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Ryan N. Contreras

Platycladus orientalis (L.) Franco (syn. Thuja orientalis L.), Thuja occidentalis L., and T. plicata D. Don. are conifers often used in the landscape. Most of the available cultivars of these species share the character of having foliage that turns an off-color during winter as a result of photoinhibition. Tetraploids of the related japanese-cedar [Cryptomeria japonica (L. f.) D. Don.] have exhibited greener color retention than diploids during winter and a recent report described a simple technique to double its chromosomes. The technique used to double the chromosome number of C. japonica was applied to the three species mentioned to determine if it would be effective for inducing polyploidy and, if so, optimal duration of treatment. Seedlings were treated at the cotyledon stage for 0 (control), 10, 20, or 30 days with an aqueous solution containing 150 μM oryzalin + 0.1% Tween® 20 using a standard household spray bottle that created a fine mist. No tetraploids were observed for any species in control treatments, indicating all recovered tetraploids resulted from applying oryzalin. Tetraploids were observed for all other treatments except T. plicata at 30 days. Efficacy ranged from 0% to 27.1% of transplanted seedlings being tetraploid. There was a quadratic relationship between duration of treatment and percent tetraploids in T. occidentalis and T. plicata and a linear relationship for P. orientalis. Based on regression analysis, the optimal duration of treatment was 20.5 days for T. occidentalis and 13.9 days for T. plicata. The highest percent tetraploids recovered for P. orientalis was at 30 days and it is unclear if increasing duration beyond this would continue increasing percent tetraploids recovered. Morphology was not useful in early identification of tetraploids for any species.

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Ryan N. Contreras and Kimberly Shearer

Acer is a large and important genus of woody plants most commonly encountered as small to large trees in urban landscapes. Considerable investigation has been devoted to addressing the taxonomy of maples, but little is known about genome sizes across the genus. Relatively more work has been conducted to determine chromosome numbers and ploidy of more species, but much could be gained by expanding knowledge of genome sizes in combination with traditional cytology. Furthermore, base pair (bp) composition may have implications for a species’ adaptability and also impacts nucleic acid stability at high temperatures. Our objectives were to determine the genome size of 195 accessions of maples, assign ploidy to each using inference as well as cytology, and determine base composition of a subset of 48 accessions. Most species had small genome sizes (1.4–3.5 pg) with the exception of section Rubra, which contains many polyploids. Holoploid genome sizes ranged from 1.39 to 6.10 pg, with the latter being interpreted as 9x. The mean monoploid genome sizes (1Cx) ranged from 0.43 pg in A. carpinifolium (section Indivisa) to 1.66 pg in A. caudatifolium (section Macrantha); mean monoploid genome sizes were significantly different among sections. Forty-four of the 48 accessions measured using both fluorochromes had greater estimates with 4′,6-diamidino-2-phenylindole (DAPI) than propidium iodide (PI). The proportion of the genome composed of guanosine and cytosine (GC%) among the taxa evaluated in this study ranged from just 38.61% to 43.96% and did not appear to be related to ecological adaptability or urban tolerance among these taxa.

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Ryan N. Contreras and Luigi Meneghelli

Prunus laurocersaus L. (2n = 22x = 176), common cherrylaurel, is an evergreen shrub generally used as a hedge or screen. This species produces large drupes that are a nuisance when they drop on walkways or deposited by birds and also has escaped cultivation in parts of northwestern United States, which has raised concern about the invasive potential of common cherrylaurel. Therefore, a fruitless and sterile form of common cherrylaurel is desirable. As part of our efforts to develop sterile common cherrylaurel cultivars, we conducted two experiments to induce chromosome doubling using in vitro exposure of ‘Otto Luyken’ and ‘Schipkaensis’ cherrylaurel to oryzalin. For ‘Otto Luyken’ (Expt. 1), we tested the effects of treatment duration (1, 2, 14, or 28 days) and oryzalin concentration (0, 6.25, 12.5, 25, 50, 100, or 150 μm) applied in a liquid phase over explants. In Expt. 2, we treated ‘Schipkaensis’ cherrylaurel shoots with a single duration of 28 days and exposed explants to the same varying concentrations of oryzalin incorporated into the solidified medium. In Expt. 1, the 14-day treatment had reduced survival compared with 1- and 2-day treatments and there was still greater mortality in the 28-day treatment. Duration of the treatment affected mortality more than oryzalin concentration. Sixteen treatment combinations resulted in 44x plants. The percentage of 44x plants increased with concentration in the 1- and 2-day treatments up to 30% of treated shoots at 150 μm. Overall, the longer duration treatments in Expt. 1 were less efficient for inducing 44x plants. Expt. 2 was less effective for inducing homogenous 44x plants. It is unclear if this is due to treatment or cultivar differences but the highest concentration was 8% in the 6.25 μm treatment.

