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 N. Contreras, John M. Ruter, and Wayne W. Hanna
Ryan N. Contreras, Jim Owen, Wayne Hanna, and Brian Schwartz
Ornamental grasses such as fountaingrass or napiergrass, collectively called pennisetums, belong to the genus Pennisetum, which is a diverse genus with over 80 species adapted to a wide range of climatic regions and known for its drought tolerance. Breeding efforts have led to improvements such as more intense purple foliage color, disease resistance, and apparent sterility. These improved forms have been developed and tested in the eastern United States. The objective of this research was to evaluate container and field performance of seven new complex hybrid pennisetums in the Pacific northwestern United States. Two completely randomized experiments with three replications were conducted over 2 years (2010 and 2011) at two locations. We selected seven trispecific hybrid pennisetums resulting from interploid and interspecific crossing that were given accessions Tift 5, Tift 6, Tift 10, Tift 11, Tift, 13, Tift 15, and Tift 26. Experiment 1 evaluated container performance in Corvallis, OR, while Expt. 2 evaluated field performance in Aurora, OR. Size index (SI), growth form rating, and color rating were collected and analyzed separately by location. In the container study, significant differences were observed among selections for growth form in 2010 and color ratings in both 2010 and 2011. In 2010, Tift 6, Tift 11, Tift 13, and Tift 15 had the highest growth form rating. For color rating, Tift 5, Tift 10, and Tift 26 were among the four highest rated selections in both years. In the field study, Tift 5, Tift 10, Tift 11, and Tift 26 had the highest SI when data were pooled over the 2 years, but all selections reached acceptable size for landscape use during both years of the study. Similarly, there were color differences among selections with Tift 5, Tift 10, Tift 15, and Tift 26 being highest rated. None of the selections survived below winter temperatures of −5 °C at either location during either year of the study. Our evaluations indicate that these selections have potential in the Pacific northwestern United States as annuals. Differences in complex hybrid pennisetums were observed in SI, growth form rating, and color rating. These differences demonstrated the variation among selections and will allow producers to choose desired traits based on market preference.
Ryan N. Contreras, John M. Ruter, and David A. Knauft
American beautyberry (Callicarpa americana) is a deciduous shrub native to the southeast United States and is grown primarily for its metallic-purple fruit that develop in the fall. There are also pink- and white-fruiting and variegated forms but these traits are rare in nature and there is no information available regarding their inheritance. Also, there is confusion regarding self-compatibility and the presence of apomixis in Callicarpa L. Crosses were performed to investigate the genetics of fruit color, self-compatibility, and apomixis in american beautyberry. Test crosses between C. americana (CA) and C. americana ‘Lactea’ (CAL) suggested that white fruit is recessive to purple. White fruit appears to be controlled by a single recessive gene for which we propose the name white fruit and the gene symbol wft. Although there were only a limited number of progeny grown, crosses between CA and ‘Welch’s Pink’ suggest that purple is dominant to pink. Test crosses between CAL and ‘Welch’s Pink’ are needed to draw conclusions; however, we propose that purple, pink, and white fruit are controlled by an allelic series for which we suggest the gene symbols Wft > wft p > wft. Segregation ratios suggested that all progeny in the study developed through sexual hybridization. All genotypes used in the current study were self-compatible.
Justin A. Schulze, Jason D. Lattier, and Ryan N. Contreras
A tissue culture protocol was developed to germinate immature Prunus lusitanica seeds in vitro. The study was conducted by first identifying the best media for germination, followed by investigating effects of seed conditioning. In Expt. I, seeds were collected 12 weeks after pollination (WAP) ± 1 week and placed on media after removing the pericarp. Eight different MS media (Murashige and Skoog, 1962) were tested (M1–M8) containing two concentrations each of 6-benzylaminopurine (BA), gibberellic acid (GA3), and sucrose. The longest shoots resulted from M4 (1.45 µm GA3, 6 µm BA, and 30 g·L−1 sucrose), followed by M1 (0 µm GA3, 3 µm BA, and 30 g·L−1 sucrose). Radicle and shoot emergence was greater than or equal to 90% for M1, M3, and M4 after a stratification treatment. In Expt. II, M1 was used to test root and shoot emergence at 6, 9, and 12 WAP, with and without cold stratification. Little success was seen 6 and 9 WAP, with only callus development in 6 WAP, nonstratified seed. Cold stratification increased shoot emergence in the 12 WAP group from 4% to 28%, appearing to be critical for shoot emergence. If the cotyledons are retained on the seed, future efforts to expedite breeding of P. lusitanica using in vitro germination should not be collected before 12 WAP and will benefit from cold stratification before germinating on M1 or M4. Chemical names: 6-benzylaminopurine (BA), gibberellic acid (GA3).
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.
Ryan N. Contreras, Thomas G. Ranney, and Shyamalrau P. Tallury
Wide hybridization can lead to recombination of diverse traits and creation of unique phenotypes, but the resultant hybrids are often sterile as is the case with the intersubgeneric hybrid Rhododendron L. ‘Fragrant Affinity’. Sterility in wide hybrids can either be genic or chromosomal; the latter may be overcome by induction of polyploidy, which can restore chromosome homology and fertility. Cytologic studies of ‘Fragrant Affinity’ appear to support the presence of bridges between bivalents in metaphase I and laggard chromosomes in anaphase I. In the current study, an allotetraploid form of R. ‘Fragrant Affinity’ was developed using oryzalin (4-(dipropylamino)-3, 5-dinitro-benzenesulfonamide) as a mitotic inhibitor and chromosomal doubling agent. Genome sizes (2C) were determined using flow cytometry and found to be ≈1.6 and 3.2 pg for the diploid and allotetraploid, respectively. 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 used as a pistillate parent when pollinated with pollen from viable diploid and tetraploid parents. The allotetraploid produced viable seeds and seedlings when pollinated with pollen from either diploid or tetraploid parents, including self-pollination, demonstrating restored male and female fertility.
Killian Melsen, Mark van de Wouw, and Ryan Contreras
The promising possibilities of mutation breeding in ornamental plants have led to a great interest in effective mutagenic treatment protocols for various species. This review discusses mutagenic treatments of a large number of ornamental genera, the advantages and disadvantages of various techniques, and the possibilities of improving the associated protocols. A number of nontargeted mutagenesis methods are available, ranging from chemical treatment with alkylating agents to irradiation with X-rays, gamma rays, and neutron or heavy ion beams at various doses. These are all relatively inexpensive and have been proven to be effective mutagens in a large number of diverse species. Genetic engineering, however, remains mostly impractical for many ornamental breeding operations because of the high cost and lack of knowledge necessary to successfully transform and regenerate ornamental crops. Of the available nontargeted mutagens, irradiation with gamma rays is still the most popular. It provides high consistency compared with chemical mutagens, albeit at a seemingly lower mutagenic efficiency. Changes in the radiation dose rate may increase the efficiency, although chronic irradiation over a longer period causes fewer deleterious mutations than the commonly used acute irradiation protocols. Heavy ion beam irradiation may also provide highly consistent mutation induction at higher efficiencies because of the high particle energy associated with these treatments. There are also opportunities to improve chemical mutagenesis. Although the required knowledge of specific gene functions in many ornamentals is still lacking, combination mutagenesis with ethyl methanesulfontate with genetic screening in a process known as TILLING (Targeting Induced Local Lesions IN Genomes) may lead to a powerful mutation breeding tool in the future. Mutation breeding is still very useful, and many opportunities are available to improve the existing methods.
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.
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.