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Using atmospheric plasma to increase wettability, imbibition and germination of physically dormant seeds of Mimosa Caesalpiniafolia
Colloids and Surfaces B: Biointerfaces 157 (2017) 280–285
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Protocols
Using atmospheric plasma to increase wettability, imbibition and germination of physically dormant seeds of Mimosa Caesalpiniafolia A.R.M. da Silva a , M.L. Farias a , D.L.S. da Silva a , J.O. Vitoriano a , R.C. de Sousa b , C. Alves-Junior a,∗ a b
LABPLASMA- Department of Exact and Natural Sciences, Federal Rural University of Semiarid, Mossoró, RN, CEP: 59625-900 Brazil, LCMM – Physics Department, Federal University of Maranhão São Luís, MA, CEP: 65080-805, Brazil
a r t i c l e
i n f o
Article history: Received 22 January 2017 Received in revised form 22 May 2017 Accepted 25 May 2017 Available online 1 June 2017 Keywords: Atmospheric plasma DBD Seed dormancy Impermeable integument
a b s t r a c t In this study, we analyzed seed wettability as well as imbibition and germination after treatment with atmospheric pressure cold plasma (APCP) using dielectric barrier discharge (DBD) in seeds that have very low germination rates. To aid industrial applications, several seeds were simultaneously treated with plasma within a space between two coaxial glass tubes sandwiched by two metal mesh screens that produced high-voltage pulses at 17.5 kV with a frequency of 990 Hz. Three treatment times (3 min, 9 min and 15 min) as well as untreated seeds were used to conduct the wettability, imbibition and germination tests. The wettability and imbibition were found to be directly related to the treatment duration, but saturation of the imbibition was found for treatment durations greater than 9 min. Plasma treatment was also effective in improving germination, but shorter treatment duration presented greater germination. This apparent contradiction is explained by the cell damage caused by the increased exposure to plasma, as observed in other studies. The results suggest that there must be an optimal wettability and imbibition condition that ensures that excessive moisture does not harm the germination process. © 2017 Elsevier B.V. All rights reserved.
1. Introduction The large-scale cultivation of species such as Mimosa caesalpiniafolia Benth is very important for ecosystems and agricultural areas in maintaining soil fertility and restoring degraded areas [1,2]. However, due to integument dormancy, viability is very low (less than 10%), making the culture unfeasible. The methods used to overcome seed dormancy and to improve germination rate generally consist of increasing wetting and soaking [3]. Physical (scarification) and chemical (use of sulfuric acid and hot water) treatments are commonly used in agriculture and have high environmental costs [4]. Reduction in vigor, higher infection rate due to structural damage, increased probability of abnormality in seedling growth shoots and production of poor fitomass, in addition to residues resulting from chemical treatments, are some examples of these problems [5–7]. In recent years, there has been a growing number of agricultural applications of plasma produced by dielectric barrier discharge (DBD), especially related to the inactivation of microorganisms and overcoming seed dormancy [8–10]. Since seed tissue rehydration is essential for metabolic activities, which
∗ Corresponding author. E-mail address: [email protected] (C. Alves-Junior). http://dx.doi.org/10.1016/j.colsurfb.2017.05.063 0927-7765/© 2017 Elsevier B.V. All rights reserved.
result in germination, plasma techniques must be able to modify the surface to increase seed soaking. The surface of Mimosa seeds has a complex structure that consists of an impermeable tegument (exotesta) containing small slits that communicate with mesotesta [11]. It has been reported that plasma species that are ‘attenuated’ by the coat do not have sufficient energy for the modification of the wettability of the internal sides of the biological surfaces [12]. However, several studies show that plasma can efficiently increase imbibition and germination [9,10]. The levels of GA3 hormone and mRNA expression (biochemical processes required for germination) of an amylolytic enzyme-related gene in seeds increased after treatment with high-voltage plasma pulses [13]. However, it is still unclear whether the seed viability can be changed when only the exotesta is modified by plasma. For most seeds, germination begins with a rapid initial water uptake that is higher on surfaces with more wettability. However, excessive moisture can decrease the absorption of oxygen and affect the germination process [14]. Strategies to enhance germination might involve wettability control on seed exotesta. In this paper, we investigate the response with respect to wettability, imbibition and germination after treating Mimosa caesalpiniafolia seeds using different durations of DBD plasma treatment. To facilitate future industrial applications, several seeds were simultaneously treated with plasma in a space between two
A.R.M. da Silva et al. / Colloids and Surfaces B: Biointerfaces 157 (2017) 280–285
coaxial glass tubes sandwiched by two metal mesh screens that were connected to a power source. 2. Materials and methods 2.1. Seeds morpho-anatomy Microstructures of the integument were analyzed using a scanning electron microscope and an optical microscope. For the analysis with the optical microscope, untreated seeds were soaked in a formalina-acetic acid alcohol (FAA) solution for 24 h, dehydrated in an ethanol process and embedded into paraffin blocks. The blocks were then cut into sections 10-m thick with a rotary microtome. Histochemical testing was performed using toluidine blue dye to reveal lignin and/or cellulose [15]. The images were recorded with a digital camera attached to the optical microscope.
