MATERIALS AND METHODS

The seeds of Crotalaria retusa were obtained from disturbed forests in coastal dunes, Municipality of Carmen, Campeche State, Mexico (18° 38' 36" N and 91° 49' 51" W), at 2 m above sea level, on slopes of 2-4%. The mature seeds were collected from 400 plants on December 2009.

The laboratory assays were done in the Seed laboratory of Natural Sciences Faculty, Universidad Autónoma del Carmen, Campeche State, Mexico, between January and February 2010. A day/night regime of 32/18°C was taken into consideration along with a 12 h photoperiod. Pregerminative treatment considered the immersion of seeds in 98% Sulphuric acid (H₂SO₄), for 0 (control), 5, 10, 20 and 30 min, after which seeds were washed with distilled water.

A total of 300 seeds (ten seeds with 6 replications for treatments) were sown in each experimental unit (petri dishes with Agrolite as substrate). The germination was observed daily early in the morning for a period 30 days. The seeds were considered germinated when the radicle reached the length of the seed. Seeds with roots or cotyledons malformed were not considered for the test. The seeds were irrigated with distilled water and a 3% Captan solution.

The experimental design used was completely randomized blocks with six repetitions. Germination was registered by replica per treatment during he duration of experiment. Germination frequencies for the same treatment were grouped and expressed as percentage. Results per treatments were analyzed through Kruskal-Wallis ANOVA by ranks and Wilcoxon Matched pairs test with significance level p<0.05.

Final results are shown as germination percentages per treatment considering cumulative frequency for each treatment. Excel was used for the data primary treatments and Statistica (v 8.0, Statsoft) for the rest of analysis.

RESULTS

All treatment applied were statistically significant (p = 0.05). The C. retusa radicles started to emerge two days after the establishment of the experiment. The seeds with immersion in 98% H₂SO₄ treatment responded positively in all cases compared with the control when the temperature was under 32/18°C day/night Fig. 1. When the H₂SO4 is used, germination percentage is over 64% at day 29 in the all seed treated in contrast with 35% obtained at the control Fig. 2.

Fig. 1:	Frequency of germination by chemical scarification (H2SO4) treatment expressed as mean percentage. Error bar denote range of germination percentage including 6 replicates. Different letters means significant difference

Fig. 2:	Accumulated germination frequency (%) of C. retusa seeds with chemical scarification during 30 days of experiment

In concordance with statistical analysis, seeds exposed to immersion for 30, 20 or 5 min show similar behavior at the end of the experiment, however when the seed are treated for 30 min, the range of germination percentages by replica are very short in contrast with the other treatments and germination rate is increased, reaching more than 20% at day 3 while the same result were obtained after 9 days for the rest of the assays. The other treatments get no more than 68% of germination at the end of the assay. All treated seeds had significantly higher germination percentages than the control which only obtained 35% of germination at the end of the assays (Fig. 2).

The trend observed at different treatments shown that conditions proposed for the experiments are adequate for germination and the application of 98% H₂SO₄ for 30 and 20 min, significantly increased the germination of C. retusa.

DISCUSSION

The seeds of Crotalaria obtained from a natural environment and the H₂SO₄ scarification treatments simulated pass of the seeds through the digestive tract of animals (birds and rodents) (Chavez et al., 2010; Righini et al., 2004), which under natural conditions execute chemical scarification (Carpinellia et al., 2005).

Seed dormancy is an innate seed property that defines the environmental conditions in which the seed is able to germinate (Finch-Savage and Leubner-Metzger, 2006). Otherwise, characteristics of seeds as coat thickness and coat hardness, time and conditions where they are stored and treatment previously offered are involved at sprouting.

Effect of dormancy breaking method on germination rate depends on the plant species, thus it can be suggested that the most effective method in breaking the dormancy of forage legumes should be determined (Balouchi and Sanavy, 2006) by the application or each method with different period, concentration or degree combinations to the seeds in order to maximum germination rate. Our results demonstrate that H₂SO₄ treating were able to enhance germination because seed dormancy in C. retusa is reduced when the acid act over the seeds coat improving germination percentage.

Similar trends were found by Acosta-Percastegui and Rodriguez-Trejo (2005) in L. montanus, who reported higher percentage of germination with chemical scarification (Sulphuric acid) for 15 min at temperatures of 20/15°C with light obtaining 100% germination. Chavez et al. (2010), reported higher percentage of germination with chemical scarification (Sulphuric acid) for 15 min at temperatures of 25/15°C with light (82.3% of germination) in L. leptophyllus. Chemical germination was found in seeds of Calystegia soldanella pretreated with H₂SO₄ for 3 h (Ko et al., 2004). Chemical applications also improve germination in such species as S. obtusifolia. A positive effect of H₂SO₄ are seen when the seeds were pretreatments with reaching about 90% germination. (Baskin et al., 1998). Applications also improve germination in such species as Capparis spinosa, where positive effects are seen with pretreatments of either H₂SO₄ for 20 (Sozzi and Chiesa, 1995).

From the results of this research that simulates the effects of the pass throughout animal digestive tract in germination and from the literature reviewed it is inferred that C. retusa have about 35% germinative efficiency in natural conditions (without treatment) and when the seeds are consumed by rodents and other animals, the digestion process helps scarification and defecation the dispersion of the seeds throughout the ecosystem.

The positive response to sulfuric acid treatments indicates that the impermeable coat is responsible for a low germination rates from intact seeds as seen at experimental untreated controls. By overcoming the physical dormancy with chemical pretreatment, seed coats are softened and water uptake is enabled. Both actions are crucial to sustaining the life cycle of hard-seeded species.

CONCLUSIONS

Germination was affected by all H₂SO₄ scarification treatments. The treatment of immersion in sulphuric acid for 30 min gave 98.3% of germination after 30 days seeds were sown but germination rate was the highest during the two first weeks of experiment. For purposes of re-colonization in forest areas is recommended the use of these treatments.

ACKNOWLEDGMENT

Special thanks to PROMEP granting the internal project number PROMEP/103.5/09/4162, without who’s financing the development of this research would not be possible.