Effect of seed coat rupture on the germination of Mimosa flocculosa Burkart (Leguminosae) seeds 1

Corsato, J.M. & Fortes, A.M.T. 2020. Effect of seed coat rupture germination (Leguminosae) seeds. Hoehnea 47: e072020. http://dx.doi.org/10.1590/2236-8906-07/2020 ABSTRACT – (Effect of seed coat rupture on the germination of Mimosa flocculosa Burkart (Leguminosae) seeds). Mimosa flocculosa Burkart is a native species used for the recovery of degraded areas. Large scale seedling production in nurseries is hindered due to seed physical dormancy. This study evaluated the germination and seed coat anatomy of M. flocculosa seeds subjected to dormancy overcome treatments. Seeds of M. flocculosa were subjected to: control with distilled water, heated water at 50, 80 and 100 °C, and H 2 SO 4 for 1, 3 and 5 minutes. Germination tests were conducted with four replicates of 25 seeds in an entirely casualized delineation, analyzing germination percentage, mean germination time and germination speed index. Moreover, anatomical analysis of the seed coat was conducted. The treatments with heated water at 80 ºC, and sulfuric acid for 3 and 5 minutes promoted benefic anatomical alterations in the seed integument, reflecting in higher and faster in germination rates. Heated water at 80 ºC is recommended foram: germinação, tempo médio e índice de velocidade de germinação. Foram realizadas ainda análises anatômicas do tegumento das sementes. Os tratamentos usando água a 80 ºC, e ácido sulfúrico por 3 e 5 minutos promoveram mudanças anatômicas benéficas no tegumento da semente, refletindo no aumento da porcentagem e velocidade de germinação. Recomendamos a água aquecida a 80 ºC pela


Introduction
Native Brazilian forests constantly undergoes deforestation caused by socioeconomic growth. In 1890, forests covered 85% of the whole territory of Paraná State, while a hundred years later, this size decreased to only 3.5% (Meneghello & Mattei 2004, Rodrigues 2015.
From that perspective, there has been an increase in studies aiming to identify efficient methods to promote the recovery of degraded areas. Planting of seedlings and direct seed sowing using seeds of native species of different successional stages are among the most common methods, aiming to promote natural ecological succession and reestablishment of the local biodiversity (Fonseca et al. 2001, José et al. 2005, Pereira & Rodrigues 2012. In order to apply these methods efficiently, native seeds with high vigor are needed (Kageyama et al. 2003). However, more than half of the native species present seed dormancy (Baskin & Baskin 2005, Souza et al. 2015.
Seed dormancy is caused by factors within the seed, preventing seeds from germinating even during suitable ecological conditions. Physical dormancy is the most reported type of dormancy for native species and is also known as integumentary dormancy, which is presented as a physical barrier that controls gas changes and water uptake in the seeds (Alexandre et al. 2009, Marcos-Filho 2015. This type of dormancy is considered common among Leguminosae species, at which the physical barrier is presented as a layer of palisade thick-walled cells called macrosclereids (Baskin & Baskin 2000, Souza et al. 2015, Marcos-Filho 2015.
Ecologically, dormancy is a beneficial evolutionary adaptation because it delays germination, allowing seed dispersal throughout space and time. On the other hand, since it impedes efficient nursery seedling production, methods to overcome dormancy are essential. Several methods can be used, but the efficiency of each varies among species (Baskin & Baskin 2001, Baskin & Baskin 2005.
Mimosa flocculosa Burkart (Leguminosae), popularly known as Bracatinga de Campo-Mourão, is a native species indicated and highly used in degraded area recovery that presents physical dormancy and occurs in Mixed Ombrophilous and Stationary Semideciduous Forests (Carvalho 2002, Carvalho 2003.
Mimosa flocculosa is considered a pioneer species of small size and fast growth that is pollinized by bees and other insects, and therefore has potential for honey production (Carvalho 2002). The species has a short life cycle, which also promotes accumulation of litter and consequently benefits the ecological succession and arrival of new species in the area (Shibata et al. 2014, Shibata et al. 2016. Several studies are conducted in order to find the method that better increases productivity and simultaneous germination for each species, and the most common treatments use either heated water, stratification, chemical or mechanical scarification (Vivian et al. 2008). Recent studies with M. flocculosa seeds were conducted by Shibata et al. 2014 andShibata et al. 2016. In 2014, the authors compared two different lots and divided seeds in two categories, light and dark seeds, to verify if there was a difference in the dormancy overcome. The highest germination (59%) was presented by the treatment with sulfuric acid for 1 minute in dark seeds. In 2016, Shibata et al. verified the most adequate substrate and temperature for germination of the species, which stand out sand at 20 and 30 ºC.
In order to increase native seedling production in nurseries and consequently improve the recovery of degraded areas, this study verified the efficiency of treatments of physical dormancy overcome on the seed coat and germination of M. flocculosa seeds.

