Causes of dormancy in Ilex paraguariensis

Ilex parguariensis pyrenes, popularly known as “yerba mate”, were classified as dormant. This study aim to investigated the causes of dormancy in Ilex paraguariensis ’ pyrenes. Hence, the following tests were performed: a) Physical dormancy: rate of absorption in water and methylene blue; detection of lignin and lipophilic compounds in the endocarp and integument; b) Physiological dormancy: bioassays in lettuce seeds, detection, and quantification of phenolic compounds; c) Morphological dormancy: embryo analysis. For the absorption rate, an increase was observed in the mass of the pyrenes, however, when imbibition was performed in methylene blue, absorption only occurred in the endocarp. Lignin was also observed in the form of a sclerenchyma layer next to the seed coat. Similarly, lipophilic compounds were observed in a layer, external to the endosperm. The bioassays with lettuce seeds indicated the presence of chemical inhibitors. In the morphological evaluation of the pyrenes, only 55.5% of the embryos were visualized and they were in the globular or heart stages. Ilex paraguariensis pyrenes have combined dormancy: physical (not water absorption), morphological (due to the underdeveloped embryo), and there are shreds of evidence about physiological dormancy (presence of inhibitors); however, it is recommended to investigate the inhibitory agent.


Introduction
The yerba mate (Ilex paraguariensis A. St. -Hil.) is a tree species from Brazil, Argentina, Uruguay, and Paraguay, belonging to the Aquifoliaceae family (Carvalho 2003).The leaves are dried to prepare a traditional tea (mate tea), which is consumed by millions of people in South America (Oliveira & Waquil 2014;Bergottini et al. 2015).In recent years, the use and export of yerba mate by new sectors, such as the pharmaceutical and cosmetics industry, have been intensified; it means that many studies have been carried out on this species, due to its economic importance (Horbach et al. 2011;Santin et al. 2013).Although it has been used for several decades, some silvicultural problems persist, including low and distributed germination over time (Medeiros et al. 1999;Fowler & Sturion 2000).
The dormancy is an adaptation that prevents seed germination even when environmental conditions are favorable (Baskin & Baskin 2004;Finch-Savage & Leubner-Metzger 2006).Although dormancy is considered a survival mechanism of the species (Bewley et al. 2013), for plant production, it can be a problem -since it is challenging to obtain seedlings.
In the literature, the I. paraguariensis pyrenes, were classified as dormant; however, there is still no consensus on the causes of this dormancy.Grigoletti Júnior et al. (1999) affirm that the pyrenes have physical dormancy, which is overcome by saprophytic fungi's action, under natural conditions.Physical dormancy is defined as that in which the integument and / or the endocarp are impermeable (Baskin & Baskin 2004).According to Dolce et al. (2010), the integument and pyrenes of the I. paraguariensis are resistance to the expansion of the tissues (mechanical dormancy), making the germination difficult.
Other authors describe the dormancy of I. paraguariensis pyrenes as a morphological dormancy (Niklas 1987;Sansberro et al. 1998;Menegueti et al. 2004).This classification is due to their embryo immaturity, which requires an additional period to complete their development and germinate.The mature pyrenes are detached from the parent plant before the embryos are morphologically mature, with the maturation of embryos ending after dispersion (Malavasi 1988).
Combined dormancy was also reported for Ilex genus by Baskin & Baskin (2014), and specifically for I. paraguariensis, as morphological and physiological (morphophysiological) dormancy (Cuquel et al. 1994;Galíndez et al. 2018).Physiological dormancy occurs when there are inhibitory substances or absence of substances that promote germination, preventing it from occurring (Baskin & Baskin 2004).Thus, the germination only happens after the inhibitory substances have been removed, for example, through the digestive tract of animals (Zaidan & Barbedo 2004).It was also reported morphological and physical dormancy in these pyrenes (Medeiros 1998).
The definition of the cause(s) of dormancy is fundamental for the efficient methods to overcome it.The objective of this study was to investigate the cause(s) of dormancy in Ilex paraguariensis pyrenes.

Material and Methods
Mature fruits with a dark purple coloration (color 2,5/1 F: 5Y of the Munsell Color Chart) of Ilex paraguariensis were harvested in 2016, under screens installed below the matrices.The harvest was carried out in the altitude of 906 m, where the climate is warm and temperate, with a Cfb classification -according to Köppen and Geiger -and 16.7 °C average temperature.The average annual rainfall is 2045 mm (Climate 2016).
After the harvest, the pyrenes were extracted, using a strainer and running water, benefited by using a blower to remove impurities; later, they were used in the analyses.Initially, the water content of the pyrenes was determined by the greenhouse method at 105 ± 3 °C for 24 h, according to ISTA (2007).
To identify the causes of dormancy, the following tests were performed:

