GERMINATION AND POST-SEMINAL DEVELOPMENT OF Melaleuca alternifolia (MAIDEN & BETCHE) CHEEL

There is little information regarding the germination pattern, seed characterization, and seedling development of Melaleuca alternifolia. This study aimed to determine the ideal temperature for the germination of M. alternifolia seeds, as well as to characterize the seeds and the post-seminal development of the species. Three lots of M. alternifolia seeds were placed to germinate at 20, 25, 20-30, 20-35, 30, and 35 °C, with daily evaluations to determine germination rate, germination speed index (GSI), speed of germination (SG), and mean time for germination of 50% (t50) and 100% (t100) of seeds. The inner morphology of the seeds was evaluated through X-ray images and seedling morphology by photographs. Alternating temperatures (20-30 and 20-35 °C) led to germination rates higher than the other temperatures. The GSI had the lowest values at the constant temperature of 20 °C and the highest values at the constant 30 °C for all the lots. The SG was lowest at a temperature of 20 °C and the highest at the temperature of 35 °C. At alternating temperatures (20-30 and 20-35 °C), t50 was around 5 days and t100 reached 16 days. In conclusion, the alternating temperatures of 20-30 °C and 20-35 °C are recommended for germination of M. alternifolia, and fi nal evaluation can be performed at 16 days after sowing. The inner structures of seeds could be visualized by X-ray analysis, and full seeds could be distinguished from empty seeds and impurities. Seeds exhibit epigeal germination and seedling development is stabilized after 30 days.


1.INTRODUCTION
The Melaleuca genus belongs to the Myrtaceae family and is made up of around 230 species (Amri et al., 2012). Among them, Melaleuca alternifolia (Maiden & Betche) Cheel, native to Australia, is considered one of the most important species of the genus, due to the extensive length of time it has been used at its place of origin (Sharifi -Rad et al., 2017). It is an arboreal species that can reach a mean height of six meters and is mainly used for extraction of essential oil from its leaves, popularly known as "tea tree oil" (Chen et al., 2016). Recent studies cite diverse traits of and uses for "tea tree oil", such as antifungal and antibacterial activity , insect repellence (Yim et al., 2016), healing properties (Han and Parker, 2017), and others.
Germination is considered a fundamental step in crop establishment within the context of a whole plant cycle. During this process, a mature seed can be highly sensitive to environmental factors, which will aff ect mobilization of reserves, gene regulation, protein synthesis, and other important components of the process (Bewley et al., 2013;Finch-Savage and Bassel, 2016). Temperature is considered one of the main factors in germination, since it aff ects both germination percentage and speed. The thermal limits for germination are defi ned by the cardinal temperatures (optimum, maximum, and minimum), which determine the ecological limitations for geographic distribution and establishment of the species (Dürr et al., 2015;Marcos-Filho, 2016;Daibes and Cardoso, 2018). In general, temperatures outside the range considered optimal for a species promote or inhibit important biochemical processes, such as glycolysis and the tricarboxylic acid cycle, as well as the activity of various enzymes and other processes that are directly related to the germination capacity of dormant and nondormant seeds (Bewley et al., 2013;Xia et al., 2018).
Since M. alternifolia seeds are extremely small and diffi cult to handle, information regarding the pattern of their germination and the eff ects of temperature on this process is very limited in the literature. M. alternifolia germination has been evaluated without, however, evaluating the eff ect of diff erent temperatures (Anselmini et al., 2010). The eff ect of temperature and of light on the species Melaleuca quinquenervia has also been evaluated, in which the temperature of 27.3 °C allowed maximum germination in the shortest period of time, and it is considered ideal for that species (Martins et al., 2013).
