‘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.
Justin A. Schulze, Ryan N. Contreras, and Carolyn F. Scagel
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.
Jason D. Lattier, Hsuan Chen, and Ryan N. Contreras
Althea (Hibiscus syriacus) is a shrub prized for its winterhardiness and colorful summer flowers. Altheas are tetraploids (2n = 4x = 80); however, breeders have developed hexaploids and octoploids. Previous studies report anatomical variation among polyploids, including stomata size. The purpose of this study was 4-fold. First, identify genome size and ploidy variation in cultivars via flow cytometry and chromosome counts. Second, create a ploidy series consisting of 4x, 5x, 6x, and 8x cytotypes. Third, investigate the ploidy series for variation in stomatal guard cell lengths, stomatal density, and copy number of fluorescent ribosomal DNA (rDNA) signals. Fourth, investigate segregation patterns of rDNA signals in a subset of pentaploid seedlings. Flow cytometry revealed most cultivars to be tetraploid with holoploid 2C genome sizes from 4.55 ± 0.02 to 4.78 ± 0.06 pg. Five taxa (‘Aphrodite’, ‘Pink Giant’, ‘Minerva’, Azurri Satin®, and Raspberry Smoothie™) were hexaploids (6.68 ± 0.13 to 7.05 ± 0.18 pg). Peppermint Smoothie™ was a cytochimera with tetraploid cells (4.61 ± 0.06 pg) and octoploid cells (8.98 ± 0.13 pg). To create pentaploids, reciprocal combinations were made between hexaploid ‘Pink Giant’ and tetraploid cultivars. To create octoploids, seedlings were treated with agar solutions containing 0.2% colchicine or 125 μM oryzalin. Guard cell lengths were significantly different among the four cytotypes: 4x (27.36 ± 0.04 μm), 5x (30.35 ± 1.28 μm), 6x (35.59 ± 0.63 μm), and 8x (40.48 ± 1.05 μm). Measurements of stomatal density revealed a precipitous decline in average density from the 4x cytotype (398.22 ± 15.43 stomata/mm2) to 5x cytotype (194.06 ± 38.69 stomata/mm2) but no significant difference among 5x, 6x, and 8x cytotypes. Fluorescent in situ hybridization (FISH) revealed an increase in 5S and 45S rDNA signals that scaled with ploidy: 4x (two 5S + four 45S), 6x (three 5S + six 45S), and 8x (four 5S + eight 45S). However, pentaploid (5x) seedlings exhibited random segregation of rDNA signals between the 4x and 6x cytotypes, including all six possible combinations (two 5S, three 5S) × (four 45S, five 45S, six 45S).
Hsuan Chen, Lan Xue, Tong Li, and Ryan N. Contreras
Hibiscus syriacus is a woody shrub in the Malvaceae family that is common in landscapes due to its broad adaptability and variable ornamental characteristics. Interspecific hybridization has been used to improve Hibiscus by building novel floral traits, hybrid vigor, and hybrid infertility. A few interspecific hybrid Hibiscus cultivars (H. syriacus × H. paramutabilis), such as Lohengrin and Resi, are notable because of their vigorous vegetative growth, female infertility, and large flowers. However, little is known about the male fertility and breeding potential of these hybrid cultivars, which could increase flower size by backcrossing to H. syriacus. In this study, we estimated male fertility of the two hybrid cultivars by acetocarmine staining and in vivo pollination and assessed selection methods for floral traits, specifically flower size and petal number. A BC1F1 population of 294 individuals was developed by crossing hybrid cultivars Lohengrin or Resi with a variety of double-flowered H. syriacus cultivars. A negative correlation between petal number and petal area was detected by quantile regression, which is a method that circumvents the problem of simple linear regression, which violates statistical assumptions. Quantile regression was used to build simultaneous selection thresholds for different levels of required stringency. As expected, the female fertility of hybrid cultivars was extremely low or zero; however, the male fertility of hybrid cultivars was not reduced compared with H. syriacus cultivars. A negative linear correlation between the petal number and petal area of the BC1F1 individuals was observed. In addition, quantile regression was recommended to set a single selection threshold to be applied to the selection of two negatively correlated traits, which was more effective than independent selection of petal numbers and petal areas among progeny.
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.
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.
