Introduction

Brachiaria brizantha (Hochst. ex A. Rich.) Stapf is a perennial plant native to tropical Africa belonging to the Poaceae family. In Mexico it was introduced as an Insurgent variety and due to its high performance as a good quality feed and excellent adaptation by livestock, it is one of the fodder species most used by ranches in tropical areas (^{Peralta, 1990}). However, the low availability and quality of the seed are limiting factors to the expansion and renewal of cultivated areas of this fodder species. In addition, the presence of seed dormancy (^{Humphreys and Riveros, 1986}; ^{García and Cícero, 1992}; ^{Meschede et al., 2004}) is another factor to the minimal success obtained in the establishment of meadows of this important species.

Dormancy is the state in which the seeds, although having normal environmental conditions for their germination, do not germinate due to physical and physiological mechanisms (^{Copeland, 2001}). This characteristic is the most important factor that affects the germination of seeds of the Brachiaria genus and, consequently, limits the adequate establishment of meadows (^{Quero-Carrillo et al., 2014}). The main causes for dormancy in grass seeds include the presence of immature embryos (^{Hopkinson et al., 1998}), impermeability of the seed cover to water and gasses, special temperature and light requirements, the presence of inhibitors (in accessory bracts and embryos) and mechanical restrictions of the embryo on the growth and development or exertion and extension of the radicle during germination. Nevertheless, seed dormancy is eliminated in a natural manner during a “training” period, through its storage of three to eight months (Enríquez and Quero, 2007), depending on the climatic conditions of the storage place. Therefore, if the seed is used for the establishment of meadows immediately after harvest, it is possible that it will have low or null germination and, thus, lead to failure in the establishment of the meadow (Enríquez and Quero, 2007). However, this limiting factor of the seeds can be improved through artificial means with the use of scarification methods prior to cultivation (^{García and Cícero, 1992}; ^{Camacho, 1994}; ^{Enríquez and Quero, 2006}).

Among the methods to interrupt seed dormancy are the following: pre-refrigeration, different temperature combinations, 0.2% nitrate solution, gibberellic acid, pre-washing, pre-drying, sulfuric acid, among others (^{Faria et al., 1996}). Temperatures raised to 70 ºC for 15 hours have resulted in the reduction of seed dormancy in the B. brizantha Marandú variety, with improvements on germination and no viability deterioration (^{Martins and da Silva, 2001}). Likewise in Aeluropus lagopoides, it has been shown that alternating average temperatures (20/30 ºC) improves germination in this species of temperate climate; thus, temperature is an important factor for this purpose (^{Gulzar and Kahn, 2001}).

When the Brachiaria hybrid Mulato variety is stored in a protected warehouse chamber in Thailand (^{Hare et al., 2008}), it has been indicated that germination rises to 78% at four months of warehouse storage and all the seeds die at 20 months, contrary to its storage in a protected chamber and at cool room temperature, reaching 80% germination at 14 months after harvest and maintaining these levels of germination for three years. In Buffel grass (Cenchrus ciliaris L.), alternate temperatures of 25 and 35 ºC resulted in better viability, vigor, speed of germination and total germination (^{Machado et al., 2013}). The germination temperature for B. brizantha has been reported at 25 ºC, with no photoperiod (^{Chiodini and Araujo, 2013}).

Sulfuric acid is one of the most recommended chemical methods for the rupture of dormancy in recalcitrant seeds of tropical fodder species. Various studies have demonstrated the effectiveness of sulfuric acid in improving the germination of B. brizantha seeds (^{Faria et al., 1996}; ^{Martins and Da Silva, 2003}; ^{Usberti and Martins, 2007}), given that it dissolves, cracks, and weakens the coverings of the spikelets and mainly the leathery palea fused to the caryopsis in the Panicoideae subfamily of the Brachiaria genus, which allows for the entrance of water and the exchange of gasses necessary for the germination process, thus facilitating the expansion of the embryo and the exit of the radicle (^{Ramos, 1975}; ^{Zulay et al., 1998}). Nevertheless, in practice, the use of this treatment is scant with one of the reasons being that it has security risks involved during its handling and application.

