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RESPONSE OF Lupinus bracteolarisSEEDS TO PRE-GERMINATIVE TREATMENTS AND EXPERIMENTAL CONDITIONS
Abstract
Contextualization: Lupinus bracteolaris is a heliophile species that vegetates lowlands, open slopes and sandstone hills.
Knowledge gap: Despite this great rusticity, there is little information about the species and its propagation, especially regarding the germination of its seeds.
Purpose: Evaluate the seeds of Lupinus bracteolaris, through pre-germinative treatments, substrate, temperature, luminosity, and water absorption, to understand the initial germination processes of the species.
Methodology: Fruits were collected from 20 matrix plants, then the seeds were processed and submitted to the following tests: Pre-germinative methods (six treatments at 25 °C); substrates and temperatures (sand and paper at 20, 25 and 30 °C); photoblast test (white light, diffuse-green light and continuous dark under 20 °C) and the imbibition curve (control and scarification between sandpaper under 20 °C). Evaluations: Percentage of germination and formed seedlings, germination speed index, average germination time and seedling formation, using a completely randomized design.
Results and conclusions: The results of the analyzed variables showed relevance for the pre-germinative treatment between sandpapers for 20 seconds, in addition, the substrate paper Germibox and the temperature of 20 °C were the most suitable during the germination of the seeds of Lupinus bracteolaris, which were classified as neutral photoblastics and without dormancy.
Main Text
1. INTRODUCTION
Lupinus bracteolaris Desr. (= L. czermakii Micheli) (Fabaceae) is an annual species popularly known as native lupine, with erect growth of 30 to 40 cm in height. It vegetates in places in full sun, granite and sandstone hills, lowlands and road slopes (Pinheiro and Miotto, 2001). It presents stem and leaf structures with many trichomes, favoring their development in places with low water availability, in addition to having active nodules in the roots; therefore, it shows the symbiosis with nitrogen-fixing bacteria. Due to these characteristics, it is assumed that the species has developed important strategies to survive in poor-sandy soils and adjacent to areas affected by sanding, which makes it promising for revegetation projects.
Similar to the species Lupinus albescens Hook. & Arn., also native to the region of Rio Grande do Sul State (Paim et al., 2019), Lupinus bracteolaris can also be found in its natural habitat under different phenological phases; that is, some plants with the presence of flowers or fruits and/or seeds in the same period, which according to Cremonez et al. (2013), results in the formation of irregular populations. There is little information available in the literature about this species, especially on its propagation and regarding seed germination. To allow recommendations for the application of plants of this species in the field, or for seedling production, studies on the need for pre-germinative treatments and environmental conditions that accelerate and standardize the initial germination processes are necessary.
Lupinus bracteolaris presents seeds with a hard and resistant tegument, therefore, it is believed that the use of pre-germinative treatments may favor the increase of their permeability to water and gases, according to Guedes et al. (2013), allowing the beginning of germination processes. Pre-germination treatments contribute not only to rapid seed germination, but also to a higher efficiency in the uniformity of seedling establishment in adverse environments (Santos et al., 2011). In addition, the proper use of substrate and temperature in seed germination may favor the speed and percentage of germinated seeds, because the substrate acts to retain sufficient amounts of water and oxygen, and the temperature activates certain biochemical reactions during germination processes (Ministério da Agricultura, 2009; Taiz et al., 2017). According to Marcos Filho (2015), the correct knowledge of these factors will result in the expression of the maximum germination potential in a shorter period of time.
Besides humidity and temperature, the presence and intensity of light is of great influence on the life cycle of plants. Those factors directly affect the percentage and speed of germinated seeds (Cosmo et al., 2017). The plants can be positively or negatively influenced by light. Thus, the seeds can be classified as aphotoblastic or neutral (germination indifferent to light), positive photoblastic (greater germination under light) and negative photoblastic (greater germination in the dark) (Kerbauy, 2008). Therefore, it is essential to use methods adjusted for each species, since this may optimize propagation, reducing emergence failures and/or maximizing seedling formation (Lessa et al., 2013)
Specific studies on pre-germination methods, substrates, temperatures, luminosity and water absorption by seeds are extremely important, especially in species that are little explored and that may act in the restoration of degraded environments, as most native species. As a result, the objective of this work was to evaluate the seeds of Lupinus bracteolaris, through pre-germinative treatments, substrate, temperature, luminosity, and water absorption, to understand the initial germination processes of the species.
