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- Author or Editor: Ryan N. Contreras x
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.
Cape hyacinth (Galtonia candicans) is a geophytic herbaceous perennial from South Africa. It produces large inflorescences of pendulous white flowers during mid to late summer, followed by capsules filled with copious amounts of seed. The species has potential as a low-water-use landscape plant, but lodging and excessive seed production, which pose a risk of escape or invasion, are issues that should be addressed before marketing. Ethyl methanesulfonate (EMS) is a chemical mutagen known to induce usable mutations including dwarfing and sterility. We exposed seeds of cape hyacinth to increasing concentrations of EMS (0%, 0.2%, 0.4%, 0.6%, 0.8%, and 1%). Increased concentrations of EMS resulted in a linear decrease in seed germination when not exposed to a presoak treatment in water before exposure to EMS. No seedlings survived or were viable to field plant at 0.6%, 0.8%, or 1%. Resulting plants were field planted in 2013 and evaluated during 2014 and 2015. In both years, the inflorescence height at first flower, average seed number per capsule, and percent lodging were reduced in EMS-treated plants compared with controls. In 2015, pollen staining was evaluated and was reduced from 83% in control to less than 3% in the 0.4% treatment. Our study demonstrated that EMS is a viable option to reduce height and decrease seed set in cape hyacinth.
Sweetbox (Sarcococca confusa) is an evergreen shrub valued for attractive foliage, winter fragrance, black fruit, as well as shade and drought tolerance. The high degree of apomixis in Sarcococca limits the effectiveness of conventional breeding practices. However, mutation breeding may be a valuable tool to induce variation in seedling crops. As such, a study was conducted to expose seeds of S. confusa to ethyl methanesulfonate (EMS) at varying concentrations and exposure durations, and to evaluate the effects on seed germination, growth, and relative frequency of polyembryony. In 2010, seeds of S. confusa were treated with 0%, 0.2%, 0.4%, 0.8%, and 1.2% EMS for 24 and 48 hours. Seeds were sown and the relative germination and occurrence of polyembryony were recorded. Seedlings were later evaluated for size and phenotypic variation in the subsequent growing seasons. Percent germination was found to decrease with increasing EMS rates in the 24- and 48-hour treatments. The occurrence of polyembryony also decreased with increasing EMS rates in the 24-hour treatment but was observed to be greatest at 0.2% EMS. No significant differences in plant size index (SI) were found after four growing seasons. When mature, seeds were collected from any plants that bore fruit in each treatment and were then sown. The percentage of plants that bore fruit in each treatment decreased with increasing EMS rates, although the germination rate of these seeds did not differ. A chlorophyll mutant and several dwarf forms were identified in this population as well. They have been propagated asexually for future evaluation as new cultivars. This study demonstrates the utility of chemical mutagenesis to induce phenotypic variation in S. confusa while reducing the rate of polyembryony.
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.
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).
Vaccinium ovatum (evergreen huckleberry) is an evergreen shrub native to the Pacific Northwest. Evergreen huckleberry is diploid (2n = 2x = 24), but unreduced gametes have been reported that facilitated in interspecific tetraploids. To our knowledge, tetraploid forms of evergreen huckleberry have not previously been evaluated. There is interest in this species as a native, edible, evergreen landscape shrub, but it requires improvement of the fruit and plant qualities for an eventual cultivar release. To obtain variation in plant qualities, we induced polyploidy in a collection of plants in 2013. The purpose of this study was to assess the impacts of polyploidy on the fruit and plant qualities of V. ovatum. This fruit and plant quality study provides a contribution to the scientific knowledge base that is currently lacking for evergreen huckleberries. Plant qualities were determined by measuring plant height and width, obtained in Fall 2017. The fruit volume (mm3) and for soluble solids content (SSC, °Brix) were measured using a digital caliper and a digital refractometer, respectively. Measurements were taken on diploid, mixoploid, and tetraploid (2x, 2x + 4x, 4x) cytotypes, once in 2017, five times over 9 weeks in 2018, and three times over 9 weeks in 2019. Tetraploids had larger fruit than diploids in 2017 (P < 0.0001), suggesting there was a gigas effect from polyploidy in evergreen huckleberries. However, during 2018 and 2019, tetraploid fruit was smaller than that of diploid and mixoploid genotypes. Differences were observed in diploid fruit volume among all years (P < 0.0001) such that 2019 was largest and 2017 was smallest. It is unclear what led to this variation. In tetraploids, SSC was statistically significant among years (P = 0.0002) such that 2017 was highest and 2019 was lowest. Although our preliminary data suggested that induced polyploidy may result in larger fruit, this was not observed in subsequent years, and it does not appear that tetraploids necessarily will have larger or sweeter fruit. However, these tetraploids may facilitate crossing with other species at the tetraploid level as a means for improvement of various traits.
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.
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.
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.
Althea (Hibiscus syriacus) is a popular shrub known for its vibrant summer blooms and winterhardiness; however, althea produces capsules with numerous seeds that germinate and cause a nuisance in production and the home landscape. Breeding for sterile forms has long been a goal of Hibiscus breeders, yet many popular “sterile” cultivars have been reported as weedy. The purpose of this study was to evaluate female and male fertility of tetraploid and hexaploid cultivars, and to evaluate the female fertility of pentaploid progeny resulting from 4x × 6x and 6x × 4x crosses. More than 600 self-pollinations were performed on 21 cultivars, yet only 24% of self-pollinations resulted in filled capsules, for an overall rate of four seeds per pollination. Significant differences were observed among taxa for seeds per capsule and seeds per pollination. Highest capsule set was observed on self-pollinated White Chiffon® and Pink Chiffon®. Anecdotally, we observed reduced vigor in the S1 generation of most taxa. However, ‘Woodbridge’ produced vigorous seedlings through the S2 generation. More than 2000 cross-pollinations were also performed, resulting in more than 15,000 seeds. To evaluate female fertility, 28 taxa were pollinated with a variety of male parents. Fertility was measured as seeds per capsule and seeds per pollination. Significant differences were found among taxa within and among flower forms (single, semidouble, and double) for seeds per capsule and seeds per pollination. Double-flowered forms had reduced female fertility. Taxa previously reported to be sterile were found to be fertile, including ‘Aphrodite’, ‘Diana’, ‘Helene’, and ‘Minerva’. Two hexaploids, ‘Pink Giant’ and Raspberry Smoothie™, had reduced female fertility compared with tetraploids. Male fertility was estimated for 20 cultivars by pollinating between one and 23 cultivars. For male fertility, significant differences were found among taxa for seeds per capsule and seeds per pollination; however, no significant differences in male fertility were observed among flower forms. Four taxa had relatively high fertility with more than 10 seeds per capsule and seeds per pollination, including Blue Satin®, Lil’ Kim™, Bali™, and Tahiti™. In addition to the significant differences among female and male fertility of each taxon, capsule set varied widely among individual cross combinations. Significant differences of female fertility were found in pairwise comparisons between almost all pentaploid taxa and the mean of tetraploid control cultivars. No difference in percent seed germination was observed between 4x × 6x and 6x × 4x crosses (45% and 45%, respectively) but both were significantly lower than seeds from open-pollinated tetraploids (89%). The reduced fertility of pentaploids will likely lead to new reduced fertility or sterile cultivars for the nursery industry, especially if combined with double flowers.