Another important factor for seedling production and plant establishment under natural conditions is knowledge regarding the morphological aspects of seeds and seedlings (Abud et al., 2010). In regard to seeds, the use of non-destructive analyses, such as the X-ray test, has been documented as eff ective for studies of internal morphology of seeds of diverse species, such as Moquiniastrum polymorphum (Faria et al., 2019), Moringa oleifera (Noronha et al., 2018), and Leucaena leucocephala , among others. This technique assists in identifi cation of full seeds, empty seeds, mechanical damage, and spots that indicate deterioration, characteristics that can aff ect seed physiological potential (Marchi and Gomes Junior, 2017), and, consequently, the quality of seed lots.
Information regarding morphology of seedlings in the initial stages of development are also relevant for interpretation and standardization for correct evaluation of seed physiological quality (Abud et al., 2010), and they also contribute to identifi cation of the species under natural conditions.
In light of the above and since information for this species is quite limited, the aim of this study was to evaluate the pattern of germination and defi ne adequate temperature for germination of M. alternifolia seeds, as well as characterize the seeds and the post-seminal development of the species.

2.MATERIALS AND METHODS
The study was conducted in 2017 in the Seed Analysis Laboratory of the Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. Three lots of M. alternifolia seeds were used, collected in the municipality of Coimbra, Minas Gerais, Brazil (20º51'24"S, 42º48'10"W, 720 m). Climate in the region is classifi ed as hot and temperate, with mean temperature of 20.1°C and mean rainfall of 1283 mm. The seed lots corresponded to three groups located in diff erent areas of the municipality and were constituted by seeds from at least fi ve diff erent mother plants each. After harvest, the seeds were dried in the shade and processed using sieves for removal of larger impurities. After that, they were stored at 20 °C for approximately three months, up to the time of evaluations.
For evaluation of the eff ect of temperature on germination, the seeds of the three lots were distributed on two sheets of paper towel in 15-cm-diameter Petri dishes. The paper was moistened with distilled water at the volume of 2.5 times the weight of the dry paper.
Due to the diffi culty of defi ning a standard for normal seedlings for evaluation of the test, the number of germinated seeds (root protrusion) was considered through daily counts, up to stabilization of the counts of all the treatments. The daily germination evaluations were performed with the aid of a tabletop magnifying glass with 8x magnifi cation, and the results were expressed in germinated seeds per 0.0250 g of seeds.
The data of daily germination counts carried out for the best temperatures were evaluated to obtain the following variables: germination speed index (GSI) (Maguire, 1962), speed of germination (SG) (Edmond and Drapala, 1958), t50 (time for 50% germination of the lot) and t100 (time for 100% germination of the lot).
For analyses of inner seed morphology, radiographic images were generated using the Faxitron MX-20 device (Faxitron X-ray Corp. Wheeling, IL, U.S.A). Seeds were placed under radiation for 10 seconds at 32 kV and a focal distance of 14 cm. The digital images generated were saved on a computer and used for descriptive identifi cation of the physical components (full seeds, empty seeds, and impurities) contained in the sample.
Evaluation of post-seminal development was conducted together with germination. Seeds and seedlings in diff erent stages were selected and photographed under high resolution. The main structures of the seeds and seedlings were identifi ed through the photographs.
Each experimental unit was composed of a Petri dish containing 0.0250 g of seed. Analysis of variance was performed on the data. The mean values were compared by the Tukey test at 5% probability with assistance of the R 3.5.1 software.

3.RESULTS
Analysis of germination of M. alternifolia over time showed that, for all the lots, alternating temperatures (20-30 and 20-35 °C) led to germination rates higher than the rates from the other treatments, especially from the fi fth day after sowing on. In contrast, the constant temperatures of 20 °C and 35 °C generally led to lower germination rates. The temperatures of 25 °C and 30 °C generally exhibited intermediate germination rates compared to the other treatments (Figure 1).
The temperature of 20 °C resulted in slower germination in all the seed lots, with germination mainly increasing as of the eighth day after sowing. At the other temperatures, this accentuated increase occurred around the fourth day. In lot 1, the temperature of 20-30 °C tended to be superior to 20-35 o C ( Figure 1A). In lot 2, this response was the opposite (greater germination at the temperature of 20-35 o C) ( Figure 1B), and in lot 3, these treatments led to a similar response ( Figure 1C).
