Use of phytoregulators in overcoming macaw palm seed dormancy

The macaw palm is a tropical palm with significant potential for biofuel production; however, seed dormancy is a major factor limiting its agro-industrial use. The present study evaluated the effects of phytoregulators in overcoming macaw palm seed dormancy. We evaluated the effects of immersion in solutions of gibberellic acid (GA3) (0, 2000 and 5000 mg L ) during two exposure times (24 and 48 hours), associated with the removal or maintenance of the opercular tegument, as well as the effects of the associations between GA3 (2000 mg L ), indole-3-butyric acid (IBA) and benzylaminopurine (BAP) and the effects of repeated applications (one, two or five) of combinations of these phytoregulators. The seeds were sown in vermiculite and incubated in a humid growth chamber at 95 ± 5% relative humidity and 30°C for 18 weeks in all experiments. GA3 application and removal of the opercular tegument had positive effects on germination, but no significant differences were observed in immersion times for this phytoregulator. The application of IBA and BAP did not influence germination. The application of GA3 on five separate occasions gave the best results, with 41% germination at the end of the experiment.


Introduction
Acrocomia aculeata (Jacq.)Lodd.ex.Mart.(macaw palm) is an oleaginous palm tree that is native to the tropical Americas and has a wide geographical distribution; it is especially common in south-eastern Brazil (LORENZI et al., 2004;MOTTA et al., 2002).This species has significant potential for producing industrial quantities of biofuel because of its high productivity and tolerance to dry environments (CLEMENT et al., 2005;MOURA et al., 2009).Seed dormancy, however, restricts large-scale commercial plantings and its extensive agro-industrial use (RIBEIRO et al., 2011).Dormancy is the intrinsic blockage of the germination of viable seeds under conditions in which non-dormant seeds germinate (FINCH-SAVAGE; LEUBNER-METZER, 2006;LINKIES et al., 2010).Delayed seed germination in palms has been associated with morphological dormancy due to embryo immaturity (BASKIN; BASKIN, 1998;OROZCO-SEGOVIA et al., 2003) as well as physiological dormancy.Gibberellic acids (GAs) promote the germination of macaw palm seeds, indicating the existence of physiological dormancy (RIBEIRO et al., 2011).Additionally, embryos of this species that are cultivated in vitro germinate rapidly, indicating that their degree of maturation is not a limiting factor (RIBEIRO et al., 2012) and that their physiological dormancy is of the not deep type (BASKIN; BASKIN, 2004).
Numerous studies have demonstrated the efficiency of gibberellins in stimulating germination and overcoming physiological dormancy in palm seeds (CHIN et al., 1988;DEWIR et al., 2011;HERRERA et al., 1998;NAGAO et al., 1980;PÉREZ et al., 2008;ROBERTO;HABERMANN, 2010;YANG et al., 2007), but other factors can also influence the effectiveness of this hormone treatment.The quantity of gibberellin that is necessary to promote germination depends on the degree of restriction of embryo elongation imposed by the endosperm and tegument (DEBEAUJON; KOORNNEEF, 2000); the removal of either the operculum or opercular tegument is efficient in promoting germination in numerous palm species (CARPENTER et al., 1993;HUSSEY, 1958;PÉREZ et al., 2008).On the other hand, Nagao et al. (1980) demonstrated that the external tissues in Archontophoenix alexandrae (F.Muell.)H. Wendl.Drude and Ptychosperma macarthurii H. Wendl.restricted GA 3 penetration and that the removal of these tissues significantly increased the effects of this phytoregulator.Ribeiro et al. (2011) reported that immersing macaw palm seeds in 2000 mg L -1 of GA 3 for 24 hours in association with removal of the opercular tegument resulted in germination indices of 52% with the re-application of the phytoregulator.However, the effects of higher doses of this phytoregulator and the effects of longer periods of seed immersion were not evaluated; such treatments could increase germination and contribute to the establishment of protocols for commercial propagation programs.
There are many examples of the effects of plant hormones on germination and/or their interaction with gibberellins (BEWLEY; BLACK, 1994;KUCERA et al., 2005), and several studies have evaluated the effects of cytokinins on palm seed germination (VILLALOBOS et al., 1992;ROBERTO;HABERMANN, 2010).Ledo et al. (2001) demonstrated that the association of 1naphthaleneacetic acid (NAA) and BAP had positive effects on the in vitro germination of zygotic embryos of Euterpe oleraceae Mart.Studies evaluating the interactions between GA 3 and other phytoregulators may enhance our knowledge of A. aculeata seed germination.
The objective of this study was to evaluate the effect of the application of phytoregulators, the removal of the opercular tegument and the association between these treatments on macaw palm seed germination.

