Online version: ISSN 2317-1545 JOURNAL OF SEED SCIENCE

Continuation of the “Revista Brasileira de Sementes / Brazilian Seed Journal”

Volume 42

Collection of published articles from April to June, 2020

This Journal’s articles are indexed in: - BINAGRI / Ministério da Agricultura, Pecuária e Abastecimento www.agricultura.gov.br - SCIELO (Scientific Eletronic Library on line) www.scielo.br - SCOPUS - AGROBASE - AGRIS Coordenadoria Geral e Informação Documental Agrícola BINAGRI/MAPA - EBSCO Information Services

Associação Brasileira de Tecnologia de Sementes Brazilian Association of Seed Technology GENERAL INFORMATION

The Journal of Seed Science (JSS) is the official publication of the Brazilian Association of Seed Technology, and publishes original articles and revisions in the field of Seed Science and Technology and related areas of the agricultural services. The JSS is a publication in continuation of the “Revista Brasileira de Sementes” (Brazilian Seed Journal), from January/2013. The mission of JSS is to publish scientific papers in the area of Seed Science and Technology, providing to the national and international agricultural sectors the knowledge for producing high quality seeds and the benefits from its use. Besides, JSS aims to contribute for the development and improvement of technologies that would aid in the economic and social improvement of the population, guaranteeing the basic input of the agricultural production and the preservation of the vegetable species. The JSS is published four times a year, although special numbers can be published. The Editorial Board is composed by one or more editors, Associated Editors and a Scientists Committee formed by scientists working with Seed Science and Technology. The annual subscription rate of JSS is US$ 200.00 abroad. The price of each number is US$ 60.00. Subscriptions and or individual number should be requested by sending a money order to ABRATES, at the following address:

JOURNAL OF SEED SCIENCE Universidade Federal de Viçosa - Departamento de Agronomia Avenida PH Rolfs, s/n°, 36570-900, Viçosa - MG – Brasil Fone: +055(31) 3612-4490 / Email: [email protected] ABRATES Londrina - Fone/FAX: +055(43) 3025-5120 Email: [email protected]

INFORMAÇÕES GERAIS

A revista ”Journal of Seed Science” (JSS) é uma publicação oficial da Associação Brasileira de Tecnologia de Sementes (ABRATES) e destina-se a divulgar trabalhos científicos originais sobre Ciência e Tecnologia de Sementes e áreas correlatas. O JSS é a publicação que dará continuidade à Revista Brasileira de Sementes (Brasilian Seed Journal) a partir de Janeiro de 2013. A missão do JSS é publicar trabalhos científicos na área de Ciência e Tecnologia de Sementes, divulgando ao setor agrícola nacional e internacionals avanços do conhecimento para a obtenção de sementes de alta qualidade e informações relativas aos benefícios resultantes da sua utilização. Além disso, contribuir para o desenvolvimento e aprimoramento de tecnologias que auxiliem no desenvolvimento econômico e social da população, garantindo o insumo básico da produção agrícola e a preservação das espécies vegetais. A periodicidade do JSS é trimestral, podendo, no entanto, serem publicadas edições especiais. O Comitê Editorial é composto por um ou mais Editores, por Editores Associados e um corpo de Assessores Científicos, formado por cientistas que trabalham em Tecnologia de Sementes. O preço da assinatura, ao ano, é de US$ 85,00 (oitenta e cinco dólares americanos) para o território nacional e de US$ 200,00 (duzentos dólares americanos) para o exterior. Os pedidos acompanhados de cheque nominal à ABRATES deverão ser encaminhados à Diretoria Técnica e de Divulgação no seguinte endereço:

JOURNAL OF SEED SCIENCE Universidade Federal de Viçosa - Departamento de Agronomia Avenida PH Rolfs, s/n°, 36570-900, Viçosa - MG – Brasil Fone: +055(31) 3612-4490 / Email: [email protected] ABRATES Londrina - Fone/FAX: +055(43) 3025-5120 Email: [email protected]

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Layout Editor and Design: Jéssica Akemi Ychisawa ASSOCIAÇÃO BRASILEIRA DE TECNOLOGIA DE SEMENTES (BRAZILIAN ASSOCIATION OF SEED TECHNOLOGY) EXECUTIVE BOARD - 2017/2020

President: Francisco Carlos Krzyzanowski (EMBRAPA SOJA) 1st Vice-President: Fernando Augusto Henning (EMBRAPA SOJA) 2nd Vice-President: Maria Laene Moreira de Carvalho (UFLA) Financial Director: José de Barros França Neto (EMBRAPA SOJA) Vice-Financial Director: Alessandro Lucca Braccini (UEM) Technical and Communication Director: Denise Cunha Fernandes dos Santos Dias (UFV) Vice-Technical and Communication Director: Gilda Pizzolante de Pádua (EMBRAPA)

Supervisory Board - holders: Francisco Guilhien Gomes Junior (USP/ESALQ) Luiz Eduardo Panozzo (UFPel)

Alternates: Claudio José Barbedo (Instituto de Botânica, SP) Géri Eduardo Meneghello (UFPel) Laércio Junio da Silva (UFV)

JOURNAL OF SEED SCIENCE

EDITORS Denise Cunha Fernandes dos Santos Dias - Universidade Federal de Viçosa Gilda Pizzolante de Pádua - Empresa Brasileira de Pesquisa Agropecuária

Associated Editors: Alek Sandro Dutra (UFC) José de Barros França-Neto (EMBRAPA SOJA) Alessandro Lucca Braccini (UEM) José da Cruz Machado (UFLA) Augusto César Pereira Goulart (EMBRAPA) Julio Marcos-Filho (USP/ESALQ) Ceci Castilho Custódio (UNOESTE) João Almir Oliveira (UFLA) Claudemir Zucareli (UEL) Laércio Junio da Silva (UFV) Claudio José Barbedo (Instituto de Botânica) Luciana Magda de Oliveira (UDESC) Denise Cunha Fernandes dos Santos Dias (UFV) Marcela Carlota Nery (UFVJM) Eduardo Euclydes de Lima e Borges (UFV) Maria Laene Moreira de Carvalho (UFLA) Eduardo Fontes Araújo (UFV) Maria Luiza Nunes Costa (UFMS) Elisa Serra Negra Vieira (EMBRAPA FLORESTAS) Norimar D’Ávila Denardin (UPF) Fatima Conceição Márquez Piña Rodrigues (UFSCar) Raquel Maria de Oliveira Pires (UFLA) Fernando Augusto Henning (EMBRAPA SOJA) Renato Delmondez de Castro (UFBA) Francisco Carlos Krzyzanowski (EMBRAPA SOJA) Salvador Barros Torres (EMPARN) Francisco Guilhien Gomes Júnior (USP/ESALQ) Silvio Moure e Cícero (USP/ESALQ) Gilda Pizzolante de Pádua (EMBRAPA/EPAMIG) Sttela Dellyzete Veiga Franco da Rosa (EMBRAPA CAFÉ) Heloísa Oliveira Santos (UFLA) Warley Marcos Nascimento (EMBRAPA HORTALIÇAS) International Associated Editors: Peter Toorop (Kew Garden/England) Omar Bazzigalupi (EEA INTA/Argentina)

English Language Reviewers: Editora Genesis Infoservice Ltda. Lloyd John Friedrich Fausto Bahia Teixeira

Reviewers André Dantas de Medeiros André Luiz dos Santos Bruno Gomes de Noronha Denise Cunha Fernandes dos Santos Dias Edgard Augusto Toledo Picoli Eduardo Euclydes de Lima e Borges Everson Reis Carvalho Hugo Tiago Ribeiro Amaro Julia Abati Larissa Alexandra Cardoso Moraes Leonardo Gonçalves Bastos Marcone Moreira Santos Maria Luiza Nunes Costa Marília Lazarotto JOURNAL OF SEED SCIENCE ISSN 2317-1545 v. 42, Apr./Jun. 2020

CONTENTS

ARTICLES

Biochemical changes stimulated by accelerated aging in safflower seeds (Carthamus tinctoriusL.). Sercan Önder, Muhammet Tonguç , Damla Güvercin, Yaşar Karakurt.

Use of near infrared spectroscopy in cotton seeds physiological quality evaluation. Lívia Giro Mayrinck, Juliana Maria Espíndola Lima, Gabriel Castanheira Guimarães, Cleiton Antônio Nunes, João Almir Oliveira.

Relationships between substrate and the mobilization of reserve with temperature during seed germination of Ormosia coarctata Jack. Luciane Pereira Reis, Eduardo Euclydes de Lima e Borges, Genaina Aparecida de Souza, Danielle S. Brito.

Morphoanatomic and histochemical aspects of Elaeis oleifera (Kunth) Cortés seed. Suelen Cristina de Sousa Lima, Poliana Roversi Genovese-Marcomini, Regina Caetano Quisen, Maria Silvia de Mendonça.

Cowpea yield and quality after application of desiccating herbicides. Jeovane Nascimento Silva, Estevam Matheus Costa, Leandro Spíndola Pereira, Elaine Cristina Zuquetti Gonçalves, Jacson Zuchi, Adriano Jakelaitis.

Induction of seed coat water impermeability during maturation of Erythrina speciosa seeds. Debora Manzano Molizane, Sandra Maria Carmello-Guerreiro, Claudio José Barbedo.

Microbiolization of organic cotton seeds withTrichoderma sp. and Saccharomyces cerevisiae. José Manoel Ferreira de Lima Cruz, Eliane Cecília de Medeiros, Otília Ricardo de Farias, Edcarlos Camilo da Silva, Luciana Cordeiro do Nascimento.

Sowing dates and densities on physiological potential of seeds of white oat cultivars. José Henrique Bizzarri Bazzo, Klever Márcio Antunes Arruda, Inês Cristina de Batista Fonseca, Claudemir Zucareli.

NOTES

Osmotic treatment, growth regulator and rooter in Tabebuia roseoalba (RIDL.) Sandwith seeds for direct sowing. Alexandre Carneiro da Silva, Maiara Pilar Palmeira da Silva, Rayssa Zamith, Gustavo Galetti, Fatima Conceição Márquez Piña-Rodrigues. JOURNAL OF SEED SCIENCE ISSN 2317-1545 v. 42, Abr./Jun. 2020

CONTEÚDO

ARTIGOS

Alterações bioquímicas estimuladas pelo envelhecimento acelerado em sementes de cártamo (Carthamus tinctorius L.). Sercan Önder, Muhammet Tonguç, Damla Güvercin, Yaşar Karakurt.

Uso da espectroscopia no infravermelho próximo na avaliação da qualidade fisiológica de sementes de algodão. Lívia Giro Mayrinck, Juliana Maria Espíndola Lima, Gabriel Castanheira Guimarães, Cleiton Antônio Nunes , João Almir Oliveira.

Relações do substrato e da mobilização de reservas com a temperatura na germinação de sementes de Ormosia coarctata Jack. Luciane Pereira Reis, Eduardo Euclydes de Lima e Borges, Genaina Aparecida de Souza, Danielle S. Brito.

Aspectos morfoanatômicos e histoquímicos da semente de Caiaué (Elaeis oleifera (Kunth) Cortés). Suelen Cristina de Sousa Lima, Poliana Roversi Genovese-Marcomini, Regina Caetano Quisen, Maria Silvia de Mendonça.

Rendimento e qualidade de sementes de feijão-caupi após a aplicação de herbicidas dessecantes. Jeovane Nascimento Silva, Estevam Matheus Costa , Leandro Spíndola Pereira, Elaine Cristina Zuquetti Gonçalves, Jacson Zuchi, Adriano Jakelaitis.

Indução da impermeabilidade do tegumento em relação a água durante a maturação em sementes de Erythrina speciosa. Debora Manzano Molizane, Sandra Maria Carmello-Guerreiro, Claudio José Barbedo.

Microbiolização de sementes de algodoeiro orgânico com Trichoderma sp. e Saccharomyces cerevisiae. José Manoel Ferreira de Lima Cruz, Eliane Cecília de Medeiros, Otília Ricardo de Farias, Edcarlos Camilo da Silva, Luciana Cordeiro do Nascimento.

Épocas e densidades de semeadura no potencial fisiológico de sementes de cultivares de aveia branca. José Henrique Bizzarri Bazzo, Klever Márcio Antunes Arruda, Inês Cristina de Batista Fonseca, Claudemir Zucareli.

NOTAS

Tratamento osmótico, reguladores de crescimento e enraizador em sementes deTabebuia roseoalba (RIDL.) Sandwith para semeadura direta. Alexandre Carneiro da Silva, Maiara Pilar Palmeira da Silva, Rayssa Zamith, Gustavo Galetti, Fatima Conceição Márquez Piña-Rodrigues. Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Biochemical changes stimulated by accelerated aging in Journal of Seed Science, v.42, e202042015, 2020 safflower seeds (Carthamus tinctoriusL.) http://dx.doi.org/10.1590/2317- 1545v42227873 Sercan Önder1* , Muhammet Tonguç1 , Damla Güvercin2 , Yaşar Karakurt1

ABSTRACT: Seed vigor tests are used to estimate their quality. One of the most commonly used is the accelerated aging test (AA). The aim of the present study was to study the biochemical changes caused in the seeds and to determine their germination status after the AA. Six safflower genotypes were tested at 43 °C and 45 °C for 0, 48, 72, 96 and 120 h, and germination percentage (GP), mean germination time (MGT) and normal seedling percentage (NSP) were evaluated to determine the aging reactions of the genotypes. During the AA at 45 °C, the seeds quickly lost their germination ability after 48 h; after 120 h, the seeds lost their viability, remaining, however, still viable at 43 °C. Two genotypes that aged more (Linas and Olas) and less (Bayer-6 and Bayer-12) were chosen to examine the biochemical changes during the AA at 43 °C. Eleven biochemical analysis were performed to understand physiological changes associated with the test. Total caratone, xanthophyll, phenolics, flavonoid, soluble protein, soluble sugars, oil and malondialdehyde contents were lower after 120 h, compared to 0 h. Reducing sugars and free fatty acids contents increased in the least and most aging genotypes. However, the total tocopherol content increased in the least aging genotypes and decreased in the most aging genotypes after 120 h, compared to 0 h. The results showed that the AA at 43 °C was suitable to study the aging process in the safflower seeds. Besides, understanding the chemical changes was useful to elucidate the physiological basis of seed aging.

Index terms: germination, lipid peroxidation, oil seed, seed deterioration.

Alterações bioquímicas estimuladas pelo envelhecimento acelerado em sementes de cártamo (Carthamus tinctorius L.)

RESUMO: Testes de vigor de sementes são usados para estimar sua qualidade. Um dos mais utilizados é o teste de envelhecimento acelerado (EA). O objetivo do presente estudo foi estudar as alterações bioquímicas provocadas nas sementes e determinar seu status de germinação após o EA. Seis genótipos de cártamo foram testados a 43 °C e 45 °C por 0, 48, 72, 96 e 120 h, e a porcentagem de germinação (PG), o tempo médio de germinação (TMG) e a porcentagem normal de plântulas *Corresponding author (PNP) foram avaliados para determinar as reações de envelhecimento dos genótipos. Durante o EA E-mail: [email protected] a 45 °C, as sementes perderam rapidamente a capacidade de germinação após 48 h; após 120 h, as sementes perderam a viabilidade, mantendo-se, porém, ainda viáveis ​​a 43 °C. Dois genótipos que Received: 8/25/2019 Accepted: 3/16/2020 envelheceram mais (Linas e Olas) e menos (Bayer-6 e Bayer-12) foram escolhidos para examinar as alterações bioquímicas durante o EA a 43 °C. Onze análises bioquímicas foram realizadas para entender as alterações fisiológicas associadas ao teste. Os teores totais de caratona, xantofila, fenólicos, flavonoides, proteínas solúveis, açúcares solúveis, óleo e malondialdeído foram menores 1Isparta University of Applied Science, após 120 h, em comparação a 0 h. O teor de açúcares redutores e de ácidos graxos livres aumentou Faculty of Agricultural, Department of nos genótipos menos e mais envelhecidos. No entanto, o teor total de tocoferol aumentou nos Agricultural Biotechnology – 32200, genótipos com menor envelhecimento e diminuiu nos genótipos com maior envelhecimento após Isparta, Turkey. 120 h, em comparação a 0 h. Os resultados mostraram que o EA a 43 °C foi adequado para estudar o processo de envelhecimento nas sementes de cártamo. Além disso, compreender as alterações 2Suleyman Demirel University, químicas foi útil para elucidar a base fisiológica de envelhecimento das sementes. Faculty of Arts and Sciences, Department of Biology – 32260, Termos para indexação: germinação, peroxidação lipídica, oleaginosas, deterioração de sementes. Isparta, Turkey.

Journal of Seed Science, v.42, e202042015, 2020 2 S. Önder et al.

INTRODUCTION

Safflower (Carthamus tinctorius L.) is an important oilseed plant belonging to the Asteraceae. Safflower was used to obtain edible oil, biodiesel production, natural dyes and pharmaceuticals. In recent years, safflower started to become popular and world safflower acreage reached to 967.742 ha with a total production of 738.477 tons in 2017 (FAO, 2019). Safflower has a regular flowering pattern with the formation of side branches on the main stem (Baydar and Ülger, 1998). Flowering period lasts between 10-40 days and, consequently, seeds of different sizes and quality are obtained from the plant. Since the seeds are stored after harvesting, deterioration and aging events occur within the seeds (Priestley, 1986). Seed degradation and aging affect their viability and vigor, leading to a decrease in their quality and even to death. Seed vigor tests are used to estimate seed quality. One of the most commonly used is the accelerated aging (AA). In this test, seeds are artificially aged by exposure to high temperatures and high humidity for a certain period of time. The AA method was useful for estimating the vigor of various plants such as wheat (Ghahfarokhi et al., 2014), maize (Dias et al., 2015), soybean (Usha and Dadlani, 2015), canola (Yin et al., 2015), safflower (Demir, 2014) and sunflower (Kibinza et al., 2006; Balešević-Tubić et al., 2007), and the percent of emergence. The main components of the seeds are lipids, carbohydrates, proteins and vitamins. The existence of these compounds in the seeds varies according to the plant species, environmental conditions and variety. Seed aging at the cellular level affects these reserves, resulting in cell membrane degradation, changes in energy metabolism, changes in the structure of proteins and enzymes, reduction in seed reserve utilization, degradation in lipid and carbohydrates and production of reactive oxygen species (ROS) and toxic compounds (Priestley, 1986). All these physiological reactions can lead to the deterioration in DNA structure and to the inhibition of RNA and protein synthesis. The loss of seed viability is the cumulative result of the deterioration effects in the cell. Components that accumulate in degraded or aged seeds also negatively affect the germination of non-degraded seeds (Priestley, 1986). A large proportion of oilseed plants, such as safflower, are at risk of lipid peroxidation. During seed aging, the lipids in cells are degraded by enzymatic (lipases) lipid peroxidation or non-enzymatic (oxidative peroxidation) lipid peroxidation (Berger et al., 2001). Free radicals resulting from stress in seeds cause lipid peroxidation by acting on polyunsaturated fatty acids in membranes. Oxidative peroxidation is observed when the seed moisture content is low, but the ambient temperature is high. In enzymatic lipid peroxidation, lipases are activated when the water content of the seed is high and increases the effect of oxidative peroxidation. Although safflower is becoming an important oilseed species, few studies were conducted to investigate safflower seed aging, vigor and viability. Although the seeds are classified as high or low vigor by using different vigor tests in seed groups of some plants, the information published in vigor tests used on safflower is still not enough. In recent years, the safflower cultivation increased the need of research on this plant. The aim of the present study was to determine the germination responses of safflower genotypes after the accelerated aging test (AA), conducted under two different temperatures and periods, and to study the biochemical changes during the AA to elucidate the physiological basis of the seed aging in this species.

MATERIAL AND METHODS

Plant material Six safflower genotypes (Bayer-6, Bayer-12, Dinçer, Montola 2000, Linas, Olas) was kindly provided by Sabri Erbaş from the Department of Field Crops. Seeds of all genotypes were stored at 4 °C until the experiments. 1000-seed weights and moisture contents (MC) of the genotypes were determined according to ISTA (2009) . Surface sterilization of the seeds was carried out in 1% sodium hypochlorite for ten minutes.

Journal of Seed Science, v.42, e202042015, 2020 Accelerated aging and accompanied chemical changes in safflower seeds 3

Accelerated aging and germination test The AA was performed as indicated by ISTA (2009) after the MC of all genotypes was adjusted to 12%. Briefly, the AA was carried out in sealed plastic boxes at 100% relative humidity under five time periods (0, 48, 72, 95 and 120 h) and two different temperatures (43 °C and 45 °C) after the seeds were placed on a sieve. Each plastic box was used to include only one genotype and a single combination of temperature and time. After the test, two hundred seeds (50 x 4) from each genotype was put into filter papers, and the standard germination tests was conducted in a germination cabinet at 25 ± 1 °C for fourteen days (ISTA, 2009). The rest of the seeds were stored in the refrigerator at 4 °C for biochemical analyses. Radicle formation of at least 2 mm in length was used as a germination criterion. Seeds were counted everyday to determine the germination percentage (GP), normal seedling percentage (NSP) and mean germination time (MGT) for fourteen days (ISTA, 2009). According to the results of the AA, the two most and the least aging genotypes were chosen and all biochemical analyses were performed using them.

Biochemical analysis Total amount of carotene and xanthophylls contents were determined according to AOAC (1984). Absorbance values at 436 nm and 474 nm for carotenes and xanthophylls, respectively, were used to determine the total carotene and xanthophilic contents of the samples. The extraction and quantification of total soluble phenolics and flavonoids was done according to Sakanaka et al. (2005). The absorbance value of the samples for total soluble phenolics was determined at 760 nm, and 0.01-0.02-0.04-0.06-0.08 mM gallic acid standard was used for the standard curve. Absorbance at 510 nm was measured, and 0.2-0.4-0.6-0.8-1.0 mM (+) - catechin concentrations were used as the standard curve for flavonoid content determination. Total soluble protein content was determined by Lowry-Hartree method (Hartree, 1972). 0.03-0.06-0.09- 0.12-0.15 mg.mL-1 BSA was used to obtain the standard curve, and the absorbance values of the samples were determined at 650 nm. Total tocopherol content was determined according to modified Emmerie-Engel method as described in Backer et al. (1980). The absorbance values of the samples were determined at 522 nm after 60 s, and 0.02-0.04-0.06-0.08-0.1 mM α-tocopherol was used to obtain the standard curve. Total soluble sugars (TSS) and reducing sugars (RS) extraction was done as described in Tonguç et al. (2012). For TSS determination, phenol sulfiric acid assay (DuBois et al., 1956) and RS Nelson-Somogyi (Somogyi, 1952) methods were used. Standard curve was prepared using glucose at concentrations of 0.005-0.01-0.015-0.020-0.030 mg.mL-1. Free fatty acids content determination was carried out as recommended by Lowry and Tinsley (1976). The standard curve was prepared using 2-4-6-8-10 mg.mL-1 oleic acid, and the absorbance of the samples were read at 715 nm with a spectrophometre. The total oil content of the genotypes was determined by nuclear magnetic resonance (NMR,

Brükermqone), and the results were expressed as %. Malondialdehyde (MDA) analysis was performed as described by Jiang et al. (2018). Absorbance at 532 nm was measured and subtracted from the absorbance at 600 nm. The amount of MDA was calculated with an extinction coefficient of 155 mm.cm-1.

Statistical analysis The transformation was performed to normalize the germination test results. The obtained germination and biochemical data were analyzed with the Proc GLM procedure using SAS (1999) program. Each analysis was performed in tree replicates, and Duncan multiple comparison tests (p < 0.05) was used to discriminate the differences between the means.

Journal of Seed Science, v.42, e202042015, 2020 4 S. Önder et al.

RESULTS AND DISCUSSION

Seed characteristics There were significant differences between the one thousand seed weight and the initial moisture content of the seeds (Table 1). 1000 g weight was found to be the highest in Bayer-6, while the lowest seed weight was found in Bayer-12. Montola 2000 and Dinçer had higher MC than the other genotypes. Olas had the lowest MC in the study, but there was no significant difference with Bayer-12.

The decline of seed germination, vigor and viability during AA The germination tests were performed in seeds exposed to AA for 0, 48, 72, 96 and 120 h, at both 43 °C and 45 °C. Increased temperature and time led to significant reductions in the GPs of the genotypes (Table 2). Aging at 43 °C for 48 h only reduced GP of Olas, whereas AA at 45 °C for 48 h reduced GPs of Dinçer, Linas and Olas significantly. Exposure to the test at 43 °C for 72 h reduced GP values of all genotypes, with the exception of Bayer-12, whose GP only declined significantly after 120 h exposure. On the other hand, exposure to the test at 45 °C after 72 h reduced GP of all genotypes, and after 120 h exposure time, all genotypes lost their germination ability. The increase in temperature and time during the AA caused increased MGT and decreased NSP values (Table 2). Regardless of time and temperature, MGT values increased in the test conditions. In all genotypes, the highest MGT was observed at 43 °C for 120 h. Most of the abnormal seedlings did not have roots, or primary roots were short, or hypocotyls were deformed. Exposure to 43 °C for 48 h did not significantly affect NSP of the genotypes. However, exposure to 45 °C temperature for 48 h reduced NSP of Dinçer, Olas and Linas. After 48 h duration time, NSP values of all genotypes reduced significantly for both temperatures. Due to different MC of the seeds, seed MC was adjusted to approximately 12% to not to cause any difference during the AA. Seed MC and temperature are the most important factors affecting seed deterioration. As the seed MC and temperature increase, the rate of deterioration reactions in the seeds increase (McDonald and Kwong, 2005). A 1% decrease in seed MC increases the seed life 2-fold, likewise 5 °C reduction at the storage temperature increases the seed life twice (Harrington, 1972). In the current study, increase in MC of seeds aged at high temperature had a detrimental effect on germination and viability. High MC together with high temperatures could lead to a faster deterioration of the seeds during aging, leading to rapid viability decline in safflower (Demir, 2014) and sunflower (Kibinza et al., 2006). Our results show that as the temperature and time increased in the AA, the seeds’ vigor and viability decreased. When all germination index data were evaluated, seed vigor and viability were lost very quickly at 45 °C. Therefore, the AA at 45 °C was not suitable for determining biochemical changes in seeds. All biochemical analyses were performed on genotypes exposed to the AA at 43 °C. Two genotypes that aged more (Linas and Olas) and less (Bayer-6 and Bayer-12) were chosen to examine the biochemical changes during the AA at 43 °C.

Table 1. One thousand seeds weight and seeds moisture content of the investigated safflower genotypes.

Genotype 1000 seed weight (g) Seeds MC (%) Seeds MC before AA (%) Bayer- 6 48.99 a 4.65 b 12.00 Bayer-12 34.15 e 4.45 cd 11.99 Dinçer 40.76 c 4.79 a 11.96 Montola 2000 38.57 d 4.81 a 12.02 Linas 44.32 b 4.51 c 12.06 Olas 37.73 d 4.38 d 12.00 a,b,cMeans followed by the same letter (s) are not significantly different at p ≤ 0.05.

Journal of Seed Science, v.42, e202042015, 2020 Accelerated aging and accompanied chemical changes in safflower seeds 5

Table 2. Germination percentage (GP, %), mean germination time (MGT, day) and normal seedling percentage (NSP, %) after accelerated aging test (AA) and standard germination (SG) of safflower genotypes.

Bayer-6 Bayer-12 Dinçer Temp. Time (hour) GP MGT NSP GP MGT NSP GP MGT NSP 48 88.0 a 2.12 cd 78.0 a 88.0 a 2.11 c 81.0 a 94.0 a 1.84 d 86.5 a 72 70.5 b 2.20 cd 55.5 bc 80.0 a 2.14 c 72.0 b 80.5 b 1.52 d 57.5 d 43 °C 96 62.0 c 3.62 b 52.0 c 78.0 a 3.65 b 69.5 b 73.5 c 2.99 b 67.5 c 120 39.0 e 4.38 ab 31.5 d 43.5 c 5.0 a 35.0 c 39.5 d 3.13 b 21.5 e 48 80.5 a 2.24 cd 73.0 a 88.0 a 2.08 cd 77.5 ab 86 b 1.47 d 79.5 b 72 53.25 d 3.07 bc 38.0 d 60.0 b 3.22 b 38.5 c 42 d 2.29 c 17.5 e 45 °C 96 16.5 f 5.00 a 7.0 e 8.0 d 5.46 a 3.0 d 3.5 e 3.9 a 0.0 f 120 0.5 g 1.5 d 0.0 e 0.0 d 0.0 d 0.0 d 0.0 e 0.0 e 0.0 f Control 0 89.5 a 1.96 d 79.0 a 89.0 a 1.80 c 82.5 a 94.0 a 1.58 d 87.0 a

Montola 2000 Linas Olas Temp. Time (hour) GP MGT NSP GP MGT NSP GP MGT NSP 48 91.0 a 2.10 bc 86.0 a 82.5 a 2.36 cd 71.0 a 87.0 b 2.4 cd 77.0 ab 72 79.0 b 1.86 bc 67.5 b 67.0 b 2.61 cd 54.5 b 78.5 c 2.7 cd 72.0 b 43 °C 96 79.0 b 3.16 b 72.5 b 50.5 c 3.74 b 45.0 c 47.0 e 4.7 b 42.5 d 120 36.0 d 3.79 b 26.0 c 19.0 e 6.02 a 9.5 e 15.5 g 6.03 a 12.5 f 48 92.0 a 1.91 bc 81.5 a 69.0 b 2.54 cd 58.5 b 67.5 d 2.88 c 59.5 c 72 46.5 c 3.25 b 27.5 c 41.0 d 3.2 bc 21.0 d 33.0 f 4.14 b 22.0 e 45 °C 96 13.5 e 5.61 a 5.5 d 4.5 f 5.45 a 1.0 f 5.5 h 6.06 a 3.5 g 120 0.5 f 1.5 c 0.0 d 0.0 f 0.0 e 0.0 f 0.0 h 0.0 e 0.0 g Control 0 93.0 a 1.47 c 86.5 a 83.0 a 1.71 d 71.0 a 93.0 a 1.86 d 81.5 a a,b,cMeans followed by the same letter (s) are not significantly different at p ≤ 0.05.

Biochemical analysis in relation to seed deterioration Total carotene content of the seeds of the least and most aging genotypes decreased during the test compared to control. Carotene content of Bayer-6 and Bayer-12 decreased steadily until 96 h of aging, but increased at 120 h of aging (Table 3). In Linas and Olas, the decrease in carotene content during aging was not regular compared to control. Total carotene content in the Bayer-6 and Bayer-12 decreased by 47 and 32%, respectively, after 120 h of aging, compared to control. In Linas and Olas, the decrease in carotene content was 37 and 23%, respectively. Biochemical changes in the seeds at the molecular level are related to seed degradation (Rejeendran et al., 2018). Seed aging causes production of ROS, enzyme inactivation, degradation of cell membranes and genetic changes. Seeds have non-enzymatic defense mechanisms to reduce the effect of ROS species. The non-enzymatic detoxifying compounds in the seeds are carotenes, vitamins and phenolic substances. Carotenoids contribute to antioxidant systems to prevent production of free radicals, leading to membrane degradation and seed aging. In addition, carotenoids protect the seed oil against oxidation. Smolikova and Medvedev (2015) reported a 2.5-fold increase in carotenoid level, after aging for six days at 40 °C in cabbage seeds. Carotene content gradually decreased in seeds aged in 100% relative humidity at 40 °C, ROS level increased 2.5 times, and wheat seeds lost their germination ability completely (Galleschi et al., 2002). In this study, carotenoid contents in four genotypes decreased gradually after the AA, showing that the carotenoid levels in safflower seeds contribute to protect

Journal of Seed Science, v.42, e202042015, 2020 6 S. Önder et al. seeds from degradation and to prevent the loss of seed viability. Xanthophylls are the oxygen carrying forms of carotenes, and significant decreases in the total amount of xanthophylls were also observed along with carotenes. The change in the total xanthophyll content of the least and most aging genotypes was different from the change in carotene content, and the xanthophyll content decreased at different rates in different aging times compared to the control. The total xanthophyll content of the genotypes decreased, but there was a burst in the total amounts of xanthophyll after 96 h or 120 h of aging. Bayer-6 and Linas decreased by 49 and 53%, respectively, compared to control after 96 h of aging. A correlation between xanthophyll content and aging in wheat seeds was reported: the determination of carotene and xanthophyll content can be effective in rapidly evaluating the aging of wheat seeds (Pinzino et al., 1999). The total soluble phenolics content of the genotypes after 0 h was highest, phenolics content decreased in various amounts during AA, and it was the lowest at 120 h. The phenolics content in Bayer-12, Linas and Olas decreased regularly until 72 h, but increased after 96 h. Reduction of phenolics content in Bayer-6 during the AA was not regular. After 120 h of aging, the highest reduction in the total phenolics content was observed in Bayer-12 (33%), and the lowest in the Bayer-6 (12%) (Table 3). The total flavonoid content of the genotypes decreased regularly during the AA. The total flavonoid content of the Bayer-6 and Bayer-12 genotypes decreased by 8% and 14%, respectively, after 120 h of aging, compared to the control. In Linas and Olas, the decrease in flavonoid content was 12% and 11%, respectively (Table 3). Phenolics and flavonoids prevent lipid peroxidation by giving electrons to ROS species, which are formed due to

Table 3. Effect of accelerated aging on carotene, xanthophyll, total phenolic, total flavonoid, total soluble protein and tocopherol content in safflower seeds.

