Common cherrylaurel (Prunus laurocerasus L.) and Portuguese cherrylaurel (P. lusitanica L.), collectively referred to as cherrylaurels, are highly adaptable and important ornamental species in the United States and Europe. Cherrylaurels are drought-tolerant broadleaf-evergreens commonly used in hedging. Common cherrylaurel is a mainstay in industrial and residential landscapes alike, and an important nursery crop, particularly in the Pacific northwest of the U.S. Per industry professionals; however, these popular plants have one unanimous flaw, shothole disease.
Shothole disease affects multiple stone-fruit species. Symptoms present as numerous small holes in the leaves caused by fungal and/or bacterial pathogens (De Boer, 1980; Pscheidt and Ocamb, 2014; Williams-Woodward, 1998). Infection can decrease yield in fruit crops and esthetic qualities in landscape plants. Shothole disease has plagued growers of the P. laurocerasus across the country, a major contributing factor being that disease severity is often intensified by overhead irrigation (Dirr, 2009; Williams-Woodward, 1998). Prunus lusitanica, by contrast, rarely expresses symptoms of shothole disease (Dirr, 2009).
Interspecific hybridization of these two species could provide a path to introgress shothole disease resistance from P. lusitanica into P. laurocerasus (Dirr, 2009). There have been no reports of successful hybridization of these two species, and we have not had success after more than 10,000 attempts using hand pollination. Fruit resulting from wide, interploid crosses, as in this case [P. laurocerasus: 2n = 22x = 176 (Meurman, 1929), and P. lusitanica: 2n = 8x = 64 (Darlington and Wylie, 1956)], often abort postfertilization (Ramsey and Schemske, 1998). One technique for recovering viable seedlings from interspecific or even intergeneric hybrids is embryo rescue.
In vitro embryo rescue has been employed in numerous genera of woody shrubs and trees including Hibiscus spp. (Van Laere et al., 2007), Prunus spp. (Kukharchyk and Kastrickaya, 2006; Liu et al., 2007), Rhododendron spp. (Eeckhaut et al., 2007), and Rosa spp. (Gudin, 1993). Liu et al. (2007) described a technique used to rescue young embryos of peach–apricot, and peach–plum hybrids. They found the highest germination percentage occurred with the cytokinin 6-benzylaminopurine (BA), in half-strength MS basal salt media (Murashige and Skoog, 1962). Kukharchyk and Kastrickaya (2006) recovered over 500 Prunus hybrids in their study, and BA performed better than other cytokinins tested. Multiple sources report chilling is required in any Prunus species to overcome physiological seed dormancy (Kukharchyk and Kastrickaya, 2006; Moore and Janick, 1983).
Prunus species exhibit intermediate physiological dormancy with a chilling requirement typically 60–90 d, and seeds that do not receive the required dormancy period have poor, if any, germination (Hartmann et al., 2011; Moore and Janick, 1983). In the case of embryo rescue, chilling requirements will likely depend on the point in development at which abortion occurs. Seeds that have intermediate physiological dormancy accumulate abscisic acid (ABA) as they develop, which triggers the onset of dormancy (Bewley, 1997). Immature seeds low in ABA may not require cold stratification, although dormancy period is even required for immature Prunus seeds (Kukharchyk and Kastrickaya, 2006). Cold stratification has been avoided in Prunus species by removal of the cotyledon and testa (Şan et al., 2014). However, it is unclear at what point in development that these parts are distinct enough for identification and removal.
Giberellic acid (GA3) is a plant hormone often associated with overcoming dormancy. This hormone was used in successful in vitro germination (Şan et al., 2014) and embryo rescue (Kukharchyk and Kastrickaya, 2006) media in Prunus and could be an essential component to successful germination of immature seed. Sucrose concentration is also an important media component to be considered when performing embryo rescue, as immature embryos often require lower osmotic potential than mature embryos (Trigiano and Gray, 2011).
Once in vitro germination has occurred, seedlings often require a transfer medium to continue growth (Trigiano and Gray, 2011). The stage of root and shoot development will dictate whether an in vitro seedling can be directly transplanted or first transferred to a shoot proliferation and/or rooting media. Prior research has described effective shoot proliferation and rooting media in P. laurocerasus (Kalinina and Brown, 2007; Sulusoglu and Cavusoglu, 2013). We have found no information on protocols for in vitro propagation of P. lusitanica.
During our research to improve disease resistance of P. laurocerasus by attempted hybridization with P. lusitanica minimal and brief fruit development was observed in P. lusitanica as the pollen recipient (personal observation), and no fruit set has been observed in the reciprocal cross. Therefore, we focused our preliminary efforts to identify the optimal medium for germination of open-pollinated P. lusitanica seed. The goals of this study were to identify a tissue culture medium that would support germination of immature P. lusitanica seed and determine the effect of collection time and cold stratification on seedling germination without removal of the cotyledon.
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