Introduction
In the production of native tree seeds, it is important to determine some of the essential characteristics for propagation, likewise germination, vigor and storage potential [1]. In this sense, environmental factors are essential and have a direct influence on germination potential, in which the availability of water, temperature, oxygen and the type of substrate are considered to be the most important external factors linked to quality, viability and dormancy [2][3]. Likewise, determining the physiological behavior of seeds during storage is important for forestry, as it provides useful data for making decisions about when to sow seeds in nurseries or direct plantings, avoiding the unnecessary loss of viability and vigor of the propagules [4].
In this context, Myrtaceae is a family of plants found in tropical and subtropical environments, which plays a significant role in the natural regeneration of ecosystems [5]. The dispersal and propagation of this group is carried out primarily by seeds, and it is essential to know the characteristics of the propagules both for the conservation of biodiversity and for reforestation initiatives [6][7].
Among Myrtaceae with scarce information on their reproductive and silvicultural behavior, Siphoneugena reitzii D. Legrand, popularly known as camboim, is a specie with a tree-like habit, heliophytic, early secondary/late secondary that can reach a height of up to 15 m. The bark is smooth with a greyish or yellowish coriaceous lamina [8][9]. It occurs in high-altitude areas of southern and southeastern Brazil, such as the dense ombrophilous forest and the high-montane mixed ombrophilous forest [10] and has been classified by Gomes [11] as a “relic species,” as it thrives in high-altitude areas above 1,800 m.
The species produces very hard and heavy wood, locally used for fence posts or as firewood [8] and despite the apparent uniqueness of the establishment of this species, S. reitzii have been gaining attention due to their potential for use in the food, cosmetics and pharmaceutical industries [11] due to the presence of volatile oils with anti-inflammatory activity [12] and antimicrobial action [13]. In addition to their ecological importance in restoration, they act as bioindicator plants for dystrophic soils with high acidity [11].
Despite the proven potential of this species, due to its specific occurrence, little is known about its silvicultural characteristics. It is known that its fruits are small circular glabrous berries, reddish in color when immature and purple when ripe, which contain 1 to 2 seeds with eugenioid embryos [10] that are usually part of the seed bank in mixed ombrophilous forests at high altitudes [14][15]. However, there are no conclusive studies on germination, seed quality and physiological classification for S. reitzii. In this way, the aim of this study was to describe germination in diferents substrates and temperatures, quality and physiological classification of Siphoneugena reitzii seeds.
Material and Methods
Collection area
The study was carried out using S. reitzii seeds obtained from five matrices located at the “Fazenda das Nascentes – RPPN Serra da Farofa –owned by the Klabin SA company, located on the border between the municipalities of Painel and Urupema, Santa Catarina, Brazil, at an altitude of 1,425 m and with a Cfb climate (humid subtropical mesothermal), according to the Köppen classification [16][17]. The region’s climatic conditions include annual temperatures of around 12 to 14ºC, with mild summers, no dry season, recurrent severe frosts and well-distributed rainfall throughout the year, ranging from 1,600 to 1,900 mm per year. Snow is also common in the coldest months [17][16]. The phytoecological region is classified as araucaria forest (mixed ombrophilous forest) subclassification highmontane [17].
The trees were selected at a minimum distance of 200 m apart, in order to avoid obtaining parents with genetic similarities. Initially, the ripe fruits (dark purple in color) were collected using a lopper and then taken to the Forest Seed Laboratory (LASF) at the “Centro de Ciências Agroveterinárias (CAV)” of the “Universidade do Estado de Santa Catarina (UDESC)”, where they were pulped under running water. The seeds remained distributed on trays with paper towels for around 8 hours to dry on the surface and were then subjected to characterization and the other tests. It should be noted that the seeds from the matrices were analyzed as a single batch.
