X-Ray Test to Evaluate the Physiological Potencial of Platypodium Elegans Vog

Scientia Agropecuaria 7 (3): 305 – 311 (2016)

Facultad de Ciencias Agropecuarias Scientia Agropecuaria Universidad Nacional de Website: http://revistas.unitru.edu.pe/index.php/scientiaagrop Trujillo

X-ray test to evaluate the physiological potencial of Platypodium elegans Vog. Seeds (Fabaceae)

Kever Bruno Paradelo Gomes¹, *; Juliana Martins Mesquita Matos²; Ildeu Soares Martins²; Rosana de Carvalho Cristo Martins²

1 Instituto Federal de Educação, Ciência e Tecnologia de Brasília – Campus Gama. Brasília – DF, Brasil. ² Universidade de Brasília. Departamento de Engenharia Florestal.

Received March 11, 2016. Accepted Jun 30, 2016.

Abstract The objective of this work was to evaluate the internal morphology of Platypodium elegans Vog. seeds through the X-ray tests and to verify their relation with germination. 200 radiographic images were taken of each group and the tests were taken with the seeds set at 28.0 cm from the source of x-rays. Next, the seeds were classified according to their internal morphology as seen in the radiographic images. Three categories of seeds were established: full, empty and partially full. For each seed category, germination tests were performed. Statistical design in factorial 3x3, with three groups and three categories. From the results obtained in this work, the use of x-ray test with Platypodium elegans Vog. seeds is promising in quality of seed groups detection, assisting in empty and partially full seeds separation, invalidating their immediate use or storage. The x-ray test, in 26 kV for 1.2 seconds intensity was efficient in Platypodium elegans Vog. internal morpholy evaluation. There is a relationship between the internal morphology of seeds and the results of the germination test. The importance of a methodology of easy implementation, aiming at estimating the germinative power of the groups of seeds, was proved by the x-ray tests in seeds of Platypodium elegans Vog. and can also be used for the analysis of the internal morphology of other forest species.

Key words: Cerrado, image analysis, forest management, germination.

1. Introduction contributing for the maintenance and qua- Platypodium elegans Vog. popularly lity enhancement of this input. (de Melo et known as uruvalheira, jacarandá-do- al., 2009; Albuquerque and Guimarães, campo, faveiro, belongs to the Fabaceae 2008). The environmental compensations, family. It is a species which naturally like the compulsory replacement of native occurs in the Cerrado and in the forest- forests in rural properties, and the recovery cerrado transition zone and is usually of degraded areas, aiming at facing federal found in well-drained soils. It is widely and state laws, prompted an increase in used for commercial and ornamental seed demands of native arbor forest purposes with its planting being more species, which constitute basic input in the widely recommended in the agroforestry replacement and conservation programs of system in degraded areas recovery the ecosystem. programs (Lorenzi, 2008; Pacheco et al., In this context, the use of non-destructive 2007; Lopes et al., 2010). procedures which provide efficiency in the The morphophysiological quality evalua- identification of empty, partially full and tion of the seed groups is an essential full seeds, like the x-ray testing may be process, which is of great importance for considered an indicative of physiological several phases of the production chain of quality of the seeds of these forestry the sector of seed production, significantly species.

