky, 2007}). However, in some cases plants and soils may also respond to the treatment with higher dynamizations or power, such as 200CH or higher (^{Solange, 2011}). During the development of this study, the best effects on the majority of the variables were obtained with the 7CH treatments, which agrees with that previously mentioned. With this respect, ^{Mazón-Suástegui et al. (2018)} observed better results in the morphometric variables assessed in basil plants using Natrum muriaticum 7CH as homeopathic treatment. Likewise, an increase in radicle length was observed with 7CH power in basil plants subjected to abiotic stress (^{Ojeda-Silvera et al., 2015}); these authors concluded that the plants increased their radicle length as a defense mechanism facing such salinity stress situation.