2. MATERIALSAND METHODS
Seed collection and processing
In December 2015, Lupinus bracteolaris fruits (Figure 1A) were collected from approximately 20 matrix plants in situ, close to an arenization core (Figure 1B) in the municipality of São Francisco de Assis, State of Rio Grande do Sul (29°35’29.19” S latitude and 55°22’10.41” W longitude) (Figure 1C). The weather in the region is characterized as humid subtropical of the Cfa-type, according to the classification of Köppen, with an average annual rainfall of 1,750 mm, well distributed. The average temperature of the coldest month varies between -3 and 18 °C, and the warmest month it exceeds 22 °C (Alvares et al., 2013).
After the collection, the fruits were taken to the Horticulture Biotechnology Laboratory of the Department of Horticulture and Silviculture of the Federal University of Rio Grande do Sul (UFRGS) in Porto Alegre, RS. They were placed on a greenhouse bench with 70 % of light interception, without temperature and relative humidity control, for drying and finishing dehiscence. At the end of the process, the seeds were processed through manual collection and homogenization in only one batch.
This batch of seeds was submitted to water content evaluation, in three 0.5 g replicates, using the method of percentage difference in mass after drying in an oven at 105 ± 3 ºC until constant weight (Ministério da Agricultura, 2009). Later, during the execution of the tests, the seeds were packed in 2.0 L transparent polyethylene bags, with a zip-type closure system, each bag with about 500 mL of seeds. Then, the seeds remained at a temperature of 5 to 8 °C, in a household fridge.
In all tests, before sowing, the seeds were submitted to the disinfestation process through immersion in 70 % (v/v) ethanol for 30 seconds, followed by sodium hypochlorite 2.0 % (a.i.), with 0.01 % concentrated detergent (Tween 20®), for 10 minutes. To remove residues from these disinfestation agents, three rinses were performed in autoclaved deionized water.
Pre-germinative treatments
The seeds were subjected to six treatments: Control (T1); mechanical scarification between sandpaper No. 120 for 20 and 40 seconds (T2 and T3); immersion in water (500 ml) for 24 hours at an ambient temperature of ± 25 ºC (T4); thermal shock in water at 50 ºC and 100 ºC for 10 minutes until cool in an ambient temperature of ± 25 ºC (T5 and T6). Subsequently, the seeds were placed to germinate in a BOD-type incubator chamber with a constant temperature of 25 °C.
The pre-germinative treatment by the method of mechanical scarification between sandpaper No. 120 followed the methodology recommended for Lupinus albescens native species in the work of Paim et al. (2019).
Substrates and temperatures
The seeds used in this test were previously treated with mechanical scarification between sandpaper No. 120 for 20 seconds, due to its easy execution and the highlight for most of the analyzed variables, after which they were sown on the substrates sand and paper. These treatments were carried out at temperatures of 20, 25 and 30 ºC, in a BOD-type incubator chamber.
Photoblastic test
Seeds scarified between sandpaper No. 120 for 20 seconds were sown on paper and kept at a constant temperature of 20 ºC in a BOD-type incubator chamber. The light conditions were: White light, diffuse green light (with an intensity of 1,300 lux) and absence of light. In light treatments, the irradiance was approximately 30 μmolm-2s-1. The white light was provided by fluorescent lamps (30 W), the diffuse green light was obtained by wrapping the transparent-plastic Gerboxes with green cellophane paper (double layer, juxtaposed), while in the treatment with absence of light, the boxes were wrapped in aluminum foil and packed in a cardboard box, aiming to totally prevent the incidence of light in the seeds during the germination process.