Characterisation of the seed lots
Fruits of A. aculeata were harvested soon after natural abscission from the wild population in the municipality of Montes Claros, Minas Gerais State,Brazil (16º42'34"S;43º52'48"W) and were transported to the Micropropagation Laboratory of State University of Montes Claros, where the experiments were conducted.Two different lots of seeds that were collected at different times were used in the experiments; these lots were identified as lots 1 and 2. The lots were stored in the shade for five and four months, respectively.To obtain the seeds, the fruits were broken with the aid of a bench-top vice; damaged seeds were discarded.The seeds were surface-sterilised in a 6% chlorine solution for 10 min., followed by three rinses in running water.The water contents of the seeds in each lot were determined by drying them for 24 hours at 105ºC (BRASIL, 2009).
Effects of the removal of the opercular tegument, GA 3 dose and immersion time The seeds were maintained in polystyrene trays containing vermiculite for seven days in a humid growth chamber at 95 ± 5% relative humidity at 30°C.The opercular tegument was removed from half of the tested seeds using a razor blade under a stereomicroscope (RIBEIRO et al., 2011).The seeds were subsequently immersed for 24 or 48 hours in aqueous solutions containing 0, 2000 or 5000 mg L -1 of GA 3 (PROGIBB ® ) and then returned to the humid growth chamber.The seeds were aerated using an air pump during immersion.All possible combinations of the presence or absence of the opercular tegument, GA 3 concentrations, and immersion times were tested, with five repetitions of 20 seeds per treatment.The seeds were evaluated on a weekly basis for 18 weeks for germination (or deterioration) to calculate the germination percentage (%G) and germination velocity index (GVI).Germination was defined as the visible protrusion of the cotyledon petiole (BEWLEY; BLACK, 1994); the presence of necrosis or fungal mycelia was indicative of deterioration.

Effects of interactions between phytoregulators and the number of applications
The opercular teguments were removed from the seeds, and the effects of seed immersion in 2000 mg L -1 GA 3 (PROGIBB ® ), or its absence along with four combinations of IBA and BAP (Sigma ® ) were evaluated (Table 1).Seed immersion was performed in plastic cups containing 100 mL of aqueous solutions of phytoregulator combinations, with alternating applications of GA 3 and IBA/BAP combinations applied each week.Each combination of GA 3, IBA, and BAP was applied one, two or five times.These experiments were conducted under the same conditions as the previous experiment, testing all possible combinations of GA 3 , IBA and BAP concentrations with five repetitions of 20 seeds per treatment.Seed germination was evaluated as described earlier, and seeds that did not germinate until the end of the evaluation period were treated with 0.5% tetrazolium to determine their viability, following the protocol of Ribeiro et al. (2010).
The data from both experiments were submitted to analyses of variance, and the Tukey test was used to compare their averages (SAS INSTITUTE, 1990).The values corresponding to per cent germination and deterioration were arc sine converted (x 100 -1 ) 0.5 for comparison.

