2004 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Environmental horticulture ranks third in the nation in gross agricultural cash receipts with an estimated $49 billion economic impact. This industry, consisting primarily of small family businesses, creates nearly half a million jobs. The introduction of new ornamental plants has long been a driving force behind the nursery and landscape industries in the U.S. Landscape shrubs are increasingly important resources in the enhancement of residential, recreational, public, and industrial plantings. As suburban areas continue to expand, so does the need for landscape plants that are tolerant to a wide range of environmental stresses and that resist diseases and pests without chemical spraying or treatment. To resolve the issue of the continual need for improved landscape plants by the nursery and landscape industries, the objective of this project is to develop landscape trees and shrubs that are pest resistant, tolerant of environmental stresses, are of superior ornamental value, and have compact growth habits. Genera currently under investigation include Cercis, Gaylussacia, Halesia, Ilex, Hamamelis, Lagerstroemia, Malus, Prunus, Stewartia, and Viburnum. Specific germplasm enhancement methods include intra- and interspecific hybridizations followed by backcrossing or selfing; identification of hybrid progeny by molecular markers; evaluation of genetic relationships by molecular markers; and genetic engineering with genes for ornamental or disease resistance traits. This project also involves a cooperative evaluation and stock increase program with the nursery industry to ensure transfer of this research to the end users. This research falls primarily under National Program 301 - Plant, Microbial and Insect Germplasm, Conservation, and Development, as it involves an ongoing process of acquisition, maintenance, evaluation, management, and use of woody ornamental germplasm from diverse genera. Germplasm is characterized with respect to ornamental, disease resistance, hardiness, and/or molecular traits so that it may be used effectively in germplasm enhancement and manipulation. Specifically, aspects of this project serve to safeguard threatened genetic resources, conserve and document genetic resources, expand germplasm evaluations, and transfer this information and resulting germplasm releases to the end user. 2.List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2003) Germinate Cercis chinensis x racemosa F2 hybrids. Create more interspecific F1 hybrids using C. racemosa. Perform interspecific crosses with Prunus maackii, P. campanulata, and other flowering cherry species. Release new flowering cherry cultivar, Prunus `First Lady' Optimize regeneration systems for Prunus incisa. Make initial selections of crapemyrtle hybrids from 2001 pollinations. Release red-flowering crapemyrtle cultivars `Arapaho' and `Cheyenne'. Establish crapemyrtle selection(s) in vitro. Create segregating F2 population by self-pollination of `Betsy Ross' or backcross `Betsy Ross' to S. oblate var. dilatata. Perform controlled hybridizations with Gaylussacia brachycera accessions. Year 2 (FY2004) Obtain genes for genetic transformation of Cercis yunnanensis with antifungal protein. Perform interspecific crosses with Prunus maackii, P. campanulata, and other flowering cherry species. Transform Prunus incisa with marker gene or other contructs as available. Continue to make selections selections of crapemyrtle hybrids from 2001 pollinations. Rogue mature crapemyrtles in back nursery to select for purple and unique (fluorescent or salmon) pink flower color. If in-vitro crapemyrtle cultures are well-established, attempt regeneration using leaf pieces or other explant material. Obtain evaluation feedback information from cooperators for two Syringa hybrids that are currently under evaluation. Germinate Syringa seedlings from 2003 pollinations. Conduct controlled experiments to optimize propagation of Gaylussacia brachycera. Use AFLP markers to determine genetic distances among Gaylussacia brachycera accessions. Use AFLP markers to determine genetic relationships among accessions of American elm (with Townsend CRIS). Year 3 (FY2005) Attempt to transform Cercis yunnanensis with antifungal protein. Use SSR markers to determine genetic relationships among ornamental Prunus taxa. Propagate promising selections of crapemyrtle for further evaluation by cooperators. Evaluate Halesia seedlings from 2001 crosses and plant to field. Attempt to transform crapemyrtle if in-vitro establishment and regeneration works. Begin work on molecular diversity study