^{Meschede et al. (2004)} indicate that the removal of the glumes from the caryopsis presented the best germination results in three lots of B. brizantha cv. Marandú seeds, with an average of 60.3%. In this regard, it has been noted that the presence of leathery lemma and palea tied to the caryopses and inhibitors are the causes of seed dormancy of those belonging to the Brachiaria genus, hindering germination (^{Quero et al., 2007}). Hormonal substances such as gibberellic acid have been recommended in order to improve the germination of seeds of tropical and temperate fodder grasses; however, information that would allow for its practical application is required for its use in the improvement of the germination of fodder seeds, and for the establishment of meadows in Mexico. The objective of this study was to evaluate the effect of various treatments that stimulate the germination of the Brachiaria brizantha cv. Insurgent seed.

Materials and methods

The study was developed in the Seed Analysis Laboratory of the Instituto de Recursos Genéticos y Productividad (IREGEP) of the Postgraduate College, Campus Montecillo. Two lots of Brachiaria brizantha cv. Insurgent seeds were used, harvested by hand on September 28 (fall; lot 1) and December 21 (winter; lot 2), 2007, from the same meadow in the Experimental Field of the Universidad del Papaloapan located in Loma Bonita, Oaxaca.

Seed harvesting was done by hand using the traditional technique for tropical grasses (^{INIFAP, 1989}), which consists in cutting the inflorescences and, subsequently, subjecting them to a natural drying process; this increases the maturity of the seeds with a proper development of the embryo. The high humidity content and the accelerated desiccation of the plant facilitate the detachment of the seeds. In order to simulate the drying process, the harvested inflorescences were placed on plastic in the B. brizantha meadow and were covered with the remaining plant material after having cut the inflorescences. The drying period lasted four days. Subsequently, a dusting process of the inflorescences was carried out, cleaning and drying the seeds in the shade in a natural manner. The seeds obtained were placed in a kraft paper bag and stored in laboratory environmental conditions (darkness at a constant temperature of 18 ºC) in Montecillo, Texcoco, Estado de México.

At the start of the study, the physical and physiological qualities of both lots of seeds were determined in terms of: physical purity; weight per 1 000 seeds; and viability, using the tetrazolium test (Table 1). Seven manual and chemical scarification treatments were evaluated, with sulfuric acid (H₂SO₄) at a concentration of 98% and gibberellic acid AG₃ (Table 2). A completely random design was utilized, with four repetitions of 100 seeds per treatment. Following the application of the scarification treatments, the germination percentage was evaluated for four, five, and six months after harvest (lot 1) and for two, three, four, five, and six months after harvest (lot 2), using the standard germination test.

The seeds were placed in 14 x 14 x 5.5 cm “sandwich” boxes with lids, fitted with absorbent paper and placed inside a germinating chamber at a temperature of 25 ± 1 ºC, 8 and 16 light: darkness hours, respectively, and 100% relative humidity for a duration of 21 days (ISTA, 2005). The seeds and the material used in the germination test were disinfected with 5% chlorine for 5 minutes and, subsequently, were washed with distilled water (^{Meschede et al., 2004}).

After the immersion period of the seeds in a sulfuric acid based treatment, the seeds were washed in running water for 5 minutes in order to remove any sulfuric acid residues (^{García and Cícero, 1992}). The sapling count began on the eighth day after planting and ended on the 20th day after planting, i.e. a period of 12 days for potential germination for all treatments. The data obtained was submitted to an analysis of variance in order to detect any differences between treatments. The comparison of treatment means was carried out using the Tukey test with a level of significance of 0.05 (^{SAS, 1998}). All the values were transformed prior to the arc sine (%)/100 in order to normalize distribution and, subsequently, transformed to their original value for discussion.

Results and discussion

The seed germination percentages obtained from the different scarification treatments for lot 1 with four, five, and six months of storage presented differences (p< 0.05; Table 3). Significant differences were observed between treatments (p> 0.01) at four months of storage, with the largest germination value (58%) being obtained with the immersion treatment of the caryopses in AG3 at a concentration of 400 ppm, for 5 minutes (T4). A similar value (p> 0.05) to those was obtained with treatments T3 and T2 (55 and 52%, respectively), but different and superior (p> 0.05) to treatment T1 (control), which presented a germination value of 11.5%. A similar behavior to the aforementioned was observed at five and six months of storage, where the highest values were obtained with treatments T4, T3, and T2.

