Atmospheric Plasma Overcomes Seed Dormancy in Pityrocarpa Moniliformis (Benth) Luckow & R.W

Nº 073

Atmospheric plasma overcomes seed dormancy in Pityrocarpa moniliformis (Benth) Luckow & R.W. Jobson

JOSEFA PATRÍCIA BALDUINO NICOLAU

Macaíba/RN July of 2020 JOSEFA PATRÍCIA BALDUINO NICOLAU

Atmospheric plasma overcomes seed dormancy in Pityrocarpa moniliformis (Benth.) Luckow & R. W. Jobson

Dissertation presented to Programa de Pós-Graduação em Ciências Florestais da Universidade Federal do Rio Grande do Norte to obtain the degree of Master in Forest Science (Area of Concentration in Forest Sciences - Research Line: Seeds, Propagation and Physiology of Forest Species).

Advisor: Prof. Dra. Poliana Coqueiro Dias

Co-Advisor: Prof. Dr. Márcio Dias Pereira

Macaíba/RN July of 2020

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Atmospheric plasma overcomes seed dormancy in Pityrocarpa moniliformis (Benth.) Luckow & R. W. Jobson

Josefa Patrícia Balduino Nicolau

Macaíba/RN July of 2020

To my family and my fiance for the support and encouragement during this trajectory.

DEDICATION

ACKNOWLEDGMENTS ______

Appreciate

To my God, for his immense love and care, for all the protection and strength given at all times, especially in times of difficulty.

My greatest supporter and friend, Lúcia Aparecida, for believing in me and never letting me give up, you without a doubt are my greatest example.

To the groom, Alex Dantas Farias, for all the help and companionship, thank you for embarking with me on this new journey, for every incentive, for always being willing to accompany me, for spending hours waiting for me on school days, for always calming down with each new one. challenge, you made everything easier and more fun.

To my advisor Márcio Dias Pereira, for all the welcome, and above all for having believed in my potential. You are inspiration, as a professional and as a human being. Thank you for all your help so far, without your valuable contributions this work could not be developed.

The teacher Dinnara Layza Sousa da Silva, for the co-guidance and help to carry out this work. He was present at all stages, did not go to great lengths to make this work possible, always accessible and ready to help.

To the teachers, Riselane de Lucena, Charline Zaratin and Alek Dutra, for generously composing the examining board and contributing to this work.

André Medeiros for all his help with analysis, tips and partnership, his contributions were valuable.

To Labplasma, in particular to the Victorian Jussier who was always available to help with the various applications. To friends, Francisco Eudes, Bruno Guirra, Janikely Buriti, Jaynne Karine, Jéssica Sabrina and Gean Carlos for all their help and companionship. You were essential, this partnership will obviously go beyond UFRN.

To the Graduate Program in Forest Sciences, for the opportunity and for all the learning.

This work was carried out with the support of the Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) - Financing Code 001Finally, to everyone who believed in my potential and pushed me along the way

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GENERAL ABSTRACT ______

Atmospheric plasma overcomes seed dormancy in Pityrocarpa moniliformis (Benth.) Luckow & R. W. Jobson

Plasma technology consists of a fast, clean method with great potential for use in agriculture and forestry, especially in modifying the cutaneous surface of dormant seeds. The objective of this study was to evaluate the effect of applying atmospheric cold plasma onto Pityrocarpa moniliformis seeds, verifying its implications on the seed coat permeability, as well as its germination and vigor. The seeds were submitted to atmospheric cold plasma for 1.5; 2.0; 3.0; 4.0; and 5.0 minutes, using seeds without any treatment as the control. After application, the integument’s wettability, imbibition curve and electrical conductivity of the imbibition solution were determined. The seeds were also submitted to the germination test which determined their viability, median, uniformity and asymmetry. The germination speed index, the first germination count and the seedling length were concomitantly evaluated with the germination test. The seeds subjected to plasma for 4.0 and 5.0 minutes had the smallest apparent contact angles of 64 and 61º, respectively, characterizing greater wettability of the integument among the tested treatments. The highest imbibition rate and solute release was observed in seeds treated by 0.5; 1.5 and 4.0 minutes. The plasma provided an increase in germination and seed vigor in all tested treatments when compared with the control. The use of atmospheric plasma applied to Pityrocarpa moniliformis seeds proved to be efficient as a method to overcome integumentary dormancy, increasing the integument’s wettability and improving the germination percentage and vigor

Key words: Forest seeds, cutaneous dormancy, wettability, cold plasma.

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RESUMO GERAL ______

Plasma atmosférico supera a dormência de sementes de Pityrocarpa moniliformis (Benth.) Luckow & R. W. Jobson

A tecnologia do plasma consiste em um método rápido, limpo e com grande potencial de uso na agricultura, em especial na modificação da superfície tegumentar de sementes dormentes. Objetivou-se nesta pesquisa avaliar o efeito da aplicação do plasma frio atmosférico em sementes de Pityrocarpa moniliformis, verificando suas implicações sobre a permeabilidade do tegumento, a germinação e o vigor. As sementes foram submetidas ao plasma frio atmosférico por 1,5; 2,0; 3,0; 4,0 e 5,0 minutos, utilizando-se como controle, sementes sem nenhum tratamento. Após aplicação, determinou-se a molhabilidade do tegumento, a curva de embebição e a condutividade elétrica da solução de embebição. As sementes também foram submetidas ao teste de germinação, a partir do qual determinou-se a viabilidade, mediana, uniformidade e assimetria. Concomitante ao teste de germinação avaliou-se o índice de velocidade de germinação, a primeira contagem de germinação e o comprimento de plântulas. As sementes submetidas ao plasma por 4,0 e 5,0 minutos apresentaram os menores ângulos de contato aparente, 64º e 61º, respectivamente, caracterizando maior molhabilidade do tegumento, entre os tratamentos testados. A maior taxa de embebição e liberação de solutos foram observadas nas sementes tratadas por 0,5; 1,5 e 4 minutos. O plasma proporcionou incremento na germinação e no vigor das sementes em todos os tratamentos testados, quando comparados com o controle. O uso do plasma atmosférico, aplicado em sementes da espécie em estudo, mostrou-se eficiente como método de superação da dormência tegumentar, aumentando a molhabilidade do tegumento e melhorando o percentual de germinação e o vigor.