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The seed analysis through the radiographic groups was made considering the charac- images obtained through the x-ray testing teristic of the fragments of the vegetation is a relatively recent alternative in order to found in urban centers (Group 1); Environ- immediately classify the several internal mental protection area (EPA) addressed to morphological aspects of the seeds the conservation and protec-tion of the (Gomes, 2010). In this sense, the capture ecosystem (Água Limpa Farm – University and processing of the image which has of Brasília) (Group 2) and along road BR been x-rayed has allowed the establi- 040 which connects Brasília (DF) and shment of relations between integrity, Cristalina (GO) in degraded environ-ments morphology and determination of the (Group 3). Fruits were harvested from 20 physiological potential of the seeds mother plants of each group. (Marcos et al., 2010). The seeds were improved extracting the The forest species, particularly the ones aliforms and submitted to the mechanical from Cerrado, are characterized by the scarification, promoting a short cut on the high incidence of predation, empty fruit side of the fruit, with the use of a pocket and deficiency in the formation of the knife in the laboratory of seeds and forest embryo. This way, the x-ray test is nurseries belonging to the University of recommended as it is a viable technique in Brasília. The seeds were homogenized and the quality control of seeds (Gomes et al., stored in paper bags in B.O.D. type growth 2014). Considering the economical inte- chamber under room temperature condi- rests and the conservation of the tree tions of 25 ºC until the tests had been species of the Cerrado, it becomes conducted in a period of 30 days. The necessary to intensify the research which is samples for the tests were randomly concerning the evaluation of the internal extracted from each group of seeds. In morphology of the seeds in order to help each group, three samples of 20 seeds were the identification of the seeds which hold extracted for the moisture content potential on germination. determination. The germination test may become an The samples were weighed with a digital obstacle for the evaluation of the physio- precision scale of 0.001 g and after that logical potential on forest seeds, procedure, the moisture content was analy- considering the genetic elasticity that these zed with the standard greenhouse method species present and the long period at 105 °C ± 3 °C, for 24 hours (MAPA, demanded for the obtention of the results 2009). (Masetto et al., 2008). The x-ray test was conducted in the image Therefore, the objective of this paper was analysis laboratory of the Department of to evaluate the internal morphology of the vegetal production of the Superior School seeds of the Platypodium elegans Vog. of Agriculture “Luiz de Queiroz”, Univer- through the x-ray tests and verify their sity of São Paulo (ESALQ/USP), in relation to the germination process. Piracicaba, SP. For the obtention of radiographic images of the seeds, we counted on the digital equipment Faxitron 2. Materials and methods X-ray, model MX-20 DC-12, attached to a The fruits of Platypodium elegans Vog. Core 2 Duo computer (3.16 GHz, 2 GB were collected in sensu stricto areas of the RAM memory, 160 GB Hard Disk) and Federal District Region Cerrado at the MultiSync screen (LCD1990SX, of 17 point of physiological maturation directly inches). This way, 200 seeds of each group on the tree, considering its vigor, stature were x-rayed and the images were obtained and the sanity of the mother plants selected with the seeds placed at 28 cm far from the in September, 2014. x-ray emission source. The radiation inten- The choice of the region for the mother sity and the time of exposure of the seeds plants selection and formation of the seed to the x-rays were automatically determi-

-306- K.B. Gomes et al. / Scientia Agropecuaria 7 (3) 305 – 311 (2016) ned by the x-ray machine. For the adequate Gerais. The low level of moisture found in positioning of the seeds during the expo- the seeds of Platypodium elegans was sition of the x-rays, transparent double- fundamental to allow the adequate visuali- face sellotape was used on a rear projec- zation of the internal parts of the seeds. tion transparency (29.7 x 21.0 x 0.1 cm). Gomes et al. (2014), evaluating the inter- After they were x-rayed, the seeds were nal morphology of the seeds of Terminalia placed in individual cells of a plastic tray argentea, verified that the low level of for later germination. The seeds were moisture in the seeds (9 - 10%) led to a classified according to the internal mor- bigger optical density, which allowed a phology visualized in the radiographic better visualization of the internal structu- images. Three categories of seeds were res of the seeds which had been x-rayed. considered: empty, partially full and full. The exposure of seeds of Platypodium The result of the x-rays was interpreted elegans Vog. to the x-rays, in radiation through the confront of the radiographic intensity of 26 kV for 1,2 seconds, was image and the respective image of the nor- efficient for the visualization of the mal seedling, abnormal or dead or seed in internal morphology of the seeds. The state of dormancy after the germination images of the radiographed seeds allowed test. For each category of seeds obtained for an identification of the current internal through the x-ray test, the germination test state of the seeds, as well as the identi- was conducted in the Laboratory of seeds fication of seeds without embryonic and forest nurseries belonging to the Forest tissues. In the same way, de Carvalho et al. Engineering Department of the University (2009) verified that the potence of 25 kV, of Brasília, Brasília-DF. for 2 minutes, was enough to diagnose the The previously numbered seeds of internal structures of the seeds of Ocotea Platypodium elegans Vog. were placed in pulchella and of Persea pyrifolia, both filter paper roll (20 seeds/roll), stored in belonging to the Lauraceae family. B.O.D. type growth chamber under 25 ºC In seeds of sucupira (Bowdichia and photoperiod of 8 hours/day. For soa- virgilioides Kunth.) submitted to a radio- king, distilled water was used in the pro- graphic analysis, the intensity of 30 kV for portion of 2.5 times its mass. The germi- 45 seconds was efficient to verify the nation analysis was carried out in 30 days. internal parts of the seeds (Albuquerque The statistical delineation used was in a and Guimarães 2008). The image analysis factorial scheme, with three groups and of the seeds of Platypodium elegans Vog., three categories (3x3), with the treatment obtained through the tests of x-ray, was replicated 20 times and the 10 experi- efficient for the evaluation of the internal mental units placed totally at random. The condition of the sedes (Figure 1). analysis of variation (ANOVA) was carried out and the averages compared with the use of the Turkey’s test, with probability at 5%.

