Abstract
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.
Key words
combined dormancy; Ilex paraguariensis; morphological dormancy; physical dormancy; physiological dormancy
Resumo
Pirênios de Ilex parguariensis, popularmente conhecida como erva-mate, foram classificados como dormentes. O objetivo deste estudo foi investigar as causas da dormência dos pirênios de Ilex paraguariensis. Para isso, foram realizados os seguintes testes: a) Dormência física: taxa de absorção em água e em azul de metileno; detecção de lignina e compostos lipofílicos no endocarpo e tegumento; b) Dormência fisiológica: bioensaios em sementes de alface, detecção e quantificação de compostos fenólicos; c) Dormência morfológica: análise embrionária. Para a taxa de absorção, observou-se um aumento na massa dos pirênios, porém, quando a embebição foi realizada em azul de metileno, a absorção ocorreu apenas no endocarpo. A lignina também foi observada na forma de uma camada de esclerênquima próxima ao tegumento da semente. Semelhantemente compostos lipofílicos foram observados em uma camada externa ao endosperma. Os bioensaios com sementes de alface indicaram a presença de inibidores químicos. Na avaliação morfológica dos pirênios, apenas 55,5% dos embriões foram visualizados e estavam nos estágios globular ou coração. Pirênios de Ilex paraguariensis possuem dormência combinada: física (não há absorção de água), morfológica (devido ao subdesenvolvimento do embrião) e há evidências de dormência fisiológica (presença de inibidores); Contudo, recomenda-se investigar os agentes inibidores.
Palavras-chave
dormência combinada; Ilex paraguariensis; dormência morfológica; dormência física; dormência fisiológica
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 2003Carvalho PER (2003) Espécies arbóreas brasileiras. Embrapa Florestas, Colombo 1: 455-466.). The leaves are dried to prepare a traditional tea (mate tea), which is consumed by millions of people in South America (Oliveira & Waquil 2014Oliveira SV & Waquil PD (2014) Dinâmica de produção e comercialização da erva-mate no Rio Grande do Sul, Brasil. Ciência Rural: 1-7. DOI: 10.1590/0103-8478cr20140276 ; Bergottini et al. 2015Bergottini VM, Otegui MB, Sosa DA, Zapata PD, Mulot M, Rebord M, Zopfi J, Wiss F, Benrey B & Pilar Junier (2015) Bio-inoculation of yerba mate seedlings (Ilex paraguariensis St. Hill.) with native plant growth-promoting rhizobacteria: a sustainable alternative to improve crop yield. Biology and Fertility of Soils 51: 749-755. DOI: 10.1007/s00374-015-1012-5
https://doi.org/10.1007/s00374-015-1012-...
). 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. 2011Horbach MA, Bisognin DA, Kielse P, Quadros KM & Fick TA (2011) Micropropagação de plântulas de erva-mate obtidas de embriões zigóticos. Ciência Rural 41: 113-119. DOI: 10.1590/S0103-84782011000100018
https://doi.org/10.1590/S0103-8478201100...
; Santin et al. 2013Santin D, Benedetti EL, Kaseker JF, Bastos MC, Reissmann CB, Wedling I & Barros NF (2013) Nutrição e crescimento da erva-mate submetida à calagem. Ciência Florestal 23: 55-66. DOI: 10.5902/198050988439
https://doi.org/10.5902/198050988439...
). Although it has been used for several decades, some silvicultural problems persist, including low and distributed germination over time (Medeiros et al. 1999Medeiros ACS, Nogueira AC, Amazonas MALA, Grigoletti Junior A & Urbean AF (1999) Superação da dormência e germinação de sementes de erva-mate (Ilex paraguariensis St. Hil.). Embrapa 41: 1-3.; Fowler & Sturion 2000Fowler JAP & Sturion JA (2000) Aspectos da formação do fruto e da semente na germinação da erva-mate. Embrapa 45: 1-5. ).
