Ryan N. Contreras

In Vivo Chromosome Doubling of Prunus lusitanica and Preliminary Morphological Observations

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

Intraspecific, Interspecific, and Interseries Cross-compatibility in Lilac

N, long. 123 17#14

Ploidy and Genome Size in Lilac Species, Cultivars, and Interploid Hybrids

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

Ploidy Levels, Relative Genome Sizes, and Base Pair Composition in Cotoneaster

Lilacs (Syringa sp.) are a group of ornamental trees and shrubs in the Oleaceae composed of 22–30 species from two centers of diversity: the highlands of East Asia and the Balkan-Carpathian region of Europe. There are six series within the genus Syringa: Pubescentes, Villosae, Ligustrae, Ligustrina, Pinnatifoliae, and Syringa. Intraspecific and interspecific hybridization are proven methods for cultivar development. However, reports of interseries hybridization are rare and limited to crosses among taxa in series Syringa and Pinnatifoliae. Although hundreds of lilac cultivars have been introduced, fertility and cross-compatibility have yet to be formally investigated. Over 3 years, a cross-compatibility study was performed using cultivars and species of shrub-form lilacs in series Syringa, Pubescentes, andVillosae. A total of 114 combinations were performed at an average of 243 ± 27 flowers pollinated per combination. For each combination, we recorded the number of inflorescences and flowers pollinated as well as number of capsules, seed, seedlings germinated, and albino seedlings. Fruit and seed were produced from interseries crosses, but no seedlings were recovered. A total of 2177 viable seedlings were recovered from interspecific and intraspecific combinations in series Syringa, Pubescentes, and Villosae. Albino progeny were produced only from crosses with Syringa pubescens ssp. patula ‘Miss Kim’. In vitro germination was attempted on 161 seed from interseries crosses, resulting in three germinations from S. pubescens Bloomerang x Syringa vulgaris ‘Ludwig Spaeth’. None survived, yet cotyledons produced callus for future efforts to induce embryogenic shoots. This study is a comprehensive investigation of lilac hybridization, and the knowledge gained will aid future efforts in lilac cultivar development. Syringa is a diverse genus in the olive family (Oleaceae) representing 22–30 species from two centers of diversity: the highlands of East Asia and the Balkan-Carpathian region of Europe (Kochieva et al., 2004). Most lilacs are native to Asia, whereas S. vulgaris and S. josikaea are native to southeastern Europe (Kim and Jansen, 1998). Hundreds of cultivars have been produced and are ubiquitous in temperate gardens around the world. Historically, the most popular cultivars originated S. vulgaris, primarily grown for its fleeting spring blooms of purple, pink, blue, or white fragrant flowers. Previous phylogenies have divided lilacs into subgenera and four series (Rehder, 1945) which were later confirmed as monophyletic groups using plastid DNA (Kim and Jansen, 1998). The current phylogeny by Li et al. (2012) based on nuclear and plastid DNA sequences recognizes six series: Pubescentes, Villosae, Ligustrina, Ligustrae, Pinnatifoliae, and Syringa (Vulagares). Each series has distinguishing morphological features. Series Syringa is unique by having simple, glabrous leaves while series Pubescentes has pubescent leaves (Li et al., 2012). Series Villosae is distinct by having inflorescences develop from a single terminal bud with lateral, vegetative buds (Kim and Jansen, 1998). Ligustrina differs by its privet-like flowers (short, white corolla tubes with exerted anthers) and growth habit as a tree (Kim and Jansen, 1998). Pinnatifoliae is distinguished by having pinnately compound leaves (Li et al., 2012). Ligustrae contains several privets (Ligustrum sp.) nested within the lilacs (Li et al., 2012). Lilacs are of major economic importance to the United States nursery industry. In 2014, nationwide sales topped 1.8 million generating more than

Evaluating Soil and Foliar Fertilization of Abies nordmanniana Under Container and Field Production