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2.5. Imbibition test The seeds were soaked in plastic Gerbex-type boxes (capacity of 250 ml, dimension 11.0 × 11.0 × 3,5 cm3). The imbibition test began by placing the seeds between two germination papers, where a proportion of 3.0 ml of water was added to each gram of paper. For each test, 40 seeds were divided into 4 replicates of 10 seeds. The seeds were removed after 0, 4, 12, 24 and 36 h and weighed; the percentage increase in seed mass variation was determined by Eq. (2): %Mass =
mf − mi mi
x100
(2)
where mf is the mass of the imbibed seeds, and mi is the mass of the dry seeds. The test was conducted in a BOD type of germination chamber, temperature controlled at 25 ◦ C and a photoperiod of 8 h.
2.2. Plasma experimental apparatus
2.6. Germination test
The experimental apparatus is illustrated in Fig. 1A. The source plasma was equipped with two coaxial glass tubes, which were externally and internally coated by a metal mesh screen, and each glass tube had a wall that was 1-mm thick. The metal mesh screen was connected to a high-voltage power source with a peak value of 17.5 kV that pulsed at a frequency of 990 Hz. The power density (p) was determined with the aid of the Lissajous figure area (SL ), which was obtained by an oscilloscope using a 1000:1 probe (Tektronix P6015A, Tektronix, Beaverton, USA), by Eq. (1) [16]:
The germination capacity test was also carried out in plastic Gerbox boxes filled with sterilized sand. Prior to sowing the seeds, each box was prepared with 400 g of sand and 60 ml of distilled water. In each test, 100 seeds were divided into 4 replicates of 25 seeds. The germination of the DBD plasma-treated seeds were compared to the untreated seeds after 12 days. The experimental design was entirely carried out at random. The description of germination kinetics was obtained by fitting the experimental points to the Richard curve [18,19]. The Richards function (Yt), which has a variable inflection point, is represented by the Eq. (3):
p=
Pd f = A
V (t)dQ (t) A
f =
SL f A
(1)
where Pd is the dissipated electrical power; A is the area irradiated by plasma the area; SL is the area under the curve of the Lissajous; and V, Q, and f are the voltage, charge and frequency of the circuit, respectively. An optical fiber was used to transfer the light spectrum from the plasma to the CCD spectrometer (HR4000, Ocean Optics, Dunedin, FL, USA) – the spectral range is from 200 to 1000 nm – to measure the reactive species generated in the plasma. 2.3. Plasma treatment The slightly elliptical seeds, which have an average diameter of 6.0 mm, were placed between the two glass tubes within the plasma reactor. For the plasma treatment, around 100 seeds were uniformly distributed between the coaxial tubes at a time. Under these conditions, the plasma filled the spaces between the two glass tubes and evenly coated all of the seeds’ surfaces (Fig. 1B). The treatments were performed for durations of 3, 9 or 15 min, depending on the treatment group. After treatment, the seeds were stored in dissectors to test the wettability, imbibition and germination.
˛
Yt =
[1 + b + dx exp(−cxt)]
1⁄d
(3)
From this equation, the following parameters are obtained: Vi (viability), which is the total percentage of germination; Me (Median), which is the time of occurrence of 50% of the total germination. The Me parameter is very important for the description of the germination process and the initial growth of seedlings, as it distinguishes the behavior of the species or plant variety during germination. Qu indicates the dispersion of Me, and Sk (asymmetry) indicates the asymmetry in the frequency of distribution of the germination time, which represents the asymmetry of the Richards’ function relative to the inflection point [19,20]. 2.7. Statistical analysis The experimental design was completely randomized (DIC) for the analysis of variance (ANOVA). The Tukey (P < 0.05) average com® parison test was performed using Sisvar software. 3. Results and discussion
2.4. Wettability test
3.1. Dissipated power and active plasma species
Wettability tests were conducted using the sessile drop technique to measure the apparent contact angle of distilled water droplets. It was measured on 3 seeds under each condition [17]. The test was performed by dripping 20 l of distilled water once on each seed surface. The images were captured and recorded by a camera attached to a computer and processed to determine the ® contact angles using the Surftens 3.5 program. Descriptive statistics (arithmetic mean and standard deviation) were calculated for the values of the contact angles formed by the liquid and the integument.
