X-ray microtomography in comparison to radiographic analysis of mechanically damaged maize seeds and its effect on seed germination

Acta Scientiarum http://periodicos.uem.br/ojs/acta ISSN on-line: 1807-8621 Doi: 10.4025/actasciagron.v41i1.42608 CROP PRODUCTION X-ray microtomography in comparison to radiographic analysis of mechanically damaged maize seeds and its effect on seed germination Francisco Guilhien Gomes-Junior1* Renato Orlandi Lasso2 , Silvio Moure Cicero1, Carlos Manoel Pedro Vaz2 and Paulo Departamento de Produção Vegetal, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, Av, Pádua Dias, 11, Cx. Postal 09, 13418-900, Piracicaba, São Paulo, Brazil. 2Empresa Brasileira de Pesquisa Agropecuária, Embrapa Instrumentação, São Carlos, São Paulo, Brazil. *Author for correspondence. E-mail: 1 ABSTRACT. Among the most relevant aspects of seed production, mechanical damage may affect seed germination and reduce health and vigor. This study introduces a noninvasive high-resolution imaging procedure for evaluating the mechanical damage to maize seeds and the effects on seed germination. Seeds with different levels of mechanical damage were evaluated using a benchtop micro-computed tomography system (micro-CT) and digital X-ray equipment. The two-dimensional transaxial, coronal and sagittal micro-CT sections were used to inspect the seed anatomy and the mechanical injuries in the internal seed tissue. Germination tests were performed using paper towel rolls (25°C for 7 days) in which the seedling length was evaluated on a daily basis, and the seedling dry biomass was measured at the seventh germination day. The micro-CT cross-sectional images allowed an efficient spatial characterization of the mechanical damage inside the seeds. On average, mechanically damaged seeds produced seedlings with a length 24% shorter and a dry biomass 65% less than that of the undamaged seeds. We concluded that the micro-CT technique provides an efficient means to inspect mechanically damaged maize seeds and allows for a reliable association with germination response. Keywords: Zea mays; seed internal morphology; 3D X-ray imaging; computed tomography. Received on October 21, 2017. Accepted on March 16, 2018. Introduction Mechanical damage affects seed quality (Bewley & Black, 1994) along with harvesting, transporting, drying and handling (Carvalho & Nakagawa, 2012). Mechanical damage is associated with a loss in physical integrity, higher susceptibility to microorganism infection, sensitivity to chemical treatment, and decrease in physiological quality and seed lot storability (Bewley & Black, 1994). An improvement in the evaluation of mechanically damaged maize seeds occurred in late 1990s when X-ray radiography was introduced as a non-invasive technique for analyzing internal injuries of seeds along with their physiological quality (Cicero, Heijden, Van der Burg, & Bino, 1998; Carvalho, Aelst, Eck, & Hoekstra, 1999). Research evaluating pre-harvesting cracks on maize seeds using X-ray radiography and low-temperature scanning electron microscopy showed that internal injuries or those perpendicular to the embryonic axis affect seed germination and vigor (Carvalho et al., 1999). Nevertheless, further research has shown that in some situations, radiographic analysis was not satisfactory in identifying injuries associated with the poor germination. Because radiographic analysis is a two-dimensional (2D) projection of a seed, it is not effective in determining depth of cracks. Cicero and Banzatto-Junior (2003) showed that 23% of maize seeds with mechanical damage on the embryo produced normal seedlings and concluded that the X-ray radiography technique did not allow for an accurate evaluation of fractures. Several imaging techniques, such as magnetic resonance imaging (MRI) (Ghosh, Jayas, Gruwel, & White, 2006), synchrotron X-ray fluorescence (De Jonge & Vogt, 2010), X-ray absorption (Staedler, Masson, & Schönenberger, 2013), phase contrast (Cloetens, Mache, Schlenker, & Lerbs-Mache, 2006) and neutron tomography (Cleveland IV et al., 2008), have been applied to plant and seed analysis. Among the techniques, high-resolution desktop X-ray absorption tomography (micro-CT) is highly recommended because of its excellent contrast, which allows flexibility to analyze different biological and mineral materials and it increases commercial instrumentation availability (Stuppy, Maisano, Colbert, Rudall, & Rowe, 2003; Pittia et al., 2011; Milien, Renault-Spilmont, Cookson, Sarrazin, & Verdeil, 2012; Dawson, Francis, & Carpenter, 2014; Friis, Acta Scientiarum. Agronomy, v. 41, e42608, 2019 Page 2 of 12 Gomes-Junior et al. Marone, Pedersen, Crane, & Stampanoni, 2014). Unlike radiography, which is a projection image, micro-CT produces a pack of two-dimensional cross-sectional images that can be combined into a three-dimensional image allowing internal inspection and measurement (Stuppy et al., 2003). The objective of the present study was to establish an optimized procedure to acquire high-resolution X-ray microtomographic images of dry damaged maize seeds to evaluate their internal morphology and the damage’s effect on seed germination. Material and methods Available equipment to induce seed damage The seeds were mechanically damaged using the S-injure equipment (Figure 1). The equipment is a box (made of medium-density fiberboard) measuring 40 × 25 × 37 cm. The seeds were thrown against a stainless-steel plate (3 mm in thickness) via a pressure regulator (Arprex, model AF1) operating up to a pressure of 828 kPa. A seed sample of 400 g was launched three times against the stainless-steel plate positioned 13 cm from the exit of the seed ejector tube with a pressure of 414 kPa, resulting in an average speed of 40 m s-1. The seeds were released into a hopper and thrown against the steel plate. Subsequently, the seeds were collected in a drawer positioned at the base of the equipment, and the process was repeated two more times. Figure 1. Schematic diagram of the S-injure equipment used to induce seed damage. Superior view (A) and 3D view (B). 1 - entrance of compressed air; 2 - controlled compressed air injection; 3 - manometer; 4 - pressure controller; 5 - seed hopper; 6 - seed ejector tube; 7 - steel plate; 8 - cover; and 9 - catch drawer. Maize seed selection We investigated seeds with 12% moisture content (wet basis) of the hybrid 2B604PW (flat seeds classified in sieves with oblong screens measuring 20/64" × 3/4" = 7.9 mm × 19.0 mm; 1000 seed mass = 330.29 ± 12.05 g) with greater than 95% germination. After mechanical damage was induced, a cabinet X-ray system for specimen radiography (Faxitron X-ray, model MX-20 DC12, Tucson, USA), operating from 10 to 35 kV, 300 A and with a maximum pixel resolution of 7 m, was employed to image and select 25 seeds of different mechanical damage levels (based on injury extension in the embryo and/or endosperm) for micro-CT image analysis and germination tests using the same set of seeds. Seeds were stored in individual and numbered plastic cell trays, and all (...truncated)