20 million in total revenues (U.S. Department of Agriculture, 2016). Intraspecific and interspecific hybridization have proven to be valuable methods for the development of lilac cultivars. Interspecific hybridization has been particularly useful at producing cultivars with improved flowering and new foliar phenotypes (Table 1). Lilac breeding was scarce before the 1800s, a time when selections focused on improved form, flower color, or spring flush in chance seedlings (Fiala and Vrugtman, 2008). Early advancements in breeding produced vigorous interspecific hybrids including S. ·hyacinthiflora from crosses between S. oblata and S. vulgaris by the Lemoine nursery (Lemoine, 1878; Sax, 1930). This nursery was responsible for 214 cultivars and caused a spike in popularity of lilacs in the 1900s (Fiala and Vrugtman, 2008). Many breeders emerged to produce cultivars with a wide range of ornamental traits. Descanso Gardens in southern California and the United States National Arboretum focused on improving S. ·hyacinthiflora hybrids for southern climates by incorporating low chilling requirements and powdery mildew resistance (Fiala and Vrugtman, 2008). Cultivar improvement in series Villosae began its ascendancy with complex interspecific hybridization involving S. reflexa by Isabella Preston in Ottawa, ON, Canada (Fiala and Vrugtman, 2008). A total of 47 cultivars were introduced Received for publication 3 May 2017. Accepted for publication 5 June 2017. This research was funded in part by the Oregon Department of Agriculture Nursery Research Grant Program. Wewish to thankMara Friddle, Kim Shearer, Aleen Haddad, and the entire staff of the Ornamental Plant Breeding Lab at Oregon State University for technical support. Graduate Research Assistant. Corresponding author. E-mail: ryan.contreras@oregonstate.edu. J. AMER. SOC. HORT. SCI. 142(4):279–288. 2017. 279 from the interspecific hybrids S. ·prestoniae and S. ·josiflexa, which were created by crossing several species in series Villosae (S. villosa, S. reflexa, and S. josikaea) (Table 1) (Fiala and Vrugtman, 2008). One of Preston’s contemporaries, Frank Skinner, produced similar interspecific hybrids in Villosae, several of which are still available in the trade (Fiala and Vrugtman, 2008). Ornamental traits in series Pubescentes have been noted since the early 1900s when director of the Arnold Arboretum, Charles Sargent, noted in a wild-collected specimen of S. pubescens, ‘‘.if it keeps up its habit of flowering a second time in autumn, it will at least be interesting even if other lilacs are more beautiful.’’ Remontancy (or reblooming) as noted by Sargent would become one of the most pursued traits by modern lilac breeders (Fiala and Vrugtman, 2008). Early introductions in series Pubescentes exhibited improved form and flowers in addition to cold hardiness from wild-collected S. pubescens ssp. patula from E.H. Wilson’s Diamond Mountain expedition in Korea (Fiala and Vrugtman, 2008). Most new cultivars in series Pubescentes are prolific flowering, compact, and disease resistant with several cultivars exhibiting summer remontancy. In contrast to the success of interspecific hybridization, interseries hybridization has proven more difficult with the only successful hybrids from crosses between taxa in series Syringa and series Pinnatifoliae (Pringle, 1981). Interseries hybridization has been a goal of breeders for nearly a century, as illustrated by early reports: ‘‘.combinations of the early blooming Syringa vulgaris varieties with the late Villosae species would undoubtedly be of value if they could be made.’’ (Sax, 1930). Previous attempts to create interseries hybrids resulted in abortive fruit with no germination of recovered seed (Pringle, 1981). Abortive seed in lilacs has been explored in previous research. Anatomical studies on S. villosa, a species with high rates of seed abortion, found that after cross-pollination, embryos developed normally through the globular, heart, torpedo, and cotyledon stages before embryo and endosperm degradation (Chen et al., 2012). Few embryo rescue studies have been attempted in lilacs. However, Zhou et al. (2003) successfully cultured immature embryos on Monnier’s medium (Monnier, 1990) supplemented with 1-naphthaleneacetic acid, 6-benzylaminopurine (BAP), glutamine, and a high concentration of sucrose, indicating that tissue culture may be a platform for recovering hybrid lilacs. Even if in vitro germination fails, callus developed from the hybrid tissue may provide another source for producing interseries hybrids. Lilac somatic embryogenesis protocols using cotyledons have recently been developed for S. reticulata var. mandshurica (Liu et al., 2013). Although hundreds of improved lilac cultivars have been introduced, fertility and cross-compatibility among cultivars, species, and series have yet to be investigated in a formal study. The objectives of this study were to 1) investigate cross-compatibility of elite cultivars in intraspecific, interspecific, and interseries combinations and 2) investigate the potential for interseries hybridization and in vitro embryo rescue of abortive embryos. Materials and Methods PARENT MATERIAL. Parents were collected from nurseries, gardens, and arboreta from 2009 to 2014 (Table 2) that provided cultivar and trademark names. Full scientific names, cultivars, and trademarks are reported (Table 2), but for simplicity only market names (cultivar or trademark) are used hereafter. Taxonomic designations reflect current phylogenies and revisions, including the use of subspecies designations in Pubescentes (Chen et al., 2009). Representative species and cultivars were obtained from series Syringa, Pubescentes, and Villosae focusing on elite cultivars improved for ornamental traits including flower colors and forms, leaf pigments, and novel growth habits including dwarf habits. Flower colors included white, pink, blue, and purple, with one taxon, S. vulgaris ‘Sensation’, having picotee flowers in which the petal edges lack pigment. Flower forms included single and double flowers, with some exhibiting hose-in-hose flowers. Double flowers in lilac often represent a case of neoheterotrophy where additional floral whorls lead to supernumerary petals (Dadpour et al., 2011). Double flowers can also arise from mutations leading to petaloid sepals (Fiala, 1988). Both cas