Integrating the Lissajous figure in Fig. 2 and considering the area irradiated by plasma (A), which is equal to 0.006 m2 , the dissipated power density for a frequency of 990 Hz can be obtained using Eq. (3): −6
p = 1.07×10 ∗ 990 = 0.18w/m2 (3) A The optical spectra of the plasma (Fig. 3) was obtained using an optical fiber in front of the coaxials tubes, which transmitted to an optical emission spectrometer (OES) diagnostic of plasma species. The main peaks result from the excitation of the nitrogen molecules (N2) present in the air, which are all from the second pos-
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Fig. 1. (A) Experimental apparatus used for this experiment, (B) Details of the top view showing positioning of the seeds within the reactor.
itive system of N2, with different energetic levels. Peaks from the first positive system were also observed: N2 + apart from OH and O. The most intense peaks occur at 337 nm, 357 nm and 380 nm, corresponding, respectively, to the transitions (v’ v”) of (0,0), (0,1) and (0,2). 3.2. Seed morpho-anatomy
Fig. 2. Lissajous figure for peak-to-peak voltage of 35 kV in a DBD plasma coaxial configuration, illustrating the integrated hysteresis area.
The optical microscopy analysis (Fig. 4A) revealed that Mimosa caesalpiniafolia seeds have a structure that is characteristic of seeds that belong to the Fabaceae Family—that is, they present a hard and smooth integument with fractures almost over their entire extension, as observed by electron microscopy (Fig. 4C, D). On one side of the seed, there is a region called the pleurogram (Fig. 4A), which is marked by a U-shaped fracture, whose opening faces show the hilar region, as shown in the electron micrograph (Fig. 4B). The fractures are more densely distributed at the entrance and central part of the pleurogram (Fig. 4C,D). A cross section of the seed, which is stained with blue toluidine, shows that the cracks in the integument propagate to the palisade layer (Fig. 4E). The presence of cellulose in the integument’s layers of tissues is indicated by the purple coloration. A similar result was obtained in Schizolobium parahyba, Senna multijuga and Leucaena leucocephala seeds [21–23]. 3.3. Wettability test
Fig. 3. Emission spectrum of the plasma discharge between two glass tubes in the reactor.
While the untreated seeds presented a hydrophobic surface with an apparent contact angle of 98◦ ± 6◦ , the treated seeds presented angles of 70 ± 2◦ , 47 ± 1◦ and 42±◦ under treatment durations of 3, 9 and 15 min, respectively (Fig. 3). These results corroborate with other literature validations, in which air plasma or atmospheres containing N2 result in more hydrophilic seed surfaces.The increase in surface wettability of the treated seeds, as observed through the contact angle measurements, may be related to chemical mechanisms of surface modification, which are caused by the absorption of radicals such as OH and N2 + , making it more hydrophilic and resulting in a higher water absorption rate [24,25]. According to Vander Wielen et al. [26,27], the treatment of DBD plasma can alter the wettability of cellulosic fibers, making
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Figura 4. (A) Optical Micrograph of the Mimosa caesalpiniaefolia benth seed presenting the pleurogram and Hilar regions; (B) Details of the micropyle; (C,D) Details of the pleurogram, which presents a higher density of cracks and (E) cross-section showing the crack in the external layer of the palisade.
them hydrophobic or hydrophilic, depending upon the treatment potency, which is produced by oxidation of the surface fibers and also due to the covalent crosslinks of inter fibers generated by plasma. The presence of micro and macro fissures in the integument facilitates plasma penetration. This has been observed in porous polymeric materials where the treatment may penetrate to a depth of 1.0 mm, reaching surfaces and volumes not directly exposed to the plasma; however, its effectiveness will depend on the plasma type, treatment time, pressure, pore size, material thickness, amongst other factors [12,28–31]. In this study, it was confirmed that plasma treatment hydrophilized the seed surface; however, no micro-topographic changes were observed. Therefore, it may be assumed that the wettability increase in these seeds is only due to the changes in the chemical structure (Fig. 5).
These results confirm a direct relationship between wettability and inbibition and suggest that plasma saturation affects the mechanisms responsible for imbibition after 9 min of application. Considering that the contact angle value for samples treated for 9 min was very close to those treated for 15 min, it is plausible to attribute the change in wettability to the change in imbibition. Results from the literature have shown that Fabaceaes seeds have a lipid outer layer that impedes water absorption. As mentioned above, chemical alterations due to the surface absorption of radicals and the possible oxidation of this layer may have promoted greater wettability and, as a consequence, an increase in water absorption. Furthermore, this effect is enhanced by the micro and macro fissures on the integument. After a sufficient time span, in this case 9 min, the seed surface has been fully chemically altered; therefore, its water absorption up until this moment remains constant. After this, for times between 9 and 15 min, no further alterations were noted.
3.4. Imbibition test 3.5. Germination test Seed imbibition testing followed a defined 3-stage standard, through which it is possible to indicate the activation of physiological tools when referring to the germinative process and the resulting emission of the primary root. According to the literature, in stage 1, there is a rapid water uptake (stage I), followed by a marked reduction (Stage II) and, finally, a relevant increase (Stage III). Stage II is the longest stage in the process, where basically no weight variation occurs [32,33]. All of the plasma-treated seeds in this study presented an abrupt mass gain in the first 3 h of soaking; a smoother increase occurred up to 12 h, and then the mass remained approximately constant until the end of the experiment (Fig. 6). Seeds treated for 9 and 15 min presented the same behavior in imbibition—that is, their curves completely overlapped.