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Jason D. Lattier and Ryan N. Contreras

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 Grévy’. 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 Grévy’ (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 Grévy’ (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.

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Jason D. Lattier and Ryan N. Contreras

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, and Villosae. 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.

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Justin A. Schulze and Ryan N. Contreras

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 125 µM oryzalin 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 125 µM oryzalin 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 µM 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.

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Ryan N. Contreras and Mara W. Friddle

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Ryan N. Contreras and John M. Ruter

Genome size estimates and chromosome number information can be useful for studying the evolution or taxonomy of a group and also can be useful for plant breeders in predicting cross-compatibility. Callicarpa L. is a group of ≈140 species with nearly worldwide distribution. There are no estimates of genome size in the literature and the information on chromosome numbers is limited. Genome size estimates based on flow cytometry are reported here for 16 accessions of Callicarpa comprising 14 species in addition to chromosome counts on six species. Chromosome counts were conducted by staining meristematic cells of roots tips using modified carbol fuchsin. Holoploid genome size estimates ranged from 1.34 pg to 3.48 pg with a mean of 1.74 pg. Two tetraploids (2n = 4x = 68; C. salicifolia P'ei & W. Z. Fang and C. macrophylla Vahl GEN09-0081) were identified based on holoploid genome size and confirmed by chromosome counts. There was little variation among species for monoploid genome size. 1Cx-values ranged from 0.67 pg to 0.88 pg with a mean of 0.77 pg. Chromosome counts for six species revealed a base chromosome number of x = 17. Callicarpa chejuensis Y. H. Chung & H. Kim, C. japonica Thunb. ‘Leucocarpa’, C. longissima Merr., and C. rubella Lindl. were confirmed as diploids (2n = 2x = 34). Cytology supported flow cytometry data that C. salicifolia and C. macrophylla GEN09-0081 were tetraploids. The two accessions of C. macrophylla included in the study were found to be of different ploidy levels. The presence of two ploidy levels among and within species indicates that polyploidization events have occurred in the genus.

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Ryan N. Contreras, Ron Determann and Mara Friddle

There is a great deal of variation among japanese-cedar cultivars with regard to growth form, foliar characteristics, and winter browning. Differences in winter browning have been observed and documented by a number of authors. Previous research has established that there are differences in winter foliage color between cultivars included in the current study; however, no quantitative analysis under standardized conditions was conducted. Because of a previous report that tetraploid forms of japanese-cedar remain green during winter as a result of increased antioxidant enzyme activity, we hypothesized that cultivars that exhibit reduced winter browning were polyploids. We screened 56 accessions of japanese-cedar using flow cytometry analysis of 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei and performed chromosome counts on three cultivars. All accessions were diploid (2n = 2x = 22), although there were significant differences in genome sizes among the cultivars. Holoploid genome sizes ranged from 18.9 pg for var. sinensis JCRA to 22.3 pg for ‘Viridis’ with a mean of 20.1 pg. Chromosome counts for cultivars Ogon, Oye Keme, and Viridis supported the flow cytometry results. Although the underlying cause of the variability in morphology and winter browning among cultivars is unclear, our results show that differences in ploidy level are not responsible, because all tested genotypes were diploid. Chemical name: 4′,6-diamidino-2-phenylindole (DAPI).

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Ryan N. Contreras and Thomas G. Ranney

Wide hybridization can potentially lead to the combination of diverse traits, but these hybrids are often sterile as is the case with the inter-subgeneric hybrid Rhododendron `Fragrant Affinity'. Induction of polyploidy can restore chromosome homology and fertility in wide hybrids. In this study we successfully developed an allopolyploid form of R. `Fragrant Affinity' using oryzalin as a mitotic inhibitor and chromosome doubling agent. Approximate genome size (2C), determined using flow cytometry, was 1.6 pg for the diploid and 3.2 pg for the allotetraploid. Pollen viability, determined by staining and germination tests, was 4% and 0%, respectively for the diploid and 68% and 45%, respectively for the allotetraploid. No seeds were produced when the diploid R. `Fragrant Affinity' was crossed with pollen from viable diploid and tetraploid parents. The allotetraploid produced viable seeds and seedlings when crossed with viable pollen from either diploid or tetraploid parents, including self pollination, demonstrating restored fertility. Additional crosses were successfully completed using the allotetraploid as part of an ongoing breeding program to develop new fragrant, cold hardy, evergreen rhododendron.