Influence of the methanol extract and fractions of Smilax brasiliensis Sprengel on development in vitro of Nicotiana tabacum and Alllium cepa Influência do extrato metanólico e das frações de Smilax brasiliensis Sprengel no desenvolvimento in vitro de Nicotiana tabacum e Allium cepa Influencia del extracto de metanol y fracciones de Smilax brasiliensis Sprengel en el desarrollo in vitro de Nicotiana tabacum y Alllium cepa

Smilax brasiliensis (Smilacaceae) is a native Brazilian plant found in the Cerrado biome and commonly used in folk medicine. The aim of this study was to evaluate the influence of the methanol extract and fractions from S. brasiliensis leaves on development in vitro of Nicotiana tabacum (tobacco) and Allium cepa (onion) seeds. In vitro germination protocol of onion seeds was established. Tobacco and onion seeds were placed to germinate on basal medium added of extract or fractions of S. brasiliensis leaves and dichlorophenoxyacetic acid (2,4-D), 6benzylaminopurine (BAP) and gibberellic acid (GA) at different concentrations in the presence of light. The germination ranged from 40 to 100% for tobacco and from 60 to 100% for onion seeds. The results indicated that the extract and fractions promoted higher growth than 2,4-D and GA when analyzed number of nodes, leaves, root and root size for germinated tobacco seeds, and higher growth when analyzed number roots and stem size for onion seeds. Regarding BAP, the extract and fractions inhibited leaf growth, root and root size of tobacco seeds. Further studies are needed to evaluate the possible use of the methanol extract and fractions from S. brasiliensis leaves as natural sources of hormones and/or bioherbicides.


Introduction
Allelopathy is defined as a chemical-ecological phenomenon, in which secondary metabolites produced by a plant species are released and interfere with the germination and/or development of other plants in the same environment; this type of interference can be beneficial or harmful (Cheema, Farooq & Wahid, 2013;Soares et al., 2002;Weston & Mathesius, 2013).
Seed germination is a complex process characterized by radicle protrusion through water absorption (Weitbrecht, Muller & Leubner-Metzger, 2011). The germination process can be influenced by physical factors such as water, temperature and light, and by the presence of chemicals that can prevent this process from starting or finishing (Finch-Savage & Leubner-Metzger, 2006;Sirová et al., 2011).
Plants produce signaling molecules known as hormones, responsible for marked effects on development at very small concentrations. Phytohormones or plant hormones are naturally occurring organic compounds, which, at low concentrations, have a deep influence on plant physiology. These are chemical messengers that are produced in small quantities at a specific location and induce responses at other plant locations (Fagan et al., 2015).
Smilax brasiliensis Sprengel (Smilacaceae) is a medicinal species of the Brazilian Cerrado. Previous study showed the allelopathic effect of ethanol extract and fractions from S. brasiliensis leaves on growth of hypocotyls and radicles of Allium cepa and Lactuca sativa (Fonseca et al., 2017). Allelopathic, cytotoxic, genotoxic and antigenotoxic effects were observed in the studies of Amado et al. (2019Amado et al. ( , 2020a. The phytotoxic effects of the hexane and dichloromethane fractions from the leaves of S. brasiliensis was also demonstrated (Barbosa et al., 2021;Fonseca et al., 2019).
This work aimed to evaluate the influence of the extract and fractions S. brasiliensis leaves on development in vitro of Nicotiana tabacum and Allium cepa seeds.

Methodology
This work is an experimental research, with a quantitative approach. According to Pereira et al. (2018), a quantitative survey, translates opinions and information into numbers to classify and analyze them, using statistical techniques. In this type of research, the relationship between the variables must be formulated and classified to ensure the accuracy of the results, thus avoiding contradictions in the analysis and interpretation process.

Preparation of S. brasiliensis leaves extract and fractions
Petroleum ether and methanol were used as solvents to obtain the extracts from 300 g of dried leaves using a Soxhlet extractor. The extracts were concentrated in a rotary evaporator at 40 °C under reduced pressure to yield petroleum ether (EE, 11.121 g) and methanol (ME, 32.829 g) extracts (Amado et al., 2018).

Protocols test for A. cepa seed disinfestation
Allium cepa seeds were tested for different methods of disinfestation: 1. Disinfecting with NaOCl (1.0% commercial sodium hypochlorite) for 5 minutes and after washed three times in autoclaved distilled water; 2. Disinfecting with NaOCl (1.0% commercial sodium hypochlorite) for 3 minutes and then washed three times in autoclaved distilled water; 3. Disinfecting with NaOCl (1.0% commercial sodium hypochlorite) for 1 minutes and then washed three times in autoclaved distilled water; 4. Disinfecting with NaOCl (0.5% commercial sodium hypochlorite) for 5 minutes and then washed three times in autoclaved distilled water; 5. Disinfecting with NaOCl (0.5% commercial sodium hypochlorite) for 3 minutes and then washed three times in autoclaved distilled water; 6. Disinfecting with NaOCl (0.5% commercial sodium hypochlorite) for 1 minutes and then washed three times in autoclaved distilled water; 7. Treatment with Captan 5% for 10 minutes; 8. Disinfecting three times with autoclaved distilled water; 9. Without any disinfestation treatment.
The parameters analyzed at the end of the test were number root size and stem size.
The experimental design was completely randomized, with ten tubes and each tube containing one seed.