The cumulative germination percentages obtained in this study fit well to the Richard curve (Fig. 7). The population parameters (Vi, Me, Qu and Sk) are shown in Table 1. Vi had a significant increase in plasma-treated samples when compared to the control condition. While untreated samples presented a 6% germination rate, seeds treated for 3 min presented values of 50%. Contrary to the imbibition values for treated seeds, the Vi values were inversely proportional to treatment duration. A possible explanation for the apparent contradiction is because high levels of moisture can decrease the absorption of oxygen and affect the germination process [14]. Although samples treated for 9 min and 15 min presented similar wetting behavior, the Vi results were significantly different with 46% and 26%, respectively. It is probable
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Fig. 5. Effect of DBD plasma treatment on the water contact angle on the surface of Mimosa caesalpiniafolia benth seeds. (A) Apparent contact angle values for different treatment conditions and (B) Photograph of the sessile drop on the surface not treated (control) and surface-treated for 15 min. Error bars represent the standard deviation of the mean (n = 3). Comparison of data between different treatment conditions were analyzed using a one-way ANOVA), p < 0.05 by Student’s t-test.
Fig. 6. Percentage of imbibition (%) of Mimosa caesalpiniafolia Benth DBD plasma-treated seeds and untreated seeds. Error bars represent the standard deviation of the mean for 40 seeds (10 × 4 replicates). Comparison of data between different treatment conditions for the same imbibition duration were analyzed using a one-way ANOVA, p < 0.05 by Student’s t-test. Table 1 Population parameters Vi (viability), Me (median germination time), Qu (dispersion) and Sk (skewness) of the Richards’ function for germination of Mimosa Caesalpiniafolia. Parameters
Vi Me Qu Sk
Treatment Control
3 min
9 min
15 min
P value
6.0d ± 0.3 1.03c ± 0.43 0.93c ± 0.01c −0.07a ± 0.01
50.0a ± 1.5 3.53a ± 1.03 1.81a ± 0.03 −0.19c ± 0.03
46.0b ± 0.4 2.54b ± 0.56 1.84a ± 0.50 −0.06c ± 0.03
26.0c ± 0.3 1.53c ± 1.50 1.58b ± 1.00 0.03b ± 0.03
0.01** 0.01** 0.01** 0.04**
Different lowercase letters denote statistical differences between treatments groups at the 5% level according to Tukey test.
that seeds treated for 15 min suffered from other damages that only interfered in their germination. The durations for 50% of the total germination (Me) for the three plasma treatment conditions were higher than the control condition. According to Será et al. [17], this result suggests that plasma
treatment causes changes in the germination characteristics and alters the biological characteristics of the seeds. Also, the Me results under the treatment conditions presented a decrease with increasing treatment duration. That is, germination is delayed as treatment time increases, resulting in a decrease in viability.
A.R.M. da Silva et al. / Colloids and Surfaces B: Biointerfaces 157 (2017) 280–285
Fig. 7. Germination curves calculated using the Richard fitting function. All data points represent mean ± standard deviation for 100 seeds (25 × 4 replicates).
In Table 1, the Qu values show that under treatment conditions, there is a significant increase when compared to the control condition. Although the 3-min and 9-min conditions present similar results, there is a relatively large deviation for the 9-min value, as per the 15-min condition. Therefore, the differences in the Qu parameter values confirm the irregular dormancy and the random gradual germination of M. caesalpiniafolia seeds, which is caused by their integument. An elevated Sk value expresses that the lot has a smaller amount of quickly growing seeds, while a low Sk value expresses that the lot has a smaller amount of slowly growing seeds [19]. When comparing Sk values of treated and non-treated seeds, there is a reduction in value for the seeds treated for 3 min. Thus, there are a smaller number of seeds that sprout in the first days. 4. Conclusions An experimental apparatus for treating seeds with DBD plasma was tested and approved when used on elliptical and small seeds (∼6 mm2 ). The apparatus allowed the simultaneous treatment of 100 seeds, but this number may be increased if a continuos feed of seed is coupled to the process.The best treatment duration was 3 min, resulting in a germination rate that was 8 times higher than that of the untreated seeds. Higher treatment times resulted in lower wettability as well as lower imbibition and germination values. While wettability and imbibition presented similar behaviors for these times, their germination values were significantly different. Acknowledgements This work was partially supported by the Brazilian funding agencies CNPq and CAPES. References [1] Legumes of the World, in: G. Lewis, B. Schrire, B. Mackinder, M. Lock (Eds.), Royal Botanic Gardens, Kew, 2005, 577 pp. [2] M. Simon, R. Grether, L. Queiroz, T. Särkinen, T.V. Dutra, C. Hughes, The evolutionary history of mimosa (leguminosae): toward a phylogeny of the sensitive plants, Am. J. Bot. 98 (2011) 1201–1221. [3] E. Evans, F. Blazich, Overcoming Seed Dormancy: Trees and Shrubs Department of Horticultural Science. Horticulture Information Leaflet, 1999, pp. 8704.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Protocols
Using atmospheric plasma to increase wettability, imbibition and germination of physically dormant seeds of Mimosa Caesalpiniafolia A.R.M. da Silva a , M.L. Farias a , D.L.S. da Silva a , J.O. Vitoriano a , R.C. de Sousa b , C. Alves-Junior a,∗ a b