Sulfuric acid scarification of Callicarpa americana L. (Lamiaceae) seeds improves germination

Genome size variation can be used to investigate biodiversity, genome evolution, and taxonomic relationships among related taxa. Plant breeders use genome size variation to identify parents useful for breeding sterile or improved ornamentals. Lilacs (Syringa) are deciduous trees and shrubs valued for their fragrant spring and summer flowers. The genus is divided into six series: Syringa (Vulgares), Pinnatifoliae, Ligustrae, Ligustrina, Pubescentes, and Villosae. Reports conflict on genome evolution, base chromosome number, and polyploidy in lilac. The purpose of this study was to investigate genome size and ploidy variation across a diverse collection. Flow cytometry was used to estimate monoploid (1Cx) and holoploid (2C) genome sizes in series, species, cultivars, and seedlings from parents with three ploidy combinations: 2x x 2x, 2x x 3x, and 3x x 2x. Pollen diameter was measured to investigate the frequency of unreduced gametes in diploid and triploid Syringa vulgaris cultivars. Three triploids of S. vulgaris were observed: ‘Aucubaefolia’, ‘Agincourt Beauty’, and ‘President Gr evy’. Across taxa, significant variations in 1Cx genome size were discovered. The smallest and largest values were found in the interspecific hybrids S. ·laciniata (1.32 ± 0.04 pg) and S. ·hyacinthiflora ‘Old Glory’ (1.78 ± 0.05), both of which are in series Syringa. Series Syringa (1.68 ± 0.02 pg) had a significantly larger 1Cx genome size than the other series. No significant differences were found within series Pubescentes (1.47 ± 0.01 pg), Villosae (1.55 ± 0.02 pg), Ligustrina (1.49 ± 0.05 pg), and Pinnatifoliae (1.52 ± 0.02 pg). For S. vulgaris crosses, no significant variation in 2C genome size was discovered in 2x x 2x crosses. Interploid crosses between ‘Blue Skies’ (2x) and ‘President Gr evy’ (3x) produced an aneuploid population with variable 2C genome sizes ranging from 3.41 ± 0.03 to 4.35 ± 0.03 pg. Only one viable seedling was recovered from a cross combination between ‘President Gr evy’ (3x) and ‘Sensation’ (2x). This seedling had a larger 2C genome size (5.65 ± 0.02 pg) than either parent and the largest 2C genome size currently reported in lilac. ‘Sensation’ produced 8.5% unreduced pollen, which we inferred was responsible for the increased genome size. No unreduced pollen was discovered in the other diploids examined. Increased ploidy may provide a mechanism for recovering progeny from incompatible taxa in lilac breeding. Genome size variation can be used to investigate biodiversity, taxonomic relationships, and genome evolution among related taxa (Greilhuber, 1998; Rounsaville and Ranney, 2010; Shearer and Ranney, 2013; Zonneveld and Duncan, 2010; Zonneveld et al., 2005). Studies on genome evolution focus on large, structural changes in sequence or fluctuations in genome size resulting from natural phenomena including polyploidy, chromosome fission/fusion, and interploid hybridization (Soltis and Soltis, 2012). Genome size variation can also be used by plant breeders to identify parents for wide hybrids among parent taxa. Interspecific hybrids have been shown to have genome sizes intermediate between their parents in other woody ornamentals such as Cornus (Shearer and Ranney, 2013) and Magnolia (Parris et al., 2010). When