Ageing Carotene Xanthophyll Total soluble Total flavonoid Total soluble Tocopherol Genotype -1 -1 phenolics -1 -1 -1 time (h) (µg.g ) (µg.g ) (mg.g-1) (mg.g ) protein (mg.g ) (mg.g ) 0 (Control) 7.02 a 6.65 a 0.807 a 0.734 a 5.20 a 0.654 ab 48 3.69 bc 3.72 c 0.653 c 0.736 ab 5.03 a 0.636 b Bayer-6 72 3.57 b 3.44 d 0.724 b 0.739 ab 4.73 b 0.625 b 96 3.49 c 3.39 d 0.611 d 0.718 b 4.06 c 0.683 a 120 3.72 b 4.14 b 0.712 b 0.680 c 3.41 d 0.688 a 0 (Control) 6.19 a 5.49 a 0.865 a 0.782 a 4.89 a 0.581 ba 48 4.24 b 4.22 b 0.606 b 0.703 b 4.19 b 0.544 bc Bayer-12 72 3.90 c 3.70 b 0.547 b 0.697 b 3.99 c 0.538 c 96 3.84 cd 3.97 c 0.588 b 0.680 bc 3.94 c 0.613 a 120 4.22 b 3.69 b 0.576 b 0.668 c 3.81 c 0.619 a 0 (Control) 7.32 a 6.69 a 0.736 a 0.739 a 4.44 a 0.694 a 48 4.80 c 3.62 b 0.626 c 0.711 b 4.21 b 0.688 a Linas 72 4.55 d 3.52 b 0.591 c 0.712 b 4.07 b 0.685 a 96 5.10 b 3.12 c 0.686 b 0.699 bc 4.03 b 0.678 ab 120 4.64 d 3.54 b 0.526 d 0.647 c 3.83 c 0.653 b 0 (Control) 6.24 a 5.35 a 0.855 a 0.728 a 4.58 a 0.675 a 48 4.29 c 3.39 d 0.754 b 0.724 a 4.53 a 0.587 b Olas 72 4.67 b 3.72 c 0.561 d 0.714 a 4.45 ab 0.584 bc 96 4.40 c 3.89 c 0.668 c 0.657 b 4.39 b 0.545 c 120 4.77 b 4.25 b 0.579 d 0.647 b 3.90 c 0.528 c a,b,cMeans followed by the same letter (s) are not significantly different at p ≤ 0.05.

Journal of Seed Science, v.42, e202042015, 2020 Accelerated aging and accompanied chemical changes in safflower seeds 7 aging, and found in testa, endosperm and embryo. There is a high correlation between total phenolic content and seed viability, and phenolic substances play an important role in preventing seed aging processes (Debeaujon et al., 2000; Pukacka and Ratajczak, 2007). Total phenolic content of the clover seeds aged 42 years under natural conditions was greatly reduced compared to the control seeds (Cakmak et al., 2010). Storing Fagus sylvatica L. seeds for 2, 5, 7 and 10 years at -10 °C reduced the total phenolics content of cotyledon and embryonic axes, as the storage time increased (Pukacka and Ratajczak, 2007). Total soluble protein content decreased regularly during the AA (Table 3). Reduction in soluble protein content started after 48 h in Bayer-12 and Linas, and after 72 h in Bayer-6 and Olas. After 120 h, the highest decrease in protein content was in Bayer-6 (34%), while the lowest decrease was in Linas (14%). High protein content in the seeds helps to support vigor and viability (Rejeendran et al., 2018). The oxidation in proteins contribute to the decrease in the seeds’ germination rate (Sano et al., 2016). Decrease in protein content is considered as a common indicator and index of oxidative stress. Moori and Eisvand (2017) reported that seed degradation after AA is an indicator of the decrease in enzymatic activity and in total soluble protein content in wheat. The total soluble protein content decrease dependend on storage period in Jatropha curcas seeds (Silva et al., 2018). Safflower seeds contained 4.44-5.20 mg.g-1 protein. Bayer-6 and Bayer-12 had higher soluble protein content. In the present study, the total soluble protein content in all genotypes decreased regularly during the AA, but reduction in soluble protein content was higher in Bayer-6 and Bayer-12. Changes in the total tocopherol content between the genotypes were different throughout the test. The tocopherol content of Bayer-6 and Bayer-12 decreased until 72 h of aging, but increased after 96 h of aging. The tocopherol content was higher after 120 h, compared to 0 h. On the other hand, Linas and Olas did not show an increase in tocopherol content, and its level dropped untill 120 h (Table 3). One of the most important antioxidants stored in seeds is tocopherol. Tocopherols are considered the most important non-enzymatic inhibitors of lipid peroxidation. The main function of tocopherols in seeds is to remove ROS resulting from lipid peroxidation during aging, storage, germination and seedling development (Sattler et al., 2004). Decrease in germination was related to the decrease in the total tocopherol content in the seeds of Pinus sylvestris L. (Tammela et al., 2005), Fagus sylvatica L. (Pukacka and Ratajczak, 2007), Suaeda maritima L. (Seal et al., 2010) and sunflower (Draganić et al., 2011). Arabidopsis mutants deficient in tocopherol synthesis showed decreased GP when exposed for 72 h at 40 °C (Sattler et al., 2004). In addition, the tocopherol deficiency caused lipid peroxidation in mutant lines during seed aging. These studies confirmed that tocopherols prolonged seed life. In the present study, the total tocopherol content in the most aging genotypes decreased dramatically. The total tocopherol content increased after aging for 96 and 120 h in the least aging genotypes. It was observed that tocopherol content in safflower seeds prolongs seed life during the AA. While the total soluble sugars content decreased, reducing sugars content increased during the AA period for all genotypes. Changes in total soluble sugar content between the genotypes differed depending on aging times. Just as the changes in tocopherol content, the soluble sugar content in Bayer-6 and Bayer-12 decreased steadily until 72 h of aging, but increased after 96 h of aging. Linas and Olas also showed decrease in the soluble sugars content, but it was not regular with AA time (Table 4). The reducing sugars content in Bayer-6, Bayer-12, Linas and Olas increased by 63%, 47%, 77% and 59%, respectively, after 120 h of aging (Table 4). Sugars are usually stored as starch. In the seeds, the soluble sugars are used for respiration, followed by lipids or starches. During the storage and AA, the starch content of the seeds decreases at different rates, while the amounts of monosaccharides and disaccharides increase (Murthy et al., 2003). Monosaccharides are combined with aldehyde, ketone, and amino group of proteins, and are converted into reducing sugars. Reducing sugars can cause damage to DNA, RNA and changes the structure of proteins, reducing the viability (McDonald, 1999; Sun and Leopold, 1995). Reduction in the total soluble sugar content and increase in the reducing sugars content was reported for different species during the aging, such as mung bean (Murthy et al., 2003), canola (Wang et al., 2018) and Brassica campestris (Jiang et al., 2018).

Journal of Seed Science, v.42, e202042015, 2020 8 S. Önder et al.

Table 4. Effect of AA on total reducing sugars, total soluble sugars, oil content, total free acids, and malondialdehyde content in safflower seeds.

Ageing time Total reducing sugars Total soluble Oil content Total free fatty Malondialdehyde Genotype (h) (mg.g-1) sugars (mg.g-1) (%) Acids (mg.g-1) (nmol.g-1) 0 (Control) 0.30 c 1.16 a 30.80 a 0.68 c 31.52 a 48 0.40 b 0.62 b 30.53 a 0.85 b 28.73 b Bayer-6 72 0.41 b 0.46 de 29.72 ab 0.98 a 19.07 c 96 0.48 a 0.54 c 29.27 ab 0.68 c 17.83 c 120 0.49 a 0.50 d 28.15 b 0.69 c 14.36 d 0 (Control) 0.38 c 1.12 a 32.86 a 0.49 d 41.59 a 48 0.39 c 0.55 b 32.80 ab 0.78 b 39.52 a Bayer-12 72 0.48 b 0.49 c 31.70 b 1.11 a 37.07 b 96 0.55 a 0.55 b 31.69 b 0.69 c 31.77 c 120 0.56 a 0.52 bc 31.25 b 0.79 b 31.65 c 0 (Control) 0.34 d 1.23 a 35.31 ab 0.50 d 35.50 a 48 0.44 c 0.47 c 35.02 ab 0.66 c 26.40 b Linas 72 0.48 b 0.61 b 34.48 b 0.89 a 24.34 bc 96 0.50 b 0.34 d 34.21 bc 0.67 c 23.30 c 120 0.60 a 0.14 e 32.72 c 0.77 b 17.93 d 0 (Control) 0.34 d 1.38 a 36.37 a 0.52 d 37.85 a 48 0.39 c 0.49 bc 36.18 a 0.94 a 28.67 b Olas 72 0.46 b 0.54 b 35.63 a 0.83 b 27.25 bc 96 0.48 b 0.51 b 34.44 b 0.59 cd 25.70 c 120 0.54 a 0.45 c 33.94 b 0.65 c 22.86 d a,b,cMeans followed by the same letter (s) are not significantly different at p ≤ 0.05.

The oil content of the genotypes decreased regularly during the AA (Table 4). The oil content in Bayer-6 and Bayer-12 decreased by 9 and 5%, respectively, compared to the control after 120 h of aging. The decrease in oil content was 7% in both Linas and Olas. The total free fatty acids content was the lowest at the beginning of the experiment for all genotypes, and free fatty acids in the seeds of the least and most aging genotypes increased at different rates. Free fatty acids contents in Bayer-6, Bayer-12, and Linas increased until 72 h of aging, but decreased after 96 h of aging. Olas had the highest free fatty acids content at 48 h aging (Table 4). Oilferous seeds age very rapidly due to their high content of polyunsaturated fatty acids. The oil content in the seeds had a significant effect on the seeds’ storage life, and it was significantly affected by the storage conditions. It was reported that oil content in the seeds of Jatropha curcas (Lozano-Isla et al., 2018), Arabidopsis thaliana (Oenel et al., 2017) and sunflower (Balešević-Tubić et al., 2007) decreased during storage and AA. Free fatty acids are formed as a result of the triacylglycerols degradation in the seeds. Free fatty acids accumulation in the cytoplasm reduces pH of the oil and conversion of free fatty acids to different molecules, and the permeability of the membranes increase (Tammela et al., 2005). Free fatty acids are formed during the storage of seeds and AA, and the oil quality and germination rate decreases depending on the amount of free fatty acids formed (Balešević-Tubić et al., 2007). The total free fatty acids content in the seeds of Jatropha curcas (Akowuah et al., 2012), Arabidopsis (Oenel et al., 2017), sesame (Kavitha et al., 2017) and cotton (Nik et al., 2011) increased as the aging or storage time increased. Studies on MDA level after aging or during storage showed that MDA accumulation varies in plant species. It was suggested that lipid peroxidation in oilseed plants is an important factor in seed degradation, and MDA level could be

Journal of Seed Science, v.42, e202042015, 2020 Accelerated aging and accompanied chemical changes in safflower seeds 9 used as an indicator of lipid peroxidation in seeds during aging. MDA amount increased in plant species such as: wheat (Moori and Eisvand., 2017), oat (Xia et al., 2015), canola (Yin et al., 2015), soybean (Sharma et al., 2013), Arabidopsis (Devaiah et al., 2007), and sunflower (Kibinza et al., 2006), during aging. Despite these assumptions, Lehner et al. (2008) reported that the amount of MDA decreased after AA in two wheat varieties, and MDA levels in Jatropha curcas decreased after storage (Silva et al., 2018). MDA levels after AA decreased in four different species of Brassicaceae, and lipid peroxidation was not the main cause of seed degradation (Mira et al., 2011). The MDA content was higher at 0 h for all genotypes than the AA applied seeds. The MDA content of the genotypes after AA decreased regularly with increased aging time. The MDA content in Bayer-6, Bayer-12, Linas and Olas decreased by 54%, 24%, 50% and 40%, respectively, after 120 h of aging, indicating that lipid peroxidation was not the main cause of seed aging in safflower during the AA (Table 4).

CONCLUSIONS

Safflower genotypes showed different responses to AA, and the AA at 43 °C was suitable to asses effects of aging in safflower. Carotene, xanthophyll, soluble phenolics, flavonoid, soluble protein, soluble sugars, oil contents and MDA levels decreased, and reducing sugars and total free fatty acids increased varying amounts among the genotypes during the AA. The total tocopherol content increased in aging resistant genotypes and decreased in aging sensitive genotypes. The lipid peroxidation as indicated by the MDA levels in safflower seeds was not the main cause of seed degradation during the AA.

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Journal of Seed Science, v.42, e202042015, 2020 Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Use of near infrared spectroscopy in cotton seeds physiological Journal of Seed Science, v.42, e202042016, 2020 quality evaluation http://dx.doi.org/10.1590/2317- 1545v42227169 Lívia Giro Mayrinck*1 , Juliana Maria Espíndola Lima1 , Gabriel Castanheira Guimarães1 , Cleiton Antônio Nunes1 , João Almir Oliveira1

ABSTRACT: This study aimed to evaluate the near-infrared spectroscopy potential in analyzing the quality of cottonseed regarding different physiological quality levels, noting the need for faster techniques and tools to aid decision making. It was used eight samples of cottonseed with and without lint, presenting different physiological quality. The “high” (lots 1, 4, 5, 6 and 7) and “low” (lots 2, 3 and 8) vigor levels were defined based on vigor tests carried out and on the Normative Instruction 45/2013. The near infrared spectroscopy spectra was obtained from four types of sample preparations: whole seeds, cut in a half, without tegument and grounded seeds. Using the spectra and the grouping of lots in high and low vigor, cross validation models were optimized, built using the PLS - DA method, making it possible to predict seed classes. Grounded seeds were the best type of sample preparation, with 95% of correct predictions for high vigor seeds and 100% of low vigor (both for seeds with lint) and with 100% correct predictions for high vigor seeds and 91.7% low vigor (without lint).

Index terms: Gossypium hirsutum L., vigor, chemometrics.

Uso da espectroscopia no infravermelho próximo na avaliação da qualidade fisiológica de sementes de algodão

RESUMO: Este trabalho foi conduzido com o objetivo de avaliar o potencial da espectrometria no infravermelho próximo para analisar a qualidade de sementes de algodão em função de diferentes níveis de vigor, visto a necessidade por técnicas e ferramentas mais rápidas para o auxílio na tomada de decisão . Foram utilizadas oito amostras referentes a oito lotes de semente de algodão, com e sem línter, de qualidades fisiológicas diferentes. Foram definidos os níveis “alto” (lotes 1, 4, 5, 6 e 7) e “baixo” (lotes 2, 3 e 8) de vigor, baseados em diferentes testes de vigor realizados e na Instrução Normativa 45/2013. Os espectros obtidos pelo equipamento de espectroscopia no infravermelho próximo foram provenientes de quatro preparos de amostra: sementes inteiras, cortadas, sem tegumento e maceradas. Utilizando- *Corresponding author se dos espectros gerados e o agrupamento dos lotes em níveis, foram otimizados modelos E-mail: [email protected] de validação cruzada, construídos a partir do método PLS – DA, predizendo as classes de sementes. O melhor preparo de amostra foi o de sementes maceradas, com 95% de Received: 10/21/2019. Accepted: 4/15/2020. predições corretas para sementes de alto vigor e 100% das de baixo vigor (com línter) e com 100% de predições corretas para sementes de alto vigor e 91,7% de baixo vigor (sem línter).

Termos para indexação: Gossypium hirsutum L., vigor, quimiometria. 1Agrônomos, Universidade Federal de Lavras, Campus Universitário, Caixa Postal 3037, 37200-000, Lavras, Minas Gerais, Brasil,

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INTRODUCTION

The cotton crop represents an important part of generating income and employment on a national and global scale. Its cultivation is mainly destined to the production of fiber, in which, the harvest of 2019/2020 the production estimate was about 2,853.7 thousand tons of cotton plume. The total area planted in Brazil was 1,670.80 thousand hectares, which is the largest of the last five harvest years (CONAB, 2020). Considering all the technology used for cotton production, it is important to use high quality seeds in order to obtain satisfactory results in the field. Mattioni et al. (2012) observed that the use of seeds of medium and low quality originate plants that cannot be equated, in development and productivity, with those of high-quality seeds. In the post-harvest stages of cottonseed, the evaluation of the seed lots allows estimating their aggregate value for both reception, delineation and commercialization purposes, as well as to predict the number of seeds required for planting. Thus, vigor tests that differentiate the physiological potential of the materials are carried out so that complementary information is obtained for the internal quality control of the companies producing cottonseed, but are generally, they are more time-consuming evaluations. Seeking faster responses with consistent results with the actual quality of cottonseed, the near-infrared spectroscopy combined with chemometric methods can represent a promising alternative for such information. The advantages of this equipment are the ability of the analyzes to be carried out successively, in a short period of time. Capable of generating a large quantity of information, with less need of labor, speed, less cost, not polluting, does not use chemicals or reagents and it can be non-destructive (Amorim, 1996). Seed research has been successful in evaluating quality, such as soybeans, cotton and tomato (Bazoni et al., 2017; Gaitán-Jurado et al., 2008; Huang et al., 2013; Shrestha et al., 2016), viability in maize and spinach (Ambrose et al., 2016; Shetty et al., 2012), composition and prediction of phytic acid in Vigna radiata (Pande and Mishra, 2015), nitrogen content in Vigna unguiculata (Ishikawa et al., 2017), oil composition in sunflower and canola (Grunvald et al., 2014; Rossato et al., 2013), as well as the classification of genotypes in cotton, barley, castor beans and maize (Cui et al., 2012; Jia et al., 2015; Ringsted et al., 2016; Santos et al., 2014; Soares et al., 2016). This research aimed to evaluate the potential of the near-infrared spectroscopy using different seed sample procedure to determine the physiological quality of cottonseed with and without lint.

MATERIAL AND METHODS

The experiment was conducted at the Central Laboratory of Seed Analysis, and in the Laboratory of Seed Pathology of the Universidade Federal de Lavras – UFLA, Lavras, Minas Gerais. The same was conducted in two parts. In the first, physiological tests were carried out to determine the physiological quality levels of cottonseed samples. In the second, the samples were analyzed in the near-infrared spectroscopy equipment for the detection of these different levels of cotton seed quality. The seeds used were supplied by the company Bayer®, in which these samples were from eight lots of different physiological seed qualities, in which each sample was divided into seeds with and without lint, and to identify each sample it was given a numbering from 1 to 8.

Physiological tests The experimental design used to determine the physiological quality of the cottonseed was completely randomized with factorial 2 x 8 (with and without lint x samples) and four replications. At the reception of the samples of cottonseed were evaluated according to: Germination: four replications of 50 seeds, sow on a roll of Germitest® paper moistened 2.5 times the weight of the substrate in distilled water and kept in a germinator at 25 °C, a single count was carried out at 5th day after sowing, counting the normal seedlings higher than 5 cm. The results were expressed as a percentage (Brasil, 2009a).

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Seedling emergence: four replications of 50 seeds per treatment were used under uncontrolled environmental conditions. The substrate of the bed was composed of sand and soil in a ratio of 2:1. The counting of emerged seedlings was made on the 12th after sowing. The results were expressed as a percentage. Seedling stand: conducted together with the seedling emergence test, counting was registered on the 7th and 21st day after sowing, with the results expressed as a percentage of the number of emerged seedlings on the respective counting days. Speed of seedling emergence: also conducted with the seedling emergence test, where the number of seedlings emerged was counted daily until day 14th, using the counting parameter of Maguire (1962). Tetrazolium: four replications of 50 seeds were used for each treatment. The seeds were soaked between germitest paper moistened with distilled water at 25 °C for 16 hours. Then the seed tegument was removed and stained in 0.075% tetrazolium salt for 4 hours at 30 °C, in the absence of light. The results were expressed as a percentage of vigorous and viable seeds, according to Vieira and Von Pinho (1999). Seed health: eight replications of 25 seeds arranged on two sheets of filter paper were used in Petri dishes, then 2.4-D, distilled water, and agar were added for soaking the filter paper. The Petri dishes were incubated seven days in a chamber for 12 hours at 20 °C. Each seed was analyzed individually with the aid of a microscope to identify the incidence of fungus. The results were expressed in percentage. The filter paper method was made, as described in Brasil (2009b). The data were submitted to analysis of variance by the SISVAR software and the means were compared by the Tukey test (p < 0.05) (Ferreira, 2011), except for the sanity test, due to the large data variation. Based on the results of the physiological tests, the quality of the cotton samples was classified using the marketing standard used by the Ministério da Agricultura Pecuária e Abastecimento (MAPA), dated 09/17/2013 -IN 45/2013 (Brasil, 2013). Where cottonseed to be commercialized, must be without lint and have at least 75% of germination. Thus, two levels of physiological quality (high and low) were considered, being then, samples with values above 75% of germination considered of high quality and below this value of low quality. This classification was performed for later comparison with the data obtained in the analysis of near-infrared spectroscopy.

Near-Infrared Spectroscopy For each sample of cottonseed with and without lint, four types of seed preparation were tested: Whole seed: did not have any pre-treatment, which was exposed directly to the output of the infrared light. The number of seeds used was 100 units, each representing one replication. Seed cut in half: each seed was sectioned longitudinally with a scalpel and exposed to the infrared light, where the seed cut band was faced the light right after cutting to avoid oxidation. The number of seeds used was 100 units, each representing one replication. Seed without tegument: the tegument was removed from the seed using a scalpel and exposed directly to the infrared light right after the procedure to avoid oxidation of the sample. The number of seeds used was 100 units, each representing one replication. Grounded seed: a mill was used, with the addition of liquid nitrogen and polyvinylpyrrolidone into 40 seeds per replication, to avoid oxidation of the sample, and then the powder was placed in cuvettes for exposure to the infrared light. It was used 20 replications in the total of each treatment. Near-infrared Spectroscopy: the spectra were obtained by placing the samples directly or buckets (ground seeds), at the output of the infrared source of the tensioner device 27 Bruker®, generating spectra by FT-IR detector (Fourier- Transform Near-Infrared ) coupled with the help of OPUS_ Spectroscopy software version 6 from the same equipment manufacturer. To compile the reading database, the spectrometer collected 48 scans at each measurement of absorbance, with a resolution of 8 cm-1, in the range 10000 to 4000 cm-1, per replication. Multivariate analysis: a cross-validation model was optimized from the grouping of the cottonseed samples in high and low quality, using 3/4 of the samples for calibration and 1/4 for the test, constructed from the method of multivariate classification by partial least squares regression with discriminant analysis (PLS-DA) with multiplicative

Journal of Seed Science, v.42, e202042016, 2020 4 L. G. Mayrinck et al. scatter correction preprocessing of the data. The wavelength between 8000 cm-1 and 10000 cm-1 were removed from the analysis due to noise. It was used the Pirouette® statistical software, with the Y classes being the dependent variables and the obtained spectra constituting the independent variables X (Abdi, 2003). The sensitivity and specificity of the optimized models were obtained by dividing the number of predicted samples of the class by the total number of predictions of the class, and dividing the number of samples predicted as not being of the class by the total number of samples that are not of the class, respectively (Szymanska et al., 2011), in the validation stage.

RESULTS AND DISCUSSION

The variance analysis of the physiological tests showed significant differences between the cottonseed samples studied, which was possible to determine the two levels of seed quality desired for this work, as high and low physiological quality levels. The results of germination (Table 1) for cottonseed with lint presented better quality to the samples one, two, four, and six; otherwise, the samples three, five, seven, and eight showed lower quality. Cottonseed without lint had a higher percentage of germination for samples one, four, five, six, and seven compared to samples two, three, and eight. The presence and absence of the lint in the seed could affect the physiological quality in different ways. Seeds with lint can have low quality when the lint makes the perfect media for fungus proliferation (samples four, five and seven); otherwise, it can also help to protect the seed from fungus as a barrier (samples one, two and six). Seeds without lint can have a decrease of germination if the sulfuric acid treatment passes through the tegument damaging the embryo, and this could happen if the seed has physical damage or the sulfuric acid damages the tegument (samples two and three). Thus, if the treatment is successful in only removing the lint, percentages of germination can be increased (samples four, five, six and seven), besides making it easier for seed sow in the field. According to the results of cottonseed germination between the treatments with and without lint, the authors Queiroga et al. (2009) found that seeds without lint had better physiological qualities. For the commercialization of cottonseed, it is required that the seeds are without lint and present above 75% germination, as indicated in the Normative Instruction 45 of Brazil’s Ministry of Agriculture – (Brasil, 2013), which is routinely used by the cottonseed producing companies. From this premise, it was defined based on the germination

Table 1. Means of germination (GERM, %) of samples 1 to 8 of cotton seeds, with and without lint. GERM Samples With Without 1 95 aA* 94 aA 2 86 abA 74 bB 3 56 deA 46 cB 4 81 bcB 93 aA 5 74 cB 91 aA 6 90 abA 95 aA 7 61 dA 86 aA 8 45 eA 46 cA C.V. (%)** 6.94 Mean (%) 75.93 *Means followed by the same letter, upper case in the line and lower case in the column, do not differ by Tukey test, 5% of probability. **C. V. – coefficient of variation.

Journal of Seed Science, v.42, e202042016, 2020 Near-infrared spectroscopy in cottonseed for physiological quality evaluation 5 test (Table 1) of cottonseed without lint that samples one, four, five, six and seven were eligible for commercialization, and samples two, three and eight did not fit in this category, which was used as a parameter to determine high and low-quality seed samples. In the evaluation of seedling emergence (Table 2) from seed with lint, it was possible to observe superiority for sample 1 and inferiority for samples four, seven and eight, with the others presenting intermediate values of vigor. In seeds without lint samples, three and eight had low vigor compared to the others. When it was compared the vigor of seeds with and without lint, only sample two lowered vigor after the delinting. The speed of seedling emergence (Table 2) seedlings with lint showed to be similar to most of the samples, differentiating the sample one with the highest emergence speed and the sample eight the lowest. For seeds without lint, there was more significant variation between samples, but sample one remained with higher value, samples three and eight as inferior, and the others presented intermediate values for this variable. The results of the stand (7 and 21 days) for cotton seeds without lint were similar to those observed for germination, with samples three and eight having the lower stand, which differed statistically from the others; the stand (7 and 21 days) of seeds with lint had sample eight showing the lower percentage. Observing the results obtained from seedling emergence in the stand of 7 and 21 days (Table 2), it could not be seen any relevant variation from the seventh to the twenty-first counting day. It could be inferred that the physiological quality and the presence or absence of lint did not influence the establishment of the seedling stand. Comparing the results of germination, seedling emergence, speed of seedling emergence and stand of 7 and 21 days, it could be seen that sample eight presented inferiority in all the tests, for presence or absence of lint. These tests were also able to differentiate the sample three as inferior physiological quality, except when evaluated seed with lint (Table 2). However, sample two showed no differentiation from the samples with higher vigor. That might have occurred because this sample has intermediate vigor between high and low-quality seeds, with no differences detected in these tests (Steiner et al., 2011). The last evaluation made it identify the quality of the eight cottonseed samples was the tetrazolium test, and cotton seeds with lint presented for most of it higher or similar percentages of vigorous and viable seeds compared to the seeds without lint (Table 3). In the seed production system, cotton seeds undergo a long period of processing. The physical processes that these

Table 2. Mean results of seedling emergence (SE, %), speed of seedling emergence (SSE, index), stand of 7 and 21 days (%) of cottonseed obtained from different samples, with and without lint.

SE SSE stand 7 stand 21 Samples With Without With Without With Without With Without 1 91.0 aA 94.,5 aA 10.22 aB 14.30 aA 88 aA 94 aA 90 aA 94 aA 2 82.0 abA 80.0 aA 8.98 abB 11.77 abA 80 aA 79 aA 81 abA 80 aA 3 84.0 abA 55.5 bB 9.35 abA 7.65 cdA 82 aA 53 bB 84 abA 55 bB 4 74.0 bcB 88.5 aA 8.04 abB 11.21 bA 73 abB 87 aA 74 bcB 90 aA 5 75.0 abcB 95.0 aA 7.88 abB 12.68 abA 74 abB 92 aA 74 abcB 94 aA 6 83.5 abA 83.0 aA 8.98 abA 10.03 bcA 81 aA 81 aA 83 abA 85 aA 7 72.0 bcB 83.3 aA 8.52 abB 11.81 abA 72 abA 81 aA 72 bcA 81 aA 8 62.0 cA 56.0 bA 6.35 bA 6.43 dA 60 bA 50 bA 65 cA 54 bB C.V. (%) 9.26 14.07 9.88 8.99 Mean (%) 78.7 9.64 76.89 78.73 *Means followed by the same letter, upper case line and lower case in column, do not differ by Tukey test, 5% probability.

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Table 3. Mean results of tetrazolium evaluated according to vigorous and viable (%) cottonseed, obtained from different samples, with and without lint.

Vigorous Viable Samples With Without With Without 1 88 abA 87 aA 97 aA 95 abA 2 81 abcA 59 bB 95 aA 90 abA 3 88 abA 52 bB 96 aA 79 cB 4 62 dB 80 aA 91 aA 95 aA 5 79 bcA 83 aA 95 aA 98 aA 6 94 aA 85 aA 99 aA 97 aA 7 92 abA 89 aA 97 aA 98 aA 8 73 cdA 58 bB 90 aA 84 bcA C.V. (%) 8.18 5.5 Mean (%) 77.93 93.31 *Means followed by the same letter, upper case in the line and lower case in the column, do not differ by Tukey test, 5% probability. seeds undergo (harvest, fiber withdrawal, delinting) cause irreversible damages, reducing the physiological quality that can be detected in germination and vigor tests. The tetrazolium can be used in different stages of the processing, which make it able to monitor the origin of the damage, the severity and its extension, representing an important tool for the evaluation of the cottonseed quality (Mattioni et al., 2012; Zorzal et al., 2015). As for the separation of the vigorous seed samples without lint, the same results were observed as the germination test and similarities with the other tests, samples two, three and eight were statistically inferior to the others. The mechanical damages represented the main causes of the low vigor of the evaluated seed with and without lint. In the results of viable seeds, similarities to the other tests were also observed, as samples three and eight having the lower means. Due to the high amplitude of the seed health test, the data was not presented, no correlation that could have influence directly physiological quality was found. The fungus incidence in the seeds with and without lint was: Colletotrichum gossypii, Fusarium oxysporum, Penicillium sp., and Aspergillus sp. The presence of pathogens in the seeds implies a lower health quality of the seeds and may impair the storage and performance of the plants in the field (Gama et al., 2012; Pedroso et al., 2010). According to the results of the physiological tests, basing the NI 45 of cottonseeds commercialization, the samples were classified in high (samples one, four, five, six and seven) and low quality (samples two, three and eight) to be used as a parameter for the near-infrared spectroscopy test. Means for NIR spectra from samples of high and low physiological quality, with and without lint, are shown in Figures 1 and 2. The data were compared by using multiplicative scatter correction (MSC), using the Chemoface® software (Nunes et al., 2012). Molecular absorptions in NIR spectroscopy are not intense, which may result in overlapped bands, making them wide, lowering the sensitivity, and making it necessary to use calibration models. Thereafter, statistical tools may be associated with chemical data. The PLS-DA method used seeks the linear relationship between the dependent variables “Y” (estimation of the variables of interest) and independent variables “X” (spectra), through the regression by partial least squares (PLS), which creates correlations between similarities and structural differences between the compounds, allowing the interpretation of a series of complex data with a large number of variables. Although only minor visual differences for spectra means were observed (Figures 1 and 2), both for high and low physiological quality, such difference were detected by the models. Furthermore, spectra means were similar for cotton seed samples with and without lint from the same preparation type, as well as among different preparation types

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Figure 1. Spectra in near-infrared region corresponding to diferents preparation types of samples after mean and aplication of multiplicative scatter correction preprocessing of samples with and without lint to high and low physiological quality. (a) Whole cottonseeds with lint, (b) Whole cottonseeds without lint, (c) Cut in a half cottonseed with lint, (d) Cut in a half cottonseed without lint. The x-axis corresponds to the wave number (cm-1) and the y-axis to absorbance (u.a.-1).

Figure 2. Spectra in near-infrared region corresponding to diferents preparation types of samples after mean and aplication of multiplicative scatter correction preprocessing of samples with and without lint to high and low physiological quality. (e) Cottonseeds without tegument with lint, (f) Cottonseeds without tegument without lint, (g) Grounded cottonseeds with lint, (h) Grounded cottonseeds without lint. The x-axis corresponds to the wave number (cm-1) and the y-axis to absorbance (u.a.-1). Journal of Seed Science, v.42, e202042016, 2020 8 L. G. Mayrinck et al.

