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Prunus lusitanica (2n = 8x) and Prunus laurocerasus (2n = 22x) are evergreen woody shrubs commonly used in landscapes across the United States and Europe. To reduce the difference in ploidy between these species and with the expectation of successful hybridization, an experiment was performed to double the chromosome number of P. lusitanica. Colchicine was applied at 0%, 0.2%, 0.4%, and 0.8% (w/v), and 125mMoryzalin as a viscous liquid to the apical meristem of open-pollinated P. lusitanica seedlings. Solutions were semisolidified using 0.55% agar (w/v). Cellular penetration was increased by adding 1%dimethyl sulfoxide (v/v) in all groups except oryzalin. As a result, three chromosome doubled (2n = 16x) plants, one 2n = 12x plant, and 14 cytochimeras (2n = 8x + 16x) were recovered. Application of 125mMoryzalin had a meristem-survival rate of 17%, statistically lower than all other treatments. The oryzalin treatment also produced the highest number of altered ploidy seedlings. Oryzalin at 125 mM was the most effective chromosome doubling agent in this experiment. Phenotypic examination indicated that chromosome doubled (2n = 16x) plants displayed shorter stems, thicker leaves, and fewer but larger guard cells than the untreated controls. 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, Xanthomonasarboricolapv.pruni,Wisonomyces carpophilum,Microgloeumpruni, andCercospora 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 mM 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. Materials and Methods Plant material. Open-pollinated fruit were collected from oneP. lusitanica on the Oregon State University campus (lat. 44 34#04$N, long. 123 17#14$W) in Corvallis, OR, on 5 Sept. 2014. The exocarp and mesocarp were removed to expose the stony endocarp. Seeds were then cold-stratified the in moist perlite for 60 d at 5 C. After cold-stratification, seeds were planted in soilless substrate (Metro-Mix 840PC; SunGro Horticulture, Agawam, MA) in 50.6 · 21.6 · 5.2 cm flats (T.O. Plastics, Clearwater, MN) and grown in a glasshouse with 24 C day/9 C night temperatures. Trays were hand-watered as needed and germination began to occur 19 d after sowing. Germinated seedlings were transferred each day to 36-cell trays (T.O. Plastics), and grown under soft fluorescent lights at 90 mmol·m·s at 22 to 25 C with a 16-h photoperiod (0600 to 0000 HR) covered with clear humidity domes (T.O. Plastics). Received for publication 17 Nov. 2016. Accepted for publication 6 Jan. 2017. Wewould like to acknowledgeOregonAssociation of Nurseries for partial funding of this research.We also thank Shawn Mehlenbacher, Carolyn Scagel, John Lambrinos, and three anonymous reviewers for critical review to improve the manuscript. This article is a portion of a thesis submitted by J. Schulze in partial fulfillment of the requirements for an MS. Associate Professor of Horticulture Corresponding author. E-mail: ryan.contreras@ oregonstate.edu. 332 HORTSCIENCE VOL. 52(3) MARCH 2017 Inducing polyploidy. Seedlings were randomly placed into groups of 12 as they germinated, and each group was assigned a treatment. This occurred sequentially over time until each treatment was replicated seven times. A total of 420 seedlings (12 seedlings per replicate · 7 replicates · 5 treatments) were treated. Colchicine at a rate of 0% (control), 0.2%, 0.4%, 0.8% (SigmaAldrich, St. Louis, MO) (w/v), and 125 mM oryzalin (Surflan AS; United Phosphorus, Trenton, NJ) were applied as mitotic spindle inhibitors. One percent dimethyl sulfoxide (Sigma-Aldrich) (v/v) was added to the colchicine and control treatments to improve cellular penetration, and 0.55% agar (SigmaAldrich) (w/v) was added to all the treatments as a congealing agent. All treatments were heated to 38 C to dissolve the agar before application. Once the first true leaves opened, then 25 mL of treatment solution was applied to the exposed meristem. Treatments were maintained on the meristem for 10 d. Reapplication was conducted as droplets dried out, about every 3 to 4 d. After treatments, meristems were manually cleaned with running water to remove solution remains. The plants were then moved to a glasshouse equipped with high-wattage lamps with the ambient environmental conditions maintained as 12-h photoperiod and 24 C day/ 19 C night. Apical meristem survival rate was recorded one month after the completion of all treatments. In many instances, if the apical meristem was killed by the treatment, adventitious shoots would emerge just above the cotyledon, but such adventitious shoots were not counted. Ploidy analysis. A flow cytometer (CyFlow Ploidy Analyzer; Partec, M€unster, Germany) was used to screen all the surviving seedlings. Pisum sativum ‘Ctirad’ (2C = 8.76 pg) was used as an internal standard for genome size estimation of treated plants (Greilhuber et al., 2007). About 0.5 cm of young leaf tissue from each sample and the internal standard was finely cochopped in 0.4 mL of extraction buffer (CyStain Ultraviolet Precise P Nuclei Extraction Buffer; Partec) with a double-sided razor blade to extract nuclei. The nuclei suspension was passed through a 30 mm filter (Partec), and then stained with 1.6 mL of 4’,6-diamidino2-phenylindole (CyStain ultraviolet Precise P Staining Buffer; Partec). The stained nuclei solutions were incubated for 60 s before being fed into the flow cytometer. Genome size of P. lusitanica samples was calculated according to a formula: sample genome size = genome size of internal standard · (mean fluorescence value of sample O mean fluorescence value of

In Vivo Chromosome Doubling of Prunus lusitanica and Preliminary Morphological Observations