Palavras-chave: sementes florestais, dormência tegumentar, molhabilidade, plasma frio.

SUMMARY ______

Página 1.GENERAL INTRODUCTiON ...... 1 2.GENERAL OBJECTIVE ...... 4 3. LITERATURE REVIEW ...... 6 3.1.1. The studied species ...... 6 3.1.2.Seed dormancy ...... 7 3.1.3. Integumentary dormancy ...... 8 3.1.4. Methods for overcoming dormancy ...... 8 3.1.5. Plasma ...... 9 3.1.6. The use of plasma and its potential in agriculture...... 11 3.1.7. Plasma application to evercome seed dormancy...... 12 3.1.8. Factors which interfere with plasma efficiency...... 13

4. MATERIAL AND METHODS ...... 15 4.1. Seed procurement and processing ...... 15 4.2. Plasma application ...... 15 4.3. Determination of wettability ...... 16 4.4. Water absorption and electrical conductivity curve ...... 16 4.4.1. Germination test ...... 17 4.4.2. Seedling length ...... 18 4.4.3. Study design and statistical analysis ...... 18 5. RESULTS AND DISCUSSION ...... 20 6. CONCLUSIONS...... 32 7. ACKNOWLEDGMENTS ...... 34 8. LITERATURE CITED ...... 36

FIGURE LIST ______

Figure 1. (A) Pityrocarpa moniliformis tree; (B) inflorescence; (C) pod-shaped fruits; and (D) seeds...... 19

Figure 2. Schematic diagram of plasma formation...... 20

Figure 3. (A) Schematic representation of the system used for applying plasma with a high voltage source; (B) Detail of the seeds arranged inside the glass plate under the coplanar plate; (C) Diagram indicating the position of the electrodes and the dielectric material inside the plate in relation to the seeds…………………………………………………………………...21

Figure 4. Apparent contact angle (°) formed by the water drop and the surface of P. moliniformis seeds submitted to different exposure periods of atmospheric cold plasma by dielectric barrier discharge………………………………………………………………………….21

Figure 5. Increase in the mass of P. moniliformis seeds (%) submitted to different exposure periods to cold atmospheric plasma by dielectric barrier discharge during the imbibition process ...... 23

Figure 6. Electrical conductivity (mS/Cm) of P. moniliformis seeds subjected to different exposure periods to atmospheric cold plasma by dielectric barrier discharge during the imbibition process………………………………………………………………………………….24

Figure 7. Cumulative germination of Pityrocarpa moniliformis seeds according to the application time of atmospheric cold plasma by dielectric barrier discharge determined by the Richards’ method (1959)……………………………………………………………………………25

Figure 8. Population parameters of the (A) Richards Viability-Vi equation; (B) Median-Me; (C) Uniformity-Qu; and (D) Asymmetry-SK for the germination of P. moniliformis seeds submitted to plasma by dielectric barrier discharge ...... 26

Figure 9. (a) Germination percentage, (b) germination speed index, and (c) first germination count in P. moniliformis seeds ...... 27

Figure10. (A) Hypocotyl and (B) root length of P. moniliformis seeds submitted to cold plasma during 0 (control); 1.0; 2.0; 3.0; 4.0; and 5.0 minutes…………………………………..27

Figure11. Principal component analysis for the germination variable (G%), germination speed index (GSI), first germination count (FGC), hypocotyl length (HL) and root length (RL) of P. moniliformis seeds……………………………………………………………………………..27

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TABLES LIST ______

Table 1.Plasma classification according formation ...... 18

Table 2. The Richards function (Richards, 1959) ...... 19

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General Introduction

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1. GENERAL INTRODUCTION ______

The Pityrocarpa moniliformis (Benth.) Luckow & R. W. Jobson species is found in the Caatinga and Atlantic Forest, and is commonly used for manufacturing drugs, wood, forage and as a component in agroforestry systems. This species belongs to the Fabaceae family and produces seeds with physical dormancy, being characterized by the resistance imposed by the integument structure which is rich in lignin, cutin, suberin and has a superficial wax layer. This integumentary constitution implements hydrophobicity and restricts water absorption by the seed, hindering the germination process (MAIA-SILVA et al., 2012; MARCOS FILHO, 2015; AZEREDO et al., 2016) Chemical scarification with acids and immersion in hot water have been the most used methods to overcome the physical dormancy of this species and others of the same family which present this type of impediment (AZEREDO et al., 2010). However, such procedures can cause damage to the seed structure, reduced vigor, in addition to increased infections by microorganisms (PEREIRA, 2011). Thus, the plasma application technology in seeds has been highlighted as an alternative to these pre-germination treatments, which according to Alves Junior et al. (2017) can act as a modifier of the germination dynamics, thereby reducing the effects of dormancy and promoting germination. This use is justified because it is a faster, less invasive technology which does not produce waste or damage the seed, allowing greater economic profitability. The plasma produced in the laboratory is an ionized and electrically neutral gas composed of electrons, positive and negative ions, photons, excited and unexcited molecules, UV radiation and heat (MISRA et al., 2014; YUSAF and AL-JUBOORI, 2014; PANKAJ et al., 2015; OH et al., 2016), being able to modify surfaces in the most diverse spectra. For example, its ability to alter chemical bonds is indicative of its potential to eliminate or reduce integumentary dormancy in some seeds, which may promote the partial breakdown of polymer chains, and provide inclusion of new functional groups (DE GROOT et al., 2018; LOS et al., 2019; YODPITAKA et al., 2019). Therefore, previously hydrophobic surfaces may have a greater affinity for water, as suggested in research which used plasma for this purpose in Mimosa caesalpiniafolia (GUIMARÃES et al., 2015), Erythrina velutina Willd (ALVES JUNIOR et al., 2016), and Leucaena leucocephala (ALVES JUNIOR et al., 2020). The challenge for studies involving the use of plasma on living surfaces is the adequacy of the settings and the application conditions, as well as the materials to be changed. Research shows that factors related to plasma application, such as the exposure

1 time of the seeds, can have a decisive influence on the obtained results (PATANGE, 2019). However, these conditions are still unclear for most species.

