EFFECT OF GIBBERELLIC ACID AND TEMPERATURE ON GERMINATION OF Vitex montevidensis Cham

This work aims to evaluate treatments that may help improve speed and uniformity of germination in fruit tree species tarumã. Data were obtained from pyrenes (stones) of ripe fruits which in turn were picked from adult trees in the municipality of Vera Cruz do Oeste-PR. Treatments consisted of applying GA3 solutions at various concentrations (zero, 50 mg L -1, 100 mg L-1 or 200 mg L-1) to pyrenes via immersion for 47 hours, and thermoperiods of alternate 20oC and 30oC (8 hours at 20oC in light conditions, and 16 hours at 30oC in dark conditions) and constant 30oC with the same photoperiod. The combination of alternate temperatures of 20oC and 30oC with a 200 mg L-1 GA3 solution resulted in an increase both in germination percentage, from 19.2% to 56.2%, and in the germination speed index, from 0.19 to 3.12, with resulting normal, uniform seedlings.


INTRODUCTION
Literature has demonstrated that endogenous compounds that promote and inhibit growth are directly involved in the germination process (ARAGÃO et al., 2003;PASSOS et al., 2004;SCALON et al., 2004). These substances may act alone or in combination with others, not only during the germination process but also in postgermination events such as energy reserve mobilization. Studies on the effect of gibberellic acid on seed germination of nonfruit woody species are limited to Guarea guidonea (CASTRO et al., 1999), Cassia excelsa (JÉLLER; PEREZ, 1999), Jacaranda cuspidifolia (SCALON et al., 2006), Senna spectabilis (JÉLLER; , and Talisia esculenta (VIEIRA; GUSMÃO, 2006b).
Seed germination may demand gibberellins for one of the following stages: activation of the embryo's vegetative growth, weakening of the endosperm layer that involves the embryo and obstructs its growth, and mobilization of the energy reserves of the endosperm (TAIZ; ZIGER, 2008). According to these authors, applying gibberellins (GA 3 ) also stimulates the production of various hydrolases, including α-amylase, by aleurone layer cells of germinating cereal grains. During germination and initial growth of seedlings, endosperm reserves, particularly starch and protein, are hydrolized by various hydrolytic enzymes, and the solubilized sugars, aminoacids and other products are transported to the developing embryo. Gibberellin can be a limiting factor to α-amylase production during seed germination (CASTRO et al., 2004).

Malavasi, M. de M. et al.
in seed germination is not as well disseminated with forest species as it is with fruit-bearing, vegetable, and medicinal species. While working with seeds of Passifl ora alata, Rosseto et al. (2000) observed that using GA 3 at a concentration of 300 mg L -1 was more effective in promoting germination. Still with Passifl ora alata, Ferreira (1998) observed that GA 3 at 100 mg L -1 promoted better germination, while with Passifl ora nitida Passos et al. (2004) reported that for in vitro germination the most suitable gibberellic acid concentration was 1000 mg L -1 , with or without luminosity. Botelho and Perez (2001) reported signifi cant effects on the germination percentage and speed with seeds of Peltophorum dubium Spreng (Taubert). With seeds of Jatropha elliptica, Añez et al. (2006) found higher germination percentages in treatments using gibberellic acid at concentrations of 300 to 500 ppm. In contrast, Vieira and Gusmão (2006a) argued that GA 3 failed to stimulate germination in a study with seeds of Genipa americana.
Studying Caryocar brasiliense, Souza and Salviano (2002) recommended using 1000 mg of GA 3 to two liters of water in order to increase germination and improve seedling growth. With the same species, Pereira et al. (2004) reported that, from 250 ppm of GA 3 onward, seed germination increased signifi cantly. Similarly, Fuentes Fiallo et al. (1996) reported an increase in germination of oregano seeds (Ocimum gratissimum) with exogenous application of gibberellic acid starting at 250 ppm and as recommended for economic reasons, whereas applying the same substance for germination of the same species partially reverted light requirements (FACTOR et al., 2008).
In germination, response to temperature is dependent on species, variety, originating location and storage period. Some species require alternate temperatures to germinate while others are indifferent to that stimulus (CETNARSKI FILHO;NOGUEIRA, 2005).
Vitex montevidensis occurs in Atlantic Forest environment, from Bahia down to Rio Grande do Sul, and also in Florestas de Pinhais, Cerradão and Florestas Semideciduas of Paraná River basin. Fruits are consumed fresh or used for making candies and liqueur. The species can be used in conservation-oriented reforestation practices, providing both fruits to feed the local fauna and good quality wood (CARDOSO, 2004). However, the germination rate of tarumã seeds is less than 10% (LORENZI, 2000) and seed emergence is diffi cult where no-till planting is involved (CARRASCO et al., 2007).
The objective of this work is to evaluate the effects of GA 3 application and temperature on the germination of tarumã seeds.