(whole seeds, cut in a half, without tegument and grounded seeds). As a supervised pattern recognition method, PLS-DA requires some type of information to build the model in advance, thus the “high quality” and “low quality” classes were added as the dependent variables (y) for the construction of the calibration model. In Table 4 is presented multivariate analyses of the near-infrared spectroscopy test, and better results were obtained for the grounded seeds with and without lint, which separated samples with a high percentage. This type of sampling method has a larger contact surface to the penetration of the radiation light to the compounds of the integument and embryo at the same time, representing a greater quantity of sources of discrimination, for better predictions between the different seed quality. This type of sampling method also homogenizes the analytical material, since even in good seed sample; there may be lower quality seeds, which could influence the result. The models obtained for the grounded seeds with lint (Table 4), reached 95% and 100% of correct answers in the external validations for high and low quality, respectively. It is important to observe the results of the external validation, since it indicates the predictive capacity of the model for seeds that did not participate in the construction of the same, indicating that it is robust and capable of evaluating different samples. Models for the high-quality grounded seeds without lint it was obtained 92.3% of hits answers, 4.6% of error and 3.1% of seeds not recognized in the cross-validation, otherwise, for the external validation, there was 100% of correctness. For the low-quality samples, there was 97.4% accuracy in the cross-validation and 91.7% in the external validation. In the evaluation of the performance of the optimized models, values of sensibility and specificity had 1.00 in both values for a model (Table 4); for Forina et al. (1991), this is a perfect model. In the sampling method that used whole seeds for the modeling, it was observed a great percentage of seeds

Table 4. Mean percentages of hits and error in the crosses and external validations, and values of sensibility and specificity of the optimized models for seed sampling (whole, cut in half, without tegument and ground) of cotton seeds, with and without lint, correlated with the high (samples 1, 4, 5, 6 and 7) and low (samples 2, 3, and 8) physiological quality classification using the technique of near-infrared spectroscopy, with the aid of PLS-DA.

Cross-validation External validation Seed Sampling Sample Hits Hits Sensibility Specificity Quality Error Error High Low High Low High 86.9 10.7 2.4 69.6 25.6 4.8 Whole (with) 0.73 0.71 Low 26.7 71.1 2.2 25.3 74.7 0.0 High 95.7 3.5 0.8 92.0 8.0 0.0 Whole (without) 0.83 0.91 Low 5.3 94.2 0.4 15.3 82.4 2.4 High 86.7 11.2 2.1 76.0 20.0 4.0 Cut in half (with) 0.56 0.64 Low 20.0 76.4 3.6 54.7 42.7 2.7 High 86.1 10.7 3.2 85.6 10.4 4.0 Cut in half (without) 0.71 0.82 Low 20.9 75.6 3.6 27.6 65.8 6.6 High 80.8 12.0 7.2 49.6 38.4 12.0 Without tegument (with) 0.58 0.56 Low 30.2 60.0 9.8 21.3 65.3 13.3 High 92.3 6.4 1.3 84.8 12.8 2.4 Without tegument 0.63 0.76 (without) Low 13.3 80.4 6.2 44.0 49.3 6.7 High 89.2 7.7 3.1 95.0 5.0 0.0 Ground (with) 1.00 0.95 Low 10.3 84.6 5.1 0.0 100.0 0.0 High 92.3 4.6 3.1 100.0 0.0 0.0 Ground (without) 0.92 1.00 Low 2.6 97.4 0 8.3 91.7 0

Journal of Seed Science, v.42, e202042016, 2020 Near-infrared spectroscopy in cottonseed for physiological quality evaluation 9 classified correctly. Similar the sampling method of grounded seeds. The separation of seed samples with lint obtained lower percentages of accuracy compared to seed the samples without lint, both for high and low-quality samples (Table 4). This difference may have occurred when the lint influenced the scattering of the infrared lights into the sample, acting as a thin physical barrier for the penetration of the radiation. Using the near-infrared spectroscopy allied to chemometrics, Soares et al. (2016) classified cotton seed four high genetic quality cultivars (no lint) by PLS-DA method, obtaining 96.91% correctly classified cultivars at the first validation, and 88.66% in the second validation. Thus, it is observed a potential use of the whole cottonseed without lint for the classification between different parameters by the PLS-DA method allied to NIR spectroscopy. The values of sensibility and specificity obtained in the models optimized for the whole cottonseed were more dissimilar than the grounded seed values when comparing the results of the seeds with and without lint. From the cut in half cottonseed, less efficient models were obtained than those previously presented. It was observed the occurrence of a super fit model for the cut in half seed of low vigor with lint, as it presented good results for the samples belonging to the calibration set, but failed to predict external samples (Table 4). The values of sensitivity and specificity obtained in these optimized models were also more discrepant comparing the results of the seeds with and without lint. Oxidation of the seed after the cut could have been the main issue in this sampling method. The models generated for cottonseed without tegument did not present a high percentage of correct answers. There was disagreement between the classifications of the high and low-quality samples, counting on super adjusted models and the lower values of sensitivity and specificity obtained among the different modes of preparation. The damages caused when the tegument was removed could be an issue making such a large variation for the validations.

CONCLUSIONS

The chemometric analysis (PLS-DA), allied to near-infrared spectroscopy, made it possible to predict the physiological quality of cottonseed with good accuracy, representing a promising technique. The models optimized for grounded cottonseed, with and without lint, promote the percentage of correctness and higher values of performance among the other models.

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Journal of Seed Science, v.42, e202042016, 2020 Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Relationships between substrate and the mobilization Journal of Seed Science, v.42, e202042017, 2020 of reserve with temperature during seed germination of http://dx.doi.org/10.1590/2317- Ormosia coarctata Jack. 1545v42223509

Luciane Pereira Reis1 , Eduardo Euclydes de Lima e Borges1* , Genaina Aparecida de Souza2 , Danielle S. Brito3

ABSTRACT: Seed germination studies provide essential information for biodiversity conservation and ecological restoration programs. This work aimed to investigate the relationship between the substrates and the mobilization of reserves during germination of Ormosia coarctata seeds under different temperatures. Samples were collected every 48 h for up to 240 h for quantification of lipids, soluble sugars, starch, and soluble proteins. The optimum temperature range for germination was 25 to 35 °C. The highest germination percentages were obtained using sand or paper roll. Carbohydrate, lipid, and protein contents decreased during germination, regardless of temperature.

Index terms: temperature change, hydration, substrates, Fabaceae.

Relações do substrato e da mobilização de reservas com a temperatura na germinação de sementes de Ormosia coarctata Jack.

RESUMO: Estudos da germinação de sementes fornecem informações essenciais para a conservação da biodiversidade e programas de restauração ecológica. Este trabalho teve como objetivo investigar as relações entre os substratos e a mobilização de reservas durante a germinação de sementes de Ormosia coarctata sob diferentes temperaturas. As amostras foram coletadas a cada 48 h por até 240 h para quantificação de lipídios, açúcares solúveis, amido e proteínas solúveis. A faixa ótima de temperatura para germinação foi de *Corresponding author 25 a 35 °C. As maiores porcentagens de germinação foram obtidas com areia ou rolo de E-mail: [email protected] papel. Os teores de carboidratos, lipídios e proteínas diminuíram durante a germinação, Received: 05/03/2019. independentemente da temperatura. Accepted: 04/20/2020. Termos para indexação: mudança de temperatura, hidratação, substratos, Fabaceae.

1Departamento de Engenharia Florestal, Universidade Federal de Viçosa, 36570-900 – Viçosa, MG, Brasil.

2Empresa de Pesquisa Agropecuária de Minas Gerais (EPAMIG), 36571-000 – Viçosa, MG, Brasil.

3Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-900 – Viçosa, MG, Brasil.

Journal of Seed Science, v.42, e202042017, 2020 2 L. P. Reis et al.

INTRODUCTION

Ormosia coarctata Jacks. belongs to the family Fabaceae, subfamily Papilionoideae. The tree is distributed throughout the states of Amazonas, Pará, Roraima, and Mato Grosso in Brazil, where it is popularly known as “tento” or “olho de cabra” (Campos Filho and Sartorelli, 2015). Ormosia sp has a diversity of chemical compounds important for the industry such as alkaloids, isoflavones, lecithins, and proanthocyanidins (Fernandes et al., 2011; Pouny et al., 2014). It also presents a potential for use in landscape projects, recovery of degraded areas, and handicrafts (Carneiro et al., 1998; Lorenzi, 2002; Frausin et al., 2008). Germination is a biochemical and physiological process that begins with water uptake by the seed and the activation of metabolism. From this stage onwards, germination is influenced by seed viability and environmental factors. The ecophysiological responses of plants vary according to the conditions to which they are exposed, and one of the environmental factors that most affect germination is temperature. It influences germination speed and percentage, imbibition rate, and mobilization of storage reserves (Bewley et al., 2013). The effect of temperature on germination, however, varies among species. Acacia caven, Amburana cearensis, and Erythrina crista-galli seeds have higher germination at a constant temperature of 30 °C (Escobar et al., 2010; Guedes et al., 2010; Mello et al., 2016). Poecilanthe parviflora, Dimorphandra mollis, and Clitoria fairchildiana seeds germinate better at alternate temperatures of 25 and 30 °C (Valadares and Paula, 2008; Pacheco et al., 2010; Alves et al., 2013). During germination, carbohydrates, lipids, and proteins are mobilized for energy production and tissue growth. These processes are influenced by temperature regimes (Tesfay et al., 2016), as temperature affects the rate of metabolic reactions. In Melanoxylon brauna, α-amylase activity was shown to increase during imbibition at temperatures between 25 and 30 °C (Ataíde et al., 2016). Low germination temperatures were shown to decrease the concentration of linoleic acid in Helianthus annuus seeds (Belo et al., 2014), suggesting that the synthesis of this fatty acid was inhibited. Sugar, starch, lipid, and total protein concentrations were higher in Carica papaya seeds imbibed at constant temperature (25 °C) than at alternating temperatures (20 and 30 °C) (Mengarda et al., 2015). Brazilian biomes are threatened by continued deforestation. The implementation of ecosystem rehabilitation and restoration programs requires knowledge on tree propagation, particularly via seeds, which is the main mode of propagation of forest species. Information on physiological and biochemical responses of O. coarctata seeds to environmental conditions becomes even more relevant in the context of climate change. Therefore, this study aimed to evaluate the effect of different germinative substrates and mobilization of reserve during the germination of O. coarctata seeds at different temperatures.

MATERIAL AND METHODS

O. coarctata seeds were collected in Alta Floresta, Mato Grosso, Brazil. Average temperatures vary from 23 to 26 °C, with maximum temperatures exceeding 40 °C (Caires and Castro, 2002). After collection, seeds were sent to the laboratory, carefully selected, and stored in fiber drums in a cold room at 5 °C and 60% relative humidity for seven months, after which the experiments were performed. Dormancy was broken by mechanical scarification of the hilum or by chemical scarification with concentrated sulfuric acid (H2SO4) for 45 min under constant agitation. Seeds were germinated in sand, between papers, and in paper rolls at 15, 25, 30, 35, and 40 °C. The sand substrate was autoclaved for 2 h at 120 °C before use. Sand germination tests were conducted in transparent polystyrene boxes. The treatment with two paper was carried out in 9 cm diameter Petri dishes. The following parameters were evaluated during germination: germination percentage, germination speed index, imbibition curve, and cotyledon reserve levels.

Germination percentage Germination was recorded daily. Seeds were considered germinated upon radicle emergence. These data were

Journal of Seed Science, v.42, e202042017, 2020 Relationships between substrate and the mobilization of reserve 3 used to calculate the germination percentage (%G) and germination speed index.

Germination speed index The germination speed index (GSI) was determined according to Maguire (1962).

Imbibition curve Seeds were weighed before imbibition, at 1 h intervals for the first 12 h of imbibition, at 24 h, and then at 24 h intervals until 50% of the seeds germinated. At these pre-determined times, seeds were dried with paper towels, weighed, and then placed back in the wet substrate. Each treatment consisted of five replications of 20 seeds.

Determination of cotyledon reserves Samples were collected every 48 h until radicle emergence was observed. Seeds germinated at 15 °C were analyzed for up to 240 h, whereas seeds germinated at 40 °C were analyzed for up to 96 h. Prior to each analysis, seeds were oven dried at 45 °C for 24 h. The cotyledons were ground in a mill, and the dry powder was stored in hermetically sealed glass containers.

Determination of lipids Lipid content was determined using 1 g of dry material. Samples were placed in filter paper thimbles, weighed, and extracted for 24 h with hexane in a Soxhlet apparatus. Then, thimbles were oven dried at 45 °C for 24 h and weighed. Lipid content was calculated as the difference between initial and final dry weights and expressed as a percentage of the total dry weight. Four replications were used for each treatment.

Determination of soluble sugars Soluble sugars were determined according to Buckeridge and Dietrich (1990), with modifications. After extraction, samples were oven dried at 45 °C for 24 h and resuspended in 1.0 mL of distilled water. An aliquot of the solution (10 μL) was diluted in distilled water and used for quantification of soluble sugars by the phenol-sulfuric colorimetric method (Dubois et al., 1956). A standard curve was constructed using 0.01% (w/v) glucose. Five replications were used for each treatment.

Determination of starch The solution prepared for quantification of soluble sugars was also used for starch extraction and quantification. The sample was oven dried, and 20 mg of the resulting powder was digested for 15 min with 1.0 mL of 35% (w/v) perchloric acid. After digestion, the sample was centrifuged at 10,000 g for 5 min (Passos, 1996). Starch quantification was performed using 5 μL aliquots of the supernatant, according to the colorimetric method of Dubois et al. (1956). A standard curve was constructed using 0.01% (w/v) glucose. Five replications were used for each treatment.

Determination of total proteins Total protein content was determined by the micro-Kjeldahl method (CUNNIFF, 1995), with modifications. Defatted samples (200 mg) were mixed with 1.0 g of digestion mixture and 5 mL of sulfuric acid and placed in a digestion block at 350 °C. Upon completion of the digestion process, 10 mL of distilled water was added to the solution. Distillation was performed in the presence of sodium hydroxide (NaOH, 1:1 v/v). The distillate was collected in an Erlenmeyer flask containing 10 mL of 5% (w/v) boric acid and titrated with 0.05 N hydrochloric acid. The total protein content was estimated using a factor of 6.25. Five replications were used for each treatment.

Determination of monosaccharides Monosaccharides were extracted using the method of Blackig et al. (1996), with modifications. The dry powder

Journal of Seed Science, v.42, e202042017, 2020 4 L. P. Reis et al. was incubated with 80% (v/v) ethanol for 30 min at 75 °C, and the mixture was centrifuged at 10,000 g for 5 min. The supernatants were combined and taken to dryness. Then, the powder was resuspended in 1.0 mL of ultrapure water, and 0.5 mL of this solution was used to prepare an alditol acetate solution. Quantification was performed according to Englyst and Cummings (1985) on a Shimadzu GC 14-a gas chromatograph equipped with a flame ionization detector (FID) and a Shimadzu C-R8A Chromatopac integrator. A moderately polar column coated with 50% cyanopropylphenyl and 50% dimethylsiloxane was used. The gas flow was set at 0.25 mL.min−1. The injector, detector, and column temperatures were respectively 2500, 2200, and 2750 °C. Samples were injected with a split ratio of 1/40; 1.0 μL of alditol acetate was used. Each treatment consisted of four replications.

Statistical analysis The experiment was carried out in a 5 × 3 completely randomized factorial design, with five temperatures and three substrates. Treatments consisted of five replications of 20 seeds each, totaling 100 seeds per treatment. Germination percentages and GSI values were compared using Tukey’s test. The level of significance was set at p < 0.05. Data were analyzed using R version 3.4.1 (R Core Team, 2017) and ExpDes package version 1.1.2 (Ferreira et al., 2013).

RESULTS AND DISCUSSION

The highest germination percentages were observed in O. coarctata seeds kept at 25, 30, or 35 °C in paper roll or sand; germination in these substrates did not differ significantly (p < 0.05) (Figure 1A). Seeds germinated between papers had low germination percentage and GSI at all temperatures (Figure 1B). GSI was highest at 30 and 35 °C (Figure 1B). There was no interaction effect between temperature and substrate. The highest germination percentages and GSIs were obtained using paper roll or sand as substrate (Figures 1A and 1B), shows that their large contact area enabled O. coarctata seeds to absorb high amounts of water. A small contact area between substrate and seed might result in the rate of water loss being higher than the rate of absorption. The between paper method was unsuitable, probably because of the smaller area of contact between substrate and seed. When choosing the substrate, the shape and size of the seed must be taken into account (Brasil, 2009). M. brauna seeds (Flores et al., 2014), Inga laurina (Barrozo et al., 2013), Eugenia involucrate and Eugenia pyriformis (Gomes et al., 2016) presented higher percentages of germination in paper roll and sand. Thus, the contact area between the substrate and the seed influenced the greater water absorption and, consequently, higher germination values.

Figure 1. Germination percentage (A) and germination speed index (B) of Ormosia coarctata seeds grown in different substrates. Different letters indicate significant differences by Tukey’s test (p < 0.05). Journal of Seed Science, v.42, e202042017, 2020 Relationships between substrate and the mobilization of reserve 5

Seeds were not able to germinate at 15 °C during the 240 h experimental period. Seeds kept at 40 °C began to deteriorate and showed no signs of root protrusion after 96 h of imbibition. The theoretical optimal temperature was 27.8–27.9 °C, which is in agreement with the results. The highest GSIs were achieved at 25, 30, and 35 °C. This temperature range is the same as that of the environment where seeds were collected. Oliveira et al. (2016) verified GSI values of Ormosia arborea seeds similar to those of the present study. According to the authors, 25–35 °C is the optimal germination temperature range for the species. Germination performance at a specific temperature range reflects the species adaptation to its native ecosystem, which in forest seeds may vary according to succession stage, biome, and environmental conditions (Wood and Prichard, 2003). The rate of water uptake increased significantly with temperature, as shown by the fresh mass curves in Figure 2A. At 15 °C, imbibition was slow and continuous. At all temperatures, water content increased in the first 72 h, as water uptake at this stage is temperature independent. Imbibition occurs because of the difference in water potential between seed and substrate. In dry seeds, the matric potential is associated with the binding of water to the structural components of the cell wall and other macromolecules (Taiz et al., 2017). Imbibition initially results in the hydration of cell components, such as the cell wall and reserve polymers. The number of hydrated cells within the seed increases as imbibition proceeds (Nonogaki et al., 2010). Prior to hydration, the water potential of seed cells is more negative than that of the substrate. Upon contact, seeds rapidly absorb water. The low and steady imbibition rate observed at 15 °C (Figure 2) might be associated with the basic metabolism of the seed or with reserve degradation. Storage reserves are used during phase 2 of imbibition, increasing the osmotic potential. However, this phenomenon did not occur because water uptake was not sufficient. Low temperatures reduce imbibition rate, enzyme activity, and energy metabolism (Luo et al., 2019). It is possible that membrane-level changes are only part of the process. Most aquaporin plasma membrane intrinsic protein (PIP) genes are down-regulated in seeds under low-temperature stress (Luo et al., 2019). Reduction in the expression of these genes results in a low number of aquaporins, thereby affecting the water absorption rate of seeds. Low temperatures affect the reorganization of cell membranes by making the process difficult and slow (Carvalho et al., 2009). The degree of fatty acid unsaturation of membrane phospholipids is altered at low temperature. This

Figure 2. Imbibition curve of Ormosia coarctata seeds at different temperatures. Arrows indicate when radicle emergence occurred in 50% of the seeds.

Journal of Seed Science, v.42, e202042017, 2020 6 L. P. Reis et al. change affects the permeability of the membrane and its fluidity properties (Zheng et al., 2016; Noblet et al., 2017). In Zea mays seeds, low temperatures induce membrane disorganization, resulting in increased release of electrolytes into the medium and delayed germination (Noblet et al., 2017). A three-phase imbibition pattern was observed at 25, 30, and 35 °C, with rapid water absorption at the beginning, followed by a plateau and primary root growth. Radicle emergence occurred after 192 h at 25 °C and after 144 h at 30 and 35 °C (Figure 2). Phase I occurs both in viable and nonviable seeds, as this physical process is independent of metabolic activity (Bewley et al., 2013) and temperature. During phase I, the energy metabolism is resumed, and the energy needed for enzyme activation and metabolite turnover is released (Nonogaki et al., 2010). Similar results were reported for seeds of Bowdichia virgilioides and D. nigra (Albuquerque et al., 2009; Ataíde et al., 2014). Little variation in fresh seed mass was observed from 72 to 144 h of imbibition at 25, 30, and 35 °C. This stabilization probably occurred because of the equilibrium between seed and substrate water potentials. Guimarães et al. (2008) reported that after tissues and organelles are hydrated, membranes and enzymes become functional (phase II) and storage reserves are broken down to be used for radicle elongation. Seeds placed to germinate at 40 °C had a higher weight gain than seeds placed at other temperatures but were deteriorated after 96 h. The high temperature may have altered cell membrane permeability, leading to extravasation of cellular content. According to Badea and Basu (2009), high temperatures can modify components of the phospholipid bilayer. Microorganism contamination might be related to the loss of metabolites (Bewley et al., 2013), as exudates can serve as a substrate for microorganism development. In addition, the kinetic energy of particles is affected by the increase in temperature: particles become more accelerated and hydrogen bonds in macromolecules are weakened (Źróbek-Sokolnik, 2012). High temperatures can alter the tertiary structure of enzymes, which in turn reduces the rate of reactions (Źróbek-Sokolnik, 2012). Mobilization of reserves was lowest at 15 °C because of the low metabolic activity of seeds. This condition results in low respiration rates, affecting the breakdown of seed reserves, especially of galactose, which is the main source of energy. Consequently, germination is affected, as it requires stable and functional mitochondria (Luo et al., 2019). Seeds at 40 °C had the highest consumption rate of reserves in 96 h of imbibition. Even though seeds germinated at other temperatures were imbibed for a longer period, 96 h of imbibition at 40 °C was sufficient for metabolic activation and reserve mobilization. However, these processes were interrupted at 96 h because seeds deteriorated. Changes in the respiratory activity of cells occur with seed deterioration, such as the release of high levels of energy (Horbach et al., 2018) caused by loss of metabolic control over the electron transport chain and oxidative phosphorylation. Soluble sugar levels decreased during imbibition at all temperatures except 15 °C (Figure 3A). The highest reductions occurred after 48 h of imbibition at 25, 30, and 35 °C. At 40 °C, the content of soluble sugars decreased by 50% in 96 h. The consumption of soluble sugars during germination processes is attributed to energy expenditure during respiration, energy generation, and supply of embryo growth (Koch, 2004; Souza et al., 2018). Moreover, sugar consumption is also due to its metabolic signaling role. Many sugars act as a signal for the synthesis of enzymes and phytohormones (Souza et al., 2018). Therefore, its consumption may be related to molecular signaling for the different stages of tissue development (Koch, 2004). The total content of soluble sugars and reducers from two sunflower cultivars decreased during the first 24 hours (Erbaş et al., 2016). According to the authors, these reserves are the first to be used as an energy source during the initial germination period. Xylose levels did not change significantly during imbibition at 15 °C (Figure 3C). At 25, 30, and 35 °C, xylose concentration decreased from 0.00126 to 0.00081 mg.g−1 dry matter after 48 h. At 40 °C, xylose was rapidly consumed. Galactose levels decreased at all temperatures, although at a slower rate at 15 and 40 °C (Figure 3D). At 25, 30, and 35 °C, galactose was depleted after 144 h of imbibition. Seeds germinated at 25, 30, and 35 °C showed similar variation in the levels of xylose and galactose. Galactose was completely depleted toward the end of imbibition, whereas xylose was still detected. These results demonstrate that monosaccharides are preferentially broken down, regardless of the

Journal of Seed Science, v.42, e202042017, 2020 Relationships between substrate and the mobilization of reserve 7 presence of oligosaccharides. Xylose and galactose were consumed or released at a higher rate in seeds imbibed at 40 °C, probably because of the degradation of cell membranes toward the end of imbibition. Thus, two processes occurred during hydration of O. coarctata seeds at 40 °C. First, seed reserves were broken down, as shown by the increase in metabolic activity; then, reserves were released to the medium as a result of thermal damage. Glucose was not detected at 25, 30, or 35 °C (data not shown). However, this monosaccharide was present in seeds imbibed for 144 h at 15 °C and for 48 h at 40 °C (data not shown). These results may be explained by the partial isomerization of galactose. At 15 °C, increased glucose levels were attributed to low metabolic activity to and, at 40 °C seed death. The not detection of glucose during the germination of O. coarctata seeds may be related to its use for the production of sucrose in embryonic tissues. In Cedrela fissilis, glucose was detected only during the early stages of

Figure 3. Soluble sugar (A), starch (B), xylose (C), galactose (D), lipid (E), and total protein contents (F) in Ormosia coarctata seeds during germination at different temperatures. Journal of Seed Science, v.42, e202042017, 2020 8 L. P. Reis et al. seedling development (Aragão et al., 2015). In Sebastina virgata seeds, glucose is formed during the development of the seedlings in relatively high amounts (Tonini et al., 2010). With the development of the primary root, the degradation of wall polysaccharides is released through the degradation of ABA, consequently sucrose and glucose increase (Tonini et al., 2010). Alternatively, monosaccharides may leave the embryo or cotyledons and are retained in the membrane or seed coat, as observed by Borges et al. (2002) in Platymiscium pubescens seeds. The mobilization pattern of starch differed according to the germination temperature. Seeds imbibed at 15 °C showed no changes in starch concentration with time (Figure 3B). At 40 °C, starch levels decreased rapidly after 48 h. The decrease in carbohydrate levels in seeds germinated at optimal temperatures (25–35 °C) indicates that there was a high mobilization of starch. Thus, soluble sugars together with starch reserves indicate degradation to obtain fast energy for embryo growtn. Lipid levels varied with time of imbibition at all temperatures (Figure 3E). O. coarctata seeds initially contained 8.2% lipids. Lipid mobilization was slower at 15 °C. At 25, 30, and 35 °C, lipid levels decreased by 2.6, 7.9, and 11.1%, respectively. At 40 °C, the lipid content decreased continuously as the cotyledons deteriorated. The oil stored in the seeds in the form of triacylglycerols plays an important role in the growth of the embryo, providing energy and carbon skeletons (Knauer et al., 2013). Most seeds consume first the reserves of lower metabolic expenditure for your use (Bewley et al. 2013). However, this behavior depends on the type of seed, the amount of reserves, and the speed of germination (Bewley et al. 2013). Lipid mobilization was relatively lower than that of the other reserves. Variation in lipid levels was similar among the different germination temperatures. Mobilization of lipid reserves is more intense after germination (Hooks et al., 2010). Free fatty acids were probably used for germination instead of triglycerides. Low lipid mobilization can be associated with the low energy requirement during germination. To be converted into carbohydrates, lipids must first be broken down by lipases and then degraded through the β-oxidation and glyoxylate pathways (Taiz et al., 2017). Because they are highly reduced molecules, lipids have high energy density and can easily supply the energy needed in the early phases of seed hydration. The initial protein content was 16.6%. Protein levels were affected by imbibition time and temperature (Figure 3F). At 15 °C, the total protein content remained relatively stable for up to 72 h. After 96 h of imbibition, protein levels decreased by 2.3, 16.6, 17.2, and 17.5% in seeds soaked at 15, 25, 30, and 35 °C, respectively. At 40 °C, seeds showed a continuous reduction in protein content. The total protein level was 36.8% lower after 96 h of imbibition. During imbibition, stored proteins are proteolyzed by different enzymes (Bewley et al., 2013) resulting in free amino acids that are used for the synthesis of protein components and, therefore, essential for the development of the embryo (Erbaş et al., 2016). Unfavorable temperature conditions can negatively affect reserve mobilization, thereby impairing germination (Mengarda et al., 2015). Ataíde et al. (2016), however, observed no differences in reserve mobilization in seeds germinated at 15 or 25 °C. The authors reported that soluble carbohydrates, starch, and protein levels decreased while lipid levels showed little variation. O. coarctata seeds are composed mainly of cotyledons, as the embryo has a reduced size. Thus, we evaluated the levels of cotyledon reserves. Considering that the embryo has little demand for reserves at the beginning of germination, we assumed that the decrease in reserve levels was due to cotyledon respiration or exudation.

CONCLUSIONS

Paper roll and sand were the best substrates for O. coarctata seeds germination. The optimal temperature range was 25–35 °C. Carbohydrate, lipid, and protein reserves decreased at all temperatures. Mobilization patterns of soluble sugars, xylose, and galactose were similar in seeds germinated at 25, 30, and 35 °C. Glucose was detected in O. coarctata seeds at 15 and 40 °C, temperatures that are not optimal temperatures for germination. This monosaccharide was not present in seeds germinated at other temperatures. Journal of Seed Science, v.42, e202042017, 2020 Relationships between substrate and the mobilization of reserve 9

ACKNOWLEDGMENTS

This work was supported by CNPQ, CAPES (Pro-Amazon Project) and FAPEMIG.

REFERENCES

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Journal of Seed Science, v.42, e202042017, 2020 Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Morphoanatomic and histochemical aspects of Elaeis oleifera Journal of Seed Science, v.42, (Kunth) Cortés seed e202042018, 2020 http://dx.doi.org/10.1590/2317- 1545v42230138 Suelen Cristina de Sousa Lima1* , Poliana Roversi Genovese-Marcomini2 , Regina Caetano Quisen3 , Maria Silvia de Mendonça4

ABSTRACT: Elaeis oleifera is an oleaginous palm tree native to America. The fruit contains unsaturated fatty acid extracted from the mesocarp. The species is mainly used in breeding programs of E. guineensis in development of interspecific hybrids with higher oil yield and resistance to lethal yellowing. E. oleifera is propagated by seed, which requires the adoption of methods for breaking dormancy and increasing the germination rate. However, there are no studies on the morphology and anatomy of the seed and its ergastic substances; knowing its structure makes it possible to improve planting methods and make them more effective. The aim of the present study was to describe the morpho-anatomy and histochemistry of the seed, characterizing it and contributing information that assists in understanding dormancy. In seeds collected in the experimental area of Embrapa Amazônia Ocidental (Amazonas), morpho-anatomical and histochemical analysis was conducted to detect metabolites (starch, protein, lipids, carbohydrates, and phenolic compounds). The seeds vary in shape, oblong and ovate. The embryo has an oblique embryonic axis, composed of root apical meristem and shoot apical meristem with three leaf primordia. Phenolic compounds were found throughout *Corresponding author the seed coat; there are lipids, protein, and pectin in the embryo and endosperm. E-mail: suelen.biologa23@gmail. Index terms: morphology, anatomy, palm tree. com

Received: 10/18/2019. Accepted: 01/08/2020. Aspectos morfoanatômicos e histoquímicos da semente de Caiaué (Elaeis oleifera (Kunth) Cortés)

1 RESUMO: Elaeis oleifera é uma palmeira oleaginosa nativa da América. O fruto contém ácido Universidade Federal do Amazonas, Av. Rodrigo Otávio, 6200 - Setor graxo insaturado, extraído do mesocarpo. A espécie é explorada principalmente em programas Sul, Coroado, 69080-900 - Manaus, de melhoramento genético de E. guineensis, no desenvolvimento de híbridos interespecíficos Amazonas, Brasil. com maior produtividade de óleo e resistência ao amarelecimento fatal. A propagação de E. oleifera é realizada via semente que requer a adoção de metodologias para superação da 2Instituto Nacional de Pesquisa do dormência, e aumento da taxa de germinação. No entanto, não há estudos sobre a morfologia Amazonas, Av. André Araújo, 2.936 - e anatomia da semente e suas substâncias ergásticas, conhecer a estrutura permite aprimorar Petrópolis, Caixa Postal: 2223, 69067- e tornar efetivas as metodologias de plantio. O presente trabalho teve como objetivo 375 - Manaus, Amazonas, Brasil. descrever a morfologia, anatomia e histoquímica da semente caracterizando-a e contribuindo 3 com informações que auxiliem na compreensão da dormência. Em sementes coletadas na Embrapa Florestas, Estrada da Ribeira, Km 111 - Guaraituba, Caixa área experimental da Embrapa Amazônia Ocidental (Amazonas), foram realizados estudos Postal: 319, 83411-000 - Colombo, morfoanatômicos e histoquímicos para detecção de metabólitos (amido, proteína, lipídeo, Paraná, Brasil. carboidrato e compostos fenólicos). As sementes apresentaram variação na forma, oblongas e ovadas. O embrião apresenta eixo embrionário obliquo composto por polo radicular e caulinar 4LABAF/FCA/UFAM – Universidade com três primórdios foliares. No tegumento verificou-se a presença de compostos fenólicos Federal do Amazonas, Av. Rodrigo em toda extensão, no embrião e endosperma há presença de lipídeo, proteína e pectina. Otávio, 6200 - Setor Sul, Coroado, 69080-900 - Manaus, Amazonas, Termos para indexação: morfologia, anatomia, palmeira. Brasil.

Journal of Seed Science, v.42, e202042018, 2020 2 S. C. S. Lima et al.

