Browsing by Author "Dennis J. Werner, Committee Member"

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Azaleodendrons: Investigating Parentage, Fertility, and Effects of Polyploidy Among Hybrids of Deciduous Azaleas and Evergreen Rhododendrons
(2007-06-26) Contreras, Ryan Nelson; Thomas G. Ranney, Committee Chair; G. Craig Yencho, Committee Member; Dennis J. Werner, Committee Member
Studies were conducted in order to develop a fertile form of the azaleodendron cultivar Rhododendron L. 'Fragrant Affinity' and determine parentage of this and two other azaleodendron cultivars. Wide hybridization can potentially lead to recombination of diverse traits and creation of unique phenotypes, but these hybrids are often sterile as is the case with the inter-subgeneric hybrid R. '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. Initial cytological studies of 'Fragrant Affinity' showed bridges between bivalents in metaphase I and laggard chromosomes in anaphase I. In this study we successfully developed an allotetraploid form of R. 'Fragrant Affinity' 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 approximately 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 maternal parent when crossed with pollen from viable diploid and tetraploid parents. The allotetraploid produced viable seeds and seedlings when crossed with pollen from either diploid or tetraploid parents, including self pollination, demonstrating restored male and female fertility. Additional crosses were successfully completed using the allotetraploid as part of an ongoing breeding program to develop new fragrant, cold hardy, evergreen rhododendron. Morphological analysis has historically been used to determine parentage of unknown hybrids. This can be difficult when the purported parental taxa have very similar morphology as in the case of the three azaleodendron cultivars R. 'Fragrans', 'Fragrans Affinity', and 'Fragrant Affinity'. The three cultivars are purported hybrids of R. catawbiense Michx. or R. ponticum L. and R. viscosum (L.) Torr., the three cultivars are very similar morphologically as are the purported parents. In this study, morphological and AFLP analyses were conducted to determine if these are unique cultivars or clones and to elucidate the parents. The three cultivars, potential parents from subgenera Hymenanthes (Blume) K.Koch (evergreen rhododendrons) and Pentanthera (G.Don) Pojarkova (deciduous azaleas), and related taxa from each subgenus were evaluated using 31 primer combinations. Morphological comparison indicated that R. ponticum is likely the evergreen rhododendron parent of R. 'Fragrant Affinity' based on intermediate petiole and leaf blade lengths and floral characteristics. Genetic similarity calculated using Jaccard's coefficient of similarity was highest between the hybrids and R. ponticum among the evergreen rhododendrons and R. viscosum among the deciduous azaleas, respectively. Genetic similarities among the three azaleodendrons indicated that they are unique cultivars and not synonyms, but likely share the same parental species. A dendrogram generated using the genetic similarity matrix grouped taxa into generally accepted taxonomic groups and nested R. ponticum with the hybrids suggesting it is the evergreen rhododendron parent. Additionally, a plot of the first three principle components also showed R. ponticum to be grouped more closely with the hybrids. Furthermore, there were 18 fragments unique to R. ponticum and the hybrids, however no unique bands were found for the purported deciduous azalea parent, suggesting the original parent may have been a hybrid.