LABPLASMA- Department of Exact and Natural Sciences, Federal Rural University of Semiarid, Mossoró, RN, CEP: 59625-900 Brazil, LCMM – Physics Department, Federal University of Maranhão São Luís, MA, CEP: 65080-805, Brazil
a r t i c l e
i n f o
Article history: Received 22 January 2017 Received in revised form 22 May 2017 Accepted 25 May 2017 Available online 1 June 2017 Keywords: Atmospheric plasma DBD Seed dormancy Impermeable integument
a b s t r a c t In this study, we analyzed seed wettability as well as imbibition and germination after treatment with atmospheric pressure cold plasma (APCP) using dielectric barrier discharge (DBD) in seeds that have very low germination rates. To aid industrial applications, several seeds were simultaneously treated with plasma within a space between two coaxial glass tubes sandwiched by two metal mesh screens that produced high-voltage pulses at 17.5 kV with a frequency of 990 Hz. Three treatment times (3 min, 9 min and 15 min) as well as untreated seeds were used to conduct the wettability, imbibition and germination tests. The wettability and imbibition were found to be directly related to the treatment duration, but saturation of the imbibition was found for treatment durations greater than 9 min. Plasma treatment was also effective in improving germination, but shorter treatment duration presented greater germination. This apparent contradiction is explained by the cell damage caused by the increased exposure to plasma, as observed in other studies. The results suggest that there must be an optimal wettability and imbibition condition that ensures that excessive moisture does not harm the germination process. © 2017 Elsevier B.V. All rights reserved.
1. Introduction The large-scale cultivation of species such as Mimosa caesalpiniafolia Benth is very important for ecosystems and agricultural areas in maintaining soil fertility and restoring degraded areas [1,2]. However, due to integument dormancy, viability is very low (less than 10%), making the culture unfeasible. The methods used to overcome seed dormancy and to improve germination rate generally consist of increasing wetting and soaking [3]. Physical (scarification) and chemical (use of sulfuric acid and hot water) treatments are commonly used in agriculture and have high environmental costs [4]. Reduction in vigor, higher infection rate due to structural damage, increased probability of abnormality in seedling growth shoots and production of poor fitomass, in addition to residues resulting from chemical treatments, are some examples of these problems [5–7]. In recent years, there has been a growing number of agricultural applications of plasma produced by dielectric barrier discharge (DBD), especially related to the inactivation of microorganisms and overcoming seed dormancy [8–10]. Since seed tissue rehydration is essential for metabolic activities, which
∗ Corresponding author. E-mail address: [email protected] (C. Alves-Junior). http://dx.doi.org/10.1016/j.colsurfb.2017.05.063 0927-7765/© 2017 Elsevier B.V. All rights reserved.
result in germination, plasma techniques must be able to modify the surface to increase seed soaking. The surface of Mimosa seeds has a complex structure that consists of an impermeable tegument (exotesta) containing small slits that communicate with mesotesta [11]. It has been reported that plasma species that are ‘attenuated’ by the coat do not have sufficient energy for the modification of the wettability of the internal sides of the biological surfaces [12]. However, several studies show that plasma can efficiently increase imbibition and germination [9,10]. The levels of GA3 hormone and mRNA expression (biochemical processes required for germination) of an amylolytic enzyme-related gene in seeds increased after treatment with high-voltage plasma pulses [13]. However, it is still unclear whether the seed viability can be changed when only the exotesta is modified by plasma. For most seeds, germination begins with a rapid initial water uptake that is higher on surfaces with more wettability. However, excessive moisture can decrease the absorption of oxygen and affect the germination process [14]. Strategies to enhance germination might involve wettability control on seed exotesta. In this paper, we investigate the response with respect to wettability, imbibition and germination after treating Mimosa caesalpiniafolia seeds using different durations of DBD plasma treatment. To facilitate future industrial applications, several seeds were simultaneously treated with plasma in a space between two
A.R.M. da Silva et al. / Colloids and Surfaces B: Biointerfaces 157 (2017) 280–285
coaxial glass tubes sandwiched by two metal mesh screens that were connected to a power source. 2. Materials and methods 2.1. Seeds morpho-anatomy Microstructures of the integument were analyzed using a scanning electron microscope and an optical microscope. For the analysis with the optical microscope, untreated seeds were soaked in a formalina-acetic acid alcohol (FAA) solution for 24 h, dehydrated in an ethanol process and embedded into paraffin blocks. The blocks were then cut into sections 10-m thick with a rotary microtome. Histochemical testing was performed using toluidine blue dye to reveal lignin and/or cellulose [15]. The images were recorded with a digital camera attached to the optical microscope.