General Objective

2. GENERAL OBJECTIVE ______

In view of the above, the objective of this study was to evaluate the effect of the applying atmospheric cold plasma onto Pityrocarpa moniliformis seeds, verifying its implications on the seed coat permeability, as well as its germination and vigor.

Literature Review

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3. LITERATURE REVIEW ______

3.1.1. The studied species

Pityrocarpa moniliformis (Benth.) Luckow& R. W. Jobson is popularly known in Brazil as angico-de-bezerro, carrasco, catanduba, catanduva, muquém, rama-de-bezerro, surucucu and quipembé. It belongs to the Fabaceae family, and occurs naturally in the states of Piauí, Ceará, Maranhão, Rio Grande do Norte and Bahia, being frequent in the São Francisco valley (AZEREDO et al., 2011). With a characteristically rounded crown, the species has a tortuous trunk with fine and rough bark, bipinate leaves and flowers arranged in cylindrical ears, as well as flat and dehiscent pods which can reach up to 13 centimeters in length (MAIA, 2004), as shown in Figure 1.

Figure 1. (A) Pityrocarpa moniliformis tree; (B) inflorescence; (C) pod-shaped fruits; and (D) seeds.

P. moniliformis leaves, peels and fruits have high antioxidant content, being used in phytotherapeutic medicines to fight diseases and to control the growth of cancer cells (ALVES et al., 2014). In addition, they also contain high levels of phenolic components which act as a bioactive substance, and can be used in food, manufacturing cosmetics and drug

6 preparation (SILVA et al., 2011). Flowering occurs in the transition from the dry and rainy seasons (between the months of December to April), when the species becomes the main supplier of pollen during climatic seasonality, which is even used by Melipona subnitida bees which is currently threatened with extinction (MAIA-SILVA et al., 2012; DANTAS, 2016). The species has a large volume of floral structures in the reproductive phase, with about 70% of flowers among these being hermaphrodites and 30% of staminate or unisexual male flowers (polygamous plant) (FERREIRA, 2009). The seeds are orthodox and of ovoid morphology, gray in color with a smooth and shiny surface, while the pleurogram is closed and visible on both sides of the seed (AZEREDO, 2009). The seeds also have dormancy mechanisms, in which germination only occurs under favorable ecological conditions (KIILL, 2012).

3.1.2. Seed dormancy

The evolutionary process of living beings constitutes one of the greatest challenges for permanently establishing species in natural ecosystems, so that metabolic strategies are created by organisms in order to adapt to the constant changes in the environment in which they are inserted (SEPÚLVEDA and EL-HANI, 2014). Dormancy is seen as an adaptation by plants to perpetuate their species. It can be defined as a natural phenomenon in which the seeds of a specific species are viable and have all the environmental conditions favorable to germination (water, oxygen, temperature and absence of inhibitors), but do not germinate; this adaptation mechanism is even more frequent in species of forest plants which produce structural layers in their seeds which restrict water passage through envelopes (LOPES et al., 2006). Dormancy is seen in an undesirable trait for agriculture, since rapid germination and initial growth of the plant are necessary characteristics for plant production, as well as for nurseries as dormancy can cause problems such as growth inequality between seedlings, exposure of the seeds and seedlings in the initial development stage to unfavorable conditions for a longer time, diseases, insects and deterioration, causing the loss of seeds (PEREIRA, 2011). It is more frequent to find external physical impediment in seeds in tropical regions with cutaneous dormancy, and the occurrence of this barrier can be seen in all environments from the tropics to the arctic, so that this phenomenon only occurs in orthodox seeds, meaning those which tolerate desiccation at low water levels (between 5 and 15%) without decreasing their viability (BASKIN and BASKIN, 2014). Still according to the same authors, dormancy can be classified based on the way in which germination is prevented and thus divided into five types: physiological (presence of

7 substances which hinder the occurrence of essential physiological reactions for triggering germination), morphological (seeds with a still immature or not fully formed embryo), morphophysiological (immature embryo combined with the presence of inhibitors), physical (seed coat is impermeable to water), and physical plus physiological (seeds with combined impervious seed coat with the appearance of inhibitors).

3.1.3. Integumentary dormancy

Integumentary hardness is caused by a number of factors which alone or combined can result in impermeability to water, such as: the existence of a waxy layer on the protective surface of the seed; suberin and cutin in the superficial layers of the integument; appearance of acid grease in the intercellular spaces of the palisade layer; oxidation of phenolic compounds present in pigmented cells of the integument; lignin degradation at the base of the cells; among others (MARCOS FILHO, 2015). Lignin is among the substances found in these integumentary layers, and corresponds to a specific group that is directly linked to the integument protection of the embryo in the dispersive process of seeds, acting as a facilitator in water conduction, as well as a conductor in the flow of nutrients related to the germination process (COSTA et al., 2011). The seeds form a single or double palisade layer of macrosclereids in order to protect such substances responsible for this displacement to the embryo, in addition to a lucid line composed by the juxtaposition of the suberin or cutin with the cellulose (MARCOS FILHO, 2015). Thus, the variation of this seed protection mechanism is an inherited characteristic attributed to the palisade layer of cells in which the cell walls are thick and protected externally by a waxy cuticular layer (NASCIMENTO et al., 2009). In summary, the seed tegument forehead region is normally hard, and has specific tissues which block water entry into the seeds, inhibiting imbibition and thereby restricting the germination process (BEWLEY et al., 2013).