The dormancy is an adaptation that prevents seed germination even when environmental conditions are favorable (Baskin & Baskin 2004Baskin JM & Baskin CC (2004) A classification system for seed dormancy. Seed Science Research 14: 1-16. DOI: 10.1079/SSR2003150
https://doi.org/10.1079/SSR2003150...
; Finch-Savage & Leubner-Metzger 2006Finch-Savage WE & Leubner-Metzger G (2006) Seed dormancy and the control of germination. New Phytologist 171: 501-523. DOI: 10.1111/j.1469-8137.2006.01787.x
https://doi.org/10.1111/j.1469-8137.2006...
). Although dormancy is considered a survival mechanism of the species (Bewley et al. 2013Bewley JD, Bradford KJ, Hilhorst HWM & Nonogaki H (2013) Seeds: physiology of development, germination and dormancy. 3rd ed. Springer, New York. 392p.), 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)Grigoletti Júnior A, Zanon A, Auer CG & Fowler JAP (1999) Efeito de fungicidas aplicados nas sementes, na emergência de plântulas de erva-mate (Ilex paraguariensis St. Hil.). Boletim de Pesquisa Florestal 39: 31-39. 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 2004Baskin JM & Baskin CC (2004) A classification system for seed dormancy. Seed Science Research 14: 1-16. DOI: 10.1079/SSR2003150
https://doi.org/10.1079/SSR2003150...
). According to Dolce et al. (2010)Dolce NR, Mroginski LA & Rey HY (2010) Endosperm and endocarp effects on the Ilex paraguariensis A. St.-HiL. (Aquifoliaceae) seed germination. Seed Science and Technology 38: 441-448. DOI: 10.15258/sst.2010.38.2.17
https://doi.org/10.15258/sst.2010.38.2.1...
, 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 1987Niklas CO (1987) Estudios embriologicos y citologicos en la yerba mate Ilex paraguariensis (Aquifoliaceae). Instituto de Botánica Del Nordeste 6: 45-56. ; Sansberro et al. 1998Sansberro PA, Rey HY, Mroginski LA & Collavino MM (1998) In vitro culture of rudimentary embryos of Ilex paraguariensis: responses to exogenous cytokinins. Journal of Plant Growth Regulation 17: 101-105. DOI: 10.1007/PL00007014
https://doi.org/10.1007/PL00007014...
; Menegueti et al. 2004Menegueti JCB, Nóbrega LHP, Smanhotto A & Silveira JCM (2004) Superação da dormência de sementes de erva-mate (Ilex paraguariensis St. Hill) por estratificação. Revista Varia Scientia 4: 157-182. ). 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 1988Malavasi MM (1988) Germinação de sementes. In: Rodrigues FMC (ed.) Manual de análise de sementes florestais. Fundação Cargil, Campinas. Pp. 25-40.).
Combined dormancy was also reported for Ilex genus by Baskin & Baskin (2014)Baskin CC & Baskin JM (2014) Seeds: ecology, biogeography and evolution of dormancy and germination. 2nd ed. Elsevier, San Diego. 1602p., and specifically for I. paraguariensis, as morphological and physiological (morphophysiological) dormancy (Cuquel et al. 1994Cuquel FL, Carvalho MLM & Chamma HMCP (1994) Avaliação de métodos de estratificação para a quebra de dormência em sementes de erva-mate. Scientia Agricola 51: 415- 421. DOI: 10.1590/S0103-90161994000300006
https://doi.org/10.1590/S0103-9016199400...
; Galíndez et al. 2018Galíndez G, Ceccato D, Bubillo R, Lindow-López L, Malagrina G, Ortega-Baes P & Baskin CC (2018) Three levels of simple morphophysiological dormancy in seeds of Ilex (Aquifoliaceae) species from Argentina. Seed Science Research 28: 131-139. DOI: 10.1017/S0960258518000132
https://doi.org/10.1017/S096025851800013...