Physical dormancy
Absorption in water and methylene blue, lignin and lipophilic compounds detection The water absorption was performed in four replicates of 100 pyrenes, which had their mass measured and submitted to imbibition in a transparent plastic germination boxes (11 × 11 × 3,5 cm) with three layers of paper towel, moistened with water 2.5 times the mass of the paper and at a temperature of 25 °C.The pyrenes' mass was measured every hour in the first 24 hours, every twelve hours on the second day, every twenty-four hours from the third to the fifth day.From the sixth day on, the measurement occurred every 48 hours.The absorption was calculated by the difference between the initial mass and the obtained mass.
The absorption also was evaluated in methylene blue solution 1% (Orozco-Segovia et al. 2007), in full pyrenes and scarified pyrenes with sandpaper, until the visualization of the endosperm.The pyrenes were immersed in a solution during 48 hours; later, they were longitudinally sectioned and analysed with stereomicroscope -zoomed up to 60x.
For the detection of lignin and lipophilic compounds, histochemical methods were used.The pyrenes were fixed in FAA70 for 72 hours and stored in 70% alcohol.After that, dehydration was carried out in an ethanolic series of 2 hours each (80%, 90% and 100%).The pyrenes were included in Leica® meta-acrylate glycol resin and placed in a pre-infiltration solution for 24 hours and 15 minutes in a vacuum chamber (-20 mbar).Afterwards, the pyrenes were packed in the infiltration solution for 72 hours, at 10 °C, and placed in the inclusion solution.After 48 hours, longitudinal anatomical sections were made with disposable blades in Leica RM 2025 microtome and 7 μm thickness.
For the lignin staining, 2% Phloroglucinol was used and for the reaction of lipophilic compounds, Sudam III; then, semi-permanent blades were made with stained glass.Later, the slides were evaluated to investigate the presence of staining reaction in lignin and lipophilic compounds -under an optical camera microscope.

Physiological dormancy
Bioassay on lettuce seeds, detection and quantification of phenols.Lettuce seeds were submitted to germination tests in substrates moistened with hydroalcoholic and aqueous extracts of Ilex paraguariensis pyrenes.For the extracts preparation (Matos 1988), the pyrenes were separated in two parts: 1) endocarp; and 2) seed (integument, endosperm, and embryo).Then, they were dehydrated in a forced air circulation oven for 48 hours at 40 °C, until they became dry fragments with constant mass.The fragments were triturated in a knife mill, until the generation of powder; hence, from the powder, the extracts were made.In the hydroalcoholic extracts, 70% ethyl alcohol was added and left in the dark for seven days.Later, they were filtered in filter paper and the alcohol was evaporated in a rotavaporator at 100 rpm and 55 °C, until constant volume.Dimethyl sulfoxide (DMSO) was added to the extract and diluted with distilled water, with concentration of 15% for the seeds and 20% for the endocarps.
Water was added at 80 °C, from the powder to the aqueous extracts, and closed in a bottle for 24 hours.The extract was diluted with distilled water until the concentration of 30% for the seeds and 20% for the endocarp.
The germination test and treatments of lettuce seeds were performed, according to recommendations previously described by ISTA (2008).The lettuce germination was conducted in a BOD germination chamber, at 20 °C, with 4 replicates of 50 seeds, in paper filter substrate, which was moistened with 2.5 times the weight of the paper with the extracts.The first germination count was performed 4 days after the beginning of the test and the final count was 7 days after.
Germination tests of lettuce seeds on substrates moistened with distilled water (Witness-water) and DMSO (Witness-DMSO) were also carried out to evaluate the product's influence on seed germination.The germination was evaluated daily, to determine the germination speed index (IVG), until the final count; thus, the value was obtained using the Maguire (1962) equation.
The phenolic compounds detection were performed by histochemical analysis, using the inclusion of the pyrenes in resin (as described in the previous item for lignin and lipophilic compounds) and reaction with ferric chloride (Johansen 1940).
The phenolics compounds quantification was made from the seed (integument, endosperm, and embryo) and endocarp.The samples were macerated in a porcelain mortar using methanol and water (1:1).The solution was filtered, and the total phenol compound content was determined in a spectrophotometer with absorbance of 760 nm.Folin-Ciocalteu reagent was used for the staining reaction.Data were expressed as mg of Gallic acid, equivalent per gram.A calibration curve of Gallic acid (standard curve) was constructed to determine the total phenolic compounds content (Fig. 1).