combining genome sizes with their corresponding chromosome counts, genome size data can be used to discover ploidy variation among related taxa (Contreras et al., 2013; Lattier, 2016; Parris et al., 2010; Shearer and Ranney, 2013). Polyploidy, or whole genome duplication, is a driving force in evolution and occurs naturally through somatic mutations in meristematic cells and through unreduced gametes (Harlan and deWet, 1975; Ranney, 2006). There are two broad categories of polyploidy; autopolyploidy is the duplication of a single genome, whereas allopolyploidy is the combination of two or more different genomes and an associated duplication event (Chen and Ni, 2006). The identification and induction of polyploidy can be valuable tools for plant breeding. Irregular meiosis in gametes can result in sterility, whereas ‘‘gigas’’ effects of somatic cells can result in thicker, glossier cuticles, enlarged flowers, or enlarged fruit (Ranney, 2006). In addition, polyploids have been used to overcome interploid hybridization barriers (Ranney, 2006) and to restore fertility in wide hybrids of ornamentals such as Rhododendron ‘Fragrant Affinity’ and ·Chitalpa tashkentensis (Contreras et al., 2007; Olsen et al., 2006). Syringa is a genus of deciduous, woody trees, and shrubs grown primarily for their heavy spring and summer blooms of fragrant flowers. Syringa comprised 21–28 species that are part of the monophyletic subfamily Oleoideae in family Oleaceae and are closely allied with Ligustrum (Li et al., 2002;Wallander and Albert, 2000). Recent taxonomic studies divide the genus into six series: Syringa, Pinnatifoliae, Ligustrae, Ligustrina, Pubescentes, and Villosae (Li et al., 2012). Most species are native to eastern Asia while two species, S. vulgaris and S. josikaea, are native to southeastern Europe (Kim and Jansen, 1998). Most cultivar development over centuries of breeding has focused on improvements of common lilac (S. vulgaris) within series Syringa. Lilacs have proven to be important ornamental crops, yet little is known about how nuclear genome varies among series, species, hybrids, and cultivars. A survey of genome Received for publication 14 June 2017. Accepted for publication 13 July 2017. This research was funded in part by the Oregon Department of Agriculture. We acknowledge the assistance of Mara Friddle, Kim Shearer, and Aleen Haddad in this research. Graduate Research Assistant. Corresponding author. E-mail: ryan.contreras@oregonstate.edu. J. AMER. SOC. HORT. SCI. 142(5):355–366. 2017. 355 size (C-value) and ploidy level within Syringa would contribute to the call for a global census of angiosperm C-values (Galbraith et al., 2011). Although genome sequencing is a powerful tool for studying gene function, C-values calculated from sequencing data tend to underestimate true genome size (relative to flow cytometry) because of misassembly and the inability to sequence through repetitive regions of the genome (Bennett and Leitch, 2011). Flow cytometry measurements of genome size have proven useful for the identification of species, hybrids, polyploids, and polyploid series (Galbraith et al., 2011). In genera such as lilac with a long history of breeding and cultivation, variation in genome size and chromosome number can occur from interspecific hybridization, unreduced gametes, and the induction of