3.1.4. Methods for overcoming dormancy

When seed dormancy is instigated by factors inherent to the tegument, it can be overcome by some treatments with the objective to facilitate the seed water imbibition process. Tegument impermeability is present in many botanical species, with an emphasis on Fabaceae (CARVALHO and NAKAGAWA, 2012). As a result, some mechanisms are used to imbibition dormancy consisting of rupturing or weakening the integument, thereby allowing water passage and initiating the

8 germinative process. Chemical, mechanical scarification or immersion in hot water stand out among the methods used to overcome dormancy in forest specie seeds, with the efficiency depending on factors such as dormancy intensity, which varies greatly between species, as well as its origin and year of collection (SANTOS et al., 2013). It is important to note that the success of these methods depends on the degree of dormancy and the characteristics of the species. Mechanical scarification has a low cost and is considered a simple and widely used method to promote dormancy in seeds of many species; however, the scarification intensity can cause damage to the integument and decrease germination. Another method used is hot or boiling water, which is effective in overcoming seed dormancy of some forest species. However, despite being a cheap and efficient method, this method can result in lower germination values (URSULINO et al., 2019). Although there are different methods used to overcome integumentary dormancy in seeds, they can cause environmental problems such as in the use of chemical scarification, or even those which cause damage to the embryo such as the use of punctures and files. Thus, cold plasma has been shown as an efficient alternative to remove this type of dormancy quickly, economically and in an environmentally friendly way.

3.1.5. Plasma

Matter is made up of atoms, and these in turn are composed of electrons, neutrons and protons, while the interaction between these particles determines the formation of the three states: solid, liquid and gas (ALVES JUNIOR, 2001). An increase in the energy level in matter causes a transition from the solid to the liquid state and then to the gaseous state. Therefore, if more energy is added in the gas phase, the constituent atoms/molecules start a collision process causing the breakdown of the gas molecules, resulting in the formation of charged and neutral species, and the release of radiation at variable wavelengths; this mixture of species and radiation characterizes plasma (D’AGOSTINO et al., 2005; LIÃO et al., 2017), as shown in Figure 2.

Energy Energy Energy

Solid Liquid Gaseous Plasma

Source: LIAO et al., 2017 Figure 2: Schematic diagram of plasma formation.

Plasma makes up about 99% of the matter in the universe, with 1% referring to the other states of matter aggregation (solid, liquid and gas); there are some natural forms of plasma in the universe, such as lightning and the northern lights (D’AGOSTINO et al., 2005). In order to form plasma in the laboratory it is necessary to use two electrodes and a neutral gas and subject them to a potential difference which will generate an electric field by the excitation of electrons and ions, which will then collide with neutral particles transferring energy in parallel, and more electrons and ions are released due to the electric field, and this collision with other particles causes gas ionization (ALVES JUNIOR et al., 2016). Plasma can be classified into two main categories depending on the thermodynamic temperature balance of the constituents and based on their temperature, being low temperature (LTP - Low Temperature Plasma) or high temperature (HTP - High Temperature Plasma) (NEHRA et al., 2008; RUTSCHER, 2008), as observed in Table 1. Table 1. Plasma classification according to temperature

In which: Tg= Temperature of the gas molecules; Ti = Temperature of the ions; Te = Temperature of the electrons.

Cold atmospheric pressure plasma (CAPP) is also known as low temperature non- thermal plasma because it has a low temperature in its system (≤ 105 K). In this case, there is no frequent collision between the charged and neutral particles, meaning LTP is the result of partial gas ionization. Thermal plasma can be observed in much of the universe, for example in the sun and other stars. Its application has been turned to the metallurgical area due to its ability to heat, melt and, in some cases, vaporize metals and metal alloys (FELIPINI; LIEBERMAN and LICHTENBERG, 2005; FERNÁNDEZ-GUTIERREZ et al., 2010). CAPP has been used for several purposes in recent years due to its practicality and low cost as it does not require a vacuum system. In addition, it can be generated in different ways, but the most common ways are through dielectric barrier discharge (DBD), plasma jet and corona discharge (LU et al., 2014). The plasma generated through dielectric barrier discharge has advantages over other systems, mainly because it can use different combinations of gases. Moreover, another positive point of this system is its flexibility to adapt the plasma generation in different geometric shapes of the electrodes (EHLBECK et al., 2011; CHIZOBA EKEZIE et al., 2017). DBD is generated by an alternating current emitted from a potential difference applied between two electrodes, in which the dielectric layer is inserted between them. When there is high voltage application between the electrodes, electrical charges accumulate on the surface of the dielectric until it breaks the dielectric strength of the gas and cause micro- discharges, which are short-lived and are governed by ionization processes and atomic and molecular excitations (BÁRDOS and BARÁNKOVÁ; FERNÁNDEZ-GUTIERREZ et al., 2010; BHIDE, 2016). Dielectrics are responsible for limiting the amount of charge and the energy transmitted to an individual micro discharge, distributing the micro discharge over the entire length of the electrode (KOGELSCHATZ, 2003). Due to its adaptability of configurations for application, it is possible to use planar, coplanar or surface configurations. Plasma has gained prominence in various science fields in recent years as it is a highly innovative technique and at the same time challenging in several areas, including with good support for the agricultural and forestry sector.

3.1.6. The use of plasma and its potential in agriculture

Promising studies on plasma application have been reported in the agricultural and forestry industries, mainly because it has great benefits including low energy consumption, the non-use of chemicals, and having versatility providing work with different sample sizes and not producing polluting residues (MASAFUMI et al., 2012).