Initial characterization and testing of temperatures and substrates
For the initial characterization of the lots, germination, vigor and water content tests were carried out. The water content was determined according to the Rules for Seed Analysis [18], with the samples in an oven at 105±3°C for ٢٤ hours with two repeti-tions of ٣ g per repetition. The seeds were weighed on an analytical balance in aluminum capsules with a determined weight and placed in an oven. The water content was calculated based on the weight of the wet seeds. To assess predated and empty seeds, ١٠٠ seeds of each species were cut lengthways with a scalpel and analyzed under a stereomicroscope.
S. reitzii seeds were exposed to different treatments to assess the influence of different substrates and temperatures on seed germination. The germination tests were carried out in a 2 x 3 factorial design (temperature x substrate) in a B.O.D. chamber. The seeds were placed to germinate in three substrates: in acrylic gerboxes with autoclaved sand (SA), blotting paper (mt) and paper rolls (RP) moistened to 2.5 (mL) times the weight of the paper (G), and at temperatures of 20 and 25ºC in constant light [18][19]. Evaluations were carried out daily from the 4th day until the 40th day of the test. After the end of the experiment, the ungerminated seeds were opened with a scalpel to check if they had rotted.
Four replicates of 25 seeds were used per batch/treatment, where the percentages of normal, abnormal, dead and hard seedlings were assessed, in accordance with the Instructions for Seed Analysis of Forest Species [20], and the seeds were considered germinated when they had more than 2 mm of primary root. At the same time, the germination percentage index (G%) [18], the germination speed index (GVI) [21] and the mean germination time (MGT) [22] were evaluated.
Physiological classification of Siphoneugena reitzii seeds
For the physiological classification of S. reitzii, the protocol proposed by Hong and Ellis [23] and adapted by Sacandé et al. [24] was used. The first stage, before drying, a seed germination test carried out at 20ºC on blotting paper. On the second stage, the rapid drying method was carried out by placing the seeds in airtight recipients exposed to silica gel at a temperature of 23ºC, until they reached appro-ximately 10% humidity. The weight was checked on an analytical balance every hour for the first 8 to 124 hours, and every 12 hours thereafter. The loss of water from the seeds was monitored using the mass difference calculation proposed by Sacandé et al [24].
In the third stage, the seeds that reached low water contents were stored in a freezer set at -20ºC for three months in airtight plastic bags. All stages were tested for viability using a germination test in a B.O.D. set at 20ºC for S. reitzii, both on blotting paper, according to the results of the previous tests. Based on the germination rate obtained, the seeds were classified as orthodox, intermediate or recalcitrant.
Data analysis
The germination and seed physiology tests were analyzed using the R statistical program with the “ExpDesp” package [25], which tested the normality and homogeneity of the data obtained in each of the analysis stages. The Entirely Randomized Design (ICD) was used for the germination analysis. After obtaining the data, normality was assessed using the Shapiro-Wilk test. Analyses of variance were carried out and mean tests were conducted (Tukey at 5% probability). Given the assumptions of normality for physiological quality in storage and germination, the data was submitted to analysis of variance and the means to the t-test at 5% probability.
Results
Initial characterization and testing of temperatures and substrates
The moisture content obtained for the S. reitzii seeds was 34.2%. It should be noted that all the seeds evaluated showed no apparent predation, damage, parasites or empty propagules and all the germinated seeds formed normal seedlings, while the non-germinated seeds showed rot (Figure 1).
The test with S. reitzii seeds showed an interaction between germination and the substrates tested. The highest average germination occurred on blotting paper at 20ºC, followed by sand at 20 and 25ºC and blotting paper at 25ºC (Table 1; Figure 1D).
The treatments using roll paper rolls did not show satisfactory germination results (Table1). The MGT showed that germination occurs in 11 days, while the germination speed index (GVI) was not significant for all treatments.
Physiological classification
S. reitzii seeds had a water content of 34.2% and a germination percentage of up to 69% before drying. After drying (to approximately 11% humidity in 4 days) the seeds completely lost their viability, as indicated by the germination test (Tab. 2).