autopolyploids. Interspecific hybridization has been a valuable tool for producing many new cultivars of lilac (Table 1). Two reports on genome size estimates in lilac focused on two European species, S. vulgaris and S. josikaea. Siljak-Yakovlev et al. (2010) reported S. vulgaris to have a 2C genome size of 2.4 pg based on propidium iodide flow cytometry. Olszewska and Osiecka (1984) reported S. josikaea to have a 2C genome size of 2.6 pg based on Feulgen cytophotometry. Despite the paucity of genome size estimates in lilac, much effort has been dedicated to studying chromosome number variation in lilac and the Oleaceae. Phylogenetic analysis has determined the ancestral state of the Oleaceae to be diploid (Taylor, 1945). Cyto-taxonomy divides the Oleaceae into two groups according to basic chromosome number with the first group consisting ofMendora (x = 11), Jasminum (x = 13), Fontanesia (x = 13), Forsythia (x = 14), and Abeliophyllum (x = 14). The second group (originally designated as subfamily Oleoideae) consists of genera with x= 23, includingOlea, Syringa,Ligustrum,Fraxinus,Osmanthus, Forestiera, Phillyrea, Osmarea, and Chionanthus (Taylor, 1945). Lilacs are primarily diploids with basic chromosome numbers reported at x = 22, 23, or 24 (Darlington and Wylie, 1956). Sax (1930) reported the ‘‘fundamental’’ chromosome number in lilac to be x = 12 and hypothesized that ancestral polyploidization of an x = 11 or x = 12 cytotype was responsible for the variation in chromosome numbers, with the x = 23 cytotype resulting from the loss of a pair of chromosomes. By contrast, Taylor (1945) reported that most wild-type lilac specimens are x = 23 cytotypes, not x = 22 or x = 24. The prevalence and stability of the x = 23 cytotype throughout the Oleaceae, illustrated by Taylor (1945) and Stebbins (1940), indicates that the x = 23 cytotype likely predates other cytotypes in Syringa and originated as the result of allopolyploidy between ancestral Oleaceae taxa of two cytotypes, x = 11 and x = 12. Therefore, the variation in chromosome number observed in common lilac is likely the result of aneuploidy over centuries of plant collection and wide hybridization. Aside from theories of ancestral allopolyploidy, no reports exist to confirm polyploidy in wild or cultivated lilac populations. In addition, no reports of natural polyploidy exist for the closely related genus Ligustrum. However, natural polyploidy has been discovered in other related genera in Oleaceae. Taylor (1945) reported tetraploids in Mendora, tetraploids and triploids in Jasminum, tetraploids and hexaploids in Fraxinus, and hexaploids in Osmanthus. In white ash (Fraxinus), the tetraploid F. smallii and hexaploids, such as F. biltmoreana and F. profunda, are hypothesized to have allopolyploid origins (Miller, 1955; Nesom, 2010; Santamour, 1962). Early efforts producing artificial polyploids in lilac were reported to be successful. In the middle of the 20th century, Karl Sax produced colchicine-induced autopolyploids of S. vulgaris at the Arnold Arboretum (Fiala and Vrugtman, 2008). Fiala reportedly produced tetraploid forms of S. julianae, S. komarowii, S. ·prestoniae, S. wolfii, S. yunnanensis, S. vulgaris, S. oblata, and S. ·hyacinthiflora (Fiala and Vrugtman, 2008). Despite these previous reports of induced polyploidy, no cytological evidence exist

