Priming and stress under high humidity and temperature on the physiological quality of Brachiaria brizantha cv . MG-5 seeds

Palisade grass is a forage plant that is widely used in pasture cropping in the Brazilian savannah. The aim of this experiment was to evaluate palisade grass (Brachiaria brizantha cv. MG–5) seeds subjected to priming and stress at high humidity and temperature (before and after conditioning). The experimental design was completely randomized in a 2x5 factorial arrangement. Seeds were exposed to stress under high humidity and temperature (before and after conditioning) and five priming treatments [Water (Control), potassium nitrate (KNO3) at 0.2%, calcium nitrate Ca(NO3)2 at 0.2%, gibberellin (GA3) at 0.2% and glucose at 10%] with four replications. Two experiments were performed: Experiment I seed with chemical scarification using H2SO4 and Experiment II without scarification. The stress on the seed was applied using artificial aging at 41°C for 96 hours. Seed priming was accomplished by immersion at 25°C for 2 hours. Thereafter, the seeds were oven-dried at 35°C until they regained hygroscopic equilibrium. Seed germination and vigor were evaluated. Priming using KNO3 and Ca(NO3)2 produced seeds with high tolerance to stress under high temperature, and this process is efficient to overcome dormancy.


Introduction
Plants of the Brachiaria genus are widely used in pasture establishment in the Brazilian savannah.Because pasture implantation occurs through seed, it is imperative that high-quality seed is sown.
Seed dormancy occurs in B. brizantha, which is a limiting factor to crop establishment.Dormancy in this species is linked to several factors, especially physical attributes that are related to seed coat restrictions imposed by the glumes (lemma and palea), pericarp and tegument (Binotti, Sueda Junior, Cardoso, Haga, & Nogueira, 2014).Under laboratory conditions (Ministério da Agricultura, Pecuária e Abastecimento [Mapa], 2009), seed technologists recommend chemical scarification using sulfuric acid to overcome dormancy.In addition, supplying the germination substratum with potassium nitrate (KNO 3 ) and oven-dry thermal treatment can be used for dormancy release.Munhoz, Zonetti and Roman (2009) found that five minutes of chemical scarification is effective to overcome the dormancy of Brachia brizantha cv.MG-5.Meschede, Sales, Braccini, Scapim and Schuab (2004) reported that dormancy can be overcome by artificial aging, but the results depend on the physiological quality of the seed lot.However, this practice can expose the seeds to conditions that reduce their physiological potential (Cardoso et al., 2014).
Priming is an alternative method to stress processes (Paparella, Araújo, Rossi, Wijayasinghe, Carbonera and Balestrazzi (2015).Seeds are often subjected to priming, as reported by Oliveira and Gomes Filho (2010) for sorghum seeds.These authors reported that priming partially reversed the negative effects of aging.Priming can improve the seed quality of Brachiaria and other grasses, e.g., Bonome, Guimarães, Oliveira, Andrade and Cabral (2006) reported the effects of osmoconditioning on the uniformity of germination after applying KNO 3 to the seeds of B. brizantha cv.Marandu for 12 hours, and Aragão et al. (2003) reported increased germination and expression of vigor when supersweet sweet corn seeds were subjected to presoaking using gibberellic acid.Among the techniques used for priming, Soltani & Soltani (2015) highlights that hidropriming (direct immersion) is the most likely application.
The objective of this experiment was to evaluate the physiological quality of Brachiaria brizantha cv.MG-5 using seed priming and stress under high humidity and temperature conditions.This objective was based on the abovementioned factors, the need to overcome seed dormancy of Brachiaria, the benefits of priming on the quality of the seed lot and the limited number of studies on the effects of these practices on Brachiaria seeds.