Effect of Fertilizer Nitrogen Rate and Time of Application on Growth and Performance of Six Herbaceous Perennials and Soil Solution Nitrogen Concentration in a Simulated Landscape
(2006-01-09) Proctor, Cynthia Lambert; Dennis J. Werner, Committee Member; Rob Mikkelsen, Committee Member; Stuart L. Warren, Committee Chair; Dennis J. Werner, Committee Member; Rob Mikkelsen, Committee Member; Stuart L. Warren, Committee Chair; Dennis J. Werner, Committee Member; Rob Mikkelsen, Committee Member; Stuart L. Warren, Committee Chair
Herbaceous perennials are planted worldwide in public gardens, and commercial and home landscapes. Little research based information exists on response of herbaceous perennials to fertilizer nitrogen (FN) rate and timing and the potential for loss of applied N via leaching. Therefore, we constructed simulated landscapes and installed canna lily (Canna L. 'President'), coreopsis (Coreopsis verticillata L. 'Moonbeam' ), purple coneflower (Echinacea purpurea L. 'Magnus'), iris (Iris siberica L. 'Caesar's Brother'), switchgrass (Panicum virgatum L. 'Shenandoah'), sedum (Sedum L. 'Herbstfreude'), and sage (Salvia x sylvestris L. 'East Friesland') to determine how FN rate and timing affected growth and performance of herbaceous perennials and potential N leaching. Porous ceramic cup lysimeters were installed in situ in each landscape plot 38 cm below the soil surface to examine the effects of FN rate and timing on soil solution N concentrations. The experiment was a 4 x 4 factorial in a randomized complete block design with four replications. Four rates of FN: 0, 7, 14, and 28 g•m-2 N were divided equally into two applications and applied at the following times: 1) winter (Jan. 15 and Feb.15), 2) spring (Apr. 15 and May 15), 3) summer (June 15 and July 15), or 4) fall (Sept. 15 and Oct.15) beginning 2001. Soil solution samples were collected approximately every 2 weeks from Jan. 30, 2002 to Nov. 3, 2003 and analyzed to determine soil N (NO3- and NH4+) concentrations. Data collected in 2002 and 2003 for each species included the following number of flowers, plant visual evaluations, growth index (GI), and top dry weight. In 2003, mineral nutrient concentration was determined for each species. Plant response to treatments varied in 2002 and 2003. Our results indicated that despite statistical significance differences, many differences were small and FN treatments had little impact on the growth and ornamental qualities of these species. However, applying no N may not advisable, as some perennials species did benefit from an FN application during a specific time. Soil N concentrations remained above 10 mg•L-1 for 110 days, 62 days, 52 days, and 192 days when applied during January/February, April/May, June/July, 2002 and September/October in 2001 respectively. In 2003, soil N concentrations from FN rates of 7 g•m-2 N (1.5 lbs•1000 ft-2) and 14 g•m-2 N (3.0 lbs•1000 ft-2) applied in January/February, June/July, April/May and September/October remained below 10 mg•L-1 until the end of the study (Oct. 14, 2003). Nitrogen concentrations increased to 19 mg•L-1, 38 mg•L-1, and 21 mg•L-1, after 28 g•m-2 N (6.0 lbs•1000 ft-2) was applied in January/February, April/May, and June/July, respectively. To cover the needs of a wide variety of perennial species that usually exists in one landscape as well as minimize N concentrations in the soil solutions, we recommend a low to moderate rate of FN (5 g•m-2 to 15 g•m-2 ) be applied in split applications in spring and early fall.
Investigating Prevalence, Induction, and Fertility of Polyploid Rhododendron L. and the Development of Protocols for Vegetative Propagation
(2008-04-25) Jones, Jeffrey Robert; Thomas G, Ranney, Committee Chair; Anthony V. LeBude, Committee Member; Dennis J. Werner, Committee Member