Cold plasma has also gained increasing attention in recent years among conventional methods for inactivating microorganisms due to low operational risks, not presenting toxicity and also for providing a significant reduction in undesirable microorganisms (SONG et al. 2009; CHIANG et al; KORACHI et al., 2010). Corroborating the results reported by Kordas et al. (2015) in working with Triticum sp. seeds treated with atmospheric plasma for different exposure periods, they observed a decrease in the formation of fungi colonies of the Alternaria sp., Aspergillus sp. and Rhizopus sp. genera. The use of plasma can be seen as an efficient alternative to decrease the contamination present in Allium cepa L., Raphanus sativus L. and Lepidium sativum L. seeds (BUTSCHER et al., 2016). Furthermore, Puligundla et al. (2017) found a considerable reduction in the microbial load present in Brassica napus L. seeds, and this treatment did not affect germination or seedling development. In addition, studies involving dielectric barrier discharge (DBD) have shown that the plasma produced from this methodology provides greater benefit to seed quality, improving water permeability and seed germination which present dormancy, meaning those which show difficulties to germinate even under ideal conditions, especially forest specie seeds.

3.1.7. Plasma application to overcome seed dormancy

The cold plasma technology produced by dielectric barrier discharge (DBD) can improve seed performance and germination, and consequently crop productivity (SELCUK et al; SERÁ et al., 2008). In this sense, some studies on applying cold plasma as a method to overcome dormancy and improve germination have been reported in the literature. For example, in working with Cassia torosa and Saphora flavescens seeds, Yamauchi et al. (2012) found that there was an increase in the seed germination percentage after the plasma irradiation and consequently greater imbibition. A similar result was obtained by Bormashenko et al. (2015) in Phaseolus vulgaris L. seeds in which the plasma provided greater wettability and therefore increased germination. Moreover, an increase in the emergence percentage was observed in Leucaena leucocephala Lam. seeds treated with DBD plasma (GUIMARÃES et al., 2015). These works with using plasma to break dormancy in seeds present significant results, which are probably due to chemical reactions from the interaction of the gas with the seed surface causing the layer which was previously hydrophobic to become hydrophilic, thus reducing the tegument impermeability and consequently promoting water absorption in the seeds (SERÁ et al., 2008). As a result, it provides metabolism acceleration during germination (FILATOVA et al., 2015).

Such technology very much depends on operational conditions, and the information from other research serves as support for new studies with forest species, making it possible to test and adapt the methodologies according to the characteristics of the species, as well as the system to be used, taking into account the factors which may influence this process.

3.1.8. Factors which interfere with plasma efficiency

Cold plasma systems are different in configuration, structure, power supply and working conditions, thus enabling a wide range of applications. It has potential for application in the processing and packaging of food products, cleaning waste water, medical treatment (coagulating blood, inducing apoptosis in malignant tissues) and surface disinfection/sterilization of medical equipment and heat sensitive materials, as well as in agriculture and forestry (PATANGE, 2019). However, some factors are decisive for this technology to be efficient, among which the composition, the injected gas, as well as the flow and conduction time during the process are highlighted. Regarding gas (atmospheric air, O2, H2, N2, He, Ar), the main influence is on the plasma constituents to be originated. In turn, the gas flow and conduction time are directly responsible for the absorption rate of reactive species in the plasma to which the sample will be subjected (GUO et al., 2015; PANKAJ et al., 2015; MIR et al., 2016).

Material and Methods

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4. MATERIAL AND METHODS ______

4.1.1. Seed procurement and processing

P. moniliformis fruits were harvested at the Experimental Farm of the Federal Rural University of Semiárido - Mossoró (5º 03’ S and 37º 24’ W), Rio Grande do Norte, Brazil. Then, they were placed to dry in the shade and manual processing was performed, extracting and selecting the healthy and well-formed seeds. The selected seeds were taken to the laboratory where they were then subjected to atmospheric plasma and evaluations.

4.1.2. Plasma application

The seeds were subjected to an application of atmospheric cold plasma for different exposure periods (1.5; 2.0; 3.0; 4.0 and 5.0 minutes) and seeds without any treatment were used as a control for comparison. Plasma was applied at the Plasma Laboratory at the Federal Rural University of the Semi-Arid (UFERSA). Plasma generated from a dielectric barrier was used, produced in a coplanar plate (Figure 3).

Figure 3. (A) Schematic representation of the system used for applying plasma with a high voltage source; (B) Detail of the seeds arranged inside the glass plate under the coplanar

15 plate; (C) Diagram indicating the position of the electrodes and the dielectric material inside the plate in relation to the seeds.

The coplanar plate system was powered by a high voltage source with a power of 10 kv and a frequency of 400 khz (Figure 1A). A total of 50 seeds were used in each experimental condition at a time in the Petri dish with dimensions of 90 mm in diameter and 15 cm in height, and dispersed so as not to overlap (Figure 1B). The system interior consisted of a phenolite plate with copper-clad electrodes (Figure 1C).

4.1.3. Determination of wettability

Both the treated seeds and those used in the control had the apparent contact angle of the integument with water deposited in the form of a drop, being measured by the sessile drop method in order to indirectly assess the integument’s wettability. To do so, seven seeds of each treatment were used which were deposited on a flat surface, applying a drop of distilled water on the integument using a 100µl micropipette. The image of the drop contact with the seed surface was then captured 30 seconds after the water drop was applied, according to the methodology described by Alves Junior et al. (2016). After image acquisition, the images were transferred to a computer and processed in the Surftens® 3.5 software program (Frankfurt, Germany) (Yanling et al., 2014), in which the values of the apparent contact angles obtained by the drop of water and the integument were obtained. These values were submitted to descriptive statistics (arithmetic mean and standard deviation). The smaller the contact angle, the greater the wettable surface, and therefore the greater the wettability of the seeds.