of Lagerstroemia species (with C. Pounders, Poplarville). Notify cooperators to begin stock increase of two Syringa selections. Use sequence analysis of ChsA gene to determine evolutionary relationships among Gaylussacia brachycera accessions. Complete elm AFLP project from FY2004. Year 4 (FY2006) Send promising crapemyrtle selections to cooperators for evaluation. Complete work on molecular diversity study of Lagerstroemia species (with C. Pounders, Poplarville). Propagate promising Halesia seedling selections, if any. Evaluate Stewartia interspecific hybrid seedlings from 2000, 2001 crosses. Evaluate Syringa seedlings from 2003 pollinations for mildew resistance. Year 5 (FY2007) Evaluate Cercis F2 hybrids from 2003 and earlier for vigor, disease resistance, and possibly flower traits. Begin evaluation and selection for vigor and flower characteristics of Prunus hybrids created in 2003 and 2004. Send promising Halesia selections for cooperative evaluation. Release two new Syringa selections, if not done in FY2006. Evaluate Syringa seedlings from 2003 pollinations for flower traits. 3.Milestones: A. Obtain genes for genetic transformation of Cercis yunnanensis with antifungal protein. This milestone was not completed due to our inability to identify genes that had no restrictive IPR associated with them, or to find genes that other scientists were ready to test on our material. We will continue to contact other scientists, particularly within ARS, to identify genes that may be useful. Perform interspecific crosses with Prunus maackii, P. campanulata, and other flowering cherry species. Transform Prunus incisa with marker gene or other contructs as available. Continue to make selections selections of crapemyrtle hybrids from 2001 pollinations. Rogue mature crapemyrtles in back nursery to select for purple and unique (fluorescent or salmon) pink flower color. If in-vitro crapemyrtle cultures are well-established, attempt regeneration using leaf pieces or other explant material. This milestone was not achieved since the crapemyrtle cultures did not thrive. We will not pursue this milestone further. Obtain evaluation feedback information from cooperators for two Syringa hybrids that are currently under evaluation. Germinate Syringa seedlings from 2003 pollinations. Conduct controlled experiments to optimize propagation of Gaylussacia brachycera. This milestone was only partially achieved. Preliminary experiments were conducted to determine timing of cuttings. We probably will not perform controlled propagation/production experiments due to the retirement of one of the investigators on this project. Use AFLP markers to determine genetic distances among Gaylussacia brachycera accessions. Use AFLP markers to determine genetic relationships among accessions of American elm (with Townsend CRIS). B. Year 3 (FY2005) Transformation of Cercis yunnanensis with antifungal protein or marker genes will be attempted. If successful, this will represent the first published report of Cercis transformation and would offer unique opportunities to develop canker resistant germplasm. Genetic relationships among ornamental Prunus taxa will be determined using SSR markers. This data will help clarify the confusing taxonomy of the ornamental flowering cherries and will be useful in helping to guide decisions about pollinations and germplasm preservation. Promising selections of crapemyrtle will be propagated for further evaluation by cooperators, thus helping ARS to make well-informed decisions as to which selections to release. Halesia seedlings from 2001 crosses will be evaluated and planted to field. These seedlings represent the first of our Halesia pollinations. A molecular diversity study of Lagerstroemia species (with C. Pounders, Poplarville) will begin. This study will help predict crossability among species to aid in broadening the genetic base of cultivated crapemyrtle to include other underutilized species. Cooperators will be notified to begin stock increase of two Syringa selections. These selections will be released one to two years later. Sequence analyais of the ChsA gene will be used to determine evolutionary relationships among Gaylussacia brachycera accessions. Our previous study with AFLP markers could only estimate genetic distances, while this study will provide insight into the evolutionary history of this species. The American elm AFLP project from FY2004 will be completed. This information will be used to identify or verify hybrids and cultivars and to assess how diverse the American elm germplasm is. Year 4 (FY2006) Promising crapemyrtle selections propagated in FY2005 