Seeds of Caesalpinia echinata Lam. Under Water Stress at Different Temperatures

The Caesalpinia echinata Lam. species, belongs to the Fabaceae family, popularly known as Pau Brazil, is a large tree, native to Brazil, that can be found from Ceará to Rio de Janeiro, with a relevant national historic and economic value. The objective of this study was to evaluate the effects of water stress at different temperatures on germination and seed vigor. The experiment was carried out at the Laboratório de Análise de Sementes of the Centro de Ciências Agrárias of the Universidade Federal da Paraíba, Areia-Paraíba. The water stress was formulated with solutions of polyethylene glycol 6000 (PEG 6000) in different osmotic potentials of 0.0 (control), -0.2; -0.4; -0.6 MPa at constant temperatures of 25, 30 and 35 °C, in a completely randomized design. The percentage of germination, first counting of germination, germination speed index, length and dry matter of seedlings were evaluated to determine the effects of the treatments. Decreases of the osmotic potential negatively influenced the germination and vigor of C. echinata seeds at the three evaluated temperatures (25 ºC, 30 ºC, 35 °C).

. For the germination of seeds of tropical species, the optimum temperature varies from 15 to 30 °C (Marcos Filho, 2015).
The water stress is a situation that the plants can find in the field. In a laboratory conditions it is possible to simulate water stress for seed germination (Pelegrini, Borcioni, Nogueira, Koehler, & Quoirin, 2013). For this, the use of osmotically active chemicals as a way of inducing the water stress in the seed has been widely diffused and is usually done by the addition of solutes such as calcium chloride (CaCl 2 ), sodium chloride (NaCl) and polyethylene glycol (Oliveira & Gomes-Filho, 2009).
For each osmotic solution, there are chemical differences that can lead to different results in seed germination, even in similar water potentials (Souza & Cardoso, 2000). Polyethylene glycol 6000 (PEG 6000) has been used to identify the osmotic potential that is less damaging to the seeds various species. It has a high molecular weight, being non-toxic to the seed (Moraes, Freitas, & Menezes 2003), providing slow and controlled imbibition of the seeds (Villela, Doni Filho, & Siqueira, 1991).
Based on the above and the importance of this species, the objective of this study was to evaluate the effect of water stress at different temperatures on the germination and vigor of seeds of Caesalpina echinata Lam.

Method
The experiment was carried out at the Laboratório de Análise de Sementes (LAS) belonging to the Departamento de Fitotecnia e Ciências Ambientais (DFCA) of the Centro de Ciências Agrárias (CCA) of the Universidade Federal da Paraíba (UFPB), located in Areia -Paraíba. The fruits of C. echinata were harvested in mother trees located in the CCA/UFPB and then taken to the LAS for processing by manual opening for the extraction of the seeds.
For the formulation of water stress, polyethylene glycol (PEG 6000) was used as the solute, which concentrations were formulated according to the specifications of Vilela et al. (1991) to obtain osmotic potential levels of -0.2; -0.4 and -0.6 MPa, the zero level (0.0) was used as control, using only distilled water to moisten the substrate.

Germination Test
The test was performed on germinators Biological Oxygen Demand (BOD) type at constant temperatures of 25, 30 and 35 °C with photoperiod of 8/16 hours of light and dark, respectively, using fluorescent lamps day-light type (4 × 20 W), using 100 seeds divided into four replicates of 25, treated with the fungicide Captan ® in the dose of 240 g for 100 kg of seeds. The seeds were then distributed over two sheets of germinating paper, covered with a third layer, arranged in a roll, the paper was moistened with the PEG 6000 solutions in the previous mentioned potentials, with an amount equivalent to 2.5 times the paper dry weight without further addition of the solution, only distilled water was used for the control. The rolls were stored in transparent plastic bags to avoid loss of water by evaporation and the evaluations were carried out daily, from the third to the twelfth day after the installation of the experiment, the criteria for evaluation used were normal seedlings, as described by Brasil (2009), with results expressed in percentage.

First Counting of Germination
Determined concurrently with the germination test, by counting the normal seedlings in the third day after installation of the test, the results were expressed in percentage.

Germination Speed Index
Performed by the daily counting of germinated seeds, at the same time, for 12 days, the index was calculated by the formula proposed by Maguire (1962).

Length and Dry Matter of Roots and Shoots
At the end of the germination test, the normal seedlings of each treatment and replication were measured (root and shoot) using a ruler graduated in centimeters, with the results expressed in cm. After the measurements, the roots and shoots of the seedlings, without the cotyledon leaves, were placed in paper bags and placed in a lab stove at 80 °C for 24 hours (Nakagawa, 1999). After this period the samples were weighed on an analytical scale with an accuracy of 0.001 g and the results expressed in grams. Below is a figure containing the total number of four repetitions in each osmotic potential of normal seedlings, which were put to dry in an oven, thus obtaining their dry mass.

Experimental Design and Statistical Analysis
The experimental design was completely randomized, with the treatments distributed in a 4 × 3 factorial scheme (osmotic potentials × temperatures). Data were submitted to analysis of variance using the F test (p < 0.05) for comparison of squares. For the quantitative effects, polynomial regression analysis was performed by testing the linear and quadratic models and selecting the one with the highest significant degree. The SISVAR software was used (Ferreira, 2007).

Results and Discussion
On Figure 2, it can be verified that the water potentials influenced the germination of C. echinata seeds regardless of temperature. At the temperature of 25 °C, the maximum germination (93%) was obtained in the osmotic potential of 0.0, with a decrease following the more negative water potentials. However, at this temperature, seed germination was higher in all osmotic potentials when compared to temperatures of 30 and 35 °C. The most abrupt decrease in germination was observed in seeds submitted to a temperature of 35 °C. Germination and vigor are negatively affected if temperature extremes tolerated by seeds (cardinal temperatures) are exceeded (Felix et al., 2018).
When associated with other factors, such as water deficit, temperature increase may reduce germination, as found for Caesalpinia echinata. Unlike C. echinata, germination and initial development of Combretum leprosum Mart. was impaired already at a temperature of 25 °C in the osmotic potentials from -0.2 to -0.5 Mpa (Leal et al., 2020). The decrease in germination of seeds under water stress is a response of the enzymatic activity (Santos, Calil, Ruiz, Alvarenga & Santos, 1992) and, for each species, there is a critical value of water potential, where in many cases germination does not occur (Carvalho, 2005).
In several studies with species of the Fabaceae family similar results were found. According to Silva, Aguiar & Rodrigues (2001), for Bowdichia virgiloides Kunth. after the potential level of -0.5 MPa of PEG 6000 the germination was reduced in a critical way to a point that in smaller potentials such as -0.9 MPa the seeds did not germinate.      Vol. 12, No. 7;