). Physiological dormancy occurs when there are inhibitory substances or absence of substances that promote germination, preventing it from occurring (Baskin & Baskin 2004Baskin JM & Baskin CC (2004) A classification system for seed dormancy. Seed Science Research 14: 1-16. DOI: 10.1079/SSR2003150
https://doi.org/10.1079/SSR2003150...
). Thus, the germination only happens after the inhibitory substances have been removed, for example, through the digestive tract of animals (Zaidan & Barbedo 2004Zaidan LBP & Barbedo CJ (2004) Quebra de dormência em sementes. In: Ferreira AG & Borghetti F (eds.) Germinação: do básico ao aplicado. Artmed, Porto Alegre. Pp. 134-146.). It was also reported morphological and physical dormancy in these pyrenes (Medeiros 1998Medeiros ACS (1998) Dormência em sementes de erva-mate. Embrapa-CNPF 36: 1-25.).
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 2016Climate (2016) Climate data for world cities. Available at <http://pt.climate-data.org/>. Access on 9 September 2016.
http://pt.climate-data.org/...
).
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)ISTA - International Seed Testing Association (2007) Determination of moisture content. In: International rules for seed testing. Cap. 9. Seed Testing Association, Bassersdorf. Pp. 9.1-9.20..
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. 2007Orozco-Segovia A, Márquez-Guzmán J, Sánchez-Coronado ME, Gamboa De Buen A, Baskin JM & Baskin CC (2007) Seed anatomy and water uptake in relation to seed dormancy in Opuntia tomentosa (Cactaceae, Opuntioideae). Annals of Botany 99: 581-592. DOI: 10.1093/aob/mcm001
https://doi.org/10.1093/aob/mcm001...
), 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 1988Matos FJA (1988) Introdução a fitoquímica experimental. UFC, Fortaleza. Pp. 1-128.), 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)ISTA - International Seed Testing Association (2008) Testing coated seeds. In: International rules for seed testing. Cap. 11. Seed Testing Association, Bassersdorf. Pp. 11.1-11.8. . 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)Maguire JD (1962) Speed of germination-aid in selection and evaluation for seedling emergence and vigor. Crop Science, Madison 2: 176-177. 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 1940Johansen DA (1940) Plant microtechnique. McGraw-Hill, New York. 523p.).
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).
4. Torpedo: cotyledons are differentiated (occupies approximately 20% of the total length).
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 2016Silva FAS & Azevedo CAV (2016) The assistat software version 7.7 and its use in the analysis of experimental data. African Journal of Agricultural Research 11: 3733-3740. DOI: 10.5897/AJAR2016.11522
https://doi.org/10.5897/AJAR2016.11522...
).
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).
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.
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.
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.
Anatomical sections: we can observe lignin’s presence in the sclerenchyma layer next to the Ilex paraguariensis seed wrappers (Fig. 4).
Presence of lignin in Ilex paraguariensis pyrenees. Scale bar: 0.5 mm. EN = endosperm; LI = lignin; EC = endocarp.
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).
Presence of lipophilic compounds in Ilex paraguariensis seeds. Scale bar: 0.5 mm. EN = endosperm; CL = lipophilic compounds; EC = endocarp.
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).
– Germination (%) and Germination Speed Index (IVG) of Lactuca sativa (lettuce), with extracts: aqueous 30% seeds (integument, endosperm, and embryo) and 20% endocarp; hydroalcoholic 15% seeds (integument, endosperm, and embryo) and 20% endocarp of the Ilex paraguariensis A. St. Hil.
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).
Quantification of phenolic compounds in the endocarp and seed (integument, endosperm and embryo) of Ilex paraguariensis.
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 %).
a-b. Ilex paraguariensis pyrenees sectioned longitudinally with embryos – a. at the globular stage; b. at the heart stage. Scale bar: 0.5 mm.
