Ryan N. Contreras and Mara W. Friddle
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).
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.
Tyler C. Hoskins, Jason D. Lattier, and Ryan N. Contreras
Common lilac is an important flowering shrub that accounts for ≈$20 million of sales in the U.S. nursery industry. Cultivar improvement in common lilac has been ongoing for centuries, yet little research has focused on shortening the multiple-year juvenility period for lilacs and the subsequent time required between breeding cycles. The practice of direct-sowing of immature “green” seed has been shown to reduce juvenility in some woody plants, but it has not been reported for common lilac. This study investigated the effects of seed maturity [weeks after pollination (WAP)], pregermination seed treatment (direct-sown vs. cold-stratified), and postgermination seedling chilling on the germination percentage, subsequent plant growth, and time to flower on lilac seedlings. All seedlings were derived from the female parent ‘Ludwig Spaeth’ and the male parent ‘Angel White’. Seeds harvested at 15 and 20 WAP resulted in 58% (sd ± 9.9%) and 80% (sd ± 9.0%) germination, respectively, which were similar to that of dry seed collected at 20 WAP with stratification (62% ± 4.2%). Seedlings from the green seed collected at 15 and 20 WAP were also approximately three-times taller than those of dry seed groups DS1, DS2, and DS3 after the first growing season. Over the next two growing seasons, there were no differences in seedling height across all treatments. Flowering occurred at the beginning of the fourth season and without differences among treatments. These results indicate that the collection and direct sowing of immature, green seed can be used to successfully grow lilac seedlings, but that they do not reduce the juvenility period. However, this method can provide more vegetative growth in year one to observe early vegetative traits such as leaf color, and it can provide more material for DNA extraction to support molecular research.
Jason D. Lattier, Hsuan Chen, and Ryan N. Contreras
Chromosome numbers are an important botanical character for multiple fields of plant sciences, from plant breeding and genetics to systematics and taxonomy. Accurate chromosome counts in root tips of woody plants are often limited by their small, friable roots with numerous, small chromosomes. Current hydrolysis and enzyme digestion techniques require handling of roots before the root squash. However, optimum chromosome spread occurs when the cell walls have degraded past the point of easy handling. Here, we present a new enzyme digestion protocol that is fast, efficient, and flexible. This protocol reduces handling of the roots allowing for long-duration enzyme digestion. Digestions are performed on a microscope slide, eliminating the need for handling digested cells with forceps or pipettes. To illustrate the flexibility of this method across woody plant taxa, we performed chromosome counts on five angiosperms and one gymnosperm. Ploidy levels included diploids, triploids, and tetraploids with chromosome numbers ranging from 2n = 16 to 2n = 80. The range of holoploid 2C genome sizes spanned 1.54–24.71 pg. This protocol will provide a useful technique for plant cytologists working with taxa that exhibit a wide range of genome size and ploidy levels.
Hsuan Chen, Jason D. Lattier, Kelly Vining, and Ryan N. Contreras
Lilacs (Syringa sp.) have been used as ornamental plants since the mid-16th century and remain important in modern gardens due to their attractive and fragrant flowers. However, a short flowering season is a critical drawback for their ornamental value. Breeders have identified remontancy (reblooming) in dwarf lilac (Syringa pubescens), and have tried to introgress this trait into related species by interspecific hybridization. Molecular tools for lilac breeding are limited because of the shortage of genome sequence knowledge and currently no molecular markers are available to use in breeding for remontancy. In this study, an F1 population from crossing Syringa meyeri ‘Palibin’ × S. pubescens ‘Penda’ Bloomerang® Purple was created and subjected to genotyping-by-sequencing (GBS) analysis and phenotyped for remontancy. Plants were categorized as remontant, semi-remontant, and nonremontant based on the relative quantity of inflorescences during the second flush of flowers. A total of 20,730 single-nucleotide polymorphism (SNP) markers from GBS were used in marker-trait association to find remontant-specific marker(s) without marker position information. Two SNP markers, TP70580 (A locus) and TP82604 (B locus), were correlated with remontancy. The two loci showed a partial epistasis and additive interaction effects on the level of remontancy. Accumulation of recessive alleles at the two loci was positively correlated with increased reblooming. For example, 87% of aabb plants were remontant, and only 9% were nonremontant. In contrast, 100% of AaBB plants were nonremontant. These two SNP markers associated with remontancy will be useful in developing markers for future breeding and demonstrate the feasibility of developing markers for breeding woody ornamental taxa that lack a reference genome or extensive DNA sequence information.