Material and Methods
The experiment was conducted between January and March 2018 in the Plant Physiology Laboratory of the Universidade Estadual do Oeste do Paraná, Cascavel, Paraná State, Brazil. The seeds of Mimosa flocculosa Burkart used in this study were collected in 2017. After pod collection and seed extraction, initial water content was evaluated and seeds were stored in a cold chamber at 4 ºC.
Dormancy overcome essay -Seeds were submersed in distilled water at several temperatures or concentrate sulfuric acid 98% for different periods of time as described on table 1. After the application of the following treatments, seeds were rinsed in running water for 3 minutes to wash out any remnant of the solutions.
Germination essay -After the application of the treatments described on table 1, M. flocculosa seeds were placed in "Germitest" roll papers moistened with 2.5 times of the paper weight with distilled water and stored in a germination chamber (TE-4020 TECNAL) at 25 ºC and 12h photoperiod (Brasil 2013). Each treatment consisted of four replicates of 25 seeds, organized in a completely randomized delineation. Although we evaluated seeds for 45 days, after 14 days of germination tests seeds were highly deteriorated by fungi and presented no differences in germination. Therefore, as other studies (Shibata et al. 2014, Shibata et al. 2016, we opted to present the results of the two-week evaluation period, as recommended by Brasil (2013). Seeds were considered germinated when they presented at least 2 mm of primary root protrusion (Hadas 1976). The parameters analyzed were germination percentage, mean germination time (MGT) (Edmond & Drapala 1958) and germination speed index (GSI) (Silva & Nakagawa 1995).
For statistical analysis, Shapiro-Wilk test for normality and the Levene's test for homogeneity of variances were performed, followed by the one-way ANOVA and Tukey tests. For each analysis a 5% significance level was used. Software used was RStudio 3.2.2.
Seed Coat Anatomical analysis -The seeds subjected to different treatments (CW, W50, W80, W100, SA1, SA3 and SA5) were fixed with FAA 50 (Johansen 1940), preserved in ethanol 70%, dehydrated in ethanolic series and included in Leica historesin (following fabricant orientations). Before fixation, in order to optimize the historesin infiltration, seeds were cut transversely in the median hilum-region and longitudinally in the median raphe-region. We analyzed the integument structure opposed to where the infiltration cuts were made, by producing transversal and longitudinal sections of 5 µm width using a rotary microtome (Leica RM 2245). Sections were stained with toluidine blue 0.05% (O'Brien et al. 1964) and Entellan was used to mount permanent slides. Section images were captured using a photomicroscope Olympus Bx70 by the program DP Controller. The terminology of the structure used were according to the revision proposal of Souza & Marcos-Filho (2001).

Results
The treatments that promoted the highest germination rates and also higher GSI were W80 (84%), SA5 (82%) and SA3 (77%). SA1 also increased the germination percentage (50%), while W100 and W50 presented the same germination rates as the control (Table 2). Only 21% of the CW seeds germinated. Seeds took between 2 and 4 days to germinate and no difference was observed for MGT between the treatments.
The seed coat of M. flocculosa is constituted externally by one layer of elongated and juxtaposed cells (macrosclereids (Mc) or palisade layer), with thick-walled and reduced lumen covered by cuticle (Ct), as observed in the figure 1 a. The light line (Ll) is also observed in the distal region of palisade layer. Internally, there is a single layer of shorter cells and with a wider lumen than in the palisade layer, called the hourglass cells (Hc). These cells also presented thick walls, but they are separated by intercellular spaces. The exotesta (Ex) region is composed by the palisade layer and the hourglass cells (figure 1 a). The hourglass layer is followed by several layers (4-8) of parenchymatic cells (Pc) tangentially elongated, with thinwalled and dense protoplasm, known as mesotesta (Me) region.
In the treatments for dormancy overcome using either heated water or with acid caused damage mainly in the palisade layer ( figure 1 b-d). In the W80, we observed fissures mainly in the anticlinal cell-walls of the macrosclereids (figure 1 b). In contrast, in the SA3 (figure 1 c) and SA5 (figure 1 d) we noticed corrosion of the superficial region of the palisade layer, with removal of the cuticle (figure 1 c-d). Table 1. Treatments applied for dormancy overcome. Lines indicate the abbreviations used for each treatment throughout the text and their corresponding solutions, temperatures and immersion times. CW: control with no immersion. W50: immersion in distilled water at 50 ºC for 10 min. W80: immersion in distilled water at 80 ºC for 10 min. W100: immersion in distilled water at 100 ºC for 10 min. SA1: immersion in concentrated sulfuric acid for 1 min. SA3: immersion in concentrated sulfuric acid for 3 min. SA5: immersion in concentrated sulfuric acid for 5 min. Values followed by the same letter in the column do not differ by Tukey test at 5% probability. C.V.: coefficient of variation.