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2.5. Imbibition test The seeds were soaked in plastic Gerbex-type boxes (capacity of 250 ml, dimension 11.0 × 11.0 × 3,5 cm3). The imbibition test began by placing the seeds between two germination papers, where a proportion of 3.0 ml of water was added to each gram of paper. For each test, 40 seeds were divided into 4 replicates of 10 seeds. The seeds were removed after 0, 4, 12, 24 and 36 h and weighed; the percentage increase in seed mass variation was determined by Eq. (2): %Mass =
mf − mi mi
x100
(2)
where mf is the mass of the imbibed seeds, and mi is the mass of the dry seeds. The test was conducted in a BOD type of germination chamber, temperature controlled at 25 ◦ C and a photoperiod of 8 h.
2.2. Plasma experimental apparatus
2.6. Germination test
The experimental apparatus is illustrated in Fig. 1A. The source plasma was equipped with two coaxial glass tubes, which were externally and internally coated by a metal mesh screen, and each glass tube had a wall that was 1-mm thick. The metal mesh screen was connected to a high-voltage power source with a peak value of 17.5 kV that pulsed at a frequency of 990 Hz. The power density (p) was determined with the aid of the Lissajous figure area (SL ), which was obtained by an oscilloscope using a 1000:1 probe (Tektronix P6015A, Tektronix, Beaverton, USA), by Eq. (1) [16]:
The germination capacity test was also carried out in plastic Gerbox boxes filled with sterilized sand. Prior to sowing the seeds, each box was prepared with 400 g of sand and 60 ml of distilled water. In each test, 100 seeds were divided into 4 replicates of 25 seeds. The germination of the DBD plasma-treated seeds were compared to the untreated seeds after 12 days. The experimental design was entirely carried out at random. The description of germination kinetics was obtained by fitting the experimental points to the Richard curve [18,19]. The Richards function (Yt), which has a variable inflection point, is represented by the Eq. (3):
p=
Pd f = A
V (t)dQ (t) A
f =
SL f A
(1)
where Pd is the dissipated electrical power; A is the area irradiated by plasma the area; SL is the area under the curve of the Lissajous; and V, Q, and f are the voltage, charge and frequency of the circuit, respectively. An optical fiber was used to transfer the light spectrum from the plasma to the CCD spectrometer (HR4000, Ocean Optics, Dunedin, FL, USA) – the spectral range is from 200 to 1000 nm – to measure the reactive species generated in the plasma. 2.3. Plasma treatment The slightly elliptical seeds, which have an average diameter of 6.0 mm, were placed between the two glass tubes within the plasma reactor. For the plasma treatment, around 100 seeds were uniformly distributed between the coaxial tubes at a time. Under these conditions, the plasma filled the spaces between the two glass tubes and evenly coated all of the seeds’ surfaces (Fig. 1B). The treatments were performed for durations of 3, 9 or 15 min, depending on the treatment group. After treatment, the seeds were stored in dissectors to test the wettability, imbibition and germination.
˛
Yt =
[1 + b + dx exp(−cxt)]
1⁄d
(3)
From this equation, the following parameters are obtained: Vi (viability), which is the total percentage of germination; Me (Median), which is the time of occurrence of 50% of the total germination. The Me parameter is very important for the description of the germination process and the initial growth of seedlings, as it distinguishes the behavior of the species or plant variety during germination. Qu indicates the dispersion of Me, and Sk (asymmetry) indicates the asymmetry in the frequency of distribution of the germination time, which represents the asymmetry of the Richards’ function relative to the inflection point [19,20]. 2.7. Statistical analysis The experimental design was completely randomized (DIC) for the analysis of variance (ANOVA). The Tukey (P < 0.05) average com® parison test was performed using Sisvar software. 3. Results and discussion
2.4. Wettability test
3.1. Dissipated power and active plasma species
Wettability tests were conducted using the sessile drop technique to measure the apparent contact angle of distilled water droplets. It was measured on 3 seeds under each condition [17]. The test was performed by dripping 20 l of distilled water once on each seed surface. The images were captured and recorded by a camera attached to a computer and processed to determine the ® contact angles using the Surftens 3.5 program. Descriptive statistics (arithmetic mean and standard deviation) were calculated for the values of the contact angles formed by the liquid and the integument.