Ryan N. Contreras, John M. Ruter, and Brian M. Schwartz
Japanese-cedar [Cryptomeria japonica (L.f.) D. Don] represents an alternative to leyland cypress [×Cuprocyparis leylandii (A.B. Jacks. & Dallim.) Farjon] as an evergreen screen or specimen plant for landscapes. It performs well under a range of soil and environmental conditions but has been underused attributable, in part, to unsightly winter browning caused by photoinhibition. In previous studies, chance seedlings that did not exhibit winter browning were identified as tetraploids. The current study was conducted to induce polyploidy in japanese-cedar. Approximately 600 seedlings were sprayed with 150 μM oryzalin + 0.1% SilEnergy™ for 30 consecutive days under laboratory conditions. Two hundred thirty-seven seedlings with thickened and twisted leaves were selected, transplanted, and grown in a glasshouse for 120 days. Seedling ploidy levels were analyzed using flow cytometry 180 days after treatment (DAT), identifying 197 (83.1%) tetraploids, 22 (9.3%) cytochimeras, and 18 (7.6%) diploids. Morphology of induced tetraploids was similar to that previously described and provided a phenotypic marker during selection that was over 92% accurate. A random subset of 20 tetraploid individuals was analyzed 270 DAT and were found to contain only tetraploid cells in the leaves analyzed, confirming stability over this period. This study demonstrated the use of oryzalin for inducing tetraploids in japanese-cedar, which we predict will be effective in other gymnosperms.
Ryan N. Contreras, Jonathan J. Velez, and Rob Golembiewski
New technologies such as online databases, interactive dichotomous keys, and online courses have changed the way some plant identification courses are delivered. These changing resources may create discrepancies between traditional instruction of landscape plant materials courses and the way modern students learn, which may result in students not meeting their potential. However, what resources students are using to study plant materials is unclear. We investigated the relationship between learning styles, study habits, and performance of students during two terms of woody landscape plant materials courses. To assess these relationships, we determined the characteristics of the participants and their preferred study method throughout the duration of the term as well as correlations between 1) preferred learning styles and performance, 2) preferred learning styles and preferred study method, and 3) performance and preferred study method. The participants in this study (n = 31) consisted of 14 males and 17 females. Of the 31 participants, 3 were freshmen, 3 were sophomores, 16 were juniors, 7 were seniors, and 2 were graduate students. Based on preference scores for learning style, 15 students were identified as visual learners, 3 as auditory, and 13 as kinesthetic learners. No significant relationships were observed between preferred learning style and performance or between preferred learning style and preferred study method. The two preferred study methods were using branch samples collected by the instructor and notecards created by students. No relationship existed between preferred study method and performance in the course. Our study provides information on study methods of woody plant identification students enrolled in a site-based course. We did not observe statistically significant relationships among preferred learning style, preferred study method, and course grade, but anecdotal evidence indicated students who prepared their own study aids by making notecards scored better in these courses.
Justin A. Schulze, Ryan N. Contreras, and Carolyn F. Scagel
‘Schipkaensis’ common cherrylaurel (Prunus laurocerasus) is an important nursery crop across the United States. In our breeding efforts to reduce shot-hole symptoms and weediness, we have created chromosome doubled forms of this cultivar. Vegetative propagation is an important factor in nursery production, and we have found no studies that have looked at comparative adventitious rooting of stem cuttings using induced polyploids. The objective of this research was to determine if rooting ability varied between these two ploidy levels. Semihardwood stem cuttings from wild-type (22x) and polyploid (44x) ploidy levels were taken at the end of July 2015 and the beginning of July 2016. Cuttings were dipped in 1030 ppm (0.10%) indole-3-butyric acid (IBA) and 660 ppm (0.066%) 1-naphthaleneacetic acid (NAA) before being set in rooting substrate. After 1 month, cuttings were removed from substrate and data collected. Data included; rooting percentage, root number per rooted cutting, average root length, and total root length. In 2015, 88% of the cuttings from the 44x plants and 63% of the cuttings from the 22x plants rooted. In 2016, 100% of cuttings from both ploidy levels rooted. In both years, average root length and total root length were similar between ploidy levels; however, cuttings from 22x plants generally had more roots than those from 44x. Chromosome-doubled ‘Schipkaensis’ common cherrylaurel rooted effectively, and produce transplantable cuttings similar to the standard ploidy.
Joseph J. Rothleutner, Mara W. Friddle, and Ryan N. Contreras
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.