Discussion
The structure of the seed coat observed in M. flocculosa CW seeds are similar to those described for other species from the same genus, as Mimosa daleoides Benth., M. dolens Vell. var. anisitsii (Lindm.) Barneby and M. orthacantha Benth, and also from other Leguminosae genera, specially the waxy cuticle that cover the macrosclereids (Iwasaki 2008, Molizane et al. 2018. These characteristics are responsible for the rigidity and impermeability of the integument, hampering gas changes and water uptake by the seed and consequently resulting in the physical dormancy (Perez 2004, Marcos-Filho 2015. The occurrence of physical dormancy in M. flocculosa was confirmed by the efficacy of some treatments (W80, SA3 and SA5) to promote higher germination rates (84, 77 and 82%, respectively) than the control (21%). The results are reinforced by the structural changes observed in the seeds exposed to the dormancy overcome treatments, largely related to the cuticle removal and the damage to macrosclereid cells, which caused ruptures in the impermeable palisade layer. These ruptures promoted water uptake, which promotes tissue rehydration, and oxygen entrance, which intensifies the metabolic activities and the degradation of reserves within the seed, essential processes for embryonic axis growth (Rodrigues et al. 2019).
Similar results were described by Sperandio et al. (2013) using seeds of Mimosa setosa Benth, at which by using sulfuric acid for 1 and 3 min or water at 70 ºC and 80 ºC during 1, 3 and 5 min obtained higher germination rates for the species (> 90%). When studying Mimosa caesalpiniifolia Benth seeds, Passos et al. (2007) observed increases in germination (89.5%) by using sulfuric acid P.A. during 4 min, which was similar to results found in this study for M. flocculosa.
The other treatments to dormancy overcome presented in this study that presented lower increase in germination (SA1) or the same percentages as the control (W50 and W100) were also tested similarly by Shibata et al. (2014) for M. flocculosa seeds of different colors, and the immersion in water at 50 °C and sulfuric acid for 1 min, also presented low germination (22% and 7%, respectively). The dormancy overcome study conducted by Alencar et al. (2009) for Stylosanthes SW, another Leguminosae genus, also showed relatively low germination (40%) when seeds were exposed to water at 50 ºC during 5, 10 and 15 hours.
The low germination percentage presented by M. flocculosa seeds in W50 may be explained by the fact that this treatment was not able to loosen the integument cell layers, and hence not being enough to increase water intake and consequently increase seed germination rates. On the other hand, W100 increased germination when compared to control, although not as efficiently as the other treatments (W80, SA3 and SA5). This might be attributed to the high temperature, which we suggest that caused damage in vital structures and therefore the loss of important functions, as the stability and permeability of cell membranes and the activity of enzymes, irreversibly compromising germination. Same results were found by Ribeiro et al. (2016) in Peltophorum dubium dormant seeds, in which seeds were unable to germinate when seeds were soaked in water at 100 ºC for 10 min.
We also observed an increase in GSI for the treatments with highest germination (W80, SA3 and SA5), proving that the physical dormancy was the obstacle for a high and fast germination. Several authors highlight the importance of analyzing GSI in dormancy overcome treatments, especially because high GSI represents seed vigor and a trend of accelerating the germinative process (Lima & Meiado 2017).
Mimosa flocculosa seeds presented the same MGT in all treatments, taking 2 to 4 days to start germination. Monteiro & Castilho (2014) studied treatments for overcoming dormancy of Peltophorum dubium seeds, and also found no difference in MGT between the analyzed treatments.
In accordance with the results found in our study, Santos et al. (2014) observed that in seeds of Piptadenia viridiflora (Kunth) Benth the highest GSI values were found in dormancy overcome treatments using sulfuric acid. The acceleration of the germination process is considered beneficial, since the faster seeds germinate, the less they will be exposed to adverse conditions and pathogen incidence, which may lead to tissue deterioration. Moreover, faster germination tends to be more synchronized, a desirable characteristic in seedling production (Joshi & Chinnusamy 2014, Rodrigues et al. 2019. Similar to our results, Sperandio et al. (2013) obtained the greatest germination rates applying sulfuric acid and heated water in overcoming dormancy of Mimosa setosa Benth seeds, but stated the risk of handling sulfuric acid, since it is highly corrosive. Therefore, the authors suggested heated water as a better treatment, since it is cheaper and presents less risk for the nurseries. Moreover, after 14 days of evaluation, the only seeds that were not deteriorated by fungi were the CW seeds. To verify if the viability of these seeds was intact, we subjected the CW seeds to the best overcome dormancy treatment, i.e. distilled water at 80 ºC for 10 min. All seeds germinated, confirming the efficacy of the W80 treatment and also the importance of the seed coat for seed integrity and vigor.
Finally, our study corroborates with several authors, confirming that applying sulfuric acid for 3 and 5 minutes is efficient to overcome the physical dormancy of Mimosa flocculosa seeds. On the other hand, water heated at 80 ºC has the same efficiency and is less expensive and safer to use, which are beneficial characteristics for seedling production in nurseries.

Conclusion
Among all treatments tested, the best for overcoming Mimosa flocculosa seed dormancy is heated water at 80 ºC and sulfuric acid for 3 and 5 minutes, since they promoted beneficial morphoanatomical changes in the integument that reflected in faster and higher germination rates.

Aknowledgements
We thank CAPES, for the financial support and Instituto Ambiental do Paraná, for seed collection and donation.