Discussion
The water content of the pyrenes was 8.7%, similar to what was observed in other studies with this species (Souza & Silva 2001Souza AC & Silva L (2001) Efeitos da secagem na viabilidade das sementes de Ilex paraguariensis St. Hil. Boletim de Pesquisa Florestal 42: 35-46. ; Menegueti et al. 2004Menegueti JCB, Nóbrega LHP, Smanhotto A & Silveira JCM (2004) Superação da dormência de sementes de erva-mate (Ilex paraguariensis St. Hill) por estratificação. Revista Varia Scientia 4: 157-182. ).
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. 2013Tezuka T, Yokoyam H, Tanaka H, Shiozaki S & Oda M (2013) Factors affecting seed germination of Ilex latifolia and I. rotunda. HortScience 48: 352-356. DOI: 10.21273/HORTSCI.48.3.352
https://doi.org/10.21273/HORTSCI.48.3.35...
). 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)Grigoletti Júnior A, Zanon A, Auer CG & Fowler JAP (1999) Efeito de fungicidas aplicados nas sementes, na emergência de plântulas de erva-mate (Ilex paraguariensis St. Hil.). Boletim de Pesquisa Florestal 39: 31-39.. The seeds integuments can cause dormancy, what is called physical dormancy (Baskin & Baskin 2004Baskin JM & Baskin CC (2004) A classification system for seed dormancy. Seed Science Research 14: 1-16. DOI: 10.1079/SSR2003150
https://doi.org/10.1079/SSR2003150...
). The integument protects the embryo, besides regulating the absorption of water and oxygen (Zeng et al. 2004Zeng CL, Wang JB, Liu AH & Wu XM (2004) Seed coat microsculpturing changes during seed development in diploid and amphidiploid brassica species. Annals of Botany 93: 555-566. DOI: 10.1093/aob/mch080
https://doi.org/10.1093/aob/mch080...
); however, the coating can infer dormancy by preventing the passage of water (Debeaujon et al. 2000Debeaujon I, León- Kloosterziel KM & Koorneef M (2000) Influence of the Testa on seed dormancy, germination and longevity in Arabidopsis. Plant Physiology 122: 403-413. DOI: 10.1104/pp.122.2.403
https://doi.org/10.1104/pp.122.2.403...
) and gas exchanges or mechanically restrict the output of the radicle (Bewley 1997Bewley JD (1997) Seed germination and dormancy. The Plant Cell 9: 1055-1066. DOI: 10.1105/tpc.9.7.1055
https://doi.org/10.1105/tpc.9.7.1055...
). 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 2004Perez SCJGA (2004) Envoltórios. In: Ferreira AG & Borghetti F (eds.) Germinação: do básico ao aplicado. Artmed, Porto Alegre. Pp. 125-133.). 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)Ives SA (1923) Maturation and germination of seeds of Ilex opaca. Botanical Gazette 76: 60-77. DOI: 10.1086/333226
https://doi.org/10.1086/333226...
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 2002Nawrath C (2002) The Biopolymers cutin and suberin. The Arabidopsis Book 4: 1-14.). Suberin is composed of two distinct types of insoluble polyesters: fatty acid and glycerol (Spurný 1964Spurný M (1964) Changes in the permeability of the seed coat in connection with the development of Suberin Adcrustations of the Macrosclereids from the seed coat of the pea (Pisum sativum L.). Flora 154: 547-567. DOI: 10.1016/S0367-1615(17)32709-X
https://doi.org/10.1016/S0367-1615(17)32...
). Genetic evidence show that suberin deposition controls seed permeability (Beisson et al. 2007Beisson FLIY, Bonaventure G, Pollard M & Ohlrogge JB (2007) The Acyltransferase GPAT5 is required for the synthesis of suberin in seed coat and root of Arabidopsis. The Plant Cell 19; 351-368. DOI: 10.1105/tpc.106.048033).