INTRODUCTION

Palm trees are among the most diversified groups of plants; there are more than 150 species, with extraordinary variations in geographic distributions, regional patterns of abundance, and growth forms, and palm trees provide countless ecosystem services (Balslev et al., 2019). They are important for maintaining native fauna and flora; they interact with other plant species and serve as food and shelter for animals (Oliveira et al., 2010). For traditional populations, they are highly useful, as a food source – fruit and heart of palm; in craft production – the seed and vegetable matter; and in construction of dwellings - the stipe and the leaves. In addition, palm oils are widely used in the food, cosmetics, and medication industries (Oliveira and Rios, 2014). Elaeis oleifera (Kunth) Cortés, popularly known as Caiaué in Brazil, belongs to the same genus as oil palm (Elaeis guineensis). It is native to the Americas, occurring in Mexico, Brazil, Colombia, Costa Rica, Ecuador, French Guiana, Honduras, Nicaragua, Panama, Peru, Suriname, and Venezuela (Meunier, 1975; Corley and Tinker, 2003; Cunha et al., 2012; Leitman et al., 2015). In Brazil, it more frequently occurs in the central region of the state of Amazonas, in wet and higher locations on firm soil (Oliveira and Rios, 2014). In addition to natural populations, areas are planted to this species for experimental purposes and to maintain germplasm, especially because it represents a genetic resource of great interest for oil palm breeding programs (Arias et al., 2015). Traits of interest in the species include resistance to pests and diseases (especially lethal yellowing), a lower rate of vertical growth of the stipe, and oil of notable quality (Rios et al., 2012). Various breeding programs have already made interspecific hybrids between American oil palm and African oil palm (HIE OxG) available on the market, with commercial planted areas found mainly in Colombia, Brazil, and Ecuador, among other countries. The oil palm breeding program conducted by Embrapa Amazônia Ocidental released the first HIE OxG, called BRS Manicoré (Cunha and Lopes, 2010), in 2009, which is grown mainly in the state of Pará, the area of occurrence of lethal yellowing (LY), an anomaly of yet unknown etiology that makes growing of oil palm unviable in that region. Populations or progenies of E. oleifera are sexually reproduced, i.e., by seeds, which is an indispensable tool for ex situ maintenance and conservation of the germplasm of the species, in conducting breeding programs, and in establishing fields of parents for commercial production of hybrid seeds. In general, under natural conditions, germination of Elaeis genus seeds is low and uneven, and may take years (Hussey, 1958). Various methods have been used with the aim of breaking seed dormancy in this crop. Among them, protocols with the use of thermal treatment by Hussey (1958) and Rees (1962) predominate, who observed the need to subject the seeds to a thermal treatment by heating. Green et al. (2013) and Lima et al. (2014) found that different genotypes respond in a particular manner in relation to moisture content and to the period of exposure of seeds in the thermal treatment. Lima et al. (2017) obtained germination rates from 72% to 76% through adjusting the seed moisture content of Caiaué from 20% to 23%. Myint et al. (2010) scarified the seed, with removal of the operculum, and observed 88% germinated seeds in the period from three to eight days. The studies conducted are only aimed at breaking dormancy, seeking to accelerate germination. Nevertheless, factors related to the morphology of the seed coat, such as number of cells, thickness of the cell wall, and presence of flavonoids (Debeaujon et al., 2007), as well as differentiation and maturation of the embryo, can affect germination (Hilhorst, 2007). According to Bewley (1997), complex interactions between the embryo and seed coat determine seed germination. It is necessary to study the morphology, anatomy, and histochemistry of the seed coat to verify the level of dormancy and germination of the seed and to identify the factors that determine it (Debeaujon et al., 2007). The elements in the chemical composition of the seed can also affect germination, due to the presence of products of primary metabolism, such as carbohydrates, proteins, and lipids, which are consumed during germination by the embryo for formation of new cell structures (Carvalho and Nakagawa, 2012). Due to the absence of studies on the structure of the Elaeis oleifera seed and the biological and economic contribution that knowledge of its morphology and anatomy will provide, the aim of the present study was to describe

Journal of Seed Science, v.42, e202042018, 2020 Morphoanatomy and histochemistry of Elaeis oleifera 3 aspects of the morphoanatomy and histochemistry of the Elaeis oleifera seed, assisting understanding of its structure, and seeking to identify barriers that may affect germination.

MATERIALS AND METHODS

Fruit from E. oleifera was collected from plants of the Caiaué Active Germplasm Bank (Banco Ativo de Germoplasma de Caiaué - BAG) established at the Rio Urubu Experimental Station of Embrapa Amazônia Ocidental, Rio Preto da Eva, Amazonas (state), at 2º35’ S and 59º28’ W. Initially, controlled pollinations were carried out as described by Cunha et al. (2007) using Elaeis oleifera plants of Manicoré origin as male and female parents. In 2016 and 2017, five months after pollination, bunches were collected upon reaching physiological maturity, determined indirectly by natural detachment of three to five ripe coconuts, as has been adopted in commercial seed production. Fruit was processed to obtain seeds according to the procedures presented by Lima et al. (2014). After obtaining the diaspores, formed by the endocarp and the true seed (endosperm + embryo) (Lima et al., 2014), they were placed in polyethylene bags and taken to the Agroforestry Botanical Laboratory of the School of Agrarian Sciences of the Universidade Federal do Amazonas.

Morphology, anatomy, histochemistry, and proximate analysis of the seed To study morphology, the diaspores were squeezed in a vice to break the endocarp, remove the seed and make the longitudinal and transversal cuts. The color, consistency, and shape of the seed coat, endosperm, and embryo were observed in these segments with the aid of a magnifying glass and microscope. For biometric analysis, seeds from three identified plants were mixed (one hundred seeds each) from which length and width (basal and apical) were measured with a digital caliper rule, and the weight of each seed was determined with a precision (0.0001 g) balance (Brasil, 2009). For anatomical study of the seed, fresh material in the seed coat and endosperm was cut transversally on a benchtop microtome, according to normal techniques in plant anatomy (Kraus and Arduin, 1997). The embryos were removed from the seeds and fixed in FAA 70% (formalin, acetic acid, and ethyl alcohol) (Kraus and Arduin, 1997), dehydrated in gradations of ethyl alcohol (70–95%), soaked in 2-hydroxyethyl-methacrylate (Historesin® Leica), and suspended in blocks, according to manufacturer’s instructions. The blocked material was cut in a manual rotary microtome with thickness from 5 to 7 µm and stained with 0.5% toluidine blue in a citrate buffer, pH 4.0 (O’Brien et al., 1964), and slides were set up in water (Genovese-Marcomini et al., 2013). The parameters used in the morpho-anatomical descriptions and the terms placed on the structures are in accordance with the studies of Dransfield et al. (2008), Werker (1997), Tomlinson (1990), and Martin (1946). The histochemical tests of the seed coat, endosperm, and embryo were applied on sections of fresh material obtained from a benchtop microtome from fragments of the seed coat and of the endosperm, and from the entire embryo, since it is small and does not require reduction in the size of the sample. This material was then stained with specific reagents for each substance, namely, Lugol’s solution for detection of starch (Jensen, 1962), Xilidine Ponceau for detection of total proteins (O’Brien and McCully, 1981), Sudan Red III for detection of total lipids (Brundrett et al., 1991), ruthenium red for detection of pectins and mucilages (Johansen, 1940), Iron(III) chloride for detection of general phenolic compounds (Gabe, 1968), Wagner reagent for detection of alkaloids (Furr and Mahlberg, 1981), and phloroglucinol for detection of lignin (Johansen, 1940). The material was then examined under a microscope and images were registered. Observation and photomicrographs in regard to the anatomical and histochemical study were obtained through the optical microscope Zeiss Primo Star coupled to the Canon Power Shot A650 IS digital photographic camera. Proximate composition analyses for determination of percentage of moisture, ash, lipids, proteins, and carbohydrates were carried out according to the method described by Horwitz and Latimer (2000) and Lutz (1985).

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RESULTS AND DISCUSSION

The seeds had a mean length of 14.15 mm, apical diameter of 10.12 mm, basal diameter of 10.75 mm, and 0.93 g of fresh matter (Table 1). These measurements are near those of another palm tree of the same genus, Elaeis guineensis, which had mean diameter of 0.93 mm and weight of 0.99 g – mean values observed by Camillo et al. (2014) upon analyzing the biometry of seeds from eighteen bunches from different plants. Seed moisture was 25.92% (Table 2). In studies conducted on Elaeis oleifera seeds and seeds from an oil palm with Caiaué hybrid, moisture content was found to affect germination. In seeds with a moisture content from 20% to 23% for Elaeis oleifera and from 19% to 22% for hybrid seeds, germination was greater than 70% (Lima et al., 2014; 2017). Moisture content is of utmost importance since it is associated with the physiological quality of the seeds, which may favor their germination, and it can indicate the best period for collection and storage of the seeds from different species. The longitudinal and transversal measurements in Elaeis oleifera seeds indicated variation in shape – seeds dorsoventrally flattened were observed, ovate or oblong (Figure 1A). The seed coat is brown, with vascular extensions on its surface of a lighter shade and punctiform micropylar pore (Figure 1A). In Oenocarpus minor seeds there is variation in its shape and the same color of the seed coat (Mendonça et al., 2008). The endosperm is white, with an oily, solid, and abundant appearance and is in direct contact with the seed coat (Figure 1B), a characteristic also observed in Butia capitata (Oliveira et al., 2013) and in Acrocomia aculeata (Souza et al., 2017). The embryo is straight and occupies an apical position in the seed. It has two distinct regions, separated by a slight constriction: the proximal region, the shorter part of whitish yellow color, and the distal region, the more elongated part of pale white color (Figure 1C). According to Tomlinson (1990), embryos of palm seeds have a proximal region that accommodates the embryonic axis and a distal region that corresponds to the cotyledon blade. In some palm species, the proximal and distal regions of the embryo can be differentiated by a difference in coloring, as in Acrocomia aculeata (Moura et al., 2010), Mauritia flexuosa (Silva et al., 2014), and Butia capitata (Oliveira et al., 2013). In the Bactris gasipaes palm, the embryo has a homogeneous color, with the distal and proximal regions morphologically distinct (Nazário et al., 2013). Anatomically, the Elaeis oleifera seed has a seed coat consisting of various juxtaposed layers of cells, from 12 to 15 (Figures 2A and 2B), which lend rigidity and impede the passage of light, acting as a regulator of germination. The arrangement of the seed coat cells reduces the intercellular spaces, contributing to impermeability to water, hindering gas diffusion, and affecting germination (Esau, 1977).

Table 1. Biometric characteristics of the Elaeis oleifera (Kunth) Cortés seed: mean values, amplitude, standard deviation (σ), and coefficient of variation (CV) of the apical and basal width, length, and fresh matter.

Variable Mean Amplitude Standard Deviation CV (%) Apical width (mm) 10.12 5.4±15.2 1.97 19.5 Basal width (mm) 10.75 5.4±17.5 2.96 27.6 Length (mm) 14.15 8.6±18.9 2.13 15.1 Weight (g) 0.93 0.4±1.9 0.39 41.9

Table 2. Proximate analysis percentages of Elaeis oleifera (Kunth) Cortés seeds.

Estimate (%) Moisture Ash Lipids Proteins Carbohydrates Mean 25.92 1.82 24.4 8.35 39.51 Standard Deviation 0.29 0.04 0.01 0.3 1.31

Journal of Seed Science, v.42, e202042018, 2020 Morphoanatomy and histochemistry of Elaeis oleifera 5

Figure 1. Seed morphology of Elaeis oleifera (Kunth) Cortés. a) Different shapes of the seed with micropylar pore and vascular extensions. b) Seed section in longitudinal cut showing seed coat, endosperm, and embryo. c) Embryo with proximal and distal region (pm: micropylar pore; ev: vascular extensions; en: endocarp; tg: tegument; ed: endosperm; em: embryo; rp: proximal region; rd: distal region).

Figure 2. Seed of Elaeis oleifera (Kunth) Cortés in transversal cut. a) Seed coat and endosperm. b) Seed coat cells. c) Endosperm cells (te: tegument; en: endosperm).

The endosperm is formed of rounded cells, with dense walls, without intercellular spaces, and it occupies nearly all the inside of the seed (Figure 2C), characteristics also observed in Euterpe edulis, Washingtonia filiferaand Phoenix dactylifera (Panza et al., 2004; DeMason, 1986; DeMason and Thomson, 1981). In Acrocomia aculeata, the cell walls are not overly dense, but pectins are noted in the cell walls, as well as in the middle lamella, which are also identified in small amounts in the inner wall (Moura et al., 2010). The embryo is lined by the protoderm formed by juxtaposed cells, straight walls, and evident nucleus (Figures 3A and 4A-B). The embryonic axis of oblique position is in the proximal region, composed by two meristematic regions, the

Journal of Seed Science, v.42, e202042018, 2020 6 S. C. S. Lima et al. shoot meristem (plumule) and the root meristem. In the region of the root meristem, cells of smaller diameter, intensely ruddy, and in the shape of a shell are observed, the radicle; in the shoot meristem, which corresponds to the plumule, there are three leaf primordia that will form the first and second cataphyll and the eophyll, sequentially (Figures 3B-D and 4A), lined by a protoderm, as also observed in Acrocomia aculeata, Euterpe precatoria, and Syagrus inajai (Souza et al., 2017; Aguiar and Mendonça, 2003; Genovese-Marcomini et al., 2013). The distal region, which corresponds to the cotyledon blade, has a haustorial function, with an irregular surface and provascular bundles (Figures 3E and 4C-D). The cotyledonary gap appears in the direction of the apex of the leaf primordia, through which plumule emergence will occur at the time of germination (Figure 3B). The fundamental meristem is composed by cells with greater diameter than those of the proximal region, with various provascular bundles (Figure 4D) directed toward the periphery, near the protoderm, as observed in Syagrus inajai (Genovese-Marcomini et al., 2013). The embryo is found with its regions defined, as observed in Acrocomia aculeata, Bactris gasipaes, and Syagrus inajai (Ribeiro et al., 2012; Nazário et al., 2013; Genovese-Marcomini et al., 2013), thus discarding the idea of seed immaturity. In relation to seed chemical composition, the greatest content was found for carbohydrates, with 39.51%, followed by lipids, with 24.40% (Table 2). Carbohydrates and lipids are the nutritional compounds most found in palm. According to Barton and Crocker (1953), in Elaeis guineenses seeds, such compounds were found in different proportions: carbohydrates 28%, lipids 49%, and proteins 0.9%. The presence of lipids does not refer only to species of Elaeis, but also to two subspecies of Acrocomia: A. aculeata sclereocarpa and A. aculeata sub. totai, exhibiting 55.42% and 47.76%, respectively (Machado et al., 2015). Carbohydrates, lipids, and proteins are the main reserve substances in seeds, and the proportion varies according to the species (Marcos-Filho, 2015). They are synthesized in the seeds as reserve materials, to be used by the embryo during germination to constitute new cell structures (Henderson, 2002).

Figure 3. Embryo of Elaeis oleifera (Kunth) Cortés. a) Longitudinal section, showing the proximal region (embryonic axis) and the distal region (cotyledon blade or haustorium) protoderm (arrow). b) Cotyledon gap shown directed toward the apex of the leaf primordia (arrow). c) Embryonic axis, showing the radicle and plumule. d) Embryonic axis, showing the plumule. e) Distribution of the procambium in the distal region of the cotyledon blade (circle) (rp: proximal region; rd: distal region; ra: radicle; pl: plumule).

Journal of Seed Science, v.42, e202042018, 2020 Morphoanatomy and histochemistry of Elaeis oleifera 7

Figure 4. Embryo of Elaeis oleifera (Kunth) Cortés. a) Embryonic axis showing the radicle and the plumule. b) Cells of the fundamental meristem and protoderm (arrow) in the proximal region. c) Distal region showing the provascular bundles in dotted line. d) Procambium (arrow) near the protoderm in the distal region (pl: plumule; mf: fundamental meristem; pr: protoderm; ra: radicle).

In histochemical analyses performed in the seed, alkaloids appeared in the seed coat (Figure 5C). These metabolites play an important role in defense against herbivore insects and pathogens, in addition to allelopathic activity (Taiz and Zeiger, 2009). Phenolic compounds were found in the seed coat of Elaeis oleifera. These inhibitors can restrict the entry of oxygen in the seed, impeding germination (Tokuhisa et al., 2007). Studies performed in Bactris maraja and Bactis gasipaes seeds indicated that organization of the seed coat cells, together with their phenolic composition, act as germination regulators, impeding germination (Rodrigues et al., 2015; Nazário et al., 2013). In Acrocomia aculeata (Moura et al., 2010), the presence of this compound and arrangement of the cells are likewise factors that hinder germination. Pectins were identified in the cell walls of the endosperm cells (Figure 5A). They are generally found in the cell content in other species of the same family, such as Bactris maraja (Rodrigues et al., 2015), Bactris gasipaes (Nazário et al., 2013), and Attalea microcarpa (Melo et al., 2017). Pectins are polysaccharides, components of the cell wall that contain sugar and acids that contribute to adhesion among cells and mechanical resistance of the cell wall (Taiz and Zeiger, 2009). Protein bodies were observed in large number of endosperm cells (Figure 5B), a component required to provide initial support of the seedlings until they become autotrophic organisms. Proteins are molecules constituted by nitrogen; they act as an energy source during seed germination (Buckeridge et al., 2004) and are commonly found in the endosperm of seeds of Attalea microcarpa (Melo et al., 2017), Bactris maraja (Rodrigues et al., 2015), Bactris gasipaes (Nazário et al., 2013), Euterpe oleraceae (Gonçalves et al., 2010), Acrocomia aculeata (Moura et al., 2010), and Euterpe edulis (Panza et al., 2004). Lipids in the form of corpuscles were detected in the endosperm cells (Figure 5D). This compound is metabolized in the initial stages of germination and seedling establishment (Bewley and Black, 2012).

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Figure 5. Histochemical tests applied on the Elaeis oleifera (Kunth) Cortés seed; a-e: seed coat and endosperm; f-i: embryo. a) Endosperm cell wall with pectin (ruthenium red). b) Endosperm stained with Xilidine Ponceau showing protein bodies (arrow). c) Endosperm cells with alkaloid concentration (Wagner). d) Lipids within the endosperm cells (Sudan). e) Seed coat with phenolic compounds (iron(III) chloride). f) Reaction for lipids within the protoderm cells and fundamental meristem. g) Protoderm cells and fundamental meristem with reaction for alkaloids. h) Reaction for pectin. i) Protein bodies within the cells of the fundamental meristem (arrow) (en: endosperm, mf: fundamental meristem, pr: protoderm, te: tegument).

The presence of lipids (Figure 5F), alkaloids (Figure 5G), pectin (Figure 5H), and proteins (Figure 5I) was identified in the protoderm and fundamental meristem of the embryo of E. oleifera. The presence of these compounds is a characteristic common to palm trees of different genera, such as Acrocomia aculeata (Moura et al., 2010) and Euterpe oleracea (Scherwinski-Pereira et al., 2012), and its existence contributes to embryo growth during the germination process as a food reserve.

CONCLUSIONS

The dense seed coat, of juxtaposed cells with phenolic composition, may contribute to impermeability to water, impeding gas diffusion and affecting germination. There is no embryonic immaturity in the seed, because the embryo has a conspicuous embryonic axis, composed of a plumule and radicle and distal region with differentiated haustorium without signs of morphological dormancy.

Journal of Seed Science, v.42, e202042018, 2020 Morphoanatomy and histochemistry of Elaeis oleifera 9

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Journal of Seed Science, v.42, e202042018, 2020 Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Cowpea yield and quality after application of desiccating Journal of Seed Science, v.42, herbicides e202042019, 2020 http://dx.doi.org/10.1590/ 2317-1545v42228204 Jeovane Nascimento Silva1 , Estevam Matheus Costa1 , Leandro Spíndola Pereira1 , Elaine Cristina Zuquetti Gonçalves1 , Jacson Zuchi1 , Adriano Jakelaitis1*

ABSTRACT: The aim of this study was to evaluate the effects of pre-harvest desiccant herbicides on the yield and the physiological and technological quality of cowpea seeds after harvest and after storage. The experiment was conducted in a randomized block design with four replications. A split-plot design (6 × 2) was composed of the desiccant herbicides flumioxazin (30 g ai.ha-1), glufosinate ammonium (500 g ai.ha-1), paraquat (400 g ai.ha-1), saflufenacil (70 g ai.ha-1), and carfentrazone (24 g ai.ha-1) and an untreated control, as well as two seed evaluation periods, at harvest and six months after h arvest. Desiccants were applied at the R5 stage. The desiccants affected the yield, classification, color, and physiological quality of seeds of ‘BRS Guariba’. The glufosinate ammonium and paraquat herbicides compromised seed physiological quality. Flumioxazin did not affect seed yield components, color, and physiological quality. Storage at 20 °C for six months affected seed physiological quality.

Index terms: Vigna unguiculata L., vigor, storage.

Rendimento e qualidade de sementes de feijão-caupi após a aplicação de herbicidas dessecantes

RESUMO: O objetivo deste estudo foi avaliar os efeitos de herbicidas dessecantes aplicados em pré-colheita sobre o rendimento, a qualidade fisiológica e tecnológica de sementes de feijão-caupi após a colheita e o armazenamento. O experimento foi conduzido em delineamento de blocos ao acaso, com quatro repetições. Adotou-se o esquema de parcelas subdivididas (6 x 2), compostas pelos herbicidas dessecantes: flumioxazim (30 g i.a.ha-1), glufosinato de amônio (500 g i.a.ha-1), paraquate (400 g i.a.ha-1), saflufenacil (70 g i.a.ha-1), carfentrazone (24 g i.a.ha-1) e uma testemunha não tratada, por duas épocas de avaliação das sementes: colheita e seis meses após a colheita. A aplicação dos dessecantes ocorreu no *Corresponding author estágio R5. Os dessecantes afetaram o rendimento, a classificação, a coloração e a qualidade E-mail: adriano.jakelaitis@ifgoiano. fisiológica das sementes da cultivar BRS Guariba. Os herbicidas glufosinato de amônio e edu.br paraquate comprometeram a qualidade fisiológica das sementes. O flumioxazin não afetou Received: 3/09/2019. os componentes de rendimento, a coloração e a qualidade fisiológica. O armazenamento a Accepted: 17/02/2020. 20 °C por seis meses afetou a qualidade fisiológica das sementes.

Termos para indexação: Vigna unguiculata L., vigor, armazenamento. 1Diretoria de Pós-Graduação, Pesquisa e Inovação, Instituto Federal Goiano Campus Rio Verde, Caixa Postal 66, Cep: 75901-970, Rio Verde, Goiás, Brasil.

Journal of Seed Science, v.42, e202042019, 2020 2 J. N. Silva et al.

INTRODUCTION

In Brazil, cowpea (Vigna unguiculata L.) occupied a planted area of 1516 thousand hectares, with production of 789.8 thousand (metric) tons of grain in 2018 (CONAB, 2019). In the state of Goias, average yield of cowpea for the 2019 crop season is estimated to be 1260 kg.ha-1, whereas the average yield for Brazil is 521 kg.ha-1 (CONAB, 2019). Due to development of varieties with traits that favor mechanized harvest, the crop is expanding in the Center-West region of Brazil, although production is still mainly concentrated in the Northeast region (CONAB, 2019). Modernization of Brazilian agriculture has required changes to improve the production process and ensure seed quality. High quality seeds assume a fundamental role for production companies. Plants generally remain in the field for some time beyond their physiological maturity, exposed to environmental variations that can compromise their physiological quality (Paiva et al., 2018). One of the significant difficulties during harvest of cowpea seeds is their lack of uniform physiological maturity. High quality seeds depend on harvest occurring at the ideal time, frequently when physiological maturity is reached, coinciding with maximum accumulation of seed dry matter, high vigor, and high germination (Lima et al., 2018). One of the alternatives is desiccant herbicide prior to harvest, which can reduce exposure of seeds to unfavorable environmental conditions. Desiccant herbicides has been adopted in some dry edible bean (common bean) producing regions (Kappes et al., 2012). Desiccant herbicides applied in an appropriate manner promote uniformity in crop maturation, allow earlier harvest, and do not cause yield loss; and seeds of high physiological quality can be obtained (Lamego et al., 2013). Nevertheless, some important aspects should be taken into consideration in desiccant herbicide, such as the mode of action of the product, the phenological stage the crop is in, and the effect on seed yield, germination, and vigor (Finoto et al., 2017). In addition, desiccation can interfere in the physiological quality of cowpea seeds during storage. How seed quality is affected depends on the genotype, the edaphic and climatic conditions, and biotic factors. Deterioration in quality can occur during storage under inadequate temperature and moisture conditions (Zuchi et al., 2013). Such conditions cannot be prevented, only minimized. In this context, the aim of this study was to evaluate the effects of desiccant herbicides applied prior to harvest on the yield and physiological and technological quality of cowpea seeds at the time of harvest and after six months of storage.

MATERIALS AND METHODS

The ‘BRS Guariba’ cowpea crop was grown in a field in Rio Verde, GO, at the coordinates 17°48’67” S and 50°54’18” W and altitude of 754 m. Soil in the area (Latossolo Vermelho distroférrico) at the depth of 0-20 cm had the following physical-chemical composition: pH 6.2 (SMP), Ca 4.64 cmolc.dm-3, Mg 2.50 cmolc.dm-3, Al3+ 0.04 cmolc.dm-3, H+Al 4.5 cmolc.dm-3, CEC 12.1 cmolc.dm-3, K 0.46 cmolc.dm-3, P (Mehlich) 13.1 mg.dm-3, organic matter 3.62 mg.dm-3, Zn 4.5 mg.dm-3, base saturation 62.8%, aluminum saturation 0.5%, clay 64.5%, silt 10%, and sand 25.5%. A randomized block experimental design was adopted in split plots (6 × 2) with four replications. The first factor was constituted by application of the desiccant herbicides flumioxazin (Flumyzin 500, 500 g a.e.L-1 WP, Sumito Chemical do Brasil) at 60 g.ha-1, glufosinate ammonium (Liberty®, 200 g a.e.L-1 SL, Basf) at 2.5 L.ha-1, paraquat (Gramoxone®, 200 g a.e.L-1 SL, Syngenta) at 2 L.ha-1, saflufenacil (Heat®, 700 g a.e.kg-1 WG, Basf) at 100 g.ha-1, and carfentrazone (Aurora® 400 EC, 400 g a.e.L-1 EC, FMC) at 60 mL.ha-1, and an untreated control. The second factor was constituted by the time of seed quality evaluation – after harvest and six months after harvest (6 MO). Each plot had an area of 24.5 m2, with four 7-m-length rows and between-row spacing of one half meter. The area of the plot used for data collection was the central five meters of the two central rows.

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The cowpea seeds were treated with 100 g de pyraclostrobin + methyl thiophanate + fipronil (Standak® Top) for 100 kg of seed and then inoculated with 300 mL of Bradyrhizobium spp. for 100 kg of seed. Seeds were sown on March 17, 2018, with twelve seeds per linear meter at a depth of 3 cm. Fertilization at planting was 300 kg.ha-1 of the formulation 04-14-08 (Souza and Lobato, 2004). At thirteen days after emergence (DAE), the commercial mixture of bentazon + imazamox (Amplo®) was applied at the rate of 1 L of the commercial product per hectare, and, at seventeen DAE, haloxyfop-p-methyl (Verdict®R) was applied at the rate of 0.4 L of the commercial product per hectare. During the crop cycle, one application was made of the insecticide lambda-cyhalothrin + chlorantraniliprole (Ampligo®) at the rate of 0.16 L of the commercial product per hectare for control of Spodoptera eridania (Cramer) and another of the fungicide fluxapyroxad + pyraclostrobin (Orkestra®SC) at the rate of 0.3 L of commercial product per hectare for control of Erysiphe polygoni.

The herbicides were applied 78 days after sowing (DAS) at the R5 stage, when the pods were at physiological maturity with brown to tan color and seeds had 70% moisture content (Carvalho and Nakagawa, 2012). The herbicides were applied with a CO2 pressurized backpack sprayer, model TT110°03 with four spray tips, at a constant pressure of 2.5 bar and spray volume of 200 L.ha-1. The seed moisture content in each plot was monitored, establishing the point of harvest when seeds had moisture levels near 11%. Upon reaching this level, plants were cut at soil level and the pods were removed and threshed manually. The seeds were dried in a laboratory oven at 35 °C until reaching 11% moisture. The number of days of anticipation of harvest (earlier harvest in days) was determined by counting the days from application of desiccation herbicides up to when the plants were completely defoliated and grain moisture was in the range of 13–15% moisture compared to the day of harvest of the untreated control (Brasil, 2009; Krzyzanowski et al., 2015). One thousand seed weight was determined as recommended in the Rules for Seed Testing (Brasil, 2009), with eight replications of 100 seeds, extrapolating the values to 1000 seeds. In a parallel manner, yield in kg.ha-1 was determined by weighing all the seeds obtained from the area used for data collection and extrapolating. The seeds collected from the area used for data collection were cleaned in sieves and classified through determination of the proportion of seeds per sieve. The samples from each plot were weighed, passed through a set of metallic sieves with mesh sizes of 7.5 mm oval, 4.5 mm oblong, 3.5 mm oblong, 3.5 mm oval, and bottom collector (Brasil, 2009). The seeds that passed through a sieve with oblong mesh of 3 mm width and 19 mm length were considered immature, with incomplete physiological development. Those that were retained in sieves with meshes of 4.5 mm or more were considered healthy, with complete physiological development (Knabben and Costa, 2012). The samples were placed in plastic bags and then in BOD (20 °C), where they remained throughout the period of evaluations. Relative humidity (RH) and temperature were registered by a digital data logger (accuracy: 0.1 °C; 5.0% RH). Physiological quality was checked after harvest and at six months of storage through the following tests: germination, seedling dry matter, root and shoot length, accelerated aging, emergence in sand substrate, emergence speed index, electrical conductivity, hydration coefficient, and grain color. Evaluations were conducted with duplicate sets of fifty seeds for each replication. Germination (G): First, seeds were treated with 45 g a.i. of carbendazim + 105 g a.i. of thiram for 100 kg of seed and, after that, the seeds were sown in germitest paper moistened with distilled water in the amount of 2.5 times the weight of the dry paper. The rolls were placed separately by field replication in plastic bags and kept at 25 °C in a germination chamber for eight days, at which time the percentage of normal seedlings was determined (Brasil, 2009). First germination count (FGC): This was performed together with the germination tests. One count was performed on the fifth day after setting up the germination test (Brasil, 2009), and the second germination count was performed on the eighth day after setting up the germination test. After counting, calculations of germination percentage [as recommended by the Brazilian Ministry of Agriculture (Ministério da Agricultura, Pecuária e Abastecimento – MAPA)] were carried out for percentage values greater than 80 (Brasil, 2009).

Journal of Seed Science, v.42, e202042019, 2020 4 J. N. Silva et al.

Seedling dry matter (SDM): Fourteen seedlings per experimental unit were used. Cotyledons were extracted and the seedlings were then placed in Kraft paper bags in an air circulation laboratory oven at 80 °C for 24 hours. After drying, they were weighed, and results were expressed in mg per seedling (Vieira and Krzyzanowski, 1999). Seedling length (SL): Fourteen seedlings from the germination test of each experimental unit were used. Evaluation was carried out on the eighth day after setting up the germination test, and seedlings classified as normal in the germination test were selected. Total length of the seedling was determined from the tip of the main root up to the point of connection of the cotyledons with the aid of a millimeter ruler at eight days after sowing. The mean total length of fourteen seedlings was obtained. The shoot (SHL) and root (RL) were also evaluated (Vieira and Krzyzanowski, 1999). Accelerated aging test: The seeds were placed in a gerbox transparent plastic box with an aluminum screen inside. Distilled water (40 mL) was added to each gerbox, and the seeds were arranged on the screen. Lids were placed on the boxes and they were placed in a germination chamber kept at 41 °C for 48 hours (Marcos-Filho, 1999). After this aging period, the germination test was performed on the seeds, as described above. Five days after sowing in germitest paper, the results of percentage of normal seedlings were obtained (Brasil, 2009). Emergence test: This was conducted in a greenhouse, with four replications of fifty seeds. Seeds were sown in sand at a depth of 3 cm. The environment was irrigated by an automatic sprinkler four times a day. Emerged seedlings were counted daily up to numerical stabilization, which occurred at eight days after emergence. Seedlings with cotyledons in the horizontal position were considered to have emerged. The results of the emergence speed index were calculated according to Maguire (1962). Conductivity test: This was carried out with eight replications of fifty seeds from each treatment. Seeds were first weight on a precision balance (0.01 g) and placed in plastic cups containing 75 mL of distilled and deionized water; they were kept in imbibition in BOD at 25 °C. After 24 hours, electrical conductivity was read using a Technal TEC-4MP digital conductivity meter. Results were expressed in µS cm-1.g-1 of seed (Vieira and Krzyzanowski, 1999). Determination of hydration coefficient: In this test, 15 g of seeds soaked in 60 mL of distilled water (1:4 proportion) at ambient temperature (25 °C) were used. After twelve hours, maceration water was removed, followed by removal of free water, leaving each sample for two minutes on absorbent paper before weighing. Weight gain was considered as the amount of water absorbed, and expressed as the hydration coefficient (HC), calculated by the following equation: HC = WW/WS × 100, where HC is the hydration coefficient, WW is the weight of the seed after hydration, and WS is the weight of the seed before hydration (El-Refai et al., 1988; Nasar-Abbas et al., 2008). Color of seed coat of whole, uniform seeds: This was determined using a ColorFlex EZ colorimeter with a Hunter color system, which indicates colors in a three-dimensional system. The vertical L* axis indicates the color of the sample from white to black, the “a” axis from the color red to green, and the “b” axis from yellow to blue (Afonso- Júnior and Corrêa, 2003). For better characterization, the seeds were evaluated in the resting position at two different points, subsequently calculating the mean value of each seed. The difference in color (ΔE*) was obtained by the equation ΔE* = [(ΔL*)2 + (Δa*)2] 0.5, where ΔE* = value for color difference; ΔL* = difference between the L* of the initial sample and the L* of the stored sample; Δa* = difference between the a* of the initial sample and the a* of the stored sample. The Shapiro-Wilk test was performed on the data and, when significant (p < 0.05), they were transformed in square root (x + 0.5) for analysis. After that, analysis of variance was performed by the F test (p < 0.05) and, when significant, they were compared by the Tukey test (p < 0.05).

