Prunus azorica (Hort. ex Mouillef.) Rivas Mart., Lousã, Fern. Prieto, E. Dias, J.C. Costa, and C. Aguiar, commonly named Azorean cherry or “ginja-do-mato,” is an endangered tree endemic to the Azores, Portugal. It is currently found in São Miguel, Terceira, São Jorge, Pico, Faial, and the Flores islands (Cardoso et al., 2008; Martins et al., 2008; Schäfer, 2005; Silva et al., 2009). It is usually a small tree or shrub, rarely above 4 m tall. However, in sheltered locations (such as water stream margins), it is able to attain height greater than 10 m. It occurs at altitudes above 500 m, mainly in craters and deep narrow ravines, or scattered in undisturbed hyperhumid native forest (Silva et al., 2009). This species has become very rare and recently, only scattered individuals were found in São Miguel and Pico, whereas in Flores, only one individual is known to exist. Considered as one of the top 100 priority taxa for conservation in Macaronesia (Martins et al., 2008), it is one of the constituents of the middle altitude laurel forest species, a type of forest that was largely replaced by other land uses. Moreover, in São Miguel Island, it is also one of the main food sources for the endangered bird Pyrrhula murina. Flowering occurs from the end of March to July with white flowers developing in racemes. The number of developed flowers by inflorescence is not regular, because in general it ranges from 10 to 35. Fruits mature from the base to the apex of the raceme and are dark red drupes with ≈0.8 to 1.2 cm diameter, which become ripe from July to October. Long periods of overlapping flowering and fructification occur with flowers and ripe fruits occurring concomitantly. Dispersal occurs mainly by endozoochory (Silva et al., 2009).
To control further spread of invasive species in the Azores islands, there is a growing demand for conservation and reforestation projects that require the implementation of active propagation measures for native and endemic species. Recently, Moreira et al. (2009) established an efficient protocol for the propagation of P. azorica by air-layering and by cuttings. However, in cases of depauperated populations and to maintain the genetic variability of the population, propagation by seeds might be preferable (Barrett and Kohn, 1991). Until recently, germination of P. azorica was based on empirical knowledge, leading to relatively low germination percentages and to the demand for large numbers of seeds to produce a desired quantity of growing stocks, which further deplete the natural populations’ seed stock. Previous studies by the Azorean Forest Service using seeds collected in September/October and germinated in trays inside a greenhouse reported a germination percentage of ≈50% (Fagundo and Isidoro, 2004). Additionally, there was no knowledge on the different factors that might affect the germination of this species. Maciel (1995, 1996), when failing to promote germination of P. azorica seeds, suggested the occurrence of a dormancy connected to inhibitory mechanisms located at the embryo and seedcoat; however, no further experiments were conducted.
Dormancy is a condition in which seeds do not germinate even when the environmental conditions are favorable for germination (Baskin and Baskin, 2004; Bewley and Black, 1994; Hartmann et al., 1997; Nicolaeva, 1977). In several Prunus species, seed dormancy is considered as an adaptive mechanism to protect fruit trees from freeze damage during the winter (Martynez-Gómez and Dicenta, 2001). However, the occurrence of seed dormancy can become a significant hindrance in cases of endangered taxa, preventing the possibility of fast production of a large number of seedlings for reintroduction in the native populations (Moura and Silva, 2010).
Some prunoid species have fruits with a stony endocarp and embryos with deep physiological dormancy, requiring long periods of cold stratification for dormancy break (Crocker and Barton, 1931; Flemion, 1934; Nicolaeva, 1969, 1977). There is a considerable variety of methods that can be used for dormancy-breaking and seed germination among different Prunus species (Ellis et al., 1985; Finch-Savage et al., 2002; Grisez, 1974; Suszka, 1962). Cherries generally have deep dormant seeds and in wild cherry (P. avium), a deep and variable embryo dormancy is the most significant germination inhibiting factor, which requires a very complex moist stratification regime with consecutive warm and cold periods, imitating the end of summer or fall, to break dormancy (Esen et al., 2006; Finch-Savage, 2001; Grisez, 1974; Suszka, 1967, 1990). Inversely, long periods at constant high temperatures during stratification and germination tests induced secondary seed dormancy in the same species (Esen et al., 2009). Grisez (1974) referred a requirement of 3 to 4 months of cold stratification to overcome embryo dormancy in cherry seeds. Experimental comparisons among various stratification temperatures for several species showed that constant temperatures from 2 to 5 °C are more favorable than those below 1.7 °C or above 8 °C (Coe and Gerber, 1934; Crocker and Barton, 1931; Haut, 1938). However, warm stratification before cold stratification might be needed in cases of underdeveloped embryos (Finch-Savage, 2001; Finch-Savage et al., 2002).
Addition of gibberellins was also shown to increase germination percentage in Prunus species (Carrera et al., 1988; Giba et al., 1993; Karam and Al-Salem, 2001). Gibberellin (GA3) treatments can replace a portion of the stratification period in several Prunus spp., but only after endocarp removal (Grisez et al., 2003). Likewise, GA3 addition promoted the germination of non-stratified P. campanulata seeds after endocarp removal (Chen et al., 2007) and significantly increased the germination percentages of P. avium and P. serotina seeds (Çetinbas and Koyuncu, 2006; Phartyal et al., 2009).
Regarding incubation temperature, an alternating temperature regime (daily oscillation between a high and a low value) during germination trials has enhanced seed germination percentages not only in Prunus spp. (Suszka, 1962), but also in other Azorean woody species (Martins et al., 2012; Moura and Silva, 2010).
The present study aims to determine which treatments are able to break the dormancy of P. azorica seeds and to increase the germination percentage and speed, improving the efficiency of its propagation. Besides testing those factors that might affect germination and lead to dormancy break, following seedling development after radicle emergence might also be important to determine the type of seed dormancy, namely the epicotyl dormancy classification (Baskin and Baskin, 1998). For that, 1) the effect of several pretreatments on seed germination percentage was evaluated, including endocarp removal, stratification regime, growth regulator concentration, and incubation temperature; and 2) initial seedling development was followed.
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