Morphological dormancy
Morphological analysis of embryos.Four replicates with 100 pyrenes were used, which were immersed in water for 24 hours, and later longitudinally sectioned, to evaluate the embryos' development.The evaluations were performed in Stemi-305 stereomicroscope with zoom up to 60x.
The embryos were classified considering the stages: 1. Globular: when the embryo is composed of a mass of cells; it is not possible to identify cotyledonary primordial.
2. Heart: it is possible identify cotyledonary primordial, where the shape resembles a heart (occupies approximately 10% of the length of the seed).
3. Post-heart: the cotyledons are already larger (occupies approximately 15% of the length of the seed).
5. Mature: the embryo is developed and completely differentiated (occupying approximately 40% of the length of the seed).
The experiments were conducted in a completely randomized design.To compare the means between treatments, the Tukey 5% probability test was used, using the software Assistat (Silva & Azevedo 2016).

Results
The water content of the pyrenes was 8.7%.During the experiment related to physical dormancy, an increase in the mass of the pyrenes exposed to the moistened substrate was observed, probably due to the absorption of water (Fig. 2).
Although the absorption in water was verified, it was observed, in the integral pyrenes immersed in methylene blue, that it is related only to the entrance of water in the endocarp (Fig. 3a).In the pyrenes that were scarified with sandpaper (Fig. 3b), the pigment entry in the endosperm and embryo were observed during the 48 hours of imbibition.
Anatomical sections: we can observe lignin's presence in the sclerenchyma layer next to the Ilex paraguariensis seed wrappers (Fig. 4).
Using the reagent Sudam III, in the histochemical tests, it was possible to identify a layer -external to the endosperm -of lipophilic compounds (Fig. 5).
Test of lettuce seeds germination with the aqueous extract: there was germination; however, it was slow compared to the control, differing statistically both from the IVG and from the first germination count.With the hydroalcoholic extract from the seeds and the endocarp, germination did not occur; hence, it may indicate the presence of chemical inhibitors (Tab.1).
The control, with water or DMSO, in the first count, during four days, showed high germination (99% and 98%).This germination was statistically different from the aqueous extract of endocarp (90%), and different from the aqueous extract of seeds (integument, endosperm and embryo), that was 69% in the first count.In the final count -seven days after the beginning  of the test -the germination of the control with water or DMSO did not change.Seed germination with aqueous seed extract increased to 91% and endocarp extract to 98%.Both hydroalcoholic extracts did not encourage germination.The best number of IVG was the two controls, after aqueous extract of endocarp (8.87) and aqueous extract of seeds (6.25).
The presence of phenols in the endocarp and in the seed (integument, endosperm and embryo) was verified in the Ilex paraguariensis pyrenes, with the highest amount present in the endocarp (Fig. 6).
In the morphological evaluation was verified in that, in 55.5 % of the pyrenes, the embryos were in the globular or heart stage (Fig. 7); furthermore, the embryos were not visualized in the other stages (44.5 %).