4.1.4. Water absorption and electrical conductivity curve

Four repetitions with 50 seeds of each treatment were used to characterize the imbibition curve. The weight of each repetition was initially measured on a precision analytical scale (0.001 g). Next, the seeds were placed in properly identified plastic cups containing 75 ml of distilled water and placed in a Biochemical Oxygen Demand (BOD) germinator at 25°C with a 12h photoperiod (PEREIRA et al., 2015). The relative imbibition percentage (I%) was obtained from the variation in the seed mass after the periods of 0, 2, 4, 6, 12, 24, 36, 48, 60, 72 and 96 h of imbibition. To do so, a sieve was used to separate the seeds from the liquid, which were then deposited on paper towels in order to remove excess water, and the weight was measured afterwards. In conjunction with determining the imbibition curve, the liquid in which the seeds were

16 immersed was evaluated to quantify the leaching of the substances from the seeds into the imbibition medium by electrical conductivity. The evaluations were carried out at the respective intervals, in which the seed weights were measured during the imbibition period, as previously described. After evaluation in each period, the seeds were put back in the same liquid in which they were immersed during the imbibition process. The electrical conductivity was determined using a Digimed DM conductivity meter and the average values obtained for each treatment expressed in µS.cm-1.g-1 of seeds

4.1.5. Germination test

For the germination test, a Germitest® paper roll was used as a substrate, moistened with distilled water in an amount corresponding to 2.5 times the mass of the dry substrate in a Biochemical Oxygen Demand (BOD) chamber at a temperature of 25ºC and 12h photoperiod. The results were expressed as a percentage of normal seedlings 21 days after the test was installed (BRASIL, 2013). The first count of normal seedlings was jointly performed with the germination test, counted on the seventh day after installing the test. Germination speed was also evaluated by determining the germination speed index (GSI), calculated according to the equation proposed by Maguire (1962), based on the daily count of the number of seeds that emitted the primary root (greater than or equal to mm) from the 1st to the 21st day after installing the germination test. The modeling of the accumulated germination curve was adjusted according to the model proposed by Richards (1959) which determines the population growth represented by the function presented in Table 2. Table 2: The Richards’ function (Richards, 1959).

Function Richards Population 훼 parameters 훾 = 휏 *1 + 푏 ∙ 푑 ∙ 푒푥푝(−푐 ∙ 푡)+ 1⁄푑 Viability (Vi ) = 훼 1 푏∙푑 Median (Me) = ∙ 퐼푛 푐 2푑−1 1 4푑 − 1 Dispersal (Qu ) ∙ 퐼푛 2푐 (4⁄3)푑−1 푏 ∙ 푑 4푑 − 1 Asymmetry (Sk ) 2 ∙ {퐼푛 푑 /퐼푛 } 2 − 1 (4⁄3)푑−1

These were associated with population parameters of germination, where: viability (Vi), the maximum germination of a seed lot; median (Me), the time in which 50% of germination is reached; dispersion (Qu), uniformity of germination; and asymmetry (Sk), which represents asymmetry.

4.1.6. Seedling length

First, four replicates of 10 seedlings were used to determine the seedlings’ length (Nakagawa et al., 1999), being removed at random from the germination substrate and placed side by side without touching one another on a blue background. The images were subsequently acquired using a cell phone camera to capture the images, and then transferred to a computer and saved in different folders, identified according to the treatment and repetition. The ImageJ® software program was used to measure seedlings, adopting the methodology described by Noronha et al. (2018). The hypocotyl and root lengths of the seedlings were obtained through this program.

4.1.7. Study design and statistical analysis

The study design used was completely randomized with four replicates per treatment. Analysis of variance was performed using the F-test (5%), and the treatment means were compared using the Tukey test when significant. Regression analysis was used for quantitative data. A multivariate principal components analysis was also carried out. The analyzes were performed using the R statistical software program (R Development Core Team, 2010).

Results and Discussion

______

5. RESULTS AND DISCUSSION ______

5.1.1. Wettability

The seeds submitted to plasma showed a significant effect in the wettability analysis. There was a decrease in the apparent contact angle values as the exposure time of the seeds to plasma was increased, with those being treated for 4.0 and 5.0 minutes presenting smaller angles of 64 and 61°, respectively (Figure 4). These results enable us to infer that the plasma promoted chemical interactions in the surface components of the integumentary structure, capable of altering its relationship with water, reducing the apparent contact angle between the integument and the water drop (SILVA et al., 2018).

The smallest apparent contact angle for the treated seeds was that obtained when there was longer exposure to plasma (4.0 and 5.0 minutes), which implies a greater wettable surface in these seeds. This result can be justified by the chemical action that cold plasma obtained at atmospheric pressure has on the structural characteristics of biomaterials, such as seeds, as the plasma effect accumulates with an increase in the application time. The untreated seeds had the highest contact angle, differing from all other treatments, which

20 show that the plasma reduced the contact angle of the seeds with the water droplets in all exposure periods tested, increasing their wettability. The contact angle between a drop of water and the seed surface is directly linked to the hydrophobicity of this surface, so the apparent contact angle can be used to estimate the hydrophobic changes on the seed surface after treatment with plasma. According to Silva et al. (2018), it is possible that the outer composition layer of the seed undergoes chemical modifications with the increased exposure of seeds to plasma in terms of dysfunctions of functional groups, capable of altering the hydrophobic profile of the integument, and eventually providing an increase in water absorption. Hydrophobicity generally occurs in seeds which have a hard coat and have waxy surface layers formed by specific substances which are able to block water entry into the seeds, inhibiting imbibition, and thus delaying or preventing the germination process (BEWLEY et al., 2013; SERÝ et al., 2020). The results found in this study are in agreement with those obtained by Alves Junior et al. (2016), in which they verified that the apparent contact angle of Erythrina velutina seeds was also reduced when exposed to plasma, which indicates that other seeds that present tegumentary hardness also had their surfaces modified after plasma treatment.