Material and methods
The experiment was carried out from November 2013 to February 2014 using Brachiaria brizantha cv.MG-5 seeds from the 2013/14 growing season.
The experimental design was completely randomized using a 2 x 5 factorial arrangement that corresponded to seeds exposed to stress under high humidity and temperature (before and after conditioning) and five priming treatments [Water (Control), potassium nitrate (KNO 3 ) at 0.2%, calcium nitrate Ca(NO 3 ) 2 at 0.2%, gibberellin (GA 3 ) at 0.2% and glucose at 10%] with four replications.Two experiments were performed: Experiment I -seed with chemical scarification using H 2 SO 4 and Experiment II -without scarification.
In Experiment I, the seeds were subjected to chemical scarification using concentrated sulfuric acid for 5 min.Thereafter, they were rinsed using running deionized water and placed on paper towels to dry.
Stress at high humidity and temperature was induced at 41ºC for 96 hours using the Gerbox method for artificial seed aging (adapted Marcos Filho, 1999).Seeds were primed using hydration at 25°C for 2 hours.The conditioning products were diluted in deionized water by preparing 100 mL of the solutions in plastic recipients placed in germination BOD chamber.For every treatment, we evaluated 9 g of B. brizantha seeds (subsamples were collected from each lot).Thereafter, the seeds were oven-dried at 35°C until they reached the same hygroscopic equilibrium as that before the priming application.
After treating the seeds, the following evaluations were conducted using pure seeds: 'Germination test' -This evaluation was based on 4 subsamples of 50 seeds per treatment that were distributed in plastic boxes (11.0 x 1.0 x 3.5 cm).Counting of normal seedlings occurred on the 7 th ('First count of percent seed germination') and 21 st days after sowing, based on the Rules for Seed Testing (Mapa, 2009).These results were expressed as a percentage; 'Dormant seeds' -The presence of seed dormancy was evaluated using the tetrazolium test described by the Rules for Seed Testing (Mapa, 2009) for Brachiaria seeds and was calculated using the number of remaining viable seeds from the germination test; 'Electrical conductivity' -The electrical conductivity of the seed soaking solution was measured using the 'Bulk Conductivity' test that was carried out using four subsamples of 50 seeds, weighed to at least two decimal places.The seeds were imbibed in 75 mL of deionized water and maintained at 25ºC for 24 hours.Then, the electrical conductivity of the soaking solution was measured and the results are expressed as μS cm -1 g -1 ; 'Seedling Emergence test' -Seedling emergence was evaluated under greenhouse conditions using 4 subsamples of 50 seeds per treatment sown at a depth of 1 cm in perforated trays to allow water drainage from the vermiculite.The percentage of seedling emergence was evaluated within 28 days of sowing; emerged seedlings were those with a shoot length longer than 20 mm.
'Statistical Analysis': The data were evaluated using analysis of variance (F test; p ≤ 0.05).When significant, we used the Tukey test to compare treatments.These analyses were performed using the Sanest program.