Results and discussion
Effects of the removal of the opercular tegument, GA 3 dose and immersion time The seed lot used in these experiments (lot 1) had a water content of 6.6% before preimbibition.After the treatment, the water content of the seed lot reached 22%.The start of germination was observed in the second week after the GA 3 immersion treatments.The duration of immersion in GA 3 did not influence the germination percentage.Significant effects were observed resulting from the removal of the opercular tegument, different doses of GA 3 , and in the interactions among these variables (Figure 1A).The removal of the opercular tegument increased germination independent of the GA 3 concentration; thus, this phytoregulator was effective only in seeds with intact opercular teguments.The %G of seeds immersed in 2000 and 5000 mg L -1 were not significantly different, but they were both higher than the control.Positive effects were observed with respect to the GVI, resulting from the removal of the opercular tegument, immersion in GA 3 , and interactions between the variables (Figure 1B).The GVI of seeds immersed in 2000 and 5000 mg L -1 were not significantly different but were higher than those of the control.Immersion in GA 3 for 48 hours resulted in the highest GVI, but there were no significant interactions between the immersion duration and the other variables.Seed deterioration was approximately 18% and was significantly influenced by immersion in GA 3 (p = 0.0002).
The removal of the opercular tegument results in increases in germination percentages in other palm GA 3 (mg L -1 ) GA 3 (mg L -1 ) species (CARPENTER et al., 1993;HUSSEY, 1958;PÉREZ et al., 2008).The efficacy of this treatment is most likely related to the decreased resistance to embryonic elongation that is normally imposed by the opercular tegument.
Gibberellins are known to aid in overcoming seed dormancy by stimulating embryo development and by the production of hydrolases that weaken seed structures adjacent to the embryo (HOOLEY, 1994;YAMAGUCHI;KAMIYA, 2002).Increased macaw palm seed germination after exposure to GA 3 was reported by Ribeiro et al. (2011), and other studies have shown similar effects in other palm trees (CHIN et al., 1988;DEWIR et al., 2011;HERRERA et al., 1998;NAGAO et al., 1980;PÉREZ et al., 2008;ROBERTO;HABERMANN, 2010;YANG et al., 2007).Ribeiro et al. (2011) reported that the removal of the opercular tegument associated with a single application of 2000 mg L -1 GA 3 resulted in increased germination, as was observed in the present study.These authors also noted that repeated exposure to GA 3 for four weeks after sowing resulted in even higher germination percentages, and they suggested that the positive effects of the reapplication of this hormone were due to an increasing sensitivity of the seeds to GA 3 over time; alternatively, 24 hours of exposure was insufficient to promote seed germination.Our results indicate that the effects of immersion in GA 3 for 24 hours on seed germination were not significantly different from the results of 48 hours of immersion.Likewise, immersion in 2000 or 5000 mg L -1 GA 3 did not result in significant differences in germination.As such, the positive effects of the re-application of GA 3 does not indicate that either the 2000 mg L -1 concentration or immersion for 24 hours were insufficient, in agreement with the hypothesis of increased sensitivity to GA 3 over time.
The levels of seed deterioration observed in experiments involving exogenous GA 3 (17.8%)were very close to those reported by Ribeiro et al. (2011), who considered this problem a roadblock to the commercial propagation of macaw palm.