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 2000Ferreira AG & Aquila MEA (2000) Alelopatia: Uma área emergente da ecofisiologia. Revista Brasileira de Fisiologia Vegetal 12: 175-204. ). 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 1993Durigan JC & Almeida FS (1993) Noções sobre alelopatia. UNESP/FUNEP, Jaboticabal. 28p.).
In the same genus Ilex, Tezuka et al. (2013)Tezuka T, Yokoyam H, Tanaka H, Shiozaki S & Oda M (2013) Factors affecting seed germination of Ilex latifolia and I. rotunda. HortScience 48: 352-356. DOI: 10.21273/HORTSCI.48.3.352
https://doi.org/10.21273/HORTSCI.48.3.35...
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 2002Taiz L & Zeiger E (2002) Plant physiology. Sinauer, Sunderland. 690p.). 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. 2000Debeaujon I, León- Kloosterziel KM & Koorneef M (2000) Influence of the Testa on seed dormancy, germination and longevity in Arabidopsis. Plant Physiology 122: 403-413. DOI: 10.1104/pp.122.2.403
https://doi.org/10.1104/pp.122.2.403...
; Marcos Filho 2015Marcos Filho J (2015) Fisiologia de plantas cultivadas. Fealq, Piracicaba. 659p.). These compounds are secondary and chemically heterogeneous - from the shikimic acid and malonic acid routes - with varying solubility, according to the compound (Taiz & Zeiger 2002Taiz L & Zeiger E (2002) Plant physiology. Sinauer, Sunderland. 690p.).
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. 1992Maciel AS, Borges EEL & Borges RCG (1992) Determinação da presença de fenóis em sementes de espécies florestais e sua relação com inibidores de germinação. Revista Brasileira de Sementes 14: 1-4. DOI: 10.17801/0101-3122/rbs.v14n1p1-4
https://doi.org/10.17801/0101-3122/rbs.v...
). 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 1987Niklas CO (1987) Estudios embriologicos y citologicos en la yerba mate Ilex paraguariensis (Aquifoliaceae). Instituto de Botánica Del Nordeste 6: 45-56. ; Fowler et al. 2007Fowler JAP, Sturion JÁ & Zuffellato-Ribas KC (2007) Variação do desenvolvimento embrionário das sementes de erva-mate. Pesquisa Florestal Brasileira 54: 105-108.).
Immature embryos were also reported in other species of Ilex (Hu et al. 1979Hu CY, Rogalski F & Ward C (1979) Factors maintaining Ilex rudimentary embryos in the quiescent state and the ultrastructural changes during in vitro activation. Chicago Journals 140: 272-279.; Tsang & Corlett 2005Tsang ACW & Corlett RT (2005) Reproductive biology of the Ilex species (Aquifoliaceae) in Hong Kong, China. Canadian Journal of Botany 83: 1645-1654. DOI: 10.1139/b05-131
https://doi.org/10.1139/b05-131...
; Chien et al. 2011Chien CT, Chen SY, Chien TY, Baskin JM & Baskin CC (2011) Nondeep simple morphophysiological dormancy in seeds of Ilex maximowicziana from northern (subtropical) and southern (tropical) Taiwan. Ecological Research 26: 163-171. DOI: 10.1007/s11284-010-0773-4
https://doi.org/10.1007/s11284-010-0773-...
), 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)Ferreira AG, Cunha GG, Silveira TS & Hu CY (1991) In vitro germination of immature embryos of Ilex paraguariensis St. Hil. Phyton 54: 27-32. 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)Dolce NR, Mroginski LA & Rey HY (2010) Endosperm and endocarp effects on the Ilex paraguariensis A. St.-HiL. (Aquifoliaceae) seed germination. Seed Science and Technology 38: 441-448. DOI: 10.15258/sst.2010.38.2.17
https://doi.org/10.15258/sst.2010.38.2.1...
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.
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.
Acknowledgements
The authors acknowledge the financial support given by Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (Fapesc) (2017TR639). The authors also would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), for awarding doctoral scholarship to the first author.