will be sent to cooperators for evaluation, thus helping ARS to make well-informed decisions as to which selections to release. Molecular diversity study of Lagerstroemia species (with C. Pounders, Poplarville) will be completed. Data in the resulting publication will help predict crossability among species to aid in broadening the genetic base of cultivated crapemyrtle to include other underutilized species. Any promising Halesia seedling selections from 2001 will be propagated in order to evaluate them in replicated trials. Stewartia interspecific hybrid seedlings from 2000, 2001 crosses will be evaluated initially for vigor and horticultural attributes. Syringa seedlings from 2003 `Betsy Ross' pollinations will be evaluated for mildew resistance with the aim of capturing more of the desirable traits of Syringa oblata var. dilatata for adaptation to southern climates. Year 5 (FY2007) The Cercis F2 hybrids from 2003 and earlier will be evaluated for vigor, disease resistance, and possibly flower traits. These selections will represent the first advanced generation interspecific hybrids to incorporate C. racemosa. Evaluation and selection for vigor and flower characteristics of Prunus hybrids created in 2003 and 2004 will begin. These hybrids represent new combinations of Prunus species that have not created before. Promising Halesia selections will be sent for cooperative evaluation. These selections will be the first Halesia germplasm to come from any ARS breeding program. Two new Syringa selections will be released (if not done in FY2006). These selections have received favorable evaluations from cooperators, especially for flower color and/or disease resistance. Syringa seedlings from 2003 pollinations will be evaluated for flower traits so that only disease tolerant, superior selections will be propagated for advanced trials. 4.What were the most significant accomplishments this past year? A. The genetic diversity of 24 accessions of Gaylussacia brachycera (box huckleberry) was determined using molecular (AFLP) markers. This plant is a slow-growing, dwarf evergreen groundcover that is native to eight states in the Eastern U.S., with a global conservation status of G3 (rare). The information gained from determining the genetic relationships among accessions can be used to guide decisions regarding conservation, preservation, breeding, and re-introduction of this species. D. In late 2003, Specific Cooperative Agreements were established between ARS and two cooperating institutions, North Carolina State University (NCSU) and Oregon State University (OSU), to develop non-invasive nursery plants. Invasive species are a primary threat to biodiversity on the planet, and some plants that are currently grown as ornamentals are considered to be invasive. The objective of these SCAs is to develop sterile or highly infertile cultivars of important nursery crops so that growers have alternatives to producing invasive or potentially invasive plants. This portion of the report serves to document research conducted under a Specific Cooperative Agreement between ARS and North Carolina State University to develop non-invasive nursery plants. One common approach for developing seedless plants is to create triploids - plants with an extra set of chromosomes. Although triploids typically grow and function normally, they have an inherent reproductive, in that the 3 sets of chromosomes cannot be divided evenly during meiosis, thereby yielding unequal segregation of the chromosomes (aneuploids) or complete meiotic failure. Triploids can occur naturally or can be bred by hybridizing a tetraploid with a diploid. Natural polyploids frequently occur in nature. Tetraploids can be identified in existing populations or induced using mitotic inhibitors. These tetraploids are then used to develop triploids that combine infertility with other desirable traits using traditional breeding approaches. We are currently working on developing sterile triploids of a wide range of important nursery crops. We have confirmed tetraploids of Acer ginnala (amur maple), Acer platanoides (Norway maple), Albizia julibrissan (mimosa), Campsis x tagliabuana (trumpet vine), Hypercium androsaemum (tutsan St. Johnswort), Koelreuteria paniculata (goldenraintree), Ligustrum spp. (privet), Pyrus calleryana (callery pear), and Ulmus parvifolia (lacebark elm). We have confirmed triploids of Pyrus calleryana and we have begun working on Cytissus scoparius (scotch broom), Hedera helix (English ivy), and Miscanthus sinensis (maiden grass). As triploids are developed and begin to flower, we will begin evaluating fertility, seed production, seed viability, and other characteristics of interest. Ultimately, these plants will be released for production by the nursery industry. This portion of the report serves to document research conducted under a Specific Cooperative Agreement between ARS and Oregon State University to develop non-invasive nursery plants. This project focuses on the on the Worplesdon variety of sweetgum (Liquididambar) as a model for genetic engineering of sterility in a shade tree. This year we. 1)imposed a number of flower induction treatments in a greenhouse to determine if flowering could be induced in juvenile plants. Results are still being evaluated. . 