Integrating the Lissajous figure in Fig. 2 and considering the area irradiated by plasma (A), which is equal to 0.006 m2 , the dissipated power density for a frequency of 990 Hz can be obtained using Eq. (3): −6
p = 1.07×10 ∗ 990 = 0.18w/m2 (3) A The optical spectra of the plasma (Fig. 3) was obtained using an optical fiber in front of the coaxials tubes, which transmitted to an optical emission spectrometer (OES) diagnostic of plasma species. The main peaks result from the excitation of the nitrogen molecules (N2) present in the air, which are all from the second pos-
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Fig. 1. (A) Experimental apparatus used for this experiment, (B) Details of the top view showing positioning of the seeds within the reactor.
itive system of N2, with different energetic levels. Peaks from the first positive system were also observed: N2 + apart from OH and O. The most intense peaks occur at 337 nm, 357 nm and 380 nm, corresponding, respectively, to the transitions (v’ v”) of (0,0), (0,1) and (0,2). 3.2. Seed morpho-anatomy
Fig. 2. Lissajous figure for peak-to-peak voltage of 35 kV in a DBD plasma coaxial configuration, illustrating the integrated hysteresis area.
The optical microscopy analysis (Fig. 4A) revealed that Mimosa caesalpiniafolia seeds have a structure that is characteristic of seeds that belong to the Fabaceae Family—that is, they present a hard and smooth integument with fractures almost over their entire extension, as observed by electron microscopy (Fig. 4C, D). On one side of the seed, there is a region called the pleurogram (Fig. 4A), which is marked by a U-shaped fracture, whose opening faces show the hilar region, as shown in the electron micrograph (Fig. 4B). The fractures are more densely distributed at the entrance and central part of the pleurogram (Fig. 4C,D). A cross section of the seed, which is stained with blue toluidine, shows that the cracks in the integument propagate to the palisade layer (Fig. 4E). The presence of cellulose in the integument’s layers of tissues is indicated by the purple coloration. A similar result was obtained in Schizolobium parahyba, Senna multijuga and Leucaena leucocephala seeds [21–23]. 3.3. Wettability test
Fig. 3. Emission spectrum of the plasma discharge between two glass tubes in the reactor.
While the untreated seeds presented a hydrophobic surface with an apparent contact angle of 98◦ ± 6◦ , the treated seeds presented angles of 70 ± 2◦ , 47 ± 1◦ and 42±◦ under treatment durations of 3, 9 and 15 min, respectively (Fig. 3). These results corroborate with other literature validations, in which air plasma or atmospheres containing N2 result in more hydrophilic seed surfaces.The increase in surface wettability of the treated seeds, as observed through the contact angle measurements, may be related to chemical mechanisms of surface modification, which are caused by the absorption of radicals such as OH and N2 + , making it more hydrophilic and resulting in a higher water absorption rate [24,25]. According to Vander Wielen et al. [26,27], the treatment of DBD plasma can alter the wettability of cellulosic fibers, making
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Figura 4. (A) Optical Micrograph of the Mimosa caesalpiniaefolia benth seed presenting the pleurogram and Hilar regions; (B) Details of the micropyle; (C,D) Details of the pleurogram, which presents a higher density of cracks and (E) cross-section showing the crack in the external layer of the palisade.
them hydrophobic or hydrophilic, depending upon the treatment potency, which is produced by oxidation of the surface fibers and also due to the covalent crosslinks of inter fibers generated by plasma. The presence of micro and macro fissures in the integument facilitates plasma penetration. This has been observed in porous polymeric materials where the treatment may penetrate to a depth of 1.0 mm, reaching surfaces and volumes not directly exposed to the plasma; however, its effectiveness will depend on the plasma type, treatment time, pressure, pore size, material thickness, amongst other factors [12,28–31]. In this study, it was confirmed that plasma treatment hydrophilized the seed surface; however, no micro-topographic changes were observed. Therefore, it may be assumed that the wettability increase in these seeds is only due to the changes in the chemical structure (Fig. 5).
These results confirm a direct relationship between wettability and inbibition and suggest that plasma saturation affects the mechanisms responsible for imbibition after 9 min of application. Considering that the contact angle value for samples treated for 9 min was very close to those treated for 15 min, it is plausible to attribute the change in wettability to the change in imbibition. Results from the literature have shown that Fabaceaes seeds have a lipid outer layer that impedes water absorption. As mentioned above, chemical alterations due to the surface absorption of radicals and the possible oxidation of this layer may have promoted greater wettability and, as a consequence, an increase in water absorption. Furthermore, this effect is enhanced by the micro and macro fissures on the integument. After a sufficient time span, in this case 9 min, the seed surface has been fully chemically altered; therefore, its water absorption up until this moment remains constant. After this, for times between 9 and 15 min, no further alterations were noted.
3.4. Imbibition test 3.5. Germination test Seed imbibition testing followed a defined 3-stage standard, through which it is possible to indicate the activation of physiological tools when referring to the germinative process and the resulting emission of the primary root. According to the literature, in stage 1, there is a rapid water uptake (stage I), followed by a marked reduction (Stage II) and, finally, a relevant increase (Stage III). Stage II is the longest stage in the process, where basically no weight variation occurs [32,33]. All of the plasma-treated seeds in this study presented an abrupt mass gain in the first 3 h of soaking; a smoother increase occurred up to 12 h, and then the mass remained approximately constant until the end of the experiment (Fig. 6). Seeds treated for 9 and 15 min presented the same behavior in imbibition—that is, their curves completely overlapped.