3. Results and discussion

The seeds of Platypodium elegans Vog. Figure 1. Seeds of Platypodium elegans Vog. presented low level of moisture (9 - 11%). classified as partially full (1), full (2) and The values which were obtained were empty (3) through the analysis of radiographic similar to those presented by de Souza et images. al. (2008), when they found a 6 - 9% moisture level of the seeds of Platypodium Making use of the previous works on seeds elegans recently harvested in the region of of Tabebuia serratifolia debe-loped by de Northern Cerrado of the state of Minas Oliveira et al. (2004) and through the

-307- K.B. Gomes et al. / Scientia Agropecuaria 7 (3) 305 – 311 (2016) indication of the rules for seed analysis lowest values observed for all the groups (MAPA, 2009), it was possible to establish (4 – 10%) (Table 1), these seeds must be the criteria according to which we deter- discarded before they are stored or sown, mined if the seeds could be considered full due to the high probability of producing (totally formed), empty or partially full. abnormal seedling or non-germinating According to Table 1, it is possible to seeds and/or dead, inappropriate for the identify that groups 2 and 3 had a higher field development. In radiographed seeds percentage of seeds that were perfectly of Cedrela fissilis Masetto et al. (2008) morphologically formed (full) than group found around 30% of partially full seeds. 1. This analysis was possible with the use According to the results of these authors, of radiographic tests of x-ray. the deformation of the embryo precluded The category full for all the groups was the the normal development of the seedlings. one that presented the highest percentage of seeds of Platypodium elegans Vog., Table 1 statistically differing from the categories Percentage of seeds of Platypodium elegans empty and partially full. There was a Vog. obtained in each category according to the x-ray tests and the percentage of moisture significant statistic difference (p < 0.05) between the categories empty and partially full in all of the observations. The Mois- X-ray category (%)* Group ture percentage of full seeds of the three groups Partially (%) Full Empty varied between 65 and 80.5% (Table 1). Full These values were higher than the ones 1 9.3 65.0 bA 30.0 B 5.0 C found by Sturião et al. (2012), when they 2 10.4 85.0 aA 7.0 B 8.0 C evaluated the internal morphology of seeds 3 11.2 80.5 aA 15.5 B 4.0 C * The averages that were followed by the same short of Syagrus romanzoffiana through the x- letter in the horizontal line for the x-ray categories ray tests; these authors found around and capital letters in the vertical line for the groups 60.5% of viable seeds (full). According to do not statistically differ among one another MAPA (2009), fruit-seeds or seeds according to the Turkey’s test at 5% probability. classified as full or totally formed by the x- ray tests contain all the essential tissues for Forest arbor species like the Platypodium the germination and, because of this, they elegans Vog. which are recommended for are considered viable. the recovery of degraded areas, must hold The percentage between 15.5 and 25.5% groups of seeds with good genetic forma- (Table 1) of the seeds classified in the tion, as the seedlings that have been category empty is a sign that the process of formed need good resistance and adapta- classification of the seeds can be enhanced, bility to poor soils; therefore, seeds that are avoiding unnecessary costs with the pro- partially full, damaged and/or empty, duction of seedling and tests of laboratory. according to x-ray tests, must be elimi- According to Gomes et al. (2014), the nated from the programs of production of occurrence of empty seeds directly influen- native seedlings. This aims at assuring ces the storage, the efficiency of the good germination performance of the sowing and the commercialization of groups. The discard of seeds that are groups of seeds. This fact is observed with partially full, detected by the analysis of some frequency in forest species where the the radio-graphed seeds was also structure of dispersion is not a true seed recommended by Machado and Cícero (de Souza et al., 2005) (2003) for seeds of Lithraea molleoides Seeds that are partially full of Platypodium and by de Oliveira et al. (2003) for seeds elegans Vog. were also found through the of Peltophorum dubium, both resulted in x-ray tests, confirming the efficiency of the death of the seeds in the germination this method in the detection of embryonic test. Therefore, as Amaral et al. (2011) abnormalities. Even though they were the have observed in seeds of Tabebuia