2)Tested different transformation parameters and methods to improve the production rate of transgenic plants. . 3)Cocultivated more than 8,000 explants with 10 different gene constructs, including 9 that may induce male and/or female sterility. More than two dozen transgenic plantlets are now being propagated for further testing, and many more are under selection. As these plants mature, we will begin evaluating fertility, seed production, seed viability, and other characteristics of interest. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. In order to provide novel landscape plants to the nursery and landscape industry and the public, two new crapemyrtles `Arapaho' and `Cheyenne', were introduced. Both cultivars have bright red flowers, are easy to propagate, show outstanding tolerance to powdery mildew, and will offer nurseries and the public, especially in the Southeastern U.S., a greater diversity of well-adapted landscape material from which to choose. These plants were released from the Floral and Nursery Plants Research Unit at the U.S. National Arboretum, and were evaluated and propagated by nursery cooperators in locations throughout the U.S prior to their introduction. In July, 2003, release notices and color fact sheets for each of these cultivars were distributed to cooperators, and the cultivars were exhibited at the annual SNA Trade Show in August 2003. Also in 2003, a flowering cherry cultivar `First Lady' was released to the public. This new ornamental cherry is characterized by exceptionally dark pink blooms and an upright, almost columnar growth habit. Although it is difficult to propagate by means of rooted cuttings, it can be grafted or budded. It is currently available through a limited number of wholesale and retail nurseries. We have established a molecular marker system using automated sequencing equipment with fluorescently labeled primers as an efficient and reliable system to fingerprint woody plant germplasm to aid in genetic diversity estimates and to identify hybrid progeny. These markers have been used successfully in hemlock, crapemyrtle, hamelia, and box huckleberry. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Two new crapemyrtle cultivars (`Arapaho' and `Cheyenne') and one flowering cherry cultivar (`First Lady') were introduced to the nursery industry in 2003. The crapemyrtles are currently in production at the wholesale level, and are expected to be available at the retail level (i.e. to the public) as early as 2005. The flowering cherry is available in limited quantities at the retail level. There are no known constraints on the adoption and durability of these products. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. Lagerstroemia `Arapaho', (p.32), 'Lagerstroemia `Cheyenne', (p.34), 'Prunus `First Lady', (p.36). American Nurseryman 198(12). December 15, 2003. 'Beyond the Tidal Basin, there's a cherry jubilee', Adrian Higgins, Washington Post, 4/8/2004, Home Section, p. H1 (Discusses flowering cherry collection at the Arboretum). 'Enjoy the four-season beauty of crape myrtle', Dora Galitzki, Martha Stewart Living, July 2004, pp. 56-59. (Discusses the breeding program at the Arboretum). Presented a poster about the Arboretum's ornamental cherry breeding program at the Rosaceae Genomics Conference, Clemson, SC. (5/2004). Presented invited talk about the Arboretum's shrub breeding program at the Woody Plant Conference in Swarthmore, PA (7/2004). Presented posters at about Gaylussacia brachycera work, crapemyrtle breeding, and Hamelia project at ASHS meetings (10/03 and 7/04). Review Publications Cheong, E., Pooler, M.R. 2003. Micropropagation of Chinese Redbud (Cercis Yunnanensis)Through Axillary Bud Breading and Induction of Adventitious Shoots From Leaf Pieces. In Vitro Cell Dev Biol - Plant. 2003. v:39, pp:455-458. Cheong, E., Pooler, M.R. 2004. Factors Affecting Somatic Embryogenesis in Prunus Incisa cv. February Pink. Plant Cell Reports. 2004. v:22, pp:810-815.