The storage period (considered the normal preparation time for tropical grass seeds) had a positive effect on germination. The seed with six months of storage showed the greatest germination, independent of the scarification treatment; the highest values (80 and 76%) were registered at six months of storage with treatments T4 and T3, respectively. It is important to note that the use of concentrated sulfuric acid did not surpass the usage of AG3. Also, the fact that the caryopsis without treatment showed the least germination indicates the absence of this stimuli in the embryo, which can be interpreted as dormancy imposed by the embryo’s condition in this species: presence of inhibitors and lack of adequate promoters due to immaturity in order to reach germination.

The seed germination percentages obtained from the different scarification treatments for lot 2 with two, three, four, five, and six months of storage showed significant differences between treatments p> 0.01 (Table 4). At two months of storage, the greatest germination value (55.6%) was observed with treatment T4, a value similar (p> 0.05) to those obtained with treatments T3 and T2 (51 and 46%, respectively), but different and superior (p> 0.05) to the other treatments, mainly with regard to the control which presented a germination value of 3%. A similar behavior to the aforementioned was observed at three, four, five, and six months of storage, where the highest germination values were obtained with treatments T4, T3, and T2; the answer to the germination promoters is not affected by the post-harvest age of the seed. As with lot 1 (fall lot), an increase was observed in the germination percentage, independent to the scarification process, as the seed’s storage time was increased. The highest values (92.5, 92.4, and 86.4%) were registered at six months after harvest with treatments T4, T3 and T2, respectively.

The results found in this study indicate that all the evaluated scarification treatments, independent of the lot, increased the germination percentage of the Insurgent grass seeds in comparison with the control, with the best scarification treatments being the combination of manual removal of the structures (accessory bracts - glume, lemma and palea) that encase the caryopsis and the subsequent immersion of the caryopsis in gibberellic acid solution with a concentration of 300 and 400 ppm, for 5 minutes.

The increase in the seed germination percentage by removing its structures is due to it allowing the entrance of water and the exchange of gasses, which facilitates the expansion of the embryo and the resulting exertion of the radicle (Zualy et al., 1998; ^{Mérola and Díaz, 2012}). In the case of the subsequent immersion of the caryopsis in gibberellic acid solution, its penetration towards the embryo is promoted through the cracked head towards the embryo, advancing its growth, resulting in higher germination.

Similar results were reported by other researchers (^{Meschede et al., 2004}) who, when evaluating treatments to break dormancy in three lots of B. brizantha cv. Marandú seeds, found that the removal of glumes was the treatment with the greatest germination values, at 60%. This same answer was reported by other authors (^{Vieira et al., 1998}) who, when evaluating different scarification treatments on caryopses of B. brizantha cv. Insurgent, found that the evaluated treatments were always superior to the control.

In this same species, other authors (^{Faria et al., 1996}; ^{Martins and Da Silva, 2003}; ^{Usberti and Martins, 2007}) have reported that the immersion of the seeds in sulfuric acid, for 5 to 15 minutes, improves germination by more than 30% in comparison with the control. On the other hand, Brachiaria brizantha cv. Marandú seeds were used, subjected to immersion in H2SO4 diluted to 50% for 4 minutes, finding a germination of 34%, with this treatment providing the best response in comparison to seven other treatments (^{Martinez et al., 2013}). The increase in germination was due to the fact that the acid dissolved part of the palea and lemma of the spikelet, causing cracks and weakening the coverings of the spikelet, which permitted water to enter and the exchange of gasses to occur, facilitating the expansion of the embryo and the exit of the radicle (^{Ramos, 1975}; Sulay et al., 1998). It has been noted that in seeds belonging to the Brachiaria genus, the presence of leathery lemma and palea tied to the caryopsis hampers germination (^{Quero et al., 2007}), given that it hinders water absorption causing failures that result in the recalcitrance of the seed (^{Jiménez, 1990}).

It was observed in both lots of seeds that the differences in germination percentages obtained with the various scarification treatments in comparison with the control were broader at two (lot 1) and four (lot 2) months of storage of the seed, which indicates a decisive effectiveness of the scarification treatments evaluated in incrementing the germination of the recently harvested B. brizantha cv. Insurgent seeds and, similarly, a different response between seeds harvested in fall and those harvested in winter.