Woody Ornamentals of the Temperate Zone

ABSTRACT. The genus Cotoneaster (Rosaceae, Maloideae) is highly diverse, containing ’400 species. Like other maloids, there is a high frequency of naturally occurring polyploids within the genus, with most species being tetraploid or triploid. Apomixis is also prevalent and is associated with polyploidy. The objective of this study was to estimate genome sizes and infer ploidy levels for species that had not previously been investigated as well as compare estimates using two fluorochromes and determine base pair (bp) composition. Chromosome counts of seven species confirmed ploidy levels estimated from flow cytometric analysis of nuclei stained with 4#,6-diamidino-2-phenylindole (DAPI). Monoploid (1Cx) genome sizes ranged from 0.71 to 0.96 pg. Differences in monoploid genome size were not related to current taxonomic treatment, indicating that while chromosome sizes may vary among species, there are no clear differences related to subgeneric groups. A comparison of DAPI and propidium iodide (PI) showed a difference in DNA staining in Cotoneaster comparable to other rosaceous species. Base pair composition (AT%) in Cotoneaster ranged from 58.4% to 60.8%, which led to overestimation of genome size estimates in many cases—assuming the estimates of the DNA intercalator are accurate. Our findings will inform breeders with regard to the reproductive behavior of potential parents and may be used to confirm hybrids from interploid crosses.

Comparing Vegetative Propagation of Two ‘Schipkaensis’ Common Cherrylaurel Ploidy Levels

Abstract Considerable debates exist about the efficacy of foliar application of nutrients to Christmas trees, but little research has been conducted to determine whether this method of fertilization is beneficial. In this study, standard foliar and soil-applied fertilization products were applied to Nordmann fir Christmas trees under greenhouse and field-grown management regimes. On both sites, foliar nitrogen (N) and boron (B) concentrations, color, and normalized difference vegetation index (NDVI) were evaluated. Plant growth and needle chlorophyll/carotenoids were also monitored at the greenhouse site and sulfur (S) at the field site. At all sites, the soil-applied fertilizers were effective in increasing foliar N% compared to untreated and foliar applications. The foliar-applied products did not improve foliar N% compared to untreated trees. Foliar B concentrations were correlated with foliar fertilizer applications, indicating that B can be absorbed via foliar application. A second part of this study investigated alternate or complementary methods of assessing foliar N%. We addressed whether plant color, spectral reflectance, chlorophyll measures, or NDVI measurements could serve as surrogates for foliar N%. Color, chlorophyll/carotenoid, and foliar N% were closely correlated. However, NDVI evaluations showed no relationship with foliar N%, color, or chlorophyll/carotenoid levels.

Improved Method of Enzyme Digestion for Root Tip Cytology

An experiment was conducted to determine if sulfuric acid scarification improved seed germination of Callicarpa americana L. (Lamiaceae). Treatments included a control (0 min), 15-min, and 30-min soaks in concentrated (18N) sulfuric acid followed by a 15-min rinse in tap water. The 30-min treatment had the earliest germination with seedlings appearing 18 d after treatment (DAT). The 15-min treatment had seedlings emerge at 26 DAT while seedlings in the control did not begin to emerge until 60 DAT. After 60 d, seeds from the acid treatments had approximately 50% germination while the control had less than 10%. At the conclusion of the study, the control, 15-min, and 30-min acid treatments germinated at 8.9%, 57.8%, and 48.9%, respectively. The results of this study show the benefit of sulfuric acid scarification in the germination of Callicarpa americana. Recommendations should be amended to include a 15- to 30-min soak in concentrated sulfuric acid to promote rapid and more uniform germination for this species.Contreras RN, Ruter JM. 2009. Sulfuric acid scarification of Callicarpa americana L. (Lamiaceae) seeds improves germination. Native Plants Journal 10 (3): 283–286.

In Vitro Germination of Immature Prunus lusitanica Seed

Woody ornamental plants comprise a large number of genera, species, and cultivars that display a huge amount of phenotypic diversity. New introductions mainly result from natural existing variation or from selections in open-pollinated populations. Systematic commercial breeding mainly takes place in a few important genera. More advanced techniques such as interspecific hybridization and polyploidization have been successfully adopted to broaden the possibilities of woody plant improvement. Because of increased sophistication in marketing and branding, visual attractiveness at the point of sale has become one of the major breeding goals. But the real challenges continue to be biotic (disease and pest) and abiotic stress resistance (winter hardiness, heat, drought, pH, and flooding tolerance). In addition, some new trends are observed, such as breeding for sterility, breeding for multipurpose, and breeding and selecting of native plants with an ecological function. This review also provides an overview of the current status of genomics and genetic engineering in woody plants and the potential of application of genome-editing technologies to enable faster and more precise breeding.