RESULTS AND DISCUSSION

Significant effects were observed for the variables earlier harvest in days (EHD), thousand seed weight (1000SW), seed yield (SY), sieve of 4.5 mm mesh (S4.5) and sieve of 3.5 mm mesh with oblong shape (S3.5 OB) for the herbicides, and significant effects were not found for seeds classified in the sieve of 7.5 mm oval mesh (S7.5) and the sieve of 3.5 mm oval

Journal of Seed Science, v.42, e202042019, 2020 Cowpea yield and quality with desiccant herbicides 5 mesh (S3.5 O) and bottom collector (Table 1). With application of paraquat, harvest was ten days earlier compared to the untreated control; however, the more intense effect of the herbicide led to lower SY and 1000SW (Table 1). The degree of desiccation is related to the damage the herbicide causes to cell membranes, bringing about drying, leaf drop, and, simultaneously, water loss in seeds and low seed weight (Tarumoto et al., 2015). In the specific case of materials of indeterminate growth habit and lack of uniform maturation, such as ‘BRS Guariba’, this effect can be more intense, as observed for paraquat, for which the decline in SY values was approximately 46% compared to the control. For the saflufenacil and glufosinate ammonium herbicides, which also promoted EHD, the same response was observed, but in lower intensity in relation to the others (Table 1). The intensity of the effects of the herbicides on cowpea plants also affected seed classification. In the treatments with herbicides that led to greater EHD in cowpea plants and, consequently, lower SY and 1000SW, seeds were mostly retained in the 3.5 mm oblong mesh sieve (Table 1). In the sieves with larger meshes, such as the 4.5 mm mesh with oblong shape, a larger number of seeds were found coming from plants that did not receive herbicides or coming from treatments with flumioxazin (Table 1). This result is important for seed production because seeds retained in sieves of higher numbering generally exhibit greater vigor in relation to the soybean varieties retained in sieves of greater size (Mathias and Coelho, 2018). The herbicides used for plant desiccation generally affected germination, shoot length (SHL) and root length (RL) of seedlings, and seedling dry matter (SDM), but did not affect percentage of emergence (E) (Tables 2 and 3). The use of paraquat and glufosinate ammonium reduced germination of the cowpea seeds evaluated after harvest in relation to the untreated control and to the other herbicides (Table 2). Studies indicate that herbicides recommended for pre-harvest desiccation of crops have reduced seed germination due to their harmful effects on seed formation. For soybean, glufosinate ammonium was the treatment least recommended for desiccation, due to lower values of seed germination percentage (Delgado et al., 2015; Souza et al., 2017). Desiccating products have different mechanisms and degrees of desiccation, intrinsically related to the injury caused to cell membranes, allowing rapid water loss. The process of seed and plant water loss should be known for the specific crop. The particular characteristics of each product and application rates can bring about water losses during harvest that can directly affect seed physical and chemical quality (Lamego et al., 2013). The use of desiccants in pre-harvest did not affect the shoots and roots of seedlings grown from the seeds harvested. However, after six months of storage, the shoot length of seeds from all the treatments increased, which consequently

Table 1. Earlier harvest in days (EHD), seed yield (SY), 1000 seed weight (1000SW), and classification of seeds by means of sieves of 7.5 mm oval mesh (S7.5), 4.5 mm oblong mesh (S4.5), 3.5 mm oblong mesh (S3.5 OB), 3.5 mm oval mesh (S3.5 O), and bottom collector of cowpea as a result of desiccant herbicide treatments.

SY1/ 1000SW S7.51/ S4.51/ S3.5 OB1/ S3.5 O1/ BOTTOM1/ Desiccant EHD1/ (kg.ha-1) ------( g ) ------Control 0.00 e2/ 1315.65 a 194.62 a 4.35 a 573.75 a 55.47 bc 3.82 a 0.52 a Flumioxazin 2.00 d 1219.15 ab 197.68 a 6.92 a 531.25 ab 55.70 bc 6.75 a 1.90 a

Glufosinate ammonium 4.00 c 855.45 abc 176.04 ab 4.32 a 303.32 cd 101.10 ab 11.40 a 0.17 a

Paraquat 10.00 a 704.75 c 164.38 b 0.65 a 207.62 d 120.30 a 15.35 a 1.80 a Saflufenacil 6.00 b 791.65 bc 184.92 ab 3.90 a 339.97 bcd 62.85 bc 18.15 a 0.65 a Carfentrazone 0.00 e 1015.75 abc 195.13 a 2.87 a 433.90 abc 45.15 c 3.52 a 0.00 a CV (%) 0.00 10.02 2.66 50.31 11.53 15.77 40.10 40.34 1Data transformed in square root (x + 0.5). 2Mean values followed by the same letters are statistically equal by the Tukey test (p < 0.05).

Journal of Seed Science, v.42, e202042019, 2020 6 J. N. Silva et al. affected root growth. The desiccant herbicide treatments that were most harmful were paraquat and saflufenacil. Consequently, these treatments had the greatest effect on root growth of seeds after storage, and paraquat was the herbicide that most affected germination and root growth (Table 3). Tests related to length of seedlings or their parts are effective for detecting subtle differences in seed vigor (Vanzolini et al., 2007). After storage of the cowpea seeds, the effects of the desiccants on seed germination were observed (Table 2). Pre-harvest desiccation with glufonisate ammonium carried out on soybean cultivars harmed the physiological quality of their seeds after six months of storage (Silva et al., 2016). Marcos-Filho (2015) confirms that the use of herbicides with contact action on plants reduces the seed germination. Prolonging these effects does not interfere in transfer of dry matter during the maturation process, and this leads to more vigorous seeds with the energy necessary to maintain vital functions during storage.

Table 2. Percentage of emergence (E), germination of cowpea seeds evaluated after harvest and after six months of storage (6 MO) as a result of desiccant herbicide treatments.

E1/ (%) Germination Desiccant Harvest 6 MO Harvest 6 MO Control 912/ 81 97 aA 80 aA Flumioxazin 85 82 98 aA 93 aA Glufosinate ammonium 61 67 66 bA 72 aA Paraquat 67 74 63 bB 83 aA Saflufenacil 83 81 88 abA 72 aA Carfentrazone 87 84 81 abA 84 aA CV A (%) 11.78 9.15 CV B (%) 4.43 6.93 1Data transformed in square root (x + 0.5). 2Mean values followed by the same lowercase letters in the columns and uppercase letters in the rows are statistically equal by the Tukey test (p < 0.05).

Table 3. Shoot length (SHL), root length (RL), and dry matter (SDM) of seedlings from cowpea seeds evaluated after harvest and after six months of storage (6 MO) as a result of desiccant herbicide treatments.

SHL1/ (cm) RL1/ (cm) SDM 1/ (g) Desiccant Harvest 6 MO Harvest 6 MO Harvest 6 MO Control 3.33 aB2/ 6.58 aA 7.54 aA 5.29 abA 0.53 aA 0.74 aA Flumioxazin 3.40 aB 8.53 aA 7.79 aA 6.49 aA 0.52 aB 0.83 aA Glufosinate ammonium 4.03 aB 6.60 aA 4.02 aA 6.37 abA 0.43 aB 0.81 aA Paraquat 4.41 aB 8.05 aA 7.99 aA 2.70 bB 0.55 aA 0.56 aA Saflufenacil 3.12 aB 6.86 aA 6.69 aA 2.63 bB 0.44 aA 0.65 aA Carfentrazone 2.56 aB 6.10 aA 5.73 aA 3.75 abA 0.35 aB 0.69 aA CV A (%) 18.30 19.55 9.15 CV B (%) 14.53 14.57 9.85 1Data transformed in square root (x + 0.5). 2Mean values followed by the same lowercase letters in the columns and uppercase letters in the rows are statistically equal by the Tukey test (p < 0.05). Journal of Seed Science, v.42, e202042019, 2020 Cowpea yield and quality with desiccant herbicides 7

For the seeds evaluated at crop harvest, effects from the herbicides were not observed on SHL, RL, and SDM of the seedlings; effects were observed only after storage (Table 3). An increase in SHL values was found due to storage for all the treatments tested, whereas an increase in SDM only appeared for the flumioxazin, glufosinate ammonium, and carfentrazone herbicide treatments. Inhibitory effects were observed for RL as a result of seed storage in the paraquat and saflufenacil treatments. The changes observed in this variable stem from the cell disorder in seeds coming from these treatments from the time of early harvest on, bringing about damage to cell components, as found in the electrical conductivity test. Seed physiological quality is mainly compromised after storage periods, a natural deterioration process inherent to a seed production program (Moussa et al., 2011). In addition, the BRS Guariba cultivar showed greater sensitivity to the storage period, with a lower germination percentage and lower vigor, which hurt initial establishment of seedlings in the field through having less developed roots and, consequently, lower initial biomass accumulation (Boiago et al., 2013). The accelerated aging test (normal seedlings after accelerated aging – NSAA) was performed on the seeds, which provided the percentage of normal seedlings, confirming the significant interaction between herbicides and the period of evaluation (Table 4). At the time of crop harvest, the seeds coming from the treatments with paraquat, glufosinate ammonium, and saflufenacil had lower NSAA values compared to the other treatments, and the decrease in NSAA values intensified with storage both for the seeds coming from herbicide treatments and for the seeds from the untreated control. Seeds coming from the treatment with glufosinate ammonium had a percentage of normal seedlings after accelerated aging lower than the percentages from the other treatments. According to Lima et al. (2018), seeds from common bean desiccated with glufosinate ammonium at different time periods have lower NSAA values in relation to the untreated control. Tavares et al. (2016) also observed effects after storage of seeds coming from a treatment with application of paraquat for desiccation of azuki bean. The greatest rate of water absorption (HC) by seeds was observed in seeds coming from the treatment with paraquat, at both periods of evaluation (Table 4), and effects from storage were not observed. In contrast, effects from storage were observed on reduction in seed moisture. The values were 11.51% and 10.61% in the evaluations conducted before and after the six months of storage, respectively.

Table 4. Normal seedlings from accelerated aging (NSAA), water absorption rate (hydration coefficient - HC), and electrical conductivity (EC) of cowpea seeds evaluated after harvest and after six months of storage (6 MO) as a result of desiccant herbicide treatments.

NSAA (%) HC (%) EC (µS cm-1.g-1) Desiccant Harvest 6 MO Harvest 6 MO Harvest 6 MO Control 90 aA2/ 40 aB 225 bA 229.2 bA 133.1 bB 164.8 aA Flumioxazin 89 aA 34 aB 223 bA 230.2 bA 118.5 bB 163.9 aA G. ammonium1 66 bcA 23 aB 230 bA 235.6 bA 169.9 aB 196.0 aA Paraquat 56 cA 32 aB 249 aA 254.1 aA 124.1 bB 179.9 aA Saflufenacil 57 cA 37 aB 231 bA 236.2 bA 116.5 bB 172.5 aA Carfentrazone 78 abA 33 aB 227 bA 213.7 bA 128.7 bB 184.3 aA CV A (%) 17.29 4.22 11.30 CV B (%) 16.90 3.98 9.95 1Glufosinate ammonium 2Mean values followed by the same lowercase letters in the columns and uppercase letters in the rows are statistically equal by the Tukey test (p < 0.05). Journal of Seed Science, v.42, e202042019, 2020 8 J. N. Silva et al.

For both evaluation periods, the seeds from the treatment with glufosinate ammonium exhibited changes in membrane integrity, whereas the seeds from the treatments with the other herbicides did not differ from each other for EC (Table 4). After storage, the EC values were higher than those found at the time of harvest. According to Moura et al. (2017), the greater the EC, the lower seed vigor is, because conductivity is a direct result of leachates. The increase in electrical conductivity after storage may be related to damage, such as cracks, microfissures, and disorganization in seed cells, factors which contribute to an increase in EC values (Zucareli et al., 2015). Information associated with the intensity, hue, and lightness of color is important for evaluation of cowpea. The added value of the product is related to the quality of the grain that is recently harvested and stored for a certain period. The chroma “C” defines the intensity of the color. In this respect, there was difference among the treatments at the time of harvest and after storage (Table 5). The treatments under saflufenacil had the greatest chroma; all the other treatments led to color of lesser intensity. Thus, saflufenacil can change cowpea seed color. After storage, there was reduction in “C” values in all the treatments. The hue angle “°h” is used to define the hue of the seeds, and significant interaction was observed among the treatments (Table 5). Statistical differences in this aspect of seed color were not observed after harvest for the different herbicide treatments, though after storage, lower values were observed for the saflufenacil and glufosinate ammonium treatments (Table 5). After storage, there was change in the hue of the seeds from the control and from the seeds coming from plants treated with flumioxazin and carfentrazone. Change in the hue of seeds is possibly associated with biochemical changes that occurred in the seeds, and these changes were able to affect the other technological variables, such as seed color intensity and lightness, which define seed coat color. Lightness in color of the grain, expressed by the “L” coordinate, is desired by the consumer and implies acceptance of the product. When harvested, regardless of the desiccant treatments, the seeds had “L” values near 50. After storage, there was reduction to values from 26.12 to 28.03 (Table 5). This is related to reduction in quality and to the need for longer cooking time. The nearer the value is to 50, the better the quality (Ganascini et al., 2014). Environmental and genetic factors are responsible for bean seed coat darkening (Coutin et al., 2017). The low yield of the cowpea crop is still one of the problems faced by growers, and this limitation may be related to the low physiological and genetic quality of the seeds. In this respect, studies on the time of desiccation after plant physiological maturity, spray volume for desiccation, and the spray tips used are fundamental for maintaining physiological quality of seeds for formation of viable seedlings.

Table 5. Chroma (C), hue angle (°h), and L coordinate of cowpea seeds evaluated after harvest and after six months of storage (6 MO) as a result of desiccant herbicide treatments.

C °h L Desiccant Harvest 6 MO Harvest 6 MO Harvest 6 MO Control 20.31 abA1/ 4.27 aB 1.30 aB 1.40 aA 47.99 abcA 28.03 aB Flumioxazin 21.81 abA 4.11 aB 1.31 aB 1.40 aA 50.10 aA 27.06 aB Glufosinate ammonium 22.02 abA 4.74 aB 1.28 aA 1.30 bA 46.45 abcA 26.47 aB Paraquat 20.39 abA 5.10 aB 1.32 aA 1.36 abA 44.77 cA 26.16 aB Saflufenacil 22.35 aA 4.95 aB 1.29 aA 1.31 bA 45.49 bcA 26.72 aB Carfentrazone 19.41 bA 4.11 aB 1.32 aB 1.40 aA 48.90 abA 27.48 aB CV A (%) 9.76 2.31 4.23 CV B (%) 8.23 2.43 4.71 1Mean values followed by the same lowercase letters in the columns and uppercase letters in the rows are statistically equal by the Tukey test (p < 0.05).

Journal of Seed Science, v.42, e202042019, 2020 Cowpea yield and quality with desiccant herbicides 9

CONCLUSIONS

Desiccant herbicides applied on ‘BRS Guariba’ cowpea in the R5 maturity stage allow harvest to occur earlier, reduce yield and seed weight, and affect seed classification. Desiccant herbicides affect seed quality. Glufosinate ammonium compromises seedling vigor, and flumioxazin affects cowpea seed quality.

ACKNOWLEDGMENTS

This study was carried out with the support of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) under funding code 001, and the support of the Instituto Federal de Goiano, Campus Rio Verde, GO, Brazil.

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Journal of Seed Science, v.42, e202042019, 2020 Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Induction of seed coat water impermeability during maturation Journal of Seed Science, v.42, of Erythrina speciosa seeds e202042020, 2020 http://dx.doi.org/10.1590/ 2317-1545v42228614 Debora Manzano Molizane1 , Sandra Maria Carmello-Guerreiro2 , Claudio José Barbedo3*

ABSTRACT: Dormancy is a physiological process that allows seeds to survive in unfavorable environments by preventing their germination. For a large number of species, seed desiccation at the end of maturation is common, and for some of these seeds, this includes seed coat water impermeability (SCWI). The environmental conditions in which the mother plant develops affect the seed maturation process, causing variations in both seed physiological quality and the onset of physical dormancy. In this study, we analyzed the induction of SCWI in immature seeds of Erythrina speciosa by artificial drying. Seeds at three stages of immaturity were dried gradually for subsequent evaluation of their germination. At each level of drying, the anatomical structure of the seed coat was also analyzed. Artificial drying was able to induce SCWI in immature seeds. Furthermore, environmental conditions affected at which stage of maturity SCWI began, and they affected development of desiccation tolerance. However, unlike other species, there were no anatomical differences related to this SCWI (whether by natural drying or artificial drying) and, therefore, in E. speciosa seeds, SCWI may be related to biochemical differences in the seed coat.

Index terms: dormancy, drying, desiccation tolerance, tropical tree species.

Indução da impermeabilidade do tegumento em relação a água durante a maturação em sementes de Erythrina speciosa

RESUMO: A dormência é um processo fisiológico que permite que as sementes sobrevivam em ambientes desfavoráveis, impedindo sua germinação. Para grande número de espécies, a dessecação de sementes no final da maturação é comum e, em algumas delas, instala- *Corresponding author se a impermeabilidade do tegumento à água (IT). As condições ambientais sob as quais a E-mail: [email protected] planta-mãe se desenvolve interferem no processo de maturação das sementes, causando variações tanto na qualidade fisiológica quanto na instalação da dormência física. Neste Received: 12/09/2019. trabalho analisou-se a instalação de IT em sementes imaturas de Erythrina speciosa Accepted: 27/02/2020. por secagem artificial. Sementes de três estádios imaturos foram secadas em intervalos graduais e sua germinação foi analisada. O tegumento das sementes de cada nível de 1 secagem também foi analisado anatomicamente. Concluiu-se que a secagem artificial foi Instituto de Biociências (UNESP), Caixa Postal 510, 18618-970 – capaz de promover a instalação da IT em sementes imaturas. Além disso, as condições Botucatu, SP, Brasil. ambientais influenciaram o estádio de maturidade no qual a IT foi instalada, bem como o desenvolvimento da tolerância à dessecação. No entanto, diferente de outras espécies, não 2Instituto de Biologia (UNICAMP), houve diferenças anatômicas relacionadas a essa IT (tanto pela secagem natural quanto 13083-862 – Campinas, SP, Brasil. pela artificial) e, portanto, em sementes de E. speciosa, a impermeabilidade pode estar relacionada a diferenças bioquímicas no tegumento da semente. 3Instituto de Botânica, Núcleo de Pesquisa em Sementes, 04301-902 – Termos para indexação: dormência, secagem, tolerância à dessecação, espécie arbórea tropical. São Paulo, SP, Brasil.

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INTRODUCTION

Dormancy is a physiological process that allows seeds to survive in adverse environments by preventing immediate and/or synchronized germination, thus allowing temporal dispersion of the seeds. Among the various types of dormancy, physical dormancy, imposed by impermeability of the seed coat to water, is commonly found in Fabaceae. Seeds often exhibit dormancy by impermeability of the seed coat to water (SCWI) at the end of the maturation process, and this impermeability may be triggered by desiccation (Baskin and Baskin, 2000; Jayasuriya et al., 2007a, 2007b; Nakagawa et al., 2007). The environmental conditions that the mother plant is exposed to during the seed maturation process can affect the induction of physical dormancy. Thus, seeds of the same species can exhibit considerable differences in regard to physical dormancy, both in relation to its intensity and to the time at which it is induced. These variations are frequently linked to environmental conditions and therefore depend on the location or year of production of the seeds (Hay et al., 2010; Gama-Arachchige et al., 2011; Lamarca et al., 2013). In Trifolium ambiguum seeds, for example, artificial drying led to the induction of physical dormancy in still immature seeds, simultaneous to the process of acquisition of desiccation tolerance. Immature seeds of Geranium carolinianum dried down to 11% moisture became dormant, while dormancy was not induced in those dried to 13% (Hay et al., 2010; Gama-Arachchige et al., 2011). Therefore, the onset of dormancy represented by seed coat water impermeability may not necessarily be connected with completing the maturation process, but may occur as a result of natural drying at the end of maturation. Therefore, there is the need for more studies in this respect to understand the drying process and the onset of dormancy. Erythrina speciosa Andrews (Leguminosae, Faboideae) has seeds that are tolerant to desiccation and can be stored for several years. When these seeds are mature, they frequently have a seed coat impermeable to water absorption (Mello et al., 2010; Lima and Martins, 2014). Thus, they are interesting materials for studying induction of dormancy in immature seeds. The aim of this study was to analyze the relationship between the impermeability of the testa of mature seeds of E. speciosa and the impermeability induced by artificial drying in immature seeds.

MATERIALS AND METHODS

Plant material: Erythrina speciosa fruit was collected from thirty mother plants in the Catavento Cultural Park in an urban area of the municipality of São Paulo, SP (23°32’44”S and 46°37’40”W) from August to October of 2013 and 2014. The fruit was broken and seeds were removed manually. These seeds were separated into three maturity stages according to their size and the morphological characteristics of the seed coat, seeking to follow the classification of Hell et al. (2019): Stage III (S3) – light green color with light brown spots, mean length of 1.6 cm; Stage IV (S4) – green color and covered by brown spots, mean length of 1.88 cm; Stage V (S5) – brown color with green spots, mean length of 1.52 cm. That way, the following sets of seeds were obtained, according to maturity stage and year of collection: S3/2013, S3/2014, S4/2013, S4/2014, S5/2013, and S5/2014. Immediately after this separation, seeds from each stage, from each year, were analyzed regarding moisture content, dry matter content, and germination, as described below. Physical and physiological analyses: moisture content and dry matter content of the seeds were evaluated by the gravimetric method in a laboratory oven at 103 °C for seventeen hours (Brasil, 2009) using four replications of five seeds, and the results were presented in percentage (moisture content, wet basis) and in mg.seed-1 (dry matter content). The germination test was set up in rolls of paper previously moistened with water (2.5 times the weight of the paper) (Brasil, 2009) and placed in a germination room at 25 °C and 70% relative humidity. Evaluations were carried out every two days over a period of thirty days, registering the number of seeds with primary root emergence with a size greater than or equal to 0.5 cm (for calculation of germination percentage). Drying: seeds from each stage were dried in a forced air circulation laboratory oven at 40 °C, reducing the initial moisture content of the seeds (69%, 50%, 37% for S3, S4, S5, respectively, in 2013; 70%, 63%, and 36% for S3, S4, and S5, respectively, in 2014) in gradual intervals (reduction of ca. 2.0% to 2.5%.h-1) up to six levels of drying (D1, D2, D3,

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D4, D5, and D6), aiming to reach 10% moisture intervals between the drying levels to finally arrive at only 7% moisture content. After each step of drying, the moisture content and germination were evaluated. The seeds that had an impermeable testa were then mechanically scarified with sandpaper for wood (number 60) and then tested once more for germination, as already described. Anatomical analyses: the seed coat of the S3/2013, S4/2013, S4/2014, S5/2013, and S5/2014 seeds were analyzed anatomically. Samples were fixed in FAA70 (formalin, 37% glacial acetic acid, 70% ethanol – 1:1:8 v/v, Johansen, 1940) and stored in 70% ethanol, according to manufacturer’s recommendations. The samples were embedded in plastic resin (Leica Historesin®), cut in transversal sections with a disposable blade in a manual rotary microtome RM2245 (Leica®) at a thickness of 7 μm, and stained with 0.05% toluidine blue (O’Brien et al., 1964). Slides were analyzed in an Olympus BX51 photomicroscope, obtaining images by a coupled Olympus DP71 camera, on the DPManager program, and the scales were obtained under the same conditions as the photomicrographs. From these images, detailed analyses were performed with the aid of the ImageJ program, measuring the following structures: height of each layer in the seed coat, i.e., palisade cells, hourglass cells, and parenchyma, as well as measurements of the thickness of the light line and cuticle. Experimental design and statistical analysis: a completely randomized experimental design was used with four replications. Three replications for each treatment were made for the measurements in each structure of the testa. Analysis of variance (F test) was performed on the data and the means were compared by the Tukey test at the level of 5% by the R statistical program.

RESULTS AND DISCUSSION

The seeds from S3/2013 and S3/2014 initially had 70% moisture (Figures 1 and 2). Drying brought these values to 7% (S3/2013, Figure 1a) and 12% (S3/2014, Figure 2a). In S4, the seeds from 2013 had 50% moisture and, after the last drying period, reached 7% (Figure 1a); those from 2014, which initially had 60% moisture, arrived at 14% after the last drying (Figure 2a). In S5, the seed lots had similar initial moisture content, 38% for 2013 and 36% for 2014, and maximum drying brought moisture content to 7% (2013, Figure 1a) and 12% (2014, Figure 2a). In the S3/2013 set, the seeds became impermeable upon reaching 15% moisture (D3 and D3sc, Figures 1a and 1b). The seeds of S4/2013, for their part, already became impermeable upon reaching 31% moisture (D2 and D2sc, Figures 1a and 1b), but those of S5/2013 only became impermeable when they reached 7% moisture (D3 and D3sc, Figures 1a and 1b). In the seeds of 2014, impermeability to water was established only in S5, when drying brought moisture content to 13% (Figures 1a and 1b). The seeds of S3/2014 had not yet developed desiccation tolerance; more than 50% of them lost viability when drying brought moisture content to 25%, and all lost viability when moisture content was reduced to 12% (Figures 2a and 2b). The seeds of S4/2014 did not become impermeable even when their moisture content was reduced to 14% (Figures 2a and 2b). However, it is probable that impermeability to water was developing in these seeds because, in addition to showing a small reduction in germination percentage (Figure 2b), this germination was delayed, as shown by the mean time for germination (Figure 2c). The E. speciosa seeds did not exhibit anatomical structural changes in the testa during drying of the immature stages, since the same structures present in the mature seeds (cuticle, palisade cells, light line, hourglass cells, and parenchyma) were present in the immature seeds (Figures 3 and 4). Differences were observed only in the tickness measurements of the layers in the testa (Figure 5) in S4 of 2013 and of 2014, as for example, the hourglass cells and also palisade cells, but without a clear relationship with drying, since neither layer exhibited a decrease in the measurements in accordance with drying. The differences in thickness of the light line and of the cuticle (Figures 5c and 5d) also did not exhibit a relationship to drying. The tickness of the hourglass cells (Figures 5e and 5f) of S4/2013 was the only one that exhibited results related to drying. The measurement observed for the palisade cells (Figure 5a) was near 100 µm for both years of collection, while the parenchyma cells were between 50 and 200 µm for the collection of 2013, and between 50 and 100 µm for the collection of 2014. The cuticle had measurements ranging from 1 to 2.5 µm for the collection of 2013 and from 1 to 2 µm for 2014. The hourglass cells, for their part, ranged from 10 to 33 µm for the 2013 collection, and from 8 to 18 µm for 2014. Journal of Seed Science, v.42, e202042020, 2020 4 D. M. Molizane et al.

Columns with the same letter within each maturity stage represent values that do not differ from each other by the Tukey test (5%). D2sc, D3sc, and D4sc: scarified seeds; D: intact seeds.

Figure 1. Moisture content (a), germination (b), and mean germination time (c) of immature seeds of Stage 3 (S3), Stage 4 (S4), and Stage 5 (S5) of Erythrina speciosa collected in 2013 under four levels of drying (D1, D2, D3, and D4).

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Columns with the same letter within each maturity stage represent value that do not differ from each other by the Tukey test (5%). D2sc and D3sc: scarified seeds; D: intact seeds.

Figure 2. Moisture content (a), germination (b), and mean germination time (c) of immature seeds of Stage 3 (S3), Stage 4 (S4), and Stage 5 (S5) of Erythrina speciosa collected in 2014 under six levels of drying (D1, D2, D3, D4, D5, and D6).

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PC: palisade cells; HC: hourglass cells; Pa: parenchyma; PaC: collapsed parenchyma. →: cuticle. *: light line. Scale: 100 µm. Figure 3. Transversal section of the testa of intact immature seeds of Erythrina speciosa collected in 2013 in Stage 3 (a, b, c, d, e), Stage 4 (f, g, h, i), and Stage 5 (j, k, l, m), without drying (a, f, j), or dried to the first (b, g, k), second (c, h, l), third (d, i, m), and fourth (e) levels.

The only tissue present in the seed testa that exhibited differences correlated with drying was the parenchyma. In S3 and S4 in the seeds of 2013, there was reduction in height consonant with reduction in seed moisture content. The parenchyma did not exhibit a height reduction in S5 in either year, possibly because the water removed during drying was in the cotyledons. The results of germination before and after the drying of S3 and S4 seeds of 2013 and 2014 showed the proximity between the events of acquiring desiccation tolerance (DT) and the onset of dormancy by seed coat water impermeability (SCWI) when placed under drying. Considering that in orthodox seeds (tolerant to desiccation) moisture content is directly associated with the degree of seed maturation (Barbedo et al., 2013), the seeds of S3 of the two years could be in degrees of maturity quite near each other, because they exhibited the same moisture content (70%). However, those of 2013 were tolerant to desiccation, but not those of 2014. The S4 seeds, also through moisture content, show a small

Journal of Seed Science, v.42, e202042020, 2020 Induction of impermeability inE. speciosa seeds 7 variation in degree of maturation from 2013 to 2014, the former a little more mature than the latter (moisture content of 50% for 2013 and 60% for 2014, Figures 1a and 2a). Although both already had DT, this difference was enough so that the seeds of 2013 acquired SCWI after drying, but not those of 2014 (Figures 1b and 2b). These variations within the same species may have occurred as a result of the environmental conditions in which the seeds developed, as indicated by Barbedo (2018). Germination differences in seeds coming from the same location but from different years are expected since the climate conditions the mother plant is exposed to can have an effect on maturation and vigor. E. speciosa seeds exhibited differences in SCWI, showing the effect of climate conditions both on the onset of SCWI and on the sensitivity of seeds to breaking this dormancy (Molizane et al., 2018). At any rate, the results of moisture content and germination in the present study confirm that immature seeds of E. speciosa can become impermeable, just as was observed by Gama-Arachchige et al. (2011) in Geranium carolinianum seeds. The results of DT and SCWI in seeds of different ages and different years suggest the need for a minimum level of development so that seeds can independently develop the ability to germinate, tolerate desiccation, and establish seed coat impermeability. The seeds of S3 and S4 of 2014 did not achieve minimum development to tolerate drying or to develop SCWI, even though the seeds exhibited morphological and anatomical traits and moisture contents similar to those of 2013. Germination capacity and desiccation tolerance are not always acquired at the same time, and neither are desiccation tolerance and longevity (Leprince et al., 2017).

PC: palisade cells; HC: hourglass cells; Pa: parenchyma; PaC: collapsed parenchyma. →: cuticle. *: light line. Scale: 100 µm. Figure 4. Transversal section of the testa of intact immature seeds of Erythrina speciosa collected in 2014 in Stage 4 (a, b, c, d) and Stage 5 (e, f, g, h), without drying (a, e), or dried to the first (b, f), second (c, g), or third (d, h) level.

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Figure 5. Measurements of tickness of the layers of palisade cells and parenchyma cells (a, b), cuticle and light line (c, d), and hourglass cells (e, f) of the testa of Erythrina speciosa in intact immature seeds collected in 2013 (a, c, e) and 2014 (b, d, f) and artificially dried.

Barbedo et al. (2013) cite dormancy as a factor that extends seed longevity, as was also observed by Brancalion et al. (2010). In addition, Barbedo et al. (2013) suggest that the degree of dormancy, as well as the development of desiccation tolerance, is strongly affected by climatic conditions during maturation. This was observed in the present study with E. speciosa seeds produced in different years, which had different capacities for surviving drying and different degrees of dormancy. Richard et al. (2018) likewise observed a relationship between seed origin and dormancy in Desmanthus virgatus; the seeds coming from arid locations had a thicker seed coat and higher level of physical dormancy. Nevertheless, the author also suggests that biochemical analyses should be conducted on the species to confirm these observations. The seed coat protects the embryo from injuries and regulates exchanges of gases and water between the embryo and the environment. Some cell layers present in the seed coat are considered to signal its impermeability. The seed

Journal of Seed Science, v.42, e202042020, 2020 Induction of impermeability inE. speciosa seeds 9 coat of the immature seed in the present study had the same cell layers as the mature seeds observed by Molizane et al. (2018). Figures 3 and 4 show the layers of the palisade cells (PC) and the presence of the light line (*) in the upper part of the cells near the cuticle (arrow). The cuticle and PC in Ormosia paraenses Ducke, for example, were considered to be associated with SCWI (Silva et al., 2018). The seed coat of E. speciosa also had hourglass cells (HC), a layer of parenchymal cells (Pa), and collapsed parenchyma cells (PaC). Many authors consider that this set of structures indicates the presence of seed coat impermeability in mature seeds (Manning and Van Staden, 1985; Lazarevićet al., 2017). In the treatments of induction of SCWI by drying, there was reduction in the thickness of the parenchyma and of the collapsed parenchyma. It was possible to differentiate the two types of parenchyma found in Erythrina seeds, but this differentiation cannot be made after the drying treatments. The parenchyma was the only tissue that showed statistical differences in its measurements related to induction from drying (Figures 5a and 5b). In addition, various authors indicate that the light line present in the palisade cells and in the hilum may also signal the onset of physical dormancy. Manning and Van Staden (1985) describe the light line as being a hydrophobic zone composed of suberin and cellulose, which leads to light refraction when observed in the microscope. The authors furthermore describe that the light line in Papilionoideae is located near the cuticle, but that in Mimosoideae and Caesalpinioideae it can be observed in the center of the palisade layer. However, the relationship between the presence of the light line and SCWI is controversial (Ferreira et al., 2011; Smýkal et al., 2014). The seeds in this study show that even in immature seeds of the three stages of 2013 (Figures 3a, 3f, and 3j) and the two stages of 2014 (Figures 4a and 4e) analyzed, without drying, the light line was evident, but without changes in its thickness. Therefore, it was not possible to establish a relationship of this structure with the onset of SCWI. Chai et al. (2016) observed a relationship between the light line, together with the cuticle, and the onset of SCWI. Unlike that observation, as well as the observation made by Silva et al. (2018) for Ormosia paraenses, the measurements observed in the cuticle present above the layer of palisade cells in the seeds of E. speciosa in the present study did not show significant changes related to the onset of dormancy or to induction from drying. All the treatments in the two years of collection had a visible cuticle (Figures 3 and 4); therefore, the presence of the cuticle and light line for this species is not related to dormancy. There were no changes in the layer of the palisade cells in the seeds of the immature stages before and after drying (Figures 3, 4, 5a, and 5b). Therefore, changes in this cell layer also appear not to be related to the onset of SCWI, unlike that observed by Silva et al. (2018) in Ormosia paraenses. In Desmanthus virgatus changes were found in the measurements of the palisade cells according to the year and location of collection (Richard et al., 2018). In E. speciosa of the present study, differences in the thickness of the testa in different years of collection were not observed. Thus, the anatomical observations and the measurements of the seed coat in this study did not identify changes during induction of SCWI by drying that would confirm the relationship of dormancy with the palisade cells, light line, and the cuticle, as was observed in seeds of Glycine max and Vicia sativa (Harris, 1987; Chai et al., 2016). Therefore, for E. speciosa, SCWI may be related to biochemical differences in the testa of seeds brought about by environmental conditions, as observed in D. virgatu seeds studied by Vijayambika et al. (2011).