References
- Baskin CC & Baskin JM (2014) Seeds: ecology, biogeography and evolution of dormancy and germination. 2nd ed. Elsevier, San Diego. 1602p.
- Baskin JM & Baskin CC (2004) A classification system for seed dormancy. Seed Science Research 14: 1-16. DOI: 10.1079/SSR2003150
» https://doi.org/10.1079/SSR2003150 - Beisson FLIY, Bonaventure G, Pollard M & Ohlrogge JB (2007) The Acyltransferase GPAT5 is required for the synthesis of suberin in seed coat and root of Arabidopsis. The Plant Cell 19; 351-368. DOI: 10.1105/tpc.106.048033
- Bergottini VM, Otegui MB, Sosa DA, Zapata PD, Mulot M, Rebord M, Zopfi J, Wiss F, Benrey B & Pilar Junier (2015) Bio-inoculation of yerba mate seedlings (Ilex paraguariensis St. Hill.) with native plant growth-promoting rhizobacteria: a sustainable alternative to improve crop yield. Biology and Fertility of Soils 51: 749-755. DOI: 10.1007/s00374-015-1012-5
» https://doi.org/10.1007/s00374-015-1012-5 - Bewley JD (1997) Seed germination and dormancy. The Plant Cell 9: 1055-1066. DOI: 10.1105/tpc.9.7.1055
» https://doi.org/10.1105/tpc.9.7.1055 - Bewley JD, Bradford KJ, Hilhorst HWM & Nonogaki H (2013) Seeds: physiology of development, germination and dormancy. 3rd ed. Springer, New York. 392p.
- Carvalho PER (2003) Espécies arbóreas brasileiras. Embrapa Florestas, Colombo 1: 455-466.
- Chien CT, Chen SY, Chien TY, Baskin JM & Baskin CC (2011) Nondeep simple morphophysiological dormancy in seeds of Ilex maximowicziana from northern (subtropical) and southern (tropical) Taiwan. Ecological Research 26: 163-171. DOI: 10.1007/s11284-010-0773-4
» https://doi.org/10.1007/s11284-010-0773-4 - Climate (2016) Climate data for world cities. Available at <http://pt.climate-data.org/>. Access on 9 September 2016.
» http://pt.climate-data.org/ - Cuquel FL, Carvalho MLM & Chamma HMCP (1994) Avaliação de métodos de estratificação para a quebra de dormência em sementes de erva-mate. Scientia Agricola 51: 415- 421. DOI: 10.1590/S0103-90161994000300006
» https://doi.org/10.1590/S0103-90161994000300006 - Debeaujon I, León- Kloosterziel KM & Koorneef M (2000) Influence of the Testa on seed dormancy, germination and longevity in Arabidopsis. Plant Physiology 122: 403-413. DOI: 10.1104/pp.122.2.403
» https://doi.org/10.1104/pp.122.2.403 - Dolce NR, Mroginski LA & Rey HY (2010) Endosperm and endocarp effects on the Ilex paraguariensis A. St.-HiL. (Aquifoliaceae) seed germination. Seed Science and Technology 38: 441-448. DOI: 10.15258/sst.2010.38.2.17
» https://doi.org/10.15258/sst.2010.38.2.17 - Durigan JC & Almeida FS (1993) Noções sobre alelopatia. UNESP/FUNEP, Jaboticabal. 28p.
- Ferreira AG & Aquila MEA (2000) Alelopatia: Uma área emergente da ecofisiologia. Revista Brasileira de Fisiologia Vegetal 12: 175-204.
- Ferreira AG, Cunha GG, Silveira TS & Hu CY (1991) In vitro germination of immature embryos of Ilex paraguariensis St. Hil. Phyton 54: 27-32.
- Finch-Savage WE & Leubner-Metzger G (2006) Seed dormancy and the control of germination. New Phytologist 171: 501-523. DOI: 10.1111/j.1469-8137.2006.01787.x
» https://doi.org/10.1111/j.1469-8137.2006.01787.x - Fowler JAP & Sturion JA (2000) Aspectos da formação do fruto e da semente na germinação da erva-mate. Embrapa 45: 1-5.