Imbibition curve
Seeds submitted to the control treatments (without scarification) and mechanical scarification between sandpaper No. 120 for 20 seconds were sown on paper and taken to the environment with a constant temperature of 20 ºC in a BOD-type incubator chamber.
During the imbibition monitoring, the seeds were evaluated by means of successive weighing every 2 hours, for the first 12 hours and every 12 hours until 50 % of the seeds of each repetition showed radicle protrusion. At each weighing, the seeds were removed from the Gerboxes, placed on paper towels to remove the excess of water and weighed on a precision scale (0.001 g capacity). After weighing, the seeds returned to their respective boxes and remained under a controlled environment. After the last weighing, the water content of the seeds was determined (Ministério da Agricultura, 2009).
The percentage increase in mass in relation to the initial mass, for each evaluation time was calculated through the formula:
Formula 1
The sowing of the tests above occurred on Germibox paper, with the exception of the substrate by temperature test, which was on sand. In each replicate, a sheet (previously sterilized in an autoclave for 20 minutes) was used, moistened with autoclaved deionized water at the proportion of 2.5 times the dry paper mass (Ministério da Agricultura, 2009).
The papers were packed in transparent plastic Gerbox, which were individually arranged in transparent polyethylene bags, that is, to avoid excessive respiration. When sand was used as a substrate, it was previously autoclaved and at the time of sowing, hydrated with autoclaved deionized water up to 60 % of the water holding capacity (Ministério da Agricultura, 2009).
All experiments were conducted under a 16-hour photoperiod of light in incubator chambers of the BOD type (Biochemical Oxygen Demand). The tests described below are in the chronological phase of execution.
The evaluations of all the tests described above consisted of the daily count of the number of germinated seeds and normal seedlings formed, except in the treatment with absence of light from the photoblast test, which was developed at the end of the experiment. The parameter used in germination was the protrusion of the radicle and in the formation of seedlings, those that had a normal aerial part and root system formed (Ministério da Agricultura, 2009).
At the end of the analysis period, when in three consecutive evaluations there was no emergence and seedling formation, approximately on the 18th day, after the installation of the experiment, the following variables were calculated: Germination percentage (G) and formed seedlings (FS), according to the total of normal seedlings; the germination speed index (GSI), according to the formula suggested by Maguire (1962); the average germination time (AGT) and the average seedling formation time (SFT), according to the formula proposed by Silva and Nakagawa (1995). The calculated variables were as follows:
Formula 2
Where: N = Number of seeds germinated at the end of the test. Unity: %.
Formula 3
Where: ni = number of seeds that germinated in time 'i'; ti = time after installation of the test; i = 1 → 18 days. Unity: Dimensionless.
Formula 4:
Where: ni = number of seedsgerminated per day; ti = incubation time; i = 1 → 18 days. Unit: Days.
The experimental design used in this study was the completely randomized (CRD), in a single-factor scheme for the tests of pre-germinative treatments, photoblastism and water absorption curve. The substrate and temperature test were performed in a two-factor arrangement (2 × 3), where the first factor was the substrates sand and paper, and the second the temperatures of 20, 25 and 30 ºC. For all the tests above, four replicates of 25 seeds were used, totaling 100 seeds per treatment, with the exception of the photoblast test, in which five replicates were used.
Statistical Analysis
The data were submitted to Bartlett's normality test and, after analysis of variance (ANOVA), followed by comparison of means through the MSD test (Minimum Significant Difference) at the level of 5 % probability of error, using the features of CoStat 6.4 software. Being that, when the tests were performed with two factors, the analysis of variance was tested to verify the interaction between the factors.
3. RESULTS AND DISCUSSION
On the day following the collection, the seeds of Lupinus bracteolaris had a water content of 6.82 %. Thus, the seeds from the field showed a very low value, possibly indicating that the degree of seed maturity was nearing completion. In the laboratory, the completion of the seed maturation process was observed in only 15 days, by changing the color of the vegetables from gray-yellow to totally dark gray, in addition to opening all the vegetables and releasing the seeds. These results show the importance of determining the water content, in order to assist in the interpretation of the maturation phases of the seeds collected in the field. Particularly, in the case of species with little information available in the literature, as is the case of Lupinus bracteolaris, and mainly because the seeds collected in natural populations are in different phenological stages, some plants with the presence of flowers or fruits and/or seeds, providing irregular phases within the same population.