CONCLUSIONS

Drying of immature seeds of Erythrina speciosa can induce the onset of dormancy as long as a minimum level of maturity has been reached. Anatomical changes are not necessarily associated with the onset of this dormancy.

ACKNOWLEDGMENTS

The authors thank the Graduate Studies Program in Plant Biodiversity and Environment from Botanic Institute for the opportunity granted to the first author for doctoral studies; to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the doctoral scholarship granted to D.M. Molizane; and to the Parque Cultural Catavento for allowing seed collection. Journal of Seed Science, v.42, e202042020, 2020 10 D. M. Molizane et al.

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Journal of Seed Science, v.42, e202042020, 2020 Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Microbiolization of organic cotton seeds with Trichoderma Journal of Seed Science, v.42, sp. and Saccharomyces cerevisiae e202042021, 2020 http://dx.doi.org/10.1590/ 2317-1545v42229182 José Manoel Ferreira de Lima Cruz1* , Eliane Cecília de Medeiros1 , Otília Ricardo de Farias1 , Edcarlos Camilo da Silva1 , Luciana Cordeiro do Nascimento1

ABSTRACT: Seed microbiolization is an alternative to chemical pesticides for seed treatment in organic agriculture. Thus, this study aimed at evaluating the initial growth and control of fungi associated with organic cotton seeds, through seed microbiolization with Trichoderma sp. and Saccharomyces cerevisiae. Certified cotton seeds (cultivar Aroeira), whose linter was removed with sulfuric acid, were analyzed in a completely randomized design in a 5 x 2 + 1 factorial scheme, corresponding to five concentrations (0, 0.5, 1.0, 1.5, and 2.0), two biocontrol agents (Trichoderma sp. and S. cerevisiae), and an additional treatment composed of a fungicide (Captan®). Seed health, germination, and emergence tests were conducted to confirm the hypotheses. The microbiolization of seeds is efficient in reducing both incidence and initial growth of fungi in organic cotton cv. Aroeira. The appraised biocontrol agents proved to be superior to the chemical treatment regarding the initial seedling growth. Trichoderma sp. is the most effective agent and provides a high initial seedling growth and a significant reduction in fungal incidence.

Index terms: Gossypium hirsutum, biocontrol agents, initial growth, biological control, growth stimulation.

Microbiolização de sementes de algodoeiro orgânico com Trichoderma sp. e Saccharomyces cerevisiae

RESUMO: A microbiolização de sementes é uma alternativa na substituição dos defensivos químicos para o tratamento de sementes na agricultura orgânica. Assim, objetivou-se avaliar o crescimento inicial e o controle de fungos associados a sementes de algodoeiro orgânico, através da microbiolização de sementes com Trichoderma sp. e Saccharomyces cerevisiae. Sementes certificadas da cultivar Aroeira, cujo línter havia sido removido com ácido sulfúrico, foram analisadas em delineamento inteiramente casualizado em arranjo fatorial de 5 x 2 + 1, com cinco concentrações (0; 0,5; 1,0; 1,5 e 2,0), dois agentes biocontroladores *Corresponding author (Trichoderma sp. e S. cerevisiae) e o fungicida como tratamento adicional (Captan®). Os E-mail: [email protected] testes de sanidade, germinação e emergência das sementes foram avaliados para confirmar as hipóteses. A microbiolização das sementes é eficiente na redução da incidência de Received: 9/25/2019. 3/18/2020. fungos e no crescimento inicial de plântulas de algodoeiro orgânico cv. Aroeira. Os agentes Accepted: biocontroladores avaliados são superiores ao tratamento químico no crescimento inicial de plântulas. Trichoderma sp. é o agente mais eficaz, proporcionando alto crescimento inicial de plântulas e redução significativa da incidência de fungos. 1Departamento de Fitotecnia e Ciências Ambientais, Universidade Termos para indexação: Gossypium hirsutum, biocontroladores, crescimento inicial, controle Federal da Paraíba, Caixa Postal 66, biológico, promoção de crescimento. 58397-000 – Areia, PB, Brasil. Journal of Seed Science, v.42, e202042021, 2020 2 J. M. F. L. Cruz et al.

INTRODUCTION

One of the biggest obstacles to organic production is finding alternatives for controlling diseases that have as similar effectiveness as the usual agrochemicals. Among the limitations of such production system when applied to cotton culture, Kikuti et al. (2002) point out the difficulty in obtaining seeds with high physical, physiological, and sanitary qualities. These aspects can guarantee the establishment of crops with plant stands that are uniform, vigorous, and free from diseases, directly reflecting on the final yield. In organic cotton cultivation, as in many other cultures, the seeds are the most efficient object for survival and dissemination of several phytopathogens of economic importance. Therefore, the seeds can introduce such microorganisms in former clean areas, thus establishing the initial cycle of the disease and permanently infesting the field (Silva Flávio et al., 2014). Among the pathogenic fungi associated with cotton seeds, the most important are Fusarium oxysporum f. sp. vasinfectum, Colletotrichum gossypii, C. g. var. cephalosporioides, Rhizoctonia solani, Alternaria spp., Aspergillus spp., Penicillium spp. By affecting seed quality directly, they can cause the death of seedlings before and after emergence, leading to loss of vigor, reduction in germination, and rotting. In this context, performing seed microbiolization with biocontrol agents as part of the integrated disease management is a valuable strategy for organic agriculture, due to its potential for controlling pathogenic microorganisms. The biocontrol agents help to minimize production losses related to phytopathogens through multiple natural processes, such as mycoparasitism, antibiosis, competition, and induced resistance (Xu et al., 2011). Considering the biocontrol agents, Trichoderma spp. is the most researched and studied fungal genus worldwide. It is considered a key biocontrol microorganism due to its active antagonism against several phytopathogens, which can positively influence seed germination and vegetative growth (Saito et al., 2009). Yeasts have also been used as biocontrol agents, as they are part of the epiphytic and endophytic microbiota, and compete with phytopathogenic agents, through several antagonistic mechanisms (Mello et al., 2011). Among them, research has drawn attention to the enormous biocontrol potential of the genus Saccharomyces spp. (Heling et al., 2017). Therefore, the microbiotization of seeds emerges as a feasible alternative for controlling phytopathogens in organic production systems. Nevertheless, studies are still on demand to contrast the effect of these biocontrol agents with agrochemicals traditionally used in seed treatment. On that account, the present work aimed at evaluating the initial growth and control of fungi associated with organic cotton seeds (cv. Aroeira), after their microbiotization with Trichoderma sp. and Saccharomyces cerevisiae.

MATERIAL AND METHODS

The experiment was carried out in the Laboratory of Phytopathology (LAFIT) of the Department of Plant Sciences and Environmental Sciences at the Federal University of Paraíba (UFPB) — city of Areia, state of Paraíba, Brazil. Certified organic cotton seeds of the cultivar Aroeira were provided by the Brazilian Agricultural Research Corporation

(EMBRAPA). First, they had the linter removed though wet sulfuric acid delinting (H2SO4). Next, they were analyzed in compliance with a completely randomized design, in a 5 x 2 + 1 factorial scheme, corresponding to five concentrations, two biocontrol agents, and the fungicide (additional treatment). The biocontrol agents tested were the yeast Saccharomyces cerevisiae and the fungus Trichoderma sp. The former came from the commercial dry yeast Blaupan®, whose instantaneous formula is based on S. cerevisiae. In turn, the isolate of Trichoderma sp. (CMLT005) belonged to the microorganism collection of the LAFIT — UFPB, and it had been first obtained from seeds of Leucaena leucocephala. The selection of this isolate was based on previous studies. For the experiments, pure cultures of the fungus were reactivated and multiplied in a potato-dextrose-agar medium (PDA). They were maintained inside an incubation chamber for eight days, at 25 ± 2 °C, and a 12-hour photoperiod. The action of seed microbiotization on fungal incidence was appraised by the blotter test (Brasil, 2009) using 200

Journal of Seed Science, v.42, e202042021, 2020 Microbiolization of organic cotton seeds 3 seeds. They were divided into 10 replications of 20 units, which were individually distributed on Petri dishes (Ø 15 cm), following aseptic conditions. Each dish contained a double-layer filter paper that was previously sterilized and moistened with 10 mL sterile distilled water (SDW). The inoculation of the biocontrol agents was done in suspension for five minutes. Trichoderma sp. suspensions were attained by adding 10 mL of SDW in each Petri dish containing the pure colonies. Then, with the aid of a soft brush, the conidia were released and filtered through a sterilized double gauze. The final conidial concentration was determined in a Neubauer chamber set at 0.5, 1.0, 1.5, and 2.0 x 107 conidia.mL-1. The suspension of S. cerevisiae was concocted by weighing the biological product in an analytical balance (0.001 g accuracy), and then diluting it in SDW to the concentrations of 0.5, 1.0, 1.5, and 2.0 g.L-1. The control treatment comprised seeds submerged in a 1% sodium hypochlorite solution for three minutes. For comparison purposes, an additional treatment was performed, with the application of the fungicide Captan® at the dose of 240 g of the product per 100 kg of seeds. The dishes were incubated in a growth chamber, Biochemical Oxygen Demand type (BOD), for eight days, at 25 ± 2 °C temperature, and a 12-hour photoperiod. Ultimately, an optical microscope and specialized literature (Seifert et al., 2011) were used to identify and determine the incidence of fungi in the seeds. The results were expressed in percentage of fungal occurrence. The initial seedling growth was assessed through the germination and emergence tests, performed on four replications of 50 microbiolized seeds, according to Brasil (2009). The emergence test was conducted in a greenhouse, and the seeds were sown in plastic trays dimensioned 66 x 33 x 6 cm, containing washed, previously sterilized sand. These sets were watered twice a day. For the germination test, the seeds were distributed on sterilized germitest paper sheets, which were moistened at 2.5 times the dry paper weight. After sowing, the papers were wrapped in rolls, put inside transparent plastic bags, and then placed in a BOD germination chamber, set at 25 ± 2 °C and a 12-hour photoperiod. The number of seed germinated and emerged (cotyledon above the ground) were recorded daily from the 4th to the 12th day after sowing — the germination test considered both the normal seedlings (root and hypocotyl) and the abnormal ones. The results were expressed in percentage. The first germination and emergence counts were done together with the tests, which allowed to calculate the germination and emergence speed indices (GSI and ESI, respectively), according to the formula proposed by Maguire (1962). The length and dry mass of seedlings were assessed in both tests individually. With a digital caliper (0.001 mm accuracy), the length of the shoot and root of the seedlings was gauged, and the results were delivered in centimeters. Subsequently, shoots and roots were separately packed in Kraft paper bags, and then taken to a forced air convection oven set at 65 °C for 48 h. After that time, having the samples reached a constant weight, the dry mass of each seedling part was weighed in an analytical balance (0.001 g accuracy), and the results were expressed in grams. The statistical analysis was handled with the software R (R Core Team, 2019), and the results were submitted to analysis of variance and polynomial regression, in which the linear and quadratic models were tested. The mean values of each biocontrol agent were contrasted at each concentration level by the F-test (p ≤ 0.05), and the treatments were compared with the fungicide by the Dunnett’s test (p ≤ 0.05). The values of fungus incidence were transformed in (√x + 1) beforehand.

RESULTS AND DISCUSSION

Table 1 reports the effect of the biocontrol agents on fungal incidence. The following fungus genera were identified: Alternaria sp., Aspergillus sp., Chaetomium sp., Cladosporium sp., Colletotrichum sp., Fusarium sp., Macrophomina sp., Nigrospora sp., and Phoma sp. The high occurrence of phytopathogenic fungi in the organic cotton seeds evidences that the treatments were crucial for minimizing the damages these microorganisms might cause in all culture phases. As stated by Machado et al. (2012), the primary advantage of employing biocontrol agents is their contribution to a steadier regulation of diseases. Over the years, this can alter the agroecosystem equilibrium, making it inhospitable for pathogen development, without causing, however, significant impacts on the overall environment.

Journal of Seed Science, v.42, e202042021, 2020 4 J. M. F. L. Cruz et al.

Table 1. Incidence of fungi associated with organic cotton seeds treated with Trichoderma sp. (TCH) and Saccharomyces cerevisiae (SCH).

Fusarium sp. (%) Cladosporium sp. (%) Chaetomium sp. (%) Concentration TCH SCH TCH SCH TCH SCH (107 conidia.mL-1) (g.L-1) (107 conidia.mL-1) (g.L-1) (107 conidia.mL-1) (g.L-1) 0 52 a* 52 a* 33 a* 33 a* 20 a* 20 a* 0.5 7 a* 3 b 2 a 3 a 0 a 0 a 1.0 4 a 2 a 0 a 1 a 0 a 0 a 1.5 0 a 2 a 0 a 2 a 0 a 0 a 2.0 0 a 1 a 0 a 1 a 0 a 0 a Captan® 0 a 0 a 0 a 0 a 0 a 0 a CV (%) 25.80 25.09 34.44 Aspergillus sp. (%) Nigrospora sp. (%) Phoma sp. (%) Concentration TCH SCH TCH SCH TCH SCH (107 conidia.mL-1) (g.L-1) (107 conidia.mL-1) (g.L-1) (107 conidia.mL-1) (g.L-1) 0 42 a* 42 a* 13 a* 13 a* 11 a* 11 a* 0.5 23 b* 34 a* 0 a 0 a 0 a 0 a 1.0 10 b* 23 a* 0 a 0 a 0 a 0 a 1.5 5 b 21 a* 0 a 0 a 0 a 0 a 2.0 5 b 12 a* 0 a 0 a 0 a 0 a Captan® 0 a 0 a 0 a 0 a 0 a 0 a CV (%) 31.37 21.73 23.52 Macrophomina sp. (%) Colletotrichum sp. (%) Alternaria sp. (%) Concentration TCH SCH TCH SCH TCH SCH (107 conidia.mL-1) (g.L-1) (107 conidia.mL-1) (g.L-1) (107 conidia.mL-1) (g.L-1) 0 10 a* 10 a* 10 a* 10 a* 9 a* 9 a* 0.5 0 a 0 a 0 a 0 a 0 a 0 a 1.0 0 a 0 a 0 a 0 a 0 a 0 a 1.5 0 a 0 a 0 a 0 a 0 a 0 a 2.0 0 a 0 a 0 a 0 a 0 a 0 a Captan® 0 a 0 a 0 a 0 a 0 a 0 a CV (%) 28.12 23.52 22.77

Means followed by the same letter in the row do not differ statistically, according to the F-test (p ≤ 0.05); and *: mean statistically differs from the commercial fungicide, according to Dunnett’s test (p ≤ 0.05). Means transformed in √x + 1. CV: Coeffcient of variation.

The microbiolization with Trichoderma sp. (TCH) and S. cerevisiae (SCH), at the levels tested, proved to be efficient at lowering the incidence of the identified fungus genera. Except for Fusarium sp. and Aspergillus sp., all concentrations proved to be as successful as the agrochemical. Although the antagonistic effect of the yeast can be attributed to the competition for nutrients, an antibiosis relationship might occur with the fungi. Such relation, based on chemical interference, may include the production of enzymes that affect the fungal cell wall, such as glucanases. Antibiosis can also occur as a result of predatory activity, in which case the yeast produces extracellular glycolipids or glycoproteins with fungicidal or fungistatic activity (Golubev, 2006).

Journal of Seed Science, v.42, e202042021, 2020 Microbiolization of organic cotton seeds 5

S. cerevisiae at 0.5 g.L-1 produced a greater decrease in Fusarium sp. incidence in comparison to Trichoderma sp. at 0.5 x 107 conidia.mL-1. The latter treatment also performed worse than the commercial product. Differently, the other concentrations acted similarly to the agrochemical, and the bioagents significantly reduced the pathogenic fungus alike. As for the control of Aspergillus sp., Trichoderma sp. was significantly more efficient than S. cerevisiae. In fact, at 1.5 and 2.0 x 107 conidia.mL-1, the biocontrol fungus caused 88% reduction, a rate similar to the agrochemical. Trichoderma sp. can exert a biocontrol activity directly (through predation or production of growth inhibitors) or indirectly. The latter case happens when the colonized plants have high endogenous levels of auxins, ethylene, gibberellins, plant enzymes, antioxidants, solutes, and compatible compounds, such as phytoalexins and phenols. These substances furnish them with tolerance to induced biotic and abiotic stresses (López-Bucio et al., 2015). All concentrations tested of Trichoderma sp. and S. cerevisiae decreased by 100% the incidence of Alternaria sp., Chaetomium sp., Colletotrichum sp., Macrophomina sp., Nigrospora sp., and Phoma sp. Regarding the initial growth variables, neither the emergence nor the germination and emergence speed indices showed significant alteration with the application of the biocontrol agents. In the first germination count (FGC), no significant difference was observed between the biocontrol agents at the levels tested. However, for Trichoderma sp., this rate gradually increased, as the concentrations increased. Particularly, at 2.0 x 107 conidia.mL-1, it even surpassed the chemical treatment and scored 98.5% (Figure 1A).

SCH (g.L-1) TCH (107 conidia.mL-1) A SCH (g.L-1) TCH (107 conidia.mL-1) B 100 80 a* 98 75 a* a* a* a 96 a a 70 a* a* a a 94 a 65 a* a* b* 92 60

FEC (%) FEC b* FGC (%) FGC 90 55 88 ŷ( ) = 89.20 + 5.00x R² = 90 % 50 ŷ( ) = 69.43 + 7.19x - 4.14x² R² = 26 % a ŷ( ) = 88.36 + 11.97x - 4.29x² R² = 85 % ŷ( ) = 71.72 - 19.55x + 9.50x² R² = 98 % 86 45 0 0.5 1 1.5 2 Captan® 0 0.5 1 1.5 2 Captan®

C SCH (g.L-1) TCH (107 conidia.mL-1) D SCH (g.L-1) TCH (107 conidia.mL-1) 105 30 a* 100 a a a 25 95 a a b 20 ŷ( ) = 25.34 - 41.47x + 15.29x² R² = 93 % 90 b ŷ( ) = 26.63 - 52.75x + 25.50x² R² = 97 % 85 15 NS (%) 80 AS (%) 10 75 5 a 70 ŷ( ) = 69.33 + 48.39x - 18.14x² R² = 95 % a a a* ŷ( ) = 68.60 + 58.70x - 29.00x² R² = 95 % a b 65 0 a 0 0.5 1 1.5 2 Captan® 0 0.5 1 1.5 2 Captan® Concentrations Concentrations Means accompanied by the same letter, at a given concentration, do not differ statistically according to the F-test (p ≤ 0.05); and *: mean statistically differs from the commercial fungicide, according to Dunnett’s test (p ≤ 0.05). Figure 1. Results of first germination count (FGC), first emergence count (FEC), normal seedlings (NS), and abnormal seedlings (AS) obtained in the germination test of organic cotton seeds treated with Trichoderma sp. (TCH) and Saccharomyces cerevisiae (SCH).

Journal of Seed Science, v.42, e202042021, 2020 6 J. M. F. L. Cruz et al.

All levels of both Trichoderma sp. and S. cerevisiae exhibited values of first emergence count (FGC) significantly higher than the fungicide. Also, when the control agents were contrasted, only the concentrations of 1.0 and 1.5 x 107 conidia.mL-1 of the fungus showed a statistical difference from the yeast. The highest (76.5%) and lowest (61%) average values of FGC corresponded to 1.0 x 107 conidia.mL-1 of Trichoderma sp. and 1.0 g.L-1 of S. cerevisiae, respectively (Figure 1B). In the analyses of normal (NS) and abnormal (AS) seedlings, the concentrations of both Trichoderma sp. and S. cerevisiae attained effects similar to the chemical product — except for the control treatment, which presented the lowest NS and the highest AS values (67.5% and 27.5%, respectively) (Figures 1C and 1D). As their concentrations increased, the biocontrol agents exhibited meaningful differences between them, as for the production of NS (Figure 1C). Considering the formation of AS, only S. cerevisiae 1.5 g.L-1 showed a significant difference in comparison with Trichoderma sp. at 1.5 x 107 conidia.mL-1 (Figure 1D). Overall, the concentrations of Trichoderma sp. showed attribute numbers statistically superior to those of S. cerevisiae. The only exception concerns the shoot dry mass obtained in germination (SDMG), in which the fungus at 2.0 x 107 conidia.mL-1 was no different from the yeast at 2.0 g.mL-1. Moreover, the biocontrol agents, at all concentrations tested, performed better than the conventional agrochemical (Figure 2). The vegetative growth response triggered by Trichoderma spp. has been acknowledged, as these microorganisms are capable of solubilizing phosphate and other minerals, making them available to plants, and also producing auxin

SCH (g.L-1) TCH (107 conidia.mL-1) A SCH (g.L-1) TCH (107 conidia.mL-1) B 25 14 12 a* a* a* 20 a* a* a* a* a* 10 b* 15 8 b* b* b* b* b* b* 10 b* 6 a SLE (cm) SLE SLG (cm) SLG a* 4 5 ŷ( ) = 2.89 + 2.85x - 3.16x² R² = 97 % ŷ( ) = 9.76 + 15.46x - 6.57x² R² = 93% 2 ŷ( ) = 6.98 + 7.61x - 0.85x² R² = 95 % ŷ( ) = 9.23 + 5.27x - 1.268x² R² = 99 % 0 0 0 0.5 1 1.5 2 Captan® 0 0.5 1 1.5 2 Captan®

SCH (g.L-1) TCH (107 conidia.mL-1) C SCH (g.L-1) TCH (107 conidia.mL-1) D 0.15 0.12 a* a* ) )

a* 1 1

- a* - a* 0.12 a* 0.10 a* 0.08 a* 0.09 b* b* b*

b* seedling seedling b* 0.06 b* b* a* 0.06 a 0.04 ŷ( ) = 0.08 + 0.11x - 0.05x² R² = 96% a ŷ( ) = 0.04 + 0.10x - 0.04x² R² = 99 % 0.03 0.02

SDMG (g. SDME (g. ŷ( ) = 0.08 - 0.04x - 0.02x² R² = 94 % ŷ( ) = 0.04 + 0.06x - 0.03x² R² = 93 % 0.00 0.00 0 0.5 1 1.5 2 Captan® 0 0.5 1 1.5 2 Captan® Concentrations Concentrations Means accompanied by the same letter, at a given concentration, do not differ statistically according to the F-test (p ≤ 0.05); and *: mean statistically differs from the commercial fungicide, according to Dunnett’s test (p ≤ 0.05). Figure 2. Shoot length found in the germination (SLG) and emergence (SLE) tests and shoot dry mass obtained in the germination (SDMG) and emergence (SDME) tests of organic cotton seedlings treated with Trichoderma sp. (TCH) and Saccharomyces cerevisiae (SCH).

Journal of Seed Science, v.42, e202042021, 2020 Microbiolization of organic cotton seeds 7 analogs (Vinale et al., 2008). The performance of the plant can also be enhanced as a result of the release of plant growth regulators by yeasts (El-Tarabily and Sivasithamparam, 2006), including indole-3-acetic acid (IAA), indole-3- pyruvic acid (IPA), gibberellins, and polyamines (El-Tarabily and Sivasithamparam, 2006; Cloete et al., 2009). The Trichoderma sp. suspension at 1.5 x 107 conidia.mL-1 delivered the highest values of shoot length in germination (SLG; 11.35 cm) and shoot dry mass in both germination (SDMG; 0.097 g) and emergence (SDME; 0.132 g) (Figures 2B, 2C, and 2D). However, the concentration of 2.0 x 107 conidia.mL-1 was responsible for the highest shoot length in the emergence (SLE; 18.17 cm) (Figure 2A). Regarding the variables related to root development, Trichoderma sp. proved to be more effective than the agrochemical in all concentrations tested (Figure 3). This effect was equally observed in S. cerevisiae for the attributes root length (RLG) and root dry mass (RDMG), both in germination (Figures 3B and 3D). According to López-Bucio et al. (2015), the radicular development prompted by Trichoderma sp. is connected with a series of compounds released by the mycelium. Some of these elements stimulate root branching, thus improving the absorption of nutrients and water, which ultimately leads to root growth. Another beneficial factor is the exudation of substances, such as siderophores and organic acids, which increase the availability of nutrients (Zhao et al., 2014). The RLG and RLE (root length in germination and emergence, respectively) allowed to verify that, at all concentrations, Trichoderma sp. caused a significantly better development than S. cerevisiae (Figures 3A and 3B). Regarding the RDME (root dry mass in the emergence), the concentrations of Trichoderma sp. also performed statistically better than S.

SCH (g.L-1) TCH (107 conidia.mL-1) A SCH (g.L-1) TCH (107 conidia.mL-1) B 18 12 a* a* a* a* 15 a* 10 a* a* a* 12 b* b* 8 b* b* b* 9 6 b*

b* (cm) RLG RLE (cm) RLE 6 b 4 a* a* ŷ( ) = 4.63 + 4.91x - 2.03x² R² = 99 % 3 ŷ( ) = 6.67 + 14.03x - 5.58x² R² = 97% 2 ŷ( ) = 4.43 + 10.06x - 4.14x² R² = 95 % ŷ( ) = 6.50 - 1.07x + 3.22x² R² = 94 % 0 0 0 0.5 1 1.5 2 Captan® 0 0.5 1 1.5 2 Captan®

SCH (g.L-1) TCH (107 conidia.mL-1) C SCH (g.L-1) TCH (107 conidia.mL-1) D 0.04 a* 0.04

a* ) ) a* a* 1 1 a* - - a* a* 0.03 0.03 a* b* b b b a* b* b* 0.02 a 0.02 (g.seedling (g.seedling

0.01 0.01 a ŷ( ) = 0.02 + 0.01x - 0.002x² R² = 92% ŷ( ) = 0.01 + 0.03x - 0.01x² R² = 97 % RDME ŷ( ) = 0.02 - 0.02x + 0.01x² R² = 95% RDMG ŷ( ) = 0.01 + 0.02x - 0.01x² R² = 93 % 0 0.00 0 0.5 1 1.5 2 Captan® 0 0.5 1 1.5 2 Captan® Concentrations Concentrations Means accompanied by the same letter, at a given concentration, do not differ statistically according to the F-test (p ≤ 0.05); and *: mean statistically differs from the commercial fungicide, according to Dunnett’s test (p ≤ 0.05). Figure 3. Root length in germination (RLG) and emergence (RLE) tests and root dry mass in germination (RDMG) and emergence (RDME) of organic cotton seedlings treated with Trichoderma sp. (TCH) and Saccharomyces cerevisiae (SCH). Journal of Seed Science, v.42, e202042021, 2020 8 J. M. F. L. Cruz et al. cerevisiae. However, the treatments at 1.5 x 107 conidia.mL-1 of the fungus and 1.5 g.L-1 of the yeast led to a similar result (Figure 3C). The RDMG showed a gradual accumulation of mass, as the concentrations of S. cerevisiae increased. Moreover, at 2.0 g.L-1, the yeast surpassed the treatment with 2.0 x 107 conidia.mL-1 of the fungus, reaching an RDMG value of 0.03 g (Figure 3D). An increasing number of studies indicate that the growth of roots can be enhanced directly or indirectly by yeasts existing in the rhizosphere (El-Tarabily and Sivasithamparam, 2006; Cloete et al., 2009). Besides, organic and inorganic fertilizers, made up by a combination of yeasts, are capable of reestablishing the sustainability of ecosystems, as well as boosting the productivity of farmlands. The suspension of Trichoderma sp. at 1.5 x 107 conidia.mL-1 resulted in the most expressive values of RLG (11.10 cm), RLE (14.75 cm), RDMG (0.034 g), and RDME (0.035 g). Nevertheless, at 1.5 g.L-1, S. cerevisiae also provided a high RDME (0.036 g).

CONCLUSIONS

Seed microbiolization is effective at diminishing both incidence and initial growth of fungi in organic cotton seedlings cv. Aroeira. The biocontrol agents considered in this study perform better than the chemical treatment, regarding the initial seedling growth. Trichoderma sp. is the most effective agent, as it provides high initial seedling growth and a significant decrease in fungal occurrence.

REFERENCES

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Journal of Seed Science, v.42, e202042021, 2020 Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Sowing dates and densities on physiological potential of Journal of Seed Science, v.42, e202042023, 2020 seeds of white oat cultivars http://dx.doi.org/10.1590/ 2317-1545v42232390 José Henrique Bizzarri Bazzo1* , Klever Márcio Antunes Arruda2 , Inês Cristina de Batista Fonseca1 , Claudemir Zucareli1

ABSTRACT: The aim of this study was to evaluate the physiological potential of seeds of white oat cultivars grown at different sowing dates and densities. Two independent experiments were conducted with two different sowing dates in a randomized block experimental design in a 4 × 2 factorial arrangement, with four replications. The treatments consisted of four sowing densities (180, 240, 300, and 360 viable seed.m-2) and two cultivars (IPR Afrodite and IPR Artemis). The following evaluations were made: thousand seed weight, germination percentage, first germination count, seedling length, seedling dry matter, emergence speed index, and seedling emergence in sand. Joint analysis of variance was carried out on the data regarding sowing dates separately for the cultivars. The mean values for sowing dates were compared by the F test; and polynomial regression analysis up to the second degree at 5% probability was conducted on the densities. The seeds produced by the plants grown from the first sowing date had better vigor than those produced by plants from the later sowing date. The increase in sowing density reduces the weight and vigor of the seeds produced by plants from the second sowing date. For the IPR Afrodite and IPR Artemis cultivars, the use of 180 seed.m-2 in the first growing period allows production of seeds with greater physiological potential.

Index terms: plant population, Avena sativa L., growing environments, vigor, germination.

Épocas e densidades de semeadura no potencial fisiológico de sementes de cultivares de aveia branca

RESUMO: Objetivou-se avaliar o potencial fisiológico de sementes de cultivares de aveia branca cultivadas em diferentes épocas e densidades de semeadura. Dois experimentos independentes foram conduzidos em duas épocas de semeadura, utilizando-se o delineamento experimental de blocos casualizados em esquema fatorial 4 x 2, com quatro repetições. Os tratamentos constaram de quatro densidades de semeadura (180, 240, 300 e 360 sementes viáveis.m-2) e duas cultivares (IPR Afrodite e IPR Artemis). Foram avaliados: massa de mil sementes, geminação, *Corresponding author primeira contagem da germinação, comprimento e massa seca de plântula, índice de velocidade E-mail: [email protected] de emergência e emergência de plântulas em areia. Os dados foram submetidos à análise de variância conjunta para épocas de semeadura, separadamente para as cultivares. As médias de Received: 12/19/2019 Accepted: 03/02/2020 épocas foram comparadas pelo teste F e as de densidades submetidas à análise de regressão polinomial até segundo grau, a 5% de probabilidade. As sementes produzidas na primeira época de semeadura apresentam melhor vigor. O aumento da densidade de semeadura reduz a massa e o vigor das sementes produzidas na segunda época de cultivo. Para as cultivares IPR Afrodite 1Departamento de Agronomia, e IPR Artemis, a utilização de 180 sementes.m-2, na primeira época de semeadura, possibilita a Universidade Estadual de Londrina (UEL), Caixa Postal 6001, 86057-970 – produção de sementes de melhor potencial fisiológico. Londrina, PR, Brazil. Termos para indexação: população de plantas, Avena sativa L., ambientes de cultivo, vigor, 2Instituto Agronômico do Paraná, germinação. 86047-902 – Londrina, PR, Brazil. Journal of Seed Science, v.42, e202042023, 2020 2 J. H. B. Bazzo et al.