Results and discussion
Priming and stress interactions at high humidity and temperature were evaluated for the first count of germination, total germination, and percent dormancy of chemically scarified seeds (Table 1).The results showed the highest germination percentage (94%) at 21 days was obtained with priming using KNO 3 or Ca(NO 3 ) 2 followed by stress.These treatments represented a significant gain of 42% compared with the germination (52%) obtained in response to water and gibberellin (priming followed by stress).Priming using KNO 3 or Ca(NO 3 ) 2 followed by stress resulted in rapid germination based on the first germination count (7 days).The B. brizantha seeds conditioned with KNO 3 or Ca(NO 3 ) 2 showed higher tolerance to stress at high humidity and temperature, indicating that the expression of their physiological potential with the use of these agents was not affected even after stress (Table 1).
These results are similar to those presented by Binotti, Sueda Junior, Cardoso, Haga and Nogueira (2014), who found an improvement in the germination rate of B. brizantha cv.MG-5 seeds resulting from a pre-germinative treatment using KNO 3 at 0.2%.Reis, Silva, Neves and Guimarães (2013) also confirmed a positive effect on the physiological quality of gherkin seeds in response to KNO 3 .Pereira, Borges, Oliveira, Leite and Gonçalves (2010) verified that soaking P. reticulate seeds in nitric oxide reduced the germination inhibition caused by accelerated aging.
We found that priming using KNO 3 and Ca(NO 3 ) 2 , even after the stress process, was able to break dormancy in B. brizantha cv.MG-5 seeds.In the treatments with these agents, the percentage of dormant seeds was 0 and 1% for KNO 3 and Ca(NO 3 ) 2 , respectively, compared with 39 (water), 38 (gibberellin) and 32% (glucose).Priming using water, Ca(NO 3 ) 2 or gibberellin after stress promoted lower number of seeds dormancy with 22% of water and 12% of Ca(NO 3 ) 2 and gibberellin against 39 (water), 1% Ca(NO 3 ) 2 and 38% (gibberellin) before stress (Table 1).We concluded that after overcoming dormancy, the entrance of water and diffusion of gases occurred, caused by the chemical removal of the seed coat through scarification, and the secondary dormancy experienced by the B. brizantha seeds, which was probably associated with physiological processes, was released with the use of KNO 3 and Ca(NO 3 ) 2 .
Gibberellin is a hormone that acts directly in germination and dormancy breaking (Cardoso, 2013).When applied after stress, gibberellin was able to improve seedling emergence; however, when applied after stress, this effect did not occur even within the germination and dormancy breaking patterns, pointing to the sensitivity of this hormone to deterioration processes (high humidity and temperature).Câmara and Stacciarini-Seraphin (2002) reported that gibberellin applied to B. brizantha cv.Marandu showed reduced action under application conditions used for the seeds and during subsequent storage.
Priming using Ca(NO 3 ) 2 or KNO 3 resulted in a lower electrical conductivity (30.14 μS cm -1 g -1 ) than that obtained using glucose (38.34 μS cm -1 g -1 ) but did not differ from the other treatments.Stress after priming resulted in a lower rate of 31.94 versus 34.61 μS cm -1 g -1 for stress before priming (Table 2).Thus, we concluded that primed seeds under conditions that lead to deterioration tend to have less damage caused to their cell membranes, which results in less loss of cellular components.The effects of priming and stress at high humidity and temperature were determined based on the first germination counts, total germination, percent dormant seeds and electrical conductivity of non-scarified seeds (Table 3).Means followed by different lowercase letters within columns and uppercase letters within rows for priming and for stress at high humidity and temperature, respectively, vary at 5% probability based on the F test and the Tukey test; C.V., coefficient of variation.
A higher total germination percentage (80% at 21 days) was observed through the application of KNO 3 and Ca(NO 3 ) 2 before stress in contrast to 38% (water and gibberellin) and 41% (glucose), i.e., a significant gain of 42 and 39%, respectively.A higher first germination count (7 days) was also observed with the use of KNO 3 (of the 84% available seeds, 75% had already germinated) and Ca(NO 3 ) 2 (of the 88% available seeds, 63% had already germinated).Similar responses were observed for the scarified seeds, which confirm the effectiveness of KNO 3 and Ca(NO 3 ) 2 application before stress at high humidity and temperature (Table 3).
There was a decrease in the number of dormant seeds with the use of KNO 3 and Ca(NO 3 ) 2 before stress, i.e., 4 and 8%, respectively, in contrast to 44 (water and gibberellin) and 37% (glucose).KNO 3 application after stress resulted in a lower number of dormant seeds, i.e., 23% compared with 42 (water), 41 (gibberellin) and 44% (glucose) (Table 3).This result is different from that obtained by Cardoso et al. (2014) who did not observe dormancy breaking using priming with 0.2% KNO 3 in nonscarified B. brizantha cv.MG -5 seeds that went through artificial aging (stress at high humidity and temperature) before priming.
However, Cardoso et al. (2014) observed that increasing periods of aging (stress at high humidity and temperature) positively influenced dormancy breaking, which was also verified by Meschede et al. (2004).However, these authors also highlighted that this effect depends on the physiological quality of the initial lot because aging (stress at high humidity and temperature) can break physical dormancy in non-scarified B. brizantha seeds.
When applied before stress, these treatments reduced the number of dormant seeds, e.g., the application of KNO 3 before stress resulted in 4% dormancy compared with 23% after stress.However, the results obtained with the application of Ca(NO 3 ) 2 (8% before stress and 36% after stress) showed a significant decrease in the number of secondary dormant seeds, i.e., 19 and 28%, respectively, when applied before stress (Table 3).According to Cardoso (2013), nitrate stimulates seed germination by reducing seed dormancy.Seshu and Dadlani (1991) observed that dormancy in rice seed is controlled by nonanoic acid and abscisic acid in the hull and pericarp.The presence of these compounds have negative effects on the activity of amylase, which is an enzyme related to starch break down.Thus, the percentage of seed germination is improved, and pre-soaking in KNO 3 reduces the seed dormancy imposed by the nonanoic acid.
There was better organization in the membrane system (after stress) in response to the application of water, KNO 3 and gibberellin, based on the average values of 8.78, 12.75 and 9.86 μS cm -1 g -1 , respectively, compared with glucose (20.04 μS cm -1 g -1 ).The application of Ca(NO 3 ) 2 before stress promoted higher seedling emergence (74%), which indicates a significant gain of 28 and 17% over the 46 and 57% seedling emergence measured with the use of water and gibberellin, respectively (Table 3).

Conclusion
Seeds conditioned using KNO 3 and Ca(NO 3 ) 2 show higher tolerance under adverse conditions such as high humidity and temperature.
Priming using KNO 3 and Ca(NO 3 ) 2 followed by artificial aging treatments is efficient in breaking dormancy.
Gibberellin application through priming provides beneficial effects to seeds that are not exposed to deterioration processes.

M
Means followed by different letters within columns differ significantly at 5% probability based on the Tukey test and the F test; ** significant at 1% probability; C.V., coefficient of variation.

Table 1 .
The effects of priming and stress under high humidity and temperature on the first germination count, total germination, and percent dormancy of scarified B. brizantha MG-5 seeds.different lowercase letters within columns and uppercase letters within rows for priming and for stress at high humidity and temperature, respectively, vary at 5% probability based on the F test and the Tukey test; C.V., coefficient of variation.
MMeans followed by

Table 2 .
Electrical conductivity in B. brizantha MG-5 in response to priming and stress under high humidity and temperature.

Table 3 .
The effects of priming and stress under high humidity and temperature on the first germination count, total germination, and percent dormancy of non-scarified B. brizantha MG-5 seeds.