2 Materials and Methods

2.1 Plant material

The experiment was carried out with 2 kg of Jatropha curcas seeds its were collected in a commercial plantation from the Atlantic rain forest region (09°28’S; 35°51’W m.a.s.l.). The plantation consisted of plants that were at least 8 years of age, and the spacing between plants was 2 m × 2 m. Fruits of J. curcas were randomly collected during the rainy season from 2015. The seeds presented 72% viability and stored as recommended by Moncaleano-Escandon et al. (2013).

2.2 Seed imbibiton test and water relation

The seeds were distributed in 52 frasks (400 mL) with 25 seed for each replication treatment in a controlled room chamber at 25°C. For each frask, 100 ml deionized water was applied and the seeds were soaked according to the imbibition treatments (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 hours). The pH (W3B, Bel Engineering, Italy) and electrical conductivity (CD-4306, Lutron, Taiwan) were evaluated with 20 mL of soaking solution for each imbibition treatment. For seed water content, 50 mL deionized water was applied in frasks of 100 mL and were added 10 seed previously weighted in an analytic scale, according to different imbibition time. After each treatment was take the seeds imbibition weight and putted in papers bags for oven at 105°C for 24 hours and determinate seeds dry weight. The water relation variables were calculated according the following formulas: \(SMT(\%) = 1 - (S_{dw}/S_{fw})*100\) and \(SWC(\%) = (S_{tw} - S_{fw})/S_{fw} *100\). Where \(SMT(\%)\), Seed moisture; \(SWC(\%)\) Seed Water content; \(S_{dw} (g)\), Seed dry weight; \(S_{fw} (g)\), Seed fresh weight and \(S_{tw} (g)\), Seed turgid weight.

2.3 Germination test

For each replication treatment, 25 seeds were uniformly distributed in polyethylene trays content 1000 g of river sand and covered with 200 g of the substrate. The germination experiment was carried out in greenhouse condition with average temperature of 27.5°C and 78% relative humidity. Seed germination was evaluated daily according to agronomic criteria consider germinated seed when the radicle had emerged about 10 mm above the soil surface. When no additional germination was observed in all treatments at least in five consecutive days, the germination was considered completed (Moncaleano-Escandon et al., 2013).

2.4 Data analysis

The experiment was carried out in a completely randomized design with 13 treatments of imbibition times (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 hours) with four replications with seeds of J. curcas. Statistical analysis were performed in the statistical software R (R Core Team, 2017). The analysis of variance (ANOVA) was performed to evaluate the differences between the factors and the comparison of the means with the Student-Newman-Keuls test (p < 0.05) (de Mendiburu, 2017). The germination variables and graphs were performed according the GerminaR R package (Lozano Isla et al., 2017). For the multivariate analysis, correlation analysis (de Mendiburu, 2017; Wei and Simko, 2017) and principal component analysis were conducted (Husson et al., 2017).

References

Moncaleano-Escandon, J., Silva, B.C., Silva, S.R., Granja, J.A., Alves, M.C.J., Pompelli, M.F., 2013. Germination responses of jatropha curcas l. seeds to storage and aging. Industrial Crops and Products 44, 684–690. https://doi.org/10.1016/j.indcrop.2012.08.035

R Core Team, 2017. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.

de Mendiburu, F., 2017. Agricolae: Statistical procedures for agricultural research.

Lozano Isla, F., Benites Alfaro, O., Pompelli, M.F., 2017. GerminaR: Germination indexes for seed germination variables for ecophysiological studies.

Wei, T., Simko, V., 2017. Corrplot: Visualization of a correlation matrix.

Husson, F., Josse, J., Le, S., Mazet, J., 2017. FactoMineR: Multivariate exploratory data analysis and data mining.