INTRODUCTION

Seed quality is one of the main factors that affect establishment and performance of crops, and it is related to the sum of the genetic, physical, physiological, and health aspects that affect the capacity of seeds to give rise to high- yielding plants (Marcos-Filho, 2015). Physiological potential is related to the ability of the seed in performing its vital functions, bringing together information regarding seed germination and vigor (Carvalho and Nakagawa, 2012). The quantity and quality of seeds produced can be maximized by sowing at periods of the year that favor the growth, development, and yield performance of the plant. This management practice is characterized by changing the relationships among the meteorological elements available to the crop during its cycle (Silva et al., 2011; Tafernaberri- Júnior et al., 2012; Bornhofen et al., 2015). This plant growing strategy combines the different phenological stages of the crop with the environmental conditions most favorable to the plant, which has a positive impact on the production and distribution of assimilates and, consequently, on seed yield and quality (Toledo et al., 2009). Understanding the relationship between time elements of the crop environments and the yield performance of cultivars is essential for producing seeds of superior quality at satisfactory yield levels (Gomes et al., 2012; Silva et al., 2014). According to Caron et al. (2017), the characterization of phenological modifications that occur in plants as a result of contrasting sowing dates is important for defining the adoption of crop practices that assist in taking better advantage of environmental conditions and in maximizing the yield of better quality seeds from each sowing/growing period. One of the important management techniques that can be combined with the temporal aspect of crop seasons, aiming to increase seed yield and physiological performance, is sowing density (Carvalho and Nakagawa, 2012; Abati et al., 2017), since it modifies inter- and intraspecific competition for resources from the growth medium and, consequently, the number and composition of the seeds (Almeida and Mundstock, 2001). Variations in sowing densities in the oat crop modify the number of tillers (Almeida and Mundstock, 2001) and bring about significant modifications in yield components (Schuch et al., 2000a). In addition, sowing density leads to morphophysiological changes in plants (Zagonel et al., 2002), which can affect their growth and development and, consequently, seed yield and quality. Costa et al. (2013) report that cultivars can differ in capacity to produce tillers, in their cycle, and in seed yield potential. Thus, the effort of establishing appropriate sowing density should also take the genotypes and the sowing/ growing period into consideration, to reduce competition and promote an increase in seed yield and seed quality from the cultivars (Freitas et al., 2010; Tavares et al., 2014). Thus, the sowing densities associated with the genotype and with the sowing/growing period can have an effect on yield components, such as number of tillers, number of panicles, and the number of seeds per area, with a direct effect on plant potential for seed filling and, consequently, the weight and number of seeds per panicle. Therefore, appropriate management can result in larger and heavier seeds that may have a greater amount of reserves, which would affect their physiological performance. Thus, the aim of this study was to evaluate the physiological potential of seeds of white oat cultivars grown at different sowing dates and densities.

MATERIALS AND METHODS

Two independent experiments, with two different sowing dates (May 5 and June 24), were carried out in Londrina, PR, at the Experimental Station of the Instituto Agronômico do Paraná (IAPAR) in a Latossolo Vermelho eutroférrico at 23°23’S and 51°11’W, at an altitude of 610 m. Climate in the region, according to the Köppen classification is type Cfa, described as humid subtropical with hot summers, few frosts, and a tendency for concentration of rains in the summer months, though without a defined dry season. Rainfall and temperature data were obtained from records of the IAPAR meteorological stations (Figure 1).

Journal of Seed Science, v.42, e202042023, 2020 Sowing dates and densities on physiological potential of oat seeds 3

P1 P2 H1 40 H2 90.00

35 75.00 30

C) 60.00 ƒ 25

20 45.00

Temperature (

Rainfall (mm)

Temperature (°C) Temperature 15 30.00 10 15.00 5

0 0.00 5/1 5/16 5/31 6/15 6/30 7/15 7/30 8/14 8/29 9/13 9/28 10/13 10/28 PeriodPeriod ofof the yearyear P1: seedling emergence period 1 (May, 13); P2: seedling emergence period 2 (July, 1); H1: harvest period 1 (September, 23); H2: harvest period 2 (October, 21). Figure 1. Daily data of maximum and minimum temperatures (lines) and rainfall (bars) for the period of conducting the experiments.

The chemical characteristics of the soil at the depths of 0–10 cm and 10–20 cm, determined before setting up the -3 3+ -3 2+ -3 experiment, were as follows: 0–10 cm - pH (CaCl2) 4.80, 5.76 cmolc.dm H + Al , 4.42 cmolc.dm Ca , 1.56 cmolc.dm 2+ -3 + -3 -3 Mg , 0.35 cmolc.dm K , 36.3 mg.dm P, and 19.09 g.dm organic matter; and for 10-20 cm - pH (CaCl2) 4.90, 5.76 -3 3+ -3 2+ -3 2+ -3 + -3 -3 cmolc.dm H + Al , 4.57 cmolc.dm Ca , 1.52 cmolc.dm Mg , 0.27 cmolc.dm K , 15.1 mg.dm P, and 16.59 g.dm organic matter. The trials were conducted using the cultivars IPR Afrodite (medium cycle, moderate resistance to lodging, medium stature, released in 2012 by IAPAR) and IPR Artemis (medium cycle, moderate resistance to lodging, medium statute, released in 2016 by IAPAR). In both experiments, at the two sowing dates, a randomized block experimental design was used in a 4 × 2 factorial arrangement, with four replications. Treatments consisted of four sowing densities (180, 240, 300, and 360 viable seed.m-2) and two cultivars (IPR Afrodite and IPR Artemis). The plots were composed of six 5-meter- length rows at a spacing of 0.17 m between rows; and an area of 5.1 m2 was used for data collection. The experiments were conducted under a conventional soil management system in an area previously planted to soybean. The chemical characteristics of the soil of the experimental area were the basis for calculation of base mineral -1 -1 fertilization in the planting furrow, constant for all the treatments, which was 20 kg.ha N, 60 kg.ha P2O5, and 20 -1 -1 kg.ha K2O, using 200 kg.ha of the formulation 10-30-10. The plant health treatments for disease control and other crop treatments were carried out according to need and recommendations for the crop. Seeds were harvested upon reaching maturity for harvest, a stage characterized by hardening of the caryopsis, plants with a dry appearance, and seeds with moisture below 20%. The following evaluations were carried out for determination of seed physiological potential: Thousand seed weight: obtained through counting and weighing of eight replications of one hundred seeds of oats per plot. The mean of these values was multiplied by ten to obtain the value of thousand seed weight (Brasil, 2009). Germination percentage: performed with eight replications of fifty seeds in Germitest® paper towel moistened with distilled water in the amount of 2.5 times the weight of the substrate. The rolls of paper towel were kept in a seed

Journal of Seed Science, v.42, e202042023, 2020 4 J. H. B. Bazzo et al. germinator at a temperature of 20 °C. Evaluation consisted of two counts, at five (first count) and at ten (final count) days after setting up the test, and then calculating the percentage of normal seedlings (Brasil, 2009). Seedling length: performed by sowing four replications of twenty seeds in the upper third of the sheet of Germitest® paper, moistened with distilled water at the rate of 2.5 times the weight of the dry paper. The rolls of paper containing the seeds remained for five days in a seed germinator at a temperature of 20 °C, at which time the length of normal seedlings was evaluated using a millimeter ruler (Nakagawa, 1999). Results were expressed in centimeters per seedling. Seedling dry matter: the normal seedlings, coming from the test of seedling length, were placed in paper bags and then in a forced air circulation laboratory oven regulated to a temperature of 80 °C to constant weight (Nakagawa, 1999). Dry matter was evaluated and results were expressed in mg.seedling-1. Seedling emergence in sand: performed in a greenhouse with four replications of fifty seeds per treatment, sown at a depth of 3 cm. The sand used was previously washed and then placed in plastic trays. Moisture was maintained through irrigation according to need. Evaluation of the number of normal seedlings that emerged was performed on the fifteen day after sowing (Nakagawa, 1999). Seedling emergence speed index: performed together with the seedling emergence in sand test through daily counts of the number of normal seedlings emerged, up to stabilization of emergence, according to the formula proposed by Maguire (1962). The data were analyzed for normality and homogeneity of errors, and then joint analysis of variance was performed for sowing dates separately for the cultivars. The mean values from sowing dates were compared by the F test, and the mean values of densities were analyzed through second order polynomial regression, at 5% probability.

RESULTS AND DISCUSSION

For the IPR Afrodite cultivar, there was an effect of interaction between the sowing date and sowing density factors for the thousand seed weight and seedling dry matter traits. For the variables first germination count, seedling length, and emergence speed index, an isolated effect of sowing date was found. Only the first germination count exhibited a significant isolated effect of sowing densities. No significant effect of sowing dates, densities, and interaction between the factors was found for the germination percentage and seedling emergence in sand variables. For the IPR Artemis cultivar, a significant interaction was found between the sowing date and sowing density factors for the thousand seed weight, first germination count, seedling length, and seedling dry matter traits. An isolated effect of sowing/growing periods was found only for the emergence speed index. A significant effect of sowing dates, sowing densities, and interaction between the factors was not found for the germination percentage and seedling emergence in sand variables. Rainfall during the cycle of plants grown from the first sowing date was 622.70 mm, whereas in the second sowing date it was 336.80 mm (Figure 1). In the later growing period, the amount of rainfall was below the minimum required by the crop, with the period of most accentuated water restriction during the vegetative growth phase. However, for both sowing dates, the rainfall distribution was uneven, which may have altered the agronomic performance and, possibly, seed formation and physiological potential. One thousand seed weight of the IPR Afrodite and IPR Artemis cultivars in the first sowing date (period 1) did not exhibit an effect of sowing density. In the second sowing date (period 2), this trait fit quadratic equations, with minimum values at 264 seed.m-2 for IPR Afrodite and 258 seed.m-2 for IPR Artemis (Figures 2A and 2B). For both cultivars, at all sowing densities, period 1 resulted in production of seeds with greater weight. This can be explained by the better meteorological conditions (better rain distribution and milder temperatures) during the vegetative growth and development phase of the plants grown in this period (Figure 1), thus allowing them to accumulate a greater amount of dry matter, increasing seed weight. Caron et al. (2017), working with two maize hybrids (Dekalb 240 and Dow AgroSciences 2A106) and six sowing

Journal of Seed Science, v.42, e202042023, 2020 Sowing dates and densities on physiological potential of oat seeds 5

dates (9/15, 9/30, 10/15, 10/30, 11/16, and 12/3) in Frederico Westphalen, Brazil, also found that both cultivars had lower thousand seed weight when sown later. Similar results were found also for the rice crop, in which Venske et al. (2016) compared the effect of two sowing dates (10/18 and 11/9) on the yield components and seed yield of the cultivar IRGA 424 and found that thousand seed weight decreased in the second growing period. In relation to sowing density, results similar to those found for thousand seed weight of the IPR Afrodite and IPR Artemis cultivars were obtained by Almeida et al. (2003), who worked with four oat cultivars (UFRGS 14, UFRGS 18, UPF 16, and UPF 17), five plant densities (50, 185, 320, 455, and 590 plant.m-2) and two crop years (1998 and 1999). They found that, in 1998, thousand seed weight of the cultivars UPF 17, UFRGS14, and UFRGS 18 did not exhibit a significant effect for plant density, and found that the same variable for the UPF 16 cultivar fit a quadratic equation with a minimum value of 390.90 plant.m-2. A study performed by Tavares et al. (2014), with the aim of evaluating the response of wheat genotypes (PF 014384, BRS Tangará, and BRS Pardela) grown at different sowing densities (150, 250, 350, and 450 viable seed.m-2) in Londrina, PR, and Ponta Grossa, PR, in two crop years in regard to yield components and seed yield, also exhibited results similar to those obtained in the present study for seed weight. In that study, the authors concluded that thousand seed weight was not affected by sowing densities in either location in the first year.

A

B

Figure 2. Thousand seed weight of seeds from the white oat cultivars IPR Afrodite (A) and IPR Artemis (B) grown under different sowing dates and sowing densities.

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In contrast, in Ponta Grossa and Londrina in the second year, they found that a quadratic equation fit thousand seed weight, as a result of the increase in the number of seeds per unit area. Although seed germination of the IPR Afrodite and IPR Artemis cultivars did not exhibit an isolated effect or an effect of interaction among the factors analyzed, in this study, it nevertheless resulted in a percentage of normal seedlings above the standard established for sale of oat seeds by the Brazilian Ministry of Agriculture (Ministério da Agricultura, Pecuária e Abastecimento - MAPA) in all the treatments, a standard which requires minimum germination of 80%. Results for the IPR Afrodite cultivar showed that period 1 favored first germination count of seeds (Table 1), just as observed by Venske et al. (2015) who evaluated the effect of two sowing dates (10/18 and 11/19) on the physiological quality of rice seeds produced in Capão do Leão, Brazil, and also found that the percentage of normal seedlings in first germination count was higher in the first growing period. In relation to sowing density, the first germination count of seeds of the IPR Afrodite cultivar, regardless of the sowing date, fit a quadratic equation with a minimum value of 279.78 seed.m-2 (Figure 3A). In this case, it is noteworthy that the amplitude of change in the number of normal seedlings found in the first germination count was low among the sowing densities. For the IPR Artemis cultivar, the first germination count, of seed germination, in period 1, was not significantly altered by the increase in sowing density. However, in period 2, this trait fit a decreasing linear function in response to increase in seed.m2 (Figure 3B). At the density of 180 seed.m-2, there was no significant difference between period 1 and period 2. However, at the densities of 300 and 360 seed.m-2, the second plant growth period exhibited lower values for the trait evaluated, through the decreasing linear function. Different results were obtained in studies conducted by Salau et al. (2017) and Schuch et al. (2000b), who evaluated the effect of different populations of plants on the physiological potential of barley and black oat seeds, respectively. They found no effect from the increase in the number of plants per unit area on seed vigor expressed through the first germination count test. Seedling length for the IPR Afrodite cultivar was greater for seeds coming from period 1 (Table 1). This result corroborates that found by Venske et al. (2015) who, working with rice growing, also found that sowing at a later date caused a reduction in seed quality, shown by lower performance of seeds in the seedling length test. The IPR Artemis cultivar did not have a significantly different response to the increase in seed.m-2 in terms of the length of seedlings coming from its seeds produced in period 1. However, for the seeds coming from period 2, this trait fit a decreasing linear function as a response to an increase in seed.m-2 (Figure 4). At the density of 180 seed.m-2, there was no significant difference between period 1 and period 2 for the variable analyzed. However, for the other seed densities, period 1 led to production of seeds that produced seedlings of greater length.

Table 1. Mean values of first germination count (FGC), seedling length (SL), and emergence speed index (ESI) of the seeds from two oat cultivars grown under two different sowing dates (Period 1: May 5; Period 2: June 24).

IPR Afrodite Sowing date/growing period FGC (%) SL (cm) ESI (%) Period 1 99 A 16.52 A 10.44 A Period 2 97 B 15.18 B 9.84 B CV (%) 1.95 7.24 3.75 IPR Artemis Sowing date/growing period ESI (%) Period 1 10.45 A Period 2 9.87 B CV (%) 3.87 Mean values followed by the same letter in the column do not differ from each other by the F test (p < 0.05).

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Dry matter of seedlings coming from seeds of the IPR Afrodite cultivar produced in period 1 did not show a significant effect from sowing density. However, for seeds from period 2, this variable fit a quadratic equation with a minimum value of 284 seeds.m-2 (Figure 5A). The densities of 180 and 360 seed.m-2 did not lead to a significant difference between period 1 and period 2 for the variable in question. However, seeds from the densities of 240 and 300 seed.m2 in period 1 showed higher values for seedling dry matter.

A

B

Figure 3. First germination count of seeds from the white oat cultivar IPR Afrodite (A) grown under different sowing densities, and IPR Artemis (B) grown under different sowing dates and sowing densities.

Figure 4. Length of seedlings from seeds from the white oat cultivar IPR Artemis grown under different sowing dates and sowing densities. Journal of Seed Science, v.42, e202042023, 2020 8 J. H. B. Bazzo et al.

A

B

Figure 5. Dry matter of seedlings from seeds from the white oat cultivars IPR Afrodite (A) and IPR Artemis (B) grown under different sowing dates and sowing densities.

For the IPR Artemis cultivar, the dry matter of seedlings coming from seeds grown in period 1 and 2 fit increasing linear equations in response to the increase in sowing density (Figure 5B). The densities of 180 and 300 seed.m-2 did not exhibit significant difference between period 1 and period 2 for the variable in question. However, the densities of 240 and 360 seed.m-2 in period 1 led to greater and lower oat seedling dry matter, respectively. Results that contrast with those found in the present study regarding the effect of sowing dates on the results of seedling dry matter were obtained by Venske et al. (2015), who found that shoot and root dry matter of rice seedlings were not different for seeds grown in different periods. Regarding the response of this same variable to sowing density, Salau et al. (2017), working with four populations of barley plants (44, 66, 88, and 110 plant.m-2) in Pelotas, RS, found that shoot dry matter was not modified by an increase in plant.m-2; however, they found that root dry matter fit a quadratic equation with minimum value at the density of 70 plant.m-2. The seedling emergence speed index for seeds from both cultivars was greater for seeds grown in period 1 (Table 1). However, for seedling emergence in sand, there was no significant effect from growing periods, densities, and interaction between the factors for either of the cultivars analyzed in the present study. The results obtained show that delay in sowing of both cultivars caused reduction in seed quality. Among the meteorological elements, those of most prominence and that seem to explain the lower physiological performance of seeds as a result of later sowing were rainfall and air temperature, especially during the crop vegetative growth period (Figure 1). In that period, lower rainfall and higher temperatures favored accelerated vegetative development, which

Journal of Seed Science, v.42, e202042023, 2020 Sowing dates and densities on physiological potential of oat seeds 9 resulted in contraction of the cycle of plants sown/grown in period 2, reducing their growth. These morphophysiological changes may have limited yield capacity and distribution of photoassimilates of the plants, affecting the development and accumulation of reserves in the seeds and, consequently, their quality. Temperatures rose to beyond 32 °C during the final phase of seed maturation of the plants grown in period 2. According to Marcos-Filho (2015), higher temperature, reaching values higher than 30 °C during the seed development period, can cause severe losses in seed yield and quality. The same author affirms that these losses are related to the significant reduction in filling time and in the photosynthetic rate after flowering. In addition, Castro et al. (2012) report that in winter cereal crops, temperatures greater than or equal to 32 °C during one or more days in the maturation phase stop seed development and lead to early seed maturation. This situation may also have contributed to reduction in the vigor of seeds produced in period 2. In this same context, high temperatures during maturation also lead to reduction in translocation of photoassimilates to the seeds. Under these conditions, maturation is “forced”, producing low vigor seeds, because natural deposition of carbohydrates, lipids, and proteins is limited (Castro et al., 2012). Another factor that may have contributed to improve the physiological performance of seeds produced by the IPR Afrodite e IPR Artemis cultivars in period 1 was the greater weight of these seeds (Figures 2A and 2B). According to Carvalho and Nakagawa (2012), seeds of greater weight usually have well-formed embryos with more reserves, and thus potentially have greater vigor. Albrecht et al. (2008) report that the choice of the sowing date is the crop factor that by itself most affects plant development, crop yield, and seed quality. In this respect, Marcos-Filho (2015) affirms that the effect of the environment on seed development is mainly reflected in variations in the size, weight, and physiological quality of the seeds, which is confirmed by the results obtained in the present study. The response of seed physiological potential in relation to the increase in seed sowing density varied considerably between the IPR Afrodite and IPR Artemis cultivars. According to Marcos-Filho (2015), the physiological response of seeds is based on the genotype, as a consequence of genetic and/or morphophysiological traits, which make them more or less susceptible to damage during the formation period or after physiological maturity of the seeds. However, the interaction between the genetic potential of the cultivar and crop management techniques can result in changes in the expression of yield components, yield, and seed quality (Silva et al., 2015). Just as in the present study, variations in seed physiological quality as a result of modifications in the sowing density of the plants that produced them were also found by Salau et al. (2017), Barbieri et al. (2013), and Schuch et al. (2000b) working with barley, wheat, and black oat crops, respectively. In general, increase in sowing density for both cultivars, though especially in the second growing period, resulted in little change or in reduction of the vigor of the seeds the cultivars produced. Greater competition among oat plants for resources from their environment as a result of the greater number of seeds per area may explain the results obtained in these cases. In this regard, the increase in sowing density in association with worse meteorological conditions during the cycle of plants grown in period 2 led to losses in the traits related to seed vigor. This study shows that the potential for producing quality seeds can be optimized by growing plants in periods that favor plant growth and development through adequate choice of sowing time, a management practice characterized by the changes it effects in the relationships among the meteorological elements available to the crop throughout its cycle. According to Ramos et al. (2013), this strategy allows different phenological stages of the crop to occur at times in which the climate conditions are more favorable for the plant, which has a positive effect on production of quality seeds. Furthermore, an increase in sowing density reduces the vigor of the seeds produced by the IPR Afrodite and IPR Artemis cultivars in the later growing period. Thus, combined use of the results of the various tests that evaluated seed vigor in the present study confirms the possibility of using 180 seed.m-2 in setting up the crop in period 1, which would ensure production of high quality seeds and would allow reduction in the quantity of seeds used.

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CONCLUSIONS

The two sowing dates exhibit potential for production of oat seeds, resulting in seeds with germination that meets the standards for commercialization of seeds of the species. The seeds produced by plants grown in the first plant growing period exhibit greater vigor than those produced by plants grown in the later growing period. The increase in sowing density reduces the weight and vigor of seeds produced in the second growing period. For the IPR Afrodite and IPR Artemis cultivars, the use of 180 seed.m-2 in the first growing period allows production of seeds with greater physiological potential.

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Journal of Seed Science, v.42, e202042023, 2020 Journal of Seed Science ISSN 2317-1545 NOTE www.abrates.org.br/revista

Osmotic treatment, growth regulator and rooter inTabebuia Journal of Seed Science, v.42, e202042022, 2020 roseoalba (RIDL.) Sandwith seeds for direct sowing http://dx.doi.org/10.1590/2317- 1545v42226945 Alexandre Carneiro da Silva1* , Maiara Pilar Palmeira da Silva3 , Rayssa Zamith4 , Gustavo Galetti4 , Fatima Conceição Márquez Piña-Rodrigues2

ABSTRACT: Direct seeding is a technology that reduces the costs of forest restoration projects and favors species which are difficult to establish for seedlings. The seeds osmotic treatment to accelerate and standardize germination and induce tolerance to environmental stresses may favor seedling establishment in field through direct sowing and contribute to the greater efficiency of this technique. With the purpose of favor seed germination and seedling establishment under direct seeding conditions in the field, Tabebuia roseoalba osmoprimed seeds in polyethylene glycol (PEG) solution and unconditioned seeds were treated with isolated and/or combined solutions of plant growth regulators (PGR) and rooting (RTG). These seeds were submitted to germination test and evaluated for percentage germination rate, germination speed index and normal seedlings, and to seedling emergence test by direct field seeding. RTG had a toxic effect on T. roseoalba seeds. Osmoconditioning induced stress tolerance by RTG during germination and on seedling establishment. PGR treatment favors seedling emergence in field conditions and alleviates the toxicity effect caused by RTG. These treatments have great potential for use in direct sowing of T. roseoalba seeds.

Index terms: osmopriming, phytohormones, forest restoration, seedling establishment, stress tolerance.

*Corresponding author: E-mail: [email protected] Tratamento osmótico, reguladores de crescimento e enraizador em sementes de Tabebuia roseoalba (RIDL.) Sandwith para semeadura direta Received: 8/1/2019. Accepted: 2/21/2020. RESUMO: A semeadura direta é uma tecnologia que reduz os custos de projetos de restauração florestal e favorece espécies de difícil estabelecimento por mudas. O tratamento osmótico de sementes para acelerar, uniformizar e induzir tolerância a estresses ambientais durante a 1Instituto Federal de Educação, germinação pode favorecer o estabelecimento de plântulas em campo, por meio da semeadura Ciência e Tecnologia do Acre, 69970- direta e contribuir para a maior eficiência dessa técnica. Com o objetivo de favorecer a germinação 000 – Tarauacá, AC, Brasil. de sementes e o estabelecimento de plântulas em condições de semeadura direta em campo, Tabebuia roseoalba sementes de osmocondiocionadas em solução de polietilenoglicol (PEG) 2Universidade Federal de São e sementes não condicionadas foram tratadas com soluções isoladas e/ou combinadas de Carlos, Departamento de Ciências reguladores de crescimento vegetal (PGR) e de enraizantes (RTG). Essas sementes foram Ambientais, 18052-780 – Sorocaba, submetidas ao teste de germinação e avaliadas quanto à porcentagem de germinação, índice de SP, Brasil. velocidade de germinação e plântulas normais e ao teste de emergência de plântula, por meio de semeadura direta em campo. O RTG teve um efeito tóxico nas sementes de T. roseoalba. 3Universität zu Köln, Botanisches O osmocondicionamento induziu tolerância ao estresse por RTG durante a germinação e o Institut – 50674 Köln, NRW, estabelecimento de plântulas. O tratamento com PGR favorece a emergência de plântulas em Germany. condições de campo e alivia o efeito da toxicidade causada pelo RTG. Esses tratamentos têm grande potencial de uso na semeadura direta de sementes de T. roseoalba. 4Universidade Federal de São Carlos, Programa de Pós-Graduação em Termos para indexação: osmocondicionamento, fitormônios, restauração florestal, Ciências Ambientais, 13565-905 – estabelecimento de plântulas, tolerância a estresse. São Carlos, SP, Brasil.

Journal of Seed Science, v.42, e202042022, 2020 2 A. C. Silva et al.

INTRODUCTION

Seedling establishment is one of the most critical and vulnerable processes for plant life and often represents a challenge after emerging from the protected environment of the seed (Gommers and Monte, 2018). It is a critical step for the regeneration of plant populations, because of the common high mortality rates during this stage (Grubb, 1977; Harper, 1977; Clark et al., 1999). In plant species with a wide distribution, the populations are usually subjected to different biotic and abiotic conditions and, therefore, to environments with contrasting limitations on the seedling establishment (Castro et al., 2004). Tabebuia roseoalba (Ridl.) Sandwith (Bignoniaceae) is an endangered tropical tree species, with a wide geographic distribution in South America. In Brazil, it is mostly found in Seasonal Semideciduous Atlantic Forest and in Savanna (Feres et al., 2012). It has large commercial, medicinal and ornamental value, with widespread use in urban forestry and plant recovery of degraded areas. Their seeds, currently classified as orthodox, displays a strong variation in quality during storage and a short period of viability, which hampers its natural dispersal as well as the development of cropping techniques for this tree (Lorenzi, 2009). Reforestation usually falls into three main categories: assisted natural regeneration, direct seeding, and planting seedlings by tubestock (Greening Australia Victoria, 2003). The choice between different restoration strategies depends in part on a compromise between effectiveness and cost (Prieto-Rodao et al., 2019). In recent years, direct seeding of native trees has been used as a low-cost alternative for restoration (Engel and Parrotta, 2001; Wallin et al., 2009; Ceccon et al., 2016). Direct seeding involves applying seed propagules directly into the soil where reforestation is required without first germinating seeds off-site (Schirmer and Field, 2000; Summers et al., 2015). Direct seeding is considered a feasible alternative for large-scale forest restoration (Freitas et al., 2019). It has as advantages the reducing costs in the implementation of forest restoration projects by exempting the use of nurseries, providing successful establishment of species with difficulty to produce seedlings, growing in definitive location, and reducing losses of sensitive species to transplant shock (Pancel, 1993; Ferreira et al., 2007; Cole et al., 2011). However, the chances of survival of the plant in direct seeding are less than planting seedlings, because they are unprotected against lethal agents controlled in the greenhouse stage (Smith, 1986). Osmopriming is a pre-sowing treatment defined as the controlled hydration of seeds by exposure to osmotic solutions up to a limit that allows pre-germination metabolic events to proceed without visible protrusion of the radicle (Heydecker and Coolbear, 1977; Bradford, 1986; Jisha et al., 2013). This treatment improves germination efficiency and seedling emergence under adverse environmental conditions as drought (Wang et al., 2003; Jisha et al., 2013) and salinity (Sivritepe et al., 2003; Jisha et al., 2013), and improve the resistance or tolerance to elevated temperatures (Ligterink et al., 2007). Plant growth regulators (PGRs) were used to improve seeds germination, seedling establishment and induce tolerance to diverse environmental stress in these stages and during plant growth (Riefler et al., 2006; Wen et al., 2010; Peleg and Blumwald, 2011; Shinohara et al., 2017). PGRs were associated with micronutrients, which act primarily as enzyme catalysts by seed coating, seeking higher germination values and better establishment of plants in the field (Silva et al., 2008). The aim of this work was to evaluate osmopriming combined with the use of plant growth regulators, rooting and seed coating with polymers in the treatment of Tabebuia roseoalba seeds to favor seed germination and the establishment of seedlings under direct sowing conditions in the field.

MATERIAL AND METHODS

Freshly harvested T. roseoalba seeds from forest fragment located in the municipality of Promissão, São Paulo State, were used. The water content in wet basis and a mass of one thousand seeds were obtained (Brasil, 2009). During the experiment the seeds were stored at 5 °C in hermetic plastic packaging. The osmopriming was performed by seeds imbibition in osmotic polyethylene glycol (PEG) 6000 solution with hydric Journal of Seed Science, v.42, e202042022 2020 Tabebuia roseoalba seeds treatment for direct sowing 3 potential (ψw) of -0.8 MPa at 25 °C, calculated according to the equation proposed by Michel and Kaufmann (1973), during 18 h. The choice of hydric potential and treatment time was determined based on previous imbibition curves established with 0.0 MPa (deionized water) and -0.8 MPa (PEG solution) at 25 °C. Each curve’s point was obtained of three independent replicates of fifteen seeds, seeded in Petri dishes (9 cm diameter), between two sheets of paper, moistened with 12 mL of water or PEG solution with the photoperiod of sixteen hours of light. After different imbibition times, the seeds were weighed (0.0001 g precision) to obtain the wet mass (gravimetric method), and the water content was calculated. After osmopriming, the seeds were washed in running water to remove the PEG residues and placed to dry for 72 h in controlled atmosphere of 42% RH, at 25 °C (Buitink et al., 2003; Silva et al., 2017). Osmoprimed and unprimed seeds were submitted to treatments with plant growth regulators (PGRs) (0.009% kinetin, 0.005% gibberellic acid, 0.005% indolbutyric acid) and/or rooting (RTG) (1.5% water soluble nitrogen, 6% organic carbon, 0.2 % boron). The following treatments were established: i – control (without PGRs or RTG); ii – RTG; iii – PGRs; iv – RTG + PGRs. For each treatment, 2 mL of PRGs and/or 2 mL of RTG were used, and 1 mL of a polymer (Polifix G5®) and water were added until the final solution volume of 10 mL was complete. The proportion of 10 mL of the final solution per 100 g of seed was applied. After that, the seeds and solutions were placed in a plastic bag and mixed by gentle shaking for three minutes, and then the seeds were left to dry at room temperature for 24 hours. The germination test was performed by four replicates of thirty seeds seeded in a roll of germinating paper, moistened with 2.5 times the volume of water in relation to the weight of the dry paper, at 25 °C. The percentage of germination (G%) and normal seedlings (NS%) was evaluated. The seeds were considered germinated with root protrusion equal to or greater than 1 mm. Seedlings exhibiting well-formed primary root and hypocotyl, cotyledons and leaves were classified as normal. From the daily germination count, the germination speed index (GSI) was calculated according to Maguire (1962). The direct seedling was conducted in March 13th, 2016, in an experimental area (23,5944S and 47,5234W UTM), in Sorocaba, São Paulo State. The area was a pasture covered by Brachiaria decumbens L., totally removed by manual weeding one week prior to the experiment. The soil was uniform with Eutrophic Red Latosol (LVe). The climatic conditions during the test are shown in Figure 1.

Source: Brazilian National Institute of Meteorology (BRASIL, 2016). Figure 1. Minimum temperature (T. min), maximum temperature (T. max), relative air humidity (RU) and precipitation (Prec). Sorocaba, São Paulo, Brazil. March 2016. Journal of Seed Science, v.42, e202042022, 2020 4 A. C. Silva et al.

Four experimental blocks were established, represented by four beds of 2 x 3 m, 70 cm apart. In each block eight lines of 3 m spaced at 20 cm were established, in which one replication of thirty seeds for each treatment was distributed. The seeds were seeded every 10 cm, at a depth of 1 cm. The lines were covered with Brachiaria grass straw, 5 cm high (Silva et al., 2015). At fifteen days after seeded, when the emergence of seedlings ceased, the number of emerged seedlings above the straw was counted, and the percentage of seedling emergence (SE%) was calculated. For the G%, NS% and GSI analyzes, a completely randomized experimental design was adopted. For the SE%, the randomized block design was used. Treatments were evaluated by a factorial arrangement of 2 (osmoprimed and unprimed seeds) x 4 (3 treatments + control), with four replicates per treatment. The data normality was evaluated using the Shapiro-Wilk’s test, and analysis of variance (ANOVA) was performed. The means were compared by the Tukey test (p < 0.05). All analyses were performed using software R (2015).

RESULTS AND DISCUSSION

The water seeds content found in the work lot was 9.4%, while the mass of one thousand seeds was 0.6897g.seed-1. The beginning of germination was observed after 41 hours of imbibition in water. Seeds imbibed in osmotic solution did not germinate and maintained water content without significant variation after reaching phase II (Figure 2). There was no significative differences in G%, GSI, NS% and SE% among osmoprimed seeds. For unprimed seeds, the G%, NS% and GSI of the control (untreated seed) was higher than the seeds treated with PGRs and with RTG, while for SE% the best result was observed for seeds treated with PGRs and PGRs + RTG (Figure 3). There was no difference in G% between unprimed and osmoprimed seeds when treated with PGRs or with PGRs + RTG. For untreated seeds (control), the osmopriming reduced G% and GSI, whereas for seeds treated with RTG, the osmopriming provided a higher G% and GSI. The NS% differed when treated with PGRs + RTG, while the SE% differed only when treated with PGRs, with best results for unprimed seeds (Figure 3). According to imbibition curves (Figure 2), the imbibition time of the seed in osmotic solution (-0.8 MPa) during 18 h meets the requirements for application of the osmopriming technique. In this condition, the imbibition of the seeds in osmotic solutions of enough hydric potential is low enough to prevent germination, but allows to occur some pre- germinating physiological and biochemical processes (Heydecker and Coolbear, 1977; Bradford, 1986; Bewley et al., 2013).