Since the seeds do not germinate, they lost viability after drying and were not subjected to freezing according to the protocol.
Discussion
The satisfactory germination percentage of S. reiztii reinforces the fact that its seeds germinate quickly, with no major impediments to radicle emission, such as dormancy or low physiological quality, in a pattern similar to that observed in Myrceugenia euosma [26]. Its water content is similar to that obtained by other Myrtaceae, such as Eugenia rostrifolia D. Legrand (34%) and Campomanesia xanthocarpa (Mart.) O.Berg (33%) [27]. It should also be noted that the humidity found for its seeds is a common characteristic in other Myrtaceae [19].
The temperature of 20ºC favored a higher germination percentage when blotting paper was used, indicating a possible relationship with the availability of moisture provided by this substrate, which also contributed to higher germination in Blepharocalyx salicifolius (Kunth) O.Berg (Myrtaceae) seeds [28]. The use of sand had no statistically significant effect on the germination percentage at 20 and 25ºC. This suggests that sand may have varied humidity between treatments due to its drainage capacity, which is unsuitable for fast-germinating seeds, as observed in other Myrtaceae species [29][19][30][31].
On the other hand, the use of paper rolls is not recommended for this species due to the low germination values observed, possibly due to the low light intensity and the availability of moisture for the seeds, which also affected the germination of Eugenia umbellata Spreng. seeds [29]. In addition, at a temperature of 25ºC, a decrease in germination was observed compared to 20ºC, perhaps related to the physiological characteristics of the seeds in the face of the exposed temperature. This species is native to places with high altitudes and low temperatures [١٠]. In fact, the municipality of Urupema, Santa Cataria State, where the seeds were collected, has average temperatures that oscillate around 18ºC during the summer, when the ripe fruits were collected. Therefore, it can be inferred that the ideal temperature for the germination of S. reitzii is related to its region of origin, as observed for Eugenia involucrata [19].
The ability to germinate at temperatures close to local averages, the quality of the seeds and the adaptation to different types of substrates suggest an ecological plasticity that can be exploited in conservation and propagation programs for the species [26]. Furthermore, understanding the optimum germination conditions can facilitate the production of seedlings and expand the cultivation of S. reitzii in nurseries, contributing to initiatives on its conservation and sustainable use [1][2].
The low tolerance of S. reitzii seeds to desiccation at 10-12% moisture allows them to be classified as recalcitrant according to their physiological performance in storage, and Myrtaceae often includes several recalcitrant species [32] [33]. The causes of desiccation intolerance may be related to several factors, including the accumulation of insoluble reserves, cellular dedifferentiation, the efficiency of the antioxidant system and the accumulation of protective proteins and oligosaccharides, as well as a disorder in membrane repair metabolism [34][32]. This initial parameter can inform future research into the critical and lethal degree of humidity and points to the need for specific precautions when storing these seeds.
Conclusion
Siphoneugena reitzii seeds showed a response to the substrates and temperatures tested, with the highest germination on blotting paper at 20ºC, followed by moderate germination in sand at 20 and 25ºC. The seeds germinate quickly, have no apparent problems with empty seeds or predation and form normal seedlings, demonstrating the viability and potential of this species for propagation. However, the seeds are recalcitrant, losing their viability completely during desiccation at approximately 10% humidity.
Acknowledgements
The authors would like to thank CAPES/PROAP-AUXPE, CNPq, FAPESC - 2021TR787, FIEPE CAV, FUMDES/UNIEDU, and Klabin SA for their financial support. The first author would like to thank the “Programa de Pós-graduação em Produção Vegetal (PPGPV) of “Universidade do Estado de Santa Catarina (UDESC)”, Mireli Moura Pitz Floriani (RPPN Serra da Farofa) and professors Dr. Adriano Valentin, Dr. Christopher Thomas Blum, Dr. Jaçanan Eloisa de Freitas Milani for their comments. The authors are also grateful to the editors and anonymous reviewers.
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