Portuguese cherrylaurel (Prunus lusitanica) and common cherrylaurel (Prunus laurocerasus) are popular landscape plants throughout the northern temperate zone. They are densely growing evergreen shrubs, commonly used in hedging. An important difference between the two species is that P. lusitanica is resistant to shot-hole disease, while P. laurocerasus is susceptible (Dirr, 2009; Williams-Woodward, 1998). Shot-hole disease refers collectively to a number of bacterial and fungal pathogens (e.g., Pseudomonas syringae pv. syringae, Xanthomonas arboricola pv. pruni, Wisonomyces carpophilum, Microgloeum pruni, and Cercospora sp.) which detract from ornamental appearance of leaves and may eventually kill diseased trees if cankers girdle stems (De Boer, 1980; Marchi et al., 2014; Pscheidt and Ocamb, 2014; Williams-Woodward, 1998). Symptoms typically present as numerous small holes in the leaves of affected plants through the loss of necrotic leaf tissue.
So far, there have been no reports of successful hybridization between P. lusitanica and P. laurocerasus. We believe this apparent sexual incompatibility is likely due, at least in part, to the difference in ploidy level. Prunus laurocerasus is a 22-ploid with a chromosome number of 2n = 22x = 176 (Meurman, 1929). P. lusitanica is an octoploid with a chromosome number of 2n = 8x = 64 (Darlington and Wylie, 1956).
We theorize that if we can double the ploidy level of P. lusitanica to 2n = 16x, an interspecific cross might be possible. Similar approaches have been successfully applied in Rhododendron (Kehr, 1996), Rosa (Debener et al., 2003), and Vaccinium (Lyrene, 2011) by doubling the chromosomes of one of the parents. Theoretically, the resulting hybrid would have a chromosome number of 2n = 19x = 152. This odd ploidy level, in tandem with being an interspecific hybrid, could produce a low-fertility plant.
In recent years, increasing attention has been given to the level of fertility in nursery and landscape plants (Niemiera and Von Holle, 2009). Legislation and regulation of weedy plants is becoming commonplace and some of these species are economically important for nursery growers. As such, reducing fertility has become a goal of breeders and may be regarded as a value-added trait since sterile or nearly sterile plants are less likely to escape from cultivation. A common goal of ornamental plant breeders is to create plants with odd ploidy levels (i.e., triploids). This typically reduces a plant’s fertility and ability to develop seed. For example, this technique was used in the development of low-fertility Hypericum androsaemum without losing its ornamental appearances (Olsen et al., 2006).
Induction of polyploidy, or chromosome doubling, can be accomplished in several ways. Commonly, seedlings or shoots tips are treated with colchicine (in vitro or in vivo). Colchicine, a mitotic spindle inhibitor affecting chromosome separation during mitosis, has been used for chromosome doubling since the late 1930s (Blakeslee and Avery, 1937). The effectiveness of colchicine treatment in chromosome doubling has been seen in many woody species including Acacia crassicarpa (Lam et al., 2014), Lagerstroemia indica (Ye et al., 2010), Platanus acerifolia (Liu et al., 2007), Pyrus pyrifolia (Kadota and Niimi, 2002), and Ziziphus jujuba (Gu et al., 2005).
Oryzalin is another effective mitotic inhibitor for chromosome doubling in many woody plants including Acacia crassicarpa (Lam et al., 2014), P. laurocerasus (Contreras and Meneghelli, 2016), Platycladus orientalis (Contreras, 2012), Rhododendron (Jones et al., 2008), Rosa (Kermani et al., 2003), Thuja occidentalis (Contreras, 2012), and Thuja plicata (Contreras, 2012). Oryzalin is now commonly used in plants due to its binding affinity to plant tubulin rather than mammalian (Hugdahl and Morejohn, 1993), therefore reducing toxicity to humans. Oryzalin is also effective at much lower concentrations than colchicine. Colchicine is typically applied at a rate of 0.1% to 1%, while in one recent example, where treatment was applied to the meristem in vivo, Jones et al. (2008) doubled the chromosomes of Rhododendron using only 50 µM oryzalin.
A shot-hole disease resistant cherrylaurel hybrid with low fertility could have the potential to be widely adopted by the nursery industry. Establishing an effective chromosome doubling method in P. lusitanica is the first step in developing hybrids that realize these traits. The objectives of this study were to 1) develop methods for generating 2n = 16x P. lusitanica that can be used in future breeding projects and 2) assess morphological variation among different cytotypes.
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