- Fowler JAP, Sturion JÁ & Zuffellato-Ribas KC (2007) Variação do desenvolvimento embrionário das sementes de erva-mate. Pesquisa Florestal Brasileira 54: 105-108.
- Galíndez G, Ceccato D, Bubillo R, Lindow-López L, Malagrina G, Ortega-Baes P & Baskin CC (2018) Three levels of simple morphophysiological dormancy in seeds of Ilex (Aquifoliaceae) species from Argentina. Seed Science Research 28: 131-139. DOI: 10.1017/S0960258518000132
» https://doi.org/10.1017/S0960258518000132 - Grigoletti Júnior A, Zanon A, Auer CG & Fowler JAP (1999) Efeito de fungicidas aplicados nas sementes, na emergência de plântulas de erva-mate (Ilex paraguariensis St. Hil.). Boletim de Pesquisa Florestal 39: 31-39.
- Horbach MA, Bisognin DA, Kielse P, Quadros KM & Fick TA (2011) Micropropagação de plântulas de erva-mate obtidas de embriões zigóticos. Ciência Rural 41: 113-119. DOI: 10.1590/S0103-84782011000100018
» https://doi.org/10.1590/S0103-84782011000100018 - Hu CY, Rogalski F & Ward C (1979) Factors maintaining Ilex rudimentary embryos in the quiescent state and the ultrastructural changes during in vitro activation. Chicago Journals 140: 272-279.
- ISTA - International Seed Testing Association (2007) Determination of moisture content. In: International rules for seed testing. Cap. 9. Seed Testing Association, Bassersdorf. Pp. 9.1-9.20.
- ISTA - International Seed Testing Association (2008) Testing coated seeds. In: International rules for seed testing. Cap. 11. Seed Testing Association, Bassersdorf. Pp. 11.1-11.8.
- Ives SA (1923) Maturation and germination of seeds of Ilex opaca Botanical Gazette 76: 60-77. DOI: 10.1086/333226
» https://doi.org/10.1086/333226 - Johansen DA (1940) Plant microtechnique. McGraw-Hill, New York. 523p.
- Maciel AS, Borges EEL & Borges RCG (1992) Determinação da presença de fenóis em sementes de espécies florestais e sua relação com inibidores de germinação. Revista Brasileira de Sementes 14: 1-4. DOI: 10.17801/0101-3122/rbs.v14n1p1-4
» https://doi.org/10.17801/0101-3122/rbs.v14n1p1-4 - Maguire JD (1962) Speed of germination-aid in selection and evaluation for seedling emergence and vigor. Crop Science, Madison 2: 176-177.
- Malavasi MM (1988) Germinação de sementes. In: Rodrigues FMC (ed.) Manual de análise de sementes florestais. Fundação Cargil, Campinas. Pp. 25-40.
- Marcos Filho J (2015) Fisiologia de plantas cultivadas. Fealq, Piracicaba. 659p.
- Matos FJA (1988) Introdução a fitoquímica experimental. UFC, Fortaleza. Pp. 1-128.
- Medeiros ACS (1998) Dormência em sementes de erva-mate. Embrapa-CNPF 36: 1-25.
- Medeiros ACS, Nogueira AC, Amazonas MALA, Grigoletti Junior A & Urbean AF (1999) Superação da dormência e germinação de sementes de erva-mate (Ilex paraguariensis St. Hil.). Embrapa 41: 1-3.
- Menegueti JCB, Nóbrega LHP, Smanhotto A & Silveira JCM (2004) Superação da dormência de sementes de erva-mate (Ilex paraguariensis St. Hill) por estratificação. Revista Varia Scientia 4: 157-182.
- Nawrath C (2002) The Biopolymers cutin and suberin. The Arabidopsis Book 4: 1-14.