Evaluation of the fi nal germination of the three lots analyzed shows that the performance or response of each one of the lots was maintained at all the temperatures evaluated -lot 1 had germination greater than the others and lot 3 was lower than lots 1 and 2 ( Figure 2).
Alternating temperatures (20-30 and 20-35 °C) led to higher germination rates and did not diff er from each other. In the three seed lots analyzed, germination at the temperature of 35 °C was statistically lower than at the other temperatures analyzed (Figure 2A), contributing to reduction in M. alternifolia seed germination and indicating that 35 °C is above the temperature considered optimal for the species (Figure 2).
The germination speed index (GSI) had the lowest values at the constant temperature of 20 °C and the highest values at the constant temperature of 30°C for the three lots analyzed ( Figure 3A). Speed of germination (SG), which represents the number of days necessary to germinate, was generally lower (greater number of days) at the temperature of 20 °C and greater (fewer number of days) at the temperature of 35 °C, respectively ( Figure 3B).
From analysis of internal morphology of M. alternifolia seeds by the X-ray test, it can be seen that power adjusted to 32 kV, exposure time of 10 seconds, and focal distance of 14 cm allowed visualization of internal seed structures and diff erentiation of full seed and empty seed groups, as well as identifi cation of impurities in the sample ( Figure 5).
Seedlings of M. alternifolia in their initial stage of development up to 30 days after sowing (when development stabilized) under the alternating temperatures of 20-30 °C are shown in Figure 5D. Seed germination was found to be of the epigeal type, and the seed coat adheres to the cotyledons in the initial phase of seedling growth, where the hypocotyl-radicle axis emerges from the substrate. The two cotyledons emerge and expand. The normal seedling has a robust main root and few secondary roots. The hypocotyl is of a greenishwhite color and cylindrical, and the partially expanded cotyledons expose the convex surface, exhibiting epigeal germination.

4.DISCUSSION
The lower germination rates observed at the temperatures of 20 °C and 35 °C indicate that these temperatures inhibited germination of M. alternifolia, probably for being outside the optimum range for this species. Similar results were observed in Dalbergia odorifera seeds, for which the germination rate declined both at 15 °C and at 35 °C (Liu et al., 2017). In contrast, the ideal temperature for germination of Melaleuca ericifolia was approximately 20 °C, showing the diff erence in response even between species of the same genus (Robinson et al., 2006).
In general, temperatures below those considered optimal reduce speed of germination and lead to  physiological changes in the seeds, such as deactivation of gibberellin (GA), abscisic acid (ABA) synthesis, reduction in starch hydrolysis, and other processes that, in combination, contribute to reduction in germination . In germination of the species Paeonia ostii, it was observed that in addition to eff ects related to ABA induction and lower starch degradation, low temperature induced lower protein synthesis and transcription of genes involved in the germination process (Ren et al., 2018). In addition, lower speed of germination induced by low temperatures increases the possibility of infestation by fungi, due to greater exposure time of seeds to the environment they are in (Souza et al., 2016). In a way similar to the results observed in this study, Melaleuca quinquenervia exhibited reduction in germination under 35 °C and 40 °C (Martins et al., 2013). In general, temperatures above the ideal lead to greater speed of germination, yet, at the same time, reduce the percentage of germination and vigor through diverse physiological, biochemical, and genetic processes (Bita and Gerats, 2013). Among their deleterious eff ects, high temperatures can suppress germination of dormant or non-dormant seeds through thermoinhibition, which, for its part, is related to expression of genes involved in ABA biosynthesis (Kendall and Penfi eld, 2012). In addition, high temperatures contribute to an increase in respiratory rates and, consequently, production of reactive species, which can cause irreversible physiological damage, such as denaturation of proteins and enzymatic inhibition (Mittler, 2017). High temperatures may also aff ect seedling development in relation to stomatal density, as observed in Melaleuca lanceolata plants (Hill et al., 2014).