Pre-germinative treatments
The first study carried out to accelerate the germination phase of Lupinus bracteolaris seeds showed superior results in the T2 treatment (mechanical scarification with sandpaper No. 120, for 20 seconds), for the variables: Germination percentage, seedling formation and speed index germination (Table 1) but did not differ statistically from treatment T3 (mechanical scarification with sandpaper No. 120 for 40 seconds).
In other species from the same family, the method of mechanical scarification with sandpaper also showed superior results in the seed germination processes, as well as a high rate of germination speed, as for Schizolobium parahyba (Vell.) Blake (Pereira et al., 2011) and Lupinus albescens (Paim et al., 2019). This is mainly due to the fact that mechanical scarification causes cracks in the tegument of the seeds, therefore increasing the permeability to water and gases, promoting the beginning of imbibition processes and, consequently, germination (Carvalho and Nakagawa, 2012; Guedes et al., 2013).
Thus, seed scarification becomes a highly relevant method to standardize and increase germination speed. In addition, it is an important technique in the germination of other native species, such as Macroptilium martii Benth. (Araújo et al., 2014a) and Lupinus albescens (Paim et al., 2019), Fabaceae species that inhabit extreme areas of low water availability and poor soils in the Caatinga and Pampa biomes, respectively.
The comparison between the results of the seed treatments, with the locals of occurrence of the species Lupinus bracteolaris and Lupinus albescens (Paim et al., 2019), that is, sandy soils of arenitic origin, shows that the results found, under the ecophysiological aspect, corroborate the work of Rovedder et al. (2010), in which the authors mention that the propagules dispersed in these environments are susceptible to the action of wind and water erosive processes, which contributes to the rupture in the seed tegument, simulating a mechanical scarification resulting from the sand abrasion effects.
The average time of germination and seedling formation showed shorter periods for treatment T4 (immersion in water for 24 hours at room temperature) (Table 1); nevertheless, the application of this treatment becomes impracticable, since the average of germinated seeds was only 20 %. A shorter average germination time is justified in the T4 treatment due to the fact that the few seeds that germinated in a short period of time had a permeable tegument. Because the treatment was not effective in overcoming the physical barrier to germination, the seeds able to germinate were pre-soaked and started the germination process during the treatment.
According to the results obtained in the pre-germinative treatment test, where, for most of the variables analyzed, the treatment regarding scarification between sandpapers was superior, in addition to its easy execution, it was adopted as the standard for further experiments.
Substrates and temperatures
The analysis of variance showed interaction between the two factors for the variables of germination percentage and seedling formation, as well as for the average germination time (Table 2). For the germination speed index and the average time of seedling formation, there was no interaction between temperatures and substrates, with a significant effect only for these factors analyzed separately (Table 2).
The germination percentage did not differ significantly in the sand substrate for temperatures of 20 and 25 ºC, reaching 79 and 70 % of germinated seeds, respectively; this is also observed in the variable seedling formation, at both temperatures (Table 3). It was observed that the lower temperatures promoted the germination and the formation of seedlings in the sand substrate, because the highest temperature tested (30 °C) resulted in non-germinated seeds and seedlings with malformation; specifically, essential structures of the aerial part and root with reduced size.
In the case of the paper substrate, no significant differences were found between the temperatures tested, with an overall mean of 84 % for germination and seedling formation (Table 3). Likewise, the species Dioclea violaceae Mart. (Zucareli et al., 2010) presented similar results when the seeds were submitted to temperatures of 20, 25 and 30 ºC, using the paper substrate. As a result, the paper stood out as an important substrate for these species, as it provided germination at all temperatures evaluated, with no identification of critical temperatures at this stage. According to Pilau et al. (2012), the knowledge of temperature and substrate factors are fundamental in the beginning of germination processes, due to the peculiar characteristics among the species.