-308- K.B. Gomes et al. / Scientia Agropecuaria 7 (3) 305 – 311 (2016) heptaphylla, the radiographic images of the the categories of x-ray, the variation seeds of Platypodium elegans Vog. were analysis allowed for the separation of the fundamental to visua-lize the embryonic groups, however, when the results of the abnormalities, which were probably tests of germination were obtained, there originated during the formation and was no significant effect among the maturation of the fruit. groups, which did not prompt the choice of When the results obtained in this work are the group with highest vigor. This fact may taken into consideration, the use of x-ray be explained through the approximation of tests in seeds of Platypodium elegans Vog. the values of percentage of germination of is promising in the quality detection of the the categories full (60.4 – 72.64%) and due groups of seeds, helping the separation of to the fact that the categories empty and empty seeds and with embryonic abnor- partially full do not obtain germinated malities unfeasible for an immediate use or seeds for all the groups. Pacheco et al. for storage. (2007) when evaluated pre-germination For the percentage of germination, the treatments in seeds of Platypodium elegans analysis of the variation demonstrated Vog., observed that the removal of part of significant effect among the categories the pericarp through the longitudinal obtained by the x-ray tests (Table 2), cutting increased the permeability to water, which resulted in no difference among the which can, this way, induce a higher expo- groups of seeds. sure to light and temperature, positively influencing the germination process of the Table 2 seeds. Percentage of germination of three groups of According to the results obtained in the seeds of the species Platypodium elegans Vog. germination test, the morphological perfect found for the categories full, empty and seeds (full) originated normal seedlings partially full obtained through the analysis of the images of radiographic seeds and non-germinated seeds. This kind of result is expected, as, in the radiography, Group X-ray categories Germination (%)* the images indicate if there are or not Full 60.40 a formed tissues; however, they do not 1 Empty 0.00 b necessarily establish direct relation to the Partially full 0.00 b physiological processes of the seed. This Full 70.23 a analysis parameter was also observed by 2 Empty 0.00 b Partially full 0.00 b Pupim et al. (2008), in which some seeds Full 72.64 a with adequate characteristics, according to 3 Empty 0.00 b the x-ray test, did not germinate, probably, Partially full 0.00 b due to the infection by microorganisms, * The averages followed by the same letter do not mechanical damages and possible dor- statistically differ among one another according to the Turkey’s test at 5% probability. mancy. Along this work, it was possible to observe For the three groups analyzed, the that the morphological conditions may percentage of germination of the categories influence the quality of the seeds. The x- full statistically differed from the other ray applied in this work revealed the categories. For the categories empty and unfeasibility of the seeds of Platypodium partially full, 100% of non-germinating elegans Vog. which had imperfect or seeds were observed at the end of the empty embryonic axes, as all the seeds of germination test, which attests a corre- these categories (partially full and empty) lation between the radiographic images by did not germinate. This reality was also the x-ray test and the results obtained observed by Machado and Cícero (2003) in through the germination test. radiographed seeds of Lithraea molleoides It is also important to mention that in the submitted to the germination test. Accor- observation of the frequencies of seeds of ding to Carvalho and Oliveira (2006), the