In vitro germination
Fruits containing N. tabacum seeds were collected in Santana do Jacaré, Minas Gerais, Brazil (20°50"44.22'S and 45°0"48.87'W) in August 2018. The fruits were dried at room temperature and after drying, were tainted (streaked), separating the seeds, using 30 mesh sieves. After cleaning, the seeds were submitted to germination test.
N. tabacum seeds were disinfected with 70% alcohol for 1 minute and washed once in autoclaved distilled water.
After, the seeds were treated with NaOCl (2% commercial sodium hypochlorite) and a drop of Tween 20 for 30 minutes while stirring. Finally, they were washed six times in autoclaved distilled water.
A. cepa seeds were disinfected with NaOCl (1% commercial sodium hypochlorite) for 1 minute, washed three times in autoclaved distilled water.
N. tabacum and A. cepa seeds were placed on basal medium MS (Murashige & Skoog, 1962) supplemented with 30 g/L sucrose and solidified with 5 g/L. The pH was adjusted to 5.7 ± 0.1 with 0.1 N NaOH, and the medium was sterilized at 120 °C (1.37 × 10 5 Pa) for 20 minutes.
Methanol extract and dichloromethane, ethyl acetate and hydromethanol fractions were tested at concentrations of 250, 500, 750 and 1000 µg/mL. After inoculation, N. tabacum and A. cepa seeds were kept in a growth chamber at 27 ± 1 °C, 16 hours photoperiod and radiation of 45 µmol/m 2 s for 60 and 30 days, respectively. The percentages of germinated seeds, oxidation and contamination of the medium and seeds were observed at 7 days intervals.
The parameters analyzed at the end of the test were number of leaves, knots, shoots, root, stem size and root in centimeters for N. tabacum seedlings and stem and root size for A. cepa seedlings.
The experimental design was completely randomized, with fifteen tubes and each tube containing one seed.

Statistical analysis
The data are presented as the mean ± SE of experiments with ten doses. Statistical differences were determined by analysis of variance (ANOVA) followed by Student's t-test using GraphPad Prism 5.0 software. Values of p < 0.05 were considered statistically significant.

Protocols test for A. cepa seed disinfestation
Results for the disinfestation protocols tested are shown in Table 1. The seeds without treatment or when disinfected with distilled water only presented 100% of contamination. The best result was observed for seeds disinfected with 1% sodium Research, Society and Development, v. 10, n. 8, e26410817199, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i8.17199 5 hypochlorite for 1 minute, being this protocol chosen for disinfection of seeds during in vitro germination test with the extract and fractions, due the better growth and absence of contamination. Results were expressed as mean ± standard error (n = 10). Standard error values were omitted when they are less than 0.099. Source: Authors.

In vitro germination
The germination percentage varied from 40 to 100% for tobacco seeds and from 60 to 100% for onion seeds ( Figure 1 and Figure 2). The percentage of contamination observed shows that onion seeds had greater contamination with percentages ranging from 10 to 60%, while the contamination percentage for tobacco ranged from 10 to 30% (Table 2). There was statistical difference when compared to germination and contamination of the tested samples with the positive control and negative control. The Figure 1 shows the seedlings of N. tabacum after seed germination. The greatest growth can be observed in (G), when they were treated with the HM fraction at concentration of 500 µg/mL. Meanwhile, the smallest growth was observed in (E), when they were treated with the AC fraction at the same concentration.  Research, Society and Development, v. 10, n. 8, e26410817199, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i8.17199 Table 2. Germination and contamination percentage of N. tabacum (tobacco) and A. cepa (onion) seeds.
Results were expressed as mean ± standard error (n = 15). Standard error values were omitted when they are less than 0.099. There was no statistical difference in relation to 2,4-D, GA, BAP and negative control. 2,4 -D: 2,4-dichlorophenoxyacetic acid, BAP: 6-benzylaminopurine, GA: gibberellic acid, NC: negative control, ME: methanol extract, DCM: dichloromethane fraction, AC: ethyl acetate fraction, HM: hydromethanol fraction. Source: Authors. Table 3 shows the number of nodes, shoots, leaves, root, stem size and root size for germinated tobacco seeds. The results indicated that the tested samples of S. brasiliensis that presented statistical difference in relation to 2,4-D and BAP, when evaluated number of nodes, leaves, root and root size, presented higher averages than the tested control.

Samples
Treatments (µg/mL) The results indicated that the growth of seeds was inhibited by gibberellic acid, and the seeds treated with the extract and the fractions also had lower average number of leaves, root and root size (Table 3).