The average germination time is an easily measured variable that can be used to determine the vigor of the seeds, as according to Carvalho et al. (2012) a rapid germination and seedling emergence are important attributes for the formation of quality seedlings. For Lupinus bracteolaris seeds, there was a shorter average germination time in the sand substrate at 20 ºC (6.85 days), while on paper, the shortest time was for temperatures of 20 and 25 ºC, with an average of 5.91 and 6.15 days, respectively (Table 3).
In both substrates (sand and paper), it was found that the lowest temperatures (20 and 25 °C) promoted accelerated germination of seeds. It is assumed that mild temperatures may have triggered certain biochemical reactions during the germination process, which boosted specific enzyme systems (Marcos Filho, 2015).
The highest rate of germination speed of 5.79 was found for the paper substrate, in addition, this substrate also provided a shorter average time for seedling formation, with an average of 11.64 days (Table 4). The paper substrate also provided satisfactory germination speed index results in works with other species, such as for Albizia edwallii (Hoehne) Barneby & J. W. Grimes (Duarte et al., 2015) and Anadenanthera peregrina (L.) Speg. (Miranda et al., 2012).
Therefore, the paper substrate was more successful for a greater number of variables during the germination and seedling formation of the Lupinus bracteolaris species. It should be observed that the use of an appropriate substrate has a great influence on the germination of species, as is the case with paper, which is the most used in the tests and it has the advantage of maintaining constant humidity for long periods (Ministério da Agricultura, 2009). This is very important as the size of the seed can change the requirement for the amount of water, as well as the sensitivity or not to light and the ease in the course of seedling analysis (Ministério da Agricultura, 2009).
In relation to temperatures, the treatment of 20 ºC showed superior results for the variable germination speed index with a value of 5.40, moreover, this temperature also had a shorter average time of seedling formation, this being 11.25 days (Table 4), resulting in suitable conditions for Lupinus bracteolaris seeds to express their vigor. Knowing the optimal temperature in relation to the maximum or minimum becomes important, since extreme temperatures imply a reduction in the speed of germination processes, causing the exposure of seedlings to adverse factors for long periods, which can lead to reduction of the germination rate (Baskin and Baskin, 2014).
Low temperatures delay the germination and growth of seedlings, due to the reduction in the activities of enzymes involved in respiration and metabolism (Marcos Filho, 2015); However, in relation to Lupinus bracteolaris, laboratory results have shown that mild temperatures benefit the species, so it appears that in the field, its germination phase coincides naturally with a period of lower temperatures.
It was found that the temperature of 20 ºC was the most relevant and promising in the germination of Lupinus bracteolaris, in the same way as the use of the paper substrate. These two factors showed greater adequacy in the germination of the species, according to most of the variables analyzed and the visualization of fewer fungi in the seeds, resulting in the formation of healthy seedlings and of uniform size (aerial part and roots).
Photoblast
The results of the photoblast test did not highlight significant differences between treatments for the percentage of seed germination (Table 5). It is likely that the characteristics of the species promoted its germination in wide light conditions, thus providing the development of phenotypic plasticity, allowing to classify Lupinus bracteolaris seeds as neutral photoblastic. Similar results were also found for Lupinus albescens, another species native to the sands region (Paim et al., 2019).
Water absorption curve
Imbibition was observed in both the control and the pre-germinative treatments with mechanical scarification for 20 seconds; however, only the scarified seeds showed a higher mass gain in less time in the three stages of germination (Figure 2). Therefore, it was observed that only the seeds submitted to the pre-germinative treatment followed the three-phase pattern mentioned by Marcos Filho (2015), but the imbibition in the control treatment indicates the absence of physical dormancy in the seeds of the species.
The three-phase pattern presents important phases for the germination process, from water imbibition by the seeds to the beginning of the growth of the embryo. Thus, it was observed that after soaking, the scarified seeds absorbed water rapidly, reaching a level of approximately 12 % increase in mass, after 8 hours (phase I), about 67 % more than the control in this same period. This first imbibition phase is a consequence of the difference in water potential of the seed and the medium, this is a physical process in which the seed absorbs water for its rehydration until it reaches the equilibrium point, which does not depend on the viability of the seeds and the length of stay varies from species to species (Marcos Filho, 2015).