Ryan N. Contreras, John M. Ruter, and Wayne W. Hanna
Hibiscus acetosella Welw. ex Hiern. ‘Panama Red’ PP20,121 (Malvaceae) has generated public and grower interest due to its attractive red foliage and vigorous growth, however, a horticultural goal is to develop more compact forms. Even though organs of induced polyploids are often larger than the wild type, whole plants are often shorter in stature. Three studies were conducted to induce polyploidy and to evaluate the growth and reproductive potential of the resulting polyploids. In study 1, seeds were soaked for 24 hours in aqueous solutions of 0%, 0.2%, 0.4%, or 0.5% colchicine (w/v) plus 0.5% dimethyl sulfoxide. In studies 2 and 3, apical meristems of seedlings at the cotyledon stage were treated for 1 or 3 days with 0, 50, 100, or 150 μm oryzalin solidified with 0.8% agar. Visual observations and measurement of guard cells were used to identify plants that potentially had their chromosome number doubled. Flow cytometry of nuclei stained with DAPI was used for confirmation of polyploidy. No induced polyploidy was observed following seed treatment with colchicine at the rates and duration used in this study. One-time application of 50 μm oryzalin resulted in a single mixoploid (4x + 8x) in which the ploidy of the L-I, L-II, and L-III histogenic layers were identified as a 4–4-4 + 8, respectively. Three-day applications with 100 and 150 μm oryzalin resulted in an octoploid (8x) and a mixoploid (4x + 8x), respectively. The mixoploid from the 3-day treatment stabilized at the 8x level before flowering, but was identified as a 4 + 8-x-4 cytochimera. Plant height was reduced, leaves were smaller, internodes were shorter, and canopy volume was reduced in the octoploid (8x) form compared with the tetraploid (4x) form. Furthermore, in contrast to the tetraploid, the octoploid produced no self-pollinated seed and performed poorly as a staminate and pistillate parent in controlled crosses. This represents the first time oryzalin has been reported to induce polyploidy in Hibiscus L. section Furcaria DC. H. acetosella is an allotetraploid species with the genome composition AABB. The resulting autoallooctoploid (AAAABBBB) form of ‘Panama Red’ exhibits a more compact habit and reduced production of seed.
Ryan C. Graebner, Hsuan Chen, Ryan N. Contreras, Kathleen G. Haynes, and Vidyasagar Sathuvalli
Conventional wisdom regarding potato breeding indicates that a strong triploid block prevents the development of viable triploid seeds from crosses between tetraploid and diploid clones. However, in a recent set of crosses between elite tetraploid potatoes and an improved diploid hybrid population derived from group Stenotomum and group Phureja, 61.5% of the resulting clones were found to be triploid. If clones derived from one diploid parent suspected of producing a high frequency of unreduced gametes were excluded, then the frequency of triploid clones increased to 74.4%. Tubers of these triploids are generally intermediates of the two parental groups. Our findings indicate the possibility of using triploid potatoes in potato variety development programs and in genetic and genomic studies.
Kelly J. Vining, Ryan N. Contreras, Martin Ranik, and Steven H. Strauss
Because cultivation of exotic woody ornamental plants has led to establishment of a number of invasive species, there is considerable interest in breeding methods to reduce the propensity for spread. We review progress in conventional breeding and transgenic biotechnology approaches to producing sterile forms of ornamental woody plants. Conventional forms of inducing sterility, including induction of polyploidy, interspecific hybridization, and mutagenesis, are generally inexpensive and can be applied to a diversity of species at low to moderate cost. They have also been shown to be capable of producing commercially successful cultivars. In contrast, despite a variety of highly promising and rapidly developing approaches using transgenic methods, the inability to efficiently regenerate and genetically transform most ornamental species makes application of these innovations highly problematic. Moreover, because of the fragmented pattern of ornamental nursery ownership, the numerous species and varieties used, and the high regulatory cost for permits to sell most types of transgenic varieties (even when their environmental risk of spread has been reduced by sterility), application of transgenic methods is largely infeasible. A combination of fundamental regulatory reform and expanded biological research on generalized transformation and sterility methods is needed to overcome these barriers.
Ryan N. Contreras, John M. Ruter, James S. Owen Jr., and Andy Hoegh
Japanese-cedar has been underused in landscapes of the United States until recent years. There are now over 100 cultivars, many of which are grown in the southeast of the United States. Performance of cultivars has been described from U.S. Department of Agriculture (USDA) Zone 6b to USDA Zone 7b; however, there are no reports on how cultivars perform in USDA Zone 8. The current study was conducted to measure chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid content and assign visual color ratings to determine if there was a relationship between pigment values and perceived greenness, which generally is regarded as a desirable and potentially heritable trait. Total chlorophyll (P = 0.0051), carotenoids (P = 0.0266), and the ratio of total chlorophyll to carotenoids (P = 0.0188) exhibited a positive relationship with greenness after accounting for season and tree effects. In contrast, the ratio of chlorophyll a to chlorophyll b did not have an effect on greenness. There was a linear relationship between total chlorophyll and carotenoid regardless of season (summer R 2 = 0.94; winter R 2 = 0.88) when pooled across 2 years. The observed correlation between chlorophyll and carotenoid content suggests they could be used interchangeably as predictors of greenness. There were large differences in rainfall between the 2 years that may have resulted in additional variation. Furthermore, the climate in which the evaluation was conducted differs greatly from the native distribution of japanese-cedar occurring in China and Japan.