MATERIAL AND METHODS
Reproductive structures of tarumã were collected in March 2005 from adult plants located in the municipality of Vera Cruz do Oeste (24 o 43'S and 53 o 48'W). Pyrenes were manually extracted using a surgical knife and choosing ripe fruits whose epicarp was color 1N according to Munsell color chart (MUNSELL, 1976). Pyrenes were subjected to treatment immediately after being extracted.
Variables being measured included germination percentage in sterilized sand arranged in plastic trays (40 x 25 x 8 cm) and germination speed index (MAGUIRE, 1962), in four replicates of 25 pyrenes each.
The quantifi cation of gibberellic acid (GA 3 ) and temperature effects on germination used a completely randomized design in a 4x2 factorial consisting of four GA 3 concentrations and two thermoperiods. GA 3 concentrations being tested included zero, 50 mg L -1 , 100 mg L -1 or 200 mg L -1 , applied to pyrenes via immersion for 47 hours, according to preliminary data. Thermoperiods using BOD chambers (Fanen®, 347G model) consisted of alternate temperatures of 20ºC and 30ºC and constant 30ºC, both with 8 hours of light being provided by daylight fl uorescent lamps (4x20 W) according to procedures suggested by Brasil (1992).
Different GA 3 concentrations were applied to the solutions containing pyrenes in 500 mL of distilled water, in beaker glassware with a capacity of 1 L, which were subjected to artifi cial air circulation using an aquarium pump (1.5 L min -1 pressure, 0.01Mpa) and water volume maintenance so as to prevent oxygen shortage in the solutions.
For the purpose of statistical analysis, data on germination percentage were transformed into arcsin √ x/100 and germination speed index into x + 0.5 (SANTANA; RANAL, 2004). Results were subjected to analysis of variance and, when a statistical difference was found, means were compared using the Tukey test at the 5% probability level.

RESULTS AND DISCUSSION
Analyses of variance of germination percentage and germination speed index (IVG) data resulted in signifi cant interaction (P<0.05) for the effects of applying GA 3 and temperature on the germination process (Table 1). Effct of gibberellic acid and temperature ...
Results of growth regulator and temperature effects indicate that the use of GA 3 promoted an average germination increase in tarumã seeds from 17.7% (control) to more than 50% in treatments using 200 mg L -1 with any of the thermoperiods. The combination of alternate temperatures of 20ºC and 30ºC and a 200 mg L -1 GA 3 solution resulted in the highest germination percentage. When using a constant 30ºC thermoperiod, higher germination values were observed for GA 3 concentrations of 100 mg L -1 and 200 mg L -1 , which did not differ statistically.
Typically, initial species in secondary succession require temperature alternation in order to germinate (BRANCALION et al., 2010). The experimental results corroborate results reported by Santos and Aguiar (2005) with seeds of tropical shrub branquilho (Sebastiania commersoniana Bailon) using alternate temperatures of 20ºC and 30ºC, and also results found by Lopes et al. (2002) with seeds of calabura (Muntingia calabura L.), and Medeiros Filho et al. (2002) with seeds of Operculina macrocarpa (L.) Farwel and Operculina alata (Ham.) Urban.
In a study with seeds of tarumã-branco (Citharexylum myrianthum Cham.), Zanon et al. (1997) reported that seed germination values at 20ºC were statistically lower than germination values found at a constant temperature of 25ºC or 30ºC. However, these authors also reported that a temperature of 30ºC caused damage to seedling cotyledons and hypocotyls when vermiculite substrate was being used.
A higher germination speed index (IVG) was obtained from observations resulting from the combination of alternate 20ºC and 30ºC temperatures with 200 mg L -1 of GA 3 , which did not signifi cantly differ (P<0.05) from the combination between constant 30ºC temperature and 100 mg L -1 of GA 3 . Results found for the germination speed index are similar to results found for germination percentage.
These results are similar to those found by Agustin and Alviter (1996) with cherimoya seeds (Annona cherimola Mill.), who reported fast seed germination using gibberellic acid solutions of 150 mg L -1 to 200 mg L -1 for a period of twelve hours. Similarly, Ferreira et al. (2002) observed with seeds of sugar apple (Anona squamosa L.) that the highest concentration being tested (200 mg L -1 ) provided the best germination results. Still on the subject of gibberellic acid for seed germination, other authors obtained better germination results under conditions similar to those described in this work (AÑEZ et al., 2006;FERREIRA, 1996;FUENTES FIALLO et al., 1996;GARCIA et al., 2006;ROSSETO et al., 2000).
Experimental results lead to the conclusion that improved germination percentage and germination speed index can be obtained with tarumã seeds if using alternate temperatures of 20ºC and 30ºC with 200 mg L -1 of GA 3 or a constant temperature of 30ºC with 100 mg L -1 or 200 mg L -1 of GA 3 . Atividades amilolítica e qualidade fi siológica de sementes armazenadas de milho super doce tratadas com ácido giberélico. Revista Brasileira de Sementes, Brasília, v. 25, n. 1, p. 43-48, 2003.