The effectiveness of the scarification treatments was less as the storage period of the seed increased. The aforementioned has been reported for B. humidicola, where scarification with concentrated sulfuric acid had a major effect during the first nine months of storage of the seed (^{Costa et al., 2011}). For example in lot 1, with the best scarification treatment (immersion of the caryopsis in gibberellic acid solution at a concentration of 400 ppm) at four to five months of storage, a 33% germination increase was obtained; whereas at five to six months of storage, there was only a 4% increase. A similar behavior to the aforementioned was observed for lot 2, where during the period of two to three months of storage, germination increased 179%; whereas at five to six months of storage, there was only a 1% increase. This confirms the maturity process of the embryo and lesser need for external stimuli such as gibberellic acid and/or aggressive sulfuric acid treatments beyond opening the exchange of gasses and the weakening of the structures for the exertion of the radicle and plumule. On the other hand, the low germination percentages (11.5 and 3%) obtained with the control treatment at four months (lot 1) and two months (lot 2) of storage, respectively, corroborate the high seed dormancy levels in this fodder species, same that is imposed by the embryo.

It has been noted that the B. brizantha seed shows high dormancy (^{Humphreys and Riveros, 1986}; ^{García and Cícero, 1992}; ^{Enríquez and Quero, 2006}; ^{Ascorra and Lara, 2003}), which is eliminated in a natural manner during a storage period of two to eight months; or rather, in an artificial manner, through the application of mechanical and chemical scarification treatments (^{Enríquez and Quero, 2006}).When evaluating the effect of the time of storage on the germination of B. brizantha seeds, ^{Ascorra and Lara (2003)} found germination values of 2, 3, and 48% at 0, 3.5, and 6.5 months of storage, respectively.

The low seed germination during the first months after having been harvested is due to it being locked by the vigorous structures that cover the caryopsis (palea and lemma); similarly, due to the presence of inhibitors and/ or the lack of response under germination conditions and stimulants of this on the part of the immature embryo or with an excess of inhibitors. ^{Vieira et al. (1998)} indicated that dormancy is not only due to the hard structures of the seed, but rather there is also another dormancy mechanism attributed to the presence of substances that inhibit germination within the embryo, or rather, due to the absence of growth promoting substances, which implies that in order for them to germinate, it is necessary to eliminate the inhibiting components through the manual separation of the covers or through the use of chemical substances such as sulfuric acid and growth promoting substances such as gibberellic acid.

This study also revealed better germination in lot 1 (fall) compared with lot 2 (winter). In lot 1, the seeds without scarification treatment at four months of storage showed a germination of 11.55%, whereas for lot 2 with the same storage period, the seeds showed a germination of 22.9%. The aforementioned is in response to the ripening environment of the seed in the parent plant. Two aspects are notably different at these stages: decreasing night temperatures and greater photoperiods, which are difficult to document without adequate strategies for their discernment.

Likewise for lot 1 (fall harvest) with the best scarification treatment (immersion of the caryopsis in gibberellic acid solution at a concentration of 400 ppm) and at four months of storage, a 58% germination was obtained; whereas for lot 2 (winter harvest) at four months of storage, a 90% germination was observed. This result is attributed to the better physical and physiological qualities of the seeds in lot 2, as a response to the fertilizing, development, and ripening environmental conditions on the parent plant. In this regard, it has been noted that the low germination of the recently harvested seeds is due to the presence of embryos that have not yet fully developed (^{Hopkinson et al., 1998}; ^{Enríquez and Quero, 2006}).

Another implicated factor can be the rate of development of the callus from abscission that is under the glumes (^{Enríquez et al., 2005}), which can be slower at a lower temperature. The aforementioned would allow better nutrition of the embryo by photosynthates of the parent plant, during its development, given that the callose (carbohydrate that forms a barrier between the parent plant and the in-development seed) reduces the communication between the seed and the parent plant, provokes the fall of the seed and is formed more slowly at a lower temperature and in response to the hours of light. The best nutrition for a greater period of time can result in better developed embryos, which in turn result in seeds with embryos more resistant to the environment and responsive to conditions or germination promoters, improving the embryo: endosperm relation, among other aspects.

Conclusions

The scarification treatments improve the germination of the Insurgent grass seed with regard to the full spikelet. The highest germination values were obtained with the elimination of floral bracts (glume, lemma and palea) through the immersion of the caryopsis in gibberellic acid solution at a concentration of 300 and 400 ppm for 5 minutes. The removal of the accessory bracts from the caryopsis and the effect of the gibberellic acid improve germination on Brachiaria brizantha. The seed with the best germination quality was harvested in winter; therefore, the handling of floral induction could program them so that they ripen during the second fortnight of December. It is recommended that this study is continued in order to determine with greater precision the best method to promote germination in seeds belonging to the Brachiaria genus.