-0.8 MPa 0.0 MPa

Figure 2. Imbibition curves of T. roseoalba seeds in water (0.0 MPa) and osmotic solution (-0.8 MPa) at 25 °C.

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Considering that RTG treatment in unprimed seeds resulted in lower G%, GSI%, NS% and SE% in relation to untreated seeds (control), it is possible to infer that the rooting treatment had a toxic effect on T. roseoalba seeds, in the concentration and form utilized in this study. In favorable conditions, the osmopriming did not benefit seed germination, since for untreated seeds (control) the G% was higher in unprimed seeds (Figure 3A). However, assuming that rooting had a toxic effect on seed germination, it follows that the osmotic conditioning induced tolerance to stress caused by rooting treatment, once the G% and GSI for seeds treated with RTG were higher in seeds previously submitted to osmopriming (Figures 3A and 3B). During the osmopriming, the seeds are subjected to hydric stress (Ashraf et al., 2018), which produces a memory mediated by proteins and transcription factors (Bruce et al., 2007), activating networks of physiological and metabolic mechanisms for defense against the stress initially imposed. Thus, there may be an tolerance induction to other types of stresses (Genoud and Métraux, 1999; Kranner et al., 2010). Seeds unprimed treated with RTG + PGRs showed the best results compared to osmoprimed seeds treated only with RTG for all avaliations (Figure 3), indicating that PGRs treatment alleviate the effect to rooting toxicity. Due to the potential of the osmopriming to induce stress tolerance during germination and seedling establishment, and because Seedlings emergence (%) Seedlings emergence

Means ± SDs, n = 4. Different capital letters represent significant differences at (p < 0.05) unprimed and osmoprimed seeds, submitted to the same treatment. Lowercase letters represent significant differences at (p < 0.05) between unprimed or osmoprimed seeds, submitted to different treatments. Figure 3. Germination (A), germination speed index (B), normal seedlings (C), and seedlings emergence (D) of T. roseoalba unprimed and osmoprimed seeds, treated with plant growth regulators (PGRs) and rooting (RTG). Journal of Seed Science, v.42, e202042022, 2020 6 A. C. Silva et al. the PGRs treatment increased the seedling emergence and also alleviated the toxicity effect of rooting treatment observed in this study, there is great potential for use of the osmopriming and PGRs in seeds treatment for direct seedling of T. rosealba. The present study provides a basis for the development of new researches to improve the techniques of osmotic conditioning, including variation in potential osmotic of the solution, time of imbibition in osmotic solution and temperature associated or not to PGRs treatments, using different doses per mass of seeds for the direct seedling of T. roseoalba in forest restoration.

CONCLUSIONS

The RTG had an apparent toxic effect on the seeds. Osmopriming induces stress tolerance to toxicity during T. roseoalba seed germination. PGR treatment accelerates germination and induces greater seedling emergence, favoring seedling establishment under field conditions.

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SILVA, T.T.A.; VON PINHO, E.V.R.; CARDOSO, D.L.; FERREIRA, C.A.; ALVIM, P.O.; COSTA, A.A.F. Qualidade fisiológica de sementes de milho na presença de bioestimulantes. Ciência e Agrotecnologia, v.32, n.3, p.840-846, 2008. http://dx.doi.org/10.1590/S1413- 70542008000300021

SIVRITEPE, N.; SIVRITEPE, H.O.; ERIS, A. The effects of NaCl priming on salt tolerance in melon seedling grown under saline conditions. Scientia Horticulturae, v.97, n.3-4, p.229-237, 2003. https://doi.org/10.1016/S0304-4238(02)00198-X

SMITH, D.M. The practice of silviculture. 8. ed. New York: John Wiley, 1986. 610p.

SUMMERS, D.M.; BRYAN, B.A.; NOLAN, M.; HOBBS, T.J. The costs of reforestation: a spatial model of the costs of establishing environmental and carbon plantings. Land Use Policy, v.44, p.110-121, 2015. https://doi.org/10.1016/j.landusepol.2014.12.002

WALLIN, L.; SVENSSON, B.M.; LÖNN, M. Artificial dispersal as a restoration tool in meadows: sowing or planting? Restoration Ecology, v.17, n.2, p.270-279, 2009. https://doi.org/10.1111/j.1526-100X.2007.00350.x

WANG, W.; VINOCUR, B.; ALTMAN, A. Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance. Planta, v.218, p.1-14, 2003. https://doi.org/10.1007/s00425-003-1105-5

WEN, F.; ZHANG, Z.; BAI, T.; XU, Q.; PAN, Y. Proteomics reveals the effects of gibberellic acid (GA3) on salt-stressed rice (Oryza sativa L.) shoots. Plant Science, v.178, n.2, p.170-175, 2010. https://doi.org/10.1016/j.plantsci.2009.11.006

This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Journal of Seed Science, v.42, e202042022 2020 INSTRUCTIONS TO AUTHORS

1. SCOPE AND POLICY From 2017 the Journal of Seed Science (JSS) will circulate on-line version only. Original scientific studies and communications, not yet published or submitted to another journal for publication and written in Portuguese or English, will be accepted for publication. For manuscripts submitted in English, the authors should provide an adequated version. The SCIENTIFIC COMMUNICATION is a category of scientific manuscript which describes a technique, an equipment, new species or observations and surveys of limited results. It has the same scientific rigor as the “Scientific Articles” and the same value as a publication. The classification of a manuscript as a SCIENTIFIC COMMUNICATION is based on its content and scientific merit but it can be a preliminary study, simple and not definitive on a certain subject, with publication justified by its uniqueness and contribution to the area. The Editorial Board of the JSS may invite leading authors of recognized reputation to compose specific Review Articles covering topics of their specialization that will convey to the scientific community the state-of-the-art knowledge related to the specific theme.

Its aims are: - To publish original articles in thematic areas relevant to Seed Science and Technology; - To publish articles that significantly contribute towards the knowledge of this area, featuring a scientific stance and in-depth knowledge on themes and trends within Seed Science and Technology; - To provide a strict evaluation policy with articles sent for publication; each manuscript is evaluated by two reviewers who are carefully selected from the scientific community. The acceptance of article is the result of the revisers’ assessment and of the editors concerned; - To maintain a high ethical behavior with regard to the review and its collaborators; - To maintain strict quality in scientific article to be published.

The articles will be published in the order they are approved. The Editorial Committee will make a preliminary evaluation of the manuscript submitted and accept it for publication or not, according to the journal’s policy and criteria of relevance. After initial acceptance, the EDITOR will designate an ASSOCIATE EDITOR (of the area), who will proceedv to edit with the help of at least two SCIENTIFIC ASSESSORS of JSS, who has the same prerogatives to accept the manuscript for publication or not. The whole editorial process will be accompanied by the authors, assessors or associate editors, through the use of an access code (login) and password, given at the beginning of the submission process. The data, opinions and concepts emitted in the articles, as well as the accuracy of the bibliographic references, are the entire responsibility of the author(s). The eventual quoting of products and commercial brands does not mean that ABRATES recommends their use. However, the EDITOR, with the help of the Editorial Committee and the Scientific Assessors, reserves the right to suggest or request advisable or necessary changes.

Creative Commons Licence All content published by the Journal of Seed Science is licensed by Creative Commons Attribution 4.0 International. This license allow others to share, remix, tweak and create from your article, even for commercial purposes, and although the new articles have to award it due credit, users do not have to license these derivative articles under the same terms.

Open Access Policy The JSS provides immediate open access to its content on the principle that scientific knowledge offered freely to the public, supports a greater global democratization of knowledge.

Publication Fees The payment of an article publication fee is obligatory for all authors, including members of ABRATES (one of the authors should be member), and will be as follows: For members of ABRATES - Up to six diagrammed pages in final form: US$ 10.00 per page + US$ 27.00 per additional page. For non-members - Up to six diagrammed pages in final form: US$ 20.00 per page + US$ 54.00 per additional page. In the case of more than one author, including members, the total value will be divided between the number of authors, but only the members whose dues are paid up-to-date will have a 50% discount of their part. Payment of a submission or administrative fee will no longer be necessary.

For readers: There is not any fees for readers.

2. PREPARATION OF MANUSCRIPT The advice given in these instructions should be followed in full by the author(s). The manuscripts should be organized into a SHORT TITLE (centrally placed at the beginning of the first page), TITLE (in english), AUTHORS, ABSTRACT (up to 200 words), TITLE (in portuguese), RESUMO (abstract in Portuguese - up to 200 words), INTRODUCTION, MATERIAL AND METHODS, RESULTS AND DISCUSSION, CONCLUSIONS, ACKNOWLEDGEMENTS AND REFERENCES. The ABSTRACT and the RESUMO must also contain no more than five “Index Terms” (“Termos para indexação”), which are not cited in the title. The following norms should be adhered to when preparing the manuscript: The manuscripts should be typed in Word text editor (DOC or RTF) in numbered lines (up to 30 lines per page) with double spacing and 2cm margins, with Times New Roman 14 font for the title and 12 for the text, without the inclusion of tables and figures, which will be annexed at the end of the manuscript. The figures should be in programs compatible with WINDOWS, such as EXCEL, and the image format: Figures (GIF or TIFF) and Photos (JPEG) with 300 dpi resolution. The manuscript should not be longer than 20 pages, including figures, tables and references. Manuscripts longer than 20 pages will be returned. The writing of the manuscripts should be concise, objective and clear, written impersonally in the past, except for the conclusions which should be written in the present tense. Paragraphs will not be permitted in the ABSTRACT or the RESUMO nor the presentation of data in columns or tables or the inclusion of bibliographic references. The full name(s) of the author(s) should be mentioned immediately below the title. The corresponding author has to be identified by an asterisk and an e-mail must be provided. The notes should be placed on the foot of the page, which has the respective superscripted number of each author indicating his/her institutional affiliation and address - Department or Section, Institution, Post office box, Zip code, Municipality and Country. A maximum of six (6) authors per article is recommended.

Text Citations:the citations of authors in the text will be by the surname with only the first letter as a capital, followed by the year of publication. In the case of two authors, both surnames will be included, separated by “and”; if there are more than two authors, only the first will be cited, followed by “et al.”. In the case of the citation of two or more publications of the same author(s), published in the same year, they should be identified by small letters (a,b,c, etc.), placed immediately after the year of publication.

References: sixty percent (60%) of the references should have been listed on the database ISI Web of Knowledge, Scopus or SciELO during the last 10 years. Citations from theses, dissertations, monographs, proceedings or annals of congress, abstracts, and magazines will not be accepted.

Avoid: - excessive citations of textbooks; - obsolete citations and informative magazines which are not scientific. Citations of recent articles published in the JSS can be accessed at the site: www.scielo.br/jss The references should be given in alphabetical order by the surname of the first author, without numbering; mention all the authors of the article separated by “;”. Follow the norms of the ABNT NBR6023. The references should have hyperlinks to permit access to any Web page on the internet. With the cursor placed on the desired spot of a text or spreadsheet, type the address of the page e.g.:www.abrates.org.br and press the space bar. The hyperlink will be automatically created. Position the cursor on one of the letters of the hyperlink which was created, press Shift F10 to open the menu, move the arrow down until the option to open the hyperlink, press enter and the page will open.

Some examples are shown as follows:

Journal Articles: (it will not be necessary to mention where the journal was published) LIMA, L.B.; MARCOS-FILHO, J. Condicionamento fisiológico de sementes de pepino e germinação sob diferentes temperaturas. Revista Brasileira de Sementes, v.32, n.1, p.138-147, 2010. http://www.scielo.br/scielo.php?script=sci_ pdf&pid=S0101-31222010000100017&lng=en&nrm=iso&tlng=pt

OLIVEIRA, A.S.; CARVALHO, M.L.M.; NERY, M.C.; OLIVEIRA, J.A.; GUIMARÃES, R.M. Seed quality and optimal spatial arrangement of fodder radish. Scientia Agricola, v.68, n.4, p.417-423, 2011. http://www.scielo.br/scielo.php?script=sci_ pdf&pid=S0103-90172011000400005&lng=en&nrm=iso&tlng=en

Books: MARCOS-FILHO, J. Fisiologia de sementes de plantas cultivadas. 2 ed. Londrina: ABRATES, 2015. 660p.

BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Regras para análise de sementes. Ministério da Agricultura, Pecuária e Abastecimento. Secretaria de Defesa Agropecuária. Brasília: MAPA/ACS, 2009. 395p. http:// www.agricultura. gov.br/arq_editor/file/2946_regras_analise__sementes.pdf

Book Chapter: VIEIRA, R.D.; KRZYZANOWSKI, F.C. Teste de condutividade elétrica. In: KRZYZANOWSKI, F.C.; VIEIRA, R.D.; FRANÇA-NETO, J.B. (Ed.).Vigor de sementes: conceitos e testes. Londrina: ABRATES, 1999. p.4.1-4.26.

Laws, Decrees, Directives: Country or State. Law, Decrees or Directives n. ..., of (month) (day), (year). Diário Oficial da União, place of publication, (month) (day), (year). Section ..., p. ...

BRASIL. Medida provisória nº 1.569-9, de 11 de dezembro de 1997. Diário Oficial da República Federativa do Brasil, Poder Executivo, Brasília, DF, 14 dez. 1997. Seção I, p.29514.

Technical Report: FRANÇA-NETO, J.B.; HENNING, A.A.; COSTA, N.P. Estudo da deterioração da semente de soja no solo. In: Resultados de Pesquisa de Soja, 1984/85. Londrina, 1985. p.440-445. (EMBRAPA-CNPSo. Documentos, 15).

Electronic Documents: BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. SNPC - Lista de cultivares protegidas. http://extranet. agricultura.gov.br/php/proton/cultivarweb/cultivares_protegidas.php Accessed on: Jan. 13th 2010. Tables The tables in the “picture” format numbered with arabic numerals, should be headed by a self-explanatory title, with small letters, and vertical lines should not be used to separate the columns.

Figures The figures (graphs, drawings, maps or photographs) should be numbered with arabic numerals in programs compatible with WORD FOR WINDOWS,(GIF or TIFF) inserted in the text preferentially as an object. The drawings and photographs should be digitalized in high quality (JPEG 300 dpi) and sent in the same size as they will be published in the journal. The legends typed immediately below the figure and initiated with the word Figure, should be followed by their respective number and text in small letters.

Units of Measurement: Should be typed with a space between the number and the unit. Examples: 10 oC, 10 mL, µS.cm-1.g-1. The percentage symbol should be next to the numeral without any space. E.g.: 10%.

3. SUBMISSION OF MANUSCRIPTS Start the submission process reviewing in full the Instructions for Authors to ensure that the article is in agreement with JSS standards. The submission of manuscripts to the JSS should be done exclusively on -line in the site http://www.scielo.br/ jss clicking on . The manuscript file should not be greater than 1.5 Kb. Besides this, a document with the signature and agreement of all the authors to submit and/ or publish the manuscript in the JSS, and delegating the rights to translation into English (see letter format on the site), should be sent by mail ([email protected]).

Authors of articles in the journal Journal of Seed Science profit from the following benefits:

• JSS is an open access journal. • Articles translations are free of charge. • On line submission and revision of articles. • Quick publication: average time of 7 months, in 2016. • Pdf articles freely available on the WEB. • JSS adopts the Ithenticate software against plagiarism. • All articles are published with DOI.

Send your article to http://www.scielo.br/scielo.php?script=sci_serial&pid=2317-1537&lng=pt clicking Submission on line.

Thank you for choosing the JSS.

It is recommended that the authors fully accomplish these instructions, according to the model. FORMATTING MODEL OF MANUSCRIPT TO BE FORWARDED TO JSS:

(Short title) Storage of Euterpe oleracea Mart. seeds

Conservation of Euterpe oleracea Mart. Seeds1

Walnice Maria Oliveira do Nascimento2*, Sílvio Moure Cicero3, Ana Dionísia Luz Coelho Novembre3

ABSTRACT – text (200 words)

Index terms:

Conservação de sementes de açaí (Euterpe oleracea Mart.)1

RESUMO – texto (200 palavras)

Termos para indexação:

______1Submitted on______. Accepted for publication on ______. 2Embrapa Amazônia Oriental, Caixa Postal 48, 66095-100 – Belém, PA, Brasil. 3Departamento de ProduçãoVegetal, USP/ESALQ, Caixa Postal 9, 13418-900 – Piracicaba, SP, Brasil. *Corresponding author

Introduction text

Material and Methods text Results and Discussion text

Conclusions text Acknowledgement (optional) text References (Start in another page) Follow the ABNT NBR6023 as mentioned in the item References Model for tables presentation

Table 1. Relationship between seed quality and sowing densities on field seedling emergence and speed emergence index (SEI) in corn ‘BRS201’.

Number of seeds.ha-1 (1000) Seed quality (%) 50 60 70 Emergence (%) SEI Emergence (%) SEI Emergence (%) SEI Q1 (95.0) 94.8 a* 13.3 a* 95.1 a* 13.4 a* 97.0 a* 13.6 a* Q2 (90.0) 95.6 a 13.5 a 95.1 a 13.0 a 96.0 a 13.2 a Q3 (85.0) 84.2 b 10.9 b 83.7 b 10.6 b 82.0 b 10.7 b Q4 (75.0) 72.3 c 9.4 c 76.2 c 9.6 c 74.4 c 9.5 c Mean 86.7 11.8 87.5 11.6 87.4 11.8 *Means within each column followed by the same letter do not differ by Duncan test at 5% probability.

Model for figures presentation

100

80

60

40 Germination (%) Germination 20 (%) Germination

0 0 3 6 9 12 15 StorageStorage (days) (days) 2 3 2 4040 DAA: DDA: y =y 1.76= 1.76 - 7.26x – 7.26x + 1.88x + 1.88x-0.08x2 – 0.08x R =3 0.9637 R2 = 0.9637 2 2 5050 DAA: DDA: y =y 64.28= 64.28 + 5.24x + 5.25x - 0.238x – 0.238x R2 = 0.9163 R2 = 0.9637 2 3 2 6060 DAA: DDA: y =y 82.96= 82.96 + 3.15x + 3.15x - 0.38x + 0.38x + 0.013x2 – 0.013x R 3 = R0.93062 = 0.9305 7070 DAA: DDA: y =y 91.52= 91.52 - 3.48x + 3.48x - 0.68x + 0.68x2 - 0.029x2 – 0.029x3 R2 =3 0.9039 R2 = 0.9039

Figure 1. Germination of pepper seeds extracted from fruits harvested at 40, 50, 60 and 70 DAA and stored for 0, 3, 6, 9, 12 and 15 days. INSTRUÇÕES AOS AUTORES

1. ESCOPO E POLÍTICA A partir de 2017 a revista Journal of Seed Science (JSS) circulará apenas na versão on line. Serão aceitos para publicação artigos científicos originais e notas científicas, ainda não publicados, nem encaminhados a outra revista para o mesmo fim, em idioma português ou inglês. Para artigos submetidos em inglês, os autores deverão providenciar uma versão com qualidade. Todos os artigos serão publicados em inglês. A NOTA CIENTÍFICA é uma categoria de manuscrito científico que descreve uma técnica, uma nova espécie ou observações e levantamentos de resultados limitados. Tem o mesmo rigor científico dos “Artigos Científicos” e o mesmo valor como publicação. A classificação de um trabalho como NOTA CIENTÍFICA é baseada no seu conteúdo e mérito científico, mas pode tratar-se de um trabalho preliminar, simples e não definitivo sobre determinado assunto, com publicação justificada pelo seu ineditismo e contribuição para área. Os artigos serão publicados conforme a ordem de aprovação e relevância. Artigos de revisão sobre temas relevantes e atuais poderão ser publicados por autores convidados pela Editoria do JSS.

O JSS tem como objetivos: - Publicar artigos originais em áreas temáticas relevantes da Ciência e Tecnologia de Sementes; - Publicar artigos que representem contribuição significativa para o conhecimento da área, os quais deverão ter caráter científico e buscar abordar em profundidade temas e tendências no âmbito da Ciência e Tecnologia de Sementes; - Apresentar uma política rigorosa de avaliação dos artigos submetidos à publicação, com cada manuscrito sendo avaliado por dois revisores, criteriosamente selecionados na comunidade científica. A decisão de aceite para publicação pautar-se-á sempre na recomendação do corpo de editores e de revisores ad hoc; - Manter elevada conduta ética em relação à publicação e seus colaboradores; - Manter rigor com a qualidade dos artigos científicos a serem publicados.

Os artigos serão publicados conforme a ordem de aprovação e relevância. O Comitê Editorial fará uma avaliação preliminar do manuscrito submetido podendo aceitá-lo ou não para publicação, de acordo com a política e os critérios de relevância da revista. Após aceite prévio, o EDITOR designará um EDITOR ASSOCIADO (de área), que procederá a editoração com o auxílio de pelo menos dois ASSESSORES CIENTÍFICOS do JSS, tendo as mesmas prerrogativas de aceitar ou não o trabalho para publicação. Todo processo de editoração poderá ser acompanhado pelos autores, assessores ou editores associados, mediante a utilização de código de acesso (login) e senha fornecidos no inicio do processo de submissão. Os dados, opiniões e conceitos emitidos nos artigos, bem como a exatidão das referências bibliográficas, são de inteira responsabilidade do(s) autor(es). A eventual citação de produtos e marcas comerciais não significa recomendação de seu uso pela ABRATES. Contudo, o EDITOR, com assistência da Comissão Editorial e dos Assessores Científicos, reservar-se-á o direito de sugerir ou solicitar modificações aconselháveis ou necessárias.

Licença Creative Commons Todo o conteúdo publicado pelo Journal of Seed Science é licenciado pela Licença Creative CommonsAtribuição Não Comercial 4.0 Internacional. Esta licença permite que outros remixem, adaptem e criem a partir do seu trabalho para fins não comerciais, e embo- ra os novos trabalhos tenham de lhe atribuir o devido crédito e não possam ser usados para fins comerciais, os usuários não têm de licenciar esses trabalhos derivados sob os mesmos termos.

Política de acesso livre O JSS oferece acesso livre imediato ao seu conteúdo, seguindo o princípio de que disponibilizar gratuitamente o conhecimento científico ao público proporciona maior democratização mundial do conhecimento.

Custos para publicação O pagamento da taxa de publicação de artigos é obrigatório, inclusive para sócios da ABRATES. Pelo menos um dos autores deverá ser sócio da ABRATES. O valor para publicação de artigos é: Para sócios da ABRATES - Até seis páginas diagramadas no formato final: R$ 30,00 por página + R$ 80,00 por página adicional. Para NÃO SÓCIOS - Até seis páginas diagramadas no formato final: R$ 60,00 por página + R$ 160,00 por página adicional. No caso de mais de um autor, incluindo sócios, o valor total será dividido pelo número de autores, entretanto somente os sócios que estiverem com pagamento em dia, terão desconto. Não será mais necessário o pagamento de taxa de tramitação ou submissão.

Taxas para leitores: não há cobrança de taxas aos leitores.

2. PREPARAÇÃO DE MANUSCRITOS As orientações explicitadas nessas instruções deverão ser seguidas plenamente pelo(s) autor (es). Organizar os manuscritos seguindo a ordem: TÍTULO RESUMIDO (Colocado Centralizado No Início Da Primeira Página), TÍTULO, AUTORES, RESUMO (máximo de 200 palavras), TÍTULO EM INGLÊS, ABSTRACT (máximo de 200 palavras), INTRODUÇÃO, MATERIAL E MÉTODOS, RESULTADOS E DISCUSSÃO, CONCLUSÕES, AGRADECIMENTOS (Opcional) E REFERÊNCIAS. Serão necessários no RESUMO “Termos para indexação” e no ABSTRACT “Index terms”, no máximo cinco, que não estejam citados no título. Na elaboração dos manuscritos, deverão ser atendidas as seguintes normas: Os artigos deverão ser digitados em editor de texto Word (DOC ou RTF), em linhas numeradas (máximo de 30 linhas por página), em espaço duplo e com margens de 2 cm (papel A4), fonte Times New Roman 14 para o título e 12 para o texto, sem intercalação de tabelas e figuras que serão anexadas ao final do trabalho. As figuras deverão estar em programas compatíveis com o WINDOWS, como o EXCEL, e formato de imagens: Figuras (GIF ou TIFF) e Fotos (JPEG) com resolução de 300 dpi. O manuscrito não deve exceder um total de 20 páginas, incluindo figuras, tabelas e referências. Artigos com mais de 20 páginas serão devolvidos. A redação dos trabalhos deverá apresentar concisão, objetividade e clareza, com a linguagem no passado impessoal, exceto para as conclusões que devem ser redigidas no presente. No RESUMO e no ABSTRACT não serão permitidos parágrafos, bem como a apresentação de dados em colunas ou em quadros e a inclusão de citações bibliográficas. O(s) nome(s) do(s) autor (es) deverá(ão) ser mencionado(s) por extenso logo abaixo do título. O autor para correspondência deve ser identificado por um asterisco. No rodapé da primeira página, através de chamadas apropriadas, deverá ser inserida a afiliação institucional do(s) autor (es), mencionando Departamento ou Seção, Instituição, Caixa Postal, CEP, Município e País e apenas o e-mail do autor para correspondência. Recomenda-se no máximo seis (6) autores por artigo.

Citações no Texto: as citações de autores, no texto, serão feitas pelo sobrenome com apenas a primeira letra em maiúsculo, seguida do ano de publicação. No caso de dois autores, serão incluídos os sobrenomes de ambos, intercalado por “e”; havendo mais de dois autores, será citado apenas o sobrenome do primeiro, seguindo de “et al.”. Em caso de citação, de duas ou mais obras do(s) mesmo(s) autor (es), publicadas no mesmo ano, elas deverão ser identificadas por letras minúsculas (a,b,c, etc.), colocadas imediatamente após o ano de publicação.

Referências: será exigido que 60% das referências bibliográficas sejam de artigos listados na base ISI Web of Knowledge, Scopus ou SciELO (revistas indexadas) com data de publicação inferior a 10 anos. Não serão aceitos nas referências citações de monografias, dissertações e teses, anais e resumos.

Evitar: - citações excessivas de livros textos; - citações obsoletas e revistas informativas e não cientificas. Citações de artigos recentes publicados no JSS podem ser acessadas pelo site: www.scielo.br/jss As referências deverão ser apresentadas em ordem alfabética pelo sobrenome do autor ou do primeiro autor, sem numeração; mencionar todos os autores do trabalho separados por “;”. Seguir as normas da ABNT NBR6023. As referências deverão conter hiperlinks para possibilitar acesso para qualquer página Web na Internet. Basta posicionar o cursor no local desejado de um texto ou planilha, digitar o endereço da página ex: www.abrates.org.br e teclar a barra de espaços. O hyperlink será criado automaticamente. Posicione o cursor em uma das letras do hyperlink criado, tecle Shift F10 para abrir o menu, desça com a seta até a opção abrir hyperlink e tecle enter que a página será aberta.

Alguns exemplos são apresentados a seguir:

Artigos de Periódicos:(não deverá ser mencionado o local de publicação do periódico). LIMA, L.B.; MARCOS-FILHO, J. Condicionamento fisiológico de sementes de pepino e germinação sob diferentes temperaturas. Revista Brasileira de Sementes, v.32, n.1, p.138-147, 2010. http://www.scielo.br/scielo.php?script=sci_ pdf&pid=S0101-31222010000100017&lng=en&nrm=iso&tlng=pt

OLIVEIRA, A.S.; CARVALHO, M.L.M.; NERY, M.C.; OLIVEIRA, J.A.; GUIMARÃES, R.M. Seed quality and optimal spatial arrangement of fodder radish. Scientia Agricola, v.68, n.4, p.417-423, 2011. http://www.scielo.br/scielo.php?script=sci_ pdf&pid=S0103-90172011000400005&lng=en&nrm=iso&tlng=en

Livros: MARCOS-FILHO, J. Fisiologia de sementes de plantas cultivadas. 2 ed. Londrina: ABRATES, 2015. 660p.

BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Regras para análise de sementes. Ministério da Agricultura, Pecuária e Abastecimento. Secretaria de Defesa Agropecuária. Brasília: MAPA/ACS, 2009. 395p. http:// www.agricultura. gov.br/arq_editor/file/2946_regras_analise__sementes.pdf

Capítulos de Livro: VIEIRA, R.D.; KRZYZANOWSKI, F.C. Teste de condutividade elétrica. In: KRZYZANOWSKI, F.C.; VIEIRA, R.D.; FRANÇA-NETO, J.B. (Ed.). Vigor de sementes: conceitos e testes. Londrina: ABRATES, 1999. p.4.1-4.26.

Leis, Decretos, Portarias: País ou Estado. Lei, Decreto, ou Portaria nº ..., de (dia) de (mês) de (ano). Diário Oficial da União, local de publicação, data mês e ano. Seção ..., p. ...

BRASIL. Medida provisória nº 1.569-9, de 11 de dezembro de 1997. Diário Oficial da República Federativa do Brasil, Poder Executivo, Brasília, DF, 14 dez. 1997. Seção I, p.29514.

Documentos Eletrônicos: BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. SNPC - Lista de cultivares protegidas. http://extranet. agricultura.gov.br/php/proton/cultivarweb/cultivares_protegidas.php Acesso em: 13 jan. 2010.

Tabelas As tabelas no formato “retrato” numeradas com algarismos arábicos, devem ser encabeçadas por título auto-explicativo, com letras minúsculas, não devendo ser usadas linhas verticais para separar colunas nem constar o local e data de realização do experimento.

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Unidades de medida Devem ser redigidas com espaço entre o valor numérico e a unidade. Ex: 10 ⁰C, 10 mL, µS.cm-1.g-1. O símbolo de percentagem deve ficar junto do algarismo, sem espaço. Ex: 10%. Utilizar o Sistema Internacional de Unidades em todo texto.

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(Título resumido) Armazenamento de sementes de açaí

Conservation of Euterpe oleracea Mart. Seeds1

Walnice Maria Oliveira do Nascimento2*, Sílvio Moure Cicero3, Ana Dionísia Luz Coelho Novembre3

ABSTRACT – text (200 words)

Index terms:

Conservação de sementes de açaí (Euterpe oleracea Mart.)1

RESUMO – texto (200 palavras)

Termos para indexação:

______1Submetido em______. Aceito para publicação em______. 2Embrapa Amazônia Oriental, Caixa Postal 48, 66095-100 – Belém, PA, Brasil. 3Departamento de Produção Vegetal, USP/ESALQ, Caixa Postal 9, 13418-900 – Piracicaba, SP, Brasil. *Autor correspondente

Introdução texto

Material e Métodos texto

Resultados e Discussão texto

Conclusões texto

Agradecimentos (opcional) texto Referências (iniciar em página separada) Seguir as normas da ABNT NBR6023 conforme já mencionado no item Referências. Modelo para apresentação de tabela

Tabela 1. Relação entre a qualidade de semente e a densidade de semeadura na emergência de plântulas em campo e o índice de velocidade de emergência em milho BRS 201.

Número de sementes.ha-1 (1000) Qualidade de semente 50 60 70 (%) Emergência (%) IVE Emergência (%) IVE Emergência (%) IVE Q1 (95,0) 94,8 a* 13,3 a* 95,1 a* 13,4 a* 97,0 a* 13,6 a* Q2 (90,0) 95,6 a 13,5 a 95,1 a 13,0 a 96,0 a 13,2 a Q3 (85,0) 84,2 b 10,9 b 83,7 b 10,6 b 82,0 b 10,7 b Q4 (75,0) 72,3 c 9,4 c 76,2 c 9,6 c 74,4 c 9,5 c Média 86,7 11,8 87,5 11,6 87,4 11,8 *Médias dentro de cada coluna seguidas da mesma letra não diferem entre si pelo teste de Duncan, a 5% de probabilidade,

Modelo para apresentação de figura

100

80

60

40 Germination (%) Germination

(%) Germinação 20

0 0 3 6 9 12 15 ArmazenamentoStorage (days) (dias) 2 3 2 4040 DAA: DDA: y =y 1.76= 1.76 - 7.26x – 7.26x + 1.88x + 1.88x-0.08x2 – 0.08x R =3 0.9637 R2 = 0.9637 5050 DAA: DDA: y =y 64.28= 64.28 + 5.24x + 5.25x - 0.238x – 0.238x2 R22 = 0.9163R2 = 0.9637 6060 DAA: DDA: y =y 82.96= 82.96Armazenamento + 3.15x + 3.15x - 0.38x + 0.38x2 + 0.013x2 (dias)– 0.013x3 R23 = R0.93062 = 0.9305 7070 DAA: DDA: y =y 91.52= 91.52 - 3.48x + 3.48x - 0.68x + 0.68x2 - 0.029x2 – 0.029x3 R2 3= 0.9039 R2 = 0.9039

Figura 1. Germinação de sementes de pimenta extraídas de frutos colhidos aos 40, 50, 60 e 70 DAA e armazenados por 0, 3, 6, 9, 12 e 15 dias.