Germination (%) Contamination (%) Germination (%) Contamination (%)
Regarding the negative control, the results observed for the extract and fractions showed lower mean values for number of nodes, shoots, leaves, root and root size (Table 3).
For the onion seed test, the results demonstrated that the methanol extract and the fractions were statistically different from the 2,4-D, GA, BAP and negative control only for stem size (  Table 4. Number of root, stem size and root size of germinated A. cepa seeds. Results were expressed as mean ± standard error (n = 15). Standard error values were omitted when they are less than 0.099. a Statistical difference in relation to 2,4-D (p ˂ 0,05). b Statistical difference in relation to GA (p ˂ 0,05). c Statistical difference in relation to BAP (p ˂ 0,05). d Statistical difference in relation to negative control (p ˂ 0,05). 2,4 -D: 2,4-dichlorophenoxyacetic acid, BAP: 6-benzylaminopurine, GA: gibberellic acid, NC: negative control, ME: methanol extract, DCM: dichloromethane fraction, AC: ethyl acetate fraction, HM: hydromethanol fraction. Source: Authors.

Discussion
For in vitro germination to become a reliable source of aseptic material, disinfection methods must be effective, providing the absence of pathological agents.
The most commonly used germicidal substances in seed disinfestation protocols are ethanol and sodium hypochlorite (Quisen & Angelo, 2008). The most used sodium hypochlorite concentrations in disinfestation protocols range from 0.5% to 2.0% active chlorine, with exposure times ranging from a few seconds to minutes (Torres et al., 2000). The high exposure to hypochlorite, as well as ethanol, ends up causing the opposite effect, promoting tissue oxidation and tissue death, which was not observed in this study, since the exposure time and concentration were not detrimental onion seed germination (Smith, 2000).
The onion is an excellent organism for bioassays because once rehydrated, it goes into germination process, undergoing rapid physiological changes and becomes highly sensitive, expressing any external changes to which it is submitted  (Costa & Menk, 2000;Souza et al., 2005). Tobacco, in turn, offers numerous advantages as a model plant for several studies, due to its short life cycle and large number of seeds per capsule (Brasileiro, 1998).
With the discovery of the effects of plant regulators on plants and the benefits promoted by these substances, many compounds and combinations of these products have been researched.
These natural substances can be applied directly to plants (leaves, fruits, seeds), causing changes in vital and structural processes in order to increase production, improve quality and facilitate harvesting (Castro & Melotto, 1989).
The parameters analyzed in this study indicated that the extract and fractions tested promoted higher growth and development of tobacco and onion seeds when compared to 2,4-D. According to Cordoba (1976), auxins would be involved in the germination process because it indirectly controls the transport of gibberellin from the embryonic axis to endosperm cells.
Auxin is a growth-related hormone because it promotes increased cell stretching rates. They also have the ability to promote the elongation of coleoptile, stem segments, and in the presence of cytokinins promote cell division in callus cultures, formation of adventitious roots in leaves or cut stems (Taiz & Zeiger, 2013).
Cytokinins, on the other hand, can stimulate or inhibit a variety of physiological, metabolic, biochemical processes in the context of development. They are responsible for stimulating the process of cell division or cytokinesis and cell differentiation, especially in the formation of stem buds (Kerbauy, 2008). When comparing the results obtained for BAP and the results obtained for the extract and fractions, it was observed that the best results were expressed for BAP, with the averages of the samples of S. brasiliensis being lower.
The results observed and compared with gibberellin show better results for the extract and fractions. Gibberellic acid accelerates seed germination and thus ensures uniformity in germination. When seeds begin germination (triggered by water absorption), the embryo releases gibberellins, which diffuse into the aleurone cells and then stimulate the synthesis of hydrolytic enzymes, which digest the nutrient reserves of the starch endosperm (Taiz & Zeiger, 1991). Gibberellins play an important role in breaking seed dormancy or latency and are of fundamental importance in breaking down endosperm reserve substances from seeds. These responsible for stem elongation, also acting in the transition from the juvenile to the adult stage of the plant, consequently causing the induction of flowering (Taiz & Zeiger, 2013).
The main compounds identified by Amado et al. (2020b) in the methanol extract and dichloromethane, ethyl acetate and hydromethanol fractions of S. brasiliensis were glycosylated and non-glycosylated flavonoids, especially quercetin, and phenylpropanoids, such as chlorogenic acid. According to Shirley (1998) and Gould and Lister (2006) flavonoids have significant effects in seeds germination. In the plants, flavonoids act as internal physiological messengers and for this function flavonoids are required in small amount (Samanta, Das & Das, 2011). The flavonoids are secondary metabolites present in most plant seeds and play important roles in the protection of the seeds against pathogen and herbivores and act in seeds dormancy and maturation (Shirley, 1998).
No studies on the influence of S. brasiliensis on in vitro seed germination were found.

Conclusion
In order to improve crop yield, it is suggested that bioactive compounds found in S. brasiliensis leaf may in future be used as plant hormones and/or natural herbicides, since the results indicated growth promotion and inhibition of the analyzed parameters in relation to the tested positive controls.
The results of this work suggest that future studies aim to identify which bioactive compounds present in the extract and in the fractions may have the effect of plant hormones.