-309- K.B. Gomes et al. / Scientia Agropecuaria 7 (3) 305 – 311 (2016) test of x-ray does not detect all the were similar to the ones found by de Souza problems related to the physiological et al. (2008), in seeds of Platypodium quality of the seeds, however, it enables a elegans. For these authors, the obtained quick and non-destructive diagnosis for the data attests that the assumption that the examined seeds, providing useful and internal morphology of the seeds can be an essential data for the research of laborato- indication of their viability. For Albu- ries of agriculture and forestry seed querque and Guimarães (2008), the x-ray analysis as well as for the production of test in seeds of Bowdichia virgilioides, commercial seedlings. when related to the germination test, can In order to obtain a more detailed help in the evaluation of the physical diagnosis, the technique of radiographic quality of the seeds. images analysis played an important role, The external simultaneous analysis of the as it was possible to visualize the current seeds of the Platypodium elegans Vog., internal condition of the seeds and, as this their respective radiographed images, technique is non-destructive, it was normal seedlings and in state of dormancy possible to establish cause and effect or dead seeds deriving from the test of relations, after the test of germination had germination, permitted the diagnosis for been checked (Figure 2). each studied case. Hence, if the external image of the seeds were analyzed isola- tedly, (Figure 2A), the imprecision of the diagnosis would be clear, as, this way, it would not be possible to identify internal damages or the frequency of seeds without embryonic tissues. According to de Carvalho and de Oliveira (2006), the test of x-ray does not detect all the problems related to the physiological quality of the seeds, howe-ver, it allows for a quick and non-des-tructive diagnosis for the examined seeds, providing useful and essential data for the research of laboratories of agriculture and forestry seed analysis as well as for the production of seedlings. The adequation of the x-ray test applied to the Figure 2. Seeds of Platypodium elegans Vog. native Forest seeds of the Cerrado, as the visually intact (A), classified by the radiographic analysis as Full seeds (B), Partially full one considered for this study, provides seeds (C) and Empty seeds (D); originating agility in the identification of the viability normal seedling (E) and non-germinated seeds and physio-logical quality of the groups of and/or dead (F). seeds to be used in the production of seedlings. In the Figure 2B, it can be observed in the x-ray images, full seeds, which contribute to a positive effect on the germination with 4. Conclusions the development of a normal seedling The x-ray test in radiation intensity of 26 (Figure 2E). However, in the Figure 2C kV for 1.2 seconds, is efficient in the and 2D, it is possible to observe empty and evaluation of the internal morphology of imperfect seeds that did not germinate the seeds of Platypodium elegans Vog. (Figure 2F). Full seeds also resulted in There was relation between the internal non-germinated seeds after the germination morphology of the seeds and the results of test. Abnormal seedlings were not obser- the germination test. The radiographic ved in any of the groups. These results images showed that the detected damage