The cumulative germination percentages obtained in this study fit well to the Richard curve (Fig. 7). The population parameters (Vi, Me, Qu and Sk) are shown in Table 1. Vi had a significant increase in plasma-treated samples when compared to the control condition. While untreated samples presented a 6% germination rate, seeds treated for 3 min presented values of 50%. Contrary to the imbibition values for treated seeds, the Vi values were inversely proportional to treatment duration. A possible explanation for the apparent contradiction is because high levels of moisture can decrease the absorption of oxygen and affect the germination process [14]. Although samples treated for 9 min and 15 min presented similar wetting behavior, the Vi results were significantly different with 46% and 26%, respectively. It is probable
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Fig. 5. Effect of DBD plasma treatment on the water contact angle on the surface of Mimosa caesalpiniafolia benth seeds. (A) Apparent contact angle values for different treatment conditions and (B) Photograph of the sessile drop on the surface not treated (control) and surface-treated for 15 min. Error bars represent the standard deviation of the mean (n = 3). Comparison of data between different treatment conditions were analyzed using a one-way ANOVA), p < 0.05 by Student’s t-test.
Fig. 6. Percentage of imbibition (%) of Mimosa caesalpiniafolia Benth DBD plasma-treated seeds and untreated seeds. Error bars represent the standard deviation of the mean for 40 seeds (10 × 4 replicates). Comparison of data between different treatment conditions for the same imbibition duration were analyzed using a one-way ANOVA, p < 0.05 by Student’s t-test. Table 1 Population parameters Vi (viability), Me (median germination time), Qu (dispersion) and Sk (skewness) of the Richards’ function for germination of Mimosa Caesalpiniafolia. Parameters
Vi Me Qu Sk
Treatment Control
3 min
9 min
15 min
P value
6.0d ± 0.3 1.03c ± 0.43 0.93c ± 0.01c −0.07a ± 0.01
50.0a ± 1.5 3.53a ± 1.03 1.81a ± 0.03 −0.19c ± 0.03
46.0b ± 0.4 2.54b ± 0.56 1.84a ± 0.50 −0.06c ± 0.03
26.0c ± 0.3 1.53c ± 1.50 1.58b ± 1.00 0.03b ± 0.03
0.01** 0.01** 0.01** 0.04**
Different lowercase letters denote statistical differences between treatments groups at the 5% level according to Tukey test.
that seeds treated for 15 min suffered from other damages that only interfered in their germination. The durations for 50% of the total germination (Me) for the three plasma treatment conditions were higher than the control condition. According to Será et al. [17], this result suggests that plasma
treatment causes changes in the germination characteristics and alters the biological characteristics of the seeds. Also, the Me results under the treatment conditions presented a decrease with increasing treatment duration. That is, germination is delayed as treatment time increases, resulting in a decrease in viability.
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Fig. 7. Germination curves calculated using the Richard fitting function. All data points represent mean ± standard deviation for 100 seeds (25 × 4 replicates).
In Table 1, the Qu values show that under treatment conditions, there is a significant increase when compared to the control condition. Although the 3-min and 9-min conditions present similar results, there is a relatively large deviation for the 9-min value, as per the 15-min condition. Therefore, the differences in the Qu parameter values confirm the irregular dormancy and the random gradual germination of M. caesalpiniafolia seeds, which is caused by their integument. An elevated Sk value expresses that the lot has a smaller amount of quickly growing seeds, while a low Sk value expresses that the lot has a smaller amount of slowly growing seeds [19]. When comparing Sk values of treated and non-treated seeds, there is a reduction in value for the seeds treated for 3 min. Thus, there are a smaller number of seeds that sprout in the first days. 4. Conclusions An experimental apparatus for treating seeds with DBD plasma was tested and approved when used on elliptical and small seeds (∼6 mm2 ). The apparatus allowed the simultaneous treatment of 100 seeds, but this number may be increased if a continuos feed of seed is coupled to the process.The best treatment duration was 3 min, resulting in a germination rate that was 8 times higher than that of the untreated seeds. Higher treatment times resulted in lower wettability as well as lower imbibition and germination values. While wettability and imbibition presented similar behaviors for these times, their germination values were significantly different. Acknowledgements This work was partially supported by the Brazilian funding agencies CNPq and CAPES. References [1] Legumes of the World, in: G. Lewis, B. Schrire, B. Mackinder, M. Lock (Eds.), Royal Botanic Gardens, Kew, 2005, 577 pp. [2] M. Simon, R. Grether, L. Queiroz, T. Särkinen, T.V. Dutra, C. Hughes, The evolutionary history of mimosa (leguminosae): toward a phylogeny of the sensitive plants, Am. J. Bot. 98 (2011) 1201–1221. [3] E. Evans, F. Blazich, Overcoming Seed Dormancy: Trees and Shrubs Department of Horticultural Science. Horticulture Information Leaflet, 1999, pp. 8704.
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