- Niklas CO (1987) Estudios embriologicos y citologicos en la yerba mate Ilex paraguariensis (Aquifoliaceae). Instituto de Botánica Del Nordeste 6: 45-56.
- Oliveira SV & Waquil PD (2014) Dinâmica de produção e comercialização da erva-mate no Rio Grande do Sul, Brasil. Ciência Rural: 1-7. DOI: 10.1590/0103-8478cr20140276
- Orozco-Segovia A, Márquez-Guzmán J, Sánchez-Coronado ME, Gamboa De Buen A, Baskin JM & Baskin CC (2007) Seed anatomy and water uptake in relation to seed dormancy in Opuntia tomentosa (Cactaceae, Opuntioideae). Annals of Botany 99: 581-592. DOI: 10.1093/aob/mcm001
» https://doi.org/10.1093/aob/mcm001 - Perez SCJGA (2004) Envoltórios. In: Ferreira AG & Borghetti F (eds.) Germinação: do básico ao aplicado. Artmed, Porto Alegre. Pp. 125-133.
- Sansberro PA, Rey HY, Mroginski LA & Collavino MM (1998) In vitro culture of rudimentary embryos of Ilex paraguariensis: responses to exogenous cytokinins. Journal of Plant Growth Regulation 17: 101-105. DOI: 10.1007/PL00007014
» https://doi.org/10.1007/PL00007014 - Santin D, Benedetti EL, Kaseker JF, Bastos MC, Reissmann CB, Wedling I & Barros NF (2013) Nutrição e crescimento da erva-mate submetida à calagem. Ciência Florestal 23: 55-66. DOI: 10.5902/198050988439
» https://doi.org/10.5902/198050988439 - Silva FAS & Azevedo CAV (2016) The assistat software version 7.7 and its use in the analysis of experimental data. African Journal of Agricultural Research 11: 3733-3740. DOI: 10.5897/AJAR2016.11522
» https://doi.org/10.5897/AJAR2016.11522 - Souza AC & Silva L (2001) Efeitos da secagem na viabilidade das sementes de Ilex paraguariensis St. Hil. Boletim de Pesquisa Florestal 42: 35-46.
- Spurný M (1964) Changes in the permeability of the seed coat in connection with the development of Suberin Adcrustations of the Macrosclereids from the seed coat of the pea (Pisum sativum L.). Flora 154: 547-567. DOI: 10.1016/S0367-1615(17)32709-X
» https://doi.org/10.1016/S0367-1615(17)32709-X - Taiz L & Zeiger E (2002) Plant physiology. Sinauer, Sunderland. 690p.
- Tezuka T, Yokoyam H, Tanaka H, Shiozaki S & Oda M (2013) Factors affecting seed germination of Ilex latifolia and I. rotunda HortScience 48: 352-356. DOI: 10.21273/HORTSCI.48.3.352
» https://doi.org/10.21273/HORTSCI.48.3.352 - Tsang ACW & Corlett RT (2005) Reproductive biology of the Ilex species (Aquifoliaceae) in Hong Kong, China. Canadian Journal of Botany 83: 1645-1654. DOI: 10.1139/b05-131
» https://doi.org/10.1139/b05-131 - Zaidan LBP & Barbedo CJ (2004) Quebra de dormência em sementes. In: Ferreira AG & Borghetti F (eds.) Germinação: do básico ao aplicado. Artmed, Porto Alegre. Pp. 134-146.
- Zeng CL, Wang JB, Liu AH & Wu XM (2004) Seed coat microsculpturing changes during seed development in diploid and amphidiploid brassica species. Annals of Botany 93: 555-566. DOI: 10.1093/aob/mch080
» https://doi.org/10.1093/aob/mch080
Edited by
Publication Dates
-
Publication in this collection
01 Apr 2022 -
Date of issue
2022
History
-
Received
23 Oct 2020 -
Accepted
01 Apr 2021