-310- K.B. Gomes et al. / Scientia Agropecuaria 7 (3) 305 – 311 (2016) affected the germination of the seeds of de Souza, L.A.; dos Reis, D.N.; dos Santos, J.P.; Davide, A.C. 2008. Uso de raios x na avaliação da qualidade de Platypodim elegans Vog. sementes de Platypodium elegans Vog. Revista The importance of a methodology of easy Ciência Agronômica 39(2): 343-347. implementation, aiming at estimating the de Souza, L.A.; Mesquita, H.A.; Alvarenga, M.I.N.; Davide, A.C. 2005. Utilizacao de raios-X na avaliacao germinative power of the groups of seeds, da qualidade fisiologica de sementes de sucupira was proved by the x-ray tests in seeds of branca (Pterodon emarginatus VOG). Informativo Abrates 15: 284-290. Platypodium elegans Vog. and can also be do Amaral, J.; Martins, L.; Forti, V.; Cícero, S.; Marcos, used for the analysis of the internal F.J. 2011. Teste de raios x para avaliação do potencial morphology of other forest species. fisiológico de sementes de ipê-roxo. Revista Brasileira de Sementes 33(4): 601-607. Gomes, J.F.G. 2010. Aplicação da análise de imagens para avaliação da morfologia interna de sementes. Acknowledgements Informativo Abrates 20(3): 33-51. Pro-rector of Research and Innovation of Gomes, K.; Martins, R.; Martins, I.; Gomes, J.F. 2014. Instituto Federal of Brasília (PRPI-IFB) and Avaliação da morfologia interna de sementes de University of Brasília for financial support. Terminalia argentea (Combretaceae) pelo teste de raios -X. Revista Ciência Agronômica 45(4): 752-759. Lopes, S.; Oliveira, A.; Neves, S.; Shiavini, I. 2010. References Dispersão de sementes de uruvalheira (Platypodium elegans Vog.) (Fabaceae) em um cerradão, Uberlândia Albuquerque, K.; Guimarães, R. 2008. Avaliação da – MG. Revista Arvore 34(5): 807-813. qualidade de sementes de sucupira-preta (Bowdichia Lorenzi, H. 2008. Árvores brasileiras: manual de virgilioides Kunth.) pelo teste de raios X. Revista identificação e cultivo de plantas arbóreas do Brasil. 1: Ciência e Agrotecnologia 32(6): 1713-1718. 5 ed. Nova Odessa, SP. Instituto Plantarum. MAPA - Ministério da Agricultura, Pecuária e Abasteci- Machado, C.; Cicero, S. 2003. Aroeira-branca (Lithraea mento. 2009. Regras para análise de sementes. SDA: molleoides (Vell.) Engl. – Anacardiaceae) seed quality Brasília: MAPA. 399 p. evaluation by the X-ray test. Scientia Agricola 60(2): de Carvalho, M.L.M; de Oliveira, L.M. 2006. Raios X na 393-397. avaliação da qualidade de sementes. Informativo Marcos, F.J.; Gomes, J.F.; Bennett, M.; Wells, A.; Stieve, Abrates 6(1): 93-99. S. 2010. Using tomato analyzer software to determine de Carvalho, L.R.; de Carvalho, M.L.M.; Davide, A.C. embryo size in x-rayed seeds. Revista Brasileira de 2009. Utilização do teste de raios X na avaliação da Sementes 32(2): 146-153. qualidade de sementes de espécies florestais de Masetto, T.; Faria, J.; Queiroz, S. 2008. Avaliação da Lauraceae. Revista Brasileira Sementes 31: 57-66 qualidade de sementes de cedro (Cedrella fissilis – de Melo, P.; Oliveira, J.; de Carvalho, M.; Guimarães, R.; Meliaceae) pelo teste de raios X. Ciência e Carvalho, B. 2009. Aplicação do teste de raios x no Agrotecnologia 32(6): 1-7. estudo da morfologia interna e da qualidade fisiológica Pacheco, M.; Matos, V.; Barbosa, M.; Ferreira, R.; Passos de aquênios de arnica (Lychnophora pinaster Mart.). M. 2007. Germinação de sementes de Platypodium Revista Brasileira de Sementes 31(2): 146-154. elegans Vog. submetidas a diferentes tratamentos pré- de Oliveira, L.M.; de Carvalho, M.L.M.; Davide, A.C. germinativos e substratos. Revista Brasileira de 2003. Utilização do teste de raios-X na avaliação da Engenharia Agrícola e Ambiental 11(5) 497-501. qualidade de sementes de canafístula (Peltophorum Pupim, T.; Novembre, A.; Carvalho, M.; Cicero, S. 2008. dubium (Sprengel) Taubert. Revista Brasileira Adequação do teste de raios x para avaliação da Sementes 25: 116-120. qualidade de sementes de embaúba (Cecropia de Oliveira, L.; de Carvalho, M.; Giimarães, R.; Masetto, pachystachya Trec.). Revista Brasileira de Sementes T. 2004. Avaliação da qualidade de sementes de 30(2): 28-32. Tabebuia serratifolia Vahl Nichi. e T. impetigiosa Sturião, W.; Landgraf, P.; Rosa, T. 2012. Avaliação da (Martius ex A. P. de Candolle Standley) – qualidade fisiológica de sementes de palmeira jerivá (Bignoniaceae) pelo teste de raios x. Revista Brasileira pelo teste de raios x. 2012. Semina: Ciências Agrárias de Sementes 26(2): 138-143. 33(1): 213-218.

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