The second phase is known as stationary, preparatory biochemistry or growth-inducing, which will activate the degradation of reserves that will be used as an energy source for the reactivation of the embryo (Portes et al., 2018). There is usually a reduction in water absorption and in the respiratory processes (Araújo et al., 2014a) in this phase, as occurred in the Lupinus bracteolaris seeds which gained only 3 % in a period of 4 hours. Nevertheless, water absorption and mass gain were observed due to the treatment with mechanical scarification providing the opening of channels and the removal of the waxy layer, which promoted the entry of water at a greater speed, a fact that shows the lack of dormancy in the species seeds. According to Araújo et al. (2014b), seeds with dormancy may present this phase longer and with low mass gain.
The third phase started after 12 hours, with an increase in water absorption and a 60 % mass gain in the seeds in relation to phase II, consequently, beginning the protrusion of the radicle in this phase. Oliveira and Bosco (2013) corroborate that, at this stage there is an increase in the water content of the seeds and the emission of primary root. Therefore, the three-phase process of germination of Lupinus bracteolaris seeds was completed in 120 hours, when 50 % of the seeds of each repetition germinated.
The duration of each phase of germination can vary according to the tegument permeability and the size of the seed, as well as the conditions used during the imbibition process, such as temperature and substrate (Carvalho and Nakagawa, 2012). In view of this, the absorption curve showed that there was imbibition in both treatments, which allows us to conclude that the seed coat of Lupinus bracteolaris does not present impermeability, it just needs a longer period for the imbibition process. Thus, it is evident that the use of pre-germinative treatment with mechanical scarification accelerates the absorption of water, and, consequently, the germination processes. due to the opening of channels on the tegument. This method causes small fractures in the seed tegument, favoring greater permeability during imbibition and, thus, water absorption occurs more rapidly through tissues and speeds up the depletion of endosperm reserves (Shimizu et al. 2011).
Most species belonging to the Fabaceae family commonly develop seeds of impermeable and resistant integuments, acting as barriers in the absorption of water and subsequent biochemical reactions inside it. However, it is a condition that limits the germination of many plant species. Nevertheless, seeds that germinate in less than 28 days of sowing under natural conditions of the environment are not considered with integumentary dormancy (Baskin and Baskin, 2014). Therefore, the results of this work indicate that the seeds of the native species Lupinus bracteolaris have no tegument dormancy, due to the fact that germination occurs naturally in less than 28 days.
In addition, the different species belonging to the genus Lupinus (American, European and African) are considered as highly tolerant to dry habitats and poor nutrient soils (Elbandy and Rho, 2014; Martínez Alcalá et al. 2010). Therefore, some natives of this group can be denominated pioneers, as they are the first to colonize newly-impacted and/or disturbed soils; namely, soil with little vegetation and exposed to adverse edaphoclimatic factors (light, rain, wind, soil, etc.). This information is in line with observations made in this study, as the collection sites of Lupinus bracteolaris seeds are commonly sunny, low fields, slopes of roads with exposed soil and areas adjacent to sandy soils.
4. CONCLUSIONS
Lupinus bracteolaris shows a positive response to pre-germinative treatment through mechanical scarification of its seeds, indicating a limitation in water absorption due to the semipermeable resistance of the propagule casings.
For the analysis of its seeds, the substrate paper Germibox and the temperature of 20 ºC are the most appropriate among those tested, reaching the formation of normal seedlings about 11 days after the beginning of the test.
Lupinus bracteolaris seeds can be classified as neutral photoblastic and, although there is no tegument dormancy, water absorption is slow in the absence of a scarification treatment.
Abstract
Main Text
1. INTRODUCTION
2. MATERIALSAND METHODS
3. RESULTS AND DISCUSSION
4. CONCLUSIONS