5.1.2. Water absorption curve

The dynamics of water absorption are directly linked to wettability. Thus, a variation in the imbibition of the seeds submitted to the plasma was observed when compared to the untreated (control) seeds, showing that the plasma provided greater wettability, and consequently greater mass gain by the P. moniliformis seeds (Figure 5).

Figure 5. Increase in the mass of P. moliniformis seeds (%) submitted to different exposure periods to cold atmospheric plasma by dielectric barrier discharge during the imbibition process. There is a greater variation in the mass of the seeds submitted to the plasma during the process of water entering the seeds which absorbed a greater amount of water throughout the process, as characterized by the imbibition curve. On the other hand, the control treatment seeds showed a lower increase, confirming what was described in the wettability analysis, in which it was found that the plasma modified the surface of the seeds and resulted in greater water absorption by the treated seeds. This process occurred in a three-phase manner for the species under study, as it does for most seeds. Phase I was marked by rapid imbibition, followed by a plateau effect with absorption stabilization, characterizing phase II. Next, phase III, which has its beginning marked by root protrusion, imbibition resumed as described in the proposed model by Bewley and Black (1994). Therefore, it is possible that the seeds treated with plasma underwent an increase in their hydrophilic density in the integument, thus reaching phase III more quickly. This behavior was observed in all seeds submitted to plasma (Figure 5). There was an increase in electrical conductivity in all tested conditions for the treated seeds (Figure 6). This result may indicate a greater transfer of solutes from the seed cells to the imbibition solution due to the modified physical structure of the seed coat, allowing a greater transit of water and solutes from inside it to the solution. Temporary structural changes occur during the water absorption process by the seeds, especially in the membranes, which leads to the immediate and rapid leakage of solutes and metabolites to

22 the solution in which it is immersed (Bewley, 2013). An evaluation of the release of substances and ions during seed immersion through electrical conductivity signals the chemical relationships in the most permeable membranes and indicates changes in the waxy layer, which externally surrounds the integument.

Figure 6. Electrical conductivity (mS/Cm) of P. moniliformis seeds subjected to different exposure periods to atmospheric cold plasma by dielectric barrier discharge during the imbibition process.

The seeds submitted to plasma obtained greater electrical conductivity of the imbibition liquid in relation to the other treatments; therefore, these results corroborate those obtained for wettability and water absorption curve by the seeds. The increase in electrical conductivity in seeds treated with plasma may be related to possible changes in the chemical structure of the seeds promoted by exposure to plasma. This may have enabled greater contact and water absorption, and consequently greater solute leaching due to the release of ions, organic acids and sugars during imbibition, which are responsible for increasing the acidification of the medium. This behavior was not observed in the control seeds after 48 h, as they kept the surfaces of their integuments more hydrophobic with less water contact with the seed and less and slower imbibition process, which reduced the amount of substances leachate into the imbibition solution. Similar results to this study were presented by Silva et al. (2018) with hybanthus calceolaria seeds treated with atmospheric plasma for different exposure and imbibition periods.

5.1.3. Cumulative germination

The seeds exposed to plasma showed different behaviors for the accumulated germination curve (Figure 7). The untreated seeds showed an accumulation percentage of germinated seeds which did not exceed 8% of germination at 10 days after sowing when germination stabilized. There was an increase in germination accumulation over the days for seeds treated for 4.0 minutes with higher germination percentages than the control treatment seeds, proving the beneficial effect of plasma on seed germination.

The treatment which provided the highest accumulated germination was exposure of the seeds to the plasma for 5 minutes in which an accumulation close to 40% is observed. This represents an increase of 30% in the accumulated germination in relation to the control seeds, and therefore showing that the exposure time influences plasma efficiency on seed germination, as seen in Figure 7. This germination increase in P. moniliformis seeds can be justified by the action of plasma in contact with the seed surface since greater water absorption occurs from the interaction between free radicals produced by the plasma and the elements which constitute the seed, and which favors the germination process (ZAHORANOVA et al., 2016; ALVES

JÚNIOR et al., 2016; SIVACHANDIRAN and KHACEF, 2017). Figure 8 shows the results related to population parameters obtained through the Richards’ germination curve.

Viability values for seeds treated with plasma indicated increased germination (p<0.05) compared to untreated seeds. This increase was more accentuated for seeds treated for 5.0 minutes whose viability was 38.95%, while the value in the control treatment was 12.17%. Thus, it is possible to state that the plasma treatment promoted an increase in the germination process. The median (Me) estimates the time required for 50% of the seeds to germinate and characterizes the rate of this process in the studied species (ALVES JUNIOR et al., 2017). The seeds exposed for 5.0 minutes showed the lowest average germination time in relation to the other treatments, with this characteristic being very important for seedling production since the establishment of plants in the field is directly related to the initial development of seedlings. The seeds treated for 5.0 minutes showed greater uniformity (Qu), with this parameter being of great interest for the seedling production of forest species since this characteristic will predict how these seedlings behave in the field. The values expressed by these parameters indicate changes during the biological cycle of the species after the plasma

25 treatment. This is confirmed by the value presented in the asymmetry variable (Sk) in which all experimental conditions showed changes, but which did not occur in the control seeds. Similar results were exposed by Alves Junior et al. (2016), and can be justified due to the plasma action in the structures of the seeds, providing better water entry into them

5.1.4. Physiological quality of the seeds

The results obtained from the germination test (Figure 9a) show that the germination percentage and the germination speed index gradually increased with the increase in the exposure period to plasma, confirming that it is an efficient method to overcome dormancy in seeds of this species. It is also observed that the results shown by untreated seeds indicate a low germination percentage, demonstrating that the seed coat prevented water entry and the events involved in germination without the exposure of these seeds to plasma. Lower germination values in untreated seeds can be justified by the absence of plasma action on these seeds, since it has the ability to promote structural changes in the seed coat, reducing its impermeability (SILVA et al., 2018). Such changes in the structure which covers the seed provide greater water absorption by them, which is one of the basic requirements for germination to occur (Bewley et al., 2013). According to the regression curves fitted to the quadratic polynomial model (Figure 9), there is a gradual increase in the germination speed index according to the increase in the exposure time of the seeds to the plasma, obtaining maximum value when they are exposed to the method for 5.0 minutes (Figure 9).