Studies were conducted to determine the ploidy levels of specific Rhododendron taxa, to develop a simple and effective ex-vitro method for inducing polyploidy in Rhododendron seedlings, and to evaluate the effect of increased ploidy level on pollen fertility. A diverse collection of species, hybrids, and cultivars in the Hymenanthes (elepidote rhododendrons), Rhododendron (lepidote rhododendrons), Pentanthera (deciduous azaleas), and Tsutsusi (evergreen azaleas) subgenera were surveyed to determine ploidy level and relative genome size using flow cytometry. In instances where ploidy levels were inconsistent with past literature, chromosome counts were made on young root tips to substantiate findings. Mean 2C holoploid genome sizes varied as a function of subgenus and ploidy level. Relative genome sizes (2C) within ploidy level, for a given subgenus, had a narrow range providing clear distinction among ploidy levels. Mean 1Cx monoploid genome size was conserved across ploidy levels within a subgenus. Polyploidy was found to be common in the genus Rhododendron and considerably more prevalent in the subgenus Pentanthera than previously known. Particularly noteworthy were the findings that R. occidentale includes both diploid and tetraploid individuals and that R. atlanticum and R. austrinum are predominantly tetraploid species. Induction studies were then completed with the goal of obtaining artificial tetraploids from diploid x diploid hybridizations. The effectiveness of using repeated treatments of an oryzalin suspension in a warm agar solution applied directly to apical shoots of Rhododendron seedlings to induce polyploidy was tested. Apical meristems of hybrid seedlings were subjected to 1, 2, 3, or 4 applications of oryzalin separated by 4 day intervals or left untreated (control). The results of this study demonstrated that the method of applying a suspension of oryzalin in warm, semi-solid agar to the shoots of Rhododendron seedlings was an effective method for inducing polyploidy. Although single applications resulted in some polyploid plants, multiple applications increased efficacy for some of the taxa studied. Treatments resulted in a range of ploidy levels, from 2x to 8x, including mixaploids (cytochimeras). The effect of increased ploidy level on pollen fertility and the occurrence of unreduced gametes in triploid taxa were also studied. Pollen viability was compared between corresponding progenitor and polyploid taxa by staining pollen with 1% acetocarmine (w⁄v) for 15 minutes. The existence of unreduced gametes in triploid taxa was determined by the presence of dyad and/or monad pollen grains. The results demonstrated that the fertility of polyploid Rhododendron can be highly variable and that the induction of polyploidy may either enhance or compromise fertility. Moreover, some triploids produced viable, unreduced pollen (as high as 5%), allowing for the possible utilization of these plants in breeding programs. Documentation of polyploid taxa, improved methods for inducing polyploidy, and information on fertility and reproductive biology of polyploid Rhododendron will be valuable to plant breeders. Methods for improving propagation of stem cutting of North American deciduous azaleas were also examined. Influence of dormant hedging, a range of rooting hormone concentrations (0, 2500, 5000, 7500, 10,000 ppm K-IBA), and tissue growth stage on rooting of Rhododendron austrinum (Small) Rehder and R. flammeum (Michx.) Sarg. were evaluated. Softwood stem cuttings of both species rooted from 70-90% while semi-hardwood cuttings rooted from 20-70% Cuttings from hedged stock plants rooted at higher percentages and possessed higher root system indexes compared to the unhedged counterparts in R. flammeum, but the effect of hedging was less evident in R. austrinum. Increasing IBA concentration increased rooting percentage of softwood cuttings of R. flammeum, but had no beneficial effect on rooting of semi-hardwood cuttings of R. flammeum or on R. austrinum, regardless of growth stage.
A North Carolina field study to evaluate the effect of a coastal stormwater wetland on water quality and quantity and nitrogen accumulation in five wetland plants in two constructed stormwater wetlands.