Effects of interactions between phytoregulators and the number of applications
The seeds used in this experimental procedure (lot 2) had water contents of 8% at the beginning of the experiments, but by two weeks after the tests, their water contents had reached 20%.As in the previous experiment, germination starts in the second week after exposure to GA 3 .Immersion in IBA and BAP had no effect on the germination percentages, although immersion in GA 3 resulted in a significantly higher germination percentage than that of the controls, reaching 41% after the fifth application.When applications of the other phytoregulators (AIB and BAP) were increased in the absence of GA 3 , there was no increase in the germination percentage above that of the control.On the other hand, when GA 3 was applied five times, a significant increase in %G was observed (Figure 2A).Identical lower case letters indicate the absence of any significant differences between the numbers of applications in each treatment (with or without GA 3 ).Identical upper case letters indicate the absence of any significant differences between treatments (with or without GA 3 ) with identical numbers of applications, using the Tukey test at a 5% level of probability.Bars represent the standard error of mean.
Seeds that were immersed in GA 3 demonstrated greater GVI independent of the quantities applied (Figure 2B).Immersion in IBA and BAP did not significantly influence the GVI.Seed deterioration was approximately 35% and was influenced by GA 3 treatment (p < 0.001); tetrazolium tests demonstrated that 92.5% of the seed embryos that had not deteriorated remained viable.
Increasing numbers of applications of GA 3 resulted in higher germination percentages, as was observed by Ribeiro et al. (2011), and may be related to seed  , 2006).
In addition to the differences in the degree of dormancy among seeds of the same lot, the positive effects of the reapplication of GA 3 may be related to their endogenous levels of abscisic acid (ABA).ABA is a positive regulator of dormancy induction and a negative regulator of germination (KUCERA et al., 2005;NAMBARA et al., 2010).The seeds in this study were submitted to warm stratification in a humid growth chamber, which could have provoked decreases in ABA levels over time, resulting in variations in the responses of those seeds to GA 3 .Chien et al. (1998) reported decreases in ABA concentrations in Taxus mairei (Taxodiaceae) seeds during warm stratification, and the same process could have occurred in the A. aculeata seeds tested in the present study.This hypothesis is corroborated by experiments with various other species that have demonstrated that incubating seeds at high temperatures in humid environments overcomes physiological dormancy and increases sensitivity to external applications of GA 3 (BASKIN; BASKIN, 1998;FINCH-SAVAGE;LEUBNER-METZGER, 2006).Pérez et al. (2008) reported that removal of the operculum followed by incubation at high temperatures (25-35ºC) resulted in high germination percentages in Pritchardia remota palm seeds.Additionally, high-temperature treatments promoted decreases in ABA levels in the seeds of Elaeis guineensis palms (JIMENEZ et al., 2008), making it an effective treatment for overcoming dormancy in that species (HUSSEY, 1958;REES, 1962).Another factor that should be considered is the diffusion velocity of GA 3 to the seed interior.Nagao et al. (1980) observed that gibberellin effectiveness was limited by its ability to penetrate the seed tegument.Slow diffusion would necessarily require longer exposure times or repeated hormone applications at later times, allowing the seeds to absorb sufficient quantities of GA 3 to promote germination.As such, the efficiency of the reapplication of GA 3 might be a reflection of its slow absorption by the seeds.
The effects of immersion in auxin solutions (such as IBA) on seed germination have not been well studied, and little is known about the role of auxins in seed germination at the molecular level (KUCERA et al., 2005).However, Jimenez et al. (2008) observed that the levels of indole acetic acid (IAA) in Elaeis guineensis seeds increased considerably during imbibition, suggesting that this hormone plays an important role in germination in that species.
The endosperm has been described as a source of cytokinins that are necessary to promote embryonic cell division (KUCERA et al., 2005).A number of studies have provided evidence that cytokinins, like gibberellins, might aid in overcoming ABA-imposed inhibition (FOUNTAIN;BEWLEY, 1976), so that in some species, the simple use of cytokinins is sufficient to overcome seed dormancy (KUCERA et al., 2005).On the other hand, immersion in BAP did not result in increased germination in Euterpe edulis (ROBERTO; HABERMANN, 2010) and Bactris gasipaes (VILLALOBOS et al., 1992) palms, as was observed in A. aculeata in the present study.
The present experiments resulted in even higher deterioration levels (34.8%), which might be related to the effects of the weekly immersions to which the seeds were submitted.These results highlight the importance of additional studies concerning microbial control in perfecting the propagation technologies for A. aculeata.

Conclusion
The immersion of A. aculeata seeds in 2000 or 5000 mg L -1 GA 3 and the removal of the opercular tegument result in increased germination.
The positive effects of immersion in GA 3 tended to increase with increasing numbers of applications.
Exposure to IBA and BAP was ineffective in promoting the germination of A. aculeata seeds.

Figure 1 .
Figure 1.(A) Germination percentages (%G) and (B)germination velocity index (GVI) of A. aculeata seeds with or without the opercular tegument (OT) after immersion in solutions with different concentrations of GA 3 .Identical lower case letters indicate the absence of significant differences between seeds with or without OT in each concentration of GA 3 .Identical upper case letters indicate the absence of any significant differences between GA 3 concentrations in seeds with or without OT by the Tukey test at a 5% level of probability.Bars represent the standard error of mean.

Table 1 .
Combinations of IBA and BAP applied to A. aculeata seeds in association with GA 3 .