40 8

30 6 20 4

10 2

Germination (%) 2 2 y = -0.0834x + 1.4707x + 2.3111

y = 0.5536x + 0.325x + 14.3 Germination speed index R² = 0.8431 R² = 0.8727 0 0 0 1 2 3 4 5 0 1 2 3 4 5 Exposure time (Min) Exposure time (Min)

4 y = 0.2634x2 + 0.7473x + 7.5089

R² = 0.8701 Firstgermination count 0 0 1 2 3 4 5 Exposure time (Min)

Figure 9. (a) Germination percentage, (b) germination speed index, and (c) first germination count in P. moniliformis seeds.

Research with other forest species using plasma also showed an increase in the germination rate of seeds. For example, Silva et al. (2017) found an increase in the germination percentage in Mimosa caesalpiniafolia seeds using atmospheric plasma for three minutes. In addition, the germination speed index in E. velutina seeds submitted to plasma did not show any difference between the conventionally used methods, but it indicated a positive effect of the plasma in increasing the germination speed of the treated seeds. A similar trend was observed for germination and the germination speed index (Figure 9b and 9c) for the first germination count, respectively, whose data also adjusted to the quadratic model in which the maximum value was obtained when the seeds were exposed to atmospheric plasma for a period of 5.0 minutes.

Different results to that found in this research were reported by Alves Junior et al. (2017) working with E. velutina seeds in which they compared treated and untreated seeds with plasma. These authors stated that the total number of seeds germinated in the first 5 days of sowing were similar in both tested conditions; however, the seeds submitted to plasma showed a higher percentage of normal seedlings. Results with different species and different plasma application conditions indicate the need to further study the action mechanisms and protocols for using plasma, with the objective of reducing the effects of cutaneous dormancy in seeds. Regarding seedling development, it was observed that the length data also adjusted to the quadratic model (Figure 10), with an increase in seedling length as the exposure time of the seeds to plasma increased. This result reinforces the efficiency of this method in overcoming the dormancy of P.moniliformis seeds and the influence of their exposure period to plasma. It is also important to note that the indication of a method not only has triggering the germinative process as its basic assumption, but it is also necessary to consider rapid and uniform development of seedlings, since this characteristic is desirable when the objective is to work with seedling production. There was an increase in the hypocotyl and root length as the exposure period to plasma increased (Figure 10).

6 6

)

4 4

2 2

y = -0.1196x2 + 1.6432x - 0.67 Root length (cm) R² = 0.9676 y = -0.0714x2 + 1.1086x + 0.7 0 R² = 0.9406

0 1 2 3 4 5 0 Length of the hypocotyl Length of the hypocotyl (cm 0 1 2 3 4 5 Exposure time (min) Exposure time (min)

Figure 10. (A) Hypocotyl and (B) root length of P. moniliformis seeds submitted to cold plasma during 0 (control); 1.0; 2.0; 3.0; 4.0; and 5.0 minutes. Different results from those found in this study were presented by Diógenes (2017) with E. velutina seeds, in which the exposure of the seeds to plasma for up to 9.0 minutes did not cause significant changes in the shoot length. Thus, it is reinforced that the

28 application methodology and the plasma effects may not be similar for all species, with the need for specific adjustments for each one. It is also important to note that the efficiency of seed treatment with plasma is also associated to other factors in addition to the exposure period, such as the type of plasma used and the physical and chemical characteristics of the seeds of each species.

5.1.5. Principal components analysis

The contribution of each variable was determined from the multivariate principal components analysis (PCA) using the variables obtained from the experimental conditions, with the components (PC1 and PC2) explaining 99.2% of the total data variability. Thus, it was possible to reduce all variables into just two dimensions through various linear combinations, which explained a significant percentage of the observations (Figure 11).

The multivariate analysis for the results of the physiological seed quality variables proved to be elucidating, and according to Medeiros (2018), this analysis contributes to a

29 more objective understanding and interpretation of results. There is greater dispersion between treatments according to the central ordering diagram, indicating that there was variation between the treatments which constituted the PCA. In contrast, there was a greater grouping of treatments 3.0; 4.0; and 5.0 minutes, corroborating the results previously expressed in the physiological analyzes. Thus, the treatments which were located in a distant and opposite position to the physiological quality vectors (represented in the correlation circle on the right side of the central ordering diagram) were those which presented the lowest values for these variables. In the correlations circle, it is noted that the vectors which comprise the physiological quality variables showed a high correlation for the experimental conditions with plasma. Thus, these variables were able to demonstrate the efficiency and correlation of treatments with viability and physiological quality. Several studies in the seed area have used this type of analysis involving principal components, as it enables identifying patterns in the seed quality data, and thus expresses the similarities and differences to be observed, reducing the dimensionality without losing much information (Medeiros et al., 2018).

Conclusions

6. CONCLUSION

______

The atmospheric plasma application technology is an efficient method to overcome the integumentary dormancy of P. moniliformis seeds. The exposure periods of seeds to plasma for 4.0 and 5.0 minutes provided greater wettability and physiological quality.

Acknowledgements

______

7. ACKNOWLEDGEMENTS

______

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

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