(2008-08-27) Lenhart, Hayes Austin; Dennis J. Werner, Committee Member; William F. Hunt III, Committee Chair; Michael R. Burchell II, Committee Member
Propagation by Stem Cuttings and Nitrogen Nutrition of Eastern Redbud (Cercis canadensis)
(2008-05-28) Wooldridge, John Michael; Frank A. Blazich, Committee Co-Chair; Stuart L. Warren, Committee Co-Chair; Barry Goldfarb, Committee Member; Dennis J. Werner, Committee Member
Utilizing Polyploidy for Developing Improved Nursery Crops: Restoring Fertility in Wide Hybrids, Limiting Fertility of Invasive Species, Embryo Culture of Triploids, Pest Resistance, and Inheritance of Ornamental Traits
(2007-04-13) Olsen, Richard Thomas; G. Craig Yencho, Committee Member; Dennis J. Werner, Committee Member; Thomas G. Ranney, Committee Chair; David A. Danehower, Committee Member
Multiple projects were conducted to investigate the potential for developing a breeding program utilizing species of Catalpa Scop. and Chilopsis D. Don. The efficacy of oryzalin was evaluated for inducing polyploidy and restoring fertility in the sterile, intergeneric hybrid xChitalpa tashkentensis Elias & Wisura [Catalpa bignonioides Walt. x Chilopsis linearis (Cav.) Sweet] 'Pink Dawn'. Submerging meristems in 150 μM oryzalin for up to 24 hours was effective at inducing tetraploids and cytochimeras. Pollen from the diploid cultivar was non-viable, but pollen from the polyploid stained and germinated as well as pollen from progenitor taxa. Polyploid xChitalpa were self-compatible yielding tetraploids when self pollinated and triploids when crossed with C. bignonioides, but reciprocal crosses with Chilopsis taxa failed. To increase recovery of triploids, we investigated germination of ovules and embryos at various harvest dates on Schenk and Hildebrandt (SH) basal salts supplemented with various medium components. Germination of triploid [(polyploid xChitalpa) x C. bignonioides] and tetraploid (selfed polyploid xChitalpa) embryos was greatest at 7 weeks after pollination on SH with 20 g•L⁺¹ sucrose and ≥ 1 μM gibberellic acid (GA₃). Triploids [Chilopsis linearis x (polyploid xChitalpa)] germinated < 5%. Additional studies were conducted to screen diverse taxa for resistance to powdery mildew (PM), Erysiphe elevata (Burr.) U. Braun & S. Takam, and catalpa sphinx larvae (CSL), Ceratomia catalpae (Boisduval). Twenty-four taxa from Catalpa (section Catalpa Paclt and Macrocatalpa Grisebach), Chilopsis, and xChitalpa were screened in 2004-05 for susceptibility to PM. Disease incidence and severity were recorded to calculate area under the disease progress curves (AUDPC) for each year. North American Catalpa spp. in sect. Catalpa, Chilopsis, and xChitalpa taxa were all moderate to highly susceptible to PM. Chinese Catalpa spp. in sect. Catalpa and West Indian sect. Macrocatalpa were resistant to PM. Hybrids among North American and Chinese Catalpa spp. in sect. Catalpa varied in susceptibility, indicating inheritance of partial resistance to PM. A no-choice feeding study conducted with CSL in 2005 found no differences in survival or growth of larvae reared on taxa from both sections of Catalpa, Chilopsis, and xChitalpa. Future breeding of xChitalpa can utilize the identified sources of resistance for PM; however, a source of resistance to CSL was not found. Triploids are generally infertile and may be deployed by breeders to limit invasive potential of introduced ornamentals. However, inheritance of ornamental traits can be complex at higher ploidy levels. Inheritance of two mutant foliage types, variegated and purple, was investigated for diploid, triploid and tetraploid tutsan (Hypericum androsaemum L.). Fertility of progeny was evaluated with pollen viability tests, percent fruit set, and germinative capacity of seed from specific crosses. Segregation ratios were determined for diploids in reciprocal di-hybrid F₁, F₂, BC[subscript 1P1], and BC[subscript 1P2] families and selfed F₂s with the parental phenotypes and triploid and tetraploid F₂s. Diploid di-hybrid crosses fit the expected 9:3:3:1 ratio for a single, simple recessive gene for both traits, with no evidence of linkage. A novel phenotype representing a combination of parental phenotypes was recovered. Data from backcrosses and selfing also supported the recessive model. Both traits behaved as expected at the triploid level; however, at the tetraploid level the number of variegated progeny increased, with segregation ratios between random chromosome and random chromatid assortment models. We propose the gene symbol var (variegated) and pl (purple leaf) for the variegated and purple alleles, respectively. Triploid pollen stained moderately well, but pollen germination was low. Triploid plants demonstrated extremely low male fertility and no measurable female fertility (no viable seed production). Research presented herein demonstrates the feasibility of manipulating ploidy levels for breeding desirable ornamental traits including non-invasiveness.