It is important to emphasize that seeking to maximize seedling production, as well as a satisfactory germination rate, one of the main objectives is to obtain fast and uniform germination, which, for its part, will be optimized under optimal temperature conditions (Rajjou et al., 2012;Martins et al., 2013). In this context, in spite of leading to the best results for GSI and SG (Figure 3), the constant temperatures of 30 °C and 35 °C cannot be considered ideal for the germination of M. alternifolia since they led to lower rates of germination compared to the alternating temperatures ( Figure 2). Unlike that observed here, the constant temperature of 27.3 °C was considered ideal for germination of Melaleuca quinquinervia (Martins et al., 2013). Nevertheless, these authors did not evaluate alternating temperatures and diff erent seed lots.
Thus, alternating temperatures (20-30 and 20-35 °C) were selected as most adequate for germination of M. alternifolia, since in a general context, they combined greater percentage and greater speed of germination (Figures 2 and 3). Seeds of Mauritia fl exuosa also germinated better under alternating temperatures, and their eff ects may be related to enzymatic mechanisms capable of being activated at diff erent temperatures (Almeida et al., 2018).
In addition to confi rming the similarity of results for the temperatures of 20-30 and 20-35 °C, there is an indication that fi nal evaluation of M. alternifolia germination can be carried out at around the sixteenth day after sowing, since there were no increases in germination of the three seed lots analyzed from that time on, at either of the temperatures ( Figure 4C and D).
Temperature is also considered an important environmental factor related to the diff erent dormancy mechanisms. MacGregor et al. (2015) observed that in Arabidopsis seeds, low temperatures caused an increase in gene expression of enzymes and compounds related to breaking dormancy. In other species, such as Carex diandra (Fernández-Pascual et al., 2015) and Anisantha rubens (Jiménez-Alfaro et al., 2018), it has been shown that alternating temperatures increase germination through breaking dormancy, which, for its part, is mainly related to transcription of genes involved in metabolism, plant hormones, and the circadian clock (Foley et al., 2010). However, it is important to reinforce that the aim of the present study was not to evaluate dormancy and, thus, the results observed may serve as a tool for future evaluations.
The application of the X-ray test in evaluation of seed quality is interesting as a precise method, where seeds may be examined individually in expanded images that are able to indicate, in detail, the location and extent of damaged or altered structures (Cicero, 2010). Recent studies carried out with Moquiniastrum polymorphum (Faria et al., 2019), Leucaena leucocephala , and Senna multijuga (Marchi and Gomes Junior, 2017) also showed effi cient application of the use of radiographic images for analysis of internal seed morphology. Although the option in this study was for more descriptive evaluation regarding aspects related to seed radiographic images, the study is relevant because it deals with introduction of the technique in the species in a pioneering way and can serve as a basis for future studies. The growth of M. alternifolia seedlings stabilized after 30 days under the condition of 20-30 °C, probably requiring transfer to substrate beginning at this time. This information is important mainly in regard to optimization of the seedling production process, aiming at higher rates and speed of germination.
Based on the results observed and due to the limited amount of information regarding seed characteristics and initial seedling development of M. alternifolia, this study may assist future research applied to seed technology of this species.

5.CONCLUSIONS
Alternating temperatures of 20-30 and 20-35 °C with 8-h photoperiod are recommended for germination of M. alternifolia, leading to higher rates and greater speeds of germination. Under these temperatures, fi nal evaluation of germination can be carried out at 16 days after sowing. X-ray images allow identifi cation of full seeds, empty seeds, and impurities contained in M. alternifolia seed lots and can serve as a support for development of automated analyses and assist in selection of lots with a higher level of purity. Germination of M. alternifolia seeds is epigeal, and seedlings exhibit stabilization of development after 30 days under alternating temperature of 20-30 °C.