Discussion
The water content of the pyrenes was 8.7%, similar to what was observed in other studies with this species (Souza & Silva 2001;Menegueti et al. 2004).
The increase in the mass of the pyrenes was 0.3 g in relation to the initial weight, exposed to the moistened substrate; in the first 24 hours, the mass increased 78%.In the integral pyrenes, immersed in methylene blue, the water entrance is related only in the endocarp.The absorption of the methylene blue by the pyrenes was followed during 48 hours, due to the observed results in the absorption of water (mass increase before that period).In the scarified pyrenes, the entrance of water occurred in the endocarp, tegument, and endosperm, in the first hours.
In studies with I. latifolia and I. ronduta, the pyrenes were immersed in distilled water and methylene blue (1%) for 48 hours.Staining was verified only in the endocarp and in the integument; however, the endosperm and embryo did not color (Tezuka et al. 2013).These results are similar to those observed in this study.
Physical dormancy in Ilex paraguariensis pyrenes was also observed by Grigoletti Júnior et al. (1999).The seeds integuments can cause dormancy, what is called physical dormancy (Baskin & Baskin 2004).The integument protects the embryo, besides regulating the absorption of water and oxygen (Zeng et al. 2004); however, the coating can infer dormancy by preventing the passage of water (Debeaujon et al. 2000) and gas exchanges or mechanically restrict the output of the radicle (Bewley 1997).Studies carried out by Dolce et al. (2010), about I. paraguariensis pyrenes, demonstrated that germination was only possible after the pyrenes were sectioned.Physical dormancy may be caused by lipophilic compounds, such as: serous cuticle, suberin, lignin, paliçadic tissue, cutin and mucilages in the integument and/or pericarp (Perez 2004).In the histochemical tests, using the reagent Sudam III, it was possible to identify a layer of lipophilic compounds -external to the endosperm; this layer has not been described in the literature yet, in relation to Ilex paraguariensis.In I. opaca, Ives (1923) described a layer external to the endosperm, composed of suberin, followed by integument and endocarp; thus, this layer was formed by lignin and pectic substances.
The accumulation of suberin forms a coating, a barrier that prevents the entrance of water (Nawrath 2002).Suberin is composed of two distinct types of insoluble polyesters: fatty acid and glycerol (Spurný 1964).Genetic evidence show that suberin deposition controls seed permeability (Beisson et al. 2007).
However, the physical barrier is not the only obstacle to the germination process of Ilex paraguariensis seeds, since in germination tests of lettuce seeds with the hydroalcoholic extract with seeds and endocarp, germination did not occur; thus, it may indicate the presence of chemical inhibitors.In the aqueous extract, there was germination; nevertheless, it was slow compared to the control, differing statistically both from the IVG and from the first germination count.A factor to be considered is the solubility of the inhibitor(s) in alcohol, which may be used for the future identification of such inhibitor(s).It was observed that there was 98% germination of the lettuce seeds in the substrate moistened with the extractor (DMSO), indicating that it did not influence the results.
Lettuce seeds are used to examine allelopathy, since their sensitivity to secondary metabolites functions as allelochemicals (Ferreira & Aquila 2000).Allelopathic compounds may affect processes, such as: seed germination and seedling growth, nutrient assimilation, photosynthesis, respiration, protein synthesis, enzyme activity, and nutrient loss -through the effects on cell membrane permeability (Durigan & Almeida 1993).
In the same genus Ilex, Tezuka et al. (2013) indicated the presence of inhibitors in the endosperm, integument and/or endocarp, because isolated embryos germinated 100% in 8 weeks of incubation, confirming the results obtained in this work.
The presence of inhibitors in the germination process conditions promotes the occurrence of physiological type dormancy.Physiological  dormancy has been related to phytohormones, especially abscisic acid (ABA), or to secondary metabolism products, such as terpenes and phenolic compounds (Taiz & Zeiger 2002).The phenolic compounds can influence the germinative process, because they represent an obstacle to the diffusion of gases, whose function is to protect against leakage of solutes, imbibition damage and oxidative stress (Debeaujon et al. 2000;Marcos Filho 2015).These compounds are secondary and chemically heterogeneous -from the shikimic acid and malonic acid routes -with varying solubility, according to the compound (Taiz & Zeiger 2002).
The presence of phenols was verified in the Ilex paraguariensis pyrenes, with the highest amount present in the endocarp.In forest seeds of Piptadenia macrocarpa Benth, Joannesia princeps Vell.and Dalbergia nigra Vell., there was a variation of 0.329 to 1.342 mg.g -1 of phenols in the integuments, and 0.016 to 0.495 mg.g -1 in the seed embryos, indicating a lower concentration of these compounds in the internal tissues of the seeds (Maciel et al. 1992).With the bioassays, the presence of germination inhibitors was evident; however, other studies are necessary to identify them.
In addition to the physical barrier and inhibitors of germination present in the endocarp and seed, it was verified that, during the morphological evaluation and in 55.5% of the pyrenes, the embryos were in the globular or heart stage; nevertheless, embryos were not visualized in the other stages (44.5%).The predominant stages of Ilex paraguariensis embryos development -described in the literature -range from globular to post-heart (Niklas 1987;Fowler et al. 2007).
Immature embryos were also reported in other species of Ilex (Hu et al. 1979;Tsang & Corlett 2005;Chien et al. 2011), and it may be a common phenomenon for the genus.Studying the germination of immature embryos of I. paraguariensis in vitro, of fruits of different maturation stages, Ferreira et al. (1991) reported that there was interruption of embryo growth at the stage known as heart, when the fruits were ripe and could be a consequence of the presence of inhibitors in the endosperm, and possibly in the embryos themselves.On the other hand, Dolce et al. (2010) reported that there was no significant difference between percentages of germination of excised embryos and sectioned pyrenes, suggesting that the endosperm does not inhibit the development or germination of embryos.It is believed that the different causes of dormancy, in the same pyrene, are complementary, since the physics makes gas exchanges difficult and prevents the release and assimilation of the inhibitors, which hinder the development of the embryo.

Figure 2
Figure 2 -a-b.Water absorption of the Ilex paraguariensis pyrenees based on mass increase (g) over time (h) -a.absorption in the first 5 hours; b. absorption in 600 hours.

Figure 3
Figure 3 -a-b.Ilex paraguariensis pyrenees after immersion in 1% methylene blue solution for 48 hours -a.intact pyrene; b. sanded pyrene, the red arrow indicates the scarified location.Scale bar: 1 mm.a b

Figure 6 -
Figure 6 -Quantification of phenolic compounds in the endocarp and seed (integument, endosperm and embryo) of Ilex paraguariensis.

Figure 7
Figure 7 -a-b.Ilex paraguariensis pyrenees sectioned longitudinally with embryos -a. at the globular stage; b. at the heart stage.Scale bar: 0.5 mm. a b